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Sample records for porphobilinogen deaminase pbgd

  1. Crystallization and preliminary X-ray characterization of the tetrapyrrole-biosynthetic enzyme porphobilinogen deaminase from Bacillus megaterium

    International Nuclear Information System (INIS)

    Azim, N.; Deery, E.; Warren, M. J.; Erskine, P.; Cooper, J. B.; Wood, S. P.; Akhtar, M.

    2013-01-01

    The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step in the biosynthesis of tetrapyrroles in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. PBGD from B. megaterium was expressed and the enzyme was crystallized in a form which diffracts synchrotron radiation to high resolution. The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Expression in Escherichia coli of a His-tagged form of Bacillus megaterium PBGD permitted the crystallization and preliminary X-ray analysis of the enzyme from this species at high resolution

  2. Glucose metabolism during fasting is altered in experimental porphobilinogen deaminase deficiency.

    Science.gov (United States)

    Collantes, María; Serrano-Mendioroz, Irantzu; Benito, Marina; Molinet-Dronda, Francisco; Delgado, Mercedes; Vinaixa, María; Sampedro, Ana; Enríquez de Salamanca, Rafael; Prieto, Elena; Pozo, Miguel A; Peñuelas, Iván; Corrales, Fernando J; Barajas, Miguel; Fontanellas, Antonio

    2016-04-01

    Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria, AIP) is characterized by neurovisceral attacks when hepatic heme synthesis is activated by endogenous or environmental factors including fasting. While the molecular mechanisms underlying the nutritional regulation of hepatic heme synthesis have been described, glucose homeostasis during fasting is poorly understood in porphyria. Our study aimed to analyse glucose homeostasis and hepatic carbohydrate metabolism during fasting in PBGD-deficient mice. To determine the contribution of hepatic PBGD deficiency to carbohydrate metabolism, AIP mice injected with a PBGD-liver gene delivery vector were included. After a 14 h fasting period, serum and liver metabolomics analyses showed that wild-type mice stimulated hepatic glycogen degradation to maintain glucose homeostasis while AIP livers activated gluconeogenesis and ketogenesis due to their inability to use stored glycogen. The serum of fasted AIP mice showed increased concentrations of insulin and reduced glucagon levels. Specific over-expression of the PBGD protein in the liver tended to normalize circulating insulin and glucagon levels, stimulated hepatic glycogen catabolism and blocked ketone body production. Reduced glucose uptake was observed in the primary somatosensorial brain cortex of fasted AIP mice, which could be reversed by PBGD-liver gene delivery. In conclusion, AIP mice showed a different response to fasting as measured by altered carbohydrate metabolism in the liver and modified glucose consumption in the brain cortex. Glucose homeostasis in fasted AIP mice was efficiently normalized after restoration of PBGD gene expression in the liver. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  3. Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution

    International Nuclear Information System (INIS)

    Azim, N.; Deery, E.; Warren, M. J.; Wolfenden, B. A. A.; Erskine, P.; Cooper, J. B.; Coker, A.; Wood, S. P.; Akhtar, M.

    2014-01-01

    The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step in the biosynthesis of tetrapyrroles in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. Two near-atomic resolution structures of PBGD from B. megaterium are reported that demonstrate the time-dependent accumulation of partially oxidized forms of the cofactor, including one that possesses a tetrahedral C atom in the terminal pyrrole ring. The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor, which is covalently linked by a thioether bridge to an invariant cysteine residue (Cys241 in the Bacillus megaterium enzyme). The cofactor is extended during the reaction by the sequential addition of the four substrate molecules, which are released as a linear tetrapyrrole product. Expression in Escherichia coli of a His-tagged form of B. megaterium PBGD has permitted the X-ray analysis of the enzyme from this species at high resolution, showing that the cofactor becomes progressively oxidized to the dipyrromethene and dipyrromethanone forms. In previously solved PBGD structures, the oxidized cofactor is in the dipyromethenone form, in which both pyrrole rings are approximately coplanar. In contrast, the oxidized cofactor in the B. megaterium enzyme appears to be in the dipyrromethanone form, in which the C atom at the bridging α-position of the outer pyrrole ring is very clearly in a tetrahedral configuration. It is suggested that the pink colour of the freshly purified protein is owing to the presence of the dipyrromethene form of the cofactor which, in the structure reported here, adopts the same conformation as the fully reduced dipyrromethane form

  4. Activity and immunohistochemical localization of porphobilinogen deaminase in rat tissues

    DEFF Research Database (Denmark)

    Jørgensen, P E; Erlandsen, E J; Poulsen, Steen Seier

    2000-01-01

    the activity and the immunohistochemical localization of PBGD in the following tissues of wistar female rats: brain, heart, submandibular gland, liver, kidney, pancreas, ovary, stomach, duodenum, jejunum, ileum, colon and musculature. The PBGD activity varied considerably among the tissues. It was highest...

  5. Biochemical Characterization of Porphobilinogen Deaminase–Deficient Mice During Phenobarbital Induction of Heme Synthesis and the Effect of Enzyme Replacement

    Science.gov (United States)

    Johansson, Annika; Möller, Christer; Fogh, Jens; Harper, Pauline

    2003-01-01

    Acute intermittent porphyria (AIP) is a genetic disorder caused by a deficiency of porphobilinogen deaminase (PBGD), the 3rd enzyme in heme synthesis. It is clinically characterized by acute attacks of neuropsychiatric symptoms and biochemically by increased urinary excretion of the porphyrin precursors porphobilinogen (PBG) and 5-aminolevulinic acid (ALA). A mouse model that is partially deficient in PBGD and biochemically mimics AIP after induction of the hepatic ALA synthase by phenobarbital was used in this study to identify the site of formation of the presumably toxic porphyrin precursors and study the effect of enzyme-replacement therapy by using recombinant human PBGD (rhPBGD). After 4 d of phenobarbital administration, high levels of PBG and ALA were found in liver, kidney, plasma, and urine of the PBGD-deficient mice. The administration of rhPBGD intravenously or subcutaneously after a 4-d phenobarbital induction was shown to lower the PBG level in plasma in a dose-dependent manner with maximal effect seen after 30 min and 2 h, respectively. Injection of rhPBGD subcutaneously twice daily during a 4-d phenobarbital induction reduced urinary PBG excretion to 25% of the levels found in PBGD-deficient mice administered with only phenobarbital. This study points to the liver as the main producer of PBG and ALA in the phenobarbital-induced PBGD-deficient mice and demonstrates efficient removal of accumulated PBG in plasma and urine by enzyme-replacement therapy. PMID:15208740

  6. Spectroscopic evidence for a porphobilinogen deaminase-tetrapyrrole complex that is an intermediate in the biosynthesis of uroporphyrinogen III

    International Nuclear Information System (INIS)

    Rose, S.; Frydman, R.B.; de los Santos, C.; Sburlati, A.; Valasinas, A.; Frydman, B.

    1988-01-01

    Incubation of porphobilinogen (PBG) with PBG deaminase from Rhodopseudomonas sphaeroides in carbonate buffer to total PBG consumption resulted in low yields of uroporphyrinogen I(uro'gen I). In the reaction mixture a pyrrylmethane accumulated, which at longer incubation periods was transformed into uro'gen I. The accumulated pyrrylmethane gave an Ehrlich reaction which was different from that of a 2-(aminomethyl)dipyrrylmethane or 2-(aminomethyl)tripyrrane. It resembled that of a bilane but was different from that of a 2-(hydroxymethyl)bilane. The 13 C NMR spectra of incubations carried out with [11- 13 C]PBG indicated that the pyrrylmethane was a tetrapyrrole with methylene resonances at 22.35-22.50 ppm. It was loosely bound to the deaminase, and when separated from the enzyme by gel filtration or gel electrophoresis, it immediately cyclized to uro'gen I. No enzyme-bound methylene could be detected by its chemical shift, suggesting that its line width must be very broad. When uro'gen III-cosynthase was added to the deaminase-tetrapyrrole complex, uro'gen III was formed at the expense of the latter in about 75% yield. A protonated uro'gen I structure for this intermediate was ruled out by incubations using [2,11- 13 C]PBG. Uro'gen III formation from 2-(hydroxymethyl)bilane (HMB) and from the deaminase-tetrapyrrole intermediate was compared by using deaminase-cosynthase and cosynthase from several sources. It was found that while the HMB inhibited uro'gen III formation at higher concentrations and longer incubation times, uro'gen III formation from the complex did not decrease with time

  7. The Porphobilinogen Conundrum in Prebiotic Routes to Tetrapyrrole Macrocycles

    Science.gov (United States)

    Taniguchi, Masahiko; Ptaszek, Marcin; Chandrashaker, Vanampally; Lindsey, Jonathan S.

    2017-03-01

    Attempts to develop a credible prebiotic route to tetrapyrroles have relied on enzyme-free recapitulation of the extant biosynthesis, but this process has foundered from the inability to form the pyrrole porphobilinogen ( PBG) in good yield by self-condensation of the precursor δ-aminolevulinic acid ( ALA). PBG undergoes robust oligomerization in aqueous solution to give uroporphyrinogen (4 isomers) in good yield. ALA, PBG, and uroporphyrinogen III are universal precursors to all known tetrapyrrole macrocycles. The enzymic formation of PBG entails carbon-carbon bond formation between the less stable enolate/enamine of one ALA molecule (3-position) and the carbonyl/imine (4-position) of the second ALA molecule; without enzymes, the first ALA reacts at the more stable enolate/enamine (5-position) and gives the pyrrole pseudo-PBG. pseudo-PBG cannot self-condense, yet has one open α-pyrrole position and is proposed to be a terminator of oligopyrromethane chain-growth from PBG. Here, 23 analogues of ALA have been subjected to density functional theoretical (DFT) calculations, but no motif has been identified that directs reaction at the 3-position. Deuteriation experiments suggested 5-(phosphonooxy)levulinic acid would react preferentially at the 3- versus 5-position, but a hybrid condensation with ALA gave no observable uroporphyrin. The results suggest efforts toward a biomimetic, enzyme-free route to tetrapyrroles from ALA should turn away from structure-directed reactions and focus on catalysts that orient the two aminoketones to form PBG in a kinetically controlled process, thereby avoiding formation of pseudo-PBG.

  8. Regulation of porphyrin synthesis and photodynamic therapy in heavy metal intoxication.

    Science.gov (United States)

    Grinblat, Borislava; Pour, Nir; Malik, Zvi

    2006-01-01

    Protoporphyrin IX (PpIX) synthesis by malignant cells is successfully exploited for photodynamic therapy (PDT) following administration of 5-aminolevulinic acid (ALA) and light irradiation. The influence of two environmental heavy metal poisons, lead and gallium, on PpIX-synthesis and ALA-PDT was studied in two neu-ronal cell lines, SH-SY5Y neuroblastoma and PC12 pheochromocytoma. The heavy metal intoxication affected two of the heme-synthesis enzymes, ALA-dehydratase (ALAD) and porphobilinogen deaminase (PBGD). The present results show that lead poisoning significantly decreased the PBGD cellular level and inhibited its enzymatic activity, whereas the effects of gallium were less prominent. Although, the protein levels were reduced, the mRNA levels of PBGD remained unchanged during metal intoxication. These findings show additional inhibitory activity of lead on top of its classical effect on ALAD. Proteasome activity was enhanced during lead treatment, as measured by the AMC fluorigenic proteasome assay. The reduction in PBGD levels was not a consequence of PBGD mRNA reduced synthesis, which remained unchanged as shown by RT-PCR analysis. As a result of the lead poisoning, marked alterations in the cell cycle were observed, including a decreased G1 phase and an increased number of S phase cells. The efficacy of ALA-PDT was reduced in correlation with decreased activities of the enzymes during lead intoxication. We may conclude that lead poisoning adversely affects the outcome of ALA photodynamic therapy of cancer.

  9. wALADin benzimidazoles differentially modulate the function of porphobilinogen synthase orthologs.

    Science.gov (United States)

    Lentz, Christian S; Halls, Victoria S; Hannam, Jeffrey S; Strassel, Silke; Lawrence, Sarah H; Jaffe, Eileen K; Famulok, Michael; Hoerauf, Achim; Pfarr, Kenneth M

    2014-03-27

    The heme biosynthesis enzyme porphobilinogen synthase (PBGS) is a potential drug target in several human pathogens. wALADin1 benzimidazoles have emerged as species-selective PBGS inhibitors against Wolbachia endobacteria of filarial worms. In the present study, we have systematically tested wALADins against PBGS orthologs from bacteria, protozoa, metazoa, and plants to elucidate the inhibitory spectrum. However, the effect of wALADin1 on different PBGS orthologs was not limited to inhibition: several orthologs were stimulated by wALADin1; others remained unaffected. We demonstrate that wALADins allosterically modulate the PBGS homooligomeric equilibrium with inhibition mediated by favoring low-activity oligomers, while 5-aminolevulinic acid, Mg(2+), or K(+) stabilized high-activity oligomers. Pseudomonas aeruginosa PBGS could be inhibited or stimulated by wALADin1 depending on these factors and pH. We have defined the wALADin chemotypes responsible for either inhibition or stimulation, facilitating the design of tailored PBGS modulators for potential application as antimicrobial agents, herbicides, or drugs for porphyric disorders.

  10. Adenosine deaminase activity of erythrocytes in hyperuricemia

    International Nuclear Information System (INIS)

    Krueger, W.; Richter, V.; Beenken, O.; Weinhold, D.; Hirschberg, K.; Rotzsch, W.; Akademie der Wissenschaften der DDR, Leipzig. Zentralinstitut fuer Isotopen- und Strahlenforschung)

    1982-01-01

    Erythrocytic adenosine deaminase (ADA) activity was determined in 55 patients with primary hyperuricemia and in 37 healthy control persons. Unlike the controls, the ADA activity in the patient group showed a two-peak response. Hyperuricemia patients with high ADA activity also exhibited increased uric acid excretion and elevated 15 N incorporation into uric acid. High activity values of erythrocytic ADA can be interpreted as an uric acid overproduction, giving hints for a therapeutic plan. (author)

  11. A facile synthesis of δ-aminolevulinic acid (ALA) regio-selectively labeled with 13C and direct observation of enzymatic transformation from ALA to porphobilinogen (PBG)

    International Nuclear Information System (INIS)

    Kurumaya, Katsuyuki; Okazaki, Takeo; Seido, Nobuo; Akasaka, Yuzuru; Kawajiri, Yoshiki; Kajiwara, Masahiro; Kondo, Masao

    1989-01-01

    δ-Aminolevulinic acid (ALA), labeled with 13 C at position 1, 2, 3, 4, or 5, was synthesized from 13 C-labeled glycine, Meldrum's acid, or bromoacetate. The latter compounds were prepared from 13 C-sodium acetate or 13 C-acetic acid. Enzymatic transformation from ALA to porphobilinogen (PBG) was directly observed by 13 C-NMR. (author)

  12. Primary adenosine monophosphate (AMP) deaminase deficiency in a hypotonic infant.

    Science.gov (United States)

    Castro-Gago, Manuel; Gómez-Lado, Carmen; Pérez-Gay, Laura; Eirís-Puñal, Jesús; Martínez, Elena Pintos; García-Consuegra, Inés; Martín, Miguel Angel

    2011-06-01

    The spectrum of the adenosine monophosphate (AMP) deaminase deficiency ranges from asymptomatic carriers to patients who manifest exercise-induced muscle pain, occasionally rhabdomyolysis, and idiopathic hyperCKemia. However, previous to the introduction of molecular techniques, rare cases with congenital weakness and hypotonia have also been reported. We report a 6-month-old girl with the association of congenital muscle weakness and hypotonia, muscle deficiency of adenosine monophosphate deaminase, and the homozygous C to T mutation at nucleotide 34 of the adenosine monophosphate deaminase-1 gene. This observation indicates the possible existence of a primary adenosine monophosphate deaminase deficiency manifested by congenital muscle weakness and hypotonia.

  13. Renal failure affects the enzymatic activities of the three first steps in hepatic heme biosynthesis in the acute intermittent porphyria mouse.

    Directory of Open Access Journals (Sweden)

    Carmen Unzu

    Full Text Available Chronic kidney disease is a long-term complication in acute intermittent porphyria (AIP. The pathophysiological significance of hepatic overproduction of the porphyrin precursors aminolevulinate acid (ALA and porphobilinogen (PBG in chronic kidney disease is unclear. We have investigated the effect of repetitive acute attacks on renal function and the effect of total or five-sixth nephrectomy causing renal insufficiency on hepatic heme synthesis in the porphobilinogen deaminase (PBGD-deficient (AIP mouse. Phenobarbital challenge in the AIP-mice increased urinary porphyrin precursor excretion. Successive attacks throughout 14 weeks led to minor renal lesions with no impact on renal function. In the liver of wild type and AIP mice, 5/6 nephrectomy enhanced transcription of the first and rate-limiting ALA synthase. As a consequence, urinary PBG excretion increased in AIP mice. The PBG/ALA ratio increased from 1 in sham operated AIP animals to over 5 (males and over 13 (females in the 5/6 nephrectomized mice. Total nephrectomy caused a rapid decrease in PBGD activity without changes in enzyme protein level in the AIP mice but not in the wild type animals. In conclusion, high concentration of porphyrin precursors had little impact on renal function. However, progressive renal insufficiency aggravates porphyria attacks and increases the PBG/ALA ratio, which should be considered a warning sign for potentially life-threatening impairment in AIP patients with signs of renal failure.

  14. In Vitro Optimization of Enzymes Involved in Precorrin-2 Synthesis Using Response Surface Methodology.

    Science.gov (United States)

    Fang, Huan; Dong, Huina; Cai, Tao; Zheng, Ping; Li, Haixing; Zhang, Dawei; Sun, Jibin

    2016-01-01

    In order to maximize the production of biologically-derived chemicals, kinetic analyses are first necessary for predicting the role of enzyme components and coordinating enzymes in the same reaction system. Precorrin-2 is a key precursor of cobalamin and siroheme synthesis. In this study, we sought to optimize the concentrations of several molecules involved in precorrin-2 synthesis in vitro: porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), and S-adenosyl-l-methionine-dependent urogen III methyltransferase (SUMT). Response surface methodology was applied to develop a kinetic model designed to maximize precorrin-2 productivity. The optimal molar ratios of PBGS, PBGD, UROS, and SUMT were found to be approximately 1:7:7:34, respectively. Maximum precorrin-2 production was achieved at 0.1966 ± 0.0028 μM/min, agreeing with the kinetic model's predicted value of 0.1950 μM/min. The optimal concentrations of the cofactor S-adenosyl-L-methionine (SAM) and substrate 5-aminolevulinic acid (ALA) were also determined to be 200 μM and 5 mM, respectively, in a tandem-enzyme assay. By optimizing the relative concentrations of these enzymes, we were able to minimize the effects of substrate inhibition and feedback inhibition by S-adenosylhomocysteine on SUMT and thereby increase the production of precorrin-2 by approximately five-fold. These results demonstrate the effectiveness of kinetic modeling via response surface methodology for maximizing the production of biologically-derived chemicals.

  15. Helper-dependent adenovirus achieve more efficient and persistent liver transgene expression in non-human primates under immunosuppression.

    Science.gov (United States)

    Unzu, C; Melero, I; Hervás-Stubbs, S; Sampedro, A; Mancheño, U; Morales-Kastresana, A; Serrano-Mendioroz, I; de Salamanca, R E; Benito, A; Fontanellas, A

    2015-11-01

    Helper-dependent adenoviral (HDA) vectors constitute excellent gene therapy tools for metabolic liver diseases. We have previously shown that an HDA vector encoding human porphobilinogen deaminase (PBGD) corrects acute intermittent porphyria mice. Now, six non-human primates were injected in the left hepatic lobe with the PBGD-encoding HDA vector to study levels and persistence of transgene expression. Intrahepatic administration of 5 × 10(12) viral particles kg(-1) (10(10) infective units kg(-1)) of HDA only resulted in transient (≈14 weeks) transgene expression in one out of three individuals. In contrast, a more prolonged 90-day immunosuppressive regimen (tacrolimus, mycophenolate, rituximab and steroids) extended meaningful transgene expression for over 76 weeks in two out of two cases. Transgene expression under immunosuppression (IS) reached maximum levels 6 weeks after HDA administration and gradually declined reaching a stable plateau within the therapeutic range for acute porphyria. The non-injected liver lobes also expressed the transgene because of vector circulation. IS controlled anticapsid T-cell responses and decreased the induction of neutralizing antibodies. Re-administration of HDA-hPBGD at week +78 achieved therapeutically meaningful transgene expression only in those animals receiving IS again at the time of this second vector exposure. Overall, immunity against adenoviral capsids poses serious hurdles for long-term HDA-mediated liver transduction, which can be partially circumvented by pharmacological IS.

  16. Myoadenylate deaminase deficiency, hypertrophic cardiomyopathy and gigantism syndrome.

    Science.gov (United States)

    Skyllouriotis, M L; Marx, M; Bittner, R E; Skyllouriotis, P; Gross, M; Wimmer, M

    1997-07-01

    We report a 20-year-old man with gigantism syndrome, hypertrophic cardiomyopathy, muscle weakness, exercise intolerance, and severe psychomotor retardation since childhood. Histochemical and biochemical analysis of skeletal muscle biopsy revealed myoadenylate deaminase deficiency; molecular genetic analysis confirmed the diagnosis of primary (inherited) myoadenylate deaminase deficiency. Plasma, urine, and muscle carnitine concentrations were reduced. L-Carnitine treatment led to gradual improvement in exercise tolerance and cognitive performance; plasma and tissue carnitine levels returned to normal, and echocardiographic evidence of left ventricular hypertrophy disappeared. The combination of inherited myoadenylate deaminase deficiency, gigantism syndrome and carnitine deficiency has not previously been described.

  17. A facile synthesis of. delta. -aminolevulinic acid (ALA) regio-selectively labeled with sup 13 C and direct observation of enzymatic transformation from ALA to porphobilinogen (PBG)

    Energy Technology Data Exchange (ETDEWEB)

    Kurumaya, Katsuyuki; Okazaki, Takeo; Seido, Nobuo; Akasaka, Yuzuru; Kawajiri, Yoshiki; Kajiwara, Masahiro (Meiji College of Pharmacy, Tokyo (Japan)); Kondo, Masao (Institute of Public Health, Tokyo (Japan))

    1989-02-01

    {delta}-Aminolevulinic acid (ALA), labeled with {sup 13}C at position 1, 2, 3, 4, or 5, was synthesized from {sup 13}C-labeled glycine, Meldrum's acid, or bromoacetate. The latter compounds were prepared from {sup 13}C-sodium acetate or {sup 13}C-acetic acid. Enzymatic transformation from ALA to porphobilinogen (PBG) was directly observed by {sup 13}C-NMR. (author).

  18. Discovery of a Bacterial 5-Methylcytosine Deaminase

    Science.gov (United States)

    2015-01-01

    5-Methylcytosine is found in all domains of life, but the bacterial cytosine deaminase from Escherichia coli (CodA) will not accept 5-methylcytosine as a substrate. Since significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes, this compound must eventually be catabolized and the fragments recycled by enzymes that have yet to be identified. We therefore initiated a comprehensive phylogenetic screen for enzymes that may be capable of deaminating 5-methylcytosine to thymine. From a systematic analysis of sequence homologues of CodA from thousands of bacterial species, we identified putative cytosine deaminases where a “discriminating” residue in the active site, corresponding to Asp-314 in CodA from E. coli, was no longer conserved. Representative examples from Klebsiella pneumoniae (locus tag: Kpn00632), Rhodobacter sphaeroides (locus tag: Rsp0341), and Corynebacterium glutamicum (locus tag: NCgl0075) were demonstrated to efficiently deaminate 5-methylcytosine to thymine with values of kcat/Km of 1.4 × 105, 2.9 × 104, and 1.1 × 103 M–1 s–1, respectively. These three enzymes also catalyze the deamination of 5-fluorocytosine to 5-fluorouracil with values of kcat/Km of 1.2 × 105, 6.8 × 104, and 2.0 × 102 M–1 s–1, respectively. The three-dimensional structure of Kpn00632 was determined by X-ray diffraction methods with 5-methylcytosine (PDB id: 4R85), 5-fluorocytosine (PDB id: 4R88), and phosphonocytosine (PDB id: 4R7W) bound in the active site. When thymine auxotrophs of E. coli express these enzymes, they are capable of growth in media lacking thymine when supplemented with 5-methylcytosine. Expression of these enzymes in E. coli is toxic in the presence of 5-fluorocytosine, due to the efficient transformation to 5-fluorouracil. PMID:25384249

  19. Adenosine-deaminase (ADA activity in Psoriasis (A Preliminary Study

    Directory of Open Access Journals (Sweden)

    S D Chaudhry

    1988-01-01

    Full Text Available Study of adenosine-deaminase activity ′in 23 patients hav-mg psoriasis compared with an equal number of healthy controls revealed significantly high ADA-activity in the psotiatic patients.

  20. The catalase activity of diiron adenine deaminase

    Energy Technology Data Exchange (ETDEWEB)

    Kamat S. S.; Swaminathan S.; Holmes-Hampton, G. P.; Bagaria, A.; Kumaran, D.; Tichy, S. E.; Gheyi, T.; Zheng, X.; Bain, K.; Groshong, C.; Emtage, S.; Sauder, J. M.; Burley, S. K.; Lindahl, P. A.; Raushel, F. M.

    2011-12-01

    Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn{sup 2+} before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO{sub 4}. Inductively coupled plasma mass spectrometry and Moessbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe{sup II}/Fe{sup II}]-ADE catalyzed the conversion of H{sub 2}O{sub 2} to O{sub 2} and H{sub 2}O. The values of k{sub cat} and k{sub cat}/K{sub m} for the catalase activity are 200 s{sup -1} and 2.4 x 10{sup 4} M{sup -1} s{sup -1}, respectively. [Fe{sup II}/Fe{sup II}]-ADE underwent more than 100 turnovers with H{sub 2}O{sub 2} before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g{sub ave} = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H{sub 2}O{sub 2} by [Fe{sup II}/Fe{sup II}]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.

  1. Characterization of pterin deaminase from Mucor indicus MTCC 3513

    Science.gov (United States)

    Thandeeswaran, M.; Karthika, P.; Mahendran, R.; Palaniswamy, M.; Angayarkanni, J.

    2018-03-01

    Pterin deaminase is an amidohydrolase enzyme which hydrolyses pteridines to produce lumazine derivatives and ammonia. Even though the enzyme was shown as early as 1959 for its anticancer efficacy there was a long gap in the communique after that which was in 2013. In our study we have chosen Mucor indicus MTCC 3513 which was a promising strain for production of different industrial products.The pterin deaminase enzyme was harvested and extracellular from M. indicus. The extracellular sample was partially purified by using ethanol precipitation and ion exchange column (Hi-Trap QFF) in Fast Protein Liquid Chromatography. The molecular weight of the purified pterin deaminase enzyme was apparently determined by SDS-PAGE. The purified enzyme was further biochemically characterized. Molecular docking studies with the predicted sequence showed higher binding affinity towards folic acid interaction. The structure of this protein may open the windows for new drug targets for cancer therapy.

  2. Plasma Adenosine Deaminase Enzyme Reduces with Treatment of ...

    African Journals Online (AJOL)

    olayemitoyin

    Plasma Adenosine Deaminase Enzyme Reduces with Treatment of Pulmonary Tuberculosis in Nigerian Patients: Indication for. Diagnosis and Treatment Monitoring. Ige O.a, Edem V.F.b and Arinola O.G.b,*. aDepartment of Medicine, University of Ibadan, Ibadan, Nigeria b Department of Chemical Pathology,. University of ...

  3. Contributory role of adenosine deaminase in metabolic syndrome ...

    African Journals Online (AJOL)

    Adenosine deaminase (ADA) is an enzyme of purine metabolism commonly associated with severe combined immunodeficiency disease and believed to modulate bioactivity of insulin. Its contributory role in patients with metabolic syndrome (having features such as obesity, insulin resistance, fasting hyperglycaemia, lipid ...

  4. Contributory role of adenosine deaminase in metabolic syndrome

    African Journals Online (AJOL)

    olayemitoyin

    Cytokine balance was also changed in diet induced obese mice (Mito and Hiyosin, 2002). Although Mito et al (2000) ... immunity in man (Sadasivudu et al, 1982) adenosine deaminase modulates cell growth (Lelieuve et al, .... Colgiuri, S. (2002) The Carnivore Connection- evolution aspect of insulin resistance. Eur. J. Clin.

  5. Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

    Energy Technology Data Exchange (ETDEWEB)

    S Kamat; A Bagaria; D Kumaran; G Holmes-Hampton; H Fan; A Sali; J Sauder; S Burley; P Lindahl; et. al.

    2011-12-31

    Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k{sub cat} and k{sub cat}/K{sub m} values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction

  6. Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

    Energy Technology Data Exchange (ETDEWEB)

    Kamat, S.S.; Swaminathan, S.; Bagaria, A.; Kumaran, D.; Holmes-Hampton, G. P.; Fan, H.; Sali, A.; Sauder, J. M.; Burley, S. K.; Lindahl, P. A.; Raushel, F. M.

    2011-03-22

    Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with kcat and kcat/Km values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism and the

  7. Beneficial role of spermidine in chlorophyll metabolism and D1 protein content in tomato seedlings under salinity-alkalinity stress.

    Science.gov (United States)

    Hu, Lipan; Xiang, Lixia; Li, Shuting; Zou, Zhirong; Hu, Xiao-Hui

    2016-04-01

    Polyamines are important in protecting plants against various environmental stresses, including protection against photodamage to the photosynthetic apparatus. The molecular mechanism of this latter effect is not completely understood. Here, we have investigated the effects of salinity-alkalinity stress and spermidine (Spd) on tomato seedlings at both physiological and transcriptional levels. Salinity-alkalinity stress decreased leaf area, net photosynthetic rate, maximum net photosynthetic rate, light saturation point, apparent quantum efficiency, total chlorophyll, chlorophyll a and chlorophyll a:chlorophyll b relative to the control. The amount of D1 protein, an important component of photosystem II, was reduced compared with the control, as was the expression of psbA, which codes for D1. Expression of the chlorophyll biosynthesis gene porphobilinogen deaminase (PBGD) was reduced following salinity-alkalinity stress, whereas the expression of Chlase, which codes for chlorophyllase, was increased. These negative physiological effects of salinity-alkalinity stress were alleviated by exogenous Spd. Expression of PBGD and psbA were enhanced, whereas the expression of Chlase was reduced, when exogenous Spd was included in the stress treatment compared with when it was not. The protective effect of Spd on chlorophyll and D1 protein content during stress may maintain the photosynthetic apparatus, permitting continued photosynthesis and growth of tomato seedlings (Solanum lycopersicum cv. Jinpengchaoguan) under salinity-alkalinity stress. © 2015 Scandinavian Plant Physiology Society.

  8. AID/APOBEC cytosine deaminase induces genome-wide kataegis

    Directory of Open Access Journals (Sweden)

    Lada Artem G

    2012-12-01

    Full Text Available Abstract Clusters of localized hypermutation in human breast cancer genomes, named “kataegis” (from the Greek for thunderstorm, are hypothesized to result from multiple cytosine deaminations catalyzed by AID/APOBEC proteins. However, a direct link between APOBECs and kataegis is still lacking. We have sequenced the genomes of yeast mutants induced in diploids by expression of the gene for PmCDA1, a hypermutagenic deaminase from sea lamprey. Analysis of the distribution of 5,138 induced mutations revealed localized clusters very similar to those found in tumors. Our data provide evidence that unleashed cytosine deaminase activity is an evolutionary conserved, prominent source of genome-wide kataegis events. Reviewers This article was reviewed by: Professor Sandor Pongor, Professor Shamil R. Sunyaev, and Dr Vladimir Kuznetsov.

  9. Enzymatic conformational fluctuations along the reaction coordinate of cytidine deaminase

    OpenAIRE

    Noonan, Ryan C.; Carter, Charles W.; Bagdassarian, Carey K.

    2002-01-01

    Analysis of the crystal structures for cytidine deaminase complexed with substrate analog 3-deazacytidine, transition-state analog zebularine 3,4-hydrate, and product uridine establishes significant changes in the magnitude of atomic-scale fluctuations along the (approximate) reaction coordinate of this enzyme. Differences in fluctuations between the substrate analog complex, transition-state analog complex, and product complex are monitored via changes in corresponding crystallographic tempe...

  10. The emerging role of adenosine deaminases in insects

    Czech Academy of Sciences Publication Activity Database

    Doleželová, Eva; Žurovec, Michal; Doležal, T.; Šimek, Petr; Bryant, P. J.

    2005-01-01

    Roč. 35, č. 5 (2005), s. 381-389 ISSN 0965-1748 R&D Projects: GA ČR(CZ) GA204/04/1205; GA AV ČR(CZ) IAA5007107 Grant - others:United States National Science Foundation(US) 440860-21565 Institutional research plan: CEZ:AV0Z50070508 Keywords : adenosine deaminase * ADA * growth factor Subject RIV: ED - Physiology Impact factor: 2.733, year: 2005

  11. Adenosine deaminase organic effect in normal and abnormal cerebrospinal fluid

    International Nuclear Information System (INIS)

    Hamad, A.M.; Samarai, M.A.

    2007-01-01

    To study the effect of the organic substances on adenosine deaminase (ADA) activity in normal and abnormal cerebrospinal fluid (CSF). Various concentrations of 2-mercaptopurine, Ame-tycine, Adenosine analogues (Guanine, Thymine) and ATP were tested to see their effect on ADA activity in normal and abnormal CSF. ADA activity in normal and abnormal CSF was remarkably decreased with the increasing of concentrations of substances tested. These effects may have important therapeutic implications. (author)

  12. ADENOSINE DEAMINASE ACTIVITY IN TYPE 2 DIABETES MELLITUS

    Directory of Open Access Journals (Sweden)

    Farija Peruvankuzhiyil

    2017-01-01

    Full Text Available BACKGROUND Altered blood levels of adenosine deaminase may help in predicting immunological dysfunction in diabetic individuals. But very few studies exist on ADA activity in type 2 diabetes mellitus. Aim of this study is to compare serum adenosine deaminase activity in type 2 diabetic patients with non-diabetic control. MATERIALS AND METHODS A comparative study design was used in data collection process. The study was conducted in 40 type 2 diabetes mellitus patients attending diabetic clinic or admitted in the medicine ward for metabolic control of diabetes in medical college, Calicut from January 2011 to January 2012. The adenosine deaminase (ADA level in the serum is measured by endpoint method in these patients. The results were expressed as mean and standard deviation. The statistical significance of the differences between the values was assessed by ANOVA. RESULTS Among 40 diabetic patients, mean ADA level in the serum is 38.56, SD±6.72 (min 30, max 53. Mean ADA level in the serum in the control group is 22.04±4.625 (min 13, max 29. CONCLUSION ADA level in the serum is found to be increased indicating its role as an important immunoenzyme marker in the aetiopathology of type 2 diabetes mellitus.

  13. Physiological Characterization and Comparative Transcriptome Analysis of White and Green Leaves of Ananas comosus var. bracteatus.

    Directory of Open Access Journals (Sweden)

    Xia Li

    Full Text Available Leaf coloration is one of the most important and attractive characteristics of Ananas comosus var. bracteatus. The chimeric character is not stable during the in vitro tissue culturing. Many regenerated plants lost economic values for the loss of the chimeric character of leaves. In order to reveal the molecular mechanisms involved in the albino phenotype of the leaf cells, the physiological and transcriptional differences between complete white (CWh and green (CGr leaf cells of A. comosus var. bracteatus were analyzed. A total of 1,431 differentially expressed unigenes (DEGs in CGr and CWh leaves were identified using RNA-seq. A comparison to the COG, GO and KEGG annotations revealed DEGs involved in chlorophyll biosynthesis, chloroplast development and photosynthesis. Furthermore, the measurement of main precursors of chlorophyll in the CWh leaves confirmed that the rate-limiting step in chlorophyll biosynthesis, and thus the cause of the albino phenotype of the white cells, was the conversion of pyrrole porphobilinogen (PBG to uroporphyrinogen III (Uro III. The enzyme activity of porphobilinogen deaminase (PBGD and uroporporphyrinogn III synthase (UROS, which catalyze the transition of PBG to Uro III, was significantly decreased in the CWh leaves. Our data showed the transcriptional differences between the CWh and CGr plants and characterized key steps in chlorophyll biosynthesis of the CWh leaves. These results contribute to our understanding of the mechanisms and regulation of pigment biosynthesis in the CWh leaf cells of A. comosus var. bracteatus.

  14. ACTIVATION OF A CRYPTIC D-SERINE DEAMINASE (DSD) GENE FROM PSEUDOMONAS CEPACIA 17616

    Science.gov (United States)

    D-serine inhibits growth of P. cepacia 17616; however, resistant mutants able to express an ordinarily cryptic D-serine deaminase (dsd) gene were isolated readily. The resistant strains formed high levels of a D-serine deaminase active on D-threonine as well as D-serine. IS eleme...

  15. Genotype-specific enrichment of ACC deaminase-positive bacteria in winter wheat rhizospheres

    Science.gov (United States)

    Bacteria that produce ACC deaminase promote plant growth and development by lowering levels of the stress hormone ethylene through deamination of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene. Therefore, it is hypothesized that ACC deaminase positive (ACC+) bacteri...

  16. Hydrolytic cleavage of N-6-substituted adenine derivatives by eukaryotic adenine and adenosine deaminases

    Czech Academy of Sciences Publication Activity Database

    Pospíšilová, H.; Šebela, M.; Novák, Ondřej; Frébort, I.

    2008-01-01

    Roč. 28, č. 6 (2008), s. 335-347 ISSN 0144-8463 R&D Projects: GA ČR(CZ) GA522/06/0022 Institutional research plan: CEZ:AV0Z50380511 Keywords : adenine deaminase * adenosine deaminase (ADA) * aminohydrolase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.525, year: 2008

  17. Evaluation of usefulness of pleural fluid adenosine deaminase in diagnosing tuberculous pleural effusion from empyema

    Directory of Open Access Journals (Sweden)

    Vijetha Shenoy

    2014-02-01

    Full Text Available Objective: To evaluate the utility of adenosine deaminase activity in the pleural fluid for the diagnosis of tuberculous pleural effusion from empyema of non-tubercular origin. Method: A retrospective analysis of data was performed on patients who were diagnosed to have tuberculous pleural effusion and empyema of non tubercular origin. Among 46 patients at Kasturba Hospital, Manipal University, Manipal, Karnataka, India, from November 201 2 to February 2013 who underwent pleural fluid adenosine deaminase estimation, 25 patients with tuberculous pleural effusion and 21 patients with empyema were diagnosed respectively. Adenosine deaminase in pleural fluid is estimated using colorimetric, Galanti and Guisti method. Results: Pleural fluid Adenosine Deaminase levels among tuberculous pleural effusion(109.38依 53.83 , empyema (141.20依71.69 with P=0.27. Conclusion: Pleural fluid adenosine deaminase alone cannot be used as a marker for the diagnosis of tuberculous pleural effusion.

  18. Diagnostic significance of adenosine deaminase in pleural tuberculosis

    International Nuclear Information System (INIS)

    Khurshid, R.; Shore, N.; Saleem, M.; Zameer, N.

    2009-01-01

    Tuberculosis (TB) is a major cause of pleural effusion, which in TB usually has lymphocytic and exudative characteristics. Analysis of adenosine deaminase (ADA) activity is a very useful diagnostic approach to achieve a more rapid and precise diagnosis in cases of Pleural TB (pTB). Fifty male and fifty female patients presenting with tuberculosis pleural effusion was included in the study. The patients were taken from the medical ward of Sir Ganga Ram Hospital between September 2001 and September 2002. Activity of Adenosine Deaminase (ADA) was estimated by the technique of Sodium dodecyl sulphate electrophoresis (SDS-EF) using 10% polyacrylamide gel. Mean age of males was 45.72+-19.22 years and of female was 43.74+-16.09 years. Mean protein level was 3.39+-0.24 g/dl in males, and it was 3.02+-0.26 g/dl in females. Mean specific gravity both in males and females was 1.020+-0.01. The results show an increased level of enzyme ADA in patients as compared to normal subjects. Estimation of ADA activity may provide basis for rapid and efficient diagnosis of pleural TB in different clinical settings. However study should be extended to larger number of patients to reach a better conclusion. (author)

  19. Mass spectrometric characterisation of a condensation product between porphobilinogen and indolyl-3-acryloylglycine in urine of patients with acute intermittent porphyria.

    Science.gov (United States)

    Marcos, Josep; Ibañez, Maria; Ventura, Rosa; Segura, Jordi; To-Figueras, Jordi; Pozo, Oscar J

    2015-07-01

    We document the presence of a previously unknown species in the urine of patients with acute intermittent porphyria (AIP). The compound was fully characterised by liquid chromatography tandem mass spectrometry. Interpretation of both full spectrum acquisition and product ion spectra acquired in positive and negative ionisation modes by quadrupole time of flight MS allowed for the identification of a condensation product arising from porphobilinogen (PBG, increased in the urine of AIP patients) and indolyl-3-acryloylglycine (IAG, derived from indolylacrylic acid and present in human urine). The structure was unequivocally confirmed through comparison between the selected reaction monitoring chromatograms obtained from the urinary species and the condensation product qualitatively synthesised in the laboratory. Owing to the large amounts of both PBG and IAG in urine of AIP patients, the possible ex vivo formation of PBG-IAG in urine samples was evaluated. The product was spontaneously formed at room temperature, at 4 °C and even during storage at -20 °C when spiking a control sample with PBG. A positive correlation was found between PBG and PBG-IAG in samples collected from AIP patients. However, no correlation was found between PBG-IAG and IAG. Purified PBG-IAG did not form the characteristic chromogen after application of p-dimethylaminobenzaldehyde in HCl, thus suggesting that the current techniques used to measure PBG in urine of AIP patients based on Ehlrich's reaction do not detect this newly characterised PBG-IAG fraction. Copyright © 2015 John Wiley & Sons, Ltd.

  20. Hypermutation by intersegmental transfer of APOBEC3G cytidine deaminase.

    Science.gov (United States)

    Nowarski, Roni; Britan-Rosich, Elena; Shiloach, Tamar; Kotler, Moshe

    2008-10-01

    Deamination of cytidine residues in single-stranded DNA (ssDNA) is an important mechanism by which apolipoprotein B mRNA-editing, catalytic polypeptide-like (APOBEC) enzymes restrict endogenous and exogenous viruses. The dynamic process underlying APOBEC-induced hypermutation is not fully understood. Here we show that enzymatically active APOBEC3G can be detected in wild-type Vif(+) HIV-1 virions, albeit at low levels. In vitro studies showed that single enzyme-DNA encounters result in distributive deamination of adjacent cytidines. Nonlinear translocation of APOBEC3G, however, directed scattered deamination of numerous targets along the DNA. Increased ssDNA concentrations abolished enzyme processivity in the case of short, but not long, DNA substrates, emphasizing the key role of rapid intersegmental transfer in targeting the deaminase. Our data support a model by which APOBEC3G intersegmental transfer via monomeric binding to two ssDNA segments results in dispersed hypermutation of viral genomes.

  1. Discovery and structure determination of the orphan enzyme isoxanthopterin deaminase .

    Science.gov (United States)

    Hall, Richard S; Agarwal, Rakhi; Hitchcock, Daniel; Sauder, J Michael; Burley, Stephen K; Swaminathan, Subramanyam; Raushel, Frank M

    2010-05-25

    Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin 6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a ( gi|44585104 ) and NYSGXRC-9236b ( gi|44611670 ), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 A resolution (Protein Data Bank entry 2PAJ ). This protein folds as a distorted (beta/alpha)(8) barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosylhomocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin 6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s(-1), 8.0 muM, and 1.3 x 10(5) M(-1) s(-1) (k(cat), K(m), and k(cat)/K(m), respectively). The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (Protein Data Bank entry 2UZ9 ). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed on the basis of the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that form hydrogen bonds with the carbonyl oxygen at C4, a conserved threonine residue that forms hydrogen bonds with N5, and another conserved threonine residue that forms hydrogen bonds with the carbonyl group at C7. These conserved active site residues were used to identify 24 other genes

  2. Discovery and Structure Determination of the Orphan Enzyme Isoxanthopterin Deaminase

    Energy Technology Data Exchange (ETDEWEB)

    Hall, R.S.; Swaminathan, S.; Agarwal, R.; Hitchcock, D.; Sauder, J. M.; Burley, S. K.; Raushel, F. M.

    2010-05-25

    Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin 6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a (gi|44585104) and NYSGXRC-9236b (gi|44611670), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 {angstrom} resolution (Protein Data Bank entry 2PAJ). This protein folds as a distorted ({beta}/{alpha}){sub 8} barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosylhomocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin 6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s{sup -1}, 8.0 {micro}M, and 1.3 x 10{sup 5} M{sup -1} s{sup -1} (k{sub cat}, K{sub m}, and k{sub cat}/K{sub m}, respectively). The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (Protein Data Bank entry 2UZ9). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed on the basis of the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that form hydrogen bonds with the carbonyl oxygen at C4, a conserved threonine residue that forms hydrogen bonds with N5, and another conserved threonine residue that forms hydrogen bonds with the carbonyl group at C7. These conserved active site

  3. Mechanish of dTTP Inhibition of the Bifunctional dCTP Deaminase:dUTPase Encoded by Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Helt, Signe Smedegaard; Thymark, Majbritt; Harris, Pernille

    2008-01-01

    Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme to be cha......Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme...

  4. Myeloprotection by Cytidine Deaminase Gene Transfer in Antileukemic Therapy

    Directory of Open Access Journals (Sweden)

    Nico Lachmann

    2013-03-01

    Full Text Available Gene transfer of drug resistance (CTX-R genes can be used to protect the hematopoietic system from the toxicity of anticancer chemotherapy and this concept recently has been proven by overexpression of a mutant O6-methylguaninemethyltransferase in the hematopoietic system of glioblastoma patients treated with temozolomide. Given its protection capacity against such relevant drugs as cytosine arabinoside (ara-C, gemcitabine, decitabine, or azacytidine and the highly hematopoiesis-specific toxicity profile of several of these agents, cytidine deaminase (CDD represents another interesting candidate CTX-R gene and our group recently has established the myeloprotective capacity of CDD gene transfer in a number of murine transplant studies. Clinically, CDD overexpression appears particularly suited to optimize treatment strategies for acute leukemias and myelodysplasias given the efficacy of ara-C (and to a lesser degree decitabine and azacytidine in these disease entities. This article will review the current state of the art with regard to CDD gene transfer and point out potential scenarios for a clinical application of this strategy. In addition, risks and potential side effects associated with this approach as well as strategies to overcome these problems will be highlighted.

  5. Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase

    Energy Technology Data Exchange (ETDEWEB)

    R Hall; A Fedorov; C Xu; E Fedorov; S Almo; F Raushel

    2011-12-31

    Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a K{sub i} of 52 nM. The zinc- and iron-containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pKa of 6.0, and Zn-CDA has a kinetic pKa of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on k{sub cat} and k{sub cat}/K{sub m}, consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed.

  6. Moonlighting adenosine deaminase: a target protein for drug development.

    Science.gov (United States)

    Cortés, Antoni; Gracia, Eduard; Moreno, Estefania; Mallol, Josefa; Lluís, Carme; Canela, Enric I; Casadó, Vicent

    2015-01-01

    Interest in adenosine deaminase (ADA) in the context of medicine has mainly focused on its enzymatic activity. This is justified by the importance of the reaction catalyzed by ADA not only for the intracellular purine metabolism, but also for the extracellular purine metabolism as well, because of its capacity as a regulator of the concentration of extracellular adenosine that is able to activate adenosine receptors (ARs). In recent years, other important roles have been described for ADA. One of these, with special relevance in immunology, is the capacity of ADA to act as a costimulator, promoting T-cell proliferation and differentiation mainly by interacting with the differentiation cluster CD26. Another role is the ability of ADA to act as an allosteric modulator of ARs. These receptors have very general physiological implications, particularly in the neurological system where they play an important role. Thus, ADA, being a single chain protein, performs more than one function, consistent with the definition of a moonlighting protein. Although ADA has never been associated with moonlighting proteins, here we consider ADA as an example of this family of multifunctional proteins. In this review, we discuss the different roles of ADA and their pathological implications. We propose a mechanism by which some of their moonlighting functions can be coordinated. We also suggest that drugs modulating ADA properties may act as modulators of the moonlighting functions of ADA, giving them additional potential medical interest. © 2014 Wiley Periodicals, Inc.

  7. Distribution of adenosine deaminase complexing protein (ADCP) in human tissues.

    Science.gov (United States)

    Dinjens, W N; ten Kate, J; van der Linden, E P; Wijnen, J T; Khan, P M; Bosman, F T

    1989-12-01

    The normal distribution of adenosine deaminase complexing protein (ADCP) in the human body was investigated quantitatively by ADCP-specific radioimmunoassay (RIA) and qualitatively by immunohistochemistry. In these studies we used a specific rabbit anti-human ADCP antiserum. In all 19 investigated tissues, except erythrocytes, ADCP was found by RIA in the soluble and membrane fractions. From all tissues the membrane fractions contained more ADCP (expressed per mg protein) than the soluble fractions. High membrane ADCP concentrations were found in skin, renal cortex, gastrointestinal tract, and prostate. Immunoperoxidase staining confirmed the predominant membrane-associated localization of the protein. In serous sweat glands, convoluted tubules of renal cortex, bile canaliculi, gastrointestinal tract, lung, pancreas, prostate gland, salivary gland, gallbladder, mammary gland, and uterus, ADCP immunoreactivity was found confined to the luminal membranes of the epithelial cells. These data demonstrate that ADCP is present predominantly in exocrine glands and absorptive epithelia. The localization of ADCP at the secretory or absorptive apex of the cells suggests that the function of ADCP is related to the secretory and/or absorptive process.

  8. Adenosine deaminase complexing protein (ADCP) immunoreactivity in colorectal adenocarcinoma.

    Science.gov (United States)

    ten Kate, J; van den Ingh, H F; Khan, P M; Bosman, F T

    1986-04-15

    Immunoreactive adenosine deaminase complexing protein (ADCP) was studied in 91 human colorectal adenocarcinomas. The expression of ADCP was correlated with that of secretory component (SC) and carcinoembryonic antigen (CEA), with the histological grade and the Dukes' stage of the carcinomas. The histological grade was scored semi-quantitatively according to 5 structural and 4 cytological variables. ADCP expression was observed in 3 different staining patterns, namely: (1) diffuse cytoplasmic (77% of the carcinomas); (2) granular cytoplasmic (13%); and (3) membrane-associated (66%). These patterns were observed alone or in combination. Eleven percent of the carcinomas exhibited no ADCP immunoreactivity. Linear regression analysis showed that the expression of ADCP correlates with that of SC and CEA. However, no significant correlation emerged between the histological parameters or the Dukes' stage and any of the immunohistological parameters. Comparison of the histological characteristics of carcinomas exhibiting little or no ADCP immunoreactivity with those showing extensive immunoreactivity, showed that membranous ADCP immunoreactivity occurs more frequently in well-differentiated carcinomas. Structural parameters showed a better correlation with membranous ADCP expression than the cytological variables. It is concluded that membranous expression of ADCP and CEA are indicators of a high level of differentiation as reflected primarily in the structural characteristics of the tumor.

  9. Overproduction, Purification and Characterization of Adenylate Deaminase from Aspergillus oryzae.

    Science.gov (United States)

    Li, Shubo; Qian, Yi; Liang, Yunlong; Chen, Xinkuan; Zhao, Mouming; Guo, Yuan; Pang, Zongwen

    2016-12-01

    Adenylate deaminase (AMPD, EC 3.5.4.6) is an aminohydrolase that widely used in the food and medicine industries. In this study, the gene encoding Aspergillus oryzae AMPD was cloned and expressed in Escherichia coli. Induction with 0.75 mM isopropyl β-D-l-thiogalactopyranoside resulted in an enzyme activity of 1773.9 U/mL. Recombinant AMPD was purified to electrophoretic homogeneity using nickel affinity chromatography, and its molecular weight was calculated as 78.6 kDa. Purified AMPD exhibited maximal activity at 35 °C, pH 6.0 and 30 mM K + , with apparent K m and V max values of 2.7 × 10 -4  M and 77.5 μmol/mg/min under these conditions. HPLC revealed that recombinant AMPD could effectively catalyse the synthesis of inosine-5'-monophosphate (IMP) with minimal by-products, indicating high specificity and suggesting that it could prove useful for IMP production.

  10. Adenosine deaminase production by an endophytic bacterium (Lysinibacillus sp.) from Avicennia marina.

    Science.gov (United States)

    Kathiresan, Kandasamy; Saravanakumar, Kandasamy; Sahu, Sunil Kumar; Sivasankaran, Muthu

    2014-06-01

    The present study was carried out with the following objectives: (1) to isolate the endophytic bacilli strains from the leaves of mangrove plant Avicennia marina, (2) to screen the potential strains for the production of adenosine deaminase, (3) to statistically optimize the factors that influence the enzyme activity in the potent strain, and (4) to identify the potent strain using 16S rRNA sequence and construct its phylogenetic tree. The bacterial strains isolated from the fresh leaves of a mangrove A. marina were assessed for adenosine deaminase activity by plating method. Optimization of reaction process was carried out using response surface methodology of central composite design. The potent strain was identified based on 16S rRNA sequencing and phylogeny. Of five endophytic strains, EMLK1 showed a significant deaminase activity over other four strains. The conditions for maximum activity of the isolated adenosine deaminase are described. The potent strain EMLK1 was identified as Lysinibacillus sp. (JQ710723) being the first report as a mangrove endophyte. Mangrove-derived endophytic bacillus strain Lysinibacillus sp. EMLK1 is proved to be a promising source for the production of adenosine deaminase and this enzyme deserves further studies for purification and its application in disease diagnosis.

  11. [Isolation, identification and characterization of ACC deaminase-containing endophytic bacteria from halophyte Suaeda salsa].

    Science.gov (United States)

    Teng, Songshan; Liu, Yanping; Zhao, Lei

    2010-11-01

    We Isolated and characterized 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing endophytic bacteria from halophyte Suaeda salsa to understand the interactions between endophytes and halophyte. ACC deaminase-containing endophytic bacteria were isolated from root, stalk and leaf of Suaeda salsa and were identified based on morphological, physiological-biochemical properties, API and 16S rRNA sequence analysis. Isolates were evaluated for their ACC deaminase, antifungal, protease activity, siderophores and phytohormones, such as indole-3-acetic acid, gibberellic acid and abscisic acid production, as well as atmospheric nitrogen fixation and phosphate solubilization. Four ACC deaminase-containing endophytic bacteria strains named as LP11, SS12, TW1 and TW2 were isolated and identified as Pseudomonas oryzihabitans, Pseudomonas sp., Pantoea agglomerans and Pseudomonas putida respectively. All the strains possessed the phosphate-solubilizing ability and could produce siderophores and phytohormones more or less. None of them could fix atmospheric nitrogen or produce protease. Only strain SS12 showed antagonism against two phytopathogenic fungi viz Fusarium oxysporum f. sp. conglutinans and F. oxysporum f. sp. cucumerinum. ACC deaminase-containing endophytic bacteria of Pseudomonas sp. and Pantoea sp. isolated from halophyte Suaeda salsa have abundant biological characteristics related to plant growth promotion, stress homeostasis regulation and biocontrol activity.

  12. Biological function of activation-induced cytidine deaminase (AID

    Directory of Open Access Journals (Sweden)

    Ritu Kumar

    2014-10-01

    Full Text Available Activation-induced Cytidine Deaminase (AID is an essential regulator of B cell diversification, but its full range of action has until recently been an enigma. Based on homology, it was originally proposed to be an RNA-editing enzyme, but so far, no RNA substrates are known. Rather, it functions by deaminating cytidine, and in this manner, coupled with base-excision repair or mismatch repair machinery, it is a natural mutator. This allows it to play a central role in adaptive immunity, whereby it initiates the processes of class switch recombination and somatic hypermutation to help generate a diverse and high-affinity repertoire of immunoglobulin isotypes. More recently, it has been appreciated that methylated cytidine, already known as a key epigenetic mark on DNA controlling gene expression, can also be a target for AID modification. Coupled with repair machinery, this can facilitate the active removal of methylated DNA. This activity can impact the process of cellular reprogramming, including transition of a somatic cell to pluripotency, which requires major reshuffling of epigenetic memory. Thus, seemingly disparate roles for AID in controlling immune diversity and epigenetic memory have a common mechanistic basis. However, the very activity that is so useful for B cell diversity and cellular reprogramming is dangerous for the integrity of the genome. Thus, AID expression and activity is tightly regulated, and deregulation is associated with diseases including cancer. Here, we review the range of AID functions with a focus on its mechanisms of action and regulation. Major questions remain to be answered concerning how and when AID is targeted to specific loci and how this impacts development and disease.

  13. Human adenosine deaminase: properties and turnover in cultured T and B lymphoblasts

    International Nuclear Information System (INIS)

    Daddona, P.E.

    1981-01-01

    In this study, the properties and rate of turnover of adenosine deaminase are compared in cultured human T and B lymphoblast cell lines. 1) Relative to B lymphoblasts, the level of adenosine deaminase activity in extracts of T lymphoblast cell lines (MOLT-4, RPMI-8402, CCRF-CEM, and CCRF-HSB-2) is elevated 7-14-fold and differs by 2-fold between the C cell lines. 2) In both T and B lymphoblast extracts, the enzyme is apparently identical, based on K/sub m/ for adenosine and deoxyadenosine, K/sub i/ for inosine, V/sub max/ for adenosine, /sub S20,w/, isoelectric pH, and heat stability. Furthermore, by radioimmunoassay, the quantity of adenosine deaminase-immunocreative protein is proportional to the level of enzyme activity in all cell lines studies. 3) Using a purification and selective immunoprecipitation technique, the enzyme turnover could be assessed in cell lines labeled with [ 35 S]methionine. The apparent rate of adenosine deaminase synthesis, relative to total protein, is 2-fold faster in both T cell lines (RPMI-8402 and CCRF-CEM) than in the B cell lines (MGL-8 and GM-130). The apparent half-life (tsub1/2) for the enzyme degradation is 19 and 39 h, respectively, in CCFR-CEM and RPMI-8402, while the tsub1/2 in both B cell lines is 7-9 h. From the net rate of synthesis and degradation, the T cell lines, respectively, exhibit approximately a 6- and 12-fold difference in adenosine deaminase turnover relative to B cells, consistent with the observed differences in enzyme activity. This study suggests that while adenosine deaminase is apparently identical in both T and B lymphoblast cell lines, alterations in both the rate of enzyme synthesis and degradation contribute to its high steady state level in T cells

  14. Altered activity of heme biosynthesis pathway enzymes in individuals chronically exposed to arsenic in Mexico

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez-Zavala, A.; Del Razo, L.M.; Garcia-Vargas, G.G.; Aguilar, C.; Borja, V.H.; Albores, A.; Cebrian, M.E. [CINVESTAV-IPN, Mexico (Mexico). Dept. de Farmacologia y Toxicologica

    1999-03-01

    Our objective was to evaluate the activities of some enzymes of the heme biosynthesis pathway and their relationship with the profile of urinary porphyrin excretion in individuals exposed chronically to arsenic (As) via drinking water in Region Lagunera, Mexico. We selected 17 individuals from each village studied: Benito Juarez, which has current exposure to 0.3 mg As/l; Santa Ana, where individuals have been exposed for more than 35 years to 0.4 mg As/l, but due to changes in the water supply (in 1992) exposure was reduced to its current level (0.1 mg As/l), and Nazareno, with 0.014 mg As/l. Average arsenic concentrations in urine were 2058, 398, and 88 {mu}g As/g creatinine, respectively. The more evident alterations in heme metabolism observed in the highly exposed individuals were: (1) small but significant increases in porphobilinogen deaminase (PBG-D) and uroporphyrinogen decarboxylase (URO-D) activities in peripheral blood erythrocytes; (2) increases in the urinary excretion of total porphyrins, mainly due to coproporphyrin III (COPROIII) and uroporphyrin III (UROIII); and (3) increases in the COPRO/URO and COPROIII/COPROI ratios. No significant changes were observed in uroporphyrinogen III synthetase (UROIII-S) activity. The direct relationships between enzyme activities and urinary porphyrins, suggest that the increased porphyrin excretion was related to PBG-D, whereas the increased URO-D activity would enhance coproporphyrin synthesis and excretion at the expense of uroporphyrin. None of the human studies available have reported the marked porphyric response and enzyme inhibition observed in rodents. In conclusion, chronic As exposure alters human heme metabolism; however the severity of the effects appears to depend on characteristics of exposure not yet fully characterized. (orig.) With 1 fig., 3 tabs., 20 refs.

  15. The Effect of Acute Exercise upon Adenosin Deaminase Oxidant and Antioxidant Activity

    Science.gov (United States)

    Kafkas, M. Emin; Karabulut, Aysun Bay; Sahin, Armagan; Otlu, Onder; Savas, Seyfi; Aytac, Aylin

    2012-01-01

    The purpose of this study was to determine the changes of MDA, glutation (GSH), Adenozine deaminase (ADA) and superoxidase dismutaze (SOD) levels with exercise training in obese middle-aged women (body mass index, MMI [greater than or equal to] 30.0). Twelve obese middle-aged women participated in this study. The descriptive statistics of some of…

  16. APOBEC3 cytidine deaminases in double-strand DNA break repair and cancer promotion.

    Science.gov (United States)

    Nowarski, Roni; Kotler, Moshe

    2013-06-15

    High frequency of cytidine to thymidine conversions was identified in the genome of several types of cancer cells. In breast cancer cells, these mutations are clustered in long DNA regions associated with single-strand DNA (ssDNA), double-strand DNA breaks (DSB), and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs, and clustered mutations. Cancer Res; 73(12); 3494-8. ©2013 AACR. ©2013 AACR.

  17. Adenosine deaminase complexing protein (ADCP): a transformation sensitive protein with potentials of a cancer marker.

    Science.gov (United States)

    Herbschleb-Voogt, E; Ten Kate, J; Meera Khan, P

    1983-01-01

    Several observations by independent investigators in the past have indicated that adenosine deaminase complexing protein (ADCP), present in considerable quantities in certain human tissues, was absent or decreased in the cancers originated from them. During the present study, electrophoretic analysis of adenosine deaminase (ADA) isozymes and radioimmunoassay for ADCP in the primary fibroblasts and the transformed as well as certain tumor derived cell lines have demonstrated that ADCP present in large quantities in the primary cells was absent or nearly absent in the transformed or tumor-derived cell lines. Though the mechanisms involved are not yet clear, the above observations indicate that ADCP has the potentials of a useful marker in the studies on transformed cells and cancer tissues.

  18. Regulation of adenosine deaminase (ADA) on induced mouse experimental autoimmune uveitis (EAU) ?

    OpenAIRE

    Liang, Dongchun; Zuo, Aijun; Zhao, Ronglan; Shao, Hui; Kaplan, Henry J.; Sun, Deming

    2016-01-01

    Adenosine is an important regulator of the immune response and adenosine deaminase (ADA) inhibits this regulatory effect by converting adenosine into functionally inactive molecules. Studies have shown that adenosine receptor (AR) agonists can be either anti- or pro-inflammatory. Clarification of the mechanisms that cause these opposing effects should provide a better guide for therapeutic intervention. In this study, we investigated the effect of ADA on the development of experimental autoim...

  19. Radio-chromatographic determination of plasmatic adenosine deaminase (A.D.)

    International Nuclear Information System (INIS)

    Chivot, J.J.; Depernet, D.; Caen, J.

    1970-01-01

    We were able, by using a radio-chromatographic method, to measure an adenosine deaminase activity in normal human heparinized platelet-poor plasma, which can degrade 0.016 μM adenosine. This activity suppressed by heating 56 C for 30 minutes is inhibited by high concentrations of urea and is proportional to the amount of plasma, source of enzyme, in the systems. (authors) [fr

  20. Inducing salt tolerance in maize through ACC-deaminase biotechnology (abstract)

    International Nuclear Information System (INIS)

    Shahroona, B.; Arshad, M.; Zahir, Z.A.

    2005-01-01

    Ethylene is one of the five established classes of phytohormones. Its involvement in evoking physiological responses in plants exposed to any kind of environmental stresses (such as salinity, drought and metal toxicity stresses) is well established, thus it has also been designated as 'stress' hormone. It is generally believed that stress induces accelerated synthesis of ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), which subsequently results in the out burst of ethylene production and plant responds to this higher level of ethylene. Thus any check on this accelerated ethylene production in plants exposed to salinity stress could help in minimizing the negative impact of this stress and plants might survive better. There are some soil bacteria which carry ACC-deaminase enzyme and their presence on root surface results in lowering the accelerated production of ethylene. Trials were conducted under axenic condition to study the effect of inoculation with ACC-deaminase containing rhizobacteria to ameliorate the effect of salinity on seedling growth. Maize seedlings were exposed to different salinity levels and results indicated that inoculation with ACC-deaminase containing rhizobacteria significantly increased root elongation, shoot length, fresh and dry weight of seedlings at all the salinity levels. (author)

  1. A halotolerant Enterobacter sp. displaying ACC deaminase activity promotes rice seedling growth under salt stress.

    Science.gov (United States)

    Sarkar, Anumita; Ghosh, Pallab Kumar; Pramanik, Krishnendu; Mitra, Soumik; Soren, Tithi; Pandey, Sanjeev; Mondal, Monohar Hossain; Maiti, Tushar Kanti

    2018-01-01

    Agricultural productivity is proven to be hampered by the synthesis of reactive oxygen species (ROS) and production of stress-induced ethylene under salinity stress. One-aminocyclopropane-1-carboxylic acid (ACC) is the direct precursor of ethylene synthesized by plants. Bacteria possessing ACC deaminase activity can use ACC as a nitrogen source preventing ethylene production. Several salt-tolerant bacterial strains displaying ACC deaminase activity were isolated from rice fields, and their plant growth-promoting (PGP) properties were determined. Among them, strain P23, identified as an Enterobacter sp. based on phenotypic characteristics, matrix-assisted laser desorption ionization-time of flight mass spectrometry data and the 16S rDNA sequence, was selected as the best-performing isolate for several PGP traits, including phosphate solubilization, IAA production, siderophore production, HCN production, etc. Enterobacter sp. P23 was shown to promote rice seedling growth under salt stress, and this effect was correlated with a decrease in antioxidant enzymes and stress-induced ethylene. Isolation of an acdS mutant strain enabled concluding that the reduction in stress-induced ethylene content after inoculation of strain P23 was linked to ACC deaminase activity. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  2. Replication protein A (RPA) hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA.

    Science.gov (United States)

    Lada, Artem G; Waisertreiger, Irina S-R; Grabow, Corinn E; Prakash, Aishwarya; Borgstahl, Gloria E O; Rogozin, Igor B; Pavlov, Youri I

    2011-01-01

    Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G), restricts retroviruses, and Activation Induced Deaminase (AID) generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G. We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the "hit and run" single base substitution events observed in yeast. Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.

  3. Replication protein A (RPA hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA.

    Directory of Open Access Journals (Sweden)

    Artem G Lada

    Full Text Available Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G, restricts retroviruses, and Activation Induced Deaminase (AID generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA, the eukaryotic single-stranded DNA (ssDNA binding protein, severely inhibits the deamination activity and processivity of A3G.We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the "hit and run" single base substitution events observed in yeast.Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.

  4. Regulation of Expression of the prb-1b / ACC Deaminase gene by UV-B in Transgenic tomatoes

    International Nuclear Information System (INIS)

    Tamot, B.K.; Pauls, K.P.; Glick, R.

    2003-01-01

    Transgenic tomato plants with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UWA4 under the control of a pathogenesis-related promoter (prb-1b) from tobacco were challenged by abiotic stresses to determine the expression patterns of the transgene. No ACC deaminase RNA or protein was detected bu RT-PCR and in western blots prepared from leaf proteins of transgenic plants after wounding or treatment with alpha-amino butyric acid, xylanase, ethephon, salicylic acid, jasmonic acid , ethylene, or ethylene plus jasmonic acid. However, expression of the ACC deaminase transgene was observed in leaves and roots of transformed tomato lines exposed to UV light. The UV response required a minimum of 48 h of exposure and was specific to UV-B light

  5. Threonine deaminase from extremely halophilic bacteria - Cooperative substrate kinetics and salt dependence.

    Science.gov (United States)

    Lieberman, M. M.; Lanyi, J. K.

    1972-01-01

    The effect of salt on the activity, stability, and allosteric properties of catabolic threonine deaminase from Halobacterium cutirubrum was studied. The enzyme exhibits sigmoidal kinetics with the substrate, threonine. The Hill slope is 1.55 at pH 10. The enzyme is activated by ADP at low substrate concentrations. In the presence of this effector, sigmoidal kinetics are no longer observed. At pH 10, in the absence of ADP, enzyme activity increases with increasing NaCl concentration from 0 to 4 M.

  6. Optimising the utility of pleural fluid adenosine deaminase for the diagnosis of adult tuberculous pleural effusion in Hong Kong.

    Science.gov (United States)

    Chang, K C; Chan, M C; Leung, W M; Kong, F Y; Mak, C M; Chen, S Pl; Yu, W C

    2018-02-01

    Pleural fluid adenosine deaminase level can be applied to rapidly detect tuberculous pleural effusion. We aimed to establish a local diagnostic cut-off value for pleural fluid adenosine deaminase to identify patients with tuberculous pleural effusion, and optimise its utility. We retrospectively reviewed the medical records of consecutive adults with pleural fluid adenosine deaminase level measured by the Diazyme commercial kit (Diazyme Laboratories, San Diego [CA], United States) during 1 January to 31 December 2011 in a cluster of public hospitals in Hong Kong. We considered its level alongside early (within 2 weeks) findings in pleural fluid and pleural biopsy, with and without applying Light's criteria in multiple scenarios. For each scenario, we used the receiver operating characteristic curve to identify a diagnostic cut-off value for pleural fluid adenosine deaminase, and estimated its positive and negative predictive values. A total of 860 medical records were reviewed. Pleural effusion was caused by congestive heart failure, chronic renal failure, or hypoalbuminaemia caused by liver or kidney diseases in 246 (28.6%) patients, malignancy in 198 (23.0%), non-tuberculous infection in 168 (19.5%), tuberculous pleural effusion in 157 (18.3%), and miscellaneous causes in 91 (10.6%). All those with tuberculous pleural effusion had a pleural fluid adenosine deaminase level of ≤100 U/L. When analysis was restricted to 689 patients with pleural fluid adenosine deaminase level of ≤100 U/L and early negative findings for malignancy and non-tuberculous infection in pleural fluid, the positive predictive value was significantly increased and the negative predictive value non-significantly reduced. Using this approach, neither additionally restricting analysis to exudates by Light's criteria nor adding closed pleural biopsy would further enhance predictive values. As such, the diagnostic cut-off value for pleural fluid adenosine deaminase is 26.5 U/L, with a

  7. Discovery of a cAMP Deaminase That Quenches Cyclic AMP-Dependent Regulation

    Science.gov (United States)

    Goble, Alissa M.; Feng, Youjun; Raushel, Frank M.; Cronan, John E.

    2013-01-01

    An enzyme of unknown function within the amidohydrolase superfamily was discovered to catalyze the hydrolysis of the universal second messenger, cyclic-3’, 5’-adenosine monophosphate (cAMP). The enzyme, which we have named CadD, is encoded by the human pathogenic bacterium Leptospira interrogans. Although CadD is annotated as an adenosine deaminase, the protein specifically deaminates cAMP to cyclic-3’, 5’-inosine monophosphate (cIMP) with a kcat/Km of 2.7 ± 0.4 × 105 M−1 s−1 and has no activity on adenosine, adenine, or 5’-adenosine monophosphate (AMP). This is the first identification of a deaminase specific for cAMP. Expression of CadD in Escherichia coli mimics the loss of adenylate cyclase in that it blocks growth on carbon sources that require the cAMP-CRP transcriptional activator complex for expression of the cognate genes. The cIMP reaction product cannot replace cAMP as the ligand for CRP binding to DNA in vitro and cIMP is a very poor competitor of cAMP activation of CRP for DNA binding. Transcriptional analyses indicate that CadD expression represses expression of several cAMP-CRP dependent genes. CadD adds a new activity to the cAMP metabolic network and may be a useful tool in intracellular study of cAMP-dependent processes. PMID:24074367

  8. Ecto- and cytosolic 5'-nucleotidases in normal and AMP deaminase-deficient human skeletal muscle

    DEFF Research Database (Denmark)

    Hanisch, Frank; Hellsten, Ylva; Zierz, Stephan

    2006-01-01

    homogenate 5'-nucleotidase and ectoN, or in cN-I expression on Western blots. No correlation for age, fibre type distribution and AMPD1 genotype was found for whole homogenate nucleotidase, total cN and cN-I using multiple linear regression analysis. There was no gender-specific difference in the activities...... with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with different...... AMPD1 genotypes [homozygotes for C34T mutation (TT); heterozygotes for C34T mutation (CT); and homozygotes for wild type (CC): diseased controls CC; and normal controls CC]. AMP deaminase activity showed genotype-dependent differences. Total cN activity in normal controls accounted for 57...

  9. Curcumin inhibits adenosine deaminase and arginase activities in cadmium-induced renal toxicity in rat kidney

    Directory of Open Access Journals (Sweden)

    Ayodele Jacob Akinyemi

    2017-04-01

    Full Text Available In this study, the effect of enzymes involved in degradation of renal adenosine and l-arginine was investigated in rats exposed to cadmium (Cd and treated with curcumin, the principal active phytochemical in turmeric rhizome. Animals were divided into six groups (n = 6: saline/vehicle, saline/curcumin 12.5 mg/kg, saline/curcumin 25 mg/kg, Cd/vehicle, Cd/curcumin 12.5 mg/kg, and Cd/curcumin 25 mg/kg. The results of this study revealed that the activities of renal adenosine deaminase and arginase were significantly increased in Cd-treated rats when compared with the control (p < 0.05. However, co-treatment with curcumin inhibits the activities of these enzymes compared with Cd-treated rats. Furthermore, Cd intoxication increased the levels of some renal biomarkers (serum urea, creatinine, and electrolytes and malondialdehyde level with a concomitant decrease in functional sulfhydryl group and nitric oxide (NO. However, co-treatment with curcumin at 12.5 mg/kg and 25 mg/kg, respectively, increases the nonenzymatic antioxidant status and NO in the kidney, with a concomitant decrease in the levels of malondialdehyde and renal biomarkers. Therefore, our results reinforce the importance of adenosine deaminase and arginase activities in Cd poisoning conditions and suggest some possible mechanisms of action by which curcumin prevent Cd-induced renal toxicity in rats.

  10. APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair.

    Science.gov (United States)

    Nowarski, Roni; Wilner, Ofer I; Cheshin, Ori; Shahar, Or D; Kenig, Edan; Baraz, Leah; Britan-Rosich, Elena; Nagler, Arnon; Harris, Reuben S; Goldberg, Michal; Willner, Itamar; Kotler, Moshe

    2012-07-12

    APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy.

  11. How We Manage Adenosine Deaminase-Deficient Severe Combined Immune Deficiency (ADA SCID).

    Science.gov (United States)

    Kohn, Donald B; Gaspar, H Bobby

    2017-05-01

    Adenosine deaminase-deficient severe combined immune deficiency (ADA SCID) accounts for 10-15% of cases of human SCID. From what was once a uniformly fatal disease, the prognosis for infants with ADA SCID has improved greatly based on the development of multiple therapeutic options, coupled with more frequent early diagnosis due to implementation of newborn screening for SCID. We review the various treatment approaches for ADA SCID including allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen-matched sibling or family member or from a matched unrelated donor or a haplo-identical donor, autologous HSCT with gene correction of the hematopoietic stem cells (gene therapy-GT), and enzyme replacement therapy (ERT) with polyethylene glycol-conjugated adenosine deaminase. Based on growing evidence of safety and efficacy from GT, we propose a treatment algorithm for patients with ADA SCID that recommends HSCT from a matched family donor, when available, as a first choice, followed by GT as the next option, with allogeneic HSCT from an unrelated or haplo-identical donor or long-term ERT as other options.

  12. Diagnostic Value of Serum Adenosine Deaminase (ADA) Level for Pulmonary Tuberculosis.

    Science.gov (United States)

    Salmanzadeh, Shokrollah; Tavakkol, Heshmatollah; Bavieh, Khalid; Alavi, Seyed Mohammad

    2015-03-01

    Diagnosis of tuberculosis (TB) is not always easy, thus employing methods with a short duration and acceptable sensitivity and specificity is necessary to diagnose TB. The aim of this study was to investigate the diagnostic value of serum adenosine deaminase (ADA) level for diagnosis of pulmonary tuberculosis. A total of 160 sex and age-matched subjects were included in this study, and were divided to four groups; forty patients with pulmonary tuberculosis (PTB) diagnosed based on the national TB program (NTP), forty patients with non-tuberculosis bacterial pneumonia, forty patients with lung cancer and forty people who were healthy in every respect. Serum adenosine deaminase activity in patients of each group was measured by the Giusti and Galanti calorimetry method using a commercial kit (Diazyme, USA). The ANOVA analysis was used to compare groups for quantitative variables. Mean serum ADA level in the PTB group was clearly higher than the mean serum ADA in the other three groups. Mean serum ADA was 26 IU/L in PTB patients, 19.48 IU/L in patients with pneumonia, 15.8 IU/L in patients with lung cancer, and 10.7 IU/L in the control group (P ADA in patients with PTB sensitivity and specificity was defined as 35% and 91%, respectively. Serum ADA activity with high specificity percentage may be a useful alternative test in restricted resource areas to rule out diagnosis of PTB. However, serum ADA activity is not a useful tool for TB diagnosis.

  13. Development of gene therapy: potential in severe combined immunodeficiency due to adenosine deaminase deficiency

    Directory of Open Access Journals (Sweden)

    Claudia A Montiel-Equihua

    2009-12-01

    Full Text Available Claudia A Montiel-Equihua, Adrian J Thrasher, H Bobby GasparCentre for Immunodeficiency, Molecular Immunology Unit, UCL Institute of Child Health, London, UKAbstract: The history of stem cell gene therapy is strongly linked to the development of gene therapy for severe combined immunodeficiencies (SCID and especially adenosine deaminase (ADA-deficient SCID. Here we discuss the developments achieved in over two decades of clinical and laboratory research that led to the establishment of a protocol for the autologous transplant of retroviral vector-mediated gene-modified hematopoietic stem cells, which has proved to be both successful and, to date, safe. Patients in trials in three different countries have shown long-term immunological and metabolic correction. Nevertheless, improvements to the safety profile of viral vectors are underway and will undoubtedly reinforce the position of stem cell gene therapy as a treatment option for ADA-SCID.Keywords: adenosine deaminase, severe combined immunodeficiency, gene therapy, hematopoietic stem cell, retrovirus, clinical trial

  14. Pleural Fluid Adenosine Deaminase (ADA) Predicts Survival in Patients with Malignant Pleural Effusion.

    Science.gov (United States)

    Terra, Ricardo Mingarini; Antonangelo, Leila; Mariani, Alessandro Wasum; de Oliveira, Ricardo Lopes Moraes; Teixeira, Lisete Ribeiro; Pego-Fernandes, Paulo Manuel

    2016-08-01

    Systemic and local inflammations have been described as relevant prognostic factors in patients with cancer. However, parameters that stand for immune activity in the pleural space have not been tested as predictors of survival in patients with malignant pleural effusion. The objective of this study was to evaluate pleural lymphocytes and Adenosine Deaminase (ADA) as predictors of survival in patients with recurrent malignant pleural effusion. Retrospective cohort study includes patients who underwent pleurodesis for malignant pleural effusion in a tertiary center. Pleural fluid protein concentration, lactate dehydrogenase, glucose, oncotic cytology, cell count, and ADA were collected before pleurodesis and analyzed. Survival analysis was performed considering pleurodesis as time origin, and death as the event. Backwards stepwise Cox regression was used to find predictors of survival. 156 patients (out of 196 potentially eligible) were included in this study. Most were female (72 %) and breast cancer was the most common underlying malignancy (53 %). Pleural fluid ADA level was stratified as low (Pleural fluid cell count and lymphocytes number and percentage did not correlate with survival. Pleural fluid Adenosine Deaminase levels (pleural effusion who undergo pleurodesis.

  15. [Isolation and identification of hydrogen-oxidizing bacteria producing 1-aminocyclopropane-1-carboxylate deaminase and the determination of enzymatic activity].

    Science.gov (United States)

    Fu, Bo; Wang, Weiwei; Tang, Ming; Chen, Xingdu

    2009-03-01

    We used Medicago sativa rhizosphere in Shaanxi province of China to isolate and identify hydrogen-oxidizing bacteria that produced ACC (1-aminocyclopropane-1-carboxylate) deaminase, and then studied the mechanism why they can promote the growth of plants. Hydrogen-oxidizing bacteria were isolated by gas-cycle incubation system. We studied the morphological character, physiological characteristics, 16S rDNA sequence analysis and built the phylogenic tree. Thin layer chromatography was used to isolate the strain that produced ACC deaminase. Ninhydrin reaction was used to test the enzyme activity. In total 37 strains were isolated, 8 of which could oxidize H2 strongly and grow chemolithoautotrophically. We initially identified them as hydrogen-oxidizing bacteria. Only strain WMQ-7 produced ACC deaminase among these 8 strains. Morphological and physiological characteristics analysis showed that strain WMQ-7 was essentially consistent with Pseudomonas putida. The 16S rDNA sequence analysis (GenBank accession number EU807744) suggested that strain WMQ-7 was clustered together with Pseudomonas putida in phylogenetic tree, with the sequence identity of 99%. Based on all these results, strain WMQ-7 was identified as Pseudomonas putida. The enzyme activity of strain WMQ-7 was 0.671 U/microg. A strain producing ACC deaminase was identified and tested.

  16. Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig Loci in activated B cells

    NARCIS (Netherlands)

    Staszewski, Ori; Baker, Richard E.; Ucher, Anna J.; Martier, Raygene; Stavnezer, Janet; Guikema, Jeroen E. J.

    2011-01-01

    After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading

  17. Characterization of 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase-Containing Pseudomonas spp. in the Rhizosphere of Salt-Stressed Canola

    NARCIS (Netherlands)

    Akhgar, A.; Arzanlou, M.; Bakker, Peter; Hamidpour, M.

    2014-01-01

    When exposed to biotic or abiotic stress conditions, plants produce ethylene from its immediate precursor 1-aminocyclopropane-1- carboxylate (ACC), leading to retarded root growth and senescence. Many plant growth-promoting rhizobacteria contain the enzyme ACC deaminase and this enzyme can cleave

  18. [The influence of fasting, of a hyperprotein diet and of nicotinamide on hepatic L-threonine deaminase].

    Science.gov (United States)

    Aleo, M F; Casella, A; Marinello, E

    1981-09-15

    The induction of L-threonine deaminase, following nicotinamide injection has been studied: the effect of fasting and of hyperproteic diet have been also taken in consideration. Maximal induction is observed after 5 days hyperproteic diet, and is additional only with nicotinamide treatment. Results are interpreted assuming a different hepatic content and behavior of multiple forms of the enzyme.

  19. Isolation and characterization of novel bacteria containing acc deaminase from the rhizosphere resource on dry-farming lands

    International Nuclear Information System (INIS)

    Zheng, P.; Tian, L.; Chen, F.; Cui, Z.

    2014-01-01

    Soil-microbe-plant interactions are known to be intricate and they can greatly influence the crop vigor and yield. Plant growth promoting rhizobacteria (PGPR) containing ACC deaminase can markedly affect plant metabolic processes under stress conditions. In the present study, we isolated 300 bacterial strains from the rhizosphere of maize or apple grown in drought-hit soil including four different locations of the Loess Plateau, China. Of all isolated strains, four with ACC deaminase activity (ranging from 28.88 to 155.12 nmol alpha-ketobutyrate mg-1 h-1) were further studied by determining their biological characters and sequencing the 16S rRNA gene. All four strains showed positive performance in terms of arabinose, citrate utilization, urease, indol, glucose and melibiose. In connection with the results of biochemical characters and phylogenetic analysis, these strains commonly belong to three different genera: Klebsiella, Pseudomonas and Raoultella and four different species: Klebsiella oxytoca, Klebsiella variicola, Pseudomonas fluorescens and Raoultella planticola. Although some researchers have reported their performance under stress conditions, we are the first to report Klebsiella oxytoca, Klebsiella variicola and Raoultella planticola containing ACC deaminase under drought stress. These findings are a reasonable explanation to their superb ability of causing stress-resistance in maize (Zea mays) or apple (Malus domestica) plants. The presence of diverse PGPR possessing potential ACC deaminase activity may be beneficial for enhancing crop production under different stress conditions. (author)

  20. Effect of alginate microencapsulation on the catalytic efficiency and in vitro enzyme-prodrug therapeutic efficacy of cytosine deaminase and of recombinant E. coli expressing cytosine deaminase.

    Science.gov (United States)

    Funaro, Michael G; Nemani, Krishnamurthy V; Chen, Zhihang; Bhujwalla, Zaver M; Griswold, Karl E; Gimi, Barjor

    2016-02-01

    Cytosine deaminase (CD) catalyses the enzymatic conversion of the non-toxic prodrug 5-fluorocytosine (5-FC) to the potent chemotherapeutic form, 5-fluorouracil (5-FU). Intratumoral delivery of CD localises chemotherapy dose while reducing systemic toxicity. Encapsulation in biocompatible microcapsules immunoisolates CD and protects it from degradation. We report on the effect of alginate encapsulation on the catalytic and functional activity of isolated CD and recombinant E. coli engineered to express CD (E. coli(CD)). Alginate microcapsules containing either CD or Escherichia coli(CD) were prepared using ionotropic gelation. Conversion of 5-FC to 5-FU was quantitated in unencapsulated and encapsulated CD/E. coli(CD) using spectrophotometry, with a slower rate of conversion observed following encapsulation. Both encapsulated CD/5-FC and E. coli(CD)/5-FC resulted in cell kill and reduced proliferation of 9 L rat glioma cells, which was comparable to direct 5-FU treatment. Our results show that encapsulation preserves the therapeutic potential of CD and E. coli(CD) is equally effective for enzyme-prodrug therapy.

  1. Radio-chromatographic determination of plasmatic adenosine deaminase (A.D.); Determination radiochromatographique de l'adenosine deaminase (A.D.)

    Energy Technology Data Exchange (ETDEWEB)

    Chivot, J J; Depernet, D; Caen, J [Commissariat a l' Energie Atomique, Bruyeres-le-Chatel (France). Centre d' Etudes

    1970-07-01

    We were able, by using a radio-chromatographic method, to measure an adenosine deaminase activity in normal human heparinized platelet-poor plasma, which can degrade 0.016 {mu}M adenosine. This activity suppressed by heating 56 C for 30 minutes is inhibited by high concentrations of urea and is proportional to the amount of plasma, source of enzyme, in the systems. (authors) [French] Nous avons pu, en utilisant une methode radiochromatographique, mesurer une activite adenosine deaminasique dans le plasma humain pauvre en plaquettes heparine qui peut degrader 0,016 {mu}M d'adenosine. Cette activite qui est supprimee par chauffage a 56 degres pendant 30 minutes, est reduite par conservation a -20 C pendant une semaine, est inhibee par d'importantes concentrations d'uree et ne l'est pas, ni par le dipyridamol, ni par le pHMB. Cette activite est proportionnelle a la quantite de plasma, source d'enzyme, mise dans les differents systemes reactifs. (auteur)

  2. Serum adenosine deaminase activity and its isoenzyme in patients treated for tuberculosis

    International Nuclear Information System (INIS)

    Rokayan, S.A

    2003-01-01

    Objective: Increased serum adenosine deaminase (ADA) activity, mainly associated with tuberculosis can also occur in a number of other diseases thus negatively affecting the diagnostic utility of ADA measurements in tuberculosis. The aim of the study was to determine whether or not the combined use of the activity of ADA, its isoenzymes and differential cell counts would provide a more efficient means of diagnosing tuberculosis than the use of ADA levels alone. Results: Data suggested significant (p 0.75) of ADA/sub 2/ADA was found to be better indicator of tuberculosis. Lymphocyte neutrophil ratio (L/N)> 0.69 gave additional benefit to increase the sensitivity and specificity for the use of ADA as marker in diagnosing tuberculosis. Conclusion: The combined use of activity of ADA, its isoenzymes and total and differential cell counts is a better indicator and gives better understanding to diagnose and evaluate tuberculosis and response to therapy. (author)

  3. Acquisition of Genetic Aberrations by Activation-Induced Cytidine Deaminase (AID) during Inflammation-Associated Carcinogenesis

    International Nuclear Information System (INIS)

    Takai, Atsushi; Marusawa, Hiroyuki; Chiba, Tsutomu

    2011-01-01

    Genetic abnormalities such as nucleotide alterations and chromosomal disorders that accumulate in various tumor-related genes have an important role in cancer development. The precise mechanism of the acquisition of genetic aberrations, however, remains unclear. Activation-induced cytidine deaminase (AID), a nucleotide editing enzyme, is essential for the diversification of antibody production. AID is expressed only in activated B lymphocytes under physiologic conditions and induces somatic hypermutation and class switch recombination in immunoglobulin genes. Inflammation leads to aberrant AID expression in various gastrointestinal organs and increased AID expression contributes to cancer development by inducing genetic alterations in epithelial cells. Studies of how AID induces genetic disorders are expected to elucidate the mechanism of inflammation-associated carcinogenesis

  4. Nitric oxide - an activating factor of adenosine deaminase 2 in vitro.

    Science.gov (United States)

    Sargisova, Ye G; Andreasyan, N A; Hayrapetyan, H L; Harutyunyan, H A

    2012-01-01

    In this study we have investigated the effect of reactive oxygen species produced by some chemicals in aqueous solutions on activity of adenosine deaminase 2 (ADA2) purified from human blood plasma. An activating effect on ADA2 was observed in vitro with sodium nitroprusside (SNP), the source of NO (nitrosonium ions NO(-) in aqueous solutions). Not SH-groups of cysteine but other amino acid residues sensitive to NO were responsible for ADA2 activation. The SNP-derived activation was more pronounced when purified ADA2 was preincubated with heparin and different proteins as an experimental model of the protein environment in vivo. The most effective was heparin, which is known for its ability to regulate enzyme and protein functions in extracellular matrix. We conclude that ADA2 is a protein with flexible conformation that is affected by the protein environment, and it changes its activity under oxidative (nitrosative) stress.

  5. A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

    Science.gov (United States)

    Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

    2011-05-01

    A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

  6. Adenosine Deaminase (ADA)-Deficient Severe Combined Immune Deficiency (SCID): Molecular Pathogenesis and Clinical Manifestations.

    Science.gov (United States)

    Bradford, Kathryn L; Moretti, Federico A; Carbonaro-Sarracino, Denise A; Gaspar, Hubert B; Kohn, Donald B

    2017-10-01

    Deficiency of adenosine deaminase (ADA, EC3.5.4.4), a housekeeping enzyme of purine metabolism encoded by the Ada gene, is a cause of human severe combined immune deficiency (SCID). Numerous deleterious mutations occurring in the ADA gene have been found in patients with profound lymphopenia (T - B - NK - ), thus underscoring the importance of functional purine metabolism for the development of the immune defense. While untreated ADA SCID is a fatal disorder, there are multiple life-saving therapeutic modalities to restore ADA activity and reconstitute protective immunity, including enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) with autologous gene-corrected hematopoietic stem cells (HSC). We review the pathogenic mechanisms and clinical manifestations of ADA SCID.

  7. Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells

    International Nuclear Information System (INIS)

    Wilson, J.M.; Danos, O.; Grossman, M.; Raulet, D.H.; Mulligan, R.C.

    1990-01-01

    Recombinant retroviruses encoding human adenosine deaminase have been used to infect murine hematopoietic stem cells. In bone marrow transplant recipients reconstituted with the genetically modified cells, human ADA was detected in peripheral blood mononuclear cells of the recipients for at least 6 months after transplantation. In animals analyzed in detail 4 months after transplantation, human ADA and proviral sequences were detected in all hematopoietic lineages; in several cases, human ADA activity exceeded the endogenous activity. These studies demonstrate the feasibility of introducing a functional human ADA gene into hematopoietic stem cells and obtaining expression in multiple hematopoietic lineages long after transplantation. This approach should be helpful in designing effective gene therapies for severe combined immunodeficiency syndromes in humans

  8. Conformational change of adenosine deaminase during ligand-exchange in a crystal.

    Science.gov (United States)

    Kinoshita, Takayoshi; Tada, Toshiji; Nakanishi, Isao

    2008-08-15

    Adenosine deaminase (ADA) perpetuates chronic inflammation by degrading extracellular adenosine which is toxic for lymphocytes. ADA has two distinct conformations: open form and closed form. From the crystal structures with various ligands, the non-nucleoside type inhibitors bind to the active site occupying the critical water-binding-position and sustain the open form of apo-ADA. In contrast, substrate mimics do not occupy the critical position, and induce the large conformational change to the closed form. However, it is difficult to predict the binding of (+)-erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), as it possesses characteristic parts of both the substrate and the non-nucleoside inhibitors. The crystal structure shows that EHNA binds to the open form through a novel recognition of the adenine base accompanying conformational change from the closed form of the PR-ADA complex in crystalline state.

  9. IgG4-related Pleuritis with Elevated Adenosine Deaminase in Pleural Effusion: A Case Report.

    Science.gov (United States)

    Nagayasu, Atsushi; Kubo, Satoshi; Nakano, Kazuhisa; Nakayamada, Shingo; Iwata, Shigeru; Miyagawa, Ippei; Fukuyo, Shunsuke; Saito, Kazuyoshi; Tanaka, Yoshiya

    2018-03-09

    An 81-year-old man was admitted with bilateral pleural effusion. A clinical examination showed lymphocytic pleura effusion and elevated serum IgG4 levels, so that IgG4-related disease was suggested, whereas tuberculous pleurisy was suspected because of high adenosine deaminase (ADA) levels in the pleural effusion. A surgical pleural biopsy revealed that there were large numbers of IgG4-positive cells and IgG4/IgG positive cell ratio exceeded 40% in several sites. Accordingly, we diagnosed IgG4-related pleuritis and treated with the patient with glucocorticoid therapy. The ADA levels in pleural effusion can increase in IgG4-related pleuritis, and it is therefore important to perform a pleural biopsy.

  10. ACC deaminase and IAA producing growth promoting bacteria from the rhizosphere soil of tropical rice plants.

    Science.gov (United States)

    Bal, Himadri Bhusan; Das, Subhasis; Dangar, Tushar K; Adhya, Tapan K

    2013-12-01

    Beneficial plant-associated bacteria play a key role in supporting and/or promoting plant growth and health. Plant growth promoting bacteria present in the rhizosphere of crop plants can directly affect plant metabolism or modulate phytohormone production or degradation. We isolated 355 bacteria from the rhizosphere of rice plants grown in the farmers' fields in the coastal rice field soil from five different locations of the Ganjam district of Odisha, India. Six bacteria producing both ACC deaminase (ranging from 603.94 to 1350.02 nmol α-ketobutyrate mg(-1)  h(-1) ) and indole acetic acid (IAA; ranging from 10.54 to 37.65 μM ml(-1) ) in pure cultures were further identified using polyphasic taxonomy including BIOLOG((R)) , FAME analysis and the 16S rRNA gene sequencing. Phylogenetic analyses of the isolates resulted into five major clusters to include members of the genera Bacillus, Microbacterium, Methylophaga, Agromyces, and Paenibacillus. Seed inoculation of rice (cv. Naveen) by the six individual PGPR isolates had a considerable impact on different growth parameters including root elongation that was positively correlated with ACC deaminase activity and IAA production. The cultures also had other plant growth attributes including ammonia production and at least two isolates produced siderophores. Study indicates that presence of diverse rhizobacteria with effective growth-promoting traits, in the rice rhizosphere, may be exploited for a sustainable crop management under field conditions. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Effects of an induced adenosine deaminase deficiency on T-cell differentiation in the rat

    International Nuclear Information System (INIS)

    Barton, R.W.

    1985-01-01

    Inherited deficiency of the enzyme adenosine deaminase (ADA) has been found in a significant proportion of patients with severe combined immunodeficiency disease and inherited defect generally characterized by a deficiency of both B and T cells. Two questions are central to understanding the pathophysiology of this disease: (1) at what stage or stages in lymphocyte development are the effects of the enzyme deficiency manifested; (2) what are the biochemical mechanisms responsible for the selective pathogenicity of the lymphoid system. We have examined the stage or stages of rat T-cell development in vivo which are affected by an induced adenosine deaminase deficiency using the ADA inhibitors, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and 2'-deoxycoformycin (DCF). In normal rats given daily administration of an ADA inhibitor, cortical thymocytes were markedly depleted; peripheral lymphocytes and pluripotent hemopoietic stem cells (CFU-S) all were relatively unaffected. Since a deficiency of ADA affects lymphocyte development, the regeneration of cortical and medullary thymocytes and their precursors after sublethal irradiation was used as a model of lymphoid development. By Day 5 after irradiation the thymus was reduced to 0.10-0.5% of its normal size; whereas at Days 9 and 14 the thymus was 20-40% and 60-80% regenerated, respectively. When irradiated rats were given daily parenteral injections of the ADA inhibitor plus adenosine or deoxyadenosine, thymus regeneration at Days 9 and 14 was markedly inhibited, whereas the regeneration of thymocyte precursors was essentially unaffected. Thymus regeneration was at least 40-fold lower than in rats given adenosine or deoxyadenosine alone. Virtually identical results were obtained with both ADA inhibitors, EHNA and DCF

  12. A putative antiviral role of plant cytidine deaminases [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Susana Martín

    2017-06-01

    Full Text Available Background: A mechanism of innate antiviral immunity operating against viruses infecting mammalian cells has been described during the last decade.  Host cytidine deaminases (e.g., APOBEC3 proteins edit viral genomes, giving rise to hypermutated nonfunctional viruses; consequently, viral fitness is reduced through lethal mutagenesis.  By contrast, sub-lethal hypermutagenesis may contribute to virus evolvability by increasing population diversity.  To prevent genome editing, some viruses have evolved proteins that mediate APOBEC3 degradation.  The model plant Arabidopsis thaliana genome encodes nine cytidine deaminases (AtCDAs, raising the question of whether deamination is an antiviral mechanism in plants as well. Methods: Here we tested the effects of expression of AtCDAs on the pararetrovirus Cauliflower mosaic virus (CaMV. Two different experiments were carried out. First, we transiently overexpressed each one of the nine A. thaliana AtCDA genes in Nicotiana bigelovii plants infected with CaMV, and characterized the resulting mutational spectra, comparing them with those generated under normal conditions.  Secondly, we created A. thaliana transgenic plants expressing an artificial microRNA designed to knock-out the expression of up to six AtCDA genes.  This and control plants were then infected with CaMV.  Virus accumulation and mutational spectra where characterized in both types of plants. Results:  We have shown that the A. thaliana AtCDA1 gene product exerts a mutagenic activity, significantly increasing the number of G to A mutations in vivo, with a concomitant reduction in the amount of CaMV genomes accumulated.  Furthermore, the magnitude of this mutagenic effect on CaMV accumulation is positively correlated with the level of AtCDA1 mRNA expression in the plant. Conclusions: Our results suggest that deamination of viral genomes may also work as an antiviral mechanism in plants.

  13. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Wei, Shi-Cheng, E-mail: kqsc-wei@bjmu.edu.cn [Peking University School of Stomatology, Beijing 100081 (China); Liang, Yu-He, E-mail: kqsc-wei@bjmu.edu.cn; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China)

    2007-09-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.

  14. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    International Nuclear Information System (INIS)

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong; Wei, Shi-Cheng; Liang, Yu-He; Su, Xiao-Dong

    2007-01-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å

  15. One adenosine deaminase allele in a patient with severe combined immunodeficiency contains a point mutation abolishing enzyme activity.

    OpenAIRE

    Valerio, D; Dekker, B M; Duyvesteyn, M G; van der Voorn, L; Berkvens, T M; van Ormondt, H; van der Eb, A J

    1986-01-01

    We have cloned and sequenced an adenosine deaminase (ADA) gene from a patient with severe combined immunodeficiency (SCID) caused by inherited ADA deficiency. Two point mutations were found, resulting in amino acid substitutions at positions 80 (Lys to Arg) and 304 (Leu to Arg) of the protein. Hybridization experiments with synthetic oligonucleotide probes showed that the determined mutations are present in both DNA and RNA from the ADA-SCID patient. In addition, wild-type sequences could be ...

  16. Increased activity of vascular adenosine deaminase in atherosclerosis and therapeutic potential of its inhibition.

    Science.gov (United States)

    Kutryb-Zajac, Barbara; Mateuszuk, Lukasz; Zukowska, Paulina; Jasztal, Agnieszka; Zabielska, Magdalena A; Toczek, Marta; Jablonska, Patrycja; Zakrzewska, Agnieszka; Sitek, Barbara; Rogowski, Jan; Lango, Romuald; Slominska, Ewa M; Chlopicki, Stefan; Smolenski, Ryszard T

    2016-11-01

    Extracellular nucleotides and adenosine that are formed or degraded by membrane-bound ecto-enzymes could affect atherosclerosis by regulating the inflammation and thrombosis. This study aimed to evaluate a relation between ecto-enzymes that convert extracellular adenosine triphosphate to adenine dinucleotide phosphate, adenosine monophosphate, adenosine, and inosine on the surface of the vessel wall with the severity or progression of experimental and clinical atherosclerosis. Furthermore, we tested whether the inhibition of adenosine deaminase will block the development of experimental atherosclerosis. Vascular activities of ecto-nucleoside triphosphate diphosphohydrolase 1, ecto-5'-nucleotidase, and ecto-adenosine deaminase (eADA) were measured in aortas of apolipoprotein E-/- low density lipoprotein receptor (ApoE-/-LDLR-/-) and wild-type mice as well as in human aortas. Plaques were analysed in the entire aorta, aortic root, and brachiocephalic artery by Oil-Red O and Orcein Martius Scarlet Blue staining and vascular accumulation of macrophages. The cellular location of ecto-enzymes was analysed by immunofluorescence. The effect of eADA inhibition on atherosclerosis progression was studied by a 2-month deoxycoformycin treatment of ApoE-/-LDLR-/- mice. The vascular eADA activity prominently increased in ApoE-/-LDLR-/- mice when compared with wild type already at the age of 1 month and progressed along atherosclerosis development, reaching a 10-fold difference at 10 months. The activity of eADA correlated with atherosclerotic changes in human aortas. High abundance of eADA in atherosclerotic vessels originated from activated endothelial cells and macrophages. There were no changes in ecto-nucleoside triphosphate diphosphohydrolase 1 activity, whereas ecto-5'-nucleotidase was moderately decreased in ApoE-/-LDLR-/- mice. Deoxycoformycin treatment attenuated plaque development in aortic root and brachiocephalic artery of ApoE-/-LDLR-/- mice, suppressed vascular

  17. Identification of a New Uncompetitive Inhibitor of Adenosine Deaminase from Endophyte Aspergillus niger sp.

    Science.gov (United States)

    Zhang, Xin-Guo; Liu, Jin-Wen; Tang, Peng; Liu, Zi-Yu; Guo, Guang-Jun; Sun, Qiao-Yun; Yin, Jian-Jun

    2018-05-01

    Adenosine deaminase (ADA) is an enzyme widely distributed from bacteria to humans. ADA is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Endophytes are endosymbionts, often bacteria or fungi, which live within plant tissues and internal organs or intercellular space. Endophytes have a broad variety of bioactive metabolites that are used for the identification of novel natural compounds. Here, 54 morphologically distinct endophyte strains were isolated from six plants such as Peganum harmala Linn., Rheum officinale Baill., Gentiana macrophylla Pall., Radix stephaniae tetrandrae, Myrrha, and Equisetum hyemale Linn. The isolated strains were used for the search of ADA inhibitors that resulted in the identification of the strain with the highest inhibition activity, Aspergillus niger sp. Four compounds were isolated from this strain using three-step chromatography procedure, and compound 2 was determined as the compound with the highest inhibition activity of ADA. Based on the results of 1 H and 13 C NMR spectroscopies, compound 2 was identified as 3-(4-nitrophenyl)-5-phenyl isoxazole. We showed that compound 2 was a new uncompetitive inhibitor of ADA with high cytotoxic effect on HepG2 and SMCC-7721 cells (the IC 50 values were 0.347 and 0.380 mM, respectively). These results suggest that endophyte strains serve as promising sources for the identification of ADA inhibitors, and compound 2 could be an effective drug in the cancer treatment.

  18. Activation-induced cytidine deaminase deficiency causes organ-specific autoimmune disease.

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    Koji Hase

    Full Text Available Activation-induced cytidine deaminase (AID expressed by germinal center B cells is a central regulator of somatic hypermutation (SHM and class switch recombination (CSR. Humans with AID mutations develop not only the autosomal recessive form of hyper-IgM syndrome (HIGM2 associated with B cell hyperplasia, but also autoimmune disorders by unknown mechanisms. We report here that AID-/- mice spontaneously develop tertiary lymphoid organs (TLOs in non-lymphoid tissues including the stomach at around 6 months of age. At a later stage, AID-/- mice develop a severe gastritis characterized by loss of gastric glands and epithelial hyperplasia. The disease development was not attenuated even under germ-free (GF conditions. Gastric autoantigen -specific serum IgM was elevated in AID-/- mice, and the serum levels correlated with the gastritis pathological score. Adoptive transfer experiments suggest that autoimmune CD4+ T cells mediate gastritis development as terminal effector cells. These results suggest that abnormal B-cell expansion due to AID deficiency can drive B-cell autoimmunity, and in turn promote TLO formation, which ultimately leads to the propagation of organ-specific autoimmune effector CD4+ T cells. Thus, AID plays an important role in the containment of autoimmune diseases by negative regulation of autoreactive B cells.

  19. The diagnostic value of procalcitonin, adenosine deaminase for tuberculous pleural effusions

    International Nuclear Information System (INIS)

    Sun Jia; Jing Xiufeng; Hui Fuxin

    2010-01-01

    Objective: To explore differential diagnostic value of procalcitonin (PCT), adenosine deaminase (ADA) in pleural fluid and serum for tuberculous pleural effusions. Methods: The concentrations of PCT and ADA both in serum and pleural fluid in one hundred and twenty-eight patients with pleural effusion were detected. These patients were divided into three groups. Fifty-two patients with tuberculous plueral effusion were composed of the tuberculous group. Twenty-two patients with parapneumonic effusion composed the pneumonic group and forty patients with malignant pleural effusion and fourteen patients with heart faliure composed of the control group. Results: There were no statistically significant differences in serum PCT among the three groups (P > 0.05). PCT of pleural fluid was significantly increased in tuberculous and parapneumonic groups compared to the control group (P < 0.05). ADA activities in tuberculous serum and pleural fluid were both higher than those in the parapneumonic and the control groups (P < 0.01). The ratio of ADA in pleural fluid and serum (P /S) was calculated. The diagnostic sensitivity and specificity of P /S (cut-off value 1.27) were 92.3% and 100% respectively for tuberculous pleural effusions calcuted by receiver operating curve. Conclusion: Combined measurements of PCT and ADA in pleural fluid are useful in diagnosing tuberculous pleural effusions. (authors)

  20. Raised Serum Adenosine Deaminase Level in Nonobese Type 2 Diabetes Mellitus

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    Vineet Kumar Khemka

    2013-01-01

    Full Text Available The role of inflammation being minimal in the pathogenesis of type 2 diabetes mellitus (T2DM in nonobese patients; the aim of the study was to investigate the role of adenosine deaminase (ADA and see its association with diabetes mellitus. The preliminary case control study comprised of 56 cases and 45 healthy controls which were age and sex matched. 3 mL venous blood samples were obtained from the patients as well as controls after 8–10 hours of fasting. Serum ADA and routine biochemical parameters were analyzed. Serum ADA level was found significantly higher among nonobese T2DM subjects with respect to controls (38.77±14.29 versus 17.02±5.74 U/L; P<0.0001. Serum ADA level showed a significant positive correlation with fasting plasma glucose (r=0.657; P<0.0001 level among nonobese T2DM subjects, but no significant correlation was observed in controls (r=-0.203; P=0.180. However, no correlation was observed between serum ADA level compared to BMI and HbA1c levels. Our study shows higher serum ADA, triglycerides (TG and fasting plasma glucose (FPG levels in nonobese T2DM patients, and a strong correlation between ADA and FPG which suggests an association between ADA and nonobese T2DM subjects.

  1. AMP Deaminase 3 Deficiency Enhanced 5′-AMP Induction of Hypometabolism

    Science.gov (United States)

    Daniels, Isadora Susan; O′Brien, William G.; Nath, Vinay; Zhao, Zhaoyang; Lee, Cheng Chi

    2013-01-01

    A hypometabolic state can be induced in mice by 5′-AMP administration. Previously we proposed that an underlying mechanism for this hypometabolism is linked to reduced erythrocyte oxygen transport function due to 5′-AMP uptake altering the cellular adenylate equilibrium. To test this hypothesis, we generated mice deficient in adenosine monophosphate deaminase 3 (AMPD3), the key catabolic enzyme for 5′-AMP in erythrocytes. Mice deficient in AMPD3 maintained AMPD activities in all tissues except erythrocytes. Developmentally and morphologically, the Ampd3−/− mice were indistinguishable from their wild type siblings. The levels of ATP, ADP but not 5′-AMP in erythrocytes of Ampd3−/− mice were significantly elevated. Fasting blood glucose levels of the Ampd3−/− mice were comparable to wild type siblings. In comparison to wild type mice, the Ampd3−/− mice displayed a deeper hypometabolism with a significantly delayed average arousal time in response to 5′-AMP administration. Together, these findings demonstrate a central role of AMPD3 in the regulation of 5′-AMP mediated hypometabolism and further implicate erythrocytes in this behavioral response. PMID:24066180

  2. AMP deaminase histochemical activity and immunofluorescent isozyme localization in rat skeletal muscle

    Science.gov (United States)

    Thompson, J. L.; Sabina, R. L.; Ogasawara, N.; Riley, D. A.

    1992-01-01

    The cellular distribution of AMP deaminase (AMPda) isozymes was documented for rat soleus and plantaris muscles, utilizing immunofluorescence microscopy and immunoprecipitation methods. AMPda is a ubiquitous enzyme existing as three distinct isozymes, A, B and C, which were initially purified from skeletal muscle, liver (and kidney), and heart, respectively. AMPda-A is primarily concentrated subsarcolemmally and intermyofibrillarly within muscle cells, while isozymes B and C are concentrated within non-myofiber elements of muscle tissue. AMPda-B is principally associated with connective tissues surrounding neural elements and the muscle spindle capsule, and AMPda-C is predominantly associated with circulatory elements, such as arterial and venous walls, capillary endothelium, and red blood cells. These specific localizations, combined with documented differences in kinetic properties, suggest multiple functional roles for the AMPda isozymes or temporal segregation of similar AMPda functions. Linkage of the AMPda substrate with adenosine production pathways at the AMP level and the localization of isozyme-C in vascular tissue suggest a regulatory role in the microcirculation.

  3. Adenosine Deaminase Inhibitor EHNA Exhibits a Potent Anticancer Effect Against Malignant Pleural Mesothelioma

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    Yasuhiro Nakajima

    2015-01-01

    Full Text Available Background/Aims: Malignant pleural mesothelioma (MPM is an aggressive malignant tumor and an effective therapy has been little provided as yet. The present study investigated the possibility for the adenosine deaminase (ADA inhibitor EHNA as a target of MPM treatment. Methods: MTT assay, TUNEL staining, monitoring of intracellular adenosine concentrations, and Western blotting were carried out in cultured human MPM cell lines without and with knocking-down ADA. The in vivo effect of EHNA was assessed in mice inoculated with NCI-H2052 MPM cells. Results: EHNA induced apoptosis of human MPM cell lines in a concentration (0.01-1 mM- and treatment time (24-48 h-dependent manner, but such effect was not obtained with another ADA inhibitor pentostatin. EHNA increased intracellular adenosine concentrations in a treatment time (3-9 h-dependent manner. EHNA-induced apoptosis of MPM cells was mimicked by knocking-down ADA, and the effect was neutralized by the adenosine kinase inhibitor ABT-702. EHNA clearly suppressed tumor growth in mice inoculated with NCI-H2052 MPM cells. Conclusion: The results of the present study show that EHNA induces apoptosis of MPM cells by increasing intracellular adenosine concentrations, to convert to AMP, and effectively prevents MPM cell proliferation. This suggests that EHNA may be useful for treatment of the tragic neoplasm MPM.

  4. Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

    Science.gov (United States)

    Senavirathne, Gayan; Bertram, Jeffrey G.; Jaszczur, Malgorzata; Chaurasiya, Kathy R.; Pham, Phuong; Mak, Chi H.; Goodman, Myron F.; Rueda, David

    2015-12-01

    Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ~5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.

  5. ADENOSINE DEAMINASE ACTIVITY AND SERUM C-REACTIVE PROTEIN AS PROGNOSTIC MARKERS OF CHAGAS DISEASE SEVERITY

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    Iván Darío BRAVO-TOBAR

    2015-10-01

    Full Text Available SUMMARY Chagas disease is a public health problem worldwide. The availability of diagnostic tools to predict the development of chronic Chagas cardiomyopathy is crucial to reduce morbidity and mortality. Here we analyze the prognostic value of adenosine deaminase serum activity (ADA and C-reactive protein serum levels (CRP in chagasic individuals. One hundred and ten individuals, 28 healthy and 82 chagasic patients were divided according to disease severity in phase I (n = 35, II (n = 29, and III (n = 18. A complete medical history, 12-lead electrocardiogram, chest X-ray, and M-mode echocardiogram were performed on each individual. Diagnosis of Chagas disease was confirmed by ELISA and MABA using recombinant antigens; ADA was determined spectrophotometrically and CRP by ELISA. The results have shown that CRP and ADA increased linearly in relation to disease phase, CRP being significantly higher in phase III and ADA at all phases. Also, CRP and ADA were positively correlated with echocardiographic parameters of cardiac remodeling and with electrocardiographic abnormalities, and negatively with ejection fraction. CRP and ADA were higher in patients with cardiothoracic index ≥ 50%, while ADA was higher in patients with ventricular repolarization disturbances. Finally, CRP was positively correlated with ADA. In conclusion, ADA and CRP are prognostic markers of cardiac dysfunction and remodeling in Chagas disease.

  6. A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1.

    Science.gov (United States)

    Wang, Yuru; Havel, Jocelyn; Beal, Peter A

    2015-11-20

    Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. ADAR1 and ADAR2 are two members of the family that have been shown to be catalytically active. Earlier, we reported a phenotypic screen for the study of human ADAR2 using budding yeast S. cerevisiae as the host system. While this screen has been successfully applied to the study of ADAR2, it failed with ADAR1. Here, we report a new reporter that uses a novel editing substrate and is suitable for the study of ADAR1. We screened plasmid libraries with randomized codons for two important residues in human ADAR1 (G1007 and E1008). The screening results combined with in vitro deamination assays led to the identification of mutants that are more active than the wild type protein. Furthermore, a screen of the ADAR1 E1008X library with a reporter construct bearing an A•G mismatch at the editing site suggests one role for the residue at position 1008 is to sense the identity of the base pairing partner for the editing site adenosine. This work has provided a starting point for future in vitro evolution studies of ADAR1 and led to new insight into ADAR's editing site selectivity.

  7. Significance of determination of serum cytidine deaminase (CD) levels for diagnosis of active rheumatoid arthritis (RA)

    International Nuclear Information System (INIS)

    Xiao Chuangqing; Jang Xiaogong; He Yunnan

    2005-01-01

    Objective: To determine the clinical value of measurement of serum cytidine deaminase (CD) levels in patients with active rheumatoid arthritis (RA). Methods: Serum levels of CD were detected with spectrophotometry, in 33 patients with active RA and 60 controls. The erythrocyte sedimentation rate (ESR) and CRP content were also determined in both groups. Results: The ser- um CD contents in patients with active RA(14.80 ± 2.11U/ml) were significantly higher than those in controls(4.86±1.86 U/ml,P<0.01). The CRP contents (51.46 ± 20.43mg/L) and ESR readings(85.03 ± 27.6mm/h) in the patients were also significantly higher than those in the controls(3.40 ± 2.21mg/L and 13.04 ± 4.89mm/h respectively, all P<0.01). In the patients, the serum CD contents were linearly positively correlated with the ESR contents and CRP readings (r=0.6324 and 0.8013 respectively, P <0.01). Conclusion: Serum CD is an early biochemical marker for diagnosis of active rheumatoid arthritis and is also of prognostic value. (authors)

  8. Drought response of Mucuna pruriens (L. DC. inoculated with ACC deaminase and IAA producing rhizobacteria.

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    Aansa Rukya Saleem

    Full Text Available Drought is one of the major constraints limiting agricultural production worldwide and is expected to increase in the future. Limited water availability causes significant effects to plant growth and physiology. Plants have evolved different traits to mitigate the stress imposed by drought. The presence of plant growth-promoting rhizobacteria (PGPR could play an important role in improving plant performances and productivity under drought. These beneficial microorganisms colonize the rhizosphere of plants and increase drought tolerance by lowering ethylene formation. In the present study, we demonstrate the potential to improve the growth of velvet bean under water deficit conditions of two different strains of PGPR with ACCd (1-Aminocyclopropane-1-Carboxylate deaminase activity isolated from rainfed farming system. We compared uninoculated and inoculated plants with PGPR to assess: a photosynthetic performance and biomass; b ACC content and ethylene emission from leaves and roots; c leaf isoprene emission. Our results provided evidence that under drought conditions inoculation with PGPR containing the ACCd enzyme could improve plant growth compared to untreated plants. Ethylene emission from roots and leaves of inoculated velvet bean plants was significantly lower than uninoculated plants. Moreover, isoprene emission increased with drought stress progression and was higher in inoculated plants compared to uninoculated counterparts. These findings clearly illustrate that selected PGPR strains isolated from rainfed areas could be highly effective in promoting plant growth under drought conditions by decreasing ACC and ethylene levels in plants.

  9. Drought response of Mucuna pruriens (L.) DC. inoculated with ACC deaminase and IAA producing rhizobacteria.

    Science.gov (United States)

    Saleem, Aansa Rukya; Brunetti, Cecilia; Khalid, Azeem; Della Rocca, Gianni; Raio, Aida; Emiliani, Giovanni; De Carlo, Anna; Mahmood, Tariq; Centritto, Mauro

    2018-01-01

    Drought is one of the major constraints limiting agricultural production worldwide and is expected to increase in the future. Limited water availability causes significant effects to plant growth and physiology. Plants have evolved different traits to mitigate the stress imposed by drought. The presence of plant growth-promoting rhizobacteria (PGPR) could play an important role in improving plant performances and productivity under drought. These beneficial microorganisms colonize the rhizosphere of plants and increase drought tolerance by lowering ethylene formation. In the present study, we demonstrate the potential to improve the growth of velvet bean under water deficit conditions of two different strains of PGPR with ACCd (1-Aminocyclopropane-1-Carboxylate deaminase) activity isolated from rainfed farming system. We compared uninoculated and inoculated plants with PGPR to assess: a) photosynthetic performance and biomass; b) ACC content and ethylene emission from leaves and roots; c) leaf isoprene emission. Our results provided evidence that under drought conditions inoculation with PGPR containing the ACCd enzyme could improve plant growth compared to untreated plants. Ethylene emission from roots and leaves of inoculated velvet bean plants was significantly lower than uninoculated plants. Moreover, isoprene emission increased with drought stress progression and was higher in inoculated plants compared to uninoculated counterparts. These findings clearly illustrate that selected PGPR strains isolated from rainfed areas could be highly effective in promoting plant growth under drought conditions by decreasing ACC and ethylene levels in plants.

  10. Diagnostic value of adenosine deaminase in ascites for tuberculosis ascites: a meta-analysis.

    Science.gov (United States)

    Tao, Lin; Ning, Hong-Jian; Nie, Hai-Ming; Guo, Xiao-Yun; Qin, Shan-Yu; Jiang, Hai-Xing

    2014-05-01

    The diagnosis of tuberculosis (TB) ascites using standard diagnostic tools is difficult. The aim of the present meta-analysis was to establish the overall diagnostic accuracy of adenosine deaminase (ADA) levels in ascites for diagnosing TB ascites. A systematic review was performed of English language publications prior to April 2013. Sensitivity, specificity, and other measures of the accuracy of ADA for the diagnosis of TB ascites using ascites fluid were summarized using a random-effects model or a fixed-effects model. Receiver operating characteristic curves were used to summarize overall test performance. Seventeen studies involving 1797 subjects were eligible for the analysis. The summary estimates of sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and the area under cure of overall analysis were: 0.93, 0.94, 13.55, 0.11, 169.83, and 0.976, respectively; the results of sensitivity analysis of studies that used Giusti method were 0.94, 0.94, 12.99, 0.08, 183.18, and 0.977, respectively. Our results suggest that ADA in the ascites can be a sensitive and specific target and a critical criterion for the diagnosis of TB ascites. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Activation-induced cytidine deaminase (AID) is localized to subnuclear domains enriched in splicing factors

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    Hu, Yi, E-mail: yihooyi@gmail.com; Ericsson, Ida, E-mail: ida.ericsson@ntnu.no; Doseth, Berit, E-mail: berit.doseth@ntnu.no; Liabakk, Nina B., E-mail: nina.beate.liabakk@ntnu.no; Krokan, Hans E., E-mail: hans.krokan@ntnu.no; Kavli, Bodil, E-mail: bodil.kavli@ntnu.no

    2014-03-10

    Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation. - Highlights: • AID and its interaction partner CTNNBL1 localize to Cajal bodies and nuclear speckles. • AID associates with its activating kinase PKA in nuclear speckles. • AID is linked to the splicing machinery in switching B-cells. • Our findings suggest a transcription-coupled splicing associated mechanism for AID targeting and activation.

  12. Restoration of adenosine deaminase-deficient human thymocyte development in vitro by inhibition of deoxynucleoside kinases.

    Science.gov (United States)

    Joachims, Michelle L; Marble, Patrick A; Laurent, Aletha B; Pastuszko, Peter; Paliotta, Marco; Blackburn, Michael R; Thompson, Linda F

    2008-12-01

    Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture to study ADA-deficient human thymocyte development in an "in vivo-like" environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular dATP. Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients.

  13. Involvement of activation-induced cytidine deaminase in skin cancer development.

    Science.gov (United States)

    Nonaka, Taichiro; Toda, Yoshinobu; Hiai, Hiroshi; Uemura, Munehiro; Nakamura, Motonobu; Yamamoto, Norio; Asato, Ryo; Hattori, Yukari; Bessho, Kazuhisa; Minato, Nagahiro; Kinoshita, Kazuo

    2016-04-01

    Most skin cancers develop as the result of UV light-induced DNA damage; however, a substantial number of cases appear to occur independently of UV damage. A causal link between UV-independent skin cancers and chronic inflammation has been suspected, although the precise mechanism underlying this association is unclear. Here, we have proposed that activation-induced cytidine deaminase (AID, encoded by AICDA) links chronic inflammation and skin cancer. We demonstrated that Tg mice expressing AID in the skin spontaneously developed skin squamous cell carcinoma with Hras and Trp53 mutations. Furthermore, genetic deletion of Aicda reduced tumor incidence in a murine model of chemical-induced skin carcinogenesis. AID was expressed in human primary keratinocytes in an inflammatory stimulus-dependent manner and was detectable in human skin cancers. Together, the results of this study indicate that inflammation-induced AID expression promotes skin cancer development independently of UV damage and suggest AID as a potential target for skin cancer therapeutics.

  14. Adenosine deaminase complexing protein (ADCP) expression and metastatic potential in prostatic adenocarcinomas.

    Science.gov (United States)

    Dinjens, W N; Ten Kate, J; Kirch, J A; Tanke, H J; Van der Linden, E P; Van den Ingh, H F; Van Steenbrugge, G J; Meera Khan, P; Bosman, F T

    1990-03-01

    The expression of the adenosine deaminase complexing protein (ADCP) was investigated by immunohistochemistry in the normal and hyperplastic human prostate, in 30 prostatic adenocarcinomas, and in seven human prostatic adenocarcinoma cell lines grown as xenografts in athymic nude mice. In the normal and hyperplastic prostate, ADCP was localized exclusively in the apical membrane and the apical cytoplasm of the glandular epithelial cells. In prostatic adenocarcinomas, four distinct ADCP expression patterns were observed: diffuse cytoplasmic, membranous, both cytoplasmic and membranous, and no ADCP expression. The expression patterns were compared with the presence of metastases. We found an inverse correlation between membranous ADCP immunoreactivity and metastatic propensity. Exclusively membranous ADCP immunoreactivity occurred only in non-metastatic tumours. In contrast, the metastatic tumours showed no or diffuse cytoplasmic ADCP immunoreactivity. This suggests that immunohistochemical detection of ADCP might predict the biological behaviour of prostatic cancer. However, the occurrence of membranous ADCP immunoreactivity in the xenograft of a cell line (PC-EW), derived from a prostatic carcinoma metastasis, indicates that not only the tendency to metastasize modulates ADCP expression.

  15. Assessment of adenosine deaminase (ADA) activity and oxidative stress in patients with chronic tonsillitis.

    Science.gov (United States)

    Garca, Mehmet Fatih; Demir, Halit; Turan, Mahfuz; Bozan, Nazım; Kozan, Ahmet; Belli, Şeyda Bayel; Arslan, Ayşe; Cankaya, Hakan

    2014-06-01

    To emphasize the effectiveness of adenosine deaminase (ADA) enzyme, which has important roles in the differentiation of lymphoid cells, and oxidative stress in patients with chronic tonsillitis. Serum and tissue samples were obtained from 25 patients who underwent tonsillectomy due to recurrent episodes of acute tonsillitis. In the control group, which also had 25 subjects, only serum samples were taken as obtaining tissue samples would not have been ethically appropriate. ADA enzyme activity, catalase (CAT), carbonic anhydrase (CA), nitric oxide (NO) and malondialdehyde (MDA) were measured in the serum and tissue samples of patients and control group subjects. The serum values of both groups were compared. In addition, the tissue and serum values of patients were compared. Serum ADA activity and the oxidant enzymes MDA and NO values of the patient group were significantly higher than those of the control group (p ADA activity (p > 0.05). Elevated ADA activity may be effective in the pathogenesis of chronic tonsillitis both by impairing tissue structure and contributing to SOR formation.

  16. Regulation of adenosine deaminase (ADA) on induced mouse experimental autoimmune uveitis (EAU) ‡

    Science.gov (United States)

    Liang, Dongchun; Zuo, Aijun; Zhao, Ronglan; Shao, Hui; Kaplan, Henry J.; Sun, Deming

    2016-01-01

    Adenosine is an important regulator of the immune response and adenosine deaminase (ADA) inhibits this regulatory effect by converting adenosine into functionally inactive molecules. Studies have shown that adenosine receptor (AR) agonists can be either anti- or pro-inflammatory. Clarification of the mechanisms that cause these opposing effects should provide a better guide for therapeutic intervention. In this study, we investigated the effect of ADA on the development of experimental autoimmune uveitis (EAU) induced by immunizing EAU-prone mice with a known uveitogenic peptide, IRBP1–20. Our results showed that the effective time to administer a single dose of ADA to suppress induction of EAU was 8–14 days post-immunization, shortly before EAU expression, but ADA treatment at other time points exacerbated disease. ADA preferentially inhibited Th17 responses and this effect was γδ T cell-dependent. Our results demonstrated that the existing immune status strongly influences the anti- or proinflammatory effects of ADA. Our observations should help improve the design of ADA- and AR-targeted therapies. PMID:26856700

  17. Diagnostic Utility of Pleural Fluid Adenosine Deaminase Level in Tuberculousis Pleural Effusion

    International Nuclear Information System (INIS)

    Suleman, A.; Abbasi, M. A.; Anwar, S. A.; Kamal, M.; Khan, H.

    2016-01-01

    Background: Early diagnosis and management of tuberculosis is essential for decreasing the disease burden. Pakistan is one of the few countries of world with a very high burden of tuberculosis. Many diagnostic tests are available for detection of tuberculosis but each is fraught with certain limitations of its own. Methods: This study was a cross sectional validation study that sought to determine the validity of pleural fluid adenosine deaminase levels for diagnosis of tuberculous pleural effusion. Results: A total of 160 patients with exudative lymphocytic pleural effusions were enrolled in this study. The mean pleural fluid ADA level was 52.18±1.98 U/L. The mean pleural fluid ADA level in patients diagnosed to have tuberculosis on pleural biopsy/histopathology was higher as compared to patients who did not have tuberculous pleural effusion 52.16±2.4 U/L vs 38.6±3.14 U/L. The difference was found to be statistically significant between the two groups (p<0.05). The sensitivity, specificity, ppv and npv of pleural fluid ADA level were 88.88 percent, 77.04 percent, 86.28 percent and 81.04 percent respectively. Conclusion: Despite wide variations in the reported sensitivity and specificity of pleural fluid ADA level, it can be used as a surrogate for pleural biopsy when the latter is not feasible. (author)

  18. Radiochromatographic determination of activity of adenosine deaminase and purine nucleoside phosphorylase in blood cells

    International Nuclear Information System (INIS)

    Pechan, I.; Rendekova, V.; Pechanova, E.; Krizko, J.

    1982-01-01

    Expeditious and sensitive methods are described for determining the activities of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in human lymphocytes and erythrocytes. ADA and PNP activity is determined on the basis of the reaction of (U- 14 C)adenosine or (8- 14 C)inosine with the lysate of human blood cells. Reaction products are separated using paper chromatography. Following the measurement of the radioactivity of spots of adenosine, inosine and hypoxanthine, a calculation is made of ADA and PNP activity from the results of the said measurements. On a sample of 52 clinically healthy people average ADA and PNP activity in isolated lymphocytes was found to be (51.6+-18.8) and (185.6+-94.7) pcat/10 6 cells and in erythrocytes (9.8+-2.98) and (17.1+-3.19) pcat/mg of proteins, respectively. The advantage of the method is the small amount of sample needed (1 to 2 ml) which allows its application in pediatrics. (Ha)

  19. Investigations into the origin of the molecular recognition of several adenosine deaminase inhibitors.

    Science.gov (United States)

    Gillerman, Irina; Fischer, Bilha

    2011-01-13

    Inhibitors of adenosine deaminase (ADA, EC 3.5.4.4) are potential therapeutic agents for the treatment of various health disorders. Several highly potent inhibitors were previously identified, yet they exhibit unacceptable toxicities. We performed a SAR study involving a series of C2 or C8 substituted purine-riboside analogues with a view to discover less potent inhibitors with a lesser toxicity. We found that any substitution at C8 position of nebularine resulted in total loss of activity toward calf intestinal ADA. However, several 2-substituted-adenosine, 8-aza-adenosine, and nebularine analogues exhibited inhibitory activity. Specifically, 2-Cl-purine riboside, 8-aza-2-thiohexyl adenosine, 2-thiohexyl adenosine, and 2-MeS-purine riboside were found to be competitive inhibitors of ADA with K(i) values of 25, 22, 6, and 3 μM, respectively. We concluded that electronic parameters are not major recognition determinants of ADA but rather steric parameters. A C2 substituent which fits ADA hydrophobic pocket and improves H-bonding with the enzyme makes a good inhibitor. In addition, a gg rotamer about C4'-C5' bond is apparently an important recognition determinant.

  20. Diagnostic value of pleural fluid adenosine deaminase activity in tuberculosis pleurisy

    Directory of Open Access Journals (Sweden)

    Abbas ali Niazi

    2009-09-01

    Full Text Available Background: Diagnosis of tuberculosis pleurisies is difficult because of its nonspecific clinical presentation and insufficient traditional diagnostic methods. We investigated the use of adenosine deaminase (ADA activity in tuberculosis pleurisies. Methods: A number of 85 patients were analyzed with exudative pleural effusions. Using the ROC curve, we determined the optimal cutoff for TB pleurisy. Results: A number of 58 exudative samples were nontuberculous (non-TB and 27 were tuberculosis (TB. There was statistically significant difference (p<0.0001 between the means of pleural fluid ADA levels among the TB and non-TB populations. The prevalence of TB pleurisy in the studied population was 31%. Using the cutoff point equal to 35 for diagnosing TB effusions the sensitivity and specificity 70.3% and 91.3%, respectively. The positive predictive value (PPV was 79.1% and the negative predictive value (NPV was 86.8%. A pleural fluid ADA value <19 IU/L suggests that a tuberculosis effusion is highly unlikely. Conclusion: Pleural fluid total ADA assay is a sensitive and specific test suitable for rapid diagnosis of TB pleurisy.

  1. Adaptive evolution of threonine deaminase in plant defense against insect herbivores

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    Gonzales-Vigil, Eliana; Bianchetti, Christopher M.; Phillips, Jr., George N.; Howe, Gregg A. (MSU); (UW)

    2011-11-07

    Gene duplication is a major source of plant chemical diversity that mediates plant-herbivore interactions. There is little direct evidence, however, that novel chemical traits arising from gene duplication reduce herbivory. Higher plants use threonine deaminase (TD) to catalyze the dehydration of threonine (Thr) to {alpha}-ketobutyrate and ammonia as the committed step in the biosynthesis of isoleucine (Ile). Cultivated tomato and related Solanum species contain a duplicated TD paralog (TD2) that is coexpressed with a suite of genes involved in herbivore resistance. Analysis of TD2-deficient tomato lines showed that TD2 has a defensive function related to Thr catabolism in the gut of lepidopteran herbivores. During herbivory, the regulatory domain of TD2 is removed by proteolysis to generate a truncated protein (pTD2) that efficiently degrades Thr without being inhibited by Ile. We show that this proteolytic activation step occurs in the gut of lepidopteran but not coleopteran herbivores, and is catalyzed by a chymotrypsin-like protease of insect origin. Analysis of purified recombinant enzymes showed that TD2 is remarkably more resistant to proteolysis and high temperature than the ancestral TD1 isoform. The crystal structure of pTD2 provided evidence that electrostatic interactions constitute a stabilizing feature associated with adaptation of TD2 to the extreme environment of the lepidopteran gut. These findings demonstrate a role for gene duplication in the evolution of a plant defense that targets and co-opts herbivore digestive physiology.

  2. Development of Highly Sensitive and Specific mRNA Multiplex System (XCYR1) for Forensic Human Body Fluids and Tissues Identification

    Science.gov (United States)

    Xu, Yan; Xie, Jianhui; Cao, Yu; Zhou, Huaigu; Ping, Yuan; Chen, Liankang; Gu, Lihua; Hu, Wei; Bi, Gang; Ge, Jianye; Chen, Xin; Zhao, Ziqin

    2014-01-01

    The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples. PMID:24991806

  3. A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase.

    Science.gov (United States)

    Li, Z; Chang, S; Lin, L; Li, Y; An, Q

    2011-08-01

    1-Aminocyclopropane-1-carboxylate (ACC) deaminase activity is an efficient marker for bacteria to promote plant growth by lowering ethylene levels in plants. We aim to develop a method for rapidly screening bacteria containing ACC deaminase, based on a colorimetric ninhydrin assay of ACC. A reliable colorimetric ninhydrin assay was developed to quantify ACC using heat-resistant polypropylene chimney-top 96-well PCR plates, having the wells evenly heated in boiling water, preventing accidental contamination from boiling water and limiting evaporation. With this method to measure bacterial consumption of ACC, 44 ACC-utilizing bacterial isolates were rapidly screened out from 311 bacterial isolates that were able to grow on minimal media containing ACC as the sole nitrogen source. The 44 ACC-utilizing bacterial isolates showed ACC deaminase activities and belonged to the genus Burkholderia, Pseudomonas or Herbaspirillum. Determination of bacterial ACC consumption by the PCR-plate ninhydrin-ACC assay is a rapid and efficient method for screening bacteria containing ACC deaminase from a large number of bacterial isolates. The PCR-plate ninhydrin-ACC assay extends the utility of the ninhydrin reaction and enables a rapid screening of bacteria containing ACC deaminase from large numbers of bacterial isolates. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

  4. Diagnostic significance of pleural fluid adenosine deaminase activity in tuberculous pleurisy

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    Sharmeen Ahmed

    2011-01-01

    Full Text Available Diagnosis of tuberculous pleural effusion (TPE is difficult because of its non-specific clinical presentation and insufficient efficiency of conventional diagnostic methods. The study was carried out to evaluate the utility of adenosine deaminase (ADA activity in pleural fluid for the diagnosis of TPE. ADA activity was measured in pleural fluid of 103 pleural effusion patients by colorimetric method using a commercial ADA assay kit. The diagnosis of TPE was made from pleural fluid examinations (including cytology, biochemistry, and bacteriology and pleural biopsy. Patient with negative result of this methods were diagnosed by response of empirical treatment. Out of 130 cases, 62 (61.1% had TPE and the remaining 41 (39.8% had pleural effusion due to non tuberculous diseases. There was statistically significant difference (p < 0.001 between the mean of pleural fluid ADA levels (70.82±22.54 U/L in TPE group and (30.07±22.93 U/L in non-TPE group. Of 62 TPE cases, microscopy for AFB and culture for M.tuberculosis in pleural fluid revealed positivity in 9.6% and 22.5% cases respectively, and biopsy of pleura showed typical epithelioid granuloma in only 43.5% cases. The cut-off value of ADA for diagnosing TPE was 40 U/L using a ROC curve, with a sensitivity of 94% and specificity of 88%. Positive and negative predictive value of ADA assay were 92% and 90% respectively. The overall test accuracy was 90%. Pleural fluid ADA assay is therefore a simple, rapid, highly sensitive and specific adjunct test for diagnosis of TPE. Ibrahim Med. Coll. J. 2011; 5(1: 1-5

  5. Cerebrospinal fluid adenosine deaminase levels as a diagnostic marker in tuberculous meningitis in adult Nepalese patients

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    Anil Chander

    2013-02-01

    Full Text Available Objective: To study the cerebrospinal fluid (CSF adenosine deaminase (ADA levels in tuberculous meningitis (TBM and non-TBM -viral meningitis cases and to determine its diagnostic significance as a biochemical marker of TBM infection.Methods: The study population comprised two different patient groups. TBM - group I - 28 cases and non-TBM-viral meningitis - 22 cases. These were enrolled consecutively in the study and CSF specimens were collected from them. ADA estimation was carried out by spectrophotometry.Results: ADA levels (mean依 SD in the TBM and non-TBM groups were 16.46依6.24 U/L and 5.13依2.96 U/L, respectively (highly significant P10 IU/L, the test showed a good sensitivity of 82.14% (95% CI 64.41-92.12 and a high specificity of 90.91% (95% CI 72.19-97.47. Positive and negative predictive value and positive and negative likelihood ratios and accuracy of the test in TBM cases were 92% (95% CI 75.03-97.77, 80% (95% CI 60.86-91.13, 9.03 (95% CI 2.38- 34.25, 0.19 (95% CI 0.09-0.44 and 86%, respectively.Conclusion: CSF ADA levels are elevated in the TBM cases as compared to the non-TBM - viral meningitis cases with a good sensitivity and a high specificity. It is a simple and inexpensive diagnostic adjunctive test in the rapid and early diagnosis of TBM.

  6. Autoimmune dysregulation and purine metabolism in adenosine deaminase (ADA-deficiency

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    Aisha Vanessa Sauer

    2012-08-01

    Full Text Available Genetic defects in the adenosine deaminase (ADA gene are among the most common causes for severe combined immunodeficiency (SCID. ADA-SCID patients suffer from lymphopenia, severely impaired cellular and humoral immunity, failure to thrive and recurrent infections. Currently available therapeutic options for this otherwise fatal disorder include bone marrow transplantation (BMT, enzyme replacement therapy with bovine ADA (PEG-ADA or hematopoietic stem cell gene therapy (HSC-GT. Although varying degrees of immune reconstitution can be achieved by these treatments, breakdown of tolerance is a major concern in ADA-SCID. Immune dysregulation such as autoimmune hypothyroidism, diabetes mellitus, hemolytic anemia, and immune thrombocytopenia are frequently observed in milder forms of the disease. However, several reports document similar complications also in patients on long-term PEG-ADA and after BMT or GT treatment.A skewed repertoire and decreased immune functions have been implicated in autoimmunity observed in certain B-cell and/or T-cell immunodeficiencies, but it remains unclear to what extent specific mechanisms of tolerance are affected in ADA deficiency. Herein we provide an overview about ADA-SCID and the autoimmune manifestations reported in these patients before and after treatment. We also assess the value of the ADA-deficient mouse model as a useful tool to study both immune and metabolic disease mechanisms. With focus on regulatory T and B cells we discuss the lymphocyte subpopulations particularly prone to contribute to the loss of self-tolerance and onset of autoimmunity in ADA deficiency. Moreover we address which aspects of immune dysregulation are specifically related to alterations in purine metabolism caused by the lack of ADA and the subsequent accumulation of metabolites with immunomodulatory properties.

  7. Cerebrospinal fluid adenosine deaminase activity: A complimentary tool in the early diagnosis of tuberculous meningitis

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    Taori Girdhar M

    2006-03-01

    Full Text Available Abstract Background Tuberculous meningitis (TBM is the commonest form of neurotuberculosis caused by Mycobacterium tuberculosis bacilli (MTB. The diagnosis of TBM is often difficult. A reliable, cost-effective and rapid diagnostic test, which can be performed in any standard pathology laboratory, could be of help in the diagnosis of TBM. In the present study we measured the adenosine deaminase (ADA activity in cerebrospinal fluid (CSF of TBM and non-TBM patients. Method ADA activity in CSF was determined according to a method based on the Berthlot reaction, which is the formation of a colored indophenol complex from ammonia liberated from adenosine, and quantified spectrophotometrically. Results The CSF ADA activity from TBM patients was compared with CSF ADA from non-TBM infectious meningitis patients, and from patients with non-infectious neurological disorders. The mean CSF ADA activity was found to be significantly higher in CSF of TBM patients, 14.31 ± 3.87 (2.99–26.94, mean ± SD with range, than in the CSF from non-TBM infectious meningitis, 9.25 ± 2.14 (4.99–13.96 and from the non-infectious neurological disorders group, 2.71 ± 1.96 (0.00–7.68, P Conclusion This study demonstrated that ADA activity in the CSF of TBM patients, using a cut-off value 11.39 U/L/min, can be useful for the early differential diagnosis of TBM. This test can be performed in any pathology laboratory where more sophisticated methods are not available.

  8. Diagnostic Algorithm for Glycogenoses and Myoadenylate Deaminase Deficiency Based on Exercise Testing Parameters: A Prospective Study.

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    Fabrice Rannou

    Full Text Available Our aim was to evaluate the accuracy of aerobic exercise testing to diagnose metabolic myopathies.From December 2008 to September 2012, all the consecutive patients that underwent both metabolic exercise testing and a muscle biopsy were prospectively enrolled. Subjects performed an incremental and maximal exercise testing on a cycle ergometer. Lactate, pyruvate, and ammonia concentrations were determined from venous blood samples drawn at rest, during exercise (50% predicted maximal power, peak exercise, and recovery (2, 5, 10, and 15 min. Biopsies from vastus lateralis or deltoid muscles were analysed using standard techniques (reference test. Myoadenylate deaminase (MAD activity was determined using p-nitro blue tetrazolium staining in muscle cryostat sections. Glycogen storage was assessed using periodic acid-Schiff staining. The diagnostic accuracy of plasma metabolite levels to identify absent and decreased MAD activity was assessed using Receiver Operating Characteristic (ROC curve analysis.The study involved 51 patients. Omitting patients with glycogenoses (n = 3, MAD staining was absent in 5, decreased in 6, and normal in 37 subjects. Lactate/pyruvate at the 10th minute of recovery provided the greatest area under the ROC curves (AUC, 0.893 ± 0.067 to differentiate Abnormal from Normal MAD activity. The lactate/rest ratio at the 10th minute of recovery from exercise displayed the best AUC (1.0 for discriminating between Decreased and Absent MAD activities. The resulting decision tree achieved a diagnostic accuracy of 86.3%.The present algorithm provides a non-invasive test to accurately predict absent and decreased MAD activity, facilitating the selection of patients for muscle biopsy and target appropriate histochemical analysis.

  9. Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins.

    Science.gov (United States)

    Moreno, Estefanía; Canet, Júlia; Gracia, Eduard; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Cortés, Antoni; Casadó, Vicent

    2018-01-01

    Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A 2A R present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A 2A R and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A 2A R involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A 2A R-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A 2A R). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits.

  10. Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

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    Monica K Akre

    Full Text Available Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

  11. Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

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    Estefanía Moreno

    2018-02-01

    Full Text Available Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26 and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET, we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26 and dendritic cells (expressing A2AR. This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector without partitioning these functions in different subunits.

  12. Diagnostic value of sputum adenosine deaminase (ADA) level in pulmonary tuberculosis.

    Science.gov (United States)

    Binesh, Fariba; Jalali, Hadi; Zare, Mohammad Reza; Behravan, Farhad; Tafti, Arefeh Dehghani; Behnaz, Fatemah; Tabatabaee, Mohammad; Shahcheraghi, Seyed Hossein

    2016-06-01

    Tuberculosis is still a considerable health problem in many countries. Rapid diagnosis of this disease is important, and adenosine deaminase (ADA) has been used as a diagnostic test. The aim of this study was to assess the diagnostic value of ADA in the sputum of patients with pulmonary tuberculosis. The current study included 40 patients with pulmonary tuberculosis (culture positive, smear ±) and 42 patients with non tuberculosis pulmonary diseases (culture negative). ADA was measured on all of the samples. The median value of ADA in non-tuberculosis patients was 2.94 (4.2) U/L and 4.01 (6.54) U/L in tuberculosis patients, but this difference was not statistically significant (p=0.100). The cut-off point of 3.1 U/L had a sensitivity of 61% and a specificity of 53%, the cut-off point of 2.81 U/L had a sensitivity of 64% and a specificity of 50% and the cut-off point of 2.78 U/L had a sensitivity of 65% and a specificity of 48%. The positive predictive values for cut-off points of 3.1, 2.81 and 2.78 U/L were 55.7%, 57.44% and 69.23%, respectively. The negative predictive values for the abovementioned cut-off points were 56.75%, 57.14% and 55.88%, respectively. Our results showed that sputum ADA test is neither specific nor sensitive. Because of its low sensitivity and specificity, determination of sputum ADA for the diagnosis of pulmonary tuberculosis is not recommended.

  13. Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing

    International Nuclear Information System (INIS)

    Akeson, A.L.; Wiginton, D.A.; States, C.J.; Perme, C.M.; Dusing, M.R.; Hutton, J.J.

    1987-01-01

    Adenosine deaminase deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, the authors synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNA showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These change do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency

  14. Diagnosis of tuberculosis pleurisy with adenosine deaminase (ADA): a systematic review and meta-analysis.

    Science.gov (United States)

    Gui, Xuwei; Xiao, Heping

    2014-01-01

    This systematic review and meta-analysis was performed to determine accuracy and usefulness of adenosine deaminase (ADA) in diagnosis of tuberculosis pleurisy. Medline, Google scholar and Web of Science databases were searched to identify related studies until 2014. Two reviewers independently assessed quality of studies included according to standard Quality Assessment of Diagnosis Accuracy Studies (QUADAS) criteria. The sensitivity, specificity, diagnostic odds ratio and other parameters of ADA in diagnosis of tuberculosis pleurisy were analyzed with Meta-DiSC1.4 software, and pooled using the random effects model. Twelve studies including 865 tuberculosis pleurisy patients and 1379 non-tuberculosis pleurisy subjects were identified from 110 studies for this meta-analysis. The sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnosis odds ratio (DOR) of ADA in the diagnosis of tuberculosis pleurisy were 45.25 (95% CI 27.63-74.08), 0.86 (95% CI 0.84-0.88), 0.88 (95% CI 0.86-0.90), 6.32 (95% CI 4.83-8.26) and 0.15 (95% 0.11-0.22), respectively. The area under the summary receiver operating characteristic curve (SROC) was 0.9340. Our results demonstrate that the sensitivity and specificity of ADA are high in the diagnosis of tuberculosis pleurisy especially when ADA≥50 (U/L). Thus, ADA is a relatively sensitive and specific marker for tuberculosis pleurisy diagnosis. However, it is cautious to apply these results due to the heterogeneity in study design of these studies. Further studies are required to confirm the optimal cut-off value of ADA.

  15. Clinical efficacy of gene-modified stem cells in adenosine deaminase-deficient immunodeficiency.

    Science.gov (United States)

    Shaw, Kit L; Garabedian, Elizabeth; Mishra, Suparna; Barman, Provaboti; Davila, Alejandra; Carbonaro, Denise; Shupien, Sally; Silvin, Christopher; Geiger, Sabine; Nowicki, Barbara; Smogorzewska, E Monika; Brown, Berkley; Wang, Xiaoyan; de Oliveira, Satiro; Choi, Yeong; Ikeda, Alan; Terrazas, Dayna; Fu, Pei-Yu; Yu, Allen; Fernandez, Beatriz Campo; Cooper, Aaron R; Engel, Barbara; Podsakoff, Greg; Balamurugan, Arumugam; Anderson, Stacie; Muul, Linda; Jagadeesh, G Jayashree; Kapoor, Neena; Tse, John; Moore, Theodore B; Purdy, Ken; Rishi, Radha; Mohan, Kathey; Skoda-Smith, Suzanne; Buchbinder, David; Abraham, Roshini S; Scharenberg, Andrew; Yang, Otto O; Cornetta, Kenneth; Gjertson, David; Hershfield, Michael; Sokolic, Rob; Candotti, Fabio; Kohn, Donald B

    2017-05-01

    Autologous hematopoietic stem cell transplantation (HSCT) of gene-modified cells is an alternative to enzyme replacement therapy (ERT) and allogeneic HSCT that has shown clinical benefit for adenosine deaminase-deficient (ADA-deficient) SCID when combined with reduced intensity conditioning (RIC) and ERT cessation. Clinical safety and therapeutic efficacy were evaluated in a phase II study. Ten subjects with confirmed ADA-deficient SCID and no available matched sibling or family donor were enrolled between 2009 and 2012 and received transplantation with autologous hematopoietic CD34+ cells that were modified with the human ADA cDNA (MND-ADA) γ-retroviral vector after conditioning with busulfan (90 mg/m2) and ERT cessation. Subjects were followed from 33 to 84 months at the time of data analysis. Safety of the procedure was assessed by recording the number of adverse events. Efficacy was assessed by measuring engraftment of gene-modified hematopoietic stem/progenitor cells, ADA gene expression, and immune reconstitution. With the exception of the oldest subject (15 years old at enrollment), all subjects remained off ERT with normalized peripheral blood mononuclear cell (PBMC) ADA activity, improved lymphocyte numbers, and normal proliferative responses to mitogens. Three of nine subjects were able to discontinue intravenous immunoglobulin replacement therapy. The MND-ADA vector was persistently detected in PBMCs (vector copy number [VCN] = 0.1-2.6) and granulocytes (VCN = 0.01-0.3) through the most recent visits at the time of this writing. No patient has developed a leukoproliferative disorder or other vector-related clinical complication since transplant. These results demonstrate clinical therapeutic efficacy from gene therapy for ADA-deficient SCID, with an excellent clinical safety profile. ClinicalTrials.gov NCT00794508. Food and Drug Administration Office of Orphan Product Development award, RO1 FD003005; NHLBI awards, PO1 HL73104 and Z01 HG000122; UCLA

  16. Evaluation of the adenosine deaminase (ADA) G22A gene polymorphism with recurrent spontaneous abortion among Egyptian patients

    Science.gov (United States)

    Abu-Gabal, Khadiga; Katta, Maha; Ibrahim, Raghda

    2017-01-01

    Introduction Adenosine and deoxyadenosine metabolism is influenced by adenosine deaminase (ADA) enzyme. ADA increases in different diseases and is considered as one of the markers for cell-mediated immunity. Pregnancy is associated with depressed cell-mediated immunity. The level of ADA expression, which seems to play a key role in maintaining pregnancy, is influenced by adenosine deaminase G22A gene polymorphism. We aimed in our study to evaluate the association of ADA G22A gene polymorphism with recurrent spontaneous abortion (RSA) in Egyptian women. Material and methods Adenosine deaminase G22A gene polymorphism was genotyped in 40 patients (age range 22-39 years) with a history of RSA, selected from those attending the Gynaecology and Obstetrics Clinic of Beni-Suef University Hospital, and 20 age-matched healthy women as a control group, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Results In our study, no statistically significant difference was found between RSA patients and control group as regards ADA G22A genotypes (p = 0.653) and alleles (p = 0.697). A comparison of the frequencies of ADA alleles in RSA patients as regards the below-35-years-old age group revealed that ADA 2(A) allele was associated with a low risk for RSA in patients aged 35 years old or younger (p = 0.008). Conclusions In conclusion, our study revealed an age-dependent protective value of ADA 2(A) allele in recurrent spontaneous abortions among the Egyptian population. PMID:29204093

  17. Diversification of AID/APOBEC-like deaminases in metazoa: multiplicity of clades and widespread roles in immunity.

    Science.gov (United States)

    Krishnan, Arunkumar; Iyer, Lakshminarayan M; Holland, Stephen J; Boehm, Thomas; Aravind, L

    2018-04-03

    AID/APOBEC deaminases (AADs) convert cytidine to uridine in single-stranded nucleic acids. They are involved in numerous mutagenic processes, including those underpinning vertebrate innate and adaptive immunity. Using a multipronged sequence analysis strategy, we uncover several AADs across metazoa, dictyosteliida, and algae, including multiple previously unreported vertebrate clades, and versions from urochordates, nematodes, echinoderms, arthropods, lophotrochozoans, cnidarians, and porifera. Evolutionary analysis suggests a fundamental division of AADs early in metazoan evolution into secreted deaminases (SNADs) and classical AADs, followed by diversification into several clades driven by rapid-sequence evolution, gene loss, lineage-specific expansions, and lateral transfer to various algae. Most vertebrate AADs, including AID and APOBECs1-3, diversified in the vertebrates, whereas the APOBEC4-like clade has a deeper origin in metazoa. Positional entropy analysis suggests that several AAD clades are diversifying rapidly, especially in the positions predicted to interact with the nucleic acid target motif, and with potential viral inhibitors. Further, several AADs have evolved neomorphic metal-binding inserts, especially within loops predicted to interact with the target nucleic acid. We also observe polymorphisms, driven by alternative splicing, gene loss, and possibly intergenic recombination between paralogs. We propose that biological conflicts of AADs with viruses and genomic retroelements are drivers of rapid AAD evolution, suggesting a widespread presence of mutagenesis-based immune-defense systems. Deaminases like AID represent versions "institutionalized" from the broader array of AADs pitted in such arms races for mutagenesis of self-DNA, and similar recruitment might have independently occurred elsewhere in metazoa. Copyright © 2018 the Author(s). Published by PNAS.

  18. Pleural effusion adenosine deaminase: a candidate biomarker to discriminate between Gram-negative and Gram-positive bacterial infections of the pleural space

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    Ruolin Li

    2016-05-01

    Full Text Available OBJECTIVES: Delay in the treatment of pleural infection may contribute to its high mortality. In this retrospective study, we aimed to evaluate the diagnostic accuracy of pleural adenosine deaminase in discrimination between Gram-negative and Gram-positive bacterial infections of the pleural space prior to selecting antibiotics. METHODS: A total of 76 patients were enrolled and grouped into subgroups according to Gram staining: 1 patients with Gram-negative bacterial infections, aged 53.2±18.6 years old, of whom 44.7% had empyemas and 2 patients with Gram-positive bacterial infections, aged 53.5±21.5 years old, of whom 63.1% had empyemas. The pleural effusion was sampled by thoracocentesis and then sent for adenosine deaminase testing, biochemical testing and microbiological culture. The Mann-Whitney U test was used to examine the differences in adenosine deaminase levels between the groups. Correlations between adenosine deaminase and specified variables were also quantified using Spearman’s correlation coefficient. Moreover, receiver operator characteristic analysis was performed to evaluate the diagnostic accuracy of pleural effusion adenosine deaminase. RESULTS: Mean pleural adenosine deaminase levels differed significantly between Gram-negative and Gram-positive bacterial infections of the pleural space (191.8±32.1 U/L vs 81.0±16.9 U/L, p<0.01. The area under the receiver operator characteristic curve was 0.689 (95% confidence interval: 0.570, 0.792, p<0.01 at the cutoff value of 86 U/L. Additionally, pleural adenosine deaminase had a sensitivity of 63.2% (46.0-78.2%; a specificity of 73.7% (56.9-86.6%; positive and negative likelihood ratios of 2.18 and 0.50, respectively; and positive and negative predictive values of 70.6% and 66.7%, respectively. CONCLUSIONS: Pleural effusion adenosine deaminase is a helpful alternative biomarker for early and quick discrimination of Gram-negative from Gram-positive bacterial infections of the

  19. Selection of efficient salt-tolerant bacteria containing ACC deaminase for promotion of tomato growth under salinity stress

    Directory of Open Access Journals (Sweden)

    Kannika Chookietwattana* and Kedsukon Maneewan

    2012-05-01

    Full Text Available For successful application of plant growth promoting bacteria (PGPB in salt-affected soil, bioinoculant with salt-tolerant property is required in order to provide better survival and perform well in the field. The present study aimed to select the most efficient salt-tolerant bacterium containing 1-aminocyclopropane-1-carboxylic acid (ACC deaminase from eighty four bacterial strains and to investigate the effects of the selected bacterium on the germination and growth of tomato (Licopersicon esculentum Mill. cv. Seeda under saline conditions. The Bacillus licheniformis B2r was selected for its ability to utilize ACC as a sole nitrogen source under salinity stress. It also showed a high ACC deaminase activity at 0.6 M NaCl salinity. Tomato plants inoculated with the selected bacterium under various saline conditions (0, 30, 60, 90 and 120 mM NaCl revealed a significant increase in the germination percentage, germination index, root length, and seedling dry weight especially at salinity levels ranging from 30-90 mM NaCl. The work described in this report is an important step in developing an efficient salt-tolerant bioinoculant to facilitate plant growth in saline soil.

  20. Crystallization and preliminary X-ray crystallographic analysis of the tRNA-specific adenosine deaminase from Streptococcus pyogenes

    Energy Technology Data Exchange (ETDEWEB)

    Ku, Min-Je [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Won-Ho [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biotechnology and Genetic Engineering, Korea University, Seoul 136-701 (Korea, Republic of); Nam, Ki-hyun; Rhee, Kyeong-hee [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Ki-Seog [Biotechnology and Genetic Engineering, Korea University, Seoul 136-701 (Korea, Republic of); Kim, Eunice EunKyung [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Yu, Myung-Hee [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Hwang, Kwang Yeon, E-mail: hwangky@kist.re.kr [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of)

    2005-04-01

    The tRNA-specific adenosine deaminase from the pathogenic bacteria S. pyogenes has been overexpressed and crystallized. The tRNA-specific adenosine deaminase from the pathogenic bacteria Streptococcus pyogenes (spTAD) has been overexpressed in Escherichia coli and crystallized in the presence of Zn{sup 2+} ion at 295 K using ammonium sulfate as a precipitant. Flash-cooled crystals of spTAD diffracted to 2.0 Å using 30%(v/v) glycerol as a cryoprotectant. X-ray diffraction data have been collected to 2.0 Å using synchrotron radiation. The crystal belongs to the tetragonal space group P4{sub 2}2{sub 1}2, with unit-cell parameters a = b = 81.042, c = 81.270 Å. The asymmetric unit contains one subunit of spTAD, with a corresponding crystal volume per protein weight (V{sub M}) of 3.3 Å{sup 3} Da{sup −1} and a solvent content of 62.7%.

  1. Regulation of 5'-adenosine monophosphate deaminase in the freeze tolerant wood frog, Rana sylvatica.

    Science.gov (United States)

    Dieni, Christopher A; Storey, Kenneth B

    2008-04-22

    The wood frog, Rana sylvatica, is one of a few vertebrate species that have developed natural freeze tolerance, surviving days or weeks with 65-70% of its total body water frozen in extracellular ice masses. Frozen frogs exhibit no vital signs and their organs must endure multiple stresses, particularly long term anoxia and ischemia. Maintenance of cellular energy supply is critical to viability in the frozen state and in skeletal muscle, AMP deaminase (AMPD) plays a key role in stabilizing cellular energetics. The present study investigated AMPD control in wood frog muscle. Wood frog AMPD was subject to multiple regulatory controls: binding to subcellular structures, protein phosphorylation, and effects of allosteric effectors, cryoprotectants and temperature. The percentage of bound AMPD activity increased from 20 to 35% with the transition to the frozen state. Bound AMPD showed altered kinetic parameters compared with the free enzyme (S0.5 AMP was reduced, Hill coefficient fell to approximately 1.0) and the transition to the frozen state led to a 3-fold increase in S0.5 AMP of the bound enzyme. AMPD was a target of protein phosphorylation. Bound AMPD from control frogs proved to be a low phosphate form with a low S0.5 AMP and was phosphorylated in incubations that stimulated PKA, PKC, CaMK, or AMPK. Bound AMPD from frozen frogs was a high phosphate form with a high S0.5 AMP that was reduced under incubation conditions that stimulated protein phosphatases. Frog muscle AMPD was activated by Mg.ATP and Mg.ADP and inhibited by Mg.GTP, KCl, NaCl and NH4Cl. The enzyme product, IMP, uniquely inhibited only the bound (phosphorylated) enzyme from muscle of frozen frogs. Activators and inhibitors differentially affected the free versus bound enzyme. S0.5 AMP of bound AMPD was also differentially affected by high versus low assay temperature (25 vs 5 degrees C) and by the presence/absence of the natural cryoprotectant (250 mM glucose) that accumulates during freezing

  2. Regulation of epithelial and lymphocyte cell adhesion by adenosine deaminase-CD26 interaction.

    Science.gov (United States)

    Ginés, Silvia; Mariño, Marta; Mallol, Josefa; Canela, Enric I; Morimoto, Chikao; Callebaut, Christian; Hovanessian, Ara; Casadó, Vicent; Lluis, Carmen; Franco, Rafael

    2002-01-01

    The extra-enzymic function of cell-surface adenosine deaminase (ADA), an enzyme mainly localized in the cytosol but also found on the cell surface of monocytes, B cells and T cells, has lately been the subject of numerous studies. Cell-surface ADA is able to transduce co-stimulatory signals in T cells via its interaction with CD26, an integral membrane protein that acts as ADA-binding protein. The aim of the present study was to explore whether ADA-CD26 interaction plays a role in the adhesion of lymphocyte cells to human epithelial cells. To meet this aim, different lymphocyte cell lines (Jurkat and CEM T) expressing endogenous, or overexpressing human, CD26 protein were tested in adhesion assays to monolayers of colon adenocarcinoma human epithelial cells, Caco-2, which express high levels of cell-surface ADA. Interestingly, the adhesion of Jurkat and CEM T cells to a monolayer of Caco-2 cells was greatly dependent on CD26. An increase by 50% in the cell-to-cell adhesion was found in cells containing higher levels of CD26. Incubation with an anti-CD26 antibody raised against the ADA-binding site or with exogenous ADA resulted in a significant reduction (50-70%) of T-cell adhesion to monolayers of epithelial cells. The role of ADA-CD26 interaction in the lymphocyte-epithelial cell adhesion appears to be mediated by CD26 molecules that are not interacting with endogenous ADA (ADA-free CD26), since SKW6.4 (B cells) that express more cell-surface ADA showed lower adhesion than T cells. Adhesion stimulated by CD26 and ADA is mediated by T cell lymphocyte function-associated antigen. A role for ADA-CD26 interaction in cell-to-cell adhesion was confirmed further in integrin activation assays. FACS analysis revealed a higher expression of activated integrins on T cell lines in the presence of increasing amounts of exogenous ADA. Taken together, these results suggest that the ADA-CD26 interaction on the cell surface has a role in lymphocyte-epithelial cell adhesion. PMID

  3. Restriction of Equine Infectious Anemia Virus by Equine APOBEC3 Cytidine Deaminases ▿ †

    Science.gov (United States)

    Zielonka, Jörg; Bravo, Ignacio G.; Marino, Daniela; Conrad, Elea; Perković, Mario; Battenberg, Marion; Cichutek, Klaus; Münk, Carsten

    2009-01-01

    The mammalian APOBEC3 (A3) proteins comprise a multigene family of cytidine deaminases that act as potent inhibitors of retroviruses and retrotransposons. The A3 locus on the chromosome 28 of the horse genome contains multiple A3 genes: two copies of A3Z1, five copies of A3Z2, and a single copy of A3Z3, indicating a complex evolution of multiple gene duplications. We have cloned and analyzed for expression the different equine A3 genes and examined as well the subcellular distribution of the corresponding proteins. Additionally, we have tested the functional antiretroviral activity of the equine and of several of the human and nonprimate A3 proteins against the Equine infectious anemia virus (EIAV), the Simian immunodeficiency virus (SIV), and the Adeno-associated virus type 2 (AAV-2). Hematopoietic cells of horses express at least five different A3s: A3Z1b, A3Z2a-Z2b, A3Z2c-Z2d, A3Z2e, and A3Z3, whereas circulating macrophages, the natural target of EIAV, express only part of the A3 repertoire. The five A3Z2 tandem copies arose after three consecutive, recent duplication events in the horse lineage, after the split between Equidae and Carnivora. The duplicated genes show different antiviral activities against different viruses: equine A3Z3 and A3Z2c-Z2d are potent inhibitors of EIAV while equine A3Z1b, A3Z2a-Z2b, A3Z2e showed only weak anti-EIAV activity. Equine A3Z1b and A3Z3 restricted AAV and all equine A3s, except A3Z1b, inhibited SIV. We hypothesize that the horse A3 genes are undergoing a process of subfunctionalization in their respective viral specificities, which might provide the evolutionary advantage for keeping five copies of the original gene. PMID:19458006

  4. Expression of activation-induced cytidine deaminase is confined to B-cell non-Hodgkin's lymphomas of germinal-center phenotype

    NARCIS (Netherlands)

    Smit, Laura A.; Bende, Richard J.; Aten, Jan; Guikema, Jeroen E. J.; Aarts, Wilhelmina M.; van Noesel, Carel J. M.

    2003-01-01

    Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation and class switch recombination of the immunoglobulin (IG) genes in B cells. It has recently been proposed that AID, as the newly identified DNA mutator in man, may be instrumental in initiation and progression of

  5. An Escherichia coli strain deficient for both exonuclease 5 and deoxycytidine triphosphate deaminase shows enhanced sensitivity to ionizing radiation

    International Nuclear Information System (INIS)

    Estevenon, A.M.; Kooistra, J.; Sicard, N.

    1995-01-01

    An Escherichia coli mutant lacking deoxycytidine triphosphate deaminase (Dcd) activity and an unknown function encoded by a gene designated ior exhibits sensitivity to ionizing radiation whereas dcd mutants themselves are not sensitive. A DNA fragment from an E. coli genomic library that restores the wild type level of UV and gamma ray resistance to this mutant has been cloned in the multicopy vector pBR322. Comparison of its restriction map with the physical map of the E. coli chromosome revealed complete identity to the recBD genes. ior affects ATP-dependent exonuclease activity, suggesting that it is an allele of recB. This mutation alone does not confer sensitivity to UV and gamma radiation, indicating that lack of Dcd activity is also required for expression of radiation sensitivity

  6. Non-linear quantitative structure-activity relationship for adenine derivatives as competitive inhibitors of adenosine deaminase

    International Nuclear Information System (INIS)

    Sadat Hayatshahi, Sayyed Hamed; Abdolmaleki, Parviz; Safarian, Shahrokh; Khajeh, Khosro

    2005-01-01

    Logistic regression and artificial neural networks have been developed as two non-linear models to establish quantitative structure-activity relationships between structural descriptors and biochemical activity of adenosine based competitive inhibitors, toward adenosine deaminase. The training set included 24 compounds with known k i values. The models were trained to solve two-class problems. Unlike the previous work in which multiple linear regression was used, the highest of positive charge on the molecules was recognized to be in close relation with their inhibition activity, while the electric charge on atom N1 of adenosine was found to be a poor descriptor. Consequently, the previously developed equation was improved and the newly formed one could predict the class of 91.66% of compounds correctly. Also optimized 2-3-1 and 3-4-1 neural networks could increase this rate to 95.83%

  7. Hyperbilirubinemia and rapid fatal hepatic failure in severe combined immunodeficiency caused by adenosine deaminase deficiency (ADA-SCID).

    Science.gov (United States)

    Kühl, J S; Schwarz, K; Münch, A; Schmugge, M; Pekrun, A; Meisel, C; Wahn, V; Ebell, W; von Bernuth, H

    2011-03-01

    Adenosin deaminase (ADA) deficiency is the cause for Severe Combined Immunodeficiency (SCID) in about 15% of patients with SCID, often presenting as T (-)B (-)NK (-)SCID. Treatment options for ADA-SCID are enzyme replacement, bone marrow transplantation or gene therapy. We here describe the first patient with ADA-SCID and fatal hepatic failure despite bone marrow transplantation from a 10/10 HLA identical related donor. As patients with ADA-SCID may be at yet underestimated increased risk for rapid hepatic failure we speculate whether hepatitis in ADA-SCID should lead to the immediate treatment with enzyme replacement by pegylated ADA. © Georg Thieme Verlag KG Stuttgart · New York.

  8. Cytidine deaminases from B. subtilis and E. coli: compensating effects of changing zinc coordination and quaternary structure.

    Science.gov (United States)

    Carlow, D C; Carter, C W; Mejlhede, N; Neuhard, J; Wolfenden, R

    1999-09-21

    Cytidine deaminase from E. coli is a dimer of identical subunits (M(r) = 31 540), each containing a single zinc atom. Cytidine deaminase from B. subtilis is a tetramer of identical subunits (M(r) = 14 800). After purification from an overexpressing strain, the enzyme from B. subtilis is found to contain a single atom of zinc per enzyme subunit by flame atomic absorption spectroscopy. Fluorescence titration indicates that each of the four subunits contains a binding site for the transition state analogue inhibitor 5-fluoro-3,4-dihydrouridine. A region of amino acid sequence homology, containing residues that are involved in zinc coordination in the enzyme from E. coli, strongly suggests that in the enzyme from B. subtilis, zinc is coordinated by the thiolate side chains of three cysteine residues (Cys-53, Cys-86, and Cys-89) [Song, B. H., and Neuhard, J. (1989) Mol. Gen. Genet. 216, 462-468]. This pattern of zinc coordination appears to be novel for a hydrolytic enzyme, and might be expected to reduce the reactivity of the active site substantially compared with that of the enzyme from E. coli (His-102, Cys-129, and Cys-132). Instead, the B. subtilis and E. coli enzymes are found to be similar in their activities, and also in their relative binding affinities for a series of structurally related inhibitors with binding affinities that span a range of 6 orders of magnitude. In addition, the apparent pK(a) value of the active site is shifted upward by less than 1 unit. Sequence alignments, together with model building, suggest one possible mechanism of compensation.

  9. Piracetam prevents scopolamine-induced memory impairment and decrease of NTPDase, 5'-nucleotidase and adenosine deaminase activities.

    Science.gov (United States)

    Marisco, Patricia C; Carvalho, Fabiano B; Rosa, Michelle M; Girardi, Bruna A; Gutierres, Jessié M; Jaques, Jeandre A S; Salla, Ana P S; Pimentel, Víctor C; Schetinger, Maria Rosa C; Leal, Daniela B R; Mello, Carlos F; Rubin, Maribel A

    2013-08-01

    Piracetam improves cognitive function in animals and in human beings, but its mechanism of action is still not completely known. In the present study, we investigated whether enzymes involved in extracellular adenine nucleotide metabolism, adenosine triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase and adenosine deaminase (ADA) are affected by piracetam in the hippocampus and cerebral cortex of animals subjected to scopolamine-induced memory impairment. Piracetam (0.02 μmol/5 μL, intracerebroventricular, 60 min pre-training) prevented memory impairment induced by scopolamine (1 mg/kg, intraperitoneal, immediately post-training) in the inhibitory avoidance learning and in the object recognition task. Scopolamine reduced the activity of NTPDase in hippocampus (53 % for ATP and 53 % for ADP hydrolysis) and cerebral cortex (28 % for ATP hydrolysis). Scopolamine also decreased the activity of 5'-nucleotidase (43 %) and ADA (91 %) in hippocampus. The same effect was observed in the cerebral cortex for 5'-nucleotidase (38 %) and ADA (68 %) activities. Piracetam fully prevented scopolamine-induced memory impairment and decrease of NTPDase, 5'-nucleotidase and adenosine deaminase activities in synaptosomes from cerebral cortex and hippocampus. In vitro experiments show that piracetam and scopolamine did not alter enzymatic activity in cerebral cortex synaptosomes. Moreover, piracetam prevented scopolamine-induced increase of TBARS levels in hippocampus and cerebral cortex. These results suggest that piracetam-induced improvement of memory is associated with protection against oxidative stress and maintenance of NTPDase, 5'-nucleotidase and ADA activities, and suggest the purinergic system as a putative target of piracetam.

  10. The genetics of feto-placental development: A study of acid phosphatase locus 1 and adenosine deaminase polymorphisms in a consecutive series of newborn infants

    Directory of Open Access Journals (Sweden)

    Bergamaschi Antonio

    2008-09-01

    Full Text Available Abstract Background Acid phosphatase locus 1 and adenosine deaminase locus 1 polymorphisms show cooperative effects on glucose metabolism and immunological functions. The recent observation of cooperation between the two systems on susceptibility to repeated spontaneous miscarriage prompted us to search for possible interactional effects between these genes and the correlation between birth weight and placental weight. Deviation from a balanced development of the feto-placental unit has been found to be associated with perinatal morbidity and mortality and with cardiovascular diseases in adulthood. Methods We examined 400 consecutive newborns from the Caucasian population of Rome. Birth weight, placental weight, and gestational length were registered. Acid phosphatase locus 1 and adenosine deaminase locus 1 phenotypes were determined by starch gel electrophoresis and correlation analysis was performed by SPSS programs. Informed verbal consent to participate in the study was obtained from the mothers. Results Highly significant differences in birth weight-placental weight correlations were observed among acid phosphatase locus 1 phenotypes (p = 0.005. The correlation between birth weight and placental weight was markedly elevated in subjects carrying acid phosphatase locus 1 phenotypes with medium-low F isoform concentration (A, CA and CB phenotypes compared to those carrying acid phosphatase locus 1 phenotypes with medium-high F isoform concentration (BA and B phenotypes (p = 0.002. Environmental and developmental variables were found to exert a significant effect on birth weight-placental weight correlation in subjects with medium-high F isoform concentrations, but only a marginal effect was observed in those with medium-low F isoform concentrations. The correlation between birth weight and placental weight is higher among carriers of the adenosine deaminase locus 1 allele*2, which is associated with low activity, than in homozygous adenosine

  11. Partial resolution of bone lesions. A child with severe combined immunodeficiency disease and adenosine deaminase deficiency after enzyme-replacement therapy

    International Nuclear Information System (INIS)

    Yulish, B.S.; Stern, R.C.; Polmar, S.H.

    1980-01-01

    A child with severe combined immunodeficiency disease and adenosine deaminase deficiency, with characteristic bone dysplasia, was treated with transfusions of frozen irradiated RBCs as a means of enzyme replacement. This therapy resulted in restoration of immunologic competence and partial resolution of the bone lesions. Although the natural history of these lesions without therapy is not known, enzyme-replacement therapy may have played a role in the resolution of this patient's bone lesions

  12. Asymmetric Modification of Hepatitis B Virus (HBV) Genomes by an Endogenous Cytidine Deaminase inside HBV Cores Informs a Model of Reverse Transcription.

    Science.gov (United States)

    Nair, Smita; Zlotnick, Adam

    2018-05-15

    Cytidine deaminases inhibit replication of a broad range of DNA viruses by deaminating cytidines on single-stranded DNA (ssDNA) to generate uracil. While several lines of evidence have revealed hepatitis B virus (HBV) genome editing by deamination, it is still unclear which nucleic acid intermediate of HBV is modified. Hepatitis B virus has a relaxed circular double-stranded DNA (rcDNA) genome that is reverse transcribed within virus cores from a RNA template. The HBV genome also persists as covalently closed circular DNA (cccDNA) in the nucleus of an infected cell. In the present study, we found that in HBV-producing HepAD38 and HepG2.2.15 cell lines, endogenous cytidine deaminases edited 10 to 25% of HBV rcDNA genomes, asymmetrically with almost all mutations on the 5' half of the minus strand. This region corresponds to the last half of the minus strand to be protected by plus-strand synthesis. Within this half of the genome, the number of mutations peaks in the middle. Overexpressed APOBEC3A and APOBEC3G could be packaged in HBV capsids but did not change the amount or distribution of mutations. We found no deamination on pregenomic RNA (pgRNA), indicating that an intact genome is encapsidated and deaminated during or after reverse transcription. The deamination pattern suggests a model of rcDNA synthesis in which pgRNA and then newly synthesized minus-sense single-stranded DNA are protected from deaminase by interaction with the virus capsid; during plus-strand synthesis, when enough dsDNA has been synthesized to displace the remaining minus strand from the capsid surface, the single-stranded DNA becomes deaminase sensitive. IMPORTANCE Host-induced mutation of the HBV genome by APOBEC proteins may be a path to clearing the virus. We examined cytidine-to-thymidine mutations in the genomes of HBV particles grown in the presence or absence of overexpressed APOBEC proteins. We found that genomes were subjected to deamination activity during reverse transcription

  13. SU-C-303-01: Activation-Induced Cytidine Deaminase Confers Cancer Resistance to Radiation Therapy

    International Nuclear Information System (INIS)

    Yi, S; La Count, S; Liu, J; Bai, X; Lu, L

    2015-01-01

    Purpose: To study the role of activation-induced cytidine deaminase (AID) in malignant cell resistance to radiation therapy. Methods: We first developed several small devices that could be used to adopt radiation beams from clinical high dose rate brachy therapy (HDR) or linac-based megavoltage machines to perform pre-clinical cell and mouse experiments. Then we used these devices to deliver radiation to AID-positive and AID-silenced cancer cells or tumors formed by these cells in mice. Cells and mice bearing tumors received the same dose under the same experimental conditions. For cells, we observed the apoptosis and the cell survival rate over time. For mice bearing tumors, we measured and recorded the tumor sizes every other day for 4 weeks. Results: For cell experiments, we found that the AID-positive cells underwent much less apoptosis compared with AID-silenced cells upon radiation. And for mouse experiments, we found that AID-positive tumors grew significantly faster than the AID-silenced tumors despite of receiving the same doses of radiation. Conclusion: Our study suggests that AID may confer cancer resistance to radiation therapy, and AID may be a significant biomarker predicting cancer resistance to radiation therapy for certain cancer types

  14. Single nucleotide editing without DNA cleavage using CRISPR/Cas9-deaminase in the sea urchin embryo.

    Science.gov (United States)

    Shevidi, Saba; Uchida, Alicia; Schudrowitz, Natalie; Wessel, Gary M; Yajima, Mamiko

    2017-12-01

    A single base pair mutation in the genome can result in many congenital disorders in humans. The recent gene editing approach using CRISPR/Cas9 has rapidly become a powerful tool to replicate or repair such mutations in the genome. These approaches rely on cleaving DNA, while presenting unexpected risks. In this study, we demonstrate a modified CRISPR/Cas9 system fused to cytosine deaminase (Cas9-DA), which induces a single nucleotide conversion in the genome. Cas9-DA was introduced into sea urchin eggs with sgRNAs targeted for SpAlx1, SpDsh, or SpPks, each of which is critical for skeletogenesis, embryonic axis formation, or pigment formation, respectively. We found that both Cas9 and Cas9-DA edit the genome, and cause predicted phenotypic changes at a similar efficiency. Cas9, however, resulted in significant deletions in the genome centered on the gRNA target sequence, whereas Cas9-DA resulted in single or double nucleotide editing of C to T conversions within the gRNA target sequence. These results suggest that the Cas9-DA approach may be useful for manipulating gene activity with decreased risks of genomic aberrations. Developmental Dynamics 246:1036-1046, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. Circulating type 1 vaccine-derived poliovirus may evolve under the pressure of adenosine deaminases acting on RNA.

    Science.gov (United States)

    Liu, Yanhan; Ma, Tengfei; Liu, Jianzhu; Zhao, Xiaona; Cheng, Ziqiang; Guo, Huijun; Xu, Ruixue; Wang, Shujing

    2015-01-01

    Poliovirus, the causative agent of poliomyelitis, is a human enterovirus and member of the Picornaviridae family. An effective live-attenuated poliovirus vaccine strain (Sabin 1) has been developed and has protected humans from polio. However, a few cases of vaccine virulence reversion have been documented in several countries. For instance, circulating type 1 vaccine-derived poliovirus is a highly pathogenic poliovirus that evolved from an avirulent strain, but the mechanism by which vaccine strains undergo reversion remains unclear. In this study, vaccine strains exhibited A to G/U to C and G to A/C to U hypermutations in the reversed evolution of Sabin 1. Furthermore, the mutation ratios of U to C and C to U were higher than those of other mutation types. Dinucleotide editing context was then analyzed. Results showed that A to G and U to C mutations exhibited preferences similar to adenosine deaminases acting on RNA (ADAR). Hence, ADARs may participate in poliovirus vaccine evolution.

  16. First Occurrence of Plasmablastic Lymphoma in Adenosine Deaminase-Deficient Severe Combined Immunodeficiency Disease Patient and Review of the Literature

    Directory of Open Access Journals (Sweden)

    Maddalena Migliavacca

    2018-02-01

    Full Text Available Adenosine deaminase-deficient severe combined immunodeficiency disease (ADA-SCID is a primary immune deficiency characterized by mutations in the ADA gene resulting in accumulation of toxic compounds affecting multiple districts. Hematopoietic stem cell transplantation (HSCT from a matched donor and hematopoietic stem cell gene therapy are the preferred options for definitive treatment. Enzyme replacement therapy (ERT is used to manage the disease in the short term, while a decreased efficacy is reported in the medium-long term. To date, eight cases of lymphomas have been described in ADA-SCID patients. Here we report the first case of plasmablastic lymphoma occurring in a young adult with ADA-SCID on long-term ERT, which turned out to be Epstein–Barr virus associated. The patient previously received infusions of genetically modified T cells. A cumulative analysis of the eight published cases of lymphoma from 1992 to date, and the case here described, reveals a high mortality (89%. The most common form is diffuse large B-cell lymphoma, which predominantly occurs in extra nodal sites. Seven cases occurred in patients on ERT and two after haploidentical HSCT. The significant incidence of immunodeficiency-associated lymphoproliferative disorders and poor survival of patients developing this complication highlight the priority in finding a prompt curative treatment for ADA-SCID.

  17. A case of red-cell adenosine deaminase overproduction associated with hereditary hemolytic anemia found in Japan.

    Science.gov (United States)

    Miwa, S; Fujii, H; Matsumoto, N; Nakatsuji, T; Oda, S; Asano, H; Asano, S

    1978-01-01

    A case of red cell adenosine deaminase (ADA) overproduction associated with hereditary hemolytic anemia is reported here. This appears to be the second report. Proband is a 38-year-old Japanese male who had hemoglobin, 15.8 g/100 ml; reticulocyte count, 4.5%; serum indirect bilirubin, 4.9 mg/100 ml; 51Cr-labeled red cell half-life, 12 days; red cells showed moderate stomatocytosis. His red cell ADA activity showed 40-fold increase while that of the mother showed 4-fold increase. The mother was hematologically normal. The father had a normal enzyme activity. The proband and the mother showed slightly high serum uric acid levels. The proband's red cell showed: ATP, 628 nmoles/ml (normal, 1,010--1,550); adenine nucleotide pool, 46% of the normal mean; 2,3-diphosphoglycerate content, 3,782 nmoles/ml (normal 4,170--5,300); increased oxygen affinity of hemoglobin, P50 of intact erythrocytes being 21.8 mmHg (normal, 24.1--26.1). Red cell glycolytic intermediates in the proband were low in general, and the rate of lactate production was low. Kinetic studies using crude hemolysate revealed a normal Km for adenosine, normal electrophoretic mobility but slightly abnormal pH curve and slightly low utilization of 2-deoxyadenosine. The ADA activity of lymphocytes was nearly normal.

  18. 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

  19. Can pleural adenosine deaminase (ADA) levels in pleural tuberculosis predict the presence of pulmonary tuberculosis? A CT analysis

    International Nuclear Information System (INIS)

    Koh, Myung Je; Lee, In Jae; Kim, Joo-Hee

    2016-01-01

    Aim: To assess the relationship between imaging features of pulmonary tuberculosis at computed tomography (CT) and adenosine deaminase (ADA) values via pleural fluid analysis in patients with pleural tuberculosis. Materials and methods: This retrospective study enrolled 60 patients who underwent fluid analysis for ADA and chest CT and were diagnosed with tuberculosis by culture or polymerase chain reaction of pleural fluid and sputum. The presence of centrilobular nodules, consolidation, cavitation, and mediastinal lymphadenopathy at CT were evaluated. The relationship between ADA values and the pattern of pulmonary involvement of tuberculosis was analysed. Results: Pulmonary involvement was seen in 42 of the 60 patients. A centrilobular nodular pattern was seen in 37 and consolidation in 22. In 17 patients, both findings were identified. A centrilobular nodular pattern was more common than consolidation or cavitary lesions. When ADA values were high, pulmonary involvement was more frequent (p=0.002). Comparing low and high ADA groups using an obtained cut-off value of 80 IU/l, the high group had more frequent pulmonary involvement (p<0.001). Conclusion: Patients with tuberculous pleurisy who had high ADA values had a higher probability of manifesting pulmonary tuberculosis. High ADA values may help predict contagious pleuroparenchymal tuberculosis. The most common pulmonary involvement of tuberculous pleurisy showed a centrilobular nodular pattern. - Highlights: • To know the relationship of ADA values and pulmonary involvement pattern of pleural tuberculosis. • To help exact diagnosis of pleuroparenchymal tuberculosis in clinical setting. • The imaging findings of pleuroparenchymal tuberculosis.

  20. Utility of adenosine deaminase (ADA), PCR & thoracoscopy in differentiating tuberculous & non-tuberculous pleural effusion complicating chronic kidney disease.

    Science.gov (United States)

    Kumar, Sravan; Agarwal, Ritesh; Bal, Amanjit; Sharma, Kusum; Singh, Navneet; Aggarwal, Ashutosh N; Verma, Indu; Rana, Satyawati V; Jha, Vivekanand

    2015-03-01

    Pleural effusion is a common occurrence in patients with late-stage chronic kidney disease (CKD). In developing countries, many effusions remain undiagnosed after pleural fluid analysis (PFA) and patients are empirically treated with antitubercular therapy. The aim of this study was to evaluate the role of adenosine deaminase (ADA), nucleic acid amplification tests (NAAT) and medical thoracoscopy in distinguishing tubercular and non-tubercular aetiologies in exudative pleural effusions complicating CKD. Consecutive stage 4 and 5 CKD patients with pleural effusions underwent PFA including ADA and PCR [65 kDa gene; multiplex (IS6110, protein antigen b, MPB64)]. Patients with exudative pleural effusion undiagnosed after PFA underwent medical thoracoscopy. All 107 patients underwent thoracocentesis with 45 and 62 patients diagnosed as transudative and exudative pleural effusions, respectively. Twenty six of the 62 patients underwent medical thoracoscopy. Tuberculous pleurisy was diagnosed in six while uraemic pleuritis was diagnosed in 20 subjects. The sensitivity and specificity of pleural fluid ADA, 65 kDa gene PCR, and multiplex PCR were 66.7 and 90 per cent, 100 and 50 per cent, and 100 and 100 per cent, respectively. Thoracoscopy was associated with five complications in three patients. Uraemia remains the most common cause of pleural effusion in CKD even in high TB prevalence country. Multiplex PCR and thoracoscopy are useful investigations in the diagnostic work-up of pleural effusions complicating CKD while the sensitivity and/or specificity of ADA and 65 kDa gene PCR is poor.

  1. Can pleural adenosine deaminase (ADA) levels in pleural tuberculosis predict the presence of pulmonary tuberculosis? A CT analysis.

    Science.gov (United States)

    Koh, Myung Je; Lee, In Jae; Kim, Joo-Hee

    2016-06-01

    To assess the relationship between imaging features of pulmonary tuberculosis at computed tomography (CT) and adenosine deaminase (ADA) values via pleural fluid analysis in patients with pleural tuberculosis. This retrospective study enrolled 60 patients who underwent fluid analysis for ADA and chest CT and were diagnosed with tuberculosis by culture or polymerase chain reaction of pleural fluid and sputum. The presence of centrilobular nodules, consolidation, cavitation, and mediastinal lymphadenopathy at CT were evaluated. The relationship between ADA values and the pattern of pulmonary involvement of tuberculosis was analysed. Pulmonary involvement was seen in 42 of the 60 patients. A centrilobular nodular pattern was seen in 37 and consolidation in 22. In 17 patients, both findings were identified. A centrilobular nodular pattern was more common than consolidation or cavitary lesions. When ADA values were high, pulmonary involvement was more frequent (p=0.002). Comparing low and high ADA groups using an obtained cut-off value of 80 IU/l, the high group had more frequent pulmonary involvement (pADA values had a higher probability of manifesting pulmonary tuberculosis. High ADA values may help predict contagious pleuroparenchymal tuberculosis. The most common pulmonary involvement of tuberculous pleurisy showed a centrilobular nodular pattern. Copyright © 2016 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

  2. Long-term expression of human adenosine deaminase in mice transplanted with retrovirus-infected hematopoietic stem cells

    International Nuclear Information System (INIS)

    Lim, B.; Apperley, J.F.; Orkin, S.H.; Williams, D.A.

    1989-01-01

    Long-term stable expression of foreign genetic sequences transferred into hematopoietic stem cells by using retroviral vectors constitutes a relevant model for somatic gene therapy. Such stability of expression may depend on vector design, including the presence or absence of specific sequences within the vector, in combination with the nature and efficiency of infection of the hematopoietic target cells. The authors have previously reported successful transfer of human DNA encoding adenosine deaminase (ADA) into CFU-S (colony-forming unit-spleen) stem cells using simplified recombinant retroviral vectors. Human ADA was expressed in CFU-S-derived spleen colonies at levels near to endogenous enzyme. However, because of the lack of an efficient dominant selectable marker and low recombinant viral titers, stability of long-term expression of human ADA was not examined. They report here the development of an efficient method of infection of hematopoietic stem cells (HSC) without reliance on in vitro selection. Peripheral blood samples of 100% of mice transplanted with HSC infected by this protocol exhibit expression of human ADA 30 days after transplantation. Some mice (6 of 13) continue to express human ADA in all lineages after complete hematopoietic reconstitution (4 months). The use of recombinant retroviral vectors that efficiently transfer human ADA cDNA into HSC leading to stable expression of functional ADA in reconstituted mice, provides an experimental framework for future development of approaches to somatic gene therapy

  3. Gene therapy for adenosine deaminase-deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans.

    Science.gov (United States)

    Candotti, Fabio; Shaw, Kit L; Muul, Linda; Carbonaro, Denise; Sokolic, Robert; Choi, Christopher; Schurman, Shepherd H; Garabedian, Elizabeth; Kesserwan, Chimene; Jagadeesh, G Jayashree; Fu, Pei-Yu; Gschweng, Eric; Cooper, Aaron; Tisdale, John F; Weinberg, Kenneth I; Crooks, Gay M; Kapoor, Neena; Shah, Ami; Abdel-Azim, Hisham; Yu, Xiao-Jin; Smogorzewska, Monika; Wayne, Alan S; Rosenblatt, Howard M; Davis, Carla M; Hanson, Celine; Rishi, Radha G; Wang, Xiaoyan; Gjertson, David; Yang, Otto O; Balamurugan, Arumugam; Bauer, Gerhard; Ireland, Joanna A; Engel, Barbara C; Podsakoff, Gregory M; Hershfield, Michael S; Blaese, R Michael; Parkman, Robertson; Kohn, Donald B

    2012-11-01

    We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34(+) cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m(2)). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.

  4. Adenovirus-assisted lipofection: efficient in vitro gene transfer of luciferase and cytosine deaminase to human smooth muscle cells.

    Science.gov (United States)

    Kreuzer, J; Denger, S; Reifers, F; Beisel, C; Haack, K; Gebert, J; Kübler, W

    1996-07-01

    Smooth muscle cells (SMC) are a central cell type involved in multiple processes of coronary artery diseases including restenosis and therefore are major target cells for different aspects of gene transfer. Previous attempts to transfect primary arterial cells using different techniques like liposomes, CaPO4 and electroporation resulted in only low transfection efficiency. The development of recombinant adenoviruses dramatically improved the delivery of foreign genes into different cell types including SMC. However, cloning and identification of recombinants remain difficult and time-consuming techniques. The present study demonstrates that a complex consisting of reporter plasmid encoding firefly luciferase (pLUC), polycationic liposomes and replication-deficient adenovirus was able to yield very high in vitro transfection of primary human smooth muscle cells under optimized conditions. The technique of adenovirus-assisted lipofection (AAL) increases transfer and expression of plasmid DNA in human smooth muscle cells in vitro up to 1000-fold compared to lipofection. To verify the applicability of AAL for gene transfer into human smooth muscle cells we studied a gene therapy approach to suppress proliferation of SMC in vitro, using the prokaryotic cytosine deaminase gene (CD) which enables transfected mammalian cells to deaminate 5-fluorocytosine (5-FC) to the highly toxic 5-fluorouracil (5-FU). The effect of a transient CD expression on RNA synthesis was investigated by means of a cotransfection with a RSV-CD expression plasmid and the luciferase reporter plasmid. Western blot analysis demonstrated high expression of CD protein in transfected SMC. Cotransfected SMC demonstrated two-fold less luciferase activity in the presence of 5-FC (5 mmol/l) after 48 h compared to cells transfected with a non-CD coding plasmid. The data demonstrate that a transient expression of CD could be sufficient to reduce the capacity of protein synthesis in human SMC. This simple and

  5. Regulated production and anti-HIV type 1 activities of cytidine deaminases APOBEC3B, 3F, and 3G.

    Science.gov (United States)

    Rose, Kristine M; Marin, Mariana; Kozak, Susan L; Kabat, David

    2005-07-01

    APOBEC3G and 3F (A3G and A3F) cytidine deaminases incorporate into retroviral cores where they lethally hypermutate nascent DNA reverse transcripts. As substantiated here, the viral infectivity factor (Vif) encoded by human immunodeficiency virus type-1 (HIV-1) binds A3G and A3F and induces their degradation, thereby precluding their incorporation into viral progeny. Previous evidence suggested that A3G is expressed in H9 and other nonpermissive cells that contain this antiviral defense but not in several permissive cells, and that overexpression of A3G or A3F makes permissive cells nonpermissive. Using a broader panel of cell lines, we confirmed a correlation between A3G and cellular abilities to inactivate HIV-1(Deltavif). However, there was a quantitative discrepancy because several cells with weak antiviral activities had similar amounts of wild-type A3G mRNA and protein compared to H9 cells. Antiviral activity of H9 cells was also attenuated in some conditions. These quantitative discrepancies could not be explained by the presence of A3F or other A3G paralogs in some of the cell lines. Thus, A3A, A3B, and A3C had weak but significant anti-HIV-1 activities and did not dominantly interfere with A3G or A3F antiviral functions. Control of A3G synthesis by the protein kinase C/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway was also similar in permissive and nonpermissive cells. A3G in highly permissive cells is degraded by Vif, suggesting that it is not in a sequestered site, and is specifically incorporated in low amounts into HIV-1(Deltavif). Although A3G and/or A3F inactivate HIV-1(Deltavif) and are neutralized by Vif, the antiviral properties of cell lines are also influenced by other cellular and viral factors.

  6. Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA.

    Directory of Open Access Journals (Sweden)

    Simon Gemble

    2015-07-01

    Full Text Available Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at "difficult-to-replicate" sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS, a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3'-5' DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.

  7. Potential benefits of combining cytosine deaminase/5-fluorocytosine gene therapy and irradiation for prostate cancer. Experimental study

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Hiroaki; Koshida, Kiyoshi; Yokoyama, Kunihiko; Mizokami, Atsushi; Namiki, Mikio [Kanazawa Univ. (Japan). School of Medicine

    2002-10-01

    The purpose of this study was to investigate the potential of combining cytosine deaminase/5-fluorocytosine (CD/5-FC) gene therapy and radiation therapy (either external beam radiation or radioimmunotherapy [RIT]), for the treatment of prostate cancer. Tumor xenografts of CD-transduced LNCaP cells grown in the testes of severe combined immunodeficiency (SCID) mice were used to evaluate antitumor effect. The mice were injected intraperitoneally with 500 mg/kg of 5-FC, or with 5, 15 or 30 mg/kg of 5-fluorouracil (5-FU), for 9 days. The tumors were treated with fractionated radiation at a dose of 1 or 3 Gy/day for 3 days, or I-131 labelled anti-prostate specific antigen (anti-PSA) monoclonal antibody (mAb) administration at a subtherapeutic dose of 20 or 80 {mu}Ci. Intratumoral and serum concentrations of 5-FU were measured using high performance liquid chromatography. Mice treated with CD/5-FC gene therapy presented a significant tumor growth inhibition comparable to that obtained with 15 mg/kg, 5-FU systemic administration without marked weight loss. Treatment with CD/5-FC gene therapy resulted in higher tumor but lower serum concentrations of 5-FU than treatment with systemic 5-FU chemotherapy. An additive antitumor effect was obtained when CD/5-FC therapy was combined with 1 Gy irradiation, which by itself did not produce a significant antitumor effect. However, the efficacy of CD/5-FC therapy was not enhanced when combined with RIT, probably due to poor accumulation of the mAb as the tumor/blood ratio never exceeded 1. These findings indicate that CD/5-FC gene therapy for prostate cancer may function with enhanced antitumor effect when combined with external beam radiation. However, combining CD/5-FC gene therapy and RIT using an anti-PSA mAb may not be effective because of insufficient accumulation of the mAb at the target tumors. (author)

  8. Potential benefits of combining cytosine deaminase/5-fluorocytosine gene therapy and irradiation for prostate cancer. Experimental study

    International Nuclear Information System (INIS)

    Kato, Hiroaki; Koshida, Kiyoshi; Yokoyama, Kunihiko; Mizokami, Atsushi; Namiki, Mikio

    2002-01-01

    The purpose of this study was to investigate the potential of combining cytosine deaminase/5-fluorocytosine (CD/5-FC) gene therapy and radiation therapy (either external beam radiation or radioimmunotherapy [RIT]), for the treatment of prostate cancer. Tumor xenografts of CD-transduced LNCaP cells grown in the testes of severe combined immunodeficiency (SCID) mice were used to evaluate antitumor effect. The mice were injected intraperitoneally with 500 mg/kg of 5-FC, or with 5, 15 or 30 mg/kg of 5-fluorouracil (5-FU), for 9 days. The tumors were treated with fractionated radiation at a dose of 1 or 3 Gy/day for 3 days, or I-131 labelled anti-prostate specific antigen (anti-PSA) monoclonal antibody (mAb) administration at a subtherapeutic dose of 20 or 80 μCi. Intratumoral and serum concentrations of 5-FU were measured using high performance liquid chromatography. Mice treated with CD/5-FC gene therapy presented a significant tumor growth inhibition comparable to that obtained with 15 mg/kg, 5-FU systemic administration without marked weight loss. Treatment with CD/5-FC gene therapy resulted in higher tumor but lower serum concentrations of 5-FU than treatment with systemic 5-FU chemotherapy. An additive antitumor effect was obtained when CD/5-FC therapy was combined with 1 Gy irradiation, which by itself did not produce a significant antitumor effect. However, the efficacy of CD/5-FC therapy was not enhanced when combined with RIT, probably due to poor accumulation of the mAb as the tumor/blood ratio never exceeded 1. These findings indicate that CD/5-FC gene therapy for prostate cancer may function with enhanced antitumor effect when combined with external beam radiation. However, combining CD/5-FC gene therapy and RIT using an anti-PSA mAb may not be effective because of insufficient accumulation of the mAb at the target tumors. (author)

  9. A decision tree for the genetic diagnosis of deficiency of adenosine deaminase 2 (DADA2): a French reference centres experience.

    Science.gov (United States)

    Rama, Mélanie; Duflos, Claire; Melki, Isabelle; Bessis, Didier; Bonhomme, Axelle; Martin, Hélène; Doummar, Diane; Valence, Stéphanie; Rodriguez, Diana; Carme, Emilie; Genevieve, David; Heimdal, Ketil; Insalaco, Antonella; Franck, Nathalie; Queyrel-Moranne, Viviane; Tieulie, Nathalie; London, Jonathan; Uettwiller, Florence; Georgin-Lavialle, Sophie; Belot, Alexandre; Koné-Paut, Isabelle; Hentgen, Véronique; Boursier, Guilaine; Touitou, Isabelle; Sarrabay, Guillaume

    2018-04-23

    Deficiency of adenosine deaminase 2 (DADA2) is a recently described autoinflammatory disorder. Genetic analysis is required to confirm the diagnosis. We aimed to describe the identifying symptoms and genotypes of patients referred to our reference centres and to improve the indications for genetic testing. DNA from 66 patients with clinically suspected DADA2 were sequenced by Sanger or next-generation sequencing. Detailed epidemiological, clinical and biological features were collected by use of a questionnaire and were compared between patients with and without genetic confirmation of DADA2. We identified 13 patients (19.6%) carrying recessively inherited mutations in ADA2 that were predicted to be deleterious. Eight patients were compound heterozygous for mutations. Seven mutations were novel (4 missense variants, 2 predicted to affect mRNA splicing and 1 frameshift). The mean age of the 13 patients with genetic confirmation was 12.7 years at disease onset and 20.8 years at diagnosis. Phenotypic manifestations included fever (85%), vasculitis (85%) and neurological disorders (54%). Features best associated with a confirmatory genotype included fever with neurologic or cutaneous attacks (odds ratio [OR] 10.71, p = 0.003 and OR 10.9, p < 0.001), fever alone (OR 8.1, p = 0.01), and elevated C-reactive protein (CRP) level with neurologic involvement (OR 6.63, p = 0.017). Our proposed decision tree may help improve obtaining genetic confirmation of DADA2 in the context of autoinflammatory symptoms. Prerequisites for quick and low-cost Sanger analysis include one typical cutaneous or neurological sign, one marker of inflammation (fever or elevated CRP level), and recurrent or chronic attacks in adults.

  10. Expression of activation-induced cytidine deaminase gene in B lymphocytes of patients with common variable immunodeficiency.

    Science.gov (United States)

    Abolhassani, Hassan; Farrokhi, Amir Salek; Pourhamdi, Shabnam; Mohammadinejad, Payam; Sadeghi, Bamdad; Moazzeni, Seyed-Mohammad; Aghamohammadi, Asghar

    2013-08-01

    Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by reduced serum level of IgG, IgA or IgM and recurrent bacterial infections. Class switch recombination (CSR) as a critical process in immunoglobulin production is defective in a group of CVID patients. Activation-induced cytidine deaminase (AID) protein is an important molecule involving CSR process. The aim of this study was to investigate the AID gene mRNA production in a group of CVID patients indicating possible role of this molecule in this disorder. Peripheral blood mononuclear cells (PBMC) of 29 CVID patients and 21 healthy controls were isolated and stimulated by CD40L and IL-4 to induce AID gene expression. After 5 days AID gene mRNA production was investigated by real time polymerase chain reaction. AID gene was expressed in all of the studied patients. However the mean density of extracted AID mRNA showed higher level in CVID patients (230.95±103.04 ng/ml) rather than controls (210.00±44.72 ng/ml; P=0.5). CVID cases with lower level of AID had decreased total level of IgE (P=0.04) and stimulated IgE production (P=0.02); while cases with increased level of AID presented higher level of IgA (P=0.04) and numbers of B cells (P=0.02) and autoimmune disease (P=0.02). Different levels of AID gene expression may have important roles in dysregulation of immune system and final clinical presentation in CVID patients. Therefore investigating the expression of AID gene can help in classifying CVID patients.

  11. A Hospital Based Study on Estimation of Adenosine Deaminase Activity (ADA) in Cerebrospinal Fluid (CSF) in Various Types of Meningitis.

    Science.gov (United States)

    Agarwal, Ashok Kumar; Bansal, Sonia; Nand, Vidya

    2014-02-01

    Tuberculosis kills 3.70 lakh patients in India every year,out of which 7-12 % are meningeal involvement. Delay in its diagnosis and initiation of treatment results in poor prognosis and squeal in up to 25% of cases. The aim of the present study is to look for a simple, rapid, cost effective, and fairly specific test in differentiating tubercular aetiology from other causes of meningitis. In the present study we measured the adenosine deaminase activity (ADA) in Cerebrospinal Fluid (CSF) of Tubercular Meningitis (TBM) and non-TBM patients. Fifty six patients attending hospital with symptoms and signs of meningitis were selected and divided into three groups: tubercular, pyogenic, and aseptic meningitis, depending upon the accepted criteria. CSF was drawn and ADA estimated. Out of 32 tubercular patients, 28 had CSF-ADA at or above the cut-off value while four had below. Out of 24 non-tuberculous patients (pyogenic and aseptic meningitis), two aseptic meningitis (AM) patient had ADA levels at or above the cut-off value while 22 had below this value. RESULTS of our study indicate that ADA level estimation in CSF is not only of considerable value in the diagnosis of TBM, CSF, and ADA level 10 U/L as a cut-off value with sensitivity 87.5% and specificity 83.33% and positive predictive value of the test was 87.5%.and 83.3% negative predictive value. It can be concluded that ADA estimation in CSF is not only simple, inexpensive and rapid but also fairly specific method for making a diagnosis of tuberculous aetiology in TBM, especially when there is a dilemma of differentiating the tuberculous aetiology from non-tuberculous ones. For this reason ADA estimation in TBM may find a place as a routine investigation.

  12. Autosomal recessive hyper IgM syndrome associated with activation-induced cytidine deaminase gene in three Turkish siblings presented with tuberculosis lymphadenitis - Case report.

    Science.gov (United States)

    Patiroglu, Turkan; Akar, H Haluk; van der Burg, Mirjam; Unal, Ekrem

    2015-09-01

    The hyper-immunoglobulin M (HIGM) syndrome is a heterogeneous group of genetic disorders characterized by recurrent infections, decreased serum levels of immunoglobulin G (IgG) and IgA, and normal/increased serum levels of IgM. Herein, we describe three Turkish siblings with HIGM syndrome who had a homozygous missense mutation (c.70C>T, p.Arg24Trp) in the activation-induced cytidine deaminase gene which results in autosomal recessive HIGM syndrome. Two of the siblings, sibling 1 and sibling 3, presented with cervical deep abscess and cervical tuberculosis lymphadenitis, respectively.

  13. Effect of repeated pesticide applications on soil properties in cotton fields: II. Insecticide residues and impact on dehydrogenase and arginine deaminase activities

    International Nuclear Information System (INIS)

    Vig, K.; Singh, D.K.; Agarwal, H.C.; Dhawan, A.K.; Dureja, P.

    2001-01-01

    Insecticides were applied sequentially at recommended dosages post crop emergence in cotton fields and soil was sampled at regular intervals after each treatment. Soil was analysed for insecticide residues and activity of the enzymes dehydrogenase and arginine deaminase. Insecticide residues detected in the soil were in small quantities and they did not persist for long. Only endosulfan leached below 15 cm. Insecticides had only temporary effects on enzyme activities which disappeared either before the next insecticide treatment or by the end of the experimental period. (author)

  14. Targeted cytosine deaminase-uracil phosphoribosyl transferase suicide gene therapy induces small cell lung cancer-specific cytotoxicity and tumor growth delay

    DEFF Research Database (Denmark)

    Christensen, Camilla L; Gjetting, Torben; Poulsen, Thomas Tuxen

    2010-01-01

    Small cell lung cancer (SCLC) is a highly malignant cancer for which there is no curable treatment. Novel therapies are therefore in great demand. In the present study we investigated the therapeutic effect of transcriptionally targeted suicide gene therapy for SCLC based on the yeast cytosine...... deaminase (YCD) gene alone or fused with the yeast uracil phosphoribosyl transferase (YUPRT) gene followed by administration of 5-fluorocytosine (5-FC) prodrug. Experimental design: The YCD gene or the YCD-YUPRT gene was placed under regulation of the SCLC-specific promoter insulinoma-associated 1 (INSM1...

  15. Crystallization and preliminary X-ray crystallographic analysis of adenosine 5′-monophosphate deaminase (AMPD) from Arabidopsis thaliana in complex with coformycin 5′-phosphate

    International Nuclear Information System (INIS)

    Han, Byung Woo; Bingman, Craig A.; Mahnke, Donna K.; Sabina, Richard L.; Phillips, George N. Jr

    2005-01-01

    Adenosine 5′-monophosphate deaminase from A. thaliana has been crystallized in complex with coformycin 5′-phosphate. Diffraction data have been collected to 3.34 Å resolution. Adenosine 5′-monophosphate deaminase (AMPD) is a eukaryotic enzyme that converts adenosine 5′-monophosphate (AMP) to inosine 5′-monophosphate (IMP) and ammonia. AMPD from Arabidopsis thaliana (AtAMPD) was cloned into the baculoviral transfer vector p2Bac and co-transfected along with a modified baculoviral genome into Spodoptera frugiperda (Sf9) cells. The resulting recombinant baculovirus were plaque-purified, amplified and used to overexpress recombinant AtAMPD. Crystals of purified AtAMPD have been obtained to which coformycin 5′-phosphate, a transition-state inhibitor, is bound. Crystals belong to space group P6 2 22, with unit-cell parameters a = b = 131.325, c = 208.254 Å, α = β = 90, γ = 120°. Diffraction data were collected to 3.34 Å resolution from a crystal in complex with coformycin 5′-phosphate and to 4.05 Å resolution from a crystal of a mercury derivative

  16. Targeted inhibition of osteosarcoma tumor growth by bone marrow-derived mesenchymal stem cells expressing cytosine deaminase/5-fluorocytosine in tumor-bearing mice.

    Science.gov (United States)

    NguyenThai, Quynh-Anh; Sharma, Neelesh; Luong, Do Huynh; Sodhi, Simrinder Singh; Kim, Jeong-Hyun; Kim, Nameun; Oh, Sung-Jong; Jeong, Dong Kee

    2015-01-01

    Mesenchymal stem cells (MSCs) are considered as an attractive approach for gene or drug delivery in cancer therapy. In the present study, the ability of human bone marrow-derived MSCs expressing the cytosine deaminase/5-fluorocytosine prodrug (CD/5-FC MSCs) to target the human osteosarcoma cell line Cal72 was evaluated. The stable CD/5-FC MSC cell line was established by transfection of pEGFP containing the cytosine deaminase gene into MSCs with G418 selection. The anti-tumor effect was verified by a bystander effect assay in vitro and co-injection of Cal72 and CD/5-FC MSCs in cancer-bearing mice. The therapeutic CD/5-FC MSCs retained the characteristics of multipotent cells, such as differentiation into adipocytes/osteocytes and expression of mesenchymal markers (CD90 and CD44), and showed migration toward Cal72 cells to a greater extent than the native MSCs. The bystander effect assay showed that the CD/5-FC MSCs significantly augmented Cal72 cytotoxicity in direct co-culture and in the presence of 5-FC through the application of conditioned medium. In osteosarcoma-bearing mice, the CD/5-FC MSCs inhibited tumor growth compared to control mice subcutaneously injected with only Cal72 cells. Taken together, these findings suggest that CD/5-FC MSCs may be suitable for targeting human osteosarcoma. Copyright © 2015 John Wiley & Sons, Ltd.

  17. Feed-Forward Inhibition of CD73 and Upregulation of Adenosine Deaminase Contribute to the Loss of Adenosine Neuromodulation in Postinflammatory Ileitis

    Directory of Open Access Journals (Sweden)

    Cátia Vieira

    2014-01-01

    Full Text Available Purinergic signalling is remarkably plastic during gastrointestinal inflammation. Thus, selective drugs targeting the “purinome” may be helpful for inflammatory gastrointestinal diseases. The myenteric neuromuscular transmission of healthy individuals is fine-tuned and controlled by adenosine acting on A2A excitatory receptors. Here, we investigated the neuromodulatory role of adenosine in TNBS-inflamed longitudinal muscle-myenteric plexus of the rat ileum. Seven-day postinflammation ileitis lacks adenosine neuromodulation, which may contribute to acceleration of gastrointestinal transit. The loss of adenosine neuromodulation results from deficient accumulation of the nucleoside at the myenteric synapse despite the fact that the increases in ATP release were observed. Disparity between ATP outflow and adenosine deficit in postinflammatory ileitis is ascribed to feed-forward inhibition of ecto-5′-nucleotidase/CD73 by high extracellular ATP and/or ADP. Redistribution of NTPDase2, but not of NTPDase3, from ganglion cell bodies to myenteric nerve terminals leads to preferential ADP accumulation from released ATP, thus contributing to the prolonged inhibition of muscle-bound ecto-5′-nucleotidase/CD73 and to the delay of adenosine formation at the inflamed neuromuscular synapse. On the other hand, depression of endogenous adenosine accumulation may also occur due to enhancement of adenosine deaminase activity. Both membrane-bound and soluble forms of ecto-5′-nucleotidase/CD73 and adenosine deaminase were detected in the inflamed myenteric plexus. These findings provide novel therapeutic targets for inflammatory gut motility disorders.

  18. Crystallization and preliminary X-ray crystallographic analysis of adenosine 5′-monophosphate deaminase (AMPD) from Arabidopsis thaliana in complex with coformycin 5′-phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Han, Byung Woo [Department of Biochemistry, University of Wisconsin-Madison, WI 53706-1544 (United States); Center for Eukaryotic Structural Genomics (CESG), University of Wisconsin-Madison, WI 53706-1549 (United States); Bingman, Craig A. [Center for Eukaryotic Structural Genomics (CESG), University of Wisconsin-Madison, WI 53706-1549 (United States); Mahnke, Donna K.; Sabina, Richard L. [Department of Biochemistry, The Medical College of Wisconsin, Milwaukee, WI 53226-4801 (United States); Phillips, George N. Jr, E-mail: phillips@biochem.wisc.edu [Department of Biochemistry, University of Wisconsin-Madison, WI 53706-1544 (United States); Center for Eukaryotic Structural Genomics (CESG), University of Wisconsin-Madison, WI 53706-1549 (United States)

    2005-08-01

    Adenosine 5′-monophosphate deaminase from A. thaliana has been crystallized in complex with coformycin 5′-phosphate. Diffraction data have been collected to 3.34 Å resolution. Adenosine 5′-monophosphate deaminase (AMPD) is a eukaryotic enzyme that converts adenosine 5′-monophosphate (AMP) to inosine 5′-monophosphate (IMP) and ammonia. AMPD from Arabidopsis thaliana (AtAMPD) was cloned into the baculoviral transfer vector p2Bac and co-transfected along with a modified baculoviral genome into Spodoptera frugiperda (Sf9) cells. The resulting recombinant baculovirus were plaque-purified, amplified and used to overexpress recombinant AtAMPD. Crystals of purified AtAMPD have been obtained to which coformycin 5′-phosphate, a transition-state inhibitor, is bound. Crystals belong to space group P6{sub 2}22, with unit-cell parameters a = b = 131.325, c = 208.254 Å, α = β = 90, γ = 120°. Diffraction data were collected to 3.34 Å resolution from a crystal in complex with coformycin 5′-phosphate and to 4.05 Å resolution from a crystal of a mercury derivative.

  19. 5-Aza-2'-deoxycytidine synergistic action with thymidine on leukemic cells and interaction of 5-aza-dCMP with dCMP deaminase

    International Nuclear Information System (INIS)

    Momparler, R.L.; Bartolucci, S.; Bouchard, J.; Momparler, L.F.; Raia, C.A.; Rossi, M.

    1986-01-01

    The authors observe a synergistic antineoplastic effect between 5-AZA-dCR and dTR on leukemia cells in culture. In order to understand the mechanism behind this interaction the authors investigate the effects of dTTP on the deamination of 5-aza-2'-deoxycytidine-5'-monophosphate (5-AZA-dCMP) by dCMP deaminase. The effects of 5-AZA-dCTP on this enzyme is also studied. The incorporation of tritium-5-AZA-Cdr into DNA of leukemic cells was performed. The amount of radioactivity incorproated into DNA was determined by trapping the cells on GF/C glass fiber filters and washing with cold TCA. It is shown that the modulation of the atieoplastic activity of deoxycytidine analogs by allosteric effectors such as dTTP may have the potential to increase the effectiveness of the chemotherapy for acute leukemia

  20. Comparative effectiveness of ACC-deaminase and/or nitrogen-fixing rhizobacteria in promotion of maize (Zea mays L.) growth under lead pollution.

    Science.gov (United States)

    Hassan, Waseem; Bano, Rizwana; Bashir, Farhat; David, Julie

    2014-09-01

    Lead (Pb) pollution is appearing as an alarming threat nowadays. Excessive Pb concentrations in agricultural soils result in minimizing the soil fertility and health which affects the plant growth and leads to decrease in crop production. Plant growth promoting rhizobacteria (PGPR) are beneficial bacteria which can protect the plants against many abiotic stresses, and enhance the growth. The study aimed to identify important rhizobacterial strains by using the 1-aminocyclopropane-1-carboxylate (ACC) enrichment technique and examine their inoculation effects in the growth promotion of maize, under Pb pollution. A pot experiment was conducted and six rhizobacterial isolates were used. Pb was added to 2 kg soil in each pot (with 4 seeds/pot) using Pb(NO3)2 at the rate of 0, 100, 200, 300, and 400 mg kg(-1) Pb with three replications in completely randomized design. Rhizobacterial isolates performed significantly better under all Pb levels, i.e., 100 to 400 Pb mg kg(-1) soil, compared to control. Comparing the efficacy of the rhizobacterial isolates under different Pb levels, rhizobacterial isolates having both ACC-deaminase and nitrogen-fixing activities (AN8 and AN12) showed highest increase in terms of the physical, chemical and enzymatic growth parameters of maize, followed by the rhizobacterial isolates having ACC-deaminase activity only (ACC5 and ACC8), and then the nitrogen-fixing rhizobia (Azotobacter and RN5). However, the AN8 isolate showed maximum efficiency, and highest shoot and root length (14.2 and 6.1 cm), seedling fresh and dry weights (1.91 and 0.14 g), chlorophyll a, b, and carotenoids (24.1, 30.2 and 77.7 μg/l), protein (0.82 mg/g), proline (3.42 μmol/g), glutathione S-transferase, peroxidase and catalase (12.3, 4.2 and 7.2 units/mg protein), while the lowest Pb uptake in the shoot and root (0.83 and 0.48 mg/kg) were observed under this rhizobial isolate at the highest Pb level (i.e., 400 Pb mg kg(-1) soil). The results revealed that PGPR

  1. An Insight into the Environmental Effects of the Pocket of the Active Site of the Enzyme. Ab initio ONIOM-Molecular Dynamics (MD) Study on Cytosine Deaminase

    International Nuclear Information System (INIS)

    Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

    2008-01-01

    We applied the ONIOM-molecular dynamics (MD) method to cytosine deaminase to examine the environmental effects of the amino acid residues in the pocket of the active site on the substrate taking account of their thermal motion. The ab initio ONIOM-MD simulations show that the substrate uracil is strongly perturbed by the amino acid residue Ile33, which sandwiches the uracil with His62, through the steric contact due to the thermal motion. As a result, the magnitude of the thermal oscillation of the potential energy and structure of the substrate uracil significantly increases. TM and MA were partly supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.MD was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, and by the Office of Biological and Environmental Research of the U.S. Department of Energy DOE. Battelle operates Pacific Northwest National Laboratory for DOE

  2. Analysis of serum adenosine deaminase (ADA) and ADA1 and ADA2 isoenzyme activities in HIV positive and HIV-HBV co-infected patients.

    Science.gov (United States)

    Khodadadi, Iraj; Abdi, Mohammad; Ahmadi, Abbas; Wahedi, Mohammad Saleh; Menbari, Shahoo; Lahoorpour, Fariba; Rahbari, Rezgar

    2011-08-01

    To determine adenosine deaminase (ADA) activity as a possible diagnostic marker in HIV and HIV-HBV co-infected patients. Blood samples were collected from 72 healthy, 33 HIV positive and 30 HIV-HBV co-infected subjects. Blood CD4+ cell count was recorded and serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total ADA, and ADA1 and ADA2 isoenzyme activities were determined. Serum ALT, AST, total ADA and ADA2 isoenzyme activities were significantly higher in HIV positive and HIV-HBV co-infected groups compare to the control (pADA activities (R(2)=0.589, pADA was significantly increased in HIV and HIV-HBV co-infections. Therefore, because of its low cost and simplicity to perform, ADA activity might be considered as a useful diagnostic tool among the other markers in these diseases. Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  3. Ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) and adenosine deaminase (ADA) activities in prostate cancer patients: influence of Gleason score, treatment and bone metastasis.

    Science.gov (United States)

    Battisti, Vanessa; Maders, Liési D K; Bagatini, Margarete D; Battisti, Iara E; Bellé, Luziane P; Santos, Karen F; Maldonado, Paula A; Thomé, Gustavo R; Schetinger, Maria R C; Morsch, Vera M

    2013-04-01

    The relation between adenine nucleotides and cancer has already been described in literature. Considering that the enzymes ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) and adenosine deaminase (ADA) act together to control nucleotide levels, we aimed to investigate the role of these enzymes in prostate cancer (PCa). E-NPP and ADA activities were determined in serum and platelets of PCa patients and controls. We also verified the influence of the Gleason score, bone metastasis and treatment in the enzyme activities. Platelets and serum E-NPP activity increased, whereas ADA activity in serum decreased in PCa patients. In addition, Gleason score, metastasis and treatment influenced E-NPP and ADA activities. We may propose that E-NPP and ADA are involved in the development of PCa. Moreover, E-NPP and ADA activities are modified in PCa patients with distinct Gleason score, with bone metastasis, as well as in patients under treatment. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  4. Good Laboratory Practice Preclinical Safety Studies for GSK2696273 (MLV Vector-Based Ex Vivo Gene Therapy for Adenosine Deaminase Deficiency Severe Combined Immunodeficiency) in NSG Mice.

    Science.gov (United States)

    Carriglio, Nicola; Klapwijk, Jan; Hernandez, Raisa Jofra; Vezzoli, Michela; Chanut, Franck; Lowe, Rhiannon; Draghici, Elena; Nord, Melanie; Albertini, Paola; Cristofori, Patrizia; Richards, Jane; Staton, Hazel; Appleby, Jonathan; Aiuti, Alessandro; Sauer, Aisha V

    2017-03-01

    GSK2696273 (autologous CD34+ cells transduced with retroviral vector that encodes for the human adenosine deaminase [ADA] enzyme) is a gamma-retroviral ex vivo gene therapy of bone marrow-derived CD34+ cells for the treatment of adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCID). ADA-SCID is a severe monogenic disease characterized by immunologic and nonimmunologic symptoms. Bone-marrow transplant from a matched related donor is the treatment of choice, but it is available for only a small proportion of patients. Ex vivo gene therapy of patient bone-marrow CD34+ cells is an alternative treatment. In order to prepare for a marketing authorization application in the European Union, preclinical safety studies in mice were requested by the European Medicines Agency (EMA). A pilot study and a main biodistribution study were performed according to Good Laboratory Practice (GLP) at the San Raffaele Telethon Institute for Gene Therapy test facility. In the main study, human umbilical cord blood (UCB)-derived CD34+ cells were transduced with gamma-retroviral vector used in the production of GSK2696273. Groups of 10 male and 10 female NOD-SCID gamma (NSG) mice were injected intravenously with a single dose of transduced- or mock-transduced UCB CD34+ cells, and they were observed for 4 months. Engraftment and multilineage differentiation of blood cells was observed in the majority of animals in both groups. There was no significant difference in the level of chimerism between the two groups. In the gene therapy group, vector was detectable in lymphohemopoietic and nonlymphohemopoietic tissues, consistent with the presence of gene-modified human hematopoietic donor cells. Given the absence of relevant safety concerns in the data, the nonclinical studies and the clinical experience with GSK2696273 supported a successful application for market authorization in the European Union for the treatment of ADA-SCID patients, for whom no suitable human leukocyte

  5. The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism.

    Science.gov (United States)

    Stenmark, Pål; Moche, Martin; Gurmu, Daniel; Nordlund, Pär

    2007-10-12

    We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.

  6. A Novel Regulator of Activation-Induced Cytidine Deaminase/APOBECs in Immunity and Cancer: Schrödinger’s CATalytic Pocket

    Directory of Open Access Journals (Sweden)

    Mani Larijani

    2017-04-01

    Full Text Available Activation-induced cytidine deaminase (AID and its relative APOBEC3 cytidine deaminases boost immune response by mutating immune or viral genes. Because of their genome-mutating activities, AID/APOBECs are also drivers of tumorigenesis. Due to highly charged surfaces, extensive non-specific protein–protein/nucleic acid interactions, formation of polydisperse oligomers, and general insolubility, structure elucidation of these proteins by X-ray crystallography and NMR has been challenging. Hence, almost all available AID/APOBEC structures are of mutated and/or truncated versions. In 2015, we reported a functional structure for AID using a combined computational–biochemical approach. In so doing, we described a new regulatory mechanism that is a first for human DNA/RNA-editing enzymes. This mechanism involves dynamic closure of the catalytic pocket. Subsequent X-ray and NMR studies confirmed our discovery by showing that other APOBEC3s also close their catalytic pockets. Here, we highlight catalytic pocket closure as an emerging and important regulatory mechanism of AID/APOBEC3s. We focus on three sub-topics: first, we propose that variable pocket closure rates across AID/APOBEC3s underlie differential activity in immunity and cancer and review supporting evidence. Second, we discuss dynamic pocket closure as an ever-present internal regulator, in contrast to other proposed regulatory mechanisms that involve extrinsic binding partners. Third, we compare the merits of classical approaches of X-ray and NMR, with that of emerging computational–biochemical approaches, for structural elucidation specifically for AID/APOBEC3s.

  7. Adenovirus-mediated interleukin-12 gene transfer combined with cytosine deaminase followed by 5-fluorocytosine treatment exerts potent antitumor activity in Renca tumor-bearing mice

    International Nuclear Information System (INIS)

    Hwang, Kyung-Sun; Cho, Won-Kyung; Yoo, Jinsang; Yun, Hwan-Jung; Kim, Samyong; Im, Dong-Soo

    2005-01-01

    Therapeutic gene transfer affords a clinically feasible and safe approach to cancer treatment but a more effective modality is needed to improve clinical outcomes. Combined transfer of therapeutic genes with different modes of actions may be a means to this end. Interleukin-12 (IL-12), a heterodimeric immunoregulatory cytokine composed of covalently linked p35 and p40 subunits, has antitumor activity in animal models. The enzyme/prodrug strategy using cytosine deaminase (CD) and 5-fluorocytosine (5-FC) has been used for cancer gene therapy. We have evaluated the antitumor effect of combining IL-12 with CD gene transfer in mice bearing renal cell carcinoma (Renca) tumors. Adenoviral vectors were constructed encoding one or both subunits of murine IL-12 (Ad.p35, Ad.p40 and Ad.IL-12) or cytosine deaminase (Ad.CD). The functionality of the IL-12 or CD gene products expressed from these vectors was validated by splenic interferon (IFN)-γ production or viability assays in cultured cells. Ad.p35 plus Ad.p40, or Ad.IL-12, with or without Ad.CD, were administered (single-dose) intratumorally to Renca tumor-bearing mice. The animals injected with Ad.CD also received 5-FC intraperitoneally. The antitumor effects were then evaluated by measuring tumor regression, mean animal survival time, splenic natural killer (NK) cell activity and IFN-γ production. The inhibition of tumor growth in mice treated with Ad.p35 plus Ad.p40 and Ad.CD, followed by injection of 5-FC, was significantly greater than that in mice treated with Ad.CD/5-FC, a mixture of Ad.p35 plus Ad.p40, or Ad.GFP (control). The combined gene transfer increased splenic NK cell activity and IFN-γ production by splenocytes. Ad.CD/5-FC treatment significantly increased the antitumor effect of Ad.IL-12 in terms of tumor growth inhibition and mean animal survival time. The results suggest that adenovirus-mediated IL-12 gene transfer combined with Ad.CD followed by 5-FC treatment may be useful for treating cancers

  8. Mixed Inhibition of Adenosine Deaminase Activity by 1,3-Dinitrobenzene: A Model for Understanding Cell-Selective Neurotoxicity in Chemically-Induced Energy Deprivation Syndromes in Brain

    Science.gov (United States)

    Wang, Yipei; Liu, Xin; Schneider, Brandon; Zverina, Elaina A.; Russ, Kristen; Wijeyesakere, Sanjeeva J.; Fierke, Carol A.; Richardson, Rudy J.; Philbert, Martin A.

    2012-01-01

    Astrocytes are acutely sensitive to 1,3-dinitrobenzene (1,3-DNB) while adjacent neurons are relatively unaffected, consistent with other chemically-induced energy deprivation syndromes. Previous studies have investigated the role of astrocytes in protecting neurons from hypoxia and chemical injury via adenosine release. Adenosine is considered neuroprotective, but it is rapidly removed by extracellular deaminases such as adenosine deaminase (ADA). The present study tested the hypothesis that ADA is inhibited by 1,3-DNB as a substrate mimic, thereby preventing adenosine catabolism. ADA was inhibited by 1,3-DNB with an IC50 of 284μM, Hill slope, n = 4.8 ± 0.4. Native gel electrophoresis showed that 1,3-DNB did not denature ADA. Furthermore, adding Triton X-100 (0.01–0.05%, wt/vol), Nonidet P-40 (0.0015–0.0036%, wt/vol), or bovine serum albumin (0.05 mg/ml or changing [ADA] (0.2 and 2nM) did not substantially alter the 1,3-DNB IC50 value. Likewise, dynamic light scattering showed no particle formation over a (1,3-DNB) range of 149–1043μM. Kinetics revealed mixed inhibition with 1,3-DNB binding to ADA (KI = 520 ± 100μM, n = 1 ± 0.6) and the ADA-adenosine complex (KIS = 262 ± 7μM, n = 6 ± 0.6, indicating positive cooperativity). In accord with the kinetics, docking predicted binding of 1,3-DNB to the active site and three peripheral sites. In addition, exposure of DI TNC-1 astrocytes to 10–500μM 1,3-DNB produced concentration-dependent increases in extracellular adenosine at 24 h. Overall, the results demonstrate that 1,3-DNB is a mixed inhibitor of ADA and may thus lead to increases in extracellular adenosine. The finding may provide insights to guide future work on chemically-induced energy deprivation. PMID:22106038

  9. Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein

    Directory of Open Access Journals (Sweden)

    A.R.M. Sabbatini

    2011-02-01

    Full Text Available Histidine-rich glycoprotein (HRG is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD. We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown. In the present paper we investigate the origin of skeletal muscle HRG. The screening of a human skeletal muscle cDNA expression library using an anti-HRG antibody failed to reveal any positive clone. The RT-PCR analysis, performed on human skeletal muscle RNA as well as on RNA from the rhabdomyosarcoma (RD cell line, failed to show any mRNA specific for the plasma HRG or for the putative muscle variant. When the RD cells were incubated with human plasma HRG, a time-dependent increase of the HRG immunoreactivity was detected both at the plasma membrane level and intracellularly. The internalisation of HRG was inhibited by the addition of heparin. The above data strongly suggest that skeletal muscle cells do not synthesize the muscle variant of HRG but instead can actively internalise it from plasma.

  10. G22A Polymorphism of Adenosine Deaminase and its Association with Biochemical Characteristics of Gestational Diabetes Mellitus in an Iranian Population

    Directory of Open Access Journals (Sweden)

    Mohammad Ali Takhshid

    2015-03-01

    Full Text Available Adenosine deaminase (ADA is an important regulator of insulin action. The single nucleotide polymorphism (SNP G22A in the ADA gene decreases enzymatic activity of ADA. The aim of this study was to investigate the relationship between the SNP G22A and blood glycemic control, insulin resistance, and obesity of gestational diabetes mellitus (GDM patients in an Iranian population. SNP G22A was determined in women with GDM (N=70 and healthy pregnant women (control, N=70 using polymerase chain reaction-restriction fragment length polymorphism. Fasting plasma glucose (FPG, hemoglobin A1C (HbA1c, plasma insulin levels and plasma lipids were measured using commercial kits. Homeostasis model of assessment for insulin resistance (HOMA-IR was calculated. The distribution of genotypes and alleles among GDM patients was similar to that of the control group. FPG and HbA1c were significantly higher in GDM patients with GG genotype compared with GDM patients with GA+AA genotype and non-GDM patients. The frequency of GG genotype was significantly higher in obese GDM patients compared to lean GDM patients. The SNP G22A in the ADA gene was not associated with the risk of GDM in our population. GG genotype was associated with poor glycemic control and obesity in GDM patients.

  11. Selective in vivo radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells transduced with the E. coli cytosine deaminase (CD) gene

    International Nuclear Information System (INIS)

    Gabel, M.; Kim, J.H.; Kolozsvary, A.; Khil, M.; Freytag, S.

    1998-01-01

    Purpose: The E. coli cytosine deaminase (CD) gene encodes an enzyme capable of converting the nontoxic prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a known radiosensitizer. Having previously shown that combined CD suicide gene therapy and radiation (RT) results in pronounced radiosensitization in vitro, we progressed to in vivo studies of combined therapy. Methods and Materials: WiDr human colon cancer cells were transduced in vitro with the CD gene and cells expressing CD were selected for use as xenografts in a nude mouse model. After administration of 5-FC, tumors received 10-30 Gy local field radiation (RT) and tumor growth delay was compared to control animals receiving either 5-FU, 5-FC, or RT alone. Results: Maximal growth delay was seen in mice treated with 5-FC for 6 consecutive days prior to RT. Combined treatment with 15 Gy radiation resulted in a dose-modifying factor (DMF) of 1.50, and a greater DMF was observed with higher doses of radiation. There was no appreciable toxicity using this new approach. In contrast, a similar treatment of combined 5-FU and radiation resulted in considerable toxicity and no appreciable radiosensitization. Conclusion: The present results show that combined suicide gene therapy and RT results in pronounced antitumor effect without any notable toxicity. This indicates that the CD gene may be useful in the development of novel treatment strategies combining radiation and gene therapy in the treatment of locally advanced cancers

  12. Ethylene emission and PR protein synthesis in ACC deaminase producing Methylobacterium spp. inoculated tomato plants (Lycopersicon esculentum Mill.) challenged with Ralstonia solanacearum under greenhouse conditions.

    Science.gov (United States)

    Yim, Woojong; Seshadri, Sundaram; Kim, Kiyoon; Lee, Gillseung; Sa, Tongmin

    2013-06-01

    Bacteria of genus Methylobacterium have been found to promote plant growth and regulate the level of ethylene in crop plants. This work is aimed to test the induction of defense responses in tomato against bacterial wilt by stress ethylene level reduction mediated by the ACC deaminase activity of Methylobacterium strains. Under greenhouse conditions, the disease index value in Methylobacterium sp. inoculated tomato plants was lower than control plants. Plants treated with Methylobacterium sp. challenge inoculated with Ralstonia solanacearum (RS) showed significantly reduced disease symptoms and lowered ethylene emission under greenhouse condition. The ACC and ACO (1-aminocyclopropane-1-carboxylate oxidase) accumulation in tomato leaves were significantly reduced with Methylobacterium strains inoculation. While ACC oxidase gene expression was found higher in plants treated with R. solanacearum than Methylobacterium sp. treatment, PR proteins related to induced systemic resistance like β-1,3-glucanase, PAL, PO and PPO were increased in Methylobacterium sp. inoculated plants. A significant increase in β-1,3-glucanase and PAL gene expression was found in all the Methylobacterium spp. treatments compared to the R. solanacearum treatment. This study confirms the activity of Methylobacterium sp. in increasing the defense enzymes by modulating the ethylene biosynthesis pathway and suggests the use of methylotrophic bacteria as potential biocontrol agents in tomato cultivation. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  13. Molecular characterization of adenosine 5'-monophosphate deaminase--the key enzyme responsible for the umami taste of nori (Porphyra yezoensis Ueda, Rhodophyta).

    Science.gov (United States)

    Minami, Seiko; Sato, Minoru; Shiraiwa, Yoshihiro; Iwamoto, Koji

    2011-12-01

    The enzyme adenosine 5'-monophosphate deaminase (AMPD, EC 3.5.4.6) catalyzes the conversion of adenosine 5'-monophosphate to inosine 5'-mononucleotide (IMP). IMP is generally known as the compound responsible for the umami taste of the edible red alga Porphyra yezoensis Ueda that is known in Japan as nori. Therefore, we suspect that AMPD plays a key role in providing a favorable nori taste. In this study, we undertake the molecular characterization of nori-derived AMPD. The nori AMPD protein has a molecular mass of 55 kDa as estimated from both gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The calculated molecular mass from the amino acid sequence deduced from cDNA is 57.1 kDa. The isoelectric point is 5.71. The coding region of AMPD consists of 1,566 bp encoding 522 amino acids and possesses a transmembrane domain and two N-glycosylation sites. The sequence identity of nori AMPD in human and yeast AMPDs was found to be less than 50% and 20% in DNA and amino acid sequences, respectively. Proline in the conserved motif of [SA]-[LIVM]-[NGS]-[STA]-D-D-P was found to be converted to glutamate. These results indicate that nori AMPD is a novel type of AMPD.

  14. Methylobacterium oryzae sp. nov., an aerobic, pink-pigmented, facultatively methylotrophic, 1-aminocyclopropane-1-carboxylate deaminase-producing bacterium isolated from rice.

    Science.gov (United States)

    Madhaiyan, Munusamy; Kim, Byung-Yong; Poonguzhali, Selvaraj; Kwon, Soon-Wo; Song, Myung-Hee; Ryu, Jeoung-Hyun; Go, Seung-Joo; Koo, Bon-Sung; Sa, Tong-Min

    2007-02-01

    A pink-pigmented, facultatively methylotrophic bacterium, strain CBMB20T, isolated from stem tissues of rice, was analysed by a polyphasic approach. Strain CBMB20T utilized 1-aminocyclopropane 1-carboxylate (ACC) as a nitrogen source and produced ACC deaminase. It was related phylogenetically to members of the genus Methylobacterium. 16S rRNA gene sequence analysis indicated that strain CBMB20T was most closely related to Methylobacterium fujisawaense, Methylobacterium radiotolerans and Methylobacterium mesophilicum; however, DNA-DNA hybridization values were less than 70 % with the type strains of these species. The DNA G+C content of strain CBMB20T was 70.6 mol%. The study presents a detailed phenotypic characterization of strain CBMB20T that allows its differentiation from other Methylobacterium species. In addition, strain CBMB20T is the only known member of the genus Methylobacterium to be described from the phyllosphere of rice. Based on the data presented, strain CBMB20T represents a novel species in the genus Methylobacterium, for which the name Methylobacterium oryzae sp. nov. is proposed, with strain CBMB20T (=DSM 18207T=LMG 23582T=KACC 11585T) as the type strain.

  15. Comparative effectiveness of Pseudomonas and Serratia sp. containing ACC-deaminase for improving growth and yield of wheat (Triticum aestivum L.) under salt-stressed conditions.

    Science.gov (United States)

    Zahir, Zahir Ahmad; Ghani, Usman; Naveed, Muhammad; Nadeem, Sajid Mahmood; Asghar, Hafiz Naeem

    2009-05-01

    Ethylene synthesis is accelerated in response to various environmental stresses like salinity. Ten rhizobacterial strains isolated from wheat rhizosphere taken from different salt affected areas were screened for growth promotion of wheat under axenic conditions at 1, 5, 10 and 15 dS m(-1). Three strains, i.e., Pseudomonas putida (N21), Pseudomonas aeruginosa (N39) and Serratia proteamaculans (M35) showing promising performance under axenic conditions were selected for a pot trial at 1.63 (original), 5, 10 and 15 dS m(-1). Results showed that inoculation was effective even in the presence of higher salinity levels. P. putida was the most efficient strain compared to the other strains and significantly increased the plant height, root length, grain yield, 100-grain weight and straw yield up to 52, 60, 76, 19 and 67%, respectively, over uninoculated control at 15 dS m(-1). Similarly, chlorophyll content and K(+)/Na(+) of leaves also increased by P. putida over control. It is highly likely that under salinity stress, 1-aminocyclopropane-1-carboxylic acid-deaminase activity of these microbial strains might have caused reduction in the synthesis of stress (salt)-induced inhibitory levels of ethylene. The results suggested that these strains could be employed for salinity tolerance in wheat; however, P. putida may have better prospects in stress alleviation/reduction.

  16. Deficiency in L-serine deaminase interferes with one-carbon metabolism and cell wall synthesis in Escherichia coli K-12.

    Science.gov (United States)

    Zhang, Xiao; El-Hajj, Ziad W; Newman, Elaine

    2010-10-01

    Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes L-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S L-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of L-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C(1) units and interferes with cell wall synthesis. We suggest that at high concentrations, L-serine decreases synthesis of UDP-N-acetylmuramate-L-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high L-serine is overcome in several ways: by a large concentration of L-alanine, by overproducing MurC together with a low concentration of L-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide.

  17. Deficiency in l-Serine Deaminase Interferes with One-Carbon Metabolism and Cell Wall Synthesis in Escherichia coli K-12▿

    Science.gov (United States)

    Zhang, Xiao; El-Hajj, Ziad W.; Newman, Elaine

    2010-01-01

    Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes l-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S l-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of l-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C1 units and interferes with cell wall synthesis. We suggest that at high concentrations, l-serine decreases synthesis of UDP-N-acetylmuramate-l-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high l-serine is overcome in several ways: by a large concentration of l-alanine, by overproducing MurC together with a low concentration of l-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide. PMID:20729359

  18. Assignment of adenosine deaminase complexing protein (ADCP) gene(s) to human chromosome 2 in rodent-human somatic cell hybrids.

    Science.gov (United States)

    Herbschleb-Voogt, E; Grzeschik, K H; Pearson, P L; Meera Khan, P

    1981-01-01

    The experiments reported in this paper indicate that the expression of human adenosine deaminase complexing protein (ADCP) in the human-rodent somatic cell hybrids is influenced by the state of confluency of the cells and the background rodent genome. Thus, the complement of the L-cell derived A9 or B82 mouse parent apparently prevents the expression of human ADCP in the interspecific somatic cell hybrids. In the a3, E36, or RAG hybrids the human ADCP expression was not prevented by the rodent genome and was found to be proportional to the degree of confluency of the cell in the culture as in the case of primary human fibroblasts. An analysis of human chromosomes, chromosome specific enzyme markers, and ADCP in a panel of rodent-human somatic cell hybrids optimally maintained and harvested at full confluency has shown that the expression of human ADCP in the mouse (RAG)-human as well as in the hamster (E36 or a3)-human hybrids is determined by a gene(s) in human chromosome 2 and that neither chromosome 6 nor any other of the chromosomes of man carry any gene(s) involved in the formation of human ADCP at least in the Chinese hamster-human hybrids. A series of rodent-human hybrid clones exhibiting a mitotic separation of IDH1 and MDH1 indicated that ADCP is most probably situated between corresponding loci in human chromosome 2.

  19. The Role of G22 A Adenosine Deaminase 1 Gene Polymorphism and the Activities of ADA Isoenzymes in Fertile and Infertile Men.

    Science.gov (United States)

    Fattahi, Amir; Khodadadi, Iraj; Amiri, Iraj; Latifi, Zeinab; Ghorbani, Marzieh; Tavilani, Heidar

    2015-10-01

    To evaluate frequency distribution of adenosine deaminase 1 (ADA1) G22 A alleles and genotypes in fertile and infertile men. In this study we evaluate frequency distribution of ADA1 G22 A alleles and genotypes in 200 fertile and 200 infertile men. The polymerase chain reaction-restriction fragment length polymorphism technique was used for determining ADA1 G22 A variants. In addition, ADA isoenzymes activities (ADA1 and ADA2) were measured using colorimetric method. The frequency of GG genotype was significantly higher and GA genotype was lower in infertile males compared with fertile men (P = .048 and P = .045, respectively). However, there was not any noticeable difference in allele distribution between groups (P >.05). Based on logistic regression analysis, the GA genotype has a protective role and can decrease the risk of male infertility 1.7 times (P = .046). There were significantly higher activities of ADAT and its isoenzymes in infertile males compared with fertile men (P ADA1 activity with GG genotype was higher than GA carriers in all population (P = .001). Our results revealed that the activity of ADA isoenzymes and distribution of ADA1 G22 A genotypes were different among fertile and infertile men and more likely the GA genotype, which had lower ADA1 activity and was higher in fertile men is a protective factor against infertility. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Serum Adenosine Deaminase (ADA) Activity: A Novel Screening Test to Differentiate HIV Monoinfection From HIV-HBV and HIV-HCV Coinfections.

    Science.gov (United States)

    Abdi, Mohammad; Rahbari, Rizgar; Khatooni, Zahed; Naseri, Nima; Najafi, Adel; Khodadadi, Iraj

    2016-05-01

    CD4(+) cell count, the common HIV infection screening test, is costly and unable to differentiate HIV monoinfection from its concurrent infection with hepatitis B or C virus. We aimed to ascertain diagnostic value of serum adenosine deaminase (ADA) activity as a useful tool to differentiate HIV mono- and co-infection. Blood samples were collected from 30 HIV-HBV and 30 HIV-HCV coinfected patients, 33 HIV positive subjects, and 72 controls. CD4(+) cell count, serum total ADA (tADA), and ADA1, and ADA2 isoenzyme activities were determined and their sensitivity and specificity were computed. tADA and ADA2 activities were significantly higher and CD4(+) counts were markedly lower in all patients compared with controls. Strong inverse agreements between CD4(+) cell counts and both tADA and ADA2 activities were observed. Serum tADA and ADA1 activities showed the highest specificity and the highest sensitivity, respectively, for differentiating HIV monoinfection from HIV-HBV and HIV-HCV coinfections. We showed strong agreement and correlation between CD4(+) cell count and ADA enzyme activity. Based on high ADA sensitivity and specificity, it is concluded that determination of ADA activity might be a novel diagnostic tool to distinguish of HIV monoinfection from its coinfection with HBV or HCV. © 2015 Wiley Periodicals, Inc.

  1. Exploiting the pyrazolo[3,4-d]pyrimidin-4-one ring system as a useful template to obtain potent adenosine deaminase inhibitors.

    Science.gov (United States)

    La Motta, Concettina; Sartini, Stefania; Mugnaini, Laura; Salerno, Silvia; Simorini, Francesca; Taliani, Sabrina; Marini, Anna Maria; Da Settimo, Federico; Lavecchia, Antonio; Novellino, Ettore; Antonioli, Luca; Fornai, Matteo; Blandizzi, Corrado; Del Tacca, Mario

    2009-03-26

    A number of pyrazolo[3,4-d]pyrimidin-4-ones bearing either alkyl or arylalkyl substituents in position 2 of the nucleus were synthesized and tested for their ability to inhibit adenosine deaminase (ADA) from bovine spleen. The 2-arylalkyl derivatives exhibited excellent inhibitory activity, showing Ki values in the nanomolar/subnanomolar range. The most active compound, 1-(4-((4-oxo-4,5-dihydropyrazolo[3,4-d]pyrimidin-2-yl)methyl)phenyl)-3-(4-(trifluoromethyl)phenyl)urea, 14d, was tested in rats with colitis induced by 2,4-dinitrobenzenesulfonic acid to assess its efficacy to attenuate bowel inflammation. The treatment with 14d induced a significant amelioration of both systemic and intestinal inflammatory alterations in animals with experimental colitis. Docking simulations of the synthesized compounds into the ADA catalytic site were also performed to rationalize the structure-activity relationships observed and to highlight the key pharmacophoric elements of these products, thus prospectively guiding the design of novel ADA inhibitors.

  2. ANESTHETIC MANAGEMENT FOR A PATIENT WITH ACUTE INTERMITTENT PORPHYRIA

    Directory of Open Access Journals (Sweden)

    Nenad Savić

    2010-09-01

    Full Text Available Acute intermittent porphyria is a rare metabolic disorder resulting from a partial deficiency of porphobilinogen deaminase, enzyme in the heme biosynthetic pathway. Its inheritance is autosomal dominant. A deficiency of porphobilinogen deaminase is not sufficient by its self to produce acute intermittent porphyria, and other activating factors must also be present. These include some drugs, hormones, infection, injury and alcohol. Besides others, anesthetics have been implicated in the triggering of a number of severe porphyric reactions. Although there is no clinical evidence, the fear of hypothesized porphyrinogenicity of repetitive anesthetics exposures still remains. Despite these doubts, we report here the case of uneventful repeated exposure to anesthetics in a patient suffering from acute intermittent porphyria, within a fifteen- month period. On both occasions, the patient was safely exposed to certain anesthetics included: propofol, sevoflurane, rocuronium, midazolam and fentanyl.

  3. Of the Nine Cytidine Deaminase-Like Genes in Arabidopsis, Eight Are Pseudogenes and Only One Is Required to Maintain Pyrimidine Homeostasis in Vivo.

    Science.gov (United States)

    Chen, Mingjia; Herde, Marco; Witte, Claus-Peter

    2016-06-01

    CYTIDINE DEAMINASE (CDA) catalyzes the deamination of cytidine to uridine and ammonia in the catabolic route of C nucleotides. The Arabidopsis (Arabidopsis thaliana) CDA gene family comprises nine members, one of which (AtCDA) was shown previously in vitro to encode an active CDA. A possible role in C-to-U RNA editing or in antiviral defense has been discussed for other members. A comprehensive bioinformatic analysis of plant CDA sequences, combined with biochemical functionality tests, strongly suggests that all Arabidopsis CDA family members except AtCDA are pseudogenes and that most plants only require a single CDA gene. Soybean (Glycine max) possesses three CDA genes, but only two encode functional enzymes and just one has very high catalytic efficiency. AtCDA and soybean CDAs are located in the cytosol. The functionality of AtCDA in vivo was demonstrated with loss-of-function mutants accumulating high amounts of cytidine but also CMP, cytosine, and some uridine in seeds. Cytidine hydrolysis in cda mutants is likely caused by NUCLEOSIDE HYDROLASE1 (NSH1) because cytosine accumulation is strongly reduced in a cda nsh1 double mutant. Altered responses of the cda mutants to fluorocytidine and fluorouridine indicate that a dual specific nucleoside kinase is involved in cytidine as well as uridine salvage. CDA mutants display a reduction in rosette size and have fewer leaves compared with the wild type, which is probably not caused by defective pyrimidine catabolism but by the accumulation of pyrimidine catabolism intermediates reaching toxic concentrations. © 2016 American Society of Plant Biologists. All Rights Reserved.

  4. Effects of the inoculations using bacteria producing ACC deaminase on ethylene metabolism and growth of wheat grown under different soil water contents.

    Science.gov (United States)

    Zhang, Guozhuang; Sun, Yonglin; Sheng, Hao; Li, Haichao; Liu, Xiping

    2018-04-01

    Crop growth and productivity are often impacted by the increased ethylene content induced by adverse environmental conditions such drought. Inoculations with bacteria producing ACC deaminase is considered as a potential biological approach to improve the growth and tolerance of stressed plants by lowering endogenous ethylene level. In this study, germinated wheat seeds were inoculated using three species of the rhizobacteria, which were isolated from the rhizosphere of wheat growing in dryland, and sown in pots. After three weeks, wheat seedlings were exposed to non-limiting water condition, medium drought and severe drought, respectively, for six weeks. The results showed that, irrespective of rhizobacterial inoculations, decreased soil water contents stimulated wheat ethylene metabolism, which was reflected by the significantly increased activity of ACC synthetase and ACC oxidase, besides an increased content of ACC both in the roots and leaves, and an enhanced capacity of leaves to release ethylene, concomitant with a significant decline in shoot and roots biomass. The inoculations of all three rhizobacterial species under each water condition reduced ACC content in wheat leaves, but effects of the inoculations on ACC synthase and ACC oxidase activity in the leaves and roots, ACC content in the roots, the capacity of leaves to release ethylene, and wheat growth varied with water conditions and bacterial species. Hence, both soil water conditions and rhizobacterial inoculations acted on all the processes of ethylene metabolism, with the former being dominant. The inoculations under non-limiting water condition and medium drought promoted shoot and root growth of wheat plants. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  5. Changes in Adenosine Deaminase Activity in Patients with Type 2 Diabetes Mellitus and Effect of DPP-4 Inhibitor Treatment on ADA Activity

    Science.gov (United States)

    Lee, Jae-Geun; Kang, Dong Gu; Yu, Jung Re; Kim, Youngree; Kim, Jinsoek; Koh, Gwanpyo

    2011-01-01

    Background Dipeptidyl peptidase 4 (DPP-4, also known as CD26) binds with adenosine deaminase (ADA) to activate T lymphocytes. Here, we investigated whether ADA activity is specifically affected by treatment with DPP-4 inhibitor (DPP4I) compared with other anti-diabetic agents. Methods Fasting ADA activity, in addition to various metabolic and biochemical parameters, were measured in 262 type 2 diabetes mellitus (T2DM) patients taking various anti-diabetic agents and in 46 non-diabetic control subjects. Results ADA activity was increased in T2DM patients compared with that in non-diabetic control subjects (mean±standard error, 23.1±0.6 U/L vs. 18.6±0.8 U/L; PADA activity was correlated with fasting plasma glucose (r=0.258, P9%) showed significantly increased ADA activity (21.1±0.8 U/L vs. 25.4±1.6 U/L; PADA activity in T2DM patients did not differ from those of other oral anti-diabetic agents or insulin. T2DM patients on metformin monotherapy showed a lower ADA activity (20.9±1.0 U/L vs. 28.1±2.8 U/L; PADA activity is increased in T2DM patients compared to that in non-diabetic patients, is positively correlated with blood glucose level, and that DPP4I has no additional specific effect on ADA activity, except for a glycemic control- or HbA1c-dependent effect. PMID:21738897

  6. Curcumin improves episodic memory in cadmium induced memory impairment through inhibition of acetylcholinesterase and adenosine deaminase activities in a rat model.

    Science.gov (United States)

    Akinyemi, Ayodele Jacob; Okonkwo, Princess Kamsy; Faboya, Opeyemi Ayodeji; Onikanni, Sunday Amos; Fadaka, Adewale; Olayide, Israel; Akinyemi, Elizabeth Olufisayo; Oboh, Ganiyu

    2017-02-01

    Curcumin, the main polyphenolic component of turmeric (Curcuma longa) rhizomes has been reported to exert cognitive enhancing potential with limited scientific basis. Hence, this study sought to evaluate the effect of curcumin on cerebral cortex acetylcholinesterase (AChE) and adenosine deaminase (ADA) activities in cadmium (Cd)-induced memory impairment in rats. Animals were divided into six groups (n = 6): saline/vehicle, saline/curcumin 12.5 mg/kg, saline/curcumin 25 mg/kg, Cd/vehicle, Cd/curcumin 12.5 mg/kg, and Cd/curcumin 25 mg/kg. Rats received Cd (2.5 mg/kg) and curcumin (12.5 and 25 mg/kg, respectively) by gavage for 7 days. The results of this study revealed that cerebral cortex AChE and ADA activities were increased in Cd-poisoned rats, and curcumin co-treatment reversed these activities to the control levels. Furthermore, Cd intoxication increased the level of lipid peroxidation in cerebral cortex with a concomitant decreased in functional sulfuhydryl (-SH) group and nitric oxide (NO), a potent neurotransmitter and neuromodulatory agent. However, the co-treatment with curcumin at 12.5 and 25 mg/kg, respectively increased the non-enzymatic antioxidant status and NO in cerebral cortex with a decreased in malondialdehyde (MDA) level. Therefore, inhibition of AChE and ADA activities as well as increased antioxidant status by curcumin in Cd-induced memory dysfunction could suggest some possible mechanism of action for their cognitive enhancing properties.

  7. Epstein-Barr Virus Lytic Reactivation Activates B Cells Polyclonally and Induces Activation-Induced Cytidine Deaminase Expression: A Mechanism Underlying Autoimmunity and Its Contribution to Graves' Disease.

    Science.gov (United States)

    Nagata, Keiko; Kumata, Keisuke; Nakayama, Yuji; Satoh, Yukio; Sugihara, Hirotsugu; Hara, Sayuri; Matsushita, Michiko; Kuwamoto, Satoshi; Kato, Masako; Murakami, Ichiro; Hayashi, Kazuhiko

    2017-04-01

    Graves' disease is an autoimmune disease that results in and is the most common cause of hyperthyroidism, and the reactivation of persisting Epstein-Barr virus (EBV) in B lymphocytes induces the differentiation of host B cells into plasma cells. We previously reported that some EBV-infected B cells had thyrotropin receptor antibodies (TRAbs) as surface immunoglobulins (Igs), and EBV reactivation induced these TRAb+EBV+ cells to produce TRAbs. EBV reactivation induces Ig production from host B cells. The purpose of the present study was to examine total Ig productions from B cell culture fluids and to detect activation-induced cytidine deaminase (AID), nuclear factor kappa B (NF-κB), and EBV latent membrane protein (LMP) 1 in culture B cells during EBV reactivation induction and then we discussed the mechanisms of EBV reactivation-induced Ig production in relation to autoimmunity. We showed that the EBV reactivation induces the production of every isotype of Ig and suggested that the Ig production was catalyzed by AID through LMP1 and NF-κB. The results that the amount of IgM was significantly larger compared with IgG suggested the polyclonal B cell activation due to LMP1. We proposed the pathway of EBV reactivation induced Ig production; B cells newly infected with EBV are activated by polyclonal B cell activation and produce Igs through plasma cell differentiation induced by EBV reactivation. LMP1-induced AID enabled B cells to undergo class-switch recombination to produce every isotype of Ig. According to this mechanism, EBV rescues autoreactive B cells to produce autoantibodies, which contribute to the development and exacerbation of autoimmune diseases.

  8. Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1.

    Science.gov (United States)

    Kanehiro, Yuichi; Todo, Kagefumi; Negishi, Misaki; Fukuoka, Junji; Gan, Wenjian; Hikasa, Takuya; Kaga, Yoshiaki; Takemoto, Masayuki; Magari, Masaki; Li, Xialu; Manley, James L; Ohmori, Hitoshi; Kanayama, Naoki

    2012-01-24

    Somatic hypermutation (SHM) of Ig variable region (IgV) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.

  9. Bioconversion of l-glutamic acid to α-ketoglutaric acid by an immobilized whole-cell biocatalyst expressing l-amino acid deaminase from Proteus mirabilis.

    Science.gov (United States)

    Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-dong; Chen, Rachel R; Du, Guocheng; Liu, Long; Chen, Jian

    2014-01-01

    The goal of this work was to develop an immobilized whole-cell biocatalytic process for the environment-friendly synthesis of α-ketoglutaric acid (α-KG) from l-glutamic acid. We compared the suitability of Escherichia coli and Bacillus subtilis strains overexpressing Proteus mirabilisl-amino acid deaminase (l-AAD) as potential biocatalysts. Although both recombinant strains were biocatalytically active, the performance of B. subtilis was superior to that of E. coli. With l-glutamic acid as the substrate, α-KG production levels by membranes isolated from B. subtilis and E. coli were 55.3±1.73 and 21.7±0.39μg/mg protein/min, respectively. The maximal conversion ratio of l-glutamic acid to α-KG was 31% (w/w) under the following optimal conditions: 15g/L l-glutamic acid, 20g/L whole-cell biocatalyst, 5mM MgCl2, 40°C, pH 8.0, and 24-h incubation. Immobilization of whole cells with alginate increased the recyclability by an average of 23.33% per cycle. This work established an efficient one-step biotransformation process for the production of α-KG using immobilized whole B. subtilis overexpressing P. mirabilisl-AAD. Compared with traditional multistep chemical synthesis, the biocatalytic process described here has the advantage of reducing environmental pollution and thus has great potential for the large-scale production of α-KG. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Evaluation of a diagnostic flow chart applying medical thoracoscopy, adenosine deaminase and T-SPOT.TB in diagnosis of tuberculous pleural effusion.

    Science.gov (United States)

    He, Y; Zhang, W; Huang, T; Wang, X; Wang, M

    2015-10-01

    To evaluate a diagnostic flow chart applying medical thoracoscoy (MT), adenosine deaminase (ADA) and T-SPOT.TB in diagnosis of tuberculous pleural effusion (TPE) at a high TB burden country. 136 patients with pleural effusion (PE) were enrolled and divided into TPE and Non-TPE group. MT (histology), PE ADA and T-SPOT.TB were conducted on all patients. ROC analysis was performed for the best cut-off value of PE ADA in detection of TPE. The diagnostic flow chart applying MT, ADA and T-SPOT.TB was evaluated for improving the limitations of each diagnostic method. ROC analysis showed that the best cut-off value of PE ADA was 30U/L. The sensitivity and specificity of these tests were calculated respectively to be: 71.4% (58.5%-81.6%) and 100% (95.4-100.0%) for MT, 92.9% (83.0-97.2%) and 68.8% (57.9-77.9%) for T-SPOT.TB, and 80.0% (69.6-88.1%) and 92.9% (82.7-98.0%) for PE ADA. The sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, positive predictive value and negative predictive value of the diagnostic flow chart were 96.4% (87.9-99.0%), 96.3% (89.6-98.7%), 25.714, 0.037, 97.4 and 94.9, respectively. The diagnostic flow chart applying MT, ADA and T-SPOT.TB is an accurate and rapid diagnostic method in detection of TPE.

  11. Double-stranded-RNA-specific adenosine deaminase 1 (ADAR1) is proposed to contribute to the adaptation of equine infectious anemia virus from horses to donkeys.

    Science.gov (United States)

    Tang, Yan-Dong; Zhang, Xiang; Na, Lei; Wang, Xue-Feng; Fu, Li-Hua; Zhu, Chun-Hui; Wang, Xiaojun; Zhou, Jian-Hua

    2016-10-01

    Equine infectious anemia virus (EIAV) is a member of the genus Lentivirus of the family Retroviridae. Horses are the most susceptible equids to EIAV infection and are therefore the primary hosts of this virus. In contrast, infected donkeys do not develop clinically active equine infectious anemia (EIA). This phenomenon is similar to what has been observed with HIV-1, which fails to induce AIDS in non-human primates. Interestingly, Shen et al. developed a donkey-tropic pathogenic virus strain (EIAVDV117, DV117) by serially passaging a horse-tropic pathogenic strain, EIAVLN40 (LN40), in donkeys. LN40, which was generated by passaging a field isolate in horses, displayed enhanced virulence in horses but caused no clinical symptoms in donkeys. Infection with DV117 induced acute EIA in nearly 100 % of donkeys. Genomic analysis of DV117 revealed a significantly higher frequency of A-to-G substitutions when compared to LN40. Furthermore, detailed analysis of dinucleotide editing showed that A-to-G mutations had a preference for 5'TpA and 5'ApA. These results strongly implicated the activity of the adenosine deaminase, ADAR1, in this type of mutation. Further investigation demonstrated that overexpression of donkey ADAR1 increased A-to-G mutations within the genome of EIAV. Together with our previous finding that multiple mutations in multiple genes are generated in DV117 during its adaptation from horses to donkeys, the present study suggests that ADAR1-induced A-to-G mutations occur during virus adaption to related new hosts contributing to the alteration of EIAV host tropism.

  12. Changes in Adenosine Deaminase Activity in Patients with Type 2 Diabetes Mellitus and Effect of DPP-4 Inhibitor Treatment on ADA Activity.

    Science.gov (United States)

    Lee, Jae-Geun; Kang, Dong Gu; Yu, Jung Re; Kim, Youngree; Kim, Jinsoek; Koh, Gwanpyo; Lee, Daeho

    2011-04-01

    Dipeptidyl peptidase 4 (DPP-4, also known as CD26) binds with adenosine deaminase (ADA) to activate T lymphocytes. Here, we investigated whether ADA activity is specifically affected by treatment with DPP-4 inhibitor (DPP4I) compared with other anti-diabetic agents. Fasting ADA activity, in addition to various metabolic and biochemical parameters, were measured in 262 type 2 diabetes mellitus (T2DM) patients taking various anti-diabetic agents and in 46 non-diabetic control subjects. ADA activity was increased in T2DM patients compared with that in non-diabetic control subjects (mean±standard error, 23.1±0.6 U/L vs. 18.6±0.8 U/L; PADA activity was correlated with fasting plasma glucose (r=0.258, P9%) showed significantly increased ADA activity (21.1±0.8 U/L vs. 25.4±1.6 U/L; PADA activity in T2DM patients did not differ from those of other oral anti-diabetic agents or insulin. T2DM patients on metformin monotherapy showed a lower ADA activity (20.9±1.0 U/L vs. 28.1±2.8 U/L; PADA activity is increased in T2DM patients compared to that in non-diabetic patients, is positively correlated with blood glucose level, and that DPP4I has no additional specific effect on ADA activity, except for a glycemic control- or HbA1c-dependent effect.

  13. Identification of pleural effusion with low levels of adenosine deaminase but without signs of acute inflammation or pleural thickening to diagnose early malignant pleural mesothelioma

    International Nuclear Information System (INIS)

    Moriyama, Satoru; Tanahashi, Masayuki; Suzuki, Eriko

    2012-01-01

    We reviewed the clinical findings and diagnostic methods used in the diagnosis of malignant pleural mesothelioma (MPM) in patients with pleural effusion with low levels of adenosine deaminase (ADA), but without signs of acute inflammation or pleural thickening. The hospital records of 40 patients with pleural effusion of unknown origin or pleural thickening were retrospectively investigated. In all of those studies, pleural effusion was exudative, lymphocyte-dominant, no mycobacteria or other bacteria, and low levels of ADA. There were 30 men and 10 women with an average age of 62.8 years old. The diagnosis of MPM was obtained by cytology of the pleural effusion in 3 patients and by core-needle biopsy of the thickened pleura in 3. Thoracoscopic pleural biopsy under general anesthesia was performed for the other 34 patients. The pathologic diagnosis of pleural biopsy was MPM in 20 patients, inflammatory change in 12, and pleural dissemination of cancer in 2. All of the 7 patients with more than 100 μg/ml of hyaluronic acid in their pleural effusion received a diagnosis of MPM. A total of 20 of 23 patients with irregular or nodular pleural thickening on computed tomography findings were confirmed to have MPM. Moreover, 6 of 17 patients with smooth pleural thickening were confirmed to have MPM. In patients with nodular pleural thickening it was easy to make the diagnosis. However, in those with smooth thickening, careful observation was required to select the appropriate biopsy site and resection margins of full-thickness pleura. As the rate of MPM in the patients with pleural effusion with low levels of ADA, but without signs of acute inflammation or pleural thickening is high (65%), an early thoracoscopic pleural biopsy is strongly recommended. (author)

  14. Increased Number of Circulating CD8/CD26 T Cells in the Blood of Duchenne Muscular Dystrophy Patients Is Associated with Augmented Binding of Adenosine Deaminase and Higher Muscular Strength Scores

    Directory of Open Access Journals (Sweden)

    Jonathan H. Soslow

    2017-12-01

    Full Text Available Duchenne muscular dystrophy (DMD is an X-linked disorder that leads to cardiac and skeletal myopathy. The complex immune activation in boys with DMD is incompletely understood. To better understand the contribution of the immune system into the progression of DMD, we performed a systematic characterization of immune cell subpopulations obtained from peripheral blood of DMD subjects and control donors. We found that the number of CD8 cells expressing CD26 (also known as adenosine deaminase complexing protein 2 was increased in DMD subjects compared to control. No differences, however, were found in the levels of circulating factors associated with pro-inflammatory activation of CD8/CD26 cells, such as tumor necrosis factor-α (TNFα, granzyme B, and interferon-γ (IFNγ. The number of CD8/CD26 cells correlated directly with quantitative muscle testing (QMT in DMD subjects. Since CD26 mediates binding of adenosine deaminase (ADA to the T cell surface, we tested ADA-binding capacity of CD8/CD26 cells and the activity of bound ADA. We found that mononuclear cells (MNC obtained from DMD subjects with an increased number of CD8/CD26 T cells had a greater capacity to bind ADA. In addition, these MNC demonstrated increased hydrolytic deamination of adenosine to inosine. Altogether, our data demonstrated that (1 an increased number of circulating CD8/CD26 T cells is associated with preservation of muscle strength in DMD subjects, and (2 CD8/CD26 T cells from DMD subjects mediated degradation of adenosine by adenosine deaminase. These results support a role for T cells in slowing the decline in skeletal muscle function, and a need for further investigation into contribution of CD8/CD26 T cells in the regulation of chronic inflammation associated with DMD.

  15. The tertiary origin of the allosteric activation of E. coli glucosamine-6-phosphate deaminase studied by sol-gel nanoencapsulation of its T conformer.

    Directory of Open Access Journals (Sweden)

    Sergio Zonszein

    Full Text Available The role of tertiary conformational changes associated to ligand binding was explored using the allosteric enzyme glucosamine-6-phosphate (GlcN6P deaminase from Escherichia coli (EcGNPDA as an experimental model. This is an enzyme of amino sugar catabolism that deaminates GlcN6P, giving fructose 6-phosphate and ammonia, and is allosterically activated by N-acetylglucosamine 6-phosphate (GlcNAc6P. We resorted to the nanoencapsulation of this enzyme in wet silica sol-gels for studying the role of intrasubunit local mobility in its allosteric activation under the suppression of quaternary transition. The gel-trapped enzyme lost its characteristic homotropic cooperativity while keeping its catalytic properties and the allosteric activation by GlcNAc6P. The nanoencapsulation keeps the enzyme in the T quaternary conformation, making possible the study of its allosteric activation under a condition that is not possible to attain in a soluble phase. The involved local transition was slowed down by nanoencapsulation, thus easing the fluorometric analysis of its relaxation kinetics, which revealed an induced-fit mechanism. The absence of cooperativity produced allosterically activated transitory states displaying velocity against substrate concentration curves with apparent negative cooperativity, due to the simultaneous presence of subunits with different substrate affinities. Reaction kinetics experiments performed at different tertiary conformational relaxation times also reveal the sequential nature of the allosteric activation. We assumed as a minimal model the existence of two tertiary states, t and r, of low and high affinity, respectively, for the substrate and the activator. By fitting the velocity-substrate curves as a linear combination of two hyperbolic functions with Kt and Kr as KM values, we obtained comparable values to those reported for the quaternary conformers in solution fitted to MWC model. These results are discussed in the

  16. [Diagnostic performance of T-SPOT.TB on peripheral blood in combination with adenosine deaminase on pleural fluid for the diagnosis of tuberculous pleurisy within different age group].

    Science.gov (United States)

    Xu, H Y; Zhang, D Q; Ye, J R; Su, S S; Xie, Y P; Chen, C S; Li, Y P

    2017-06-27

    Objective: To evaluate the performance of T cell enzyme-linked immuno-spot assay (T-SPOT) on peripheral blood in combination with adenosine deaminase (ADA) on pleural fluid for diagnosis of tuberculous (TB) pleurisy within different age groups. Methods: The data of patients with pleural effusion from the Department of Pulmonary and Critical Care Medicine of the First Affiliated Hospital of Wenzhou Medical University from April 2012 to November 2016 were retrospectively analyzed, and the diagnoses of these patients were histopathologically confirmed through medical thoracoscopy. The cases who had confirmed diagnosis, in the same time, received peripheral blood T-SPOT.TB were enrolled. The performance of peripheral blood T-SPOT.TB in combination with pleural fluid ADA on diagnosing TB pleurisy in the younger patients (16-59 years old) and elderly patients (≥60 years old) were analyzed respectively. The sensitivity, specificity and the receiver operating characteristic (ROC) curve were adopted for statistical analysis. Results: A total of 448 cases were finally enrolled, 341(76.1%) confirmed with TB pleurisy, 224 males, 117 females, (47±19) years old; and 107 (23.9%) classified as non-TB pleurisy, 65 males, 42 females, (61±14) years old. There were 285 cases who were classified as younger group, and the other 163 cases were classified as elderly group. The sensitivity and specificity of peripheral blood T-SPOT.TB were 85.4% (204/239) and 71.7% (33/46) in the younger patients, 76.5% (78/102) and 59.0% (36/61) respectively in the elderly patients. The sensitivity of peripheral blood T-SPOT.TB in the younger patients was significantly higher than that in the elderly patients ( P =0.047). The sensitivity and specificity were 99.2% and 95.7% in combination with peripheral blood T-SPOT.TB and pleural fluid ADA respectively in the younger patients. The area under ROC curve (AUC) of T-SPOT.TB in the younger patients was 0.833, AUC of T-SPOT.TB combined with ADA was 0

  17. Production of phenylpyruvic acid from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis: comparison of enzymatic and whole-cell biotransformation approaches.

    Science.gov (United States)

    Hou, Ying; Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng

    2015-10-01

    Phenylpyruvic acid (PPA) is an important organic acid that has a wide range of applications. In this study, the membrane-bound L-amino acid deaminase (L-AAD) gene from Proteus mirabilis KCTC 2566 was expressed in Escherichia coli BL21(DE3) and then the L-AAD was purified. After that, we used the purified enzyme and the recombinant E. coli whole-cell biocatalyst to produce PPA via a one-step biotransformation from L-phenylalanine. L-AAD was solubilized from the membrane and purified 52-fold with an overall yield of 13 %, which corresponded to a specific activity of 0.94 ± 0.01 μmol PPA min(-1)·mg(-1). Then, the biotransformation conditions for the pure enzyme and the whole-cell biocatalyst were optimized. The maximal production was 2.6 ± 0.1 g·L(-1) (specific activity of 1.02 ± 0.02 μmol PPA min(-1)·mg(-1) protein, 86.7 ± 5 % mass conversion rate, and 1.04 g·L(-1)·h(-1) productivity) and 3.3 ± 0.2 g L(-1) (specific activity of 0.013 ± 0.003 μmol PPA min(-1)·mg(-1) protein, 82.5 ± 4 % mass conversion rate, and 0.55 g·L(-1)·h(-1) productivity) for the pure enzyme and whole-cell biocatalyst, respectively. Comparative studies of the enzymatic and whole-cell biotransformation were performed in terms of specific activity, production, conversion, productivity, stability, need of external cofactors, and recycling. We have developed two eco-friendly and efficient approaches for PPA production. The strategy described herein may aid the biotransformational synthesis of other α-keto acids from their corresponding amino acids.

  18. A Ser29Leu substitution in the cytosine deaminase Fca1p is responsible for clade-specific flucytosine resistance in Candida dubliniensis.

    LENUS (Irish Health Repository)

    McManus, Brenda A

    2009-11-01

    The population structure of the opportunistic yeast pathogen Candida dubliniensis is composed of three main multilocus sequence typing clades (clades C1 to C3), and clade C3 predominantly consists of isolates from the Middle East that exhibit high-level resistance (MIC(50) > or = 128 microg\\/ml) to the fungicidal agent flucytosine (5FC). The close relative of C. dubliniensis, C. albicans, also exhibits clade-specific resistance to 5FC, and resistance is most commonly mediated by an Arg101Cys substitution in the FUR1 gene encoding uracil phosphoribosyltransferase. Broth microdilution assays with fluorouracil (5FU), the toxic deaminated form of 5FC, showed that both 5FC-resistant and 5FC-susceptible C. dubliniensis isolates exhibited similar 5FU MICs, suggesting that the C. dubliniensis cytosine deaminase (Fca1p) encoded by C. dubliniensis FCA1 (CdFCA1) may play a role in mediating C. dubliniensis clade-specific 5FC resistance. Amino acid sequence analysis of the CdFCA1 open reading frame (ORF) identified a homozygous Ser29Leu substitution in all 12 5FC-resistant isolates investigated which was not present in any of the 9 5FC-susceptible isolates examined. The tetracycline-inducible expression of the CdFCA1 ORF from a 5FC-susceptible C. dubliniensis isolate in two separate 5FC-resistant clade C3 isolates restored susceptibility to 5FC, demonstrating that the Ser29Leu substitution was responsible for the clade-specific 5FC resistance and that the 5FC resistance encoded by FCA1 genes with the Ser29Leu transition is recessive. Quantitative real-time PCR analysis showed no significant difference in CdFCA1 expression between 5FC-susceptible and 5FC-resistant isolates in either the presence or the absence of subinhibitory concentrations of 5FC, suggesting that the Ser29Leu substitution in the CdFCA1 ORF is the sole cause of 5FC resistance in clade C3 C. dubliniensis isolates.

  19. Imaging Expression of Cytosine Deaminase-Herpes Virus Thymidine Kinase Fusion Gene (CD/TK Expression with [124I]FIAU and PET

    Directory of Open Access Journals (Sweden)

    Trevor Hackman

    2002-01-01

    Full Text Available Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CDherpes simplex virus type 1 thymidine kinase (HSV1-tk fusion gene (CD/TK with 5-fluorocytosine (5FC, ganciclovir (GCV, and radiotherapy is currently under evaluation for treatment of different tumors. We assessed the efficacy of noninvasive imaging with [124I]FIAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodo-uracil and positron emission tomography (PET for monitoring expression of the CD/TK fusion gene. Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells. The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively. A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels. Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats. PET imaging of HSV1-TK subunit activity with [124I]FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively. CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay. A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation. Knowing this relationship, the parametric images of CD subunit activity were generated. Imaging with [124I]FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials.

  20. Tandem mass spectrometry, but not T-cell receptor excision circle analysis, identifies newborns with late-onset adenosine deaminase deficiency.

    Science.gov (United States)

    la Marca, Giancarlo; Canessa, Clementina; Giocaliere, Elisa; Romano, Francesca; Duse, Marzia; Malvagia, Sabrina; Lippi, Francesca; Funghini, Silvia; Bianchi, Leila; Della Bona, Maria Luisa; Valleriani, Claudia; Ombrone, Daniela; Moriondo, Maria; Villanelli, Fabio; Speckmann, Carsten; Adams, Stuart; Gaspar, Bobby H; Hershfield, Michael; Santisteban, Ines; Fairbanks, Lynette; Ragusa, Giovanni; Resti, Massimo; de Martino, Maurizio; Guerrini, Renzo; Azzari, Chiara

    2013-06-01

    Adenosine deaminase (ADA)-severe combined immunodeficiency (SCID) is caused by genetic variants that disrupt the function of ADA. In its early-onset form, it is rapidly fatal to infants. Delayed or late-onset ADA-SCID is characterized by insidious progressive immunodeficiency that leads to permanent organ damage or death. Quantification of T-cell receptor excision circles (TRECs) or tandem mass spectrometry (tandem-MS) analysis of dried blood spots (DBSs) collected at birth can identify newborns with early-onset ADA-SCID and are used in screening programs. However, it is not clear whether these analyses can identify newborns who will have delayed or late-onset ADA-SCID before symptoms appear. We performed a retrospective study to evaluate whether tandem-MS and quantitative TREC analyses of DBSs could identify newborns who had delayed-onset ADA-SCID later in life. We tested stored DBSs collected at birth from 3 patients with delayed-onset ADA-SCID using tandem-MS (PCT EP2010/070517) to evaluate levels of adenosine and 2'-deoxyadenosine and real-time PCR to quantify TREC levels. We also analyzed DBSs from 3 newborns with early-onset ADA-SCID and 2 healthy newborn carriers of ADA deficiency. The DBSs taken at birth from the 3 patients with delayed-onset ADA-SCID had adenosine levels of 10, 25, and 19 μmol/L (normal value, <1.5 μmol/L) and 2'-deoxyadenosine levels of 0.7, 2.7, and 2.4 μmol/L (normal value, <0.07 μmol/L); the mean levels of adenosine and 2'-deoxyadenosine were respectively 12.0- and 27.6-fold higher than normal values. DBSs taken at birth from all 3 patients with delayed-onset ADA deficiency had normal TREC levels, but TRECs were undetectable in blood samples taken from the same patients at the time of diagnosis. Tandem-MS but not TREC quantification identifies newborns with delayed- or late-onset ADA deficiency. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  1. Rapid, reliable, and sensitive detection of adenosine deaminase activity by UHPLC-Q-Orbitrap HRMS and its application to inhibitory activity evaluation of traditional Chinese medicines.

    Science.gov (United States)

    Qi, Shenglan; Guan, Huida; Deng, Gang; Yang, Tao; Cheng, Xuemei; Liu, Wei; Liu, Ping; Wang, Changhong

    2018-05-10

    Adenosine deaminase (ADA), which is a key enzyme in the metabolism of purine nucleosides, plays important roles in diverse disorders, such as tuberculosis, diabetes, liver disorders, and cancer. Determination of the activities of ADA and its isoenzymes in body fluids has received considerable attention in the diagnosis and treatment of relative diseases. Ultraviolet spectroscopy with adenosine (AD) as a substrate is a classical approach for screening potential ADA inhibitors by measuring the decrease in substrate (AD) at 265 nm or increase in the product (inosine) at 248 nm. However, AD and inosine share a very close maximum absorption wavelength, and the reaction is uncertain and is frequently interfered by the background color of matrix compounds or plant extracts. Thus, the method usually yields false positive or negative results. In this study, a novel, rapid, sensitive, and accurate ultra-high-performance liquid chromatography-Q exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometric (UHPLC-Q-Orbitrap HRMS) method was developed for determining and screening ADA inhibitors by directly determining the deamination product of AD, inosine. A proper separation was achieved for inosine and chlormequat (internal standard) within 2 min via isocratic elution (0.1% formic acid:methanol = 85:15, v/v) at a flow rate of 0.3 mL min -1 on a Waters ACQUITY HSS T3 column (2.1 mm × 100 mm, 1.8 μm) following a simple precipitation of proteins. The intra- and inter-day precisions of the developed method were below 7.17% and 8.99%, respectively. The method exhibited advantages of small total reaction volume (60 μL), short running time (2 min), high sensitivity (lowest limit of quantification of 0.02 μM for inosine), and low cost (small enzyme consumption of 0.007 unit mL -1 for ADA and substrate of 3.74 μM for AD in individual inhibition), and no matrix effects (101.64%-107.12%). Stability results showed that all

  2. Value of combined detection of interferon-γ, vascular endothelial growth factor, C-reactive protein and adenosine deaminase in differential diagnosis of tuberculous and malignant pleural effusion

    International Nuclear Information System (INIS)

    Li Guo'an; Han Sugui; Zhou Xiuyan; He Weishe; Sun Fangchu

    2012-01-01

    Objective: To explore the value of interferon II, vascular endothelial growth factor, C-reactive protein and adenosine deaminase in differential diagnosis of tuberculous and malignant pleural effusion. Methods: 122 cases with tuberculous pleurisy, 56 cases of malignant pleural effusion, 48 cases of tuberculous pleural effusion, 18 cases of inflammatory and other pleural fluid were studied. The serum and pleural fluid levels of IFN-γ, VEGF-C, CRP and ADA serum in those patients were detected. Results: The IFN-γ, CRP and ADA levels in tuberculous pleural effusion were higher than in malignant pleural effusion(P<0.01). According to the receiver operator characteristic (ROC) curve, when 100 ng/L was regarded as critical value of IFN-γ, the sensitivity and specificity of IFN-γ in diagnosing tuberculous pleural effusion were 83.1% and 92.3% respectively. When 45 U/L ADA was regarded as critical value of ADA, the sensitivity and specificity of ADA in diagnosing tuberculous pleural effusion were 85.6% and 96.3% respectively. When 110 mg/L was regarded as critical value of CRP, the sensitivity and specificity of CRP were 79.1% and 84.2% respectively. When combine detection of three markers, the diagnosis sensitivity and specificity were 87.8% and 86.0% respectively. The VEGF-C concentration in malignant pleural effusion was higher than that in tuberculous pleural effusion and inflammatory and other pleural effusion (P<0.01). When the ratio of VEGF-C to ADA≥8, the sensitivity and specificity in diagnosis of malignant pleural effusion were 86.3% and 82.6% respectively, and the ration VEGF-C to ADA≤3, the sensitivity and specificity in diagnosis of tuberculous pleural effusion were 85.1% and 87.1% respectively. Conclusion: The combined detection of IFN-γ, CRP and ADA could improve sensitivity and specificity in diagnosing tuberculous pleurisy. The concentration ratios of VEGF-C to ADA have clinical value in differential diagnosis of pleural effusions. (authors)

  3. Clinical value of jointly detection serum lactate dehydrogenase/pleural fluid adenosine deaminase and pleural fluid carcinoembryonic antigen in the identification of malignant pleural effusion.

    Science.gov (United States)

    Zhang, Fan; Hu, Lijuan; Wang, Junjun; Chen, Jian; Chen, Jie; Wang, Yumin

    2017-09-01

    Limited data are available for the diagnostic value, and for the diagnostic sensitivity and specificity of joint detection of serum lactate dehydrogenase (sLDH)/pleural fluid adenosine deaminase (pADA) and pleural fluid carcinoembryonic antigen (pCEA) in malignant pleural effusion (MPE). We collected 987 pleural effusion specimens (of which 318 were malignant pleural effusion, 374 were tubercular pleural effusion, and 295 were parapneumonic effusion specimens) from the First Affiliated Hospital of Wenzhou Medical University from July 2012 to March 2016. The pADA, sLDH, pleural fluid LDH (pLDH), serum C-reactive protein (sCRP), pleural fluid protein, pCEA, white blood cell (WBC), and red blood cell (RBC) were analyzed, and the clinical data of each group were collected for statistical analysis. The level of sLDH/pADA, pCEA, and RBC from the MPE group was markedly higher than the tuberculosis pleural effusion (TB) group (Mann-Whitney U=28422.000, 9278.000, 30518, P=.000, .000, .000) and the parapneumonic pleural fluid group (Mann-Whitney U=5972.500, 7113.000, 36750.500, P=.000, .000, .000). The receiver operating characteristic curve ROC showed that the area under the ROC curve (AUC) (=0.924, 0.841) of pCEA and sLDH/pADA (cutoff=4.9, 10.6) were significantly higher than other markers for the diagnosis of MPE. Thus, joint detection of pCEA and sLDH/pADA suggested that the sensitivity, specificity, and AUC was 0.94, 81.70, and 94.32 at the cutoff 0.16 and diagnostic performance was higher than pCEA or sLDH/pADA. Joint detection of sLDH/pADA and pCEA can be used as a good indicator for the identification of benign and MPE with higher sensitivity and specificity than pCEA or sLDH/pADA. © 2016 Wiley Periodicals, Inc.

  4. Avaliação da adenosina desaminase no diagnóstico da tuberculose pleural: uma metanálise brasileira Evaluation of adenosine deaminase in the diagnosis of pleural tuberculosis: a Brazilian meta-analysis

    Directory of Open Access Journals (Sweden)

    Patrizio Morisson

    2008-04-01

    Full Text Available OBJETIVO: Avaliar trabalhos brasileiros resumindo a acurácia da adenosina desaminase no diagnóstico da tuberculose pleural, com o intuito de contribuir para a concretização do exame como rotina na investigação dos derrames pleurais. MÉTODOS: Depois de realizada uma busca por trabalhos brasileiros referentes à dosagem da adenosina desaminase no líquido pleural, estes foram avaliados e incluídos no estudo. A análise dos dados foi feita por meio da curva summary receiver operating characteristic (SROC que possibilitou a reunião dos estudos quanto a acurácia para o diagnóstico. Com os valores globais de sensibilidade e especificidade foi aplicado o teorema de Bayes para calcular as probabilidades pós-teste em diferentes prevalências da doença. RESULTADOS: Entre 1987 e 2005 foram encontrados 25 estudos contendo informações suficientes que poderiam ser utilizadas na metanálise. Após avaliação, foram incluídos nove estudos, totalizando 1.674 pacientes. De acordo com a curva SROC, foi encontrada uma sensibilidade de 91,8% (IC95%: 89,8-93,6% e uma especificidade de 88,4% (IC95%: 86,0-90,5%, com uma área abaixo da curva de 0,969. O odds ratio global foi de 112,0 (IC95%: 51,6-243,2. Considerando uma prevalência da tuberculose pleural de 50% (considerada neutra, a probabilidade do diagnóstico pós-teste positivo é de 88,7% e sua exclusão após resultado negativo de 91,5%. CONCLUSÕES: Apesar das diferenças encontradas entre os estudos, é possível concluir que a adenosina desaminase possui alta acurácia no diagnóstico da tuberculose pleural, devendo ser utilizada de rotina em sua investigação.OBJECTIVE: To evaluate Brazilian studies by summarizing the accuracy of adenosine deaminase in the diagnosis of pleural tuberculosis, with the objective of lending support to the movement to make the test part of the routine investigation of pleural effusions. METHODS: A search for Brazilian studies related to the determination of

  5. Carrier frequency of a nonsense mutation in the adenosine deaminase (ADA) gene implies a high incidence of ADA-deficient severe combined immunodeficiency (SCID) in Somalia and a single, common haplotype indicates common ancestry

    DEFF Research Database (Denmark)

    Sanchez Sanchez, Juan Jose; Monaghan, Gemma; Børsting, Claus

    2007-01-01

    Inherited adenosine deaminase (ADA) deficiency is a rare metabolic disorder that causes immunodeficiency, varying from severe combined immunodeficiency (SCID) in the majority of cases to a less severe form in a small minority of patients. Five patients of Somali origin from four unrelated families......, with severe ADA-SCID, were registered in the Greater London area. Patients and their parents were investigated for the nonsense mutation Q3X (ADA c7C>T), two missense mutations K80R (ADA c239A>G) and R142Q (ADA c425G>A), and a TAAA repeat located at the 3' end of an Alu element (AluVpA) positioned 1.1 kb...... upstream of the ADA transcription start site. All patients were homozygous for the haplotype ADA-7T/ADA-239G/ADA-425G/AluVpA7. Among 207 Somali immigrants to Denmark, the frequency of ADA c7C>T and the maximum likelihood estimate of the frequency of the haplotype ADA-7T/ADA-239G/ADA-425G/AluVpA7 were both...

  6. Urtica dioica inhibits cell growth and induces apoptosis by targeting Ornithine decarboxylase and Adenosine deaminase as key regulatory enzymes in adenosine and polyamines homeostasis in human breast cancer cell lines.

    Science.gov (United States)

    Fattahi, Sadegh; Ghadami, Elham; Asouri, Mohsen; Motevalizadeh Ardekanid, Ali; Akhavan-Niaki, Haleh

    2018-02-28

    Breast cancer is a heterogeneous and multifactorial disease with variable disease progression risk, and treatment response. Urtica dioica is a traditional herb used as an adjuvant therapeutic agent in cancer. In the present study, we have evaluated the effects of the aqueous extract of Urtica dioica on Adenosine deaminase (ADA) and Ornithine decarboxylase (ODC1) gene expression in MCF-7, MDA-MB-231, two breast cancer cell lines being estrogen receptor positive and estrogen receptor negative, respectively.  Cell lines were cultured in suitable media. After 24 h, different concentrations of the extract were added and after 72 h, ADA and ODC1 gene expression as well as BCL2 and BAX apoptotic genes were assessed by Taqman real time PCR assay. Cells viability was assessed by MTT assay, and apoptosis was also evaluated at cellular level. The intra and extracellular levels of ODC1 and ADA enzymes were evaluated by ELISA. Results showed differential expression of ADA and ODC1 genes in cancer cell lines. In MCF-7 cell line, the expression level of ADA was upregulated in a dose-dependent manner but its expression did not change in MDA-MB cell line. ODC1 expression was increased in both examined cell lines. Also, increased level of the apoptotic BAX/BCL-2 ratio was detected in MCF-7 cells. These results demonstrated that Urtica dioica induces apoptosis in breast cancer cells by influencing ODC1 and ADA genes expression, and estrogen receptors. The different responses observed with these cell lines could be due to the interaction of Urtica dioica as a phytoestrogen with the estrogen receptor.

  7. Differential diagnosis of acute abdominal pain – acute intermittent porphyria

    Directory of Open Access Journals (Sweden)

    Mislav Klobučić

    2011-08-01

    Full Text Available Acute intermittent porphyria (AIP is a rare autosomal dominant disorder of heme biosynthesis in liver due to deficiency of porphobilinogen deaminase enzyme. Clinically, AIP is dominatedby a colicky type pain, which does not subside after taking usual analgesics. Additional frequent symptoms are vomiting, hypertension, peripheral neuropathy, seizures, depression, delirium and coma. This paper reported a case of a twenty-fi- ve-year-old female patient, who had undergone a period of six days between the first presentationto the medical department and the diagnosis confirmation. It has accentuated possible mistakes in symptomatic therapy administration as well as dangers of a delayed diagnosis.

  8. A rare case of acute intermittent porphyria with ichthyosis vulgaris in a young boy

    Directory of Open Access Journals (Sweden)

    Garima Agrawal Varshney

    2018-01-01

    Full Text Available Acute intermittent porphyria (AIP and ichthyosis vulgaris both are autosomal dominant disorders with incomplete penetrance caused by the deficiency of porphobilinogen deaminase enzyme and filaggrin protein, respectively. We report a rare case of a 9-year-old boy having two genetic diseases with an unclear association. An acute attack of AIP is characterized by gastrointestinal symptoms and neuropsychiatric manifestations. Although rare in the first decade of life, the presence of reddish urine with a typical presentation such as abdominal pain, hypertension, seizure, and paresthesias lead us to the diagnosis of AIP. The precipitating factor in the present case was prolonged fasting in Ramadan.

  9. Genetics Home Reference: adenosine deaminase deficiency

    Science.gov (United States)

    ... Combined Immunodeficiency (SCID) and Conditions Associated with T Cell Lymphoneia (PDF) Genetic Testing (1 link) Genetic Testing Registry: Severe ... Diseases Immune Deficiency Foundation Jeffrey Modell Foundation National Organization for Rare ... OMIM (1 link) SEVERE COMBINED IMMUNODEFICIENCY, AUTOSOMAL RECESSIVE, T CELL-NEGATIVE, B CELL-NEGATIVE, NK CELL-NEGATIVE, DUE ...

  10. 胸腔积液腺苷脱氨酶对内科胸腔镜检查临床病例选择的意义%Clinical significance of adenosine deaminase in selection patient for medical thoracoscopy

    Institute of Scientific and Technical Information of China (English)

    李玺; 黄小艳; 刘斌; 荣福

    2017-01-01

    Objective To evaluate clinical significance of adenosine deaminase (ADA) in selection patient for medical thoracoscopy.Methods A retrospective analysis of January 2013 to April 2016,198 cases unexplained pleural effusion patients were divided into youth group,middle-aged and elderly groups.To determine the sensitivity and specificity of ADA≥45 U/L or ADA≥45 U/L combine percentage of lymphocytes in white blood cells ≥50% as the diagnosis criteria of tuberculous pleurisy,and analysis gender and age influence on ADA.Results The diagnosis of medical thoracoscopy in unexplained pleural effusion was 98.9 %.The sensitivity and specificity of ADA≥45 U/L in diagnosis of tuberculous pleurisy were 73.3% and 89.2%;The sensitivity and specificity of ADA≥ 45 U/L combine percentage of lymphocytes in white blood cells≥ 50% in diagnosis of tuberculous pleurisy were 71% and 98.5%.Especially in youth group,the sensitivity and specificity of ADA ≥ 45 U/L combine percentage of lymphocytes in white blood cells≥50% in diagnosis of tuberculous pleurisy was 100%.Conclusions If young patients have typical clinical manifestations of tuberculous pleurisy and ADA≥45 U/L percentage of lymphocytes in white blood cells ≥ 50% in pleural effusion,anti-TB treatment without further thoracoscopy is appropriate;For middle-aged and elderly patients of unexplained pleural liquid,thoracoscopy is recommended to avoid misdiagnosis.%目的 探讨胸腔积液腺苷脱氨酶(ADA)对内科胸腔镜检查临床病例选择的意义.方法 回顾性分析2013年1月至2016年4月经内科胸腔镜检查的不明原因胸腔积液患者198例,分为青年组、中年组和老年组,以胸腔积液ADA≥45 U/L或ADA≥45 U/L联合淋巴细胞占白细胞比例≥50%作为诊断结核性胸膜炎的标准,确定其敏感度和特异度,并分析性别、年龄对ADA的影响.结果 内科胸腔镜对不明原因胸腔积液的诊断率为98.9%.胸腔积液ADA≥45 U/L诊

  11. Investigation on the diagnosis significance of C reactive protein and adenosine deaminase in cerebrospinal fluid among children with meningitis%脑脊液C反应蛋白和腺苷脱氨酶检测在小儿脑膜炎中的诊断价值探讨

    Institute of Scientific and Technical Information of China (English)

    徐仁荣; 张慧华; 朱华丽

    2015-01-01

    Objective To determine C reactive protein and adenosine deaminase in cerebrospinal fluid,and to investigate the clinical diagnosis significance for tuberculosis meningitis,purulent meningitis and viral meningitis. Methods A total of 31 5 children with meningitis (1 02 cases of tuberculosis meningitis,1 08 cases of purulent meningitis and 1 05 cases of viral meningitis)were enrolled,96 children undergoing operation without meningitis were enrolled as control group,and their cerebrospinal fluid samples were collected.The levels of C reactive protein and adenosine deaminase were determined,and the results were compared.Results C reactive protein and adenosine deaminase in control and viral meningitis groups were significantly lower than those in tuberculosis and purulent meningitis groups (P 0.05 ).C reactive protein in purulent meningitis group was higher than that in tuberculosis meningitis group(P <0.05 ),and adenosine deaminase was lower than that in tuberculosis meningitis group (P <0.05).C reactive protein in purulent meningitis group was positive,and the positive rate was 1 00%.There were 63 positive cases in tuberculosis meningitis group,and the positive rate was 61 .76%.That in viral meningtis was negative.Conclusions In cerebrospinal fluid,C reactive protein and adenosine deaminase determinations have important reference significance for the differential diagnosis of bacterial meningitis (purulent meningitis and tuberculosis meningitis)and viral meningitis.Adenosine deaminase may be a good indicator for the diagnosis of tuberculosis meningitis,in order to provide the reference for the early diagnosis of various types of meningitis.%目的:探讨小儿脑脊液C反应蛋白和腺苷脱氨酶在结核性脑膜炎、化脓性脑膜炎和病毒性脑膜炎早期诊断中的临床意义。方法分别检测315例小儿脑膜炎患儿(包括结核性脑膜炎102例、化脓性脑膜炎108例、病毒性脑膜炎105例)及96例非脑膜炎外科手术

  12. 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

  13. The role of DNA methylation in catechol-enhanced erythroid differentiation of K562 cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiao-Fei; Wu, Xiao-Rong; Xue, Ming; Wang, Yan; Wang, Jie; Li, Yang; Suriguga,; Zhang, Guang-Yao; Yi, Zong-Chun, E-mail: yizc@buaa.edu.cn

    2012-11-15

    Catechol is one of phenolic metabolites of benzene in vivo. Catechol is also widely used in pharmaceutical and chemical industries. In addition, fruits, vegetables and cigarette smoke also contain catechol. Our precious study showed that several benzene metabolites (phenol, hydroquinone, and 1,2,4-benzenetriol) inhibited erythroid differentiation of K562 cells. In present study, the effect of catechol on erythroid differentiation of K562 cells was investigated. Moreover, to address the role of DNA methylation in catechol-induced effect on erythroid differentiation in K562 cells, methylation levels of erythroid-specific genes were analyzed by Quantitative MassARRAY methylation analysis platform. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation in K562 cells in concentration- and time-dependent manners. The mRNA expression of erythroid specific genes, including α-globin, β-globin, γ-globin, erythroid 5-aminolevulinate synthase, erythroid porphobilinogen deaminase, and transcription factor GATA-1 genes, showed a significant concentration-dependent increase in catechol-treated K562 cells. The exposure to catechol caused a decrease in DNA methylation levels at a few CpG sites in some erythroid specific genes including α-globin, β-globin and erythroid porphobilinogen deaminase genes. These results indicated that catechol improved erythroid differentiation potency of K562 cells at least partly via up-regulating transcription of some erythroid related genes, and suggested that inhibition of DNA methylation might be involved in up-regulated expression of some erythroid related genes. -- Highlights: ► Catechol enhanced hemin-induced hemoglobin accumulation. ► Exposure to catechol resulted in up-regulated expression of erythroid genes. ► Catechol reduced methylation levels at some CpG sites in erythroid genes.

  14. The role of DNA methylation in catechol-enhanced erythroid differentiation of K562 cells

    International Nuclear Information System (INIS)

    Li, Xiao-Fei; Wu, Xiao-Rong; Xue, Ming; Wang, Yan; Wang, Jie; Li, Yang; Suriguga,; Zhang, Guang-Yao; Yi, Zong-Chun

    2012-01-01

    Catechol is one of phenolic metabolites of benzene in vivo. Catechol is also widely used in pharmaceutical and chemical industries. In addition, fruits, vegetables and cigarette smoke also contain catechol. Our precious study showed that several benzene metabolites (phenol, hydroquinone, and 1,2,4-benzenetriol) inhibited erythroid differentiation of K562 cells. In present study, the effect of catechol on erythroid differentiation of K562 cells was investigated. Moreover, to address the role of DNA methylation in catechol-induced effect on erythroid differentiation in K562 cells, methylation levels of erythroid-specific genes were analyzed by Quantitative MassARRAY methylation analysis platform. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation in K562 cells in concentration- and time-dependent manners. The mRNA expression of erythroid specific genes, including α-globin, β-globin, γ-globin, erythroid 5-aminolevulinate synthase, erythroid porphobilinogen deaminase, and transcription factor GATA-1 genes, showed a significant concentration-dependent increase in catechol-treated K562 cells. The exposure to catechol caused a decrease in DNA methylation levels at a few CpG sites in some erythroid specific genes including α-globin, β-globin and erythroid porphobilinogen deaminase genes. These results indicated that catechol improved erythroid differentiation potency of K562 cells at least partly via up-regulating transcription of some erythroid related genes, and suggested that inhibition of DNA methylation might be involved in up-regulated expression of some erythroid related genes. -- Highlights: ► Catechol enhanced hemin-induced hemoglobin accumulation. ► Exposure to catechol resulted in up-regulated expression of erythroid genes. ► Catechol reduced methylation levels at some CpG sites in erythroid genes.

  15. Exogenous spermidine is enhancing tomato tolerance to salinity-alkalinity stress by regulating chloroplast antioxidant system and chlorophyll metabolism.

    Science.gov (United States)

    Li, Jianming; Hu, Lipan; Zhang, Li; Pan, Xiongbo; Hu, Xiaohui

    2015-12-29

    Salinity-alkalinity stress is known to adversely affect a variety of processes in plants, thus inhibiting growth and decreasing crop yield. Polyamines protect plants against a variety of environmental stresses. However, whether exogenous spermidine increases the tolerance of tomato seedlings via effects on chloroplast antioxidant enzymes and chlorophyll metabolism is unknown. In this study, we examined the effect of exogenous spermidine on chlorophyll synthesis and degradation pathway intermediates and related enzyme activities, as well as chloroplast ultrastructure, gene expression, and antioxidants in salinity-alkalinity-stressed tomato seedlings. Salinity-alkalinity stress disrupted chlorophyll metabolism and hindered uroorphyrinogen III conversion to protoporphyrin IX. These effects were more pronounced in seedlings of cultivar Zhongza No. 9 than cultivar Jinpengchaoguan. Under salinity-alkalinity stress, exogenous spermidine alleviated decreases in the contents of total chlorophyll and chlorophyll a and b in seedlings of both cultivars following 4 days of stress. With extended stress, exogenous spermidine reduced the accumulation of δ-aminolevulinic acid, porphobilinogen, and uroorphyrinogen III and increased the levels of protoporphyrin IX, Mg-protoporphyrin IX, and protochlorophyllide, suggesting that spermidine promotes the conversion of uroorphyrinogen III to protoporphyrin IX. The effect occurred earlier in cultivar Jinpengchaoguan than in cultivar Zhongza No. 9. Exogenous spermidine also alleviated the stress-induced increases in malondialdehyde content, superoxide radical generation rate, chlorophyllase activity, and expression of the chlorophyllase gene and the stress-induced decreases in the activities of antioxidant enzymes, antioxidants, and expression of the porphobilinogen deaminase gene. In addition, exogenous spermidine stabilized the chloroplast ultrastructure in stressed tomato seedlings. The tomato cultivars examined exhibited different

  16. Maintaining Genome Stability: The Role of Helicases and Deaminases

    Science.gov (United States)

    2008-07-01

    Errors in duplicating DNA can result in genomic instability, leading to various human diseases, such as cancer, immune system disorder, muscle dystrophy ...as cancer, immune system disorder, muscle dystrophy , and neurodegenerations. Thus, maintaining genomic integrity is vital to the normal growth of...31–38. Eberharter, A., R. Ferreira and P. Becker , 2005 Dynamic chro- matin: concerted nucleosome remodelling and acetylation. Biol. Chem. 386: 745

  17. High pleural fluid adenosine deaminase levels: A valuable tool for ...

    African Journals Online (AJOL)

    To determine the positive predictive value (PPV) of FADA, the frequent causes of FPs in our laboratory and the demographic characteristics of tuberculous pleural effusions (TPEs) and non-tuberculous pleural effusions (NTPEs). Methods. High FADA results generated in the past year were extracted with corresponding TB ...

  18. Validity of serum Adenosine deaminase in diagnosis of tuberculosis

    African Journals Online (AJOL)

    abp

    2013-08-14

    Aug 14, 2013 ... culture methods take several weeks to become positive where the diagnosis of .... Indian. Pediatr. 1995; 32 (10): 1087-8. PubMed | Google Scholar. 12. Mishra OP ... patients. Southeast Asian J Trop Med Public Health. 2007.

  19. deaminase from plant growth promoting rhizobacteria in Striga

    African Journals Online (AJOL)

    Experiments were conducted in pots to determine the growth effect of different rhizobacteria on maize under Striga hermonthica infestation. Three bacteria were selected based on their plant growth promoting effects. Whole bacterial cells of the rhizobacteria were used to amplify 1-amino-cyclopropane-1-carboxylic acid ...

  20. A role for adenosine deaminase in Drosophila larval development

    Czech Academy of Sciences Publication Activity Database

    Doležal, T.; Doleželová, Eva; Žurovec, Michal; Bryant, P. J.

    2005-01-01

    Roč. 3, č. 7 (2005), s. 1213-1224 ISSN 1544-9173 R&D Projects: GA ČR(CZ) GA204/04/1205; GA AV ČR(CZ) IAA5007107 Grant - others:United States National Science Foundation(US) 440860-21565 Institutional research plan: CEZ:AV0Z50070508 Keywords : Drosophila Subject RIV: ED - Physiology Impact factor: 14.672, year: 2005

  1. 脑脊液免疫球蛋白、乳酸脱氢酶及腺苷脱氨酶检测在成人颅内感染鉴别诊断中的临床意义%Clinical significance of immunoglobulins, lactatedehydrogenase ,and adenosine deaminase detection in differential diagnosis of intracranial infections in adults

    Institute of Scientific and Technical Information of China (English)

    张丽琴

    2012-01-01

    OBJECTIVE To investigate the changes of immunoglobulin (Ig), lactate dehydrogenase(LDH), and adenosine deaminase(ADA) in cerebrospinal fluid (CSF)of the adults with intracranial infections and explore the clinical significance.METHODS Totally 17 patients with tuberculous meningitis, 19 patients with purulent meningitis, and 21 patients with viral meningitis were recruited in this study.The levels of LDH, ADA, and Ig in CSF from.each patient were measured and compared with the control group.RESULTS The concentrations of LDH, ADA, IgM, IgG.and IgA in CSF of the patients with purulent meningitis were(85.60±18.11)U/L,(2.59 ±0.52)U/L,(63.85±13.44)mg/L,(92.55±35.18)mg/L,and (20.60±8.75)mg/L,respectively ; the levels of LDA, ADA, IgM, IgG, and IgA in CSF of the patients with tuberculous meningitis were(78.80±22.38)U/L, (11.83±2.65)U/L, (18.90±8.62)mg/L, (180.69±42.28)mg/L, and(41.63±ll.42)mg/L, respectively) the levels of LDA, ADA, IgM, IgG, and IgA in CSF of the patients with viral meningitis were(22.43±9.56)U/L, (2.66±0.64)U/L, (5.83±1.34)mg/L,(20.90±10.35)mg/L,and(7.20±2.30)mg/L, respectively; of the three groups of adults with intracranial infectious diseases, the levels of IgM, IgG, IgA, and LDH in CSF of the patients with tuberculous meningitis or purulent meningitis were significantly higher than those in the patients with viral meningitis(P<0.01), the levels of IgG and IgA in CSF of the patients with tuberculous meningitis increased most significantly, the level of IgM in CSF of the patients with purulent meningitis increased most significantly, the activity of ADA of the tuberculous meningitis group was significantly higher than control group and other two groups(P<0.01 ); no statistical significance was found when between the indexes of viral meningitis group and control group.CONCLUSION The blood brain barriers of adult patients have been injured in varying degreest the detection of Ig, LDH in CSF is helpful to clinical diagnosis and

  2. Comprehensive analysis of the tryptophan metabolome in urine of patients with acute intermittent porphyria.

    Science.gov (United States)

    Gomez-Gomez, Alex; Marcos, Josep; Aguilera, Paula; To-Figueras, Jordi; Pozo, Oscar J

    2017-08-15

    Acute intermittent porphyria (AIP) is a rare metabolic disorder due to a deficiency of porphobilinogen deaminase, the third enzyme of the heme biosynthetic pathway. This low enzymatic activity may predispose to the appearance of acute neurological attacks. Seminal studies suggested that AIP was associated with changes in tryptophan homeostasis with inconclusive results. Therefore, the aim of this study was to analyze the urinary metabolome of AIP patients focusing on tryptophan metabolism using state-of-the-art technology. This was a case-control study including a group of 25 AIP patients with active biochemical disease and increased excretion of heme-precursors and 25 healthy controls. Tryptophan and related compounds and metabolites including: large neutral amino acids (LNAAs), serotonin, kynurenine, kynurenic acid and anthranilic acid were quantified in urine by liquid chromatography tandem-mass spectrometry (LC-MS/MS). Twenty-nine biological markers (including metabolic ratios and absolute concentrations) were compared between patients and controls. Significant differences were found in the tryptophan-kynurenine metabolic pathway. Compared to controls, AIP patients showed: (a) increased urinary excretion of kynurenine and anthranilic acid (Pmetabolome of hepatic porphyrias. Copyright © 2017. Published by Elsevier B.V.

  3. A porphodimethene chemical inhibitor of uroporphyrinogen decarboxylase.

    Directory of Open Access Journals (Sweden)

    Kenneth W Yip

    Full Text Available Uroporphyrinogen decarboxylase (UROD catalyzes the conversion of uroporphyrinogen to coproporphyrinogen during heme biosynthesis. This enzyme was recently identified as a potential anticancer target; its inhibition leads to an increase in reactive oxygen species, likely mediated by the Fenton reaction, thereby decreasing cancer cell viability and working in cooperation with radiation and/or cisplatin. Because there is no known chemical UROD inhibitor suitable for use in translational studies, we aimed to design, synthesize, and characterize such a compound. Initial in silico-based design and docking analyses identified a potential porphyrin analogue that was subsequently synthesized. This species, a porphodimethene (named PI-16, was found to inhibit UROD in an enzymatic assay (IC50 = 9.9 µM, but did not affect porphobilinogen deaminase (at 62.5 µM, thereby exhibiting specificity. In cellular assays, PI-16 reduced the viability of FaDu and ME-180 cancer cells with half maximal effective concentrations of 22.7 µM and 26.9 µM, respectively, and only minimally affected normal oral epithelial (NOE cells. PI-16 also combined effectively with radiation and cisplatin, with potent synergy being observed in the case of cisplatin in FaDu cells (Chou-Talalay combination index <1. This work presents the first known synthetic UROD inhibitor, and sets the foundation for the design, synthesis, and characterization of higher affinity and more effective UROD inhibitors.

  4. Two Cases of Severe Combined Immunodeficiency Caused By Adenosine Deaminase Deficiency

    Directory of Open Access Journals (Sweden)

    Turkan Patiroglu

    2014-08-01

    Full Text Available Severe Combined Immune Deficiency (SCID is a primary immune deficiency disorder manifested with severe infections upon first months of life, which is characterized by diverse genetic defects in T and B lymphocyte functions and occasionally in NK cells. ADA deficiency is a form of SCID progressing with severe lymphopenia and immune deficiency caused by toxic metabolites of ADA. Bone marrow transplantation (BMT is the only curative treatment although prophylactic anti-microbial therapy, intravenous immunoglobulin (IVIG and enzyme replacement can achieve transient improvements. Early diagnosis before development of severe infections and organ injury and referral to pediatric immunology clinics will make considerable contributions to prognosis. Here, we presented 2 cousins with SCID who had positive family history with deceased sibling; presented with tanning at skin, severe neonatal infections and Q246X (c736C>T non-sense mutation in exon 8 in ADA gene  in order to emphasize this rare mutation and pediatric emergencies associated with this disorder.

  5. Evolution of vertebrate adaptive immunity: immune cells and tissues, and AID/APOBEC cytidine deaminases.

    Science.gov (United States)

    Hirano, Masayuki

    2015-08-01

    All surviving jawed vertebrate representatives achieve diversity in immunoglobulin-based B and T cell receptors for antigen recognition through recombinatorial rearrangement of V(D)J segments. However, the extant jawless vertebrates, lampreys and hagfish, instead generate three types of variable lymphocyte receptors (VLRs) through a template-mediated combinatorial assembly of different leucine-rich repeat (LRR) sequences. The clonally diverse VLRB receptors are expressed by B-like lymphocytes, while the VLRA and VLRC receptors are expressed by lymphocyte lineages that resemble αβ and γδ T lymphocytes, respectively. These findings suggest that three basic types of lymphocytes, one B-like and two T-like, are an essential feature of vertebrate adaptive immunity. Around 500 million years ago, a common ancestor of jawed and jawless vertebrates evolved a genetic program for the development of prototypic lymphoid cells as a foundation for an adaptive immune system. This acquisition preceded the convergent evolution of alternative types of clonally diverse receptors for antigens in all vertebrates, as reviewed in this article. © 2015 WILEY Periodicals, Inc.

  6. Synthesis of [5-14C]pentostatin, an antileukemic agent and potent adenosine deaminase inhibitor

    International Nuclear Information System (INIS)

    Woo, P.W.K.; Lee, H.T.

    1990-01-01

    Reaction of triethyl ortho[ 14 C]formate (2) with 2-amino-1-(5-amino-1H-imidazol-4-yl)ethanone dihydrochloride (1) in the presence of molecular sieves 4A gave 6,7-dihydro[5- 14 C]imidazo[4,5-d]{1,3]diazepin-8(3H)-one hydrochloride monodimethyl sulfoxide (3) (radiochemical yield, 60%). The latter was persilylated with bis(trimethylsilyl)trifluroacetamide (4) and glycosylated with 2-deoxy-3,5-di-0-p-toluoyl-α-D-erythro-pentufuranosyl chloride (6) to give a mixture from which the 3-N-β-glycosylated product 8 was isolated by chromatography and crystallization (13%). Deprotective saponification with methanolic sodium methoxide and subsequent sodium borohydride reduction of the 8-keto function gave a (R,S)-mixture from which the desired (R)-isomer, [5- 14 C]pentostatin (11), was isolated by preparative HPLC over a C18 column, desalting with Diaion-HP20, and subsequent crystallization (39%). (author)

  7. Pleural effusion: Role of pleural fluid cytology, adenosine deaminase level, and pleural biopsy in diagnosis

    Directory of Open Access Journals (Sweden)

    Biswajit Biswas

    2016-01-01

    Full Text Available Objective: The present study is designed to evaluate the role of pleural fluid analysis in diagnosing pleural diseases and to study the advantages and disadvantages of thoracocentasis and pleural biopsy. Materials and Methods: We prospectively included 66 consecutive indoor patients over a duration of 1 year. Pleural fluid was collected and cytological smears were made from the fluid. Plural biopsy was done in the same patient by Cope needle. Adequate pleural biopsy tissue yielding specific diagnosis was obtained in 47 (71.2% cases. Results: Tuberculosis was the commonest nonneoplastic lesion followed by chronic nonspecific pleuritis comprising 60% and 33.3% of the nonneoplastic cases respectively and tuberculosis was predominantly diagnosed in the younger age group. Majority (70.8% of malignancy cases were in the age group of >50-70. Adenocarcinoma was found to be the commonest (66.7% malignant neoplasm in the pleurae followed by small-cell carcinoma (20.8%. Conclusion: Pleural biopsy is a useful and minimally invasive procedure. It is more sensitive and specific than pleural fluid smears.

  8. Modulation of microRNA editing, expression and processing by ADAR2 deaminase in glioblastoma.

    Science.gov (United States)

    Tomaselli, Sara; Galeano, Federica; Alon, Shahar; Raho, Susanna; Galardi, Silvia; Polito, Vinicia Assunta; Presutti, Carlo; Vincenti, Sara; Eisenberg, Eli; Locatelli, Franco; Gallo, Angela

    2015-01-13

    ADAR enzymes convert adenosines to inosines within double-stranded RNAs, including microRNA (miRNA) precursors, with important consequences on miRNA retargeting and expression. ADAR2 activity is impaired in glioblastoma and its rescue has anti-tumoral effects. However, how ADAR2 activity may impact the miRNome and the progression of glioblastoma is not known. By integrating deep-sequencing and array approaches with bioinformatics analyses and molecular studies, we show that ADAR2 is essential to edit a small number of mature miRNAs and to significantly modulate the expression of about 90 miRNAs in glioblastoma cells. Specifically, the rescue of ADAR2 activity in cancer cells recovers the edited miRNA population lost in glioblastoma cell lines and tissues, and rebalances expression of onco-miRNAs and tumor suppressor miRNAs to the levels observed in normal human brain. We report that the major effect of ADAR2 is to reduce the expression of a large number of miRNAs, most of which act as onco-miRNAs. ADAR2 can edit miR-222/221 and miR-21 precursors and decrease the expression of the corresponding mature onco-miRNAs in vivo and in vitro, with important effects on cell proliferation and migration. Our findings disclose an additional layer of complexity in miRNome regulation and provide information to better understand the impact of ADAR2 editing enzyme in glioblastoma. We propose that ADAR2 is a key factor for maintaining edited-miRNA population and balancing the expression of several essential miRNAs involved in cancer.

  9. Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila by depleting extracellular adenosine

    Czech Academy of Sciences Publication Activity Database

    Žurovec, Michal; Doležal, Tomáš; Gaži, Michal; Pavlová, Eva; Bryant, P. J.

    2002-01-01

    Roč. 99, č. 7 (2002), s. 4403-4408 ISSN 0027-8424 R&D Projects: GA ČR GA204/01/1022; GA AV ČR IAA5007107 Institutional research plan: CEZ:AV0Z5007907 Keywords : adenosine daminase * minimal medium Subject RIV: CE - Biochemistry Impact factor: 10.701, year: 2002

  10. Characterization of the Escherichia coli codBA operon encoding cytosine permease and cytosine deaminase

    DEFF Research Database (Denmark)

    Danielsen, S; Kilstrup, M; Barilla, K

    1992-01-01

    . A two-codon overlap between the two reading frames indicates that they constitute an operon. Transcription of the operon was found to be regulated by exogenous purines. Polypeptides specified by each of the two reading frames were expressed in minicells, and the codB gene product was found to be highly...

  11. Cloning, expression, and purification of cytidine deaminase from Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Vincenzetti, Silvia; Cambi, Alessandra; Neuhard, Jan

    1999-01-01

    -optical emission spectroscopy analysis indicated that the enzyme contains 1 mol of zinc atom per mole of subunit. The kinetic properties of AT-CDA1 both toward the natural substrates and with analogs indicated that the catalytic mechanism of the plant enzyme is probably very similar to that of the human the...

  12. Molecular Analysis of Activation-Induced Cytidine Deaminase Gene in Immunoglobulin-E Deficient Patients

    Directory of Open Access Journals (Sweden)

    Sergio Roa

    2008-01-01

    Full Text Available Understanding how class switch recombination (CSR is regulated to produce immunoglobulin E (IgE has become fundamental because of the dramatic increase in the prevalence of IgE-mediated hypersensitivity reactions. CSR requires the induction of the enzyme AICDA in B cells. Mutations in AICDA have been linked to Hyper-IgM syndrome (HIGM2, which shows absence of switching to IgE as well as to IgG and IgA. Although isolated IgE deficiency is a rare entity, here we show some individuals with normal serum IgM, IgG, and IgA levels that had undetectable total serum IgE levels. We have analyzed the AICDA gene in these individuals to determine if there are mutations in AICDA that could lead to selective IgE deficiency. Conformational sensitive gel electrophoresis (CSGE and sequencing analysis of AICDA coding sequences demonstrated sequence heterogeneity due to 5923A/G and 7888C/T polymorphisms, but did not reveal any novel mutation that might explain the selective IgE deficit.

  13. Structural, kinetic, and mutational studies of the zinc ion environment in tetrameric cytidine deaminase

    DEFF Research Database (Denmark)

    Johansson, Eva; Neuhard, Jan; Willemoës, Martin

    2004-01-01

    with the dipole moments from two alpha-helices partially neutralizes the additional negative charge in the active site, leading to a catalytic activity similar to D-CDA. Arg56 has been substituted by a glutamine (R56Q), the corresponding residue in D-CDA, an alanine (R56A), and an aspartate (R56D). Moreover, one...

  14. Mutational analysis of the nucleotide binding site of Escherichia coli dCTP deaminase

    DEFF Research Database (Denmark)

    Thymark, Majbritt; Johansson, Eva; Larsen, Sine

    2007-01-01

    detectable activity with a 30- and 140-fold reduction in k(cat), respectively. Furthermore, S111T and E138D both showed altered dTTP inhibition compared to wild-type enzyme. S111T was almost insensitive to the presence of dTTP. With the E138D enzyme the dTTP dependent increase in cooperativity of d...... of E138D in complex with dUTP showed a hydrogen bonding network in the active site similar to wild-type enzyme. However, changes in the hydrogen bond lengths between the carboxylate and a catalytic water molecule as well as a slightly different orientation of the pyrimidine ring of the bound nucleotide...

  15. Role of Glutamate 64 in the Activation of the Prodrug 5-Fluorocytosine by Yeast Cytosine Deaminase

    Czech Academy of Sciences Publication Activity Database

    Wang, J.; Sklenák, Štěpán; Liu, A.; Felczak, K.; Wu, Y.; Li, Y.; Yan, H.

    2012-01-01

    Roč. 51, č. 1 (2012), s. 475-486 ISSN 0006-2960 R&D Projects: GA AV ČR IAA400400812; GA AV ČR IAA400400908; GA ČR GA203/09/1627 Institutional research plan: CEZ:AV0Z40400503 Keywords : transition-state analog * barrier hydrogen-bond * side-chain amides Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.377, year: 2012

  16. The effect of cyclophosphamide and gamma irradiation on adenosine deaminase and purine nucleoside phosphorylase in mice

    International Nuclear Information System (INIS)

    Hosek, B.; Bohaecek, J.; Sikulova, J.

    1991-01-01

    Changes in ADA and PNP activities in the spleens and thymuses of mice were studied after a single administration of cyclophosphamide and after whole-body gamma irradiation, applied alone or three days after CY application, In the first days after the treatment the enzyme activities were significantly depressed with the exception of ADA in the spleen, where a high elevation in relation to controls was observed. During the regeneration period a pronounced rise of PNP activity in the spleen occurred mainly after a combined application of CY and irradiation. In the thymus the regeneration was manifested by a mild increase of both ADA and PNP activities towards control values. The findings suggest that the expressive changes of ADA and PNP activities, participating in the purine salvage pathway, may, after a cytotoxic treatment, influence the nucleotide pool and DNA synthesis in lymphoid organs

  17. Organic Nitrogen Utilization by Phytoplankton: The Role of Cell-Surface Deaminases

    Science.gov (United States)

    1989-06-01

    determination is the absorbance at 550 nm duction of ferricytochrome C does not rep- minus absorbance at 500 nm (defined as resent cellular release of O-, and...or large suggests that biota are the most likely sources procaryotic organisms. It is possible, of of dark H,O, production. The production course

  18. One-Pot Enzymatic Synthesis of D-Arylalanines Using Phenylalanine Ammonia Lyase and L-Amino Acid Deaminase.

    Science.gov (United States)

    Zhu, Longbao; Feng, Guoqiang; Ge, Fei; Song, Ping; Wang, Taotao; Liu, Yi; Tao, Yugui; Zhou, Zhemin

    2018-06-08

    The phenylalanine ammonia-lyase (AvPAL) from Anabaena variabilis catalyzes the amination of substituent trans-cinnamic acid (t-CA) to produce racemic D,L-enantiomer arylalanine mixture owing to its low stereoselectivity. To produce high optically pure D-arylalanine, a modified AvPAL with high D-selectivity is expected. Based on the analyses of catalytic mechanism and structure, the Asn347 residue in the active site was proposed to control stereoselectivity. Therefore, Asn347 was mutated to construct mutant AvPAL-N347A, the stereoselectivity of AvPAL-N347A for D-enantiomer arylalanine was 2.3-fold higher than that of wild-type AvPAL (WtPAL). Furthermore, the residual L-enantiomer product in reaction solution could be converted into the D-enantiomer product through stereoselective oxidation by PmLAAD and nonselective reduction by reducing agent NH 3 BH 3 . At optimal conditions, the conversion rate of t-CA and optical purity (enantiomeric excess (ee D )) of D-phenylalanine reached 82% and exceeded 99%, respectively. The two enzymes displayed activity toward a broad range of substrate and could be used to efficiently synthesize D-arylalanine with different groups on the phenyl ring. Among these D-arylalanines, the yield of m-nitro-D-phenylalanine was highest and reached 96%, and the ee D exceeded 99%. This one-pot synthesis using AvPAL and PmLAAD has prospects for industrial application.

  19. Role of ascitic fluid adenosine deaminase (ADA) and serum CA-125 in the diagnosis of tuberculous peritonitis.

    Science.gov (United States)

    Ali, N; Nath, N C; Parvin, R; Rahman, A; Bhuiyan, T M; Rahman, M; Mohsin M N

    2014-12-01

    This cross sectional study was carried out in the department of gastroenterology, BIRDEM, Dhaka from January 2010 to May 2011 to determine the role of ascitic fluid ADA and serum CA-125 in the diagnosis of clinically suspected tubercular peritonitis. Total 30 patients (age 39.69 ± 21.26, 18M/12F) with clinical suspicion of tuberculosis peritonitis were included in this study after analyzing selection criteria. Laparoscopic peritoneal biopsy with 'histopathological diagnosis' was considered gold standard against which accuracics of two biomarkers (ADA & CA-125) were compared. Cut off value of ADA and CA-125 are 24 u/l, 35 U/ml respectively. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of ADA as a diagnostic modality in tuberculos peritonitis were 87.5%, 83.33%, 95.45%, 62.5% and 86.67% respectively where as CA-125 was found to have 83.33% sensitivity, 50% specificity, 86.9% positive predictive value, 42.85% negative predictive value and 76.6% accuracy. Both biomarkers are simple, non-invasive, rapid and relatively cheap diagnostic test where as laparoscopy is an invasive procedure, costly & requires trained staff and not without risk and also not feasible in all the centre in our country. So ascitic fluid ADA and serum CA-125 are important diagnostic test for peritoneal tuberculosis.

  20. Chlorophyll loss associated with heat-induced senescence in bentgrass.

    Science.gov (United States)

    Jespersen, David; Zhang, Jing; Huang, Bingru

    2016-08-01

    Heat stress-induced leaf senescence is characterized by the loss of chlorophyll from leaf tissues. The objectives of this study were to examine genetic variations in the level of heat-induced leaf senescence in hybrids of colonial (Agrostis capillaris)×creeping bentgrass (Agrostis stolonifera) contrasting in heat tolerance, and determine whether loss of leaf chlorophyll during heat-induced leaf senescence was due to suppressed chlorophyll synthesis and/or accelerated chlorophyll degradation in the cool-season perennial grass species. Plants of two hybrid backcross genotypes ('ColxCB169' and 'ColxCB190') were exposed to heat stress (38/33°C, day/night) for 28 d in growth chambers. The analysis of turf quality, membrane stability, photochemical efficiency, and chlorophyll content demonstrated significant variations in the level of leaf senescence induced by heat stress between the two genotypes, with ColXCB169 exhibiting a lesser degree of decline in chlorophyll content, photochemical efficiency and membrane stability than ColXCB190. The assays of enzymatic activity or gene expression of several major chlorophyll-synthesizing (porphobilinogen deaminase, Mg-chelatase, protochlorophyllide-reductase) and chlorophyll-degrading enzymes (chlorophyllase, pheophytinase, and chlorophyll-degrading peroxidase) indicated heat-induced decline in leaf chlorophyll content was mainly due to accelerated chlorophyll degradation, as manifested by increased gene expression levels of chlorophyllase and pheophytinase, and the activity of pheophytinase (PPH), while chlorophyll-synthesizing genes and enzymatic activities were not differentially altered by heat stress in the two genotypes. The analysis of heat-induced leaf senescence of pph mutants of Arabidopsis further confirmed that PPH could be one enzymes that plays key roles in regulating heat-accelerated chlorophyll degradation. Further research on enzymes responsible in part for the loss of chlorophyll during heat

  1. Influence of Estradiol-17beta on Progesterone and Estrogen Receptor mRNA Expression in Porcine Follicular Granulosa Cells during Short-Term, In Vitro Real-Time Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Sylwia Ciesiółka

    2016-01-01

    Full Text Available Progesterone (P4 and estradiol (E2 play a significant role in mammalian reproduction. Our study demonstrated that separated porcine cumulus cells (CCs and/or granulosa cells (GCs might proliferate in vitro during short-term, real-time primary culture. The GCs were analyzed according to gene expression of the progesterone receptor (nuclear form (pgr, progesterone receptor membrane component 1 (pgrmc1, and estrogen-related receptor beta 3 (esrrb3 in relation to two housekeeping genes: actb and pbgd. GCs were cultivated in medium with the E2. Both pgr/actb and pgr/pbgd revealed higher expression between 24 and 168 h of IVC of prolonged E2 treatment and at 48 h of IVC after acute E2 administration. The pgrmc1/actb and pgrmc1/pbgd displayed increased expression after prolonged E2 treatment between 24 and 120 h of IVC. The highest level of esrrb3/actb at 120 and 144 h, as well as esrrb3/pbgd at 120 h, in untreated controls as compared to the hormone-stimulated group, was observed. We suggest that E2 significantly influences the upregulation of pgr, pgrmc1, and esrrb3 expression in porcine GCs during real-time cell proliferation. Since esrrb3 expression is stimulated by E2 in both an acute and prolonged manner, estradiol may be recognized as a potential estrogen receptor agonist in GCs.

  2. Increased extracellular adenosine in Drosophila that are deficient in adenosine deaminase activates a release of energy stores leading to wasting and death

    Czech Academy of Sciences Publication Activity Database

    Zuberová, M.; Fencková, M.; Šimek, Petr; Janečková, L.; Doležal, T.

    2010-01-01

    Roč. 3, 11/12 (2010), s. 773-784 ISSN 1754-8403 R&D Projects: GA ČR(CZ) GD204/09/H058 Grant - others:AV ČR(CZ) KJB501410602; GA ČR(CZ) GA204/09/1463 Institutional research plan: CEZ:AV0Z50070508 Keywords : central nervous system * glycogen phosphorylase * immune response Subject RIV: CE - Biochemistry Impact factor: 4.584, year: 2010

  3. A Pilot Feasibility Study of Oral 5-Fluorocytosine and Genetically-Modified Neural Stem Cells Expressing E.Coli Cytosine Deaminase for Treatment of Recurrent High Grade Gliomas

    Science.gov (United States)

    2017-11-07

    Adult Anaplastic Astrocytoma; Recurrent Grade III Glioma; Recurrent Grade IV Glioma; Adult Anaplastic Oligodendroglioma; Adult Brain Tumor; Adult Giant Cell Glioblastoma; Adult Glioblastoma; Adult Gliosarcoma; Adult Mixed Glioma; Recurrent Adult Brain Tumor; Adult Anaplastic Oligoastrocytoma; Recurrent High Grade Glioma

  4. Recombination and horizontal transfer of nodulation and ACC deaminase (acdS) genes within Alpha- and Betaproteobacteria nodulating legumes of the Cape Fynbos biome.

    Science.gov (United States)

    Lemaire, Benny; Van Cauwenberghe, Jannick; Chimphango, Samson; Stirton, Charles; Honnay, Olivier; Smets, Erik; Muasya, A Muthama

    2015-11-01

    The goal of this work is to study the evolution and the degree of horizontal gene transfer (HGT) within rhizobial genera of both Alphaproteobacteria (Mesorhizobium, Rhizobium) and Betaproteobacteria (Burkholderia), originating from South African Fynbos legumes. By using a phylogenetic approach and comparing multiple chromosomal and symbiosis genes, we revealed conclusive evidence of high degrees of horizontal transfer of nodulation genes among closely related species of both groups of rhizobia, but also among species with distant genetic backgrounds (Rhizobium and Mesorhizobium), underscoring the importance of lateral transfer of symbiosis traits as an important evolutionary force among rhizobia of the Cape Fynbos biome. The extensive exchange of symbiosis genes in the Fynbos is in contrast with a lack of significant events of HGT among Burkholderia symbionts from the South American Cerrado and Caatinga biome. Furthermore, homologous recombination among selected housekeeping genes had a substantial impact on sequence evolution within Burkholderia and Mesorhizobium. Finally, phylogenetic analyses of the non-symbiosis acdS gene in Mesorhizobium, a gene often located on symbiosis islands, revealed distinct relationships compared to the chromosomal and symbiosis genes, suggesting a different evolutionary history and independent events of gene transfer. The observed events of HGT and incongruence between different genes necessitate caution in interpreting topologies from individual data types. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Bacillus subtilis guanine deaminase is encoded by the yknA gene and is induced during growth with purines as the nitrogen source

    DEFF Research Database (Denmark)

    Nygaard, P.; Bested, S. M.; Andersen, K. A. K.

    2000-01-01

    amino acid polypeptide and was preceded by a promoter sequence that is recognized by the A form of RNA polymerase. High levels of GDEase were found in cells grown with purines and intermediary compounds of the purine catabolic pathway as nitrogen sources. Allantoic acid, most likely, is a low molecular...

  6. Trichoderma harzianum containing 1-aminocyclopropane-1-carboxylate deaminase and chitinase improved growth and diminished adverse effect caused by Fusarium oxysporum in soybean.

    Science.gov (United States)

    Zhang, Fuli; Chen, Can; Zhang, Fan; Gao, Lidong; Liu, Jidong; Chen, Long; Fan, Xiaoning; Liu, Chang; Zhang, Ke; He, Yuting; Chen, Chen; Ji, Xiue

    2017-03-01

    An isolate, named Trichoderma harzianum T-soybean, showed growth-promoting for soybean seedlings and induced resistance to Fusarium oxysporum under greenhouse. Compared to control soybean seedlings, fresh weight, dry weight, lateral root number, chlorophyll content, root activity and soluble protein of plants pretreated with T-soybean increased, but initial pod height reduced. Furthermore, we found that T-soybean inhibited the growth of F. oxysporum by parasitic function. In addition, plate test results showed that culture filtrates of T-soybean also inhibited significantly F. oxysporum growth. Meanwhile, T-soybean treatment obviously reduced disease severity and induced quickly the H 2 O 2 and O 2 - burst as well as pathogenesis related protein gene (PR3) expression after F. oxysporum inoculation, and subsequently diminished the cell damage in soybean caused by the pathogen challenge. Reactive oxygen species (ROS) scavenging enzymes activity analysis showed that the activities of peroxidase (POD), polyphenol oxidase (PPO) and superoxide dismutase (SOD) increased significantly in T-soybean pretreated plants. These results suggested that T-soybean treatment induced resistance in soybean seedlings to F. oxysporum by companying the production of ROS and the increasing of ROS scavenging enzymes activity as well as PR3 expression. Copyright © 2016 Elsevier GmbH. All rights reserved.

  7. Cholangiocarcinomas associated with long-term inflammation express the activation-induced cytidine deaminase and germinal center-associated nuclear protein involved in immunoglobulin V-region diversification.

    Science.gov (United States)

    Chan-On, Waraporn; Kuwahara, Kazuhiko; Kobayashi, Naoya; Ohta, Kazutaka; Shimasaki, Tatsuya; Sripa, Banchob; Leelayuwat, Chanvit; Sakaguchi, Nobuo

    2009-08-01

    Cholangiocarcinoma (CCA) represents a model of tumor development after long-term inflammation which causes DNA damage or impairs DNA repair mechanism. AID and GANP, both appearing in antigen-driven B cells, are involved in affinity maturation of the immunoglobulin V-region with increased somatic mutation. A normal cholangiocyte line showed the induction of AID transcripts after stimulation with TNF-alpha, whereas ganp transcripts appeared constitutively in this cell line. Next, we examined the expression of AID and GANP in clinical CCA specimens to obtain information whether their expression levels are associated with the malignant grade of CCA. AID expression was similarly detected in the clinical cases of both well-differentiated and poorly-differentiated CCAs. On the contrary, GANP expression was detected in CCA cells at a higher level in the nucleus of poorly-differentiated CCAs with shorter survivals than in that of well-differentiated CCAs. The high and low cases of nuclear GANP expression showed no change in the frequency of the TP53 mutations, however, further investigation by in vitro experiment demonstrated that the high GANP expression caused the increased number of gammaH2AX foci after DNA damage by ionizing-irradiation. These results suggest that GANP is involved in regulation of DNA repair mechanism and the abnormal over-expression of GANP together with AID might be associated with rigorous DNA damage, potentially causing the malignant development of CCAs during long-term inflammation.

  8. Regulation of dCTP deaminase from Escherichia coli by nonallosteric dTTP binding to an inactive form of the enzyme

    DEFF Research Database (Denmark)

    Johansson, Eva; Thymark, Majbritt; Bynck, Julie H

    2007-01-01

    -maximal activity and the cooperativity of dCTP saturation. Likewise, increasing concentrations of dCTP increase the cooperativity of dTTP inhibition. Previous structural studies showed that the complexes of inactive mutant protein, E138A, with dUTP or dCTP bound, and wild-type enzyme with dUTP bound were all...... highly similar and characterized by having an ordered C-terminal. When comparing with a new structure in which dTTP is bound to the active site of E138A, the region between Val120 and His125 was found to be in a new conformation. This and the previous conformation were mutually exclusive within...

  9. Diagnostic value of combined determination of serum and chest fluid adenosine deaminase (ADA), IL-2, IL-6, IL-10 contents for differentiation of tuberculous from malignant pleural effusion

    International Nuclear Information System (INIS)

    Wu Jiaming; Wang Limin

    2005-01-01

    Objective: To investigate the possible diagnostic value of combined determination of serum and chest fluid contents of ADA, IL-2, IL-6, IL-10 in patients with tuberculous and malignant pleural effusion. Methods: Serum and chest fluid ADA (with biochemical method), IL-2, IL-6, IL-10 (with ELISA) contents were measured in 56 patients with tuberculosis pleural effusion, 53 patients with malignant effusion and 30 controls (in serum only). The receiving operative characteristic (ROC) curve for each parameter was analyzed for study of respective area under curse (Auc). Results: The serum IL-6 levels in both groups of patients were significantly higher than those in the controls (P<0.05). The chest fluid contents of ADA, IL-2, IL-6 and IL-10 in patients with tuberculous effusion were all significantly higher than those in patients with malignancies (P<0.05). The Auc in the ROC was largest in the case of ADA, followed by IL-10, IL-6 with IL-2 the least. Conclusion: Determination of chest fluid ADA, IL-2, IL-6, IL-10 contents was helpful in the differentiation of tuberculous from malignant pleural effusion. Combined determination of chest fluid ADA and IL-10 provided the highest accuracy rate for differentional diagnosis. (authors)

  10. Study on the clinical value of determination of serum adenosine deaminase (ADA) and HA, PIIIP levels for diagnosis of chronic hepatitis

    International Nuclear Information System (INIS)

    Ma Yun

    2007-01-01

    Objective: To explore the clinical value of determination of serum ADA and HA, PIIIP levels for diagnosis of chronic hepatitis. Methods: Serum ADA levels were determined with biochemistry and HA, PIIIP levels were detected with RIA in 36 patients with chronic hepatitis and 35 controls. Results: The levels of serum ADA, HA and PIIIP were significantly higher in the patients than those in controls (P<0.01). Serum ADA, HA, PIIIP levelers were mutually positively correlated (r=0.6117, 0.7134, P<0.01). Conclusion: Detection of serum ADA levels could reflect the liver reserve function and status of fibrosis. (authors)

  11. Measurement of urinary porphyrins and porphyrin precursors in Dutch hospital laboratories: a review of quality control over 5 years.

    NARCIS (Netherlands)

    Zuijderhoudt, F.M.; Weykamp, C.W.; Willems, J.L.

    2003-01-01

    BACKGROUND: We evaluated a quality control scheme for the measurement of urinary uroporphyrin, coproporphyrin, total urinary porphyrins and precursors of urinary porphyrins, delta-aminolevulinic acid and porphobilinogen that was performed in The Netherlands during a period of 5 years. METHODS: Six

  12. ORF Alignment: NC_000913 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available amine-6-Phosphate ... Deaminase From E.Coli, T Conformer, At 1.9a Resolution ... pdb|1FS6|A Ch...ain A, Glucosamine-6-Phosphate Deaminase ... From E.Coli, T Conformer, At ...phate Isomerase pdb|1FRZ|B Chain B, ... Glucosamine-6-Phosphate Deaminase From E.Coli, R ... C...lution ... pdb|1FRZ|A Chain A, Glucosamine-6-Phosphate Deaminase ... From E.Coli, R Conformer....hate At 2.2 A ... Resolution pdb|1CD5|A Chain A, Glucosamine-6-Phosphate ... Deaminase From E.Coli

  13. Analysis of 6912 unselected somatic hypermutations in human VDJ rearrangements reveals lack of strand specificity and correlation between phase II substitution rates and distance to the nearest 3' activation-induced cytidine deaminase target

    DEFF Research Database (Denmark)

    Ohm-Laursen, Line; Barington, Torben

    2007-01-01

    -23*01) from blood B lymphocytes enriched for CD27-positive memory cells. Analyses of 6,912 unique, unselected substitutions showed that in vivo hot and cold spots for the SHM of C and G residues corresponded closely to the target preferences reported for AID in vitro. A detailed analysis of all possible four......-nucleotide motifs present on both strands of the V(H) gene showed significant correlations between the substitution frequencies in reverse complementary motifs, suggesting that the SHM machinery targets both strands equally well. An analysis of individual J(H) and D gene segments showed that the substitution...... rates in G and T residues correlated inversely with the distance to the nearest 3' WRC AID hot spot motif on both the nontranscribed and transcribed strands. This suggests that phase II SHM takes place 5' of the initial AID deamination target and primarily targets T and G residues or, alternatively...

  14. A study on the porphirin metabolism in rats in conditions of acute uranium intoxication

    International Nuclear Information System (INIS)

    Khadzhirusev, S.; Pavlova, V.; Mikhajlov, M.A.

    1975-01-01

    The changes of urine porphyrin content are studied in albino rats with experimental acute uranium intoxication (single intraperitoneal injection of uranyl acetate, 7.0 mg/kg bodyweight). The observations have been conducted in the course of 10 days. It is found that both in control and in treated animals the urine is practically free from uroporphyrin. The delta-aminolevulinic acid content varied within broad limits, but the differences from control animals is statistically insignificant. A significant increase in urine porphobilinogen is observed, with a maximum on the second and eigth day after treatment. Coproporphyrin was significantly reduced since the first day of the experiment. All these changes seem to be due to impaired excretory capacity of the kidneys against the background of developing nitrogen retention and overall intoxication of the animal organism. Another possible explanation is that uranyl acetate inhibits some enzymes responsible for the transformation of porphobilinogen into uroporphyrin. (Ch.K.)

  15. Prenatal detection of a probable heterozygote for ADA deficiency and severe combined immunodeficiency disease using a microradioassay

    International Nuclear Information System (INIS)

    Aitken, D.A.; Kleijer, W.J.; Niermeijer, M.F.; Galjaard, H.; Herbschleb-Voogt, E.

    1980-01-01

    A pregnancy at risk for adenosine deaminase deficiency and severe combined immunodeficiency disease has been investigated by assay of adenosine deaminase activity in cultured amniotic fluid cells using a microradioassay. A low-normal level of consistent with heterozygote status in the foetus was found and confirmed after birth by assay of red cell and fibroblast adenosine deaminase activities. It is suggested that the radioassay method offers significant advantages in sensitivity and specificity over the standard spectrophotometric procedure. (author)

  16. ORF Alignment: NC_002932 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available ytidylate ... deaminase family protein [Chlorobium tepidum TLS] ... Length = 155 ... Query: 30 ... EARMRFV...VGASRKSVEEVSGGPFAAAVFEIESGRLVSLGVNLVLTQNSSILHAEMVAIVL 89 ... EARMRFV...VGASRKSVEEVSGGPFAAAVFEIESGRLVSLGVNLVLTQNSSILHAEMVAIVL Sbjct: 1 ... EARMRFVVGASRKSVEEVSGGPFAAAVFEIESGRLVSLGVNLV

  17. mRNA profiling for the identification of blood-Results of a collaborative EDNAP exercise

    DEFF Research Database (Denmark)

    Haas, Cordula; Hanson, E; Bär, W

    2010-01-01

    of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative......A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, using...

  18. mRNA profiling for the identification of blood--Results of a collaborative EDNAP exercise

    DEFF Research Database (Denmark)

    Haas, C.; Hanson, E.; Bär, W.

    2011-01-01

    of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative......A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, using...

  19. Biochemical parameters of pregnant rats and their offspring exposed to different doses of inorganic mercury in drinking water.

    Science.gov (United States)

    Oliveira, Cláudia S; Oliveira, Vitor A; Ineu, Rafael P; Moraes-Silva, Lucélia; Pereira, Maria E

    2012-07-01

    This work investigated the effects of low and high doses of inorganic mercury in drinking water on biochemical parameters of pregnant rats and their offspring. Female Wistar rats were treated during pregnancy with 0, 0.2, 0.5, 10 or 50 μg Hg(2+)/mL as HgCl(2). Rats were euthanized on day 20 of pregnancy. Pregnant rats presented a decrease in total water intake in all doses of mercury tested. At high doses, a decrease in the total food intake and in body weight gain was observed. Pregnant rats exposed to 50 μg Hg(2+)/mL presented an increase in kidney relative weight. Mercury exposure did not change serum urea and creatinine levels in any of the doses tested. Moreover, mercury exposure did not change porphobilinogen synthase activity of kidney, liver and placenta from pregnant rats in any of the doses tested, whereas fetuses of pregnant rats exposed to 50 μg Hg(2+)/mL presented an increase in the hepatic porphobilinogen synthase activity. In general, pregnant rats presented alterations due to HgCl(2) exposure in drinking water. However, only the dose 50 μg Hg(2+)/mL appeared to be enough to cross the blood-placenta barrier, since at this dose the fetuses presented change in the porphobilinogen synthase activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Herbicide-induced experimental variegate porphyria in mice: tissue porphyrinogen accumulation and response to porphyrogenic drugs.

    Science.gov (United States)

    Krijt, J; Stranska, P; Maruna, P; Vokurka, M; Sanitrak, J

    1997-01-01

    Administration of oxadiazon or oxyfluorfen (1000 ppm in the diet) to male BALB/c mice for 9 days resulted in experimental porphyria, resembling the acute phase of human variegate porphyria. Urinary concentrations of 5-aminolevulinic acid and porphobilinogen reached 1500 and 3000 mumol/L, respectively. Both herbicides caused a decrease of protoporphyrinogen oxidase activity in liver and kidney. Brain protoporphyrinogen oxidase activity was not altered. Liver and kidney porphyrin content increased to 11 and 17 nmol/g, respectively (control mice, 2 nmol/g). Over 50% of liver and kidney porphyrins were in the reduced (porphyrinogen) form. Bile of oxadiazon-treated mice contained 700 nmol/mL of protoporphyrinogen (control mice, 15 nmol/mL). Porphyrin content of the trigeminal nerve increased from 1 nmol/g in control animals to 11 nmol/g in oxadiazon-treated animals, suggesting a possible contribution of peripheral nerve porphyrins to porphyric neuropathy. Mice treated with 125 ppm of oxadiazon in the diet for 9 days excreted moderately elevated levels of porphobilinogen in urine (control mice, less than 50 mumol/L; treated mice, 330 mumol/L). Administration of phenobarbital or phenytoin (single injections on days 7, 8, and 9) increased the urinary porphobilinogen concentration to 3500 mumol/L. This response to porphyrogenic drugs resembles the response observed in human acute porphyrias.

  1. ORF Alignment: NC_002678 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_002678 gi|13474471 >1gkpA 2 454 5 422 3e-10 ... ref|NP_106039.1| creatinine deamin...ase [Mesorhizobium loti MAFF303099] ... dbj|BAB51825.1| creatinine deaminase [Mesorhizobium loti ...

  2. Rapid determination of the hypoxanthine increase in ischemic exercise tests

    NARCIS (Netherlands)

    Bolhuis, P. A.; Zwart, R.; Bär, P. R.; de Visser, M.; van der Helm, H. J.

    1988-01-01

    After ischemic exercise tests, performed to detect glycogenoses or myoadenylate deaminase (EC 3.5.4.6) deficiency, the increases in serum lactate and ammonia usually are measured. Determination of hypoxanthine instead of ammonia can also be used to show myoadenylate deaminase deficiency, but HPLC of

  3. Regulation and function of DNA methylation in plants and animals

    KAUST Repository

    He, Xinjian; Chen, Taiping; Zhu, Jian-Kang

    2011-01-01

    ) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment

  4. Host range, symbiotic effectiveness and nodulation competitiveness ...

    African Journals Online (AJOL)

    SERVER

    2008-04-17

    Apr 17, 2008 ... ERIC-PCR DNA fingerprinting patterns were used to identify the ... Apart from cowpea where all the isolates were effective, there were significant ..... aminocyclopropane-1-carboxylate deaminase gene in Sinorhizobium.

  5. The Expression and Regulation of the Cell Adhesion Molecule CD44 in Human Breast Cancer

    National Research Council Canada - National Science Library

    Ge, Lisheng

    1997-01-01

    ... alternative splicing signals of CD44 variant exons as control elements in CEPT. In our colon cancer metastasis to liver model, we developed dual modulation vectors to increase tissue-specific expression of cytosine deaminase (CD...

  6. Isolation and characterization of novel plant growth promoting Micrococcus sp NII-0909 and its interaction with cowpea

    Digital Repository Service at National Institute of Oceanography (India)

    Dastager, S.G.; Deepa, C.K.; Pandey, A.

    , and 16S rRNA gene sequence. The strain exhibited the plant growthpromoting attributes of phosphate solubilization, auxin production, 1-aminocyclopropane-1-carboxylate deaminase activity, and siderophore production. It was able to solubilize (122.4 mg...

  7. Targeting a Novel Vector for Breast Cancer Gene Therapy

    National Research Council Canada - National Science Library

    Bzik, David

    2002-01-01

    ... in vitro and in vivo models. We found that cytosine deaminase (CD) and thymidine kinase (TK) markers expressed in T gondii produce a significant bystander killing effect on both human fibroblasts and SKBR3 tumor cells in vitro...

  8. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    oxidatively to propionate via 2-ketobutyrate by biodegradative threonine deaminase, 2-ketobutyrate formate-lyase (or ... In the recent past, extensive structural and biochemical studies have been carried out on these enzymes by various groups.

  9. Total-Body Irradiation Followed By Cyclosporine and Mycophenolate Mofetil in Treating Patients With Severe Combined Immunodeficiency Undergoing Donor Bone Marrow Transplant

    Science.gov (United States)

    2017-07-12

    Adenosine Deaminase Deficiency; Autosomal Recessive Disorder; Immune System Disorder; Purine-Nucleoside Phosphorylase Deficiency; Severe Combined Immunodeficiency; Severe Combined Immunodeficiency With Absence of T and B Cells; X-Linked Severe Combined Immunodeficiency

  10. Prodrug Therapy for Breast Cancer Targeted by Single-Chain Antibodies F19 and 3S193

    National Research Council Canada - National Science Library

    Deckert, Peter

    2003-01-01

    ... of E.coli CD with the yeast isoenzyme. In addition, a second line of fusion constructs with green fluorescent protein instead of cytosine deaminase was designed for histological and intracellular distribution studies...

  11. ATP-consuming and ATP-generating enzymes secreted by pancreas

    DEFF Research Database (Denmark)

    Yegutkin, Gennady G; Samburski, Sergei S; Jalkanen, Sirpa

    2006-01-01

    -generating enzymes in pancreatic juice, adenylate kinase, and NDP kinase, capable of sequentially phosphorylating AMP via ADP to ATP. Activities of nonspecific phosphatases, nucleotide pyrophosphatase/phosphodiesterases, and adenosine deaminase were negligible. Taken together, CCK-8 stimulation of pancreas causes...

  12. Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2 '-deoxyuridine and 5-ethynyl-2 '- deoxycytidine

    Czech Academy of Sciences Publication Activity Database

    Ligasová, A.; Liboska, Radek; Friedecký, D.; Mičová, K.; Adam, T.; Oždian, T.; Rosenberg, Ivan; Koberna, K.

    2016-01-01

    Roč. 6, č. 1 (2016), č. článku 150172. ISSN 2046-2441 R&D Projects: GA MZd NV15-31604A Institutional support: RVO:61388963 Keywords : cytidine deaminase * dCMP deaminase * 5-ethynyl-2 '-deoxyuridine * 5-ethynyl-2 '-deoxycytidine * DNA replication Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.481, year: 2016 http://rsob.royalsocietypublishing.org/content/6/1/150172

  13. Kinetics of adenylate metabolism in human and rat myocardium

    OpenAIRE

    Tavenier, M.; Skladanowski, A.C.; Abreu, R.A. de; Jong, J.W. de

    1995-01-01

    textabstractPathways producing and converting adenosine have hardly been investigated in human heart, contrasting work in other species. We compared the kinetics of enzymes associated with purine degradation and salvage in human and rat heart cytoplasm assaying for adenosine deaminase, nucleoside phosphorylase, xanthine oxidoreductase, AMP deaminase, AMP- and IMP-specific 5′-nucleotidases, adenosine kinase and hypoxanthine guanine phosphoribosyltransferase (HGPRT). Xanthine oxidoreductase was...

  14. Reference: 497 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available hal albino phenotype. Rescue of tha2 mutants and tha1 tha2 double mutants by overproduction of feedback-inse...-specific expression of feedback-insensitive Thr deaminase in both tha1 and tha2 Thr aldolase mutants greatl...nsitive Thr deaminase (OMR1) shows that Gly formation by THA1 and THA2 is not essential in Arabidopsis. Seed

  15. Insulin and adenosine regulate the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes.

    Science.gov (United States)

    Kiechle, F L; Sykes, E; Artiss, J D

    1995-01-01

    Blockade of adenosine receptors by 3-isobutyl-1-methylxanthine or degradation of endogenous adenosine with adenosine deaminase increased the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes, an effect which was suppressed by the phosphatidylethanolamine methyltransferase inhibitor, S-adenosyl-L-homocysteine, and reversed by the adenosine analogue, N6-(L-phenylisopropyl)-adenosine. For example, the addition of N6-(L-phenylisopropyl)-adenosine to adenosine deaminase pretreated plasma membranes rapidly lowered the concentration of phosphatidylcholine by 171 nmol/mg at 30 seconds compared to control. Insulin-induced stimulation of phospholipid methylation in membranes treated with 3-isobutyl-1-methylxanthine or adenosine deaminase was achieved only after the addition of N6-(L-phenylisopropyl)-adenosine. These results suggest that adenosine receptor occupancy inhibits phospholipid methylation, is required for insulin stimulation of phospholipid methylation, and may perhaps activate a phosphatidylcholine-specific phospholipase C or phospholipase D.

  16. Stemcell Information: SKIP000184 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available negtive, due to adenosine deaminase deficiency adenosine deaminase 欠損患者線維芽細胞(GM01390) 由来。常染...色体劣性adenosine deaminase 欠失。患者は重度の免疫不全、T細胞、B細胞、NK細胞陰性。 ... human ES-like -- Retroviru...deficiency 102700 ... 0-9 Male ... Yes No iPSCs derived from GM01390 Fibroblast. T cell negtive, B cell negative, NK cell...Collections/NIGMS/ipsc_list.aspx?PgId=696 ... 18691744 10.1016/j.cell.2008.07.041 Disease-Specific Induced P

  17. Enzymatically Active APOBEC3G Is Required for Efficient Inhibition of Human Immunodeficiency Virus Type 1▿

    OpenAIRE

    Miyagi, Eri; Opi, Sandrine; Takeuchi, Hiroaki; Khan, Mohammad; Goila-Gaur, Ritu; Kao, Sandra; Strebel, Klaus

    2007-01-01

    APOBEC3G (APO3G) is a cellular cytidine deaminase with potent antiviral activity. Initial studies of the function of APO3G demonstrated extensive mutation of the viral genome, suggesting a model in which APO3G's antiviral activity is due to hypermutation of the viral genome. Recent studies, however, found that deaminase-defective APO3G mutants transiently expressed in virus-producing cells exhibited significant antiviral activity, suggesting that the antiviral activity of APO3G could be disso...

  18. Effect of repeated applications of pesticides used on cotton on soil properties

    International Nuclear Information System (INIS)

    Zayed, S.M.A.D.; Farghaly, M.; Soliman, S.M.; Taha, H.

    2001-01-01

    Repeated application of monocrotophos, methomyl and carbaryl for four years considerably reduced microbial counts, iron reduction, nitrification and arginine deaminase activity in soil. The microbial activities seemed to recover several weeks following pesticide application. The inhibition of enzyme activities was in general more obvious during the second to the fourth years. The maximum inhibition of iron reduction capacity and arginine deaminase activity was observed by the end of the fourth year and amounted to about 90% of control values. No pronounced effect of the used insecticides on respiration and dehydrogenase activity could be detected over the experimental period. (author)

  19. Engineering of High Yield Production of L-serine in Escherichia coli

    DEFF Research Database (Denmark)

    Mundhada, Hemanshu; Schneider, Konstantin; Christensen, Hanne Bjerre

    2016-01-01

    by deletion of three L-serine deaminases sdaA, sdaB, and tdcG, as well as serine hydroxyl methyl transferase (SHMT) encoded by glyA. Upon overexpression of the serine production pathway, consisting of a feedback resistant version of serA along with serB and serC, this quadruple deletion strain showed a very...

  20. Antigen receptors and somatic hypermutation in B-cell chronic lymphocytic leukemia with Richter's transformation

    NARCIS (Netherlands)

    Smit, Laura A.; van Maldegem, Febe; Langerak, Anton W.; van der Schoot, C. Ellen; de Wit, Mireille J.; Bea, Silvia; Campo, Elias; Bende, Richard J.; van Noesel, Carel J. M.

    2006-01-01

    BACKGROUND AND OBJECTIVES: Activation-induced cytidine deaminase is essential for somatic hypermutation and class switch recombination of the immunoglobulin genes in B cells. It has been proposed that aberrant targeting of the somatic hypermutation machinery is instrumental in initiation and

  1. Unedited Version

    Indian Academy of Sciences (India)

    58

    Identification of RNA-specific adenosine deaminase 1 (ADAR1) gene results in ... youth male patient with a deleterious substitution of Leu1052Pro of ADAR1 ... reaction system contained 14.75 µl double-distilled water, 2.5 µl 10X buffer, 2 µl.

  2. A novel missense mutation of gene in a Chinese family leading to ...

    Indian Academy of Sciences (India)

    SHUAI-MEI LIU

    2017-12-18

    Dec 18, 2017 ... Identification of RNA-specific adenosine deaminase 1 (ADAR1) gene results in DSH. ... In this study, we found that a 28 year-old male patient harbouring a deleterious ... tion system contained 14.75 µL double-distilled water,.

  3. Assay for mutagenesis in heterozygous diploid human lymphoblasts

    Science.gov (United States)

    Skopek, Thomas R.; Liber, Howard L.; Penman, Bruce W.; Thilly, William G.; Hoppe, IV, Henry

    1981-01-01

    An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

  4. Native bacterial endophytes promote host growth in a species-specific manner; phytohormone manipulations do not result in common growth responses.

    Directory of Open Access Journals (Sweden)

    Hoang Hoa Long

    Full Text Available BACKGROUND: All plants in nature harbor a diverse community of endophytic bacteria which can positively affect host plant growth. Changes in plant growth frequently reflect alterations in phytohormone homoeostasis by plant-growth-promoting (PGP rhizobacteria which can decrease ethylene (ET levels enzymatically by 1-aminocyclopropane-1-carboxylate (ACC deaminase or produce indole acetic acid (IAA. Whether these common PGP mechanisms work similarly for different plant species has not been rigorously tested. METHODOLOGY/PRINCIPAL FINDINGS: We isolated bacterial endophytes from field-grown Solanum nigrum; characterized PGP traits (ACC deaminase activity, IAA production, phosphate solubilization and seedling colonization; and determined their effects on their host, S. nigrum, as well as on another Solanaceous native plant, Nicotiana attenuata. In S. nigrum, a majority of isolates that promoted root growth were associated with ACC deaminase activity and IAA production. However, in N. attenuata, IAA but not ACC deaminase activity was associated with root growth. Inoculating N. attenuata and S. nigrum with known PGP bacteria from a culture collection (DSMZ reinforced the conclusion that the PGP effects are not highly conserved. CONCLUSIONS/SIGNIFICANCE: We conclude that natural endophytic bacteria with PGP traits do not have general and predictable effects on the growth and fitness of all host plants, although the underlying mechanisms are conserved.

  5. AcEST: DK945559 [AcEST

    Lifescience Database Archive (English)

    Full Text Available DEC_RHILO Adenine deaminase OS=Rhizobium loti GN=ade ... 34 0.58 sp|O24006|AMP_IMPBA Antimicrobial peptides ...A E DH+GT Sbjct: 222 YASRDLGLP-FHGYVAGGPEDDHEGT 246 >sp|O24006|AMP_IMPBA Antimicrobial peptides OS=Impatiens

  6. Elevated Adenosine Induces Placental DNA Hypomethylation Independent of A2B Receptor Signaling in Preeclampsia.

    Science.gov (United States)

    Huang, Aji; Wu, Hongyu; Iriyama, Takayuki; Zhang, Yujin; Sun, Kaiqi; Song, Anren; Liu, Hong; Peng, Zhangzhe; Tang, Lili; Lee, Minjung; Huang, Yun; Ni, Xin; Kellems, Rodney E; Xia, Yang

    2017-07-01

    Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear. Using 2 independent experimental models of preeclampsia, adenosine deaminase-deficient mice and a pathogenic autoantibody-induced mouse model of preeclampsia, we demonstrate that elevated placental adenosine not only induces hallmark features of preeclampsia but also causes placental DNA hypomethylation. The use of genetic approaches to express an adenosine deaminase minigene specifically in placentas, or adenosine deaminase enzyme replacement therapy, restored placental adenosine to normal levels, attenuated preeclampsia features, and abolished placental DNA hypomethylation in adenosine deaminase-deficient mice. Genetic deletion of CD73 (an ectonucleotidase that converts AMP to adenosine) prevented the elevation of placental adenosine in the autoantibody-induced preeclampsia mouse model and ameliorated preeclampsia features and placental DNA hypomethylation. Immunohistochemical studies revealed that elevated placental adenosine-mediated DNA hypomethylation predominantly occurs in spongiotrophoblasts and labyrinthine trophoblasts and that this effect is independent of A2B adenosine receptor activation in both preeclampsia models. Extending our mouse findings to humans, we used cultured human trophoblasts to demonstrate that adenosine functions intracellularly and induces DNA hypomethylation without A2B adenosine receptor activation. Altogether, both mouse and human studies reveal novel mechanisms underlying placental DNA hypomethylation and potential therapeutic approaches for preeclampsia. © 2017 American Heart Association, Inc.

  7. Protection against herbivores

    Energy Technology Data Exchange (ETDEWEB)

    Howe, Gregg A.; Chen, Hui

    2017-10-25

    The present invention relates to genes, proteins and methods comprising molecules that alter amino acid levels. In one embodiment, the present invention relates to altering guanidino substrate hydrolysis activities in plants, arthropods and microorganisms using molecules within the arginase family and other molecules that alter an amino acid levels. In ones embodiment, the present invention relates to altering threonine substrate deamination and dehydration activities in plants, arthropods and microorganisms using molecules within the threonine deaminase family and other molecules that alter amino acid levels. In one embodiment, the present invention relates to using genes, proteins and methods comprising arginase or threonine deaminase for altering the pathophysiology of plants, arthropods and microorganisms. In a preferred embodiment, the present invention relates to altering guanidino substrate hydrolysis activity in plants, arthropods, and microorganisms using arginase. In another preferred embodiment, the invention relates to altering threonine substrated deamination and dehydration activity in plants, arthropods, and microorganisms using threonine deaminase. In some embodiments, the invention related to overexpression and increased activity of arginase, threonine deaminase and a proteinase inhibitor.

  8. Generation and analysis of cDNA library from lipopolysaccharide ...

    African Journals Online (AJOL)

    These immune-related genes include cytidine deaminase, ferritin, nonmuscle myosin essential light chain, cytochrome c oxidase subunit I, CD63 antigen-like protein and lysosomal-associated transmembrane protein. This study may contribute to the understanding of the immune mechanism of gastropod abalone Haliotis ...

  9. ADA genetic polymorphism and the effect of smoking on neonatal bilirubinemia and developmental parameters.

    Science.gov (United States)

    Gloria-Bottini, F; Magrini, A; Cozzoli, E; Bergamaschi, A; Bottini, E

    2008-11-01

    Genetic variability of metabolic enzymes may influence the effect of cigarette smoking on intrauterine development and on early neonatal events. To investigate the role of adenosine deaminase genetic polymorphism on the effect of smoking on neonatal bilirubinemia and developmental parameters. Analysis of association between adenosine deaminase phenotypes and neonatal developmental parameters. Prospective study of serum bilirubin level in relation to adenosine deaminase phenotype. We have studied 360 consecutive newborn infants from the Caucasian population of Rome. Serum bilirubin concentration was determined at birth and every 24 h for the first five days. Overall maternal smoking is associated with a slight decrease in the incidence of phototherapy (13.4% in non smoking vs 11.7% in smoking mothers) and with a reduction of birth weight (3374 g in non smoking mothers vs 3133 g in smoking mothers). There is a significant interaction between smoke and adenosine deaminase. While in non smoking mothers the incidence of phototherapy in carriers of ADA 2 allele is higher than in ADA 1 phenotype, in infants from smoking mothers the pattern is reversed and the incidence of phototherapy in carriers of ADA 2 allele is lower than in infants with ADA 1 phenotype. Other neonatal bilirubin parameters follow a similar pattern of interaction between smoking and ADA. The negative effect of smoke on birth weight is much more evident in infant with ADA 1 phenotype than in those carrying the ADA 2 allele. The data suggest that ADA phenotype modifies the effect of smoking on developmental and bilirubin parameters.

  10. Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides

    DEFF Research Database (Denmark)

    Oehlenschlæger, Christian Berg; Løvgreen, Monika Nøhr; Reinauer, Eva

    2015-01-01

    Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase...

  11. Growth inhibition of human breast cancer cells and down-regulation of ODC1 and ADA genes by Nepeta binaloudensis

    Directory of Open Access Journals (Sweden)

    Akbar Safipour Afshar

    Full Text Available ABSTRACT Nepeta binaloudensis Jamzad, Lamiaceae, is a rare medicinal plant endemic to Iran. In spite of many studies about the chemical constituents and antibacterial effects of this species, no report has been provided about its cytotoxic and anticancer activities. In this study we have evaluated the effects of EtOH 70%, hexane and aqueous extracts of N. binaloudensis on the cell proliferation and n-hexane extract on the expression of adenosine deaminase and ornithine decarboxylase 1 genes in breast cancer cell lines (MCF-7, MDA-MB-231 compared to non-cancer line (MCF-10A. The cell lines were subjected to increasing doses of the extracts ranging from 10 to 320 µg/ml. Cell viability was quantified by MTS assay. Expression of adenosine deaminase and ornithine decarboxylase 1 genes was analyzed by real time PCR. N. binaloudensis inhibited the growth of malignant cells in a time and dose-dependent manner. Among extracts of N. binaloudensis, the hexane extract was found to be more toxic compared to other extracts. Results showed a marked decrease in the expression of ornithine decarboxylase 1 and adenosine deaminase genes in cancer cell lines. At 60 µg/ml concentration of N. binaloudensis hexane extract ornithine decarboxylase 1 and adenosine deaminase mRNA expression were reduced 4.9 fold and 3.5 fold in MCF-7 cell line and 3.6 fold and 2.6 fold in MDA-MB-231 cell line compared to control, respectively. The result of our study highlights the potential influences of N. binaloudensis hexane extract on ornithine decarboxylase 1 and adenosine deaminase genes expression in breast cancer cells and its relation to inhibition of cancer cell growth.

  12. Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum*

    Science.gov (United States)

    Cassera, María B.; Hazleton, Keith Z.; Riegelhaupt, Paul M.; Merino, Emilio F.; Luo, Minkui; Akabas, Myles H.; Schramm, Vern L.

    2008-01-01

    Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum. PMID:18799466

  13. Differential evolution of antiretroviral restriction factors in pteropid bats as revealed by APOBEC3 gene complexity.

    Science.gov (United States)

    Hayward, Joshua A; Tachedjian, Mary; Cui, Jie; Cheng, Adam Z; Johnson, Adam; Baker, Michelle; Harris, Reuben S; Wang, Lin-Fa; Tachedjian, Gilda

    2018-03-29

    Bats have attracted attention in recent years as important reservoirs of viruses deadly to humans and other mammals. These infections are typically nonpathogenic in bats raising questions about innate immune differences that might exist between bats and other mammals. The APOBEC3 gene family encodes antiviral DNA cytosine deaminases with important roles in the suppression of diverse viruses and genomic parasites. Here we characterize pteropid APOBEC3 genes and show that species within the genus Pteropus possess the largest and most diverse array of APOBEC3 genes identified in any mammal reported to date. Several bat APOBEC3 proteins are antiviral as demonstrated by restriction of retroviral infectivity using HIV-1 as a model, and recombinant A3Z1 subtypes possess strong DNA deaminase activity. These genes represent the first group of antiviral restriction factors identified in bats with extensive diversification relative to homologues in other mammals.

  14. Plant Growth-Promoting Rhizobacteria Enhance Salinity Stress Tolerance in Okra through ROS-Scavenging Enzymes

    Directory of Open Access Journals (Sweden)

    Sheikh Hasna Habib

    2016-01-01

    Full Text Available Salinity is a major environmental stress that limits crop production worldwide. In this study, we characterized plant growth-promoting rhizobacteria (PGPR containing 1-aminocyclopropane-1-carboxylate (ACC deaminase and examined their effect on salinity stress tolerance in okra through the induction of ROS-scavenging enzyme activity. PGPR inoculated okra plants exhibited higher germination percentage, growth parameters, and chlorophyll content than control plants. Increased antioxidant enzyme activities (SOD, APX, and CAT and upregulation of ROS pathway genes (CAT, APX, GR, and DHAR were observed in PGPR inoculated okra plants under salinity stress. With some exceptions, inoculation with Enterobacter sp. UPMR18 had a significant influence on all tested parameters under salt stress, as compared to other treatments. Thus, the ACC deaminase-containing PGPR isolate Enterobacter sp. UPMR18 could be an effective bioresource for enhancing salt tolerance and growth of okra plants under salinity stress.

  15. Co-inoculation with Rhizobium and plant growth promoting rhizobacteria (PGPR for inducing salinity tolerance in mung bean under field condition of semi arid climate

    Directory of Open Access Journals (Sweden)

    Muhammad Aamir

    2013-04-01

    Full Text Available Salinity stress severely affects the growth, nodulation and yield of mung bean (Vigna radiata L.. However, its growth can be improved under salinity stress by inoculation/co-inoculation with rhizobia and plant growth promoting rhizobacteria (PGPR containing 1-Aminocyclopropane-1-carboxylic acid (ACC deaminase enzyme. ACC-deaminase containing bacteria regulate the stress induced ethylene production by hydrolyzing the ACC (immediate precursor of ethylene into ammonia and ketobutyric acid, thus improve plant growth by lowering the ethylene level. A study was conducted under salt affected field conditions where pre-isolated strains of Rhizobium and PGPR were used alone as well as in combination for mitigating the salinity stress on growth, nodulation and yield of mung bean by following the randomized complete block design (RCBD. The data were recorded and analyzed statistically to see the difference among treatments.

  16. Gene Therapy in Cardiac Arrhythmias

    OpenAIRE

    Praveen, S.V; Francis, Johnson; Venugopal, K

    2006-01-01

    Gene therapy has progressed from a dream to a bedside reality in quite a few human diseases. From its first application in adenosine deaminase deficiency, through the years, its application has evolved to vascular angiogenesis and cardiac arrhythmias. Gene based biological pacemakers using viral vectors or mesenchymal cells tested in animal models hold much promise. Induction of pacemaker activity within the left bundle branch can provide stable heart rates. Genetic modification of the AV...

  17. Salt and alkali stresses reduction in wheat by plant growth promoting haloalkaliphilic bacteria

    OpenAIRE

    Torbaghan, Mehrnoush Eskandari; Lakzian, Amir; Astaraei, Ali Reza; Fotovat, Amir; Besharati, Hossein

    2017-01-01

    Haloalkaliphilic bacteria have plant growth promoting characteristics that can be used to deal with different environmental stresses. To study the effect of haloalkaliphilic bacteria to reduce salinity and alkalinity stress in wheat, 48 isolates were isolated and grouped into halophiles, alkaliphiles and haloalkaliphiles based on growth characteristics. The ammonia, 3-indole acetic acid and ACC (1-aminocyclopropane-1-carboxylate) deaminase production were studied. Wheat yield was evaluated in...

  18. Relation fonctionnelle entre le pool de nucléotides et PARP-1 : une nouvelle source d'instabilité génétique

    OpenAIRE

    Gemble , Simon

    2015-01-01

    Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, w...

  19. Metabolic consequences of DNA damage: The role of poly (ADP-ribose) polymerase as mediator of the suicide response

    International Nuclear Information System (INIS)

    Berger, N.A.; Berger, S.J.

    1986-01-01

    Recent studies show that DNA damage can produce rapid alterations in steady state levels of deoxynucleoside triphosphate pools, for example, MNNG or uv-irradiation cause rapid increases in dATP and dTTP pools without significant changes in dGTP or dCTP pools. In vitro, studies with purified eukaryotic DNA polymerases show that the frequency of nucleotide misincorporation was affected by alterations in relative concentrations of the deoxynucleoside triphosphates. Thus the alterations in dNTP pool sizes that occur consequent to DNA damage may contribute to an increased mutagenic frequency. Poly(ADP-ribose) polymerase mediated suicide mechanism may participate in the toxicity of adenosine deaminase deficiency and severe combined immune deficiency disease in humans. Individuals with this disease suffer severe lymphopenia due to the toxic effects of deoxyadenosine. The lymphocytotoxic effect of adenosine deaminase deficiency can be simulated in lymphocyte cell lines from normal individuals by incubating them with the adenosine deaminase inhibitor, deoxycoformycin. Incubation of such leukocytes with deoxycoformycin and deoxyadenosine results in the gradual accumulation of DNA strand breaks and the depletion of NAD + leading to cell death over a period of several days. This depletion of NAD and loss of cell viability were effectively blocked by nicotinamide or 3-amino benzamide. Thus, persistent activation of poly(ADP-ribose) polymerase by unrepaired or recurrent DNA strand breaks may activate the suicide mechanism of cell death. This study provides a basis for the interesting suggestion that treatment with nicotinamide could block the persistent activity of poly(ADP-ribose) polymerase and may help preserve lymphocyte function in patients with adenosine deaminase deficiency. 16 refs., 3 figs., 2 tabs

  20. Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID

    OpenAIRE

    Wang, Meng; Rada, Cristina; Neuberger, Michael S.

    2010-01-01

    High-affinity antibodies are generated by somatic hypermutation with nucleotide substitutions introduced into the IgV in a semirandom fashion, but with intrinsic mutational hotspots strategically located to optimize antibody affinity maturation. The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5?-flanking nucleotide. As a critical test of whether such DNA deaminati...

  1. US Army Institute of Surgical Research Annual Research Progress Report, FY 1980.

    Science.gov (United States)

    1980-10-01

    Arabinose - - + - deaminase positive, urease negative, indole positive, ornithine nega- tive, glucose and inositol fermented, citrate utilized...portion of biopsy sample was plated on the surface of a screw-capped tissue culture bottle containing a layer of Sabouraud’s agar. Results of this series...this new system are illustrated in Figure I. The 8x4x4 foot chamber is constructed of 3/16 inch steel covered by a I inch layer of thermal insulation

  2. Characterization of Mn-resistant endophytic bacteria from Mn-hyperaccumulator Phytolacca americana and their impact on Mn accumulation of hybrid penisetum.

    Science.gov (United States)

    Zhang, Wen-Hui; Chen, Wei; He, Lin-Yan; Wang, Qi; Sheng, Xia-Fang

    2015-10-01

    Three hundred Mn-resistant endophytic bacteria were isolated from the Mn-hyperaccumulator, Phytolacca americana, grown at different levels of Mn (0, 1, and 10mM) stress. Under no Mn stress, 90%, 92%, and 11% of the bacteria produced indole acetic acid (IAA), siderophore, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase, respectively. Under Mn stress, 68-94%, 91-92%, and 21-81% of the bacteria produced IAA, siderophore, and ACC deaminase, respectively. Greater percentages of ACC deaminase-producing bacteria were found in the Mn-treated P. americana. Furthermore, the ratios of IAA- and siderophore-producing bacteria were significantly higher in the Mn treated plant leaves, while the ratio of ACC deaminase-producing bacteria was significantly higher in the Mn treated-roots. Based on 16S rRNA gene sequence analysis, Mn-resistant bacteria were affiliated with 10 genera. In experiments involving hybrid penisetum grown in soils treated with 0 and 1000mgkg(-1) of Mn, inoculation with strain 1Y31 was found to increase the root (ranging from 6.4% to 18.3%) and above-ground tissue (ranging from 19.3% to 70.2%) mass and total Mn uptake of above-ground tissues (64%) compared to the control. Furthermore, inoculation with strain 1Y31 was found to increase the ratio of IAA-producing bacteria in the rhizosphere and bulk soils of hybrid penisetum grown in Mn-added soils. The results showed the effect of Mn stress on the ratio of the plant growth-promoting factor-producing endophytic bacteria of P. americana and highlighted the potential of endophytic bacterium as an inoculum for enhanced phytoremediation of Mn-polluted soils by hybrid penisetum plants. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. ORF Alignment: NC_002695 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available aminase; ... Chain: A, B; Ec: 5.3.1.10; Engineered: Yes; Heterogen: ... 2-Deoxi-2-Amino-Glucit...Phosphate Deaminase; ... Chain: A, B; Ec: 5.3.1.10; Engineered: Yes; Heterogen: ... 2-Deoxi-2-...nase; ... Chain: A, B; Ec: 5.3.1.10; Engineered: Yes; Heterogen: ... ... Chain: A, ... B; Ec: 5.3.1.10; Engineered: Yes; Heterogen: Inorganic ... Phosphate gb|AAA2419

  4. Screening for genital tuberculosis in a limited resource country: case report

    OpenAIRE

    Namani, Sadie; Qehaja-Bu?aj, Emine; Namani, Diell?za

    2017-01-01

    Background Screening for benign or malignant process of pelvis in young females is a challenge for a physician in a limited resource country. Tuberculosis should be always considered in the differential diagnosis of a pelvic mass in countries with high prevalence of tuberculosis. Negative results of analysis of peritoneal fluid for acid-fast staining, late cultures, and unavailability of new diagnostics methods such as polymerase chain reaction and adenosine deaminase of the aspirated fluid f...

  5. The Role of CREB in CML

    Science.gov (United States)

    2008-02-01

    responses differ after stroke, seizures, hypoglycemia , and hypoxia: Blood genomic fingerprints of disease. Ann Neurol 2001;50:699–707. 62 Whitney LW...et al. Engraft- ment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase defi- ciency. Nat Med. 1995;1:1017-1023. 33...Proteolysis Targeting Chimeric molecules: Recruiting Cancer- Causing Proteins for Ubiquitination and Degradation. Modulation of Protein Stability in

  6. Moellerella wisconsensis, a new genus and species of Enterobacteriaceae found in human stool specimens.

    OpenAIRE

    Hickman-Brenner, F W; Huntley-Carter, G P; Saitoh, Y; Steigerwalt, A G; Farmer, J J; Brenner, D J

    1984-01-01

    The name Moellerella wisconsensis is proposed for a group of the family Enterobacteriaceae previously called enteric group 46. The species name, wisconsensis, was coined because six of the nine strains were isolated in Wisconsin. M. wisconsensis strains were negative for indole production, Voges-Proskauer, H2S production, urea, phenylalanine deaminase, lysine and ornithine decarboxylases, arginine dihydrolase, gas production from D-glucose, acid production from trehalose, and motility; the st...

  7. Investor Outlook: Rising from the Ashes; GSK's European Approval of Strimvelis for ADA-SCID.

    Science.gov (United States)

    Schimmer, Joshua; Breazzano, Steven

    2016-06-01

    GlaxoSmithKline's (GSK) and partner San Raffaele Telethon Institute for Gene Therapy's recent positive European approval for Strimvelis for treatment of severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) represents the second EU-approved gene therapy and the first γ-retrovirus and first ex vivo gene therapy. In this article we discuss the significance and implications of this historic approval for the broader gene therapy field.

  8. GenBank blastx search result: AK058464 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK058464 001-016-A10 U27202.1 Actinobacillus pleuropneumoniae riboflavin biosynthesis operon, riboflavin...-specific deaminase (ribG), riboflavin synthase alpha subunit (ribB), bifunctional GTP ...cyclohydrase II/3,4-dihydroxy-2-butanone-4-phosphate synthase (ribA), and riboflavin synthase beta subunit (ribH) genes, complete cds.|BCT BCT 1e-105 +2 ...

  9. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

    Directory of Open Access Journals (Sweden)

    Jing Cai

    Full Text Available Quantitative real-time PCR (qPCR is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD, an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA and nonparametric (Kruskal-Wallis tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

  10. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

    Science.gov (United States)

    Cai, Jing; Li, Tao; Huang, Bangxing; Cheng, Henghui; Ding, Hui; Dong, Weihong; Xiao, Man; Liu, Ling; Wang, Zehua

    2014-01-01

    Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

  11. Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity.

    Science.gov (United States)

    Kouno, Takahide; Silvas, Tania V; Hilbert, Brendan J; Shandilya, Shivender M D; Bohn, Markus F; Kelch, Brian A; Royer, William E; Somasundaran, Mohan; Kurt Yilmaz, Nese; Matsuo, Hiroshi; Schiffer, Celia A

    2017-04-28

    Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 Å. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A-ssDNA complex defines the 5'-3' directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics.

  12. Clinically Important Features of Porphyrin and Heme Metabolism and the Porphyrias

    Directory of Open Access Journals (Sweden)

    Siddesh Besur

    2014-11-01

    Full Text Available Heme, like chlorophyll, is a primordial molecule and is one of the fundamental pigments of life. Disorders of normal heme synthesis may cause human diseases, including certain anemias (X-linked sideroblastic anemias and porphyrias. Porphyrias are classified as hepatic and erythropoietic porphyrias based on the organ system in which heme precursors (5-aminolevulinic acid (ALA, porphobilinogen and porphyrins are chiefly overproduced. The hepatic porphyrias are further subdivided into acute porphyrias and chronic hepatic porphyrias. The acute porphyrias include acute intermittent, hereditary copro-, variegate and ALA dehydratase deficiency porphyria. Chronic hepatic porphyrias include porphyria cutanea tarda and hepatoerythropoietic porphyria. The erythropoietic porphyrias include congenital erythropoietic porphyria (Gűnther’s disease and erythropoietic protoporphyria. In this review, we summarize the key features of normal heme synthesis and its differing regulation in liver versus bone marrow. In both organs, principal regulation is exerted at the level of the first and rate-controlling enzyme, but by different molecules (heme in the liver and iron in the bone marrow. We also describe salient clinical, laboratory and genetic features of the eight types of porphyria.

  13. δ-Aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    De Siervi, Adriana; Vazquez, Elba S; Rezaval, Carolina; Rossetti, María V; Batlle, Alcira M del [Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP), Argentine National Research Council (CONICET), Department of Biological Chemistry, FCEN, University of Buenos Aires (Argentina)

    2002-01-01

    Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, δ-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines. We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations.

  14. δ-Aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines

    Directory of Open Access Journals (Sweden)

    del Batlle Alcira M

    2002-03-01

    Full Text Available Abstract Background Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, δ-aminolevulinic acid (ALA and porphobilinogen (PBG. ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines. Results We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations.

  15. δ-Aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines

    International Nuclear Information System (INIS)

    De Siervi, Adriana; Vazquez, Elba S; Rezaval, Carolina; Rossetti, María V; Batlle, Alcira M del

    2002-01-01

    Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, δ-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines. We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations

  16. Two different zinc sites in bovine 5-aminolevulinate dehydratase distinguished by extended x-ray absorption fine structure

    International Nuclear Information System (INIS)

    Dent, A.J.; Hasnain, S.S.; Beyersmann, D.; Block, C.

    1990-01-01

    The zinc coordination in 5-aminolevulinate dehydratase was investigated by extended x-ray absorption fine structure (EXAFS) associated with the zinc K-edge. The enzyme binds 8 mol of zinc/mol of octameric protein, but only four zinc ions seem sufficient for full activity. The authors have undertaken a study on four forms of the enzyme: (a) the eight-zinc native enzyme; (b) the enzyme with only the four zinc sites necessary for full activation occupied; (c) the enzyme with the vacant sites of (b) occupied by four lead ions; (d) the product complex between (b) and porphobilinogen. They have shown that two structurally distinct types of zinc sites are available in the enzyme. The site necessary for activity has an average zinc environment best described by two/three histidines and one/zero oxygen from a group such as tyrosine or a solvent molecule at 2.06 ± 0.02 angstrom, one tyrosine or aspartate at 1.91 ± 0.03 angstrom, and one cysteine sulfur at 2.32 ± 0.03 angstrom with a total coordination of five ligands. The unoccupied site in (b) is dominated by a single contribution of four cysteinyl sulfur atoms at 2.28 ± 0.02 angstrom. Spectra from samples (c) and (d) show only small changes from that of (b), reflecting a slight rearrangement of the ligands around the zinc atom

  17. Delftia tsuruhatensis WGR-UOM-BT1, a novel rhizobacterium with PGPR properties from Rauwolfia serpentina (L.) Benth. ex Kurz also suppresses fungal phytopathogens by producing a new antibiotic-AMTM.

    Science.gov (United States)

    Prasannakumar, S P; Gowtham, H G; Hariprasad, P; Shivaprasad, K; Niranjana, S R

    2015-11-01

    The bacterial strain designated as WGR-UOM-BT1 isolated from rhizosphere of Rauwolfia serpentina exhibited broad-spectrum antifungal activity and also improved early plant growth. Based on morphological, biochemical and 16S rRNA gene sequence analyses, the strain BT1 was identified as Delftia tsuruhatensis (KF727978). Under in vitro conditions, the strain BT1 suppressed the growth of wide range of fungal phytopathogens. Purified antimicrobial metabolite from the strain BT1 was identified as nitrogen-containing heterocyclic compound, 'amino(5-(4-methoxyphenyl)-2-methyl-2-(thiophen-2-yl)-2,3-dihydrofuran-3-yl)methanol' (AMTM), with molecular mass of 340•40 and molecular formula of C17 H19 NO3 S. The strain BT1 was positive for rhizosphere colonization (tomato), IAA production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity and phosphate solubilization. Under laboratory and greenhouse conditions, the strain BT1 promoted plant growth and suppressed foliar and root fungal pathogens of tomato. Therefore, antimicrobial and disease protection properties of strain BT1 could serve as an effective biological control candidate against devastating fungal pathogens of vegetable plants. Besides, the production of IAA, P solubilization and ACC deaminase activity enhance its potential as a biofertilizer and may stabilize the plant performance under fluctuating environmental conditions. In this study, we reported that Delftia tsuruhatensis WGR-UOM-BT1 strain has the plant growth promotion activities such as rhizosphere colonization (tomato), IAA production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity and phosphate solubilization. This bacterial strain was found producing an antimicrobial nitrogen-containing heterocyclic compound identified as 'amino(5-(4-methoxyphenyl)-2-methyl-2-(thiophen-2-yl)-2,3-dihydrofuran-3-yl)methanol' [C17 H19 NO3 S] (AMTM), which is new to the bacterial world. © 2015 The Society for Applied Microbiology.

  18. Effect of plant growth-promoting rhizobacteria (PGPRs) on plant growth, yield, and quality of tomato (Lycopersicon esculentum Mill.) under simulated seawater irrigation.

    Science.gov (United States)

    Shen, Min; Jun Kang, Yi; Li Wang, Huan; Sheng Zhang, Xiang; Xin Zhao, Qing

    2012-01-01

    To determine the effects of three PGPRs on plant growth, yield, and quality of tomato under simulated seawater irrigation, a two consecutive seasons' field experiment was conducted in Yancheng Teachers University plot from April to June and August to October, 2011. The results showed that Erwinia persicinus RA2 containing ACC deaminase exhibited the best ability compared with Bacillus pumilus WP8 and Pseudomonas putida RBP1 which had no ACC deaminase activity to enhance marketable yields of fresh and dried fruits in tomato under simulated seawater irrigation especially under HS condition. B. pumilus WP8 had significant effects on improving tomato fruit quality under the conditions of irrigating with 1.0% NaCl solution (MS) and with 2.0% NaCl solution (HS). Na(+) contents were generally accumulated much more in tomato plant mid-shoot leaves than in fruits whatever the salt concentration. More sodium accumulation in leaves of E. persicinus RA2 and B. pumilus WP8 treatments under HS condition were found than in control. E. persicinus RA2 and B. pumilus WP8 can promote tomato growth, improve fruit quality more firmly than P. putida RBP1 during two consecutive seasons. Our study suggested that E. persicinus RA2 and B. pumilus WP8 are considered to be promising PGPR strains which are suited for application in salt marsh planting, ACC deaminase activity was not unique index on screening for PGPRs with the aim of salt stress tolerance, and plant growth promoting activities may be relevant to different growth indices and different stress conditions.

  19. A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria.

    Directory of Open Access Journals (Sweden)

    Annika Osswald

    Full Text Available Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria.

  20. Impact of heavy repeated long term pesticide applications on soil properties in a cotton agroecosystem

    International Nuclear Information System (INIS)

    Hussain, Altaf; Asi, Muhammad Rafique; Iqbal, Zafar; Chaudhry, Jamil Anwar

    2001-01-01

    A study was conducted under field conditions to investigate the effects of heavy repeated long term pesticide applications, at their recommended doses, on some biological properties in relation to the cotton agroecosystem at NIAB, Faisalabad, Pakistan. Control, test and treated soils before (BPA) and after pesticide applications (APA) were collected and analyzed at different stages of pesticide applications. The selective tests were measurements of microbial numbers, basal as well as substrate-induced respiration, nitrification, Fe-III reduction and the activities, of dehydrogenase and arginine deaminase. Endosulfan, profenophos + alphamethrin and methamidophos inhibited while monocrotophos and bifenthrin + acetamiprid enhanced the bacterial population numbers. The fungal population was depressed with endosulfan while monocrotophos, profenophos and methamidophos stimulated it. All other applied pesticides did not cause any appreciable change in total bacterial and fungal populations throughout the study period. Monocrotophos, propargite, endosulfan alone or with dimethoate and profenophos with cypermethrin or with ethion inhibited the respiration and hence affected the biomass. All other pesticides had no effect in test and treated soils compared to control soil. No pronounced inhibition or stimulation was seen in respiration after several weeks following the applications of pesticide. Endosulfan, endosulfan with dimethoate, methamidophos stimulated while profenophos + cypermethrin and bifenthrin + endosulfan inhibited the nitrification. All other pesticide applications showed similar nitrification rates in test and treated soils compared to control soil. Iron reduction capacity was stimulated by dimethoate + endosulfan and propenophos + cypermethrin and profenphos, methamidophos, propargite and diafenthiuron + profenophos reduced it. Soil dehydrogenase activity was inhibited by methamidophos, fenpropathrin, endosulfan + dimethoate and bifenthrin + ethion

  1. A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria

    Science.gov (United States)

    Osswald, Annika; Westermann, Christina; Sun, Zhongke; Riedel, Christian U.

    2015-01-01

    Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria. PMID:26086721

  2. Effect of lead on heme synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Neuberger, A.

    1975-01-01

    Recently, a fair amount of work has been done on the effect of lead on porphobilinogen dehydratase, which has been used as a sensitive indicator of lead poisoning. How far this is in itself harmful depends on the Michaelis constants of both the aminolaevulinic synthetase and of the dehydratase, and in addition on the relative activities of the two enzymes in a cell and also on the tissue concentration of glycine. Information on some of these points is still fragmentary, and a reliable judgement is at the present not very easy. Another step in the heme synthesis, which is sensitive to low concentrations of lead, is the incorporation of iron into protoporphyrin. Inhibition of this step may be important in accounting to a large extent for the anaemia found in individuals with lead poisoning. Reduction in the tissue concentration of heme or of heme-like compounds may also explain, through the mechanism of de-repression, the excretion of increased amounts of aminolaevulinic acid in the urine observed in cases of lead poisoning. A third step in heme synthesis, which might be sensitive to lead, is the oxidative decarboxylation of coproporphyrin to protoporphyrin, and this may explain why the former derivative is excreted in the urine. Recent work of the Harvard Medical School has indicated that greatly reduced levels of ALA dehydratase may be found in most cases of severe liver damage due to alcoholism. In most of these cases the level of lead in the blood is within normal limits, and there is no history of exposure to toxic amounts of lead. We therefore have to assume that a reduction in the blood level of this enzyme is not necessarily an indication of lead poisoning.

  3. [ALLELES C282Y AND H63D HFE GENE, INSULIN RESISTANCE AND SUSCEPTIBILITY TO DISTURBANCE OF PORPHYRIN METABOLISM IN NON-ALCOHOLIC FATTY LIVER DISEASE].

    Science.gov (United States)

    Krivosheev, A B; Maximov, V N; Voevoda, M I; Kuimov, A D; Kondratova, M A; Tuguleva, T A; Koval, O N; Bezrukova, A A; Bogorianova, P A; Rybina, O V

    2015-01-01

    The aim of the present work was to study the frequency of genotypes and alleles of C282Y and H63D HFE gene that may be associated with impaired porphyrin metabolism, as well as possible reasons for the formation of dysmetabolism porphyrins with NAFLD. The study involved 65 patients (52 men and 13 women) aged 21 to 69 years (mean age 48.5±1.5 years). Excretion uroporphyrin, coproporphyrin, 6-aminolevulinic acid of porphobilinogen in urine was determined by chromatography and spectrophotometry calculated total excretion of porphyrins. Allele frequencies C282Y and H63D were determined during the molecular genetic analysis of DNA using the polymerase chain reaction followed by analysis of length polymorphism restraktsionnyh fragments. Condition of carbohydrate metabolism was evaluated by the level of fasting blood glucose and standard glucose tolerance test. Diagnosis of insulin resistance was performed according to the criteria proposed by the European Group for the Study of insulin resistance (EGIR). Skill test for the C282Y mutation carriage and H63D in the HFE gene in 65 patients with non-alcoholic fatty liver disease. Disturbances in the metabolism of porphyrins were recorded in 43 (66.2%) patients. H63D and C282Y mutations were found in 18 (27.7%) patients, of whom 13 (72.2%) people with different options dismetabolism porphyrins and signs of insulin resistance. In 47 (72.3%) patients without mutations studied porphyrin metabolism disorders were detected in 30 (63.8 %), of which insulin resistance is registered only in 16 (34.0 %). Detection of mutations C282Y and H63D in the HFE gene in combination with disorders of porphyrin metabolism on the background of insulin resistance is likely to allow such patients considered as candidates for inclusion in the higher risk of formation of diabetes.

  4. Experimental study on the effect of ingested lead shot on Estuarine crocodiles: significance for Finniss River field studies

    International Nuclear Information System (INIS)

    Hammerton, K.M.

    2002-01-01

    Lead has long been recognised as a cumulative metabolic poison in humans, domestic animals and wildlife. Because of the many industrial activities that have brought about its widespread distribution, lead is ubiquitous in the environment. For example, uranium mining at the Rum Jungle site on the Finniss River, Northern Territory, resulted in contamination of river sediments with lead. Today, lead levels remain at about 250 mg kg-1 of wet river sediment within the immediate vicinity of the mine. Another potential source of lead poisoning in wildlife is the use of lead gunshot for hunting. Mortality in wild waterfowl caused by the ingestion of spent lead shot has been recognised in North America and Europe for over a century. An experimental study was undertaken to assess the above hypothesis on the effects of lead in the environs of crocodiles. The specific effects of lead intoxication arise mainly from the interaction of lead with the enzymatic processes in the haem biosynthetic pathway. One of these enzymes, δ-aminolevulinic acid dehydratase (ALAD), catalyses the condensation of two molecules of aminolevulinic acid to produce the pyrrole, porphobilinogen, the building block of the haem molecule. ALAD is a metalloenzyme requiring zinc for activity and is inhibited by lead displacing the essential zinc. This inhibition of ALAD by lead has been used as a specific biomarker for lead poisoning in fish, birds and mammals. An assay system was developed for the measurement of ALAD activity in crocodilian blood. It was found that ALAD was inhibited by up to 90% during the first week after exposure of the crocodiles to lead shot. There was an inverse correlation between BPb and ALAD activity throughout the 42 week experimental period. BPb concentrations greater than 100 mg dL-1 produced significant inhibition of ALAD. The results indicated that ALAD inhibition could be used as a specific biomarker of lead toxicity in crocodiles

  5. The effect of haem biosynthesis inhibitors and inducers on intestinal iron absorption and liver haem biosynthetic enzyme activities

    International Nuclear Information System (INIS)

    Laftah, A.H.; Simpson, R.J.; Peters, T.J.; Raja, K.B.

    2008-01-01

    The relation between haem biosynthesis and intestinal iron absorption is not well understood, we therefore investigated the effect of compounds that alter haem metabolism on duodenal iron absorption. CD1 mice were treated with either an inhibitor (succinyl acetone (SA)) or stimulator (2-allyl-2-isopropylacetamide (AIA)) of haem biosynthesis. 5-Aminolaevulinic acid (ALA) dehydratase and urinary ALA and porphobilinogen (PBG) levels, were determined. Intestinal iron absorption was assayed with in vivo and in vitro techniques. Liver hepcidin (Hamp1) and duodenal iron transporter mRNA levels were measured using RT-PCR. AIA caused increased hepatic ALA synthase (1.6-fold) and ALA dehydratase (1.4-fold, both p < 0.005) activities and increased urinary ALA and PBG excretion (2.1- and 1.4-fold, p < 0.005, p < 0.05, respectively). In vivo intestinal iron absorption was reduced to 49% of control (p < 0.005). Mice treated with SA showed decreased urinary ALA and PBG levels (75 and 55% control, both p < 0.005) and reductions in both ALA synthase and ALA dehydratase activities (77 and 56% control, p < 0.05, p < 0.005, respectively) in the liver. Liver and duodenal haem and cytochrome oxidase levels were not significantly decreased. Iron absorption was enhanced (1.26-fold, p < 0.05) and hepatic Hamp1 mRNA was reduced (53% of control, p < 0.05). In vitro duodenal iron uptake after mice were injected with SA also demonstrated an increase in Fe(III) reduction and uptake (1.27- and 1.41-fold, p < 0.01 respectively). Simultaneous injections of SA and ALA blocked the enhancing effect on iron absorption seen with SA alone. We conclude that alterations in haem biosynthesis can influence iron absorption and in particular, the intermediate ALA seems to be an inhibitor of iron absorption

  6. Melatonin modulates drug-induced acute porphyria

    Directory of Open Access Journals (Sweden)

    Sandra M. Lelli

    Full Text Available This work investigated the modulation by melatonin (Mel of the effects of the porphyrinogenic drugs 2-allyl-2-isopropylacetamide (AIA and 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-collidine (DDC on oxidative environment, glucose biosynthesis and heme pathway parameters. Administration of Mel before rat intoxication with AIA/DDC showed a clear beneficial effect in all cases. Mel induced decreases of 42% and 35% in the excretion of the hemeprecursors 5-aminolevulinic acid (ALA and porphobilinogen (PBG, respectively, and a 33% decrease in the induction of the heme regulatory enzyme 5-aminolevulinic acid-synthase (ALA-S. The activity of the glucose metabolism enzyme phosphoenolpyruvate carboxykinase (PEPCK, which had been diminished by the porphyrinogenic treatment, was restored by 45% when animals were pre-treated with Mel. Mel abolished the modest decrease in glucose 6-phospatase (G6Pase activity caused by AIA/DDC treatment. The oxidative status of lipids was attenuated by Mel treatment in homogenates by 47%, whereas no statistically significant AIA/DDC-induced increase in thiobarbituric acid reactive substances (TBARS was observed in microsomes after Mel pre-treatment. We hypothesize that Mel may be scavenging reactive species of oxygen (ROS that could be damaging lipids, PEPCK, G6Pase and ferrochelatase (FQ. Additionally, Mel administration resulted in the repression of the key enzyme ALA-S, and this could be due to an increase in glucose levels, which is known to inhibit ALA-S induction. The consequent decrease in levels of the heme precursors ALA and PBG had a beneficial effect on the drug-induced porphyria. The results obtained open the possibility of further research on the use of melatonin as a co-treatment option in acute porphyria. Keywords: Melatonin, Glucose synthesis, Heme pathway, Acute porphyria, Oxidative stress

  7. Diversification of the Primary Antibody Repertoire by AID-Mediated Gene Conversion.

    Science.gov (United States)

    Lanning, Dennis K; Knight, Katherine L

    2015-01-01

    Gene conversion, mediated by activation-induced cytidine deaminase (AID), has been found to contribute to generation of the primary antibody repertoire in several vertebrate species. Generation of the primary antibody repertoire by gene conversion of immunoglobulin (Ig) genes occurs primarily in gut-associated lymphoid tissues (GALT) and is best described in chicken and rabbit. Here, we discuss current knowledge of the mechanism of gene conversion as well as the contribution of the microbiota in promoting gene conversion of Ig genes. Finally, we propose that the antibody diversification strategy used in GALT species, such as chicken and rabbit, is conserved in a subset of human and mouse B cells.

  8. Vif Proteins from Diverse Primate Lentiviral Lineages Use the Same Binding Site in APOBEC3G

    OpenAIRE

    Letko, Michael; Silvestri, Guido; Hahn, Beatrice H.; Bibollet-Ruche, Frederick; Gokcumen, Omer; Simon, Viviana; Ooms, Marcel

    2013-01-01

    APOBEC3G (A3G) is a cytidine deaminase that restricts human immunodeficiency virus type 1 (HIV-1) and other lentiviruses. Most of these viruses encode a Vif protein that directly binds A3G and leads to its proteasomal degradation. Both Vif proteins of HIV-1 and African green monkey simian immunodeficiency virus (SIVagm) bind residue 128 of A3G. However, this position does not control the A3G degradation by Vif variants derived from HIV-2 and SIVmac, which both originated from SIV of sooty man...

  9. SRSF1-3 contributes to diversification of the immunoglobulin variable region gene by promoting accumulation of AID in the nucleus

    OpenAIRE

    Kawaguchi, Yuka; Nariki, Hiroaki; Kawamoto, Naoko; Kanehiro, Yuichi; Miyazaki, Satoshi; Suzuki, Mari; Magari, Masaki; Tokumitsu, Hiroshi; Kanayama, Naoki

    2017-01-01

     Activation-induced cytidine deaminase (AID) is essential for diversification of the Ig variable region (IgV). AID is excluded from the nucleus, where it normally functions. However, the molecular mechanisms responsible for regulating AID localization remain to be elucidated. The SR-protein splicing factor SRSF1 is a nucleocytoplasmic shuttling protein, a splicing isoform of which called SRSF1-3, has previously been shown to contribute to IgV diversification in chicken DT40 cells. In this stu...

  10. Histone H3.3 promotes IgV gene diversification by?enhancing formation of AID?accessible single?stranded DNA

    OpenAIRE

    Romanello, Marina; Schiavone, Davide; Frey, Alexander; Sale, Julian E

    2016-01-01

    Abstract Immunoglobulin diversification is driven by activation?induced deaminase (AID), which converts cytidine to uracil within the Ig variable (IgV) regions. Central to the recruitment of AID to the IgV genes are factors that regulate the generation of single?stranded DNA (ssDNA), the enzymatic substrate of AID. Here, we report that chicken DT40 cells lacking variant histone H3.3 exhibit reduced IgV sequence diversification. We show that this results from impairment of the ability of AID t...

  11. Complex interaction of deferasirox and Pythium insidiosum: iron-dependent attenuation of growth in vitro and immunotherapy-like enhancement of immune responses in vivo.

    Directory of Open Access Journals (Sweden)

    Régis A Zanette

    Full Text Available Pythium insidiosum iron acquisition mechanisms are unknown. We previously showed that the iron chelator deferasirox had weak activity in vitro and in rabbits with experimental pythiosis. Here we show that deferasirox causes damage to P. insidiosum hyphae in vitro, but that activity is diminished in the presence of exogenous iron. The tissue activity of the proinflammatory enzyme adenosine deaminase and the histological pattern observed in pythiosis lesions of rabbits treated with deferasirox were similar to the ones in animals treated with immunotherapy.

  12. Clinical significance of combined determination of the changes of the serum ADA, HA and PIIIP levels in patients with hepatitis B

    International Nuclear Information System (INIS)

    Wang Xiaoyan

    2009-01-01

    Objective: To explore the clinical significance of the changes of serum ADA, HA and PIIIP levels in patients with hepatitis B. Methods: Serum adenosine deaminase (ADA) (with biochemistry) and HA PIIIP (with RIA) levels were determined in 102 patients with hepatitis B of various stages and 35 controls. Results: Serum ADA, HA and PIIIP levels in patients with hepatitis B were significantly higher than those in controls (P<0.01). Particularly in patients with cirrhosis (n=13) (P<0.001). Conclusion: The serum ADA, HA and PIIIP levels were closely related to the diseases process of hepatitis B and were of great clinical values. (authors)

  13. Improved negative selection protocol for Plasmodium berghei in the rodent malarial model

    Directory of Open Access Journals (Sweden)

    Orr Rachael Y

    2012-03-01

    Full Text Available Abstract An improved methodology is presented here for transgenic Plasmodium berghei lines that express the negative selectable marker yFCU (a bifunctional protein that combines yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT and substitutes delivery of selection drug 5-fluorocytosine (5FC by intraperitoneal injection for administration via the drinking water of the mice. The improved methodology is shown to be as effective, less labour-intensive, reduces animal handling and animal numbers required for successful selection thereby contributing to two of the "three Rs" of animal experimentation, namely refinement and reduction.

  14. The polydeoxyadenylate tract of Alu repetitive elements is polymorphic in the human genome

    International Nuclear Information System (INIS)

    Economou, E.P.; Bergen, A.W.; Warren, A.C.; Antonarakis, S.E.

    1990-01-01

    To identify DNA polymorphisms that are abundant in the human genome and are detectable by polymerase chain reaction amplification of genomic DNA, the authors hypothesize that the polydeoxyadenylate tract of the Alu family of repetitive elements is polymorphic among human chromosomes. Analysis of the 3' ends of three specific Alu sequences showed two occurrences, one in the adenosine deaminase gene and other in the β-globin pseudogene, were polymorphic. This novel class of polymorphism, termed AluVpA [Alu variable poly(A)] may represent one of the most useful and informative group of DNA markers in the human genome

  15. Spectrum of mutations in a cohort of UK patients with ADA deficient SCID: Segregation of genotypes with specific ethnicities.

    Science.gov (United States)

    Adams, Stuart P; Wilson, Melanie; Harb, Elissar; Fairbanks, Lynette; Xu-Bayford, Jinhua; Brown, Lucie; Kearney, Laura; Madkaikar, Manisha; Bobby Gaspar, H

    2015-12-01

    Severe combined immunodeficiency (SCID) arises from a number of different genetic defects, one of the most common being mutations in the gene encoding adenosine deaminase (ADA). In the UK, ADA deficient SCID compromises approximately 20% of all known cases of SCID. We carried out a retrospective analysis of the ADA gene in 46 known ADA deficient SCID patients on whom DNA had been stored. Here, we report a high frequency of two previously reported mutations and provide a link between the mutations and patient ethnicity within our patient cohort. We also report on 9 novel mutations that have been previously unreported. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. A high proportion of ADA point mutations associated with a specific alanine-to-valine substitution.

    OpenAIRE

    Markert, M L; Norby-Slycord, C; Ward, F E

    1989-01-01

    In 15%-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The overall goal of our research has been to identify the precise molecular defects in patients with ADA-deficient SCID. In this study, we focused on a patient whom we found to have normal sized ADA mRNA by Northern analysis and an intact ADA structural gene by Southern analysis. By cloning and sequencing this patient's ADA cDNA, we found a C-to-T point mutation ...

  17. Exceptional Antibodies Produced by Successive Immunizations.

    Directory of Open Access Journals (Sweden)

    Patricia J Gearhart

    2015-12-01

    Full Text Available Antibodies stand between us and pathogens. Viruses mutate quickly to avoid detection, and antibodies mutate at similar rates to hunt them down. This death spiral is fueled by specialized proteins and error-prone polymerases that change DNA sequences. Here, we explore how B lymphocytes stay in the race by expressing activation-induced deaminase, which unleashes a tsunami of mutations in the immunoglobulin loci. This produces random DNA substitutions, followed by selection for the highest affinity antibodies. We may be able to manipulate the process to produce better antibodies by expanding the repertoire of specific B cells through successive vaccinations.

  18. Imatinib-induced pleural effusion: A case report

    Directory of Open Access Journals (Sweden)

    R Banka

    2017-01-01

    Full Text Available Imatinib is a tyrosine kinase inhibitor and has rarely been reported to cause pleural effusion. We report the case of an 88-year-old male, known case of gastrointestinal stromal tumor on treatment with imatinib, who presented with a 2-week history of cough and dyspnea. He was diagnosed to have a right-sided pleural effusion and thoracentesis of the fluid revealed an exudate with low adenosine deaminase and negative cytology. Withdrawal of the drug lead to resolution of symptoms. We report this case to highlight the side effect profile of imatinib and warn physicians regarding this potential adverse effect which may be mistaken for metastasis or infection.

  19. X-ray structure of imidazolonepropionase from Agrobacterium tumefaciens at 1.87 Å resolution

    Energy Technology Data Exchange (ETDEWEB)

    Tyagi, Rajiv; Kumaran, Desigan; Burley, Stephen K.; Swaminathan, Subramanyam (SGX); (BNL)

    2010-01-12

    Histidine degradation in Agrobacterium tumefaciens involves four enzymes, including histidase (EC 4.3.1.3), urocanase (EC 4.2.1.49), imidazolonepropionase (EC 3.5.2.7), and N-formylglutamate amidohydrolase (EC 3.5.3.8). The third enzyme of the pathway, imidazolone-propionase, a 45.6 kDa protein, catalyzes conversion of imidazolone-5-propanoate to N-forminio-L-glutamate. Initial studies of the role of imidazolonepropionase in histidine degradation were published in 1953. Subsequent publications have been limited to enzyme kinetics, crystallization, and a recently reported structure determination. The imidazolonepropionases are members of metallodepenent-hydrolases (or amidohydroase) superfamily, which includs ureases, adenosine deaminases, phosphotriesterases, dihydroorotases, allantoinases, hydantoinases, adenine and cytosine deaminases, imidazolonepropionases, aryldial-kylphosphatases, chlorohydrolases, and formylmethanofuran dehydroases. Proteins belonging to this large group share a common three-dimensional structural motif (an eightfold {alpha}/{beta} or TIM barrel) with similar active sites. Most superfamily members also share a conserved metal binding site, involving four histidine residues and one aspartic acid. Imidazolonepropionase is one of the targets selected for X-ray crystallpgrahpic structure determination by the New York Structural GenomiX Research Consortium (NYSGXRC) Target ID: 9252b to correlate the structure function relationship of poorly studied by important enzyme. Here they report the crystal structure of imidazolonepropionase from Agrobacterium tumefaciens determined at 1.87 {angstrom} resolution.

  20. Theophylline and adenosine modulate the inflammatory functions of the human neutrophil by exerting an opposing influence on the stimulus-induced increase in intracellular calcium

    International Nuclear Information System (INIS)

    Schmeichel Morley, C.J.

    1988-01-01

    Based on evidence that endogenously-produced adenosine inhibited neutrophil responses, the influence of methylxanthine bronchodilators on neutrophil responses stimulated in vitro by n-formyl-methionyl-leucyl-phenylalanine (fMLP) was examined. At concentrations between 10/sup /minus/5/ M and 10/sup /minus/4/ M, theophylline potentiated lysosomal enzyme release by 30 to 50%, superoxide anion formation by 30 to 60%, and neutrophil aggregation. Theophylline at concentrations >10/sup /minus/4/ M inhibited the same responses by >90%. Adenosine deaminase mimicked, whereas adenosine reversed the theophylline potentiation. A potential role for calcium in the modulation of the neutrophil responses by theophylline and adenosine was explored. Theophylline enhanced by >150% the fMLP-stimulated increase in cytoplasmic calcium concentration ([Ca 2+ ]/sub i/) at time points between 5 and 90 sec as measured by Fura-2. Adenosine deaminase induced a comparable enhancement, whereas 3 /times/ 10/sup /minus/7/ M adenosine and 10/sup /minus/7/ M N-ethylcarboxamideadenosine decreased the [Ca 2+ ]/sub i/ in fMLP-stimulated neutrophils. Extracellular calcium was not required for the opposing influences of theophylline and adenosine and neither compound altered fMLP-stimulated 45 Ca uptake at the early time points

  1. Elevated Serum ADA Activity as a Marker for Diagnosis and Prognosis of Visceral Leishmaniasis and Post Kala-Azar Dermal Leishmaniasis in Indian Patients

    Science.gov (United States)

    Vijayamahantesh; Amit, Ajay; Dikhit, Manas R.; Pandey, Raj K.; Singh, Kuljit; Mishra, Ritesh; Das, V. N. R; Das, Pradeep; Bimal, Sanjiva

    2016-01-01

    Serum adenosine deaminase (ADA) activity increases in diseases where cellular immunity is involved. Since cell-mediated immune responses play a paramount role in the pathogenesis and healing of the visceral leishmaniasis, therefore, the present study was undertaken to evaluate the serum ADA activity in different pathological conditions. Adenosine deaminase was determined in sera of active visceral leishmaniasis (VL) patients (n = 39), active postkala-azar dermal leishmaniasis (PKDL) cases (n = 34) at the point of diagnosis and after treatment stages along with healthy controls (n = 30), endemic healthy subjects (n = 34) and endemic asymptomatic subjects (n = 34).Our in-vitro result revealed that monocytes secrete significant ADA level in response to Leishmania donovani (L.donovani) stimulation. The serum ADA activity in active VL and PKDL subjects were found to be significantly higher than that of respective treated cases and healthy controls. We also observed a marginal number (17.6%) of endemic asymptomatic subjects showed elevated serum ADA activity. Further, the ADA activity in PKDL was found to be decreased gradually during the different phases of treatment. Interestingly, 2 out of 32 treated VL cases found to have high serum ADA activity during follow up period were relapsed within few days. These results suggest the possibility of ADA as a marker of clinical pathogenesis and can be used as a surrogate marker in the diagnosis and prognosis of VL and PKDL. PMID:27186641

  2. Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency.

    Science.gov (United States)

    Waddell, D; Ullman, B

    1983-04-10

    From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.

  3. [Features of influence adenosine, AMP and hyperadrenalinemiya on the immune status, metabolic enzymes of purine nucleotides and the antioxidant defense system].

    Science.gov (United States)

    Tapbergenov, S O; Sovetov, B S; Tapbergenov, A T

    2016-11-01

    Administration of a large dose of adrenaline (4 mg/kg 60 min before analysis) increased blood levels of total leukocytes, lymphocytes, decreased T-cell suppressors, leukocyte migration inhibition reaction (LMIR) and NBT test, but increased the level of conjugated dienes (CD). Administration of AMPand adenosine increased levels of total leukocytes, lymphocytes, T- lymphocytes, T-helpers, decreased the level of malondialdehyde (MDA), LMIR, and T-cell suppressors. Sympathetic hyperactivation induced by administration of a large dose of adrenaline (4 mg/kg 60 min before analysis) was accompanied by an increase in heart and liver activities of glutathione peroxidase (GPx), catalase, AMP deaminase (AMPD), and adenosine deaminase (AD). Administration of AMP or adenosine caused a decrease in activities of glutathione reductase (GR), GPx, catalase, a decrease in the MDA level and an increase in activities of AMPD and AD in the heart. In the liver AMP and adenosine also caused a decrease in activities of glutathione reductase (GR), GPx, a decrease in the MDA level and an increase in activities of AMPD and AD. The data obtained suggest that administration of adrenaline, AMP, and adenosine influences activity of enzymes involved in purine nucleotide metabolism. However, in contrast to adrenaline, administration of AMP or adenosine does not provoke stress reaction.

  4. The enzymatic activity of CEM15/Apobec-3G is essential for the regulation of the infectivity of HIV-1 virion but not a sole determinant of its antiviral activity.

    Science.gov (United States)

    Shindo, Keisuke; Takaori-Kondo, Akifumi; Kobayashi, Masayuki; Abudu, Aierken; Fukunaga, Keiko; Uchiyama, Takashi

    2003-11-07

    Human immunodeficiency virus, type 1 (HIV-1) Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion. Vif functions to counteract an anti-HIV-1 cellular factor in non-permissive cells, CEM15/Apobec-3G, which shares a cytidine deaminase motif. CEM15/Apobec-3G deaminates dC to dU in the minus strand DNA of HIV-1, resulting in G to A hypermutation in the plus strand DNA. In this study, we have done the mutagenesis analysis on two cytidine deaminase motifs in CEM15/Apobec-3G and examined their antiviral functions as well as the DNA editing activity. Point mutations in the C-terminal active site such as E259Q and C291A almost completely abrogated the antiviral function, while those in the N-terminal active site such as E67Q and C100A retained this activity to a lesser extent as compared with that of the wild type. The DNA editing activities of E67Q and E259Q mutants were both retained but impaired to the same extent. This indicates that the enzymatic activity of this protein is essential but not a sole determinant of the antiviral activity. Furthermore, all the deletion mutants tested in this study lost the antiviral activity because of the loss of the activity for dimerization, suggesting that the entire protein structure is necessary for the antiviral function.

  5. Enzyme catalysis by entropy without Circe effect.

    Science.gov (United States)

    Kazemi, Masoud; Himo, Fahmi; Åqvist, Johan

    2016-03-01

    Entropic effects have often been invoked to explain the extraordinary catalytic power of enzymes. In particular, the hypothesis that enzymes can use part of the substrate-binding free energy to reduce the entropic penalty associated with the subsequent chemical transformation has been very influential. The enzymatic reaction of cytidine deaminase appears to be a distinct example. Here, substrate binding is associated with a significant entropy loss that closely matches the activation entropy penalty for the uncatalyzed reaction in water, whereas the activation entropy for the rate-limiting catalytic step in the enzyme is close to zero. Herein, we report extensive computer simulations of the cytidine deaminase reaction and its temperature dependence. The energetics of the catalytic reaction is first evaluated by density functional theory calculations. These results are then used to parametrize an empirical valence bond description of the reaction, which allows efficient sampling by molecular dynamics simulations and computation of Arrhenius plots. The thermodynamic activation parameters calculated by this approach are in excellent agreement with experimental data and indeed show an activation entropy close to zero for the rate-limiting transition state. However, the origin of this effect is a change of reaction mechanism compared the uncatalyzed reaction. The enzyme operates by hydroxide ion attack, which is intrinsically associated with a favorable activation entropy. Hence, this has little to do with utilization of binding free energy to pay the entropic penalty but rather reflects how a preorganized active site can stabilize a reaction path that is not operational in solution.

  6. Phylloplane bacteria of Jatropha curcas: diversity, metabolic characteristics, and growth-promoting attributes towards vigor of maize seedling.

    Science.gov (United States)

    Dubey, Garima; Kollah, Bharati; Ahirwar, Usha; Mandal, Asit; Thakur, Jyoti Kumar; Patra, Ashok Kumar; Mohanty, Santosh Ranjan

    2017-10-01

    The complex role of phylloplane microorganisms is less understood than that of rhizospheric microorganisms in lieu of their pivotal role in plant's sustainability. This experiment aims to study the diversity of the culturable phylloplane bacteria of Jatropha curcas and evaluate their growth-promoting activities towards maize seedling vigor. Heterotrophic bacteria were isolated from the phylloplane of J. curcas and their 16S rRNA genes were sequenced. Sequences of the 16S rRNA gene were very similar to those of species belonging to the classes Bacillales (50%), Gammaproteobacteria (21.8%), Betaproteobacteria (15.6%), and Alphaproteobacteria (12.5%). The phylloplane bacteria preferred to utilize alcohol rather than monosaccharides and polysaccharides as a carbon source. Isolates exhibited ACC (1-aminocyclopropane-1-carboxylic acid) deaminase, phosphatase, potassium solubilization, and indole acetic acid (IAA) production activities. The phosphate-solubilizing capacity (mg of PO 4 solubilized by 10 8 cells) varied from 0.04 to 0.21. The IAA production potential (μg IAA produced by 10 8 cells in 48 h) of the isolates varied from 0.41 to 9.29. Inoculation of the isolates to maize seed significantly increased shoot and root lengths of maize seedlings. A linear regression model of the plant-growth-promoting activities significantly correlated (p < 0.01) with the growth parameters. Similarly, a correspondence analysis categorized ACC deaminase and IAA production as the major factors contributing 41% and 13.8% variation, respectively, to the growth of maize seedlings.

  7. Cellular radiosensitivity in human severe-combined-immunodeficiency (SCID) syndromes

    International Nuclear Information System (INIS)

    Sproston, Anthony R.M.; West, Catharine M.L.; Hendry, Jolyon H.

    1997-01-01

    Purpose: The aim of the work was to establish to what extent a variety of human severe-combined-immunodeficiency (SCID) disorders are associated with in vitro cellular hypersensitivity to ionizing radiation. Materials and methods: A study was made of fibroblast strains established from individuals with adenosine deaminase deficiency, T(-)B(-) SCID, Omenn's syndrome and a SCID heterozygote. For comparison, an assessment was also made of the radiosensitivity of a series of fibroblast strains derived from: normal donors, a patient with ataxia-telangiectasia (A-T) and an A-T heterozygote. Radiosensitivity was determined using a clonogenic assay following both high (HDR) and low (LDR) dose-rate irradiation. Results: Following HDR irradiation, the fibroblast strains derived from the different human SCID disorders displayed a wide range of radiosensitivity: the adenosine deaminase deficiency cells were similar in radiosensitivity to normal fibroblasts, T(-)B(-) cells were as hypersensitive to radiation as A-T cells and the Omenn's syndrome cells showed intermediate radiosensitivity. However, whereas all four normal cell strains studied showed significant LDR sparing, none of the SCID fibroblasts did. Conclusions: These data indicate that human SCID is variable in terms of radiosensitivity depending on the particular defect. In addition, the lack of LDR sparing of radiation-induced damage suggests the involvement of some form(s) of DNA repair defect in all the human SCID syndromes

  8. DNA Methylation Dynamics of Germinal Center B Cells Are Mediated by AID

    Directory of Open Access Journals (Sweden)

    Pilar M. Dominguez

    2015-09-01

    Full Text Available Changes in DNA methylation are required for the formation of germinal centers (GCs, but the mechanisms of such changes are poorly understood. Activation-induced cytidine deaminase (AID has been recently implicated in DNA demethylation through its deaminase activity coupled with DNA repair. We investigated the epigenetic function of AID in vivo in germinal center B cells (GCBs isolated from wild-type (WT and AID-deficient (Aicda−/− mice. We determined that the transit of B cells through the GC is associated with marked locus-specific loss of methylation and increased methylation diversity, both of which are lost in Aicda−/− animals. Differentially methylated cytosines (DMCs between GCBs and naive B cells (NBs are enriched in genes that are targeted for somatic hypermutation (SHM by AID, and these genes form networks required for B cell development and proliferation. Finally, we observed significant conservation of AID-dependent epigenetic reprogramming between mouse and human B cells.

  9. Drought-Tolerant Plant Growth-Promoting Rhizobacteria Associated with Foxtail Millet in a Semi-arid Agroecosystem and Their Potential in Alleviating Drought Stress

    Directory of Open Access Journals (Sweden)

    Xuguang Niu

    2018-01-01

    Full Text Available The application of plant growth promoting rhizobacteria (PGPR to agro-ecosystems is considered to have the potential for improving plant growth in extreme environments featured by water shortage. Herein, we isolated bacterial strains from foxtail millet (Setaria italica L., a drought-tolerant crop cultivated in semiarid regions in the northeast of China. Four isolates were initially selected for their ability to produce ACC deaminase as well as drought tolerance. The isolates were identified as Pseudomonas fluorescens, Enterobacter hormaechei, and Pseudomonas migulae on the basis of 16S rRNA sequence analysis. All of these drought-tolerant isolates were able to produce EPS (exopolysaccharide. Inoculation with these strains stimulated seed germination and seedling growth under drought stress. Pseudomonas fluorescens DR7 showed the highest level of ACC deaminase and EPS-producing activity. DR7 could efficiently colonize the root adhering soil, increased soil moisture, and enhance the root adhering soil/root tissue ratio. These results suggest drought tolerant PGPR from foxtail millet could enhance plant growth under drought stress conditions and serve as effective bioinoculants to sustain agricultural production in arid regions.

  10. Characterization of plant-growth promoting diazotrophic bacteria isolated from field grown Chinese cabbage under different fertilization conditions.

    Science.gov (United States)

    Yim, Woo-Jong; Poonguzhali, Selvaraj; Madhaiyan, Munusamy; Palaniappan, Pitchai; Siddikee, M A; Sa, Tongmin

    2009-04-01

    Diazotrophic bacteria isolated from the rhizosphere of Chinese cabbage were assessed for other plant growth promoting characteristics viz., production of IAA, ethylene, ACC deaminase, phosphate solubilization, and gnotobiotic root elongation. Their effect on inoculation to Chinese cabbage was also observed under growth chamber conditions. A total of 19 strains that showed higher nitrogenase activity identified by 16S rRNA gene sequence analysis were found to be the members of the genera Pseudomonas and Agrobacterium belonging to alpha- and gamma-Proteobacteria groups. These strains were also efficient in producing IAA and ACC deaminase though they produced low levels of ethylene and no phosphate solubilization. In addition, inoculation of selected diazotrophic bacterial strains significantly increased seedling length, dry weight, and total nitrogen when compared to uninoculated control. The colonization of crop plants by diazotrophic bacteria can be affected by many biotic and abiotic factors, and further studies are oriented towards investigating the factors that could influence the establishment of a selected bacterial community.

  11. Combined deficiency of MSH2 and Sμ region abolishes class switch recombination.

    Science.gov (United States)

    Leduc, Claire; Haddad, Dania; Laviolette-Malirat, Nathalie; Nguyen Huu, Ngoc-Sa; Khamlichi, Ahmed Amine

    2010-10-01

    Class switch recombination (CSR) is mediated by G-rich tandem repeated sequences termed switch regions. Transcription of switch regions generates single-stranded R loops that provide substrates for activation-induced cytidine deaminase. Mice deficient in MSH2 have a mild defect in CSR and analysis of their switch junctions has led to a model in which MSH2 is more critical for switch recombination events outside than within the tandem repeats. It is also known that deletion of the whole Sμ region severely impairs but does not abrogate CSR despite the lack of detectable R loops. Here, we demonstrate that deficiency of both MSH2 and the Sμ region completely abolishes CSR and that the abrogation occurs at the genomic level. This finding further supports the crucial role of MSH2 outside the tandem repeats. It also indicates that during CSR, MSH2 has access to activation-induced cytidine deaminase targets in R-loop-deficient Iμ-Cμ sequences rarely used in CSR, suggesting an MSH2-dependent DNA processing activity at the Iμ exon that may decrease with transcription elongation across the Sμ region.

  12. ADAR RNA editing below the backbone.

    Science.gov (United States)

    Keegan, Liam; Khan, Anzer; Vukic, Dragana; O'Connell, Mary

    2017-09-01

    ADAR RNA editing enzymes ( a denosine d e a minases acting on R NA) that convert adenosine bases to inosines were first identified biochemically 30 years ago. Since then, studies on ADARs in genetic model organisms, and evolutionary comparisons between them, continue to reveal a surprising range of pleiotropic biological effects of ADARs. This review focuses on Drosophila melanogaster , which has a single Adar gene encoding a homolog of vertebrate ADAR2 that site-specifically edits hundreds of transcripts to change individual codons in ion channel subunits and membrane and cytoskeletal proteins. Drosophila ADAR is involved in the control of neuronal excitability and neurodegeneration and, intriguingly, in the control of neuronal plasticity and sleep. Drosophila ADAR also interacts strongly with RNA interference, a key antiviral defense mechanism in invertebrates. Recent crystal structures of human ADAR2 deaminase domain-RNA complexes help to interpret available information on Drosophila ADAR isoforms and on the evolution of ADARs from tRNA deaminase ADAT proteins. ADAR RNA editing is a paradigm for the now rapidly expanding range of RNA modifications in mRNAs and ncRNAs. Even with recent progress, much remains to be understood about these groundbreaking ADAR RNA modification systems. © 2017 Keegan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  13. Tolerance of transgenic canola plants (Brassica napus) amended with plant growth-promoting bacteria to flooding stress at a metal-contaminated field site

    International Nuclear Information System (INIS)

    Farwell, Andrea J.; Vesely, Susanne; Nero, Vincent; Rodriguez, Hilda; McCormack, Kimberley; Shah, Saleh; Dixon, D. George; Glick, Bernard R.

    2007-01-01

    The growth of transgenic canola (Brassica napus) expressing a gene for the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase was compared to non-transformed canola exposed to flooding and elevated soil Ni concentration, in situ. In addition, the ability of the plant growth-promoting bacterium Pseudomonas putida UW4, which also expresses ACC deaminase, to facilitate the growth of non-transformed and transgenic canola under the above mentioned conditions was examined. Transgenic canola and/or canola treated with P. putida UW4 had greater shoot biomass compared to non-transformed canola under low flood-stress conditions. Under high flood-stress conditions, shoot biomass was reduced and Ni accumulation was increased in all instances relative to low flood-stress conditions. This is the first field study to document the increase in plant tolerance utilizing transgenic plants and plant growth-promoting bacteria exposed to multiple stressors. - Using transgenic plants and plant growth-promoting bacteria as phytoremediation methods increased plant tolerance at a metal-contaminated field site under low flood conditions

  14. Pegademase bovine (PEG-ADA for the treatment of infants and children with severe combined immunodeficiency (SCID

    Directory of Open Access Journals (Sweden)

    Claire Booth

    2009-06-01

    Full Text Available Claire Booth1,2, H Bobby Gaspar1,21Centre for Immunodeficiency, Molecular Immunology Unit, UCL Institute of Child Health, London, UK; 2Dept of Clinical Immunology, Great Ormond Street Hospital NHS Trust, London, UKAbstract: Adenosine deaminase deficiency (ADA is a rare, inherited disorder of purine metabolism characterized by immunodeficiency, failure to thrive and metabolic abnormalities. A lack of the enzyme ADA allows accumulation of toxic metabolites causing defects of both cell mediated and humoral immunity leading to ADA severe combined immune deficiency (SCID, a condition that can be fatal in early infancy if left untreated. Hematopoietic stem cell transplant is curative but is dependent on a good donor match. Other therapeutic options include enzyme replacement therapy (ERT with pegademase bovine (PEG-ADA and more recently gene therapy. PEG-ADA has been used in over 150 patients worldwide and has allowed stabilization of patients awaiting more definitive treatment with hematopoietic stem cell transplant. It affords both metabolic detoxification and protective immune function with patients remaining clinically well, but immune reconstitution is often suboptimal and may not be long lived. We discuss the pharmacokinetics, immune reconstitution, effects on systemic disease and side effects of treatment with PEG-ADA. We also review the long-term outcome of patients receiving ERT and discuss the role of PEG-ADA in the management of infants and children with ADA-SCID, alongside other therapeutic options.Keywords: adenosine deaminase deficiency, PEG-ADA, enzyme replacement therapy, severe combined immune deficiency (SCID

  15. Short Communication Synergistic effect of rhizobia and plant growth promoting rhizobacteria on the growth and nodulation of lentil seedlings under axenic conditions

    Directory of Open Access Journals (Sweden)

    Muhammad Zafar-ul-Hye

    2013-05-01

    Full Text Available Plant growth promoting rhizobacteria (PGPR containing ACC-deaminase in combination with rhizobia can improve the growth and nodulation in plants by suppressing the endogenous level of ethylene. In the present study, ten strains, each of PGPR and rhizobia from the previously screened cultures were tested for their effect as co-inoculants on growth and nodulation of lentil in growth pouches under axenic conditions. Results showed that most of the combinations improved the lentil growth as compared to the un-inoculated control. Maximum increase in shoot length (1.87 fold, root length (1.97 fold and total biomass (1.98 fold over the un-inoculated control was observed in the treatment where the lentil seedlings were inoculated with the combination Z24P10. Co-inoculation also improved the nodulation in lentil and the maximum number of nodules plant-1 (24 nodules were observed in the combination Z22P10. However, there was no nodulation in few combinations. It is concluded that the co-inoculation with rhizobia and PGPR containing ACC-deaminase has improved the growth and nodulation in lentil under axenic conditions and the selected combinations may be evaluated in pot and field trials

  16. Inherited variants affecting RNA editing may contribute to ovarian cancer susceptibility

    DEFF Research Database (Denmark)

    Permuth, Jennifer B; Reid, Brett; Earp, Madalene

    2016-01-01

    RNA editing in mammals is a form of post-transcriptional modification in which adenosine is converted to inosine by the adenosine deaminases acting on RNA (ADAR) family of enzymes. Based on evidence of altered ADAR expression in epithelial ovarian cancers (EOC), we hypothesized that single nucleo......, including rs1127313 (G/A), a SNP in the 3' untranslated region. In summary, germline variation involving RNA editing genes may influence EOC susceptibility, warranting further investigation of inherited and acquired alterations affecting RNA editing.......RNA editing in mammals is a form of post-transcriptional modification in which adenosine is converted to inosine by the adenosine deaminases acting on RNA (ADAR) family of enzymes. Based on evidence of altered ADAR expression in epithelial ovarian cancers (EOC), we hypothesized that single...... nucleotide polymorphisms (SNPs) in ADAR genes modify EOC susceptibility, potentially by altering ovarian tissue gene expression. Using directly genotyped and imputed data from 10,891 invasive EOC cases and 21,693 controls, we evaluated the associations of 5,303 SNPs in ADAD1, ADAR, ADAR2, ADAR3, and SND1...

  17. Investigating the ability of Pseudomonas fluorescens UW4 to reduce cadmium stress in Lactuca sativa via an intervention in the ethylene biosynthetic pathway.

    Science.gov (United States)

    Albano, Lucas J; Macfie, Sheila M

    2016-12-01

    A typical plant response to any biotic or abiotic stress, including cadmium (Cd), involves increased ethylene synthesis, which causes senescence of the affected plant part. Stressed plants can experience reduced ethylene and improved growth if they are inoculated with bacteria that have the enzyme ACC deaminase, which metabolizes the ethylene precursor ACC (1-aminocyclopropane-1-carboxylate). We investigated whether one such bacterium, Pseudomonas fluorescens UW4, reduces the production of ethylene and improves the growth of lettuce (Lactuca sativa) sown in Cd-contaminated potting material (PRO-MIX® BX). Plants were inoculated with the wild-type P. fluorescens UW4 or a mutant strain that cannot produce ACC deaminase. Cadmium-treated plants contained up to 50 times more Cd than did control plants. In noninoculated plants, Cd induced a 5-fold increase in ethylene concentration. The wild-type bacterium prevented Cd-induced reductions in root biomass but there was no relationship between Cd treatment and ethylene production in inoculated plants. In contrast, when the concentration of ethylene was plotted against the extent of bacterial colonization of the roots, increased colonization with wild-type P. fluorescens UW4 was associated with 20% less ethylene production. Ours is the first study to show that the protective effect of this bacterium is proportional to the quantity of bacteria on the root surface.

  18. Deficits in Endogenous Adenosine Formation by Ecto-5′-Nucleotidase/CD73 Impair Neuromuscular Transmission and Immune Competence in Experimental Autoimmune Myasthenia Gravis

    Directory of Open Access Journals (Sweden)

    Laura Oliveira

    2015-01-01

    Full Text Available AMP dephosphorylation via ecto-5′-nucleotidase/CD73 is the rate limiting step to generate extracellular adenosine (ADO from released adenine nucleotides. ADO, via A2A receptors (A2ARs, is a potent modulator of neuromuscular and immunological responses. The pivotal role of ecto-5′-nucleotidase/CD73, in controlling extracellular ADO formation, prompted us to investigate its role in a rat model of experimental autoimmune myasthenia gravis (EAMG. Results show that CD4+CD25+FoxP3+ regulatory T cells express lower amounts of ecto-5′-nucleotidase/CD73 as compared to controls. Reduction of endogenous ADO formation might explain why proliferation of CD4+ T cells failed upon blocking A2A receptors activation with ZM241385 or adenosine deaminase in EAMG animals. Deficits in ADO also contribute to neuromuscular transmission failure in EAMG rats. Rehabilitation of A2AR-mediated immune suppression and facilitation of transmitter release were observed by incubating the cells with the nucleoside precursor, AMP. These findings, together with the characteristic increase in serum adenosine deaminase activity of MG patients, strengthen our hypothesis that the adenosinergic pathway may be dysfunctional in EAMG. Given that endogenous ADO formation is balanced by ecto-5′-nucleotidase/CD73 activity and that A2ARs exert a dual role to restore use-dependent neurocompetence and immune suppression in myasthenics, we hypothesize that stimulation of the two mechanisms may have therapeutic potential in MG.

  19. Potential Development of Tumor-Targeted Oral Anti-Cancer Prodrugs: Amino Acid and Dipeptide Monoester Prodrugs of Gemcitabine.

    Science.gov (United States)

    Tsume, Yasuhiro; Drelich, Adam J; Smith, David E; Amidon, Gordon L

    2017-08-10

    One of the main obstacles for cancer therapies is to deliver medicines effectively to target sites. Since stroma cells are developed around tumors, chemotherapeutic agents have to go through stroma cells in order to reach tumors. As a method to improve drug delivery to the tumor site, a prodrug approach for gemcitabine was adopted. Amino acid and dipeptide monoester prodrugs of gemcitabine were synthesized and their chemical stability in buffers, resistance to thymidine phosphorylase and cytidine deaminase, antiproliferative activity, and uptake/permeability in HFF cells as a surrogate to stroma cells were determined and compared to their parent drug, gemcitabine. The activation of all gemcitabine prodrugs was faster in pancreatic cell homogenates than their hydrolysis in buffer, suggesting enzymatic action. All prodrugs exhibited great stability in HFF cell homogenate, enhanced resistance to glycosidic bond metabolism by thymidine phosphorylase, and deamination by cytidine deaminase compared to their parent drug. All gemcitabine prodrugs exhibited higher uptake in HFF cells and better permeability across HFF monolayers than gemcitabine, suggesting a better delivery to tumor sites. Cell antiproliferative assays in Panc-1 and Capan-2 pancreatic ductal cell lines indicated that the gemcitabine prodrugs were more potent than their parent drug gemcitabine. The transport and enzymatic profiles of gemcitabine prodrugs suggest their potential for delayed enzymatic bioconversion and enhanced resistance to metabolic enzymes, as well as for enhanced drug delivery to tumor sites, and cytotoxic activity in cancer cells. These attributes would facilitate the prolonged systemic circulation and improved therapeutic efficacy of gemcitabine prodrugs.

  20. Elevated Serum ADA Activity as a Marker for Diagnosis and Prognosis of Visceral Leishmaniasis and Post Kala-Azar Dermal Leishmaniasis in Indian Patients.

    Science.gov (United States)

    Vijayamahantesh; Amit, Ajay; Dikhit, Manas R; Pandey, Raj K; Singh, Kuljit; Mishra, Ritesh; Das, V N R; Das, Pradeep; Bimal, Sanjiva

    2016-01-01

    Serum adenosine deaminase (ADA) activity increases in diseases where cellular immunity is involved. Since cell-mediated immune responses play a paramount role in the pathogenesis and healing of the visceral leishmaniasis, therefore, the present study was undertaken to evaluate the serum ADA activity in different pathological conditions. Adenosine deaminase was determined in sera of active visceral leishmaniasis (VL) patients (n = 39), active postkala-azar dermal leishmaniasis (PKDL) cases (n = 34) at the point of diagnosis and after treatment stages along with healthy controls (n = 30), endemic healthy subjects (n = 34) and endemic asymptomatic subjects (n = 34).Our in-vitro result revealed that monocytes secrete significant ADA level in response to Leishmania donovani (L.donovani) stimulation. The serum ADA activity in active VL and PKDL subjects were found to be significantly higher than that of respective treated cases and healthy controls. We also observed a marginal number (17.6%) of endemic asymptomatic subjects showed elevated serum ADA activity. Further, the ADA activity in PKDL was found to be decreased gradually during the different phases of treatment. Interestingly, 2 out of 32 treated VL cases found to have high serum ADA activity during follow up period were relapsed within few days. These results suggest the possibility of ADA as a marker of clinical pathogenesis and can be used as a surrogate marker in the diagnosis and prognosis of VL and PKDL.

  1. Analysis of the ectoenzymes ADA, ALP, ENPP1, and ENPP3, in the contents of ovarian endometriomas as candidate biomarkers of endometriosis.

    Science.gov (United States)

    Trapero, Carla; Jover, Lluis; Fernández-Montolí, Maria Eulàlia; García-Tejedor, Amparo; Vidal, August; Gómez de Aranda, Inmaculada; Ponce, Jordi; Matias-Guiu, Xavier; Martín-Satué, Mireia

    2018-02-01

    The diagnosis of endometriosis, a prevalent chronic disease with a strong inflammatory component, is usually delayed due to the lack of noninvasive diagnostic tests. Purinergic signaling, a key cell pathway, is altered in many inflammatory disorders. The aim of the present work was to evaluate the levels of adenosine deaminase (ADA), alkaline phosphatase (ALP), ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and ENPP3, elements of purinergic signaling, as biomarker candidates for endometriosis. A case-control comparative study was conducted to determine ADA, ALP, ENPP1 and ENPP3 levels in echo-guided aspirated fluids of endometriomas (case group) and simple ovarian cysts (control group) using the ELISA technique. Adenosine deaminase, ALP, ENPP1, and ENPP3 were present and quantifiable in the contents of endometriomas and simple cysts. There were significant differences in ADA and ENPP1 levels in endometriomas in comparison with simple cysts (2787 U/L and 103.9 ng/mL more in endometriomas, for ADA and ENPP1, respectively). Comparisons of ALP and ENPP3 levels between the two groups did not reveal significant differences. The ectoenzymes ADA and ENPP1 are biomarker candidates for endometriosis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. AIDing Chromatin and Transcription-Coupled Orchestration of Immunoglobulin Class-Switch Recombination

    Science.gov (United States)

    Vaidyanathan, Bharat; Yen, Wei-Feng; Pucella, Joseph N.; Chaudhuri, Jayanta

    2014-01-01

    Secondary diversification of the antibody repertoire upon antigenic challenge, in the form of immunoglobulin heavy chain (IgH) class-switch recombination (CSR) endows mature, naïve B cells in peripheral lymphoid organs with a limitless ability to mount an optimal humoral immune response, thus expediting pathogen elimination. CSR replaces the default constant (CH) region exons (Cμ) of IgH with any of the downstream CH exons (Cγ, Cε, or Cα), thereby altering effector functions of the antibody molecule. This process depends on, and is orchestrated by, activation-induced deaminase (AID), a DNA cytidine deaminase that acts on single-stranded DNA exposed during transcription of switch (S) region sequences at the IgH locus. DNA lesions thus generated are processed by components of several general DNA repair pathways to drive CSR. Given that AID can instigate DNA lesions and genomic instability, stringent checks are imposed that constrain and restrict its mutagenic potential. In this review, we will discuss how AID expression and substrate specificity and activity is rigorously enforced at the transcriptional, post-transcriptional, post-translational, and epigenetic levels, and how the DNA-damage response is choreographed with precision to permit targeted activity while limiting bystander catastrophe. PMID:24734031

  3. Inducible tertiary lymphoid structures, autoimmunity, and exocrine dysfunction in a novel model of salivary gland inflammation in C57BL/6 mice.

    Science.gov (United States)

    Bombardieri, Michele; Barone, Francesca; Lucchesi, Davide; Nayar, Saba; van den Berg, Wim B; Proctor, Gordon; Buckley, Christopher D; Pitzalis, Costantino

    2012-10-01

    Salivary glands in patients with Sjögren's syndrome (SS) develop ectopic lymphoid structures (ELS) characterized by B/T cell compartmentalization, the formation of high endothelial venules, follicular dendritic cell networks, functional B cell activation with expression of activation-induced cytidine deaminase, as well as local differentiation of autoreactive plasma cells. The mechanisms that trigger ELS formation, autoimmunity, and exocrine dysfunction in SS are largely unknown. In this article, we present a novel model of inducible ectopic lymphoid tissue formation, breach of humoral self-tolerance, and salivary hypofunction after delivery of a replication-deficient adenovirus-5 in submandibular glands of C57BL/6 mice through retrograde excretory duct cannulation. In this model, inflammation rapidly and consistently evolves from diffuse infiltration toward the development of SS-like periductal lymphoid aggregates within 2 wk from AdV delivery. These infiltrates progressively acquire ELS features and support functional GL7(+)/activation-induced cytidine deaminase(+) germinal centers. Formation of ELS is preceded by ectopic expression of lymphoid chemokines CXCL13, CCL19, and lymphotoxin-β, and is associated with development of anti-nuclear Abs in up to 75% of mice. Finally, reduction in salivary flow was observed over 3 wk post-AdV infection, consistent with exocrine gland dysfunction as a consequence of the inflammatory response. This novel model has the potential to unravel the cellular and molecular mechanisms that regulate ELS formation and their role in exocrine dysfunction and autoimmunity in SS.

  4. Structural studies of substrate and product complexes of 5-aminolaevulinic acid dehydratase from humans, Escherichia coli and the hyperthermophile Pyrobaculum calidifontis.

    Science.gov (United States)

    Mills-Davies, N; Butler, D; Norton, E; Thompson, D; Sarwar, M; Guo, J; Gill, R; Azim, N; Coker, A; Wood, S P; Erskine, P T; Coates, L; Cooper, J B; Rashid, N; Akhtar, M; Shoolingin-Jordan, P M

    2017-01-01

    A number of X-ray analyses of an enzyme involved in a key early stage of tetrapyrrole biosynthesis are reported. Two structures of human 5-aminolaevulinate dehydratase (ALAD), native and recombinant, have been determined at 2.8 Å resolution, showing that the enzyme adopts an octameric quaternary structure in accord with previously published analyses of the enzyme from a range of other species. However, this is in contrast to the finding that a disease-related F12L mutant of the human enzyme uniquely forms hexamers [Breinig et al. (2003), Nature Struct. Biol. 10, 757-763]. Monomers of all ALADs adopt the TIM-barrel fold; the subunit conformation that assembles into the octamer includes the N-terminal tail of one monomer curled around the (α/β) 8 barrel of a neighbouring monomer. Both crystal forms of the human enzyme possess two monomers per asymmetric unit, termed A and B. In the native enzyme there are a number of distinct structural differences between the A and B monomers, with the latter exhibiting greater disorder in a number of loop regions and in the active site. In contrast, the second monomer of the recombinant enzyme appears to be better defined and the active site of both monomers clearly possesses a zinc ion which is bound by three conserved cysteine residues. In native human ALAD, the A monomer also has a ligand resembling the substrate ALA which is covalently bound by a Schiff base to one of the active-site lysines (Lys252) and is held in place by an ordered active-site loop. In contrast, these features of the active-site structure are disordered or absent in the B subunit of the native human enzyme. The octameric structure of the zinc-dependent ALAD from the hyperthermophile Pyrobaculum calidifontis is also reported at a somewhat lower resolution of 3.5 Å. Finally, the details are presented of a high-resolution structure of the Escherichia coli ALAD enzyme co-crystallized with a noncovalently bound moiety of the product, porphobilinogen (PBG

  5. Involvement of A1 adenosine receptors and neural pathways in adenosine-induced bronchoconstriction in mice.

    Science.gov (United States)

    Hua, Xiaoyang; Erikson, Christopher J; Chason, Kelly D; Rosebrock, Craig N; Deshpande, Deepak A; Penn, Raymond B; Tilley, Stephen L

    2007-07-01

    High levels of adenosine can be measured from the lungs of asthmatics, and it is well recognized that aerosolized 5'AMP, the precursor of adenosine, elicits robust bronchoconstriction in patients with this disease. Characterization of mice with elevated adenosine levels secondary to the loss of adenosine deaminase (ADA) expression, the primary metabolic enzyme for adenosine, further support a role for this ubiquitous mediator in the pathogenesis of asthma. To begin to identify pathways by which adenosine can alter airway tone, we examined adenosine-induced bronchoconstriction in four mouse lines, each lacking one of the receptors for this nucleoside. We show, using direct measures of airway mechanics, that adenosine can increase airway resistance and that this increase in resistance is mediated by binding the A(1) receptor. Further examination of this response using pharmacologically, surgically, and genetically manipulated mice supports a model in which adenosine-induced bronchoconstriction occurs indirectly through the activation of sensory neurons.

  6. Bystander or No Bystander for Gene Directed Enzyme Prodrug Therapy

    Directory of Open Access Journals (Sweden)

    Adam V. Patterson

    2009-11-01

    Full Text Available Gene directed enzyme prodrug therapy (GDEPT of cancer aims to improve the selectivity of chemotherapy by gene transfer, thus enabling target cells to convert nontoxic prodrugs to cytotoxic drugs. A zone of cell kill around gene-modified cells due to transfer of toxic metabolites, known as the bystander effect, leads to tumour regression. Here we discuss the implications of either striving for a strong bystander effect to overcome poor gene transfer, or avoiding the bystander effect to reduce potential systemic effects, with the aid of three successful GDEPT systems. This review concentrates on bystander effects and drug development with regard to these enzyme prodrug combinations, namely herpes simplex virus thymidine kinase (HSV-TK with ganciclovir (GCV, cytosine deaminase (CD from bacteria or yeast with 5-fluorocytodine (5-FC, and bacterial nitroreductase (NfsB with 5-(azaridin-1-yl-2,4-dinitrobenzamide (CB1954, and their respective derivatives.

  7. Enterobacter asburiae KUNi5, a Nickel Resistant Bacterium for Possible Bioremediation of Nickel Contaminated Sites.

    Science.gov (United States)

    Paul, Anirudha; Mukherjee, Samir Kumar

    2016-01-01

    Nickel resistant bacterial strain Enterobacter asburiae KUNi5 was isolated and showed resistance up to 15 mM and could remove Ni optimally better at 37 degrees C and pH 7. Maximum removal was found at initial concentration of 0.5 to 2 mM, however, growth and Ni removal were affected by other heavy metals. Major amount of the metal was accumulated in the membrane fractions and certain negatively charged groups were found responsible for Ni binding. KUNi5 could also produce 1-aminocyclopropane-1-carboxylate deaminase, indole-acetic acid and siderophore. It seems that KUNi5 could be a possible candidate for Ni detoxification and plant growth promotion in Ni-contaminated field.

  8. Tumor targeted gene therapy

    International Nuclear Information System (INIS)

    Kang, Joo Hyun

    2006-01-01

    Knowledge of molecular mechanisms governing malignant transformation brings new opportunities for therapeutic intervention against cancer using novel approaches. One of them is gene therapy based on the transfer of genetic material to an organism with the aim of correcting a disease. The application of gene therapy to the cancer treatment had led to the development of new experimental approaches such as suicidal gene therapy, inhibition of oncogenes and restoration of tumor-suppressor genes. Suicidal gene therapy is based on the expression in tumor cells of a gene encoding an enzyme that converts a prodrug into a toxic product. Representative suicidal genes are Herpes simplex virus type 1 thymidine kinase (HSV1-tk) and cytosine deaminase (CD). Especially, physicians and scientists of nuclear medicine field take an interest in suicidal gene therapy because they can monitor the location and magnitude, and duration of expression of HSV1-tk and CD by PET scanner

  9. Antibody Affinity Maturation in Fishes—Our Current Understanding

    Directory of Open Access Journals (Sweden)

    Brad G. Magor

    2015-07-01

    Full Text Available It has long been believed that fish lack antibody affinity maturation, in part because they were thought to lack germinal centers. Recent research done on sharks and bony fishes indicates that these early vertebrates are able to affinity mature their antibodies. This article reviews the functionality of the fish homologue of the immunoglobulin (Ig mutator enzyme activation-induced cytidine deaminase (AID. We also consider the protein and molecular evidence for Ig somatic hypermutation and antibody affinity maturation. In the context of recent evidence for a putative proto-germinal center in fishes we propose some possible reasons that observed affinity maturation in fishes often seems lacking and propose future work that might shed further light on this process in fishes.

  10. An unusual case of pseudochylothorax

    Directory of Open Access Journals (Sweden)

    M Padma Priya

    2015-01-01

    Full Text Available A 25-year-old male patient presented with right-sided pleuritic chest pain and pain in the ankle. Radiological investigations revealed a right sided pleural effusion, lytic lesion in spine D10 with paravertebral abscess. Pleural fluid analysis showed elevated lactate dehydrogenase, adenosine deaminase, increased triglycerides, cholesterol, and no chylomicrons. Hence, a diagnosis of pseudochylothorax secondary to tuberculosis was made. Pleural fluid was drained by tube thoracostomy, decortication was done to improve the lung function and patient was started on anti-tuberculosis treatment (ATT. Patient improved with ATT. Pseudochylous effusion or chyliform effusions are uncommon. <200 cases has been reported in the international literature. The possibility of tuberculosis has to be considered in diagnosis and treatment of such cases. Here, we present a case of tuberculous pseudochylous effusion.

  11. AMPD2 regulates GTP synthesis and is mutated in a potentially treatable neurodegenerative brainstem disorder.

    Science.gov (United States)

    Akizu, Naiara; Cantagrel, Vincent; Schroth, Jana; Cai, Na; Vaux, Keith; McCloskey, Douglas; Naviaux, Robert K; Van Vleet, Jeremy; Fenstermaker, Ali G; Silhavy, Jennifer L; Scheliga, Judith S; Toyama, Keiko; Morisaki, Hiroko; Sonmez, Fatma M; Celep, Figen; Oraby, Azza; Zaki, Maha S; Al-Baradie, Raidah; Faqeih, Eissa A; Saleh, Mohammed A M; Spencer, Emily; Rosti, Rasim Ozgur; Scott, Eric; Nickerson, Elizabeth; Gabriel, Stacey; Morisaki, Takayuki; Holmes, Edward W; Gleeson, Joseph G

    2013-08-01

    Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acid synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH) due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a potentially treatable early-onset neurodegenerative disease. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. AMPD2 Regulates GTP Synthesis and is Mutated in a Potentially-Treatable Neurodegenerative Brainstem Disorder

    Science.gov (United States)

    Akizu, Naiara; Cantagrel, Vincent; Schroth, Jana; Cai, Na; Vaux, Keith; McCloskey, Douglas; Naviaux, Robert K.; Vleet, Jeremy Van; Fenstermaker, Ali G.; Silhavy, Jennifer L.; Scheliga, Judith S.; Toyama, Keiko; Morisaki, Hiroko; Sonmez, Fatma Mujgan; Celep, Figen; Oraby, Azza; Zaki, Maha S.; Al-Baradie, Raidah; Faqeih, Eissa; Saleh, Mohammad; Spencer, Emily; Rosti, Rasim Ozgur; Scott, Eric; Nickerson, Elizabeth; Gabriel, Stacey; Morisaki, Takayuki; Holmes, Edward W.; Gleeson, Joseph G.

    2013-01-01

    Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acids synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a new distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH), due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a new, potentially treatable early-onset neurodegenerative disease. PMID:23911318

  13. Effect of manganese on neonatal rat: manganese concentration and enzymatic alterations in brain

    Energy Technology Data Exchange (ETDEWEB)

    Seth, P K; Husain, R; Mushtaq, M; Chandra, S V

    1977-01-01

    Suckling rats were exposed for 15 and 30 days to manganese through the milk of nursing dams receiving 15 mg MnCl/sub 2/.4H/sub 2/O/kg/day orally and after which the neurological manifestations of metal poisoning were studied. No significant differences in the growth rate, developmental landmarks and walking movements were observed between the control and manganese-exposed pups. The metal concentration was significantly increased in the brain of manganese-fed pups at 15 days and exhibited a further three-fold increase over the control, at 30 days. The accumulation of the metal in the brain of manganese-exposed nursing dams was comparatively much less. A significant decrease in succinic dehydrogenase, adenosine triphosphatase, adenosine deaminase, acetylcholine esterase and an increase in monoamine oxidase activity was observed in the brain of experimental pups and dams. The results suggest that the developing brain may also be susceptible to manganese.

  14. Sustained Elevated Adenosine via ADORA2B Promotes Chronic Pain through Neuro-immune Interaction

    Directory of Open Access Journals (Sweden)

    Xia Hu

    2016-06-01

    Full Text Available The molecular mechanisms of chronic pain are poorly understood and effective mechanism-based treatments are lacking. Here, we report that mice lacking adenosine deaminase (ADA, an enzyme necessary for the breakdown of adenosine, displayed unexpected chronic mechanical and thermal hypersensitivity due to sustained elevated circulating adenosine. Extending from Ada−/− mice, we further discovered that prolonged elevated adenosine contributed to chronic pain behaviors in two additional independent animal models: sickle cell disease mice, a model of severe pain with limited treatment, and complete Freund’s adjuvant paw-injected mice, a well-accepted inflammatory model of chronic pain. Mechanistically, we revealed that activation of adenosine A2B receptors on myeloid cells caused nociceptor hyperexcitability and promoted chronic pain via soluble IL-6 receptor trans-signaling, and our findings determined that prolonged accumulated circulating adenosine contributes to chronic pain by promoting immune-neuronal interaction and revealed multiple therapeutic targets.

  15. The 'Starry Sky' in Tuberculoma

    Directory of Open Access Journals (Sweden)

    Shinjan Patra, Partha Pratim Chakraborty, Gouranga Santra

    2017-12-01

    Full Text Available A 27 year old lady was admitted with high-grade fever, severe headache along with dry-cough and she was being treated for neurocysticercosis on basis of ‘starry sky’ pattern on her magnetic resonance imaging (MRI of brain. She was found to have hemorrhagic and exudative pleural effusion (Light’s criteria with lymphocytic pleocytosis, adenosine deaminase (ADA value was 105.2 U/L, cartridge based nucleic acid amplification test (CBNAAT for tubercular bacilli from fluid came positive and Mantoux test was positive also. With a strong suspicion of tuberculosis the patient was put on anti-tubercular therapy along with anticonvulsants and oral steroid. Interestingly she showed remarkable permanent improvement.

  16. Pleurisy in tuberculosis and HIV-infected patients

    Directory of Open Access Journals (Sweden)

    A. K. Ivanov

    2014-01-01

    Full Text Available A clinical and epidemiological study for 14 years was conducted. Among TB patients, the percentage of persons with mixed infection (TB+HIV infection increased during the observation period from 10 up to 64%. About one third of them had a pleura reaction with an accumulation of fluid between pleura’s petals. Pleuritis in patients with mixed infection were characterized by special features: pleurisy complicated another form of tuberculosis more often, in one-third of patients (29,8% pleural liquid had hemorrhagic type, Mycobacterium tuberculosis in the pleural fluid was detected six times more often. The level of activity of adenosine deaminase and neopterin in the exudate of patients with tuberculosis and HIV infection remained significantly higher than in the control group of persons. These data can be useful in the diagnostics of specific diseases in HIV-infected patients.

  17. Differential programming of B cells in AID deficient mice.

    Directory of Open Access Journals (Sweden)

    Marc A Hogenbirk

    Full Text Available The Aicda locus encodes the activation induced cytidine deaminase (AID and is highly expressed in germinal center (GC B cells to initiate somatic hypermutation (SHM and class switch recombination (CSR of immunoglobulin (Ig genes. Besides these Ig specific activities in B cells, AID has been implicated in active DNA demethylation in non-B cell systems. We here determined a potential role of AID as an epigenetic eraser and transcriptional regulator in B cells. RNA-Seq on different B cell subsets revealed that Aicda(-/- B cells are developmentally affected. However as shown by RNA-Seq, MethylCap-Seq, and SNP analysis these transcriptome alterations may not relate to AID, but alternatively to a CBA mouse strain derived region around the targeted Aicda locus. These unexpected confounding parameters provide alternative, AID-independent interpretations on genotype-phenotype correlations previously reported in numerous studies on AID using the Aicda(-/- mouse strain.

  18. Visualization of the Differential Transition State Stabilization within the Active Site Environment

    Directory of Open Access Journals (Sweden)

    Jerzy Leszczynski

    2004-05-01

    Full Text Available Abstract: Increasing interest in the enzymatic reaction mechanisms and in the nature of catalytic effects in enzymes causes the need of appropriate visualization methods. A new interactive method to investigate catalytic effects using differential transition state stabilization approach (DTSS [1, 2] is presented. The catalytic properties of the active site of cytidine deaminase (E.C. 3.5.4.5 is visualized in the form of differential electrostatic properties. The visualization was implemented using scripting interface of VMD [3]. Cumulative Atomic Multipole Moments (CAMM [4,5,6] were utilized for efficient yet accurate evaluation of the electrostatic properties. The implementation is efficient enough for interactive presentation of catalytic effects in the active site of the enzyme due to transition state or substrate movement. This system of visualization of DTTS approach can be potentially used to validate hypotheses regarding the catalytic mechanism or to study binding properties of transition state analogues.

  19. Plant growth-promoting traits of yeasts isolated from the phyllosphere and rhizosphere of Drosera spatulata Lab.

    Science.gov (United States)

    Fu, Shih-Feng; Sun, Pei-Feng; Lu, Hsueh-Yu; Wei, Jyuan-Yu; Xiao, Hong-Su; Fang, Wei-Ta; Cheng, Bai-You; Chou, Jui-Yu

    2016-03-01

    Microorganisms can promote plant growth through direct and indirect mechanisms. Compared with the use of bacteria and mycorrhizal fungi, the use of yeasts as plant growth-promoting (PGP) agents has not been extensively investigated. In this study, yeast isolates from the phyllosphere and rhizosphere of the medicinally important plant Drosera spatulata Lab. were assessed for their PGP traits. All isolates were tested for indole-3-acetic acid-, ammonia-, and polyamine-producing abilities, calcium phosphate and zinc oxide solubilizing ability, and catalase activity. Furthermore, the activities of siderophore, 1-aminocyclopropane-1-carboxylate deaminase, and fungal cell wall-degrading enzymes were assessed. The antagonistic action of yeasts against pathogenic Glomerella cingulata was evaluated. The cocultivation of Nicotiana benthamiana with yeast isolates enhanced plant growth, indicating a potential yeast-plant interaction. Our study results highlight the potential use of yeasts as plant biofertilizers under controlled and field conditions. Copyright © 2016 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  20. Functional requirements of AID's higher order structures and their interaction with RNA-binding proteins.

    Science.gov (United States)

    Mondal, Samiran; Begum, Nasim A; Hu, Wenjun; Honjo, Tasuku

    2016-03-15

    Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID's structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.

  1. Advances of gene therapy for primary immunodeficiencies [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Fabio Candotti

    2016-03-01

    Full Text Available In the recent past, the gene therapy field has witnessed a remarkable series of successes, many of which have involved primary immunodeficiency diseases, such as X-linked severe combined immunodeficiency, adenosine deaminase deficiency, chronic granulomatous disease, and Wiskott-Aldrich syndrome. While such progress has widened the choice of therapeutic options in some specific cases of primary immunodeficiency, much remains to be done to extend the geographical availability of such an advanced approach and to increase the number of diseases that can be targeted. At the same time, emerging technologies are stimulating intensive investigations that may lead to the application of precise genetic editing as the next form of gene therapy for these and other human genetic diseases.

  2. Dicty_cDB: Contig-U05760-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 3 2 ( BD411378 ) Method for determining a risk of developing diabe... 44 3.9 3 ( DQ117977 ) Biomphalaria gla... gigantea 12,15,18h embr... 46 1.2 1 ( EJ531809 ) 1092955234593 Global-Ocean-Sampling_GS-29-01-01-1... 34 3....10 A87181( A87181 ) cytidine deaminase [imported] - Mycobacterium lep... 67 2e-10 CP000048_594( CP000048 |pid:none) Borreli...000852 ) Caldivirga maquilingensis IC-167, complete genome. 50 0.079 1 ( CP000771 ) Fervidobacterium nodosum Rt17-B1, compl...1-557B13 (Roswell Park Canc... 44 4.8 1 ( AC114772 ) Homo sapiens BAC clone RP11-427H3 from 2, complet... 44

  3. 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.

  4. Natural resistance to HIV infection: The Vif-APOBEC interaction.

    Science.gov (United States)

    Malim, Michael H

    2006-11-01

    Members of the APOBEC family of cellular polynucleotide cytidine deaminases (e.g., APOBEC3G) are potent inhibitors of HIV infection. Wild type viral infections are largely spared from APOBEC function through the action of the viral Vif protein. In Vif's absence, inhibitory APOBEC proteins are encapsidated by budding virus particles leading to excessive cytidine (C) to uridine (U) hypermutation of negative sense reverse transcripts in newly infected cells. This registers as guanosine (G) to adenosine (A) mutations in plus stranded cDNA. Because the functions of Vif and APOBEC proteins oppose each other, it is likely that fluctuations in the Vif/APOBEC balance can influence the natural history of HIV infection. Experimental support for this notion would further justify and stimulate drug discovery initiatives in this area.

  5. Interactive effect of biochar and plant growth-promoting bacterial endophytes on ameliorating salinity stress in maize

    DEFF Research Database (Denmark)

    Saleem Akhtar, Saqib; Andersen, Mathias Neumann; Naveed, Muhammad

    2015-01-01

    The objective of this work was to study the interactive effect of biochar and plant growth-promoting endophytic bacteria containing 1-aminocyclopropane-1-carboxylate deaminase and exopolysaccharide activity on mitigating salinity stress in maize (Zea mays L.). The plants were grown in a greenhouse...... under controlled conditions, and were subjected to separate or combined treatments of biochar (0% and 5%, w/w) and two endophytic bacterial strains (Burkholderia phytofirmans (PsJN) and Enterobacter sp. (FD17)) and salinity stress. The results indicated that salinity significantly decreased the growth...... of maize, whereas both biochar and inoculation mitigated the negative effects of salinity on maize performance either by decreasing the xylem Na+ concentration ([Na+]xylem) uptake or by maintaining nutrient balance within the plant, especially when the two treatments were applied in combination. Moreover...

  6. RNA-DNA Differences Are Generated in Human Cells within Seconds after RNA Exits Polymerase II

    Directory of Open Access Journals (Sweden)

    Isabel X. Wang

    2014-03-01

    Full Text Available RNA sequences are expected to be identical to their corresponding DNA sequences. Here, we found all 12 types of RNA-DNA sequence differences (RDDs in nascent RNA. Our results show that RDDs begin to occur in RNA chains ∼55 nt from the RNA polymerase II (Pol II active site. These RDDs occur so soon after transcription that they are incompatible with known deaminase-mediated RNA-editing mechanisms. Moreover, the 55 nt delay in appearance indicates that they do not arise during RNA synthesis by Pol II or as a direct consequence of modified base incorporation. Preliminary data suggest that RDD and R-loop formations may be coupled. These findings identify sequence substitution as an early step in cotranscriptional RNA processing.

  7. Gene Therapy in Cardiac Arrhythmias

    Directory of Open Access Journals (Sweden)

    Praveen S.V

    2006-04-01

    Full Text Available Gene therapy has progressed from a dream to a bedside reality in quite a few human diseases. From its first application in adenosine deaminase deficiency, through the years, its application has evolved to vascular angiogenesis and cardiac arrhythmias. Gene based biological pacemakers using viral vectors or mesenchymal cells tested in animal models hold much promise. Induction of pacemaker activity within the left bundle branch can provide stable heart rates. Genetic modification of the AV node mimicking beta blockade can be therapeutic in the management of atrial fibrillation. G protein overexpression to modify the AV node also is experimental. Modification and expression of potassium channel genes altering the delayed rectifier potassium currents may permit better management of congenital long QT syndromes. Arrhythmias in a failing heart are due to abnormal calcium cycling. Potential targets for genetic modulation include the sarcoplasmic reticulum calcium pump, calsequestrin and sodium calcium exchanger.Lastly the ethical concerns need to be addressed.

  8. A role for PCNA ubiquitination in immunoglobulin hypermutation.

    Directory of Open Access Journals (Sweden)

    Hiroshi Arakawa

    2006-11-01

    Full Text Available Proliferating cell nuclear antigen (PCNA is a DNA polymerase cofactor and regulator of replication-linked functions. Upon DNA damage, yeast and vertebrate PCNA is modified at the conserved lysine K164 by ubiquitin, which mediates error-prone replication across lesions via translesion polymerases. We investigated the role of PCNA ubiquitination in variants of the DT40 B cell line that are mutant in K164 of PCNA or in Rad18, which is involved in PCNA ubiquitination. Remarkably, the PCNA(K164R mutation not only renders cells sensitive to DNA-damaging agents, but also strongly reduces activation induced deaminase-dependent single-nucleotide substitutions in the immunoglobulin light-chain locus. This is the first evidence, to our knowledge, that vertebrates exploit the PCNA-ubiquitin pathway for immunoglobulin hypermutation, most likely through the recruitment of error-prone DNA polymerases.

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

    International Nuclear Information System (INIS)

    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.

    2004-01-01

    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

  10. Re-editing the paradigm of Cytidine (C) to Uridine (U) RNA editing.

    Science.gov (United States)

    Fossat, Nicolas; Tam, Patrick P L

    2014-01-01

    Cytidine (C) to Uridine (U) RNA editing is a post-trancriptional modification that until recently was known to only affect Apolipoprotein b (Apob) RNA and minimally require 2 components of the C to U editosome, the deaminase APOBEC1 and the RNA-binding protein A1CF. Our latest work has identified a novel RNA-binding protein, RBM47, as a core component of the editosome, which can substitute A1CF for the editing of ApoB mRNA. In addition, new RNA species that are subjected to C to U editing have been identified. Here, we highlight these recent discoveries and discuss how they change our view of the composition of the C to U editing machinery and expand our knowledge of the functional attributes of C to U RNA editing.

  11. C to U RNA editing mediated by APOBEC1 requires RNA-binding protein RBM47.

    Science.gov (United States)

    Fossat, Nicolas; Tourle, Karin; Radziewic, Tania; Barratt, Kristen; Liebhold, Doreen; Studdert, Joshua B; Power, Melinda; Jones, Vanessa; Loebel, David A F; Tam, Patrick P L

    2014-08-01

    Cytidine (C) to Uridine (U) RNA editing is a post-transcriptional modification that is accomplished by the deaminase APOBEC1 and its partnership with the RNA-binding protein A1CF. We identify and characterise here a novel RNA-binding protein, RBM47, that interacts with APOBEC1 and A1CF and is expressed in tissues where C to U RNA editing occurs. RBM47 can substitute for A1CF and is necessary and sufficient for APOBEC1-mediated editing in vitro. Editing is further impaired in Rbm47-deficient mutant mice. These findings suggest that RBM47 and APOBEC1 constitute the basic machinery for C to U RNA editing. © 2014 The Authors.

  12. Higher capecitabine AUC in elderly patients with advanced colorectal cancer (SWOGS0030).

    Science.gov (United States)

    Louie, S G; Ely, B; Lenz, H-J; Albain, K S; Gotay, C; Coleman, D; Raghavan, D; Shields, A F; Gold, P J; Blanke, C D

    2013-10-01

    The aging process is accompanied by physiological changes including reduced glomerular filtration and hepatic function, as well as changes in gastric secretions. To investigate what effect would aging have on the disposition of capecitabine and its metabolites, the pharmacokinetics between patients ≥70 years and AUC) was accompanied by reduction in capecitabine clearance in ≥70 years patients (PAUCs between the two age groups, suggesting that carboxylesterase and cytidine deaminase (CDA) activity was similar between the two age groups. These results suggest that metabolic enzymes involved in converting capecitabine metabolites are not altered by age. An elevation in capecitabine Cmax and reduction in clearance was seen in females, where capecitabine AUC was 40.3% higher in women. Elevation of DFUR Cmax (45%) and AUC (46%) (PAUC was observed in patients ≥70 years when compared with younger patients who were >60 years.

  13. Establishment of an in vitro system representing the chicken gut-associated lymphoid tissue.

    Science.gov (United States)

    Alitheen, Noorjahan Banu; McClure, Susan Jane; Yeap, Swee Keong; Kristeen-Teo, Ye Wen; Tan, Sheau Wei; McCullagh, Peter

    2012-01-01

    The bursa of Fabricius is critical for B cell development and differentiation in chick embryos. This study describes the production in vitro, from dissociated cell suspensions, of cellular agglomerates with functional similarities to the chicken bursa. Co-cultivation of epithelial and lymphoid cells obtained from embryos at the appropriate developmental stage regularly led to agglomerate formation within 48 hours. These agglomerates resembled bursal tissue in having lymphoid clusters overlaid by well organized epithelium. Whereas lymphocytes within agglomerates were predominantly Bu-1a(+), a majority of those emigrating onto the supporting membrane were Bu-1a(-) and IgM(+). Both agglomerates and emigrant cells expressed activation-induced deaminase with levels increasing after 24 hours. Emigrating cells were actively proliferating at a rate in excess of both the starting cell population and the population of cells remaining in agglomerates. The potential usefulness of this system for investigating the response of bursal tissue to avian Newcastle disease virus (strain AF2240) was examined.

  14. The AID-induced DNA damage response in chromatin

    DEFF Research Database (Denmark)

    Daniel, Jeremy A; Nussenzweig, André

    2013-01-01

    Chemical modifications to the DNA and histone protein components of chromatin can modulate gene expression and genome stability. Understanding the physiological impact of changes in chromatin structure remains an important question in biology. As one example, in order to generate antibody diversity...... with somatic hypermutation and class switch recombination, chromatin must be made accessible for activation-induced cytidine deaminase (AID)-mediated deamination of cytosines in DNA. These lesions are recognized and removed by various DNA repair pathways but, if not handled properly, can lead to formation...... of oncogenic chromosomal translocations. In this review, we focus the discussion on how chromatin-modifying activities and -binding proteins contribute to the native chromatin environment in which AID-induced DNA damage is targeted and repaired. Outstanding questions remain regarding the direct roles...

  15. Intramolecular epistasis and the evolution of a new enzymatic function.

    Directory of Open Access Journals (Sweden)

    Sajid Noor

    Full Text Available Atrazine chlorohydrolase (AtzA and its close relative melamine deaminase (TriA differ by just nine amino acid substitutions but have distinct catalytic activities. Together, they offer an informative model system to study the molecular processes that underpin the emergence of new enzymatic function. Here we have constructed the potential evolutionary trajectories between AtzA and TriA, and characterized the catalytic activities and biophysical properties of the intermediates along those trajectories. The order in which the nine amino acid substitutions that separate the enzymes could be introduced to either enzyme, while maintaining significant catalytic activity, was dictated by epistatic interactions, principally between three amino acids within the active site: namely, S331C, N328D and F84L. The mechanistic basis for the epistatic relationships is consistent with a model for the catalytic mechanisms in which protonation is required for hydrolysis of melamine, but not atrazine.

  16. Comprehensive analysis of RNA-Seq data reveals extensive RNA editing in a human transcriptome

    DEFF Research Database (Denmark)

    Peng, Zhiyu; Cheng, Yanbing; Tan, Bertrand Chin-Ming

    2012-01-01

    a computational pipeline that carefully controls for false positives while calling RNA editing events from genome and whole-transcriptome data of the same individual. We identified 22,688 RNA editing events in noncoding genes and introns, untranslated regions and coding sequences of protein-coding genes. Most......RNA editing is a post-transcriptional event that recodes hereditary information. Here we describe a comprehensive profile of the RNA editome of a male Han Chinese individual based on analysis of ∼767 million sequencing reads from poly(A)(+), poly(A)(-) and small RNA samples. We developed...... changes (∼93%) converted A to I(G), consistent with known editing mechanisms based on adenosine deaminase acting on RNA (ADAR). We also found evidence of other types of nucleotide changes; however, these were validated at lower rates. We found 44 editing sites in microRNAs (miRNAs), suggesting a potential...

  17. Mimicking a New 2-Phenylethanol Production Pathway from Proteus mirabilis JN458 in Escherichia coli.

    Science.gov (United States)

    Liu, Jinbin; Jiang, Jing; Bai, Yajun; Fan, Tai-Ping; Zhao, Ye; Zheng, Xiaohui; Cai, Yujie

    2018-04-04

    Bacteria rarely produce natural 2-phenylethanol. We verified a new pathway from Proteus mirabilis JN458 to produce 2-phenylethanol using Escherichia coli to coexpress l-amino acid deaminase, α-keto acid decarboxylase, and alcohol dehydrogenase from P. mirabilis. Based on this pathway, a glucose dehydrogenase coenzyme regeneration system was constructed. The optimal conditions of biotransformation by the recombinant strain E-pAEAKaG were at 40 °C and pH 7.0. Finally, the recombinant strain E-pAEAKaG produced 3.21 ± 0.10 g/L 2-phenylethanol in M9 medium containing 10 g/L l-phenylalanine after a 16 h transformation. Furthermore, when the concentration of l-phenylalanine was 4 g/L (24 mM), the production of 2-phenylethanol reached 2.88 ± 0.18 g/L and displayed a higher conversion rate of 97.38 mol %.

  18. CSF ADA Determination in Early Diagnosis of Tuberculous Meningitis in HIV-Infected Patients.

    Science.gov (United States)

    Ghosh, Gopal Chandra; Sharma, Brijesh; Gupta, B B

    2016-01-01

    Tuberculous and Cryptococcal meningitis are common in HIV patients. A highly specific and sensitive rapid test for diagnosis of Tuberculous meningitis especially in setting of HIV is not available in developing countries where the burden of disease is high. We measured ADA (adenosine deaminase) levels using spectrophotometric method in the CSF of HIV patients with meningitis to differentiate Tuberculous meningitis from meningitis due to other causes. Kruskal-Wallis test was used to compare ADA values between tuberculous meningitis (TBM) and nontuberculous (non-TB) meningitis patients and a receiver-operating characteristic (ROC) analysis curve was drawn from these values. Levels of ADA in the CSF of patients with TBM were significantly higher than those in patients with meningitis due to other causes. CSF ADA level determination with a cut-off value of 6 IU/L was found to be highly specific and fairly sensitive test for the diagnosis of TBM in HIV positive patients.

  19. A study of three polymorphic sites of ADA gene in colon cancer.

    Science.gov (United States)

    Spina, C; Saccucci, P; Cozzoli, E; Bottini, E; Gloria-Bottini, F

    2010-12-01

    Adenosine inhibits the immune response in tumors. Adenosine deaminase (ADA) controls adenosine level and as ecto-enzyme acts as costimulatory molecule of adenosine receptors and/or CD26. We examined ADA₁, ADA₂, ADA₆ polymorphic sites of ADA gene in 109 subjects with colon cancer from Rome's population and in 246 blood donors as controls from the same population. In colon cancer ADA₁*2/ADA₂*1 haplotype is more represented, while ADA₁*2/ADA₂*2 is less represented than in controls. ADA₂*2/ADA₆*2 is less represented in patients than in controls. Polymorphic sites of ADA might influence cell-mediated anti-tumor immune responses controlling adenosine level and extraenzymatic protein functions.

  20. Maternal T-cell engraftment impedes with diagnosis of a SCID-ADA patient.

    Science.gov (United States)

    Lanfranchi, Arnalda; Lougaris, Vassilios; Notarangelo, Lucia Dora; Soncini, Elena; Comini, Marta; Beghin, Alessandra; Bolda, Federica; Montanelli, Alessandro; Imberti, Luisa; Porta, Fulvio

    2018-02-02

    We describe the case of a child affected by severe combined immunodeficiency (SCID) with adenosine deaminase (ADA) deficiency showing a maternal T-cell engraftment, a finding that has never been reported before. The presence of engrafted maternal T cells was misleading. Although ADA enzymatic levels were suggestive of ADA-SCID, the child did not present the classical signs of ADA deficiency; therefore, the initial diagnosis was of a conventional SCID. However, ADA toxic metabolites and molecular characterization confirmed this diagnosis. Polyethylene glycol-modified bovine (PEG) ADA therapy progressively decreased the number of maternal engrafted T cells. The child was grafted with full bone marrow from a matched unrelated donor, after a reduced conditioning regimen, and the result was the complete immunological reconstitution. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Characteristic scapular and rib changes on chest radiographs of children with ADA-deficiency SCIDS in the first year of life.

    Science.gov (United States)

    Manson, David; Diamond, Lauren; Oudjhane, Kamaldine; Hussain, Faisal Bin; Roifman, Chaim; Grunebaum, Eyal

    2013-03-01

    We describe radiographic changes in the ribs and scapulae seen in the first 6 months of life in children with ADA (adenosine deaminase) deficiency severe combined immundeficiency syndrome (SCIDS). We suggest that these changes are reversible with appropriate enzyme replacement therapy. The purpose of this study was to describe characteristic rib and scapular radiographic changes in infants with ADA-deficiency SCIDS. This was a retrospective review of chest radiographs of nine children with ADA-deficiency SCIDS performed in the first year of life by two experienced pediatric radiologists. A control cohort of unaffected children was used for comparison. All children with ADA-deficiency SCIDS manifested unusual scapular spurring and anterior rib cupping. None of the control children manifested these changes. Characteristic and reversible scapular and rib changes in the correct clinical setting should suggest an early diagnosis of ADA deficiency, prompting appropriate diagnostic and therapeutic measures.

  2. Restoring balance to B cells in ADA deficiency.

    Science.gov (United States)

    Luning Prak, Eline T

    2012-06-01

    It is paradoxical that immunodeficiency disorders are associated with autoimmunity. Adenosine deaminase (ADA) deficiency, a cause of X-linked severe combined immunodeficiency (SCID), is a case in point. In this issue of the JCI, Sauer and colleagues investigate the B cell defects in ADA-deficient patients. They demonstrate that ADA patients receiving enzyme replacement therapy had B cell tolerance checkpoint defects. Remarkably, gene therapy with a retrovirus that expresses ADA resulted in the apparent correction of these defects, with normalization of peripheral B cell autoantibody frequencies. In vitro, agents that either block ADA or overexpress adenosine resulted in altered B cell receptor and TLR signaling. Collectively, these data implicate a B cell-intrinsic mechanism for alterations in B cell tolerance in the setting of partial ADA deficiency that is corrected by gene therapy.

  3. Improved Performance of the Potentiometric Biosensor for the Determination of Creatinine

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov; Rasmussen, Claus/Dallerup; Zachau-Christiansen, Birgit

    2007-01-01

    The development of potentiometric biosensors for the determination of creatinine is attractive because it is a frequently analysed species in clinical chemistry. Contemporary methods of analysing creatinine engage chemicals harmful to the environment and generate large volumes of waste disposals....... By introducing a membrane-based potentiometric biosensor with immobilised creatinine deaminase, the measurements can be performed by miniaturised portable devices that are easy to handle and allow rapid analysis at a minimum consumption of chemicals. Thus, the enzymatic creatinine biosensors was revisited...... performed by flow injection analysis (FIA) showed that the response time could be lowered to approx. 30 sec. using sample volumes of 30 L. Interferences were corrected for by application of the Nicolsky-Eisenman equation thus allowing determination of creatinine in matrices resembling those of clinical...

  4. AID to overcome the limitations of genomic information by introducing somatic DNA alterations.

    Science.gov (United States)

    Honjo, Tasuku; Muramatsu, Masamichi; Nagaoka, Hitoshi; Kinoshita, Kazuo; Shinkura, Reiko

    2006-05-01

    The immune system has adopted somatic DNA alterations to overcome the limitations of the genomic information. Activation induced cytidine deaminase (AID) is an essential enzyme to regulate class switch recombination (CSR), somatic hypermutation (SHM) and gene conversion (GC) of the immunoglobulin gene. AID is known to be required for DNA cleavage of S regions in CSR and V regions in SHM. However, its molecular mechanism is a focus of extensive debate. RNA editing hypothesis postulates that AID edits yet unknown mRNA, to generate specific endonucleases for CSR and SHM. By contrast, DNA deamination hypothesis assumes that AID deaminates cytosine in DNA, followed by DNA cleavage by base excision repair enzymes. We summarize the basic knowledge for molecular mechanisms for CSR and SHM and then discuss the importance of AID not only in the immune regulation but also in the genome instability.

  5. Genetic Determinants of RNA Editing Levels of ADAR Targets in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Yerbol Z. Kurmangaliyev

    2016-02-01

    Full Text Available RNA editing usually affects only a fraction of expressed transcripts and there is a vast amount of variation in editing levels of ADAR (adenosine deaminase, RNA-specific targets. Here we explore natural genetic variation affecting editing levels of particular sites in 81 natural strains of Drosophila melanogaster. The analysis of associations between editing levels and single-nucleotide polymorphisms allows us to map putative cis-regulatory regions affecting editing of 16 A-to-I editing sites (cis-RNA editing quantitative trait loci or cis-edQTLs, P < 10−8. The observed changes in editing levels are validated by independent molecular technique. All identified regulatory variants are located in close proximity of modulated editing sites. Moreover, colocalized editing sites are often regulated by same loci. Similar to expression and splicing QTL studies, the characterization of edQTLs will greatly expand our understanding of cis-regulatory evolution of gene expression.

  6. Update on gene therapy of inherited immune deficiencies.

    Science.gov (United States)

    Engel, Barbara C; Kohn, Donald B; Podsakoff, Greg M

    2003-10-01

    Gene therapy has been under development as a way to correct inborn errors for many years. Recently, patients with two forms of inherited severe combined immunodeficiency (SCID), adenosine deaminase and X-linked, treated by three different clinical investigative teams, have shown significant immune reconstitution leading to protective immunity. These advances irrefutably prove the concept that hematopoietic progenitor cell gene therapy can ameliorate these diseases. However, due to proviral insertional oncogenesis, two individuals in one of the X-SCID studies developed T-cell leukemia more than two years after the gene transfer. Depending upon the results of long-term follow-up, the successes together with the side effects highlight the relative merits of this therapeutic approach.

  7. Tuberculous Pleural Effusion

    Science.gov (United States)

    Cohen, Leah A.; Light, Richard W.

    2015-01-01

    When a patient presents with new pleural effusion, the diagnosis of tuberculous (TB) pleuritis should be considered. The patient is at risk for developing pulmonary or extrapulmonary TB if the diagnosis is not made. Between 3% and 25% of patients with TB will have TB pleuritis. The incidence of TB pleuritis is higher in patients who are human immunodeficiency virus (HIV)-positive. Pleural fluid is an exudate that usually has a predominance of lymphocytes. The easiest way to diagnose TB pleuritis in a patient with lymphocytic pleural effusion is to demonstrate a pleural fluid adenosine deaminase level above 40 IU/L. The treatment for TB pleuritis is the same as that for pulmonary TB. Tuberculous empyema is a rare occurrence, and the treatment is difficult. PMID:29404070

  8. APOBEC3A is an oral cancer prognostic biomarker in Taiwanese carriers of an APOBEC deletion polymorphism.

    Science.gov (United States)

    Chen, Ting-Wen; Lee, Chi-Ching; Liu, Hsuan; Wu, Chi-Sheng; Pickering, Curtis R; Huang, Po-Jung; Wang, Jing; Chang, Ian Yi-Feng; Yeh, Yuan-Ming; Chen, Chih-De; Li, Hsin-Pai; Luo, Ji-Dung; Tan, Bertrand Chin-Ming; Chan, Timothy En Haw; Hsueh, Chuen; Chu, Lichieh Julie; Chen, Yi-Ting; Zhang, Bing; Yang, Chia-Yu; Wu, Chih-Ching; Hsu, Chia-Wei; See, Lai-Chu; Tang, Petrus; Yu, Jau-Song; Liao, Wei-Chao; Chiang, Wei-Fan; Rodriguez, Henry; Myers, Jeffrey N; Chang, Kai-Ping; Chang, Yu-Sun

    2017-09-06

    Oral squamous cell carcinoma is a prominent cancer worldwide, particularly in Taiwan. By integrating omics analyses in 50 matched samples, we uncover in Taiwanese patients a predominant mutation signature associated with cytidine deaminase APOBEC, which correlates with the upregulation of APOBEC3A expression in the APOBEC3 gene cluster at 22q13. APOBEC3A expression is significantly higher in tumors carrying APOBEC3B-deletion allele(s). High-level APOBEC3A expression is associated with better overall survival, especially among patients carrying APOBEC3B-deletion alleles, as examined in a second cohort (n = 188; p = 0.004). The frequency of APOBEC3B-deletion alleles is ~50% in 143 genotyped oral squamous cell carcinoma -Taiwan samples (27A3B -/- :89A3B +/- :27A3B +/+ ), compared to the 5.8% found in 314 OSCC-TCGA samples. We thus report a frequent APOBEC mutational profile, which relates to a APOBEC3B-deletion germline polymorphism in Taiwanese oral squamous cell carcinoma that impacts expression of APOBEC3A, and is shown to be of clinical prognostic relevance. Our finding might be recapitulated by genomic studies in other cancer types.Oral squamous cell carcinoma is a prevalent malignancy in Taiwan. Here, the authors show that OSCC in Taiwanese show a frequent deletion polymorphism in the cytidine deaminases gene cluster APOBEC3 resulting in increased expression of A3A, which is shown to be of clinical prognostic relevance.

  9. Transfection of Babesia bovis by Double Selection with WR99210 and Blasticidin-S and Its Application for Functional Analysis of Thioredoxin Peroxidase-1.

    Directory of Open Access Journals (Sweden)

    Masahito Asada

    Full Text Available Genetic manipulation is an essential technique to analyze gene function; however, limited methods are available for Babesia bovis, a causative pathogen of the globally important cattle disease, bovine babesiosis. To date, two stable transfection systems have been developed for B. bovis, using selectable markers blasticidin-S deaminase (bsd or human dihydrofolate reductase (hdhfr. In this work, we combine these two selectable markers in a sequential transfection system. Specifically, a parent transgenic B. bovis line which episomally expresses green fluorescent protein (GFP and human dihydrofolate reductase (hDHFR, was transfected with a plasmid encoding a fusion protein consisting of red fluorescent protein (RFP and blasticidin-S deaminase (BSD. Selection with WR99210 and blasticidin-S resulted in the emergence of parasites double positive for GFP and RFP. We then applied this method to complement gene function in a parasite line in which thioredoxin peroxidase-1 (Bbtpx-1 gene was knocked out using hDHFR as a selectable marker. A plasmid was constructed harboring both RFP-BSD and Bbtpx-1 expression cassettes, and transfected into a Bbtpx-1 knockout (KO parasite. Transfectants were independently obtained by two transfection methods, episomal transfection and genome integration. Complementation of Bbtpx-1 resulted in full recovery of resistance to nitrosative stress, via the nitric oxide donor sodium nitroprusside, which was impaired in the Bbtpx-1 KO parasites. In conclusion, we developed a sequential transfection method in B. bovis and subsequently applied this technique in a gene complementation study. This method will enable broader genetic manipulation of Babesia toward enhancing our understanding of the biology of this parasite.

  10. AMP-regulated protein kinase activity in the hearts of mice treated with low- or high-fat diet measured using novel LC-MS method.

    Science.gov (United States)

    Rybakowska, I M; Slominska, E M; Romaszko, P; Olkowicz, M; Kaletha, K; Smolenski, R T

    2015-06-01

    AMP-regulated protein kinase (AMPK) is involved in regulation of energy-generating pathways in response to the metabolic needs in different organs including the heart. The activity of AMPK is mainly controlled by AMP concentration that in turn could be affected by nucleotide metabolic pathways. This study aimed to develop a procedure for measurement of AMPK activity together with nucleotide metabolic enzymes and its application for studies of mice treated with high-fat diet. The method developed was based on analysis of conversion of AMARA peptide to pAMARA by partially purified heart homogenate by liquid chromatography/mass spectrometry (LC/MS). Activities of the enzymes of nucleotide metabolism were evaluated by analysis of conversion of substrates into products by HPLC. The method was applied for analysis of hearts of mice fed 12 weeks with low- (LFD) or high-fat diet (HFD). The optimized method for AMPK activity analysis (measured in presence of AMP) revealed change of activity from 0.089 ± 0.035 pmol/min/mg protein in LFD to 0.024 ± 0.002 in HFD. This coincided with increase of adenosine deaminase (ADA) activity from 0.11 ± 0.02 to 0.19 ± 0.06 nmol/mg tissue/min and decrease of AMP-deaminase (AMPD) activity from 1.26 ± 0.35 to 0.56 ± 0.15 nmol/mg tissue/min for LFD and HFD, respectively. We have proven quality of our LC/MS method for analysis of AMPK activity. We observed decrease in AMPK activity in the heart of mice treated with high-fat diet. However, physiological consequences of this change could be modulated by decrease in AMPD activity.

  11. Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria.

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    Yim, W J; Kim, K Y; Lee, Y W; Sundaram, S P; Lee, Y; Sa, T M

    2014-07-15

    Biotic stress like pathogenic infection increases ethylene biosynthesis in plants and ethylene inhibitors are known to alleviate the severity of plant disease incidence. This study aimed to reduce the bacterial spot disease incidence in tomato plants caused by Xanthomonas campestris pv. vesicatoria (XCV) by modulating stress ethylene with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Methylobacterium strains. Under greenhouse condition, Methylobacterium strains inoculated and pathogen challenged tomato plants had low ethylene emission compared to pathogen infected ones. ACC accumulation and ACC oxidase (ACO) activity with ACO related gene expression increased in XCV infected tomato plants over Methylobacterium strains inoculated plants. Among the Methylobacterium spp., CBMB12 resulted lowest ACO related gene expression (1.46 Normalized Fold Expression), whereas CBMB20 had high gene expression (3.42 Normalized Fold Expression) in pathogen challenged tomato. But a significant increase in ACO gene expression (7.09 Normalized Fold Expression) was observed in the bacterial pathogen infected plants. In contrast, Methylobacterium strains enhanced β-1,3-glucanase and phenylalanine ammonia-lyase (PAL) enzyme activities in pathogen challenged tomato plants. The respective increase in β-1,3-glucanase related gene expressions due to CBMB12, CBMB15, and CBMB20 strains were 66.3, 25.5 and 10.4% higher over pathogen infected plants. Similarly, PAL gene expression was high with 0.67 and 0.30 Normalized Fold Expression, in pathogen challenged tomato plants inoculated with CBMB12 and CBMB15 strains. The results suggest that ethylene is a crucial factor in bacterial spot disease incidence and that methylobacteria with ACC deaminase activity can reduce the disease severity with ultimate pathogenesis-related protein increase in tomato. Copyright © 2014 Elsevier GmbH. All rights reserved.

  12. Alkyladenine DNA glycosylase (Aag) in somatic hypermutation and class switch recombination.

    Science.gov (United States)

    Longerich, Simonne; Meira, Lisiane; Shah, Dharini; Samson, Leona D; Storb, Ursula

    2007-12-01

    Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis. We investigated the possibility that deamination at adenines also initiates SHM. Adenosine deamination would generate hypoxanthine (Hx), a substrate for the alkyladenine DNA glycosylase (Aag). Aag would generate abasic sites which then are subject to error-prone repair as above for AID-deaminated cytosine processed by Ung. If the action of an adenosine deaminase followed by Aag were responsible for significant numbers of mutations at A, we would find a preponderance of A:T>G:C transition mutations during SHM in an Aag deleted background. However, this was not observed and we found that the frequencies of SHM and CSR were not significantly altered in Aag-/- mice. Paradoxically, we found that Aag is expressed in B lymphocytes undergoing SHM and CSR and that its activity is upregulated in activated B cells. Moreover, we did find a statistically significant, albeit low increase of T:A>C:G transition mutations in Aag-/- animals, suggesting that Aag may be involved in creating the SHM A>T bias seen in wild type mice.

  13. Targeting APOBEC3A to the viral nucleoprotein complex confers antiviral activity

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    Strebel Klaus

    2007-08-01

    Full Text Available Abstract Background APOBEC3 (A3 proteins constitute a family of cytidine deaminases that provide intracellular resistance to retrovirus replication and to transposition of endogenous retroelements. A3A has significant homology to the C-terminus of A3G but has only a single cytidine deaminase active site (CDA, unlike A3G, which has a second N-terminal CDA previously found to be important for Vif sensitivity and virus encapsidation. A3A is packaged into HIV-1 virions but, unlike A3G, does not have antiviral properties. Here, we investigated the reason for the lack of A3A antiviral activity. Results Sequence alignment of A3G and A3A revealed significant homology of A3A to the C-terminal region of A3G. However, while A3G co-purified with detergent-resistant viral nucleoprotein complexes (NPC, virus-associated A3A was highly detergent-sensitive leading us to speculate that the ability to assemble into NPC may be a property conveyed by the A3G N-terminus. To test this model, we constructed an A3G-3A chimeric protein, in which the N-terminal half of A3G was fused to A3A. Interestingly, the A3G-3A chimera was packaged into HIV-1 particles and, unlike A3A, associated with the viral NPC. Furthermore, the A3G-3A chimera displayed strong antiviral activity against HIV-1 and was sensitive to inhibition by HIV-1 Vif. Conclusion Our results suggest that the A3G N-terminal domain carries determinants important for targeting the protein to viral NPCs. Transfer of this domain to A3A results in A3A targeting to viral NPCs and confers antiviral activity.

  14. Trichoderma-Plant Root Colonization: Escaping Early Plant Defense Responses and Activation of the Antioxidant Machinery for Saline Stress Tolerance

    Science.gov (United States)

    Brotman, Yariv; Landau, Udi; Cuadros-Inostroza, Álvaro; Takayuki, Tohge; Fernie, Alisdair R.; Chet, Ilan; Viterbo, Ada; Willmitzer, Lothar

    2013-01-01

    Trichoderma spp. are versatile opportunistic plant symbionts which can colonize the apoplast of plant roots. Microarrays analysis of Arabidopsis thaliana roots inoculated with Trichoderma asperelloides T203, coupled with qPCR analysis of 137 stress responsive genes and transcription factors, revealed wide gene transcript reprogramming, proceeded by a transient repression of the plant immune responses supposedly to allow root colonization. Enhancement in the expression of WRKY18 and WRKY40, which stimulate JA-signaling via suppression of JAZ repressors and negatively regulate the expression of the defense genes FMO1, PAD3 and CYP71A13, was detected in Arabidopsis roots upon Trichoderma colonization. Reduced root colonization was observed in the wrky18/wrky40 double mutant line, while partial phenotypic complementation was achieved by over-expressing WRKY40 in the wrky18 wrky40 background. On the other hand increased colonization rate was found in roots of the FMO1 knockout mutant. Trichoderma spp. stimulate plant growth and resistance to a wide range of adverse environmental conditions. Arabidopsis and cucumber (Cucumis sativus L.) plants treated with Trichoderma prior to salt stress imposition show significantly improved seed germination. In addition, Trichoderma treatment affects the expression of several genes related to osmo-protection and general oxidative stress in roots of both plants. The MDAR gene coding for monodehydroascorbate reductase is significantly up-regulated and, accordingly, the pool of reduced ascorbic acid was found to be increased in Trichoderma treated plants. 1-Aminocyclopropane-1-carboxylate (ACC)-deaminase silenced Trichoderma mutants were less effective in providing tolerance to salt stress, suggesting that Trichoderma, similarly to ACC deaminase producing bacteria, can ameliorate plant growth under conditions of abiotic stress, by lowering ameliorating increases in ethylene levels as well as promoting an elevated antioxidative capacity

  15. Comparison of identical and functional Igh alleles reveals a nonessential role for Eμ in somatic hypermutation and class-switch recombination.

    Science.gov (United States)

    Li, Fubin; Yan, Yi; Pieretti, Joyce; Feldman, Danielle A; Eckhardt, Laurel A

    2010-11-15

    Somatic hypermutation (SHM), coupled with Ag selection, provides a mechanism for generating Abs with high affinity for invading pathogens. Class-switch recombination (CSR) ensures that these Abs attain pathogen-appropriate effector functions. Although the enzyme critical to both processes, activation-induced cytidine deaminase, has been identified, it remains unclear which cis-elements within the Ig loci are responsible for recruiting activation-induced cytidine deaminase and promoting its activity. Studies showed that Ig gene-transcription levels are positively correlated with the frequency of SHM and CSR, making the intronic, transcriptional enhancer Eμ a likely contributor to both processes. Tests of this hypothesis yielded mixed results arising, in part, from the difficulty in studying B cell function in mice devoid of Eμ. In Eμ's absence, V(H) gene assembly is dramatically impaired, arresting B cell development. The current study circumvented this problem by modifying the murine Igh locus through simultaneous insertion of a fully assembled V(H) gene and deletion of Eμ. The behavior of this allele was compared with that of a matched allele carrying the same V(H) gene but with Eμ intact. Although IgH transcription was as great or greater on the Eμ-deficient allele, CSR and SHM were consistently, but modestly, reduced relative to the allele in which Eμ remained intact. We conclude that Eμ contributes to, but is not essential for, these complex processes and that its contribution is not as a transcriptional enhancer but, rather, is at the level of recruitment and/or activation of the SHM/CSR machinery.

  16. Creatinine and urea biosensors based on a novel ammonium ion-selective copper-polyaniline nano-composite.

    Science.gov (United States)

    Zhybak, M; Beni, V; Vagin, M Y; Dempsey, E; Turner, A P F; Korpan, Y

    2016-03-15

    The use of a novel ammonium ion-specific copper-polyaniline nano-composite as transducer for hydrolase-based biosensors is proposed. In this work, a combination of creatinine deaminase and urease has been chosen as a model system to demonstrate the construction of urea and creatinine biosensors to illustrate the principle. Immobilisation of enzymes was shown to be a crucial step in the development of the biosensors; the use of glycerol and lactitol as stabilisers resulted in a significant improvement, especially in the case of the creatinine, of the operational stability of the biosensors (from few hours to at least 3 days). The developed biosensors exhibited high selectivity towards creatinine and urea. The sensitivity was found to be 85 ± 3.4 mAM(-1)cm(-2) for the creatinine biosensor and 112 ± 3.36 mAM(-1)cm(-2) for the urea biosensor, with apparent Michaelis-Menten constants (KM,app), obtained from the creatinine and urea calibration curves, of 0.163 mM for creatinine deaminase and 0.139 mM for urease, respectively. The biosensors responded linearly over the concentration range 1-125 µM, with a limit of detection of 0.5 µM and a response time of 15s. The performance of the biosensors in a real sample matrix, serum, was evaluated and a good correlation with standard spectrophotometric clinical laboratory techniques was found. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Induction of drought tolerance in cucumber plants by a consortium of three plant growth-promoting rhizobacterium strains.

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    Chun-Juan Wang

    Full Text Available Our previous work showed that a consortium of three plant growth-promoting rhizobacterium (PGPR strains (Bacillus cereus AR156, Bacillus subtilis SM21, and Serratia sp. XY21, termed as BBS for short, was a promising biocontrol agent. The present study investigated its effect on drought tolerance in cucumber plants. After withholding watering for 13 days, BBS-treated cucumber plants had much darker green leaves and substantially lighter wilt symptoms than control plants. Compared to the control, the BBS treatment decreased the leaf monodehydroascorbate (MDA content and relative electrical conductivity by 40% and 15%, respectively; increased the leaf proline content and the root recovery intension by 3.45-fold and 50%, respectively; and also maintained the leaf chlorophyll content in cucumber plants under drought stress. Besides, in relation to the control, the BBS treatment significantly enhanced the superoxide dismutase (SOD activity and mitigated the drought-triggered down-regulation of the expression of the genes cAPX, rbcL, and rbcS encoding cytosolic ascorbate peroxidase, and ribulose-1,5-bisphosphate carboxy/oxygenase (Rubisco large and small subunits, respectively, in cucumber leaves. However, 1-aminocyclopropane-1-carboxylate (ACC deaminase activity was undetected in none of the culture solutions of three BBS constituent strains. These results indicated that BBS conferred induced systemic tolerance to drought stress in cucumber plants, by protecting plant cells, maintaining photosynthetic efficiency and root vigor and increasing some of antioxidase activities, without involving the action of ACC deaminase to lower plant ethylene levels.

  18. Identification of arsenic resistant endophytic bacteria from Pteris vittata roots and characterization for arsenic remediation application.

    Science.gov (United States)

    Tiwari, Sarita; Sarangi, Bijaya Ketan; Thul, Sanjog T

    2016-09-15

    Mitigation of arsenic (As) pollution is a topical environmental issue of high R&D priority. The present investigation was carried out to isolate As resistant endophytes from the roots of Indian ecotype Pteris vittata and characterize their As transformation and tolerance ability, plant growth promoting characteristics and their role to facilitate As uptake by the plant. A total of 8 root endophytes were isolated from plants grown in As amended soil (25 mg As kg(-1)). These isolates were studied for minimum inhibitory concentration (MIC), arsenite As(III) - arsenate As(V) transformation ability, plant growth promoting (PGP) characteristics through siderophore, indole acetic acid (IAA) production, phosphatase, ACC deaminase activity, and presence of arsenite oxidase (aox) and arsenite transporter (arsB) genes. On the basis of 16S rDNA sequence analysis, these isolates belong to Proteobacteria, Firmicutes and Bacteroidetes families under the genera Bacillus, Enterobacter, Stenotrophomonas and Rhizobium. All isolates were found As tolerant, of which one isolates showed highest tolerance up to 1000 mg L(-1) concentration in SLP medium. Five isolates were IAA positive with highest IAA production up to 60 mg/L and two isolates exhibited siderophore activity. Phosphatase activity was shown by only one isolate while ACC deaminase activity was absent in all the isolates. The As transformation study by silver nitrate test showed that only two strains had dual characteristics of As(III) oxidation and As (V) reduction, four strains exhibited either of the characteristics while other two didn't confirmed any of the two characteristics. Presence of aox gene was detected in two strains and arsB gene in six isolates. The strain with highest As tolerance also showed highest IAA production and occurrence of arsB gene. Present investigation may open up further scope of utilizing these endophytes for up gradation of phytoextraction process. Copyright © 2016 Elsevier Ltd. All

  19. Dipeptidyl peptidase-4 (CD26): knowing the function before inhibiting the enzyme.

    Science.gov (United States)

    Matteucci, E; Giampietro, O

    2009-01-01

    Dipeptidyl peptidase-4 (DPP4) or adenosine deaminase complexing protein 2 (ADCP 2) or T-cell activation antigen CD26 (EC 3.4.14.5.) is a serine exopeptidase belonging to the S9B protein family that cleaves X-proline dipeptides from the N-terminus of polypeptides, such as chemokines, neuropeptides, and peptide hormones. The enzyme is a type II transmembrane glycoprotein, expressed on the surface of many cell types, whose physiological functions are largely unknown. Protein dimerisation should be required for catalytic activity and glycosylation of the enzyme could impact on its physiological functions. The dimeric glycoprotein ADCP has been found linked to adenosine deaminase (ADA) whose relationship with lymphocyte maturation-differentiation is well-established. Since implicated in the regulation of the biological activity of hormones and chemokines, such as glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide, DPP4 inhibition offers a new potential therapeutic approach for type 2 diabetes mellitus, as monotherapy and adjunct therapy to other oral agents. The clinical use of presently available orally active inhibitors of DPP4, however, has been associated with side effects that have been in part attributed to the inhibition of related serine proteases, such as DPP8 and DPP9. Indeed, it is noteworthy that CD26 has a key role in immune regulation as a T cell activation molecule and in immune-mediated disorder. All-cause infections were increased after sitagliptin treatment. It is noteworthy that the effects of DPP4 inhibition on the immune system have not been extensively investigated. So far, only routine laboratory safety variables have been measured in published randomised controlled trials. The review summarises present knowledge in the field and suggests some potential directions of future research.

  20. Characterization of ectonucleotidases in human medulloblastoma cell lines: ecto-5'NT/CD73 in metastasis as potential prognostic factor.

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    Angélica Regina Cappellari

    Full Text Available Medulloblastoma (MB is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5'NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy and inosine accumulation (ONS76. On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5'NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5'NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

  1. Rapid Detection of Cell-Free Mycobacterium tuberculosis DNA in Tuberculous Pleural Effusion.

    Science.gov (United States)

    Che, Nanying; Yang, Xinting; Liu, Zichen; Li, Kun; Chen, Xiaoyou

    2017-05-01

    Tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, but its diagnosis remains difficult. In this study, we report for the first time on the detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and an evaluation of a newly developed molecular assay for the detection of cell-free Mycobacterium tuberculosis DNA. A total of 78 patients with pleural effusion, 60 patients with tuberculous pleurisy, and 18 patients with alternative diseases were included in this study. Mycobacterial culture, the Xpert MTB/RIF assay, the adenosine deaminase assay, the T-SPOT.TB assay, and the cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. The cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities of 75.0% and 68.3%, respectively, than mycobacterial culture and the Xpert MTB/RIF assay, which had sensitivities of 26.7% and 20.0%, respectively ( P pleural effusion showed the highest sensitivity of 95.0% but the lowest specificity of 38.9%. The cell-free Mycobacterium tuberculosis DNA assay detected as few as 1.25 copies of IS 6110 per ml of pleural effusion and showed good accordance of the results between repeated tests ( r = 0.978, P = 2.84 × 10 -10 ). These data suggest that the cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology. Copyright © 2017 American Society for Microbiology.

  2. Five-chlorodeoxycytidine, a tumor-selective enzyme-driven radiosensitizer, effectively controls five advanced human tumors in nude mice

    International Nuclear Information System (INIS)

    Greer, Sheldon; Alvarez, Marcy; Mas, Marisol; Wozniak, Chandra; Arnold, David; Knapinska, Anna; Norris, Christina; Burk, Ronald; Aller, Alex; Dauphinee, Michael

    2001-01-01

    Purpose: The study's goals were as follows: (1) to extend our past findings with rodent tumors to human tumors in nude mice, (2) to determine if the drug protocol could be simplified so that only CldC and one modulator, tetrahydrouridine (H 4 U), would be sufficient to obtain efficacy, (3) to determine the levels of deoxycytidine kinase and dCMP deaminase in human tumors, compared to adjacent normal tissue, and (4) to determine the effect of CldC on normal tissue radiation damage to the cervical spinal cord of nude mice. Methods and Materials: The five human tumors used were as follows: prostate tumors, PC-3 and H-1579; glioblastoma, SF-295; breast tumor, GI-101; and lung tumor, H-165. The duration of treatment was 3-5 weeks, with drugs administered on Days 1-4 and radiation on Days 3-5 of each week. The biomodulators of CldC were N-(Phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartyl transcarbamoylase, 5-fluorodeoxycytidine (FdC), resulting in tumor-directed inhibition of thymidylate synthetase, and H 4 U, an inhibitor of cytidine deaminase. The total dose of focused irradiation of the tumors was usually 45 Gy in 12 fractions. Results: Marked radiosensitization was obtained with CldC and the three modulators. The average days in tumor regrowth delay for X-ray compared to drugs plus X-ray, respectively, were: PC-3 prostate, 42-97; H-1579 prostate, 29-115; glioblastoma, 5-51; breast, 50-80; lung, 32-123. Comparative studies with PC-3 and H-1579 using CldC coadministered with H 4 U, showed that both PALA and FdC are dispensable, and the protocol can be simplified with equal and possibly heightened efficacy. For example, PC-3 with X-ray and (1) no drugs, (2) CldC plus the three modulators, (3) a high dose of CldC, and (4) escalating doses of CldC resulted in 0/10, 3/9, 5/10, and 6/9 cures, respectively. The tumor regrowth delay data followed a similar pattern. After treating mice only 1((1)/(2)) weeks with CldC + H 4 U, 92% of the PC-3 tumor cells were found

  3. Dietary modulation of thymic enzymes.

    Science.gov (United States)

    Susana, Feliu María; Paula, Perris; Slobodianik, Nora

    2014-01-01

    Malnutrition is a complex syndrome caused by an inadequate intake of energy, protein, minerals and vitamins which affects the immune system. Nutritional imbalances, present in children with energy-protein malnutrition and infections, make defining the specific effects of each of them on the thymus difficult. For this reason, it is necessary to design an experimental model in animals that could define a single variable. As the thymus atrophy described in humans is similar to that observed in murines, a rat experimental model makes the extrapolation to man possible. Some authors suggest that the activity of Adenosine Deaminase (ADA) and Purine Nucleoside Phosphorylase (PNP)--involved in purine metabolism--have an influence on T lymphocyte development and the immune system, due to intracellular accumulation of toxic levels of deoxynucleotides. Studies in our group, performed in an experimental model on Wistar growing rats, have demonstrated that protein deficiency or imbalance in the profile of essential amino acids in the diet, produce loss of thymus weight, reduction in the number of thymocytes, a diminished proportion of T cells presenting the W3/13 antigenic determinant and DNA content with concomitant increase in cell size, and the proportion of immature T cells and activity of ADA and PNP, without modifying the activity of 5´Nucleotidase in the thymus. It is important to point out that there were neither differences in energy intake between experimental groups and their controls, nor clinical symptoms of deficiency of other nutrients. The increase in these thymic enzyme activities was an alternative mechanism to avoid the accumulation of high levels of deoxynucleotides, which would be toxic for T lymphocytes. On the other hand, the administration of a recovery diet, with a high amount of high quality protein, was able to reverse the mentioned effects. The quick reply of Adenosine Deaminase to nutritional disorders and the following nutritional recovery, points

  4. Effect of Lead (Pb Exposure on the Activity of Superoxide Dismutase and Catalase in Battery Manufacturing Workers (BMW of Western Maharashtra (India with Reference to Heme biosynthesis

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    Kusal K. Das

    2006-12-01

    Full Text Available The aim of this study was to estimate the activity of superoxide dismutase (SOD and catalase in erythrocytes and malondialdehyde (MDA in plasma of battery manufacturing workers (BMW of Western Maharashtra (India who were occupationally exposed to lead (Pb over a long period of time (about 15 years. This study was also aimed to determine the Pb intoxication resulted in a disturbance of heme biosynthesis in BMW group. The blood Pb level of BMW group (n = 28 was found to be in the range of 25.8 – 78.0 μg/dL (mean + SD, 53.63 + 16.98 whereas in Pb unexposed control group (n = 35 the range was 2.8 – 22.0 μg/dL (mean + SD, 12.52 + 4.08. The blood level (Pb-B and urinary lead level (Pb-U were significantly increased in BMW group as compared to unexposed control. Though activated d- aminolevulinic acid dehydratase (ALAD activities in BMW group did not show any significant change when compared to control group but activated / non activated erythrocyte – ALAD activities in BMW group showed a significant increase. Erythrocyte- zinc protoporphyrin (ZPP, urinary daminolevulinic acid (ALA-U and porphobilinogen (PBG-U of BMW groups elevated significantly as compared to control. A positive correlation (r = 0.66, p 1.0 were observed in control group. Hematological study revealed a significant decrease of hemoglobin concentration, packed cell volume (% and other blood indices and a significant increase of total leucocytes count in BMW group in comparison to control group. The serum MDA content was significantly increased (p< 0.001 and the activities of antioxidant enzymes such as erythrocyte- SOD (p< 0.001 and erythrocytecatalase (p< 0.001 were significantly reduced in BMW group as compared to control group. A positive correlation (r = 0.45, p<0.02 between Pb-B and serum MDA level was observed in BMW group (Pb-B range 25.8 – 78.0 μg / dL but such significant correlation did not notice in

  5. Error-free versus mutagenic processing of genomic uracil--relevance to cancer.

    Science.gov (United States)

    Krokan, Hans E; Sætrom, Pål; Aas, Per Arne; Pettersen, Henrik Sahlin; Kavli, Bodil; Slupphaug, Geir

    2014-07-01

    Genomic uracil is normally processed essentially error-free by base excision repair (BER), with mismatch repair (MMR) as an apparent backup for U:G mismatches. Nuclear uracil-DNA glycosylase UNG2 is the major enzyme initiating BER of uracil of U:A pairs as well as U:G mismatches. Deficiency in UNG2 results in several-fold increases in genomic uracil in mammalian cells. Thus, the alternative uracil-removing glycosylases, SMUG1, TDG and MBD4 cannot efficiently complement UNG2-deficiency. A major function of SMUG1 is probably to remove 5-hydroxymethyluracil from DNA with general back-up for UNG2 as a minor function. TDG and MBD4 remove deamination products U or T mismatched to G in CpG/mCpG contexts, but may have equally or more important functions in development, epigenetics and gene regulation. Genomic uracil was previously thought to arise only from spontaneous cytosine deamination and incorporation of dUMP, generating U:G mismatches and U:A pairs, respectively. However, the identification of activation-induced cytidine deaminase (AID) and other APOBEC family members as DNA-cytosine deaminases has spurred renewed interest in the processing of genomic uracil. Importantly, AID triggers the adaptive immune response involving error-prone processing of U:G mismatches, but also contributes to B-cell lymphomagenesis. Furthermore, mutational signatures in a substantial fraction of other human cancers are consistent with APOBEC-induced mutagenesis, with U:G mismatches as prime suspects. Mutations can be caused by replicative polymerases copying uracil in U:G mismatches, or by translesion polymerases that insert incorrect bases opposite abasic sites after uracil-removal. In addition, kataegis, localized hypermutations in one strand in the vicinity of genomic rearrangements, requires APOBEC protein, UNG2 and translesion polymerase REV1. What mechanisms govern error-free versus error prone processing of uracil in DNA remains unclear. In conclusion, genomic uracil is an

  6. Polimorfismos genéticos determinantes da performance física em atletas de elite Genetic polymorphisms determining of the physical performance in elite athletes

    Directory of Open Access Journals (Sweden)

    Rodrigo Gonçalves Dias

    2007-06-01

    Full Text Available Este artigo direciona-se à revisão de publicações sobre os "genes candidatos" e sua relação com os fenótipos de performance física humana em atletas de elite. Nosso objetivo é trazer ao conhecimento do leitor informações atualizadas sobre marcadores e variantes genéticas que podem levar certos indivíduos a sobressair-se em modalidades esportivas específicas. Além disso, serão descritos os mecanismos pelos quais um gene pode contribuir para a performance física, detalhando em cada momento as propriedades celulares, fisiológicas e moleculares do sistema em questão. Por esse motivo, limitamos nossa discussão a um número pequeno de variantes genéticas: polimorfismos R577X do gene da alfa-actinina 3 (ACTN3, C34T do gene da AMP deaminase (AMPD1, I/D da enzima conversora de angiotensina (ECA, -9/+9 do receptor beta2 de bradicinina (BDKRB2 e 985+185/1170 do gene da enzima creatina quinase M (CK-M. Esperamos com este artigo informar e sensibilizar o leitor para o fato de que a identificação de talentos e a otimização do potencial individual do atleta, com conseqüente sucesso no esporte, estão diretamente associados a variantes genéticas.This article is focused on the review of studies looking for "candidate genes" and their relationship with physical performance phenotypes in elite athletes. Our goal is to bring to readers what makes some individuals excel in some sports modalities, based on variants in genetic loci and markers. In addition, we assume the necessity to describe by what mechanisms a gene can contribute in physical performance, detailing in each part the cellular, physiological and molecular pathways involved. For this reason, we limited our discussion to a small number of genetic variants: polymorphisms R577X alpha-actinin 3 gene (ACTN3, C34T AMP deaminase gene (AMPD1, I/D angiotensin converting enzyme gene (ACE, -9/+9 beta2 bradykinin receptor gene (BDKRB2, and 985+185/ 1170 creatine kinase M gene (CK-M. We

  7. Within-Host Variations of Human Papillomavirus Reveal APOBEC-Signature Mutagenesis in the Viral Genome.

    Science.gov (United States)

    Hirose, Yusuke; Onuki, Mamiko; Tenjimbayashi, Yuri; Mori, Seiichiro; Ishii, Yoshiyuki; Takeuchi, Takamasa; Tasaka, Nobutaka; Satoh, Toyomi; Morisada, Tohru; Iwata, Takashi; Miyamoto, Shingo; Matsumoto, Koji; Sekizawa, Akihiko; Kukimoto, Iwao

    2018-03-28

    Persistent infection with oncogenic human papillomaviruses (HPVs) causes cervical cancer, accompanied with the accumulation of somatic mutations into the host genome. There are concomitant genetic changes in the HPV genome during viral infection; however, their relevance to cervical carcinogenesis is poorly understood. Here we explored within-host genetic diversity of HPV by performing deep sequencing analyses of viral whole-genome sequences in clinical specimens. The whole genomes of HPV types 16, 52 and 58 were amplified by type-specific PCR from total cellular DNA of cervical exfoliated cells collected from patients with cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC), and were deep-sequenced. After constructing a reference vial genome sequence for each specimen, nucleotide positions showing changes with > 0.5% frequencies compared to the reference sequence were determined for individual samples. In total, 1,052 positions of nucleotide variations were detected in HPV genomes from 151 samples (CIN1, n = 56; CIN2/3, n = 68; ICC, n = 27), with varying numbers per sample. Overall, C-to-T and C-to-A substitutions were the dominant changes observed across all histological grades. While C-to-T transitions were predominantly detected in CIN1, their prevalence was decreased in CIN2/3 and fell below that of C-to-A transversions in ICC. Analysis of the tri-nucleotides context encompassing substituted bases revealed that Tp C pN, a preferred target sequence for cellular APOBEC cytosine deaminases, was a primary site for C-to-T substitutions in the HPV genome. These results strongly imply that the APOBEC proteins are drivers of HPV genome mutation, particularly in CIN1 lesions. IMPORTANCE HPVs exhibit surprisingly high levels of genetic diversity, including a large repertoire of minor genomic variants in each viral genotype. Here, by conducting deep sequencing analyses, we show for the first time a comprehensive snapshot of the "within

  8. Turning up the volume on mutational pressure: Is more of a good thing always better? (A case study of HIV-1 Vif and APOBEC3

    Directory of Open Access Journals (Sweden)

    Wong Joseph K

    2008-03-01

    Full Text Available Abstract APOBEC3G and APOBEC3F are human cytidine deaminases that serve as innate antiviral defense mechanisms primarily by introducing C-to-U changes in the minus strand DNA of retroviruses during replication (resulting in G-to-A mutations in the genomic sense strand sequence. The HIV-1 Vif protein counteracts this defense by promoting the proteolytic degradation of APOBEC3G and APOBEC3F in the host cell. In the absence of Vif expression, APOBEC3 is incorporated into HIV-1 virions and the viral genome undergoes extensive G-to-A mutation, or "hypermutation", typically rendering it non-viable within a single replicative cycle. Consequently, Vif is emerging as an attractive target for pharmacological intervention and therapeutic vaccination. Although a highly effective Vif inhibitor may result in mutational meltdown of the viral quasispecies, a partially effective Vif inhibitor may accelerate the evolution of drug resistance and immune escape due to the codon structure and recombinogenic nature of HIV-1. This hypothesis rests on two principal assumptions which are supported by experimental evidence: a there is a dose response between intracellular APOBEC concentration and degree of viral hypermutation, and, b HIV-1 can tolerate an elevated mutation rate, and a true error or extinction threshold is as yet undetermined. Rigorous testing of this hypothesis will have timely and critical implications for the therapeutic management of HIV/AIDS, and delve into the complexities underlying the induction of lethal mutagenesis in a viral pathogen.

  9. Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions.

    Science.gov (United States)

    Silvas, Tania V; Hou, Shurong; Myint, Wazo; Nalivaika, Ellen; Somasundaran, Mohan; Kelch, Brian A; Matsuo, Hiroshi; Kurt Yilmaz, Nese; Schiffer, Celia A

    2018-05-14

    The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, secondary structure, and solution pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), which correlated with enzymatic activity. We also validated that A3A binds RNA in a sequence specific manner. A3A bound tighter to substrate binding motif within a hairpin loop compared to linear oligonucleotide, suggesting A3A affinity is modulated by substrate structure. Based on these findings and previously published A3A-ssDNA co-crystal structures, we propose a new model with intra-DNA interactions for the molecular mechanism underlying A3A sequence preference. Overall, the sequence and structural preferences identified for A3A leads to a new paradigm for identifying A3A's involvement in mutation of endogenous or exogenous DNA.

  10. Arabidopsis and Maize RidA Proteins Preempt Reactive Enamine/Imine Damage to Branched-Chain Amino Acid Biosynthesis in Plastids[C][W][OPEN

    Science.gov (United States)

    Niehaus, Thomas D.; Nguyen, Thuy N.D.; Gidda, Satinder K.; ElBadawi-Sidhu, Mona; Lambrecht, Jennifer A.; McCarty, Donald R.; Downs, Diana M.; Cooper, Arthur J.L.; Fiehn, Oliver; Mullen, Robert T.; Hanson, Andrew D.

    2014-01-01

    RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase. PMID:25070638

  11. DNA repair in Mycobacterium tuberculosis revisited.

    Science.gov (United States)

    Dos Vultos, Tiago; Mestre, Olga; Tonjum, Tone; Gicquel, Brigitte

    2009-05-01

    Our understanding of Mycobacterium tuberculosis DNA repair mechanisms is still poor compared with that of other bacterial organisms. However, the publication of the first complete M. tuberculosis genome sequence 10 years ago boosted the study of DNA repair systems in this organism. A first step in the elucidation of M. tuberculosis DNA repair mechanisms was taken by Mizrahi and Andersen, who identified homologs of genes involved in the reversal or repair of DNA damage in Escherichia coli and related organisms. Genes required for nucleotide excision repair, base excision repair, recombination, and SOS repair and mutagenesis were identified. Notably, no homologs of genes involved in mismatch repair were identified. Novel characteristics of the M. tuberculosis DNA repair machinery have been found over the last decade, such as nonhomologous end joining, the presence of Mpg, ERCC3 and Hlr - proteins previously presumed to be produced exclusively in mammalian cells - and the recently discovered bifunctional dCTP deaminase:dUTPase. The study of these systems is important to develop therapeutic agents that can counteract M. tuberculosis evolutionary changes and to prevent adaptive events resulting in antibiotic resistance. This review summarizes our current understanding of the M. tuberculosis DNA repair system.

  12. Significance of diazotrophic plant growth-promoting Herbaspirillum sp. GW103 on phytoextraction of Pband Zn by Zea mays L.

    Science.gov (United States)

    Praburaman, Loganathan; Park, Sung-Hee; Cho, Min; Lee, Kui-Jae; Ko, Jeong-Ae; Han, Sang-Sub; Lee, Sang-Hyun; Kamala-Kannan, Seralathan; Oh, Byung-Taek

    2017-01-01

    Microbe-assisted phytoremediation has been considered a promising measure for the remediation of heavy metal-polluted soil. The aim of this study was to assess the effect of diazotrophic plant growth-promoting Herbaspirillum sp. GW103 on growth and lead (Pb) and zinc (Zn) accumulation in Zea mays L. The strain GW103 exhibited plant growth-promoting traits such as indole-3-acetic acid, siderophores, and 1-aminocyclopropane-1-carboxylic deaminase. Treatment of Z. mays L. plants with GW103 significantly increased 19, 31, and 52% of plant biomass and 10, 50, and 126% of chlorophyll a contents in Pb, Zn, and Pb + Zn-amended soils, respectively. Similarly, the strain GW103 significantly increased Pb and Zn accumulation in shoots and roots of Z. mays L., which were 77 and 25% in Pb-amended soil, 42 and 73% in Zn-amended soil, and 27 and 84% in Pb + Zn-amended soil. Furthermore, addition of GW103 increased 8, 12, and 7% of total protein content, catalase, and superoxide dismutase levels, respectively, in Z. mays L. plants. The results pointed out that isolate GW103 could potentially reduce the phytotoxicity of metals and increase Pb and Zn accumulation in Z. mays L. plant.

  13. Somatic hypermutation of T cell receptor α chain contributes to selection in nurse shark thymus.

    Science.gov (United States)

    Ott, Jeannine A; Castro, Caitlin D; Deiss, Thaddeus C; Ohta, Yuko; Flajnik, Martin F; Criscitiello, Michael F

    2018-04-17

    Since the discovery of the T cell receptor (TcR), immunologists have assigned somatic hypermutation (SHM) as a mechanism employed solely by B cells to diversify their antigen receptors. Remarkably, we found SHM acting in the thymus on α chain locus of shark TcR. SHM in developing shark T cells likely is catalyzed by activation-induced cytidine deaminase (AID) and results in both point and tandem mutations that accumulate non-conservative amino acid replacements within complementarity-determining regions (CDRs). Mutation frequency at TcRα was as high as that seen at B cell receptor loci (BcR) in sharks and mammals, and the mechanism of SHM shares unique characteristics first detected at shark BcR loci. Additionally, fluorescence in situ hybridization showed the strongest AID expression in thymic corticomedullary junction and medulla. We suggest that TcRα utilizes SHM to broaden diversification of the primary αβ T cell repertoire in sharks, the first reported use in vertebrates. © 2018, Ott et al.

  14. Isolation and characterization of novel plant growth promoting Micrococcus sp NII-0909 and its interaction with cowpea.

    Science.gov (United States)

    Dastager, Syed G; Deepa, C K; Pandey, Ashok

    2010-12-01

    A phosphate-solubilizing bacterial strain NII-0909 isolated from the Western ghat forest soil in India was identified as Micrococcus sp on the basis of phenotypic characteristics, carbon source utilization pattern, fatty acid methyl esters analysis, and 16S rRNA gene sequence. The strain exhibited the plant growth-promoting attributes of phosphate solubilization, auxin production, 1-aminocyclopropane-1-carboxylate deaminase activity, and siderophore production. It was able to solubilize (122.4μg of Ca(3)PO(4) ml(-1)), and produce IAA (109μgml(-1)) at 30°C. P-solubilizing activity of the strain NII-0909 was associated with the release of organic acids and a drop in the pH of the NBRIP medium. HPLC analysis detected two organic acids in the course of P-solubilization. A significant increase in the growth of cow pea was recorded for inoculations under controlled conditions. Scanning electron microscopic study revealed the root colonization of strain on cow pea seedlings. These results demonstrate that isolates NII-0909 has the promising PGPR attributes to be develop as a biofertilizer to enhance soil fertility and promote the plant growth. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

  15. Effects of Vector Backbone and Pseudotype on Lentiviral Vector-mediated Gene Transfer: Studies in Infant ADA-Deficient Mice and Rhesus Monkeys

    Science.gov (United States)

    Carbonaro Sarracino, Denise; Tarantal, Alice F; Lee, C Chang I.; Martinez, Michele; Jin, Xiangyang; Wang, Xiaoyan; Hardee, Cinnamon L; Geiger, Sabine; Kahl, Christoph A; Kohn, Donald B

    2014-01-01

    Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options. PMID:24925206

  16. Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID

    Science.gov (United States)

    Sauer, Aisha V.; Brigida, Immacolata; Carriglio, Nicola; Jofra Hernandez, Raisa; Scaramuzza, Samantha; Clavenna, Daniela; Sanvito, Francesca; Poliani, Pietro L.; Gagliani, Nicola; Carlucci, Filippo; Tabucchi, Antonella; Roncarolo, Maria Grazia; Traggiai, Elisabetta; Villa, Anna

    2012-01-01

    Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)–mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA–treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA−/− Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA–treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA–treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID. Trials were registered at www.clinicaltrials.gov as NCT00598481/NCT00599781. PMID:22184407

  17. RPA accumulation during class switch recombination represents 5'-3' DNA-end resection during the S-G2/M phase of the cell cycle.

    Science.gov (United States)

    Yamane, Arito; Robbiani, Davide F; Resch, Wolfgang; Bothmer, Anne; Nakahashi, Hirotaka; Oliveira, Thiago; Rommel, Philipp C; Brown, Eric J; Nussenzweig, Andre; Nussenzweig, Michel C; Casellas, Rafael

    2013-01-31

    Activation-induced cytidine deaminase (AID) promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs) at immunoglobulin (Ig) genes and oncogenes in the G1 phase. RPA is a single-stranded DNA (ssDNA)-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR), such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here, we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, recombination-activating genes (RAG), or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in the S-G2/M phase is extensive, ATM independent, and associated with Rad51 accumulation. In the S-G2/M phase, RPA increases in nonhomologous-end-joining-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during class switch recombination represents salvage of unrepaired breaks by homology-based pathways during the S-G2/M phase of the cell cycle. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Impact of long term applications of cotton pesticides on soil biological properties, dissipation of [14C]-methyl parathion and persistence of multi-pesticide residues

    International Nuclear Information System (INIS)

    Andrea, M.M.; Peres, T.B.; Luchini, L.C.; Marcondes, M.A.; Pettinelli, A. Jr.; Nakagawa, L.E.

    2001-01-01

    Biological parameters were followed in soils from a cotton farm (Tatui) where the recommended pesticides have been used for years, and from an experimental field (Sao Paulo) which was subdivided in two areas: one received the recommended pesticides and the other was maintained untreated. The soil bioactivities monitored from 1995 to 1998, after different pesticide applications, were: basal and glucose-induced respiration; anaerobic activity; nitrification rate; activity of the enzymes: dehydrogenase, aryl sulfatase and arginine deaminase; the soil capacity to mineralize an aromatic pesticide molecule ([ 14 C]-2,4-D), fungal and bacterial contributions for soil respiration until the beginning of 1998, and fungal and bacterial numbers from the beginning of 1998. The dissipation of [ 14 C]-methyl parathion - one of the recommended pesticides - was followed by radiometric techniques only in Sao Paulo, but persistence of multi-residues was determined in both soils by gas-liquid chromatography. All the biological parameters varied each sampling time and values also varied among soil samples, being inhibited or stimulated by the different pesticide applications, but they mostly recovered the initially detected activity. Dissipation of methyl parathion was fast and not affected by the other pesticide applications. Pesticide residues varied between the two soils but were mostly low after all applications, which indicates their dissipation. (author)

  19. DNA-dependent protein kinase inhibits AID-induced antibody gene conversion.

    Directory of Open Access Journals (Sweden)

    Adam J L Cook

    2007-04-01

    Full Text Available Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID-dependent hypermutation of Ig V(DJ rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(DJ regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(DJ hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

  20. A Molecular Dynamics (MD and Quantum Mechanics/Molecular Mechanics (QM/MM Study on Ornithine Cyclodeaminase (OCD: A Tale of Two Iminiums

    Directory of Open Access Journals (Sweden)

    James W. Gauld

    2012-10-01

    Full Text Available Ornithine cyclodeaminase (OCD is an NAD+-dependent deaminase that is found in bacterial species such as Pseudomonas putida. Importantly, it catalyzes the direct conversion of the amino acid L-ornithine to L-proline. Using molecular dynamics (MD and a hybrid quantum mechanics/molecular mechanics (QM/MM method in the ONIOM formalism, the catalytic mechanism of OCD has been examined. The rate limiting step is calculated to be the initial step in the overall mechanism: hydride transfer from the L-ornithine’s Cα–H group to the NAD+ cofactor with concomitant formation of a Cα=NH2+ Schiff base with a barrier of 90.6 kJ mol−1. Importantly, no water is observed within the active site during the MD simulations suitably positioned to hydrolyze the Cα=NH2+ intermediate to form the corresponding carbonyl. Instead, the reaction proceeds via a non-hydrolytic mechanism involving direct nucleophilic attack of the δ-amine at the Cα-position. This is then followed by cleavage and loss of the α-NH2 group to give the Δ1-pyrroline-2-carboxylate that is subsequently reduced to L-proline.

  1. The absence of A-to-I editing in the anticodon of plant cytoplasmic tRNA (Arg) ACG demands a relaxation of the wobble decoding rules.

    Science.gov (United States)

    Aldinger, Carolin A; Leisinger, Anne-Katrin; Gaston, Kirk W; Limbach, Patrick A; Igloi, Gabor L

    2012-10-01

    It is a prevalent concept that, in line with the Wobble Hypothesis, those tRNAs having an adenosine in the first position of the anticodon become modified to an inosine at this position. Sequencing the cDNA derived from the gene coding for cytoplasmic tRNA (Arg) ACG from several higher plants as well as mass spectrometric analysis of the isoacceptor has revealed that for this kingdom an unmodified A in the wobble position of the anticodon is the rule rather than the exception. In vitro translation shows that in the plant system the absence of inosine in the wobble position of tRNA (Arg) does not prevent decoding. This isoacceptor belongs to the class of tRNA that is imported from the cytoplasm into the mitochondria of higher plants. Previous studies on the mitochondrial tRNA pool have demonstrated the existence of tRNA (Arg) ICG in this organelle. In moss the mitochondrial encoded distinct tRNA (Arg) ACG isoacceptor possesses the I34 modification. The implication is that for mitochondrial protein biosynthesis A-to-I editing is necessary and occurs by a mitochondrion-specific deaminase after import of the unmodified nuclear encoded tRNA (Arg) ACG.

  2. Hypoxia targeted bifunctional suicide gene expression enhances radiotherapy in vitro and in vivo

    International Nuclear Information System (INIS)

    Sun, Xiaorong; Xing, Ligang; Deng, Xuelong; Hsiao, Hung Tsung; Manami, Akiko; Koutcher, Jason A.; Clifton Ling, C.; Li, Gloria C.

    2012-01-01

    Purpose: To investigate whether hypoxia targeted bifunctional suicide gene expression-cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) with 5-FC treatments can enhance radiotherapy. Materials and methods: Stable transfectants of R3327-AT cells were established which express a triple-fusion-gene: CD, UPRT and monomoric DsRed (mDsRed) controlled by a hypoxia inducible promoter. Hypoxia-induced expression/function of CDUPRTmDsRed was verified by western blot, flow cytometry, fluorescent microscopy, and cytotoxicity assay of 5-FU and 5-FC. Tumor-bearing mice were treated with 5-FC and local radiation. Tumor volume was monitored and compared with those treated with 5-FC or radiation alone. In addition, the CDUPRTmDsRed distribution in hypoxic regions of tumor sections was visualized with fluorescent microscopy. Results: Hypoxic induction of CDUPRTmDsRed protein correlated with increased sensitivity to 5-FC and 5-FU. Significant radiosensitization effects were detected after 5-FC treatments under hypoxic conditions. In the tumor xenografts, the distribution of CDUPRTmDsRed expression visualized with fluorescence microscopy was co-localized with the hypoxia marker pimonidazole positive staining cells. Furthermore, administration of 5-FC to mice in combination with local irradiation resulted in significant tumor regression, as in comparison with 5-FC or radiation treatments alone. Conclusions: Our data suggest that the hypoxia-inducible CDUPRT/5-FC gene therapy strategy has the ability to specifically target hypoxic cancer cells and significantly improve the tumor control in combination with radiotherapy.

  3. Increased growth and root Cu accumulation of Sorghum sudanense by endophytic Enterobacter sp. K3-2: Implications for Sorghum sudanense biomass production and phytostabilization.

    Science.gov (United States)

    Li, Ya; Wang, Qi; Wang, Lu; He, Lin-Yan; Sheng, Xia-Fang

    2016-02-01

    Endophytic bacterial strain K3-2 was isolated from the roots of Sorghum sudanense (an bioenergy plant) grown in a Cu mine wasteland soils and characterized. Strain K3-2 was identified as Enterobacter sp. based on 16S rRNA gene sequence analysis. Strain K3-2 exhibited Cu resistance and produced 1-aminocyclopropane-1-carboxylate (ACC) deaminase, indole-3-acetic acid (IAA), siderophores, and arginine decarboxylase. Pot experiments showed that strain K3-2 significantly increased the dry weight and root Cu accumulation of Sorghum sudanense grown in the Cu mine wasteland soils. Furthermore, increase in total Cu uptake (ranging from 49% to 95%) of the bacterial inoculated-Sorghum sudanense was observed compared to the control. Notably, most of Cu (83-86%) was accumulated in the roots of Sorghum sudanense. Furthermore, inoculation with strain K3-2 was found to significantly increase Cu bioconcentration factors and the proportions of IAA- and siderophore-producing bacteria in the root interiors and rhizosphere soils of Sorghum sudanense compared with the control. Significant decrease in the available Cu content was also observed in the rhizosphere soils of the bacterial-inoculated Sorghum sudanense. The results suggest that the endophytic bacterial strain K3-2 may be exploited for promoting Sorghum sudanense biomass production and Cu phytostabilization in the Cu mining wasteland soils. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. RNA editing is induced by type I interferon in esophageal squamous cell carcinoma.

    Science.gov (United States)

    Zhang, Jinyao; Chen, Zhaoli; Tang, Zefang; Huang, Jianbing; Hu, Xueda; He, Jie

    2017-07-01

    In recent years, abnormal RNA editing has been shown to play an important role in the development of esophageal squamous cell carcinoma, as such abnormal editing is catalyzed by ADAR (adenosine deaminases acting on RNA). However, the regulatory mechanism of ADAR1 in esophageal squamous cell carcinomas remains largely unknown. In this study, we investigated ADAR1 expression and its association with RNA editing in esophageal squamous cell carcinomas. RNA sequencing applied to esophageal squamous cell carcinoma clinical samples showed that ADAR1 expression was correlated with the expression of STAT1, STAT2, and IRF9. In vitro experiments showed that the abundance of ADAR1 protein was associated with the induced activation of the JAK/STAT pathway by type I interferon. RNA sequencing results showed that treatment with type I interferon caused an increase in the number and degree of RNA editing in esophageal squamous cell carcinoma cell lines. In conclusion, the activation of the JAK/STAT pathway is a regulatory mechanism of ADAR1 expression and causes abnormal RNA editing profile in esophageal squamous cell carcinoma. This mechanism may serve as a new target for esophageal squamous cell carcinoma therapy.

  5. The uropathogenic species Staphylococcus saprophyticus tolerates a high concentration of D-serine.

    Science.gov (United States)

    Sakinç, Türkân; Michalski, Nadine; Kleine, Britta; Gatermann, Sören G

    2009-10-01

    Human urine contains a relatively high concentration of d-serine, which is toxic to several nonuropathogenic bacteria, but can be utilized or detoxified by uropathogenic Escherichia coli (UPEC). The sequenced genome of uropathogenic Staphylococcus saprophyticus contains a gene with homology to the d-serine deaminase gene (dsdA) of UPEC. We found the gene in several clinical isolates of S. saprophyticus; however, the gene was absent in Staphylococcus xylosus and Staphylococcus cohnii, phylogenetically close relatives of S. saprophyticus, and could also not be detected in isolates of Staphylococcus aureus, Staphylococcus epidermidis and 13 other staphylococcal species. In addition, the genomes of other sequenced staphylococci do not harbor homologues of this operon. Interestingly, S. saprophyticus could grow in media supplemented with relatively high concentrations of d-serine, whereas S. aureus, S. epidermidis and other staphylococcal species could not. The association of the dsdA gene with growth in media including d-serine was proved by introducing the gene into S. aureus Newman. Given the fact that UPEC and S. saprophyticus tolerate this compound, d-serine utilization and detoxification may be a general property of uropathogenic bacteria. © 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  6. Deoxycytidine and Deoxythymidine Treatment for Thymidine Kinase 2 Deficiency.

    Science.gov (United States)

    Lopez-Gomez, Carlos; Levy, Rebecca J; Sanchez-Quintero, Maria J; Juanola-Falgarona, Martí; Barca, Emanuele; Garcia-Diaz, Beatriz; Tadesse, Saba; Garone, Caterina; Hirano, Michio

    2017-05-01

    Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2 -/- ) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy. To test these hypotheses, we assessed two therapies in Tk2 -/- mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP. We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2 -/- animals compared to dCMP+dTMP. Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641-652. © 2017 American Neurological Association.

  7. IMP metabolism in human skeletal muscle after exhaustive exercise

    DEFF Research Database (Denmark)

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

    1995-01-01

    This study addressed whether AMP deaminase (AMPD)myosin binding occurs with deamination during intense exercise in humans and the extent of purine loss from muscle during the initial minutes of recovery. Male subjects performed cycle exercise (265 +/- 2 W for 4.39 +/- 0.04 min) to stimulate muscle...... 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...

  8. Alleviation of salt stress by halotolerant and halophilic plant growth-promoting bacteria in wheat (Triticum aestivum

    Directory of Open Access Journals (Sweden)

    Furkan Orhan

    Full Text Available ABSTRACT In the current study, 18 halotolerant and halophilic bacteria have been investigated for their plant growth promoting abilities in vitro and in a hydroponic culture. The bacterial strains have been investigated for ammonia, indole-3-acetic acid and 1-aminocyclopropane-1-carboxylate-deaminase production, phosphate solubilisation and nitrogen fixation activities. Of the tested bacteria, eight were inoculated with Triticum aestivum in a hydroponic culture. The investigated bacterial strains were found to have different plant-growth promoting activities in vitro. Under salt stress (200 mM NaCl, the investigated bacterial strains significantly increased the root and shoot length and total fresh weight of the plants. The growth rates of the plants inoculated with bacterial strains ranged from 62.2% to 78.1%.Identifying of novel halophilic and halotolerant bacteria that promote plant growth can be used as alternatives for salt sensitive plants. Extensive research has been conducted on several halophilic and halotolerant bacterial strains to investigate their plant growth promoting activities. However, to the best of my knowledge, this is the first study to inoculate these bacterial strains with wheat.

  9. Gut TFH and IgA: key players for regulation of bacterial communities and immune homeostasis.

    Science.gov (United States)

    Kato, Lucia M; Kawamoto, Shimpei; Maruya, Mikako; Fagarasan, Sidonia

    2014-01-01

    The main function of the immune system is to protect the host against pathogens. However, unlike the systemic immune system, the gut immune system does not eliminate, but instead nourishes complex bacterial communities and establishes advanced symbiotic relationships. Immunoglobulin A (IgA) is the most abundant antibody isotype in mammals, produced mainly in the gut. The primary function of IgA is to maintain homeostasis at mucosal surfaces, and studies in mice have demonstrated that IgA diversification has an essential role in the regulation of gut microbiota. Dynamic diversification and constant adaptation of IgA responses to local microbiota require expression of activation-induced cytidine deaminase by B cells and control from T follicular helper and Foxp3(+) T cells in germinal centers (GCs). We discuss the finely tuned regulatory mechanisms for IgA synthesis in GCs of Peyer's patches and emphasize the roles of CD4(+) T cells for IgA selection and the maintenance of appropriate gut microbial communities required for immune homeostasis.

  10. Malaria, Epstein-Barr virus infection and the pathogenesis of Burkitt's lymphoma.

    Science.gov (United States)

    Mawson, Anthony R; Majumdar, Suvankar

    2017-11-01

    A geographical and causal connection has long been recognized between malaria, Epstein-Barr virus (EBV) infection and Burkitt's lymphoma (BL), but the underlying mechanisms remain obscure. Potential clues are that the malaria parasite Plasmodium falciparum selectively absorbs vitamin A from the host and depends on it for its biological activities; secondly, alterations in vitamin A (retinoid) metabolism have been implicated in many forms of cancer, including BL. The first author has proposed that the merozoite-stage malaria parasite, emerging from the liver, uses its absorbed vitamin A as a cell membrane destabilizer to invade the red blood cells, causing anemia and other signs and symptoms of the disease as manifestations of an endogenous form of hypervitaminosis A (Mawson AR, Path Global Health 2013;107(3):122-9). Repeated episodes of malaria would therefore be expected to expose the tissues of affected individuals to potentially toxic doses of vitamin A. It is proposed that such episodes activate latent EBV infection, which in turn activates retinoid-responsive genes. Expression of these genes enhances viral replication and induces germinal center (GC) B cell expansion, activation-induced cytidine deaminase (AID) expression, and c-myc translocation, which in turn predisposes to BL. Thus, an endogenous form of retinoid toxicity related to malaria infection may be the common factor linking frequent malaria, EBV infection and BL, whereby prolonged exposure of lymphatic tissues to high concentrations of retinoids may combine to induce B-cell translocation and increase the risk of Burkitt's lymphoma. © 2017 UICC.

  11. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    Science.gov (United States)

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

  12. Rhizosphere bacteria of Costularia spp. from ultramafic soils in New Caledonia: diversity, tolerance to extreme edaphic conditions, and role in plant growth and mineral nutrition.

    Science.gov (United States)

    Gonin, Mathieu; Gensous, Simon; Lagrange, Alexandre; Ducousso, Marc; Amir, Hamid; Jourand, Philippe

    2013-03-01

    Rhizosphere bacteria were isolated from Costularia spp., pioneer sedges from ultramafic soils in New Caledonia, which is a hotspot of biodiversity in the South Pacific. Genus identification, ability to tolerate edaphic constraints, and plant-growth-promoting (PGP) properties were analysed. We found that 10(5) colony-forming units per gram of root were dominated by Proteobacteria (69%) and comprised 21 genera, including Burkholderia (28%), Curtobacterium (15%), Bradyrhizobium (9%), Sphingomonas (8%), Rhizobium (7%), and Bacillus (5%). High proportions of bacteria tolerated many elements of the extreme edaphic conditions: 82% tolerated 100 μmol·L(-1) chromium, 70% 1 mmol·L(-1) nickel, 63% 10 mmol·L(-1) manganese, 24% 1 mmol·L(-1) cobalt, and 42% an unbalanced calcium/magnesium ratio (1/16). These strains also exhibited multiple PGP properties, including the ability to produce ammonia (65%), indole-3-acetic acid (60%), siderophores (52%), and 1-aminocyclopropane-1-carboxylate (ACC) deaminase (39%); as well as the capacity to solubilize phosphates (19%). The best-performing strains were inoculated with Sorghum sp. grown on ultramafic substrate. Three strains significantly enhanced the shoot biomass by up to 33%. The most successful strains influenced plant nutrition through the mobilization of metals in roots and a reduction of metal transfer to shoots. These results suggest a key role of these bacteria in plant growth, nutrition, and adaptation to the ultramafic constraints.

  13. Genome Sequencing of a Mung Bean Plant Growth Promoting Strain of P. aeruginosa with Biocontrol Ability

    Directory of Open Access Journals (Sweden)

    Devaraj Illakkiam

    2014-01-01

    Full Text Available Pseudomonas aeruginosa PGPR2 is a mung bean rhizosphere strain that produces secondary metabolites and hydrolytic enzymes contributing to excellent antifungal activity against Macrophomina phaseolina, one of the prevalent fungal pathogens of mung bean. Genome sequencing was performed using the Ion Torrent Personal Genome Machine generating 1,354,732 reads (6,772,433 sequenced bases achieving ~25-fold coverage of the genome. Reference genome assembly using MIRA 3.4.0 yielded 198 contigs. The draft genome of PGPR2 encoded 6803 open reading frames, of which 5314 were genes with predicted functions, 1489 were genes of known functions, and 80 were RNA-coding genes. Strain specific and core genes of P. aeruginosa PGPR2 that are relevant to rhizospheric habitat were identified by pangenome analysis. Genes involved in plant growth promoting function such as synthesis of ACC deaminase, indole-3-acetic acid, trehalose, mineral scavenging siderophores, hydrogen cyanide, chitinases, acyl homoserine lactones, acetoin, 2,3-butanediol, and phytases were identified. In addition, niche-specific genes such as phosphate solubilising 3-phytase, adhesins, pathway-specific transcriptional regulators, a diguanylate cyclase involved in cellulose synthesis, a receptor for ferrienterochelin, a DEAD/DEAH-box helicase involved in stress tolerance, chemotaxis/motility determinants, an HtpX protease, and enzymes involved in the production of a chromanone derivative with potent antifungal activity were identified.

  14. A continuous spectrophotometric assay for monitoring adenosine 5'-monophosphate production.

    Science.gov (United States)

    First, Eric A

    2015-08-15

    A number of biologically important enzymes release adenosine 5'-monophosphate (AMP) as a product, including aminoacyl-tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5'-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5'-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD(+)) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. High frequencies of chromatid aberrations produced during G/sub 2/ in human lymphocytes by very low doses (0. 025-0. 4 Gy) of X-rays in combination with inhibitors of DNA synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Andersson, H.C.; Kihlman, B.A. (Uppsala Univ. (Sweden). Dept. of Genetics)

    1984-09-01

    Whole-blood cultures of human lymphocytes were exposed in the G/sub 2/-phase (3.5 h before harvesting) to various doses of X-rays and post-treated for 3 h with inhibitors of DNA synthesis. The inhibitors used were 2'-deoxyadenosine (dAdo), hydroxyurea (HU) and 1-..beta..-D-arabinofuranosylcytosine (ara-C). To prevent deamination of dAdo by adenosine deaminase (ADA), the dAdo treatments were carried out in the presence of the ADA inhibitor coformycin. HU and ara-C were used either alone or in combination. After the 3-h inhibitor treatments, the cultures were harvested and slides prepared and analyzed for chromatid aberrations in metaphase. When the inhibitors were used at concentrations high enough to cause marked chromosome damage by themselves, very low doses of X-rays (0.025-0.2 Gy) were sufficient to produce a dramatic increase in the frequency of chromatid aberrations. High frequencies of chromatid aberrations were also obtained when cultures that had received moderate doses of X-rays (0.4-0.8 Gy) were post-treated with low inhibitor concentrations that produce no or only a few aberrations by themselves.

  16. Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs.

    Science.gov (United States)

    Girelli Zubani, Giulia; Zivojnovic, Marija; De Smet, Annie; Albagli-Curiel, Olivier; Huetz, François; Weill, Jean-Claude; Reynaud, Claude-Agnès; Storck, Sébastien

    2017-04-03

    During somatic hypermutation (SHM) of immunoglobulin genes, uracils introduced by activation-induced cytidine deaminase are processed by uracil-DNA glycosylase (UNG) and mismatch repair (MMR) pathways to generate mutations at G-C and A-T base pairs, respectively. Paradoxically, the MMR-nicking complex Pms2/Mlh1 is apparently dispensable for A-T mutagenesis. Thus, how detection of U:G mismatches is translated into the single-strand nick required for error-prone synthesis is an open question. One model proposed that UNG could cooperate with MMR by excising a second uracil in the vicinity of the U:G mismatch, but it failed to explain the low impact of UNG inactivation on A-T mutagenesis. In this study, we show that uracils generated in the G1 phase in B cells can generate equal proportions of A-T and G-C mutations, which suggests that UNG and MMR can operate within the same time frame during SHM. Furthermore, we show that Ung -/- Pms2 -/- mice display a 50% reduction in mutations at A-T base pairs and that most remaining mutations at A-T bases depend on two additional uracil glycosylases, thymine-DNA glycosylase and SMUG1. These results demonstrate that Pms2/Mlh1 and multiple uracil glycosylases act jointly, each one with a distinct strand bias, to enlarge the immunoglobulin gene mutation spectrum from G-C to A-T bases. © 2017 Girelli Zubani et al.

  17. MSH6- or PMS2-deficiency causes re-replication in DT40 B cells, but it has little effect on immunoglobulin gene conversion or on repair of AID-generated uracils

    Science.gov (United States)

    Campo, Vanina A.; Patenaude, Anne-Marie; Kaden, Svenja; Horb, Lori; Firka, Daniel; Jiricny, Josef; Di Noia, Javier M.

    2013-01-01

    The mammalian antibody repertoire is shaped by somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) loci of B lymphocytes. SHM and CSR are triggered by non-canonical, error-prone processing of G/U mismatches generated by activation-induced deaminase (AID). In birds, AID does not trigger SHM, but it triggers Ig gene conversion (GC), a ‘homeologous’ recombination process involving the Ig variable region and proximal pseudogenes. Because recombination fidelity is controlled by the mismatch repair (MMR) system, we investigated whether MMR affects GC in the chicken B cell line DT40. We show here that Msh6−/− and Pms2−/− DT40 cells display cell cycle defects, including genomic re-replication. However, although IgVλ GC tracts in MMR-deficient cells were slightly longer than in normal cells, Ig GC frequency, donor choice or the number of mutations per sequence remained unaltered. The finding that the avian MMR system, unlike that of mammals, does not seem to contribute towards the processing of G/U mismatches in vitro could explain why MMR is unable to initiate Ig GC in this species, despite initiating SHM and CSR in mammalian cells. Moreover, as MMR does not counteract or govern Ig GC, we report a rare example of ‘homeologous’ recombination insensitive to MMR. PMID:23314153

  18. CD25+ B-1a Cells Express Aicda

    Directory of Open Access Journals (Sweden)

    Hiroaki Kaku

    2017-06-01

    Full Text Available B-1a cells are innate-like B-lymphocytes producing natural antibodies. Activation-induced cytidine deaminase (AID, a product of the Aicda gene, plays a central role in class-switch recombination and somatic hypermutation in B cells. Although a role for Aicda in B-1a cells has been suggested on the basis of experiments with knock out (KO mice, whether B-1a cells express Aicda, and if so, which B-1a cell subpopulation expresses Aicda, remains unknown. Here, we demonstrate that B-1 cells express Aicda, but at a level below that expressed by germinal center (GC B cells. We previously reported that B-1a cells can be subdivided based on CD25 expression. We show here that B-1a cell Aicda expression is concentrated in the CD25+ B-1a cell subpopulation. These results suggest the possibility that previous studies of memory B cells identified on the basis of Aicda expression may have inadvertently included an unknown number of CD25+ B-1a cells. Although B-1a cells develop normally in the absence of Aicda, a competitive reconstitution assay reveals enhanced vigor for AID KO B-1a cell bone marrow (BM progenitors, as compared with wild-type BM B-1 cell progenitors. These results suggest that AID inhibits the development of B-1a cells from BM B-1 cell progenitors in a competitive environment.

  19. Paracetamol - toxicity and microbial utilization. Pseudomonas moorei KB4 as a case study for exploring degradation pathway.

    Science.gov (United States)

    Żur, Joanna; Wojcieszyńska, Danuta; Hupert-Kocurek, Katarzyna; Marchlewicz, Ariel; Guzik, Urszula

    2018-09-01

    Paracetamol, a widely used analgesic and antipyretic drug, is currently one of the most emerging pollutants worldwide. Besides its wide prevalence in the literature only several bacterial strains able to degrade this compound have been described. In this study, we isolated six new bacterial strains able to remove paracetamol. The isolated strains were identified as the members of Pseudomonas, Bacillus, Acinetobacter and Sphingomonas genera and characterized phenotypically and biochemically using standard methods. From the isolated strains, Pseudomonas moorei KB4 was able to utilize 50 mg L -1 of paracetamol. As the main degradation products, p-aminophenol and hydroquinone were identified. Based on the measurements of specific activity of acyl amidohydrolase, deaminase and hydroquinone 1,2-dioxygenase and the results of liquid chromatography analyses, we proposed a mechanism of paracetamol degradation by KB4 strain under co-metabolic conditions with glucose. Additionally, toxicity bioassays and the influence of various environmental factors, including pH, temperature, heavy metals at no-observed-effective-concentrations, and the presence of aromatic compounds on the efficiency and mechanism of paracetamol degradation by KB4 strain were determined. This comprehensive study about paracetamol biodegradation will be helpful in designing a treatment systems of wastewaters contaminated with paracetamol. Copyright © 2018 Elsevier Ltd. All rights reserved.

  20. Involvement of purinergic signaling on nitric oxide production by neutrophils stimulated with Trichomonas vaginalis.

    Science.gov (United States)

    Frasson, Amanda Piccoli; De Carli, Geraldo Attilio; Bonan, Carla Denise; Tasca, Tiana

    2012-03-01

    Trichomonas vaginalis is a parasite from the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. The neutrophil infiltration has been considered to be primarily responsible for cytological changes observed at infection site, and the chemoattractants can play an important role in this leukocytic recruitment. Nitric oxide (NO) is one of the most widespread mediator compounds, and it is implicated in modulation of immunological mechanisms. Extracellular nucleotides and nucleosides are signaling molecules involved in several processes, including immune responses and control of leukocyte trafficking. Ectonucleoside triphosphate diphosphohydrolase members, ecto-5'-nucleotidase, and adenosine deaminase (ectoADA) have been characterized in T. vaginalis. Herein, we investigated the effects of purinergic system on NO production by neutrophils stimulated with T. vaginalis. The trophozoites were able to induce a high NO synthesis by neutrophils through iNOS pathway. The extracellular nucleotides ATP, ADP, and ATPγS (a non-hydrolyzable ATP analog) showed no significant change in NO secretion. In contrast, adenosine and its degradation product, inosine, promoted a low production of the compound. The immunosuppressive effect of adenosine upon NO release by neutrophils occurred due to adenosine A(2A) receptor activation. The ecto-5'-nucleotidase activity displayed by T. vaginalis was shown to be important in adenosine generation, indicating the efficiency of purinergic cascade. Our data suggest the influence of purinergic signaling, specifically adenosinergic system, on NO production by neutrophils in T. vaginalis infection, contributing to the immunological aspects of disease.

  1. Hypoxia-targeted suicidal gene therapy system enhances antitumor effects of radiotherapy

    International Nuclear Information System (INIS)

    Liu Junye; Guo Yao; Guo Guozhen

    2006-01-01

    Objective: To explore the effects of hypoxia-targeted suicidal gene therapy system combined with radiotherapy on pancreatic cancer. Methods: The recombinant adenovirus Ad-5HRE/hCMVmp-BCD was constructed by DNA recombinant technique. Western blot was used to detect hypoxia-induced expression of bacterial cytosine deaminase (BCD). Cell growth inhibition assay was used to determine the sensitivity of human pancreatic cancer cells MIA-PACA2 to 5-fluorocytosine (5-FC). Tumor xenograft growth delay assays was used to evaluate the effects of Ad-5HRE/hCMVmp-BCD/5-FC combined with radiotherapy on pancreatic cancer. Results: Western blot analysis demonstrated that hypoxia-induced BCD protein expression was achieved in MIA-PACA2 cells infected with Ad-5HRE/hCMVmp-BCD. With hypoxia treatment, the sensitivity of MIA-PACA2 cells infected with Ad-5HRE/hCMVmp-BCD to 5-FC significantly increased. Administration of either Ad-5HRE/hCMVmp-BCD/5-FC or radiotherapy could inhibit the growth of MIA-PACA2 xenografts in nude mice. Moreover, combination of Ad-5HRE/hCMVmp-BCD/5-FC could significantly enhance suppressing effects of radiotherapy on MIA-PACA2 xenografts. Conclusion: Hypoxia-targeted suicidal gene therapy system Ad-5HRE/hCMVmp-BCD/5-FC could enhance antitumor effects of radiotherapy on pancreatic cancer and can be used as a powerful adjunct to conventional radiotherapy. (authors)

  2. Gene Directed Enzyme Prodrug Therapy Using Rabbit Cytochrome P450 4B1 in Murine Colon Adenocarcinoma

    International Nuclear Information System (INIS)

    Kim, Sung Joo; Kang, Joo Hyun; Lee, Tae Sup; Kim, Kyeong Min; Woo, Kwang Sun; Chung, Wee Sup; Cheon, Gi Jeong; Choi, Chang Woon; Lim, Sang Moo

    2007-01-01

    The conventional cancer therapy is chemotherapy, surgical resection and/or radiotherapy. Chemotherapy using cytotoxic drug has some problems with lack of tumor selectivity resulting in toxicity to normal tissues. To enhance the tumor selectivity of cytotoxic drug, the application of suicidal gene therapy technology was designed. Suicidal gene therapy is based on the expression in tumor cells of a gene encoding an enzyme that converts a non-toxic prodrug into a cytotoxic product. Representative suicidal genes are Herpes simplex virus type 1 thymidine kinase (HSV1- tk) and cytosine deaminase (cd). Recently, a new prodrug-converting enzyme based on rabbit cytochrome P450 4B1 gene (cyp4B1) has been reported for therapy of experimental brain tumor. This enzyme activates the prodrugs such as 4-ipomeanol (4-IM) and 2- aminoanthracene (2-AA) to highly reactive furane epoxide and unsaturated dialdehyde intermediate, respectively. DNA alkylation seems to be the main mechanism of cytotoxicity of these activated drugs. In this study, we isolated cyp4B1 cDNA from rabbit lung, transduced cyp4B1 expression vector into murine colon cancer cell, and then analyzed the cytotoxic properties of cyp4b1-activated 2-AA in cyp4B1 transduced cells to verify the cyp4B1 enzyme system for gene directed enzyme prodrug therapy

  3. APOBEC3G inhibits elongation of HIV-1 reverse transcripts.

    Directory of Open Access Journals (Sweden)

    Kate N Bishop

    2008-12-01

    Full Text Available APOBEC3G (A3G is a host cytidine deaminase that, in the absence of Vif, restricts HIV-1 replication and reduces the amount of viral DNA that accumulates in cells. Initial studies determined that A3G induces extensive mutation of nascent HIV-1 cDNA during reverse transcription. It has been proposed that this triggers the degradation of the viral DNA, but there is now mounting evidence that this mechanism may not be correct. Here, we use a natural endogenous reverse transcriptase assay to show that, in cell-free virus particles, A3G is able to inhibit HIV-1 cDNA accumulation not only in the absence of hypermutation but also without the apparent need for any target cell factors. We find that although reverse transcription initiates in the presence of A3G, elongation of the cDNA product is impeded. These data support the model that A3G reduces HIV-1 cDNA levels by inhibiting synthesis rather than by inducing degradation.

  4. Effect of pesticide applications on soil microbial activity and on 14C-methyl parathion dissipation

    International Nuclear Information System (INIS)

    Peres, Terezinha Bonanho

    2000-01-01

    Some crops, as cotton, need different pesticide application to control pests and diseases. These compounds reach soil and may affect the soil microbial activity. As the microorganisms play important role on the nutrient cycling, changes in their activities may affect the soil fertility. The influence of several pesticides on soil microbial activity of the 0-15 cm and 15-30 cm depth of the soil profile, and the 14 C-methyl parathion dissipation was studied under influence of other pesticide applications. The influence of pesticides on the microorganisms was followed in an experimental area of the Instituto Biologico, that was divided in two subareas, both under cotton crop. Columns of PVC was buried in both subareas and a solution of 14 C-methyl parathion diluted in the technical compound was applied on the soil surface of each column. One subarea received all the recommended pesticides for the cotton crop besides the 14 C-methyl parathion. The other subarea received only 14 C-methyl parathion solution on the columns soil surface. The soil microbial activity of both subareas was estimated by measurements of dehydrogenase, arylsulfatase and arginine deaminase enzymes. Further, the availability of total nitrogen in the soil was also measured. The dissipation of 14 C-methyl parathion was studied by radiocarbon recovery in soil extracts and combustion of extracted soil and quantification by radiometric techniques. (author)

  5. Plant Growth Promoting Rhizobacteria in Amelioration of Salinity Stress: A Systems Biology Perspective

    Directory of Open Access Journals (Sweden)

    Gayathri Ilangumaran

    2017-10-01

    Full Text Available Salinity affects plant growth and is a major abiotic stress that limits crop productivity. It is well-understood that environmental adaptations and genetic traits regulate salinity tolerance in plants, but imparting the knowledge gained towards crop improvement remain arduous. Harnessing the potential of beneficial microorganisms present in the rhizosphere is an alternative strategy for improving plant stress tolerance. This review intends to elucidate the understanding of salinity tolerance mechanisms attributed by plant growth promoting rhizobacteria (PGPR. Recent advances in molecular studies have yielded insights into the signaling networks of plant–microbe interactions that contribute to salt tolerance. The beneficial effects of PGPR involve boosting key physiological processes, including water and nutrient uptake, photosynthesis, and source-sink relationships that promote growth and development. The regulation of osmotic balance and ion homeostasis by PGPR are conducted through modulation of phytohormone status, gene expression, protein function, and metabolite synthesis in plants. As a result, improved antioxidant activity, osmolyte accumulation, proton transport machinery, salt compartmentalization, and nutrient status reduce osmotic stress and ion toxicity. Furthermore, in addition to indole-3-acetic acid and 1-aminocyclopropane-1-carboxylic acid deaminase biosynthesis, other extracellular secretions of the rhizobacteria function as signaling molecules and elicit stress responsive pathways. Application of PGPR inoculants is a promising measure to combat salinity in agricultural fields, thereby increasing global food production.

  6. The inhibitory effect of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) and its family members on the activity of cellular microRNAs.

    Science.gov (United States)

    Zhang, Hui

    2010-01-01

    The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or APOBEC3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. However, the cellular function of APOBEC3G remains to be further clarified. It has been reported that APOBEC3s can restrict the mobility of endogenous retroviruses and LTR-retrotransposons, suggesting that they can maintain stability in host genomes. However, APOBEC3G is normally cytoplasmic. Further studies have demonstrated that it is associated with an RNase-sensitive high molecular mass (HMM) and located in processing bodies (P-bodies) of replicating T-cells, indicating that the major cellular function of APOBEC3G seems to be related to P-body-related RNA processing and metabolism. As the function of P-body is closely related to miRNA activity, APOBEC3G could affect the miRNA function. Recent studies have demonstrated that APOBEC3G and its family members counteract miRNA-mediated repression of protein translation. Further, APOBEC3G enhances the association of miRNA-targeted mRNA with polysomes, and facilitates the dissociation of miRNA-targeted mRNA from P-bodies. As such, APOBEC3G regulate the activity of cellular miRNAs. Whether this function is related to its potent antiviral activity remains to be further determined.

  7. CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

    Directory of Open Access Journals (Sweden)

    Shweta Jain

    Full Text Available Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID, signifying class switch recombination (CSR. Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

  8. CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

    Science.gov (United States)

    Jain, Shweta; Chodisetti, Sathi Babu; Agrewala, Javed N

    2011-01-01

    Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

  9. Suppression of the E. coli SOS response by dNTP pool changes.

    Science.gov (United States)

    Maslowska, Katarzyna H; Makiela-Dzbenska, Karolina; Fijalkowska, Iwona J; Schaaper, Roel M

    2015-04-30

    The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30(+)-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction. Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  10. In vitro and in vivo studies of pharmacokinetics and antitumor efficacy of D07001-F4, an oral gemcitabine formulation.

    Science.gov (United States)

    Hao, Wei-Hua; Wang, Jong-Jing; Hsueh, Shu-Ping; Hsu, Pei-Jing; Chang, Li-Chien; Hsu, Chang-Shan; Hsu, Kuang-Yang

    2013-02-01

    The chemotherapy agent gemcitabine is currently administered intravenously because the drug has poor oral bioavailability. In order to assess the pharmacokinetics and antitumor activity of D07001-F4, a new self-microemulsifying oral drug delivery system preparation of gemcitabine, this study was performed to compare the effect of D07001-F4 with administered gemcitabine in vitro and in vivo. D07001-F4 pharmacokinetics was examined by evaluation of in vitro deamination of D07001-F4 and gemcitabine hydrochloride by recombinant human cytidine deaminase (rhCDA) and in vivo evaluation of D07001-F4 pharmacokinetics in mice. Antitumor activity was evaluated by comparing the effect of D07001-F4 and gemcitabine hydrochloride in inhibiting growth in nine cancer cell lines and by examining the effect of D07001-F4 and gemcitabine in two xenograft tumor models in mice. In vitro deamination of D07001-F4 by rhCDA was 3.3-fold slower than deamination of gemcitabine hydrochloride. Growth inhibition by D07001-F4 of 7 of the 8 cancer cell lines was increased compared with that seen with gemcitabine hydrochloride, and D07001-F4 inhibited the growth of pancreatic and colon cancer xenografts. In vivo pharmacokinetics showed the oral bioavailability of D07001-F4 to be 34%. D07001-F4 was effective against several cancer types, was metabolized more slowly than gemcitabine hydrochloride, and exhibited enhanced oral bioavailability.

  11. AZIN1 RNA editing confers cancer stemness and enhances oncogenic potential in colorectal cancer.

    Science.gov (United States)

    Shigeyasu, Kunitoshi; Okugawa, Yoshinaga; Toden, Shusuke; Miyoshi, Jinsei; Toiyama, Yuji; Nagasaka, Takeshi; Takahashi, Naoki; Kusunoki, Masato; Takayama, Tetsuji; Yamada, Yasuhide; Fujiwara, Toshiyoshi; Chen, Leilei; Goel, Ajay

    2018-06-21

    Adenosine-to-inosine (A-to-I) RNA editing, a process mediated by adenosine deaminases that act on the RNA (ADAR) gene family, is a recently discovered epigenetic modification dysregulated in human cancers. However, the clinical significance and the functional role of RNA editing in colorectal cancer (CRC) remain unclear. We have systematically and comprehensively investigated the significance of the expression status of ADAR1 and of the RNA editing levels of antizyme inhibitor 1 (AZIN1), one of the most frequently edited genes in cancers, in 392 colorectal tissues from multiple independent CRC patient cohorts. Both ADAR1 expression and AZIN1 RNA editing levels were significantly elevated in CRC tissues when compared with corresponding normal mucosa. High levels of AZIN1 RNA editing emerged as a prognostic factor for overall survival and disease-free survival and were an independent risk factor for lymph node and distant metastasis. Furthermore, elevated AZIN1 editing identified high-risk stage II CRC patients. Mechanistically, edited AZIN1 enhances stemness and appears to drive the metastatic processes. We have demonstrated that edited AZIN1 functions as an oncogene and a potential therapeutic target in CRC. Moreover, AZIN1 RNA editing status could be used as a clinically relevant prognostic indicator in CRC patients.

  12. Inoculation of Brassica oxyrrhina with plant growth promoting bacteria for the improvement of heavy metal phytoremediation under drought conditions.

    Science.gov (United States)

    Ma, Ying; Rajkumar, Mani; Zhang, Chang; Freitas, Helena

    2016-12-15

    The aim of this study was to investigate the effects of drought resistant serpentine rhizobacteria on plant growth and metal uptake by Brassica oxyrrhina under drought stress (DS) condition. Two drought resistant serpentine rhizobacterial strains namely Pseudomonas libanensis TR1 and Pseudomonas reactans Ph3R3 were selected based on their ability to stimulate seedling growth in roll towel assay. Further assessment on plant growth promoting (PGP) parameters revealed their ability to produce indole-3-acetic acid, siderophore and 1-aminocyclopropane-1-carboxylate deaminase. Moreover, both strains exhibited high resistance to various heavy metals, antibiotics, salinity and extreme temperature. Inoculation of TR1 and Ph3R3 significantly increased plant growth, leaf relative water and pigment content of B. oxyrrhina, whereas decreased concentrations of proline and malondialdehyde in leaves under metal stress in the absence and presence of DS. Regardless of soil water conditions, TR1 and Ph3R3 greatly improved organ metal concentrations, translocation and bioconcentration factors of Cu and Zn. The successful colonization and metabolic activities of P. libanensis TR1 and P. reactans Ph3R3 represented positive effects on plant development and metal phytoremediation under DS. These results indicate that these strains could be used as bio-inoculants for the improvement of phytoremediation of metal polluted soils under semiarid conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID.

    Science.gov (United States)

    Wang, Meng; Rada, Cristina; Neuberger, Michael S

    2010-01-18

    High-affinity antibodies are generated by somatic hypermutation with nucleotide substitutions introduced into the IgV in a semirandom fashion, but with intrinsic mutational hotspots strategically located to optimize antibody affinity maturation. The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5'-flanking nucleotide. As a critical test of whether such DNA deamination activity underpins antibody diversification and to gain insight into the extent to which the antibody mutation spectrum is dependent on the intrinsic substrate specificity of AID, we investigated whether it is possible to change the IgV mutation spectrum by altering AID's active site such that it prefers a pyrimidine (rather than a purine) flanking the targeted deoxycytidine. Consistent with the DNA deamination mechanism, B cells expressing the modified AID proteins yield altered IgV mutation spectra (exhibiting a purine-->pyrimidine shift in flanking nucleotide preference) and altered hotspots. However, AID-catalyzed deamination of IgV targets in vitro does not yield the same degree of hotspot dominance to that observed in vivo, indicating the importance of features beyond AID's active site and DNA local sequence environment in determining in vivo hotspot dominance.

  14. Histone H3.3 promotes IgV gene diversification by enhancing formation of AID-accessible single-stranded DNA.

    Science.gov (United States)

    Romanello, Marina; Schiavone, Davide; Frey, Alexander; Sale, Julian E

    2016-07-01

    Immunoglobulin diversification is driven by activation-induced deaminase (AID), which converts cytidine to uracil within the Ig variable (IgV) regions. Central to the recruitment of AID to the IgV genes are factors that regulate the generation of single-stranded DNA (ssDNA), the enzymatic substrate of AID Here, we report that chicken DT40 cells lacking variant histone H3.3 exhibit reduced IgV sequence diversification. We show that this results from impairment of the ability of AID to access the IgV genes due to reduced formation of ssDNA during IgV transcription. Loss of H3.3 also diminishes IgV R-loop formation. However, reducing IgV R-loops by RNase HI overexpression in wild-type cells does not affect IgV diversification, showing that these structures are not necessary intermediates for AID access. Importantly, the reduction in the formation of AID-accessible ssDNA in cells lacking H3.3 is independent of any effect on the level of transcription or the kinetics of RNAPII elongation, suggesting the presence of H3.3 in the nucleosomes of the IgV genes increases the chances of the IgV DNA becoming single-stranded, thereby creating an effective AID substrate. © 2016 MRC Laboratory of Molecular Biology. Published under the terms of the CC BY 4.0 license.

  15. GANP regulates the choice of DNA repair pathway by DNA-PKcs interaction in AID-dependent IgV region diversification.

    Science.gov (United States)

    Eid, Mohammed Mansour Abbas; Maeda, Kazuhiko; Almofty, Sarah Ameen; Singh, Shailendra Kumar; Shimoda, Mayuko; Sakaguchi, Nobuo

    2014-06-15

    RNA export factor germinal center-associated nuclear protein (GANP) interacts with activation-induced cytidine deaminase (AID) and shepherds it from the cytoplasm to the nucleus and toward the IgV region loci in B cells. In this study, we demonstrate a role for GANP in the repair of AID-initiated DNA damage in chicken DT40 B cells to generate IgV region diversity by gene conversion and somatic hypermutation. GANP plays a positive role in IgV region diversification of DT40 B cells in a nonhomologous end joining-proficient state. DNA-PKcs physically interacts with GANP, and this interaction is dissociated by dsDNA breaks induced by a topoisomerase II inhibitor, etoposide, or AID overexpression. GANP affects the choice of DNA repair mechanism in B cells toward homologous recombination rather than nonhomologous end joining repair. Thus, GANP presumably plays a critical role in protection of the rearranged IgV loci by favoring homologous recombination of the DNA breaks under accelerated AID recruitment. Copyright © 2014 by The American Association of Immunologists, Inc.

  16. Random-walk enzymes

    Science.gov (United States)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C →U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  17. Sampling protein motion and solvent effect during ligand binding

    Science.gov (United States)

    Limongelli, Vittorio; Marinelli, Luciana; Cosconati, Sandro; La Motta, Concettina; Sartini, Stefania; Mugnaini, Laura; Da Settimo, Federico; Novellino, Ettore; Parrinello, Michele

    2012-01-01

    An exhaustive description of the molecular recognition mechanism between a ligand and its biological target is of great value because it provides the opportunity for an exogenous control of the related process. Very often this aim can be pursued using high resolution structures of the complex in combination with inexpensive computational protocols such as docking algorithms. Unfortunately, in many other cases a number of factors, like protein flexibility or solvent effects, increase the degree of complexity of ligand/protein interaction and these standard techniques are no longer sufficient to describe the binding event. We have experienced and tested these limits in the present study in which we have developed and revealed the mechanism of binding of a new series of potent inhibitors of Adenosine Deaminase. We have first performed a large number of docking calculations, which unfortunately failed to yield reliable results due to the dynamical character of the enzyme and the complex role of the solvent. Thus, we have stepped up the computational strategy using a protocol based on metadynamics. Our approach has allowed dealing with protein motion and solvation during ligand binding and finally identifying the lowest energy binding modes of the most potent compound of the series, 4-decyl-pyrazolo[1,5-a]pyrimidin-7-one. PMID:22238423

  18. Involvement of cholinergic and adenosinergic systems on the branchial immune response of experimentally infected silver catfish with Streptococcus agalactiae.

    Science.gov (United States)

    Baldissera, M D; Souza, C F; Doleski, P H; Moreira, K L S; da Veiga, M L; da Rocha, M I U M; Santos, R C V; Baldisserotto, B

    2018-01-01

    It has been recognized that the cholinergic and adenosinergic systems have an essential role in immune and inflammatory responses during bacterial fish pathogens, such as the enzymes acetylcholinesterase (AChE) and adenosine deaminase (ADA), which are responsible for catalysis of the anti-inflammatory molecules acetylcholine (ACh) and adenosine (Ado) respectively. Thus, the aim of this study was to investigate the involvement of the cholinergic and adenosinergic systems on the immune response and inflammatory process in gills of experimentally infected Rhamdia quelen with Streptococcus agalactiae. Acetylcholinesterase activity decreased, while ACh levels increased in gills of infected animals compared to uninfected animals. On the other hand, a significant increase in ADA activity with a concomitant decrease in Ado levels was observed in infected animals compared to uninfected animals. Based on this evidence, we concluded that infection by S. agalactiae in silver catfish alters the cholinergic and adenosinergic systems, suggesting the involvement of AChE and ADA activities on immune and inflammatory responses, regulating the ACh and Ado levels. In summary, the downregulation of AChE activity exerts an anti-inflammatory profile in an attempt to reduce or prevent the tissue damage, while the upregulation of ADA activity exerts a pro-inflammatory profile, contributing to disease pathophysiology. © 2017 John Wiley & Sons Ltd.

  19. Chemovirotherapy for head and neck squamous cell carcinoma with EGFR-targeted and CD/UPRT-armed oncolytic measles virus.

    Science.gov (United States)

    Zaoui, K; Bossow, S; Grossardt, C; Leber, M F; Springfeld, C; Plinkert, P K; Kalle, C von; Ungerechts, G

    2012-03-01

    First-line treatment of recurrent and/or refractory head and neck squamous cell carcinoma (HNSCC) is based on platinum, 5-fluorouracil (5-FU) and the monoclonal antiEGFR antibody cetuximab. However, in most cases this chemoimmunotherapy does not cure the disease, and more than 50% of HNSCC patients are dying because of local recurrence of the tumors. In the majority of cases, HNSCC overexpress the epidermal growth factor receptor (EGFR), and its presence is associated with a poor outcome. In this study, we engineered an EGFR-targeted oncolytic measles virus (MV), armed with the bifunctional enzyme cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT). CD/UPRT converts 5-fluorocytosine (5-FC) into the chemotherapeutic 5-FU, a mainstay of HNSCC chemotherapy. This virus efficiently replicates in and lyses primary HNSCC cells in vitro. Arming with CD/UPRT mediates efficient prodrug activation with high bystander killing of non-infected tumor cells. In mice bearing primary HNSCC xenografts, intratumoral administration of MV-antiEGFR resulted in statistically significant tumor growth delay and prolongation of survival. Importantly, combination with 5-FC is superior to virus-only treatment leading to significant tumor growth inhibition. Thus, chemovirotherapy with EGFR-targeted and CD/UPRT-armed MV is highly efficacious in preclinical settings with direct translational implications for a planned Phase I clinical trial of MV for locoregional treatment of HNSCC.

  20. Transformation of pWWO in Rhizobium leguminosarum DPT to Engineer Toluene Degrading Ability for Rhizoremediation.

    Science.gov (United States)

    Goel, Garima; Pandey, Piyush; Sood, Anchal; Bisht, Sandeep; Maheshwari, D K; Sharma, G D

    2012-06-01

    Rhizoremediation of organic xenobiotics is based on interactions between plants and their associated micro-organisms. The present work was designed to engineer a bacterial system having toluene degradation ability along with plant growth promoting characteristics for effective rhizoremediation. pWWO harboring the genes responsible for toluene breakdown was isolated from Pseudomonas putida MTCC 979 and successfully transformed in Rhizobium DPT. This resulted in a bacterial strain (DPT(T)) which had the ability to degrade toluene as well as enhance growth of host plant. The frequency of transformation was recorded 5.7 × 10(-6). DPT produced IAA, siderophore, chitinase, HCN, ACC deaminase, solubilized inorganic phosphate, fixed atmospheric nitrogen and inhibited the growth of Fusarium oxysporum and Macrophomina phaseolina in vitro. During pot assay, 50 ppm toluene in soil was found to inhibit the germination of Cajanus cajan seeds. However when the seeds bacterized with toluene degrading P. putida or R. leguminosarum DPT were sown in pots, again no germination was observed. Non-bacterized as well as bacterized seeds germinated successfully in toluene free soil as control. The results forced for an alternative mode of application of bacteria for rhizoremediation purpose. Hence bacterial suspension was mixed with soil having 50 ppm of toluene. Germination index in DPT treated soil was 100% while in P. putida it was 50%. Untreated soil with toluene restricted the seeds to germinate.