Sample records for yeast cytosine deaminase

  1. Oncolytic virotherapy for ovarian carcinomatosis using a replication-selective vaccinia virus armed with a yeast cytosine deaminase gene

    Microsoft Academic Search

    S Chalikonda; M H Kivlen; M E O'Malley; J A McCart; M C Gorry; X-Y Yin; C K Brown; H J Zeh; Z S Guo; D L Bartlett

    2008-01-01

    In this study, we assessed the ability of a highly tumor-selective oncolytic vaccinia virus armed with a yeast cytosine deaminase gene to infect and lyse human and murine ovarian tumors both in vitro and in vivo. The virus vvDD-CD could infect, replicate in and effectively lyse both human and mouse ovarian cancer cells in vitro. In two different treatment schedules

  2. Oncolytic Virotherapy for Ovarian Carcinomatosis Using a Replication-Selective Vaccinia Virus Armed with a Yeast Cytosine Deaminase Gene*

    PubMed Central

    Chalikonda, Sricharan; Kivlen, Maryann H.; O’Malley, Mark E.; Dong, Xiang Da (Eric); McCart, J. Andrea; Gorry, Michael C.; Yin, Xiao-Yu; Brown, Charles K.; Zeh, Herbert J.; Guo, Z. Sheng; Bartlett, David L.

    2010-01-01

    In this study, we assessed the ability of a highly tumor-selective oncolytic vaccinia virus armed with a yeast cytosine deaminase gene to infect and lyse human and murine ovarian tumors both in vitro and in vivo. The virus vvDD-CD could infect, replicate in and effectively lyse both human and mouse ovarian cancer cells in vitro. In two different treatment schedules involving either murine MOSEC or human A2780 ovarian carcinomatosis models, regional delivery of vvDD-CD selectively targeted tumor cells and ovarian tissue, effectively delaying the development of either tumor or ascites and leading to significant survival advantages. Oncolytic virotherapy using vvDD-CD in combination with the prodrug 5-fluorocytosine (5-FC) conferred an additional long-term survival advantage upon tumor-bearing immunocompetent mice. These findings demonstrate that a tumor-selective oncolytic vaccinia combined with gene-directed enzyme prodrug therapy (GDEPT) is a highly effective strategy for treating advanced ovarian cancers in both syngeneic mouse and human xenograft models. Given the biological safety, tumor selectivity and oncolytic potency of this armed oncolytic virus, this dual therapy merits further investigation as a promising new treatment for metastatic ovarian cancer. PMID:18084242

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

    PubMed Central

    2012-01-01

    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. PMID:23249472

  4. Usefulness of Repeated Direct Intratumoral Gene Transfer Using Hemagglutinating Virus of Japan-Liposome Method for Cytosine Deaminase Suicide Gene Therapy

    Microsoft Academic Search

    Hiroki Kanyama; Naohiro Tomita; Tomoki Yamano; Tomohiko Aihara; Yasuo Miyoshi; Masayuki Ohue; Mitsugu Sekimoto; Isao Sakita; Yasuhiro Tamaki; Yasufumi Kaneda; Peter D. Senter; Morito Monden

    To investigate the feasibility of repeated gene transfection in suicide gene therapy against human solid tumors by a combination of 5- fluoro- cytosine (5-FC) and its converting enzyme, cytosine deaminase (CD), we repeatedly transfected the yeast CD gene into the human pancreatic cancer cell line BXPC3 using the hemagglutinating virus of Japan-lipo- some in a new gene transfer method. The

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

    SciTech Connect

    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. Cytosine Deaminase/5-Fluorocytosine Exposure Induces Bystander and Radiosensitization Effects in Hypoxic Glioblastoma Cells in vitro

    SciTech Connect

    Chen, Jennifer K. [Brain Tumor Research Center of the Department of Neurological Surgery, University of California-San Francisco, San Francisco, CA (United States); Hu, Lily J. [Brain Tumor Research Center of the Department of Neurological Surgery, University of California-San Francisco, San Francisco, CA (United States); Wang Dongfang [Brain Tumor Research Center of the Department of Neurological Surgery, University of California-San Francisco, San Francisco, CA (United States); Lamborn, Kathleen R. [Brain Tumor Research Center of the Department of Neurological Surgery, University of California-San Francisco, San Francisco, CA (United States); Deen, Dennis F. [Brain Tumor Research Center of the Department of Neurological Surgery, University of California-San Francisco, San Francisco, CA (United States)]. E-mail: dennisdeen@juno.com

    2007-04-01

    Purpose: Treatment of glioblastoma (GBM) is limited by therapeutic ratio; therefore, successful therapy must be specifically cytotoxic to cancer cells. Hypoxic cells are ubiquitous in GBM, and resistant to radiation and chemotherapy, and, thus, are logical targets for gene therapy. In this study, we investigated whether cytosine deaminase (CD)/5-fluorocytosine (5-FC) enzyme/prodrug treatment induced a bystander effect (BE) and/or radiosensitization in hypoxic GBM cells. Methods and Materials: We stably transfected cells with a gene construct consisting of the SV40 minimal promoter, nine copies of a hypoxia-responsive element, and the yeast CD gene. During hypoxia, a hypoxia-responsive element regulates expression of the CD gene and facilitates the conversion of 5-FC to 5-fluorouracil, a highly toxic antimetabolite. We used colony-forming efficiency (CFE) and immunofluorescence assays to assess for BE in co-cultures of CD-expressing clone cells and parent, pNeo- or green fluorescent protein-stably transfected GBM cells. We also investigated the radiosensitivity of CD clone cells treated with 5-FC under hypoxic conditions, and we used flow cytometry to investigate treatment-induced cell cycle changes. Results: Both a large BE and radiosensitization occurred in GBM cells under hypoxic conditions. The magnitude of the BE depended on the number of transfected cells producing CD, the functionality of the CD, the administered concentration of 5-FC, and the sensitivity of cell type to 5-fluorouracil. Conclusion: Hypoxia-inducible CD/5-FC therapy in combination with radiation therapy shows both a pronounced BE and a radiosensitizing effect under hypoxic conditions.

  7. Tumors Expressing the Cytosine Deaminase Suicide Gene Can Be Eliminated \\/\\/\\/ Vivo with 5-Fluorocytosine and Induce Protective Immunity to Wild Type Tumor

    Microsoft Academic Search

    Craig A. Mullen; Melissa M. Coale; Robert Lowe; R. Michael Blaese

    Successful expression of the cytosine deaminase (CD) suicide gene in vivo is demonstrated in three weakly immunogenic murine tumor models: the 102 and 205 fibrosarcomas and the 38 adenocarcinoma. Normal mam malian cells do not contain cytosine deaminase, but tumor cells transduced with retroviral vectors containing the CD gene metabolize the relatively nontoxic prodrug 5-fluorocytosine to the highly toxic 5-fluorouracil.

  8. Genetic immunotherapy for hepatocellular carcinoma by endothelial progenitor cells armed with cytosine deaminase.

    PubMed

    Chen, Rong; Yu, Hui; An, Yan-Li; Yu-Jia, Zhen; Teng, Gao-Jun

    2014-02-01

    Endothelial progenitor cells (EPCs) serve as cellular vehicles for targeting cancer cells and are a powerful tool for delivery of therapeutic genes. Cytosine deaminase (CD), a kind of frequent suicide gene which can kill carcinoma cells by converting a non-poisonous pro-drug 5-flucytosine (5-FC) into a poisonous cytotoxic 5-fluorouracil (5-FU). We combined super-paramagnetic iron oxide (SPIO) nanoparticles labeled EPCs with CD gene to treat grafted liver carcinomas and tracked them with 7.0 T Magnetic resonance imaging (MRI). Results showed that the therapeutic EPCs loaded with CD plus 5-Fc provided stronger carcinoma growth suppression compared with treatment using CD alone. The CD/5-Fc significantly inhibited the growth of endothelial cells and induced carcinoma cells apoptosis. These results indicate that EPCs transfected with anti-carcinoma genes can be used in carcinoma therapy as a novel therapeutic modality. PMID:24738335

  9. Retrovirus-mediated transduction of a cytosine deaminase gene preserves the stemness of mesenchymal stem cells

    PubMed Central

    Sung Park, Jin; Chang, Da-Young; Kim, Ji-Hoi; Hwa Jung, Jin; Park, JoonSeong; Kim, Se-Hyuk; Lee, Young-Don; Kim, Sung-Soo; Suh-Kim, Haeyoung

    2013-01-01

    Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles to deliver therapeutic genes for ex-vivo therapy of diverse diseases; this is, in part, because they have the capability to migrate into tumor or lesion sites. Previously, we showed that MSCs could be utilized to deliver a bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we assessed whether transduction with a retroviral vector encoding CD gene altered the stem cell property of MSCs. MSCs were transduced at passage 1 and cultivated up to passage 11. We found that proliferation and differentiation potentials, chromosomal stability and surface antigenicity of MSCs were not altered by retroviral transduction. The results indicate that retroviral vectors can be safely utilized for delivery of suicide genes to MSCs for ex-vivo therapy. We also found that a single retroviral transduction was sufficient for sustainable expression up to passage 10. The persistent expression of the transduced gene indicates that transduced MSCs provide a tractable and manageable approach for potential use in allogeneic transplantation. PMID:23429359

  10. First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G

    SciTech Connect

    Li, Ming; Shandilya, Shivender M.D.; Carpenter, Michael A.; Rathore, Anurag; Brown, William L.; Perkins, Angela L.; Harki, Daniel A.; Solberg, Jonathan; Hook, Derek J.; Pandey, Krishan K.; Parniak, Michael A.; Johnson, Jeffrey R.; Krogan, Nevan J.; Somasundaran, Mohan; Ali, Akbar; Schiffer, Celia A.; Harris, Reuben S. (Pitt); (UMASS, MED); (SLUHSC); (UCSF); (UMM)

    2012-04-04

    APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. Twenty of 34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access to substrate DNA cytosines.

  11. Replication Protein A (RPA) Hampers the Processive Action of APOBEC3G Cytosine Deaminase on

    E-print Network

    Borgstahl, Gloria

    , Bethesda, Maryland, United States of America, 3 Institute of Cytology and Genetics, Novosibirsk, Russia and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, United States of America, 2: University of Vermont, Burlington, Vermont, United States of America Introduction Deaminases of the AID

  12. Mutator Effects and Mutation Signatures of Editing Deaminases Produced in Bacteria and Yeast

    PubMed Central

    Lada, A. G.; Krick, C. Frahm; Kozmin, S. G.; Mayorov, V. I.; Karpova, T. S.; Rogozin, I. B.

    2014-01-01

    Enzymatic deamination of bases in DNA or RNA leads to an alteration of flow of genetic information. Adenine deaminases edit RNA (ADARs, TADs). Specialized cytidine deaminases are involved in RNA/DNA editing in lipid metabolism (APOBEC1) and in innate (APOBEC3 family) and humoral (AID) immunity. APOBEC2 is required for proper muscle development and, along with AID, was implicated in demethylation of DNA. The functions of APOBEC4, APOBEC5, and other deaminases recently discovered by bioinformatics approaches are unknown. What is the basis for the diverse biological functions of enzymes with similar enzyme structure and the same principal enzymatic reaction? AID, APOBEC1, lamprey CDA1, and APOBEC3G enzymes cause uracil DNA glycosylase-dependent induction of mutations when overproduced ectopically in bacteria or yeast. APOBEC2, on the contrary, is nonmutagenic. We studied the effects of the expression of various deaminases in yeast and bacteria. The mutagenic specificities of four deaminases, hAID, rAPOBEC1, hAPOBEC3G, and lamprey CDA1, are strikingly different. This suggests the existence of an intrinsic component of deaminase targeting. The expression of yeast CDD1 and TAD2/TAD3, human APOBEC4, Xanthomonas oryzae APOBEC5, and deaminase encoded by Micromonas sp. gene MICPUN_56782 was nonmutagenic. A lack of a mutagenic effect for Cdd1 is expected because the enzyme functions in the salvage of pyrimidine nucleotides, and it is evolutionarily distant from RNA/DNA editing enzymes. The reason for inactivity of deaminases grouped with APOBEC2 is not obvious from their structures. This can not be explained by protein insolubility and peculiarities of cellular distribution and requires further investigation. PMID:21568845

  13. Low-Dose Etoposide Enhances Telomerase-Dependent Adenovirus Mediated Cytosine Deaminase Gene Therapy through Augmentation of Adenoviral Infection and Transgene Expression in a Syngeneic Bladder Tumor Model

    Microsoft Academic Search

    Gia-Shing Shieh; Ai-Li Shiau; Yi-Te Yo; Chao-Ching Chang; Tzong-Shin Tzai; Chao-Liang Wu

    The human telomerase reverse transcriptase (hTERT) promot- er can selectively drive transgene expression in many telomer- ase-positive human cancer cells. Here we evaluated combination therapy of adenoviral vector Ad-hTERT-CD encoding E. coli cytosine deaminase (CD) driven by the hTERT promoter and low-dose etoposide (0.1 Mg\\/mL) for treating bladder cancer. Ad-hTERT-CD conferred sensitivity to 5- fluorocytosine (5-FC) in bladder cancer cells,

  14. Computational modeling and functional analysis of Herpes simplex virus type-1 thymidine kinase and Escherichia coli cytosine deaminase fusion protein

    SciTech Connect

    Zhang, Jufeng [Central Experimental Laboratory, the First People's Hospital, Shanghai Jiaotong University, Shanghai 200080 (China); Wang, Zhanli [Technology Center, NeoTrident Technology Ltd., Beijing 100080 (China); Wei, Fang [Central Experimental Laboratory, the First People's Hospital, Shanghai Jiaotong University, Shanghai 200080 (China); Qiu, Wei [Central Experimental Laboratory, the First People's Hospital, Shanghai Jiaotong University, Shanghai 200080 (China); Zhang, Liangren [School of Pharmaceutical Science, Peking University, Beijing 100083 (China); Huang, Qian [Central Experimental Laboratory, the First People's Hospital, Shanghai Jiaotong University, Shanghai 200080 (China)]. E-mail: qhuang@sjtu.edu.cn

    2007-08-17

    Herpes simplex virus type-1 thymidine kinase (HSV-1TK) and Escherichia coli cytosine deaminase (CD) fusion protein was designed using InsightII software. The structural rationality of the fusion proteins incorporating a series of flexible linker peptide was analyzed, and a suitable linker peptide was chosen for further investigated. The recombinant plasmid containing the coding regions of HSV-1TK and CD cDNA connected by this linker peptide coding sequence was generated and subsequently transfected into the human embryonic kidney 293 cells (HEK293). The Western blotting indicated that the recombinant fusion protein existed as a dimer with a molecular weight of approximately 90 kDa. The toxicity of the prodrug on the recombinant plasmid-transfected human lung cancer cell line NCIH460 was evaluated, which showed that TKglyCD-expressing cells conferred upon cells prodrug sensitivities equivalent to that observed for each enzyme independently. Most noteworthy, cytotoxicity could be enhanced by concurrently treating TKglyCD-expressing cells with prodrugs GCV and 5-FC. The results indicate that we have successfully constructed a HSV-1TKglyCD fusion gene which might have a potential application for cancer gene therapy.

  15. Cytosine deaminase-expressing human neural stem cells inhibit tumor growth in prostate cancer-bearing mice.

    PubMed

    Lee, Hong Jun; Doo, Seung Whan; Kim, Dae Hong; Cha, Young Joo; Kim, Jae Heon; Song, Yun Seob; Kim, Seung U

    2013-07-10

    Prostate cancer is the most common malignancy among men. Prostate cancer-related deaths are largely attributable to the development of hormone resistance in the tumor. No effective chemotherapy has yet been developed for advanced prostate cancer. It is desirable if a drug can be delivered directly and specifically to prostate cancer cells. Stem cells have selective migration ability toward cancer cells and therapeutic genes can be easily transduced into stem cells. In one form of gene therapy for cancer, the stem cells carry a gene encoding an enzyme that transforms an inert prodrug into a toxic product. Cytosine deaminase (CD) transforms the pro-drug 5-fluorocytosine into highly cytotoxic 5-fluorouracil (5-FU). The migration of the genetically modified stem cells was monitored by molecular magnetic resonance imaging, after labeling the stem cells with fluorescent magnetic nanoparticles (MNPs). Human neural stem cells encoding CD (HB1.F3.CD) were prepared and labeled with MNP. In tumor-bearing C57B mice, systemically transplanted HB1.F3.CD stem cells migrated toward the tumor and in combination with prodrug 5-FC, the volume of tumor implant was significantly reduced. These findings may contribute to development of a new selective chemotherapeutic strategy against prostate cancer. PMID:23391716

  16. Breast cancer-specific expression of the Candida albicans cytosine deaminase gene using a transcriptional targeting approach.

    PubMed

    Anderson, L M; Krotz, S; Weitzman, S A; Thimmapaya, B

    2000-06-01

    We constructed a series of adenoviral (Ad) vectors that express the Candida albicans cytosine deaminase (CD) suicide gene under the transcriptional control of either the human alpha-lactalbumin (ALA) or ovine beta-lactoglobulin (BLG) promoter (Ad.ALA.CD and Ad.BLG.CD, respectively). The Ad.ALA.CD and the Ad.BLG.CD vectors converted the prodrug 5-fluorocytosine (5-FC) to the toxic nucleotide analog 5-fluorouracil in a breast cancer cell-specific manner, with a conversion rate of 40% and 52% in T47D cells and 50% and 41% in MCF7 cells, respectively. No significant conversion (< or =3%) was observed in an immortalized nontumorigenic breast epithelial cell line (MCF10A) and a human osteosarcoma cell line (U2OS). Adenovirus vector-based prodrug conversion of the 5-FC in T47D and MCF7 in the presence of 1 mg/mL of 5-FC led to cytotoxicity that resulted in a nearly complete cell death (> or =90%) after 5 days, whereas MCF10A and U2OS cells remained resistant (< or =10%). Nude mice harboring T47D-derived breast tumors that were injected intratumorally (i.t.) with therapeutic adenovirus vectors at a dose of 2 x 10(8) plaque-forming units and treated systemically with 5-FC at a concentration of 500 mg/kg/day showed a marked reduction in tumor mass within 30 days when compared with animals that received vector alone. Animal survival was significantly prolonged after 72 days in mice treated with therapeutic vectors in conjunction with prodrug when compared with control animals. These preclinical data are sufficiently promising to warrant further studies of this transcriptional targeting approach to breast cancer treatment. PMID:10880014

  17. Transcriptional regulatory sequences of carcinoembryonic antigen: identification and use with cytosine deaminase for tumor-specific gene therapy.

    PubMed

    Richards, C A; Austin, E A; Huber, B E

    1995-07-01

    The 5' sequences from the human carcinoembryonic antigen gene (CEA) were analyzed using luciferase reporter gene assays. This analysis identified important cis-acting sequences needed for selective expression in CEA-positive cells. Over 50 CEA/luciferase reporter clones were constructed and analyzed in two CEA-positive and two CEA-negative cell lines. The CEA sequences analyzed extended from the translational start to 14.5 kb 5' of the CEA gene. A 408-bp region from the CEA 3' untranslated region was also examined for its effect on reporter gene activity. The CEA promoter was located between bases -90 and +69 of the transcriptional start site. Sequences between -41 and -18 were essential for expression from the CEA promoter. Multimerization of sequences between -89 and -40 resulted in copy number-related increases in both expression level and selectivity for CEA-positive cells. Two upstream regions of CEA, -13.6 to -10.7 kb or -6.1 to -4.0 kb, when linked to the multimerized promoter led to high-level, selective expression in CEA-positive cell lines. Several CEA/luciferase constructs demonstrated 80- to 120-fold higher expression in CEA-positive cell lines compared to expression in CEA-negative Hep3B cells. The expression from these constructs was quite strong in CEA-positive cells, being two- to four-fold higher than an SV40 enhancer/promoter construct. The most promising CEA transcriptional regulatory sequences were used to regulate the expression of cytosine deaminase (CD) in stable cell lines. The expression of CD was assessed directly by an enzymatic assay and indirectly by determining the in vitro IC50 to 5-fluorocytosine (5FC). The chimeric gene pCEA/CD-145 displayed the desired expression spectrum--high-level expression in the CEA-positive cells and low-level expression in CEA-negative cells. CD expression from this chimera correlated well with the expression of the endogenous CEA gene. Treatment of mice bearing NCI H508 pCEA/CD-145 tumor xenografts with 5FC lead to significant antitumor effects in vivo. The CEA/CD chimeric gene should be useful for tumor-specific suicide gene therapy of CEA-positive tumors. PMID:7578407

  18. Non-invasive molecular and functional imaging of cytosine deaminase and uracil phosphoribosyltransferase fused with red fluorescence protein

    PubMed Central

    XING, LIGANG; DENG, XUELONG; KOTEDIA, KHUSHALI; ACKERSTAFF, ELLEN; PONOMAREV, VLADIMIR; LING, C. CLIFTON; KOUTCHER, JASON A.; LI, GLORIA C.

    2014-01-01

    Introduction Increased expression of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) may improve the antitumoral effect of 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC), and thereby enhance the potential of gene-directed enzyme prodrug therapy. For the applicability of gene-directed enzyme prodrug therapy in a clinical setting, it is essential to be able to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using magnetic resonance spectroscopy and optical imaging to measure non-invasively CD and UPRT expression and function. Materials and methods Expression vectors of CD or CD/UPRT fused to monomeric DsRed (mDsRed) were constructed and rat prostate carcinoma (R3327-AT) cell lines stably expressing either CD/mDsRed or CD/UPRT/mDsRed were generated. The expression of the fusion proteins was evaluated by flow cytometry, fluorescence microscopy, and Western blot analysis. The function of the fusion protein was confirmed in vitro by assessing 5-FC and 5-FU cytotoxicity. In vivo fluorine-19 magnetic resonance spectroscopy (19F MRS) was used to monitor the conversion of 5-FC to 5-FU in mice bearing the R3327-CD/mDsRed and R3327-CD/UPRT/mDsRed tumor xenografts. Results Sensitivity to 5-FC and 5-FU was higher in cells stably expressing the CD/UPRT/mDsRed fusion gene than in cells stably expressing CD/mDsRed alone or wild-type cells. Whole tumor 19F MRS measurements showed rapid conversion of 5-FC to 5-FU within 20 min after 5-FC was administered intravenously in both CD/mDsRed and CD/UPRT/mDsRed tumors with subsequent anabolism to cytotoxic fluoronucleotides (FNucs). CD/UPRT/mDsRed tumor was more efficient in these processes. Conclusion This study demonstrates the utility of these tumor models stably expressing CD or CD/UPRT to non-invasively evaluate the efficacy of the transgene expression/activity by monitoring drug metabolism in vivo using MRS, with potential applications in preclinical and clinical settings. PMID:18661431

  19. 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

    SciTech Connect

    Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

    2008-02-01

    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.

  20. Cytosine DNA Methylation Is Found in Drosophila melanogaster but Absent in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Other Yeast Species

    PubMed Central

    2014-01-01

    The methylation of cytosine to 5-methylcytosine (5-meC) is an important epigenetic DNA modification in many bacteria, plants, and mammals, but its relevance for important model organisms, including Caenorhabditis elegans and Drosophila melanogaster, is still equivocal. By reporting the presence of 5-meC in a broad variety of wild, laboratory, and industrial yeasts, a recent study also challenged the dogma about the absence of DNA methylation in yeast species. We would like to bring to attention that the protocol used for gas chromatography/mass spectrometry involved hydrolysis of the DNA preparations. As this process separates cytosine and 5-meC from the sugar phosphate backbone, this method is unable to distinguish DNA- from RNA-derived 5-meC. We employed an alternative LC–MS/MS protocol where by targeting 5-methyldeoxycytidine moieties after enzymatic digestion, only 5-meC specifically derived from DNA is quantified. This technique unambiguously identified cytosine DNA methylation in Arabidopsis thaliana (14.0% of cytosines methylated), Mus musculus (7.6%), and Escherichia coli (2.3%). Despite achieving a detection limit at 250 attomoles (corresponding to <0.00002 methylated cytosines per nonmethylated cytosine), we could not confirm any cytosine DNA methylation in laboratory and industrial strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Saccharomyces boulardii, Saccharomyces paradoxus, or Pichia pastoris. The protocol however unequivocally confirmed DNA methylation in adult Drosophila melanogaster at a value (0.034%) that is up to 2 orders of magnitude below the detection limit of bisulphite sequencing. Thus, 5-meC is a rare DNA modification in drosophila but absent in yeast. PMID:24640988

  1. Cytosine DNA methylation is found in Drosophila melanogaster but absent in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other yeast species.

    PubMed

    Capuano, Floriana; Mülleder, Michael; Kok, Robert; Blom, Henk J; Ralser, Markus

    2014-04-15

    The methylation of cytosine to 5-methylcytosine (5-meC) is an important epigenetic DNA modification in many bacteria, plants, and mammals, but its relevance for important model organisms, including Caenorhabditis elegans and Drosophila melanogaster, is still equivocal. By reporting the presence of 5-meC in a broad variety of wild, laboratory, and industrial yeasts, a recent study also challenged the dogma about the absence of DNA methylation in yeast species. We would like to bring to attention that the protocol used for gas chromatography/mass spectrometry involved hydrolysis of the DNA preparations. As this process separates cytosine and 5-meC from the sugar phosphate backbone, this method is unable to distinguish DNA- from RNA-derived 5-meC. We employed an alternative LC-MS/MS protocol where by targeting 5-methyldeoxycytidine moieties after enzymatic digestion, only 5-meC specifically derived from DNA is quantified. This technique unambiguously identified cytosine DNA methylation in Arabidopsis thaliana (14.0% of cytosines methylated), Mus musculus (7.6%), and Escherichia coli (2.3%). Despite achieving a detection limit at 250 attomoles (corresponding to <0.00002 methylated cytosines per nonmethylated cytosine), we could not confirm any cytosine DNA methylation in laboratory and industrial strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Saccharomyces boulardii, Saccharomyces paradoxus, or Pichia pastoris. The protocol however unequivocally confirmed DNA methylation in adult Drosophila melanogaster at a value (0.034%) that is up to 2 orders of magnitude below the detection limit of bisulphite sequencing. Thus, 5-meC is a rare DNA modification in drosophila but absent in yeast. PMID:24640988

  2. Increased sensitivity of glioma cells to 5-fluorocytosine following photo-chemical internalization enhanced nonviral transfection of the cytosine deaminase suicide gene

    PubMed Central

    Zamora, Genesis; Sun, Chung-Ho; Trinidad, Anthony; Chun, Changho; Kwon, Young Jik; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

    2014-01-01

    Despite advances in surgery, chemotherapy and radiotherapy, the outcomes of patients with GBM have not significantly improved. Tumor recurrence in the resection margins occurs in more than 80 % of cases indicating aggressive treatment modalities, such as gene therapy are warranted. We have examined photochemical internalization (PCI) as a method for the non-viral transfection of the cytosine deaminase (CD) suicide gene into glioma cells. The CD gene encodes an enzyme that can convert the nontoxic antifungal agent, 5-fluorocytosine, into the chemotherapeutic drug, 5-fluorouracil. Multicell tumor spheroids derived from established rat and human glioma cell lines were used as in vitro tumor models. Plasmids containing either the CD gene alone or together with the uracil phosphoribosyl transferase (UPRT) gene combined with the gene carrier protamine sulfate were employed in all experiments. PCI was performed with the photosensitizer AlPcS2a and 670 nm laser irradiance. Protamine sulfate/CD DNA polyplexes proved nontoxic but inefficient transfection agents due to endosomal entrapment. In contrast, PCI mediated CD gene transfection resulted in a significant inhibition of spheroid growth in the presence of, but not in the absence of, 5-FC. Repetitive PCI induced transfection was more efficient at low CD plasmid concentration than single treatment. The results clearly indicate that AlPcS2a-mediated PCI can be used to enhance transfection of a tumor suicide gene such as CD, in malignant glioma cells and cells transfected with both the CD and UPRT genes had a pronounced bystander effect. PMID:24610460

  3. Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes

    PubMed Central

    Taylor, Benjamin JM; Wu, Yee Ling; Rada, Cristina

    2014-01-01

    Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors. DOI: http://dx.doi.org/10.7554/eLife.03553.001 PMID:25237741

  4. Discovery of Deaminase Activities in COG1816

    E-print Network

    Goble, Alissa M

    2013-04-24

    related to guanine and cytosine deaminase from COG0402. The deamination of 6-aminofutalosine is part of the alternative menaquinone biosynthetic pathway that involves the formation of futalosine. 6-Aminofutalosine is deaminated with a catalytic effeciency...

  5. Diffusion MRI detects early events in the response of a glioma model to the yeast cytosine deaminase gene therapy strategy

    Microsoft Academic Search

    LD Stegman; A Rehemtulla; DA Hamstra; DJ Rice; SJ Jonas; KL Stout; TL Chenevert; BD Ross

    2000-01-01

    Detection of a therapeutic response early in the course of cancer treatment, before tumor growth delay or regression, is not currently possible in experimental models or clinical medicine. New interim measures of therapeutic response would be particularly useful in the development of cancer chemosensitization gene therapy by facilitating optimization of gene transfer protocols and prodrug dosing schedules. Diffusion MRI is

  6. A novel fusion suicide gene yeast CDglyTK plays a role in radio-gene therapy of nasopharyngeal carcinoma

    Microsoft Academic Search

    Kun Xia; Desheng Liang; Aifa Tang; Yong Feng; Junyi Zhang; Qian Pan; Zhigao Long; Heping Dai; Fang Cai; Lingqian Wu; Suping Zhao; Zhuchu Chen; Jiahui Xia

    2004-01-01

    To investigate a novel suicide gene for nasopharyngeal carcinoma (NPC) therapy, the yCDglyTK gene was constructed by fusing yeast cytosine deaminase (CD) and herpes simplex type 1 thymidine kinase. The expression of the yCDglyTK gene was detected by RT-PCR and Western blotting, and its bioactivity was demonstrated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. An animal study was carried out

  7. Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1.

    PubMed

    Sharma, Sunny; Langhendries, Jean-Louis; Watzinger, Peter; Kötter, Peter; Entian, Karl-Dieter; Lafontaine, Denis L J

    2015-02-27

    The function of RNA is subtly modulated by post-transcriptional modifications. Here, we report an important crosstalk in the covalent modification of two classes of RNAs. We demonstrate that yeast Kre33 and human NAT10 are RNA cytosine acetyltransferases with, surprisingly, specificity toward both 18S rRNA and tRNAs. tRNA acetylation requires the intervention of a specific and conserved adaptor: yeast Tan1/human THUMPD1. In budding and fission yeasts, and in human cells, we found two acetylated cytosines on 18S rRNA, one in helix 34 important for translation accuracy and another in helix 45 near the decoding site. Efficient 18S rRNA acetylation in helix 45 involves, in human cells, the vertebrate-specific box C/D snoRNA U13, which, we suggest, exposes the substrate cytosine to modification through Watson-Crick base pairing with 18S rRNA precursors during small subunit biogenesis. Finally, while Kre33 and NAT10 are essential for pre-rRNA processing reactions leading to 18S rRNA synthesis, we demonstrate that rRNA acetylation is dispensable to yeast cells growth. The inactivation of NAT10 was suggested to suppress nuclear morphological defects observed in laminopathic patient cells through loss of microtubules modification and cytoskeleton reorganization. We rather propose the effects of NAT10 on laminopathic cells are due to reduced ribosome biogenesis or function. PMID:25653167

  8. Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1

    PubMed Central

    Sharma, Sunny; Langhendries, Jean-Louis; Watzinger, Peter; Kötter, Peter; Entian, Karl-Dieter; Lafontaine, Denis L.J.

    2015-01-01

    The function of RNA is subtly modulated by post-transcriptional modifications. Here, we report an important crosstalk in the covalent modification of two classes of RNAs. We demonstrate that yeast Kre33 and human NAT10 are RNA cytosine acetyltransferases with, surprisingly, specificity toward both 18S rRNA and tRNAs. tRNA acetylation requires the intervention of a specific and conserved adaptor: yeast Tan1/human THUMPD1. In budding and fission yeasts, and in human cells, we found two acetylated cytosines on 18S rRNA, one in helix 34 important for translation accuracy and another in helix 45 near the decoding site. Efficient 18S rRNA acetylation in helix 45 involves, in human cells, the vertebrate-specific box C/D snoRNA U13, which, we suggest, exposes the substrate cytosine to modification through Watson–Crick base pairing with 18S rRNA precursors during small subunit biogenesis. Finally, while Kre33 and NAT10 are essential for pre-rRNA processing reactions leading to 18S rRNA synthesis, we demonstrate that rRNA acetylation is dispensable to yeast cells growth. The inactivation of NAT10 was suggested to suppress nuclear morphological defects observed in laminopathic patient cells through loss of microtubules modification and cytoskeleton reorganization. We rather propose the effects of NAT10 on laminopathic cells are due to reduced ribosome biogenesis or function. PMID:25653167

  9. Molecular Structure of Cytosine

    NSDL National Science Digital Library

    2004-10-12

    Cytosine is one of the five main nitrogenous bases used in storing and transporting genetic information within a cell. Cytosine is a pyrimidine base that is found in both DNA and RNA and pairs with guanine. It was isolated from the nucleic acid of calf thymus tissue in 1894. A suggested structure for cytosine, published in 1903, was confirmed in the same year when that base was synthesized in the laboratory.

  10. Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase

    E-print Network

    Levin, Judith G.

    is not required for inhibition of recombinant AAV production. We also found that deaminase-deficient A3A mutantsDeaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase In~ igo of cytidine deaminases with inhibitory activity against viruses and retrotransposons. In contrast to APOBEC3G

  11. Genetics Home Reference: Adenosine deaminase deficiency

    MedlinePLUS

    ... PubMed Recent literature OMIM Genetic disorder catalog Conditions > Adenosine deaminase deficiency (often shortened to ADA deficiency ) On this page: ... other health problems. How common is ADA deficiency? Adenosine deaminase deficiency is very rare and is estimated to occur ...

  12. High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase

    PubMed Central

    Gajula, Kiran S.; Huwe, Peter J.; Mo, Charlie Y.; Crawford, Daniel J.; Stivers, James T.; Radhakrishnan, Ravi; Kohli, Rahul M.

    2014-01-01

    Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9–11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function. PMID:25064858

  13. Arxula adeninivorans recombinant guanine deaminase and its application in the production of food with low purine content.

    PubMed

    Trautwein-Schult, Anke; Jankowska, Dagmara; Cordes, Arno; Hoferichter, Petra; Klein, Christina; Matros, Andrea; Mock, Hans-Peter; Baronian, Keith; Bode, Rüdiger; Kunze, Gotthard

    2014-01-01

    Purines of exogenous and endogenous sources are degraded to uric acid in human beings. Concentrations >6.8 mg uric acid/dl serum cause hyperuricemia and its symptoms. Pharmaceuticals and the reduction of the intake of purine-rich food are used to control uric acid levels. A novel approach to the latter proposition is the enzymatic reduction of the purine content of food by purine-degrading enzymes. Here we describe the production of recombinant guanine deaminase by the yeast Arxula adeninivorans LS3 and its application in food. In media supplemented with nitrogen sources hypoxanthine or adenine, guanine deaminase (AGDA) gene expression is induced and intracellular accumulation of guanine deaminase (Agdap) protein occurs. The characteristics of the guanine deaminase isolated from wild-type strain LS3 and a transgenic strain expressing the AGDA gene under control of the strong constitutive TEF1 promoter were determined and compared. Both enzymes were dimeric and had temperature optima of 55°C with high substrate specificity for guanine and localisation in both the cytoplasm and vacuole of yeast. The enzyme was demonstrated to reduce levels of guanine in food. A mixture of guanine deaminase and other purine degradation enzymes will allow the reduction of purines in purine-rich foods. PMID:24481069

  14. Epigenetic targeting of activation-induced cytidine deaminase.

    PubMed

    Wang, Qiao; Oliveira, Thiago; Jankovic, Mila; Silva, Israel T; Hakim, Ofir; Yao, Kaihui; Gazumyan, Anna; Mayer, Christian T; Pavri, Rushad; Casellas, Rafael; Nussenzweig, Michel C; Robbiani, Davide F

    2014-12-30

    Activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) and somatic hypermutation (SHM) by deaminating cytosine residues in immunoglobulin genes (Igh, Ig?, and Ig?). At a lower frequency, AID also causes collateral DNA damage at non-Ig loci, including genes that are rearranged or mutated in B-cell lymphoma. Precisely how AID is recruited to these off-target sites is not entirely understood. To gain further insight into how AID selects its targets, we compared AID-mediated translocations in two different cell types, B cells and mouse embryonic fibroblasts (MEFs). AID targets a distinct set of hotspots in the two cell types. In both cases, hotspots are concentrated in highly transcribed but stalled genes. However, transcription alone is insufficient to recruit AID activity. Comparison of genes similarly transcribed in B cells and MEFs but targeted in only one of the two cell types reveals a common set of epigenetic features associated with AID recruitment in both cells. AID target genes are enriched in chromatin modifications associated with active enhancers (such as H3K27Ac) and marks of active transcription (such as H3K36me3) in both fibroblasts and B cells, indicating that these features are universal mediators of AID recruitment. PMID:25512519

  15. Replication Fork Collapse and Genome Instability in a Deoxycytidylate Deaminase Mutant

    PubMed Central

    Sánchez, Arancha; Sharma, Sushma; Rozenzhak, Sophie; Roguev, Assen; Krogan, Nevan J.; Chabes, Andrei

    2012-01-01

    Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1+ dCMP deaminase gene (SPBC2G2.13c) increases dCTP ?30-fold and decreases dTTP ?4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1? mutant, fission yeast dcd1? cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1? cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the ?H2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1? cells. We propose that replication forks stall and collapse in dcd1? cells, burdening DNA damage and checkpoint responses to maintain genome integrity. PMID:22927644

  16. Genetics Home Reference: Adenosine deaminase 2 deficiency

    MedlinePLUS

    ... diseases Additional NIH Resources National Institutes of Health Educational resources Information pages Patient support For patients and families ... or management of adenosine deaminase 2 deficiency in Educational resources and Patient support . General information about the diagnosis ...

  17. Specific and Modular Binding Code for Cytosine Recognition in Pumilio/FBF (PUF) RNA-binding Domains

    SciTech Connect

    Dong, Shuyun; Wang, Yang; Cassidy-Amstutz, Caleb; Lu, Gang; Bigler, Rebecca; Jezyk, Mark R.; Li, Chunhua; Tanaka Hall, Traci M.; Wang, Zefeng (NIH); (Beijing U); (UNC)

    2011-10-28

    Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.

  18. Radioimmunochemical quantitation of human adenosine deaminase.

    PubMed Central

    Daddona, P E; Frohman, M A; Kelley, W N

    1979-01-01

    Markedly reduced or absent adenosine deaminase activity in man is associated with an autosomal recesive form of severe conbined immunodeficiency disease. To further define the genetic nature of this enzyme defect, we have quantitated immunologically active adenosine deaminase (CRM) in the hemolysate of homozygous deficient patients and their heterozygous parents. A highly specific radioimmunoassay was developed capable of detecting 0.05% of normal erythrocyte adenosine deaminase. Hemolysates from nine heterozygotes (five families) showed a wide range in CRM (32--100% of normal) and variable absolute specific activities with several being at least 1 SD BELOW THE NORMAL MEAN. Hemolysates from four unrelated patients showed less than 0.09% adenosine deaminase activity with CRM ranging from less than 0.06 to 5.6% of the normal mean. In conclusion, heterozygote and homozygote hemolysates from five of the eight families analyzed revealed variable levels of CRM suggesting heterogeneous genetic alteration or expression of the silent or defective allele(s) of adenosine deaminase. PMID:468994

  19. Characterization of a novel resistance-related deoxycytidine deaminase from Brassica oleracea var. capitata.

    PubMed

    Shibu, Marthandam Asokan; Yang, Hsueh-Hui; Lo, Chaur-Tsuen; Lin, Hong-Shin; Liu, Shu-Ying; Peng, Kou-Cheng

    2014-02-26

    Brassica oleracea deoxycytidine deaminase (BoDCD), a deoxycytidine deaminase (DCD, EC 3.5.4.14) enzyme, is known to play an important role in the Trichoderma harzianum ETS 323 mediated resistance mechanism in young leaves of B. oleracea var. capitata during Rhizoctonia solani infection. BoDCD potentially neutralizes cytotoxic products of host lipoxygenase activity, and thereby BoDCD restricts the hypersensitivity-related programmed cell death induced in plants during the initial stages of infection. To determine the biochemical characteristics and to partially elucidate the designated functional properties of BoDCD, the enzyme was cloned into an Escherichia coli expression system, and its potential to neutralize the toxic analogues of 2'-deoxycytidine (dC) was examined. BoDCD transformants of E. coli cells were found to be resistant to 2'-deoxycytidine analogues at all of the concentrations tested. The BoDCD enzyme was also overexpressed as a histidine-tagged protein and purified using nickel chelating affinity chromatography. The molecular weight of BoDCD was determined to be 20.8 kDa as visualized by SDS-PAGE. The substrate specificity and other kinetic properties show that BoDCD is more active in neutralizing cytotoxic cytosine ?-d-arabinofuranoside than in deaminating 2'-deoxycytinde to 2'-deoxyuridine in nucleic acids or in metabolizing cytidine to uridine. The optimal temperature and pH of the enzyme were 27 °C and 7.5. The Km and Vmax values of BoDCD were, respectively, 91.3 ?M and 1.475 mM for its natural substrate 2'-deoxycytidine and 63 ?M and 2.072 mM for cytosine ?-d-arabinofuranoside. The phenomenon of neutralization of cytotoxic dC analogues by BoDCD is discussed in detail on the basis of enzyme biochemical properties. PMID:24475736

  20. A Role for Adenosine Deaminase in Drosophila Larval Development

    E-print Network

    Gibson, Matt

    A Role for Adenosine Deaminase in Drosophila Larval Development Tomas Dolezal1 , Eva Dolezelova2 Budejovice, Czech Republic Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth

  1. Wobble Inosine tRNA Modification Is Essential to Cell Cycle Progression in G1/S and G2/M Transitions in Fission Yeast*

    E-print Network

    Gibson, Matt

    of a genomically encoded adenosine (A). The enzyme catalyzing this reaction, termed tRNA A:34 deaminase adenosine (A), and the enzyme catalyzing this reaction is termed tRNA A:34 deaminase. In budding yeast, inosine can pair degener- ately with U, C, or adenosine, enabling a single tRNA to recog- nize up to three

  2. The stability of adenosine deaminase and adenosine monophosphate (AMP) deaminase as potential spoilage indicators for postmortem ice stored shrimp

    E-print Network

    Cheuk, Wai Lun

    1978-01-01

    THE STABILITY OF ADENOSINE DEAMINASE AND ADENOSINE MONOPHOSPHATE (AMP) DEAMINASE AS POTENTIAL SPOILAGE INDICATORS FOR POSTMORTEM ICE STORED SHRIMP A Thesis by WAI LUN CHEUK Submitted to the Graduate College of Texas A&M University in partial... fulfillment of the requirement for the degree of MASTER OF SCIENCE December 1978 Major Subject: Food Science and Technology THE STABILITY OF ADENOSINE DEAMINASE AND ADENOSINE MONOPHOSPHATE (AMP) DEAMINASE AS POTENTIAL SPOILAGE INDICATORS FOR POSTMORTEM...

  3. Prenatal diagnosis for adenosine deaminase deficiency

    Microsoft Academic Search

    J B Ziegler; M B Van der Weyden; C H Lee; A Daniel

    1981-01-01

    Amniocentesis was performed in two successive pregnancies of the mother of a child with adenosine deaminase (ADA) deficient severe combined immunodeficiency. Assay of ADA in amniotic fluid fibroblasts showed the pregnancies to be normal and homozygous deficient, respectively. These findings were confirmed by the demonstration of a normal level of erythrocyte ADA in the cord blood of the healthy male

  4. Adenosine deaminase deficiency with mosaicism for a \\

    Microsoft Academic Search

    Francisco X. Arredondo-Vega; Ines Santisteban; Eva Richard; Pawan Bali; Majed Koleilat; Michael Loubser; Abdulaziz Al-Ghonaium; Mariam Al-Helali; Michael S. Hershfield

    2002-01-01

    Four patients from 3 Saudi Arabian fami- lies had delayed onset of immune defi- ciency due to homozygosity for a novel intronic mutation, g.31701T>A, in the last splice acceptor site of the adenosine deaminase (ADA) gene. Aberrant splicing mutated the last 4 ADA amino acids and added a 43-residue \\

  5. Specific interactions between silver(I) ions and cytosine-cytosine pairs in DNA duplexes.

    PubMed

    Ono, Akira; Cao, Shiqi; Togashi, Humika; Tashiro, Mitsuru; Fujimoto, Takashi; Machinami, Tomoya; Oda, Shuji; Miyake, Yoko; Okamoto, Itaru; Tanaka, Yoshiyuki

    2008-10-21

    Very specific binding of the Ag(i) ion unexpectedly stabilized DNA duplexes containing the naturally occurring cytosine-cytosine (C-C) mismatch-base pair; because the C-C pair selectively binds to the Ag(i) ion, we developed a DNA-based Ag(i) sensor that employed an oligodeoxyribonucleotide containing C-C pairs used for Ag(i) binding sites. PMID:18830506

  6. Single Molecule Investigation of Ag+ Interactions with Single Cytosine-, Methylcytosine- and Hydroxymethylcytosine-Cytosine Mismatches in a Nanopore

    PubMed Central

    Wang, Yong; Luan, Bin-Quan; Yang, Zhiyu; Zhang, Xinyue; Ritzo, Brandon; Gates, Kent; Gu, Li-Qun

    2014-01-01

    Both cytosine-Ag-cytosine interactions and cytosine modifications in a DNA duplex have attracted great interest for research. Cytosine (C) modifications such as methylcytosine (mC) and hydroxymethylcytosine (hmC) are associated with tumorigenesis. However, a method for directly discriminating C, mC and hmC bases without labeling, modification and amplification is still missing. Additionally, the nature of coordination of Ag+ with cytosine-cytosine (C-C) mismatches is not clearly understood. Utilizing the alpha-hemolysin nanopore, we show that in the presence of Ag+, duplex stability is most increased for the cytosine-cytosine (C-C) pair, followed by the cytosine-methylcytosine (C-mC) pair, and the cytosine-hydroxymethylcytosine (C-hmC) pair, which has no observable Ag+ induced stabilization. Molecular dynamics simulations reveal that the hydrogen-bond-mediated paring of a C-C mismatch results in a binding site for Ag+. Cytosine modifications (such as mC and hmC) disrupted the hydrogen bond, resulting in disruption of the Ag+ binding site. Our experimental method provides a novel platform to study the metal ion-DNA interactions and could also serve as a direct detection method for nucleobase modifications. PMID:25103463

  7. Cytosine modifications in neurodevelopment and diseases

    PubMed Central

    Yao, Bing; Jin, Peng

    2013-01-01

    DNA methylation has been studied comprehensively and linked to both normal neurodevelopment and neurological diseases. The recent identification of several new DNA modifications, including 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), has given us a new perspective on the previously observed plasticity in 5mC-dependent regulatory processes. Here we review the latest research into these cytosine modifications, focusing mainly on their roles in neurodevelopment and diseases. PMID:23912899

  8. Hot Spot Mutations in Adenosine Deaminase Deficiency

    Microsoft Academic Search

    Rochelle Hirschhorn; Stephanie Tzall; Amy Ellenbogen

    1990-01-01

    We have previously characterized mutant adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) enzymes in seven children with partial ADA deficiency. Six children shared common origins, suggesting a common progenitor. However, we found evidence for multiple phenotypically different mutant enzymes. We hypothesized that many of the mutations would be at CpG dinucleotides, hot spots at which spontaneous deamination of 5-methylcytosine results

  9. Purine Metabolism in Adenosine Deaminase Deficiency

    Microsoft Academic Search

    Gordon C. Mills; Frank C. Schmalstieg; K. Bryan Trimmer; Armond S. Goldman; Randall M. Goldblum

    1976-01-01

    Purine and pyrimidine metabolites were measured in erythrocytes, plasma, and urine of a 5-month-old infant with adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. Adenosine and adenine were measured using newly devised ion exchange separation techniques and a sensitive fluorescence assay. Plasma adenosine levels were increased, whereas adenosine was normal in erythrocytes and not detectable in urine. Increased amounts of adenine

  10. Gene therapy for adenosine deaminase deficiency.

    PubMed

    Cappelli, Barbara; Aiuti, Alessandro

    2010-05-01

    In the last decade, gene therapy for adenosine deaminase deficiency has been developed as a successful alternative strategy to allogeneic bone marrow transplant and enzyme replacement therapy. Infusion of autologous hematopoietic stem cells, corrected ex vivo by retroviral vectors and combined to low-intensity conditioning regimen, has resulted in immunologic improvement, metabolic correction, and long-term clinical benefits. These findings have opened the way to applications of gene therapy in other primary immune deficiencies using novel vector technology. PMID:20493400

  11. Arabidopsis MET1 cytosine methyltransferase mutants.

    PubMed Central

    Kankel, Mark W; Ramsey, Douglas E; Stokes, Trevor L; Flowers, Susan K; Haag, Jeremy R; Jeddeloh, Jeffrey A; Riddle, Nicole C; Verbsky, Michelle L; Richards, Eric J

    2003-01-01

    We describe the isolation and characterization of two missense mutations in the cytosine-DNA-methyltransferase gene, MET1, from the flowering plant Arabidopsis thaliana. Both missense mutations, which affect the catalytic domain of the protein, led to a global reduction of cytosine methylation throughout the genome. Surprisingly, the met1-2 allele, with the weaker DNA hypomethylation phenotype, alters a well-conserved residue in methyltransferase signature motif I. The stronger met1-1 allele caused late flowering and a heterochronic delay in the juvenile-to-adult rosette leaf transition. The distribution of late-flowering phenotypes in a mapping population segregating met1-1 indicates that the flowering-time phenotype is caused by the accumulation of inherited defects at loci unlinked to the met1 mutation. The delay in flowering time is due in part to the formation and inheritance of hypomethylated fwa epialleles, but inherited defects at other loci are likely to contribute as well. Centromeric repeat arrays hypomethylated in met1-1 mutants are partially remethylated when introduced into a wild-type background, in contrast to genomic sequences hypomethylated in ddm1 mutants. ddm1 met1 double mutants were constructed to further our understanding of the mechanism of DDM1 action and the interaction between two major genetic loci affecting global cytosine methylation levels in Arabidopsis. PMID:12663548

  12. Crystal Structure of Staphylococcus aureus tRNA Adenosine Deaminase TadA in Complex with RNA

    SciTech Connect

    Losey,H.; Ruthenburg, A.; Verdine, G.

    2006-01-01

    Bacterial tRNA adenosine deaminases (TadAs) catalyze the hydrolytic deamination of adenosine to inosine at the wobble position of tRNA(Arg2), a process that enables this single tRNA to recognize three different arginine codons in mRNA. In addition, inosine is also introduced at the wobble position of multiple eukaryotic tRNAs. The genes encoding these deaminases are essential in bacteria and yeast, demonstrating the importance of their biological activity. Here we report the crystallization and structure determination to 2.0 A of Staphylococcus aureus TadA bound to the anticodon stem-loop of tRNA(Arg2) bearing nebularine, a non-hydrolyzable adenosine analog, at the wobble position. The cocrystal structure reveals the basis for both sequence and structure specificity in the interactions of TadA with RNA, and it additionally provides insight into the active site architecture that promotes efficient hydrolytic deamination.

  13. Zinc-binding domain-dependent, deaminase-independent actions of apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 2 (Apobec2), mediate its effect on zebrafish retina regeneration.

    PubMed

    Powell, Curtis; Cornblath, Eli; Goldman, Daniel

    2014-10-17

    The Apobec/AID family of cytosine deaminases can deaminate cytosine and thereby contribute to adaptive and innate immunity, DNA demethylation, and the modification of cellular mRNAs. Unique among this family is Apobec2, whose enzymatic activity has been questioned and whose function remains poorly explored. We recently reported that zebrafish Apobec2a and Apobec2b (Apobec2a,2b) regulate retina regeneration; however, their mechanism of action remained unknown. Here we show that although Apobec2a,2b lack cytosine deaminase activity, they require a conserved zinc-binding domain to stimulate retina regeneration. Interestingly, we found that human APOBEC2 is able to functionally substitute for Apobec2a,2b during retina regeneration. By identifying Apobec2-interacting proteins, including ubiquitin-conjugating enzyme 9 (Ubc9); topoisomerase I-binding, arginine/serine-rich, E3 ubiquitin protein ligase (Toporsa); and POU class 6 homeobox 2 (Pou6f2), we uncovered that sumoylation regulates Apobec2 subcellular localization and that nuclear Apobec2 controls Pou6f2 binding to DNA. Importantly, mutations in the zinc-binding domain of Apobec2 diminished its ability to stimulate Pou6f2 binding to DNA, and knockdown of Ubc9 or Pou6f2 suppressed retina regeneration. PMID:25190811

  14. RNA EDITING BY ADENOSINE DEAMINASES THAT ACT ON RNA

    E-print Network

    Bass, Brenda L.

    RNA EDITING BY ADENOSINE DEAMINASES THAT ACT ON RNA Brenda L. Bass Department of Biochemistry-mail: bbass@howard.genetics.utah.edu Key Words double-stranded RNA, inosine, deaminase, neurotransmission f An Overview of RNA Editing by Adenosine Deamination . . . . . . . . . . . . . . 818 THE ENZYME FAMILY

  15. ORAL PRESENTATION Open Access Double-stranded RNA adenosine deaminase

    E-print Network

    Paris-Sud XI, Université de

    ORAL PRESENTATION Open Access Double-stranded RNA adenosine deaminase ADAR1 enhances both T cell. This family of cytokines activates the expression of antiviral proteins, such as the protein kinase R (PKR), an inhibitor of viral mRNA translation, and the double-stranded RNA adenosine deaminase ADAR1. ADAR1 has

  16. Adenosine deaminase in disorders of purine metabolism and in immune deficiency

    SciTech Connect

    Tritsch, G.L.

    1985-01-01

    This book consists of five parts and a section of poster papers. Some of the selection titles are: Adenosine Deaminase Impairment and Ribonucleotide Reductase in Human Cells; Adenosine Deaminase and Malignant Cells; Inhibition of Adenosine Deaminase to Increase the Antitumor Activity of Adenine Nucleoside Analogues; and Molecular Biology of the Adenosine Deaminase Gene and Messenger RNA.

  17. Silver(I) ions and cysteine detection based on photoinduced electron transfer mediated by cytosine-Ag(+)-cytosine base pairs.

    PubMed

    Xie, Wan Yi; Huang, Wei Tao; Li, Nian Bing; Luo, Hong Qun

    2011-10-21

    Upon formation of cytosine-Ag(+)-cytosine base pairs as a mediator for the photoinduced electron transfer, the fluorescence of FAM-labeled DNA was quenched and the fluorescence emission wavelength exhibited a red shift. Based on these phenomena a novel dual-output fluorescent DNA sensor for Ag(+) ions and cysteine detection was developed. PMID:21863169

  18. Expression of Human Adenosine Deaminase after Fusion of Adenosine Deaminase-Deficient Cells with Mouse Fibroblasts

    Microsoft Academic Search

    Michael J. Siciliano; Mary R. Bordelon; Peter O. Kohler

    1978-01-01

    Two human choriocarcinoma cell lines were shown to be deficient in adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) such that they did not produce bands on starch gels after electrophoresis and histochemical staining. Radiometric assay indicated that their ADA specific activity was approximately 2% that of HeLa (human) cell controls. Subclone analysis of one of the lines indicated that this

  19. Renal Deoxyadenosine Transport and Immunodeficiency Associated with Adenosine Deaminase Deficiency

    Microsoft Academic Search

    J. Arly Nelson

    1989-01-01

    Accumulation of deoxyadenosine (or possibly adenosine) is thought to mediate the immune defect associated with adenosine deaminase deficiency. It is postulated that deoxyadenosine is particularly immunosuppressive in the neonate due to an undeveloped renal secretory mechanism.

  20. Neurologic abnormalities in patients with adenosine deaminase deficiency.

    PubMed

    Nofech-Mozes, Yehuda; Blaser, Susan I; Kobayashi, Jeff; Grunebaum, Eyal; Roifman, Chaim M

    2007-09-01

    Defects in adenosine deaminase enzyme cause severe immunodeficiency. Without enzyme replacement or allogeneic bone marrow transplantation, patients often suffer fatal infection in infancy. Adenosine deaminase is expressed ubiquitously; deficiency may affect various organs, including the brain. Neurologic abnormalities occur in some adenosine deaminase-deficient patients, mostly in association with infection or after bone marrow transplantation. Three cases with significant neurologic abnormalities, including hypotonia, head lag, nystagmus, difficulty in focusing gaze, seizure disorder, and moderate-severe developmental delay but with no evidence of infection or transplant-related medication toxicity are presented. Computed tomographic scans and cranial MRI revealed volume loss and abnormalities of basal ganglia and thalamus, which may reflect accelerated nerve cell death or altered stimulation of adenosine receptors. Detailed neurologic and neuroimaging evaluation should be performed for all patients with adenosine deaminase deficiency upon diagnosis, to identify potentially significant brain lesions. PMID:17765813

  1. Transcription-coupled mutagenesis by the DNA deaminase AID

    PubMed Central

    Larson, Erik D; Maizels, Nancy

    2004-01-01

    Activation-induced deaminase (AID) initiates switch recombination and somatic hypermutation of immunoglobulin genes in activated B cells. Compelling evidence now shows that AID travels with RNA polymerase II to deaminate actively transcribed DNA. PMID:15003109

  2. Transcription-coupled mutagenesis by the DNA deaminase AID

    Microsoft Academic Search

    Erik D Larson; Nancy Maizels

    2004-01-01

    Activation-induced deaminase (AID) initiates switch recombination and somatic hypermutation of immunoglobulin genes in activated\\u000a B cells. Compelling evidence now shows that AID travels with RNA polymerase II to deaminate actively transcribed DNA.

  3. [Adenosine deaminase in severe combined immunodeficiency syndrome].

    PubMed

    Pérez-Aguilar, Mary Carmen; Goncalves, Loredana; Bonfante-Cabarcas, Rafael

    2012-09-01

    Adenosine deaminase is an enzyme of the purine metabolism whose function is to convert adenosine to inosine and deoxyadenosine to deoxyinosine. The ecto-ADA1 binding to the cell surface through CD26 contributes to the regulation of cytokines and stimulates the proliferation of T cells by activating CD45. The deficiency of this enzyme generates the severe combined immunodeficiency syndrome, characterized by the accumulation of deoxyadenosine and adenine metabolites, which have toxic effects on lymphocytes, affecting DNA synthesis and consequently, clonal expansion. Early diagnosis of this immunodeficiency is essential, as it significantly reduces morbidity and mortality associated with recurrent infections. Recent advances in molecular biology and genetics have led to the identification of genetic defects of many primary immunodeficiencies and the development of promising diagnostic tools and treatment. PMID:23248974

  4. Serum adenosine deaminase activity in cutaneous anthrax

    PubMed Central

    Sunnetcioglu, Mahmut; Karadas, Sevdegul; Aslan, Mehmet; Ceylan, Mehmet Resat; Demir, Halit; Oncu, Mehmet Resit; Karahocagil, Mustafa Kas?m; Sunnetcioglu, Aysel; Aypak, Cenk

    2014-01-01

    Background Adenosine deaminase (ADA) activity has been discovered in several inflammatory conditions; however, there are no data associated with cutaneous anthrax. The aim of this study was to investigate serum ADA activity in patients with cutaneous anthrax. Material/Methods Sixteen patients with cutaneous anthrax and 17 healthy controls were enrolled. We measured ADA activity; peripheral blood leukocyte, lymphocyte, neutrophil, and monocyte counts; erythrocyte sedimentation rate; and C reactive protein levels. Results Serum ADA activity was significantly higher in patients with cutaneous anthrax than in the controls (p<0.001). A positive correlation was observed between ADA activity and lymphocyte counts (r=0.589, p=0.021) in the patient group. Conclusions This study suggests that serum ADA could be used as a biochemical marker in cutaneous anthrax. PMID:24997584

  5. The catalase activity of diiron adenine deaminase

    PubMed Central

    Kamat, Siddhesh S; Holmes-Hampton, Gregory P; Bagaria, Ashima; Kumaran, Desigan; Tichy, Shane E; Gheyi, Tarun; Zheng, Xiaojing; Bain, Kevin; Groshong, Chris; Emtage, Spencer; Sauder, J Michael; Burley, Stephen K; Swaminathan, Subramanyam; Lindahl, Paul A; Raushel, Frank M

    2011-01-01

    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 Mn2+ 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 FeSO4. Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [FeII/FeII]-ADE catalyzed the conversion of H2O2 to O2 and H2O. The values of kcat and kcat/Km for the catalase activity are 200 s?1 and 2.4 × 104 M?1 s?1, respectively. [FeII/FeII]-ADE underwent more than 100 turnovers with H2O2 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 gave = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H2O2 by [FeII/FeII]-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. PMID:21998098

  6. Guanine-deaminase activity in rat brain and liver

    PubMed Central

    Kumar, S.; Tewari, K. K.; Krishnan, P. S.

    1965-01-01

    1. Guanine deaminase in rat brain and liver was distributed among all the subcellular fractions: nuclei, `heavy' mitochondria, `light' mitochondria, microsomes and the supernatant fluid. The greater part of the activity passed into the soluble fraction. Among the particulate components, the `light' mitochondria constituted the richest fraction. 2. The sum of the enzymic activities of the component fractions obtained on differential centrifugation was considerably greater than the activity of guanine deaminase in the whole homogenate. 3. The `heavy'-mitochondrial fraction had a powerful inhibitory effect on the guanine-deaminase activity of the supernatant fraction. 4. All the sedimented fractions, except the microsomes, gave rise to higher guanine-deaminase activity on treatment with Triton X-100. 5. The inhibitory capacity of the `heavy' mitochondria increased on treatment with Triton X-100; the detergent-treated nuclear fraction also brought about inhibition of the 5000g supernatant. 6. Guanine-deaminase inhibitor from the `heavy' mitochondria was solubilized by high-speed grinding of the particles, followed by treatment with Triton X-100. The inhibitor appeared to be protein in nature, since it was precipitated by trichloroacetic acid and by half-saturation with ammonium sulphate, and was non-diffusible. It was inactivated by heating at 50° for 5min. 7. It is possible that the guanine deaminase associated with particles differs from the soluble enzyme in its response to inhibitor. PMID:14342518

  7. Studies on guanine deaminase and its inhibitors in rat tissue

    PubMed Central

    Kumar, S.; Josan, V.; Sanger, K. C. S.; Tewari, K. K.; Krishnan, P. S.

    1967-01-01

    1. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, as in brain and liver, the enzymic activity recovered in the supernatant was higher than that in the whole homogenate. The particulate fractions of kidney, especially the heavy mitochondria, brought about powerful inhibition of the supernatant guanine-deaminase activity. 2. In spleen, as in kidney, guanine-deaminase activity was localized in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, the particulate fractions did not inhibit the activity of the supernatant. 3. Guanine-deaminase activity in rat brain was absent from the cerebellum and present only in the cerebral hemispheres. The inhibitor of guanine deaminase was located exclusively in the cerebellum, where it was associated with the particles sedimenting at 5000g from sucrose homogenates. 4. Homogenates of cerebral hemispheres, the separated cortex or the remaining portion of the hemispheres had significantly higher guanine-deaminase activity than homogenates of whole brain. The enzymic activity of the subcellular particulate fractions was nearly the same. 5. Guanine deaminase was purified from the 15000g supernatant of sucrose homogenates of whole brain. The enzyme separated as two distinct fractions, A and B, on DEAE-cellulose columns. 6. The guanine-deaminase activity of the light-mitochondrial fraction of whole brain was fully exposed and solubilized by treatment with Triton X-100, and partially purified. 7. Tested in the form of crude preparations, the inhibitor from kidney did not act on the brain and liver supernatant enzymes and the inhibitor from cerebellum did not act on kidney enzyme, but the inhibitor from liver acted on both brain and kidney enzyme. 8. The inhibitor of guanine deaminase was purified from the heavy mitochondria of whole brain and liver and the 5000g residue of cerebellum, isolated from iso-osmotic homogenates. The inhibitor appeared to be protein in nature and was heat-labile. The inhibition of the enzyme was non-competitive. 9. Kinetic, immunochemical and electrophoretic studies with the preparations purified from brain revealed that the enzyme from light mitochondria was distinct from enzyme B from the supernatant. A distinction between the two forms of supernatant enzyme was less certain. 10. Guanine deaminase isolated from light mitochondria of brain did not react with 8-azaguanine or with the inhibitor isolated from heavy mitochondria. PMID:16742482

  8. Hot spot mutations in adenosine deaminase deficiency

    SciTech Connect

    Hirschhorn, R.; Tzall, S.; Ellenbogen, A. (New York Univ. Medical School, NY (USA))

    1990-08-01

    The authors have previously characterized mutant adenosine deaminase enzymes in seven children with partial ADA deficiency. Six children shared common origins, suggesting a common progenitor. However, they found evidence for multiple phenotypically different mutant enzymes. They hypothesized that many of the mutations would be at CpG dinucleotides, hot spots at which spontaneous deamination of 5-methylcytosine results in C to T or G to A transitions. Digestion of DNA from these children with Msp I and Taq I, enzymes recognizing CpG dinucleotides, identified three different mutations, each correlating with expression of a different mutant enzyme. Sequencing of cDNA clones and genomic DNA amplified by polymerase chain reaction confirmed the presence of C to T or G to A transitions at CpG dinucleotides. To determine the true frequency of hot spot mutation in these children, consecutively ascertained through a newborn screening program, they sequenced cDNA from the remaining alleles. Two others were hot spot mutations each again resulting in expression of a phenotypically different mutant enzyme. These seven mutations account for all 14 chromosomes in these children. There is thus a very high frequency of hot spot mutations in partial ADA deficiency. They were able to correlate genotype and phenotype and to dissect the activity of individual mutant alleles.

  9. Genetic and biochemical consequences of adenosine deaminase deficiency in humans.

    PubMed

    Bose, Rahul; Nandagopal, Krishnadas

    2013-10-01

    Adenosine deaminase deficiency accounts for approximately 15-20% of severe combined immunodeficiency in humans. The gene for adenosine deaminase is located on chromosome 20q12-q13.11 and codes for an aminohydrolase that catalyzes the deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. Absence of the enzyme causes a build-up of the substrates in addition to excess deoxyadenosine triphosphate, thereby compromising the regenerative capacity of the immune system. Due to underlying allelic heterogeneity, the disorder manifests as a spectrum, ranging from neonatal onset severe combined immunodeficiency to apparently normal partial adenosine deaminase deficiency. Tandem mass spectrometry coupled with high efficiency separation systems enables postnatal diagnosis of the disorder, while prenatal diagnosis relies on assaying enzyme activity in cultured amniotic fibroblasts or chorionic villi sampling. Screening of adenosine deaminase deficiency for relatives-at-risk may reduce costs of treatment and ensure timely medical intervention as applicable. This article reviews the genetic, biochemical and clinical aspects of adenosine deaminase deficiency. PMID:24772956

  10. Altered AMP deaminase activity may extend postmortem glycolysis.

    PubMed

    England, E M; Matarneh, S K; Scheffler, T L; Wachet, C; Gerrard, D E

    2015-04-01

    Postmortem energy metabolism drives hydrogen accumulation in muscle and results in a fairly constant ultimate pH. Extended glycolysis results in adverse pork quality and may be possible with greater adenonucleotide availability postmortem. We hypothesized that slowing adenonucleotide removal by reducing AMP deaminase activity would extend glycolysis and lower the ultimate pH of muscle. Longissimus muscle samples were incorporated into an in vitro system that mimics postmortem glycolysis with or without pentostatin, an AMP deaminase inhibitor. Pentostatin lowered ultimate pH and increased lactate and glucose 6-phosphate with time. Based on these results and that AMPK ?3(R200Q) mutated pigs (RN(-)) produce low ultimate pH pork, we hypothesized AMP deaminase abundance and activity would be lower in RN(-) muscle than wild-type. RN(-) muscle contained lower AMP deaminase abundance and activity. These data show that altering adenonucleotide availability postmortem can extend postmortem pH decline and suggest that AMP deaminase activity may, in part, contribute to the low ultimate pH observed in RN(-) pork. PMID:25498483

  11. Demethylation of 6-O-Methylinosine by an RNA-Editing Adenosine Deaminase

    E-print Network

    Beal, Peter A.

    Demethylation of 6-O-Methylinosine by an RNA-Editing Adenosine Deaminase LaHoma M. Easterwood Lake City, Utah 84112 ReceiVed August 25, 2000 ADARs are adenosine deaminases that act on RNA-modifying enzyme adenosine deaminase (ADA).5-8 These experiments illustrate significant mechanistic similarities

  12. Substrate Analogues for an RNA-Editing Adenosine Deaminase: Mechanistic Investigation and Inhibitor Design

    E-print Network

    Beal, Peter A.

    Substrate Analogues for an RNA-Editing Adenosine Deaminase: Mechanistic Investigation and Inhibitor@chem.utah.edu Abstract: ADARs are adenosine deaminases that act on RNA and are responsible for RNA-editing reactions,6-Diaminopurine ribonucleoside in RNA is not a substrate for ADAR, in contrast to adenosine deaminase (ADA), which

  13. Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila

    E-print Network

    Â?urovec, Michal

    Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila by depleting-A and ADGF-D are active adenosine deaminases (ADAs), and they cause polarization and serum: the imaginal disk growth factors (IDGFs) (4), which are related to chitinases, and the adenosine deaminase

  14. Double-Stranded RNA Adenosine Deaminases ADAR1 and ADAR2 Have Overlapping Specificities

    E-print Network

    Bass, Brenda L.

    Double-Stranded RNA Adenosine Deaminases ADAR1 and ADAR2 Have Overlapping Specificities Katrina A, 2000 ABSTRACT: Adenosine deaminases that act on RNA (ADARs) deaminate adenosines to produce inosines adenosine in vivo. Adenosine deaminases that act on RNA (ADARs)1 deami- nate adenosines to produce inosines

  15. Delayed onset adenosine deaminase deficiency associated with acute disseminated encephalomyelitis.

    PubMed

    Nakaoka, Hideyuki; Kanegane, Hirokazu; Taneichi, Hiromichi; Miya, Kazushi; Yang, Xi; Nomura, Keiko; Takezaki, Shunichiro; Yamada, Masafumi; Ohara, Osamu; Kamae, Chikako; Imai, Kohsuke; Nonoyama, Shigeaki; Wada, Taizo; Yachie, Akihiro; Hershfield, Michael S; Ariga, Tadashi; Miyawaki, Toshio

    2012-06-01

    Acute disseminated encephalomyelitis (ADEM) is a monophasic, immune-mediated demyelinating disorder that can appear after either immunizations or, more often, infections. Magnetic resonance imaging of patients shows inflammatory lesions in the brain and spinal cord. An immune-mediated mechanism may play a role in this disease, although its precise pathogenesis remains unclear. In this study, a 2-year-old boy presented with ADEM, and he showed improvement on treatment with high-dose intravenous corticosteroids. At the age of 3 years, the presence of recurrent bronchitis, bronchiectasia, and lymphopenia suggested that the patient was suffering from combined immunodeficiency. The patient was finally diagnosed with delayed onset adenosine deaminase deficiency. Delayed onset adenosine deaminase deficiency is frequently associated with autoimmune diseases, including thyroiditis and cytopenia, both of which were observed in the patient. The ADEM in this patient may be a presentation of delayed onset adenosine deaminase deficiency. PMID:22447032

  16. Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation

    PubMed Central

    Li, Li; Wu, Jun; Deng, Zixin; Zabriskie, T. Mark

    2013-01-01

    Blasticidin S is a peptidyl nucleoside antibiotic produced by Streptomyces griseochromogenes that exhibits strong fungicidal activity. To circumvent an effective DNA uptake barrier system in the native producer and investigate its biosynthesis in vivo, the blasticidin S biosynthetic gene cluster (bls) was engrafted to the chromosome of Streptomyces lividans. However, the resulting mutant, LL2, produced the inactive deaminohydroxyblasticidin S instead of blasticidin S. Subsequently, a blasticidin S deaminase (SLBSD, for S. lividans blasticidin S deaminase) was identified in S. lividans and shown to govern this in vivo conversion. Purified SLBSD was found to be capable of transforming blasticidin S to deaminohydroxyblasticidin S in vitro. It also catalyzed deamination of the cytosine moiety of cytosylglucuronic acid, an intermediate in blasticidin S biosynthesis. Disruption of the SLBSD gene in S. lividans LL2 led to successful production of active blasticidin S in the resultant mutant, S. lividans WJ2. To demonstrate the easy manipulation of the blasticidin S biosynthetic gene cluster, blsE, blsF, and blsL, encoding a predicted radical S-adenosylmethionine (SAM) protein, an unknown protein, and a guanidino methyltransferase, were individually inactivated to access their role in blasticidin S biosynthesis. PMID:23377931

  17. Streptomyces lividans blasticidin S deaminase and its application in engineering a blasticidin S-producing strain for ease of genetic manipulation.

    PubMed

    Li, Li; Wu, Jun; Deng, Zixin; Zabriskie, T Mark; He, Xinyi

    2013-04-01

    Blasticidin S is a peptidyl nucleoside antibiotic produced by Streptomyces griseochromogenes that exhibits strong fungicidal activity. To circumvent an effective DNA uptake barrier system in the native producer and investigate its biosynthesis in vivo, the blasticidin S biosynthetic gene cluster (bls) was engrafted to the chromosome of Streptomyces lividans. However, the resulting mutant, LL2, produced the inactive deaminohydroxyblasticidin S instead of blasticidin S. Subsequently, a blasticidin S deaminase (SLBSD, for S. lividans blasticidin S deaminase) was identified in S. lividans and shown to govern this in vivo conversion. Purified SLBSD was found to be capable of transforming blasticidin S to deaminohydroxyblasticidin S in vitro. It also catalyzed deamination of the cytosine moiety of cytosylglucuronic acid, an intermediate in blasticidin S biosynthesis. Disruption of the SLBSD gene in S. lividans LL2 led to successful production of active blasticidin S in the resultant mutant, S. lividans WJ2. To demonstrate the easy manipulation of the blasticidin S biosynthetic gene cluster, blsE, blsF, and blsL, encoding a predicted radical S-adenosylmethionine (SAM) protein, an unknown protein, and a guanidino methyltransferase, were individually inactivated to access their role in blasticidin S biosynthesis. PMID:23377931

  18. Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization.

    PubMed

    Pornbanlualap, Somchai; Chalopagorn, Pornchanok

    2011-08-01

    The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s?¹ at 30 °C. Since adenine is deaminated ?10³ slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-?-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common ?/? barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism. PMID:21511036

  19. Human Papillomavirus E6 Triggers Upregulation of the Antiviral and Cancer Genomic DNA Deaminase APOBEC3B

    PubMed Central

    Vieira, Valdimara C.; Leonard, Brandon; White, Elizabeth A.; Starrett, Gabriel J.; Temiz, Nuri A.; Lorenz, Laurel D.; Lee, Denis; Soares, Marcelo A.; Lambert, Paul F.; Howley, Peter M.

    2014-01-01

    ABSTRACT Several recent studies have converged upon the innate immune DNA cytosine deaminase APOBEC3B (A3B) as a significant source of genomic uracil lesions and mutagenesis in multiple human cancers, including those of the breast, head/neck, cervix, bladder, lung, ovary, and other tissues. A3B is upregulated in these tumor types relative to normal tissues, but the mechanism is unclear. Because A3B also has antiviral activity in multiple systems and is a member of the broader innate immune response, we tested the hypothesis that human papillomavirus (HPV) infection causes A3B upregulation. We found that A3B mRNA expression and enzymatic activity were upregulated following transfection of a high-risk HPV genome and that this effect was abrogated by inactivation of E6. Transduction experiments showed that the E6 oncoprotein alone was sufficient to cause A3B upregulation, and a panel of high-risk E6 proteins triggered higher A3B levels than did a panel of low-risk or noncancer E6 proteins. Knockdown experiments in HPV-positive cell lines showed that endogenous E6 is required for A3B upregulation. Analyses of publicly available head/neck cancer data further support this relationship, as A3B levels are higher in HPV-positive cancers than in HPV-negative cancers. Taken together with the established role for high-risk E6 in functional inactivation of TP53 and published positive correlations in breast cancer between A3B upregulation and genetic inactivation of TP53, our studies suggest a model in which high-risk HPV E6, possibly through functional inactivation of TP53, causes derepression of A3B gene transcription. This would lead to a mutator phenotype that explains the observed cytosine mutation biases in HPV-positive head/neck and cervical cancers. PMID:25538195

  20. Guanine deaminase functions as dihydropterin deaminase in the biosynthesis of aurodrosopterin, a minor red eye pigment of Drosophila.

    PubMed

    Kim, Jaekwang; Park, Sang Ick; Ahn, Chiyoung; Kim, Heuijong; Yim, Jeongbin

    2009-08-28

    Dihydropterin deaminase, which catalyzes the conversion of 7,8-dihydropterin to 7,8-dihydrolumazine, was purified 5850-fold to apparent homogeneity from Drosophila melanogaster. Its molecular mass was estimated to be 48 kDa by gel filtration and SDS-PAGE, indicating that it is a monomer under native conditions. The pI value, temperature, and optimal pH of the enzyme were 5.5, 40 degrees C, and 7.5, respectively. Interestingly the enzyme had much higher activity for guanine than for 7,8-dihydropterin. The specificity constant (k(cat)/K(m)) for guanine (8.6 x 10(6) m(-1).s(-1)) was 860-fold higher than that for 7,8-dihydropterin (1.0 x 10(4) m(-1).s(-1)). The structural gene of the enzyme was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis as CG18143, located at region 82A1 on chromosome 3R. The cloned and expressed CG18143 exhibited both 7,8-dihydropterin and guanine deaminase activities. Flies with mutations in CG18143, SUPor-P/Df(3R)A321R1 transheterozygotes, had severely decreased activities in both deaminases compared with the wild type. Among several red eye pigments, the level of aurodrosopterin was specifically decreased in the mutant, and the amount of xanthine and uric acid also decreased considerably to 76 and 59% of the amounts in the wild type, respectively. In conclusion, dihydropterin deaminase encoded by CG18143 plays a role in the biosynthesis of aurodrosopterin by providing one of its precursors, 7,8-dihydrolumazine, from 7,8-dihydropterin. Dihydropterin deaminase also functions as guanine deaminase, an important enzyme for purine metabolism. PMID:19567870

  1. Cytosine as an indicator of microbial nitrogen in the rumen

    E-print Network

    Hegerle, Kelly Michael

    1984-01-01

    CYTOSINE AS AN INDICATOR OF MICROBIAL NITROGEN IN THE RUMEN A Thesis KELLY MICHAEL HEGERLE Submitted to the Graduate College of Tesas AQN University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE December l984... Ma)or Sub)ect: Nutrition CYTOSINE AS AN INDICATOR OF MICROBIAL NITROGEN IN THE RUMEN A Thesis KELLY MZCHAEL HEGERLE Approved as to style and content by: Gerald T. Schelling Chairman of Committee) . W. Greene (M er) W. ms (Member) Gary C...

  2. The hydrogen bonding properties of cytosine: a computational study of cytosine complexed with hydrogen fluoride, water, and ammonia.

    PubMed

    Hunter, Ken C; Rutledge, Lesley R; Wetmore, Stacey D

    2005-10-27

    Density functional theory is used to study the hydrogen bonding pattern in cytosine, which does not contain alternating proton donor and acceptor sites and therefore is unique compared with the other pyrimidines. Complexes between various small molecules (HF, H(2)O, and NH(3)) and four main binding sites in (neutral and (N1) anionic) cytosine are considered. Two complexes (O2(N1) and N3(N4)) involve neighboring cytosine proton acceptor and donor sites, which leads to cooperative interactions and bidendate hydrogen bonds. The third (less stable) complex (N4) involves a single cytosine donor. The final (O2-N3) complex involves two cytosine proton acceptors, which leads to an anticooperative hydrogen bonding pattern for H(2)O and NH(3). On the neutral surface, the anticooperative O2-N3 complex is less stable than those involving bidentate hydrogen bonds, and the H(2)O complex cannot be characterized when diffuse functions are included in the (6-31G(d,p)) basis set. On the contrary, the anionic O2-N3 structure is the most stable complex, while the HF and H(2)O N3(N4) complexes cannot be characterized with diffuse functions. B3LYP and MP2 potential energy surface scans are used to consider the relationship between the water N3(N4) and O2-N3 complexes. These calculations reveal that diffuse functions reduce the conversion barrier between the two complexes on both the neutral and anionic surfaces, where the reduction leads to a (O2-N3) energy plateau on the neutral surface and complete (N3(N4)) complex destabilization on the anionic surface. From these complexes, the effects of hydrogen bonds on the (N1) acidity of cytosine are determined, and it is found that the trends in the effects of hydrogen bonds on the (N1) acidity are similar for all pyrimidines. PMID:16866407

  3. Spoilage yeasts.

    PubMed

    Fleet, G

    1992-01-01

    Yeasts are best known for their beneficial contributions to society, and the literature abounds with discussions of their role in the fermentation of alcoholic beverages, bread, and other products. Yeasts also cause spoilage, but, with a few exceptions, this unwanted activity often goes unrecognized and underestimated as a major problem in the food and beverage industries. In some cases, there is only a fine line between what is perceived as either a spoilage or beneficial activity. This review examines the occurrence and growth of yeasts in foods and beverages with respect to their spoilage activities, the biochemistry of this spoilage, and technologies for the enumeration and identification of spoilage yeasts. PMID:1733519

  4. Endogenous Cytosine Damage Products Alter the Site Selectivity of Human DNA Maintenance Methyltransferase DNMT1

    Microsoft Academic Search

    Victoria Valinluck; Lawrence C. Sowers

    2007-01-01

    Alterations in cytosine methylation patterns are usually observed in human tumors. The consequences of altered cytosine methylation patterns include both inappropriate activation of transforming genes and silencing of tumor suppressor genes. Despite the biological effect of methyla- tion changes, little is known about how such changes are caused. The heritability of cytosine methylation patterns from parent to progeny cells is

  5. Assemblies of cytosine within H-bonded network of adipic acid and citric acid

    NASA Astrophysics Data System (ADS)

    Das, Babulal; Baruah, Jubaraj B.

    2011-08-01

    Adipic acid binds to cytosine to form H-bonded discrete cytosine-cytosinium assemblies embedded in 1D infinite chain of adipic acid, whereas citric acid stabilizes trimeric cytosine-cytosinium assemblies having length of 19.44 Å stabilized between layered structures of citric acid molecules.

  6. Surfactant mediated optical properties of cytosine capped CdSe quantum dots Prashant K. Sharma a,

    E-print Network

    Pandey, Ravi

    Luminescence This letter demonstrates the use of one of the nucleobases, `cytosine' as a new capping agent, rather cytosine will only act as a new capping agent for reducing the surface defect density of QDs. 2Surfactant mediated optical properties of cytosine capped CdSe quantum dots Prashant K. Sharma a

  7. Sensorineural deafness in siblings with adenosine deaminase deficiency

    Microsoft Academic Search

    Chitose Tanaka; Toshiro Hara; Ichiro Suzaki; Yoshihiro Maegaki; Kenzo Takeshita

    1996-01-01

    Two siblings with adenosine deaminase deficiency were successfully treated with allogeneic bone marrow transplantation without conditioning. Although the patients were free from infections after immumologic reconstitution, both showed sensorineural deafness at 1 year of age. Because there were no structural abnormalities in the inner and middle ears, no evidence of prenatal infections of rubella, cytomegalovirus or toxoplasma, and no postnatal

  8. Gene Therapy for Immunodeficiency Due to Adenosine Deaminase Deficiency

    Microsoft Academic Search

    Alessandro Aiuti; Federica Cattaneo; Stefania Galimberti; Ulrike Benninghoff; Barbara Cassani; Luciano Callegaro; Samantha Scaramuzza; Grazia Andolfi; Massimiliano Mirolo; Immacolata Brigida; Antonella Tabucchi; Filippo Carlucci; Martha Eibl; Memet Aker; Shimon Slavin; Hamoud Al-Mousa; Abdulaziz Al Ghonaium; Alina Ferster; Andrea Duppenthaler; Luigi Notarangelo; Uwe Wintergerst; Rebecca H. Buckley; Marco Bregni; Sarah Marktel; Maria Grazia Valsecchi; Paolo Rossi; Fabio Ciceri; Roberto Miniero; Claudio Bordignon; Maria-Grazia Roncarolo

    2009-01-01

    Background We investigated the long-term outcome of gene therapy for severe combined immu- nodeficiency (SCID) due to the lack of adenosine deaminase (ADA), a fatal disorder of purine metabolism and immunodeficiency. Methods We infused autologous CD34+ bone marrow cells transduced with a retroviral vec- tor containing the ADA gene into 10 children with SCID due to ADA deficiency who lacked

  9. Adenosine-5'-phosphate deaminase. A novel herbicide target.

    PubMed Central

    Dancer, J E; Hughes, R G; Lindell, S D

    1997-01-01

    The isolation of carbocyclic coformycin as the herbicidally active component from a fermentation of Saccharothrix species was described previously (B.D. Bush, G.V. Fitchett, D.A. Gates, D. Langley [1993] Phytochemistry 32: 737-739). Here we report that the primary mode of action of carbocyclic coformycin has been identified as inhibition of the enzyme AMP deaminase (EC 3.5.4.6) following phosphorylation at the 5' hydroxyl on the carbocyclic ring in vivo. When pea (Pisum sativum L. var Onward) seedlings are treated with carbocyclic coformycin, there is a very rapid and dramatic increase in ATP levels, indicating a perturbation in purine metabolism. Investigation of the enzymes of purine metabolism showed a decrease in the extractable activity of AMP deaminase that correlates with a strong, noncovalent association of the phosphorylated natural product with the protein. The 5'-phosphate analog of the carbocyclic coformycin was synthesized and shown to be a potent, tight binding inhibitor of AMP deaminase isolated from pea seedlings. Through the use of a synthetic radiolabeled marker, rapid conversion of carbocyclic coformycin to the 5'-phosphate analog could be demonstrated in vivo. It is proposed that inhibition of AMP deaminase leads to the death of the plant through perturbation of the intracellular ATP pool. PMID:9159944

  10. Concentration by Evaporation and the Prebiotic Synthesis of Cytosine

    NASA Technical Reports Server (NTRS)

    Nelson, Kevin E.; Robertson, Michael P.; Levy, Matthew; Miller, Stanley L.

    2001-01-01

    The efficient prebiotic synthesis of cytosine from urea and cyanoacetaldehyde (CA) has recently been claimed to be invalid on the basis of possible side reactions of the starting materials and the inapplicability of prebiotic syntheses using drying beach conditions. We therefore have investigated the synthesis of cytosine and uracil from urea and cyanoacetaldehyde at 100 C under dry-down conditions, and in solution at 4 C and -20 C. We find that cytosine is produced from the low temperature experiments more efficiently than calculated from the Arrhenius extrapolation from higher temperatures, i.e., 60-120 C. In addition, we find that CA dimer is as efficient as the monomer in cytosine synthesis. We also studied whether evaporating very dilute solutions of nonvolatile organic compounds will concentrate according to theory. Solutions as dilute as 10(exp -4) M concentrate from pure water approximately according to theory. Similar solutions in 0.5 M NaCl have less than theoretical concentrations due to absorption, but concentrations near dryness were very high.

  11. NITROSATIVE CYTOSINE DEAMINATION: A NOVEL MECHANISM OF MUTAGENESIS

    E-print Network

    Glaser, Rainer

    NITROSATIVE CYTOSINE DEAMINATION: A NOVEL MECHANISM OF MUTAGENESIS Sundeep Rayat and Rainer Glaser) Bunton, C. A.; Wolfe, B. B. J. Am. Chem. Soc. 1974, 96, 7747-7752. 3 Glaser, R.; Rayat, S.; Lewis, M.; Son, M.-S.; Meyer, S. J. Am. Chem. Soc. 1999, 121, 6108. 4 Rayat, S. and Glaser, R., submitted

  12. HYBRID MODEL OF ERYTHROPOIESIS AND LEUKEMIA TREATMENT WITH CYTOSINE ARABINOSIDE

    E-print Network

    Paris-Sud XI, Université de

    HYBRID MODEL OF ERYTHROPOIESIS AND LEUKEMIA TREATMENT WITH CYTOSINE ARABINOSIDE POLINA KURBATOVA is used to study normal and leukemic red blood cell production (erythropoiesis), and treatment of leukemia the relevance of considering chronotherapeutic treatments to cure leukemia. Key words. Hybrid model, leukemia

  13. Burkitt's lymphoma in a patient with adenosine deaminase deficiency-severe combined immunodeficiency treated with polyethylene glycol-adenosine deaminase.

    PubMed

    Husain, Maitham; Grunebaum, Eyal; Naqvi, Ahmed; Atkinson, Adelle; Ngan, Bo-Yee; Aiuti, Alessandro; Roifman, Chaim M

    2007-07-01

    We describe a patient with severe combined immunodeficiency because of aberrations in adenosine deaminase (ADA) who despite adequate replacement with polyethylene glycol-linked ADA (PEG-ADA) for 13 years developed Burkitt's lymphoma. Although treatment corrected the metabolic abnormalities caused by ADA deficiency, it failed to fully restore cellular immunity. PMID:17586199

  14. Red yeast

    MedlinePLUS

    ... problems. Other conditions. More evidence is needed to rate the effectiveness of red yeast for these uses. ... can affect the muscles. Red yeast can also affect the muscles. Taking niacin along with ... cautious with this combination.Talk with your health provider.

  15. Dry yeast

    NSDL National Science Digital Library

    Ranveig Thattai (None; )

    2005-09-27

    Yeast is a type of eukaryotic organism that can live in a dormant state. It can be activated from its dormant state by water and sugar. The yeast uses the sugar to grow and produces carbon dioxide gas as a byproduct.

  16. EPR study of a copper center in a single crystal of cytosine monohydrate.

    PubMed

    Krilov, Dubravka; Leki?, Andrica; Besi?, Erim; Herak, Janko N

    2005-03-01

    Copper is found incorporated into the crystal structure of cytosine monohydrate grown from aqueous solution of commercially available cytosine. Upon ionizing irradiation, the crystals exhibited the electron paramagnetic resonance (EPR) spectra characteristic of Cu(II) complex. Planar coordination bonding to the cupric ion, having three nitrogen atoms and an oxygen as ligands, is interpreted to bridge two cytosine molecules, replacing the two cytosine-cytosine hydrogen bonds present in pure crystals. The EPR signals are much stronger for crystals grown from the solutions to which small amount of copper powder were added. PMID:15708811

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

    SciTech Connect

    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 identity of the rate-limiting steps.

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

    SciTech Connect

    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 mechanism and the identity of the rate-limiting steps.

  19. Adenosine deaminase activity in the diagnosis of tuberculous peritonitis.

    PubMed Central

    Martinez-Vazquez, J M; Ocaña, I; Ribera, E; Segura, R M; Pascual, C

    1986-01-01

    We studied the activity of adenosine deaminase in the peritoneal fluid of 66 patients who were divided into five groups according to causes of ascites as follows: tuberculous peritonitis (group I), septic peritonitis (group II), secondary to malignant tumours (group III), miscellaneous conditions (group IV), and control subjects of transudates (group V). In patients with tuberculous peritonitis the enzyme activity was significantly higher than for the rest of the groups (p less than 0.001), and enzyme concentrations in all patients were well above the upper non-tuberculous value. Adenosine deaminase activity in the peritoneal fluid has proved to be a simple and reliable method for early diagnosis of tuberculous peritonitis. PMID:3758818

  20. Participation of an extracellular deaminase in amino acid utilization by Neurospora crassa.

    PubMed Central

    DeBusk, R M; Ogilvie, S

    1984-01-01

    A strain of Neurospora crassa defective in amino acid transport can utilize a variety of amino acids for growth when readily metabolizable nitrogen is limiting. Growth is accompanied by the production of an extracellular deaminase that converts the amino acid to its respective keto acid plus equimolar quantities of utilizable nitrogen in the ammonium ion form. Production of the deaminase is subject to ammonium repression. The relationship between the ability of an amino acid to trigger deaminase production and the presence of particular amino acid permease deficiencies is complex. Four classes of amino acids have been defined with respect to this relationship. The existence of multiple extracellular deaminases is discussed. PMID:6235210

  1. Capillary Electrophoresis in Diagnosis and Monitoring of Adenosine Deaminase Deficiency

    Microsoft Academic Search

    Filippo Carlucci; Antonella Tabucchi; Alessandro Aiuti; Francesca Rosi; Federica Floccari; Roberto Pagani; Enrico Marinello

    Background: The diagnosis and monitoring of severe combined immunodeficiency disease (SCID) attribut- able to adenosine deaminase (ADA) deficiency requires measurements of ADA, purine nucleoside phosphory- lase (PNP), and S-adenosyl-L-homocysteine-hydrolase (SAHH) activity and of deoxyadenosine metabolites. We developed capillary electrophoresis (CE) methods for the detection of key diagnostic metabolites and evalua- tion of enzyme activities. Methods: Deoxyadenosine metabolites were separated in

  2. Gene therapy for adenosine-deaminase-deficient severe combined immunodeficiency

    Microsoft Academic Search

    Alessandro Aiuti

    2004-01-01

    Adenosine-deaminase-deficient SCID was the first inherited disease to be treated with gene therapy. This life-threatening disorder is characterized by a purine defect that leads to impaired immune functions, recurrent infections and systemic metabolic abnormalities. The early gene therapy trials showed the safety and feasibility of engineering haematopoietic stem cells and peripheral blood lymphocytes using retroviral vectors. However, all patients were

  3. Severe combined immunodeficiency due to adenosine deaminase deficiency.

    PubMed

    Hussain, Waqar; Batool, Asma; Ahmed, Tahir Aziz; Bashir, Muhammad Mukarram

    2012-03-01

    Severe Combined Immunodeficiency is the term applied to a group of rare genetic disorders characterised by defective or absent T and B cell functions. Patients usually present in first 6 months of life with respiratory/gastrointestinal tract infections and failure to thrive. Among the various types of severe combined immunodeficiency, enzyme deficiencies are relatively less common. We report the case of a 6 years old girl having severe combined immunodeficiency due to adenosine deaminase deficiency. PMID:22764473

  4. Animal Model for Immune Dysfunction Associated with Adenosine Deaminase Deficiency

    Microsoft Academic Search

    Antonio Tedde; M. Earl Balis; Susumu Ikehara; Rajendra Pahwa; Robert A. Good; Paul P. Trotta

    1980-01-01

    An in vivo murine model for immunodeficiency of both B and T cells is produced by continuous intraperitoneal infusion of 2'-deoxycoformycin (DCF), a specific tightly binding inhibitor of adenosine deaminase (ADase; adenosine aminohydrolase, EC 3.5.4.4). After DCF infusion, ADase of thymus, spleen, and lymph nodes was inhibited to varying degrees ranging from 57% to 100%. Immunodeficiency under these conditions was

  5. Adenosine Deaminases Acting on RNA, RNA Editing, and Interferon Action

    PubMed Central

    George, Cyril X.; Gan, Zhenji; Liu, Yong

    2011-01-01

    Adenosine deaminases acting on RNA (ADARs) catalyze adenosine (A) to inosine (I) editing of RNA that possesses double-stranded (ds) structure. A-to-I RNA editing results in nucleotide substitution, because I is recognized as G instead of A both by ribosomes and by RNA polymerases. A-to-I substitution can also cause dsRNA destabilization, as I:U mismatch base pairs are less stable than A:U base pairs. Three mammalian ADAR genes are known, of which two encode active deaminases (ADAR1 and ADAR2). Alternative promoters together with alternative splicing give rise to two protein size forms of ADAR1: an interferon-inducible ADAR1-p150 deaminase that binds dsRNA and Z-DNA, and a constitutively expressed ADAR1-p110 deaminase. ADAR2, like ADAR1-p110, is constitutively expressed and binds dsRNA. A-to-I editing occurs with both viral and cellular RNAs, and affects a broad range of biological processes. These include virus growth and persistence, apoptosis and embryogenesis, neurotransmitter receptor and ion channel function, pancreatic cell function, and post-transcriptional gene regulation by microRNAs. Biochemical processes that provide a framework for understanding the physiologic changes following ADAR-catalyzed A-to-I (?=?G) editing events include mRNA translation by changing codons and hence the amino acid sequence of proteins; pre-mRNA splicing by altering splice site recognition sequences; RNA stability by changing sequences involved in nuclease recognition; genetic stability in the case of RNA virus genomes by changing sequences during viral RNA replication; and RNA-structure-dependent activities such as microRNA production or targeting or protein–RNA interactions. PMID:21182352

  6. Free radical adducts induce alterations in DNA cytosine methylation.

    PubMed Central

    Weitzman, S A; Turk, P W; Milkowski, D H; Kozlowski, K

    1994-01-01

    Methylation of cytosines in DNA is important for the regulation of expression of many genes. During carcinogenesis, normal patterns of gene methylation can be altered. Oxygen radical injury, shown to damage DNA in a variety of ways associated with cancer development and other conditions, has been suggested to affect DNA methylation, but a mechanism has not been demonstrated. Using oligonucleotides containing the common oxygen radical adduct 8-hydroxyguanine to replace guanine, we found that the enzymatic methylation of adjacent cytosines is profoundly altered. Furthermore, there is a high degree of positional specificity with respect to this effect. Thus, free radical injury may explain some of the altered methylation observed during carcinogenesis. Images PMID:8108398

  7. Specific targeting of cytosine methylation to DNA sequences in vivo

    PubMed Central

    Smith, Alexander E.; Ford, Kevin G.

    2007-01-01

    Development of methods that will allow exogenous imposition of inheritable gene-specific methylation patterns has potential application in both therapeutics and in basic research. An ongoing approach is the use of targeted DNA methyltransferases, which consist of a fusion between gene-targeted zinc-finger proteins and prokaryotic DNA cytosine methyltransferases. These enzymes however have so far demonstrated significant and unacceptable levels of non-targeted methylation. We now report the development of second-generation targeted methyltransferase enzymes comprising enhanced zinc-finger arrays coupled to methyltransferase mutants that are functionally dominated by their zinc-finger component. Both in vitro plasmid methylation studies and a novel bacterial assay reveal a high degree of target-specific methylation by these enzymes. Furthermore, we demonstrate for the first time transient expression of targeted cytosine methyltransferase in mammalian cells resulting in the specific methylation of a chromosomal locus. Importantly, the resultant methylation pattern is inherited through successive cell divisions. PMID:17182629

  8. High-throughput sequencing of cytosine methylation in plant DNA

    PubMed Central

    2013-01-01

    Cytosine methylation is a significant and widespread regulatory factor in plant systems. Methods for the high-throughput sequencing of methylation have allowed a greatly improved characterisation of the methylome. Here we discuss currently available methods for generation and analysis of high-throughput sequencing of methylation data. We also discuss the results previously acquired through sequencing plant methylomes, and highlight remaining challenges in this field. PMID:23758782

  9. Cytosine modifications in the honey bee (Apis mellifera) worker genome.

    PubMed

    Rasmussen, Erik M K; Amdam, Gro V

    2015-01-01

    Epigenetic changes enable genomes to respond to changes in the environment, such as altered nutrition, activity, or social setting. Epigenetic modifications, thereby, provide a source of phenotypic plasticity in many species. The honey bee (Apis mellifera) uses nutritionally sensitive epigenetic control mechanisms in the development of the royal caste (queens) and the workers. The workers are functionally sterile females that can take on a range of distinct physiological and/or behavioral phenotypes in response to environmental changes. Honey bees have a wide repertoire of epigenetic mechanisms which, as in mammals, include cytosine methylation, hydroxymethylated cytosines, together with the enzymatic machinery responsible for these cytosine modifications. Current data suggests that honey bees provide an excellent system for studying the "social repertoire" of the epigenome. In this review, we elucidate what is known so far about the honey bee epigenome and its mechanisms. Our discussion includes what may distinguish honey bees from other model animals, how the epigenome can influence worker behavioral task separation, and how future studies can answer central questions about the role of the epigenome in social behavior. PMID:25705215

  10. Cytosine modifications in the honey bee (Apis mellifera) worker genome

    PubMed Central

    Rasmussen, Erik M. K.; Amdam, Gro V.

    2015-01-01

    Epigenetic changes enable genomes to respond to changes in the environment, such as altered nutrition, activity, or social setting. Epigenetic modifications, thereby, provide a source of phenotypic plasticity in many species. The honey bee (Apis mellifera) uses nutritionally sensitive epigenetic control mechanisms in the development of the royal caste (queens) and the workers. The workers are functionally sterile females that can take on a range of distinct physiological and/or behavioral phenotypes in response to environmental changes. Honey bees have a wide repertoire of epigenetic mechanisms which, as in mammals, include cytosine methylation, hydroxymethylated cytosines, together with the enzymatic machinery responsible for these cytosine modifications. Current data suggests that honey bees provide an excellent system for studying the “social repertoire” of the epigenome. In this review, we elucidate what is known so far about the honey bee epigenome and its mechanisms. Our discussion includes what may distinguish honey bees from other model animals, how the epigenome can influence worker behavioral task separation, and how future studies can answer central questions about the role of the epigenome in social behavior. PMID:25705215

  11. An efficient prebiotic synthesis of cytosine and uracil

    NASA Technical Reports Server (NTRS)

    Robertson, M. P.; Miller, S. L.

    1995-01-01

    In contrast to the purines, the routes that have been proposed for the prebiotic synthesis of pyrimidines from simple precursors give only low yields. Cytosine can be synthesized from cyanoacetylene and cyanate; the former precursor is produced from a spark discharge in a CH4/N2 mixture and is an abundant interstellar molecule. But this reaction requires relatively high concentrations of cyanate (> 0.1 M), which are unlikely to occur in aqueous media as cyanate is hydrolysed rapidly to CO2 and NH3. An alternative route that has been explored is the reaction of cyanoacetaldehyde (formed by hydrolysis of cyanoacetylene) with urea. But at low concentrations of urea, this reaction produces no detectable quantities of cytosine. Here we show that in concentrated urea solution--such as might have been found in an evaporating lagoon or in pools on drying beaches on the early Earth--cyanoacetaldehyde reacts to form cytosine in yields of 30-50%, from which uracil can be formed by hydrolysis. These reactions provide a plausible route to the pyrimidine bases required in the RNA world.

  12. Early prenatal investigation of a pregnancy at risk of adenosine deaminase deficiency using chorionic villi

    Microsoft Academic Search

    D A Aitken; D H Gilmore; C A Frew; M E Ferguson-Smith; M J Carty; W R Chatfield

    1986-01-01

    A pregnancy at risk for adenosine deaminase deficiency and severe combined immunodeficiency disease was investigated using samples of chorionic villi obtained during the eighth week of pregnancy. Adenosine deaminase levels suggested that the fetus was a probable carrier and that a diagnosis of severe combined immunodeficiency disease could be excluded. Enzyme and chromosome results were available within 24 hours of

  13. Adenosine deaminase deficiency with normal immune function. An acidic enzyme mutation.

    PubMed Central

    Daddona, P E; Mitchell, B S; Meuwissen, H J; Davidson, B L; Wilson, J M; Koller, C A

    1983-01-01

    In most instances, marked deficiency of the purine catabolic enzyme adenosine deaminase results in lymphopenia and severe combined immunodeficiency disease. Over a 2-yr period, we studied a white male child with markedly deficient erythrocyte and lymphocyte adenosine deaminase activity and normal immune function. We have documented that (a) adenosine deaminase activity and immunoreactive protein are undetectable in erythrocytes, 0.9% of normal in lymphocytes, 4% in cultured lymphoblasts, and 14% in skin fibroblasts; (b) plasma adenosine and deoxyadenosine levels are undetectable and deoxy ATP levels are only slightly elevated in lymphocytes and in erythrocytes; (c) no defect in deoxyadenosine metabolism is present in the proband's cultured lymphoblasts; (d) lymphoblast adenosine deaminase has normal enzyme kinetics, absolute specific activity, S20,w, pH optimum, and heat stability; and (e) the proband's adenosine deaminase exhibits a normal apparent subunit molecular weight but an abnormal isoelectric pH. In contrast to the three other adenosine deaminase-deficient healthy subjects who have been described, the proband is unique in demonstrating an acidic, heat-stable protein mutation of the enzyme that is associated with less than 1% lymphocyte adenosine deaminase activity. Residual adenosine deaminase activity in tissues other than lymphocytes may suffice to metabolize the otherwise lymphotoxic enzyme substrate(s) and account for the preservation of normal immune function. Images FIGURE 1 FIGURE 2 FIGURE 3 PMID:6603477

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

    EPA Science Inventory

    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. Bone marrow transplantation and alternatives for adenosine deaminase deficiency.

    PubMed

    Gaspar, H Bobby

    2010-05-01

    Adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) comprises approximately 10% to 15% of all cases of SCID. The clinical effects of ADA deficiency are manifest most dramatically in the immune system, where it leads to severe lymphopenia. Although hematopoietic stem cell transplantation remains the mainstay of treatment for ADA-deficient SCID, 2 other treatment options are available, namely enzyme replacement therapy with PEG-ADA and autologous hematopoietic stem cell gene therapy. In this article the author reviews the available data on treatment by these different options, and offers an overview on when each of the different treatment options should be used. PMID:20493398

  16. Threonine deaminase from Salmonella typhimurium. Relationship between regulatory sites.

    PubMed

    Decedue, C J; Hofler, J G; Burns, R O

    1975-02-25

    Kinetic analysis of the biosynthetic threonine deaminase, EC 4.2.1.16, from Samonella typhimurium yields hyperbolic substrate saturation curves in the absence of, and higher order substrate saturation curves in the presence of, L-isoleucine. L-Valine reverses this effect of L-isoleucine by restoring the hyperbolic substrate saturation curves. The inhibition of enzyme activity and the reversal of valine stimulation is a function of a second order concentration of L-isoleucine, whereas antagonism of inhibition is a function of first order concentration of valine. The antagonistic effects on enzyme activity of L-isoleucine and of L-valine appear as competitive in diagnostic plots. Threonine deaminase possesses two L-isoleucine binding sites (Kd equals 3.6 muM) and one L-valine binding site (Kd equals 26 muM); the binding of these ligands appear competitive. Exclusion of L-valine requires the binding of 2 molecules of L-isoleucine whereas binding of a single L-valine molecule prevents the binding of 2 L-isoleucine molecules. Cooperative binding of L-isoleucine is not observed under any of the conditions tested. Two cases, expressed in terms of modified Adair equations and based upon the assumption that L-threonine also serves as an activator ligand which binds to the L-valine site, are presented. Case I states that liganding of the activator sites must percede substrate-binding at the active site, and Case II states that the activator site liganding is required solely for reactivation of the L-isoleucine-inhibited enzyme. Analysis of kinetic data by a curve-fitting process suggests that Case II described the relationship between the activator site and the L-isoleucine sites. An enzymatically inactive derivative of threonine deaminase, prepared by reduction with borohydride, binds isoleucine and valine in a manner similar to native holoenzyme. Binding of L-threonine and L-valine to the derivatized enzyme is competitive. The Kd for threonine binding is 3 mM, which is in excellent agreement with the Kd determined by the curve fitting process. It is concluded that the modulation of threonine deaminase activity is wrought by interaction between inhibitor sites and an activator site rather than inhibitor and active sites and that induced transitions rather than concerted transitions more adequately describe the underlying regulatory principle. PMID:1089662

  17. Cytosinium–hydrogen maleate–cytosine (1/1/1)

    PubMed Central

    Benali-Cherif, Nourredine; Falek, Wahiba; Direm, Amani

    2009-01-01

    The title organic salt, C4H6N3O+·C4H3O4 ?·C4H5N3O, was synthesized from cytosine base and maleic acid. An intra­molecular O—H?O hydrogen bond occurs in the hydrogen maleate anion. The crystal packing is stabilized by inter­molecular N—H?O, N—H?N and C—H?O hydrogen bonds, giving rise to a nearly planar two-dimensional network parallel to (101). PMID:21578789

  18. Simple and efficient routes for the preparation of isoxazolidinyl nucleosides containing cytosine and 5-methyl-cytosine as new potential anti-HIV drugs

    Microsoft Academic Search

    Evelina Colacino; Antonella Converso; Angelo Liguori; Anna Napoli; Carlo Siciliano; Giovanni Sindona

    2001-01-01

    A rapid and convenient large-scale strategy for the synthesis of some new isoxazolidinyl nucleosides, as potential antiviral drugs, is reported. In particular, a multistep methodology based either on the 1,3-dipolar cycloaddition approach or on a slight modification of the convertible nucleoside concept was exploited in the preparation of 4?-aza-2?,3?-dideoxynucleoside analogues containing cytosine and 5-methyl-cytosine.

  19. Induction of guanine deaminase and its inhibitor in rodent liver and brain

    PubMed Central

    Sitaramayya, A.; Ali, Shahid; Kumar, K. Sree; Krishnan, P. S.

    1974-01-01

    1. Guanine deaminase activities in homogenates and supernatant fractions of liver and brain of rat and mouse were elevated by administration of guanine to the animals. The maximum induction in mouse tissues occurred within 24h and in rat tissues within 48h. 2. Mitochondria of rat (but not mouse) liver and brain contain an inhibitor of supernatant guanine deaminase, and this was also increased by guanine treatment. 3. Administration of ethionine, cycloheximide or actinomycin D prevented the guanine-dependent increase in deaminase activity and also the increase in mitochondrial inhibitory activity; chloramphenicol suppressed only the latter. PMID:4822729

  20. The fission yeast gene pmt1+ encodes a DNA methyltransferase homologue.

    PubMed Central

    Wilkinson, C R; Bartlett, R; Nurse, P; Bird, A P

    1995-01-01

    DNA methylation of cytosine residues is a widespread phenomenon and has been implicated in a number of biological processes in both prokaryotes and eukaryotes. This methylation occurs at the 5-position of cytosine and is catalyzed by a distinct family of conserved enzymes, the cytosine-5 methyltransferases (m5C-MTases). We have cloned a fission yeast gene pmt1+ (pombe methyltransferase) which encodes a protein that shares significant homology with both prokaryotic and eukaryotic m5C-MTases. All 10 conserved domains found in these enzymes are present in the pmt1 protein. This is the first m5C-MTase homologue cloned from a fungal species. Its presence is surprising, given the inability to detect DNA methylation in yeasts. Haploid cells lacking the pmt1+ gene are viable, indicating that pmt1+ is not an essential gene. Purified, bacterially produced pmt1 protein does not possess obvious methyltransferase activity in vitro. Thus the biological significance of the m5C-MTase homologue in fission yeast is currently unclear. Images PMID:7862522

  1. Ag Nanocluster Formation Using a Cytosine Oligonucleotide Template

    PubMed Central

    Ritchie, Caroline M.; Johnsen, Kenneth R.; Kiser, John R.; Antoku, Yasuko; Dickson, Robert M.; Petty, Jeffrey T.

    2008-01-01

    The reduction of silver cations bound to the oligonucleotide dC12 was used to form silver nanoclusters. Mass spectra show that the oligonucleotides have 2–7 silver atoms that form multiple species, as evident from the number of transitions in the fluorescence and absorption spectra. The variations in the concentrations of the nanoclusters with time are attributed to the changing reducing capacity of the solution, and the formation of oxidized nanoclusters is proposed. Via mass spectrometry and circular dichroism spectroscopy, double-stranded structures with Ag+-mediated interactions between the bases are observed, but these structures are not maintained with the reduced nanoclusters. Through variations in the pH, the nanoclusters are shown to bind with the N3 of cytosine. PMID:19079559

  2. Hypomethylation of cytosine 5-methyltransferase in human neoplasms.

    PubMed

    Butler, T L; Kay, P H; Jacobsen, P F

    2000-01-01

    Cytosine methylation is an epigenetic modification of DNA involved in control of gene expression. Neoplastic cells exhibit various alterations both in DNA methylation and activity of the enzyme responsible for this modification, 5-methyltransferase (5-MeTase). As there is little requirement for 5-methyltransferase expression in normal cells except during mitosis, we argued that the gene would be hypermethylated in normal cells. Southern analysis revealed almost complete methylation of the gene in genomic DNA from the peripheral blood leukocytes of healthy subjects and a primary fibroblast derived cell line. In contrast, in DNA from a range of tumour tissues and tumour derived cell lines, 5-MeTase exhibited marked hypomethylation. The results of this study indicate that dysregulation of the DNA methylating machinery, especially with respect to the methylation status of 5-MeTase, is a feature of a wide range of neoplasms. PMID:10928053

  3. Antibody Responses to Bacteriophage +X174 in Patients With Adenosine Deaminase Deficiency

    Microsoft Academic Search

    Hans D. Ochs; Rebecca H. Buckley; Roger H. Kobayashi; Ai Lan Kobayashi; Ricardo U. Sorensen; Steven D. Douglas; Brian L. Hamilton; Michael S. Hershfield

    1992-01-01

    ENETICALLY DETERMINED deficiency of the G enzyme adenosine deaminase (ADA) is a cause of the clinical syndrome of severe combined immunodefi- ciency (SCID) with its associated recurrent infections and failure to thrive. If untreated, the condition is usually fatal

  4. Late-onset adenosine deaminase deficiency presenting with Heck's disease.

    PubMed

    Artac, Hasibe; Göktürk, Bahar; Bozdemir, Sefika Elmas; Toy, Hatice; van der Burg, Mirjam; Santisteban, Ines; Hershfield, Michael; Reisli, Ismail

    2010-08-01

    Focal epithelial hyperplasia, also known as Heck's disease, is a rare but distinctive entity of viral etiology with characteristic clinical and histopathological features. It is a benign, asymptomatic disease of the oral mucosa caused by human papilloma viruses (HPV). Previous studies postulated an association between these lesions and immunodeficiency. Genetic deficiency of adenosine deaminase (ADA) results in varying degrees of immunodeficiency, including neonatal onset severe combined immunodeficiency (ADA-SCID), and milder, later onset immunodeficiency. We report a 12-year-old girl with the late onset-ADA deficiency presenting with Heck's disease. Our case report should draw attention to the possibility of immunodeficiency in patients with HPV-induced focal epithelial hyperplasia. PMID:20039061

  5. [Adenosine deaminase as costimulatory molecule and marker of cellular immunity].

    PubMed

    Pérez-Aguilar, Mary Carmen; Goncalves, Loredana; Ibarra, Alba; Bonfante-Cabarcas, Rafael

    2010-12-01

    Adenosine deaminase (ADA) is an enzyme of purine metabolism which has been the subject of much interest because the congenital defect of this enzyme causes severe combined immunodeficiency syndrome. One of the three isoforms of the enzyme (ecto-ADA) is capable of binding to the glycoprotein CD26 and adenosine receptors A1 and A2B. ADA-CD26 interaction produces a costimulatory signal in the events of T cell activation and secretion of IFN-gamma, TNF-alpha and IL-6. During this activation, the enzyme activity is regulated positively by IL-2 and IL-12 and negatively by IL-4, based on the mechanism of translocation. Diverse studies suggest that seric and plasmatic levels of ADA rise in some diseases caused by microorganisms infecting mainly the macrophages and in hypertensive disorders, which may represent a compensatory mechanism resulting from increased adenosine levels and the release of hormones and inflammatory mediators estimulated by hipoxia. PMID:21365880

  6. Orthologous mammalian APOBEC3A cytidine deaminases hypermutate nuclear DNA.

    PubMed

    Caval, Vincent; Suspène, Rodolphe; Vartanian, Jean-Pierre; Wain-Hobson, Simon

    2014-02-01

    The human APOBEC3 gene cluster locus encodes polynucleotide cytidine deaminases. Although many act as viral restriction factors through mutation of single-stranded DNA, recent reports have shown that human APOBEC3A was capable of efficiently hypermutating nuclear DNA and inducing DNA breaks in genomic DNA. In addition, the enzyme was unique in efficiently deaminating 5-methylcytidine in single-stranded DNA. To appreciate the evolutionary relevance of these activities, we analyzed A3A-related enzymes from the rhesus and tamarin monkey, horse, sheep, dog, and panda. All proved to be orthologous to the human enzyme in all these activities revealing strong conservation more than 148 My. Hence, their singular role in DNA catabolism is a well-established mechanism probably outweighing any deleterious or pathological roles such as genomic instability and cancer formation. PMID:24162735

  7. Activation Induced Deaminase in Antibody Diversification and Chromosome Translocation

    PubMed Central

    Gazumyan, Anna; Bothmer, Anne; Klein, Isaac A.; Nussenzweig, Michel C; McBride, Kevin M.

    2015-01-01

    DNA damage, rearrangement and mutation of the human genome are the basis of carcinogenesis and thought to be avoided at all costs. An exception is the adaptive immune system where lymphocytes utilize programmed DNA damage to effect antigen receptor diversification. Both B and T lymphocytes diversify their antigen receptors through RAG1/2 mediated recombination, but B cells undergo two additional processes – somatic hypermutation (SHM) and class switch recombination (CSR), both initiated by Activation Induced Deaminase (AID). AID deaminates cytidines in DNA resulting in U:G mismatches that are processed into point mutations in SHM or double strand breaks in CSR. Although AID activity is focused at Immunoglobulin (Ig) gene loci, it also targets a wide array of non-Ig genes including oncogenes associated with lymphomas. Here we review the molecular basis of AID regulation, targeting, and initiation of CSR and SHM, as well as AID's role in generating chromosome translocations that contribute to lymphomagenesis. PMID:22429855

  8. AMP-Deaminase from Thymus of Patients with Myasthenia Gravis.

    PubMed

    Rybakowska, I; Szyd?owska, M; Szrok, S; Baku?a, S; Kaletha, K

    2015-03-01

    Myasthenia gravis (MG) is characterized clinically by skeletal muscle fatigue following the excessive exercise. Interestingly most of MG patients manifest parallely also some abnormalities of the thymus.AMP-deaminase (AMPD) from human thymus was not a subject of studies up to now. In this paper, mRNA expression and some physico-chemical and immunological properties of AMPD purified from the thymus of MG patients were described. Experiments performed identified the liver isozyme (AMPD2) as the main isoform of AMPD expressed in this organ. The activity of AMPD found in this organ was higher than in other human non-(skeletal) muscle tissues indicating on role the enzyme may play in supplying of guanylates required for the intensive multiplication of thymocytes. PMID:25710358

  9. Alkali metal cation binding affinities of cytosine in the gas phase: revisited.

    PubMed

    Yang, Bo; Rodgers, M T

    2014-08-14

    Binding of metal cations to the nucleobases can influence base pairing, base stacking and nucleobase tautomerism. Gas-phase condensation of dc discharge generated alkali metal cations and thermally vaporized cytosine (DC/FT) has been found to produce kinetically trapped excited tautomeric conformations of the M(+)(cytosine) complexes, which influences the threshold collision-induced dissociation (TCID) behavior. In order to elucidate the effects of the size of alkali metal cation on the strength of binding to the canonical form of cytosine, the binding affinities of Na(+) and K(+) to cytosine are re-examined here, and studies are extended to include Rb(+) and Cs(+) again using TCID techniques. The M(+)(cytosine) complexes are generated in an electrospray ionization source, which has been shown to produce ground-state tautomeric conformations of M(+)(cytosine). The energy-dependent cross sections are interpreted to yield bond dissociation energies (BDEs) using an analysis that includes consideration of unimolecular decay rates, the kinetic and internal energy distributions of the reactants, and multiple M(+)(cytosine)-Xe collisions. Revised BDEs for the Na(+)(cytosine) and K(+)(cytosine) complexes exceed those previously measured by 31.9 and 25.5 kJ mol(-1), respectively, consistent with the hypothesis proposed by Yang and Rodgers that excited tautomeric conformations are accessed when the complexes are generated by DC/FT ionization. Experimentally measured BDEs are compared to theoretical values calculated at the B3LYP and MP2(full) levels of theory using the 6-311+G(2d,2p)_HW* and def2-TZVPPD basis sets. The B3LYP/def2-TZVPPD level of theory is found to provide the best agreement with the measured BDEs, suggesting that this level of theory can be employed to provide reliable energetics for similar metal-ligand systems. PMID:24967574

  10. Generation of normal lymphocyte populations following transplantation of adenosine–deaminase-deficient fetal liver cells

    Microsoft Academic Search

    AAJ Migchielsen; S Knaän-Schanzer; ML Breuer; D Valerio

    1997-01-01

    Adenosine–deaminase-deficient mice were generated to investigate the role of adenosine deaminase (ADA) in lymphocyte maturation and to test treatment options for the severe combined immunodeficiency (SCID) associated with the absence of ADA in man. Whereas either genetic absence or postnatal inhibition of ADA affect primarily the haematopoietic system in both humans and mice, ADA-deficient mice die in the perinatal period.

  11. Deoxyadenosine Triphosphate as a Potentially Toxic Metabolite in Adenosine Deaminase Deficiency

    Microsoft Academic Search

    Amos Cohen; Rochelle Hirschhorn; Sheldon D. Horowitz; Arieh Rubinstein; Stephen H. Polmar; Richard Hong; David W. Martin

    1978-01-01

    The inherited deficiency of adenosine deaminase (adenosine aminohydrolase; EC 3.5.4.4) activity in humans is associated with an immunodeficiency. Some of the immunodeficient and enzyme-deficient patients respond immunologically to periodic infusions of irradiated erythrocytes containing adenosine deaminase. It has been previously reported that erythrocytes and lymphocytes from immunodeficient and enzyme-deficient children contained increased concentrations of ATP, and in the one child

  12. A Kinetic Comparison on the Inhibition of Adenosine Deaminase by Purine Drugs

    Microsoft Academic Search

    Ghasem Ataie; Soghra Bagheri; Adeleh Divsalar; Ali Akbar Saboury; Shahrokh Safarian; Saeed Namaki

    The effects of allopurinol, acyclovir and theophylline on the activity of adenosine deaminase (ADA) were studied in 50 mM sodium phosphate buffer pH 7.5 at 27 °C, using a UV- Vis spectrophotometer. Adenosine deaminase is inhibited by these ligands, via different types of inhibition. Allopurinol, as a transition state analog of xanthine oxidase, and acyclovir competitively inhibit the catalytic activity

  13. Antinociceptive and anti-inflammatory properties of an adenosine kinase inhibitor and an adenosine deaminase inhibitor

    Microsoft Academic Search

    Anthony Poon; Jana Sawynok

    1999-01-01

    Spinal administration of an adenosine kinase inhibitor, alone or in combination with an adenosine deaminase inhibitor, produces antinociception in inflammatory pain tests. In the present study, we examined the antinociceptive and anti-inflammatory effects produced by the peripheral (intraplantar) administration of 5?-amino-5?-deoxyadenosine (an adenosine kinase inhibitor), 2?-deoxycoformycin (an adenosine deaminase inhibitor), and combinations of both agents in the carrageenan-induced thermal hyperalgesia

  14. Modulation of adenosine release from rat spinal cord by adenosine deaminase and adenosine kinase inhibitors

    Microsoft Academic Search

    Krystyna Golembiowska; Thomas D. White; Jana Sawynok

    1995-01-01

    Adenosine, a modulator of pain processing in the spinal cord, is metabolized by adenosine kinase and adenosine deaminase. In this study we determined which of these mechanisms is more important for the regulation of endogenous adenosine levels in the rat spinal cord. The effects of the adenosine kinase inhibitors, 5?-deoxyadenosine (NH2dAD) and iodotubercidin (IOT), and the adenosine deaminase inhibitor, 2?-deoxycoformycin

  15. The interconversion of ACC deaminase and d -cysteine desulfhydrase by directed mutagenesis

    Microsoft Academic Search

    Biljana Todorovic; Bernard R. Glick

    2008-01-01

    Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes\\u000a which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. These enzymes cleave ACC, the immediate\\u000a precursor of ethylene in plants, into ammonia and ?-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs\\u000a from tomato plants with the objective of establishing

  16. Expression of three stage-specific transcripts of AMP deaminase during myogenesis.

    PubMed Central

    Sabina, R L; Ogasawara, N; Holmes, E W

    1989-01-01

    AMP deaminase, a ubiquitous enzyme in eucaryotes, plays a central role in energy metabolism. In the present study, RNase protection analyses and immunoprecipitation with tissue-specific antisera were used to examine the transcripts and peptides of AMP deaminase produced during myogenesis in vivo and during myocyte differentiation in vitro. In embryonic muscle and undifferentiated myoblasts, a 3.4-kilobase (kb) transcript encoded a 78-kilodalton (kDa) AMP deaminase peptide that cross-reacted with antisera raised to the AMP deaminase isoform purified from kidney of the adult animal. In perinatal muscle and myocytes at an intermediate stage of differentiation in vitro, a 2.5-kb transcript was produced, and it encoded a 77.5-kDa AMP deaminase peptide that cross-reacted with antisera to the isoform purified from adult heart muscle. At about the time of birth, another 2.5-kb AMP deaminase transcript that encoded an 80-kDa peptide became detectable. This peptide cross-reacted with antisera to the predominant isoform purified from adult skeletal muscle. Images PMID:2568582

  17. Pentose fermentation by yeasts

    Microsoft Academic Search

    M.-L. Suihko; M. Dra?i?

    1983-01-01

    66 different yeast strains were screened for glucose, xylose and xylulose fermentation in shake flask cultures. None of the tested yeasts was able to grow or produce significant amounts of ethanol on xylose anaerobically. The best ethanol yields from xylulose were obtained with a wine yeast, two distillery yeasts, and a strain of Saccharomyces uvarum. The best conversion of xylulose

  18. Yeast-Air Balloons

    NSDL National Science Digital Library

    The Exploratorium

    2012-03-10

    In this activity, learners make a yeast-air balloon to get a better idea of what yeast can do. Learners discover that the purpose of leaveners like yeast is to produce the gas that makes bread rise. Learners discover that as yeast feeds on sugar, it produces carbon dioxide which slowly fills the balloon.

  19. A Feast for Yeast

    NSDL National Science Digital Library

    2013-07-08

    In this activity on page 6 of the PDF, learners investigate yeast. Learners prepare an experiment to observe what yeast cells like to eat. Learners feed the yeast cells various ingredients in plain bread--water, flour, sugar, and salt--to discover yeast's favorite food.

  20. Non-conventional yeasts.

    PubMed

    Spencer, J F T; Ragout de Spencer, A L; Laluce, C

    2002-02-01

    In the beginning there was yeast, and it raised bread, brewed beer, and made wine. After many not days but centuries and even millenia later, it was named Saccharomyces cerevisiae. After more years and centuries there was another yeast, and it was named Schizosaccharomyces pombe; now there were two stars in the yeast heaven. In only a few more years there were other yeasts, and then more, and more, and more. The era of the non-conventional yeasts had begun. PMID:11878307

  1. Interleukin 1 protects from cytosine arabinoside-induced alopecia in the rat model

    Microsoft Academic Search

    JOAQUIN J. JIMENEZ; GRACE H. W. WONG; ADEL A. YUNIS

    Protection from cytosine arabinoside- induced alopecia by ImuVert has recently been reported in a rat model. ImuVert, a biologic response modifier, is capable of activating mononuclear cells causing release of various cytokines. In the present study, using the young rat model, recombinant human IL 113 produced excellent protection from cytosine arabinoside-induced alopecia. Mouse recombinant tumor necrosis factor gave definite but

  2. Bacteriophage T4 alc gene product: general inhibitor of transcription from cytosine-containing DNA.

    PubMed Central

    Kutter, E M; Bradley, D; Schenck, R; Guttman, B S; Laiken, R

    1981-01-01

    The alc gene of bacteriophage T4 was originally defined on the basis of mutations which allow late protein synthesis directed by T4 DNA containing cytosine rather than hydroxymethylcytosine. The question remained whether the normal alc gene product (gpalc) also blocks the transcription of early genes from cytosine-containing DNA. Complementation experiments were performed between hydroxymethylcytosine-containing phage which direct gpalc synthesis but carry mutations in a given gene(s) and cytosine-containing phage carrying that gene(s). The required protein would then have to be directed by the cytosine-containing DNA: it is looked for directly on polyacrylamide gels or through its physiological effects or both. For all early proteins examined in this way, no synthesis was observed when 95 to 100% of the hydroxymethylcytosine was substituted by cytosine in the infecting DNA, whereas there was significant synthesis with 75% substitution or less. The results indicate that gpalc is carried in with the infecting DNA or is made very early to block transcription of all cytosine-containing DNA. Images PMID:7321103

  3. Pichia antillensis, a New Species of Yeast Associated with Necrotic Stems of Cactus in the Lesser Antilles

    Microsoft Academic Search

    WILLIAM T. STARMER; HERMAN J. PHAFF; JOANNE TREDICK; MARY MIRANDA

    1984-01-01

    We describe Pichia antillensis, a new species of yeast which is closely related to Pichia opuntiae. Pichia antillensis, 20 strains of which were isolated, is heterothallic and occurs in nature in both the haploid state and the diploid state. It produces asci with four hat-shaped spores, which are rapidly released upon maturity. The guanine-plus-cytosine content of its nuclear deoxyribonucleic acid

  4. The sequence specificity domain of cytosine-C5 methylases.

    PubMed Central

    Klimasauskas, S; Nelson, J L; Roberts, R J

    1991-01-01

    Prokaryotic DNA[cytosine-C5]methyltransferases (m5C-methylases) share a common architectural arrangement of ten conserved sequence motifs. A series of eleven hybrids have been constructed between the HpaII (recognition sequence: Cm5CGG) and HhaI (recognition sequence: Gm5CGC) DNA-methylases. The hybrids were over-expressed in E.coli and their in vivo methylation phenotypes investigated. Six were inactive by our assay while five of them retained partial methylation activity and full specificity. In all five cases the specificity matched that of the parent methylase which contributed the so-called variable region, located between conserved motifs VIII and IX. This was the only sequence held in common between the active hybrids and for the first time provides unequivocal evidence that the specificity determinants of the mono-specific m5C-methylases are located within the variable region. Correlation of the hybrid methylase structure with the efficiency of methylation suggests that conserved motif IX may interact with the variable region whereas motif X most probably interacts with the N-terminal half of the molecule. Images PMID:1659688

  5. Cytosine arabinoside induces apoptosis in cerebellar neurons in culture.

    PubMed

    Dessi, F; Pollard, H; Moreau, J; Ben-Ari, Y; Charriaut-Marlangue, C

    1995-05-01

    Cytosine arabinoside (AraC) is a pyrimidine antimetabolite that prevents cell proliferation by inhibiting DNA synthesis. We report that AraC kills cultured cerebellar neurons in a concentration-dependent fashion with an EC50 of approximately 60 microM when added shortly after seeding. This cell death has apoptotic features because we observed (1) morphology of apoptotic nuclei as judged by DNA staining with Hoechst 33258, (2) DNA fragmentation with typical ladder pattern on agarose gel, (3) positive nuclear labeling with a specific in situ DNA fragmentation staining, (4) prevention by deoxycytidine (IC50 = 1 microM), protein, and RNA synthesis inhibitors, and (5) release of DNA fragments in the incubating medium. We have also observed that several proteins were overexpressed in AraC-treated neurons by two-dimensional polyacrylamide gel electrophoresis. We conclude that AraC induces a signal that triggers a cascade of new mRNA and protein synthesis, leading to apoptotic cell death in cultured cerebellar granule cells. PMID:7536801

  6. TET proteins: on the frenetic hunt for new cytosine modifications

    PubMed Central

    Delatte, Benjamin

    2013-01-01

    Epigenetic genome marking and chromatin regulation are central to establishing tissue-specific gene expression programs, and hence to several biological processes. Until recently, the only known epigenetic mark on DNA in mammals was 5-methylcytosine, established and propagated by DNA methyltransferases and generally associated with gene repression. All of a sudden, a host of new actors—novel cytosine modifications and the ten eleven translocation (TET) enzymes—has appeared on the scene, sparking great interest. The challenge is now to uncover the roles they play and how they relate to DNA demethylation. Knowledge is accumulating at a frantic pace, linking these new players to essential biological processes (e.g. cell pluripotency and development) and also to cancerogenesis. Here, we review the recent progress in this exciting field, highlighting the TET enzymes as epigenetic DNA modifiers, their physiological roles, and their functions in health and disease. We also discuss the need to find relevant TET interactants and the newly discovered TET–O-linked N-acetylglucosamine transferase (OGT) pathway. PMID:23625996

  7. Gemcitabine and cytosine arabinoside cytotoxicity: association with lymphoblastoid cell expression

    PubMed Central

    Li, Liang; Fridley, Brooke; Kalari, Krishna; Jenkins, Gregory; Batzler, Anthony; Safgren, Stephanie; Hildebrandt, Michelle; Ames, Matthew; Schaid, Daniel; Wang, Liewei

    2008-01-01

    Two cytidine analogues, gemcitabine (dFdC) and cytosine arabinoside (AraC), show significant therapeutic effect in a variety of cancers. However, response to these drugs varies widely. Evidence from tumor biopsy samples shows that expression levels for genes involved in the cytidine transport, metabolism and bioactivation pathway contribute to this variation in response. In the present study, we set out to test the hypothesis that variation in gene expression both within and outside of this “pathway” might influence sensitivity to gemcitabine and AraC. Specifically, Affymetrix U133 Plus 2.0 GeneChip and cytotoxicity assays were performed to obtain basal mRNA expression and IC50 values for both drugs in 197 ethnically-defined Human Variation Panel lymphoblastoid cell lines. Genes with a high degree of association with IC50 values were involved mainly in cell death, cancer, cell cycle, and nucleic acid metabolism pathways. We validated selected significant genes by performing real time quantitative RT-PCR and selected two representative candidates, NT5C3 (within the pathway) and FKBP5 (outside of the pathway), for functional validation. Those studies demonstrated that down regulation of NT5C3 and FKBP5 altered tumor cell sensitivity to both drugs. Our results suggest that cell-based model system studies, when combined with complementary functional characterization, may help to identify biomarkers for response to chemotherapy with these cytidine analogues. PMID:18757419

  8. Bisulfighter: accurate detection of methylated cytosines and differentially methylated regions

    PubMed Central

    Saito, Yutaka; Tsuji, Junko; Mituyama, Toutai

    2014-01-01

    Analysis of bisulfite sequencing data usually requires two tasks: to call methylated cytosines (mCs) in a sample, and to detect differentially methylated regions (DMRs) between paired samples. Although numerous tools have been proposed for mC calling, methods for DMR detection have been largely limited. Here, we present Bisulfighter, a new software package for detecting mCs and DMRs from bisulfite sequencing data. Bisulfighter combines the LAST alignment tool for mC calling, and a novel framework for DMR detection based on hidden Markov models (HMMs). Unlike previous attempts that depend on empirical parameters, Bisulfighter can use the expectation-maximization algorithm for HMMs to adjust parameters for each data set. We conduct extensive experiments in which accuracy of mC calling and DMR detection is evaluated on simulated data with various mC contexts, read qualities, sequencing depths and DMR lengths, as well as on real data from a wide range of biological processes. We demonstrate that Bisulfighter consistently achieves better accuracy than other published tools, providing greater sensitivity for mCs with fewer false positives, more precise estimates of mC levels, more exact locations of DMRs and better agreement of DMRs with gene expression and DNase I hypersensitivity. The source code is available at http://epigenome.cbrc.jp/bisulfighter. PMID:24423865

  9. ADA (adenosine deaminase) gene therapy enters the competition

    SciTech Connect

    Culliton, B.J.

    1990-08-31

    Around the world, some 70 children are members of a select and deadly club. Born with an immune deficiency so severe that they will die of infection unless their immune systems can be repaired, they have captured the attention of would-be gene therapists who believe that a handful of these kids--the 15 or 20 who lack functioning levels of the enzyme adenosine deaminase (ADA)--could be saved by a healthy ADA gene. A team of gene therapists is ready to put the theory to the test. In April 1987, a team of NIH researchers headed by R. Michael Blaese and W. French Anderson came up with the first formal protocol to introduce a healthy ADA gene into an unhealthy human. After 3 years of line-by-line scrutiny by five review committees, they have permission to go ahead. Two or three children will be treated in the next year, and will be infused with T lymphocytes carrying the gene for ADA. If the experiment works, the ADA gene will begin producing normal amounts of ADA. An interesting feature of ADA deficiency, that makes it ideal for initial gene studies, is that the amount of ADA one needs for a healthy immune system is quite variable. Hence, once inside a patient's T cells, the new ADA gene needs only to express the enzyme in moderate amounts. No precise gene regulation is necessary.

  10. AMP deaminase 3 deficiency enhanced 5'-AMP induction of hypometabolism.

    PubMed

    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

  11. The ONIOM molecular dynamics method for biochemical applications: cytidine deaminase

    SciTech Connect

    Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

    2007-03-22

    Abstract We derived and implemented the ONIOM-molecular dynamics (MD) method for biochemical applications. The implementation allows the characterization of the functions of the real enzymes taking account of their thermal motion. In this method, the direct MD is performed by calculating the ONIOM energy and gradients of the system on the fly. We describe the first application of this ONOM-MD method to cytidine deaminase. The environmental effects on the substrate in the active site are examined. The ONIOM-MD simulations show that the product uridine is strongly perturbed by the thermal motion of the environment and dissociates easily from the active site. TM and MA were supported in part 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.

  12. Molecular epidemiology and diagnosis of PBG deaminase gene defects in acute intermittent porphyria.

    PubMed Central

    Puy, H; Deybach, J C; Lamoril, J; Robreau, A M; Da Silva, V; Gouya, L; Grandchamp, B; Nordmann, Y

    1997-01-01

    Acute intermittent porphyria (AIP) is the major autosomal dominant form of acute hepatic porphyrias. The disease is due to mutations in the gene encoding for porphobilinogen (PBG) deaminase and is characterized by life-threatening neurovisceral attacks, often precipitated by drugs, fasting, cyclical hormonal changes, or infectious diseases. This report describes a prospective study on the molecular epidemiology of PBG deaminase gene defects in AIP. It uses a sensitive, reliable, and easy-to-handle method for routine AIP molecular diagnosis and family study based on an exon-by-exon denaturing gradient gel electrophoresis (DGGE) strategy followed by direct sequencing. Fifteen genomic DNA fragments, including all the coding sequence and covering 3.35 kb of the PBG deaminase gene, were investigated in 405 subjects from 121 unrelated French Caucasian AIP families who had not been screened previously at the DNA level. PBG deaminase gene mutations were identified in 109 families, but only 78 were of different type, and each of them had a prevalence rate < 5%. Among these mutations, 33 had not been published previously. Sixty percent of these 78 mutations were located in only three exons (exons 10, 12, and 14), 44% were missense, 18% were splice defect, 19% were frameshift, and 16% were nonsense. In addition, two de novo mutational events were characterized. The evaluation of the efficiency of the standard PBG deaminase enzymatic screening method for gene-carrier detection indicated 95% of concordancy with the molecular-based diagnosis. Images Figure 1 Figure 2 PMID:9199558

  13. Chondroosseous dysplasia in severe combined immunodeficiency due to adenosine deaminase deficiency (chondroosseous dysplasia in ADA deficiency scid)

    Microsoft Academic Search

    V. S. Chakravarti; P. Borns; J. Lobell; S. D. Douglas

    1991-01-01

    Adenosine deaminase (ADA) deficiency may manifest as severe combined immunodeficiency (SCID) in early infancy. Some of these children develop radiologic changes which may be in part related to effects of this enzyme deficiency on the bony epiphysis [1]. We describe the radiologic changes in a neonate with ADA deficiency and their resolution with polyethylene glycol conjugated adenosine deaminase (PEGADA, ADAGEN;

  14. Yeast Education Network

    NSDL National Science Digital Library

    The Yeast Education Network provides a variety of resources to facilitate use of the budding yeast Saccharomyces cerevisiae in undergraduate science curricula. Laboratory, classroom, and computer-based activities can be used with college and advanced high school students.

  15. Vaginal Yeast Infections

    MedlinePLUS

    ... infection from your sexual partner. Condoms and dental dams may help prevent getting or passing yeast infections ... infection from your sexual partner. Condoms and dental dams may help prevent getting or passing yeast infections ...

  16. Yeast Based Sensors

    NASA Astrophysics Data System (ADS)

    Shimomura-Shimizu, Mifumi; Karube, Isao

    Since the first microbial cell sensor was studied by Karube et al. in 1977, many types of yeast based sensors have been developed as analytical tools. Yeasts are known as facultative anaerobes. Facultative anaerobes can survive in both aerobic and anaerobic conditions. The yeast based sensor consisted of a DO electrode and an immobilized omnivorous yeast. In yeast based sensor development, many kinds of yeast have been employed by applying their characteristics to adapt to the analyte. For example, Trichosporon cutaneum was used to estimate organic pollution in industrial wastewater. Yeast based sensors are suitable for online control of biochemical processes and for environmental monitoring. In this review, principles and applications of yeast based sensors are summarized.

  17. Molecular evolution of minisatellites in hemiascomycetous yeasts.

    PubMed

    Richard, Guy-Franck; Dujon, Bernard

    2006-01-01

    Minisatellites are DNA tandem repeats exhibiting size polymorphism among individuals of a population. This polymorphism is generated by two different mechanisms, both in human and yeast cells, "replication slippage" during S-phase DNA synthesis and "repair slippage" associated to meiotic gene conversion. The Saccharomyces cerevisiae genome contains numerous natural minisatellites. They are located on all chromosomes without any obvious distribution bias. Minisatellites found in protein-coding genes have longer repeat units and on the average more repeat units than minisatellites in noncoding regions. They show an excess of cytosines on the coding strand, as compared to guanines (negative GC skew). They are always multiples of three, encode serine- and threonine-rich amino acid repeats, and are found preferably within genes encoding cell wall proteins, suggesting that they are positively selected in this particular class of genes. Genome-wide, there is no statistically significant association between minisatellites and meiotic recombination hot spots. In addition, minisatellites that are located in the vicinity of a meiotic hot spot are not more polymorphic than minisatellites located far from any hot spot. This suggests that minisatellites, in S. cerevisiae, evolve probably by strand slippage during replication or mitotic recombination. Finally, evolution of minisatellites among hemiascomycetous yeasts shows that even though many minisatellite-containing genes are conserved, most of the time the minisatellite itself is not conserved. The diversity of minisatellite sequences found in orthologous genes of different species suggests that minisatellites are differentially acquired and lost during evolution of hemiascomycetous yeasts at a pace faster than the genes containing them. PMID:16177231

  18. AMP-deaminase in elasmobranch fish: a comparative histochemical and enzymatic study.

    PubMed

    Thébault, Marie T; Izem, Lahoucine; Leroy, Jean Paul; Gobin, Eric; Charrier, Gregory; Raffin, Jean Paul

    2005-08-01

    AMP-deaminase activity was measured in white muscle from a wide range of fish, including one cyclostome, 13 chondrosteans, and one teleost to elucidate the pattern of the AMP-deaminase activity in white muscle of fish. Compared to a mammalian (rat) muscle extract, low enzyme activities are found in the cyclostome and two elasmobranchs from two families (Scyliorhinidae, Hexanchidae). In contrast, higher AMP-deaminase activities, similar to mammals, are expressed in Squalidae, all families of skates, Chimaeridae and in the teleostean fish. We then compared AMP-deaminase activities in red and white muscles from two representative elasmobranch fish, the dogfish (Scyliorhinus canicula) and the thornback ray (Raja clavata). The fibre type composition and distribution of the locomotory musculature were determined in these two elasmobranchs to establish a relationship between the morphology, the type of fibres of the locomotion-implicated muscles and the AMP-deaminase activity. Experimental data are discussed with respect to the layout of fibres in the myotome. In both species, three fibre types were identified. In the two fish myotomes, most of the axial muscles are white fibres while red fibres constitute a thin sheet. Some differences were observed between the two species in the distribution of intermediate fibres: in dogfish, these are located between the red and white fibres; in thornback ray, some are dispersed within the white fibre region, while others form an intermediary layer like in dogfish. These results suggest that in the course of evolution, an amplification of the AMP-deaminase activity in muscle was coupled with increase of complexity of the muscular structure. PMID:15979370

  19. Xylose fermentation by yeasts

    Microsoft Academic Search

    H. Dellweg; M. Rizzi; H. Methner; D. Debus

    1984-01-01

    Utilization and fermentation of xylose by the yeasts Pachysolen tannophilus I fGB 0101 and Pichia stipitis 5773 to 5776 under aerobic and anaerobic conditions are investigated. Pa. tannophilus requires biotin and thiamine for growth, whereas Pi. stipitis does not, and growth of both yeasts is stimulated by yeast extract. Pi. stipitis converts xylose (30 g\\/l) to ethanol under anaerobic conditions

  20. Global cytosine methylation in Daphnia magna depends on genotype, environment, and their interaction.

    PubMed

    Asselman, Jana; De Coninck, Dieter I M; Vandegehuchte, Michiel B; Jansen, Mieke; Decaestecker, Ellen; De Meester, Luc; Vanden Bussche, Julie; Vanhaecke, Lynn; Janssen, Colin R; De Schamphelaere, Karel A C

    2015-05-01

    The authors characterized global cytosine methylation levels in 2 different genotypes of the ecotoxicological model organism Daphnia magna after exposure to a wide array of biotic and abiotic environmental stressors. The present study aimed to improve the authors' understanding of the role of cytosine methylation in the organism's response to environmental conditions. The authors observed a significant genotype effect, an environment effect, and a genotype?×?environment effect. In particular, global cytosine methylation levels were significantly altered after exposure to Triops predation cues, Microcystis, and sodium chloride compared with control conditions. Significant differences between the 2 genotypes were observed when animals were exposed to Triops predation cues, Microcystis, Cryptomonas, and sodium chloride. Despite the low global methylation rate under control conditions (0.49-0.52%), global cytosine methylation levels upon exposure to Triops demonstrated a 5-fold difference between the genotypes (0.21% vs 1.02%). No effects were found in response to arsenic, cadmium, fish, lead, pH of 5.5, pH of 8, temperature, hypoxia, and white fat cell disease. The authors' results point to the potential role of epigenetic effects under changing environmental conditions such as predation (i.e., Triops), diet (i.e., Cryptomonas and Microcystis), and salinity. The results of the present study indicate that, despite global cytosine methylation levels being low, epigenetic effects may be important in environmental studies on Daphnia. Environ Toxicol Chem 2015;34:1056-1061. © 2015 SETAC. PMID:25639773

  1. Autoimmune Dysregulation and Purine Metabolism in Adenosine Deaminase Deficiency

    PubMed Central

    Sauer, Aisha Vanessa; Brigida, Immacolata; Carriglio, Nicola; Aiuti, Alessandro

    2012-01-01

    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. PMID:22969765

  2. Structural and Metabolic Specificity of Methylthiocoformycin for Malarial Adenosine Deaminases

    SciTech Connect

    Ho, M.; Cassera, M; Madrid, D; Ting, L; Tyler, P; Kim, K; Almo, S; Schramm, V

    2009-01-01

    Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation. Methylthioadenosine (MTA) is a substrate for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA. The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation. Here, the crystal structure of ADA from Plasmodium vivax (PvADA) in a complex with MT-coformycin reveals an unprecedented binding geometry for 5?-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5?-methylthioribosyl groups are rotated 130 degrees. A hydrogen bonding network between Asp172 and the 3?-hydroxyl of MT-coformycin is essential for recognition of the 5?-methylthioribosyl group. Water occupies the 5?-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic, and structural analyses of PvADA and kinetic analysis of five other Plasmodium ADAs establish the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin, and is missing Asp172. Treatment of P. falciparum cultures with coformycin or MT-coformycin in the presence of MTA is effective in inhibiting parasite growth.

  3. Autoimmune dysregulation and purine metabolism in adenosine deaminase deficiency.

    PubMed

    Sauer, Aisha Vanessa; Brigida, Immacolata; Carriglio, Nicola; Aiuti, Alessandro

    2012-01-01

    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. PMID:22969765

  4. Guanine deaminase inhibitor from rat liver. Isolation and characterization

    PubMed Central

    Ali, Shahid; Sitaramayya, A.; Kumar, K. Sree; Krishnan, Padmanabhan S.

    1974-01-01

    1. An inhibitor of cytoplasmic guanine deaminase of rat liver was isolated from liver `heavy mitochondrial' fraction after freezing and thawing and treatment with Triton X-100. 2. Submitochondrial fractionation revealed that the inhibitor was localized in the outer-membrane fraction. 3. The method of purification of inhibitor, involving precipitation with (NH4)2SO4 and chromatography on DEAE-cellulose, its precipitability by trichloroacetic acid and the pattern of absorption in the u.v. indicated that the inhibitor was a protein. In confirmation, tryptic digestion of the isolated material resulted in destruction of the inhibitor activity. The inhibitor was stable to acid, but labile to heat. 4. The isolated inhibitor required phosphatidylcholine (lecithin) for activity. Phosphatidylcholine also partially protected the inhibitor against heat inactivation. 5. When detergent treatment was omitted, the inhibitor activity of frozen mitochondria was precipitated by (NH4)2SO4 in a fully active form without supplementation with phosphatidylcholine, indicating that Triton X-100 ruptured the linkage between inhibitor and lipid. 6. A reconstituted sample of inhibitor–phosphatidylcholine complex was precipitated in a fully active form by dialysis against 2-mercaptoethanol, but treatment of the precipitate with NaCl yielded an extract which was inactive unless supplemented with fresh phosphatidylcholine. 7. We interpret the results as evidence that the inhibitor was present in vivo as a lipoprotein and that once the complex was dissociated by the action of detergent and the protein precipitated, there was an absolute need for exogenous phosphatidylcholine for its activity. The manner in which inhibitor associated with the outer membrane of rat liver mitochondria might regulate the activity of the enzyme in the supernatant has been suggested. PMID:4821397

  5. Anti-adenosine deaminase antibodies in lupus erythematosus.

    PubMed

    Lee, J Y Y; Hempel, J; Deng, J S

    2002-01-01

    Adenosine deaminase (ADA) is an enzyme involved in purine metabolism and has a major role in the development and function of lymphoid cells. Congenital deficiency of ADA results in severe immunodeficiency. Patients with congenital ADA deficiency treated with polyethylene glycol-conjugated bovine ADA develop antibodies to ADA. This leads us to investigate the role of anti-ADA antibodies in patients with systemic rheumatic diseases. Commercially available ADA was used in ELISA and immunoblots for detection of anti-ADA antibodies. Four out of 100 patients examined were positive for anti-ADA antibodies. Two of them had peripheral blood lymphopenia but the antibody levels did not appear to correlate with the lymphocyte counts. Immunoblotting revealed that the antibodies recognized a 40 kDa peptide of ADA, corresponding to ADA1, the major component of ADA. Affinity-purified antibodies were used to locate the distribution of ADA on Hep-2 cells and lymphocytes by indirect immunofluorescence. Anti-ADA antibodies gave a distinct nuclear speckled pattern on acetone-fixed cells. With viable cell immunofluorescence, anti-ADA antibodies also stained the cell surface of HEp-2 cells and lymphocytes, indicating surface expression of ADA. The anti-ADA antibodies failed to gain access into the cytoplasm or nuclei when added to the cultures of HEp-2 cells. In summary, this is the first report of detection of anti-ADA1 autoantibody which is a new type of ANA with discrete, speckled nuclear staining, but which may not be associated with lymphopenia. PMID:11999881

  6. Expression and characterization of 1-aminocyclopropane-1-carboxylate deaminase from the rhizobacterium Pseudomonas putida UW4: a key enzyme in bacterial plant growth promotion.

    PubMed

    Hontzeas, Nikos; Zoidakis, Jérôme; Glick, Bernard R; Abu-Omar, Mahdi M

    2004-12-01

    The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) converts ACC, the precursor of the plant hormone ethylene, to alpha-ketobutyrate and ammonium. This enzyme has been identified in soil bacteria and has been proposed to play a key role in microbe-plant association. A soluble recombinant ACCD from Pseudomonas putida UW4 of molecular weight 41 kDa has been cloned, expressed, and purified. It showed selectivity and high activity towards the substrate ACC: K(M)=3.4+/-0.2 mM and k(cat)=146+/-5 min(-1) at pH 8.0 and 22 degrees C. The enzyme displayed optimal activity at pH 8.0 with a sharp decline to essentially no activity below pH 6.5 and a slightly less severe tapering in activity at higher pH resulting in loss of activity at pH>10. The major component of the enzyme's secondary structure was determined to be alpha-helical by circular dichroism (CD). P. putida UW4 ACCD unfolded at 60 degrees C as determined by its CD temperature profile as well as by differential scanning microcalorimetry (DSC). Enzyme activity was knocked out in the point mutant Gly44Asp. Modeling this mutation into the known yeast ACCD structure shed light on the role this highly conserved residue plays in allowing substrate accessibility to the active site. This enzyme's biochemical and biophysical properties will serve as an important reference point to which newly isolated ACC deaminases from other organisms can be compared. PMID:15588698

  7. Specific Expression of Activation-induced Cytidine Deaminase (AID), a Novel Member of the RNA-editing Deaminase Family in Germinal Center B Cells

    Microsoft Academic Search

    Masamichi Muramatsu; V. S. Sankaranand; Shrikant Anant; Manabu Sugai; Kazuo Kinoshita; Nicholas O. Davidson; Tasuku Honjo

    1999-01-01

    We have identified a novel gene referred to as activa- tion-induced deaminase (AID) by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma CH12F3-2 cells, more than 80% of which switch exclusively to IgA upon stimulation. The amino acid sequence encoded by AID cDNA is homologous to that of apolipoprotein B (apoB) mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC-1),

  8. Ionization of cytosine monomer and dimer studied by VUV photoionization and electronic structure calculations

    SciTech Connect

    Kostko, Oleg; Bravaya, Ksenia; Krylov, Anna; Ahmed, Musahid

    2009-12-14

    We report a combined theoretical and experimental study of ionization of cytosine monomers and dimers. Gas-phase molecules are generated by thermal vaporization of cytosine followed by expansion of the vapor in a continuous supersonic jet seeded in Ar. The resulting species are investigated by single photon ionization with tunable vacuum-ultraviolet (VUV) synchrotron radiation and mass analyzed using reflectron mass spectrometry. Energy onsets for the measured photoionization efficiency (PIE) spectra are 8.60+-0.05 eV and 7.6+-0.1 eV for the monomer and the dimer, respectively, and provide an estimate for the adiabatic ionization energies (AIE). The first AIE and the ten lowest vertical ionization energies (VIEs) for selected isomers of cytosine dimer computed using equation-of-motion coupled-cluster (EOM-IP-CCSD) method are reported. The comparison of the computed VIEs with the derivative of the PIE spectra, suggests that multiple isomers of the cytosine dimer are present in the molecular beam. The calculations reveal that the large red shift (0.7 eV) of the first IE of the lowest-energy cytosine dimer is due to strong inter-fragment electrostatic interactions, i.e., the hole localized on one of the fragments is stabilized by the dipole moment of the other. A sharp rise in the CH+ signal at 9.20+-0.05 eV is ascribed to the formation of protonated cytosine by dissociation of the ionized dimers. The dominant role of this channel is supported by the computed energy thresholds for the CH+ appearance and the barrierless or nearly barrierless ionization-induced proton transfer observed for five isomers of the dimer.

  9. Threonine Overproduction in Yeast Strains Carrying the HOM3-R2 Mutant Allele under the Control of Different Inducible Promoters

    PubMed Central

    Farfán, María-José; Aparicio, Luis; Calderón, Isabel L.

    1999-01-01

    The HOM3 gene of Saccharomyces cerevisiae codes for aspartate kinase, which plays a crucial role in the regulation of the metabolic flux that leads to threonine biosynthesis. With the aim of obtaining yeast strains able to overproduce threonine in a controlled way, we have placed the HOM3-R2 mutant allele, which causes expression of a feedback-insensitive enzyme, under the control of four distinctive regulatable yeast promoters, namely, PGAL1, PCHA1, PCYC1-HSE2, and PGPH1. The amino acid contents of strains bearing the different constructs were analyzed both under repression and induction conditions. Although some differences in overall threonine production were found, a maximum of around 400 nmol/mg (dry weight) was observed. Other factors, such as excretion to the medium and activity of the catabolic threonine/serine deaminase, also affect threonine accumulation. Thus, improvement of threonine productivity by yeast cells would probably require manipulation of these and other factors. PMID:9872767

  10. Global DNA cytosine methylation as an evolving trait: phylogenetic signal and correlated evolution with genome size in angiosperms

    PubMed Central

    Alonso, Conchita; Pérez, Ricardo; Bazaga, Pilar; Herrera, Carlos M.

    2015-01-01

    DNA cytosine methylation is a widespread epigenetic mechanism in eukaryotes, and plant genomes commonly are densely methylated. Genomic methylation can be associated with functional consequences such as mutational events, genomic instability or altered gene expression, but little is known on interspecific variation in global cytosine methylation in plants. In this paper, we compare global cytosine methylation estimates obtained by HPLC and use a phylogenetically-informed analytical approach to test for significance of evolutionary signatures of this trait across 54 angiosperm species in 25 families. We evaluate whether interspecific variation in global cytosine methylation is statistically related to phylogenetic distance and also whether it is evolutionarily correlated with genome size (C-value). Global cytosine methylation varied widely between species, ranging between 5.3% (Arabidopsis) and 39.2% (Narcissus). Differences between species were related to their evolutionary trajectories, as denoted by the strong phylogenetic signal underlying interspecific variation. Global cytosine methylation and genome size were evolutionarily correlated, as revealed by the significant relationship between the corresponding phylogenetically independent contrasts. On average, a ten-fold increase in genome size entailed an increase of about 10% in global cytosine methylation. Results show that global cytosine methylation is an evolving trait in angiosperms whose evolutionary trajectory is significantly linked to changes in genome size, and suggest that the evolutionary implications of epigenetic mechanisms are likely to vary between plant lineages. PMID:25688257

  11. Interleukin-1 and Tumor Necrosis Factor-? Trigger Restriction of Hepatitis B Virus Infection via a Cytidine Deaminase Activation-induced Cytidine Deaminase (AID)*

    PubMed Central

    Watashi, Koichi; Liang, Guoxin; Iwamoto, Masashi; Marusawa, Hiroyuki; Uchida, Nanako; Daito, Takuji; Kitamura, Kouichi; Muramatsu, Masamichi; Ohashi, Hirofumi; Kiyohara, Tomoko; Suzuki, Ryosuke; Li, Jisu; Tong, Shuping; Tanaka, Yasuhito; Murata, Kazumoto; Aizaki, Hideki; Wakita, Takaji

    2013-01-01

    Virus infection is restricted by intracellular immune responses in host cells, and this is typically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1? and TNF? remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-?B signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1? and TNF? in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1?, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNF?. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNF? was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNF? trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection. PMID:24025329

  12. The Electron Affinities of Deprotonated Adenine, Guanine, Cytosine, Uracil, and Thymine

    Microsoft Academic Search

    Edward C. M. Chen; John R. Wiley; Edward S. Chen

    2008-01-01

    Electron attachment rates and gas phase acidities for the canonical tautomers of the nucleobases and electron affinities for thymine, deprotonated thymine, and cytosine are reported The latter are from a new analysis of published photoelectron spectra. The values for deprotonated thymine are (all in eV) keto-N1-H, 3.327(5); enol-N3-H, 3.250(5), enol-C2OH, 3.120(5) enol-N1-H, 3.013(5), and enol-C4OH,3.123(5). The values for deprotonated cytosine,

  13. Studies on the porphobilinogen deaminase–uroporphyrinogen cosynthetase system of cultured soya-bean cells

    PubMed Central

    Llambías, Elena B. C.; Batlle, Alcira M. Del C.

    1971-01-01

    1. Porphobilinogenase was isolated and purified from soya-bean callus tissue; its components, porphobilinogen deaminase and uroporphyrinogen isomerase, were separated and purified. 2. The purified porphobilinogenase was resolved into two bands on starch-gel electrophoresis. The molecular weights of porphobilinogenase, deaminase and isomerase fractions were determined by the gel-filtration method. Porphobilinogenase activity was affected by the presence of air; uroporphyrinogens were only formed under anaerobic conditions, although substrate consumption was the same in the absence of oxygen as in its presence. 3. pH-dependence of both porphobilinogenase and deaminase was the same and a sharp optimum at pH 7.2 was obtained. Isomerase was heat-labile, but the presence of ammonium ions or porphobilinogen afforded some protection against inactivation. The action of several compounds added to the system was studied. Cysteine, thioglycollate, ammonium ions and hydroxylamine inhibited porphobilinogenase; certain concentrations of sodium and magnesium salts enhanced activity; some dicarboxylic acids and 2-methoxy-5-nitrotropone inhibited the deaminase. 4. ?-Aminolaevulate and ethionine in the culture media stimulated porphyrin synthesis and increased porphobilinogenase activity, whereas iron deficiency resulted in porphyrin accumulation. 5. The development of chlorophyll and porphobilinogenase on illumination of dark-grown callus was followed. 6. A hypothetical scheme is suggested for the enzymic synthesis of uroporphyrinogens from porphobilinogen. PMID:5165654

  14. Studies on the porphobilinogen deaminase-uroporphyrinogen cosynthetase system of cultured soya-bean cells.

    PubMed

    Llambías, E B; Battle, A M

    1971-01-01

    1. Porphobilinogenase was isolated and purified from soya-bean callus tissue; its components, porphobilinogen deaminase and uroporphyrinogen isomerase, were separated and purified. 2. The purified porphobilinogenase was resolved into two bands on starch-gel electrophoresis. The molecular weights of porphobilinogenase, deaminase and isomerase fractions were determined by the gel-filtration method. Porphobilinogenase activity was affected by the presence of air; uroporphyrinogens were only formed under anaerobic conditions, although substrate consumption was the same in the absence of oxygen as in its presence. 3. pH-dependence of both porphobilinogenase and deaminase was the same and a sharp optimum at pH 7.2 was obtained. Isomerase was heat-labile, but the presence of ammonium ions or porphobilinogen afforded some protection against inactivation. The action of several compounds added to the system was studied. Cysteine, thioglycollate, ammonium ions and hydroxylamine inhibited porphobilinogenase; certain concentrations of sodium and magnesium salts enhanced activity; some dicarboxylic acids and 2-methoxy-5-nitrotropone inhibited the deaminase. 4. delta-Aminolaevulate and ethionine in the culture media stimulated porphyrin synthesis and increased porphobilinogenase activity, whereas iron deficiency resulted in porphyrin accumulation. 5. The development of chlorophyll and porphobilinogenase on illumination of dark-grown callus was followed. 6. A hypothetical scheme is suggested for the enzymic synthesis of uroporphyrinogens from porphobilinogen. PMID:5165654

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

    Microsoft Academic Search

    R BARTON

    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

  16. Adenosine Deaminase Deficiency: Metabolic Basis of Immune Deficiency and Pulmonary Inflammation

    Microsoft Academic Search

    Michael R. Blackburn; Rodney E. Kellems

    2005-01-01

    Genetic deficiencies in the purine catabolic enzyme adenosine deaminase (ADA) in humans results primarily in a severe lymphopenia and immunodeficiency that can lead to the death of affected individuals early in life. The metabolic basis of the immunodeficiency is likely related to the sensitivity of lymphocytes to the accumulation of the ADA substrates adenosine and 2?-deoxyadenosine. Investigations using ADA-deficient mice

  17. Effects of deoxyadenosine on ribonucleotide reductase in adenosine deaminase-deficient lymphocytes

    Microsoft Academic Search

    E. Takeda; Y. Kuroda; E. Naito; I. Yokota; T. Saijo; M. Hirose; M. Miyao

    1991-01-01

    Summary To explore the relationship between ribonucleotide reductase and immunodysfunction in adenosine deaminase deficiency, the effects of deoxyadenosine on ribonucleotide reductase in ADA-deficient lymphocytes was investigated. An assay system for ribonucleotide reductase in intact permeabilized lymphocytes was developed to approximate physiological conditions. The activity of cytidine diphosphate (CDP) reductase in resting but not in proliferating lymphocytes in culture was inhibited

  18. Genotype is an important determinant of phenotype in adenosine deaminase deficiency

    Microsoft Academic Search

    Michael S Hershfield

    2003-01-01

    Adenosine deaminase (ADA) deficiency is associated with a broad clinical and mutational spectrum. Defining the relationship of genotype to phenotype among patients with different degrees of immunodeficiency has been complicated because the disease is rare, most mutations are ‘private’ and patients are often heteroallelic. In recent years, knowledge of ADA structure and systematic expression of mutant alleles have revealed that

  19. Management options for adenosine deaminase deficiency; proceedings of the EBMT satellite workshop (Hamburg, March 2006)

    Microsoft Academic Search

    Claire Booth; Mike Hershfield; Luigi Notarangelo; Rebecca Buckley; Manfred Hoenig; Nizar Mahlaoui; Marina Cavazzana-Calvo; Alessandro Aiuti; H. Bobby Gaspar

    2007-01-01

    Adenosine deaminase (ADA) deficiency is a disorder of purine salvage that has its most devastating consequences in the immune system leading to severe combined immunodeficiency (SCID). Management options for ADA SCID include hematopoietic stem cell transplantation, enzyme replacement therapy and gene therapy. Formal data on the outcome following each of the three treatment modalities are limited, and this symposium was

  20. Bilateral sensorineural deafness in adenosine deaminase-deficient severe combined immunodeficiency

    Microsoft Academic Search

    Wendy Albuquerque; Hubert B. Gaspar

    2004-01-01

    Adenosine deaminase deficiency presents with severe combined immunodeficiency and is treatable by bone marrow transplantation. With improved survival, the nonimmunologic manifestations of this condition are becoming apparent. We report a high incidence of bilateral sensorineural deafness in transplanted patients, which highlights the systemic nature of the disease.

  1. Three Novel Mutations in Porphobilinogen Deaminase Gene Identified in Russian Patients with Acute Intermittent Porphyria

    Microsoft Academic Search

    V. L. Surin; A. V. Luk'yanenko; I. V. Karpova; A. V. Misyurin; Ya. S. Pustovoit; A. V. Pivnik

    2001-01-01

    Porphobilinogen deaminase (PBGD) is a key enzyme of the heme biosynthetic pathway. Defects in the PBGD gene lead to an autosomal dominant disease, acute intermittent porphyria (AIP). Almost all AIP patients with rare exceptions are heterozygous for the defective gene. To date, at least 160 different mutations causing AIP are identified. Extensive investigations along this line are conducted in many

  2. Relevance of Adenosine Deaminase and Lysozyme Measurements in the Diagnosis of Tuberculous Pericarditis

    Microsoft Academic Search

    Constadina Aggeli; Christos Pitsavos; Stella Brili; Dimitrios Hasapis; Alexandra Frogoudaki; Christodoulos Stefanadis; Pavlos Toutouzas

    2000-01-01

    Objective: To assess the value of pericardial fluid adenosine deaminase (ADA) and pericardial lysozyme (Lys) as tools in diagnosing tuberculous pericarditis. Methods: Forty-one patients (age range 17–77 years) with significant pericardial effusion were included in the study. Diagnostic pericardiocentesis and pericardial biopsy were performed while serum and pericardial fluid ADA and Lys were measured in all patients. Grouping of patients

  3. IMMUNE FUNCTION IN MICE EXPOSED TO THE ADENOSINE DEAMINASE INHIBITOR 2'-DEOXYCOFORMYCIN DURING IMMUNE SYSTEM DEVELOPMENT

    EPA Science Inventory

    Pregnant mice were administered 2'-deoxycoformycin (2dCF), a potent inhibitor of adensoine deaminase activity, by intraperitoneal injection on day 7 or 15 of gestation or from day 8-12 or 14-18 of gestation. A total dose of 0, 0.5 or 2.0 micrograms 2dCF/g of maternal body weight ...

  4. Activation-induced deaminase cloning, localization, and protein extraction from young VH-mutant rabbit appendix

    Microsoft Academic Search

    Guibin Yang; Harold Obiakor; Rajesh K. Sinha; Barbara A. Newman; Brian L. Hood; Thomas P. Conrads; Timothy D. Veenstra; Rose G. Mage

    2005-01-01

    Studies in mouse, human, and chicken suggest that activation-induced deaminase (AID) is involved in three known processes leading to antibody diversification: somatic hypermutation, gene conversion, and class-switch recombination. Developing rabbit appendix provides a particularly good site for studying all three of these B cell maturation events. We report here successful cloning of rabbit AID and isolation of AID protein from

  5. SELECTIVE IMMUNOTOXIC EFFECTS IN MICE TREATED WITH THE ADENOSINE DEAMINASE INHIBITOR 2-DEOXYCOFORMYCIN (JOURNAL VERSION)

    EPA Science Inventory

    Mice given the adenosine deaminase inhibitor 2-deoxycoformycin, for five days were evaluated 24 h, 72 h and 6 days after the final dose. Spleen weight was decreased for up to 6 days after treatment. The number and relative percentage of circulating lymphocytes were decreased 24 a...

  6. Increases in Interstitial Adenosine and Cerebral Blood Flow with Inhibition of Adenosine Kinase and Adenosine Deaminase

    Microsoft Academic Search

    Veronica M. Sciotti; David G. L. Van Wylen

    1993-01-01

    Summary: The purpose of this study was to determine the changes in interstitial fluid (ISF) adenosine and cerebral blood flow (CBF) during inhibition of adenosine kinase or adenosine deaminase. Brain microdialysis was used to (a) measure CBF (H2 clearance), (b) sample cerebral ISF, and (c) deliver drugs locally to the brain. Microdialysis probes were implanted bilaterally in the caudate nucleus

  7. Chromosome 20: gene for adenosine deaminase, Matt RidleySite: DNA Interactive (www.dnai.org)

    NSDL National Science Digital Library

    2008-10-06

    Interviewee: Matt Ridley DNAi Location:Genome>tour>genome spots>Severe Combined Immunodeficiency (SCID) Location: chromosome 20 and X gene name: ADA (adenosine deaminase) Mutations in the sequence of the ADA gene (and another gene on the X chromosome called IL2RG) can cause severe combined immunodeficiency (SCID). People with SCID are prone to bacterial, viral, and fungal infections.

  8. An ultrasensitive electrochemical method for detection of Ag(+) based on cyclic amplification of exonuclease III activity on cytosine-Ag(+)-cytosine.

    PubMed

    Xu, Gang; Wang, Guangfeng; He, Xiuping; Zhu, Yanhong; Chen, Ling; Zhang, Xiaojun

    2013-11-21

    Ag(+) is known to bind very strongly with cytosine-cytosine (C-C) mismatches in DNA duplexes to form C-Ag(+)-C base pairs. Exonuclease III (Exo III) can catalyze the stepwise removal of mononucleotides of duplex DNA. In this work, we study Exo III activity on DNA hybrids containing C-Ag(+)-C base pairs. Our experiments show that Ag(+) ions could intentionally trigger the activity of Exo III towards a designed cytosine-rich DNA oligonucleotide (C-rich probe) by the conformational change of the probe. Our sensing strategy uses this conformation-dependent activity of Exo III, which is controlled through the cyclical shuffling of Ag(+) ions between the solid DNA hybrid and the solution phase. This interesting conversion has led to the development of an ultrasensitive detection platform for Ag(+) ions with a detection limit of 0.03 nM and a total assay time possible within minutes. This simple detection strategy could also be used for the detection of other metal ions which exhibit specific interactions with natural or synthetic bases. PMID:24071747

  9. Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations

    Microsoft Academic Search

    R. G. H. Cotton; N. R. Rodrigues; R. D. Campbell

    1988-01-01

    The chemical reactivity of thymine (T), when mismatched with the bases cytosine, guanine, and thymine, and of cytosine (C), when mismatched with thymine, adenine, and cytosine, has been examined. Heteroduplex DNAs containing such mismatched base pairs were first incubated with osmium tetroxide (for T and C mismatches) or hydroxylamine (for C mismatches) and then incubated with piperidine to cleave the

  10. AILV1 gene from the yeast Arxula adeninivorans LS3--a new selective transformation marker.

    PubMed

    Wartmann, T; Rösel, H; Kunze, I; Bode, R; Kunze, G

    1998-08-01

    The ILV1 gene of the yeast Arxula adeninivorans LS3 (AILV1) has been cloned from a genomic library, characterized and used as an auxotrophic selection marker for transformation of plasmids into this yeast. One copy of the gene is present in the Arxula genome, comprising 1653 bp and encoding 550 amino acids of the threonine deaminase. The protein sequence is similar (60.55%) to that of the threonine deaminase from Saccharomyces cerevisiae encoded by the gene ILV1. The protein is enzymatically active during the whole period of cultivation, up to 70 h. Maximal activities, as well as protein concentrations of this enzyme, were achieved after cultivation times of 20-36 h. The AILV1 gene is a suitable auxotrophic selection marker in transformation experiments using an Arxula adeninivorans ilv1 mutant and a plasmid containing this gene, which is fused into the 25S rDNA of Arxula adeninivorans. One to three copies of the linearized plasmid were integrated into the 25S rDNA by homologous recombination. Transformants resulting from complementation of the ilv1 mutation can be easily and reproducibly selected and in addition are mitotically stable. Therefore, the described system is preferred to the conventional selection for hygromycin B resistance. PMID:9730281

  11. Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively

    PubMed Central

    Sharma, Sunny; Yang, Jun; Watzinger, Peter; Kötter, Peter; Entian, Karl-Dieter

    2013-01-01

    Yeast 25S rRNA was reported to contain a single cytosine methylation (m5C). In the present study using a combination of RP-HPLC, mung bean nuclease assay and rRNA mutagenesis, we discovered that instead of one, yeast contains two m5C residues at position 2278 and 2870. Furthermore, we identified and characterized two putative methyltransferases, Rcm1 and Nop2 to be responsible for these two cytosine methylations, respectively. Both proteins are highly conserved, which correlates with the presence of two m5C residues at identical positions in higher eukaryotes, including humans. The human homolog of yeast Nop2, p120 has been discovered to be upregulated in various cancer tissues, whereas the human homolog of Rcm1, NSUN5 is completely deleted in the William's-Beuren Syndrome. The substrates and function of both human homologs remained unknown. In the present study, we also provide insights into the significance of these two m5C residues. The loss of m5C2278 results in anisomycin hypersensitivity, whereas the loss of m5C2870 affects ribosome synthesis and processing. Establishing the locations and enzymes in yeast will not only help identifying the function of their homologs in higher organisms, but will also enable understanding the role of these modifications in ribosome function and architecture. PMID:23913415

  12. Population Growth in Yeasts

    NSDL National Science Digital Library

    Engineering K-PhD Program,

    This lesson is the second of two that explore cellular respiration and population growth in yeasts. In the first lesson, students set up a simple way to indirectly observe and quantify the amount of respiration occurring in yeast-molasses cultures. Based on questions that arose during the first lesson and its associated activity, students in this lesson work in small groups to design experiments that determine how environmental factors affect yeast population growth.

  13. The role of gene body cytosine modifications in MGMT expression and sensitivity to temozolomide.

    PubMed

    Moen, Erika L; Stark, Amy L; Zhang, Wei; Dolan, M Eileen; Godley, Lucy A

    2014-05-01

    The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is known to play a role in sensitivity to temozolomide. Promoter hypermethylation of MGMT is commonly used to predict low expression levels of MGMT in gliomas, despite observed discordance between promoter methylation and protein levels. Here, we investigated the functional role of gene body cytosine modification in regulating levels of MGMT gene expression and sensitivity to temozolomide. In 91 human glioblastoma samples, we observed significant variation in MGMT expression levels in patients with an unmethylated promoter, with higher levels of gene body cytosine modification correlating with higher gene expression levels. Furthermore, inducing hypomethylation across the MGMT gene body with decitabine corresponded with decreased levels of MGMT gene expression in lymphoblastoid and glioblastoma cell lines, indicating an important functional role for gene body cytosine modifications in maintaining gene expression. We reasoned that the decrease in MGMT expression induced by decitabine may render resistant glioblastoma cell lines more sensitive to temozolomide. Consistent with this reasoning, we found that the MGMT-expressing glioblastoma cell lines exhibiting an unmethylated MGMT promoter that were pretreated with decitabine became significantly more sensitive to temozolomide. Overall, our results suggest a functional role for gene body cytosine modification in regulating gene expression of MGMT and indicate that pretreating patients whose tumors have an unmethylated MGMT promoter with decitabine before temozolomide treatment may increase their response to therapy. PMID:24568970

  14. PCNA clamp facilitates action of DNA cytosine methyltransferase 1 on hemimethylated DNA

    Microsoft Academic Search

    Tetsuo Iida; Isao Suetake; Shoji Tajima; Hiroshi Morioka; Satoshi Ohta; Chikashi Obuse; Toshiki Tsurimoto

    2002-01-01

    Background : Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein known as a processivity fac- tor of DNA polymerase ? ? ? ? . In addition to this role, PCNA interacts with a number of other proteins to increase their local concentration at replicated DNA sites. DNA cytosine methyltransferase 1 (Dnmt1), which preserves epigenetic signals by completing the methylation

  15. Characterization of resistance to cytosine arabinoside (Ara-C) in NALM-6 human B leukemia cells

    Microsoft Academic Search

    Syu-ichi Kanno; Takako Hiura; Takaharu Ohtake; Kimiko Koiwai; Hiroyoshi Suzuki; Mayuko Ujibe; Masaaki Ishikawa

    2007-01-01

    BackgroundCytosine arabinoside (1-?-d-arabinofuranosylcytosine;Ara-C) is the most important antimetabolite used for acute leukemia. We established Ara-C (0.003–1 ?mol\\/l)-resistant NALM-6 leukemia cells, and attempted the characterization of their resistance.

  16. Substitutions of a cysteine conserved among DNA cytosine methylases result in a variety of phenotypes.

    PubMed Central

    Wyszynski, M W; Gabbara, S; Bhagwat, A S

    1992-01-01

    The proposed mechanism for DNA (cytosine-5)-methyltransferases envisions a key role for a cysteine residue. It is expected to form a covalent link with carbon 6 of the target cytosine, activating the normally inactive carbon 5 for methyl transfer. There is a single conserved cysteine among all DNA (cytosine-5)-methyltransferases making it the candidate nucleophile. We have changed this cysteine to other amino acids for the EcoRII methylase; which methylates the second cytosine in the sequence 5'-CCWGG-3'. Mutants were tested for their methyl transferring ability and for their ability to form covalent complexes with DNA. The latter property was tested indirectly with the use of a genetic assay involving sensitivity of cells to 5-azacytidine. Replacement of the conserved cysteine with glycine, valine, tryptophan or serine led to an apparent loss of methyl transferring ability. Interestingly, cells carrying the mutant with serine did show sensitivity to 5-azacytidine, suggesting the ability to link to DNA. Unexpectedly, substitution of the cysteine with glycine results in the inhibition of cell growth and the mutant allele can be maintained in the cells only when it is poorly expressed. These results suggest that the conserved cysteine in the EcoRII methylase is essential for methylase action and it may play more than one role in it. Images PMID:1371346

  17. Aerobiosis Increases the Genomic Guanine Plus Cytosine Content (GC%) in Prokaryotes

    Microsoft Academic Search

    Hugo Naya; Héctor Romero; Alejandro Zavala; Beatriz Alvarez; Héctor Musto

    2002-01-01

    The huge variation in the genomic guanine plus cytosine content (GC%) among prokar-yotes has been explained by two mutually exclusive hypotheses, namely, selectionist and neutralist. The former proposals have in common the assumption that this feature is a form of adaptation to some ecological or physiological condition. On the other hand, the neutralist interpretation states that the variations are due

  18. Comments on `Concentration by Evaporation and the Prebiotic Synthesis of Cytosine'

    NASA Astrophysics Data System (ADS)

    Shapiro, Robert

    2002-06-01

    The claim by Nelson et al. (2001) that the reaction of cyanoacetaldehyde and urea provides `an efficient prebiotic synthesis' of cytosine is disputed. The authors have not dealt with the important points presented in a criticism of this reaction (Shapiro, 1999): (1) The reactants undergo side reactions with common nucleophiles that appear to proceed more rapidly than cytosine formation, and (2) No reactions have been described thus far that would produce cytosine at a rate sufficient to compensate for its decomposition by deamination, and permit accumulation over extended periods of time. Instead, Nelson et al. have conducted `drying-down' experiments, in an effort to simulate evaporations on the early Earth, but the design of these experiments is flawed. The initial reactant concentrations are much higher than might be expected in a natural setting, and potentially interfering substances such as glycine, cyanide and thiols have been excluded. `Drying beaches and drying lagoons' have been invoked as sites for such a reaction but no effort has been made to describe the characteristics of such sites or to estimate their frequency with reference to the present Earth. In the absence of contradictory data, the conclusion put forward in Shapiro (1999) remains valid: `It was quite unlikely that cytosine played a role in the origin of life'.

  19. Prebiotic Cytosine Synthesis: A Critical Analysis and Implications for the Origin of Life

    Microsoft Academic Search

    Robert Shapiro

    1999-01-01

    A number of theories propose that RNA, or an RNA-like substance, played a role in the origin of life. Usually, such hypotheses presume that the Watson-Crick bases were readily available on prebiotic Earth, for spontaneous incorporation into a replicator. Cytosine, however, has not been reported in analyses of meteorites nor is it among the products of electric spark discharge experiments.

  20. Toxicity of 1-(?-d-arabinofuranosyl)cytosine after intravitreal injection in the rabbit eye

    Microsoft Academic Search

    Joke J. A. Th. Diets-Ouwehand; Rob J. W. Keizer; Gijs Vrensen; Sylvia Groen-Jansen; Jaap A. Best

    1992-01-01

    The retinal toxicity of intravitreally injected 1-(ß-d-arabinofuranosyl)cytosine (cytarabine) was examined in 7 chinchilla rabbits to determine if cytarabine can be used as local therapy for vitreoretinal non-Hodgkin's lymphoma. Fractionated dose of 600 µg, 1500 µg, and 2700 µg cytarabine in stabilized saline were given intravitreally in one eye (2 × 300 µg, 5 × 300 µg, and 3 × 900

  1. Nitrosative Cytosine Deamination. An Exploration of the Chemistry Emanating from Deamination with

    E-print Network

    Glaser, Rainer

    with Pyrimidine Ring-Opening Sundeep Rayat, Ming Qian, and Rainer Glaser* Department of Chemistry, University to the DNA can have deleterious effects via mutagenesis, cell transformation, and cell death (1) Nitrosating (15). The spontaneous deamination is a slow reaction with a measured half-life for cytosine residues

  2. Cytosine methylation is the most common covalent modification of DNA in eukaryotes. DNA methylation has an

    E-print Network

    3959 Cytosine methylation is the most common covalent modification of DNA in eukaryotes. DNA-binding proteins and anti-methylcytosine antibodies. Combining these techniques with DNA microarrays and high- throughput sequencing has made the mapping of DNA methylation feasible on a genome-wide scale. Here we

  3. Direct observation of cytosine flipping and covalent catalysis in a DNA methyltransferase

    PubMed Central

    Gerasimait?, R?ta; Merkien?, Egl?; Klimašauskas, Saulius

    2011-01-01

    Methylation of the five position of cytosine in DNA plays important roles in epigenetic regulation in diverse organisms including humans. The transfer of methyl groups from the cofactor S-adenosyl-l-methionine is carried out by methyltransferase enzymes. Using the paradigm bacterial methyltransferase M.HhaI we demonstrate, in a chemically unperturbed system, the first direct real-time analysis of the key mechanistic events—the flipping of the target cytosine base and its covalent activation; these changes were followed by monitoring the hyperchromicity in the DNA and the loss of the cytosine chromophore in the target nucleotide, respectively. Combined with studies of M.HhaI variants containing redesigned tryptophan fluorophores, we find that the target base flipping and the closure of the mobile catalytic loop occur simultaneously, and the rate of this concerted motion inversely correlates with the stability of the target base pair. Subsequently, the covalent activation of the target cytosine is closely followed by but is not coincident with the methyl group transfer from the bound cofactor. These findings provide new insights into the temporal mechanism of this physiologically important reaction and pave the way to in-depth studies of other base-flipping systems. PMID:21245034

  4. Genomic DNA sequence and cytosine methylation changes of adult rice leaves after seeds space flight

    NASA Astrophysics Data System (ADS)

    Shi, Jinming

    In this study, cytosine methylation on CCGG site and genomic DNA sequence changes of adult leaves of rice after seeds space flight were detected by methylation-sensitive amplification polymorphism (MSAP) and Amplified fragment length polymorphism (AFLP) technique respectively. Rice seeds were planted in the trial field after 4 days space flight on the shenzhou-6 Spaceship of China. Adult leaves of space-treated rice including 8 plants chosen randomly and 2 plants with phenotypic mutation were used for AFLP and MSAP analysis. Polymorphism of both DNA sequence and cytosine methylation were detected. For MSAP analysis, the average polymorphic frequency of the on-ground controls, space-treated plants and mutants are 1.3%, 3.1% and 11% respectively. For AFLP analysis, the average polymorphic frequencies are 1.4%, 2.9%and 8%respectively. Total 27 and 22 polymorphic fragments were cloned sequenced from MSAP and AFLP analysis respectively. Nine of the 27 fragments from MSAP analysis show homology to coding sequence. For the 22 polymorphic fragments from AFLP analysis, no one shows homology to mRNA sequence and eight fragments show homology to repeat region or retrotransposon sequence. These results suggest that although both genomic DNA sequence and cytosine methylation status can be effected by space flight, the genomic region homology to the fragments from genome DNA and cytosine methylation analysis were different.

  5. Cytosine Usage Modulates the Correlation between CDS Length and CG Content in Prokaryotic Genomes

    E-print Network

    Xia, Xuhua

    Cytosine Usage Modulates the Correlation between CDS Length and CG Content in Prokaryotic Genomes empirically by prokaryotic genomes. How- ever, the correlation is weak for a number of species, with 4 species in long CDSs. Empirical data from prokaryotic genomes lend strong support for this new hypothesis

  6. Red Yeast Rice: An Introduction

    MedlinePLUS

    ... links Read our disclaimer about external links Menu Red Yeast Rice: An Introduction On this page: Key ... will help ensure coordinated and safe care. About Red Yeast Rice Red yeast rice is made by ...

  7. Cytosine methylation and hydroxymethylation mark DNA for elimination in Oxytricha trifallax

    PubMed Central

    2012-01-01

    Background Cytosine methylation of DNA is conserved across eukaryotes and plays important functional roles regulating gene expression during differentiation and development in animals, plants and fungi. Hydroxymethylation was recently identified as another epigenetic modification marking genes important for pluripotency in embryonic stem cells. Results Here we describe de novo cytosine methylation and hydroxymethylation in the ciliate Oxytricha trifallax. These DNA modifications occur only during nuclear development and programmed genome rearrangement. We detect methylcytosine and hydroxymethylcytosine directly by high-resolution nano-flow UPLC mass spectrometry, and indirectly by immunofluorescence, methyl-DNA immunoprecipitation and bisulfite sequencing. We describe these modifications in three classes of eliminated DNA: germline-limited transposons and satellite repeats, aberrant DNA rearrangements, and DNA from the parental genome undergoing degradation. Methylation and hydroxymethylation generally occur on the same sequence elements, modifying cytosines in all sequence contexts. We show that the DNA methyltransferase-inhibiting drugs azacitidine and decitabine induce demethylation of both somatic and germline sequence elements during genome rearrangements, with consequent elevated levels of germline-limited repetitive elements in exconjugant cells. Conclusions These data strongly support a functional link between cytosine DNA methylation/hydroxymethylation and DNA elimination. We identify a motif strongly enriched in methylated/hydroxymethylated regions, and we propose that this motif recruits DNA modification machinery to specific chromosomes in the parental macronucleus. No recognizable methyltransferase enzyme has yet been described in O. trifallax, raising the possibility that it might employ a novel cytosine methylation machinery to mark DNA sequences for elimination during genome rearrangements. PMID:23075511

  8. Cytosine Methylation Alteration in Natural Populations of Leymus chinensis Induced by Multiple Abiotic Stresses

    PubMed Central

    Yu, Yingjie; Yang, Xuejiao; Wang, Huaying; Shi, Fengxue; Liu, Ying; Liu, Jushan; Li, Linfeng; Wang, Deli; Liu, Bao

    2013-01-01

    Background Human activity has a profound effect on the global environment and caused frequent occurrence of climatic fluctuations. To survive, plants need to adapt to the changing environmental conditions through altering their morphological and physiological traits. One known mechanism for phenotypic innovation to be achieved is environment-induced rapid yet inheritable epigenetic changes. Therefore, the use of molecular techniques to address the epigenetic mechanisms underpinning stress adaptation in plants is an important and challenging topic in biological research. In this study, we investigated the impact of warming, nitrogen (N) addition, and warming+nitrogen (N) addition stresses on the cytosine methylation status of Leymus chinensis Tzvel. at the population level by using the amplified fragment length polymorphism (AFLP), methylation-sensitive amplified polymorphism (MSAP) and retrotransposon based sequence-specific amplification polymorphism (SSAP) techniques. Methodology/Principal Findings Our results showed that, although the percentages of cytosine methylation changes in SSAP are significantly higher than those in MSAP, all the treatment groups showed similar alteration patterns of hypermethylation and hypomethylation. It meant that the abiotic stresses have induced the alterations in cytosine methylation patterns, and the levels of cytosine methylation changes around the transposable element are higher than the other genomic regions. In addition, the identification and analysis of differentially methylated loci (DML) indicated that the abiotic stresses have also caused targeted methylation changes at specific loci and these DML might have contributed to the capability of plants in adaptation to the abiotic stresses. Conclusions/Significance Our results demonstrated that abiotic stresses related to global warming and nitrogen deposition readily evoke alterations of cytosine methylation, and which may provide a molecular basis for rapid adaptation by the affected plant populations to the changed environments. PMID:23418457

  9. Dehydration, deamination and enzymatic repair of cytosine glycols from oxidized poly(dG-dC) and poly(dI-dC)

    PubMed Central

    Tremblay, Sébastien; Wagner, J. Richard

    2008-01-01

    Cytosine glycols (5,6-dihydroxy-5,6-dihydrocytosine) are initial products of cytosine oxidation. Because these products are not stable, virtually all biological studies have focused on the stable oxidation products of cytosine, including 5-hydroxycytosine, uracil glycols and 5-hydroxyuracil. Previously, we reported that the lifetime of cytosine glycols was greatly enhanced in double-stranded DNA, thus implicating these products in DNA repair and mutagenesis. In the present work, cytosine and uracil glycols were generated in double-stranded alternating co-polymers by oxidation with KMnO4. The half-life of cytosine glycols in poly(dG-dC) was 6.5 h giving a ratio of dehydration to deamination of 5:1. At high substrate concentrations, the excision of cytosine glycols from poly(dG-dC) by purified endonuclease III was comparable to that of uracil glycols, whereas the excision of these substrates was 5-fold greater than that of 5-hydroxycytosine. Kinetic studies revealed that the Vmax was several fold higher for the excision of cytosine glycols compared to 5-hydroxycytosine. In contrast to cytosine glycols, uracil glycols did not undergo detectable dehydration to 5-hydroxyuracil. Replacing poly(dG-dC) for poly(dI-dC) gave similar results with respect to the lifetime and excision of cytosine glycols. This work demonstrates the formation of cytosine glycols in DNA and their removal by base excision repair. PMID:18032437

  10. Plant Purine Nucleoside Catabolism Employs a Guanosine Deaminase Required for the Generation of Xanthosine in Arabidopsis[W

    PubMed Central

    Dahncke, Kathleen; Witte, Claus-Peter

    2013-01-01

    Purine nucleotide catabolism is common to most organisms and involves a guanine deaminase to convert guanine to xanthine in animals, invertebrates, and microorganisms. Using metabolomic analysis of mutants, we demonstrate that Arabidopsis thaliana uses an alternative catabolic route employing a highly specific guanosine deaminase (GSDA) not reported from any organism so far. The enzyme is ubiquitously expressed and deaminates exclusively guanosine and 2’-deoxyguanosine but no other aminated purines, pyrimidines, or pterines. GSDA belongs to the cytidine/deoxycytidylate deaminase family of proteins together with a deaminase involved in riboflavin biosynthesis, the chloroplastic tRNA adenosine deaminase Arg and a predicted tRNA-specific adenosine deaminase 2 in A. thaliana. GSDA is conserved in plants, including the moss Physcomitrella patens, but is absent in the algae and outside the plant kingdom. Our data show that xanthosine is exclusively generated through the deamination of guanosine by GSDA in A. thaliana, excluding other possible sources like the dephosphorylation of xanthosine monophosphate. Like the nucleoside hydrolases NUCLEOSIDE HYDROLASE1 (NSH1) and NSH2, GSDA is located in the cytosol, indicating that GMP catabolism to xanthine proceeds in a mostly cytosolic pathway via guanosine and xanthosine. Possible implications for the biosynthetic route of purine alkaloids (caffeine and theobromine) and ureides in other plants are discussed. PMID:24130159

  11. Xylose fermentation by yeasts

    Microsoft Academic Search

    B. Weigert; C. Klein; M. Rizzi; C. Lauterbach; H. Dellweg

    1988-01-01

    Summary Furfural as a product from thermic wood hydrolysis processes may be inhibitory to growth and fermentation of yeast cells. In order to determine the influence on the aerobic growth of the yeastPichiastipitis, expermiments were conducted in stirred reactors with the addition of furfural.

  12. Yeasts: Neglected Pathogens

    Microsoft Academic Search

    Daniel Poulain; Boualem Sendid; Annie Standaert-Vitse; Chantal Fradin; Thierry Jouault; Samir Jawhara; Jean-Frederic Colombel

    2009-01-01

    Background: Current research on Crohn’s disease (CD) concerns molecular events related to loss of tolerance to microbes that could trigger or maintain inflammation in genetically susceptible individuals. CD is also associated with antimicrobial antibodies, including the antibodies we described against yeast oligomannosides (ASCA). This prompted us to investigate a role for another yeast, Candida albicans, a very common commensal of

  13. Alcoholic Fermentation in Yeast

    NSDL National Science Digital Library

    Ingrid Waldron

    Students learn about the basics of aerobic cellular respiration and alcoholic fermentation and design and carry out experiments to test how variables such as sugar concentration influence the rate of alcoholic fermentation in yeast. In an optional extension activity students can use their yeast mixture to make a small roll of bread.

  14. Characterization of Residual Enzyme Activity in Fibroblasts from Patients with Adenosine Deaminase Deficiency and Combined Immunodeficiency: Evidence for a Mutant Enzyme

    Microsoft Academic Search

    Rochelle Hirschhorn; Nicholas Beratis; Fred S. Rosen

    1976-01-01

    A proportion of patients suffering from the autosomal recessive form of severe combined immunodeficiency have an inherited deficiency of adenosine deaminase (EC 3.5.4.4; adenosine aminohydrolase) (erythrocyte isoenzyme). We have, however, found residual adenosine deaminase activity in fibroblasts derived from four such patients. The enzyme responsible for this activity is biochemically homologous with the high-molecular-weight tissue isoenzyme of adenosine deaminase found

  15. Partial characterization of the gene encoding myoadenylate deaminase from the teleost fish Platichthys flesus.

    PubMed

    Thébault, M T; Tanguy, A; Meistertzheim, A L; Raffin, J P

    2010-12-01

    AMP-deaminase (AMPD, EC 3.5.4.6), which catalyzes the irreversible hydrolytic deamination of AMP to IMP and ammonia, is an important energy-related enzyme. The partial genomic sequence of the gene encoding myoadenylate deaminase (AMPD1) from the teleost fish Platichthys flesus was determined. The amino acid sequence of P. flesus AMPD1 shows 82% homology with that of the teleost fish Danio rerio. Comparison of genomic sequences of P. flesus and Rattus norvegicus reveals a high degree of conservation of both sequence and structural organization. A phylogenetic analysis of AMPD sequences shows that bony fish and mammalian AMPD1s arise by duplication of a common primordial gene. PMID:19821138

  16. APOBEC3 Cytidine Deaminases in Double-Strand DNA Break Repair and Cancer Promotion

    PubMed Central

    Nowarski, Roni; Kotler, Moshe

    2013-01-01

    High frequency of cytidine to thymidine conversions were identified in the genome of several types of cancer cells. In breast cancer cells these mutations are clustered in long DNA regions associated with ssDNA, double-strand DNA breaks (DSBs) 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. PMID:23598277

  17. Hematopoietic stem cell gene therapy for adenosine deaminase deficient-SCID

    Microsoft Academic Search

    Alessandro Aiuti; Immacolata Brigida; Francesca Ferrua; Barbara Cappelli; Robert Chiesa; Sarah Marktel; Maria-Grazia Roncarolo

    2009-01-01

    Gene therapy is a highly attractive strategy for many types of inherited disorders of the immune system. Adenosine deaminase\\u000a (ADA) deficient-severe combined immunodeficiency (SCID) has been the target of several clinical trials based on the use of\\u000a hematopoietic stem\\/progenitor cells engineered with retroviral vectors. The introduction of a low intensity conditioning regimen\\u000a has been a crucial factor in achieving stable

  18. p53 Expression is Required for Thymocyte Apoptosis Induced by Adenosine Deaminase Deficiency

    Microsoft Academic Search

    Patricia Benveniste; Amos Cohen

    1995-01-01

    Adenosine deaminase (ADA, EC 3.5.4.4) is a ubiquitous enzyme in the purine catabolic pathway. In contrast to the widespread tissue distribution of this enzyme, inherited ADA deficiency in human results in a tissue-specific severe combined immunodeficiency. To explain the molecular basis for this remarkable tissue specificity, we have used a genetic approach to study ADA deficiency. We demonstrate that ADA

  19. Multicentric Dermatofibrosarcoma Protuberans in Patients with Adenosine Deaminase-Deficient Severe Combined Immune Deficiency

    Microsoft Academic Search

    Chimen Kesserwan; Robert Sokolic; Edward W. Cowen; Elizabeth Garabedian; Kerstin Heselmeyer-Haddad; Chyi-Chia Richard Lee; Stefania Pittaluga; Clarymar Ortiz; Kristin Baird; Dolores Lopez-Terrada; Julia Bridge; Alan S. Wayne; Fabio Candotti

    BackgroundDermatofibrosarcoma protuberans (DFSP) is a rare malignant skin tumor associated with a characteristic chromosomal translocation (t(17;22)(q22;q13)), resulting in the COL1A1-PDGFB fusion gene. This malignancy is rarely diagnosed in childhood. We observed a high incidence of this tumor in children affected with severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID)

  20. Cognitive and behavioral abnormalities in adenosine deaminase deficient severe combined immunodeficiency

    Microsoft Academic Search

    Mary Haslinger Rogers; Rebekah Lwin; Lynette Fairbanks; Bert Gerritsen; Hubert B. Gaspar

    2001-01-01

    Objective: The objective was to evaluate the cognitive, behavioral, and neurodevelopmental function in patients with adenosine deaminase deficient severe combined immunodeficiency (ADA-SCID) and to compare the findings with those of a case control group of patients without ADA-SCID. Study design: Case-matched pairs of patients with ADA-SCID (n = 11) and patients without ADA-SCID who had undergone bone marrow transplantation were

  1. Novel metabolic aspects related to adenosine deaminase inhibition in a human astrocytoma cell line.

    PubMed

    Garcia-Gil, Mercedes; Tozzi, Maria Grazia; Allegrini, Simone; Folcarelli, Serena; Della Sala, Grazia; Voccoli, Vladimir; Colombaioni, Laura; Camici, Marcella

    2012-04-01

    Adenosine deaminase, which catalyzes the deamination of adenosine and deoxyadenosine, plays a central role in purine metabolism. Indeed, its deficiency is associated with severe immunodeficiency and abnormalities in the functioning of many organs, including nervous system. We have mimicked an adenosine deaminase-deficient situation by incubating a human astrocytoma cell line in the presence of deoxycoformycin, a strong adenosine deaminase inhibitor, and deoxyadenosine, which accumulates in vivo when the enzyme is deficient, and have monitored the effect of the combination on cell viability, mitochondrial functions, and other metabolic features. Astrocytomas are the most common neoplastic transformations occurring in glial cell types, often characterized by a poor prognosis. Our experimental approach may provide evidence both for the response to a treatment affecting purine metabolism of a tumor reported to be particularly resistant to chemotherapeutic approaches and for the understanding of the molecular basis of neurological manifestations related to errors in purine metabolism. Cells incubated in the presence of the combination, but not those incubated with deoxyadenosine or deoxycoformycin alone, underwent apoptotic death, which appears to proceed through a mitochondrial pathway, since release of cytochrome c has been observed. The inhibition of adenosine deaminase increases both mitochondrial reactive oxygen species level and mitochondrial mass. A surprising effect of the combination is the significant reduction in lactate production, suggestive of a reduced glycolytic capacity, not ascribable to alterations in NAD?/NADH ratio, nor to a consumption of inorganic phosphate. This is a hitherto unknown effect presenting early during the incubation with deoxyadenosine and deoxycoformycin, which precedes their effect on cell viability. PMID:22353632

  2. Insulin-dependent diabetes mellitus and severe atopic dermatitis in a child with adenosine deaminase deficiency

    Microsoft Academic Search

    L. D. Notarangelo; G. Stoppoloni; R. Toraldo; E. Mazzolari; A. Coletta; P. Airò; C. Bordignon; A. G. Ugazio

    1992-01-01

    We report a 2.3-year-old girl with complete lack of adenosine deaminase (ADA) activity who presented with severe atopic dermatitis and insulin-dependent diabetes mellitus but only mild recurrent infections. Abnormalities of immune function included profound depletion of CD8+ lymphocytes, hyperimmunoglobulinaemia E, and very low in vitro proliferative response to mitogens. Treatment with polyethylene glycol-conjugated ADA was followed by rapid amelioration of

  3. Involvement of adenosine deaminase and adenosine kinase in regulating extracellular adenosine concentration in rat hippocampal slices

    Microsoft Academic Search

    H. G. E. Lloyd; B. B. Fredholm

    1995-01-01

    In this study the relative importance of adenosine deaminase and adenosine kinase in regulating extracellular adenosine concentration was investigated in rat hippocampal slices labelled with [3H]-adenine. The release of [3H]-purines evoked by electrical stimulation or energy depletion (oxygen and glucose deprivation) was measured and, using high-performance liquid chromatography (HPLC), the proportion of [3H]-label in the form of [3H]-adenosine, [3H]-inosine and

  4. Methylthioadenosine deaminase in an alternative quorum sensing pathway in Pseudomonas aeruginosa.

    PubMed

    Guan, Rong; Ho, Meng-Chiao; Fröhlich, Richard F G; Tyler, Peter C; Almo, Steven C; Schramm, Vern L

    2012-11-13

    Pseudomonas aeruginosa possesses an unusual pathway for 5'-methylthioadenosine (MTA) metabolism involving deamination to 5'-methylthioinosine (MTI) followed by N-ribosyl phosphorolysis to hypoxanthine and 5-methylthio-?-d-ribose 1-phosphate. The specific MTI phosphorylase of P. aeruginosa has been reported [Guan, R., Ho, M. C., Almo, S. C., and Schramm, V. L. (2011) Biochemistry 50, 1247-1254], and here we characterize MTA deaminase from P. aeruginosa (PaMTADA). Genomic analysis indicated the PA3170 locus to be a candidate for MTA deaminase (MTADA). Protein encoded by PA3170 was expressed and shown to deaminate MTA with 40-fold greater catalytic efficiency for MTA than for adenosine. The k(cat)/K(m) value of 1.6 × 10(7) M(-1) s(-1) for MTA is the highest catalytic efficiency known for an MTA deaminase. 5'-Methylthiocoformycin (MTCF) is a 4.8 pM transition state analogue for PaMTADA but causes no significant inhibition of human adenosine deaminase or MTA phosphorylase. MTCF is permeable to P. aeruginosa and exhibits an IC(50) of 3 nM on cellular PaMTADA activity. PaMTADA is the only activity in P. aeruginosa extracts to act on MTA. MTA and 5-methylthio-?-d-ribose are involved in quorum sensing pathways; thus, PaMTADA is a potential target for quorum sensing. The crystal structure of PaMTADA in complex with MTCF shows the transition state mimic 8(R)-hydroxyl group in contact with a catalytic site Zn(2+), the 5'-methylthio group in a hydrophobic pocket, and the transition state mimic of the diazepine ring in contact with a catalytic site Glu. PMID:23050701

  5. Promotion of plant growth by ACC deaminase-producing soil bacteria

    Microsoft Academic Search

    Bernard R. Glick; Zhenyu Cheng; Jennifer Czarny; Jin Duan

    2007-01-01

    Plant growth-promoting bacteria that contain the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase facilitate plant\\u000a growth and development by decreasing plant ethylene levels, especially following a variety of environmental stresses. In this\\u000a review, the physiological basis for this growth-promotion effect is examined in some detail. In addition, models are presented\\u000a that endeavour to explain (i) the seemingly paradoxical effects of ethylene on a

  6. Stable loop in the crystal structure of the intercalated four-stranded cytosine-rich metazoan telomere

    NASA Technical Reports Server (NTRS)

    Kang, C.; Berger, I.; Lockshin, C.; Ratliff, R.; Moyzis, R.; Rich, A.

    1995-01-01

    In most metazoans, the telomeric cytosine-rich strand repeating sequence is d(TAACCC). The crystal structure of this sequence was solved to 1.9-A resolution. Four strands associate via the cytosine-containing parts to form a four-stranded intercalated structure held together by C.C+ hydrogen bonds. The base-paired strands are parallel to each other, and the two duplexes are intercalated into each other in opposite orientations. One TAA end forms a highly stabilized loop with the 5' thymine Hoogsteen-base-paired to the third adenine. The 5' end of this loop is in close proximity to the 3' end of one of the other intercalated cytosine strands. Instead of being entirely in a DNA duplex, this structure suggests the possibility of an alternative conformation for the cytosine-rich telomere strands.

  7. Biosynthesis of riboflavin: characterization of the bifunctional deaminase-reductase of Escherichia coli and Bacillus subtilis.

    PubMed Central

    Richter, G; Fischer, M; Krieger, C; Eberhardt, S; Lüttgen, H; Gerstenschläger, I; Bacher, A

    1997-01-01

    The ribG gene at the 5' end of the riboflavin operon of Bacillus subtilis and a reading frame at 442 kb on the Escherichia coli chromosome (subsequently designated ribD) show similarity with deoxycytidylate deaminase and with the RIB7 gene of Saccharomyces cerevisiae. The ribG gene of B. subtilis and the ribD gene of E. coli were expressed in recombinant E. coli strains and were shown to code for bifunctional proteins catalyzing the second and third steps in the biosynthesis of riboflavin, i.e., the deamination of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (deaminase) and the subsequent reduction of the ribosyl side chain (reductase). The recombinant proteins specified by the ribD gene of E. coli and the ribG gene of B. subtilis were purified to homogeneity. NADH as well as NADPH can be used as a cosubstrate for the reductase of both microorganisms under study. Expression of the N-terminal or C-terminal part of the RibG protein yielded proteins with deaminase or reductase activity, respectively; however, the truncated proteins were rather unstable. PMID:9068650

  8. Occurrence of a catabolic L-serine (L-threonine) deaminase in Saccharomyces cerevisiae.

    PubMed

    Ramos, F; Wiame, J M

    1982-04-01

    Saccharomyces cerevisiae mutants lacking the anabolic L-threonine deaminase, the ilv1- mutants, have been found to exhibit a normal ability to grow, without auxotrophy towards isoleucine, on L-threonine of L-serine as only nitrogen nutrient. Starting from a strain carrying a ilv1- mutation, a new mutation affecting the ability to utilize L-threonine as nitrogen source was selected. This mutation, which also impairs the ability to utilize L-serine, has been denominated cha-, for 'catabolism of hydroxyamino acids' and was found to result in the lack of a catabolic L-serine (L-threonine) deaminase. This enzyme which, unlike the anabolic threonine deaminase, is more active towards serine than towards threonine, differs from the latter enzyme by a number of biochemical and regulatory properties. Whereas the anabolic enzyme is an allosteric enzyme sensitive to feedback inhibition by isoleucine, the catabolic enzyme exhibits Michaelian kinetics: no control of its activity has been detected. Its synthesis is induced by L-serine and L-threonine. These two enzymes, which thus can be easily differentiated by means of their regulations, display a limited ability to compensate for one another's absence and appear to play clearly distinct roles under normal physiological conditions. PMID:7042346

  9. Increases in interstitial adenosine and cerebral blood flow with inhibition of adenosine kinase and adenosine deaminase.

    PubMed

    Sciotti, V M; Van Wylen, D G

    1993-03-01

    The purpose of this study was to determine the changes in interstitial fluid (ISF) adenosine and cerebral blood flow (CBF) during inhibition of adenosine kinase or adenosine deaminase. Brain microdialysis was used to (a) measure CBF (H2 clearance), (b) sample cerebral ISF, and (c) deliver drugs locally to the brain. Microdialysis probes were implanted bilaterally in the caudate nucleus of halothane-anesthetized rats (n = 11). One probe was perfused with artificial cerebrospinal fluid (CSF) containing iodotubercidin (IODO), an adenosine kinase inhibitor, while the other probe was perfused with erythro-2-(2-hydroxy-3-nonyl)adenine (EHNA), an adenosine deaminase inhibitor. Both probes were subsequently perfused with EHNA+IODO. Finally, 8-(p-sulfophenyl)theophylline (SPT), an adenosine receptor antagonist, was added to EHNA + IODO in one probe, while the other probe continued to receive only EHNA + IODO. CBF and dialysate adenosine levels increased with either EHNA or IODO; however, the increases were greater with IODO. EHNA + IODO further increased CBF and dialysate adenosine. The hyperemia observed with EHNA + IODO was abolished by adenosine receptor blockade. These data suggest that basal adenosine levels are influenced to a greater extent by adenosine kinase than by adenosine deaminase. In addition, the increased CBF observed with inhibition of adenosine metabolism and the attenuation of this vasodilatory response with adenosine receptor blockade support a role for adenosine in CBF regulation. PMID:8436611

  10. Cytosine-specific DNA modification interferes with plasmid establishment in Escherichia coli K12: Involvement of rgl B

    Microsoft Academic Search

    Mario Noyer-Weidner; Ramon Diaz; Luzia Reiners

    1986-01-01

    Several chimeric pBR322\\/328 derivatives containing genes for cytosine-specific DNA methyltransferases (Mtases) can be transformed into the Escherichia coli K12\\/E. coli B hybrid strains HB101 and RR1 but not into other commonly used E. coli K12 strains. In vitro methylation of cytosine residues in pBR328 and other unrelated plasmids also reduces their potential to transform such methylation sensitive strains, albeit to

  11. Thiocytosine as a radiation energy trap in a single crystal of cytosine hydrochloride.

    PubMed

    Herak, J N; Sankovic, K; Hütterman, J

    1994-07-01

    Single crystals of cytosine hydrochloride with thiocytosine as an impurity were found suitable for the study of a possible new mechanism of long-range migration of energy deposited by ionizing radiation. In a crystal containing thiocytosine, two kinds of chlorine-containing paramagnetic centres are present that are completely absent in the pure cytosine. HCl crystals irradiated under the same condition. The thiocytosine molecules in conjunction with Cl- ions behave as hole traps. The centres have been characterized by EPR spectroscopy. One of the centres is derived from the cationic thiocytosine radical by interaction with a Cl- ion, and the other centre is formed by interaction of Cl. with a thiocytosine molecule. It is suggested that the transfer of an electron-loss site (a hole) in the Cl- network is the actual mechanism of the long-range energy transfer. PMID:8027610

  12. Understanding the disorder of the DNA base cytosine on the Au(111) surface.

    PubMed

    Kelly, Ross E A; Lukas, Maya; Kantorovich, Lev N; Otero, Roberto; Xu, Wei; Mura, Manuela; Laegsgaard, Erik; Stensgaard, Ivan; Besenbacher, Flemming

    2008-11-14

    Using ultrahigh vacuum scanning tunneling microscopy (STM) and ab initio density functional theory, we have investigated in detail structures formed by cytosine on the Au(111) surface in clean ultrahigh vacuum conditions. In spite of the fact that the ground state of this DNA base on the surface is shown to be an ordered arrangement of cytosine one-dimensional branches (filaments), this structure has never been observed in our STM experiments. Instead, disordered structures are observed, which can be explained by only a few elementary structural motifs: filaments, five- and sixfold rings, which randomly interconnect with each other forming bent chains, T junctions, and nanocages. The latter may have trapped smaller structures inside. The formation of such an unusual assembly is explained by simple kinetic arguments as a liquid-glass transition. PMID:19045423

  13. Overcoming Transcription Activator-like Effector (TALE) DNA Binding Domain Sensitivity to Cytosine Methylation*?

    PubMed Central

    Valton, Julien; Dupuy, Aurélie; Daboussi, Fayza; Thomas, Séverine; Maréchal, Alan; Macmaster, Rachel; Melliand, Kevin; Juillerat, Alexandre; Duchateau, Philippe

    2012-01-01

    Within the past 2 years, transcription activator-like effector (TALE) DNA binding domains have emerged as the new generation of engineerable platform for production of custom DNA binding domains. However, their recently described sensitivity to cytosine methylation represents a major bottleneck for genome engineering applications. Using a combination of biochemical, structural, and cellular approaches, we were able to identify the molecular basis of such sensitivity and propose a simple, drug-free, and universal method to overcome it. PMID:23019344

  14. Solution structures of oligonucleotides containing either a guanine or a cytosine in front of a gap of one nucleotide

    NASA Astrophysics Data System (ADS)

    Boulard, Y.; Faibis, V.; Fazakerley, G. V.

    1999-10-01

    We report NMR and molecular modelling studies on two DNA duplexes containing a gap of one nucleotides. The difference between the two oligonucleotides lies in the central base face to the gap, a guanine or a cytosine. For the gapG, we observed in solution a B-form conformation where the guanine stacks in the helix. For the gapC, we reveal the existence of two species, one majority where the cytosine is inside the helix and a second for which the cytosine is extrahelical. Nous présentons une étude par RMN et modélisation moléculaire sur deux duplexes d'ADN contenant une lacune de un nucléotide. La différence entre les deux oligonucléotides réside dans la base centrale en face de la lacune, une guanine ou une cytosine. Pour le duplex appelé gapG, nous observons en solution une hélice de type B dans laquelle la guanine est empilée à l'intérieur de l'hélice. Dans le cas du duplex gapC, nous montrons l'existence de deux formes, l'une où la cytosine est à l'intérieur de l'hélice; la seconde où la cytosine est extra hélicale.

  15. Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana.

    PubMed Central

    Finnegan, E J; Dennis, E S

    1993-01-01

    A plant cytosine methyltransferase cDNA was isolated using degenerate oligonucleotides, based on homology between prokaryote and mouse methyltransferases, and PCR to amplify a short fragment of a methyltransferase gene. A fragment of the predicted size was amplified from genomic DNA from Arabidopsis thaliana. Overlapping cDNA clones, some with homology to the PCR amplified fragment, were identified and sequenced. The assembled nucleic acid sequence is 4720 bp and encodes a protein of 1534 amino acids which has significant homology to prokaryote and mammalian cytosine methyltransferases. Like mammalian methylases, this enzyme has a C terminal methyltransferase domain linked to a second larger domain. The Arabidopsis methylase has eight of the ten conserved sequence motifs found in prokaryote cytosine-5 methyltransferases and shows 50% homology to the murine enzyme in the methyltransferase domain. The amino terminal domain is only 24% homologous to the murine enzyme and lacks the zinc binding region that has been found in methyltransferases from both mouse and man. In contrast to mouse where a single methyltransferase gene has been identified, a small multigene family with homology to the region amplified in PCR has been identified in Arabidopsis thaliana. Images PMID:8389441

  16. Accidental amplification and inactivation of a methyltransferase gene eliminates cytosine methylation in Mycosphaerella graminicola.

    PubMed

    Dhillon, Braham; Cavaletto, Jessica R; Wood, Karl V; Goodwin, Stephen B

    2010-09-01

    A de novo search for repetitive elements in the genome sequence of the wheat pathogen Mycosphaerella graminicola identified a family of repeats containing a DNA cytosine methyltransferase sequence (MgDNMT). All 23 MgDNMT sequences identified carried signatures of repeat induced point mutation (RIP). All copies were subtelomeric in location except for one on chromosome 6. Synteny with M. fijiensis implied that the nontelomeric copy on chromosome 6 served as a template for subsequent amplifications. Southern analysis revealed that the MgDNMT sequence also was amplified in 15 additional M. graminicola isolates from various geographical regions. However, this amplification event was specific to M. graminicola; a search for MgDNMT homologs identified only a single, unmutated copy in the genomes of 11 other ascomycetes. A genome-wide methylation assay revealed that M. graminicola lacks cytosine methylation, as expected if its MgDNMT gene is inactivated. Methylation was present in several other species tested, including the closest known relatives of M. graminicola, species S1 and S2. Therefore, the observed changes most likely occurred within the past 10,500 years since the divergence between M. graminicola and S1. Our data indicate that the recent amplification of a single-copy MgDNMT gene made it susceptible to RIP, resulting in complete loss of cytosine methylation in M. graminicola. PMID:20610411

  17. A New Nuclear Function of the Entamoeba histolytica Glycolytic Enzyme Enolase: The Metabolic Regulation of Cytosine-5 Methyltransferase 2 (Dnmt2) Activity

    PubMed Central

    Tovy, Ayala; Siman Tov, Rama; Gaentzsch, Ricarda; Helm, Mark; Ankri, Serge

    2010-01-01

    Cytosine-5 methyltransferases of the Dnmt2 family function as DNA and tRNA methyltransferases. Insight into the role and biological significance of Dnmt2 is greatly hampered by a lack of knowledge about its protein interactions. In this report, we address the subject of protein interaction by identifying enolase through a yeast two-hybrid screen as a Dnmt2-binding protein. Enolase, which is known to catalyze the conversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP), was shown to have both a cytoplasmatic and a nuclear localization in the parasite Entamoeba histolytica. We discovered that enolase acts as a Dnmt2 inhibitor. This unexpected inhibitory activity was antagonized by 2-PG, which suggests that glucose metabolism controls the non-glycolytic function of enolase. Interestingly, glucose starvation drives enolase to accumulate within the nucleus, which in turn leads to the formation of additional enolase-E.histolytica DNMT2 homolog (Ehmeth) complex, and to a significant reduction of the tRNAAsp methylation in the parasite. The crucial role of enolase as a Dnmt2 inhibitor was also demonstrated in E.histolytica expressing a nuclear localization signal (NLS)-fused-enolase. These results establish enolase as the first Dnmt2 interacting protein, and highlight an unexpected role of a glycolytic enzyme in the modulation of Dnmt2 activity. PMID:20174608

  18. Yeast expression platforms

    Microsoft Academic Search

    Erik Böer; Gerhard Steinborn; Gotthard Kunze; Gerd Gellissen

    2007-01-01

    Yeasts provide attractive expression platforms. They combine ease of genetic manipulations and the option for a simple fermentation\\u000a design of a microbial organism with the capabilities of an eukaryotic organism to secrete and to modify a protein according\\u000a to a general eukaryotic scheme. For platform applications, a range of yeast species has been developed during the last decades.\\u000a We present

  19. Nitrile Metabolizing Yeasts

    NASA Astrophysics Data System (ADS)

    Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

    Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing enzymes of yeasts.

  20. Forces in yeast flocculation.

    PubMed

    El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Vincent, Stéphane P; Abellán Flos, Marta; Hols, Pascal; Lipke, Peter N; Dufrêne, Yves F

    2015-02-01

    In the baker's yeast Saccharomyces cerevisiae, cell-cell adhesion ("flocculation") is conferred by a family of lectin-like proteins known as the flocculin (Flo) proteins. Knowledge of the adhesive and mechanical properties of flocculins is important for understanding the mechanisms of yeast adhesion, and may help controlling yeast behaviour in biotechnology. We use single-molecule and single-cell atomic force microscopy (AFM) to explore the nanoscale forces engaged in yeast flocculation, focusing on the role of Flo1 as a prototype of flocculins. Using AFM tips labelled with mannose, we detect single flocculins on Flo1-expressing cells, showing they are widely exposed on the cell surface. When subjected to force, individual Flo1 proteins display two distinct force responses, i.e. weak lectin binding forces and strong unfolding forces reflecting the force-induced extension of hydrophobic tandem repeats. We demonstrate that cell-cell adhesion bonds also involve multiple weak lectin interactions together with strong unfolding forces, both associated with Flo1 molecules. Single-molecule and single-cell data correlate with microscale cell adhesion behaviour, suggesting strongly that Flo1 mechanics is critical for yeast flocculation. These results favour a model in which not only weak lectin-sugar interactions are involved in yeast flocculation but also strong hydrophobic interactions resulting from protein unfolding. PMID:25515338

  1. Forces in yeast flocculation

    NASA Astrophysics Data System (ADS)

    El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Vincent, Stéphane P.; Abellán Flos, Marta; Hols, Pascal; Lipke, Peter N.; Dufrêne, Yves F.

    2015-01-01

    In the baker's yeast Saccharomyces cerevisiae, cell-cell adhesion (``flocculation'') is conferred by a family of lectin-like proteins known as the flocculin (Flo) proteins. Knowledge of the adhesive and mechanical properties of flocculins is important for understanding the mechanisms of yeast adhesion, and may help controlling yeast behaviour in biotechnology. We use single-molecule and single-cell atomic force microscopy (AFM) to explore the nanoscale forces engaged in yeast flocculation, focusing on the role of Flo1 as a prototype of flocculins. Using AFM tips labelled with mannose, we detect single flocculins on Flo1-expressing cells, showing they are widely exposed on the cell surface. When subjected to force, individual Flo1 proteins display two distinct force responses, i.e. weak lectin binding forces and strong unfolding forces reflecting the force-induced extension of hydrophobic tandem repeats. We demonstrate that cell-cell adhesion bonds also involve multiple weak lectin interactions together with strong unfolding forces, both associated with Flo1 molecules. Single-molecule and single-cell data correlate with microscale cell adhesion behaviour, suggesting strongly that Flo1 mechanics is critical for yeast flocculation. These results favour a model in which not only weak lectin-sugar interactions are involved in yeast flocculation but also strong hydrophobic interactions resulting from protein unfolding.

  2. Eur J Immunol. Author manuscript Splenic marginal zone B cells in humans: where do they mutate their Ig

    E-print Network

    Boyer, Edmond

    + + + ü studied the expression of the enzyme activation-induced cytidine deaminase (AID) in human spleen ; immunology ; Cytidine Deaminase ; Cytosine Deaminase ; immunology ; metabolism ; Humans ; Immunoglobulins linked with pathological situations (immunodeficiency or auto-immunity) , . In AID-deficient patients

  3. Isolation and characterization of ACC deaminase-producing fluorescent pseudomonads, to alleviate salinity stress on canola (Brassica napus L.) growth.

    PubMed

    Jalili, Farzad; Khavazi, Kazem; Pazira, Ebrahim; Nejati, Alireza; Rahmani, Hadi Asadi; Sadaghiani, Hasan Rasuli; Miransari, Mohammad

    2009-04-01

    Salinity stress is of great importance in arid and semi-arid areas of the world due to its impact in reducing crop yield. Under salinity stress, the amount of 1-aminocyclopropane-1-carboxylate (ACC), a precursor for ethylene production in plants, increases. Here, we conducted research under the hypothesis that isolated ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida can alleviate the stressful effects of salinity on canola (Brassica napus L.) growth. The experiments were conducted in the Soil and Water Research Institute, Tehran, Iran. Seven experimental stages were conducted to isolate and characterize ACC deaminase-producing Pseudomonas fluorescens strains and to determine factors enhancing their growth and, consequently, their effects on the germination of canola seeds. Under salinity stress, in 14% of the isolates, ACC deaminase activity was observed, indicating that they were able to utilize ACC as the sole N-source. Bacterial strains differed in their ability to synthesize auxin and hydrogen cyanide compounds, as well as in their ACC deaminase activity. Under salinity stress, the rate of germinating seeds inoculated with the strains of ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida, and seedling growth was significantly higher. These results indicate the significance of soil biological activities, including the activities of plant growth-promoting bacteria, in the alleviation of soil stresses such as salinity on plant growth. PMID:18829132

  4. Identification of Two Pentatricopeptide Repeat Genes Required for RNA Editing and Zinc Binding by C-terminal Cytidine Deaminase-like Domains*

    PubMed Central

    Hayes, Michael L.; Giang, Karolyn; Berhane, Beniam; Mulligan, R. Michael

    2013-01-01

    Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins are required as RNA binding specificity determinants in the RNA editing mechanism. Bioinformatic analysis has shown that most of the Arabidopsis PPR proteins necessary for RNA editing events include a C-terminal portion that shares structural characteristics with a superfamily of deaminases. The DYW deaminase domain includes a highly conserved zinc binding motif that shares characteristics with cytidine deaminases. The Arabidopsis PPR genes, ELI1 and DOT4, both have DYW deaminase domains and are required for single RNA editing events in chloroplasts. The ELI1 DYW deaminase domain was expressed as a recombinant protein in Escherichia coli and was shown to bind two zinc atoms per polypeptide. Thus, the DYW deaminase domain binds a zinc metal ion, as expected for a cytidine deaminase, and is potentially the catalytic component of an editing complex. Genetic complementation experiments demonstrate that large portions of the DYW deaminase domain of ELI1 may be eliminated, but the truncated genes retain the ability to restore editing site conversion in a mutant plant. These results suggest that the catalytic activity can be supplied in trans by uncharacterized protein(s) of the editosome. PMID:24194514

  5. Yeast killer toxins and dimorphism.

    PubMed

    Polonelli, L; Conti, S; Campani, L; Morace, G; Fanti, F

    1989-06-01

    The differential action of four selected yeast killer toxins on the mycelial and yeast forms of four isolates of the dimorphic fungus Sporothrix schenckii was comparatively evaluated. The results confirmed that the yeast killer phenomenon is present among hyphomycetes and yeasts and that both morphological forms of S. schenckii are susceptible to the action of the same yeast killer toxin. Quantitative differences in the response to the killer action of the mycelial and yeast forms in individual strains were also observed. To avoid retroconversion of the dimorphic forms, we used a modification of the conventional killer system. PMID:2754015

  6. In vivo inactivation of erythrocyte S-adenosylhomocysteine hydrolase by 2'-deoxyadenosine in adenosine deaminase-deficient patients.

    PubMed Central

    Hershfield, M S; Kredich, N M; Ownby, D R; Ownby, H; Buckley, R

    1979-01-01

    The cytotoxic nucleoside 2'-deoxyadenosine is excreted in excessive amounts by individuals with genetic deficiency of adenosine deaminase, and may be in part responsible for the severe combined immune dysfunction from which they suffer. Earlier studies from this laboratory showed that 2'-deoxyadenosine causes the irreversible inactivation of the enzyme S-adenosylhomocysteine hydrolase by an active site-directed, "suicide-like" process. In this communication we have demonstrated similar inactivation of S-adenosylhomocysteine hydrolase in hemolysate and in intact erythrocytes, as well as a striking deficiency of S-adenosylhomocysteine hydrolase activity in the erythrocytes of three adenosine deaminase-deficient patients. In vivo suicide-like inactivation of S-adenosylhomocysteine hydrolase by 2'-deoxyadenosine may contribute to the cytotoxicity of 2'-deoxyadenosine and to the immune dysfunction in adenosine deaminase deficiency. PMID:312296

  7. Somatic hypermutation of human mitochondrial and nuclear DNA by APOBEC3 cytidine deaminases, a pathway for DNA catabolism

    PubMed Central

    Suspène, Rodolphe; Aynaud, Marie-Ming; Guétard, Denise; Henry, Michel; Eckhoff, Grace; Marchio, Agnès; Pineau, Pascal; Dejean, Anne; Vartanian, Jean-Pierre; Wain-Hobson, Simon

    2011-01-01

    The human APOBEC3 (A3A–A3H) locus encodes six cytidine deaminases that edit single-stranded DNA, the result being DNA peppered with uridine. Although several cytidine deaminases are clearly restriction factors for retroviruses and hepadnaviruses, it is not known if APOBEC3 enzymes have roles outside of these settings. It is shown here that both human mitochondrial and nuclear DNA are vulnerable to somatic hypermutation by A3 deaminases, with APOBEC3A standing out among them. The degree of editing is much greater in patients lacking the uracil DNA-glycolyase gene, indicating that the observed levels of editing reflect a dynamic composed of A3 editing and DNA catabolism involving uracil DNA-glycolyase. Nonetheless, hyper- and lightly mutated sequences went hand in hand, raising the hypothesis that recurrent low-level mutation by APOBEC3A could catalyze the transition from a healthy to a cancer genome. PMID:21368204

  8. Mapping Yeast Transcriptional Networks

    PubMed Central

    Hughes, Timothy R.; de Boer, Carl G.

    2013-01-01

    The term “transcriptional network” refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face. For most yeast TFs, data have been collected on their sequence preferences, in vivo promoter occupancy, and gene expression profiles in deletion mutants. These systematic studies have led to the identification of new regulators of numerous cellular functions and shed light on the overall organization of yeast gene regulation. However, many yeast TFs appear to be inactive under standard laboratory growth conditions, and many of the available data were collected using techniques that have since been improved. Perhaps as a consequence, comprehensive and accurate mapping among TF sequence preferences, promoter binding, and gene expression remains an open challenge. We propose that the time is ripe for renewed systematic efforts toward a complete mapping of yeast transcriptional regulatory mechanisms. PMID:24018767

  9. Significance of the D-serine-deaminase and D-serine metabolism of Staphylococcus saprophyticus for virulence.

    PubMed

    Korte-Berwanger, Miriam; Sakinc, Türkan; Kline, Kimberly; Nielsen, Hailyn V; Hultgren, Scott; Gatermann, Sören G

    2013-12-01

    Staphylococcus saprophyticus is the only species of Staphylococcus that is typically uropathogenic and possesses a gene coding for a D-serine-deaminase (DsdA). As D-serine is prevalent in urine and toxic or bacteriostatic to many bacteria, it is not surprising that the D-serine-deaminase gene is found in the genome of uropathogens. It has been suggested that D-serine-deaminase or the ability to respond to or to metabolize D-serine is important for virulence. For uropathogenic Escherichia coli (UPEC), a high intracellular D-serine concentration affects expression of virulence factors. S. saprophyticus is able to grow in the presence of high D-serine concentrations; however, its D-serine metabolism has not been described. The activity of the D-serine-deaminase was verified by analyzing the formation of pyruvate from D-serine in different strains with and without D-serine-deaminase. Cocultivation experiments were performed to show that D-serine-deaminase confers a growth advantage to S. saprophyticus in the presence of D-serine. Furthermore, in vivo coinfection experiments showed a disadvantage for the ?dsdA mutant during urinary tract infection. Expression analysis of known virulence factors by reverse transcription-quantitative PCR (RT-qPCR) showed that the surface-associated lipase Ssp is upregulated in the presence of D-serine. In addition, we show that S. saprophyticus is able to use D-serine as the sole carbon source, but interestingly, D-serine had a negative effect on growth when glucose was also present. Taken together, D-serine metabolism is associated with virulence in S. saprophyticus, as at least one known virulence factor is upregulated in the presence of D-serine and a ?dsdA mutant was attenuated in virulence murine model of urinary tract infection. PMID:24082071

  10. Presence of Escherichia coli of a deaminase and a reductase involved in biosynthesis of riboflavin.

    PubMed Central

    Burrows, R B; Brown, G M

    1978-01-01

    Two enzymes have been partially purified from extracts of Escherchia coli B which together catalyze the conversion of the product of the action of GTP cyclohydrolase II, 2,5-diamino-6-oxy-4-(5'-phosphoribosylamine)pyrimidine, to 5-amino-2,6-dioxy-4-(5'-phosphoribitylamine)pyrimidine. These two compounds are currently thought to be intermediates in the biosynthesis of riboflavin. The enzymatic conversion occurs in two steps. The product of the action of GTP cyclohydrolase II first undergoes hydrolytic deamination at carbon 2 of the ring, followed by reduction of the ribosylamino group to a ribitylamino group. The enzyme which catalyzes the first step, herein called the "deaminase," has been purified 200-fold. The activity was assayed by detecting the conversion of the product of the reaction catalyzed by GTP cyclohydrolase II to a compound which reacts with butanedione to form 6,7-dimethyllumazine. The enzyme has a molecular weight of approximately 80,000 and a pH optimum of 9.1. The dephosphorylated form of the substrate is not deaminated in the presence of the enzyme. The assay for the enzyme which catalyzes the second step, referred to here as the "reductase," involves the detection of the conversion of the product of the deaminase-catalyzed reaction to a compound which, after treatment with alkaline phosphatase, reacts with butanedione to form 6,7-dimethyl-8-ribityllumazine. The reductase has a molecular weight of approximately 40,000 and a pH optimum of 7.5. Like the deaminase, the reductase does not act on the dephosphorylated form of its substrate. Reduced nicotinamide adenine dinucleotide phosphate is required as a cofactor; reduced nicotinamide adenine dinucleotide can be used about 30% as well as the phosphate form. The activity of neither enzyme is inhibited by riboflavin, FMN, or flavine adenine dinucleotide. PMID:30756

  11. Evolutionary history of Ascomyceteous Yeasts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yeasts are important for many industrial and biotechnological processes and show remarkable diversity despite morphological similarities. We have sequenced the genomes of 20 ascomyceteous yeasts of taxonomic and industrial importance including members of Saccharomycotina and Taphrinomycotina. A comp...

  12. Gene therapy for severe combined immunodeficiency due to adenosine deaminase deficiency.

    PubMed

    Montiel-Equihua, Claudia A; Thrasher, Adrian J; Gaspar, H Bobby

    2012-02-01

    The severe combined immunodeficiency caused by the absence of adenosine deaminase (SCID-ADA) was the first monogenic disorder for which gene therapy was developed. Over 30 patients have been treated worldwide using the current protocols, and most of them have experienced clinical benefit; importantly, in the absence of any vector-related complications. In this document, we review the progress made so far in the development and establishment of gene therapy as an alternative form of treatment for ADA-SCID patients. PMID:22348551

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

    NASA Technical Reports Server (NTRS)

    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.

  14. Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: A model for gene therapy

    SciTech Connect

    Lynch, C.M.; Miller, A.D. (Fred Hutchinson Cancer Research Center, Seattle, WA (United States)); Clowes, M.M.; Osborne, W.R.A.; Clowes, A.W. (Univ. of Washington, Seattle (United States))

    1992-02-01

    Gene transfer into vascular smooth muscle cells in animals was examined by using recombinant retroviral vectors containing an Escherichia coli {beta}-galactosidase gene or a human adenosine deaminase gene. Direct gene transfer by infusion of virus into rat carotid arteries was not observed. However, gene transfer by infection of smooth muscle cells in culture and seeding of the transduced cells onto arteries that had been denuded of endothelial cells was successful. Potentially therapeutic levels of human adenosine deaminase activity were detected over 6 months of observation, indicating the utility of vascular smooth muscle cells for gene therapy in humans.

  15. Long-Term Expression of Human Adenosine Deaminase in Vascular Smooth Muscle Cells of Rats: A Model for Gene Therapy

    NASA Astrophysics Data System (ADS)

    Lynch, Carmel M.; Clowes, Monika M.; Osborne, William R. A.; Clowes, Alexander W.; Dusty Miller, A.

    1992-02-01

    Gene transfer into vascular smooth muscle cells in animals was examined by using recombinant retroviral vectors containing an Escherichia coli ?-galactosidase gene or a human adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) gene. Direct gene transfer by infusion of virus into rat carotid arteries was not observed. However, gene transfer by infection of smooth muscle cells in culture and seeding of the transduced cells onto arteries that had been denuded of endothelial cells was successful. Potentially therapeutic levels of human adenosine deaminase activity were detected over 6 months of observation, indicating the utility of vascular smooth muscle cells for gene therapy in humans.

  16. Yeast killer systems.

    PubMed Central

    Magliani, W; Conti, S; Gerloni, M; Bertolotti, D; Polonelli, L

    1997-01-01

    The killer phenomenon in yeasts has been revealed to be a multicentric model for molecular biologists, virologists, phytopathologists, epidemiologists, industrial and medical microbiologists, mycologists, and pharmacologists. The surprisingly widespread occurrence of the killer phenomenon among taxonomically unrelated microorganisms, including prokaryotic and eukaryotic pathogens, has engendered a new interest in its biological significance as well as its theoretical and practical applications. The search for therapeutic opportunities by using yeast killer systems has conceptually opened new avenues for the prevention and control of life-threatening fungal diseases through the idiotypic network that is apparently exploited by the immune system in the course of natural infections. In this review, the biology, ecology, epidemiology, therapeutics, serology, and idiotypy of yeast killer systems are discussed. PMID:9227858

  17. Virtual Yeast Cell

    NSDL National Science Digital Library

    Learning about the various parts of a cell can be tricky business, but this virtual yeast cell offered by The University of Nottingham will come in handy for biology students and science instructors. This learning resource was created to help students in the brewing science program learn about yeast cytology, though just about anyone with an interest in cells will learn something from visiting the site. After entering the interactive cell, visitors can click on different parts of the cell (such as the cytoplasm or the nucleus) in order to learn more about the importance of each one. Visitors should remember that they can also download the virtual yeast cell and use it in the classroom or just with a group of friends.

  18. Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation

    PubMed Central

    Hashimoto, Hideharu; Liu, Yiwei; Upadhyay, Anup K.; Chang, Yanqi; Howerton, Shelley B.; Vertino, Paula M.; Zhang, Xing; Cheng, Xiaodong

    2012-01-01

    Cytosine residues in mammalian DNA occur in at least three forms, cytosine (C), 5-methylcytosine (M; 5mC) and 5-hydroxymethylcytosine (H; 5hmC). During semi-conservative DNA replication, hemi-methylated (M/C) and hemi-hydroxymethylated (H/C) CpG dinucleotides are transiently generated, where only the parental strand is modified and the daughter strand contains native cytosine. Here, we explore the role of DNA methyltransferases (DNMT) and ten eleven translocation (Tet) proteins in perpetuating these states after replication, and the molecular basis of their recognition by methyl-CpG-binding domain (MBD) proteins. Using recombinant proteins and modified double-stranded deoxyoligonucleotides, we show that DNMT1 prefers a hemi-methylated (M/C) substrate (by a factor of >60) over hemi-hydroxymethylated (H/C) and unmodified (C/C) sites, whereas both DNMT3A and DNMT3B have approximately equal activity on all three substrates (C/C, M/C and H/C). Binding of MBD proteins to methylated DNA inhibited Tet1 activity, suggesting that MBD binding may also play a role in regulating the levels of 5hmC. All five MBD proteins generally have reduced binding affinity for 5hmC relative to 5mC in the fully modified context (H/M versus M/M), though their relative abilities to distinguish the two varied considerably. We further show that the deamination product of 5hmC could be excised by thymine DNA glycosylase and MBD4 glycosylases regardless of context. PMID:22362737

  19. Some new reaction pathways for the formation of cytosine in interstellar space - A quantum chemical study

    NASA Astrophysics Data System (ADS)

    Gupta, V. P.; Tandon, Poonam; Mishra, Priti

    2013-03-01

    The detection of nucleic acid bases in carbonaceous meteorites suggests that their formation and survival is possible outside of the Earth. Small N-heterocycles, including pyrimidine, purines and nucleobases, have been extensively sought in the interstellar medium. It has been suggested theoretically that reactions between some interstellar molecules may lead to the formation of cytosine, uracil and thymine though these processes involve significantly high potential barriers. We attempted therefore to use quantum chemical techniques to explore if cytosine can possibly form in the interstellar space by radical-radical and radical-molecule interaction schemes, both in the gas phase and in the grains, through barrier-less or low barrier pathways. Results of DFT calculations for the formation of cytosine starting from some of the simple molecules and radicals detected in the interstellar space are being reported. Global and local descriptors such as molecular hardness, softness and electrophilicity, and condensed Fukui functions and local philicity indices were used to understand the mechanistic aspects of chemical reaction. The presence and nature of weak bonds in the molecules and transition states formed during the reaction process have been ascertained using Bader's quantum theory of atoms in molecules (QTAIMs). Two exothermic reaction pathways starting from propynylidyne (CCCH) and cyanoacetylene (HCCCN), respectively, have been identified. While the first reaction path is found to be totally exothermic, it involves a barrier of 12.5 kcal/mol in the gas phase against the lowest value of about 32 kcal/mol reported in the literature. The second path is both exothermic and barrier-less. The later has, therefore, a greater probability of occurrence in the cold interstellar clouds (10-50 K).

  20. Cytosine methylation is a conserved epigenetic feature found throughout the phylum Platyhelminthes

    PubMed Central

    2013-01-01

    Background The phylum Platyhelminthes (flatworms) contains an important group of bilaterian organisms responsible for many debilitating and chronic infectious diseases of human and animal populations inhabiting the planet today. In addition to their biomedical and veterinary relevance, some platyhelminths are also frequently used models for understanding tissue regeneration and stem cell biology. Therefore, the molecular (genetic and epigenetic) characteristics that underlie trophic specialism, pathogenicity or developmental maturation are likely to be pivotal in our continued studies of this important metazoan group. Indeed, in contrast to earlier studies that failed to detect evidence of cytosine or adenine methylation in parasitic flatworm taxa, our laboratory has recently defined a critical role for cytosine methylation in Schistosoma mansoni oviposition, egg maturation and ovarian development. Thus, in order to identify whether this epigenetic modification features in other platyhelminth species or is a novelty of S. mansoni, we conducted a study simultaneously surveying for DNA methylation machinery components and DNA methylation marks throughout the phylum using both parasitic and non-parasitic representatives. Results Firstly, using both S. mansoni DNA methyltransferase 2 (SmDNMT2) and methyl-CpG binding domain protein (SmMBD) as query sequences, we illustrate that essential DNA methylation machinery components are well conserved throughout the phylum. Secondly, using both molecular (methylation specific amplification polymorphism, MSAP) and immunological (enzyme-linked immunoabsorbent assay, ELISA) methodologies, we demonstrate that representative species (Echinococcus multilocularis, Protopolystoma xenopodis, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica and Polycelis nigra) within all four platyhelminth classes (Cestoda, Monogenea, Trematoda and ‘Turbellaria’) contain methylated cytosines within their genome compartments. Conclusions Collectively, these findings provide the first direct evidence for a functionally conserved and enzymatically active DNA methylation system throughout the Platyhelminthes. Defining how this epigenetic feature shapes phenotypic diversity and development within the phylum represents an exciting new area of metazoan biology. PMID:23837670

  1. Solvent effect on the anharmonic vibrational frequencies in guanine-cytosine base pair

    NASA Astrophysics Data System (ADS)

    Bende, A.; Muntean, C. M.

    2012-02-01

    We present an ab initio study of the vibrational properties of cytosine and guanine in the Watson-Crick and Hoogsteen base pair configurations. The results are obtained by considering the DFT method together with the Polarizable Continuum Model (PCM) using PBE and B3PW91 exchange-correlation functionals and triple-? valence basis set. We investigate the importance of anharmonic corrections for the vibrational modes taking into account the solvent effect of the water environment. In particular, the unusual anharmonic effect of the H+ vibration in the case of the Hoogsteen base pair configuration is discussed.

  2. Supramolecular hydrogen-bonding networks in cytosine salicylic acid hydrate (2 : 3 : 2) complex

    NASA Astrophysics Data System (ADS)

    Sridhar, B.; Ravikumar, K.

    2010-03-01

    Cytosine-cytosinium base pairs are interconnected by triple hydrogen bonds thereby resembling a pseudo-Watson-Crick pattern and generates two characteristic R {2/2}(8)-motifs. Both molecules of the salicylic acids interconnect the base pair and lead to the formation of one dimensional supramolecular hexameric tape along b-axis. This hexameric tape are sandwiched by the water molecules, one of the salicylic acid and salicylate anion which form one dimensional and two dimensional supramolecular hydrogen bonded networks in the crystal packing. Macrocylic rings of cavities are also noticed in the crystal structure.

  3. The Application of High-Level Iterative Coupled-Cluster Methods to the Cytosine Molecule

    SciTech Connect

    Kowalski, Karol; Valiev, Marat

    2008-06-19

    The need for inclusion higher-order correlation effects for adequate description of the excitation energies of the DNA bases became clear in the last few years. In particular, we demonstrated that there is a sizable effect of triply excited configurations estimated in a non-iterative manner on the coupled-cluster excitation energies of the cytosine molecule in DNA environment. In this paper we discuss the accuracies of the non-iterative methods for biologically relevant systems in realistic environment in comparison with interative formulations that explicitly include the effect of triply excited clusters.

  4. Cryopreservation of yeast cultures.

    PubMed

    Bond, Chris

    2007-01-01

    A method is described that allows a wide range of yeast species to be stored in liquid nitrogen while maintaining a high level of viability. Yeast cultures are sealed in commercially available polypropylene straws after having been mixed with a glycerol-based cryoprotectant. Once placed in a secondary cryotube the temperature of the sealed straws is reduced slowly to -30 degrees C in a methanol bath over a period of up to 3 h. The straws are then transferred directly to the liquid nitrogen and placed in a racking system for long-term storage. PMID:18080465

  5. Genetics of Yeasts

    NASA Astrophysics Data System (ADS)

    Querol, Amparo; Fernández-Espinar, M. Teresa; Belloch, Carmela

    The use of yeasts in biotechnology processes dates back to ancient days. Before 7000 BC, beer was produced in Sumeria. Wine was made in Assyria in 3500 BC, and ancient Rome had over 250 bakeries, which were making leavened bread by 100 BC. And milk has been made into Kefyr and Koumiss in Asia for many centuries (Demain, Phaff, & Kurtzman, 1999). However, the importance of yeast in the food and beverage industries was only realized about 1860, when their role in food manufacturing became evident.

  6. Base Flip in DNA Studied by Molecular Dynamics Simulations of Differently-Oxidized Forms of Methyl-Cytosine

    PubMed Central

    Helabad, Mahdi Bagherpoor; Kanaan, Natalia; Imhof, Petra

    2014-01-01

    Distortions in the DNA sequence, such as damage or mispairs, are specifically recognized and processed by DNA repair enzymes. Many repair proteins and, in particular, glycosylases flip the target base out of the DNA helix into the enzyme’s active site. Our molecular dynamics simulations of DNA with intact and damaged (oxidized) methyl-cytosine show that the probability of being flipped is similar for damaged and intact methyl-cytosine. However, the accessibility of the different 5-methyl groups allows direct discrimination of the oxidized forms. Hydrogen-bonded patterns that vary between methyl-cytosine forms carrying a carbonyl oxygen atom are likely to be detected by the repair enzymes and may thus help target site recognition. PMID:24995694

  7. Heat shock protein 70 (Hsp70)-stimulated deoxycytidine deaminases from a human lymphoma cell but not the activation-induced cytidine deaminase (AID) from Ramos 6.4 human Burkitt's lymphoma cells.

    PubMed

    Bases, Robert

    2011-01-01

    Deoxycytidine deaminase enzyme activity was reduced in lysates of human leukemic THP1 cells 24 h after transfection with siRNA designed to inhibit cell synthesis of heat shock protein 70 (Hsp70)1a and Hsp701b. The cytidine deaminase enzyme activity from the cell lysates was purified from an affinity column which contained bound single-stranded oligodeoxycytidylic acid. Deficient enzyme activity in certain elution fractions from the siRNA-transfected cells was restored by including recombinant HSP 70 in the assays. Enzyme activity in some other fractions was increased after siRNA transfection. Activation-induced cytidine deaminase (AID) is a central factor in the immune response. A more specific assay for AID was used to study the influence of Hsp70 on AID activity. Unlike Hsp70's ability to stimulate certain enzymes of DNA base excision repair and other cytidine deaminases, it had little effect on AID activity in vitro, or was weakly inhibitory. PMID:20680536

  8. Recognition of duplex RNA by the deaminase domain of the RNA editing enzyme ADAR2

    PubMed Central

    Phelps, Kelly J.; Tran, Kiet; Eifler, Tristan; Erickson, Anna I.; Fisher, Andrew J.; Beal, Peter A.

    2015-01-01

    Adenosine deaminases acting on RNA (ADARs) hydrolytically deaminate adenosines (A) in a wide variety of duplex RNAs and misregulation of editing is correlated with human disease. However, our understanding of reaction selectivity is limited. ADARs are modular enzymes with multiple double-stranded RNA binding domains (dsRBDs) and a catalytic domain. While dsRBD binding is understood, little is known about ADAR catalytic domain/RNA interactions. Here we use a recently discovered RNA substrate that is rapidly deaminated by the isolated human ADAR2 deaminase domain (hADAR2-D) to probe these interactions. We introduced the nucleoside analog 8-azanebularine (8-azaN) into this RNA (and derived constructs) to mechanistically trap the protein–RNA complex without catalytic turnover for EMSA and ribonuclease footprinting analyses. EMSA showed that hADAR2-D requires duplex RNA and is sensitive to 2?-deoxy substitution at nucleotides opposite the editing site, the local sequence and 8-azaN nucleotide positioning on the duplex. Ribonuclease V1 footprinting shows that hADAR2-D protects ?23 nt on the edited strand around the editing site in an asymmetric fashion (?18 nt on the 5? side and ?5 nt on the 3? side). These studies provide a deeper understanding of the ADAR catalytic domain–RNA interaction and new tools for biophysical analysis of ADAR–RNA complexes. PMID:25564529

  9. Kinetic and structural analysis of the inhibition of adenosine deaminase by acetaminophen.

    PubMed

    Ataie, G; Safarian, S; Divsalar, A; Saboury, A A; Moosavi-Movahedi, A A; Ranjbar, B; Cristalli, G; Mardanian, S

    2004-02-01

    Kinetic and thermodynamic studies have been made on the effect of acetaminophen on the activity and structure of adenosine deaminase in 50 mM sodium phosphate buffer pH 7.5, at two temperatures of 27 and 37 degrees C using UV spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. Acetaminophen acts as a competitive inhibitor at 27 degrees C (Ki = 126 microM) and an uncompetitive inhibitor at 37 degrees C (Ki = 214 microM). Circular dichroism studies do not show any considerable effect on the secondary structure of adenosine deaminase by increasing the temperature from 27 to 37 degrees C. However, the secondary structure of the protein becomes more compact at 37 degrees C in the presence of acetaminophen. Fluorescence spectroscopy studies show considerable change in the tertiary structure of the protein by increasing the temperature from 27 to 37 degrees C. Also, the fluorescence spectrum of the protein incubated with different concentrations of acetaminophen show different inhibition behaviors by the effector at the two temperatures. PMID:15202496

  10. The Role of Cytidine Deaminases on Innate Immune Responses against Human Viral Infections

    PubMed Central

    Vieira, Valdimara C.; Soares, Marcelo A.

    2013-01-01

    The APOBEC family of proteins comprises deaminase enzymes that edit DNA and/or RNA sequences. The APOBEC3 subgroup plays an important role on the innate immune system, acting on host defense against exogenous viruses and endogenous retroelements. The role of APOBEC3 proteins in the inhibition of viral infection was firstly described for HIV-1. However, in the past few years many studies have also shown evidence of APOBEC3 action on other viruses associated with human diseases, including HTLV, HCV, HBV, HPV, HSV-1, and EBV. APOBEC3 inhibits these viruses through a series of editing-dependent and independent mechanisms. Many viruses have evolved mechanisms to counteract APOBEC effects, and strategies that enhance APOBEC3 activity constitute a new approach for antiviral drug development. On the other hand, novel evidence that editing by APOBEC3 constitutes a source for viral genetic diversification and evolution has emerged. Furthermore, a possible role in cancer development has been shown for these host enzymes. Therefore, understanding the role of deaminases on the immune response against infectious agents, as well as their role in human disease, has become pivotal. This review summarizes the state-of-the-art knowledge of the impact of APOBEC enzymes on human viruses of distinct families and harboring disparate replication strategies. PMID:23865062

  11. Ab Initio ONIOM-Molecular Dynamics (MD) Study on the Deamination Reaction by Cytidine Deaminase

    SciTech Connect

    Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

    2007-08-23

    We applied the ONIOM-molecular dynamics (MD) method to the hydrolytic deamination of cytidine by cytidine deaminase, which is an essential step of the activation process of the anticancer drug inside the human body. The direct MD simulations were performed for the realistic model of cytidine deaminase calculating the energy and its gradient by the ab initio ONIOM method on the fly. The ONIOM-MD calculations including the thermal motion show that the neighboring amino acid residue is an important factor of the environmental effects and significantly affects not only the geometry and energy of the substrate trapped in the pocket of the active site but also the elementary step of the catalytic reaction. We successfully simulate the second half of the catalytic cycle, which has been considered to involve the rate-determining step, and reveal that the rate-determing step is the release of the NH3 molecule. TM and MA were supported in part 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.

  12. [Value of assaying adenosine deaminase level in patients with neuromeningeal tuberculosis].

    PubMed

    Janvier, F; Servonnet, A; Delacour, H; Fontan, E; Ceppa, F; Burnat, P

    2010-02-01

    Neuromeningeal tuberculosis is a rare extrapulmonary location in France. Delayed diagnosis can lead to therapeutic failure and severe sequels. However early diagnosis is a major challenge that requires the proper epidemiological, clinical, radiological and biological resources. Problems related to diagnosis of mycobacteria infection and to shortcomings in certain healthcare systems can hinder early diagnosis. The purpose of this review was to describe the diagnostic value of assaying adenosine deaminase activity in cerebrospinal fluid from patients with neuromeningeal tuberculosis. Evidence from studies published over the last 25 years indicate that the sensitivity and specificity of measuring adenosine deaminase activity range from 36 to 92% and 71 to 100% respectively depending of cutoff values used. Before performing this assay, it is necessary to rule out obvious or frequent etiologies such as purulent bacterial meningitis or cryptococcosis in HIV patients. Taken together these studies show that this simple, inexpensive technique is a valuable tool for early diagnosis and management of tuberculosis patients and that it can be easily implemented in hospital labs regardless of technical or financial resources. PMID:20337125

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

    PubMed

    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 ?-ketobutyrate mg(-1) ?h(-1) ) and indole acetic acid (IAA; ranging from 10.54 to 37.65??M?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. PMID:23681643

  14. Rapid degradation of Pseudomonas fluorescens 1-aminocyclopropane-1-carboxylic acid deaminase proteins expressed in transgenic Arabidopsis.

    PubMed

    Kim, Kangmin; Park, Sung-Hee; Chae, Jong-Chan; Soh, Byoung Yul; Lee, Kui-Jae

    2014-06-01

    1-Aminocyclopropane-1-carboxylate (ACC) deaminase is commonly produced by plant growth-promoting rhizobacteria (PGPR) and has been suggested to facilitate the growth and stress tolerance of hosts via a reduction in levels of ethylene. However, the regulatory mechanism of ACC deaminase (AcdS) protein within host plant cells is largely unknown. Here, we demonstrated beneficial effects and post-translational modification of PGPR-originated AcdS proteins in plants. Compared with the wild-type, transgenic Arabidopsis expressing the Pseudomonas fluorescens acdS (PfacdS) gene displayed increased root elongation and reduced sensitivity to 10 ?M exogenous ACC, an ethylene precursor. Arabidopsis expressing PfacdS also showed increased tolerance to high salinity (150 mM NaCl). PfAcdS proteins accumulated in transgenic Arabidopsis were rapidly degraded, which was potentially mediated by the 26S proteasome pathway. The degradation of PfAcdS was alleviated in the presence of exogenous ACC. In conclusion, our data suggest that the plant growth-promoting effects of bacterial AcdS proteins are potentially modulated via protein turnover inside the host plant cells. Such post-translational modification plays a physiological role in the mutualistic interactions between microorganisms and plants in the rhizospheric and/or endospheric niche. PMID:24801274

  15. L-arabinose fermenting yeast

    DOEpatents

    Zhang, Min; Singh, Arjun; Suominen, Pirkko; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric

    2014-09-23

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.

  16. Genome evolution in yeasts

    Microsoft Academic Search

    Bernard Dujon; David Sherman; Gilles Fischer; Pascal Durrens; Serge Casaregola; Ingrid Lafontaine; Jacky de Montigny; Christian Marck; Cécile Neuvéglise; Emmanuel Talla; Nicolas Goffard; Lionel Frangeul; Michel Aigle; Véronique Anthouard; Anna Babour; Valérie Barbe; Stéphanie Barnay; Sylvie Blanchin; Jean-Marie Beckerich; Emmanuelle Beyne; Claudine Bleykasten; Anita Boisramé; Jeanne Boyer; Laurence Cattolico; Fabrice Confanioleri; Antoine de Daruvar; Laurence Despons; Emmanuelle Fabre; Cécile Fairhead; Hélène Ferry-Dumazet; Alexis Groppi; Florence Hantraye; Christophe Hennequin; Nicolas Jauniaux; Philippe Joyet; Rym Kachouri; Alix Kerrest; Romain Koszul; Marc Lemaire; Isabelle Lesur; Laurence Ma; Héloïse Muller; Jean-Marc Nicaud; Macha Nikolski; Sophie Oztas; Odile Ozier-Kalogeropoulos; Stefan Pellenz; Serge Potier; Guy-Franck Richard; Marie-Laure Straub; Audrey Suleau; Dominique Swennen; Fredj Tekaia; Micheline Wésolowski-Louvel; Eric Westhof; Bénédicte Wirth; Maria Zeniou-Meyer; Ivan Zivanovic; Monique Bolotin-Fukuhara; Agnès Thierry; Christiane Bouchier; Bernard Caudron; Claude Scarpelli; Claude Gaillardin; Jean Weissenbach; Patrick Wincker; Jean-Luc Souciet

    2004-01-01

    Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity

  17. Chemical genomics in yeast

    PubMed Central

    Brenner, Charles

    2004-01-01

    Many drugs have unknown, controversial or multiple mechanisms of action. Four recent 'chemical genomic' studies, using genome-scale collections of yeast gene deletions that were either arrayed or barcoded, have presented complementary approaches to identifying gene-drug and pathway-drug interactions. PMID:15345040

  18. Yeast DNA Extraction

    NSDL National Science Digital Library

    Lana Hays

    2009-01-01

    This laboratory exercise is designed to show learners how DNA can easily be extracted from yeast using simple materials. Use this experiment to supplement any unit on genetics and to demonstrate how scientists study DNA. Adult supervision is recommended. This resource guide includes tips and suggestions for instructors as well as other DNA extraction experiments and a chart for learners to answer questions.

  19. Evolving insights on how cytosine methylation affects protein–DNA binding

    PubMed Central

    Dantas Machado, Ana Carolina; Zhou, Tianyin; Rao, Satyanarayan; Goel, Pragya; Rastogi, Chaitanya; Lazarovici, Allan; Bussemaker, Harmen J.

    2015-01-01

    Many anecdotal observations exist of a regulatory effect of DNA methylation on gene expression. However, in general, the underlying mechanisms of this effect are poorly understood. In this review, we summarize what is currently known about how this important, but mysterious, epigenetic mark impacts cellular functions. Cytosine methylation can abrogate or enhance interactions with DNA-binding proteins, or it may have no effect, depending on the context. Despite being only a small chemical change, the addition of a methyl group to cytosine can affect base readout via hydrophobic contacts in the major groove and shape readout via electrostatic contacts in the minor groove. We discuss the recent discovery that CpG methylation increases DNase I cleavage at adjacent positions by an order of magnitude through altering the local 3D DNA shape and the possible implications of this structural insight for understanding the methylation sensitivity of transcription factors (TFs). Additionally, 5-methylcytosines change the stability of nucleosomes and, thus, affect the local chromatin structure and access of TFs to genomic DNA. Given these complexities, it seems unlikely that the influence of DNA methylation on protein–DNA binding can be captured in a small set of general rules. Hence, data-driven approaches may be essential to gain a better understanding of these mechanisms. PMID:25319759

  20. Cytosine DNA methylation influences drug resistance in Escherichia coli through increased sugE expression.

    PubMed

    Militello, Kevin T; Mandarano, Alexandra H; Varechtchouk, Olga; Simon, Robert D

    2014-01-01

    Escherichia coli K-12 strains contain the orphan cytosine-5 DNA methyltransferase enzyme Dcm (DNA cytosine methyltransferase). Two recent reports indicate that Dcm has an influence on stationary phase gene expression in E. coli. Herein, we demonstrate that dcm knockout cells overexpress the drug resistance transporter SugE, which has been linked to ethidium bromide (ETBR) resistance. SugE expression also increased in the presence of the DNA methylation inhibitor 5-azacytidine, suggesting that Dcm-mediated DNA methylation normally represses sugE expression. The effect of Dcm on sugE expression is primarily restricted to early stationary phase, and RpoS is required for robust sugE expression. Dcm knockout cells are more resistant to ETBR than wild-type cells, and complementation with a plasmid-borne dcm gene restores ETBR sensitivity. SugE knockout cells are more sensitive to ETBR than wild-type cells. These data indicate that Dcm influences the sensitivity to an antimicrobial compound through changes in gene expression. PMID:24164619

  1. Approximate solution of the mode-mode coupling integral: Application to cytosine and its deuterated derivative

    NASA Astrophysics Data System (ADS)

    Rasheed, Tabish; Ahmad, Shabbir

    2010-10-01

    Ab initio Hartree-Fock (HF), density functional theory (DFT) and second-order Møller-Plesset (MP2) methods were used to perform harmonic and anharmonic calculations for the biomolecule cytosine and its deuterated derivative. The anharmonic vibrational spectra were computed using the vibrational self-consistent field (VSCF) and correlation-corrected vibrational self-consistent field (CC-VSCF) methods. Calculated anharmonic frequencies have been compared with the argon matrix spectra reported in literature. The results were analyzed with focus on the properties of anharmonic couplings between pair of modes. A simple and easy to use formula for calculation of mode-mode coupling magnitudes has been derived. The key element in present approach is the approximation that only interactions between pairs of normal modes have been taken into account, while interactions of triples or more are neglected. FTIR and Raman spectra of solid state cytosine have been recorded in the regions 400-4000 cm -1 and 60-4000 cm -1, respectively. Vibrational analysis and assignments are based on calculated potential energy distribution (PED) values.

  2. Approximate solution of the mode-mode coupling integral: Application to cytosine and its deuterated derivative.

    PubMed

    Rasheed, Tabish; Ahmad, Shabbir

    2010-10-01

    Ab initio Hartree-Fock (HF), density functional theory (DFT) and second-order Møller-Plesset (MP2) methods were used to perform harmonic and anharmonic calculations for the biomolecule cytosine and its deuterated derivative. The anharmonic vibrational spectra were computed using the vibrational self-consistent field (VSCF) and correlation-corrected vibrational self-consistent field (CC-VSCF) methods. Calculated anharmonic frequencies have been compared with the argon matrix spectra reported in literature. The results were analyzed with focus on the properties of anharmonic couplings between pair of modes. A simple and easy to use formula for calculation of mode-mode coupling magnitudes has been derived. The key element in present approach is the approximation that only interactions between pairs of normal modes have been taken into account, while interactions of triples or more are neglected. FTIR and Raman spectra of solid state cytosine have been recorded in the regions 400-4000 cm(-1) and 60-4000 cm(-1), respectively. Vibrational analysis and assignments are based on calculated potential energy distribution (PED) values. PMID:20638327

  3. Gene Therapy for Severe Combined Immunodeficiency Caused by Adenosine Deaminase Deficiency: Improved Retroviral Vectors for Clinical Trials

    Microsoft Academic Search

    Masafumi Onodera; David M. Nelson; Yukio Sakiyama; Fabio Candotti; R. Michael Blaese

    1999-01-01

    Severe combined immunodeficiency (SCID) caused by adenosine deaminase deficiency (ADA–) is the first genetic disorder to be treated with gene therapy. Since 1990 when the first trial started for 2 patients with ADA– SCID, five clinical trials enrolling 11 patients have been conducted with different clinical approaches and the results obtained from these trials have recently been reported. According to

  4. Mechanisms of apoptosis in developing thymocytes as revealed by adenosine deaminase-deficient fetal thymic organ cultures

    Microsoft Academic Search

    Linda F. Thompson; James G. Vaughn; Aletha B. Laurent; Michael R. Blackburn; C. Justin Van De Wiele

    2003-01-01

    Adenosine deaminase (ADA) catalyzes the conversion of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. ADA-deficient individuals suffer from severe combined immunodeficiency and are unable to produce significant numbers of mature T or B lymphocytes. This occurs as a consequence of the accumulation of ADA substrates or their metabolites. dATP is a candidate toxic metabolite because its concentration in RBCs

  5. Metabolic Consequences of Adenosine Deaminase Deficiency in Mice Are Associated with Defects in Alveogenesis, Pulmonary Inflammation, and Airway Obstruction

    Microsoft Academic Search

    Michael R. Blackburn; Jonathan B. Volmer; Janci L. Thrasher; Hongyan Zhong; Jeff R. Crosby; James J. Lee; Rodney E. Kellems

    Adenosine deaminase (ADA) is a purine catabolic enzyme that manages levels of the biologi- cally active purines adenosine and 2 9 -deoxyadenosine in tissues and cells. ADA-deficient mice die at 3 wk of age from severe respiratory distress. This phenotype is progressive and is linked to perturbations in pulmonary purine metabolism. The inflammatory changes found in the lungs of ADA-deficient

  6. Patients with adenosine deaminase deficiency surviving after hematopoietic stem cell transplantation are at high risk of CNS complications

    Microsoft Academic Search

    Manfred Honig; Michael H. Albert; Ansgar Schulz; Monika Sparber-Sauer; Catharina Schutz; Bernd Belohradsky; Tayfun Gungor; Markus T. Rojewski; Harald Bode; Ulrich Pannicke; Dominique Lippold; Klaus Schwarz; Klaus-Michael Debatin; Michael S. Hershfield; Wilhelm Friedrich

    2007-01-01

    Adenosine deaminase (ADA) deficiency is a systemic metabolic disease that causes an autosomal recessive variant of severe combined immunodeficiency (SCID) and less consistently other compli- cations including neurologic abnormali- ties. Hematopoietic stem cell transplanta- tion (HSCT) is able to correct the immunodeficiency, whereas control of nonimmunologic complications has not been extensively explored. We applied HSCT in 15 ADA-deficient patients con-

  7. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

    SciTech Connect

    Scott, A.I.; Roessner, C.A.; Stolowich, N.J.; Karuso, P.; Williams, H.J.; Grant, S.K.; Gonzalez, M.D.; Hoshino, T. (Texas A M Univ., College Station (USA))

    1988-10-18

    The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-(2,11-{sup 13}C{sub 2})PBG reveals that the aminomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the {alpha}-free pyrrole. NMR spectroscopy of the ES{sub 2} complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the {alpha}-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

  8. Silencing Threonine Deaminase and JAR4 in Nicotiana attenuata Impairs Jasmonic Acid-Isoleucine-Mediated Defenses against Manduca sexta

    Microsoft Academic Search

    Jin-Ho Kang; Lei Wang; Ashok Giri; Ian T. Baldwin

    2006-01-01

    Threonine deaminase (TD) catalyzes the conversion of Thr to a-keto butyrate in Ile biosynthesis; however, its dramatic upregulation in leaves after herbivore attack suggests a role in defense. In Nicotiana attenuata, strongly silenced TD transgenic plants were stunted, whereas mildly silenced TD transgenic plants had normal growth but were highly susceptible to Manduca sexta attack. The herbivore susceptibility was associated

  9. Increased 1-aminocyclopropane-1-carboxylate deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells

    PubMed Central

    Someya, Tatsuhiko; Nonaka, Satoko; Nakamura, Kouji; Ezura, Hiroshi

    2013-01-01

    Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transformation. It has been reported that increasing the number of cells transformed by T-DNA delivery can improve the frequency of stable transformation. Previously, we demonstrated that a reduction in ethylene production by plant cells during cocultivation with A. tumefaciens-expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase resulted in increased T-DNA delivery into the plant cells. In this study, to further improve T-DNA delivery by A. tumefaciens, we modified the expression cassette of the ACC deaminase gene using vir gene promoter sequences. The ACC deaminase gene driven by the virD1 promoter was expressed at a higher level, resulting in a higher ACC deaminase activity in this A. tumefaciens strain than in the strain with the lac promoter used in a previous study. The newly developed A. tumefaciens strain improves the delivery of T-DNA into Solanum lycopersicum (tomato) and Erianthus ravennae plants and thus may be a powerful tool for the Agrobacterium-mediated genetic engineering of plants. PMID:24000136

  10. GENIS: Gene Expression of Sodium Iodide Symporter for Noninvasive Imaging of Gene Therapy Vectors and Quantification of Gene Expression in Vivo

    Microsoft Academic Search

    Kenneth N. Barton; Donald Tyson; Hans Stricker; Young S. Lew; Gregory Heisey; Sweaty Koul; Alberto de la Zerda; Fang-Fang Yin; Hui Yan; Tavarekere N. Nagaraja; Kelly Ann Randall; Guk Kim Jin; Joseph D. Fenstermacher; Sissy Jhiang; Jae Ho Kim; Svend O. Freytag; Stephen L. Brown

    2003-01-01

    With the goal of optimizing adenovirus-mediated suicide gene therapy for prostate cancer, we have developed a method based on the human sodium iodide symporter (hNIS) that allows for noninvasive monitoring of adenoviral vectors and quantification of gene expression magnitude and volume within the prostate. A replication-competent adenovirus (Ad5-yCD\\/mutTKSR39rep-hNIS) coexpressing a therapeutic yeast cytosine deaminase (yCD)\\/mutant herpes simplex virus thymidine kinase

  11. Extracellular Polysaccharides Produced by Yeasts and Yeast-Like Fungi

    NASA Astrophysics Data System (ADS)

    van Bogaert, Inge N. A.; de Maeseneire, Sofie L.; Vandamme, Erick J.

    Several yeasts and yeast-like fungi are known to produce extracellular polysaccharides. Most of these contain D-mannose, either alone or in combination with other sugars or phosphate. A large chemical and structural variability is found between yeast species and even among different strains. The types of polymers that are synthesized can be chemically characterized as mannans, glucans, phosphoman-nans, galactomannans, glucomannans and glucuronoxylomannans. Despite these differences, almost all of the yeast exopolysaccharides display some sort of biological activity. Some of them have already applications in chemistry, pharmacy, cosmetics or as probiotic. Furthermore, some yeast exopolysaccharides, such as pullulan, exhibit specific physico-chemical and rheological properties, making them useful in a wide range of technical applications. A survey is given here of the production, the characteristics and the application potential of currently well studied yeast extracellular polysaccharides.

  12. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human

    SciTech Connect

    Palmer, T.D.; Hock, R.A.; Osborne, W.R.A.; Miller, A.D.

    1987-02-01

    Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. The authors studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA/sup -/ fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.

  13. Extracellular Polysaccharides Produced by Yeasts and YeastLike Fungi

    Microsoft Academic Search

    Inge N. A. Bogaert; Sofie L. Maeseneire; Erick J. Vandamme

    Several yeasts and yeast-like fungi are known to produce extracellular polysaccharides. Most of these contain D-mannose, either\\u000a alone or in combination with other sugars or phosphate. A large chemical and structural variability is found between yeast\\u000a species and even among different strains. The types of polymers that are synthesized can be chemically characterized as mannans,\\u000a glucans, phosphoman-nans, galactomannans, glucomannans and

  14. Extracellular Polysaccharides Produced by Yeasts and YeastLike Fungi

    Microsoft Academic Search

    Inge N. A. van Bogaert; Sofie L. de Maeseneire; Erick J. Vandamme

    2009-01-01

    Several yeasts and yeast-like fungi are known to produce extracellular polysaccharides. Most of these contain D-mannose, either alone or in combination with other sugars or phosphate. A large chemical and structural variability is found between yeast species and even among different strains. The types of polymers that are synthesized can be chemically characterized as mannans, glucans, phosphoman-nans, galactomannans, glucomannans and

  15. Energetics of the lattice: packing elements in crystals of four-stranded intercalated cytosine-rich DNA molecules.

    PubMed

    Berger, I; Cai, L; Chen, L; Rich, A

    1997-01-01

    Condensation of single molecules from solution into crystals represents a transition between distinct energetic states. In solution, the atomic interactions within the molecule dominate. In the crystalline state, however, a set of additional interactions are formed between molecules in close contact in the lattice--these are the packing interactions. The crystal structures of d(CCCT), d(TAACCC), d(CCCAAT), and d(AACCCC) have in common a four-stranded intercalated cytosine segment, built by stacked layers of cytosine.cytosine+ (C.C+) base pairs coming from two parallel duplexes that intercalate into each other with opposite polarity. The intercalated cytosine segments in these structures are similar in their geometry, even though the sequences crystallized in different space groups. In the crystals, adenine and thymine residues of the sequences are used to build the three-dimensional crystal lattice by elaborately interacting with symmetry-related molecules. The packing elements observed provide novel insight about the copious ways in which nucleic acid molecules can interact with each other--for example, when folded in more complicated higher order structures, such as mRNA and chromatin. PMID:9591478

  16. Energetics of the lattice: packing elements in crystals of four-stranded intercalated cytosine-rich DNA molecules

    NASA Technical Reports Server (NTRS)

    Berger, I.; Cai, L.; Chen, L.; Rich, A.

    1997-01-01

    Condensation of single molecules from solution into crystals represents a transition between distinct energetic states. In solution, the atomic interactions within the molecule dominate. In the crystalline state, however, a set of additional interactions are formed between molecules in close contact in the lattice--these are the packing interactions. The crystal structures of d(CCCT), d(TAACCC), d(CCCAAT), and d(AACCCC) have in common a four-stranded intercalated cytosine segment, built by stacked layers of cytosine.cytosine+ (C.C+) base pairs coming from two parallel duplexes that intercalate into each other with opposite polarity. The intercalated cytosine segments in these structures are similar in their geometry, even though the sequences crystallized in different space groups. In the crystals, adenine and thymine residues of the sequences are used to build the three-dimensional crystal lattice by elaborately interacting with symmetry-related molecules. The packing elements observed provide novel insight about the copious ways in which nucleic acid molecules can interact with each other--for example, when folded in more complicated higher order structures, such as mRNA and chromatin.

  17. Analysis of DNA (cytosine 5) methyltransferase mRNA sequence and expression in bovine preimplantation embryos, fetal and adult tissues

    Microsoft Academic Search

    Michael C. Golding; Mark E. Westhusin

    2003-01-01

    Mammalian preimplantation development is a critical stage for establishment of the genomic methylation pattern and proper function of the enzymes responsible for this appear essential for normal development. To date, the vast majority of work concerning the developmental expression of the DNA cytosine 5-methyltansferases (Dnmts) has been conducted in mice. Here we report the sequence and expression of the Dnmt

  18. RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis.

    PubMed

    Tuorto, Francesca; Liebers, Reinhard; Musch, Tanja; Schaefer, Matthias; Hofmann, Sarah; Kellner, Stefanie; Frye, Michaela; Helm, Mark; Stoecklin, Georg; Lyko, Frank

    2012-09-01

    The function of cytosine-C5 methylation, a widespread modification of tRNAs, has remained obscure, particularly in mammals. We have now developed a mouse strain defective in cytosine-C5 tRNA methylation, by disrupting both the Dnmt2 and the NSun2 tRNA methyltransferases. Although the lack of either enzyme alone has no detectable effects on mouse viability, double mutants showed a synthetic lethal interaction, with an underdeveloped phenotype and impaired cellular differentiation. tRNA methylation analysis of the double-knockout mice demonstrated complementary target-site specificities for Dnmt2 and NSun2 and a complete loss of cytosine-C5 tRNA methylation. Steady-state levels of unmethylated tRNAs were substantially reduced, and loss of Dnmt2 and NSun2 was further associated with reduced rates of overall protein synthesis. These results establish a biologically important function for cytosine-C5 tRNA methylation in mammals and suggest that this modification promotes mouse development by supporting protein synthesis. PMID:22885326

  19. Xylose fermentation by yeasts

    Microsoft Academic Search

    Manfred Rizzi; Petra Erlemann; Ngoc-Anh Bui-Thanh; Hanswerner Dellweg

    1988-01-01

    Xylose reductase from the xylose-fermenting yeastPichia stipitis was purified to electrophoretic homogeneity via ion-exchange, gel and affinity chromatography. At physiological pH values the thermodynamic equilibrium constant was determined to be 0.575x1010 (l·mol-1). Product inhibiton studies are reported which clearly show that the kinetic mechanism of the xylose reductase is ordered-bi-bi with isomerisation of a stable enzyme form.

  20. Mammalian Homology to Yeast

    NSDL National Science Digital Library

    1997-01-01

    This site allows researchers to retrieve a yeast-against-mammal Basic Local Alignment Search Tool (BLAST) report by entering a gene or ORF name into a search function. The supporting data were first summarized in a recent Science article which is provided via a link to the journal (Science, 22 July 1997; Issue 277: p.1259). Steve Chervitz of Stanford University maintains this site.

  1. Glutathione Production in Yeast

    NASA Astrophysics Data System (ADS)

    Bachhawat, Anand K.; Ganguli, Dwaipayan; Kaur, Jaspreet; Kasturia, Neha; Thakur, Anil; Kaur, Hardeep; Kumar, Akhilesh; Yadav, Amit

    Glutathione, ? -glutamyl-cysteinyl-glycine, is the most abundant non-protein thiol found in almost all eukaryotic cells (and in some prokaryotes). The tripeptide, which is synthesized non-ribosomally by the consecutive action of two soluble enzymes, is needed for carrying out numerous functions in the cell, most important of which is the maintenance of the redox buffer. The cycle of glutathione biosynthesis and degradation forms part of the ? -glutamyl cycle in most organisms although the latter half of the pathway has not been demonstrated in yeasts. Our current understanding of how glutathione levels are controlled at different levels in the cell is described. Several different routes and processes have been attempted to increase commercial production of glutathione using both yeast and bacteria. In this article we discuss the history of glutathione production in yeast. The current bottlenecks for increased glutathione production are presented based on our current understanding of the regulation of glutathione homeostasis, and possible strategies for overcoming these limitations for further enhancing and improving glutathione production are discussed

  2. Non-infectious lung disease in patients with adenosine deaminase deficient severe combined immunodeficiency.

    PubMed

    Booth, C; Algar, V E; Xu-Bayford, J; Fairbanks, L; Owens, C; Gaspar, H B

    2012-06-01

    Adenosine deaminase deficiency is a disorder of purine metabolism manifesting severe combined immunodeficiency (ADA-SCID) and systemic abnormalities. Increased levels of the substrate deoxyadenosine triphosphate (dATP) lead to immunodeficiency and are associated in a murine model with pulmonary insufficiency. We compared a cohort of patients with ADA-SCID and X-linked SCID and found that despite similar radiological and respiratory findings, positive microbiology is significantly less frequent in ADA-SCID patients (p?

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

    PubMed

    Montiel-Equihua, Claudia A; Thrasher, Adrian J; Gaspar, H Bobby

    2009-01-01

    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. PMID:24198507

  4. High pleural ammonia negatively interferes with the measurement of adenosine deaminase activity

    PubMed Central

    Loh, Tze Ping; Tan, Karen Mei Ling; Chew, Suru; Chan, Douglas S G

    2013-01-01

    Pleural adenosine deaminase activity (ADA) is a sensitive and specific test for tuberculous pleurisy. Here, we report a case of undetectable ADA in the pleural fluid of a man presenting with chronic cough, fever and night sweats. Subsequent laboratory investigations and review revealed that the presence of high concentrations of ammonia in pleural fluid, commonly seen in empyema, negatively interferes with ADA results when measured by the Guisti and Galanti method. The source of ammonia may come from deamination of amino acids, ammonia-producing microbes and/or leucocytes. This interference invalidates ADA results and is present in ?2% of our laboratory requests. It is important to keep this interference in mind when tuberculosis/ammonia-producing bacteria coinfections are suspected and during early phases of tuberculous pleurisy, when neutrophils predominate in the pleural fluids. PMID:23389721

  5. Epigenetic Function of Activation-Induced Cytidine Deaminase and Its Link to Lymphomagenesis

    PubMed Central

    Dominguez, Pilar M.; Shaknovich, Rita

    2014-01-01

    Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation and class switch recombination of immunoglobulin (Ig) genes during B cell maturation and immune response. Expression of AID is tightly regulated due to its mutagenic and recombinogenic potential, which is known to target not only Ig genes, but also non-Ig genes, contributing to lymphomagenesis. In recent years, a new epigenetic function of AID and its link to DNA demethylation came to light in several developmental systems. In this review, we summarize existing evidence linking deamination of unmodified and modified cytidine by AID to base-excision repair and mismatch repair machinery resulting in passive or active removal of DNA methylation mark, with the focus on B cell biology. We also discuss potential contribution of AID-dependent DNA hypomethylation to lymphomagenesis. PMID:25566255

  6. REG-? associates with and modulates the abundance of nuclear activation-induced deaminase

    PubMed Central

    Uchimura, Yasuhiro; Barton, Lance F.; Rada, Cristina

    2011-01-01

    Activation-induced deaminase (AID) acts on the immunoglobulin loci in activated B lymphocytes to initiate antibody gene diversification. The abundance of AID in the nucleus appears tightly regulated, with most nuclear AID being either degraded or exported back to the cytoplasm. To gain insight into the mechanisms regulating nuclear AID, we screened for proteins interacting specifically with it. We found that REG-?, a protein implicated in ubiquitin- and ATP-independent protein degradation, interacts in high stoichiometry with overexpressed nuclear AID as well as with endogenous AID in B cells. REG-? deficiency results in increased AID accumulation and increased immunoglobulin class switching. A stable stoichiometric AID–REG-? complex can be recapitulated in co-transformed bacteria, and REG-? accelerates proteasomal degradation of AID in in vitro assays. Thus, REG-? interacts, likely directly, with nuclear AID and modulates the abundance of this antibody-diversifying but potentially oncogenic enzyme. PMID:22042974

  7. Induction of homologous recombination between sequence repeats by the activation induced cytidine deaminase (AID) protein

    PubMed Central

    Buerstedde, Jean-Marie; Lowndes, Noel; Schatz, David G

    2014-01-01

    The activation induced cytidine deaminase (AID) protein is known to initiate somatic hypermutation, gene conversion or switch recombination by cytidine deamination within the immunoglobulin loci. Using chromosomally integrated fluorescence reporter transgenes, we demonstrate a new recombinogenic activity of AID leading to intra- and intergenic deletions via homologous recombination of sequence repeats. Repeat recombination occurs at high frequencies even when the homologous sequences are hundreds of bases away from the positions of AID-mediated cytidine deamination, suggesting DNA end resection before strand invasion. Analysis of recombinants between homeologous repeats yielded evidence for heteroduplex formation and preferential migration of the Holliday junctions to the boundaries of sequence homology. These findings broaden the target and off-target mutagenic potential of AID and establish a novel system to study induced homologous recombination in vertebrate cells. DOI: http://dx.doi.org/10.7554/eLife.03110.001 PMID:25006166

  8. Widespread genomic breaks from activation-induced cytidine deaminase are prevented by homologous recombination

    PubMed Central

    Hasham, Muneer G.; Donghia, Nina M.; Coffey, Eliot; Maynard, Jane; Snow, Kathy J.; Ames, Jacquelyn; Wilpan, Robert Y.; He, Yishu; King, Benjamin L.; Mills, Kevin D.

    2010-01-01

    Activation induced cytidine deaminase (AID) is required for somatic hypermutation and immunoglobulin class switching in activated B cells. Because AID possesses no known target site specificity, there have been efforts to identify non-immunoglobulin AID targets. We show that AID acts promiscuously, generating widespread DNA double strand breaks (DSB), genomic instability and cytotoxicity in B cells with diminished homologous recombination (HR) capability. We demonstrate that the HR factor XRCC2 suppresses AID-induced off-target DSBs, promoting B cell survival. Finally, we suggest that aberrations affecting human chromosome 7q36, including XRCC2, correlate with genomic instability in B cell cancers. Our findings demonstrate that AID has promiscuous genomic DSB-inducing activity, identify HR as a safeguard against off-target AID action, and have implications for genomic instability in B cell cancers. PMID:20657597

  9. Epigenome-wide inheritance of cytosine methylation variants in a recombinant inbred population

    PubMed Central

    Schmitz, Robert J.; He, Yupeng; Valdés-López, Oswaldo; Khan, Saad M.; Joshi, Trupti; Urich, Mark A.; Nery, Joseph R.; Diers, Brian; Xu, Dong; Stacey, Gary; Ecker, Joseph R.

    2013-01-01

    Cytosine DNA methylation is one avenue for passing information through cell divisions. Here, we present epigenomic analyses of soybean recombinant inbred lines (RILs) and their parents. Identification of differentially methylated regions (DMRs) revealed that DMRs mostly cosegregated with the genotype from which they were derived, but examples of the uncoupling of genotype and epigenotype were identified. Linkage mapping of methylation states assessed from whole-genome bisulfite sequencing of 83 RILs uncovered widespread evidence for local methylQTL. This epigenomics approach provides a comprehensive study of the patterns and heritability of methylation variants in a complex genetic population over multiple generations, paving the way for understanding how methylation variants contribute to phenotypic variation. PMID:23739894

  10. Synthesis of 1- ?- d-arabinofuranosyl-cytosine 5?-phosphate- l-1,2-diacylglycerols

    Microsoft Academic Search

    Ágnes Nyilas

    1997-01-01

    5?-O-MMTr-cytosine arabinoside was prepared on a large scale from 5?-O-MMTr-cytidine with diphenyl carbonate via 5?-protected cytidine-2?,3?-carbonate-ara-cytidine-2?,2-anhydro derivative at a 67% yield. 1,2-Dipalmitoyl-sn-glycerol, 1,2-distearoyl-sn-glycerol and 1,2-dioleoyl-sn-glycerol were phosphorylated first with 2-chlorphenyl-phosphoro-bis-triazolide quantitatively (Welch and Chattopadhyaya, 1985. Acta Chem. Scand. B39, 47–57). This method was used in order to avoid acyl migration, then the glycerophosphate intermediates were condensed with 2?,3?,N4-trileulinyl-1-?-d-arabinofuranosylcytosine in the

  11. Preparation and metal ion-binding of 4-N-substituted cytosine pairs in DNA duplexes.

    PubMed

    Sugiyama, Kumiko; Kageyama, Yoshihiko; Okamoto, Itaru; Ono, Akira

    2007-01-01

    We report the synthesis and metal ion-binding properties of DNA duplexes containing 4-N-substituted cytosine base pairs. Thermal denaturation studies of these modified DNA duplexes in the presence of various metal ions revealed that the DNA duplexes containing 4-N-carboxymethylcytosine (1) base pair(s) bound Ag (I), Ni(II), and Cu(II) ions. Moreover, ESI-TOF MS analysis of the DNA duplexes containing 1-1 base pair(s) in the presence of Cu(II) ions was consistent with one 1-1 base pair region binding one equivalent of Cu(II) ions. These results indicate that the DNA duplexes containing 1-1 base pair(s) may be useful as a new metal ion-binding motif. PMID:18029644

  12. Transfection of living HeLa cells with fluorescent poly-cytosine encapsulated Ag nanoclusters.

    PubMed

    Antoku, Yasuko; Hotta, Jun-ichi; Mizuno, Hideaki; Dickson, Robert M; Hofkens, Johan; Vosch, Tom

    2010-05-01

    The fluorescence of silver clusters encapsulated by single stranded oligo-DNA (24 cytosine base pairs, C(24):Ag(n)) was used to monitor the transfection of this new silver/DNA fluorophore inside living HeLa cells. For this, the C(24):Ag(n) molecules were complexed with a commercially available transfection reagent Lipofectamine and the internalization of C(24):Ag(n) was followed with confocal fluorescence microscopy. Bright near-infrared fluorescence was observed from inside the transfected HeLa cells, when exciting with 633 nm excitation, opening up the possibility for the use of these C(24):Ag(n) clusters for biological labelling and imaging of living cells and for monitoring the transfection process with limited harm to the living cells. PMID:20442932

  13. Epigenome-wide inheritance of cytosine methylation variants in a recombinant inbred population.

    PubMed

    Schmitz, Robert J; He, Yupeng; Valdés-López, Oswaldo; Khan, Saad M; Joshi, Trupti; Urich, Mark A; Nery, Joseph R; Diers, Brian; Xu, Dong; Stacey, Gary; Ecker, Joseph R

    2013-10-01

    Cytosine DNA methylation is one avenue for passing information through cell divisions. Here, we present epigenomic analyses of soybean recombinant inbred lines (RILs) and their parents. Identification of differentially methylated regions (DMRs) revealed that DMRs mostly cosegregated with the genotype from which they were derived, but examples of the uncoupling of genotype and epigenotype were identified. Linkage mapping of methylation states assessed from whole-genome bisulfite sequencing of 83 RILs uncovered widespread evidence for local methylQTL. This epigenomics approach provides a comprehensive study of the patterns and heritability of methylation variants in a complex genetic population over multiple generations, paving the way for understanding how methylation variants contribute to phenotypic variation. PMID:23739894

  14. The retinoblastoma gene product interacts with maintenance human DNA (cytosine-5) methyltransferase and modulates its activity.

    PubMed

    Pradhan, Sriharsa; Kim, Gun-Do

    2002-02-15

    The mammalian DNA (cytosine-5) methyltransferase (Dnmt1) is involved in the maintenance of methylation patterns in the genome during DNA replication and development. The retinoblastoma gene product, Rb, is a cell cycle regulator protein that represses transcription by recruiting histone deacetylase (HDAC1). In vivo, histone deacetylase associates with Dnmt1. Here we show that Rb itself associates with human Dnmt1 (hDnmt1) independently of its own phosphorylation status. Methyltransferase activity was co-purified with Rb. The regulatory domain of hDnmt1 binds strongly to the B and C pockets of Rb (amino acids 701-872) and inhibits methyltransferase activity by disruption of the hDnmt1-DNA binary complex. Weak interaction of Rb pockets A and B with Dnmt1 was also observed. Overexpression of Rb leads to hypomethylation of the cellular DNA, suggesting that Rb may modulate Dnmt1 activity during DNA replication in the cell cycle. PMID:11847125

  15. Intracellular Detection of Cytosine Incorporation in Genomic DNA Using 5-Ethynyl-2?-Deoxycytidine

    PubMed Central

    Guan, Lirui; van der Heijden, Godfried W.

    2012-01-01

    5-Ethynyl-2?-deoxycytidine triphosphate (EdCTP) was synthesized as a probe to be used in conjunction with fluorescent labelling to facilitate the analysis of the in vivo dynamics of DNA-centered processes (DNA replication, repair and cytosine demethylation). Kinetic analysis showed that EdCTP is accepted as a substrate by Klenow exo- and DNA polymerase ?. Incorporation of 5-ethynyl-2?-deoxycytidine (EdC) into DNA by these enzymes is at most modestly less efficient than native dC. EdC containing DNA was visualized using a click reaction with a fluorescent azide, following polymerase incorporation and T4 DNA ligase mediated ligation. Subsequent experiments in mouse male germ cells and zygotes demonstrated that EdC is a specific and reliable reporter of DNA replication in vivo. PMID:21805552

  16. DNA methylation by wheat cytosine DNA methyltransferase: modulation by protease inhibitor E-64.

    PubMed

    Vlasova, T I; Vanyushin, B F

    1998-06-01

    Cytosine DNA methyltransferase isolated from wheat seedlings and purified in the presence of metalloprotease and serine protease inhibitors has molecular mass and specific activity equal to about 85 kDa and 250 units/mg protein, respectively. Apparent K(m) for AdoMet and [I]50 for AdoHcy values are about 6 microM and 12 microM, respectively. The enzyme is active in wide pH range (pH 5.5-8.5) and is inhibited by NaCl. The enzyme rapidly loses its methyltransferase activity in the absence of substrates. Using the cysteine protease inhibitor E-64 it has been shown that rapid enzyme inactivation is caused by disappearance of essential enzyme SH-groups but is not due to proteolytic enzyme cleavage. PMID:9635138

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

    SciTech Connect

    Barton, R.W.

    1985-10-15

    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.

  18. Expression of a functional human adenosine deaminase in transgenic tobacco plants.

    PubMed

    Singhabahu, Sanjeewa; George, John; Bringloe, David

    2013-06-01

    An inherited disorder, adenosine deaminase deficiency is a form of severe combined immunodeficiency, which is ultimately caused by an absence of adenosine deaminase (ADA), a key enzyme of the purine salvage pathway. The absence of ADA-activity in sufferers eventually results in a dysfunctional immune system due to the build-up of toxic metabolites. To date, this has been treated with mixed success, using PEG-ADA, made from purified bovine ADA coupled to polyethylene glycol. It is likely, however, that an enzyme replacement therapy protocol based on recombinant human ADA would be a more effective treatment for this disease. Therefore, as a preliminary step to produce biologically active human ADA in transgenic tobacco plants a human ADA cDNA has been inserted into a plant expression vector under the control of the CaMV 35S promoter and both human and TMV 5' UTR control regions. Plant vector expression constructs have been used to transform tobacco plants via Agrobacterium-mediated transformation. Genomic DNA, RNA and protein blot analyses have demonstrated the integration of the cDNA construct into the plant nuclear genome and the expression of recombinant ADA mRNA and protein in transgenic tobacco leaves. Western blot analysis has also revealed that human and recombinant ADA have a similar size of approximately 41 kDa. ADA-specific activities of between 0.001 and 0.003 units per mg total soluble protein were measured in crude extracts isolated from transformed tobacco plant leaves. PMID:23264022

  19. Acute intermittent porphyria: expression of mutant and wild-type porphobilinogen deaminase in COS-1 cells.

    PubMed Central

    Mustajoki, S.; Laine, M.; Lahtela, M.; Mustajoki, P.; Peltonen, L.; Kauppinen, R.

    2000-01-01

    BACKGROUND: Acute intermittent porphyria (AIP) is an autosomal dominant disorder that results from the partial deficiency of porphobilinogen deaminase (PBGD) in the heme biosynthetic pathway. Patients with AIP can experience acute attacks consisting of abdominal pain and various neuropsychiatric symptoms. Although molecular biological studies on the porphobilinogen deaminase (PBGD) gene have revealed several mutations responsible for AIP, the properties of mutant PBGD in eukaryotic expression systems have not been studied previously. MATERIALS AND METHODS: Seven mutations were analyzed using transient expression of the mutated polypeptides in COS-1 cells. The properties of mutated polypeptides were studied by enzyme activity measurement, Western blot analysis, pulse-chase experiments, and immunofluorescence staining. RESULTS: Of the mutants studied, R26C, R167W, R173W, R173Q, and R225X resulted in a decreased enzyme activity (0-5%), but R225G and 1073delA (elongated protein) displayed a significant residual activity of 16% and 50%, respectively. In Western blot analysis, the polyclonal PBGD antibody detected all mutant polypeptides except R225X, which was predicted to result in a truncated protein. In the pulse-chase experiment, the mutant polypeptides were as stable as the wild-type enzyme. In the immunofluorescence staining both wild-type and mutant polypeptides were diffusely dispersed in the cytoplasm and, thus, no accumulation of mutated proteins in the cellular compartments could be observed. CONCLUSIONS: The results confirm the causality of mutations for the half normal enzyme activity measured in the patients' erythrocytes. In contrast to the decreased enzyme activity, the majority of the mutations produced a detectable polypeptide, and the stability and the intracellular processing of the mutated polypeptides were both comparable to that of the wild-type PBGD and independent of the cross-reacting immunological material (CRIM) class. PMID:11055586

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

    PubMed Central

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

    2015-01-01

    Background: 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. Objectives: The aim of this study was to investigate the diagnostic value of serum adenosine deaminase (ADA) level for diagnosis of pulmonary tuberculosis. Patients and Methods: 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. Results: 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 < 0.05). In regard to the cut off value of 26 IU/L for ADA in patients with PTB sensitivity and specificity was defined as 35% and 91%, respectively. Conclusions: 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.

  1. Nitrosative Cytosine Deamination. An Exploration of the Chemistry Emanating from Deamination with Pyrimidine Ring-Opening

    PubMed Central

    Rayat, Sundeep; Qian, Ming; Glaser, Rainer

    2008-01-01

    A discussion of nitrosative deamination of cytosine 1 is presented that argues for the formation of 6 by diazotization of 1 to cytosinediazonium ion 2 and its electrostatic complex 3, dediazoniation to 4 ? 5, and amide-bond cleavage to 6. The reaction channels available to 6 include hydrolytic deglycation to 3-isocyanatoacrylonitrile 7, water addition to carbamic acid 9 with the possibility for re-closure to uracil 13, and water addition to carbamic acid 9 and decarboxylation to 3-aminoacrylonitrile 10. With a view to the instability of the carbamic acid 9, the carbamate models ethyl (Z)-2-cyanovinyl-carbamate 14 and (Z)-2-cyano-1-t-butylvinylcarbamate 20 were studied. Acid-catalyzed hydrolysis of 14 leads to 2-amino-carbonylphenylcarbamate 15 and its cyclization yields the benzo-fused uracil quinazoline-2,4-dione 16. In contrast to the aromatic system 14, acid-catalyzed cyclization cannot compete with oligomerization in the case of 20 and 5-tert-butyluracil 22 is accessible only with base-catalysis. It is shown that 23, the parent of 10, also easily polymerizes. The experimental results provide a rational as to why 9, 10 and 12 would have escaped detection in in vitro studies: they would have oligomerized. In contrast to the in vitro experiments, the oligomerizations of 9, 10 or 12 clearly are not relevant in vivo because of low monomer concentrations. With the exclusion of recyclization and of oligomerization in vivo, attention thus needs to focus on (Z)-3-aminoacrylonitrile 10 as the most likely deamination product of cytosine aside from uracil. PMID:16097794

  2. Characterization of the Residual Adenosine Deaminating Activity in the Spleen of a Patient with Combined Immunodeficiency Disease and Adenosine Deaminase Deficiency

    Microsoft Academic Search

    William P. Schrader; Bernard Pollara; Hilaire J. Meuwissen

    1978-01-01

    A number of infants with an autosomal recessive form of combined immunodeficiency disease also lack adenosine deaminase (adenosine aminohydrolase; EC 3.5.4.4) activity in their erythrocytes. Other tissues from these infants contain only a few percent of the adenosine-deaminating activity present in corresponding normal tissue. The residual adenosine-deaminating activity in extracts from the spleen of a combined immunodeficient, adenosine deaminase-deficient patient

  3. PERFORMANCE OF PLANT GROWTH PROMOTING RHIZOBACTERIA CONTAINING ACC-DEAMINASE ACTIVITY FOR IMPROVING GROWTH OF MAIZE UNDER SALT-STRESSED CONDITIONS

    Microsoft Academic Search

    S. M. Nadeem; I. Hussain; M. Naveed; H. N. Asghar; Z. A. Zahir; M. Arshad

    2006-01-01

    Bacteria carrying 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity lower stress induced ethylene levels and may be effective to improve plant growth under salt stress conditions. Twenty strains of rhizobacteria isolated from soil samples taken from different salt affected aras were screened for plant growth promotion and ACC- deaminase enzyme activity under axenic conditions at 6 dS rn'. Three strains (S5, S15

  4. Activation-Induced Cytidine Deaminase (AID) Deficiency Causes the Autosomal Recessive Form of the Hyper-IgM Syndrome (HIGM2)

    Microsoft Academic Search

    Patrick Revy; Taro Muto; Yves Levy; Frédéric Geissmann; Alessandro Plebani; Ozden Sanal; Nadia Catalan; Monique Forveille; Rémi Dufourcq-Lagelouse; Andrew Gennery; Ilhan Tezcan; Fugen Ersoy; Hulya Kayserili; Alberto G Ugazio; Nicole Brousse; Masamichi Muramatsu; Luigi D Notarangelo; Kazuo Kinoshita; Tasuku Honjo; Alain Fischer; Anne Durandy

    2000-01-01

    The activation-induced cytidine deaminase (AID) gene, specifically expressed in germinal center B cells in mice, is a member of the cytidine deaminase family. We herein report mutations in the human counterpart of AID in patients with the autosomal recessive form of hyper-IgM syndrome (HIGM2). Three major abnormalities characterize AID deficiency: (1) the absence of immunoglobulin class switch recombination, (2) the

  5. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...ADDITION TO FOOD FOR HUMAN CONSUMPTION Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis ) and dried torula yeast (Candida utilis ) may be safely used in food...

  6. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...ADDITION TO FOOD FOR HUMAN CONSUMPTION Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis ) and dried torula yeast (Candida utilis ) may be safely used in food...

  7. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...ADDITION TO FOOD FOR HUMAN CONSUMPTION Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis ) and dried torula yeast (Candida utilis ) may be safely used in food...

  8. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...ADDITION TO FOOD FOR HUMAN CONSUMPTION Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis ) and dried torula yeast (Candida utilis ) may be safely used in food...

  9. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...ADDITION TO FOOD FOR HUMAN CONSUMPTION Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis ) and dried torula yeast (Candida utilis ) may be safely used in food...

  10. BIOSYNTHESIS OF YEAST CAROTENOIDS

    PubMed Central

    Simpson, Kenneth L.; Nakayama, T. O. M.; Chichester, C. O.

    1964-01-01

    Simpson, Kenneth L. (University of California, Davis), T. O. M. Nakayama, and C. O. Chichester. Biosynthesis of yeast carotenoids. J. Bacteriol. 88:1688–1694. 1964.—The biosynthesis of carotenoids was followed in Rhodotorula glutinis and in a new strain, 62-506. The treatment of the growing cultures by methylheptenone, or ionone, vapors permitted observations of the intermediates in the biosynthetic pathway. On the basis of concentration changes and accumulation in blocked pathways, the sequence of carotenoid formation is postulated as phytoene, phytofluene, ?-carotene, neurosporene, ?-zeacarotene, ?-carotene, torulin, a C40 aldehyde, and torularhodin. Torulin and torularhodin were established as the main carotenoids of 62-506. PMID:14240958

  11. Production of Food Grade Yeasts

    Microsoft Academic Search

    Argyro Bekatorou; Costas Psarianos; Athanasios A. Koutinas

    2006-01-01

    Summary Yeasts have been known to humans for thousands of years as they have been used in traditional fermentation processes like wine, beer and bread making. Today, yeasts are also used as alternative sources of high nutritional value proteins, enzymes and vitamins, and have numerous applications in the health food industry as food additives, conditioners and flavouring agents, for the

  12. Yeasts from ips sexdentatus (scolytidae)

    Microsoft Academic Search

    Marie-Claire Pignal; C. Chararas; Michèle Bourgeay-Causse

    1988-01-01

    Yeasts from the digestive tract of Ips sexdentatus were isolated. Four strains, representing the different identified yeast species, were chosen. Their enzymatic activity on oligosaccharides, heterosides and polysaccharides was measured. Moreover, we showed that they excrete some B group vitamins which are necessary for the insect, unable to synthesize them.

  13. New and emerging yeast pathogens.

    PubMed Central

    Hazen, K C

    1995-01-01

    The most common yeast species that act as agents of human disease are Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, and Cryptococcus neoformans. The incidence of infections by other yeasts has increased during the past decade. The most evident emerging pathogens are Malassezia furfur, Trichosporon beigelii, Rhodotorula species, Hansenula anomala, Candida lusitaniae, and Candida krusei. Organisms once considered environmental contaminants or only industrially important, such as Candida utilis and Candida lipolytica, have now been implicated as agents of fungemia, onychomycosis, and systemic disease. The unusual yeasts primarily infect immunocompromised patients, newborns, and the elderly. The role of central venous catheter removal and antifungal therapy in patient management is controversial. The antibiograms of the unusual yeasts range from resistant to the most recent azoles and amphotericin B to highly susceptible to all antifungal agents. Current routine methods for yeast identification may be insufficient to identify the unusual yeasts within 2 days after isolation. The recognition of unusual yeasts as agents of sometimes life-threatening infection and their unpredictable antifungal susceptibilities increase the burden on the clinical mycology laboratory to pursue complete species identification and MIC determinations. Given the current and evolving medical practices for management of seriously ill patients, further evaluations of the clinically important data about these yeasts are needed. PMID:8665465

  14. Effect of AMP-deaminase 3 knock-out in mice on enzyme activity in heart and other organs.

    PubMed

    Rybakowska, Iwona; Romaszko, Pawel; Zabielska, Magdalena; Turyn, Jacek; Kaletha, Krystian; Barton, Paul J; Slominska, Ewa M; Smolenski, Ryszard T

    2014-01-01

    Recent findings suggest that inhibition of AMP-deaminase (AMPD) could be effective therapeutic strategy in heart disease associated with cardiac ischemia. To establish experimental model to study protective mechanisms of AMPD inhibition we developed conditional, cardiac specific knock-outs in Cre recombinase system. AMPD3 floxed mice were crossed with Mer-Cre-Mer mice. Tamoxifen was injected to induce Cre recombinase. After two weeks, hearts, skeletal muscle, liver, kidney, and blood were collected and activities of AMPD and related enzymes were analyzed using HPLC-based procedure. We demonstrate loss of more than 90% of cardiac AMPD activity in the heart of AMPD3-/-mice while other enzymes of nucleotide metabolism such as adenosine deaminase, purine nucleoside phosphorylase were not affected. Surprisingly, activity of AMPD was also reduced in the erythrocytes and in the kidney by 20%-30%. No change of AMPD activity was observed in the skeletal muscle and the liver. PMID:24940686

  15. Regulation of Activation-Induced Cytidine Deaminase DNA Deamination Activity in B Cells by Serine-38 Phosphorylation

    PubMed Central

    Basu, Uttiya; Franklin, Andrew; Schwer, Bjoern; Cheng, Hwei-Ling; Chaudhuri, Jayanta; Alt, Frederick W.

    2012-01-01

    Human and mouse immunoglobulin (Ig) genes are diversified in mature B cells by distinct processes known as Ig heavy chain class switch recombination (CSR) and Ig variable region exon somatic hypermutation (SHM). These DNA modification processes are initiated by activation-induced cytidine deaminase (AID), a DNA cytidine deaminase predominantly expressed in activated B cells. AID is post-transcriptionally regulated via multiple mechanisms including microRNA regulation, nucleo-cytoplasmic shuttling, ubiquitination and phosphorylation. Among these regulatory processes, AID phosphorylation at Serine-38 (S38) has been a focus of particularly intense study and debate. Here, we discuss recent biochemical and mouse genetic studies that begin to elucidate the functional significance of AID S38 phosphorylation in the context of the evolution of this mode of AID regulation and the potential roles that it may play in activated B cells during a normal immune response. PMID:19442251

  16. Active DNA demethylation of the vertebrate genomes by DNA methyltransferases: deaminase, dehydroxymethylase or demethylase?

    PubMed

    Wang, Keh-Yang; Chen, Chun-Chang; Shen, Che-Kun James

    2014-06-01

    Vertebrate DNA methyltransferases (DNMTs) have been thought to primarily function to covalently add a methyl group to the 5-position of cytosine. However, recent discovery of the DNA demethylation and dehydroxymethylation activities of DNMTs in vitro suggest new routes to complete the dynamic cycle of DNA methylation-demethylation of the vertebrate genomes. The in vitro reaction conditions suggest that vertebrate DNMTs can switch from DNA methylases to DNA dehydroxymethylases under oxidative stress and to DNA demethylases in the presence of calcium ion under nonreducing conditions. These environmental parameters provide clues regarding the choices in vivo of DNMT activities utilized in different physiological systems. In particular, the nature of these parameters suggest that the DNA demethylation and dehydroxymethylation activities of the vertebrate DNMTs play essential roles in multiple biological processes including early embryo development, regulation of neuronal plasticity, tumorigenesis and hormone-regulated transcription. PMID:25111488

  17. S -Adenosylhomocysteine hydrolase activity in a lymphoblastoid cell line from a patient with adenosine deaminase deficiency disease

    Microsoft Academic Search

    S. Tsuchiya; S. Nakae; T. Konno; K. Tada

    1981-01-01

    S-Adenosylhomocysteine (S-AdoHcy) hydrolase activity in a lymphoblastoid cell line from a patient with adenosine deaminase deficiency disease (ADA(–)LCL) was found to be approximately 60% of that in ADA(+)lymphoblastoid cell lines.S-AdoHcy hydrolase of ADA(–)LCL was more sensitive to inhibition by 2-deoxyadenosine as compared with that of ADA(+)LCL. The inhibitory effect of 2-deoxyadenosine was evident in cell growth and immunoglobulin production of

  18. Lymphospecific Toxicity in Adenosine Deaminase Deficiency and Purine Nucleoside Phosphorylase Deficiency: Possible Role of Nucleoside Kinase(s)

    Microsoft Academic Search

    Dennis A. Carson; Jonathan Kaye; J. E. Seegmiller

    1977-01-01

    Inherited deficiencies of the enzymes adenosine deaminase (adenosine aminohydrolase; EC 3.5.4.4) and purine nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase; EC 2.4.2.1) preferentially interfere with lymphocyte development while sparing most other organ systems. Previous experiments have shown that through the action of specific kinases, nucleosides can be ``trapped'' intracellularly in the form of 5'-phosphates. We therefore measured the ability of newborn human tissues

  19. 861. Recombinant Adeno-Associated Virus Vector Mediated Gene Therapy of Adenosine Deaminase Deficiency in a Mouse Model

    Microsoft Academic Search

    Jennifer M. Crawford; Teh-sheng Chan; Lillian L. Chan

    2006-01-01

    Clinical trials for adenosine deaminase deficiency associated-severe combined immunodeficiency (ADA-SCID) have been ongoing for nearly two decades, yet unequivocal success has not been achieved. The use of inadequate delivery systems with severe side-effects and lack of an animal model have limited progress. Recently, our lab has generated a developmentally regulated ADA knockout mouse to study the pathogenesis of ADA-SCID and

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

    Microsoft Academic Search

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

    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 ki values. The models were trained to solve two-class problems. Unlike the previous work in which multiple linear regression was

  1. Peripheral antinociceptive effect of an adenosine kinase inhibitor, with augmentation by an adenosine deaminase inhibitor, in the rat formalin test

    Microsoft Academic Search

    Jana Sawynok; Allison Reid; Anthony Poon

    1998-01-01

    This study examined the ability of an adenosine kinase inhibitor (5?-amino-5?-deoxyadenosine; NH2dAD), an adenosine deaminase inhibitor (2?-deoxycoformycin), and combinations of these agents to produce a peripheral modulation of the pain signal in the low concentration formalin model. Drugs were administered in combination with 0.5% formalin, or into the contralateral hindpaw to test for systemic effects, and episodes of flinching behaviors

  2. Modelling the Yeast Interactome

    PubMed Central

    Janji?, Vuk; Sharan, Roded; Pržulj, Nataša

    2014-01-01

    The topology behind biological interaction networks has been studied for over a decade. Yet, there is no definite agreement on the theoretical models which best describe protein-protein interaction (PPI) networks. Such models are critical to quantifying the significance of any empirical observation regarding those networks. Here, we perform a comprehensive analysis of yeast PPI networks in order to gain insights into their topology and its dependency on interaction-screening technology. We find that: (1) interaction-detection technology has little effect on the topology of PPI networks; (2) topology of these interaction networks differs in organisms with different cellular complexity (human and yeast); (3) clear topological difference is present between PPI networks, their functional sub-modules, and their inter-functional “linkers”; (4) high confidence PPI networks have more “geometrical” topology compared to predicted, incomplete, or noisy PPI networks; and (5) inter-functional “linker” proteins serve as mediators in signal transduction, transport, regulation and organisational cellular processes. PMID:24589662

  3. Long-term expression of human adenosine deaminase in rhesus monkeys transplanted with retrovirus-infected bone-marrow cells.

    PubMed Central

    van Beusechem, V W; Kukler, A; Heidt, P J; Valerio, D

    1992-01-01

    Gene transfer into hemopoietic stem cells could offer a lasting cure for a variety of congenital disorders. As a preclinical test for such a gene therapy, rhesus monkeys were transplanted with autologous bone-marrow cells infected with helper-free recombinant retroviruses carrying the human adenosine deaminase gene. The in vivo regenerative capacity of the infected bone marrow could be conserved, suggesting survival of repopulating hemopoietic stem cells. In the hemopoietic system of transplanted animals the foreign gene could be observed for as long as the animals were analyzed (in two monkeys greater than 1 yr after transplantation). Genetically modified cell types and tissues included peripheral blood mononuclear cells, granulocytes, bone-marrow cells of various densities, and spleen and lymph nodes. The presence of the provirus in the short-living granulocytes greater than 1 yr after bone-marrow transplantation provided evidence for the transduction of very primitive hemopoietic progenitors. Moreover, the gene transfer resulted in sustained production of functional human adenosine deaminase enzyme in peripheral blood mononuclear cells. These results demonstrate the feasibility of bone-marrow gene-therapy approaches, in particular for treating adenosine deaminase deficiency. Images PMID:1502175

  4. 5-methyl-cytosine and 5-hydroxy-methyl-cytosine in the genome of Biomphalaria glabrata, a snail intermediate host of Schistosoma mansoni

    PubMed Central

    2013-01-01

    Background Biomphalaria glabrata is the mollusc intermediate host for Schistosoma mansoni, a digenean flatworm parasite that causes human intestinal schistosomiasis. An estimated 200 million people in 74 countries suffer from schistosomiasis, in terms of morbidity this is the most severe tropical disease after malaria. Epigenetic information informs on the status of gene activity that is heritable, for which changes are reversible and that is not based on the DNA sequence. Epigenetic mechanisms generate variability that provides a source for potentially heritable phenotypic variation and therefore could be involved in the adaptation to environmental constraint. Phenotypic variations are particularly important in host-parasite interactions in which both selective pressure and rate of evolution are high. In this context, epigenetic changes are expected to be major drivers of phenotypic plasticity and co-adaptation between host and parasite. Consequently, with characterization of the genomes of invertebrates that are parasite vectors or intermediate hosts, it is also essential to understand how the epigenetic machinery functions to better decipher the interplay between host and parasite. Methods The CpGo/e ratios were used as a proxy to investigate the occurrence of CpG methylation in B. glabrata coding regions. The presence of DNA methylation in B. glabrata was also confirmed by several experimental approaches: restriction enzymatic digestion with isoschizomers, bisulfite conversion based techniques and LC-MS/MS analysis. Results In this work, we report that DNA methylation, which is one of the carriers of epigenetic information, occurs in B. glabrata; approximately 2% of cytosine nucleotides are methylated. We describe the methylation machinery of B. glabrata. Methylation occurs predominantly at CpG sites, present at high ratios in coding regions of genes associated with housekeeping functions. We also demonstrate by bisulfite treatment that methylation occurs in multiple copies of Nimbus, a transposable element. Conclusions This study details DNA methylation for the first time, one of the carriers of epigenetic information in B. glabrata. The general characteristics of DNA methylation that we observed in the B. glabrata genome conform to what epigenetic studies have reported from other invertebrate species. PMID:23742053

  5. 6-Thioguanine Perturbs Cytosine Methylation at CpG Dinucleotide Site by DNA Methyltransferases in Vitro and Acts as a DNA Demethylating Agent in vivo

    PubMed Central

    Wang, Hongxia; Wang, Yinsheng

    2009-01-01

    Thiopurines are among the most successful chemotherapeutic agents for treating a number of human diseases including acute lymphoblastic leukemia. The mechanisms through which the thiopurines elicit their cytotoxic effects remain unclear. We postulate that the incorporation of 6-thioguanine into CpG site may perturb the methyltransferase-mediated cytosine methylation at this site, thereby interfering with the epigenetic pathways of gene regulation. To gain biochemical evidence for this hypothesis, we assessed, by using a restriction enzyme digestion coupled with LC-MS/MS method, the impact of 6-thioguanine on cytosine methylation mediated by two DNA methyltransferases, human DNMT1 and bacterial HpaII. Our results revealed that the incorporation of 6-thioguanine into CpG site could affect the methylation of cytosine residue by both methyltransferases and the effect on cytosine methylation is dependent on the position of 6-thioguanine with respect to the cytosine to be methylated. The presence of 6-thioguanine at methylated CpG site enhanced the DNMT1- mediated methylation of the opposing cytosine in the complementary strand, whereas the presence of 6-thioguanine at unmethylated CpG site abolished almost completely the methylation of its 5? adjacent cytosine by both DNMT1 and HpaII. We further demonstrated that the treatment of Jurkat T cells, which were derived from acute lymphoblastic leukemia, with 6- thioguanine could result in an appreciable drop in the level of global cytosine methylation. These results showed that 6-thioguanine, after being incorporated into DNA, may perturb the epigenetic pathway of gene regulation. PMID:19236003

  6. Strand displacement recognition of mixed adenine–cytosine sequences in double stranded DNA by thymine–Guanine PNA (Peptide nucleic acid)

    Microsoft Academic Search

    Peter E Nielsen; Michael Egholm

    2001-01-01

    Mixed pyrimidine–purine peptide nucleic acids (PNAs) composed of thymines and guanines are shown to form a PNA2–DNA triplex with Watson–Crick complementary adenine–cytosine oligonucleotides and to bind complementary adenine–cytosine targets in double stranded DNA by helix invasion. These results for the first time demonstrate binding of an unmodified PNA oligomer to a mixed pyrimidine–purine target in double stranded DNA and illustrate

  7. Synthesis of adenine, guanine, cytosine, and other nitrogen organic compounds by a Fischer-Tropsch-like process.

    NASA Technical Reports Server (NTRS)

    Yang, C. C.; Oro, J.

    1971-01-01

    Study of the formation of purines, pyrimidines, and other bases from CO, H2, and NH3 under conditions similar to those used in the Fischer-Tropsch process. It is found that industrial nickel/iron alloy catalyzes the synthesis of adenine, guanine, cytosine, and other nitrogenous compounds from mixtures of CO, H2, and NH3 at temperatures of about 600 C. Sufficient sample was accumulated to isolate as solid products adenine, guanine, and cytosine, which were identified by infrared spectrophotometry. In the absence of nickel/iron catalyst, at 650 C, or in the presence of this catalyst, at 450 C, no purines or pyrimidines were synthesized. These results confirm and extend some of the work reported by Kayatsu et al. (1968).

  8. Intramolecular flexibility of DNA bases in adenine–thymine and guanine–cytosine Watson–Crick base pairs

    Microsoft Academic Search

    OlegV Shishkin; Ji??? Šponer; Pavel Hobza

    1999-01-01

    The conformational flexibility of pyrimidine rings in adenine (A)–thymine (T) and guanine (G)–cytosine (C) Watson–Crick base pairs was investigated at the ab initio Hartree–Fock (HF) level using 6-31G** basis set. Transition of these rings from the planar equilibrium conformation to a distorted sofa conformation with torsion angles of 20° results in a marginal energy increase of approximately 1.3kcal\\/mol for the

  9. Cytosine arabinoside rapidly activates Bax-dependent apoptosis and a delayed Bax-independent death pathway in sympathetic neurons

    Microsoft Academic Search

    C G Besirli; T L Deckwerth; R J Crowder; R S Freeman; E M Johnson; EM Johnson Jr.

    2003-01-01

    Cytosine arabinoside (ara-C) is a nucleoside analog used in the treatment of hematologic malignancies. One of the major side effects of ara-C chemotherapy is neurotoxicity. In this study, we have further characterized the cell death induced by ara-C in sympathetic neurons. Similar to neurons undergoing trophic factor deprivation-induced apoptosis, ara-C-exposed neurons became hypometabolic before death and upregulated c-myb, c-fos, and

  10. Interaction Between Yeasts and Zinc

    NASA Astrophysics Data System (ADS)

    Nicola, Raffaele De; Walker, Graeme

    Zinc is an essential trace element in biological systems. For example, it acts as a cellular membrane stabiliser, plays a critical role in gene expression and genome modification and activates nearly 300 enzymes, including alcohol dehydrogenase. The present chapter will be focused on the influence of zinc on cell physiology of industrial yeast strains of Saccharomyces cerevisiae, with special regard to the uptake and subsequent utilisation of this metal. Zinc uptake by yeast is metabolism-dependent, with most of the available zinc translocated very quickly into the vacuole. At cell division, zinc is distributed from mother to daughter cells and this effectively lowers the individual cellular zinc concentration, which may become zinc depleted at the onset of the fermentation. Zinc influences yeast fermentative performance and examples will be provided relating to brewing and wine fermentations. Industrial yeasts are subjected to several stresses that may impair fermentation performance. Such stresses may also impact on yeast cell zinc homeostasis. This chapter will discuss the practical implications for the correct management of zinc bioavailability for yeast-based biotechnologies aimed at improving yeast growth, viability, fermentation performance and resistance to environmental stresses

  11. Lager Yeast Comes of Age

    PubMed Central

    2014-01-01

    Alcoholic fermentations have accompanied human civilizations throughout our history. Lager yeasts have a several-century-long tradition of providing fresh beer with clean taste. The yeast strains used for lager beer fermentation have long been recognized as hybrids between two Saccharomyces species. We summarize the initial findings on this hybrid nature, the genomics/transcriptomics of lager yeasts, and established targets of strain improvements. Next-generation sequencing has provided fast access to yeast genomes. Its use in population genomics has uncovered many more hybridization events within Saccharomyces species, so that lager yeast hybrids are no longer the exception from the rule. These findings have led us to propose network evolution within Saccharomyces species. This “web of life” recognizes the ability of closely related species to exchange DNA and thus drain from a combined gene pool rather than be limited to a gene pool restricted by speciation. Within the domesticated lager yeasts, two groups, the Saaz and Frohberg groups, can be distinguished based on fermentation characteristics. Recent evidence suggests that these groups share an evolutionary history. We thus propose to refer to the Saaz group as Saccharomyces carlsbergensis and to the Frohberg group as Saccharomyces pastorianus based on their distinct genomes. New insight into the hybrid nature of lager yeast will provide novel directions for future strain improvement. PMID:25084862

  12. Lager yeast comes of age.

    PubMed

    Wendland, Jürgen

    2014-10-01

    Alcoholic fermentations have accompanied human civilizations throughout our history. Lager yeasts have a several-century-long tradition of providing fresh beer with clean taste. The yeast strains used for lager beer fermentation have long been recognized as hybrids between two Saccharomyces species. We summarize the initial findings on this hybrid nature, the genomics/transcriptomics of lager yeasts, and established targets of strain improvements. Next-generation sequencing has provided fast access to yeast genomes. Its use in population genomics has uncovered many more hybridization events within Saccharomyces species, so that lager yeast hybrids are no longer the exception from the rule. These findings have led us to propose network evolution within Saccharomyces species. This "web of life" recognizes the ability of closely related species to exchange DNA and thus drain from a combined gene pool rather than be limited to a gene pool restricted by speciation. Within the domesticated lager yeasts, two groups, the Saaz and Frohberg groups, can be distinguished based on fermentation characteristics. Recent evidence suggests that these groups share an evolutionary history. We thus propose to refer to the Saaz group as Saccharomyces carlsbergensis and to the Frohberg group as Saccharomyces pastorianus based on their distinct genomes. New insight into the hybrid nature of lager yeast will provide novel directions for future strain improvement. PMID:25084862

  13. Yeasts: From genetics to biotechnology

    SciTech Connect

    Russo, S.; Poli, G. [Univ. of Milan (Italy); Siman-Tov, R.B. [Univ. of Jerusalem, Rehovot (Israel)

    1995-12-31

    Yeasts have been known and used in food and alcoholic fermentations ever since the Neolithic Age. In more recent times, on the basis of their peculiar features and history, yeasts have become very important experimental models in both microbiological and genetic research, as well as the main characters in many fermentative production processes. In the last 40 years, advances in molecular biology and genetic engineering have made possible not only the genetic selection of organisms, but also the genetic modification of some of them, especially the simplest of them, such as bacteria and yeasts. These discoveries have led to the availability of new yeast strains fit to fulfill requests of industrial production and fermentation. Moreover, genetically modified and transformed yeasts have been constructed that are able to produce large amounts of biologically active proteins and enzymes. Thus, recombinant yeasts make it easier to produce drugs, biologically active products, diagnostics, and vaccines, by inexpensive and relatively simple techniques. Yeasts are going to become more and more important in the {open_quotes}biotechnological revolution{close_quotes} by virtue of both their features and their very long and safe use in human nutrition and industry. 175 refs., 4 figs., 6 tabs.

  14. Marine yeast isolation and industrial application

    PubMed Central

    Zaky, Abdelrahman Saleh; Tucker, Gregory A; Daw, Zakaria Yehia; Du, Chenyu

    2014-01-01

    Over the last century, terrestrial yeasts have been widely used in various industries, such as baking, brewing, wine, bioethanol and pharmaceutical protein production. However, only little attention has been given to marine yeasts. Recent research showed that marine yeasts have several unique and promising features over the terrestrial yeasts, for example higher osmosis tolerance, higher special chemical productivity and production of industrial enzymes. These indicate that marine yeasts have great potential to be applied in various industries. This review gathers the most recent techniques used for marine yeast isolation as well as the latest applications of marine yeast in bioethanol, pharmaceutical and enzyme production fields. PMID:24738708

  15. Molecular cloning, expression, and characterization of starfish DNA (cytosine-5)-methyltransferases.

    PubMed

    Fujihara, Yoshihito; Miyasako, Hiroshi; Kato, Kumiko; Hayashi, Tadahiro; Toraya, Tetsuo

    2012-01-01

    To determine whether and if so how a DNA methylation-dependent epigenetic mechanism for transcriptional gene silencing functions in Echinoderms, we cloned and sequenced dnmt1 and dnmt3 cDNAs of the starfish Asterina pectinifera. Since the Strongylocentrotus purpuratus genome has only two loci of DNA (cytosine-5)-methyltransferase genes encoding Dnmt1 and Dnmt3, they might constitute a sufficient set of dnmt genes in Echinoderms. The starfish Dnmt3 whose cDNA we cloned showed highest homology to a mammalian Dnmt3a2 splicing variant. Essentially all the characteristic motifs and sequences of the mammalian counterparts were found in the starfish Dnmts as well, except that a typical PCNA binding domain motif was lacking in the starfish Dnmt1. RT-PCR analysis indicated that the dnmt1 mRNA exists in both ovary and oocytes, but its levels in other tissues were very low or almost negligible. In contrast, the dnmt3 mRNA was detected only in the ovary, and not at all in the oocytes. The size of a dnmt1 transcript was about 6.5 kb on Northern blot analysis. On heterologous expression, the starfish Dnmt1 protein was expressed in insect cells in catalytically active form. PMID:22972351

  16. ESI-MS studies of palladium (II) complexes with 1-(p-toluenesulfonyl)cytosine/cytosinato ligands.

    PubMed

    Kobeti?, Renata; Gembarovski, Dubravka; Visnjevac, Aleksandar; Zini?, Biserka; Gabelica-Markovi?, Vesna

    2010-01-01

    The mononuclear complex Pd(1-TosC-N3)(2)Cl(2) (2) containing 1-(p-toluenesulfonyl)cytosine (1) as a ligand, as well as dinuclear complexes Pd(2)(1-TosC(-)-N3,N4)(4) (3) and Pd(2)(1-TosC(-)-N3,N4)(2)DMSO(2)Cl(2) (4) containing the ligand anion (1-TosC(-)), was mass analyzed by electrospray ionization ion trap MS/MS and high resolution MS. Complexes 3 and 4 were obtained by recrystallization of 2 from DMF and DMSO, respectively. The behavior of complex 2 in different solutions was monitored by electrospray ionization mass spectrometry (ESI-MS). Under the applied ESI-MS conditions, complex 2 in methanol reorganized itself dominantly as new complex 3 and the solvent did not coordinate the formed species. In H(2)O/DMSO, CH(3)CN/DMSO and CH(3)OH/DMSO solutions, complex 2 formed several new species with solvent molecules involved in their structure, e.g. complex 4 was formed as the major product. The newly formed species were also examined by LC-MS-DAD, confirming the solvent induced reorganization and the solution instability of complex 2. PMID:19882593

  17. A computational NQR study on the hydrogen-bonded lattice of cytosine-5-acetic acid.

    PubMed

    Mirzaei, Mahmoud; Hadipour, Nasser L

    2008-04-15

    A computational study at the level of density functional theory (DFT) employing 6-311++G** standard basis set was carried out to evaluate nuclear quadrupole resonance (NQR) spectroscopy parameters in cytosine-5-acetic acid (C5AA). Since the electric field gradient (EFG) tensors are very sensitive to the electrostatic environment at the sites of quadruple nuclei, the most possible interacting molecules with the target one were considered in a five-molecule model system of C5AA using X-ray coordinates transforming. The hydrogen atoms positions were optimized and two model systems of original and H-optimized C5AA were considered in NQR calculations. The calculated EFG tensors at the sites of (17)O, (14)N, and (2)H nuclei were converted to their experimentally measurable parameters, quadrupole coupling constants and asymmetry parameters. The evaluated NQR parameters reveal that the nuclei in original and H-optimized systems contribute to different hydrogen bonding (HB) interaction. The comparison of calculated parameters between optimized isolated gas-phase and crystalline monomer also shows the relationship between the structural deformation and NQR parameters in C5AA. The basis set superposition error (BSSE) calculations yielded no significant errors for employed basis set in the evaluation of NQR parameters. All the calculations were performed by Gaussian 98 package of program. PMID:17926341

  18. Genome-wide assays that identify and quantify modified cytosines in human disease studies.

    PubMed

    Ulahannan, Netha; Greally, John M

    2015-01-01

    The number of different assays that has been published to study DNA methylation is extensive, complemented by recently described assays that test modifications of cytosine other than the most abundant 5-methylcytosine (5mC) variant. In this review, we describe the considerations involved in choosing how to study 5mC throughout the genome, with an emphasis on the common application of testing for epigenetic dysregulation in human disease. While microarray studies of 5mC continue to be commonly used, these lack the additional qualitative information from sequencing-based approaches that is increasingly recognized to be valuable. When we test the representation of functional elements in the human genome by several current assay types, we find that no survey approach interrogates anything more than a small minority of the nonpromoter cis-regulatory sites where DNA methylation variability is now appreciated to influence gene expression and to be associated with human disease. However, whole-genome bisulphite sequencing (WGBS) adds a substantial representation of loci at which DNA methylation changes are unlikely to be occurring with transcriptional consequences. Our assessment is that the most effective approach to DNA methylation studies in human diseases is to use targeted bisulphite sequencing of the cis-regulatory loci in a cell type of interest, using a capture-based or comparable system, and that no single design of a survey approach will be suitable for all cell types. PMID:25788985

  19. Dyson norms in XUV and strong-field ionization of polyatomics: Cytosine and uracil

    NASA Astrophysics Data System (ADS)

    Spanner, Michael; Patchkovskii, Serguei; Zhou, Congyi; Matsika, Spiridoula; Kotur, Marija; Weinacht, Thomas C.

    2012-11-01

    The extreme-ultraviolet (XUV) and strong-field valence ionization of cytosine and uracil is considered. We examine some simple estimates of the relative yields of the cation states populated following ionization and compare these to the results of a recently developed ab initio-type numerical model designed to compute strong-field ionization of molecules, the so-called time-dependent resolution in ionic states (TD-RIS) method. In analogy with one-photon XUV ionization, where the photoionization matrix elements can be related to the Dyson orbitals, we construct estimates for the yield of strong-field ionization (SFI) to different cation states based on the Dyson orbital norms and the Keldysh tunneling ionization rate. In the case of XUV ionization, the Dyson norms are shown to be good predictors of the relative cation yields when compared with the TD-RIS yields. The Dyson- and Keldysh-based models underestimate the yield to excited cation states in the case of SFI. The increased yield to the excited cation states in the TD-RIS results is attributed to the inclusion of multielectron effects and continuum structure not present in the simple models. The molecular Ammosov-Delone-Krainov (MO-ADK) method of calculating SFI is also considered. This later method is seen to agree more closely with the Dyson- and Keldysh-based estimates as it also fails to capture the multielectron effects and continuum structure included in the TD-RIS approach.

  20. NGSmethDB: an updated genome resource for high quality, single-cytosine resolution methylomes

    PubMed Central

    Geisen, Stefanie; Barturen, Guillermo; Alganza, Ángel M.; Hackenberg, Michael; Oliver, José L.

    2014-01-01

    The updated release of ‘NGSmethDB’ (http://bioinfo2.ugr.es/NGSmethDB) is a repository for single-base whole-genome methylome maps for the best-assembled eukaryotic genomes. Short-read data sets from NGS bisulfite-sequencing projects of cell lines, fresh and pathological tissues are first pre-processed and aligned to the corresponding reference genome, and then the cytosine methylation levels are profiled. One major improvement is the application of a unique bioinformatics protocol to all data sets, thereby assuring the comparability of all values with each other. We implemented stringent quality controls to minimize important error sources, such as sequencing errors, bisulfite failures, clonal reads or single nucleotide variants (SNVs). This leads to reliable and high-quality methylomes, all obtained under uniform settings. Another significant improvement is the detection in parallel of SNVs, which might be crucial for many downstream analyses (e.g. SNVs and differential-methylation relationships). A next-generation methylation browser allows fast and smooth scrolling and zooming, thus speeding data download/upload, at the same time requiring fewer server resources. Several data mining tools allow the comparison/retrieval of methylation levels in different tissues or genome regions. NGSmethDB methylomes are also available as native tracks through a UCSC hub, which allows comparison with a wide range of third-party annotations, in particular phenotype or disease annotations. PMID:24271385

  1. Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome

    PubMed Central

    Pedersen, Jakob Skou; Valen, Eivind; Velazquez, Amhed M. Vargas; Parker, Brian J.; Rasmussen, Morten; Lindgreen, Stinus; Lilje, Berit; Tobin, Desmond J.; Kelly, Theresa K.; Vang, Søren; Andersson, Robin; Jones, Peter A.; Hoover, Cindi A.; Tikhonov, Alexei; Prokhortchouk, Egor; Rubin, Edward M.; Sandelin, Albin; Gilbert, M. Thomas P.; Krogh, Anders; Willerslev, Eske; Orlando, Ludovic

    2014-01-01

    Epigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence data generated from hair shafts of a 4000-yr-old Paleo-Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery of the expected signals at promoter regions, exon/intron boundaries, and CTCF sites. The top-scoring nucleosome calls revealed distinct DNA positioning biases, attesting to nucleotide-level accuracy. The ancient methylation levels exhibited high conservation over time, clustering closely with modern hair tissues. Using ancient methylation information, we estimated the age at death of the Saqqaq individual and illustrate how epigenetic information can be used to infer ancient gene expression. Similar epigenetic signatures were found in other fossil material, such as 110,000- to 130,000-yr-old bones, supporting the contention that ancient epigenomic information can be reconstructed from a deep past. Our findings lay the foundation for extracting epigenomic information from ancient samples, allowing shifts in epialleles to be tracked through evolutionary time, as well as providing an original window into modern epigenomics. PMID:24299735

  2. SCAN database: facilitating integrative analyses of cytosine modification and expression QTL

    PubMed Central

    Zhang, Wei; Gamazon, Eric R.; Zhang, Xu; Konkashbaev, Anuar; Liu, Cong; Szilágyi, Keely L.; Dolan, M. Eileen; Cox, Nancy J.

    2015-01-01

    Functional annotation of genetic variants including single nucleotide polymorphisms (SNPs) and copy number variations (CNV) promises to greatly improve our understanding of human complex traits. Previous transcriptomic studies involving individuals from different global populations have investigated the genetic architecture of gene expression variation by mapping expression quantitative trait loci (eQTL). Functional interpretation of genome-wide association studies (GWAS) has identified enrichment of eQTL in top signals from GWAS of human complex traits. The SCAN (SNP and CNV Annotation) database was developed as a web-based resource of genetical genomic studies including eQTL detected in the HapMap lymphoblastoid cell line samples derived from apparently healthy individuals of European and African ancestry. Considering the critical roles of epigenetic gene regulation, cytosine modification quantitative trait loci (mQTL) are expected to add a crucial layer of annotation to existing functional genomic information. Here, we describe the new features of the SCAN database that integrate comprehensive mQTL mapping results generated in the HapMap CEU (Caucasian residents from Utah, USA) and YRI (Yoruba people from Ibadan, Nigeria) LCL samples and demonstrate the utility of the enhanced functional annotation system. Database URL: http://www.scandb.org/ PMID:25818895

  3. Variation in cytosine methylation patterns during ploidy level conversions in Eragrostis curvula.

    PubMed

    Ochogavía, Ana C; Cervigni, Gerardo; Selva, Juan P; Echenique, Viviana C; Pessino, Silvina C

    2009-05-01

    In many species polyploidization involves rearrangements of the progenitor genomes, at both genetic and epigenetic levels. We analyzed the cytosine methylation status in a 'tetraploid-diploid-tetraploid' series of Eragrostis curvula with a common genetic background by using the MSAP (Methylation-sensitive Amplified Polymorphism) technique. Considerable levels of polymorphisms were detected during ploidy conversions. The total level of methylation observed was lower in the diploid genotype compared to the tetraploid ones. A significant proportion of the epigenetic modifications occurring during the tetraploid-diploid conversion reverted during the diploid-tetraploid one. Genetic and expression data from previous work were used to analyze correlation with methylation variation. All genetic, epigenetic and gene expression variation data correlated significantly when compared by pairs in simple Mantel tests. Dendrograms reflecting genetic, epigenetic and expression distances as well as principal coordinate analysis suggested that plants of identical ploidy levels present similar sets of data. Twelve (12) different genomic fragments displaying different methylation behavior during the ploidy conversions were isolated, sequenced and characterized. PMID:19160057

  4. PMMA/polysaccharides nanofilm loaded with adenosine deaminase inhibitor for targeted anti-inflammatory drug delivery.

    PubMed

    Redolfi Riva, Eugenio; Desii, Andrea; Sartini, Stefania; La Motta, Concettina; Mazzolai, Barbara; Mattoli, Virgilio

    2013-10-29

    A novel drug delivery vector, a free-standing polymeric ultrathin film (nanofilm) composed of PMMA and a polysaccharides multilayer, is presented. Chitosan and sodium alginate are alternatively deposited by spin-assisted LbL assembly onto a plasma-treated PMMA thin film. Hydrophobic anti-inflammatory drugs, an adenosine deaminase inhibitor (APP) and its fluorescent dansyl derivate (APP-Dns), are encapsulated inside the LbL multilayer using a simple casting deposition procedure. The resulting drug loaded nanofilm can be suspended in water upon dissolution of a PVA sacrificial layer. Morphological characterization of the nanofilm shows that PMMA/LbL nanofilms possess nanometric thickness (<200 nm) and very low surface roughness (1-2 nm for drug loaded nanofilms and <1 nm for blank nanofilm). Drug loaded films exhibit a diffusion controlled release mechanism following the Korsmayer-Peppas release model, confirmed by the fit of release data with a characteristic power law. Drug release is impaired through the PMMA layer, which acts effectively as a barrier for drug transport. This ultrathin polymer film can find application as a nanopatch for targeted inflammatory drug delivery to treat localized pathologies as inflammatory bowel disease. PMID:24073802

  5. Outcome of hematopoietic stem cell transplantation for adenosine deaminase-deficient severe combined immunodeficiency.

    PubMed

    Hassan, Amel; Booth, Claire; Brightwell, Alex; Allwood, Zoe; Veys, Paul; Rao, Kanchan; Hönig, Manfred; Friedrich, Wilhelm; Gennery, Andrew; Slatter, Mary; Bredius, Robbert; Finocchi, Andrea; Cancrini, Caterina; Aiuti, Alessandro; Porta, Fulvio; Lanfranchi, Arnalda; Ridella, Michela; Steward, Colin; Filipovich, Alexandra; Marsh, Rebecca; Bordon, Victoria; Al-Muhsen, Saleh; Al-Mousa, Hamoud; Alsum, Zobaida; Al-Dhekri, Hasan; Al Ghonaium, Abdulaziz; Speckmann, Carsten; Fischer, Alain; Mahlaoui, Nizar; Nichols, Kim E; Grunebaum, Eyal; Al Zahrani, Daifulah; Roifman, Chaim M; Boelens, Jaap; Davies, E Graham; Cavazzana-Calvo, Marina; Notarangelo, Luigi; Gaspar, H Bobby

    2012-10-25

    Deficiency of the purine salvage enzyme adenosine deaminase leads to SCID (ADA-SCID). Hematopoietic cell transplantation (HCT) can lead to a permanent cure of SCID; however, little data are available on outcome of HCT for ADA-SCID in particular. In this multicenter retrospective study, we analyzed outcome of HCT in 106 patients with ADA-SCID who received a total of 119 transplants. HCT from matched sibling and family donors (MSDs, MFDs) had significantly better overall survival (86% and 81%) in comparison with HCT from matched unrelated (66%; P < .05) and haploidentical donors (43%; P < .001). Superior overall survival was also seen in patients who received unconditioned transplants in comparison with myeloablative procedures (81% vs 54%; P < .003), although in unconditioned haploidentical donor HCT, nonengraftment was a major problem. Long-term immune recovery showed that regardless of transplant type, overall T-cell numbers were similar, although a faster rate of T-cell recovery was observed after MSD/MFD HCT. Humoral immunity and donor B-cell engraftment was achieved in nearly all evaluable surviving patients and was seen even after unconditioned HCT. These data detail for the first time the outcomes of HCT for ADA-SCID and show that, if patients survive HCT, long-term cellular and humoral immune recovery is achieved. PMID:22791287

  6. The Adenosine Deaminase Gene Polymorphism Is Associated with Chronic Heart Failure Risk in Chinese

    PubMed Central

    He, Hai-Rong; Li, Yuan-Jie; He, Gong-Hao; Wang, Ya-Jun; Zhai, Ya-Jing; Xie, Jiao; Zhang, Wei-Peng; Dong, Ya-Lin; Lu, Jun

    2014-01-01

    Adenosine (Ado) is an important cardioprotective agent. Since endogenous Ado levels are affected by the enzyme Ado deaminase (ADA), polymorphisms within the ADA gene may exert some effect on chronic heart failure (CHF). This study applied a case-control investigation to 300 northern Chinese Han CHF patients and 400 ethnicity-matched healthy controls in which nine single-nucleotide polymorphisms (SNPs) of ADA were genotyped and association analyses were performed. Odds ratios (ORs) with 95% confidence intervals (CI) were used to assess the association. Overall, rs452159 polymorphism in ADA gene was significantly associated with susceptibility to CHF under the dominant model (p = 0.013, OR = 1.537, 95% CI = 1.10–2.16), after adjustment for age, sex, and traditional cardiovascular risk factors. No difference in genotype distribution and allele frequency for the rs452159 according to the functional New York Heart Association class was found. Furthermore, the values of left ventricular ejection fraction, left-ventricle end-diastolic diameter or left-ventricle end-systolic diameter did not differ significantly among the different rs452159 genotype CHF patients. Although further studies with larger cohorts and other ethnicities are required to validate the conclusions, the findings of this study potentially provide novel insight into the pathogenesis of CHF. PMID:25170811

  7. Defective B cell tolerance in adenosine deaminase deficiency is corrected by gene therapy.

    PubMed

    Sauer, Aisha V; Morbach, Henner; Brigida, Immacolata; Ng, Yen-Shing; Aiuti, Alessandro; Meffre, Eric

    2012-06-01

    Adenosine deaminase (ADA) gene defects are among the most common causes of SCID. Restoration of purine metabolism and immune functions can be achieved by enzyme replacement therapy, or more effectively by bone marrow transplant or HSC gene therapy (HSC-GT). However, autoimmune complications and autoantibody production, including anti-nuclear antibodies (ANAs), frequently occur in ADA-SCID patients after treatment. To assess whether ADA deficiency affects the establishment of B cell tolerance, we tested the reactivity of recombinant antibodies isolated from single B cells of ADA-SCID patients before and after HSC-GT. We found that before HSC-GT, new emigrant/transitional and mature naive B cells from ADA-SCID patients contained more autoreactive and ANA-expressing clones, indicative of defective central and peripheral B cell tolerance checkpoints. We further observed impaired B cell receptor (BCR) and TLR functions in B cells after ADA inhibition, which may underlie the defects in B cell tolerance. Strikingly, after HSC-GT, ADA-SCID patients displayed quasi-normal early B cell tolerance checkpoints, as evidenced by restored removal of developing autoreactive and ANA-expressing B cells. Hence, ADA plays an essential role in controlling autoreactive B cell counterselection by regulating BCR and TLR functions. PMID:22622038

  8. Myeloid dysplasia and bone marrow hypocellularity in adenosine deaminase-deficient severe combined immune deficiency.

    PubMed

    Sokolic, Robert; Maric, Irina; Kesserwan, Chimene; Garabedian, Elizabeth; Hanson, I Celine; Dodds, Margaret; Buckley, Rebecca; Issekutz, Andrew C; Kamani, Naynesh; Shaw, Kit; Tan, Ben; Bali, Pawan; Hershfield, Michael S; Kohn, Donald B; Wayne, Alan S; Candotti, Fabio

    2011-09-01

    Genetic deficiency of adenosine deaminase (ADA) can cause profound lymphopenia and result in the clinical presentation of severe combined immune deficiency (SCID). However, because of the ubiquitous expression of ADA, ADA-deficient patients often present also with nonimmunologic clinical problems, affecting the skeletal, central nervous, endocrine, and gastrointestinal systems. We now report that myeloid dysplasia features and bone marrow hypocellularity are often found in patients with ADA-SCID. As a clinical correlate to this finding, we have observed vulnerability to antibiotic-induced myelotoxicity and prolonged neutropenia after nonmyeloablative chemotherapy. We have also noted that, in the absence of enzyme replacement therapy, absolute neutrophil counts of patients with ADA deficiency vary inversely with the accumulation of deoxynucleotides. These data have significant implications for the application of standard and investigational therapies to patients with ADA-SCID and support further studies to investigate the possibility that ADA deficiency is associated with a stem cell defect. These trials were registered at www.clinicaltrials.gov as #NCT00018018 and #NCT00006319. PMID:21725047

  9. Adenosine deaminase activity modulation by some street drug: molecular docking simulation and experimental investigation

    PubMed Central

    2014-01-01

    Background Adenosine deaminase (ADA) is an enzyme that plays important roles in proliferation, maturation, function and development of the immune system. ADA activity may be altered by variety of substances including synthetic or natural products. Morphine, cocaine and their analogs exert immune suppressive activities by decreasing immune system function. The purpose of this study is to confirm that this possible effect may be modulated by interaction of these substances with ADA activity by experimental and computational method. Methods The structural changes in ADA have been studied in presence of cocaine, ethylmorphine, homatropine, morphine and thebaine by determination of ADA hydrolytic activity, circular dichroism and fluorescence spectroscopy in different concentrations. Docking study was performed to evaluate interaction method of test compound with ADA active site using AutoDock4 software. Results According to in-vitro studies all compounds inhibited ADA with different potencies, however thebaine activated it at concentration below 50 ?M, ethylmorphine inhibited ADA at 35 ?M. Moreover, fluorescence spectra patterns were differed from compounds based on structural resemblance which were very considerable for cocaine and homatropine. Conclusion The results of this study confirms that opioids and some other stimulant drugs such as cocaine can alter immune function in illegal drug abusers. These findings may lead other investigators to develop a new class of ADA activators or inhibitors in the near future. PMID:24887139

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

    PubMed Central

    Khemka, Vineet Kumar; Bagchi, Debajit; Sen, Oishimaya; Bir, Aritri; Chakrabarti, Sasanka; Banerjee, Anindita

    2013-01-01

    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. PMID:24453844

  11. Sequence requirements for transcriptional arrest in exon 1 of the human adenosine deaminase gene

    SciTech Connect

    Zhi Chen; Kellems, R.E.; Innis, J.W. (Baylor Coll. of Medicine, Houston, TX (United States)); Sun, Minghua; Wright, D.A. (Univ. of Texas, Houston (United States))

    1991-12-01

    The authors have previously demonstrated that a transcriptional arrest site exists in exon 1 of the human adenosine deaminase (ADA) gene and that this site may play a role in ADA gene expression. Sequences involved in this process are not known precisely. To further define the template requirements for transcriptional arrest within exon 1 of the human ADA gene, various ADA templates were constructed and their abilities to confer transcriptional arrest were determined following injection into Xenopus oocytes. The exon 1 transcriptional arrest signal functioned downstream of several RNA polymerase II promoters and an RNA polymerase II promoter, implying that the transcriptional arrest site in exon 1 of the ADA gene is promoter independent. They identified a 43-bp DNA fragment which functions as a transcriptional arrest signal. Additional studies showed that the transcriptional arrest site functioned only in the naturally occurring orientation. Therefore, they have identified a 43-bp DNA fragment which functions as a transcriptional arrest signal in an orientation-dependent and promoter-independent manner. On the basis of the authors findings, they hypothesize that tissue-specific expression of the ADA gene is governed by factors that function as antiterminators to promote transcriptional readthrough of the exon 1 transcriptional arrest site.

  12. Structural Insights into E. coli Porphobilinogen Deaminase during Synthesis and Exit of 1-Hydroxymethylbilane

    PubMed Central

    Bulusu, Gopalakrishnan

    2014-01-01

    Porphobilinogen deaminase (PBGD) catalyzes the formation of 1-hydroxymethylbilane (HMB), a crucial intermediate in tetrapyrrole biosynthesis, through a step-wise polymerization of four molecules of porphobilinogen (PBG), using a unique dipyrromethane (DPM) cofactor. Structural and biochemical studies have suggested residues with catalytic importance, but their specific role in the mechanism and the dynamic behavior of the protein with respect to the growing pyrrole chain remains unknown. Molecular dynamics simulations of the protein through the different stages of pyrrole chain elongation suggested that the compactness of the overall protein decreases progressively with addition of each pyrrole ring. Essential dynamics showed that domains move apart while the cofactor turn region moves towards the second domain, thus creating space for the pyrrole rings added at each stage. Residues of the flexible active site loop play a significant role in its modulation. Steered molecular dynamics was performed to predict the exit mechanism of HMB from PBGD at the end of the catalytic cycle. Based on the force profile and minimal structural changes the proposed path for the exit of HMB is through the space between the domains flanking the active site loop. Residues reported as catalytically important, also play an important role in the exit of HMB. Further, upon removal of HMB, the structure of PBGD gradually relaxes to resemble its initial stage structure, indicating its readiness to resume a new catalytic cycle. PMID:24603363

  13. PORPHOBILINOGEN DEAMINASE Deficiency Alters Vegetative and Reproductive Development and Causes Lesions in Arabidopsis

    PubMed Central

    Quesada, Víctor; Hricová, Andrea; Ponce, María Rosa; Micol, José Luis

    2013-01-01

    The Arabidopsis rugosa1 (rug1) mutant has irregularly shaped leaves and reduced growth. In the absence of pathogens, leaves of rug1 plants have spontaneous lesions reminiscent of those seen in lesion-mimic mutants; rug1 plants also express cytological and molecular markers associated with defence against pathogens. These rug1 phenotypes are made stronger by dark/light transitions. The rug1 mutant also has delayed flowering time, upregulation of the floral repressor FLOWERING LOCUS C (FLC) and downregulation of the flowering promoters FT and SOC1/AGL20. Vernalization suppresses the late flowering phenotype of rug1 by repressing FLC. Microarray analysis revealed that 280 nuclear genes are differentially expressed between rug1 and wild type; almost a quarter of these genes are involved in plant defence. In rug1, the auxin response is also affected and several auxin-responsive genes are downregulated. We identified the RUG1 gene by map-based cloning and found that it encodes porphobilinogen deaminase (PBGD), also known as hydroxymethylbilane synthase, an enzyme of the tetrapyrrole biosynthesis pathway, which produces chlorophyll, heme, siroheme and phytochromobilin in plants. PBGD activity is reduced in rug1 plants, which accumulate porphobilinogen. Our results indicate that Arabidopsis PBGD deficiency impairs the porphyrin pathway and triggers constitutive activation of plant defence mechanisms leading to leaf lesions and affecting vegetative and reproductive development. PMID:23308205

  14. QSARs and activity predicting models for competitive inhibitors of adenosine deaminase.

    PubMed

    Sadat Hayatshahi, Sayyed Hamed; Abdolmaleki, Parviz; Ghiasi, Mina; Safarian, Shahrokh

    2007-02-01

    Combinations of multiple linear regressions, genetic algorithms and artificial neural networks were utilized to develop models for seeking quantitative structure-activity relationships that correlate structural descriptors and inhibition activity of adenosine deaminase competitive inhibitors. Many quantitative descriptors were generated to express the physicochemical properties of 70 compounds with optimized structures in aqueous solution. Multiple linear regressions were used to linearly select different subsets of descriptors and develop linear models for prediction of log(k(i)). The best subset then fed artificial neural networks to develop nonlinear predictors. A committee of six hybrid models - that included genetic algorithm routines together with neural networks - was also utilized to nonlinearly select most efficient subsets of descriptors in a cross-validation procedure for nonlinear log(k(i)) prediction. The best prediction model was found to be an 8-3-1 artificial neural network which was fed by the most frequently selected descriptors among these subsets. This prediction model resulted in train set root mean sum square error (RMSE) of 0.84 log(k(i)) and prediction set RMSE of 0.85 log(k(i)) (both equivalent of 0.10 in normal range of log(k(i))) and correlation coefficient (r(2)) of 0.91. PMID:17250831

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

    NASA Technical Reports Server (NTRS)

    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.

  16. The Role of Histidine-Proline-Rich Glycoprotein as Zinc Chaperone for Skeletal Muscle AMP Deaminase

    PubMed Central

    Ranieri-Raggi, Maria; Moir, Arthur J. G.; Raggi, Antonio

    2014-01-01

    Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP deaminase (AMPD) and the metal binding protein histidine-proline-rich glycoprotein (HPRG) acts in this manner. HPRG is a major plasma protein. Recent investigations have reported that skeletal muscle cells do not synthesize HPRG but instead actively internalize plasma HPRG. X-ray absorption spectroscopy (XAS) performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (?-aqua)(?-carboxylato)dizinc(II) core with two histidine residues at each metal site. XAS on HPRG isolated from the AMPD complex showed that zinc is bound to the protein in a dinuclear cluster where each Zn2+ ion is coordinated by three histidine and one heavier ligand, likely sulfur from cysteine. We describe the existence in mammalian HPRG of a specific zinc binding site distinct from the His-Pro-rich region. The participation of HPRG in the assembly and maintenance of skeletal muscle AMPD by acting as a zinc chaperone is also demonstrated. PMID:24970226

  17. Adenosine Deaminase Acting on RNA-1 (ADAR1) Inhibits HIV-1 Replication in Human Alveolar Macrophages

    PubMed Central

    Levy, David N.; Li, Yonghua; Kumar, Rajnish; Burke, Sean A.; Dawson, Rodney; Hioe, Catarina E.; Borkowsky, William; Rom, William N.; Hoshino, Yoshihiko

    2014-01-01

    While exploring the effects of aerosol IFN-? treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL) of aerosol IFN-?-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1) in the BAL cells. IFN-? induced ADAR1 expression in monocyte-derived macrophages (MDM) but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro. Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-? suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages. PMID:25272020

  18. Integrase-defective Lentiviral Vectors as a Delivery Platform for Targeted Modification of Adenosine Deaminase Locus

    PubMed Central

    Joglekar, Alok V; Hollis, Roger P; Kuftinec, Gabriela; Senadheera, Shantha; Chan, Rebecca; Kohn, Donald B

    2013-01-01

    We investigated the use of integrase-defective lentiviral vectors (IDLVs) for transient delivery of zinc finger nucleases (ZFNs) and donor templates for site-specific modification of the human adenosine deaminase (hADA) gene. Initially, we constructed IDLVs carrying ZFN monomers (Single-IDLVs) and found them to be able to deliver their gene-editing payload to K562 cells successfully upon cotransduction, with minimal cytotoxicity. To simplify delivery, we designed an IDLV construct to deliver both ZFN monomers from the same vector (Double-IDLV). However, this construct in its original state was prone to rearrangements of the vector genome, resulting in greatly reduced functionality; this was due to recombination between highly similar ZFN monomers arranged in tandem. We modified the Double-IDLV constructs to reduce recombination and restored simultaneous delivery of both ZFNs. We also tested an IDLV construct for delivery of donor templates and demonstrated its efficacy for gene modification. In summary, we highlighted the importance of modifying vector design for co-delivery of highly similar sequences inherent to genome-editing nucleases, and demonstrated significant improvement in the use of IDLVs for delivery of ZFNs and donor templates for genome modification. PMID:23857176

  19. Trypanosoma evansi: adenosine deaminase activity in the brain of infected rats.

    PubMed

    Da Silva, Aleksandro S; Bellé, Luziane P; Bitencourt, Paula E R; Perez, Herakles A Garcia; Thomé, Gustavo R; Costa, Marcio M; Oliveira, Camila B; Teixeira, Marta M G; Moretto, Maria B; Mazzanti, Cinthia M; Lopes, Sonia T A; Monteiro, Silvia G

    2011-01-01

    The study was undertaken to evaluate changes in the activity of adenosine deaminase (ADA) in brains of rats infected by Trypanosoma evansi. Each rat was intraperitoneally infected with 10(6) trypomastigotes either suspended in fresh (group A; n = 13) and cryopreserved blood (group B; n = 13). Thirteen animals were used as control (group C). ADA activity was estimated in the cerebellum, cerebral cortex, striatum and hippocampus. No differences (P > 0.05) in ADA activity were observed in the cerebellum between infected and non-infected animals. Significant (P < 0.05) reductions in ADA activity occurred in cerebral cortex in acutely (day 4 post-infection; PI) and chronically (day 20 PI) infected rats. ADA activity was significantly (P < 0.05) decreased in the hippocampus in acutely infected rats, but significantly (P < 0.05) increased in the chronically infected rats. Significant (P < 0.05) reductions in ADA activity occurred in the striatum of chronically infected rats. Parasites could be found in peripheral blood and brain tissue through microscopic examination and PCR assay, respectively, in acutely and chronically infected rats. The reduction of ADA activity in the brain was associated with high levels of parasitemia and anemia in acute infections. Alterations in ADA activity of the brain in T. evansi-infected rats may have implications for pathogenesis of the disease. PMID:20655914

  20. Restricting activation-induced cytidine deaminase tumorigenic activity in B lymphocytes

    PubMed Central

    Casellas, Rafael; Yamane, Arito; Kovalchuk, Alexander L; Potter, Michael

    2009-01-01

    DNA breaks play an essential role in germinal centre B cells as intermediates to immunoglobulin class switching, a recombination process initiated by activation-induced cytidine deaminase (AID). Immunoglobulin gene hypermutation is likewise catalysed by AID but is believed to occur via single-strand DNA breaks. When improperly repaired, AID-mediated lesions can promote chromosomal translocations (CTs) that juxtapose the immunoglobulin loci to heterologous genomic sites, including oncogenes. Two of the most studied translocations are the t(8;14) and T(12;15), which deregulate cMyc in human Burkitt’s lymphomas and mouse plasmacytomas, respectively. While a complete understanding of the aetiology of such translocations is lacking, recent studies using diverse mouse models have shed light on two important issues: (1) the extent to which non-specific or AID-mediated DNA lesions promote CTs, and (2) the safeguard mechanisms that B cells employ to prevent AID tumorigenic activity. Here we review these advances and discuss the usage of pristane-induced mouse plasmacytomas as a tool to investigate the origin of Igh–cMyc translocations and B-cell tumorigenesis. PMID:19302140

  1. Sensitivity and specificity of adenosine deaminase in diagnosis of juvenile idiopathic arthritis

    PubMed Central

    Doudkani-Fard, Mina; Ziaee, Vahid; Moradinejad, Mohamad-Hassan; Sedaghat, Mojtaba; Haghi-Ashtiani, Mohammad-Taghi; Ahmadinejad, Zahra

    2014-01-01

    Background: Juvenile Idiopathic Arthritis (JIA) is one of the most common chronic rheumatic diseases inchildren with unknown etiology and pathogenesis. It also has no diagnostic test and its clinical diagnosis ismade through ruling out other types of arthritis. The aim of this study was to evaluate the level of ADA (AdenosineDeaminase) in the serum of JIA patients and to compare it with that of patients with Reactive Arthritis(RA). Evaluation of sensitivity and specificity of serum ADA level in JIA was another objective. Methods: The study included 120 children with JIA (mean age= 7.6 ± 4.3 years) and 40 children with RA(mean age= 5.5 ± 3.1 years). The ADA was measured in the active phase of both diseases. Results: The mean ADA serum level was obtained as 15.8 ± 11.8 U/l in JIA patients and 14.3 ± 7.5 U/l in RApatients. The difference was statistically insignificant (p= 0.4). Another finding of this study was the significantspecificity (77.5%) of this laboratory parameter for JIA in comparison with its low sensitivity (36.7%). Positivepredictive value was 83% and negative predictive value 29%. Conclusion: Determination of ADA serum levels is a noninvasive reliable and easy biomarker for diagnosis ofJIA and it can be used as alternative parameters representing disease activity. PMID:25678992

  2. Fluorescence sensing of adenosine deaminase based on adenosine induced self-assembly of aptamer structures.

    PubMed

    Feng, Tingting; Ma, Huimin

    2013-04-21

    A new approach is proposed for simple detection of adenosine deaminase (ADA) based on adenosine induced self-assembly of two pieces of single-stranded DNA (ssDNA). These ssDNA are two fragments of the aptamer that has a strong affinity for adenosine and are labeled with carboxyfluorescein and black hole quencher-1, respectively. The complementarities of the bases in the two pieces of ssDNA are insufficient to form a stable structure. In the presence of adenosine, however, the ssDNA can be assembled into the intact aptamer tertiary structure, which results in fluorescence quenching of the carboxyfluorescein-labeled aptamer fragment. As a result, the adenosine-ssDNA complex shows a low background signal, which is rather desired for achieving sensitive detection. Reaction of the complex with ADA causes a great fluorescence enhancement by converting adenosine into inosine that has no affinity for the aptamer. This behaviour leads to the development of a simple and sensitive fluorescent method for assaying ADA activity, with a detection limit of 0.05 U mL(-1), which is more sensitive than most of the existing approaches. Furthermore, the applicability of the method has been demonstrated by detecting ADA in mouse serum samples. PMID:23462984

  3. Severe combined immunodeficiency and adenosine deaminase deficiency: failure of enzyme replacement therapy.

    PubMed Central

    Ziegler, J B; Lee, C H; Van der Weyden, M B; Bagnara, A S; Beveridge, J

    1980-01-01

    A first-born baby boy presented at age 3 months with persistent diarrhoea, failure to thrive, and recurrent bacterial and fungal infections. Severe combined immunodeficiency was demonstrated. A deficiency of adenosine deaminase (ADA) activity was suggested by the presence of extensive skeletal abnormalities, and the ADA activity in erythrocyte and leucocyte lysates was < 0.005 nmol/h per mg protein. Culture of ADA-negative peripheral blood mononuclear cells, together with purified calf ADA, did not alter the absent phytohaemagglutinin response. Treatment with immunoglobulin, pentamidine, and co-trimoxazole was started and a programme of ADA enzyme replacement, with infusions of plasma and frozen irradiated erythrocytes, was begun at age 4 months and achieved blood ADA levels in excess of 30 nmol/h per mg haemoglobin. Although resolution of the interstitial pneumonitis and skeletal abnormalities was observed, there was no evidence of immunological reconstitution. The patient died at age 17 months after a parainfluenza pneumonitis. Features of importance in predicting lack of benefit from enzyme replacement by erythrocyte infusion in ADA-negative severe combined immunodeficiency appear to be early clinical presentation with associated severe skeletal abnormalities, a very low level of residual ADA activity in peripheral blood mononuclear cells, and lack of effect of exogenous ADA on the absent in vitro mitogen response of ADA-negative blood mononuclear cells. Images Fig. 1 Fig. 2 p455-a Fig. 4 PMID:7436484

  4. Expression of human adenosine deaminase in mice transplanted with hemopoietic stem cells infected with amphotropic retroviruses

    PubMed Central

    1990-01-01

    Amphotropic recombinant retroviruses were generated carrying sequences encoding human adenosine deaminase (ADA). Transcription of the human ADA gene was under control of a hybrid long terminal repeat in which the enhancer from the Moloney murine leukemia virus was replaced by an enhancer from the F101 host-range mutant of polyoma virus. Hemopoietic stem cells in murine bone marrow were infected with this virus under defined culture conditions. As a result, 59% of day-12 colony forming unit spleen (CFU-S) stem cells became infected without any in vitro selection. Infected CFU-S were shown to express human ADA before transplantation and this expression sustained upon in vivo maturation. Mice transplanted with infected bone marrow exhibited human ADA expression in lymphoid, myeloid, and erythroid cell types. Moreover, human ADA expression persisted in secondary and tertiary transplanted recipients showing that human ADA-expressing cells were derived from pluripotent stem cells. These characteristics of our amphotropic viruses make them promising tools in gene therapy protocols for the treatment of severe combined immunodeficiency caused by ADA deficiency. In this respect it is also relevant that the viral vector that served as backbone for the ADA vector was previously shown to be nonleukemogenic. PMID:1974914

  5. Kinetic characterization of adenosine deaminase activity in zebrafish (Danio rerio) brain.

    PubMed

    Rosemberg, Denis Broock; Rico, Eduardo Pacheco; Senger, Mario Roberto; Dias, Renato Dutra; Bogo, Maurício Reis; Bonan, Carla Denise; Souza, Diogo Onofre

    2008-09-01

    Adenosine deaminase (ADA; EC 3.5.4.4) activity is responsible for cleaving adenosine to inosine. In this study we described the biochemical properties of adenosine deamination in soluble and membrane fractions of zebrafish (Danio rerio) brain. The optimum pH for ADA activity was in the range of 6.0-7.0 in soluble fraction and reached 5.0 in brain membranes. A decrease of 31.3% on adenosine deamination in membranes was observed in the presence of 5 mM Zn(2+), which was prevented by 5 mM EDTA. The apparent K(m) values for adenosine deamination were 0.22+/-0.03 and 0.19+/-0.04 mM for soluble and membrane fractions, respectively. The apparent V(max) value for soluble ADA activity was 12.3+/-0.73 nmol NH(3) min(-1) mg(-1) of protein whereas V(max) value in brain membranes was 17.5+/-0.51 nmol NH(3) min(-1) mg(-1) of protein. Adenosine and 2'-deoxyadenosine were deaminated in higher rates when compared to guanine nucleosides in both fractions. Furthermore, a significant inhibition on adenosine deamination in both soluble and membrane fractions was observed in the presence of 0.1 mM of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). The presence of ADA activity in zebrafish brain may be important to regulate the adenosine/inosine levels in the CNS of this species. PMID:18582589

  6. Influence of mercury chloride on adenosine deaminase activity and gene expression in zebrafish (Danio rerio) brain.

    PubMed

    Senger, Mario Roberto; Rosemberg, Denis Broock; Seibt, Kelly Juliana; Dias, Renato Dutra; Bogo, Maurício Reis; Bonan, Carla Denise

    2010-06-01

    Mercury is a widespread environmental contaminant that is neurotoxic even at very low concentrations. In this study we investigated the effects of mercury chloride on soluble and membrane adenosine deaminase (ADA) activity and gene expression in zebrafish brain. Inhibition of ADA activity was observed in the soluble fraction at 5-250 microM HgCl(2) (84.6-92.6%, respectively), whereas inhibition occurred at 50-250 microM in membrane fractions (20.9-26%, respectively). We performed in vitro experiments with chelants (EDTA and DTT) to test if these compounds prevented or reversed the inhibition caused by HgCl(2) and found that the inhibition was partially or fully abolished. The effect on ADA activity in soluble and membrane fractions was evaluated after acute (24h) and subchronic (96h) in vivo exposure of zebrafish to 20 microg/l HgCl(2). ADA activity in the soluble fraction was decreased after both acute (24.5%) and subchronic (40.8%) exposures, whereas in brain membranes the enzyme was inhibited only after subchronic exposure (21.9%). Semiquantitative RT-PCR analysis showed that HgCl(2) did not alter ADA gene expression. This study demonstrated that ADA activity was inhibited by mercury and this effect might be related to the neurotoxicity of this heavy metal. PMID:20226812

  7. Tipping the Balance: Antagonism of PKR Kinase and ADAR1 Deaminase Functions by Virus Gene Products

    PubMed Central

    George, Cyril X.; Li, Zhiqun; Okonski, Kristina M.; Toth, Ann M.; Wang, Ying

    2009-01-01

    The protein kinase regulated by RNA (PKR) and the adenosine deaminase acting on RNA (ADAR1) are interferon-inducible enzymes that play important roles in biologic processes including the antiviral actions of interferons, signal transduction, and apoptosis. PKR catalyzes the RNA-dependent phosphorylation of protein synthesis initiation factor eIF-2?, thereby leading to altered translational patterns in interferon-treated and virus-infected cells. PKR also modulates signal transduction responses, including the induction of interferon. ADAR1 catalyzes the deamination of adenosine (A) to generate inosine (I) in RNAs with double-stranded character. Because I is recognized as G instead of A, A-to-I editing by ADAR1 can lead to genetic recoding and altered RNA structures. The importance of PKR and ADAR1 in innate antiviral immunity is illustrated by a number of viruses that encode either RNA or protein viral gene products that antagonize PKR and ADAR1 enzymatic activity, localization, or stability. PMID:19715457

  8. A gold nanoparticle-based label free colorimetric aptasensor for adenosine deaminase detection and inhibition assay.

    PubMed

    Cheng, Fen; He, Yue; Xing, Xiao-Jing; Tan, Dai-Di; Lin, Yi; Pang, Dai-Wen; Tang, Hong-Wu

    2015-03-01

    A novel strategy for the fabrication of a colorimetric aptasensor using label free gold nanoparticles (AuNPs) is proposed in this work, and the strategy has been employed for the assay of adenosine deaminase (ADA) activity. The aptasensor consists of adenosine (AD) aptamer, AD and AuNPs. The design of the biosensor takes advantage of the special optical properties of AuNPs and the interaction between AuNPs and single-strand DNA. In the absence of ADA, the AuNPs are aggregated and are blue in color under appropriate salt concentration because of the grid structure of an AD aptamer when binding to AD, while in the presence of the analyte, AuNPs remain dispersed with red color under the same concentration of salt owing to ADA converting AD into inosine which has no affinity with the AD aptamer, thus allowing quantitative investigation of ADA activity. The present strategy is simple, cost-effective, selective and sensitive for ADA with a detection limit of 1.526 U L(-1), which is about one order of magnitude lower than that previously reported. In addition, a very low concentration of the inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) could generate a distinguishable response. Therefore, the AuNP-based colorimetric biosensor has great potential in the diagnosis of ADA-relevant diseases and drug screening. PMID:25597304

  9. Combined detections of interleukin-33 and adenosine deaminase for diagnosis of tuberculous pleural effusion

    PubMed Central

    Li, Diandian; Shen, Yongchun; Fu, Xiaomin; Li, Min; Wang, Tao; Wen, Fuqiang

    2015-01-01

    Objective: To investigate the diagnostic accuracy of the combination of interleukin-33 (IL-33) and adenosine deaminase (ADA) for differentiating TPE from pleural effusions with the other etiologies. Methods: Pleural effusion samples were collected from 32 TPE patients and 55 non-TPE patients. Pleural levels of IL-33 and ADA were measured by ELISA. The corresponding biochemical indexes were also simultaneously determined. Results: The pleural levels of IL-33 and ADA in the TPE group were significantly higher than those in the non-TPE group. With a cut-off value of 68.3 pg/ml, the sensitivity and specificity for IL-33 were 83.9% and 70.9%, respectively. While for ADA, the sensitivity and specificity were 87.5% and 87.3%, respectively at a cut-off value of 10.25 U/L. Combined use of IL-33 and ADA measurements further increased the sensitivity or specificity. Conclusion: Our study suggests that the applications of new biomarker IL-33, along with ADA, may serve as efficient diagnosis strategies in the management of pleural TB. Further studies at a large scale should be performed to validate our findings. PMID:25755791

  10. Tissue culture-induced transpositional activity of mPing is correlated with cytosine methylation in rice

    PubMed Central

    Ngezahayo, Frédéric; Xu, Chunming; Wang, Hongyan; Jiang, Lily; Pang, Jinsong; Liu, Bao

    2009-01-01

    Background mPing is an endogenous MITE in the rice genome, which is quiescent under normal conditions but can be induced towards mobilization under various stresses. The cellular mechanism responsible for modulating the activity of mPing remains unknown. Cytosine methylation is a major epigenetic modification in most eukaryotes, and the primary function of which is to serve as a genome defense system including taming activity of transposable elements (TEs). Given that tissue-culture is capable of inducing both methylation alteration and mPing transposition in certain rice genotypes, it provides a tractable system to investigate the possible relationship between the two phenomena. Results mPing transposition and cytosine methylation alteration were measured in callus and regenerated plants in three rice (ssp. indica) genotypes, V14, V27 and R09. All three genotypes showed transposition of mPing, though at various frequencies. Cytosine methylation alteration occurred both at the mPing-flanks and at random loci sampled globally in callus and regenerated plants of all three genotypes. However, a sharp difference in the changing patterns was noted between the mPing-flanks and random genomic loci, with a particular type of methylation modification, i.e., CNG hypermethylation, occurred predominantly at the mPing-flanks. Pearson's test on pairwise correlations indicated that mPing activity is positively correlated with specific patterns of methylation alteration at random genomic loci, while the element's immobility is positively correlated with methylation levels of the mPing's 5'-flanks. Bisulfite sequencing of two mPing-containing loci showed that whereas for the immobile locus loss of CG methylation in the 5'-flank was accompanied by an increase in CHG methylation, together with an overall increase in methylation of all three types (CG, CHG and CHH) in the mPing-body region, for the active locus erasure of CG methylation in the 5'-flank was not followed by such a change. Conclusion Our results documented that tissue culture-induced mPing activity in rice ssp. indica is correlated with alteration in cytosine methylation patterns at both random genomic loci and the elements' flanks, while the stability of mPing positively correlates with enhanced methylation levels of both the flanks and probably the elements per se. Thus, our results implicate a possible role of cytosine methylation in maintaining mPing stability under normal conditions, and in releasing the element's activity as a consequence of epigenetic perturbation in a locus-specific manner under certain stress conditions. PMID:19604382

  11. Molecular Genetic Analysis in Yeast

    NSDL National Science Digital Library

    Daniel D. Burke (Seton Hall University; )

    1989-06-06

    The four exercises presented here use basic and advanced procedures of recombinant DNA technology to perform molecular genetic analysis in the yeast Saccharomyces cerevisiae. Their fulluse is intended for a senior-level molecular genetics (or similar) course; however, Experiments 1, 2, and 4 are appropriate for lower-level courses. It is expected that the instructor will have some familiarity with the concepts and terminology of recombinant DNA technology and with yeast genetics.

  12. Biotechnological Applications of Dimorphic Yeasts

    NASA Astrophysics Data System (ADS)

    Doiphode, N.; Joshi, C.; Ghormade, V.; Deshpande, M. V.

    The dimorphic yeasts have the equilibrium between spherical growth (budding) and polarized (hyphal or pseudohyphal tip elongation) which can be triggered by change in the environmental conditions. The reversible growth phenomenon has made dimorphic yeasts as an useful model to understand fungal evolution and fungal differentiation, in general. In nature dimorphism is clearly evident in plant and animal fungal pathogens, which survive and most importantly proliferate in the respective hosts. However, number of organisms with no known pathogenic behaviour also show such a transition, which can be exploited for the technological applications due to their different biochemical make up under different morphologies. For instance, chitin and chitosan production using dimorphic Saccharomyces, Mucor, Rhizopus and Benjaminiella, oil degradation and biotransformation with yeast-form of Yarrowia species, bioremediation of organic pollutants, exopolysac-charide production by yeast-phase of Aureobasidium pullulans, to name a few. Myrothecium verrucaria can be used for seed dressing in its yeast form and it produces a mycolytic enzyme complex in its hyphal-form for the biocontrol of fungal pathogens, while Beauveria bassiana and other entomopathogens kill the insect pest by producing yeast- like cells in the insect body. The form-specific expression of protease, chitinase, lipase, ornithine decarboxylase, glutamate dehydrogenases, etc. make Benjaminiella poitrasii, Basidiobolus sp., and Mucor rouxii strains important in bioremediation, nanobiotechnology, fungal evolution and other areas.

  13. Study of amyloids using yeast

    PubMed Central

    Wickner, Reed B.; Kryndushkin, Dmitry; Shewmaker, Frank; McGlinchey, Ryan; Edskes, Herman K.

    2012-01-01

    Summary Saccharomyces cerevisiae has been a useful model organism in such fields as the cell cycle, regulation of transcription, protein trafficking and cell biology, primarily because of its ease of genetic manipulation. This is no less so in the area of amyloid studies. The endogenous yeast amyloids described to date include prions, infectious proteins (Table 1), and some cell wall proteins (1). and amyloids of humans and a fungal prion have also been studied using the yeast system. Accordingly, the emphasis of this chapter will be on genetic, biochemical, cell biological and physical methods particularly useful in the study of yeast prions and other amyloids studied in yeast. We limit our description of these methods to those aspects which have been most useful in studying yeast prions, citing more detailed expositions in the literature. Volumes on yeast genetics methods (2–4), and on amyloids and prions (5, 6) are useful, and Masison has edited a volume of Methods on “Identification, analysis and characterization of fungal prions” which covers some of this territory (7). We also outline some useful physical methods, pointing the reader to more extensive and authoratative descriptions. PMID:22528100

  14. The cytidine deaminase signature HxE(x)n CxxC of DYW1 binds zinc and is necessary for RNA editing of ndhD-1.

    PubMed

    Boussardon, Clément; Avon, Alexandra; Kindgren, Peter; Bond, Charles S; Challenor, Michael; Lurin, Claire; Small, Ian

    2014-09-01

    In flowering plants, RNA editing involves deamination of specific cytidines to uridines in both mitochondrial and chloroplast transcripts. Pentatricopeptide repeat (PPR) proteins and multiple organellar RNA editing factor (MORF) proteins have been shown to be involved in RNA editing but none have been shown to possess cytidine deaminase activity. The DYW domain of some PPR proteins contains a highly conserved signature resembling the zinc-binding active site motif of known nucleotide deaminases. We modified these highly conserved amino acids in the DYW motif of DYW1, an editing factor required for editing of the ndhD-1 site in Arabidopsis chloroplasts. We demonstrate that several amino acids of this signature motif are required for RNA editing in vivo and for zinc binding in vitro. We conclude that the DYW domain of DYW1 has features in common with cytidine deaminases, reinforcing the hypothesis that this domain forms part of the active enzyme that carries out RNA editing in plants. PMID:25041347

  15. Metabolic regulation of yeast

    NASA Astrophysics Data System (ADS)

    Fiechter, A.

    1982-12-01

    Metabolic regulation which is based on endogeneous and exogeneous process variables which may act constantly or time dependently on the living cell is discussed. The observed phenomena of the regulation are the result of physical, chemical, and biological parameters. These parameters are identified. Ethanol is accumulated as an intermediate product and the synthesis of biomass is reduced. This regulatory effect of glucose is used for the aerobic production of ethanol. Very high production rates are thereby obtained. Understanding of the regulation mechanism of the glucose effect has improved. In addition to catabolite repression, several other mechanisms of enzyme regulation have been described, that are mostly governed by exogeneous factors. Glucose also affects the control of respiration in a third class of yeasts which are unable to make use of ethanol as a substrate for growth. This is due to the lack of any anaplerotic activity. As a consequence, diauxic growth behavior is reduced to a one-stage growth with a drastically reduced cell yield. The pulse chemostat technique, a systematic approach for medium design is developed and medium supplements that are essential for metabolic control are identified.

  16. Riboneogenesis in yeast

    PubMed Central

    Clasquin, Michelle F.; Melamud, Eugene; Singer, Alexander; Gooding, Jessica R.; Xu, Xiaohui; Dong, Aiping; Cui, Hong; Campagna, Shawn R.; Savchenko, Alexei; Yakunin, Alexander F.; Rabinowitz, Joshua D.; Caudy, Amy A.

    2011-01-01

    Summary Gluconeogenesis converts three carbon units into glucose. Here we identify an analogous pathway in Saccharomyces cerevisiae for converting three carbon units into ribose, a component of nucleic acids and nucleotides. This riboneogenic pathway involves the enzyme sedoheptulose-1,7-bisphosphatase (SHB17), whose activity was identified based on accumulation of sedoheptulose-1,7-bisphosphate in the corresponding knockout strain. We determined the crystal structure of Shb17 in complex with sedoheptulose-1,7-bisphosphate, and found that the sugar is bound in the closed furan form in the active site. Like fructose-1,6-bisphosphate, sedoheptulose-1,7-bisphosphate is produced by aldolase, in this case from erythrose 4-phosphate and dihydroxyacetone phosphate. Hydrolysis of sedoheptulose-1,7-bisphosphate by SHB17 provides an energetically favorable input to the non-oxidative pentose phosphate pathway to drive ribose production. Flux through SHB17 is enhanced under conditions when ribose demand is high relative to demand for NADPH, including during ribosome biogenesis in metabolically synchronized yeast cells. Thus, riboneogenesis provides a thermodynamically-driven route of ribose production uncoupled from formation of NADPH. PMID:21663798

  17. McrB: a prokaryotic protein specifically recognizing DNA containing modified cytosine residues.

    PubMed Central

    Krüger, T; Wild, C; Noyer-Weidner, M

    1995-01-01

    Restriction of DNA by the Escherichia coli K-12 McrBC restriction endonuclease, which consists of the two subunits McrB and McrC, depends on the presence of modified cytosine residues in a special constellation. From previous work by others it was known that restriction of 5-methylcytosine-containing DNA requires two methylated 5'-PuC sites separated by approximately 40-80 non-defined base pairs. Here we show that binding of the McrBC nuclease is mediated exclusively by the McrB subunit. McrB has a low affinity for non-methylated DNA, with which it forms low molecular weight complexes. The affinity for DNA is significantly increased, with variations depending on the sequence context, by hemi- or fully methylated 5'-PuC sites. Binding to such substrates yields high molecular weight complexes, presumably involving several McrB molecules. Methylation at unique 5'-PuC sites can be sufficient to stimulate DNA binding by McrB. As such substrates are not cleaved by the nuclease, restriction apparently requires the coordinated interaction of molecules bound to neighbouring 5'-PumC sites. The binding properties of McrB exhibit some similarities to recently identified eukaryotic proteins interacting in a non-sequence-specific manner with DNA containing methylated 5'-CpG sequences and might point to a common molecular origin of these proteins. In addition to DNA, McrB also binds GTP, an essential cofactor in DNA restriction by McrBC. McrC neither binds to DNA nor modulates the DNA binding potential of McrB. As McrC is essential for restriction it appears to predominantly function in catalysis. Images PMID:7781618

  18. Evolution of complete proteomes: guanine-cytosine pressure, phylogeny and environmental influences blend the proteomic architecture

    PubMed Central

    2013-01-01

    Background Guanine-cytosine (GC) composition is an important feature of genomes. Likewise, amino acid composition is a distinct, but less valued, feature of proteomes. A major concern is that it is not clear what valuable information can be acquired from amino acid composition data. To address this concern, in-depth analyses of the amino acid composition of the complete proteomes from 63 archaea, 270 bacteria, and 128 eukaryotes were performed. Results Principal component analysis of the amino acid matrices showed that the main contributors to proteomic architecture were genomic GC variation, phylogeny, and environmental influences. GC pressure drove positive selection on Ala, Arg, Gly, Pro, Trp, and Val, and adverse selection on Asn, Lys, Ile, Phe, and Tyr. The physico-chemical framework of the complete proteomes withstood GC pressure by frequency complementation of GC-dependent amino acid pairs with similar physico-chemical properties. Gln, His, Ser, and Val were responsible for phylogeny and their constituted components could differentiate archaea, bacteria, and eukaryotes. Environmental niche was also a significant factor in determining proteomic architecture, especially for archaea for which the main amino acids were Cys, Leu, and Thr. In archaea, hyperthermophiles, acidophiles, mesophiles, psychrophiles, and halophiles gathered successively along the environment-based principal component. Concordance between proteomic architecture and the genetic code was also related closely to genomic GC content, phylogeny, and lifestyles. Conclusions Large-scale analyses of the complete proteomes of a wide range of organisms suggested that amino acid composition retained the trace of GC variation, phylogeny, and environmental influences during evolution. The findings from this study will help in the development of a global understanding of proteome evolution, and even biological evolution. PMID:24088322

  19. A Conserved Glutamate Residue in the C-terminal Deaminase Domain of Pentatricopeptide Repeat Proteins Is Required for RNA Editing Activity.

    PubMed

    Hayes, Michael L; Dang, Kim N; Diaz, Michael F; Mulligan, R Michael

    2015-04-17

    Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins include an RNA binding domain that provides site specificity. In addition, many PPR proteins include a C-terminal DYW deaminase domain with characteristic zinc binding motifs (CXXC, HXE) and has recently been shown to bind zinc ions. The glutamate residue of the HXE motif is catalytically required in the reaction catalyzed by cytidine deaminase. In this work, we examine the activity of the DYW deaminase domain through truncation or mutagenesis of the HXE motif. OTP84 is required for editing three chloroplast sites, and transgenes expressing OTP84 with C-terminal truncations were capable of editing only one of the three cognate sites at high efficiency. These results suggest that the deaminase domain of OTP84 is required for editing two of the sites, but another deaminase is able to supply the deamination activity for the third site. OTP84 and CREF7 transgenes were mutagenized to replace the glutamate residue of the HXE motif, and transgenic plants expressing OTP84-E824A and CREF7-E554A were unable to efficiently edit the cognate editing sites for these genes. In addition, plants expressing CREF7-E554A exhibited substantially reduced capacity to edit a non-cognate site, rpoA C200. These results indicate that the DYW deaminase domains of PPR proteins are involved in editing their cognate editing sites, and in some cases may participate in editing additional sites in the chloroplast. PMID:25739442

  20. Adenosine Deaminase Acts as a Natural Antagonist for Dipeptidyl Peptidase 4-Mediated Entry of the Middle East Respiratory Syndrome Coronavirus

    PubMed Central

    Raj, V. Stalin; Smits, Saskia L.; Provacia, Lisette B.; van den Brand, Judith M. A.; Wiersma, Lidewij; Ouwendijk, Werner J. D.; Bestebroer, Theo M.; Spronken, Monique I.; van Amerongen, Geert; Rottier, Peter J. M.; Fouchier, Ron A. M.; Bosch, Berend Jan; Osterhaus, Albert D.M.E.

    2014-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) replicates in cells of different species using dipeptidyl peptidase 4 (DPP4) as a functional receptor. Here we show the resistance of ferrets to MERS-CoV infection and inability of ferret DDP4 to bind MERS-CoV. Site-directed mutagenesis of amino acids variable in ferret DPP4 thus revealed the functional human DPP4 virus binding site. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection. PMID:24257613

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

    PubMed

    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. PMID:21271505

  2. HIV-1 Vif Versus the APOBEC3 Cytidine Deaminases: An Intracellular Duel Between Pathogen and Host Restriction Factors

    PubMed Central

    Wissing, Silke; Galloway, Nicole L. K.; Greene, Warner C.

    2010-01-01

    The Vif protein of HIV is essential for the effective propagation of this pathogenic retrovirus in vivo. Vif acts by preventing virion encapsidation of two potent antiviral factors, the APOBEC3G and APOBEC3F cytidine deaminases. Decreased encapsidation in part involves Vif-mediated recruitment of a ubiquitin E3 ligase complex that promotes polyubiquitylation and proteasome-mediated degradation of APOBEC3G/F. The resultant decline in intracellular levels of these enzymes leads to decreased encapsidation of APOBECG/F into budding virions. This review discusses recent advances in our understanding of the dynamic interplay of Vif with the antiviral APOBEC3 enzymes. PMID:20538015

  3. Effects of garlic and black grape extracts on the activity of adenosine deaminase from cancerous and noncancerous human urinary bladder tissues

    Microsoft Academic Search

    ?lker Durak; Hasan Biri; ?mge B. Ergüder; Erdinç Devrim; Ça?r? ?enocak; Asl?han Avc?

    2007-01-01

    Aim  Possible effects of garlic (Allium sativum) and black grape (Fructus vitis minuta) with known antioxidant potential on adenosine deaminase (ADA) activities were investigated in cancerous and noncancerous\\u000a human bladder tissues.\\u000a \\u000a \\u000a \\u000a Methods  The effects of garlic and black grape extracts on adenosine deaminase (ADA) activities were measured in 20 pairs of cancer\\u000a and adjacent normal human bladder tissues with and without pre-incubation

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

    SciTech Connect

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

    1987-08-01

    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.

  5. Management options for adenosine deaminase deficiency; proceedings of the EBMT satellite workshop (Hamburg, March 2006).

    PubMed

    Booth, Claire; Hershfield, Mike; Notarangelo, Luigi; Buckley, Rebecca; Hoenig, Manfred; Mahlaoui, Nizar; Cavazzana-Calvo, Marina; Aiuti, Alessandro; Gaspar, H Bobby

    2007-05-01

    Adenosine deaminase (ADA) deficiency is a disorder of purine salvage that has its most devastating consequences in the immune system leading to severe combined immunodeficiency (SCID). Management options for ADA SCID include hematopoietic stem cell transplantation, enzyme replacement therapy and gene therapy. Formal data on the outcome following each of the three treatment modalities are limited, and this symposium was held in order to gather together the experience from major centers in Europe and the US. Transplantation for ADA-SCID is highly successful with survival rates of approximately 90% if a matched sibling or matched related donor is available but survival following matched unrelated donor or haploidentical procedures is 63% and 50% respectively with a significant rejection/non-engraftment rate in unconditioned procedures. Successfully transplanted patients demonstrated good immunological recovery with normal cellular and humoral function in the majority of cases. PEG-ADA has been used in over 150 patients worldwide either as an alternative to mismatched transplant or as a stabilizing measure prior to transplant. Overall, approximately two thirds of patients treated with PEG-ADA have survived with the majority of patients showing good clinical improvement. The level of immune recovery long term was less than that seen after transplant and approximately 50% of patients continued to receive immunoglobulin replacement. Gene therapy has been used as an experimental procedure in two centers in Europe. Early results from 9 patients suggest that the treatment is safe and that the majority have shown recovery of cellular immune function. Long-term follow-up of treated patients highlights a significant incidence of non-immunological problems with cognitive, neurological and audiological abnormalities most prominent. PMID:17300989

  6. Restriction of porcine endogenous retrovirus by porcine APOBEC3 cytidine deaminases.

    PubMed

    Dörrschuck, Eva; Fischer, Nicole; Bravo, Ignacio G; Hanschmann, Kay-Martin; Kuiper, Heidi; Spötter, Andreas; Möller, Ronny; Cichutek, Klaus; Münk, Carsten; Tönjes, Ralf R

    2011-04-01

    Xenotransplantation of porcine cells, tissues, and organs shows promise to surmount the shortage of human donor materials. Among the barriers to pig-to-human xenotransplantation are porcine endogenous retroviruses (PERV) since functional representatives of the two polytropic classes, PERV-A and PERV-B, are able to infect human embryonic kidney cells in vitro, suggesting that a xenozoonosis in vivo could occur. To assess the capacity of human and porcine cells to counteract PERV infections, we analyzed human and porcine APOBEC3 (A3) proteins. This multigene family of cytidine deaminases contributes to the cellular intrinsic immunity and act as potent inhibitors of retroviruses and retrotransposons. Our data show that the porcine A3 gene locus on chromosome 5 consists of the two single-domain genes A3Z2 and A3Z3. The evolutionary relationships of the A3Z3 genes reflect the evolutionary history of mammals. The two A3 genes encode at least four different mRNAs: A3Z2, A3Z3, A3Z2-Z3, and A3Z2-Z3 splice variant A (SVA). Porcine and human A3s have been tested toward their antiretroviral activity against PERV and murine leukemia virus (MuLV) using novel single-round reporter viruses. The porcine A3Z2, A3Z3 and A3Z2-Z3 were packaged into PERV particles and inhibited PERV replication in a dose-dependent manner. The antiretroviral effect correlated with editing by the porcine A3s with a trinucleotide preference for 5' TGC for A3Z2 and A3Z2-Z3 and 5' CAC for A3Z3. These results strongly imply that human and porcine A3s could inhibit PERV replication in vivo, thereby reducing the risk of infection of human cells by PERV in the context of pig-to-human xenotransplantation. PMID:21307203

  7. Pleural Fluid Adenosine Deaminase (Pfada) in the Diagnosis of Tuberculous Effusions in a Low Incidence Population

    PubMed Central

    Arnold, David T.; Bhatnagar, Rahul; Fairbanks, Lynette D.; Zahan-Evans, Natalie; Clive, Amelia O.; Morley, Anna J.; Medford, Andrew R. L.; Maskell, Nicholas A.

    2015-01-01

    Introduction Previous studies have assessed the diagnostic ability of pleural fluid adenosine deaminase (pfADA) in detecting tuberculous pleural effusions, with good specificity and sensitivity reported. However, in North Western Europe pfADA is not routinely used in the investigation of a patient with an undiagnosed pleural effusion, mainly due to a lack of evidence as to its utility in populations with low mycobacterium tuberculosis (mTB) incidence. Methods Patients presenting with an undiagnosed pleural effusion to a tertiary pleural centre in South-West England over a 3 year period, were prospectively recruited to a pleural biomarker study. Pleural fluid from consecutive patients with robust 12-month follow up data and confirmed diagnosis were sent for pfADA analysis. Results Of 338 patients enrolled, 7 had confirmed tuberculous pleural effusion (2%). All mTB effusions were lymphocyte predominant with a median pfADA of 72.0 IU/L (range- 26.7 to 91.5) compared to a population median of 12.0 IU/L (range- 0.3 to 568.4). The optimal pfADA cut off was 35 IU/L, which had a negative predictive value (NPV) of 99.7% (95% CI; 98.2-99.9%) for the exclusion of mTB, and sensitivity of 85.7% (95% CI; 42.2-97.6%) with an area under the curve of 0.88 (95% CI; 0.732–1.000). Discussion This is the first study examining the diagnostic utility of pfADA in a low mTB incidence area. The chance of an effusion with a pfADA under 35 IU/L being of tuberculous aetiology was negligible. A pfADA of over 35 IU/L in lymphocyte-predominant pleural fluid gives a strong suspicion of mTB. PMID:25647479

  8. Multicentric dermatofibrosarcoma protuberans in patients with adenosine deaminase–deficient severe combined immune deficiency

    PubMed Central

    Kesserwan, Chimen; Sokolic, Robert; Cowen, Edward W.; Garabedian, Elizabeth; Heselmeyer-Haddad, Kerstin; Lee, Chyi-Chia Richard; Pittaluga, Stefania; Ortiz, Clarymar; Baird, Kristin; Lopez-Terrada, Dolores; Bridge, Julia; Wayne, Alan S.; Candotti, Fabio

    2011-01-01

    Background Dermatofibrosarcoma protuberans (DFSP) is a rare malignant skin tumor associated with a characteristic chromosomal translocation (t[17;22][q22;q13]) resulting in the COL1A1-platelet-derived growth factor ? (PDGFB) fusion gene. This malignancy is rarely diagnosed in childhood. We observed a high incidence of this tumor in children affected with adenosine deaminase–deficient severe combined immunodeficiency (ADA-SCID). Methods Twelve patients with ADA-SCID were evaluated with a complete dermatologic examination and skin biopsy when indicated. Conventional cytogenetic and molecular analyses (fluorescence in situ hybridization, RT-PCR, or both) were performed when possible. Results Eight patients were found to have DFSP. Six patients had multicentric involvement (4-15 lesions), primarily of the trunk and extremities. Most lesions presented as 2- to 15-mm, round atrophic plaques. Nodular lesions were present in 3 patients. In all cases CD34 expression was diffusely positive, and diagnosis was confirmed either by means of cytogenetic analysis, molecular testing, or both. The characteristic DFSP-associated translocation, t(17;22)(q22;q13), was identified in 6 patients; results of fluorescence in situ hybridization were positive for fusion of the COL1A1 and PDGFB loci in 7 patients; and RT-PCR showed the COL1A1-PDGFB fusion transcript in 6 patients. Conclusions We describe a previously unrecognized association between ADA-SCID and DFSP with unique features, such as multicentricity and occurrence in early age. We hypothesize that the t(17;22)(q22;q13) translocation that results in dermal overexpression of PDGFB and favors the development of fibrotic tumors might arise because of the known DNA repair defect in patients with ADA-SCID. Although the natural course of DFSP in the setting of ADA-SCID is unknown, this observation should prompt regular screening for DFSP in patients with ADA-SCID. PMID:22153773

  9. Erythrocyte adenosine deaminase deficiency without immunodeficiency. Evidence for an unstable mutant enzyme.

    PubMed

    Hirschhorn, R; Roegner, V; Jenkins, T; Seaman, C; Piomelli, S; Borkowsky, W

    1979-10-01

    Inherited deficiency of the purine salvage enzyme adenosine deaminase (ADA) gives rise to a syndrome of severe combined immunodeficiency (SCID). We have studied a 2.5-yr-old immunologically normal child who had been found to lack ADA in his erythrocytes during New York State screening of normal newborns. His erythrocytes were not detectably less deficient in ADA than erythrocytes of ADA(-)-SCID patients. In contrast, his lymphocytes and cultured long-term lymphoid cells contained appreciably greater ADA activity than those from patients with ADA(-)-SCID. This residual ADA activity had a normal molecular weight and K(m) but was markedly unstable at 56 degrees C. His residual erythrocytes-ADA activity also appeared to have diminished stability in vivo. ADA activity in lymphoid line cells of a previously reported erythrocyte-ADA-deficient!Kung tribesman was found to contain 50% of normal activity and to exhibit diminished stability at 56 degrees C. ATP content of erythrocytes from both partially ADA-deficient individuals was detectably greater than normal (12.3 and 6.1 vs. normal of 2.6 nmol/ml packed erythrocytes). However, the dATP content was insignificant compared to that found in erythrocytes of ADA(-)-SCID patients (400-1,000 nmol/ml packed erythrocytes). The New York patient, in contrast to normals, excreted detectable amounts of deoxyadenosine, but this was <2% of deoxyadenosine excreted by ADA(-)-SCID patients. Thus, the residual enzyme in cells other than erythrocytes appears to be sufficient to almost totally prevent accumulation of toxic metabolites. PMID:479373

  10. Spatial and functional relationships among Pol V-associated loci, Pol IV-dependent siRNAs, and cytosine methylation in the Arabidopsis epigenome.

    PubMed

    Wierzbicki, Andrzej T; Cocklin, Ross; Mayampurath, Anoop; Lister, Ryan; Rowley, M Jordan; Gregory, Brian D; Ecker, Joseph R; Tang, Haixu; Pikaard, Craig S

    2012-08-15

    Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%-60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites. PMID:22855789

  11. Spatial and Functional Relationships Among Pol V-Associated loci, Pol IV-Dependent siRNAs, and Cytosine Methylation in the Arabidopsis Epigenome

    SciTech Connect

    Wierzbicki, A. T.; Cocklin, Ross; Mayampurath, Anoop; Lister, Ryan; Rowley, M. J.; Gregory, Brian D.; Ecker, Joseph R.; Tang, Haixu; Pikaard, Craig S.

    2012-08-15

    Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%-60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites.

  12. Yeast Genetics and Biotechnological Applications

    NASA Astrophysics Data System (ADS)

    Mishra, Saroj; Baranwal, Richa

    Yeast can be recognized as one of the very important groups of microorganisms on account of its extensive use in the fermentation industry and as a basic eukaryotic model cellular system. The yeast Saccharomyces cerevisiae has been extensively used to elucidate the genetics and regulation of several key functions in the cell such as cell mating, electron transport chain, protein trafficking, cell cycle events and others. Even before the genome sequence of the yeast was out, the structural organization and function of several of its genes was known. With the availability of the origin of replication from the 2 ?m plasmid and the development of transformation system, it became the host of choice for expression of a number of important proteins. A large number of episomal and integrative shuttle vectors are available for expression of mammalian proteins. The latest developments in genomics and micro-array technology have allowed investigations of individual gene function by site-specific deletion method. The application of metabolic profiling has also assisted in understanding the cellular network operating in this yeast. This chapter is aimed at reviewing the use of this system as an experimental tool for conducting classical genetics. Various vector systems available, foreign genes expressed and the limitations as a host will be discussed. Finally, the use of various yeast enzymes in biotechnology sector will be reviewed.

  13. Nuclear Transport of Yeast Proteasomes

    PubMed Central

    Enenkel, Cordula

    2014-01-01

    Proteasomes are conserved protease complexes enriched in the nuclei of dividing yeast cells, a major site for protein degradation. If yeast cells do not proliferate and transit to quiescence, metabolic changes result in the dissociation of proteasomes into proteolytic core and regulatory complexes and their sequestration into motile cytosolic proteasome storage granuli. These granuli rapidly clear with the resumption of growth, releasing the stored proteasomes, which relocalize back to the nucleus to promote cell cycle progression. Here, I report on three models of how proteasomes are transported from the cytoplasm into the nucleus of yeast cells. The first model applies for dividing yeast and is based on the canonical pathway using classical nuclear localization sequences of proteasomal subcomplexes and the classical import receptor importin/karyopherin ??. The second model applies for quiescent yeast cells, which resume growth and use Blm10, a HEAT-like repeat protein structurally related to karyopherin ?, for nuclear import of proteasome core particles. In the third model, the fully-assembled proteasome is imported into the nucleus. Our still marginal knowledge about proteasome dynamics will inspire the discussion on how protein degradation by proteasomes may be regulated in different cellular compartments of dividing and quiescent eukaryotic cells. PMID:25333764

  14. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed Central

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-01-01

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

  15. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-10-25

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

  16. Synthesis of Peptide nucleic acids containing pyridazine derivatives as Cytosine and thymine analogs, and their duplexes with complementary oligodeoxynucleotides.

    PubMed

    Tomori, Takahito; Miyatake, Yuya; Sato, Yuta; Kanamori, Takashi; Masaki, Yoshiaki; Ohkubo, Akihiro; Sekine, Mitsuo; Seio, Kohji

    2015-03-20

    Synthesis of peptide nucleic acids (PNAs) is reported with new pyridazine-type nucleobases: 3-aminopyridazine (aPz) and 1-aminophthalazine (aPh) as cytosine analogs, and pyridazin-3-one (Pz(O)) and phthalazin-1-one (Ph(O)) as thymine analogs. The PNAs having an aPz or a Pz(O) formed duplexes with each complementary oligodeoxynucleotide forming a base pair with G or A, respectively, as evaluated by using UV melting analyses and circular dichroism (CD) spectra. PMID:25753827

  17. ORIGINAL PAPER Evolutionarily engineered ethanologenic yeast detoxifies

    E-print Network

    Song, Joe

    ORIGINAL PAPER Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass with subsequent fermentation of ethanol, posing significant challenges for a sustainable cellulosic ethanol conversion industry. Numerous yeast genes were found to be associated with the inhibitor tolerance. However

  18. PHYLOGENETICS OF SACCHAROMYCETALES, THE ASCOMYCETE YEASTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ascomycete yeasts (Phylum Ascomycota: Subphylum Saccharomycotina: Class Saccharomycetes: Order Saccharomycetales) comprise a monophyletic lineage with a single order of about 1000 known species. These yeasts live as saprobes, often in association with plants, animals, and their interfaces. A few s...

  19. Bacteria, Yeast and Chemicals on Human Skin

    MedlinePLUS Videos and Cool Tools

    ... the lower right-hand corner of the player. Bacteria, Yeast and Chemicals on Human Skin HealthDay April ... the distribution and quantity of metabolites, peptides, lipids, bacteria, yeast, proteins, chemicals and more. As expected, many ...

  20. Cdc42 Oscillations in Yeasts

    NSDL National Science Digital Library

    Felipe O. Bendezu (Switzerland; University of Lausanne REV)

    2012-12-04

    A fundamental problem in cell biology is how cells define one or several discrete sites of polarity. Through mechanisms involving positive and negative feedback, the small Rho-family guanosine triphosphatase Cdc42 breaks symmetry in round budding yeast cells to define a single site of polarized cell growth. However, it is not clear how cells can define multiple sites of polarization concurrently. We discuss a study in which rod-shaped fission yeast cells, which naturally polarize growth at their two cell ends, exhibited oscillations of Cdc42 activity between these sites. We compare these findings with similar oscillatory behavior of Cdc42 detected in budding yeast cells and discuss the possible mechanism and functional outputs of these oscillations.

  1. Sorption of grape proanthocyanidins and wine polyphenols by yeasts, inactivated yeasts, and yeast cell walls.

    PubMed

    Mekoue Nguela, J; Sieczkowski, N; Roi, S; Vernhet, A

    2015-01-21

    Inactivated yeast fractions (IYFs) can be used in enology to improve the stability and mouthfeel of red wines. However, information concerning the mechanisms involved and the impact of the IYF characteristics is scarce. Adsorption isotherms were used to investigate interactions between grape proanthocyanidin fractions (PAs) or wine polyphenols (WP) and a commercial yeast strain (Y), the inactivated yeast (IY), the yeast submitted to autolyzis and inactivation (A-IY), and the cell walls obtained by mechanical disruption (CW). High affinity isotherms and high adsorption capacities were observed for grape PAs and whole cells (Y, IY, and A-IY). Affinity and adsorbed amount were lower with wine PAs, due to chemical changes occurring during winemaking. By contrast to whole cells, grape PAs and WP adsorption on CW remained very low. This raises the issue of the part played by cell walls in the interactions between yeast and proanthocyanidins and suggests the passage of the latter through the wall pores and their interaction with the plasma membrane. PMID:25575250

  2. Yeast: A Research Organism for Teaching Genetics.

    ERIC Educational Resources Information Center

    Manney, Thomas R.; Manney, Monta L.

    1992-01-01

    Explains why laboratory strains of bakers yeast, Saccharomyces cerevisiae, are particularly suited for classroom science activities. Describes the sexual life cycle of yeast and the genetic system with visible mutations. Presents an overview of activities that can be done with yeast and gives a source for teachers to obtain more information. (PR)

  3. Long-term efficacy of enzyme replacement therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID).

    PubMed

    Chan, Belinda; Wara, Diane; Bastian, John; Hershfield, Michael S; Bohnsack, John; Azen, Colleen G; Parkman, Robertson; Weinberg, Kenneth; Kohn, Donald B

    2005-11-01

    Adenosine deaminase (ADA)-deficient Severe Combined Immunodeficiency (ADA-deficient SCID) is characterized by impaired lymphocyte development and function resulting from the adenosine metabolism defect. Enzyme replacement therapy with polyethylene glycol-conjugated adenosine deaminase (PEG-ADA) minimizes infectious complications of ADA-deficient patients who have not received bone marrow transplantation. In PEG-ADA therapy, enzymatically active ADA continuously circulates to act as a metabolic sink, detoxifying the adenosine and deoxyadenosine metabolites that accumulate to high levels in the absence of ADA. Studies have shown that upon the initiation of PEG-ADA therapy, the absolute numbers of circulating T and B lymphocytes and NK cells increase and protective immune function develops. However, the long-term efficacy is unknown. This retrospective study was designed to assess the long-term effectiveness of PEG-ADA treatment, based on evaluation of the immune function of nine ADA-deficient SCID patients (age 5-15) treated over the past decade. The results showed that the lymphocyte counts of all of the PEG-ADA treated patients were below the normal range at all times, despite initial improvements. A gradual decline of mitogenic proliferative responses occurred after a few years of treatment and normal antigenic response occurred less than expected. To this date, these low numbers and functions of lymphocytes had been adequate to provide protective immunity. These patients should be followed closely to detect a premature decline in immune function with aging in future decades of PEG-ADA therapy. PMID:16112907

  4. Structural and Kinetic Characterization of Escherichia coli TadA, the Wobble-Specific tRNA Deaminase

    SciTech Connect

    Kim,J.; Malashkevich, V.; Roday, S.; Lisbin, M.; Schramm, V.; Almo, S.

    2006-01-01

    The essential tRNA-specific adenosine deaminase catalyzes the deamination of adenosine to inosine at the wobble position of tRNAs. This modification allows for a single tRNA species to recognize multiple synonymous codons containing A, C, or U in the last (3'-most) position and ensures that all sense codons are appropriately decoded. We report the first combined structural and kinetic characterization of a wobble-specific deaminase. The structure of the Escherichia coli enzyme clearly defines the dimer interface and the coordination of the catalytically essential zinc ion. The structure also identifies the nucleophilic water and highlights residues near the catalytic zinc likely to be involved in recognition and catalysis of polymeric RNA substrates. A minimal 19 nucleotide RNA stem substrate has permitted the first steady-state kinetic characterization of this enzyme (k{sub cat} = 13 {+-} 1 min{sup -1} and K{sub M} = 0.83 {+-} 0.22 {micro}M). A continuous coupled assay was developed to follow the reaction at high concentrations of polynucleotide substrates (>10 {micro}M). This work begins to define the chemical and structural determinants responsible for catalysis and substrate recognition and lays the foundation for detailed mechanistic analysis of this essential enzyme.

  5. Raman signature of the four-stranded intercalated cytosine motif in crystal and solution structures of DNA deoxycytidylates d(CCCT) and d(C8).

    PubMed

    Benevides, J M; Kang, C; Thomas, G J

    1996-05-01

    The Raman spectral signature of the four-stranded cytosine structure formed by intercalation of two hemiprotonated and parallel-stranded oligodeoxycytidylate duplexes (so-called i motif) has been obtained from the crystal structure of d(CCCT) [Kang, C.H., Berger, I., Lockshin, C., Ratliff, R., Moyzis, R., & Rich, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11636-11640]. Identification of Raman markers diagnostic of the cytosine quadruplex is complemented by results obtained in a pH titration of 2'-deoxycytosine-5'-monophosphate (5'-dCMP) to show that the spectral fingerprint associated with N3 protonation of cytosine is distinct from that of quadruplex formation. The Raman spectrum thus provides a definitive basis for evaluating quantitatively both the extent of cytosine quadruplex formation and the degree of cytosine N3 protonation in DNA. Application to aqueous d(CCCT) and d(C8) demonstrates that the four-stranded intercalated structure is formed by both of these oligodeoxycytidylates in aqueous solution. Whereas both 5'-dCMP and the d(CCCT) quadruplex exhibit a midpoint of titration (apparent pKc) of 4.5 +/- 0.2 at 10 degrees C, cytosine protonation in d(C8) is shifted significantly toward the physiological range, with pKc = 5.8 +/- 0.2. The difference in pKc between the two quadruplexes is equivalent to a free energy difference of 1.7 kcal/mol at 10 degrees C. The present findings extend the library of Raman conformation markers to deoxycytidylate residues in the novel i quadruplex. The significance of these results for probing solution conformations of telomeric DNA sequences is also considered. PMID:8639535

  6. Gene-Transferred Oligoclonal T Cells Predominantly Persist in Peripheral Blood from an Adenosine Deaminase-Deficient Patient during Gene Therapy

    Microsoft Academic Search

    Yoshikata Misaki; Ichiko Ezaki; Tadashi Ariga; Nobuaki Kawamura; Yukio Sakiyama; Kazuhiko Yamamoto

    2001-01-01

    Adenosine deaminase (ADA) deficiency is the primary cause of severe combined immunodeficiency disease and has become a focus for developing innovative approaches to gene therapy. We previously described successful treatment of a Japanese ADA-deficient patient by periodic infusions of genetically modified autologous T lymphocytes transduced with a retroviral vector containing human ADA cDNA. In order to investigate whether polyclonality was

  7. Correct splicing despite mutation of the invariant first nucleotide of a 5[prime] splice site: A possible basis for disparate clinical phenotypes in siblings with adenosine deaminase deficiency

    Microsoft Academic Search

    F. X. Arredondo-Vega; I. Santisteban; S. Kelly; M. S. Hershfield; D. T. Umetsu; C. M. Schlossman

    1994-01-01

    Adenosine deaminase (ADA) deficiency usually causes severe combined immune deficiency in infancy. Milder phenotypes also occur and are associated with less severely impaired deoxyadenosine (dAdo) catabolism. The authors have characterized the mutations responsible for ADA deficiency in siblings with disparity in clinical phenotype. Erythrocyte dAdo nucleotide pool size, which reflects total residual ADA activity, was lower in the older, more

  8. Substrate inhibition of adenosine phosphorylation in adenosine deaminase deficiency and adenosine-mediated inhibition of PP-ribose-P dependent nucleotide synthesis in hypoxanthine phosphoribosyltransferase deficient erythrocytes

    Microsoft Academic Search

    F. F. Snyder; C. Dyer; J. E. Seegmiller; R. M. Goldblum; G. C. Mills; F. C. Schmalstieg

    1988-01-01

    Summary The metabolism of adenosine and its effects on phosphoribosylpyrophosphate, PP-ribose-P, dependent nucleotide synthesis were studied using erythrocytes from patients with adenosine deaminase and hypoxanthine phosphoribosyltransferase deficiency as models. The phosphorylation of adenosine was progressively inhibited by concentrations of adenosine greater than 1 µmol L-1 for control and ADA deficient erythrocytes. There was essentially no initial rate of phosphorylation at

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

    Microsoft Academic Search

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

    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

  10. Varicella pneumonia in a bone marrow-transplanted, immune-reconstituted adenosine deaminase-deficient patient with severe combined immunodeficiency disease

    Microsoft Academic Search

    Mark Ballow; Rochelle Hirschhorn

    1985-01-01

    Bone marrow transplantation provides an important modality for “enzyme replacement” and the immune reconstitution of patients with adenosine deaminase (ADA) deficiency and severe combined immunodeficiency disease. We report a patient with ADA deficiency who develops severe varicella pneumonia 6 years after successful bone marrow transplantation and immune reconstitution. Marked abnormalities in T-cell mitogen responsiveness and pokeweed mitogen-induced polyclonal immunoglobulin synthesis

  11. HLA-haploidentical bone marrow transplantation in three infants with adenosine deaminase deficiency: Stable immunological reconstitution and reversal of skeletal abnormalities

    Microsoft Academic Search

    R. Bluetters-Sawatzki; W. Friedrich; W. Ebell; U. Vetter; H. Stoess; S. F. Goldmann; E. Kleihauer

    1989-01-01

    Three infants with severe combined immunodeficiency and adenosine deaminase (ADA) deficiency were treated by T-cell depleted bone marrow transplantation (BMT), using human leukocyte antigen (HLA)-haploidentical parents as donors. In the first patient, two initial transplants failed to engraft and no change of the immunodeficiency was observed. In order to overcome this graft resistance, cytoreductive conditioning was used prior to a

  12. Effects of Retroviral Vector Design on Expression of Human Adenosine Deaminase in Murine Bone Marrow Transplant Recipients Engrafted with Genetically Modified Cells

    Microsoft Academic Search

    Isabelle Riviere; Katja Brose; Richard C. Mulligan

    1995-01-01

    To determine which features of retroviral vector design most critically affect gene expression in hematopoietic cells in vivo, we have constructed a variety of different retroviral vectors which encode the same gene product, human adenosine deaminase (EC 3.5.4.4), and possess the same vector backbone yet differ specifically in transcriptional control sequences suggested by others to be important for gene expression

  13. Immunity 24, 779786, June 2006 2006 Elsevier Inc. DOI 10.1016/j.immuni.2006.03.021 A Role for Activation-Induced Cytidine Deaminase

    E-print Network

    Papavasiliou, F. Nina

    for Activation-Induced Cytidine Deaminase in the Host Response against a Transforming Retrovirus Polyxeni Gourzi and the deletional recom- bination between constant regions resulting in Ig isotype switching (Chaudhuri and Alt as their primary function in vivo the reduction of viral infectivity, there is no a priori reason why AID or APOBEC

  14. N4-methylation of cytosine drastically favors the formation of (6-4) photoproducts in a TCG context.

    PubMed

    Douki, Thierry; Meador, Jarah A; Bérard, Izabel; Wack, Aude

    2015-01-01

    Methylation of cytosine is a common biological process both in prokaryotic and eukaryotic cells. In addition to 5-methylcytosine (5mC), some bacterial species contain in their genome N(4) -methylcytosine (N4mC). Methylation at C5 has been shown to enhance the formation of pyrimidine dimeric photoproducts but nothing is known of the effect of N4 methylation on UV-induced DNA damage. In the present work, we compared the yield and the nature of bipyrimidine photoproducts induced in a series of trinucleotides exhibiting a TXG sequence where X is either T, C, 5mC or N4mC. HPLC associated to tandem mass spectrometry was used to quantify cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts (64PP) and their Dewar valence isomer. Methylation at position N4 was found to drastically increase the reactivity of C upon exposure to both UVC and UVB and to favor the formation of 64PP. In contrast methylation at C5 increased the yield of CPD at the expense of 64PP. In addition, enhancement of photoreactivity by C5 methylation was much higher in the UVB than in the UVC range. These results show the drastic effect of the methylation site on the photochemistry of cytosine. PMID:25319211

  15. Mutagenic effects induced by the attack of NO2 radical to the guanine-cytosine base pair

    PubMed Central

    Cerón-Carrasco, José P.; Requena, Alberto; Zúñiga, José; Jacquemin, Denis

    2015-01-01

    We investigate the attack of the nitrogen dioxide radical (NO•2) to the guanine—cytosine (GC) base pair and the subsequent tautomeric reactions able to induce mutations, by means of density functional theory (DFT) calculations. The conducted simulations allow us to identify the most reactive sites of the GC base pair. Indeed, the computed relative energies demonstrate that the addition of the NO•2 radical to the C8 position of the guanine base forms to the most stable adduct. Although the initial adducts might evolve to non-canonical structures via inter-base hydrogen bonds rearrangements, the probability for the proton exchange to occur lies in the same range as that observed for undamaged DNA. As a result, tautomeric errors in NO2-attacked DNA arises at the same rate as in canonical DNA, with no macroscopic impact on the overall stability of DNA. The potential mutagenic effects of the GC–NO•2 radical adducts likely involve side reactions, e.g., the GC deprotonation to the solvent, rather than proton exchange between guanine and cytosine basis. PMID:25798437

  16. Mutagenic Effects Induced by the Attack of NO2 Radical to the Guanine-Cytosine Base Pair

    NASA Astrophysics Data System (ADS)

    Cerón-Carrasco, José Pedro; Requena, Alberto; Zúñiga, José; Jacquemin, Denis

    2015-03-01

    We investigate the attack of the nitrogen dioxide radical (NO2) to the guanine-cytosine (GC) base pair and the subsequent tautomeric reactions able to induce mutations, by means of density functional theory (DFT) calculations. The conducted simulations allow us to identify the most reactive sites of the GC base pair. Indeed, the computed relative energies demonstrate that the addition of the NO2 radical to the C8 position of the guanine base forms to the most stable adduct. Although the initial adducts might evolve to non-canonical structures via inter-base hydrogen bonds rearrangements, the probability for the proton exchange to occur lies in the same range as that observed for undamaged DNA. As a result, tautomeric errors in NO2-attacked DNA arises at the same rate as in canonical DNA, with no macroscopic impact on the overall stability of DNA. The potential mutagenic effects of the GC-NO2 radical adducts likely involve side reactions, e.g., the GC deprotonation to the solvent, rather than proton exchange between guanine and cytosine basis.

  17. DPPA3 prevents cytosine hydroxymethylation of the maternal pronucleus and is required for normal development in bovine embryos.

    PubMed

    Bakhtari, Azizollah; Ross, Pablo J

    2014-09-01

    Dppa3 has been described in mice as an important maternal factor contributed by the oocyte that participates in protecting the maternal genome from oxidation of methylated cytosines (5mC) to hydroxymethylated cytosines (5hmC). Dppa3 is also required for normal mouse preimplantation development. This gene is poorly conserved across mammalian species, with less than 32% of protein sequence shared between mouse, cow and human. RNA-seq analysis of bovine oocytes and preimplantation embryos revealed that DPPA3 transcripts are some of the most highly abundant mRNAs in the oocyte, and their levels gradually decrease toward the time of embryonic genome activation (EGA). Knockdown of DPPA3 by injection of siRNA in germinal vesicle (GV) stage oocytes was used to assess its role in epigenetic remodeling and embryo development. DPPA3 knockdown resulted in increased intensity of 5hmC staining in the maternal pronucleus (PN), demonstrating a role for this factor in the asymmetric remodeling of the maternal and paternal PN in bovine zygotes. Also, DPPA3 knockdown decreased the developmental competence of parthenogenetic and in vitro fertilized embryos. Finally, DPPA3 knockdown embryos that reached the blastocyst stage had significantly fewer ICM cells as compared with control embryos. We conclude that DPPA3 is a maternal factor important for correct epigenetic remodeling and normal embryonic development in cattle, indicating that the role of DPPA3 during early development is conserved between species. PMID:25147917

  18. Synthesis of pyrimidin-2-one nucleosides as acid-stable inhibitors of cytidine deaminase.

    PubMed

    Kim, C H; Marquez, V E; Mao, D T; Haines, D R; McCormack, J J

    1986-08-01

    One of the problems encountered in the use of tetrahydrouridine (THU, 2) and saturated 2-oxo-1,3-diazepine nucleosides as orally administered cytidine deaminase (CDA) inhibitors is their acid instability. Under acid conditions these compounds are rapidly converted into inactive ribopyranoside forms. A solution this problem was sought by functionalizing the acid-stable but less potent CDA inhibitor 1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (1) with the hope of increasing its potency to the level achieved with THU. The selection of the hydroxymethyl substituent at C-4, which led to the synthesis of 4-(hydroxymethyl)-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (10), 3,4-dihydro-4-(hydroxymethyl)-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (7), and 3,4,5,6-tetrahydro-4-(dihydroxymethyl)-1-beta-D-ribofuranosyl-2(1H)-p yrimidinone (28) was based on the transition-state (TS) concept. The key intermediate precursor, 4-[(benzoyloxy)methyl]-1-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-2(H) -pyrimidinone (24), was obtained via the classical Hilbert-Johnson reaction between 2-methoxy-4-[(benzoyloxy)methyl]pyrimidine (20) and 2,3,5-tri-O-benzoyl-1-D-ribofuranosyl bromide (21). Deprotection of 24 afforded compound 10, while its sodium borohydride reduction products afforded compounds 7 and 28 after removal of the blocking groups. Syntheses of 3,4-dihydro-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (9) and 3,6-dihydro-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (8), which lack the hydroxymethyl substituent, was accomplished in a similar fashion. The new compounds bearing the hydroxymethyl substituent were more acid stable than THU, and their CDA inhibitory potency, expressed in terms of Ki values, spanned from 10(-4) to 10(-7) M in a manner consistent with the TS theory. Compound 7, in particular, was superior to its parent 1 and equipotent to THU (Ki = 4 X 10(-7) M) when examined against mouse kidney CDA. The superior acid stability of this compound coupled to its potent inhibitory properties against CDA should provide a means of testing oral combinations of rapidly deaminated drugs, viz. ara-C, without the complications associated with the acid instability of THU. PMID:3735306

  19. Overexpression of multisubunit replication factors in yeast.

    PubMed

    Burgers, P M

    1999-07-01

    Facile genetic and biochemical manipulation coupled with rapid cell growth and low cost of growth media has established the yeast Saccharomyces cerevisiae as a versatile workhorse. This article describes the use of yeast expression systems for the overproduction of complex multipolypeptide replication factors. The regulated overexpression of these factors in yeast provides for a readily accessible and inexpensive source of these factors in large quantities. The methodology is illustrated with the five-subunit replication factor C. Whole-cell extracts are prepared by blending yeast cells with glass beads or frozen yeast with dry ice. Procedures are described that maximize the yield of these factors while minimizing proteolytic degradation. PMID:10454996

  20. Molecular Genetic Analysis in Yeast

    NSDL National Science Digital Library

    Daniel D. Burke (Seton Hall University; )

    1990-01-01

    This resource provides techniques and protocols used in basic and advanced procedures of recombinant DNA technology to perform molecular genetic analysis in the yeast Saccharomyces cerevisiae. Students will be exposed to techniques such as transformation, restriction endonuclease digestion, electrophoresis and Southern blot analysis.

  1. Barcoding the Yeasts – Which Genes?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Old style yeast identification, as many know, is an onerous process requiring determination of growth reactions on 60-100 different media. Once completed, there is still a high degree of uncertainty about species identity. With the determination of sequences for domains 1 and 2 (D1/D2) of the nucl...

  2. Yeast Proteomics and Protein Microarrays

    PubMed Central

    Chen, Rui; Snyder, Michael

    2010-01-01

    Our understanding of biological processes as well as human diseases has improved greatly thanks to studies on model organisms such as yeast. The power of scientific approaches with yeast lies in its relatively simple genome, its facile classical and molecular genetics, as well as the evolutionary conservation of many basic biological mechanisms. However, even in this simple model organism, systems biology studies, especially proteomic studies had been an intimidating task. During the past decade, powerful high-throughput technologies in proteomic research have been developed for yeast including protein microarray technology. The protein microarray technology allows the interrogation of protein-protein, protein-DNA, protein-small molecule interaction networks as well as post-translational modification networks in a large-scale, high-throughput manner. With this technology, many groundbreaking findings have been established in studies with the budding yeast Saccharomyces cerevisiae, most of which could have been unachievable with traditional approaches. Discovery of these networks has profound impact on explicating biological processes with a proteomic point of view, which may lead to a better understanding of normal biological phenomena as well as various human diseases. PMID:20728591

  3. [Heterologous interferons synthesis in yeast Pichia pastoris].

    PubMed

    Padkina, M V; Parfenova, L V; Gradoboeva, A E; Sambuk, E V

    2010-01-01

    The HuIFNA16, HuIFNB, and BoIFNG genes encoding human [alpha]16, beta-interferons and bovine gamma-interferon were cloned under the control of the yeast Pichia pastoris AOX1 gene promoter. The yeast strains producing heterologous interferons intracellularly and extracellularly were constructed. There was no effect of high level of heterologous protein synthesis on the yeast P. pastoris cell growth, unlike yeast Saccharomyces cerevisiae. The considerable part of the heterologous interferons was detected in the yeast P. pastoris soluble protein fraction but not in the "inclusion bodies." The treatment of human beta-interferon with endoglycosidase H showed that protein was expressed in glycosilated and unglycosilated forms. On the strength of these data, the hypothesis was suggested that the more effective heterologous gene expression in yeast P. pastoris and enhanced resistance of the methylotrophic yeast to negative effects of recombinant proteins was due to the special features of its metabolism. PMID:20873170

  4. Bacterial biosynthesis of 1-aminocyclopropane-1-caboxylate (ACC) deaminase, a useful trait to elongation and endophytic colonization of the roots of rice under constant flooded conditions.

    PubMed

    Etesami, Hassan; Mirseyed Hosseini, Hossein; Alikhani, Hossein Ali

    2014-10-01

    This study was conducted to investigate the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Pseudomonas fluorescens strain REN1 and its ability to reduce ethylene levels produced during stress, endophytically colonize and promote the elongation of the roots of rice seedlings under gnotobiotic conditions. We isolated 80 bacteria from inside roots of rice plants grown in the farmers' fields in Guilan, Iran. All of the isolates were characterized for plant growth promoting (PGP) traits. In addition, the colonization assay of these isolates on rice seedlings was carried out to screen for competent endophytes. The best bacterial isolate, based on ACC deaminase production, was identified and used for further study. 16S rDNA sequence analysis revealed that the endophyte was closely related to Pseudomonas fluorescens. The results of this study showed ACC deaminase containing P. fluorescens REN1 increased in vitro root elongation and endophytically colonized the root of rice seedlings significantly, as compared to control under constant flooded conditions. The trait of low amount of indole-3-acetic acid (IAA) production (<15 ?g mL(-1)) and the high production of ACC deaminase by bacteria may be main factors in colonizing rice seedling roots compared to other PGP traits (siderophore production and phosphate solubilization) in this study. Endophytic IAA and ACC deaminase-producing bacteria may be preferential selections by rice seedlings. Therefore, it may be suggested that the utilization of ACC as a nutrient gives the isolates advantages in more endophytic colonization and increase of root length of rice seedlings. PMID:25320466

  5. The Plant Cell, Vol. 13, 17491759, August 2001, www.plantcell.org 2001 American Society of Plant Biologists Sequence Elimination and Cytosine Methylation Are Rapid and

    E-print Network

    Bhattacharyya, Madan Kumar

    Biologists Sequence Elimination and Cytosine Methylation Are Rapid and Reproducible Responses of the Genome with such genomic stress is a significant, al- beit poorly understood, question. In plants, it is estimated that polyploidy occurred in the history of 70% of flowering species (Masterson, 1994). Genome sequencing data may

  6. Cytosine Arabinoside Kills Postmitotic Neurons in a Fashion Resembling Trophic Factor Deprivation: Evidence That a Deoxycytidine-Dependent Process May Be Required for Nerve Growth Factor Signal Transduction

    Microsoft Academic Search

    David P. Martin; Thomas L. Wallace; Eugene M. Johnson

    Cytosine arabinoside (AraC) is a pyrimidine antimetabolite that kills proliferating cells by inhibiting DNA synthesis. In this paper we report that AraC kills postmitotic rat sympa- thetic neurons in a fashion similar to the neuronal death that follows nerve growth factor (NGF) deprivation. Postmitotic rat sympathetic neurons were cultured for 1 week in the presence of NGF and then treated

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

    SciTech Connect

    Sadat Hayatshahi, Sayyed Hamed [Department of Biophysics, Faculty of Science, Tarbiat Modares University, P.O. Box: 14115/175, Tehran (Iran, Islamic Republic of) ; Abdolmaleki, Parviz [Department of Biophysics, Faculty of Science, Tarbiat Modares University, P.O. Box: 14115/175, Tehran (Iran, Islamic Republic of) ]. E-mail: parviz@modares.ac.ir; Safarian, Shahrokh [Department of Biology, Faculty of Science, Tehran University, P.O. Box: 13155-6455, Tehran (Iran, Islamic Republic of) ; Khajeh, Khosro [Department of Biochemistry, Faculty of Science, Tarbiat Modares University, P.O. Box: 14115/175, Tehran (Iran, Islamic Republic of)

    2005-12-16

    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 {sub 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%.

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

    PubMed

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

    2005-12-16

    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%. PMID:16256072

  9. Random Mutagenesis MAPPIT Analysis Identifies Binding Sites for Vif and Gag in Both Cytidine Deaminase Domains of Apobec3G

    PubMed Central

    Uyttendaele, Isabel; Lavens, Delphine; Catteeuw, Dominiek; Lemmens, Irma; Bovijn, Celia

    2012-01-01

    The mammalian two-hybrid system MAPPIT allows the detection of protein-protein interactions in intact human cells. We developed a random mutagenesis screening strategy based on MAPPIT to detect mutations that disrupt the interaction of one protein with multiple protein interactors simultanously. The strategy was used to detect residues of the human cytidine deaminase Apobec3G that are important for its homodimerization and its interaction with the HIV-1 Gag and Vif proteins. The strategy is able to identify the previously described head-to-head homodimerization interface in the N-terminal domain of Apobec3G. Our analysis further detects two new potential interaction surfaces in the N-and C-terminal domain of Apobec3G for interaction with Vif and Gag or for Apobec3G dimerization. PMID:22970171

  10. Fate Mapping for Activation-Induced Cytidine Deaminase (AID) Marks Non-Lymphoid Cells During Mouse Development

    PubMed Central

    Rommel, Philipp C.; Bosque, David; Gitlin, Alexander D.; Croft, Gist F.; Heintz, Nathaniel; Casellas, Rafael; Nussenzweig, Michel C.; Kriaucionis, Skirmantas; Robbiani, Davide F.

    2013-01-01

    The Aicda gene encodes Activation-Induced cytidine Deaminase (AID), an enzyme essential for remodeling antibody genes in mature B lymphocytes. AID is also responsible for DNA damage at oncogenes, leading to their mutation and cancer-associated chromosome translocation in lymphoma. We used fate mapping and AIDGFP reporter mice to determine if AID expression in the mouse extends beyond lymphocytes. We discovered that AIDcre tags a small fraction of non-lymphoid cells starting at 10.5 days post conception (dpc), and that AIDGFP+ cells are detectable at dpc 11.5 and 12.5. Embryonic cells are tagged by AIDcre in the submandibular region, where conditional deletion of the tumor suppressor PTEN causes squamous papillomas. AIDcre also tags non-lymphoid cells in the embryonic central nervous system. Finally, in the adult mouse brain, AIDcre marks a small fraction of diverse neurons and distinct neuronal populations, including pyramidal cells in cortical layer IV. PMID:23861962

  11. Functionalized Tricyclic Cytosine Analogues Provide Nucleoside Fluorophores with Improved Photophysical Properties and a Range of Solvent Sensitivities

    PubMed Central

    Rodgers, Brittney J.; Elsharif, Nada A.; Vashisht, Nisha; Mingus, Macy M.; Mulvahill, Mark A.; Stengel, Gudrun; Kuchta, Robert D.

    2014-01-01

    Tricyclic cytosines (tC and tCO frameworks) have emerged as a unique class of fluorescent nucleobase analogues that minimally perturb the structure of B-form DNA and that are not quenched in duplex nucleic acids. Systematic derivatization of these frameworks is a likely approach to improve on and diversify photophysical properties, but has not so far been examined. Synthetic methods were refined to improve on tolerance for electron donating and electron withdrawing groups, resulting in a series of eight new, fluorescent cytidine analogues. Photophysical studies show that substitution of the framework results in a pattern of effects largely consistent across tC and tCO and provides nucleoside fluorophores that are brighter than either parent. Moreover, a range of solvent sensitivities is observed, offering promise that this family of probes can be extended to new applications that require reporting on the local environment. PMID:24311229

  12. Adjuvant properties of Cytosine-phosphate-guanosine oligodeoxynucleotide in combination with various polycations in an ovalbumin-vaccine model.

    PubMed

    Maubant, Sylvie; Banissi, Claire; Beck, Samantha; Chauvat, Anne; Carpentier, Antoine F

    2011-08-01

    Oligonucleotides containing CpG motifs (cytosine-phosphate-guanosine oligodeoxynucleotide [CpG ODN]) display strong immunostimulatory effects, and polycations have been previously reported as cellular delivery system. In the present study, we investigated the adjuvant properties of combinations of a CpG ODN with various polycations (poly-arginine, poly-lysine, poly-histidine, or chitosan) in an ovalbumin vaccination model. We showed that, when combined to CpG ODN, poly-arginine and poly-histidine, but not poly-lysine or chitosan, enhanced efficiently both the IgG antibody production and the number of splenocytes secreting interferon-gamma after stimulation with a CD8+ T cell-restricted peptide. Interestingly, CpG ODN-poly-arginine, which was the most efficient, compared favorably to the complete Freund's adjuvant and aluminium salts and induced no local toxicity, making this combination a very attractive adjuvant for vaccines. PMID:21787231

  13. Understanding the structural and dynamic consequences of DNA epigenetic modifications: Computational insights into cytosine methylation and hydroxymethylation.

    PubMed

    Carvalho, Alexandra T P; Gouveia, Leonor; Kanna, Charan Raju; Wärmländer, Sebastian K T S; Platts, Jamie A; Kamerlin, Shina Caroline Lynn

    2014-12-01

    We report a series of molecular dynamics (MD) simulations of up to a microsecond combined simulation time designed to probe epigenetically modified DNA sequences. More specifically, by monitoring the effects of methylation and hydroxymethylation of cytosine in different DNA sequences, we show, for the first time, that DNA epigenetic modifications change the molecule's dynamical landscape, increasing the propensity of DNA toward different values of twist and/or roll/tilt angles (in relation to the unmodified DNA) at the modification sites. Moreover, both the extent and position of different modifications have significant effects on the amount of structural variation observed. We propose that these conformational differences, which are dependent on the sequence environment, can provide specificity for protein binding. PMID:25625845

  14. Cumulative bone marrow stromal damage caused by X-irradiation and cytosine-arabinoside in leukemic mice.

    PubMed

    Ben-Ishay, Z; Prindull, G; Yankelev, S; Sharon, S

    1990-01-01

    A study of treated murine acute myeloid leukemia (AML) with an emphasis on the bone marrow stromal function is reported. Leukemia was induced in C57Bl mice through intraperitoneal (i.p.) inoculation of C-1498 myelogenous leukemic cells. The leukemic mice were administered: (1) total body lethal X-irradiation (t.b.i.); (2) two i.p. cytosine-arabinoside (Ara-C) injections followed by X-irradiation. Control mice received similar regimens. Bone marrow of experimental and control mice was processed for stromal cell cultures (SCC) and in vitro engraftment of hematopoietic cells onto the cultures. The results of this study indicate that the bone marrow stromal deficiency which occurs in leukemia is aggravated by Ara-C and irradiation treatments. Moreover, SCC of treated leukemic mice sustain in vitro hematopoiesis only to a limited degree. Stromal deficiency, as possible cause for graft failure in bone marrow transplanted leukemic patients, is discussed. PMID:2187123

  15. Pheromone Signaling Pathways in Yeast

    NSDL National Science Digital Library

    Henrik G. Dohlman (University of North Carolina; Department of Biochemistry and Biophysics REV)

    2006-12-05

    The actions of many extracellular stimuli are elicited by complexes of cell surface receptors, heterotrimeric guanine nucleotide–binding proteins (G proteins), and mitogen-activated protein kinase (MAPK) complexes. Analysis of haploid yeast cells and their response to peptide mating pheromones has produced important advances in the understanding of G protein and MAPK signaling mechanisms. Many of the components, their interrelationships, and their regulators were first identified in yeast. Examples include definitive demonstration of a positive signaling role for G protein ?? subunits, the discovery of a three-tiered structure of the MAPK module, development of the concept of a kinase-scaffold protein, and the discovery of the first regulator of G protein signaling protein. New and powerful genomic, proteomic, and computational approaches available in yeast are beginning to uncover new pathway components and interactions and have revealed their presence in unexpected locations within the cell. This updated Connections Map in the Database of Cell Signaling includes several major revisions to this prototypical signal response pathway.

  16. Geminivirus AL2 and L2 proteins suppress transcriptional gene silencing and cause genome-wide reductions in cytosine methylation.

    PubMed

    Buchmann, R Cody; Asad, Shaheen; Wolf, Jamie N; Mohannath, Gireesha; Bisaro, David M

    2009-05-01

    Geminiviruses replicate single-stranded DNA genomes through double-stranded intermediates that associate with cellular histone proteins. Unlike RNA viruses, they are subject to RNA-directed methylation pathways that target viral chromatin and likely lead to transcriptional gene silencing (TGS). Here we present evidence that the related geminivirus proteins AL2 and L2 are able to suppress this aspect of host defense. AL2 and L2 interact with and inactivate adenosine kinase (ADK), which is required for efficient production of S-adenosyl methionine, an essential methyltransferase cofactor. We demonstrate that the viral proteins can reverse TGS of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana when overexpressed from a Potato virus X vector and that reversal of TGS by geminiviruses requires L2 function. We also show that AL2 and L2 cause ectopic expression of endogenous Arabidopsis thaliana loci silenced by methylation in a manner that correlates with ADK inhibition. However, at one exceptional locus, ADK inhibition was insufficient and TGS reversal required the transcriptional activation domain of AL2. Using restriction-sensitive PCR and bisulfite sequencing, we showed that AL2-mediated TGS suppression is accompanied by reduced cytosine methylation. Finally, using a methylation-sensitive single-nucleotide extension assay, we showed that transgenic expression of AL2 or L2 causes global reduction in cytosine methylation. Our results provide further evidence that viral chromatin methylation is an important host defense and allow us to propose that as a countermeasure, geminivirus proteins reverse TGS by nonspecifically inhibiting cellular transmethylation reactions. To our knowledge, this is the first report that viral proteins can inhibit TGS. PMID:19279102

  17. Yeasts Diversity in Fermented Foods and Beverages

    NASA Astrophysics Data System (ADS)

    Tamang, Jyoti Prakash; Fleet, Graham H.

    People across the world have learnt to culture and use the essential microorganisms for production of fermented foods and alcoholic beverages. A fermented food is produced either spontaneously or by adding mixed/pure starter culture(s). Yeasts are among the essential functional microorganisms encountered in many fermented foods, and are commercially used in production of baker's yeast, breads, wine, beer, cheese, etc. In Asia, moulds are predominant followed by amylolytic and alcohol-producing yeasts in the fermentation processes, whereas in Africa, Europe, Australia and America, fermented products are prepared exclusively using bacteria or bacteria-yeasts mixed cultures. This chapter would focus on the varieties of fermented foods and alcoholic beverages produced by yeasts, their microbiology and role in food fermentation, widely used commercial starters (pilot production, molecular aspects), production technology of some common commercial fermented foods and alcoholic beverages, toxicity and food safety using yeasts cultures and socio-economy

  18. Biopharmaceutical discovery and production in yeast.

    PubMed

    Meehl, Michael A; Stadheim, Terrance A

    2014-12-01

    The selection of an expression platform for recombinant biopharmaceuticals is often centered upon suitable product titers and critical quality attributes, including post-translational modifications. Although notable differences between microbial, yeast, plant, and mammalian host systems exist, recent advances have greatly mitigated any inherent liabilities of yeasts. Yeast expression platforms are important to both the supply of marketed biopharmaceuticals and the pipelines of novel therapeutics. In this review, recent advances in yeast-based expression of biopharmaceuticals will be discussed. The advantages of using glycoengineered yeast as a production host and in the discovery space will be illustrated. These advancements, in turn, are transforming yeast platforms from simple production systems to key technological assets in the discovery and selection of biopharmaceutical lead candidates. PMID:25014890

  19. Metabolic engineering of malolactic wine yeast.

    PubMed

    Husnik, John I; Volschenk, Heinrich; Bauer, Jurgen; Colavizza, Didier; Luo, Zongli; van Vuuren, Hennie J J

    2006-07-01

    Malolactic fermentation is essential for the deacidification of high acid grape must. We have constructed a genetically stable industrial strain of Saccharomyces cerevisiae by integrating a linear cassette containing the Schizosaccharomyces pombe malate permease gene (mae1) and the Oenococcus oeni malolactic gene (mleA) under control of the S. cerevisiae PGK1 promoter and terminator sequences into the URA3 locus of an industrial wine yeast. The malolactic yeast strain, ML01, fully decarboxylated 5.5 g/l of malate in Chardonnay grape must during the alcoholic fermentation. Analysis of the phenotype, genotype, transcriptome, and proteome revealed that the ML01 yeast is substantially equivalent to the parental industrial wine yeast. The ML01 yeast enjoys 'Generally Regarded As Safe' status from the FDA and is the first genetically enhanced yeast that has been commercialized. Its application will prevent the formation of noxious biogenic amines produced by lactic acid bacteria in wine. PMID:16621641

  20. Physiological properties of some yeast strains.

    PubMed

    Oprean, Letitia; Gaspar, Enikö; Lengyel, Ecaterina; Cristea, V

    2006-06-01

    Twenty yeast strains have recently been isolated in pure cultures from natural and industrial sources and identified based mainly on physiological properties. The majority of the strains (15) are alcohologenic belonging to the genus Saccharomyces and comprise two brewer's (beer) yeast strains (S. carlsbergensis= S. uvarum A and B), two baker's yeast strains (S. cerevisiae CA and CP), one spirit yeast strain (S. cerevisiae CF) and ten wine yeast strains (S. cerevisiae var. ellipsoideus = S. ellipsoideus 1, 3, 4, 6, 8 and 9; S. oviformis 2, 5 and 7; and S. uvarum 10). The other 5 yeast strains belong to different species: Kloeckera apiculate, Candida mycoderma (Mycoderma vini), Pichia membranaefaciens, Rhodotorula glutinis and Torulopsis holmii, respectively. PMID:16841476

  1. Screening for l -arabinose fermenting yeasts

    Microsoft Academic Search

    Bruce S. Dien; Cletus P. Kurtzman; Badal C. Saha; Rodney J. Bothast

    1996-01-01

    Utilization of pentose sugars (d-xylose andl-arabinose) derived from hemi-cellulose is essential for the economic conversion of biomass to ethanol. Xylose-fermenting\\u000a yeasts were discovered in the 1980s, but to date, no yeasts have been found that fermentl-arabinose to ethanol in significant quantities. We have screened 116 different yeasts for the ability to fermentl-arabinose and have found the following species able to

  2. Characterisation of yeast microbial fuel cell with the yeast Arxula adeninivorans as the biocatalyst

    Microsoft Academic Search

    Nicholas D. Haslett; Frankie J. Rawson; Frèdèric Barriëre; Gotthard Kunze; Neil Pasco; Ravi Gooneratne; Keith H. R. Baronian

    2011-01-01

    Yeast microbial fuel cells have received little attention to date. Yeast should be ideal MFC catalyst because they are robust, easily handled, mostly non-pathogenic organisms with high catabolic rates and in some cases a broad substrate spectrum. Here we show that the non-conventional yeast Arxula adeninvorans transfers electrons to an electrode through the secretion of a reduced molecule that is

  3. Presence of glucosylceramide in yeast and its relation to alkali tolerance of yeast

    Microsoft Academic Search

    Katsuichi Saito; Naoya Takakuwa; Masao Ohnishi; Yuji Oda

    2006-01-01

    Glycosylceramide is a membrane lipid that has physiological functions in eukaryotic organisms. The presence of glucosylceramide has been confirmed in some yeast; however, the extent of the role of glucosylceramide in yeast is unknown. Thus, the extent of presence of glucosylceramide in yeast was surveyed using 90 strains of 24 genera. The strains were divided into two groups according to

  4. APPENDIX 4LGrowth and Manipulation of Yeast PREPARATION OF SELECTED YEAST MEDIA

    E-print Network

    Winston, Fred

    no nutritional require- ments. However, it is used most often as a basal medium to which other supplementsAPPENDIX 4LGrowth and Manipulation of Yeast PREPARATION OF SELECTED YEAST MEDIA Like Escherichia coli, yeast can be grown in either liquid media or on the surface of (or embedded in) solid agar plates

  5. Evaluation of Automated Yeast Identification System

    NASA Technical Reports Server (NTRS)

    McGinnis, M. R.

    1996-01-01

    One hundred and nine teleomorphic and anamorphic yeast isolates representing approximately 30 taxa were used to evaluate the accuracy of the Biolog yeast identification system. Isolates derived from nomenclatural types, environmental, and clinica isolates of known identity were tested in the Biolog system. Of the isolates tested, 81 were in the Biolog database. The system correctly identified 40, incorrectly identified 29, and was unable to identify 12. Of the 28 isolates not in the database, 18 were given names, whereas 10 were not. The Biolog yeast identification system is inadequate for the identification of yeasts originating from the environment during space program activities.

  6. Role of glucose signaling in yeast metabolism

    SciTech Connect

    Dam, K. van [Univ. of Amsterdam (Netherlands). E.C. Slater Inst.

    1996-10-05

    The conversion of glucose to ethanol and carbon dioxide by yeast was the first biochemical pathway to be studied in detail. The initial observation that this process is catalyzed by an extract of yeast led to the discovery of enzymes and coenzymes and laid the foundation for modern biochemistry. In this article, knowledge concerning the relation between uptake of and signaling by glucose in the yeast Saccharomyces cerevisiae is reviewed and compared to the analogous process in prokaryotes. It is concluded that (much) more fundamental knowledge concerning these processes is required before rational redesign of metabolic fluxes from glucose in yeast can be achieved.

  7. Drosophila Regulate Yeast Density and Increase Yeast Community Similarity in a Natural Substrate

    PubMed Central

    Stamps, Judy A.; Yang, Louie H.; Morales, Vanessa M.; Boundy-Mills, Kyria L.

    2012-01-01

    Drosophila melanogaster adults and larvae, but especially larvae, had profound effects on the densities and community structure of yeasts that developed in banana fruits. Pieces of fruit exposed to adult female flies previously fed fly-conditioned bananas developed higher yeast densities than pieces of the same fruits that were not exposed to flies, supporting previous suggestions that adult Drosophila vector yeasts to new substrates. However, larvae alone had dramatic effects on yeast density and species composition. When yeast densities were compared in pieces of the same fruits assigned to different treatments, fruits that developed low yeast densities in the absence of flies developed significantly higher yeast densities when exposed to larvae. Across all of the fruits, larvae regulated yeast densities within narrow limits, as compared to a much wider range of yeast densities that developed in pieces of the same fruits not exposed to flies. Larvae also affected yeast species composition, dramatically reducing species diversity across fruits, reducing variation in yeast communities from one fruit to the next (beta diversity), and encouraging the consistent development of a yeast community composed of three species of yeast (Candida californica, C. zemplinina, and Pichia kluvyeri), all of which were palatable to larvae. Larvae excreted viable cells of these three yeast species in their fecal pools, and discouraged the growth of filamentous fungi, processes which may have contributed to their effects on the yeast communities in banana fruits. These and other findings suggest that D. melanogaster adults and their larval offspring together engage in ‘niche construction’, facilitating a predictable microbial environment in the fruit substrates in which the larvae live and develop. PMID:22860093

  8. Mechanism of Interferon Action: Double-Stranded RNA-Specific Adenosine Deaminase from Human Cells Is Inducible by Alpha and Gamma Interferons

    Microsoft Academic Search

    John B. Patterson; Daniel C. Thomis; Sherrie L. Hans; Charles E. Samuel

    1995-01-01

    Treatment of human amnion U cells with interferon increased the steady state level of mRNA encoding the double-stranded (ds) RNA-specific adenosine deaminase (AdD) as measured by Northern gel-blot analysis. A single major dsRNA-specific AdD transcript of ?6.7 kb was detected; the transcript was induced by both interferon-? (IFN-?) and interferon-? (IFN-?). Likewise, Western immunoblot analysis revealed that a 150-kDa protein

  9. Characterization of an Adenosine Deaminase-deficient Human Histiocytic Lymphoma Cell Line (DHL9) and Selection of Mutants Deficient in Adenosine Kinase and Deoxycytidine Kinase1

    Microsoft Academic Search

    Masaru Kubota; Naoyuki Kamatani; Peter E. Daddona; Dennis A. Carson

    The association of adenosine deaminase (ADA) deficiency with immunodeficiency disease has emphasized the importance of this purine metabolic enzyme for human lymphocyte growth and function. This report describes the natural occurrence of ADA deficiency in a human histiocytic lymphoma cell line, DHL-9. The minimal ADA activity in DHL-9 extracts, 0.028 nmol\\/min\\/mg protein, was less than 50% of the activity in

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

    SciTech Connect

    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.

  11. A2A receptor dependent and A2A receptor independent effects of extracellular adenosine on murine thymocytes in conditions of adenosine deaminase deficiency

    Microsoft Academic Search

    Sergey Apasov; Jiang-Fan Chen; Patrick Smith; Michail Sitkovsky

    2000-01-01

    Adenosine deaminase (ADA) deficiency causes severe combined immunodefi- ciency (SCID) and is accompanied by T-cell depletion and accumulation of both intracellular and extracellular adenosine (extAdo) and deoxyadenosine. To better understand the causes of T-cell depletion in vivo and to discriminate between extracellular and intracellular effects of exogenously added adenosine in vitro, we investigated mechanisms of 2 differ- ent effects of

  12. Paclitaxel alters the expression and specific activity of deoxycytidine kinase and cytidine deaminase in non-small cell lung cancer cell lines

    Microsoft Academic Search

    Stacy S Shord; Shitalben R Patel

    2009-01-01

    BACKGROUND: We observed that paclitaxel altered the pharmacokinetic properties of gemcitabine in patients with non-small cell lung cancer (NSCLC) and limited the accumulation of gemcitabine and its metabolites in various primary and immortalized human cells. Therefore, we classified the drug-drug interaction and the effects of paclitaxel on deoxycytidine kinase (dCK) and cytidine deaminase (CDA) in three NSCLC cell lines. These

  13. The first crystal structure of a dTTP-bound deoxycytidylate deaminase validates and details the allosteric-inhibitor binding site.

    PubMed

    Marx, Ailie; Alian, Akram

    2015-01-01

    Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH2 in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed. PMID:25404739

  14. Induction of activation-induced cytidine deaminase gene expression by IL4 and CD40 ligation is dependent on STAT6 and NF B

    Microsoft Academic Search

    Fatma Dedeoglu; Bruce Horwitz; Jayanta Chaudhuri; Frederick W. Alt; Raif S. Geha

    2004-01-01

    Activation-induced cytidine deaminase (AID) is an inducible gene that plays an important role in class switch recombination, somatic hypermutation and gene conversion in B cells. We examined the regulation of AID gene expression in human and mouse B cells by IL-4 and CD40 ligation. IL-4 by itself and, to a much lesser extent, CD40 ligation induced AID mRNA expression in

  15. The splicing regulator PTBP2 interacts with the cytidine deaminase AID and promotes binding of AID to switch-region DNA

    Microsoft Academic Search

    Urszula Nowak; Allysia J Matthews; Simin Zheng; Jayanta Chaudhuri

    2010-01-01

    During immunoglobulin class-switch recombination (CSR), the cytidine deaminase AID induces double-strand breaks into transcribed, repetitive DNA elements called switch sequences. The mechanism that promotes the binding of AID specifically to switch regions remains to be elucidated. Here we used a proteomic screen with in vivo biotinylation of AID to identify the splicing regulator PTBP2 as a protein that interacts with

  16. Inhibition of Adenosine Deaminase by Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) Mimics the Effect of Inescapable Shock on Escape Learning in Rats

    Microsoft Academic Search

    James C. Woodson; Thomas R. Minor; R. F. Soames Job

    1998-01-01

    Three experiments examined the role of adenosine neuroregulation in the production of shuttle-escape deficits caused by prior exposure to inescapable electric shock in rats (learned helplessness). Intracerebroventricular administration of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), a selective adenosine deaminase inhibitor, mimicked the effect of earlier inescapable shock at a dose of 2.5 ?M in previously restrained rats. Performance deficits produced by EHNA or by

  17. YMDB: the Yeast Metabolome Database.

    PubMed

    Jewison, Timothy; Knox, Craig; Neveu, Vanessa; Djoumbou, Yannick; Guo, An Chi; Lee, Jacqueline; Liu, Philip; Mandal, Rupasri; Krishnamurthy, Ram; Sinelnikov, Igor; Wilson, Michael; Wishart, David S

    2012-01-01

    The Yeast Metabolome Database (YMDB, http://www.ymdb.ca) is a richly annotated 'metabolomic' database containing detailed information about the metabolome of Saccharomyces cerevisiae. Modeled closely after the Human Metabolome Database, the YMDB contains >2000 metabolites with links to 995 different genes/proteins, including enzymes and transporters. The information in YMDB has been gathered from hundreds of books, journal articles and electronic databases. In addition to its comprehensive literature-derived data, the YMDB also contains an extensive collection of experimental intracellular and extracellular metabolite concentration data compiled from detailed Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR) metabolomic analyses performed in our lab. This is further supplemented with thousands of NMR and MS spectra collected on pure, reference yeast metabolites. Each metabolite entry in the YMDB contains an average of 80 separate data fields including comprehensive compound description, names and synonyms, structural information, physico-chemical data, reference NMR and MS spectra, intracellular/extracellular concentrations, growth conditions and substrates, pathway information, enzyme data, gene/protein sequence data, as well as numerous hyperlinks to images, references and other public databases. Extensive searching, relational querying and data browsing tools are also provided that support text, chemical structure, spectral, molecular weight and gene/protein sequence queries. Because of S. cervesiae's importance as a model organism for biologists and as a biofactory for industry, we believe this kind of database could have considerable appeal not only to metabolomics researchers, but also to yeast biologists, systems biologists, the industrial fermentation industry, as well as the beer, wine and spirit industry. PMID:22064855

  18. Experimental evolution in budding yeast

    NASA Astrophysics Data System (ADS)

    Murray, Andrew

    2012-02-01

    I will discuss our progress in analyzing evolution in the budding yeast, Saccharomyces cerevisiae. We take two basic approaches. The first is to try and examine quantitative aspects of evolution, for example by determining how the rate of evolution depends on the mutation rate and the population size or asking whether the rate of mutation is uniform throughout the genome. The second is to try to evolve qualitatively novel, cell biologically interesting phenotypes and track the mutations that are responsible for the phenotype. Our efforts include trying to alter cell morphology, evolve multicellularity, and produce a biological oscillator.

  19. Yeast flora of grape berries during ripening

    Microsoft Academic Search

    Gianfranco Rosini; Federico Federici; Alessandro Martini

    1982-01-01

    The yeast flora associated with the surface of grapes during ripening was studied with regard to different sectors of the grape skin and the position in the bunch by means of traditional as well as more vigorous preisolation and precounting treatments. The yeast number per square centimeter of skin increases with ripening and is highest in the area immediately surrounding

  20. Chronological aging leads to apoptosis in yeast

    Microsoft Academic Search

    Eva Herker; Helmut Jungwirth; Katharina A. Lehmann; Corinna Maldener; Kai-Uwe Fröhlich; Silke Wissing; Sabrina Büttner; Markus Fehr; Stephan Sigrist; Frank Madeo

    2004-01-01

    uring the past years, yeast has been successfully established as a model to study mechanisms of apoptotic regulation. However, the beneficial effects of such a cell suicide program for a unicellular organism remained obscure. Here, we demonstrate that chronologi- cally aged yeast cultures die exhibiting typical markers of apoptosis, accumulate oxygen radicals, and show caspase activation. Age-induced cell death is

  1. Production of recombinant proteins by yeast cells

    Microsoft Academic Search

    Eda Çelik; P?nar Çal?k

    Yeasts are widely used in production of recombinant proteins of medical or industrial interest. For each individual product, the most suitable expression system has to be identified and optimized, both on the genetic and fermentative level, by taking into account the properties of the product, the organism and the expression cassette. There is a wide range of important yeast expression

  2. Growth of yeasts during wine fermentations

    Microsoft Academic Search

    Graham H. Fleet

    1990-01-01

    This article emphasises the importance of making quantitative measurements of the growth of yeast species during wine fermentations. Although such studies confirm Saccharomyces cerevisiae as the principal wine yeast, they show that indigenous species of Kloeckera and Candida make a more significant contribution to the fermentation than previously thought. Inoculation of grape juice with S. cerevisiae does not necessarily suppress

  3. Fermentation studies using Saccharomyces diastaticus yeast strains

    SciTech Connect

    Erratt, J.A.; Stewart, G.G.

    1981-01-01

    The yeast species, Saccharomyces diastaticus, has the ability to ferment starch and dextrin, because of the extracellular enzyme, glucoamylase, which hydrolyzes the starch/dextrin to glucose. A number of nonallelic genes--DEX 1, DEX 2, and dextrinase B which is allelic to STA 3--have been isolated, which impart to the yeast the ability to ferment dextrin. Various diploid yeast strains were constructed, each being either heterozygous or homozygous for the individual dextrinase genes. Using 12 (sup 0) plato hopped wort (30% corn adjunct) under agitated conditions, the fermentation rates of the various diploid yeast strains were monitored. A gene-dosage effect was exhibited by yeast strains containing DEX 1 or DEX 2, however, not with yeast strains containing dextrinase B (STA 3). The fermentation and growth rates and extents were determined under static conditions at 14.4 C and 21 C. With all yeast strains containing the dextrinase genes, both fermentation and growth were increased at the higher incubation temperature. Using 30-liter fermentors, beer was produced with the various yeast strains containing the dextrinase genes and the physical and organoleptic characteristics of the products were determined. The concentration of glucose in the beer was found to increase during a 3-mo storage period at 21 C, indicating that the glucoamylase from Saccharomyces diastaticus is not inactivated by pasteurization. (Refs. 36).

  4. Rapid and reliable protein extraction from yeast

    Microsoft Academic Search

    Vitaly V. Kushnirov

    2000-01-01

    The methods currently used for protein extraction from yeast are either laborious or insufficiently reliable. Here I report a method for protein extraction for electrophoretic analysis that is both easy and reliable. In this method, yeast cells are subjected to mild alkali treatment and then boiled in a standard electrophoresis loading buffer. The method was tested for different strains of

  5. YEAST MEIOSIS Sister kinetochores are mechanically

    E-print Network

    Asbury, Chip

    YEAST MEIOSIS Sister kinetochores are mechanically fused during meiosis I in yeast Krishna K Production of healthy gametes requires a reductional meiosis I division in which replicated sister chromatids comigrate, rather than separate as in mitosis or meiosis II. Fusion of sister kinetochores during meiosis I

  6. The wine and beer yeast Dekkera bruxellensis

    PubMed Central

    Schifferdecker, Anna Judith; Dashko, Sofia; Ishchuk, Olena P; Piškur, Jure

    2014-01-01

    Recently, the non-conventional yeast Dekkera bruxellensis has been gaining more and more attention in the food industry and academic research. This yeast species is a distant relative of Saccharomyces cerevisiae and is especially known for two important characteristics: on the one hand, it is considered to be one of the main spoilage organisms in the wine and bioethanol industry; on the other hand, it is 'indispensable' as a contributor to the flavour profile of Belgium lambic and gueuze beers. Additionally, it adds to the characteristic aromatic properties of some red wines. Recently this yeast has also become a model for the study of yeast evolution. In this review we focus on the recently developed molecular and genetic tools, such as complete genome sequencing and transformation, to study and manipulate this yeast. We also focus on the areas that are particularly well explored in this yeast, such as the synthesis of off-flavours, yeast detection methods, carbon metabolism and evolutionary history. © 2014 The Authors. Yeast published by John Wiley & Sons, Ltd. PMID:24932634

  7. Yeasts are essential for cocoa bean fermentation.

    PubMed

    Ho, Van Thi Thuy; Zhao, Jian; Fleet, Graham

    2014-03-17

    Cocoa beans (Theobroma cacao) are the major raw material for chocolate production and fermentation of the beans is essential for the development of chocolate flavor precursors. In this study, a novel approach was used to determine the role of yeasts in cocoa fermentation and their contribution to chocolate quality. Cocoa bean fermentations were conducted with the addition of 200ppm Natamycin to inhibit the growth of yeasts, and the resultant microbial ecology and metabolism, bean chemistry and chocolate quality were compared with those of normal (control) fermentations. The yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii and Kluyveromyces marxianus, the lactic acid bacteria Lactobacillus plantarum and Lactobacillus fermentum and the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateurii were the major species found in the control fermentation. In fermentations with the presence of Natamycin, the same bacterial species grew but yeast growth was inhibited. Physical and chemical analyses showed that beans fermented without yeasts had increased shell content, lower production of ethanol, higher alcohols and esters throughout fermentation and lesser presence of pyrazines in the roasted product. Quality tests revealed that beans fermented without yeasts were purplish-violet in color and not fully brown, and chocolate prepared from these beans tasted more acid and lacked characteristic chocolate flavor. Beans fermented with yeast growth were fully brown in color and gave chocolate with typical characters which were clearly preferred by sensory panels. Our findings demonstrate that yeast growth and activity were essential for cocoa bean fermentation and the development of chocolate characteristics. PMID:24462702

  8. Yeast: An Experimental Organism for Modern Biology.

    ERIC Educational Resources Information Center

    Botstein, David; Fink, Gerald R.

    1988-01-01

    Discusses the applicability and advantages of using yeasts as popular and ideal model systems for studying and understanding eukaryotic biology at the cellular and molecular levels. Cites experimental tractability and the cooperative tradition of the research community of yeast biologists as reasons for this success. (RT)

  9. Yeasts that utilize lactose in sweet whey

    SciTech Connect

    Gholson, J.H.; Gough, R.H.

    1980-01-01

    Since processing costs are usually higher for whey than for other available food or feed nutrients, only about one-third of whey produced in the US is used by food and feed industries. As a result whey disposal costs are a problem. Further; when whey is disposed of through municipal sewerage systems, the lactose present is changed by bacteria to lactic acid which tends to act as a preservative and retards further oxidation of whey constituents. This article describes a method of utilizing lactose-fermenting yeasts to produce large quantities of yeast cells, single-cell protein. Kluveromyces fragilis was found to be the most effective yeast species and the yeast cells produced could be used as a natural food or feed additive. Results of this study determined that certain methods and yeast strains could reduce whey-related pollution and thus help reduce costs of whey disposal.

  10. Production of ethanol by immobilized yeast cells

    SciTech Connect

    Williams, D.; Munnecke, D.M.

    1981-08-01

    Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to a 0.05M CaCl2 solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature,pH, ethanol concentration), cell densities, and gel concentrations. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells were examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentrations were monitored at different feedstock flow rates. (Refs. 13).

  11. Hydration properties of natural and synthetic DNA sequences with methylated adenine or cytosine bases in the R.DpnI target and BDNF promoter studied by molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Shanak, Siba; Helms, Volkhard

    2014-12-01

    Adenine and cytosine methylation are two important epigenetic modifications of DNA sequences at the levels of the genome and transcriptome. To characterize the differential roles of methylating adenine or cytosine with respect to their hydration properties, we performed conventional MD simulations and free energy perturbation calculations for two particular DNA sequences, namely the brain-derived neurotrophic factor (BDNF) promoter and the R.DpnI-bound DNA that are known to undergo methylation of C5-methyl cytosine and N6-methyl adenine, respectively. We found that a single methylated cytosine has a clearly favorable hydration free energy over cytosine since the attached methyl group has a slightly polar character. In contrast, capping the strongly polar N6 of adenine with a methyl group gives a slightly unfavorable contribution to its free energy of solvation. Performing the same demethylation in the context of a DNA double-strand gave quite similar results for the more solvent-accessible cytosine but much more unfavorable results for the rather buried adenine. Interestingly, the same demethylation reactions are far more unfavorable when performed in the context of the opposite (BDNF or R.DpnI target) sequence. This suggests a natural preference for methylation in a specific sequence context. In addition, free energy calculations for demethylating adenine or cytosine in the context of B-DNA vs. Z-DNA suggest that the conformational B-Z transition of DNA transition is rather a property of cytosine methylated sequences but is not preferable for the adenine-methylated sequences investigated here.

  12. Nucleotide excision repair in yeast.

    PubMed

    Prakash, S; Prakash, L

    2000-06-30

    In nucleotide excision repair (NER) in eukaryotes, DNA is incised on both sides of the lesion, resulting in the removal of a fragment approximately 25-30 nucleotides long. This is followed by repair synthesis and ligation. The proteins encoded by the various yeast NER genes have been purified, and the incision reaction reconstituted in vitro. This reaction requires the damage binding factors Rad14, RPA, and the Rad4-Rad23 complex, the transcription factor TFIIH which contains the two DNA helicases Rad3 and Rad25, essential for creating a bubble structure, and the two endonucleases, the Rad1-Rad10 complex and Rad2, which incise the damaged DNA strand on the 5'- and 3'-side of the lesion, respectively. Addition of the Rad7-Rad16 complex to this reconstituted system stimulates the incision reaction many fold. The various NER proteins exist in vivo as part of multiprotein subassemblies which have been named NEFs (nucleotide excision repair factors). Rad14 and Rad1-Rad10 form one subassembly called NEF1, the Rad4-Rad23 complex is named NEF2, Rad2 and TFIIH constitute NEF3, and the Rad7-Rad16 complex is called NEF4. Although much has been learned from yeast about the function of NER genes and proteins in eukaryotes, the underlying mechanisms by which damage is recognized, NEFs are assembled at the damage site, and the DNA is unwound and incised, remain to be elucidated. PMID:10915862

  13. Regulation of yeast oscillatory dynamics

    PubMed Central

    Murray, Douglas B.; Beckmann, Manfred; Kitano, Hiroaki

    2007-01-01

    When yeast cells are grown continuously at high cell density, a respiratory oscillation percolates throughout the population. Many essential cellular functions have been shown to be separated temporally during each cycle; however, the regulatory mechanisms involved in oscillatory dynamics remain to be elucidated. Through GC-MS analysis we found that the majority of metabolites show oscillatory dynamics, with 70% of the identified metabolite concentrations peaking in conjunction with NAD(P)H. Through statistical analyses of microarray data, we identified that biosynthetic events have a defined order, and this program is initiated when respiration rates are increasing. We then combined metabolic, transcriptional data and statistical analyses of transcription factor activity, identified the top oscillatory parameters, and filtered a large-scale yeast interaction network according to these parameters. The analyses and controlled experimental perturbation provided evidence that a transcriptional complex formed part of the timing circuit for biosynthetic, reductive, and cell cycle programs in the cell. This circuitry does not act in isolation because both have strong translational, proteomic, and metabolic regulatory mechanisms. Our data lead us to conclude that the regulation of the respiratory oscillation revolves around coupled subgraphs containing large numbers of proteins and metabolites, with a potential to oscillate, and no definable hierarchy, i.e., heterarchical control. PMID:17284613

  14. Silver(I)-mediated cytosine self-pairing is preferred over hoogsteen-type base pairs with the artificial nucleobase 1,3-dideaza-6-nitropurine.

    PubMed

    Megger, Dominik A; Muller, Jens

    2010-01-01

    A 2'-deoxyribonucleoside containing 1,3-dideaza-6-nitropurine was synthesized and incorporated into oligonucleotides. The acid-base properties of this nucleoside and the corresponding N9-methylated derivative were investigated by pD-dependent (1)H NMR spectroscopy. A possible formation of Hoogsteen-type base pairs with cytosine was studied by ultraviolet (UV) and circular dichroism (CD) spectroscopy in the presence and absence of Ag(I) and under neutral and acidic conditions, respectively. In each case, no indication for the formation of Hoogsteen-type base pairs was obtained, which is attributed to the higher affinity of cytosine to form self-complementary hemi-protonated base pairs under acidic conditions and metal-mediated homo base pairs in presence of Ag(I), respectively. PMID:20391190

  15. 1-[((S)-2-hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl] cytosine, an intracellular prodrug for (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine with improved therapeutic index in vivo.

    PubMed Central

    Bischofberger, N; Hitchcock, M J; Chen, M S; Barkhimer, D B; Cundy, K C; Kent, K M; Lacy, S A; Lee, W A; Li, Z H; Mendel, D B

    1994-01-01

    1-[((S)-2-hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl]cytosi ne (cyclic [cHPMPC]) was evaluated as a novel antiviral agent in comparison with (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC). Evaluation for in vitro activity against herpes simplex virus type 2 in MA-104 and MRC-5 cells showed that both cHPMPC and HPMPC have comparable activities and cytotoxicities. cHPMPC was found to be stable on incubation in human plasma and human liver homogenates. Intracellular metabolism studies revealed that cHPMPC was converted inside of the cells to HPMPC and then to the monophosphate, the diphosphate, and the monophosphate choline metabolites. In a mouse herpes simplex virus type 2 encephalitis model, both cHPMPC and HPMPC exhibited similar potencies in vivo. Nephrotoxicity, which is the dose-limiting toxicity of HPMPC, was assessed in a 14-day repeated-dose toxicity study in rats; cHPMPC has an improved safety margin of > or = 13-fold over that of HPMPC. PMID:7840575

  16. Silver(I)-Mediated Cytosine Self-Pairing is Preferred Over Hoogsteen-Type Base Pairs with the Artificial Nucleobase 1,3-Dideaza-6-Nitropurine

    Microsoft Academic Search

    Dominik A. Megger; Jens Müller

    2009-01-01

    A 2?-deoxyribonucleoside containing 1,3-dideaza-6-nitropurine was synthesized and incorporated into oligonucleotides. The acid-base properties of this nucleoside and the corresponding N9-methylated derivative were investigated by pD-dependent H NMR spectroscopy. A possible formation of Hoogsteen-type base pairs with cytosine was studied by ultraviolet (UV) and circular dichroism (CD) spectroscopy in the presence and absence of Ag(I) and under neutral and acidic conditions,

  17. Influence of the stage of advancement of leukemia L1210 in mice on the optimal schedule of treatment of cytosine arabinoside (NSC-63878).

    PubMed

    Kline, I; Woodman, R J; Gang, M; Sirica, A; Venditti, J M; Goldin, A

    1972-06-01

    Treatment with cytosine arabinoside given intraperitoneally (ip) every 3 hours for 24 hours on every fourth day (Q3h/24h/4d) from 1 day after ip implantation of 10(6) or 10(7) leukemia L1210 cells in mice, was not observed to be superior to the same treatment after implantation of 10(5) cells. Similarly, when cytosine arabinoside therapy was delayed until Day 5 after 10(5) tumor cells were implanted, treatment Q3h/24h/4d did not provide a therapeutic advantage over treatment with a single injection every second or fourth day. A single treatment of the advanced disease with cyclophosphamide did not restore the sensitivity of the disease to the Q3h/24h/4d treatment schedule. Whether treatment with cytosine arabinoside was started on Days 1, 3, or 5, the optimal total dose per day for treatment given once every fourth day was 12-20 times greater than the optimal total dose per day for treatment given in eight equal injections every fourth day. Via the oral route, the Q3h/24h/4d treatment regimen was not superior to one treatment every fourth day from Day 1. The data clearly show that when the initial inoculum is high (10(6) or 10(7) cells) or when the disease is advanced, the response to cytosine arabinoside therapy does not display treatment-schedule dependency for the Q3h/24h/4d schedule. PMID:19051491

  18. Transgene silencing in grapevines transformed with GFLV resistance genes: analysis of variable expression of transgene, siRNAs production and cytosine methylation

    Microsoft Academic Search

    Giorgio Gambino; Irene Perrone; Andrea Carra; Walter Chitarra; Paolo Boccacci; Daniela Torello Marinoni; Marco Barberis; Fatemeh Maghuly; Margit Laimer; Ivana Gribaudo

    2010-01-01

    Eight transgenic grapevine lines transformed with the coat protein gene of Grapevine fanleaf virus (GFLV-CP) were analyzed for a correlation between transgene expression, siRNAs production and DNA methylation. Bisulphite\\u000a genome sequencing was used for a comprehensive analysis of DNA methylation. Methylated cytosine residues of CpG and CpNpG\\u000a sites were detected in the GFLV-CP transgene, in the T7 terminator and in

  19. A Role for MAPK\\/ERK in Sympathetic Neuron Survival: Protection against a p53Dependent, JNK-Independent Induction of Apoptosis by Cytosine Arabinoside

    Microsoft Academic Search

    Christopher N. G. Anderson; Aviva M. Tolkovsky

    1999-01-01

    The antimitotic nucleoside cytosine arabinoside (araC) causes apoptosis in postmitotic neurons for which two mechanisms have been suggested: (1) araC directly inhibits a trophic factor- maintained signaling pathway required for survival, effectively mimicking trophic factor withdrawal; and (2) araC induces ap- optosis by a p53-dependent mechanism distinct from trophic factor withdrawal. In rat sympathetic neurons, we found that araC treatment

  20. Adsorption of Adenine, Cytosine, Thymine, and Uracil on Sulfide-Modified Montmorillonite: FT-IR, Mössbauer and EPR Spectroscopy and X-Ray Diffractometry Studies

    NASA Astrophysics Data System (ADS)

    Carneiro, Cristine E. A.; Berndt, Graciele; de Souza Junior, Ivan G.; de Souza, Cláudio M. D.; Paesano, Andrea; da Costa, Antonio C. S.; di Mauro, Eduardo; de Santana, Henrique; Zaia, Cássia T. B. V.; Zaia, Dimas A. M.

    2011-10-01

    In the present work the interactions of nucleic acid bases with and adsorption on clays were studied at two pHs (2.00, 7.00) using different techniques. As shown by Mössbauer and EPR spectroscopies and X-ray diffractometry, the most important finding of this work is that nucleic acid bases penetrate into the interlayer of the clays and oxidize Fe2+ to Fe3+, thus, this interaction cannot be regarded as a simple physical adsorption. For the two pHs the order of the adsorption of nucleic acid bases on the clays was: adenine ? cytosine > thymine > uracil. The adsorption of adenine and cytosine on clays increased with decreasing of the pH. For unaltered montmorillonite this result could be explained by electrostatic forces between adenine/cytosine positively charged and clay negatively charged. However for montmorillonite modified with Na2S, probably van der Waals forces also play an important role since both adenine/cytosine and clay were positively charged. FT-IR spectra showed that the interaction between nucleic acid bases and clays was through NH+ or NH{2/+} groups. X-ray diffractograms showed that nucleic acid bases adsorbed on clays were distributed into the interlayer surface, edge sites and external surface functional groups (aluminol, silanol) EPR spectra showed that the intensity of the line g ? 2 increased probably because the oxidation of Fe2+ to Fe3+ by nucleic acid bases and intensity of the line g = 4.1 increased due to the interaction of Fe3+ with nucleic acid bases. Mössbauer spectra showed a large decreased on the Fe2+ doublet area of the clays due to the reaction of nucleic acid bases with Fe2+.