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Sample records for yeast cytosine deaminase

  1. 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

  2. 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.

  3. Combining the Optimized Yeast Cytosine Deaminase Protein Fragment Complementation Assay and an In Vitro Cdk1 Targeting Assay to Study the Regulation of the ?-Tubulin Complex.

    PubMed

    Ear, Po Hien; Kowarzyk, Jacqueline; Booth, Michael J; Abd-Rabbo, Diala; Shulist, Kristian; Hall, Conrad; Vogel, Jackie; Michnick, Stephen W

    2016-01-01

    Cdk1 is the essential cyclin-dependent kinase in the budding yeast Saccharomyces cerevisiae. Cdk1 orchestrates cell cycle control by phosphorylating target proteins with extraordinary temporal and spatial specificity by complexing with one of the nine cyclin regulatory subunits. The identification of the cyclin required for targeting Cdk1 to a substrate can help to place the regulation of that protein at a specific time point during the cell cycle and reveal information needed to elucidate the biological significance of the regulation. Here, we describe a combination of strategies to identify interaction partners of Cdk1, and associate these complexes to the appropriate cyclins using a cell-based protein-fragment complementation assay. Validation of the specific reliance of the OyCD interaction between Cdk1 and budding yeast ?-tubulin on the Clb3 cyclin, relative to the mitotic Clb2 cyclin, was performed by an in vitro kinase assay using the ?-tubulin complex as a substrate. PMID:26254928

  4. Synthesis and characterization of a novel chitosan based E. coli cytosine deaminase nanocomposite for potential application in prodrug enzyme therapy.

    PubMed

    Yata, Vinod Kumar; Ghosh, Siddhartha Sankar

    2011-01-01

    Cytosine deaminase is a non-mammalian enzyme of widespread interest for prodrug enzyme therapy due to its ability to convert prodrug 5-fluorocytosine into anticancer drug 5-fluorouracil. Cytosine deaminase enzyme has been purified to homogeneity from E. coli K-12 MTCC 1302 strain. K(m) values for cytosine and 5-fluorocytosine were found to be 0.26 mM and 1.82 mM, respectively. We developed a chitosan-entrapped cytosine deaminase nanocomposite. Atomic force microscopy and transmission electron microscopy images showed an elongated sphere shape nanocomposite with an average size of 80 nm diameter. Fourier transform infrared spectroscopy and X-ray diffraction results confirmed gel formation and entrapment of cytosine deaminase within the nanocomposite. Sustained release of cytosine deaminase from the nanocomposite up to one week depicted its potential implication in prodrug inducted enzyme therapy. PMID:20960222

  5. Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein

    PubMed Central

    Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.

    2015-01-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzymes ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

  6. Retroviral Replicating Vectors Deliver Cytosine Deaminase Leading to Targeted 5-Fluorouracil-Mediated Cytotoxicity in Multiple Human Cancer Types.

    PubMed

    Twitty, Chris G; Diago, Oscar R; Hogan, Daniel J; Burrascano, Cindy; Ibanez, Carlos E; Jolly, Douglas J; Ostertag, Derek

    2016-02-01

    Toca 511 is a modified retroviral replicating vector based on Moloney ?-retrovirus with an amphotropic envelope. As an investigational cancer treatment, Toca 511 preferentially infects cancer cells without direct cell lysis and encodes an enhanced yeast cytosine deaminase that converts the antifungal drug 5-fluorocytosine to the anticancer drug, 5-fluorouracil. A panel of established human cancer cell lines, derived from glioblastoma, colon, and breast cancer tissue, was used to evaluate parameters critical for effective anticancer activity. Gene transfer, cytosine deaminase production, conversion of 5-fluorocytosine to 5-fluorouracil, and subsequent cell killing occurred in all lines tested. We observed >50% infection within 25 days in all lines and 5-fluorocytosine LD50 values between 0.02 and 6??g/ml. Although we did not identify a small number of key criteria, these studies do provide a straightforward approach to rapidly gauge the probability of a Toca 511 and 5-fluorocytosine treatment effect in various cancer indications: a single MTS assay of maximally infected cancer cell lines to determine 5-fluorocytosine LD50. The data suggest that, although there can be variation in susceptibility to Toca 511 and 5-fluorocytosine because of multiple mechanistic factors, this therapy may be applicable to a broad range of cancer types and individuals. PMID:26467507

  7. Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy.

    PubMed

    Nemani, Krishnamurthy V; Ennis, Riley C; Griswold, Karl E; Gimi, Barjor

    2015-06-10

    Engineered bacterial cells that are designed to express therapeutic enzymes under the transcriptional control of remotely inducible promoters can mediate the de novo conversion of non-toxic prodrugs to their cytotoxic forms. In situ cellular expression of enzymes provides increased stability and control of enzyme activity as compared to isolated enzymes. We have engineered Escherichia coli (E. coli), designed to express cytosine deaminase at elevated temperatures, under the transcriptional control of thermo-regulatory λpL-cI857 promoter cassette which provides a thermal switch to trigger enzyme synthesis. Enhanced cytosine deaminase expression was observed in cultures incubated at 42°C as compared to 30°C, and enzyme expression was further substantiated by spectrophotometric assays indicating enhanced conversion of 5-fluorocytosine to 5-fluorouracil. The engineered cells were subsequently co-encapsulated with magnetic iron oxide nanoparticles in immunoprotective alginate microcapsules, and cytosine deaminase expression was triggered remotely by alternating magnetic field-induced hyperthermia. The combination of 5-fluorocytosine with AMF-activated microcapsules demonstrated tumor cell cytotoxicity comparable to direct treatment with 5-fluorouracil chemotherapy. Such enzyme-prodrug therapy, based on engineered and immunoisolated E. coli, may ultimately yield an improved therapeutic index relative to monotherapy, as AMF mediated hyperthermia might be expected to pre-sensitize tumors to chemotherapy under appropriate conditions. PMID:25820125

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

    SciTech Connect

    Chen, Jennifer K.; Hu, Lily J.; Wang Dongfang; Lamborn, Kathleen R.; Deen, Dennis F. . 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.

  9. Reaction mechanism of zinc-dependent cytosine deaminase from Escherichia coli: a quantum-chemical study.

    PubMed

    Manta, Bianca; Raushel, Frank M; Himo, Fahmi

    2014-05-29

    The reaction mechanism of cytosine deaminase from Escherichia coli is studied using density functional theory. This zinc-dependent enzyme catalyzes the deamination of cytosine to form uracil and ammonia. The calculations give a detailed description of the catalytic mechanism and establish the role of important active-site residues. It is shown that Glu217 is essential for the initial deprotonation of the metal-bound water nucleophile and the subsequent protonation of the substrate. It is also demonstrated that His246 is unlikely to function as a proton shuttle in the nucleophile activation step, as previously proposed. The steps that follow are nucleophilic attack by the metal-bound hydroxide, protonation of the leaving group assisted by Asp313, and C-N bond cleavage. The calculated overall barrier is in good agreement with the experimental findings. Finally, the calculations reproduce the experimentally determined inverse solvent deuterium isotope effect, which further corroborates the suggested reaction mechanism. PMID:24833316

  10. The Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase

    PubMed Central

    Hall, Richard S.; Fedorov, Alexander A.; Xu, Chengfu; Fedorov, Elena V.; Almo, Steven C.; Raushel, Frank M.

    2011-01-01

    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 Ki 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 kcat and kcat/Km, 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. PMID:21545144

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

    PubMed

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

    2016-02-01

    Cytosine deaminase (CD) catalyses the enzymatic conversion of the non-toxic prodrug 5-fluorocytosine (5-FC) to the potent chemotherapeutic form, 5-fluorouracil (5-FU). Intratumoral delivery of CD localises chemotherapy dose while reducing systemic toxicity. Encapsulation in biocompatible microcapsules immunoisolates CD and protects it from degradation. We report on the effect of alginate encapsulation on the catalytic and functional activity of isolated CD and recombinant E. coli engineered to express CD (E. coli(CD)). Alginate microcapsules containing either CD or Escherichia coli(CD) were prepared using ionotropic gelation. Conversion of 5-FC to 5-FU was quantitated in unencapsulated and encapsulated CD/E. coli(CD) using spectrophotometry, with a slower rate of conversion observed following encapsulation. Both encapsulated CD/5-FC and E. coli(CD)/5-FC resulted in cell kill and reduced proliferation of 9?L rat glioma cells, which was comparable to direct 5-FU treatment. Our results show that encapsulation preserves the therapeutic potential of CD and E. coli(CD) is equally effective for enzyme-prodrug therapy. PMID:26642874

  12. Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27.

    PubMed

    Wang, Lei; Hoffmann, Jana; Watzlawick, Hildegard; Altenbuchner, Josef

    2015-01-01

    We developed a counterselectable deletion system for Thermus thermophilus HB27 based on cytosine deaminase (encoded by codA) from Thermaerobacter marianensis DSM 12885 and the sensitivity of T. thermophilus HB27 to the antimetabolite 5-fluorocytosine (5-FC). The deletion vector comprises the pUC18 origin of replication, a thermostable kanamycin resistance marker functional in T. thermophilus HB27, and codA under the control of a constitutive putative trehalose promoter from T. thermophilus HB27. The functionality of the system was demonstrated by deletion of the bglT gene, encoding a ?-glycosidase, and three carotenoid biosynthesis genes, CYP175A1, crtY, and crtI, from the genome of T. thermophilus HB27. PMID:26655764

  13. 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

  14. 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.

    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.

  15. Identification of the yeast cytidine deaminase CDD1 as an orphan C-->U RNA editase.

    PubMed

    Dance, G S; Beemiller, P; Yang, Y; Mater, D V; Mian, I S; Smith, H C

    2001-04-15

    Yeast co-expressing rat APOBEC-1 and a fragment of human apolipoprotein B (apoB) mRNA assembled functional editosomes and deaminated C6666 to U in a mooring sequence-dependent fashion. The occurrence of APOBEC-1-complementing proteins suggested a naturally occurring mRNA editing mechanism in yeast. Previously, a hidden Markov model identified seven yeast genes encoding proteins possessing putative zinc-dependent deaminase motifs. Here, only CDD1, a cytidine deaminase, is shown to have the capacity to carry out C-->U editing on a reporter mRNA. This is only the second report of a cytidine deaminase that can use mRNA as a substrate. CDD1-dependent editing was growth phase regulated and demonstrated mooring sequence-dependent editing activity. Candidate yeast mRNA substrates were identified based on their homology with the mooring sequence-containing tripartite motif at the editing site of apoB mRNA and their ability to be edited by ectopically expressed APOBEC-1. Naturally occurring yeast mRNAs edited to a significant extent by CDD1 were, however, not detected. We propose that CDD1 be designated an orphan C-->U editase until its native RNA substrate, if any, can be identified and that it be added to the CDAR (cytidine deaminase acting on RNA) family of editing enzymes. PMID:11292850

  16. Three-dimensional assessment of bystander effects of mesenchymal stem cells carrying a cytosine deaminase gene on glioma cells

    PubMed Central

    Jung, Jin Hwa; Kim, Andrew Aujin; Chang, Da-Young; Park, Yoo Ra; Suh-Kim, Haeyoung; Kim, Sung-Soo

    2015-01-01

    Stem cells carrying a suicide gene have emerged as therapeutic candidates for their cytotoxic bystander effects on neighboring cancers, while being non-toxic to other parts of the body. However, traditional cytotoxicity assays are unable to adequately assess the therapeutic effects of bystander cells. Here, we report a method to assess bystander effects of therapeutic stem cells against 3-dimensionally grown glioma cells in real time. U87 glioma cells were stably transduced to express a green fluorescence protein and co-cultivated with mesenchymal stem cells engineered to carry a bacterial cytosine deaminase gene (MSC/CD). Following addition of a 5-fluorocytine (5-FC) prodrug to the co-culture, fluorescence from U87 cells was obtained and analyzed in real time. Notably, the IC50 of 5-FC was higher when U87 cells were grown 3-dimensionally in soft agar medium for 3 weeks, as compared to those grown for one week in two-dimensional monolayer cultures. Additionally, more MSC/CD cells were required to maintain a similar level of efficacy. Since three-dimensional growth of glioma cells under our co-culture condition mimics the long-term expansion of cancer cells in vivo, our method can extend to an in vitro assay system to assess stem cell-mediated anti-cancer effects before advancing into preclinical animal studies. PMID:26609476

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

    SciTech Connect

    Zhang, Jufeng; Wang, Zhanli; Wei, Fang; Qiu, Wei; Zhang, Liangren; Huang, Qian . 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.

  18. Selection of drug-resistant transduced cells with cytosine nucleoside analogs using the human cytidine deaminase gene.

    PubMed

    Beausjour, C M; Eliopoulos, N; Momparler, L; Le, N L; Momparler, R L

    2001-09-01

    Hematopoietic toxicity produced by most anticancer drugs limits their potential for curative therapy. We have shown previously that the human cytidine deaminase (CD) gene can confer drug resistance in murine bone marrow cells (BMCs) to the nucleoside analog, cytosine arabinoside (ARA-C). In the present study, as the first objective we showed that the CD gene can also render drug resistance in BMCs to related analogs, 2',2'-difluorodeoxycytidine (dFdC) and 5-azadeoxycytidine (5-AZA-CdR). As a second objective, we investigated the potential of ex vivo selection with cytosine nucleoside analogs of CD-transduced BMC. The goal of this approach was to enrich the fraction of CD-transduced BMCs so as to increase the transgene expression and level of drug resistance before transplantation. This strategy may have the potential to circumvent the problem in clinical gene therapy of low level of gene transfer and adequate long-term gene expression. Using a bicistronic retroviral vector containing the CD and the green fluorescent protein (CDiGFP), we transduced murine L1210 leukemic cells. All three analogs, ARA-C, dFdC, and 5-AZA-CdR were demonstrated in vitro to enrich (>95%) the population of leukemic cells expressing the GFP transgene. However, with CD-transduced primary murine BMCs cultivated at high cell density we observed that in vitro selection with ARA-C was not possible due to release of CD into the culture medium at amounts that were sufficient to inactivate the analog. The CD-containing medium produced a chemoprotective effect on mock BMCs as shown by lack of significant growth inhibition in the presence of ARA-C. However, at low cell density in a cell mixture containing CD-transduced cells, the mock BMCs showed marked drug sensitivity to ARA-C as determined by clonogenic assay. Selection with ARA-C was shown to significantly increase the CD enzyme activity in transduced BMC. These results suggest that CD gene has the potential to be a good selectable marker and a possible tool for chemoprotection in cancer gene therapy. PMID:11593336

  19. Genome-Wide Mutation Avalanches Induced in Diploid Yeast Cells by a Base Analog or an APOBEC Deaminase

    PubMed Central

    Lada, Artem G.; Stepchenkova, Elena I.; Waisertreiger, Irina S. R.; Noskov, Vladimir N.; Dhar, Alok; Eudy, James D.; Boissy, Robert J.; Hirano, Masayuki; Rogozin, Igor B.; Pavlov, Youri I.

    2013-01-01

    Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis. PMID:24039593

  20. 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

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

    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.

  2. The structure of a yeast RNA-editing deaminase provides insight into the fold and function of activation-induced deaminase and APOBEC-1

    PubMed Central

    Xie, Kefang; Sowden, Mark P.; Dance, Geoffrey S. C.; Torelli, Andrew T.; Smith, Harold C.; Wedekind, Joseph E.

    2004-01-01

    Activation-induced deaminase (AID) uses base deamination for class-switch recombination and somatic hypermutation and is related to the mammalian RNA-editing enzyme apolipoprotein B editing catalytic subunit 1 (APOBEC-1). CDD1 is a yeast ortholog of APOBEC-1 that exhibits cytidine deaminase and RNA-editing activity. Here, we present the crystal structure of CDD1 at 2.0-Å resolution and its use in comparative modeling of APOBEC-1 and AID. The models explain dimerization and the need for trans-acting loops that contribute to active site formation. Substrate selectivity appears to be regulated by a central active site “flap” whose size and flexibility accommodate large substrates in contrast to deaminases of pyrimidine metabolism that bind only small nucleosides or free bases. Most importantly, the results suggested both AID and APOBEC-1 are equally likely to bind single-stranded DNA or RNA, which has implications for the identification of natural AID targets. PMID:15148397

  3. 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

  4. Enhanced EJ Cell Killing of 125I Radiation by Combining with Cytosine Deaminase Gene Therapy Regulated by Synthetic Radio-Responsive Promoter

    PubMed Central

    Li, Ling; Kang, Lei; Wang, Rong-Fu; Yan, Ping; Zhao, Qian; Yin, Lei; Guo, Feng-qin

    2015-01-01

    Abstract Aim: To investigate the enhancing effect of radionuclide therapy by the therapeutic gene placed under the control of radio-responsive promoter. Methods: The recombinant lentivirus E8-codA-GFP, including a synthetic radiation-sensitive promoter E8, cytosine deaminase (CD) gene, and green fluorescent protein gene, was constructed. The gene expression activated by 125I radiation was assessed by observation of green fluorescence. The ability of converting 5-fluorocytosine (5-FC) to 5-fluorourial (5-FU) by CD enzyme was assessed by high-performance liquid chromatography. The viability of the infected cells exposed to 125I in the presence of 5-FC was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the infected cells exposed to 125I alone served as negative control and 5-FU as positive control. Results: The recombinant lentiviral vector was constructed successfully. On exposure of infected cells to 125I, green fluorescence can be observed and 5-FU can be detected. MTT assay showed that the survival rate for infected cells treated with 125I was lower compared with the 125I control group, but higher than the positive control group. Conclusion: The synthetic promoter E8 can induce the expression of downstream CD gene under 125I radiation, and the tumor killing effect of 125I can be enhanced by combining CD gene therapy with radiosensitive promoter. PMID:26382009

  5. Absence of a gene encoding cytosine deaminase in the genome of the agaricomycete Coprinopsis cinerea enables simple marker recycling through 5-fluorocytosine counterselection.

    PubMed

    Nakazawa, Takehito; Honda, Yoichi

    2015-08-01

    Coprinopsis cinerea is a model species for molecular genetics studies of sexual development in agaricomycetes or homobasidiomycetes. Recently, efficient gene targeting was established in this fungus by generating Cc.ku70 or Cc.lig4 disruptants. To determine the molecular mechanisms underlying sexual development, which involves many genes, generating multiple gene disruptants is required. However, the number of transformation markers available for C. cinerea is limited. This problem would be solved by establishing marker recycling. In this study, we found that C. cinerea lacks a gene encoding a homolog of Saccharomyces cerevisiae cytosine deaminase (Fcy1p) in its genome, which is present in many other fungi. We also observed that C. cinerea is resistant to 5-fluorocytosine. Based on these findings, we established a simple marker recycling method in this fungus using 5-fluorocytosine counterselection after heterologous expression of FCY1 derived from Pleurotus ostreatus, together with the hygromycin resistance gene. This study proposes a simple genetic manipulation system that can be performed using wild-type strains of several fungi that lack a gene homologous to S. cerevisiae FCY1 in their genomes. PMID:26223587

  6. Apoptotic induction with bifunctional E.coli cytosine deaminase-uracil phosphoribosyltransferase mediated suicide gene therapy is synergized by curcumin treatment in vitro.

    PubMed

    Gopinath, P; Ghosh, Siddhartha Sankar

    2008-05-01

    Development of novel suicide gene therapy vector with potential application in cancer treatment has a great impact on human health. Investigation to understand molecular mechanism of cell death is necessary to evaluate the therapeutic application of suicide vectors. For example, the bifunctional E.coli cytosine deaminase & uracil phosphoribosyltransferase fusion (CD-UPRT) gene expression is known to sensitize a wide range of cells toward nontoxic prodrug 5-flurocytosine (5-FC) by converting it to toxic compounds, but the exact pathway of cell death is yet to be defined. Herein, we investigated the mechanism of cell death by 5-FC/CD-UPRT suicide system in both cancer and non-cancer cells and found that the optimum 5-FC concentration led to programmed cell death in vitro. The CD-UPRT expression of transfected cells was measured by the RT-PCR analysis. Biochemical assays, such as mitochondrial activity (MTS) and lactate dehydrogenase (LDH) measurements exhibited cell death. Microscopic experiments showed characteristic onset of apoptosis which was further supported by internucleosomal DNA cleavage of BrdU labeled cellular DNA, appearance of characteristic laddering of chromosomal DNA and involvement of caspase pathway. Furthermore, the 5-FC/CD-UPRT-mediated apoptosis was potentiated with addition of a known anticancer agent curcumin. Our in vitro studies confirmed synergistic induction of apoptotic pathway in the combination treatment. Therefore, combination of 5-FC/CD-UPRT with curcumin could be a potential chemosensitization strategy for cancer treatment. PMID:18092145

  7. CEA-negative glioblastoma and melanoma cells are sensitive to cytosine deaminase/5-fluorocytosine therapy directed by the carcinoembryonic antigen promoter.

    PubMed

    Dabrowska, Anna; Szary, Jaros?aw; Kowalczuk, Ma?gorzata; Szala, Stanis?aw; Ugorski, Maciej

    2004-01-01

    Recent studies have suggested that carcinoembryonic antigen (CEA)-promoter sequences are active only in CEA-positive cells, filing in the criteria for tumor specific targeting of suicide genes. However, the present study on gene therapy of colon cancer and cell-specificity of CEA promoter, provide evidence that CEA-positive and CEA-negative cells transfected with E. coli cytosine deaminase (CD) gene under the control of CEA promotor sequence are sensitive to enzyme/pro-drug therapy with 5-fluorocytosine (5-FC). Individual clones derived from the CEA-negative cell lines: melanoma Hs294T and glioblastoma T98G after transfection with CD differed profoundly in their sensitivity to 5-FC. The IC50 values for several clones of the CEA-negative cells were almost the same as for CEA-positive colon cancer cells. Such 5-FC-sensitive clones derived from the population of CEA-negative cells, present even in small number, because of the very effective bystender effect of this enzyme/pro-drug system can cause severe problems during therapy by efficiently killing surrounding normal cells. Safety is the major issue in gene therapy. Our data suggest that the safety of gene-directed enzyme pro-drug therapy (GDEPT) with CEA promoter driven expression of therapeutic genes is not so obvious as it has originally been claimed. PMID:15448734

  8. Mitochondrial DNA of the yeast Kluyveromyces: guanine-cytosine rich sequence clusters.

    PubMed

    Ragnini, A; Fukuhara, H

    1988-09-12

    Mitochondrial DNA from the yeast Kluyveromyces marxianus var. lactis (K.lactis) is a circular molecule of 39 kilobase-pairs. A genetic and physical map was constructed. We found that this genome contained a large number of guanine-cytosine (GC)-rich sequence clusters, many of which are characterized by the presence of SacII restriction sites (CCGCGG). The primary sequence of the GC clusters often showed a palindromic structure. These GC clusters were present in several varieties of K.marxianus, but not in others. The presence of these clusters is a major feature that distinguishes K.lactis strains from those of K.marxianus var. marxianus (including K.fragilis). PMID:2843818

  9. 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

  10. Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression

    PubMed Central

    Young, Rosanna E B; Purton, Saul

    2014-01-01

    Negative selectable markers are useful tools for forward-genetic screens aimed at identifying trans-acting factors that are required for expression of specific genes. Transgenic lines harbouring the marker fused to a gene element, such as a promoter, may be mutagenized to isolate loss-of-function mutants able to survive under selection. Such a strategy allows the molecular dissection of factors that are essential for expression of the gene. Expression of individual chloroplast genes in plants and algae typically requires one or more nuclear-encoded factors that act at the post-transcriptional level, often through interaction with the 5′ UTR of the mRNA. To study such nuclear control further, we have developed the Escherichia coli cytosine deaminase gene codA as a conditional negative selectable marker for use in the model green alga Chlamydomonas reinhardtii. We show that a codon-optimized variant of codA with three amino acid substitutions confers sensitivity to 5-fluorocytosine (5-FC) when expressed in the chloroplast under the control of endogenous promoter/5′ UTR elements from the photosynthetic genes psaA or petA. UV mutagenesis of the psaA transgenic line allowed recovery of 5-FC-resistant, photosynthetically deficient lines harbouring mutations in the nuclear gene for the factor TAA1 that is required for psaA translation. Similarly, the petA line was used to isolate mutants of the petA mRNA stability factor MCA1 and the translation factor TCA1. The codA marker may be used to identify critical residues in known nuclear factors and to aid the discovery of additional factors required for expression of chloroplast genes. PMID:25234691

  11. Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression.

    PubMed

    Young, Rosanna E B; Purton, Saul

    2014-12-01

    Negative selectable markers are useful tools for forward-genetic screens aimed at identifying trans-acting factors that are required for expression of specific genes. Transgenic lines harbouring the marker fused to a gene element, such as a promoter, may be mutagenized to isolate loss-of-function mutants able to survive under selection. Such a strategy allows the molecular dissection of factors that are essential for expression of the gene. Expression of individual chloroplast genes in plants and algae typically requires one or more nuclear-encoded factors that act at the post-transcriptional level, often through interaction with the 5' UTR of the mRNA. To study such nuclear control further, we have developed the Escherichia coli cytosine deaminase gene codA as a conditional negative selectable marker for use in the model green alga Chlamydomonas reinhardtii. We show that a codon-optimized variant of codA with three amino acid substitutions confers sensitivity to 5-fluorocytosine (5-FC) when expressed in the chloroplast under the control of endogenous promoter/5' UTR elements from the photosynthetic genes psaA or petA. UV mutagenesis of the psaA transgenic line allowed recovery of 5-FC-resistant, photosynthetically deficient lines harbouring mutations in the nuclear gene for the factor TAA1 that is required for psaA translation. Similarly, the petA line was used to isolate mutants of the petA mRNA stability factor MCA1 and the translation factor TCA1. The codA marker may be used to identify critical residues in known nuclear factors and to aid the discovery of additional factors required for expression of chloroplast genes. PMID:25234691

  12. 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

  13. Selective antitumor effect of neural stem cells expressing cytosine deaminase and interferon-beta against ductal breast cancer cells in cellular and xenograft models.

    PubMed

    Yi, Bo-Rim; Hwang, Kyung-A; Aboody, Karen S; Jeung, Eui-Bae; Kim, Seung U; Choi, Kyung-Chul

    2014-01-01

    Due to their inherent tumor-tropic properties, genetically engineered stem cells may be advantageous for gene therapy treatment of various human cancers, including brain, liver, ovarian, and prostate malignancies. In this study, we employed human neural stem cells (HB1.F3; hNSCs) transduced with genes expressing Escherichia coli cytosine deaminase (HB1.F3.CD) and human interferon-beta (HB1.F3.CD.IFN-?) as a treatment strategy for ductal breast cancer. CD can convert the prodrug 5-fluorocytosine (5-FC) to its active chemotherapeutic form, 5-fluorouracil (5-FU), which induces a tumor-killing effect through DNA synthesis inhibition. IFN-? also strongly inhibits tumor growth by the apoptotic process. RT-PCR confirmed that HB1.F3.CD cells expressed CD and HB1.F3.CD.IFN-? cells expressed both CD and IFN-?. A modified transwell migration assay showed that HB1.F3.CD and HB1.F3.CD.IFN-? cells selectively migrated toward MCF-7 and MDA-MB-231 human breast cancer cells. In hNSC-breast cancer co-cultures the viability of breast cancer cells which were significantly reduced by HB1.F3.CD or HB1.F3.CD.IFN-? cells in the presence of 5-FC. The tumor inhibitory effect was greater with the HB1.F3.CD.IFN-? cells, indicating an additional effect of IFN-? to 5-FU. In addition, the tumor-tropic properties of these hNSCs were found to be attributed to chemoattractant molecules secreted by breast cancer cells, including stem cell factor (SCF), c-kit, vascular endothelial growth factor (VEGF), and VEGF receptor 2. An in vivo assay performed using MDA-MB-231/luc breast cancer mammary fat pad xenografts in immunodeficient mice resulted in 50% reduced tumor growth and increased long-term survival in HB1.F3.CD and HB1.F3.CD.IFN-? plus 5-FC treated mice relative to controls. Our results suggest that hNSCs genetically modified to express CD and/or IFN-? genes can be used as a novel targeted cancer gene therapy. PMID:24141111

  14. Coexpression of rat glutathione S-transferase A3 and human cytidine deaminase by a bicistronic retroviral vector confers in vitro resistance to nitrogen mustards and cytosine arabinoside in murine fibroblasts.

    PubMed

    Ltourneau, S; Palerme, J S; Delisle, J S; Beausjour, C M; Momparler, R L; Cournoyer, D

    2000-05-01

    The transfer of drug resistance genes into hematopoietic cells is an experimental approach to protect patients from drug-induced myelosuppression. Because anti-cancer drugs are often administered in combination to increase their clinical efficacy, vectors that express two drug resistance genes are being developed to broaden the spectrum of chemoprotection. We have constructed a bicistronic vector, MFG/GST-IRES-CD (MFG/GIC) coexpressing rat glutathione S-transferase (GST) A3 isoform (rGST Yc1) and human cytidine deaminase (CD). Murine NIH 3T3 fibroblast cells transduced with this vector were evaluated for their resistance to nitrogen mustards and cytosine nucleoside analogs. GIC-transduced polyclonal cell populations (GIC cells) demonstrated marked increases in selenium-independent glutathione peroxidase (peroxidase) and CD activities, as well as increased resistance to melphalan (2.3-fold), chlorambucil (3.4-fold), and cytosine arabinoside (Ara-C) (8.1-fold). After selection with Ara-C, the peroxidase and CD activities of GIC cells were augmented 2.6- and 2.9-fold, respectively, in comparison with unselected cells, and the resistance to melphalan, chlorambucil, and Ara-C was further increased to 3.7-, 5.9-, and 53-fold, respectively. Melphalan selection of GIC cells likewise augmented their peroxidase (2.3-fold) and CD (1.9-fold) activities. GIC cells proliferated in the simultaneous presence of melphalan and Ara-C at drug concentrations that completely inhibited the growth of untransduced cells. The growth rate of unselected GIC cells exposed to the drug combination averaged 18% that of drug-free cultures. The growth rate of GIC cells exposed to the drug combination increased to 30% of controls after Ara-C selection and to 50% after melphalan selection. Our results suggest that retroviral transfer of MFG/GIC may be useful for chemoprotection against the toxicities of nitrogen mustards and cytosine nucleoside analogs. PMID:10830723

  15. Regional 'pro-drug' gene therapy: intravenous administration of an adenoviral vector expressing the E. coli cytosine deaminase gene and systemic administration of 5-fluorocytosine suppresses growth of hepatic metastasis of colon carcinoma.

    PubMed

    Topf, N; Worgall, S; Hackett, N R; Crystal, R G

    1998-04-01

    Direct administration of an adenoviral vector expressing the cytosine deaminase gene (AdCMV.CD) to tumors of colon carcinoma cells, with concomitant systemic administration of 5-fluorocytosine (5FC), results in local production of 5-fluorouracil (5FU) and suppression of tumor growth. Based on the demonstration that in vivo adenovirus-mediated gene transfer to intrahepatic tumors is relatively inefficient compared with in vivo gene transfer to hepatocytes, we developed a 'regional' prodrug strategy using in vivo Ad-mediated CD gene transfer to normal liver, permitting hepatocytes to convert 5FC into 5FU to treat local metastasis effectively in a 'trans' fashion. To show that hepatocytes can generate and export sufficient 5FU to achieve this goal, primary rat hepatocytes were exposed to AdCMV.CD and 5FC. Evaluation of the supernatants by spectrophotometry and by HPLC demonstrated significant conversion of 5FC into 5FU. When supernatants of hepatocytes exposed to AdCMV.CD and 5FC were transferred to cultures of CT26 mouse colon carcinoma cells, the CT26 viability was reduced by 80%. To show that this regional AdCMV.CD/5FC prodrug strategy can suppress tumor growth in vivo, a model of metastatic colon carcinoma was established by injecting CT26 cells into the left lobe of the liver of syngeneic Balb/c mice. The next day, AdCMV.CD was transferred to hepatocytes by intravenous administration, and 5FC treatment was started the following day. Evaluation of tumor growth after 15 days showed marked suppression of tumor growth in AdCMV.CD- and 5FC- treated animals compared to control groups (P < 0.007). We conclude that primary hepatocytes are capable of converting 5FC into 5FU and exporting sufficient amounts of 5FU to the local milieu to suppress the growth of liver metastases of colon carcinoma cells. PMID:9614575

  16. Rescue of the Orphan Enzyme Isoguanine Deaminase

    SciTech Connect

    D Hitchcock; A Fedorov; E Fedorov; L Dangott; S Almo; F Raushel

    2011-12-31

    Cytosine deaminase (CDA) from Escherichia coli was shown to catalyze the deamination of isoguanine (2-oxoadenine) to xanthine. Isoguanine is an oxidation product of adenine in DNA that is mutagenic to the cell. The isoguanine deaminase activity in E. coli was partially purified by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography. The active protein was identified by peptide mass fingerprint analysis as cytosine deaminase. The kinetic constants for the deamination of isoguanine at pH 7.7 are as follows: k{sub cat} = 49 s{sup -1}, K{sub m} = 72 {micro}M, and k{sub cat}/K{sub m} = 6.7 x 10{sup 5} M{sup -1} s{sup -1}. The kinetic constants for the deamination of cytosine are as follows: k{sub cat} = 45 s{sup -1}, K{sub m} = 302 {micro}M, and k{sub cat}/K{sub m} = 1.5 x 10{sup 5} M{sup -1} s{sup -1}. Under these reaction conditions, isoguanine is the better substrate for cytosine deaminase. The three-dimensional structure of CDA was determined with isoguanine in the active site.

  17. 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; Ktter, 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 WatsonCrick 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

  18. 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; Ktter, 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

  19. Cytosine methylation and DNA repair.

    PubMed

    Walsh, C P; Xu, G L

    2006-01-01

    Cytosine methylation is a common form of post-replicative DNA modification seen in both bacteria and eukaryotes. Modified cytosines have long been known to act as hotspots for mutations due to the high rate of spontaneous deamination of this base to thymine, resulting in a G/T mismatch. This will be fixed as a C-->T transition after replication if not repaired by the base excision repair (BER) pathway or specific repair enzymes dedicated to this purpose. This hypermutability has led to depletion of the target dinucleotide CpG outside of special CpG islands in mammals, which are normally unmethylated. We review the importance of C-->T transitions at non-island CpGs in human disease: When these occur in the germline, they are a common cause of inherited diseases such as epidermolysis bullosa and mucopolysaccharidosis, while in the soma they are frequently found in the genes for tumor suppressors such as p53 and the retinoblastoma protein, causing cancer. We also examine the specific repair enzymes involved, namely the endonuclease Vsr in Escherichia coli and two members of the uracil DNA glycosylase (UDG) superfamily in mammals, TDG and MBD4. Repair brings its own problems, since it will require remethylation of the replacement cytosine, presumably coupling repair to methylation by either the maintenance methylase Dnmt1 or a de novo enzyme such as Dnmt3a. Uncoupling of methylation from repair may be one way to remove methylation from DNA. We also look at the possible role of specific cytosine deaminases such as Aid and Apobec in accelerating deamination of methylcytosine and consequent DNA demethylation. PMID:16570853

  20. Discovery of a bacterial 5-methylcytosine deaminase.

    PubMed

    Hitchcock, Daniel S; Fedorov, Alexander A; Fedorov, Elena V; Almo, Steven C; Raushel, Frank M

    2014-12-01

    5-Methylcytosine is found in all domains of life, but the bacterial cytosine deaminase from Escherichia coli (CodA) will not accept 5-methylcytosine as a substrate. Since significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes, this compound must eventually be catabolized and the fragments recycled by enzymes that have yet to be identified. We therefore initiated a comprehensive phylogenetic screen for enzymes that may be capable of deaminating 5-methylcytosine to thymine. From a systematic analysis of sequence homologues of CodA from thousands of bacterial species, we identified putative cytosine deaminases where a "discriminating" residue in the active site, corresponding to Asp-314 in CodA from E. coli, was no longer conserved. Representative examples from Klebsiella pneumoniae (locus tag: Kpn00632), Rhodobacter sphaeroides (locus tag: Rsp0341), and Corynebacterium glutamicum (locus tag: NCgl0075) were demonstrated to efficiently deaminate 5-methylcytosine to thymine with values of kcat/Km of 1.4 10(5), 2.9 10(4), and 1.1 10(3) M(-1) s(-1), respectively. These three enzymes also catalyze the deamination of 5-fluorocytosine to 5-fluorouracil with values of kcat/Km of 1.2 10(5), 6.8 10(4), and 2.0 10(2) M(-1) s(-1), respectively. The three-dimensional structure of Kpn00632 was determined by X-ray diffraction methods with 5-methylcytosine (PDB id: 4R85 ), 5-fluorocytosine (PDB id: 4R88 ), and phosphonocytosine (PDB id: 4R7W ) bound in the active site. When thymine auxotrophs of E. coli express these enzymes, they are capable of growth in media lacking thymine when supplemented with 5-methylcytosine. Expression of these enzymes in E. coli is toxic in the presence of 5-fluorocytosine, due to the efficient transformation to 5-fluorouracil. PMID:25384249

  1. Discovery of a Bacterial 5-Methylcytosine Deaminase

    PubMed Central

    2015-01-01

    5-Methylcytosine is found in all domains of life, but the bacterial cytosine deaminase from Escherichia coli (CodA) will not accept 5-methylcytosine as a substrate. Since significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes, this compound must eventually be catabolized and the fragments recycled by enzymes that have yet to be identified. We therefore initiated a comprehensive phylogenetic screen for enzymes that may be capable of deaminating 5-methylcytosine to thymine. From a systematic analysis of sequence homologues of CodA from thousands of bacterial species, we identified putative cytosine deaminases where a discriminating residue in the active site, corresponding to Asp-314 in CodA from E. coli, was no longer conserved. Representative examples from Klebsiella pneumoniae (locus tag: Kpn00632), Rhodobacter sphaeroides (locus tag: Rsp0341), and Corynebacterium glutamicum (locus tag: NCgl0075) were demonstrated to efficiently deaminate 5-methylcytosine to thymine with values of kcat/Km of 1.4 105, 2.9 104, and 1.1 103 M1 s1, respectively. These three enzymes also catalyze the deamination of 5-fluorocytosine to 5-fluorouracil with values of kcat/Km of 1.2 105, 6.8 104, and 2.0 102 M1 s1, respectively. The three-dimensional structure of Kpn00632 was determined by X-ray diffraction methods with 5-methylcytosine (PDB id: 4R85), 5-fluorocytosine (PDB id: 4R88), and phosphonocytosine (PDB id: 4R7W) bound in the active site. When thymine auxotrophs of E. coli express these enzymes, they are capable of growth in media lacking thymine when supplemented with 5-methylcytosine. Expression of these enzymes in E. coli is toxic in the presence of 5-fluorocytosine, due to the efficient transformation to 5-fluorouracil. PMID:25384249

  2. Strand-biased cytosine deamination at the replication fork causes cytosine to thymine mutations in Escherichia coli.

    PubMed

    Bhagwat, Ashok S; Hao, Weilong; Townes, Jesse P; Lee, Heewook; Tang, Haixu; Foster, Patricia L

    2016-02-23

    The rate of cytosine deamination is much higher in single-stranded DNA (ssDNA) than in double-stranded DNA, and copying the resulting uracils causes C to T mutations. To study this phenomenon, the catalytic domain of APOBEC3G (A3G-CTD), an ssDNA-specific cytosine deaminase, was expressed in an Escherichia coli strain defective in uracil repair (ung mutant), and the mutations that accumulated over thousands of generations were determined by whole-genome sequencing. C:G to T:A transitions dominated, with significantly more cytosines mutated to thymine in the lagging-strand template (LGST) than in the leading-strand template (LDST). This strand bias was present in both repair-defective and repair-proficient cells and was strongest and highly significant in cells expressing A3G-CTD. These results show that the LGST is accessible to cellular cytosine deaminating agents, explains the well-known GC skew in microbial genomes, and suggests the APOBEC3 family of mutators may target the LGST in the human genome. PMID:26839411

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

    PubMed

    Stenmark, Pl; Moche, Martin; Gurmu, Daniel; Nordlund, Pr

    2007-10-12

    We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast. PMID:17765262

  4. Genetics Home Reference: Adenosine monophosphate deaminase deficiency

    MedlinePLUS

    ... understanding adenosine monophosphate deaminase deficiency? asymptomatic ; autosomal ; autosomal recessive ; cell ; deficiency ; enzyme ; gene ; inherited ; joint ; muscle cells ; mutation ; myalgia ; nucleotide ; population ; ...

  5. Genetics Home Reference: Adenosine deaminase deficiency

    MedlinePLUS

    ... providers. American Society of Gene and Cell Therapy: Gene Therapy for Genetic Disorders Baby's First Test: Severe Combined Immunodeficiency Gene Review: Adenosine Deaminase Deficiency Genetic Testing Registry: Severe ...

  6. Bacterial Ammeline Metabolism via Guanine Deaminase ?

    PubMed Central

    Seffernick, Jennifer L.; Dodge, Anthony G.; Sadowsky, Michael J.; Bumpus, John A.; Wackett, Lawrence P.

    2010-01-01

    Melamine toxicity in mammals has been attributed to the blockage of kidney tubules by insoluble complexes of melamine with cyanuric acid or uric acid. Bacteria metabolize melamine via three consecutive deamination reactions to generate cyanuric acid. The second deamination reaction, in which ammeline is the substrate, is common to many bacteria, but the genes and enzymes responsible have not been previously identified. Here, we combined bioinformatics and experimental data to identify guanine deaminase as the enzyme responsible for this biotransformation. The ammeline degradation phenotype was demonstrated in wild-type Escherichia coli and Pseudomonas strains, including E. coli K12 and Pseudomonas putida KT2440. Bioinformatics analysis of these and other genomes led to the hypothesis that the ammeline deaminating enzyme was guanine deaminase. An E. coli guanine deaminase deletion mutant was deficient in ammeline deaminase activity, supporting the role of guanine deaminase in this reaction. Two guanine deaminases from disparate sources (Bradyrhizobium japonicum USDA 110 and Homo sapiens) that had available X-ray structures were purified to homogeneity and shown to catalyze ammeline deamination at rates sufficient to support bacterial growth on ammeline as a sole nitrogen source. In silico models of guanine deaminase active sites showed that ammeline could bind to guanine deaminase in a similar orientation to guanine, with a favorable docking score. Other members of the amidohydrolase superfamily that are not guanine deaminases were assayed in vitro, and none had substantial ammeline deaminase activity. The present study indicated that widespread guanine deaminases have a promiscuous activity allowing them to catalyze a key reaction in the bacterial transformation of melamine to cyanuric acid and potentially contribute to the toxicity of melamine. PMID:20023034

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

    PubMed Central

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

    2010-01-01

    Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin-6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a (gi|44585104) and NYSGXRC-9236b (gi|44611670), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 resolution (PDB code: 2PAJ). This protein folds as a distorted (?/?)8-barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosyl homocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin-6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s?1, 8.0 ?M, and 1.3 105 M?1 s?1 for kcat, Km, and kcat/Km, respectively. The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (PDB code: 2UZ9). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed based upon the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that hydrogen bond with the carbonyl oxygen at C4, a conserved threonine residue that hydrogen bonds with N5, and another conserved threonine residue that hydrogen bonds with the carbonyl group at C7. These conserved active site residues were used to identify 24 other genes which are predicted to deaminate isoxanthopterin. PMID:20415463

  8. Theoretical study of cytosine-Al, cytosine-Cu and cytosine-Ag (neutral, anionic and cationic).

    PubMed

    Vazquez, Marco-Vinicio; Martnez, Ana

    2008-02-01

    The binding of cytosine to Al, Cu and Ag has been analyzed using the hybrid B3LYP density functional theory method. The three metals all have open shell electronic configuration, with only one unpaired valence electron. Thus it is possible to study the influence of electronic configuration on the stability of these systems. Neutral, cationic and anionic systems were analyzed, in order to assess the influence of atomic charge on bond formation. We argue that in the case of anions, nonconventional hydrogen bonds are formed. It is generally accepted that the hydrogen bond A-H...B is formed by the union of a proton donor group A-H and a proton acceptor B, which contains lone-pair electrons. In this study, we found that in the case of (Cu-cytosine)(-1) and (Ag-cytosine)(-1), N-H...Cu and N-H...Ag bonds are geometrically described as nonconventional hydrogen bonds. Their binding energies fall within the range of -20.0 to -55.4 kcal/mol (depending on the scheme of the reaction) and thus they are classified as examples of strong (>10 kcal/mol) hydrogen bonds. PMID:18193849

  9. Genetics Home Reference: Adenosine monophosphate deaminase deficiency

    MedlinePLUS

    ... that can affect the muscles used for movement (skeletal muscles). People with this condition do not make enough ... called AMP deaminase. This enzyme is found in skeletal muscle, where it plays a role in producing energy ...

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

    PubMed Central

    Oehlenschlger, Christian Berg; Lvgreen, Monika Nhr; Reinauer, Eva; Lehtinen, Emilia; Pind, Marie-Louise Lindberg; Martinussen, Jan

    2015-01-01

    Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database. PMID:25746996

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

    PubMed

    Oehlenschlger, Christian Berg; Lvgreen, Monika Nhr; Reinauer, Eva; Lehtinen, Emilia; Pind, Marie-Louise Lindberg; Harris, Pernille; Martinussen, Jan; Willemos, Martin

    2015-05-15

    Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database. PMID:25746996

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

    SciTech Connect

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

    2010-05-25

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

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

    PubMed

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

    2010-05-25

    Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin 6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a ( gi|44585104 ) and NYSGXRC-9236b ( gi|44611670 ), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 A resolution (Protein Data Bank entry 2PAJ ). This protein folds as a distorted (beta/alpha)(8) barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosylhomocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin 6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s(-1), 8.0 muM, and 1.3 x 10(5) M(-1) s(-1) (k(cat), K(m), and k(cat)/K(m), respectively). The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (Protein Data Bank entry 2UZ9 ). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed on the basis of the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that form hydrogen bonds with the carbonyl oxygen at C4, a conserved threonine residue that forms hydrogen bonds with N5, and another conserved threonine residue that forms hydrogen bonds with the carbonyl group at C7. These conserved active site residues were used to identify 24 other genes which are predicted to deaminate isoxanthopterin. PMID:20415463

  14. Dehydration of cytosine monohydrate at physiological temperatures

    SciTech Connect

    Martel, P.; Powell, B.M.

    1983-01-01

    Neutron diffraction, thermogravimetric, and mass spectrographic measurements have been used to show that cytosine monohydrate loses its water of hydration at physiological temperatures (approx. = 37/sup 0/C) and converts to cytosine. The ''activation energy'' for the dehydration process has been determined from isothermal weight curves and is 27.1 +/- 0.6 kcal . mol/sup -1/. It is suggested that pyrimidine dehydration may be involved in structural changes in DNA.

  15. 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

    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.

  16. Crystal Structure of the DNA Deaminase APOBEC3B Catalytic Domain.

    PubMed

    Shi, Ke; Carpenter, Michael A; Kurahashi, Kayo; Harris, Reuben S; Aihara, Hideki

    2015-11-20

    Functional and deep sequencing studies have combined to demonstrate the involvement of APOBEC3B in cancer mutagenesis. APOBEC3B is a single-stranded DNA cytosine deaminase that functions normally as a nuclear-localized restriction factor of DNA-based pathogens. However, it is overexpressed in cancer cells and elicits an intrinsic preference for 5'-TC motifs in single-stranded DNA, which is the most frequently mutated dinucleotide in breast, head/neck, lung, bladder, cervical, and several other tumor types. In many cases, APOBEC3B mutagenesis accounts for the majority of both dispersed and clustered (kataegis) cytosine mutations. Here, we report the first structures of the APOBEC3B catalytic domain in multiple crystal forms. These structures reveal a tightly closed active site conformation and suggest that substrate accessibility is regulated by adjacent flexible loops. Residues important for catalysis are identified by mutation analyses, and the results provide insights into the mechanism of target site selection. We also report a nucleotide (dCMP)-bound crystal structure that informs a multistep model for binding single-stranded DNA. Overall, these high resolution crystal structures provide a framework for further mechanistic studies and the development of novel anti-cancer drugs to inhibit this enzyme, dampen tumor evolution, and minimize adverse outcomes such as drug resistance and metastasis. PMID:26416889

  17. Photophysical pathways of cytosine in aqueous solution

    SciTech Connect

    Kistler, Kurt A.; Matsika, Spiridoula

    2010-02-28

    The following manuscript was reported to EMSL in accordance with the DOE Non-Proprietary User Agreement. The effects of aqueous solvation on the photophysical pathways involving the S1 excited state in cytosine have been studied with a mean-field QM/MM approach. Two main pathways with small barriers were found previously in isolated cytosine, using multireference configuration interaction (MRCI) methods, that facilitate radiationless decay to the ground state. These pathways are examined in solvated cytosine using a mean-field QM/MM combined with MRCI, and it is found that barriers in each direction increase moderately. The barriers in the presence of the solvent are 0.23 eV and 0.31 eV for the two different pathways compared to 0.15 eV and 0.14 eV in the gas phase, indicating that the aqueous environment does not make one of the two directions much more preferable.

  18. [Adenosine deaminase in experimental trypanosomiasis: future implications].

    PubMed

    Prez-Aguilar, Mary Carmen; Rondn-Mercado, Roco

    2015-09-01

    The adenosine deaminase represents a control point in the regulation of extracellular adenosine levels, thus playing a critical role in the modulation of purinergic responses to certain pathophysiological events. Several studies have shown that serum and plasma enzyme levels are elevated in some diseases caused by microorganisms, which may represent a compensatory mechanism due to the elevated levels of adenosine and the release of inflammatory mediators. Recent research indicates that adenosine deaminase activity decreases and affects hematological parameters of infected animals with Trypanosoma evansi, so that such alterations could have implications in the pathogenesis of the disease. In addition, the enzyme has been detected in this parasite; allowing the inference that it could be associated with the vital functions of the same, similar to what occurs in mammals. This knowledge may be useful in the association of chemotherapy with specific inhibitors of the enzyme in future studies. PMID:26710546

  19. 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.

  20. Single molecule investigation of Ag+ interactions with single cytosine-, methylcytosine- and hydroxymethylcytosine-cytosine mismatches in a nanopore.

    PubMed

    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

  1. Photofragmentation of guanine, cytosine, leucine and methionine

    NASA Astrophysics Data System (ADS)

    Plekan, O.; Feyer, V.; Richter, R.; Coreno, M.; de Simone, M.; Prince, K. C.

    2007-04-01

    The photofragmentation spectra of guanine, cytosine, leucine and methionine were measured with noble gas resonance radiation at energies from 8.43 to 21.2 eV, and the spectra are related to the published valence band photoemission spectra. As expected, lower photon energies lead to "softer" ionization and reduced fragmentation. For the nucleobases guanine and cytosine, photoionization below 16.67 eV leads predominantly to the parent ion. The fragmentation pattern of leucine is similar to that of published spectra of other amino acids with aliphatic side chains, and whose outer orbitals are similar, with oxygen and nitrogen lone pair character. Methionine behaves very differently because the outer orbital has sulphur lone pair character, and reduced fragmentation is observed.

  2. AID/APOBEC deaminases and cancer

    PubMed Central

    Rebhandl, Stefan; Huemer, Michael; Greil, Richard; Geisberger, Roland

    2015-01-01

    Mutations are the basis for evolution and the development of genetic diseases. Especially in cancer, somatic mutations in oncogenes and tumor suppressor genes alongside the occurrence of passenger mutations have been observed by recent deep-sequencing approaches. While mutations have long been considered random events induced by DNA-replication errors or by DNA damaging agents, genome sequencing led to the discovery of non-random mutation signatures in many human cancer. Common non-random mutations comprise DNA strand-biased mutation showers and mutations restricted to certain DNA motifs, which recently have become attributed to the activity of the AID/APOBEC family of DNA deaminases. Hence, APOBEC enzymes, which have evolved as key players in natural and adaptive immunity, have been proposed to contribute to cancer development and clonal evolution of cancer by inducing collateral genomic damage due to their DNA deaminating activity. This review focuses on how mutagenic events through AID/APOBEC deaminases may contribute to cancer development. PMID:26097867

  3. Evolution of the deaminase fold and multiple origins of eukaryotic editing and mutagenic nucleic acid deaminases from bacterial toxin systems.

    PubMed

    Iyer, Lakshminarayan M; Zhang, Dapeng; Rogozin, Igor B; Aravind, L

    2011-12-01

    The deaminase-like fold includes, in addition to nucleic acid/nucleotide deaminases, several catalytic domains such as the JAB domain, and others involved in nucleotide and ADP-ribose metabolism. Using sensitive sequence and structural comparison methods, we develop a comprehensive natural classification of the deaminase-like fold and show that its ancestral version was likely to operate on nucleotides or nucleic acids. Consequently, we present evidence that a specific group of JAB domains are likely to possess a DNA repair function, distinct from the previously known deubiquitinating peptidase activity. We also identified numerous previously unknown clades of nucleic acid deaminases. Using inference based on contextual information, we suggest that most of these clades are toxin domains of two distinct classes of bacterial toxin systems, namely polymorphic toxins implicated in bacterial interstrain competition and those that target distantly related cells. Genome context information suggests that these toxins might be delivered via diverse secretory systems, such as Type V, Type VI, PVC and a novel PrsW-like intramembrane peptidase-dependent mechanism. We propose that certain deaminase toxins might be deployed by diverse extracellular and intracellular pathogens as also endosymbionts as effectors targeting nucleic acids of host cells. Our analysis suggests that these toxin deaminases have been acquired by eukaryotes on several independent occasions and recruited as organellar or nucleo-cytoplasmic RNA modifiers, operating on tRNAs, mRNAs and short non-coding RNAs, and also as mutators of hyper-variable genes, viruses and selfish elements. This scenario potentially explains the origin of mutagenic AID/APOBEC-like deaminases, including novel versions from Caenorhabditis, Nematostella and diverse algae and a large class of fast-evolving fungal deaminases. These observations greatly expand the distribution of possible unidentified mutagenic processes catalyzed by nucleic acid deaminases. PMID:21890906

  4. DNA cytosine and methylcytosine deamination by APOBEC3B: enhancing methylcytosine deamination by engineering APOBEC3B

    PubMed Central

    Fu, Yang; Ito, Fumiaki; Zhang, Gewen; Fernandez, Braulio; Yang, Hanjing; Chen, XiaojiangS.

    2015-01-01

    APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like) is a family of enzymes that deaminates cytosine (C) to uracil (U) on nucleic acid. APOBEC3B (A3B) functions in innate immunity against intrinsic and invading retroelements and viruses. A3B can also induce genomic DNA mutations to cause cancer. A3B contains two cytosine deaminase domains (CD1, CD2), and there are conflicting reports about whether both domains are active. Here we demonstrate that only CD2 of A3B (A3BCD2) has C deamination activity. We also reveal that both A3B and A3BCD2 can deaminate methylcytosine (mC). Guided by structural and functional analysis, we successfully engineered A3BCD2 to gain over two orders of magnitude higher activity for mC deamination. Important determinants that contribute to the activity and selectivity for mC deamination have been identified, which reveals that multiple elements, rather than single ones, contribute to the mC deamination activity and selectivity in A3BCD2 and possibly other APOBECs. PMID:26195824

  5. Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells.

    PubMed

    Eliopoulos, N; Al-Khaldi, A; Beausjour, C M; Momparler, R L; Momparler, L F; Galipeau, J

    2002-04-01

    Naturally occurring drug resistance genes of human origin can be exploited for selection of genetically engineered cells co-expressing a desired therapeutic transgene. Their non-immunogenicity in clinical applications would be a major asset. Human cytidine deaminase (hCD) is a chemoresistance gene that inactivates cytotoxic cytosine nucleoside analogs, such as cytosine arabinoside (Ara-C). The aim of this study was to establish if the hCD gene can serve as an ex vivo dominant selectable marker in engineered bone marrow stromal cells (MSCs). A bicistronic retrovector comprising the hCD cDNA and the green fluorescent protein (GFP) reporter gene was generated and used for transduction of A549 cells and primary murine MSCs. Analysis of transduced cells demonstrated stable integration of proviral DNA, more than 1000-fold increase in CD enzyme activity, and drug resistance to cytosine nucleoside analogs. In a mixture of transduced and untransduced MSCs, the percentage of retrovector-expressing cells could be increased to virtual purity (>99.5%) through in vitro drug selection with 1 microM Ara-C. Increased selective pressure with 2.5 microM Ara-C allowed for enrichment of a mixed population of MSCs expressing approximately six-fold higher levels of GFP and of CD activity when compared with unmanipulated engineered MSCs. Moreover, engraftment and endothelial differentiation of these in vitro selected and enriched gene-modified marrow stromal cells was demonstrated by Matrigel assay in vivo. In conclusion, these findings outline the potential of human CD as an ex vivo selection and enrichment marker of genetically engineered MSCs for transgenic cell therapy applications. PMID:11938460

  6. 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 Mssbauer 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

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

    SciTech Connect

    Hu, Yi Ericsson, Ida Doseth, Berit Liabakk, Nina B. Krokan, Hans E. Kavli, Bodil

    2014-03-10

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

  8. A bacterial gene codA encoding cytosine deaminase is an effective conditional negative selectable marker in Glycine max

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Conditional negative selection is a powerful technique whereby the absence of a gene product allows survival in otherwise lethal conditions. In plants, the Escherichia coli gene codA has been employed as a negative selection marker. CodA is a conditionally lethal dominant gene encoding cy...

  9. Mucosal adenosine deaminase activity and gastric ulcer healing.

    PubMed

    Namiot, Z; Marcinkiewicz, M; Jaroszewicz, W; Stasiewicz, J; Gorski, J

    1993-10-26

    Adenosine deaminase activity was studied in gastric corpus mucosa close to an ulcer crater. It was found that 6 weeks of therapy with ranitidine was accompanied by a decrease in enzyme activity in the mucosa around healed ulcers and an increase around those which failed to heal. The different activities of adenosine deaminase in the vicinity of healed and unhealed ulcers may indicate its possible role in peptic ulcer healing. PMID:8276083

  10. A Screening Protocol for Identification of Functional Mutants of RNA Editing Adenosine Deaminases

    PubMed Central

    Eifler, Tristan; Chan, Dalen; Beal, Peter A.

    2013-01-01

    Genetic screens can be used to evaluate a spectrum of mutations and thereby infer the function of particular residues within a protein. The Adenosine Deaminase Acting on RNA (ADAR) family of RNA editing enzymes selectively deaminate adenosines (A) in double helical RNA, generating inosine (I). The protocol described here exploits ADAR2s editing activity in a yeast based screen by inserting an editing substrate sequence with a stop codon incorporated at the editing site upstream from sequence coding the reporter ?-galactosidase. A-to-I editing changes the stop codon to one for tryptophan, allowing normal expression of the reporter. This technique is particularly well-suited for screening ADAR and ADAR substrate mutant libraries for editing activity. PMID:23788559

  11. [Terahertz spectroscopy of DNA nucleobases: cytosine and thymine].

    PubMed

    Yan, Hui; Fan, Wen-Hui; Zheng, Zhuan-Ping; Liu, Jia

    2013-10-01

    The absorption features of DNA nucleobases cytosine and thymine were measured by terahertz time-domain spectroscopy (THz-TDS) from 0.1 to 3.5 THz. Our experimental results clearly show that these important biomolecules exhibit distinctive absorption features in THz region. To the best of our knowledge, the subtle absorption peak of cytosine at 2.53 THz is reported for the first time. Moreover, geometry optimizations and lattice dynamic calculations on cytosine crystal were also performed with the pseudo-potential plane wave method of density functional theory by taking periodic boundary conditions into account. All measured terahertz absorption features of cytosine were assigned successfully and its absorption spectrum was reproduced according to our calculations. Furthermore, our results show that absorption features of cytosine below 3.5 THz arise from external modes in translation and rotation motions, which are dominated by the intermolecular hydrogen bonds. PMID:24409701

  12. The catalase activity of diiron adenine deaminase

    SciTech Connect

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

    2011-12-01

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

  13. Formation and dissociation of protonated cytosinecytosine base pairs in i-motifs by ab initio quantum chemical calculations

    NASA Astrophysics Data System (ADS)

    Zhang, Xiao-Hu; Li, Ming; Wang, Yan-Ting; Ouyang, Zhong-Can

    2014-02-01

    Formation and dissociation mechanisms of CC+ base pairs in acidic and alkaline environments are investigated, employing ab initio quantum chemical calculations. Our calculations suggest that, in an acidic environment, a cytosine monomer is first protonated and then dimerized with an unprotonated cytosine monomer to form a CC+ base pair; in an alkaline environment, a protonated cytosine dimer is first unprotonated and then dissociated into two cytosine monomers. In addition, the force for detaching a CC+ base pair was found to be inversely proportional to the distance between the two cytosine monomers. These results provide a microscopic mechanism to qualitatively explain the experimentally observed reversible formation and dissociation of i-motifs.

  14. [Gene therapy for adenosine deaminase deficiency].

    PubMed

    Sakiyama, Yukio; Ariga, Tadashi; Ohtsu, Makoto

    2005-03-01

    A four year-old boy with adenosine deaminase (ADA-) deficient severe combined immunodeficiency(SCID) receiving PEG-ADA was treated under a gene therapy protocol targeting peripheral blood lymphocytes (PBLs) in 1995. After eleven infusions of autologous PBLs transduced with retroviral vector LASN encoding ADAcDNA, he exhibited increased levels of the CD8+ T lymphocytes, serum immunoglobulin, specific antibodies and delayed type hypersensitivity skin tests. Follow-up studies also provided evidence of long-term persistence and function of transduced PBLs with improvement in the immune function. However, the therapeutic effect of this gene therapy has been difficult to assess because of the concomitant treatment of PEG-ADA. Two ADA-SCID patients have been currently treated with autologous bone marrow CD34+ cells engineered with a retroviral vector GCsapM-ADA after discontinuation of PEG-ADA. The restoration of intracellular ADA enzymatic activity in lymphocytes and granulocytes resulted in correction of the systemic toxicity and liver function in the absence of PEG-ADA treatment. Both patients are at home where they are clinically well, and they do not experience adversed effect, with follow up being 12 months after CD34+ cells gene therapy. PMID:15773344

  15. [Gene therapy for adenosine deaminase deficiency].

    PubMed

    Sakiyama, Y

    1996-01-01

    Severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency is a fatal recessive disorder caused by mutations in the gene encoding ADA. Based on the first clinical trial of two young girls with ADA-deficient SCID by recombinant retrovirus-mediated gene transfer at the National Institute of Health of USA, we prepared to treat a four-year-old boy with ADA-deficient SCID who had been treated with PEG-ADA for 3 years. Approval to perform the clinical trial of gene therapy by using his peripheral blood T lymphocytes as the target and recombinant retroviral vector (LASN) as the vector for ADA gene transfer was obtained from both of the Ministry of Health and Welfare and the Ministry of Education, Science, Sports and Culture on 13 February, 1995. The first clinical trial of gene therapy for the patient was initiated on 1 August 1995. He received 8 x 10(8) LASN-transduced lymphocytes in an injection administered intravenously on 8 August and 2.5 x 10(9) transduced lymphocytes on 4 September without any side reactions. The procedure, safety and efficacy of clinical trial of gene therapy were discussed. PMID:8727372

  16. Information Thermodynamics of Cytosine DNA Methylation

    PubMed Central

    Sanchez, Robersy; Mackenzie, Sally A.

    2016-01-01

    Cytosine DNA methylation (CDM) is a stable epigenetic modification to the genome and a widespread regulatory process in living organisms that involves multicomponent molecular machines. Genome-wide cytosine methylation patterning participates in the epigenetic reprogramming of a cell, suggesting that the biological information contained within methylation positions may be amenable to decoding. Adaptation to a new cellular or organismal environment also implies the potential for genome-wide redistribution of CDM changes that will ensure the stability of DNA molecules. This raises the question of whether or not we would be able to sort out the regulatory methylation signals from the CDM background (“noise”) induced by thermal fluctuations. Here, we propose a novel statistical and information thermodynamic description of the CDM changes to address the last question. The physical basis of our statistical mechanical model was evaluated in two respects: 1) the adherence to Landauer’s principle, according to which molecular machines must dissipate a minimum energy ε = kBT ln2 at each logic operation, where kB is the Boltzmann constant, and T is the absolute temperature and 2) whether or not the binary stretch of methylation marks on the DNA molecule comprise a language of sorts, properly constrained by thermodynamic principles. The study was performed for genome-wide methylation data from 152 ecotypes and 40 trans-generational variations of Arabidopsis thaliana and 93 human tissues. The DNA persistence length, a basic mechanical property altered by CDM, was estimated with values from 39 to 66.9 nm. Classical methylome analysis can be retrieved by applying information thermodynamic modelling, which is able to discriminate signal from noise. Our finding suggests that the CDM signal comprises a language scheme properly constrained by molecular thermodynamic principles, which is part of an epigenomic communication system that obeys the same thermodynamic rules as do current human communication systems. PMID:26963711

  17. 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

  18. Mucosal adenosine deaminase activity and stump ulcer healing.

    PubMed

    Namiot, Z; Namiot, A; Stasiewicz, J; Marcinkiewicz, M; Jaroszewicz, W; Górski, J

    1995-06-01

    Adenosine deaminase activity was studied in endoscopically taken slices from gastric mucosa in patient after partial gastric resection performed due to complicated duodenal ulcer, and currently with peptic ulcer in the stump. The samples of gastric mucosa were taken before and after 6 weeks of treatment with ranitidine, 150 mg twice daily, at a distance within 2 cm and greater than 2 cm from the ulcer crater. Adenosine deaminase activity was measured in mucosa homogenates by determination of ammonia liberated from substrate. It was found that therapy with ranitidine was accompanied by an increase in enzyme activity in the mucosa surrounding unhealed stump ulcers, while no changes were noted in mucosa around healed stump ulcers. A possible role of mucosal adenosine deaminase activity in stump ulcer healing is postulated. PMID:7670131

  19. Surface expression of adenosine deaminase in mitogen-stimulated lymphocytes.

    PubMed Central

    Martin, M; Centelles, J J; Huguet, J; Echevarne, F; Colomer, D; Vives-Corrons, J L; Franco, R

    1993-01-01

    Adenosine deaminase (ADA) expression on the surface of mitogen-stimulated lymphocytes was studied by flow cytometry. The gate for lymphocytes was located by cell size (forward scatter), cytoplasmic complexity (side scatter) and by expression of the markers CD2, CD4, CD8 and CD19. After mitogenic proliferation two populations appeared, one corresponding to non-stimulated cells, and the other consisting of larger cells which showed relatively high expression of adenosine deaminase on their surface. The increase was similar to that observed for CD71 expression, and paralleled the increase in 3H-thymidine incorporation. There was a correlation between ADA and CD71 expression (r = 0.92 for phytohaemagglutinin (PHA) and 0.97 for pokeweed mitogen (PWM)). These results suggest a role for ecto-adenosine deaminase in lymphocyte proliferation and/or triggering. PMID:8348757

  20. 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? at 30 C. Since adenine is deaminated ?10 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

  1. Nanopores Discriminate among Five C5-Cytosine Variants in DNA

    PubMed Central

    2015-01-01

    Individual DNA molecules can be read at single nucleotide precision using nanopores coupled to processive enzymes. Discrimination among the four canonical bases has been achieved, as has discrimination among cytosine, 5-methylcytosine (mC), and 5-hydroxymethylcytosine (hmC). Two additional modified cytosine bases, 5-carboxylcytosine (caC) and 5-formylcytosine (fC), are produced during enzymatic conversion of hmC to cytosine in mammalian cells. Thus, an accurate picture of the cytosine epigenetic status in target cells should also include these C5-cytosine variants. In the present study, we used a patch clamp amplifier to acquire ionic current traces caused by phi29 DNA polymerase-controlled translocation of DNA templates through the M2MspA pore. Decision boundaries based on three consecutive ionic current states were implemented to call mC, hmC, caC, fC, or cytosine at CG dinucleotides in ?4400 individual DNA molecules. We found that the percentage of correct base calls for single pass reads ranged from 91.6% to 98.3%. This accuracy depended upon the identity of nearest neighbor bases surrounding the CG dinucleotide. PMID:25347819

  2. 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

  3. 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

  4. 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

  5. Yeast Infections

    MedlinePLUS

    ... taking antibiotics, it can multiply and cause an infection. Yeast infections affect different parts of the body in different ways: Thrush is a yeast infection that causes white patches in your mouth Candida ...

  6. Yeast Infection

    MedlinePLUS

    ... majority of vaginal yeast infections are caused by Candida Albicans ? There are some less common yeast organisms such as Torulopsis Glabrata that may cause infections that do not clear up with the ...

  7. Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif.

    PubMed

    Land, Allison M; Wang, Jiayi; Law, Emily K; Aberle, Ryan; Kirmaier, Andrea; Krupp, Annabel; Johnson, Welkin E; Harris, Reuben S

    2015-11-24

    APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy. PMID:26544511

  8. Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif

    PubMed Central

    Land, Allison M.; Wang, Jiayi; Law, Emily K.; Aberle, Ryan; Kirmaier, Andrea; Krupp, Annabel; Johnson, Welkin E.; Harris, Reuben S.

    2015-01-01

    APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy. PMID:26544511

  9. Profiling cytosine oxidation in DNA by LC-MS/MS.

    PubMed

    Samson-Thibault, Francois; Madugundu, Guru S; Gao, Shanshan; Cadet, Jean; Wagner, J Richard

    2012-09-17

    Spontaneous and oxidant-induced damage to cytosine is probably the main cause of CG to TA transition mutations in mammalian genomes. The reaction of hydroxyl radical (OH) and one-electron oxidants with cytosine derivatives produces numerous oxidation products, which have been identified in large part by model studies with monomers and short oligonucleotides. Here, we developed an analytical method based on LC-MS/MS to detect 10 oxidized bases in DNA, including 5 oxidation products of cytosine. The utility of this method is demonstrated by the measurement of base damage in isolated calf thymus DNA exposed to ionizing radiation in aerated aqueous solutions (0-200 Gy) and to well-known Fenton-like reactions (Fe(2+) or Cu(+) with H(2)O(2) and ascorbate). The following cytosine modifications were quantified as modified 2'-deoxyribonucleosides upon exposure of DNA to ionizing radiation in aqueous aerated solution: 5-hydroxyhydantoin (Hyd-Ura) > 5-hydroxyuracil (5-OHUra) > 5-hydroxycytosine (5-OHCyt) > 5,6-dihydroxy-5,6-dihydrouracil (Ura-Gly) > 1-carbamoyl-4,5-dihydroxy-2-oxoimidazolidine (Imid-Cyt). The total yield of cytosine oxidation products was comparable to that of thymine oxidation products (5,6-dihydroxy-5,6-dihydrothymine (Thy-Gly), 5-hydroxy-5-methylhydantotin (Hyd-Thy), 5-(hydroxymethyl)uracil (5-HmUra), and 5-formyluracil (5-ForUra)) as well as the yield of 8-oxo-7,8-dihydroguanine (8-oxoGua). The major oxidation product of cytosine in DNA was Hyd-Ura. In contrast, the formation of Imid-Cyt was a minor pathway of DNA damage, although it is the major product arising from irradiation of the monomers, cytosine, and 2'-deoxycytidine. The reaction of Fenton-like reagents with DNA gave a different distribution of cytosine derived products compared to ionizing radiation, which likely reflects the reaction of metal ions with intermediate peroxyl radicals or hydroperoxides. The analysis of the main cytosine oxidation products will help elucidate the complex mechanism of oxidative degradation of cytosine in DNA and probe the consequences of these reactions in biology and medicine. PMID:22725252

  10. 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.

  11. 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.

  12. Nuclear distribution of porphobilinogen deaminase (PBGD) in glioma cells: a regulatory role in cancer transformation?

    PubMed Central

    Greenbaum, L; Gozlan, Y; Schwartz, D; Katcoff, D J; Malik, Z

    2002-01-01

    Recently, considerable interest has been directed to red-fluorescence photodiagnosis of brain and other tumours during surgery using the protoporphyrin IX natural precursor, 5-aminolaevulinic acid. In the present study we focused on the role of the rate-limiting enzyme porphobilinogen deaminase in glioma C6 cell activity, differentiation and sub-cellular distribution. Over-expression of the human housekeeping porphobilinogen deaminase in the glioma cells, using the housekeeping-porphobilinogen deaminase plasmid, induced a G1 cell cycle attenuation accompanied by increases in enzyme activity and c6 differentiation toward astrocytes. Visualisation of subcellular localisation of the porphobilinogen deaminase using the independent techniques of fluorescence immuno-staining with specific anti-human porphobilinogen deaminase antibodies and cellular expression of porphobilinogen deaminase fused to green fluorescent protein, revealed (unexpectedly) a major fraction of porphobilinogen deaminase in the nucleus and only a minor fraction in the cytoplasm. Both C and N terminals of porphobilinogen deaminase fused to green fluorescent protein revealed a major fraction of the newly synthesized fused porphobilinogen deaminase in the nucleus. Furthermore, newborn rat brain cells grown in a primary culture showed the same localisation pattern of porphobilinogen deaminase in the nuclei. Stimulation of C6 glioma cell differentiation by butyrate induced a marked decrease in porphobilinogen deaminase both in the nucleus and in the cytoplasm as determined by Western blotting and fluorescence immuno-localisation. These findings suggest a possible dual role for housekeeping porphobilinogen deaminase in fast dividing glioma cells, one related to the porphyrin synthesis pathway and another coupled to nuclear function, which might be linked to tumorigenesis. British Journal of Cancer (2002) 86, 10061011. DOI: 10.1038/sj/bjc/6600173 www.bjcancer.com 2002 Cancer Research UK PMID:11953837

  13. 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 proteinRNA interactions. PMID:21182352

  14. Safety evaluation of AMP deaminase from Aspergillus oryzae.

    PubMed

    Okado, Nobuo; Sugi, Mai; Ueda, Maya; Mizuhashi, Fukutaro; Lynch, Barry S; Vo, Trung D; Roberts, Ashley S

    2015-12-01

    Adenosine-5'-monophosphate (AMP) deaminase is an enzyme used to increase concentrations of 5'-inosine monophosphate in certain foods and beverages for flavoring purposes. One commercial source of this enzyme is Aspergillus oryzae, a filamentous fungus with a history of safe use in Asia as a fermentation organism used in the production of miso sauce and sake liquors. Noting the use of the enzyme in food intended for human consumption and potential presence at trace levels in finished goods, a series of safety studies including an in vitro Ames test and chromosome aberration assay with Chinese hamster lung fibroblasts were conducted along with a 90-day oral toxicity study in rats. AMP deaminase showed no evidence of genotoxicity in the in vitro tests. Following gavage administration of Sprague-Dawley rats at dosages of 19.8, 198.4, or 1984 mg total organic solids (TOS)/kg body weight (bw)/day for 90 days, no adverse effects on body weight gain, food consumption, hematology, clinical chemistry, urinalysis, ophthalmological and histopathological examinations were observed. The no-observed-adverse-effect level was considered to be 1984 mg TOS/kg bw/day, the highest dose tested. Results of the genotoxicity studies and subchronic rat study support the safe use of AMP deaminase produced from A. oryzae in food production. PMID:26559900

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

    PubMed

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

    2015-11-20

    Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. ADAR1 and ADAR2 are two members of the family that have been shown to be catalytically active. Earlier, we reported a phenotypic screen for the study of human ADAR2 using budding yeast S. cerevisiae as the host system. While this screen has been successfully applied to the study of ADAR2, it failed with ADAR1. Here, we report a new reporter that uses a novel editing substrate and is suitable for the study of ADAR1. We screened plasmid libraries with randomized codons for two important residues in human ADAR1 (G1007 and E1008). The screening results combined with in vitro deamination assays led to the identification of mutants that are more active than the wild type protein. Furthermore, a screen of the ADAR1 E1008X library with a reporter construct bearing an AG mismatch at the editing site suggests one role for the residue at position 1008 is to sense the identity of the base pairing partner for the editing site adenosine. This work has provided a starting point for future in vitro evolution studies of ADAR1 and led to new insight into ADAR's editing site selectivity. PMID:26372505

  16. A new family of high-affinity transporters for adenine, cytosine, and purine derivatives in Arabidopsis.

    PubMed

    Gillissen, B; Brkle, L; Andr, B; Khn, C; Rentsch, D; Brandl, B; Frommer, W B

    2000-02-01

    In many organisms, including plants, nucleic acid bases and derivatives such as caffeine are transported across the plasma membrane. Cytokinins, important hormones structurally related to adenine, are produced mainly in root apices, from where they are translocated to shoots to control a multitude of physiological processes. Complementation of a yeast mutant deficient in adenine uptake (fcy2) with an Arabidopsis cDNA expression library enabled the identification of a gene, AtPUP1 (for Arabidopsis thaliana purine permease1), belonging to a large gene family (AtPUP1 to AtPUP15) encoding a new class of small, integral membrane proteins. AtPUP1 transports adenine and cytosine with high affinity. Uptake is energy dependent, occurs against a concentration gradient, and is sensitive to protonophores, potentially indicating secondary active transport. Competition studies show that purine derivatives (e.g., hypoxanthine), phytohormones (e.g., zeatin and kinetin), and alkaloids (e.g., caffeine) are potent inhibitors of adenine and cytosine uptake. Inhibition by cytokinins is competitive (competitive inhibition constant K(i) = 20 to 35 microM), indicating that cytokinins are transported by this system. AtPUP1 is expressed in all organs except roots, indicating that the gene encodes an uptake system for root-derived nucleic acid base derivatives in shoots or that it exports nucleic acid base analogs from shoots by way of the phloem. The other family members may have different affinities for nucleic acid bases, perhaps functioning as transporters for nucleosides, nucleotides, and their derivatives. PMID:10662864

  17. High-Resolution Analysis of Cytosine Methylation in Ancient DNA

    PubMed Central

    Cropley, Jennifer E.; Cooper, Alan; Suter, Catherine M.

    2012-01-01

    Epigenetic changes to gene expression can result in heritable phenotypic characteristics that are not encoded in the DNA itself, but rather by biochemical modifications to the DNA or associated chromatin proteins. Interposed between genes and environment, these epigenetic modifications can be influenced by environmental factors to affect phenotype for multiple generations. This raises the possibility that epigenetic states provide a substrate for natural selection, with the potential to participate in the rapid adaptation of species to changes in environment. Any direct test of this hypothesis would require the ability to measure epigenetic states over evolutionary timescales. Here we describe the first single-base resolution of cytosine methylation patterns in an ancient mammalian genome, by bisulphite allelic sequencing of loci from late Pleistocene Bison priscus remains. Retrotransposons and the differentially methylated regions of imprinted loci displayed methylation patterns identical to those derived from fresh bovine tissue, indicating that methylation patterns are preserved in the ancient DNA. Our findings establish the biochemical stability of methylated cytosines over extensive time frames, and provide the first direct evidence that cytosine methylation patterns are retained in DNA from ancient specimens. The ability to resolve cytosine methylation in ancient DNA provides a powerful means to study the role of epigenetics in evolution. PMID:22276161

  18. 1-Aminocyclopropane-1-Carboxylate Deaminase Enhances Agrobacterium tumefaciens-Mediated Gene Transfer into Plant Cells?

    PubMed Central

    Nonaka, Satoko; Sugawara, Masayuki; Minamisawa, Kiwamu; Yuhashi, Ken-ichi; Ezura, Hiroshi

    2008-01-01

    Agrobacterium-mediated gene transfer is widely used for plant molecular genetics, and efficient techniques are required. Recent studies show that ethylene inhibits the gene transfer. To suppress ethylene evolution, we introduced 1-aminocyclopropane-1-carboxylate (ACC) deaminase into Agrobacterium tumefaciens. The ACC deaminase enhanced A. tumefaciens-mediated gene transfer into plants. PMID:18310418

  19. 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...

  20. Red yeast

    MedlinePLUS

    ... cholesterol levels and triglycerides. However, this specific product contains large amounts of a chemical similar to "statin" ... this product and other red yeast products that contain statins to be illegal unapproved drugs. However, outside ...

  1. Counting Yeast.

    ERIC Educational Resources Information Center

    Bealer, Jonathan; Welton, Briana

    1998-01-01

    Describes changes to a traditional study of population in yeast colonies. Changes to the procedures include: (1) only one culture per student team; (2) cultures are inoculated only once; and (3) the same tube is sampled daily. (DDR)

  2. Regulation of Activation Induced Deaminase (AID) by Estrogen.

    PubMed

    Pauklin, Siim

    2016-01-01

    Regulation of Activation Induced Deaminase (AID) by the hormone estrogen has important implications for understanding adaptive immune responses as well as the involvement of AID in autoimmune diseases and tumorigenesis. This chapter describes the general laboratory techniques for analyzing AID expression and activity induced by estrogen, focusing on the isolation and preparation of cells for hormone treatment and the subsequent analysis of AID responsiveness to estrogen at the RNA level and for determining the regulation of AID activity via estrogen by analyzing Ig switch circle transcripts and mutations in switch region loci. PMID:26585164

  3. 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

  4. New Insights into 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Phylogeny, Evolution and Ecological Significance

    PubMed Central

    Nascimento, Francisco X.; Rossi, Márcio J.; Soares, Cláudio R. F. S.; McConkey, Brendan J.; Glick, Bernard R.

    2014-01-01

    The main objective of this work is the study of the phylogeny, evolution and ecological importance of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, the activity of which represents one of the most important and studied mechanisms used by plant growth–promoting microorganisms. The ACC deaminase gene and its regulatory elements presence in completely sequenced organisms was verified by multiple searches in diverse databases, and based on the data obtained a comprehensive analysis was conducted. Strain habitat, origin and ACC deaminase activity were taken into account when analyzing the results. In order to unveil ACC deaminase origin, evolution and relationships with other closely related pyridoxal phosphate (PLP) dependent enzymes a phylogenetic analysis was also performed. The data obtained show that ACC deaminase is mostly prevalent in some Bacteria, Fungi and members of Stramenopiles. Contrary to previous reports, we show that ACC deaminase genes are predominantly vertically inherited in various bacterial and fungal classes. Still, results suggest a considerable degree of horizontal gene transfer events, including interkingdom transfer events. A model for ACC deaminase origin and evolution is also proposed. This study also confirms the previous reports suggesting that the Lrp-like regulatory protein AcdR is a common mechanism regulating ACC deaminase expression in Proteobacteria, however, we also show that other regulatory mechanisms may be present in some Proteobacteria and other bacterial phyla. In this study we provide a more complete view of the role for ACC deaminase than was previously available. The results show that ACC deaminase may not only be related to plant growth promotion abilities, but may also play multiple roles in microorganism's developmental processes. Hence, exploring the origin and functioning of this enzyme may be the key in a variety of important agricultural and biotechnological applications. PMID:24905353

  5. Formation of hydrogen-bridged cytosine dimers on Cu(110)

    NASA Astrophysics Data System (ADS)

    Frankel, D. J.; Chen, Q.; Richardson, N. V.

    2006-05-01

    Cytosine was adsorbed onto a Cu(110) surface under UHV conditions. Annealing to 370K resulted in the formation of a (66)gg low energy electron diffraction (LEED) pattern, even at submonolayer coverages. Examination of this structure with scanning tunneling microscopy (STM) revealed islands of zigzag chains at low coverages and large ordered domains at monolayer saturation. Further annealing to 480K initiated a phase transition to a (62)gg structure observed both by LEED and STM. High resolution electron energy loss spectroscopy spectra for both overlayer structures exhibited mainly in-plane modes suggesting upright/tilted species on the surface. Based on the experimental data and supported by density functional theory calculations, a model is proposed for the (62)gg structure, which involves the formation of deprotonated hydrogen bridge-bonded cytosine dimers, adsorbed through the oxygen atoms.

  6. Communication: UV photoionization of cytosine catalyzed by Ag+

    NASA Astrophysics Data System (ADS)

    Taccone, Martín I.; Féraud, Geraldine; Berdakin, Matías; Dedonder-Lardeux, Claude; Jouvet, Christophe; Pino, Gustavo A.

    2015-07-01

    The photo-induced damages of DNA in interaction with metal cations, which are found in various environments, still remain to be characterized. In this paper, we show how the complexation of a DNA base (cytosine (Cyt)) with a metal cation (Ag+) changes its electronic properties. By means of UV photofragment spectroscopy of cold ions, it was found that the photoexcitation of the CytAg+ complex at low energy (315-282) nm efficiently leads to ionized cytosine (Cyt+) as the single product. This occurs through a charge transfer state in which an electron from the p orbital of Cyt is promoted to Ag+, as confirmed by ab initio calculations at the TD-DFT/B3LYP and RI-ADC(2) theory level using the SV(P) basis set. The low ionization energy of Cyt in the presence of Ag+ could have important implications as point mutation of DNA upon sunlight exposition.

  7. 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.

  8. An efficient prebiotic synthesis of cytosine and uracil

    NASA Astrophysics Data System (ADS)

    Robertson, Michael P.; Miller, Stanley L.

    1995-06-01

    IN contrast to the purines1 3, the routes that have been proposed for the prebiotic synthesis of pyrimidines from simple precursors give only low yields. Cytosine can be synthesized from cyano-acetylene and cyanate4,5; the former precursor is produced from a spark discharge in a CH4/N2 mixture4,5 and is an abundant interstellar molecule6. 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 explored7 is the reaction of cyanoacetaldehyde (formed by hydrolysis of cyanoacetylene8) with urea. But at low concentrations of urea, this reaction produces no detectable quantities of cytosine7. 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 world9.

  9. Plant growth-promoting traits of yeasts isolated from the phyllosphere and rhizosphere of Drosera spatulata Lab.

    PubMed

    Fu, Shih-Feng; Sun, Pei-Feng; Lu, Hsueh-Yu; Wei, Jyuan-Yu; Xiao, Hong-Su; Fang, Wei-Ta; Cheng, Bai-You; Chou, Jui-Yu

    2016-03-01

    Microorganisms can promote plant growth through direct and indirect mechanisms. Compared with the use of bacteria and mycorrhizal fungi, the use of yeasts as plant growth-promoting (PGP) agents has not been extensively investigated. In this study, yeast isolates from the phyllosphere and rhizosphere of the medicinally important plant Drosera spatulata Lab. were assessed for their PGP traits. All isolates were tested for indole-3-acetic acid-, ammonia-, and polyamine-producing abilities, calcium phosphate and zinc oxide solubilizing ability, and catalase activity. Furthermore, the activities of siderophore, 1-aminocyclopropane-1-carboxylate deaminase, and fungal cell wall-degrading enzymes were assessed. The antagonistic action of yeasts against pathogenic Glomerella cingulata was evaluated. The cocultivation of Nicotiana benthamiana with yeast isolates enhanced plant growth, indicating a potential yeast-plant interaction. Our study results highlight the potential use of yeasts as plant biofertilizers under controlled and field conditions. PMID:26895872

  10. Structure of the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii and its relation to other homotrimeric dUTPases.

    PubMed

    Johansson, Eva; Bjornberg, Olof; Nyman, Per Olof; Larsen, Sine

    2003-07-25

    The bifunctional dCTP deaminase-dUTPase (DCD-DUT) from Methanocaldococcus jannaschii catalyzes the deamination of the cytosine moiety in dCTP and the hydrolysis of the triphosphate moiety forming dUMP, thereby preventing uracil from being incorporated into DNA. The crystal structure of DCD-DUT has been determined to 1.88-A resolution and represents the first known structure of an enzyme catalyzing dCTP deamination. The functional form of DCD-DUT is a homotrimer wherein the subunits are composed of a central distorted beta-barrel surrounded by two beta-sheets and four helices. The trimeric DCD-DUT shows structural similarity to trimeric dUTPases at the tertiary and quaternary levels. There are also additional structural elements in DCD-DUT compared with dUTPase because of a longer primary structure. Four of the five conserved sequence motifs that create the active sites in dUTPase are found in structurally equivalent positions in DCD-DUT. The last 25 C-terminal residues of the 204-residue-long DCD-DUT are not visible in the electron density map, but, analogous to dUTPases, the C terminus is probably ordered, closing the active site upon catalysis. Unlike other enzymes catalyzing the deamination of cytosine compounds, DCD-DUT is not exploiting an enzyme-bound metal ion such as zinc or iron for nucleophile generation. The active site contains two water molecules that are engaged in hydrogen bonds to the invariant residues Ser118, Arg122, Thr130, and Glu145. These water molecules are potential nucleophile candidates in the deamination reaction. PMID:12756253

  11. Measurement of human placental 5'-AMP deaminase activity by radiometric assay

    SciTech Connect

    Maguire, M.H.; Aronson, D.M.

    1981-09-01

    The level of 5'-AMP deaminase in homogenates of human term placenta has been measured by means of a simple radiometric assay. The assay uses /sup 14/C-labeled AMP as substrate and incorporates conditions of pH and K/sup +/ concentration, which optimize the 5'-AMP deaminase activity, and inhibitors of 5'-nucleotidase and adenosine deaminase to reduce interferences from these enzymes. Assay products are separated by descending paper chromatography and quantitated by liquid scintillation counting. The activity of 5'-AMP deaminase in human term placenta determined by this assay was 474 +/- 37 nmol min/sup -1/ g/sup -1/ at 30/sup o/C and was less than the 5'-AMP phosphatase activity evident under the same assay conditions. The assay is suitable for measurement of 5'-AMP deaminase in extracts of other tissues in which high levels of phosphates and adenosine deaminase preclude assay of 5'-AMP deaminase by such techniques as ultraviolet absorption changes or ammonia estimation.

  12. Melamine Deaminase and Atrazine Chlorohydrolase: 98 Percent Identical but Functionally Different

    PubMed Central

    Seffernick, Jennifer L.; de Souza, Mervyn L.; Sadowsky, Michael J.; Wackett, Lawrence P.

    2001-01-01

    The gene encoding melamine deaminase (TriA) from Pseudomonas sp. strain NRRL B-12227 was identified, cloned into Escherichia coli, sequenced, and expressed for in vitro study of enzyme activity. Melamine deaminase displaced two of the three amino groups from melamine, producing ammeline and ammelide as sequential products. The first deamination reaction occurred more than 10 times faster than the second. Ammelide did not inhibit the first or second deamination reaction, suggesting that the lower rate of ammeline hydrolysis was due to differential substrate turnover rather than product inhibition. Remarkably, melamine deaminase is 98% identical to the enzyme atrazine chlorohydrolase (AtzA) from Pseudomonas sp. strain ADP. Each enzyme consists of 475 amino acids and differs by only 9 amino acids. AtzA was shown to exclusively catalyze dehalogenation of halo-substituted triazine ring compounds and had no activity with melamine and ammeline. Similarly, melamine deaminase had no detectable activity with the halo-triazine substrates. Melamine deaminase was active in deamination of a substrate that was structurally identical to atrazine, except for the substitution of an amino group for the chlorine atom. Moreover, melamine deaminase and AtzA are found in bacteria that grow on melamine and atrazine compounds, respectively. These data strongly suggest that the 9 amino acid differences between melamine deaminase and AtzA represent a short evolutionary pathway connecting enzymes catalyzing physiologically relevant deamination and dehalogenation reactions, respectively. PMID:11274097

  13. Adenosine deaminase and nucleoside phosphorylase activities in normal human blood mononuclear cell subpopulations

    PubMed Central

    Macdermott, R. P.; Tritsch, G. L.; Formeister, J. F.

    1980-01-01

    Adenosine deaminase and nucleoside phosphorylase activity is highest in T cells and macrophages; null cells have approximately half the amount and B cells have the least amount of both enzyme activities PMID:6781801

  14. A human-mouse somatic hybrid line selected for human deoxycytidine deaminase.

    PubMed

    Chan, T S; Long, C; Green, H

    1975-01-01

    A new selective medium has been developed for cells containing the enzyme deoxycytidine deaminase. This medium contains hypoxanthine, aminopterin, and 5-methyldeoxycytidine (HAM medium). To survive in the presence of the aminopterin, the cells must utilize deoxycytidine deaminase to convert the 5-methyldeoxycytidine to thymidine. The cells must also have thymidine kinase and hypoxanthine phosphoribosyltransferase. A mouse cell line deficient in deoxycytidine deaminase has been isolated from a deoxycytidine kinase-deficient line, using 5-bromodeoxycytidine as the selective agent. A hybrid line between this double mutant and a human diploid fibroblast was isolated in HAM medium. The hybrid line contains the chromosomes expected of a human-mouse hybrid. The deoxycytidine deaminase isozyme patterns on cellogel show that the human-mouse hybrid cell line produces an enzyme with an electrophoretic mobility intermediate between that of the human and that of the mouse. PMID:1235901

  15. Safety and Efficacy of Suicide Gene Therapy with Adenosine Deaminase 5-Fluorocytosine Silmutaneously in in Vitro Cultures of Melanoma and Retinal Cell Lines

    PubMed Central

    Sakkas, Antonios; Zarogoulidis, Paul; Domvri, Kalliopi; Hohenforst-Schmidt, Wolfgang; Bougiouklis, Dimitris; Kakolyris, Stylianos; Zarampoukas, Thomas; Kioumis, Ioannis; Pitsiou, Georgia; Huang, Haidong; Li, Qiang; Meditskou, Soultana; Tsiouda, Theodora; Pezirkianidis, Nikolaos; Zarogoulidis, Konstantinos

    2014-01-01

    Local treatment as a treatment modality is gaining increased general acceptance over time. Novel drugs and methodologies of local administration are being investigated in an effort to achieve disease local control. Suicide gene therapy is a method that has been investigated as a local treatment with simultaneously distant disease control. In our current experiment we purchased HTB-70 (melanoma cell line, derived from metastatic axillary node) and CRL-2302 (human retinal epithelium) were from ATCC LGC Standards and Ancotil, 2.5 g/250 ml (1 g/00ml) (5-Flucytosine) MEDA; Pharmaceuticals Ltd. UK. Adenosine Cytosine Deaminase (Ad.CD) was also used in order to convert the pro-drug 5-Flucytosine to the active 5-Fluoracil. Three different concentrations of 5-Flucytosine (5-FC) were administered (0.2ml, 0.8ml and 1.2ml). At indicated time-points (4h, 8h and 24h) cell viability and apoptosis were measured. Our concept was to investigate whether suicide gene therapy with Ad. CD-5-FC could be used with safety and efficiency as a future local treatment for melanoma located in the eye cavity. Indeed, our results indicated that in every 5-FC administration had mild cytotoxicity for the retinal cells, while increased apoptosis was observed for the melanoma cell line. PMID:24799955

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

    ClinicalTrials.gov

    2015-03-02

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

  17. Plant purine nucleoside catabolism employs a guanosine deaminase required for the generation of xanthosine in Arabidopsis.

    PubMed

    Dahncke, Kathleen; Witte, Claus-Peter

    2013-10-01

    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

  18. Effects of cytosine methylation on DNA charge transport

    NASA Astrophysics Data System (ADS)

    Hihath, Joshua; Guo, Shaoyin; Zhang, Peiming; Tao, Nongjian

    2012-04-01

    The methylation of cytosine bases in DNA commonly takes place in the human genome and its abnormality can be used as a biomarker in the diagnosis of genetic diseases. In this paper we explore the effects of cytosine methylation on the conductance of DNA. Although the methyl group is a small chemical modification, and has a van der Waals radius of only 2 , its presence significantly changes the duplex stability, and as such may also affect the conductance properties of DNA. To determine if charge transport through the DNA stack is sensitive to this important biological modification we perform multiple conductance measurements on a methylated DNA molecule with an alternating G:C sequence and its non-methylated counterpart. From these studies we find a measurable difference in the conductance between the two types of molecules, and demonstrate that this difference is statistically significant. The conductance values of these molecules are also compared with a similar sequence that has been previously studied to help elucidate the charge transport mechanisms involved in direct DNA conductance measurements.

  19. Epigenetic regulation of HIV-1 latency by cytosine methylation.

    PubMed

    Kauder, Steven E; Bosque, Alberto; Lindqvist, Annica; Planelles, Vicente; Verdin, Eric

    2009-06-01

    Human immunodeficiency virus type 1 (HIV-1) persists in a latent state within resting CD4+ T cells of infected persons treated with highly active antiretroviral therapy (HAART). This reservoir must be eliminated for the clearance of infection. Using a cDNA library screen, we have identified methyl-CpG binding domain protein 2 (MBD2) as a regulator of HIV-1 latency. Two CpG islands flank the HIV-1 transcription start site and are methylated in latently infected Jurkat cells and primary CD4+ T cells. MBD2 and histone deacetylase 2 (HDAC2) are found at one of these CpG islands during latency. Inhibition of cytosine methylation with 5-aza-2'deoxycytidine (aza-CdR) abrogates recruitment of MBD2 and HDAC2. Furthermore, aza-CdR potently synergizes with the NF-kappaB activators prostratin or TNF-alpha to reactivate latent HIV-1. These observations confirm that cytosine methylation and MBD2 are epigenetic regulators of HIV-1 latency. Clearance of HIV-1 from infected persons may be enhanced by inclusion of DNA methylation inhibitors, such as aza-CdR, and NF-kappaB activators into current antiviral therapies. PMID:19557157

  20. Analysis of normal and mutant forms of human adenosine deaminase - a review.

    PubMed

    Daddona, P E; Kelley, W N

    1980-02-01

    A deficiency of the enzyme adenosine deaminase is associated with an autosomal recessive form of severe combined immunodeficiency disease in man. The molecular forms of the normal human enzyme have now been well characterized in an effort to better understand the nature of the enzyme defect in affected patients. In some human tissues adenosine deaminase exists predominantly as a small molecular form while in other tissues a large form composed of adenosine deaminase (small form) and an adenosine deaminase-binding protein predominates. The small form of the enzyme purified to homogeneity by antibody affinity chromatography is a monomer of native molecular weight of 37,600. The adenosine deaminase-binding protein, purified by adenosine deaminase affinity chromatography, appears to be a dimer of native molecular weight 213,000 and contains carbohydrate. Based on direct binding measurements, chemical cross-linking studies and sedimentation equilibrium analyses, small form adenosine deaminase has been shown to combine with purified binding protein in a molar ratio of 2:1 respectively to produce the large form adenosine deaminase. Reduced, but widely ranging levels of adenosine deaminating activity, have been reported in various tissues of adenosine deaminase deficient patients. Further, the characteristics of this residual enzyme activity have been analyzed immunochemically to substantiate genetic heterogeneity in this disorder. While many types of immunodeficiency are currently recognized in man, in most cases the molecular defect is unknown. The discovery of a deficiency of the enzyme, adenosine deaminase, ADA, (EC 3.5.4.4), in some patients with severe combined immunodeficiency disease represented an early clue to the pathogenesis of immune dysfunction at the molecular level 1-4. Affected patients with markedly reduced levels of ADA exhibit a defect of both cellular and humoral immunity characterized clinically by severe recurrent infections with a fatal outcome if untreated. Attempts to elucidate the nature of the genetic mutation(s) leading to the reduction of ADA activity in these immunodeficient patients have been complicated in part by an incomplete understanding of the nature of ADA in normal tissues. In this review we will consider the structural characteristics of the normal and mutant forms of ADA as they are currently understood. PMID:6988697

  1. Detection of Cytosine Methylation in Ancient DNA from Five Native American Populations Using Bisulfite Sequencing

    PubMed Central

    Smith, Rick W. A.; Monroe, Cara; Bolnick, Deborah A.

    2015-01-01

    While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches. PMID:26016479

  2. 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 27 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

  3. Dynamics of UV-excited uracil, thymine, and cytosine

    NASA Astrophysics Data System (ADS)

    Hudock, Hanneli

    The excited state dynamics of nucleic acids has been a subject of much experimental and theoretical interest. Nucleic acid bases readily absorb UV radiation, which can lead to mutagenic dimer formation. The dynamics of UV-excited nucleic acids is an important step in understanding how these dimers form. The pyrimidine bases (uracil, thymine, and cytosine) have been studied with ab initio multiple spawning molecular dynamics and high level electronic structure methods. This work has involved both gas-phase and aqueous dynamics as well as simulation of the time-resolved photoelectron spectrum, transient absorption, fluorescence, and reaction rates. With these findings, complete relaxation mechanisms are proposed for the pyrimidines and comparisons are made directly to experimental results.

  4. Watson-Crick and sugar-edge base pairing of cytosine in the gas phase: UV and infrared spectra of cytosine·2-pyridone.

    PubMed

    Frey, Jann A; Ottiger, Philipp; Leutwyler, Samuel

    2014-01-23

    While keto-amino cytosine is the dominant species in aqueous solution, spectroscopic studies in molecular beams and in noble gas matrices show that other cytosine tautomers prevail in apolar environments. Each of these offers two or three H-bonding sites (Watson-Crick, wobble, sugar-edge). The mass- and isomer-specific S1 ← S0 vibronic spectra of cytosine·2-pyridone (Cyt·2PY) and 1-methylcytosine·2PY are measured using UV laser resonant two-photon ionization (R2PI), UV/UV depletion, and IR depletion spectroscopy. The UV spectra of the Watson-Crick and sugar-edge isomers of Cyt·2PY are separated using UV/UV spectral hole-burning. Five different isomers of Cyt·2PY are observed in a supersonic beam. We show that the Watson-Crick and sugar-edge dimers of keto-amino cytosine with 2PY are the most abundant in the beam, although keto-amino-cytosine is only the third most abundant tautomer in the gas phase. We identify the different isomers by combining three different diagnostic tools: (1) methylation of the cytosine N1-H group prevents formation of both the sugar-edge and wobble isomers and gives the Watson-Crick isomer exclusively. (2) The calculated ground state binding and dissociation energies, relative gas-phase abundances, excitation and the ionization energies are in agreement with the assignment of the dominant Cyt·2PY isomers to the Watson-Crick and sugar-edge complexes of keto-amino cytosine. (3) The comparison of calculated ground state vibrational frequencies to the experimental IR spectra in the carbonyl stretch and NH/OH/CH stretch ranges strengthen this identification. PMID:24383817

  5. 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.

  6. 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.

  7. Adenosine deaminase: functional implications and different classes of inhibitors.

    PubMed

    Cristalli, G; Costanzi, S; Lambertucci, C; Lupidi, G; Vittori, S; Volpini, R; Camaioni, E

    2001-03-01

    Adenosine deaminase (ADA) is an enzyme of the purine metabolism which catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. This ubiquitous enzyme has been found in a wide variety of microorganisms, plants, and invertebrates. In addition, it is present in all mammalian cells that play a central role in the differentiation and maturation of the lymphoid system. However, despite a number of studies performed to date, the physiological role played by ADA in the different tissues is not clear. Inherited ADA deficiency causes severe combined immunodeficiency disease (ADA-SCID), in which both B-cell and T-cell development is impaired. ADA-SCID has been the first disorder to be treated by gene therapy, using polyethylene glycol-modified bovine ADA (PEG-ADA). Conversely, there are several diseases in which the level of ADA is above normal. A number of ADA inhibitors have been designed and synthesized, classified as ground-state and transition-state inhibitors. They may be used to mimic the genetic deficiency of the enzyme, in lymphoproliferative disorders or immunosuppressive therapy (i.e., in graft rejection), to potentiate the effect of antileukemic or antiviral nucleosides, and, together with adenosine kinase, to reduce breakdown of adenosine in inflammation, hypertension, and ischemic injury. PMID:11223861

  8. Prebiotic cytosine synthesis: A critical analysis and implications for the origin of life

    PubMed Central

    Shapiro, Robert

    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. The reported prebiotic syntheses of cytosine involve the reaction of cyanoacetylene (or its hydrolysis product, cyanoacetaldehyde), with cyanate, cyanogen, or urea. These substances undergo side reactions with common nucleophiles that appear to proceed more rapidly than cytosine formation. To favor cytosine formation, reactant concentrations are required that are implausible in a natural setting. Furthermore, cytosine is consumed by deamination (the half-life for deamination at 25°C is ≈340 yr) and other reactions. No reactions have been described thus far that would produce cytosine, even in a specialized local setting, at a rate sufficient to compensate for its decomposition. On the basis of this evidence, it appears quite unlikely that cytosine played a role in the origin of life. Theories that involve replicators that function without the Watson–Crick pairs, or no replicator at all, remain as viable alternatives. PMID:10200273

  9. 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

  10. Nucleoside deaminase: an enzymatic marker for stress erythropoiesis in the mouse

    PubMed Central

    Rothman, Ivan K.; Zanjani, Esmail D.; Gordon, Albert S.; Silber, Robert

    1970-01-01

    The level of nucleoside deaminase was determined in extracts of mouse tissues obtained during a period of accelerated erythropoiesis induced by hypoxia, hemorrhage, or the injection of phenylhydrazine. Under these conditions a striking (10- to 100-fold) elevation of the enzyme activity occurred in the spleen. Similar results were obtained with the injection of purified erythropoietin. In control animals, only a trace of nucleoside deaminase activity was detected in the blood. During the reticulocyte response which followed erythropoietic stimulation, there was a sharp increase in the blood level of nucleoside deaminase, which rose up to 120 times that of control animals. By differential centrifugation, the enzyme was localized to the reticulocyte-rich fraction. Erythrocyte nucleoside deaminase remained elevated even after the reticulocyte count had fallen to normal in the phenylhydrazine-treated mice or to zero after the cessation of hypoxia. There was a very gradual decline in the enzyme activity in the blood which fell to the barely detectable control levels about 45 days after the initial reticulocyte response, a time period which corresponds to the survival of the mouse red blood cell. The persistence of high levels of nucleoside deaminase for the full life span of a generation of erythrocytes formed during stress, viewed in contrast to the virtual absence of the enzyme from normal erythrocytes of all ages, represents an enzymatic difference between the normal red blood cell and the cell produced under conditions of accelerated erythropoiesis. PMID:5475986

  11. A Cytidine Deaminase Edits C to U in Transfer RNAs in Archaea

    SciTech Connect

    Randau, L.; Stanley, B; Kohlway, A; Mechta, S; Xiong, Y; Söll, D

    2009-01-01

    All canonical transfer RNAs (tRNAs) have a uridine at position 8, involved in maintaining tRNA tertiary structure. However, the hyperthermophilic archaeon Methanopyrus kandleri harbors 30 (out of 34) tRNA genes with cytidine at position 8. Here, we demonstrate C-to-U editing at this location in the tRNA's tertiary core, and present the crystal structure of a tRNA-specific cytidine deaminase, CDAT8, which has the cytidine deaminase domain linked to a tRNA-binding THUMP domain. CDAT8 is specific for C deamination at position 8, requires only the acceptor stem hairpin for activity, and belongs to a unique family within the cytidine deaminase-like superfamily. The presence of this C-to-U editing enzyme guarantees the proper folding and functionality of all M. kandleri tRNAs.

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

    SciTech Connect

    Daddona, P.E.

    1981-12-10

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

  13. 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.

  14. 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

  15. Raman spectral study of metal-cytosine complexes: A density functional theoretical (DFT) approach

    NASA Astrophysics Data System (ADS)

    Liu, Shuanjiang; Zheng, Guimei; Li, Jianxin

    2011-09-01

    The fluctuation of surface-enhanced Raman scattering (SERS) spectra has been an obstacle to the analysis of the adsorbate on the metal surface. In this paper, we aim at using the density functional theory (DFT) to study the fluctuant Raman spectra of the cytosine molecule which interacts with a coinage metal atom or cation via N 1 and N 3 sites. The results show that the adsorption site strongly influences the Raman spectral property of cytosine molecule, especially the relative intensity of some bands. In addition, the SERS spectra of cytosine which is adsorbed on the gold, silver, and copper electrodes are measured, and the possible orientation and adsorption site of the cytosine molecule adsorbed on metal electrodes surface are proposed with the help of DFT simulations.

  16. Cytosine methylation regulates oviposition in the pathogenic blood fluke Schistosoma mansoni

    PubMed Central

    Geyer, Kathrin K.; Rodrguez Lpez, Carlos M.; Chalmers, Iain W.; Munshi, Sabrina E.; Truscott, Martha; Heald, James; Wilkinson, Mike J.; Hoffmann, Karl F.

    2011-01-01

    Similar to other metazoan pathogens, Schistosoma mansoni undergoes transcriptional and developmental regulation during its complex lifecycle and host interactions. DNA methylation as a mechanism to control these processes has, to date, been discounted in this parasite. Here we show the first evidence for cytosine methylation in the S. mansoni genome. Transcriptional coregulation of novel DNA methyltransferase (SmDnmt2) and methyl-CpG-binding domain proteins mirrors the detection of cytosine methylation abundance and implicates the presence of a functional DNA methylation machinery. Genome losses in cytosine methylation upon SmDnmt2 silencing and the identification of a hypermethylated, repetitive intron within a predicted forkhead gene confirm this assertion. Importantly, disruption of egg production and egg maturation by 5-azacytidine establishes an essential role for 5-methylcytosine in this parasite. These findings provide the first functional confirmation for this epigenetic modification in any worm species and link the cytosine methylation machinery to platyhelminth oviposition processes. PMID:21829186

  17. The CHH motif in sugar beet satellite DNA: a modulator for cytosine methylation.

    PubMed

    Zakrzewski, Falk; Schubert, Veit; Viehoever, Prisca; Minoche, Andr E; Dohm, Juliane C; Himmelbauer, Heinz; Weisshaar, Bernd; Schmidt, Thomas

    2014-06-01

    Methylation of DNA is important for the epigenetic silencing of repetitive DNA in plant genomes. Knowledge about the cytosine methylation status of satellite DNAs, a major class of repetitive DNA, is scarce. One reason for this is that arrays of tandemly arranged sequences are usually collapsed in next-generation sequencing assemblies. We applied strategies to overcome this limitation and quantified the level of cytosine methylation and its pattern in three satellite families of sugar beet (Beta vulgaris) which differ in their abundance, chromosomal localization and monomer size. We visualized methylation levels along pachytene chromosomes with respect to small satellite loci at maximum resolution using chromosome-wide fluorescent in situ hybridization complemented with immunostaining and super-resolution microscopy. Only reduced methylation of many satellite arrays was obtained. To investigate methylation at the nucleotide level we performed bisulfite sequencing of 1569 satellite sequences. We found that the level of methylation of cytosine strongly depends on the sequence context: cytosines in the CHH motif show lower methylation (44-52%), while CG and CHG motifs are more strongly methylated. This affects the overall methylation of satellite sequences because CHH occurs frequently while CG and CHG are rare or even absent in the satellite arrays investigated. Evidently, CHH is the major target for modulation of the cytosine methylation level of adjacent monomers within individual arrays and contributes to their epigenetic function. This strongly indicates that asymmetric cytosine methylation plays a role in the epigenetic modification of satellite repeats in plant genomes. PMID:24661787

  18. Benchmark Thermochemistry for Biologically Relevant Adenine and Cytosine. A Combined Experimental and Theoretical Study.

    PubMed

    Emel'yanenko, Vladimir N; Zaitsau, Dzmitry H; Shoifet, Evgeni; Meurer, Florian; Verevkin, Sergey P; Schick, Christoph; Held, Christoph

    2015-09-17

    The thermochemical properties available in the literature for adenine and cytosine are in disarray. A new condensed phase standard (p = 0.1 MPa) molar enthalpy of formation at T = 298.15 K was measured by using combustion calorimetry. New molar enthalpies of sublimation were derived from the temperature dependence of vapor pressure measured by transpiration and by the quarz-crystal microbalance technique. The heat capacities of crystalline adenine and cytosine were measured by temperature-modulated DSC. Thermodynamic data on adenine and cytosine available in the literature were collected, evaluated, and combined with our experimental results. Thus, the evaluated collection of data together with the new experimental results reported here has helped to resolve contradictions in the available enthalpies of formation. A set of reliable thermochemical data is recommended for adenine and cytosine for further thermochemical calculations. Quantum-chemical calculations of the gas phase molar enthalpies of formation of adenine and cytosine have been performed by using the G4 method and results were in excellent agreement with the recommended experimental data. The standard molar entropies of formation and the standard molar Gibbs functions of formation in crystal and gas state have been calculated. Experimental vapor-pressure data measured in this work were used to estimate pure-component PC-SAFT parameters. This allowed modeling solubility of adenine and cytosine in water over the temperature interval 278-310 K. PMID:26317826

  19. Disruption of Transcriptional Coactivator Sub1 Leads to Genome-Wide Re-distribution of Clustered Mutations Induced by APOBEC in Active Yeast Genes.

    PubMed

    Lada, Artem G; Kliver, Sergei F; Dhar, Alok; Polev, Dmitrii E; Masharsky, Alexey E; Rogozin, Igor B; Pavlov, Youri I

    2015-05-01

    Mutations in genomes of species are frequently distributed non-randomly, resulting in mutation clusters, including recently discovered kataegis in tumors. DNA editing deaminases play the prominent role in the etiology of these mutations. To gain insight into the enigmatic mechanisms of localized hypermutagenesis that lead to cluster formation, we analyzed the mutational single nucleotide variations (SNV) data obtained by whole-genome sequencing of drug-resistant mutants induced in yeast diploids by AID/APOBEC deaminase and base analog 6-HAP. Deaminase from sea lamprey, PmCDA1, induced robust clusters, while 6-HAP induced a few weak ones. We found that PmCDA1, AID, and APOBEC1 deaminases preferentially mutate the beginning of the actively transcribed genes. Inactivation of transcription initiation factor Sub1 strongly reduced deaminase-induced can1 mutation frequency, but, surprisingly, did not decrease the total SNV load in genomes. However, the SNVs in the genomes of the sub1 clones were re-distributed, and the effect of mutation clustering in the regions of transcription initiation was even more pronounced. At the same time, the mutation density in the protein-coding regions was reduced, resulting in the decrease of phenotypically detected mutants. We propose that the induction of clustered mutations by deaminases involves: a) the exposure of ssDNA strands during transcription and loss of protection of ssDNA due to the depletion of ssDNA-binding proteins, such as Sub1, and b) attainment of conditions favorable for APOBEC action in subpopulation of cells, leading to enzymatic deamination within the currently expressed genes. This model is applicable to both the initial and the later stages of oncogenic transformation and explains variations in the distribution of mutations and kataegis events in different tumor cells. PMID:25941824

  20. Disruption of Transcriptional Coactivator Sub1 Leads to Genome-Wide Re-distribution of Clustered Mutations Induced by APOBEC in Active Yeast Genes

    PubMed Central

    Dhar, Alok; Polev, Dmitrii E.; Masharsky, Alexey E.; Rogozin, Igor B.; Pavlov, Youri I.

    2015-01-01

    Mutations in genomes of species are frequently distributed non-randomly, resulting in mutation clusters, including recently discovered kataegis in tumors. DNA editing deaminases play the prominent role in the etiology of these mutations. To gain insight into the enigmatic mechanisms of localized hypermutagenesis that lead to cluster formation, we analyzed the mutational single nucleotide variations (SNV) data obtained by whole-genome sequencing of drug-resistant mutants induced in yeast diploids by AID/APOBEC deaminase and base analog 6-HAP. Deaminase from sea lamprey, PmCDA1, induced robust clusters, while 6-HAP induced a few weak ones. We found that PmCDA1, AID, and APOBEC1 deaminases preferentially mutate the beginning of the actively transcribed genes. Inactivation of transcription initiation factor Sub1 strongly reduced deaminase-induced can1 mutation frequency, but, surprisingly, did not decrease the total SNV load in genomes. However, the SNVs in the genomes of the sub1 clones were re-distributed, and the effect of mutation clustering in the regions of transcription initiation was even more pronounced. At the same time, the mutation density in the protein-coding regions was reduced, resulting in the decrease of phenotypically detected mutants. We propose that the induction of clustered mutations by deaminases involves: a) the exposure of ssDNA strands during transcription and loss of protection of ssDNA due to the depletion of ssDNA-binding proteins, such as Sub1, and b) attainment of conditions favorable for APOBEC action in subpopulation of cells, leading to enzymatic deamination within the currently expressed genes. This model is applicable to both the initial and the later stages of oncogenic transformation and explains variations in the distribution of mutations and kataegis events in different tumor cells. PMID:25941824

  1. Reduced activity of Arabidopsis chromosome-cohesion regulator gene CTF7/ECO1 alters cytosine methylation status and retrotransposon expression

    PubMed Central

    Bolaños-Villegas, Pablo; Jauh, Guang-Yuh

    2015-01-01

    Multicellular organisms such as higher plants require timely regulation of DNA replication and cell division to grow and develop. Recent work in Arabidopsis has shown that chromosome segregation during meiosis and mitosis depends on the activity of several genes that in yeast are involved in the establishment of chromosomal cohesion. In this process, proteins of the STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC) family tether chromosomes and establish inter- and intrachromosomal connections. In Arabidopsis, recruitment of SMC proteins and establishment of cohesion during key stages of the cell cycle depend on the activity of CHROMOSOME TRANSMISSION FIDELITY 7/ESTABLISHMENT OF COHESION 1 (CTF7/ECO1). Here we show that loss of CTF7/ECO1 activity alters the status of cytosine methylation in both intergenic regions and transposon loci. An increase in expression was also observed for transposon copia28, which suggests a link between CTF7/ECO1 activity, DNA methylation and gene silencing. More work is needed to determine the mechanistic relationships that intervene in this process. PMID:26039473

  2. Control of adenosine deaminase levels in human lymphoblasts.

    PubMed

    Daddona, P E; Kelley, W N

    1982-01-01

    High levels of adenosine deaminase (ADA) activity have been associated with normal T cell differentiation and T cell disease, such as acute lymphoblastic leukemia; however, possible mechanisms controlling the level of this enzyme have not been explored. In this study, the properties and rate of turnover of ADA are compared in cultured human T and B lymphoblast cell lines. (1) Relative to B lymphoblasts, the level of ADA activity in extracts of T lymphoblast cell lines (MOLT-4, RPMI-8402, CCRF-CEM and CCRF-HSB-2) is elevated 7- to 14-fold and differs by 2-fold among the T-cell lines. (2) In T and B lymphoblast extracts, the enzyme is apparently identical based on Km for adenosine and deoxyadenosine, Ki for inosine, Vmax for adenosine, S20w, isoelectric pH, and heat stability. Further, by radioimmunoassay the quantity of ADA immunoreactive protein is proportional to the level of enzyme activity in all cell lines studied. (3) Using a purification and selective immunoprecipitation technique, the enzyme turnover could be assessed in cell lines labeled with [35S]methionine. The apparent rate of ADA synthesis, relative to total protein, is 2-fold faster in both T cell lines (RPMI-8402 and CCRF-CEM) than in the B cell lines (MGL-8 and GM-130). The apparent half-life (t1/2) for the enzyme degradation is 19 and 39 hr, respectively, for CCRF-CEM and RPMI-8402, while the t1/2 for both B cell lines is 7-9 hr. From the net rate of synthesis and degradation, the T cell lines exhibit a 6- and 12-fold difference in ADA turnover relative to B cells, consistent with the observed differences in enzyme activity. (4) The level of ADA (activity and/or protein) in cultured T or B lymphoblasts is not influenced by either substrates or products of the ADA reaction or an ADA inhibitor or a selected group of immunosuppressive drugs added to these cells in culture. These studies indicate that while ADA is apparently identical in all T and B lymphoblasts, alterations in both the rate of ADA synthesis and degradation lead to its accumulation and high steady-state level in T cells. PMID:6981287

  3. Serum Adenosine Deaminase as Inflammatory Marker in Rheumatoid Arthritis

    PubMed Central

    Vinapamula, Kiranmayi S.; Bhattaram, Siddartha Kumar; Bitla, Aparna R.; Manohar, Suchitra M.

    2015-01-01

    Background Rheumatoid arthritis (RA) is a prototypical inflammatory joint disease. The degree of inflammation is reflected in the extent of joint damage, which further has influence on the quality of life of patients with RA, including risk of atherosclerosis. Hence, besides clinical indices, estimation of degree of inflammation using biochemical markers helps in effecting optimum treatment strategies. C-reactive protein (CRP) is established as an inflammatory marker in patients with RA. Adenosine deaminase (ADA), an enzyme of purine metabolism is considered as a marker of cell mediated immunity and has also been suggested as a marker of inflammatory process in RA. The present study attempts to study the efficacy of serum ADA activity as an inflammatory marker in RA. Materials and Methods Forty six RA patients and forty six age and sex matched healthy controls were included in the study. ADA activity and high sensitivity C-reactive protein (hsCRP) levels in serum were measured in all the subjects. Statistical analyses were done using Medcalc statistical software version 12.2.2. Results ADA activity and hsCRP levels were increased in RA patients compared to controls (p<0.0001 and 0.0001 respectively). Significant positive correlation was obtained between hsCRP and ADA in patients (r=0.316, p=0.033). Receiver operating characteristic (ROC) curve analysis revealed statistically significant area under curve (AUC) for ADA that is comparable to that obtained for hsCRP (0.776, p<0.0001 for ADA, 0.726, p<0.0001 for hsCRP). Similar diagnostic utility was obtained with ROC generated cut-off value of 25.3 IU/L (82.6% sensitivity and 65.2% specificity) and with control mean value of 23.48 IU/L (86.96% sensitivity and 54.35% specificity) for ADA. Conclusion Findings of the present study indicate the importance of ADA as a marker of inflammation. Considering the higher sensitivity obtained, we propose control mean (23.48 IU/L) as a cut-off for serum ADA activity as an inflammatory marker. Owing to the simplicity and also the cost effectiveness of ADA assay, ADA may be recommended as a marker of inflammation in patients with RA. However, further larger and well controlled studies are needed to establish its role as inflammatory marker. PMID:26500897

  4. Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase.

    PubMed Central

    Sheehy, R E; Honma, M; Yamada, M; Sasaki, T; Martineau, B; Hiatt, W R

    1991-01-01

    Pseudomonas sp. strain ACP is capable of growth on 1-aminocyclopropane-1-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase and the subsequent conversion of ACC to alpha-ketobutyrate and ammonia (M. Honma, Agric. Biol. Chem. 49:567-571, 1985). The complete amino acid sequence of purified ACC deaminase was determined, and the sequence information was used to clone the ACC deaminase gene from a 6-kb EcoRI fragment of Pseudomonas sp. strain ACP DNA. DNA sequence analysis of an EcoRI-PstI subclone demonstrated an open reading frame (ORF) encoding a polypeptide with a deduced amino acid sequence identical to the protein sequence determined chemically and a predicted molecular mass of 36,674 Da. The ORF also contained an additional 72 bp of upstream sequence not predicted by the amino acid sequence. Escherichia coli minicells containing the 6-kb clone expressed a major polypeptide of the size expected for ACC deaminase which was reactive with ACC deaminase antiserum. Furthermore, a lacZ fusion with the ACC deaminase ORF resulted in the expression of active enzyme in E. coli. ACC is a key intermediate in the biosynthesis of ethylene in plants, and the use of the ACC deaminase gene to manipulate this pathway is discussed. Images PMID:1885510

  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. Purification and characterization of developmentally regulated AMP deaminase from Dictyostelium discoideum.

    PubMed

    Malliaros, D P; Kozwich, D L; Jahngen, E G

    1991-04-01

    AMP deaminase, the enzyme that catalyzes the conversion of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia, was purified from the cellular slime mold, Dictyostelium discoideum in the nutrient-deprived state. The native enzyme had an apparent molecular weight of 199,000 daltons. Its apparent Km was 1.6 mM and its Vmax was 1.0 mumol min-1 mg-1, as measured by the release of IMP From AMP. The enzyme, like other AMP deaminases, was found to be activated by ATP, and inhibited either by GTP or inorganic phosphate. It was also specific for the deamination of AMP. Deaminase activity was increased either when vegetative cells were placed in a nutrient-deprived medium (for up to 6 h) or when vegetative cells were treated with the drug hadacidin. In cells actively growing in complete media, enzyme activity was more non-specific, hydrolyzing adenosine as well as AMP. AMP deaminase in D. discoideum appears to be stage-specific and developmentally regulated, possibly serving to regulate the adenylated nucleotide pool and the interconversion to guanylated nucleotides during early morphodifferentiation. PMID:1916064

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

    ERIC Educational Resources Information Center

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

    2012-01-01

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

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

    ERIC Educational Resources Information Center

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

    2012-01-01

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

  9. 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 ...

  10. Vaginal Yeast Infections (For Parents)

    MedlinePLUS

    ... I Help a Friend Who Cuts? Vaginal Yeast Infections KidsHealth > For Teens > Vaginal Yeast Infections Print A ... effect of taking antibiotics. What Is a Yeast Infection? A yeast infection is a common infection caused ...

  11. Zinc enhancement of cytidine deaminase activity highlights a potential allosteric role of loop-3 in regulating APOBEC3 enzymes

    PubMed Central

    Marx, Ailie; Galilee, Meytal; Alian, Akram

    2015-01-01

    The strong association of APOBEC3 cytidine deaminases with somatic mutations leading to cancers accentuates the importance of their tight intracellular regulation to minimize cellular transformations. We reveal a novel allosteric regulatory mechanism of APOBEC3 enzymes showing that APOBEC3G and APOBEC3A coordination of a secondary zinc ion, reminiscent to ancestral deoxycytidylate deaminases, enhances deamination activity. Zinc binding is pinpointed to loop-3 which whilst highly variable harbors a catalytically essential and spatially conserved asparagine at its N-terminus. We suggest that loop-3 may play a general role in allosterically tuning the activity of zinc-dependent cytidine deaminase family members. PMID:26678087

  12. Zinc enhancement of cytidine deaminase activity highlights a potential allosteric role of loop-3 in regulating APOBEC3 enzymes.

    PubMed

    Marx, Ailie; Galilee, Meytal; Alian, Akram

    2015-01-01

    The strong association of APOBEC3 cytidine deaminases with somatic mutations leading to cancers accentuates the importance of their tight intracellular regulation to minimize cellular transformations. We reveal a novel allosteric regulatory mechanism of APOBEC3 enzymes showing that APOBEC3G and APOBEC3A coordination of a secondary zinc ion, reminiscent to ancestral deoxycytidylate deaminases, enhances deamination activity. Zinc binding is pinpointed to loop-3 which whilst highly variable harbors a catalytically essential and spatially conserved asparagine at its N-terminus. We suggest that loop-3 may play a general role in allosterically tuning the activity of zinc-dependent cytidine deaminase family members. PMID:26678087

  13. 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.

  14. Vaginal Yeast Infection

    MedlinePLUS

    ... Skip Content Marketing Share this: Main Content Area Vaginal Yeast Infection Vaginal yeast infection, or vulvovaginal candidiasis, is a common cause of vaginal irritation. Nearly 75 percent of all adult women ...

  15. Vaginal yeast infection

    MedlinePLUS

    Yeast infection - vagina; Vaginal candidiasis; Monilial vaginitis ... Most women have a vaginal yeast infection at some time. Candida albicans is a common type of fungus. It is often found in small amounts in the vagina , mouth, ...

  16. Effectiveness of rhizobacteria containing ACC deaminase for growth promotion of peas (Pisum sativum) under drought conditions.

    PubMed

    Zahir, Z A; Munir, A; Asghar, H N; Shaharoona, B; Arshad, M

    2008-05-01

    A series of experiments were conducted to assess the effectiveness of rhizobacteria containing 1-aminocyclopropane- 1-carboxylate (ACC) deaminase for growth promotion of peas under drought conditions. Ten rhizobacteria isolated from the rhizosphere of different crops (peas, wheat, and maize) were screened for their growth promoting ability in peas under axenic condition. Three rhizobacterial isolates, Pseudomonas fluorescens biotype G (ACC-5), P. fluorescens (ACC-14), and P. putida biotype A (Q-7), were selected for pot trial on the basis of their source, ACC deaminase activity, root colonization, and growth promoting activity under axenic conditions. Inoculated and uninoculated (control) seeds of pea cultivar 2000 were sown in pots (4 seeds/pot) at different soil moisture levels (25, 50, 75, and 100% of field capacity). Results revealed that decreasing the soil moisture levels from 100 to 25% of field capacity significantly decreased the growth of peas. However, inoculation of peas with rhizobacteria containing ACC deaminase significantly decreased the "drought stress imposed effects" on growth of peas, although with variable efficacy at different moisture levels. At the lowest soil moisture level (25% field capacity), rhizobacterial isolate Pseudomonas fluorescens biotype G (ACC-5) was found to be more promising compared with the other isolates, as it caused maximum increases in fresh weight, dry weight, root length, shoot length, number of leaves per plant, and water use efficiency on fresh and dry weight basis (45, 150, 92, 45, 140, 46, and 147%, respectively) compared with respective uninoculated controls. It is highly likely that rhizobacteria containing ACC deaminase might have decreased the drought-stress induced ethylene in inoculated plants, which resulted in better growth of plants even at low moisture levels. Therefore, inoculation with rhizobacteria containing ACC deaminase could be helpful in eliminating the inhibitory effects of drought stress on the growth of peas. PMID:18633298

  17. Effects of cytosine arabinoside on human leukemia cells.

    PubMed

    Crisp, L B; Smith, S M; Mathers, M A; Young, G A; Lyons, S D; Christopherson, R I

    1996-09-01

    Cytosine arabinoside (Ara-C) is used to treat leukemias, with complete remission induced by combination chemotherapy in approximately 70% of cases of acute myelogenous leukemia (AML). Ara-CTP acts as a competitive inhibitor of DNA polymerase and may also be incorporated into DNA. Accumulation of deoxyribonucleoside triphosphates (dNTPs) induced by Ara-C may indicate disruption of DNA synthesis in susceptible leukemia cells. A procedure has been developed for the quantification of Ara-CTP and dNTPs from small samples of leukaemia cells from patients (4 x 10(7) cells) activated with concanavalin A (10 micrograms/ml, 48 hr) and grown in the presence of [32P]orthophosphate (1.1 microM, 9 x 10(6) Ci/mol, 16 hr). The susceptibilities to Ara-C of the human leukemia cell lines CCRF-CEM (IC50 = 6.30 nM), CCRF-HSB-2 (IC50 = 10.4 nM) and MOLT-4 (IC50 = 10.0 nM) may be correlated with their abilities to accumulate high concentrations of Ara-CTP (> 1000 amol/cell) with increases of between 1.3- and 3.4-fold in dATP, dGTP and dTTP for the four cell lines, while dCTP decreased between 0.23- and 0.78-fold. By contrast, an Ara-C-resistant derivative of HL-60 cells (IC50 = 400 nM) accumulated only low concentrations of Ara-CTP (71 amol/cell) without significant changes in dNTPs. High concentrations of Ara-CTP in leukemia cells induce accumulations of dATP, dGTP and dTTP due to inhibition of DNA synthesis, and depletion of dCTP. This imbalance in the pools of the four dNTPs could lead to genetic miscoding and cell death. PMID:8930129

  18. A computational study of adenine, uracil, and cytosine adsorption upon AlN and BN nano-cages

    NASA Astrophysics Data System (ADS)

    Baei, Mohammad T.; Taghartapeh, Mohammad Ramezani; Lemeski, E. Tazikeh; Soltani, Alireza

    Density-functional theory calculations are used to investigate the interaction of Al12N12 and B12N12 clusters with the adenine (A), uracil (U), and cytosine (C) molecules. The current calculations demonstrate that these hybrid adsorbent materials are able to adsorb the adenine, uracil, and cytosine molecules through exothermic processes. Our theoretical results reveal improvement in the adsorption of adenine, uracil, and cytosine on Al12N12 and B12N12. It is observed that B12N12 is highly sensitive to adenine, uracil, and cytosine compared with Al12N12 to serve as a biochemical sensor.

  19. 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. PMID:25639773

  20. Single-Cell Quantification of Cytosine Modifications by Hyperspectral Dark-Field Imaging.

    PubMed

    Wang, Xiaolei; Cui, Yi; Irudayaraj, Joseph

    2015-12-22

    Epigenetic modifications on DNA, especially on cytosine, play a critical role in regulating gene expression and genome stability. It is known that the levels of different cytosine derivatives are highly dynamic and are regulated by a variety of factors that act on the chromatin. Here we report an optical methodology based on hyperspectral dark-field imaging (HSDFI) using plasmonic nanoprobes to quantify the recently identified cytosine modifications on DNA in single cells. Gold (Au) and silver (Ag) nanoparticles (NPs) functionalized with specific antibodies were used as contrast-generating agents due to their strong local surface plasmon resonance (LSPR) properties. With this powerful platform we have revealed the spatial distribution and quantity of 5-carboxylcytosine (5caC) at the different stages in cell cycle and demonstrated that 5caC was a stably inherited epigenetic mark. We have also shown that the regional density of 5caC on a single chromosome can be mapped due to the spectral sensitivity of the nanoprobes in relation to the interparticle distance. Notably, HSDFI enables an efficient removal of the scattering noises from nonspecifically aggregated nanoprobes, to improve accuracy in the quantification of different cytosine modifications in single cells. Further, by separating the LSPR fingerprints of AuNPs and AgNPs, multiplex detection of two cytosine modifications was also performed. Our results demonstrate HSDFI as a versatile platform for spatial and spectroscopic characterization of plasmonic nanoprobe-labeled nuclear targets at the single-cell level for quantitative epigenetic screening. PMID:26505210

  1. Inheritance and Variation of Cytosine Methylation in Three Populus Allotriploid Populations with Different Heterozygosity

    PubMed Central

    Suo, Yujing; Dong, Chunbo; Kang, Xiangyang

    2015-01-01

    DNA methylation is an epigenetic mechanism with the potential to regulate gene expression and affect plant phenotypes. Both hybridization and genome doubling may affect the DNA methylation status of newly formed allopolyploid plants. Previous studies demonstrated that changes in cytosine methylation levels and patterns were different among individual hybrid plant, therefore, studies investigating the characteristics of variation in cytosine methylation status must be conducted at the population level to avoid sampling error. In the present study, an F1 hybrid diploid population and three allotriploid populations with different heterozygosity [originating from first-division restitution (FDR), second-division restitution (SDR), and post-meiotic restitution (PMR) 2n eggs of the same female parent] were used to investigate cytosine methylation inheritance and variation relative to their common parents using methylation-sensitive amplification polymorphism (MSAP). The variation in cytosine methylation in individuals in each population exhibited substantial differences, confirming the necessity of population epigenetics. The total methylation levels of the diploid population were significantly higher than in the parents, but those of the three allotriploid populations were significantly lower than in the parents, indicating that both hybridization and polyploidization contributed to cytosine methylation variation. The vast majority of methylated status could be inherited from the parents, and the average percentages of non-additive variation were 6.29, 3.27, 5.49 and 5.07% in the diploid, FDR, SDR and PMR progeny populations, respectively. This study lays a foundation for further research on population epigenetics in allopolyploids. PMID:25901359

  2. Catalysis effect of micro-hydration on the intramolecular proton transfer in cytosine

    NASA Astrophysics Data System (ADS)

    Valadbeigi, Younes; Soleiman-Beigi, Mohammad; Sahraei, Reza

    2015-06-01

    Structural and thermodynamic properties of nine isomers of cytosine were studied in gas and aqueous phases and in micro-hydrated environment employing B3LYP and MP2 methods. Also, isomerizations of cytosine were studied in gas phase and the activation energies (Ea) and Gibbs free energies (?G#) of the internal Osbnd H rotations and proton transfer processes were calculated. The calculated Osbnd H rotational barriers were smaller than 50 kJ/mol while the activation energies of intramolecular proton transfers were in the range of 110-190 kJ/mol. Effect of mono- and di-hydration of the cytosine isomers on the transition state structures and the energy barriers was investigated.

  3. DNA cytosine methylation: Structural and thermodynamic characterization of the epigenetic marking mechanism

    PubMed Central

    Yang, Jin; Lior-Hoffmann, Lee; Wang, Shenglong; Zhang, Yingkai; Broyde, Suse

    2013-01-01

    DNA cytosine methyltransferases regulate the expression of the genome through the precise epigenetic marking of certain cytosines with a methyl group, and aberrant methylation is a hallmark of human diseases including cancer. Targeting these enzymes for drug design is currently a high priority. We have utilized ab initio quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations to extensively investigate the reaction mechanism of the representative DNA methyltransferase HhaI (M.HhaI) from prokaryotes, whose overall mechanism is shared with the mammalian enzymes. We obtain for the first time full free energy profiles for the complete reaction, together with reaction dynamics in atomistic detail. Our results show an energetically preferred mechanism in which nucleophilic attack of cytosine C5 on the S-adenosyl-L-methionine (AdoMet) methyl group is concerted with formation of the Michael adduct between a conserved Cys in the active site with cytosine C6. Spontaneous and reversible proton transfer between a conserved Glu in the active site and cytosine N3 at the transition state was observed in our simulations, revealing the chemical participation of this Glu residue in the catalytic mechanism. Subsequently, the β-elimination of the C5 proton utilizes as base an OH− derived from a conserved crystal water that is part of a proton wire water channel, and this syn β-elimination reaction is the rate-limiting step. Design of novel cytosine methylation inhibitors would be advanced by our structural and thermodynamic characterization of the reaction mechanism. PMID:23528166

  4. 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.

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

    PubMed Central

    Alonso, Conchita; Prez, 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

  6. 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

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

    PubMed

    Nowarski, Roni; Kotler, Moshe

    2013-06-15

    High frequency of cytidine to thymidine conversions was identified in the genome of several types of cancer cells. In breast cancer cells, these mutations are clustered in long DNA regions associated with single-strand DNA (ssDNA), double-strand DNA breaks (DSB), and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs, and clustered mutations. Cancer Res; 73(12); 3494-8. ©2013 AACR. PMID:23598277

  8. Gas Phase Structure of Metal Mediated (Cytosine)2Ag(+) Mimics the Hemiprotonated (Cytosine)2H(+) Dimer in i-Motif Folding.

    PubMed

    Berdakin, Matias; Steinmetz, Vincent; Maitre, Philippe; Pino, Gustavo A

    2014-05-16

    The study of metal ion-DNA interaction aiming to understand the stabilization of artificial base pairing and a number of noncanonical motifs is of current interest, due to their potential exploitation in developing new technological devices and expanding the genetic code. A successful strategy has been the synthesis of metal-mediated base pairs, in which a coordinative bond to a central metal cation replaces a H-bond in a natural pair. In this work, we characterized, for the first time, the gas phase structure of the cytosineAg(+)cytosine (C-Ag(+)-C) complex by means of InfraRed-MultiPhoton-Dissociation (IR-MPD) spectroscopy and theoretical calculation. The IR-spectrum was confidently assigned to one structure with the Ag(+) acting as a bridge between the heteronitrogen atoms in each cytosine (both in the keto-amino form). This structure is biologically relevant since it mimics the structure of the hemiprotonated C-H(+)-C dimer responsible for the stabilization of the i-motif structure in DNA, with the replacement of the NHN bond by a stronger NAg(+)N bond. Moreover, since the structure of the C-Ag(+)-C complex is planar, it allows an optimum intercalation between pairs of the two antiparallel strand duplex in the DNA i-motif structure. PMID:24807048

  9. Investigation into the nature of substrate binding to the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase

    SciTech Connect

    Warren, M.J.; Jordan, P.M.

    1988-12-13

    The formation of the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was shown to depend on the presence of 5-aminolevulinic acid. A hemA/sup -/ mutant formed inactive deaminase when grown in the absence of 5-aminolevulinic acid since this strain was unable to biosynthesize the dipyrromethane cofactor. The mutant formed normal levels of deaminase, however, when grown in the presence of 5-aminolevulinic acid. Porphobilinogen, the substrate, interacts with the free ..cap alpha..-position of the dipyrromethane cofactor to give stable enzyme-intermediate complexes. Experiments with regiospecifically labeled intermediate complexes have shown that, in the absence of further substrate molecules, the complexes are interconvertible by the exchange of the terminal pyrrole ring of each complex. The formation of enzyme-intermediate complexes is accompanied by the exposure of a cysteine residue, suggesting that substantial conformational changes occur on binding substrate. Specific labeling of the dipyrromethane cofactor by growth of the E. coli in the presence of 5-amino(5-/sup 14/C)levulinic acid has confirmed that the cofactor is not subject to catalytic turnover. Experiments with the ..cap alpha..-substituted substrate analogue ..cap alpha..-bromoporphobilinogen have provided further evidence that the cofactor is responsible for the covalent binding of the substrate at the catalytic site. On the basis of these cummulative findings, it has been possible to construct a mechanistic scheme for the deaminase reaction involving a single catalytic site which is able to catalyze the addition or removal of either NH/sub 3/ or H/sub 2/O. The role of the cofactor both as a primer and as a means for regulating the number of substrates bound in each catalytic cycle is discussed.

  10. Adenosine Deaminases Acting on RNA (ADARs) are both Antiviral and Proviral Dependent upon the Virus

    PubMed Central

    Samuel, Charles E.

    2010-01-01

    A-to-I RNA editing, the deamination of adenosine (A) to inosine (I) that occurs in regions of RNA with double-stranded character, is catalyzed by a family of Adenosine Deaminases Acting on RNA (ADARs). In mammals there are three ADAR genes. Two encode proteins that possess demonstrated deaminase activity: ADAR1, which is interferon-inducible, and ADAR2 which is constitutively expressed. ADAR3, by contrast, has not yet been shown to bean active enzyme. The specificity of the ADAR1 and ADAR2 deaminases ranges from highly site-selective to non-selective, dependent on the duplex structure of the substrate RNA. A-to-I editing is a form of nucleotide substitution editing, because I is decoded as guanosine (G) instead of A by ribosomes during translation and by polymerases during RNA-dependent RNA replication. Additionally, A-to-I editing can alter RNA structure stability as I:U mismatches are less stable than A:U base pairs. Both viral and cellular RNAs are edited by ADARs. A-to-I editing is of broad physiologic significance. Among the outcomes of A-to-I editing are biochemical changes that affect how viruses interact with their hosts, changes that can lead to either enhanced or reduced virus growth and persistence dependent upon the specific virus. PMID:21211811

  11. Various effects of fluorescent bacteria of the genus Pseudomonas containing ACC deaminase on wheat seedling growth.

    PubMed

    Magnucka, Elżbieta G; Pietr, Stanisław J

    2015-12-01

    The study evaluates the effect of rhizobacteria having 1-aminocyclopropane-1-carboxylate deaminase (ACCd) on the development of wheat seedlings. This enzyme has been proposed to play a key role in microbe-plant association. Three fluorescent pseudomonads containing this deaminase were selected from 70 strains of pseudomonads isolated from rhizosphere of wheat (Triticum aestivum L.) and rape (Brassica napus L.). These bacteria, varied significantly in the ability to both biosynthesize auxins and hydrolyze ACC. Among them, Pseudomonas brassicacearum subsp. brassicacearum strain RZ310 presented the highest activities of ACC deaminase during 96h of growth in liquid Dworkin and Foster (DF) salt medium. Additionally, this rape rhizosphere strain did not produce indoles. Two other isolates, Pseudomonas sp. PO283 and Pseudomonas sp. PO366, secreted auxins only in the presence of their precursor. Phylogenetic analysis of the 16S rRNA gene and four other protein-encoding genes indicated that these wheat rhizosphere isolates belonged to the fluorescent Pseudomonas group. Moreover, the effects of these strains on wheat seedling growth under in vitro conditions were markedly dependent on both their cell suspensions used to grain inoculation and nutrient conditions. Strains tested had beneficial influence on wheat seedlings mainly at low cell densities. In addition, access to nutrients markedly changed bacteria action on cereal growth. Their presence generally favored the positive effects of pseudomonads on length and the estimated biomasses of wheat coleoptiles. Despite these general rules, impacts of each isolate on the growth parameters of cereal seedlings were unique. PMID:25983132

  12. Cytosine methylation of an ancient satellite family in the wild beet Beta procumbens.

    PubMed

    Schmidt, Martin; Hense, Sarah; Minoche, André E; Dohm, Juliane C; Himmelbauer, Heinz; Schmidt, Thomas; Zakrzewski, Falk

    2014-01-01

    DNA methylation is an essential epigenetic feature for the regulation and maintenance of heterochromatin. Satellite DNA is a repetitive sequence component that often occurs in large arrays in heterochromatin of subtelomeric, intercalary and centromeric regions. Knowledge about the methylation status of satellite DNA is important for understanding the role of repetitive DNA in heterochromatization. In this study, we investigated the cytosine methylation of the ancient satellite family pEV in the wild beet Beta procumbens. The pEV satellite is widespread in species-specific pEV subfamilies in the genus Beta and most likely originated before the radiation of the Betoideae and Chenopodioideae. In B. procumbens, the pEV subfamily occurs abundantly and spans intercalary and centromeric regions. To uncover its cytosine methylation, we performed chromosome-wide immunostaining and bisulfite sequencing of pEV satellite repeats. We found that CG and CHG sites are highly methylated while CHH sites show only low levels of methylation. As a consequence of the low frequency of CG and CHG sites and the preferential occurrence of most cytosines in the CHH motif in pEV monomers, this satellite family displays only low levels of total cytosine methylation. PMID:24994030

  13. Cytosine methylation enhances mitoxantrone-DNA adduct formation at CpG dinucleotides.

    PubMed

    Parker, B S; Cutts, S M; Phillips, D R

    2001-05-11

    Recently, we have shown that mitoxantrone can be activated by formaldehyde in vitro to form DNA adducts that are specific for CpG and CpA sites in DNA. The CpG specificity of adduct formation prompted investigations into the effect of cytosine methylation (CpG) on adduct formation, since the majority of CpG dinucleotides in the mammalian genome are methylated and hypermethylation in subsets of genes is associated with various neoplasms. Upon methylation of a 512-base pair DNA fragment (containing the lac UV5 promoter) using HpaII methylase, three CCGG sites downstream of the promoter were methylated at C5 of the internal cytosine residue. In vitro transcription studies of mitoxantrone-reacted DNA revealed a 3-fold enhancement in transcriptional blockage (and hence adduct formation) exclusively at these methylated sites. In vitro cross-linking assays also revealed that methylation enhanced mitoxantrone adduct formation by 2-3-fold, and methylation of cytosine at a single potential drug binding site on a duplex oligonucleotide also enhanced adduct levels by 3-fold. Collectively, these results indicate preferential adduct formation at methylated CpG sites. However, adducts at these methylated sites exhibited the same stability as nonmethylated sites, suggesting that cytosine methylation increases drug accessibility to DNA rather than being involved in kinetic stabilization of the adduct. PMID:11278477

  14. Five isomers of monomeric cytosine and their interconversions induced by tunable UV laser light.

    PubMed

    Lapinski, Leszek; Reva, Igor; Nowak, Maciej J; Fausto, Rui

    2011-05-28

    Photoisomerization processes involving five isomers of cytosine were induced by narrowband tunable UV irradiation of matrix-isolated monomers of the compound. Irradiation of an argon matrix containing cytosine monomers with UV ? = 313 nm laser light resulted in syn?anti photoisomerizations between the two imino-oxo forms, whereas the substantially more populated amino-hydroxy and amino-oxo forms stayed intact. Subsequent irradiation with shorter-wavelength UV ? = 311 nm laser light led to two concomitant phototautomeric processes consuming the amino-oxo isomer: (i) an oxo ? hydroxy hydrogen-atom transfer photoprocess converting the amino-oxo form into the amino-hydroxy tautomer; (ii) amino ? imino hydrogen-atom transfer converting the amino-oxo form into the imino-oxo isomers. The UV-induced phototransformations, together with mutual conversions of the two amino-hydroxy conformers induced by irradiation with narrowband NIR light, allowed positive detection and identification of the five isomeric forms of monomeric cytosine. This is the first experimental observation of all five low-energy isomers of cytosine. PMID:21499603

  15. De novo cytosine methylation in the differentiating macronucleus of the stichotrichous ciliate Stylonychia lemnae.

    PubMed

    Juranek, Stefan; Wieden, Hans-Joachim; Lipps, Hans J

    2003-03-01

    Dramatic DNA reorganization and elimination processes occur during macronuclear differentiation in ciliates. In this study we analyzed whether cytosine methylation of specific sequences plays a functional role during DNA rearrangement. Three classes of sequences, macronuclear-destined sequences (MDSs, pCE7), members from a large family of transposon-like elements and micronuclear-specific sequences (pLJ01), differing in their structure and future destiny during nuclear differentiation, were studied in the micronucleus, the developing macronucleus and, when present, in the mature macronucleus. While the MDSs become processed to a 1.1 and 1.3 kb gene-sized macronuclear DNA molecule, the family of transposon-like elements represented by MaA81 becomes removed late in the course of polytene chromosome formation. The micronuclear-specific sequence pLJ01 is eliminated together with bulk micronuclear DNA during degradation of polytene chromosomes. No methylated cytosine could be detected in the vegetative macronucleus and no difference in methylation pattern was observed either between micronucleus and developing macronucleus in MDSs or in a micronuclear-specific sequence. However, a significant percentage of the cytosines contained in the transposon-like element becomes methylated de novo in the course of macronuclear differentiation. This is the first demonstration that cytosine methylation in specific sequences occurs during macronuclear differentiation and may provide a first step towards understanding epigenetic factors involved in DNA processing. PMID:12595545

  16. Precise estimates of mutation rate and spectrum in yeast

    PubMed Central

    Zhu, Yuan O.; Siegal, Mark L.; Hall, David W.; Petrov, Dmitri A.

    2014-01-01

    Mutation is the ultimate source of genetic variation. The most direct and unbiased method of studying spontaneous mutations is via mutation accumulation (MA) lines. Until recently, MA experiments were limited by the cost of sequencing and thus provided us with small numbers of mutational events and therefore imprecise estimates of rates and patterns of mutation. We used whole-genome sequencing to identify nearly 1,000 spontaneous mutation events accumulated over ?311,000 generations in 145 diploid MA lines of the budding yeast Saccharomyces cerevisiae. MA experiments are usually assumed to have negligible levels of selection, but even mild selection will remove strongly deleterious events. We take advantage of such patterns of selection and show that mutation classes such as indels and aneuploidies (especially monosomies) are proportionately much more likely to contribute mutations of large effect. We also provide conservative estimates of indel, aneuploidy, environment-dependent dominant lethal, and recessive lethal mutation rates. To our knowledge, for the first time in yeast MA data, we identified a sufficiently large number of single-nucleotide mutations to measure context-dependent mutation rates and were able to (i) confirm strong AT bias of mutation in yeast driven by high rate of mutations from C/G to T/A and (ii) detect a higher rate of mutation at C/G nucleotides in two specific contexts consistent with cytosine methylation in S. cerevisiae. PMID:24847077

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

    PubMed

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

    2013-10-01

    Yeast 25S rRNA was reported to contain a single cytosine methylation (m(5)C). 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 m(5)C 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 m(5)C 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 m(5)C residues. The loss of m(5)C2278 results in anisomycin hypersensitivity, whereas the loss of m(5)C2870 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

  18. Single-Cell Quantification of Cytosine Modifications by Hyperspectral Dark-Field Imaging

    PubMed Central

    Wang, Xiaolei; Cui, Yi; Irudayaraj, Joseph

    2016-01-01

    Epigenetic modifications on DNA, especially on cytosine, play a critical role in regulating gene expression and genome stability. It is known that the levels of different cytosine derivatives are highly dynamic and are regulated by a variety of factors that act on the chromatin. Here we report an optical methodology based on hyperspectral dark-field imaging (HSDFI) using plasmonic nanoprobes to quantify the recently identified cytosine modifications on DNA in single cells. Gold (Au) and silver (Ag) nanoparticles (NPs) functionalized with specific antibodies were used as contrast-generating agents due to their strong Local Surface Plasmon Resonance (LSPR) properties. With this powerful platform we have revealed the spatial distribution and quantity of 5-carboxylcytosine (5caC) at the different stages in cell cycle, and demonstrated that 5caC was a stably inherited epigenetic mark. We have also shown that the regional density of 5caC on a single chromosome can be mapped due to the spectral sensitivity of the nanoprobes in relation to the inter-particle distance. Notably, HSDFI enables an efficient removal of the scattering noises from non-specifically aggregated nanoprobes, to improve accuracy in the quantification of different cytosine modifications in single cells. Further, by separating the LSPR fingerprints of AuNPs and AgNPs, multiplex detection of two cytosine modifications was also performed. Our results demonstrate HSDFI as a versatile platform for spatial and spectroscopic characterization of plasmonic nanoprobe-labeled nuclear targets at the single-cell level for quantitative epigenetic screening. PMID:26505210

  19. Linking short tandem repeat polymorphisms with cytosine modifications in human lymphoblastoid cell lines.

    PubMed

    Zhang, Zhou; Zheng, Yinan; Zhang, Xu; Liu, Cong; Joyce, Brian Thomas; Kibbe, Warren A; Hou, Lifang; Zhang, Wei

    2016-02-01

    Inter-individual variation in cytosine modifications has been linked to complex traits in humans. Cytosine modification variation is partially controlled by single nucleotide polymorphisms (SNPs), known as modified cytosine quantitative trait loci (mQTL). However, little is known about the role of short tandem repeat polymorphisms (STRPs), a class of structural genetic variants, in regulating cytosine modifications. Utilizing the published data on the International HapMap Project lymphoblastoid cell lines (LCLs), we assessed the relationships between 721 STRPs and the modification levels of 283,540 autosomal CpG sites. Our findings suggest that, in contrast to the predominant cis-acting mode for SNP-based mQTL, STRPs are associated with cytosine modification levels in both cis-acting (local) and trans-acting (distant) modes. In local scans within the ±1 Mb windows of target CpGs, 21, 9, and 21 cis-acting STRP-based mQTL were detected in CEU (Caucasian residents from Utah, USA), YRI (Yoruba people from Ibadan, Nigeria), and the combined samples, respectively. In contrast, 139,420, 76,817, and 121,866 trans-acting STRP-based mQTL were identified in CEU, YRI, and the combined samples, respectively. A substantial proportion of CpG sites detected with local STRP-based mQTL were not associated with SNP-based mQTL, suggesting that STRPs represent an independent class of mQTL. Functionally, genetic variants neighboring CpG-associated STRPs are enriched with genome-wide association study (GWAS) loci for a variety of complex traits and diseases, including cancers, based on the National Human Genome Research Institute (NHGRI) GWAS Catalog. Therefore, elucidating these STRP-based mQTL in addition to SNP-based mQTL can provide novel insights into the genetic architectures of complex traits. PMID:26714498

  20. 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

  1. DNA replication induces compositional biases in yeast.

    PubMed

    Marsolier-Kergoat, Marie-Claude; Goldar, Arach

    2012-03-01

    Asymmetries intrinsic to the process of DNA replication are expected to cause differences in the substitution patterns of the leading and the lagging strands and to induce compositional biases. These biases have been detected in the majority of eubacterial genomes but rarely in eukaryotes. Only in the human genome, the activity of a minority of replication origins seems to generate compositional biases. In this work, we provide evidence for replication-associated GC and TA skews in the genomes of two yeast species, Saccharomyces cerevisiae and Kluyveromyces lactis, whereas the data for the Schizosaccharomyces pombe genome are less conclusive. In contrast with the genomes of Homo sapiens and of the majority of eubacteria, the leading strand is enriched in cytosine and adenine in both S. cerevisiae and K. lactis. We observed significant variations across the interorigin intervals of several substitution rates in the S. cerevisiae lineage since its divergence from S. paradoxus. We also found that the S. cerevisiae genome is far from compositional equilibrium and that its present compositional biases are due to substitution rates operating before its divergence from S. paradoxus. Finally, we observed that replication and transcription tend to be cooriented in the S. cerevisiae genome, especially for genes encoding subunits of protein complexes. Taken together, our results suggest that replication-related compositional biases may be a feature of many eukaryotic genomes despite the stochastic nature of the firing of replication origins in these genomes. PMID:21948086

  2. Prions in Yeast

    PubMed Central

    Liebman, Susan W.; Chernoff, Yury O.

    2012-01-01

    The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the protein only model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-? aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in understanding cellular control of prion propagation, prion structure, mechanisms of de novo prion formation, specificity of prion transmission, and the biological roles of prions. This review summarizes what has been learned from yeast prions. PMID:22879407

  3. DNA base (cytosine) modified/capped ultrasmall Gd2S3:Eu3+ gadofluoroprobes for platelet isolation

    NASA Astrophysics Data System (ADS)

    Dutta, Ranu K.; Sharma, Prashant K.; Pandey, Avinash C.

    2010-12-01

    The present letter deals with the synthesis of ultrasmall Gd2S3:Eu3+ nanoparticles and their surface modification with "cytosine," a nucleobase present in DNA/RNA. These nanoparticles show orthorhombic (Pnma) crystal symmetry with excellent magnetic and luminescent characters simultaneously. In contrast to the previous reports, cytosine modified nanoparticles do not show a significant change in their structural and magnetic properties, whereas luminescence is enhanced slightly owing to the surface passivation. The in vitro studies show better accumulation of blood platelets with cytosine modified nanoparticles as compared to unmodified posing them a potential candidate for platelet isolation from the plasma for different applications and studies.

  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. Yin and yang of cytidine deaminase roles in clinical response to azacitidine in the elderly: a pharmacogenetics tale.

    PubMed

    Fanciullino, Raphaelle; Mercier, Cdric; Serdjebi, Cindy; Venton, Geoffroy; Colle, Julien; Fina, Frdric; Ouafik, L'Houcine; Lacarelle, Bruno; Ciccolini, Joseph; Costello, Rgis

    2015-11-01

    Azacitidine is a mainstay for treating hematological disorders. Azacitidine is metabolized by cytidine deaminase, coded by a highly polymorphic gene. Here, we present two elderly patients with opposite clinical outcomes after azacitidine treatment. First, an acute myeloid leukemia patient showed life-threatening toxicities, but outstanding complete remission, after a single round of azacitidine. Further investigations showed that this patient was cytidine deaminase 79A>C (rs2072671) homozygous with a marked deficient phenotype. Next, a chronic myelomonocytic leukemia patient displayed complete lack of response despite several cycles of azacitidine. This patient had a rapid-deaminator phenotype linked to the -31delC deletion (rs3215400). These polymorphisms lead to opposite clinical outcomes in patients with myelodysplastic syndromes treated with azacitidine, thus suggesting that determining cytidine deaminase status could help to forecast clinical outcome. PMID:26556583

  6. AMP deaminase in Dictyostelium discoideum: increase in activity following nutrient deprivation induced by starvation or hadacidin.

    PubMed

    Jahngen, E G; Rossomando, E F

    1986-06-01

    AMP deaminase, the activity that catalyzes the deamination of AMP to form IMP and NH3 has been measured in Dictyostelium discoideum. A new procedure to assay the activity of this enzyme was developed using formycin 5'-monophosphate, a fluorescent analog of AMP as the substrate, and ion-paired reverse phase HPLC to separate the reactants and products. Quantitation of the formycin containing compounds was accomplished at 290 nm. At this wavelength adenosine containing compounds were not detected and activity could be monitored in the presence of its activator ATP. The AMP deaminase activity in vegetative cells was 7.4 nmols/min/mg proteins while the activity in cells measured at 2 and 6 hrs after starvation-induced growth-arrest was 376 nmols/min/mg protein...a 51-fold increase. When vegetative cells were treated with hadacidin, a drug that restricts de novo AMP synthesis and pinocytosis, the activity of the AMP deaminase was 511 nmols/min/mg protein...a 70-fold increase compared to that in untreated vegetative cells. Smaller increases were noted following the inhibition of growth with the drugs cerulenin and vinblastine, as well as after the inhibition of de novo GMP synthesis with the drug mycophenolic acid or the partial inhibition of de novo AMP synthesis with analogs of hadacidin, N-hydroxyglycine and N-formylglycine. In addition, when the activity of two other enzymes involved in purine metabolism, namely adenosine kinase and hypoxanthine-guanine phosphoribosyl transferase, was measured in vegetative cells, and the activity of both compared to that measured in starvation and hadacidin induced growth-arrested cells, showed no significant changes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3014311

  7. Biochemistry and genetics of ACC deaminase: a weapon to “stress ethylene” produced in plants

    PubMed Central

    Singh, Rajnish P.; Shelke, Ganesh M.; Kumar, Anil; Jha, Prabhat N.

    2015-01-01

    1-aminocyclopropane-1-carboxylate deaminase (ACCD), a pyridoxal phosphate-dependent enzyme, is widespread in diverse bacterial and fungal species. Owing to ACCD activity, certain plant associated bacteria help plant to grow under biotic and abiotic stresses by decreasing the level of “stress ethylene” which is inhibitory to plant growth. ACCD breaks down ACC, an immediate precursor of ethylene, to ammonia and α-ketobutyrate, which can be further metabolized by bacteria for their growth. ACC deaminase is an inducible enzyme whose synthesis is induced in the presence of its substrate ACC. This enzyme encoded by gene AcdS is under tight regulation and regulated differentially under different environmental conditions. Regulatory elements of gene AcdS are comprised of the regulatory gene encoding LRP protein and other regulatory elements which are activated differentially under aerobic and anaerobic conditions. The role of some additional regulatory genes such as AcdB or LysR may also be required for expression of AcdS. Phylogenetic analysis of AcdS has revealed that distribution of this gene among different bacteria might have resulted from vertical gene transfer with occasional horizontal gene transfer (HGT). Application of bacterial AcdS gene has been extended by developing transgenic plants with ACCD gene which showed increased tolerance to biotic and abiotic stresses in plants. Moreover, distribution of ACCD gene or its homolog's in a wide range of species belonging to all three domains indicate an alternative role of ACCD in the physiology of an organism. Therefore, this review is an attempt to explore current knowledge of bacterial ACC deaminase mediated physiological effects in plants, mode of enzyme action, genetics, distribution among different species, ecological role of ACCD and, future research avenues to develop transgenic plants expressing foreign AcdS gene to cope with biotic and abiotic stressors. Systemic identification of regulatory circuits would be highly valuable to express the gene under diverse environmental conditions. PMID:26441873

  8. 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

  9. Significance of the d-Serine-Deaminase and d-Serine Metabolism of Staphylococcus saprophyticus for Virulence

    PubMed Central

    Sakinc, Trkan; Kline, Kimberly; Nielsen, Hailyn V.; Hultgren, Scott; Gatermann, Sren G.

    2013-01-01

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

    PubMed

    Korte-Berwanger, Miriam; Sakinc, Trkan; Kline, Kimberly; Nielsen, Hailyn V; Hultgren, Scott; Gatermann, Sren 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

  11. Flexible double-headed cytosine-linked 2'-deoxycytidine nucleotides. Synthesis, polymerase incorporation to DNA and interaction with DNA methyltransferases.

    PubMed

    Kielkowski, Pavel; Cahová, Hana; Pohl, Radek; Hocek, Michal

    2016-03-15

    New types of double-headed 2'-deoxycytidine 5'-O-triphosphates (dC(XC)TPs) bearing another cytosine or 5-fluorocytosine linked through a flexible propargyl, homopropargyl or pent-1-ynyl linker to position 5 were prepared by the aqueous Sonogashira cross-coupling reactions of 5-iodo-dCTP with the corresponding (fluoro)cytosine-alkynes. The modified dC(XC)TPs were good substrates for DNA polymerases and were used for enzymatic synthesis of cytosine-functionalized DNA by primer extension or PCR. The cytosine- or fluorocytosine-linked DNA probes did not significantly inhibit DNA methyltransferases and did not cross-link to these proteins. PMID:26899597

  12. 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.

  13. Cytotoxic activity of 2',2'-difluorodeoxycytidine, 5-aza-2'-deoxycytidine and cytosine arabinoside in cells transduced with deoxycytidine kinase gene.

    PubMed

    Beausjour, Christian M; Gagnon, Jacynthe; Primeau, Mlanie; Momparler, Richard L

    2002-05-24

    Deoxycytidine nucleoside analogs must be first phosphorylated to become active anticancer drugs. The rate-limiting enzyme in this pathway is deoxycytidine kinase (dCK). Cells deficient in this enzyme are resistant to these analogs. To evaluate the potential of dCK to be used as suicide gene for deoxycytidine nucleoside analogs, we transduced both human A-549 lung carcinoma and murine NIH3T3 fibroblast cell lines with this gene. The dCK-transduced cells showed an increase in cytotoxicity to the analogs, cytosine arabinoside (ARA-C), and 5-aza-2'-deoxycytidine (5-AZA-CdR). Unexpectedly, the related analog, 2',2'-difluorodeoxycytidine (dFdC), was less cytotoxic to the dCK-transduced cells than the wild-type cells. For the A-549-dCK cells, the phosphorylation of dFdC by dCK was much greater than control cells. In accord with the elevated enzyme activity, we observed a 6-fold increased dFdC incorporation into DNA and a more pronounced inhibition of DNA synthesis in the A-549-dCK cells. In an attempt to clarify the mechanism of dFdC, we investigated its action on A549 and 3T3 cells transduced with both cytidine deaminase (CD) and dCK. We reported previously that overexpression of CD confers drug resistance to deoxycytidine analogs. In this study, when the CD-transduced cells were also transduced with dCK they became relatively more sensitive to dFdC. In addition, we observed that dFdU, the deaminated form of dFdC, was cytotoxic to the A-549-dCK cells, but not the wild-type cells. Our working hypothesis to explain these results is that the mitochondrial thymidine kinase (TK2), an enzyme reported to phosphorylate dFdC, acts as an important modulator of dFdC-induced cell toxicity. These findings may further clarify the action of dFdC and the mechanism by which it induces cell death. PMID:12054682

  14. Disruption of the adenosine deaminase gene causes hepatocellular impairment and perinatal lethality in mice.

    PubMed Central

    Wakamiya, M; Blackburn, M R; Jurecic, R; McArthur, M J; Geske, R S; Cartwright, J; Mitani, K; Vaishnav, S; Belmont, J W; Kellems, R E

    1995-01-01

    We have generated mice with a null mutation at the Ada locus, which encodes the purine catabolic enzyme adenosine deaminase (ADA, EC 3.5.4.4). ADA-deficient fetuses exhibited hepatocellular impairment and died perinatally. Their lymphoid tissues were not largely affected. Accumulation of ADA substrates was detectable in ADA-deficient conceptuses as early as 12.5 days postcoitum, dramatically increasing during late in utero development, and is the likely cause of liver damage and fetal death. The results presented here demonstrate that ADA is important for the homeostatic maintenance of purines in mice. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7731963

  15. Use of adenosine deaminase as a diagnostic tool for tuberculous pleurisy.

    PubMed Central

    Burgess, L. J.; Maritz, F. J.; Le Roux, I.; Taljaard, J. J.

    1995-01-01

    BACKGROUND--A statistical audit of adenosine deaminase (ADA) in pleural effusions was undertaken. METHODS--ADA analysis, cytological and microbiological examinations, and differential cell counts were performed on 462 pleural fluid samples. RESULTS--ADA activity in tuberculous effusions was higher than in any other diagnostic group. At a level of 50 U/l the sensitivity and specificity for the identification of tuberculosis was 90% and 89%, respectively. CONCLUSIONS--ADA activity remains a useful test in the evaluation of pleural effusions. PMID:7638812

  16. 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

  17. 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.

  18. Sympodiomycopsis: a new yeast-like anamorph genus with basidiomycetous nature from orchid nectar.

    PubMed

    Sugiyama, J; Tokuoka, K; Suh, S O; Hirata, A; Komagata, K

    1991-02-01

    A description is provided for a new anamorph genus Sympodiomycopsis (Hyphomycetes), which is neither ballistosporogenous nor sterigmatosporogenous. The genus is typified by S. paphiopedili sp. nov. and accommodates one species which was isolated from nectar of Paphiopedilum primurinum in Japan. Phenotypically, the genus shows some similarity to Sympodiomyces because of the presence of a yeast morph with sympodial conidiogenous cell proliferation, but it is distinguished from that genus morphologically by a yeast morph with the enteroblastic-annellidic conidiogenesis and the conspicuous development of a hyphal morph with holoblastic-sympodial conidiogenous cells, and chemotaxonomically by the ubiquinone system Q-10 and 10% difference in the guanine plus cytosine (G + C) content of the nuclear DNA. Phylogenetically, the type of cell wall and septal pore ultrastructure, and the primary biochemical and chemotaxonomic characters of S. paphiopedili indicate a basidiomycetous affinity. PMID:1854191

  19. Cytosine-specific chemical probing of DNA using bromide and monoperoxysulfate.

    PubMed

    Ross, S A; Burrows, C J

    1996-12-15

    Bromination of cytosine and formation of a piperidine-labile site are observed when two simple salts, KBr and KHSO5, are allowed to react with single-stranded oligodeoxynucleotides. Selectivity for C compared with T, G or A is typically a factor of 4 or more; selectivity for Cs in a single-stranded region such as a C-bulge is nearly a factor of 10 compared with duplex Cs. Low reactivity and little base selectivity are observed using duplex DNA, although increased concentrations of reagents lead to complete degradation of the DNA. The results suggest that these conditions for in situ generation of Br2 constitute a useful tool for examination of the exposure of a non-duplex cytosine base in folded DNA structures. PMID:9016685

  20. Genome-wide, high-resolution DNA methylation profiling using bisulfite-mediated cytosine conversion

    PubMed Central

    Reinders, Jon; Delucinge Vivier, Cline; Theiler, Grgory; Chollet, Didier; Descombes, Patrick; Paszkowski, Jerzy

    2008-01-01

    Methylation of cytosines (mC) is essential for epigenetic gene regulation in plants and mammals. Aberrant mC patterns are associated with heritable developmental abnormalities in plants and with cancer in mammals. We have developed a genome-wide DNA methylation profiling technology employing a novel amplification step for DNA subjected to bisulfite-mediated cytosine conversion. The methylation patterns detected are not only consistent with previous results obtained with mC immunoprecipitation (mCIP) techniques, but also demonstrated improved resolution and sensitivity. The technology, named BiMP (for Bisulfite Methylation Profiling), is more cost-effective than mCIP and requires as little as 100 ng of Arabidopsis DNA. PMID:18218979

  1. 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. ); Clowes, M.M.; Osborne, W.R.A.; Clowes, A.W. )

    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.

  2. 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.

  3. Gas-Phase Cytosine and Cytosine-N1-Derivatives Have 0.1-1 ns Lifetimes Near the S1 State Minimum.

    PubMed

    Blaser, Susan; Trachsel, Maria A; Lobsiger, Simon; Wiedmer, Timo; Frey, Hans-Martin; Leutwyler, Samuel

    2016-03-01

    Ultraviolet radiative damage to DNA is inefficient because of the ultrafast S1 ? S0 internal conversion of its nucleobases. Using picosecond pump-ionization delay measurements, we find that the S1((1)??*) state vibrationless lifetime of gas-phase keto-amino cytosine (Cyt) is ? = 730 ps or ?700 times longer than that measured by femtosecond pump-probe ionization at higher vibrational excess energy, Eexc. N1-Alkylation increases the S1 lifetime up to ? = 1030 ps for N1-ethyl-Cyt but decreases it to 100 ps for N1-isopropyl-Cyt. Increasing the vibrational energy to Eexc = 300-550 cm(-1) decreases the lifetimes to 20-30 ps. The nonradiative dynamics of S1 cytosine is not solely a property of the amino-pyrimidinone chromophore but is strongly influenced by the N1-substituent. Correlated excited-state calculations predict that the gap between the S2((1)nO?*) and S1((1)??*) states decreases along the series of N1-derivatives, thereby influencing the S1 state lifetime. PMID:26863095

  4. The role of cytosine methylation on charge transport through a DNA strand.

    PubMed

    Qi, Jianqing; Govind, Niranjan; Anantram, M P

    2015-09-01

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Bttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit. PMID:26342369

  5. The role of cytosine methylation on charge transport through a DNA strand

    NASA Astrophysics Data System (ADS)

    Qi, Jianqing; Govind, Niranjan; Anantram, M. P.

    2015-09-01

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Bttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.

  6. Molecular energetics of cytosine revisited: a joint computational and experimental study.

    PubMed

    Gomes, Jos R B; Ribeiro da Silva, Maria D M C; Freitas, Vera L S; Ribeiro da Silva, Manuel A V

    2007-08-01

    A static bomb calorimeter has been used to measure the standard molar energy of combustion, in oxygen, at T = 298.15 K, of a commercial sample of cytosine. From this energy, the standard (p degrees = 0.1 MPa) molar enthalpy of formation in the crystalline state was derived as -(221.9 +/- 1.7) kJ.mol(-1). This value confirms one experimental value already published in the literature but differs from another literature value by 13.5 kJ.mol(-1). Using the present standard molar enthalpy of formation in the condensed phase and the enthalpy of sublimation due to Burkinshaw and Mortimer [J. Chem. Soc., Dalton Trans. 1984, 75], (155.0 +/- 3.0) kJ.mol(-1), results in a value for the gas-phase standard molar enthalpy of formation for cytosine of -66.9 kJ.mol(-1). A similar value, -65.1 kJ.mol(-1), has been estimated after G3MP2B3 calculations combined with the reaction of atomization on three different tautomers of cytosine. In agreement with experimental evidence, the hydroxy-amino tautomer is the most stable form of cytosine in the gas phase. The enthalpies of formation of the other two tautomers were also estimated as -60.7 kJ.mol(-1) and -57.2 kJ.mol(-1) for the oxo-amino and oxo-imino tautomers, respectively. The same composite approach was also used to compute other thermochemical data, which is difficult to be measured experimentally, such as C-H, N-H, and O-H bond dissociation enthalpies, gas-phase acidities, and ionization enthalpies. PMID:17616179

  7. The Role of Cytosine Methylation on Charge Transport through a DNA Strand

    SciTech Connect

    Qi, Jianqing; Govind, Niranjan; Anantram, M. P.

    2015-09-04

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modifi-cation remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Buttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. Specifically, we compare the results generated with the widely used B3LYP exchange-correlation (XC) functional and CAM-B3LYP based tuned range-separated hybrid density functional. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that with both functionals, the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and interstrand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital (HOMO) level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with both functionals. We also study the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit. Our results suggest that the effect of the two different functionals is to alter the on-site energies of the DNA bases at the HOMO level, while the transport properties don't depend much on the two functionals.

  8. Apoptosis in yeast.

    PubMed

    Madeo, Frank; Herker, Eva; Wissing, Silke; Jungwirth, Helmut; Eisenberg, Tobias; Frhlich, Kai-Uwe

    2004-12-01

    Apoptosis is a highly regulated cellular suicide program crucial for metazoan development. However, dysfunction of apoptosis also leads to several diseases. Yeast undergoes apoptosis after application of acetic acid, sugar- or salt-stress, plant antifungal peptides, or hydrogen peroxide. Oxygen radicals seem to be key elements of apoptotic execution, conserved during evolution. Furthermore, several yeast orthologues of central metazoan apoptotic regulators have been identified, such as a caspase and a caspase-regulating serine protease. In addition, physiological occurrence of cell death has been detected during aging and mating in yeast. The finding of apoptosis in yeast, other fungi and parasites is not only of great medical relevance but will also help to understand some of the still unknown molecular mechanisms at the core of apoptotic execution. PMID:15556039

  9. Anaerobic yeast killer systems.

    PubMed

    Polonelli, L; Menozzi, M G; Campani, L; Gerloni, M; Conti, S; Morace, G; Chezzi, C

    1992-05-01

    The influence of anaerobic conditions on the expression of the killer phenomenon of several yeast isolates belonging to recognized killer systems coded by different genetic determinants (Pichia spp., Kluyveromyces lactis, Saccharomyces cerevisiae) was studied. Anaerobiosis influenced the activity of killer toxins from some individual isolates of the genera Pichia and Saccharomyces on sensitive strains of P. anomala, K. lactis and Candida albicans. However, no influence was detectable on a S. cerevisiae sensitive isolate. Thus, anaerobic conditions seem to interfere more with the metabolic process of sensitive strains than with toxin production by killer yeasts. The selection of a panel of killer yeasts, able to display their activity against reference sensitive yeast isolates under anaerobic conditions in a medium that favored the growth of anaerobes, allowed the use of the killer system to type Bacteroides fragilis isolates for epidemiological purposes. PMID:1397211

  10. Yeast infections (image)

    MedlinePLUS

    Yeast infections may follow a course of antibiotics that were prescribed for another purpose. The antibiotics change the normal "balance" between organisms in the vagina by suppressing the growth of protective bacteria that normally have an antifungal effect.

  11. Pexophagy in yeasts.

    PubMed

    Oku, Masahide; Sakai, Yasuyoshi

    2016-05-01

    Pexophagy, selective degradation of peroxisomes via autophagy, is the main system for reducing organelle abundance. Elucidation of the molecular machinery of pexophagy has been pioneered in studies of the budding yeast Saccharomyces cerevisiae and the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha. Recent analyses using these yeasts have elucidated the molecular machineries of pexophagy, especially in terms of the interactions and modifications of the so-called adaptor proteins required for guiding autophagic membrane biogenesis on the organelle surface. Based on the recent findings, functional relevance of pexophagy and another autophagic pathway, mitophagy (selective autophagy of mitochondria), is discussed. We also discuss the physiological importance of pexophagy in these yeast systems. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann. PMID:26409485

  12. Yeast Infection (Candidiasis)

    MedlinePLUS

    ... pain behind the breastbone. Self-Care Guidelines Most yeast infections can be prevented by keeping body-fold areas clean and dry. Diabetics should keep their blood sugar under good control. Treat skin infection with a ...

  13. Protonation/deprotonation processes of primary products in x-irradiated cytosine derivatives: EPR and ENDOR studies at 10 K

    SciTech Connect

    Hole, E.O.; Sagstuen, E.; Nelson, W.H.; Close, D.M.

    1995-12-31

    Recently, evidence has accumulated that cytosine is the initial electron trap in irradiated DNA. It has previously been observed that the protonation/deprotonation mechanisms and sites of the pristine purine/pyrimidine electron gain and loss centers depend upon the matrix. Local variations as e.g. water of crystallization, hydrogen bond pattern, initial protonation state of the cytosine bases, and associated counter ions may be important in the selection of reaction pathways following the initial ionization event. In response to this increased interest in the primary radical production in irradiated cytosine, five different crystalline cytosine derivatives have been investigated at 10 K following X-irradiation exposure to doses between 0.8 and 30 kGy. The techniques used are K-band EPR, ENDOR, and ENDOR-induced EPR (FSE) spectroscopy. The systems studied are: cytosine monohydrate (Cm); cytosine-HCI (C:HCl); cytidine (CR); deoxycytidine:HCI (CdR:HCl); and 2{prime}-deoxycytidine 5{prime}-monophosphate (5{prime}dCMP).

  14. Effects of decreased cytosine content on rho interaction with the rho-dependent terminator trp t' in Escherichia coli.

    PubMed

    Zalatan, F; Platt, T

    1992-09-25

    We have introduced multiple cytosine-to-uracil mutations in the rho-dependent transcription terminator trp t' and have characterized a subset of the resulting mutant derivatives in vitro for termination efficiency, affinity for rho, and stimulation of rho-ATPase activity. No specific cytosine residue appears to be required for termination, and at least 13 of the 28 cytosine residues in the 104 nucleotide trp t' region can be mutated with little effect on termination efficiency. One derivative with 11 mutations is significantly less efficient than other derivatives with a similar number of mutations, implying that the pattern of alterations, as well as the number, can affect termination efficiency. Derivatives with 20 and 28 cytosines mutated are non-functional in termination, consistent with the idea that cytosines are required for rho-dependent termination. Our results are inconsistent, however, with the hypothesis that the termination efficiency is directly related to the cytosine/guanine ratio in the nascent transcript. The results support the idea that neither RNA binding affinity nor ATPase activation per se are accurate predictors of rho-dependent termination efficiency. PMID:1388163

  15. VUV photoionization of gas phase adenine and cytosine: A comparison between oven and aerosol vaporization

    NASA Astrophysics Data System (ADS)

    Touboul, D.; Gaie-Levrel, F.; Garcia, G. A.; Nahon, L.; Poisson, L.; Schwell, M.; Hochlaf, M.

    2013-03-01

    We studied the single photon ionization of gas phase adenine and cytosine by means of vacuum ultraviolet synchrotron radiation coupled to a velocity map imaging electron/ion coincidence spectrometer. Both in-vacuum temperature-controlled oven and aerosol thermodesorption were successfully applied to promote the intact neutral biological species into the gas phase. The photoion yields are consistent with previous measurements. In addition, we deduced the threshold photoelectron spectra and the slow photoelectron spectra for both species, where the close to zero kinetic energy photoelectrons and the corresponding photoions are measured in coincidence. The photoionization close and above the ionization energies are found to occur mainly via direct processes. Both vaporization techniques lead to similar electronic spectra for the two molecules, which consist of broadbands due to the complex electronic structure of the cationic species and to the possible contribution of several neutral tautomers for cytosine prior to ionization. Accurate ionization energies are measured for adenine and cytosine at, respectively, 8.267 ± 0.005 eV and 8.66 ± 0.01 eV, and we deduce precise thermochemical data for the adenine radical cation. Finally, we performed an evaluation and a comparison of the two vaporization techniques addressing the following criteria: measurement precision, thermal fragmentation, sensitivity, and sample consumption. The aerosol thermodesorption technique appears as a promising alternative to vaporize large thermolabile biological compounds, where extended thermal decomposition or low sensitivity could be encountered when using a simple oven vaporization technique.

  16. Spontaneous tunneling and near-infrared-induced interconversion between the amino-hydroxy conformers of cytosine.

    PubMed

    Reva, Igor; Nowak, Maciej J; Lapinski, Leszek; Fausto, Rui

    2012-02-14

    Spontaneous and near-infrared/infrared (NIR/IR)-induced interconversions between two amino-hydroxy conformers of monomeric cytosine have been investigated for the compound isolated in a low-temperature argon matrix. Combined use of a laser source (which provides narrowband NIR radiation) and a broadband NIR/IR source of excitation light allowed a detailed investigation of mutual conversions of the two conformers in question. The experiments carried out within the current work demonstrated that upon broadband NIR/IR irradiation (with the IR source of FTIR spectrometer) the population ratio of the two amino-hydroxy conformers changes towards a ratio corresponding to a photostationary state. Evolution of the conformer population ratio towards the photostationary ratio occurred independent of the initial ratio of conformers, which could be prepared by a population shift (in favor of one of the forms) induced by narrowband NIR excitation. Moreover, spontaneous tunneling conversion of the higher-energy conformer into a lower-energy form was observed for cytosine isolated in a low-temperature argon matrix kept in the dark. This process is slow and occurs on a time scale of days. The tunneling process, studied for matrix-isolated cytosine, clearly follows a dispersive type of kinetics rather than the classical monoexponential kinetics. PMID:22360199

  17. Spontaneous tunneling and near-infrared-induced interconversion between the amino-hydroxy conformers of cytosine

    SciTech Connect

    Reva, Igor; Fausto, Rui; Nowak, Maciej J.; Lapinski, Leszek

    2012-02-14

    Spontaneous and near-infrared/infrared (NIR/IR)-induced interconversions between two amino-hydroxy conformers of monomeric cytosine have been investigated for the compound isolated in a low-temperature argon matrix. Combined use of a laser source (which provides narrowband NIR radiation) and a broadband NIR/IR source of excitation light allowed a detailed investigation of mutual conversions of the two conformers in question. The experiments carried out within the current work demonstrated that upon broadband NIR/IR irradiation (with the IR source of FTIR spectrometer) the population ratio of the two amino-hydroxy conformers changes towards a ratio corresponding to a photostationary state. Evolution of the conformer population ratio towards the photostationary ratio occurred independent of the initial ratio of conformers, which could be prepared by a population shift (in favor of one of the forms) induced by narrowband NIR excitation. Moreover, spontaneous tunneling conversion of the higher-energy conformer into a lower-energy form was observed for cytosine isolated in a low-temperature argon matrix kept in the dark. This process is slow and occurs on a time scale of days. The tunneling process, studied for matrix-isolated cytosine, clearly follows a dispersive type of kinetics rather than the classical monoexponential kinetics.

  18. A real-time assay for CpG-specific cytosine-C5 methyltransferase activity.

    PubMed

    Wood, Robert J; McKelvie, Jennifer C; Maynard-Smith, Michael D; Roach, Peter L

    2010-05-01

    A real-time assay for CpG-specific cytosine-C5 methyltransferase activity has been developed. The assay applies a break light oligonucleotide in which the methylation of an unmethylated 5'-CG-3' site is enzymatically coupled to the development of a fluorescent signal. This sensitive assay can measure rates of DNA methylation down to 0.34 +/- 0.06 fmol/s. The assay is reproducible, with a coefficient of variation over six independent measurements of 4.5%. Product concentration was accurately measured from fluorescence signals using a linear calibration curve, which achieved a goodness of fit (R(2)) above 0.98. The oligonucleotide substrate contains three C5-methylated cytosine residues and one unmethylated 5'-CG-3' site. Methylation yields an oligonucleotide containing the optimal substrate for the restriction enzyme GlaI. Cleavage of the fully methylated oligonucleotide leads to separation of fluorophore from quencher, giving a proportional increase in fluorescence. This method has been used to assay activity of DNMT1, the principle maintenance methyltransferase in human cells, and for the kinetic characterization of the bacterial cytosine-C5 methyltransferase M.SssI. The assay has been shown to be suitable for the real-time monitoring of DNMT1 activity in a high-throughput format, with low background signal and the ability to obtain linear rates of methylation over long periods, making this a promising method of high-throughput screening for inhibitors. PMID:20139415

  19. Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability

    PubMed Central

    Ngo, Thuy T. M.; Yoo, Jejoong; Dai, Qing; Zhang, Qiucen; He, Chuan; Aksimentiev, Aleksei; Ha, Taekjip

    2016-01-01

    Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility. PMID:26905257

  20. Comprehensive gene expression analysis of the DNA (cytosine-5) methyltransferase family in rice (Oryza sativa L.).

    PubMed

    Ahmad, F; Huang, X; Lan, H X; Huma, T; Bao, Y M; Huang, J; Zhang, H S

    2014-01-01

    Cytosine DNA methylation is a conserved epigenetic regulatory mechanism in both plants and animals. DNA methyltransferases (DNA MTases) not only initiate (de novo) but also maintain the process of DNA methylation. Here, we characterized the genome-wide expression profiles of 10 cytosine DNA MTase genes belonging to 4 subfamilies, MET1, CMT, DNMT2, and DRM, in rice. Tissue-specific gene expression analysis showed that all family members varied widely in their expression and specificities and might be involved in some basic metabolic pathways. Similarly, the expression of all rice cytosine DNA MTase genes was not regulated by plant hormones except OsDRM1a and OsDRM1b, which were downregulated by jasmonic acid. The transcription level of 10 genes in rice shoots and roots was also measured under salt and osmotic stress. Meanwhile, quantitative polymerase chain reaction data of the japonica and indica rice cultivars revealed that there is large variation in the expression activities of all genes. The results provide a foundation to further explore the roles of DNA MTases and the epigenetic regulation of abiotic stress responses in rice. PMID:25061741

  1. Accidental Amplification and Inactivation of a Methyltransferase Gene Eliminates Cytosine Methylation in Mycosphaerella graminicola

    PubMed Central

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

    2010-01-01

    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

  2. A density functional theory study for the hydrogen-bonded nucleic acid base pair: cytosine dimer.

    PubMed

    Sahu, Prabhat K; Mishra, Rama K; Lee, Shyi-Long

    2005-03-31

    Theoretical investigation for the geometric and energetic properties, rotational constants, harmonic vibrational frequencies, and binding energies of nucleic acid base pair, cytosine dimer, are carried out by using the density functional theory method. The dimer structures resulting from both the keto and the enol (cis/trans) tautomers are investigated in the present study. Various isomers are considered to find the stable structures of the cytosine dimer. The planar cytosine dimer, K-K3 with C2h symmetry, resulting from nonplanar keto tautomers, is found to be thermodynamically most stable out of the four different stable isomers and having the highest binding energy value, 19.51 kcal/mol (including basis set superposition error correction). The vibrational frequency analysis also suggests a red shift of 367.97 cm(-1) for the hydrogen-bonding K-K3 symmetric dimer with two hydrogen bond lengths, each of length 1.913 angstroms. Moreover, charge distribution (ChelpG charges), Laplacian electronic density distribution, and the dimerization equilibrium for the most stable dimer, K-K3, have also been investigated using the same method and the basis set. PMID:16833606

  3. Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability.

    PubMed

    Ngo, Thuy T M; Yoo, Jejoong; Dai, Qing; Zhang, Qiucen; He, Chuan; Aksimentiev, Aleksei; Ha, Taekjip

    2016-01-01

    Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility. PMID:26905257

  4. Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability

    NASA Astrophysics Data System (ADS)

    Ngo, Thuy T. M.; Yoo, Jejoong; Dai, Qing; Zhang, Qiucen; He, Chuan; Aksimentiev, Aleksei; Ha, Taekjip

    2016-02-01

    Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility.

  5. 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.

  6. Modeling brewers' yeast flocculation

    PubMed

    van Hamersveld EH; van der Lans RG; Caulet; Luyben

    1998-02-01

    Flocculation of yeast cells occurs during the fermentation of beer. Partway through the fermentation the cells become flocculent and start to form flocs. If the environmental conditions, such as medium composition and fluid velocities in the tank, are optimal, the flocs will grow in size large enough to settle. After settling of the main part of the yeast the green beer is left, containing only a small amount of yeast necessary for rest conversions during the next process step, the lagering. The physical process of flocculation is a dynamic equilibrium of floc formation and floc breakup resulting in a bimodal size distribution containing single cells and flocs. The floc size distribution and the single cell amount were measured under the different conditions that occur during full scale fermentation. Influences on flocculation such as floc strength, specific power input, and total number of yeast cells in suspension were studied. A flocculation model was developed, and the measured data used for validation. Yeast floc formation can be described with the collision theory assuming a constant collision efficiency. The breakup of flocs appears to occur mainly via two mechanisms, the splitting of flocs and the erosion of yeast cells from the floc surface. The splitting rate determines the average floc size and the erosion rate determines the number of single cells. Regarding the size of the flocs with respect to the scale of turbulence, only the viscous subrange needs to be considered. With the model, the floc size distribution and the number of single cells can be predicted at a certain point during the fermentation. For this, the bond strength between the cells, the fractal dimension of the yeast, the specific power input in the tank and the number of yeast cells that are in suspension in the tank have to be known. Copyright 1998 John Wiley & Sons, Inc. PMID:10099210

  7. 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.

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

    PubMed

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

    2012-07-12

    APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy. PMID:22645179

  9. 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 proteinRNA 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 domainRNA interaction and new tools for biophysical analysis of ADARRNA complexes. PMID:25564529

  10. 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.

  11. A 24-Year Enzyme Replacement Therapy in an Adenosine-deaminase-Deficient Patient.

    PubMed

    Tartibi, Hana M; Hershfield, Michael S; Bahna, Sami L

    2016-01-01

    Severe combined immunodeficiency (SCID) is a fatal childhood disease unless immune reconstitution is performed early in life, with either hematopoietic stem cell transplantation or gene therapy. One of its subtypes is caused by adenosine deaminase (ADA) enzyme deficiency, which leads to the accumulation of toxic metabolites that impair lymphocyte development and function. With the development of polyethylene glycol-conjugated adenosine deaminase (PEG-ADA) enzyme replacement therapy, many ADA-deficient children with SCID who could not receive a hematopoietic stem cell transplantation or gene therapy survived and had longer and healthier lives. We report a 24-year course of treatment in a patient who was diagnosed with ADA deficiency at 4 months of age. The patient was treated with PEG-ADA, which was the only therapy available for him. The patient's plasma ADA level was regularly monitored and the PEG-ADA dose adjusted accordingly. This treatment has resulted in near-normalization of lymphocyte counts, and his clinical course has been associated with only minor to moderate infections. Thus far, he has had no manifestations of autoimmune or lymphoproliferative disorders. This patient is among the longest to be maintained on PEG-ADA enzyme replacement therapy. PMID:26684479

  12. Identification of small molecule compounds with higher binding affinity to guanine deaminase (cypin) than guanine

    PubMed Central

    Fernndez, Jos R.; Sweet, Eric S.; Welsh, William J.; Firestein, Bonnie L.

    2010-01-01

    Guanine deaminase (GDA; cypin) is an important metalloenzyme that processes the first step in purine catabolism, converting guanine to xanthine by hydrolytic deamination. In higher eukaryotes, GDA also plays an important role in the development of neuronal morphology by regulating dendritic arborization. In addition to its role in the maturing brain, GDA is thought to be involved in proper liver function since increased levels of GDA activity have been correlated with liver disease and transplant rejection. Although mammalian GDA is an attractive and potential drug target for treatment of both liver diseases and cognitive disorders, prospective novel inhibitors and/or activators of this enzyme have not been actively pursued. In this study, we employed the combination of protein structure analysis and experimental kinetic studies to seek novel potential ligands for human guanine deaminase. Using virtual screening and biochemical analysis, we identified common small molecule compounds that demonstrate a higher binding affinity to GDA than does guanine. In vitro analysis demonstrates that these compounds inhibit guanine deamination, and more surprisingly, affect GDA (cypin)-mediated microtubule assembly. The results in this study provide evidence that an in silico drug discovery strategy coupled with in vitro validation assays can be successfully implemented to discover compounds that may possess therapeutic value for the treatment of diseases and disorders where GDA activity is abnormal. PMID:20716488

  13. Oxidative stress-induced mutagenesis in single-strand DNA occurs primarily at cytosines and is DNA polymerase zeta-dependent only for adenines and guanines

    PubMed Central

    Degtyareva, Natalya P.; Heyburn, Lanier; Sterling, Joan; Resnick, Michael A.; Gordenin, Dmitry A.; Doetsch, Paul W.

    2013-01-01

    Localized hyper-mutability caused by accumulation of lesions in persistent single-stranded (ss) DNA has been recently found in several types of cancers. An increase in endogenous levels of reactive oxygen species (ROS) is considered to be one of the hallmarks of cancers. Employing a yeast model system, we addressed the role of oxidative stress as a potential source of hyper-mutability in ssDNA by modulation of the endogenous ROS levels and by exposing cells to oxidative DNA-damaging agents. We report here that under oxidative stress conditions the majority of base substitution mutations in ssDNA are caused by erroneous, DNA polymerase (Pol) zeta-independent bypass of cytosines, resulting in C to T transitions. For all other DNA bases Pol zeta is essential for ROS-induced mutagenesis. The density of ROS-induced mutations in ssDNA is lower, compared to that caused by UV and MMS, which suggests that ssDNA could be actively protected from oxidative damage. These findings have important implications for understanding mechanisms of oxidative mutagenesis, and could be applied to development of anticancer therapies and cancer prevention. PMID:23925127

  14. 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

  15. 1-Aminocyclopropane-1-Carboxylate Deaminase from Pseudomonas stutzeri A1501 Facilitates the Growth of Rice in the Presence of Salt or Heavy Metals.

    PubMed

    Han, Yunlei; Wang, Rui; Yang, Zhirong; Zhan, Yuhua; Ma, Yao; Ping, Shuzhen; Zhang, Liwen; Lin, Min; Yan, Yongliang

    2015-07-01

    1-Aminocyclopropane-1-carboxylate (ACC) deaminase, which is encoded by some bacteria, can reduce the amount of ethylene, a root elongation inhibitor, and stimulate the growth of plants under various environmental stresses. The presence of ACC deaminase activity and the regulation of ACC in several rhizospheric bacteria have been reported. The nitrogen-fixing Pseudomonas stutzeri A1501 is capable of endophytic association with rice plants and promotes the growth of rice. However, the functional identification of ACC deaminase has not been performed. In this study, the proposed effect of ACC deaminase in P. stutzeri A1501 was investigated. Genome mining showed that P. stutzeri A1501 carries a single gene encoding ACC deaminase, designated acdS. The acdS mutant was devoid of ACC deaminase activity and was less resistant to NaCl and NiCl2 compared with the wild-type. Furthermore, inactivation of acdS greatly impaired its nitrogenase activity under salt stress conditions. It was also observed that mutation of the acdS gene led to loss of the ability to promote the growth of rice under salt or heavy metal stress. Taken together, this study illustrates the essential role of ACC deaminase, not only in enhancing the salt or heavy metal tolerance of bacteria but also in improving the growth of plants, and provides a theoretical basis for studying the interaction between plant growth-promoting rhizobacteria and plants. PMID:25674802

  16. Characterization of Arabidopsis thaliana telomeres isolated in yeast.

    PubMed Central

    Richards, E J; Chao, S; Vongs, A; Yang, J

    1992-01-01

    In an effort to learn more about the genomic organization of chromosomal termini in plants we employed a functional complementation strategy to isolate Arabidopsis thaliana telomeres in the yeast, Saccharomyces cerevisiae. Eight yeast episomes carrying A. thaliana telomeric sequences were obtained. The plant sequences carried on two episomes, YpAtT1 and YpAtT7, were characterized in detail. The telomeric origins of YpAtT1 and YpAtT7 insert DNAs were confirmed by demonstrating that corresponding genomic sequences are preferentially degraded during exonucleolytic digestion. The isolated telomeric restriction fragments contain G-rich repeat arrays characteristic of A. thaliana telomeres, as well as subterminal telomere-associated sequences (TASs). DNA sequence analysis revealed the presence of variant telomeric repeats at the centromere-proximal border of the terminal block of telomere repeats. The TAS flanking the telomeric G-rich repeat in YpAtT7 corresponds to a repetitive element present at other A. thaliana telomeres, while more proximal sequences are unique to one telomere. The YpAtT1 TAS is unique in the Landsberg strain of A. thaliana from which the clone originated; however, the Landsberg TAS cross-hybridizes weakly to a second telomere in the strain Columbia. Restriction analysis with cytosine methylation-sensitive endonucleases indicated that both TASs are highly methylated in the genome. Images PMID:1508688

  17. 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. )

    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.

  18. Lymphospecific toxicity in adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency: Possible role of nucleoside kinase(s)

    PubMed Central

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

    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 to phosphorylate adenosine and deoxyadenosine, the substrate of adenosine deaminase, and also inosine, deoxyinosine, guanosine, and deoxyguanosine, the substrates of purine nucleoside phosphorylase. Substantial activities of adenosine kinase were found in all tissues studied, while guanosine and inosine kinases were detected in none. However, the ability to phosphorylate deoxyadenosine, deoxyinosine, and deoxyguanosine was largely confined to lymphocytes. Adenosine deaminase, but not purine nucleoside phosphorylase, showed a similar lymphoid predominance. Other experiments showed that deoxyadenosine, deoxyinosine, and deoxyguanosine were toxic to human lymphoid cells. The toxicity of deoxyadenosine was reversed by the addition of deoxycytidine, but not uridine, to the culture medium. Based upon these and other experiments, we propose that in adenosine deaminase and purine nucleoside phosphorylase deficiency, toxic deoxyribonucleosides produced by many tissues are selectively trapped in lymphocytes by phosphorylating enzyme(s). PMID:202960

  19. 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

  20. Oxygen requirements of yeasts.

    PubMed Central

    Visser, W; Scheffers, W A; Batenburg-van der Vegte, W H; van Dijken, J P

    1990-01-01

    Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, such as Torulaspora delbrueckii and Candida tropicalis, grew poorly mu max, 0.03 and 0.05 h-1, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth. Images PMID:2082825

  1. 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.

  2. 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

  3. Strong nucleosomes of yeasts.

    PubMed

    Trifonov, Edward N; Tripathi, Vijay

    2016-02-01

    Yeast genome lacks visibly periodic sequences characteristic of strong nucleosomes (SNs) originally discovered in A. thaliana, C. elegans, and H. sapiens. Yet, the sequences with good match to the (RRRRRYYYYY)n consensus of the SNs do show preference to centromere regions of Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Cryptococcus neoformans - property characteristic of SNs of higher eukaryotes. Candida albicans is the first exception detected so far, where their SNs do not have any affinity to the centromeres, nor pericentromeric regions. Three of the four yeast genomes analyzed possess unique repeating centromere-specific SN sequences (C. albicans, again, is an exception). The results firmly indicate that centromeres of plants, animals, and yeasts in general have special chromatin structure, favoring SNs. PMID:25893982

  4. 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...

  5. Genetics of Yeasts

    NASA Astrophysics Data System (ADS)

    Querol, Amparo; Fernndez-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. L-arabinose fermenting yeast

    SciTech Connect

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

    2010-12-07

    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. Methods of producing ethanol include utilizing these modified yeast strains. ##STR00001##

  7. 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

  8. 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

  9. 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).

  10. Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA.

    PubMed

    Briggs, Adrian W; Stenzel, Udo; Meyer, Matthias; Krause, Johannes; Kircher, Martin; Pbo, Svante

    2010-04-01

    DNA sequences determined from ancient organisms have high error rates, primarily due to uracil bases created by cytosine deamination. We use synthetic oligonucleotides, as well as DNA extracted from mammoth and Neandertal remains, to show that treatment with uracil-DNA-glycosylase and endonuclease VIII removes uracil residues from ancient DNA and repairs most of the resulting abasic sites, leaving undamaged parts of the DNA fragments intact. Neandertal DNA sequences determined with this protocol have greatly increased accuracy. In addition, our results demonstrate that Neandertal DNA retains in vivo patterns of CpG methylation, potentially allowing future studies of gene inactivation and imprinting in ancient organisms. PMID:20028723

  11. 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.

  12. Complete remission in prolymphocytic leukemia with 4-demethoxydaunorubicin and arabinosyl cytosine.

    PubMed

    Lambertenghi-Deliliers, G; Maiolo, A T; Annaloro, C; Pogliani, E; Baldini, L; Polli, E

    1984-07-15

    Prolymphocytic leukemia (PL) is a morphologically distinct disease generally characterized by unsatisfactory therapeutic response and brief survival. Aggressive chemotherapy protocols including doxorubicin (DX) have been successfully used as alternatives to treatments usually effective in chronic lymphocytic leukemia (CLL). A patient with typical PL, resistant to regimens containing DX, was treated with 4-demethoxydaunorubicin (4-dm DNR), a new anthracycline analog derived from daunorubicin (DNR). The therapeutic response was rapid and impressive; furthermore 4-dm DNR combined with arabinosyl cytosine (Ara-C) produced a complete remission which is unusual in PL. PMID:6586276

  13. Isolation and characterization of the gene coding for human cytidine deaminase.

    PubMed

    Demontis, S; Terao, M; Brivio, M; Zanotta, S; Bruschi, M; Garattini, E

    1998-12-22

    The human gene coding for cytidine deaminase (CD), the enzyme which catalyzes the deamination of cytidine and deoxycytidine to uridine and deoxyuridine, was isolated and structurally characterized. CD is a single copy gene with a length of 31 kb and consists of four exons. Exon-intron junctions do not bracket functional domains of the encoded protein as the boundary between exons 2 and 3 interrupts the catalytically important zinc-finger domain, which is well conserved along phylogenesis. 5'-RACE and RNase mapping experiments identify one major and multiple other minor transcription initiation sites, which are present in placenta as well as in the myeloid cell lines, HL-60 and U937. The 5'-flanking region of the gene contains an orientation-dependent functional promoter and is characterized by the presence of several potential sites for the binding of known transcriptional factors. PMID:9878810

  14. 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

  15. Activation-Induced Cytidine Deaminase Links Ovulation-Induced Inflammation and Serous Carcinogenesis.

    PubMed

    Sapoznik, Stav; Bahar-Shany, Keren; Brand, Hadar; Pinto, Yishay; Gabay, Orshay; Glick-Saar, Efrat; Dor, Chen; Zadok, Oranit; Barshack, Iris; Zundelevich, Adi; Gal-Yam, Einav Nili; Yung, Yuval; Hourvitz, Ariel; Korach, Jacob; Beiner, Mario; Jacob, Jasmine; Levanon, Erez Y; Barak, Michal; Aviel-Ronen, Sarit; Levanon, Keren

    2016-02-01

    In recent years, the notion that ovarian carcinoma results from ovulation-induced inflammation of the fallopian tube epithelial cells (FTECs) has gained evidence. However, the mechanistic pathway for this process has not been revealed yet. In the current study, we propose the mutator protein activation-induced cytidine deaminase (AID) as a link between ovulation-induced inflammation in FTECs and genotoxic damage leading to ovarian carcinogenesis. We show that AID, previously shown to be functional only in B lymphocytes, is expressed in FTECs under physiological conditions, and is induced in vitro upon ovulatory-like stimulation and in vivo in carcinoma-associated FTECs. We also report that AID activity results in epigenetic, genetic and genomic damage in FTECs. Overall, our data provides new insights into the etiology of ovarian carcinogenesis and may set the ground for innovative approaches aimed at prevention and early detection. PMID:26936395

  16. 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

  17. Somatic Hypermutation Is Limited by CRM1-dependent Nuclear Export of Activation-induced Deaminase

    PubMed Central

    McBride, Kevin M.; Barreto, Vasco; Ramiro, Almudena R.; Stavropoulos, Pete; Nussenzweig, Michel C.

    2004-01-01

    Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated in activated B lymphocytes by activation-induced deaminase (AID). AID is thought to make lesions in DNA by deaminating cytidine residues in single-stranded DNA exposed by RNA polymerase during transcription. Although this must occur in the nucleus, AID is found primarily in the cytoplasm. Here we show that AID is actively excluded from the nucleus by an exportin CRM1-dependent pathway. The AID nuclear export signal (NES) is found at the carboxyl terminus of AID in a region that overlaps a sequence required for CSR but not SHM. We find that AID lacking a functional NES causes more hypermutation of a nonphysiologic target gene in transfected fibroblasts. However, the NES does not impact on the rate of mutation of immunoglobulin genes in B lymphocytes, suggesting that the AID NES does not limit AID activity in these cells. PMID:15117971

  18. Analysis of a replication initiation sequence from the adenosine deaminase region of the mouse genome.

    PubMed Central

    Virta-Pearlman, V J; Gunaratne, P H; Chinault, A C

    1993-01-01

    A 4-kb HindIII fragment that supported the efficient autonomous replication of plasmid vector pDY-, a replication-defective construct based on Epstein-Barr virus sequences, in human K562 cells was rescued from amplified double-minute chromosomes containing the murine adenosine deaminase locus. Polymerase chain reaction assays of size-fractionated nascent strands demonstrated that replication initiation occurred within the same 1- to 2-kb region of this fragment in autonomously replicating plasmids containing the sequence in either orientation, in double-minute chromosomes, and in the single-copy locus at its normal chromosomal location. The complete sequence of this fragment was determined; it contains a 248-bp polypurine tract and consensus binding site sequences for several putative transcription and replication factors. Images PMID:8413198

  19. [Adenosine deaminase1 deficiency, an inborn error of metabolism underlying a severe form of combined immunodeficiency].

    PubMed

    Giraud, A; Lavocat, M-P; Cremillieux, C; Patural, H; Thouvenin, S; David, A; Perignon, J-L; Stephan, J-L

    2015-06-01

    Severe combined immune deficiencies (SCIDs) are a heterogeneous group of severe cellular immunodeficiencies. Early diagnosis is essential to allow adapted care before life-threatening systemic infections or complications associated with live vaccines. Adenosine deaminase 1 deficiency (ADA1) is an inborn error of metabolism leading to severe lymphopenia and characteristic bone lesions. Herein, we present the typical case of a child in whom ADA SCID was diagnosed at 2 months of life, revealed by lung involvement and extreme lymphopenia. Immune restoration in terms of peripheral lymphocyte count with enzyme replacement therapy, namely pegylated bovine ADA, is satisfactory so far. The search for a compatible donor is underway. Correcting the genetic defect by gene transfer is also being considered. The phenotype of this very rare condition is described. A severe peripheral lymphopenia in a young child is a finding of utmost importance for the diagnosis of a primary cellular immunodeficiency. PMID:25842197

  20. 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

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

    PubMed

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

    2009-01-01

    Gene therapy is a highly attractive strategy for many types of inherited disorders of the immune system. Adenosine deaminase (ADA) deficient-severe combined immunodeficiency (SCID) has been the target of several clinical trials based on the use of hematopoietic stem/progenitor cells engineered with retroviral vectors. The introduction of a low intensity conditioning regimen has been a crucial factor in achieving stable engrafment of hematopoietic stem cells and therapeutic levels of ADA-expressing cells. Recent studies have demonstrated that gene therapy for ADA-SCID has favorable safety profile and is effective in restoring normal purine metabolism and immune functions. Stem cell gene therapy combined with appropriate conditioning regimens might be extended to other genetic disorders of the hematopoietic system. PMID:19224139

  2. Synthesis of 5?-Methylthio Coformycins: Specific Inhibitors for Malarial Adenosine Deaminase

    PubMed Central

    Tyler, Peter C.; Taylor, Erika A.; Frhlich, Richard F. G.; Schramm, Vern L.

    2008-01-01

    Transition state theory suggests that enzymatic rate acceleration (kcat/knon) is related to the stabilization of the transition state for a given reaction. Chemically stable analogues of a transition state complex are predicted to convert catalytic energy into binding energy. Since transition state stabilization is a function of catalytic efficiency, differences in substrate specificity can be exploited in the design of tight-binding transition state analogue inhibitors. Coformycin and 2?-deoxycoformycin are natural product transition state analogue inhibitors of adenosine deaminases (ADAs). These compounds mimic the tetrahedral geometry of the ADA transition state and bind with picomolar dissociation constants to enzymes from bovine, human, and protozoan sources. The purine salvage pathway in malaria parasites is unique in that Plasmodium falciparum ADA (PfADA) catalyzes the deamination of both adenosine and 5-methylthioadenosine. In contrast, human adenosine deaminase (HsADA) does not deaminate 5-methylthioadenosine. 5?-Methylthio coformycin and 5-meththio-2?-deoxycoformycin were synthesized to be specific transition state mimics of the P. falciparum enzyme. These analogues inhibited PfADA with dissociation constants of 430 and 790 pM, respectively. Remarkably, they gave no detectable inhibition of the human and bovine enzymes. Adenosine deamination is involved in the essential pathway of purine salvage in P. falciparum and prior studies have shown that inhibition of purine salvage results in parasite death. Inhibitors of HsADA are known to cause toxicity in humans and the availability of parasite-specific ADA inhibitors may prevent this side-effect. The potent and P. falciparum-specific inhibitors described here have potential for development as antimalarials without inhibition of host ADA. PMID:17488013

  3. 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

  4. Activation-induced cytidine deaminase in B cells of hepatits C virus-related cryoglobulinaemic vasculitis.

    PubMed

    Russi, S; Dammacco, F; Sansonno, S; Pavone, F; Sansonno, D

    2015-12-01

    Immunoglobulin variable region heavy chain (IgVH ) somatic gene diversification is instrumental in the transformation process that characterizes hepatitis C virus (HCV)-related B cell lymphoproliferative disorders. However, the extent to which activation-induced cytidine deaminase (AID), an enzyme essential for IgV gene somatic hypermutation (SHM), is active in cryoglobulinaemic vasculitis (CV) remains unclear. AID mRNA expression in the peripheral blood of 102 chronically hepatitis C virus (HCV)-infected patients (58 with and 44 without CV) and 26 healthy subjects was investigated using real-time reverse transcription-polymerase chain reaction (RT-PCR). The features of activation-induced cytidine deaminase (AID) protein and mRNA transcripts were explored in liver tissue biopsies and portal tracts isolated using laser capture microdissection. In chronically HCV-infected patients, AID mRNA expression was almost threefold higher in those with than in those without CV and sevenfold higher than in healthy subjects (median-fold: 6.68 versus 2.54, P = 0.03 and versus 0.95, P = 0.0003). AID transcript levels were significantly higher in polyclonal than in clonally restricted B cell preparations in either CV or non-CV patients (median-fold, 15.0 versus 2.70, P = 0.009 and 3.46 versus 1.58, P = 0.02, respectively). AID gene expression was found to be related negatively to age and virological parameters. AID protein was found in portal tracts containing inflammatory cells that, in several instances, expressed AID mRNA transcripts. Our data indicate that the aberrant expression of AID may reflect continuous B cell activation and sustained survival signals in HCV-related CV patients. PMID:26219420

  5. 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. PMID:25861440

  6. Opportunistic Pathogenic Yeasts

    NASA Astrophysics Data System (ADS)

    Banerjee, Uma

    Advances in medical research, made during the last few decades, have improved the prophylactic, diagnostic and therapeutic capabilities for variety of infections/diseases. However, many of the prophylactic and therapeutic procedures have been seen in many instances to exact a price of host-vulnerability to an expanding group of opportunistic pathogens and yeasts are one of the important members in it. Fortunately amongst the vast majority of yeasts present in nature only few are considered to have the capability to cause infections when certain opportunities predisposes and these are termed as opportunistic pathogenic yeasts. However, the term pathogenic is quite tricky, as it depends of various factors of the host, the bug and the environment to manifest the clinical infection. The borderline is expanding. In the present century with unprecedented increase in number of immune-compromised host in various disciplines of health care settings, where any yeast, which has the capability to grow at 37 C (normal body temperature of human), can be pathogenic and cause infection in particular situation

  7. Stabilization of Mixed Frenkel-Charge Transfer Excitons Extended Across Both Strands of Guanine-Cytosine DNA Duplexes.

    PubMed

    Huix-Rotllant, Miquel; Brazard, Johanna; Improta, Roberto; Burghardt, Irene; Markovitsi, Dimitra

    2015-06-18

    The photoreactive pathways that may lead to DNA damage depend crucially upon the nature of the excited electronic states. The study of alternating guanine-cytosine duplexes by fluorescence spectroscopy and quantum mechanical calculations identifies a novel type of excited states that can be populated following UVB excitation. These states, denoted High-energy Emitting Long-lived Mixed (HELM) states, extend across both strands and arise from mixing between cytosine Frenkel excitons and guanine-to-cytosine charge transfer states. They emit at energies higher than ??* states localized on single bases, survive for several nanoseconds, are sensitive to the ionic strength of the solution, and are strongly affected by the structural transition from the B form to the Z form. Their impact on the formation of lesions of the genetic code needs to be assessed. PMID:26266599

  8. N-terminal amino acid sequences of D-serine deaminases of wild-type and operator-constitutive strains of Escherichia coli K-12.

    PubMed Central

    Heincz, M C; McFall, E

    1975-01-01

    The N-terminal amino acid sequences of the D-serine deaminases from strains of Escherichia coli K-12 that harbor wild-type and high-level constitutive catabolite-insensitive operator-initiator regions are identical: Met-Ser-GluNH2-Ser-Gly-Arg-His-Cys. This result indicates that the operator-initiator region is probably distinct from the D-serine deaminase structural gene. Images PMID:1099073

  9. The ADA*2 allele of the adenosine deaminase gene (20q13.11) and recurrent spontaneous abortions: an age-dependent association

    PubMed Central

    Nunes, Daniela Prudente Teixeira; Spegiorin, Lígia Cosentino Junqueira Franco; de Mattos, Cinara Cássia Brandão; Oliani, Antonio Helio; Vaz-Oliani, Denise Cristina Mós; de Mattos, Luiz Carlos

    2011-01-01

    OBJECTIVE: Adenosine deaminase acts on adenosine and deoxyadenosine metabolism and modulates the immune response. The adenosine deaminase G22A polymorphism (20q.11.33) influences the level of adenosine deaminase enzyme expression, which seems to play a key role in maintaining pregnancy. The adenosine deaminase 2 phenotype has been associated with a protective effect against recurrent spontaneous abortions in European Caucasian women. The aim of this study was to investigate whether the G22A polymorphism of the adenosine deaminase gene is associated with recurrent spontaneous abortions in Brazilian women. METHODS: A total of 311 women were recruited to form two groups: G1, with a history of recurrent spontaneous abortions (N = 129), and G2, without a history of abortions (N = 182). Genomic DNA was extracted from peripheral blood with a commercial kit and PCR-RFLP analysis was used to identify the G22A genetic polymorphism. Fisher's exact test and odds ratio values were used to compare the proportions of adenosine deaminase genotypes and alleles between women with and without a history of recurrent spontaneous abortion (p<0.05). The differences between mean values for categorical data were calculated using unpaired t tests. The Hardy-Weinberg equilibrium was assessed with a chi-square test. RESULTS: Statistically significant differences were identified for the frequencies of adenosine deaminase genotypes and alleles between the G1 and G2 groups when adjusted for maternal age. CONCLUSIONS: The results suggest that the adenosine deaminase *2 allele is associated with a low risk for recurrent spontaneous abortions, but this association is dependent on older age. PMID:22086524

  10. L-arabinose fermenting yeast

    DOEpatents

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

    2013-02-12

    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.

  11. 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.

  12. On the Ag(+)-cytosine interaction: the effect of microhydration probed by IR optical spectroscopy and density functional theory.

    PubMed

    Berdakin, Matias; Steinmetz, Vincent; Maitre, Philippe; Pino, Gustavo A

    2015-10-21

    The gas-phase structures of cytosine-Ag(+) [CAg](+) and cytosine-Ag(+)-H2O [CAg-H2O](+) complexes have been studied by mass-selected infrared multiphoton dissociation (IRMPD) spectroscopy in the 900-1800 cm(-1) spectral region using the Free Electron Laser facility in Orsay (CLIO). The IRMPD experimental spectra have been compared with the calculated IR absorption spectra of the different low-lying isomers (computed at the DFT level using the B3LYP functional and the 6-311G++(d,p) basis set for C, H, N and O atoms and the Stuttgart effective core potential for Ag). For the [CAg](+) complex, only one isomer with cytosine in the keto-amino (KA) tautomeric form and Ag(+) interacting simultaneously with the C(2)[double bond, length as m-dash]O(7) group and N(3) of cytosine was observed. However, the mono-hydration of the complex in the gas phase leads to the stabilization of a two quasi-isoenergetic structure of the [CAg-H2O](+) complex, in which Ag(+) interacts with the O atom of the water molecule and with the N(3) or C(2)[double bond, length as m-dash]O(7) group of cytosine. The relative populations of the two isomers determined from the IRMPD kinetics plot are in good agreement with the calculated values. Comparison of these results with those of protonated cytosine [CH](+) and its mono-hydrated complex [CH-H2O](+) shows some interesting differences between H(+) and Ag(+). In particular, while a single water molecule catalyzes the isomerization reaction in the case of [CH-H2O](+), it is found that in the case of [CAg-H2O](+) the addition of water leads to the stabilization of two isomers separated by small energy barrier (0.05 eV). PMID:26068183

  13. MSAP markers and global cytosine methylation in plants: a literature survey and comparative analysis for a wild-growing species.

    PubMed

    Alonso, Conchita; Prez, Ricardo; Bazaga, Pilar; Medrano, Mnica; Herrera, Carlos M

    2016-01-01

    Methylation of DNA cytosines affects whether transposons are silenced and genes are expressed, and is a major epigenetic mechanism whereby plants respond to environmental change. Analyses of methylation-sensitive amplification polymorphism (MS-AFLP or MSAP) have been often used to assess methyl-cytosine changes in response to stress treatments and, more recently, in ecological studies of wild plant populations. MSAP technique does not require a sequenced reference genome and provides many anonymous loci randomly distributed over the genome for which the methylation status can be ascertained. Scoring of MSAP data, however, is not straightforward, and efforts are still required to standardize this step to make use of the potential to distinguish between methylation at different nucleotide contexts. Furthermore, it is not known how accurately MSAP infers genome-wide cytosine methylation levels in plants. Here, we analyse the relationship between MSAP results and the percentage of global cytosine methylation in genomic DNA obtained by HPLC analysis. A screening of literature revealed that methylation of cytosines at cleavage sites assayed by MSAP was greater than genome-wide estimates obtained by HPLC, and percentages of methylation at different nucleotide contexts varied within and across species. Concurrent HPLC and MSAP analyses of DNA from 200 individuals of the perennial herb Helleborus foetidus confirmed that methyl-cytosine was more frequent in CCGG contexts than in the genome as a whole. In this species, global methylation was unrelated to methylation at the inner CG site. We suggest that global HPLC and context-specific MSAP methylation estimates provide complementary information whose combination can improve our current understanding of methylation-based epigenetic processes in nonmodel plants. PMID:25944158

  14. Activation of trout gill AMP deaminase by an endogenous proteinase--II. Modification of the properties of the enzyme during starvation, pollution and salinity changes.

    PubMed

    Raffin, J P

    1986-01-01

    The relative amount of modified AMP deaminase has been determined by taking advantage of the different effects of monovalent cations on the two enzymatic forms. When trout were subjected to different environmental perturbations (starvation, pollution of the water by a pesticide, transfer to sea water or reverse transfer to fresh water), modified AMP deaminase could be detected in the gill extracts. Depending on the nature of the stress and the period of experimentation, 8 to 100% of the enzyme had been modified by limited proteolysis. As a consequence of the much higher activity of the proteolyzed AMP deaminase form, a 2 to 12 times increase of the intracellular AMP deaminase activity could be expected. At the same time, limited proteolysis will modify the regulatory properties of the enzyme, since it can be estimated that 50 to 100% of the enzyme activity expressed in the cell will be an AMP deaminase form less sensitive to inhibition by inorganic phosphate and ionic strength, and to variations of the intracellular pH. Limited proteolysis will result in increased AMP deaminase activity under conditions of increased energy demand, where the concentration of inorganic phosphate is dramatically increased. The consequence should be stabilization of the adenylate energy charge. PMID:3533410

  15. Cytosine methylation changes in enhancer regions of core pro-fibrotic genes characterize kidney fibrosis development

    PubMed Central

    2013-01-01

    Background One in eleven people is affected by chronic kidney disease, a condition characterized by kidney fibrosis and progressive loss of kidney function. Epidemiological studies indicate that adverse intrauterine and postnatal environments have a long-lasting role in chronic kidney disease development. Epigenetic information represents a plausible carrier for mediating this programming effect. Here we demonstrate that genome-wide cytosine methylation patterns of healthy and chronic kidney disease tubule samples obtained from patients show significant differences. Results We identify differentially methylated regions and validate these in a large replication dataset. The differentially methylated regions are rarely observed on promoters, but mostly overlap with putative enhancer regions, and they are enriched in consensus binding sequences for important renal transcription factors. This indicates their importance in gene expression regulation. A core set of genes that are known to be related to kidney fibrosis, including genes encoding collagens, show cytosine methylation changes correlating with downstream transcript levels. Conclusions Our report raises the possibility that epigenetic dysregulation plays a role in chronic kidney disease development via influencing core pro-fibrotic pathways and can aid the development of novel biomarkers and future therapeutics. PMID:24098934

  16. 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

  17. The Role of Hydrogen Bonds in the Stabilization of Silver-Mediated Cytosine Tetramers.

    PubMed

    Espinosa Leal, Leonardo Andrs; Karpenko, Alexander; Swasey, Steven; Gwinn, Elisabeth G; Rojas-Cervellera, Victor; Rovira, Carme; Lopez-Acevedo, Olga

    2015-10-15

    DNA oligomers can form silver-mediated duplexes, stable in gas phase and solution, with potential for novel biomedical and technological applications. The nucleobase-metal bond primarily drives duplex formation, but hydrogen (H-) bonds may also be important for structure selection and stability. To elucidate the role of H-bonding, we conducted theoretical and experimental studies of a duplex formed by silver-mediated cytosine homopobase DNA strands, two bases long. This silver-mediated cytosine tetramer is small enough to permit accurate, realistic modeling by DFT-based quantum mechanics/molecular mechanics methods. In gas phase, our calculations found two energetically favorable configurations distinguished by H-bonding, one with a novel interplane H-bond, and the other with planar H-bonding of silver-bridged bases. Adding solvent favored silver-mediated tetramers with interplane H-bonding. Overall agreement of electronic circular dichroism spectra for the final calculated structure and experiment validates these findings. Our results can guide use of these stabilization mechanisms for devising novel metal-mediated DNA structures. PMID:26722777

  18. Ultrafast deactivation of an excited cytosine-guanine base pair in DNA.

    PubMed

    Groenhof, Gerrit; Schfer, Lars V; Boggio-Pasqua, Martial; Goette, Maik; Grubmller, Helmut; Robb, Michael A

    2007-05-30

    Multiconfigurational ab initio calculations and QM/MM molecular dynamics simulations of a photoexcited cytosine-guanine base pair in both gas phase and embedded in the DNA provide detailed structural and dynamical insights into the ultrafast radiationless deactivation mechanism. Photon absorption promotes transfer of a proton from the guanine to the cytosine. This proton transfer is followed by an efficient radiationless decay of the excited state via an extended conical intersection seam. The optimization of the conical intersection revealed that it has an unusual topology, in that there is only one degeneracy-lifting coordinate. This is the central mechanistic feature for the decay both in vacuo and in the DNA. Radiationless decay occurs along an extended hyperline nearly parallel to the proton-transfer coordinate, indicating the proton transfer itself is not directly responsible for the deactivation. The seam is displaced from the minimum energy proton-transfer path along a skeletal deformation of the bases. Decay can thus occur anywhere along the single proton-transfer coordinate, accounting for the remarkably short excited-state lifetime of the Watson-Crick base pair. In vacuo, decay occurs after a complete proton transfer, whereas in DNA, decay can also occur much earlier. The origin of this effect lies in the temporal electrostatic stabilization of dipole in the charge-transfer state in DNA. PMID:17488008

  19. Potential of ribozymes against deoxycytidine kinase to confer drug resistance to cytosine nucleoside analogs.

    PubMed

    Beausjour, C M; Tremblay, G; Momparler, R L

    2000-11-30

    Hematopoietic toxicity is the dose-limiting side effect produced in cancer chemotherapy with deoxycytidine nucleoside analogs. Deletion of the deoxycytidine kinase (dCK), results in a drug resistance phenotype to these analogs. An interesting gene therapy strategy to confer drug resistance to cytosine nucleoside analogs would be to specifically inactivate the dCK in normal hematopoietic stem cell. In this study, we designed hammerhead ribozymes that can specifically cut and downregulate the murine dCK mRNA. Three different ribozymes were identified and shown to cleave in vitro the dCK RNA. After introduction of ribozyme cDNA into murine L1210 leukemic cells by retroviral transfer, two of the ribozymes showed some capacity in reducing dCK activity. However, analysis of transduced L1210 clones showed that the significant reduction in the dCK mRNA was not sufficient to confer drug resistance to cytosine arabinoside. Nevertheless, these results provide a new avenue of modulating the dCK enzyme activity and with improved modifications may have the potential for use in gene therapy to confer drug resistance to deoxycytidine analogs. PMID:11095951

  20. Electron impact fragmentation of cytosine: partial ionization cross sections for positive fragments

    NASA Astrophysics Data System (ADS)

    van der Burgt, Peter J. M.

    2014-05-01

    We have measured mass spectra for positive ions produced by low-energy electron impact on cytosine using a reflectron time-of-flight mass spectrometer. The electron impact energy has been varied from 0 to 100 eV in steps of 0.5 eV. Ion yield curves of most of the fragment ions have been determined by fitting groups of adjacent peaks in the mass spectra with sequences of normalized Gaussians. The ion yield curves have been normalized by comparing the sum of the ion yields to the average of calculated total ionization cross sections. Appearance energies of the fragment ions have been determined, showing that the fragments 68 u-84 u have appearance energies between 10 and 11 eV, whereas fragments of 55 u and lower mass all have appearance energies above 12 eV. Most of the ion yields of 55 u and smaller show multiple onsets. Several groups of fragments have ion yield curves with nearly the same shape, clearly indicating the relevance of tautomerization in the fragmentation of cytosine.

  1. Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants.

    PubMed Central

    Woodcock, D M; Crowther, P J; Doherty, J; Jefferson, S; DeCruz, E; Noyer-Weidner, M; Smith, S S; Michael, M Z; Graham, M W

    1989-01-01

    Many strains of E. coli K12 restrict DNA containing cytosine methylation such as that present in plant and animal genomes. Such restriction can severely inhibit the efficiency of cloning genomic DNAs. We have quantitatively evaluated a total of 39 E. coli strains for their tolerance to cytosine methylation in phage and plasmid cloning systems. Quantitative estimations of relative tolerance to methylation for these strains are presented, together with the evaluation of the most promising strains in practical recombinant cloning situations. Host strains are recommended for different recombinant cloning requirements. These data also provide a rational basis for future construction of 'ideal' hosts combining optimal methylation tolerance with additional advantageous mutations. PMID:2657660

  2. Associations between activation-induced cytidine deaminase/apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like cytidine deaminase expression, hepatitis B virus (HBV) replication and HBV-associated liver disease (Review)

    PubMed Central

    HE, XIUTING; LI, JIE; WU, JING; ZHANG, MANLI; GAO, PUJUN

    2015-01-01

    The hepatitis B virus (HBV) infection is a major risk factor in the development of chronic hepatitis (CH) and hepa-tocellular carcinoma (HCC). The activation-induced cytidine deaminase (AID)/apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of cytidine deaminases is significant in innate immunity, as it restricts numerous viruses, including HBV, through hypermutation-dependent and -independent mechanisms. It is important to induce covalently closed circular (ccc)DNA degradation by interferon-α without causing side effects in the infected host cell. Furthermore, organisms possess multiple mechanisms to regulate the expression of AID/APOBECs, control their enzymatic activity and restrict their access to DNA or RNA substrates. Therefore, the AID/APOBECs present promising targets for preventing and treating viral infections. In addition, gene polymorphisms of the AID/APOBEC family may alter host susceptibility to HBV acquisition and CH disease progression. Through G-to-A hypermutation, AID/APOBECs also edit HBV DNA and facilitate the mutation of HBV DNA, which may assist the virus to evolve and potentially escape from the immune responses. The AID/APOBEC family and their associated editing patterns may also exert oncogenic activity. Understanding the effects of cytidine deaminases in CH virus-induced hepatocarcinogenesis may aid with developing efficient prophylactic and therapeutic strategies against HCC. PMID:26398702

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

    SciTech Connect

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

    2007-09-01

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

  4. Adenosine deaminase regulates Treg expression in autologous T cell-dendritic cell cocultures from patients infected with HIV-1.

    PubMed

    Naval-Macabuhay, Isaac; Casanova, Vctor; Navarro, Gemma; Garca, Felipe; Len, Agathe; Miralles, Laia; Rovira, Cristina; Martinez-Navio, Jos M; Gallart, Teresa; Mallol, Josefa; Gatell, Jos M; Llus, Carme; Franco, Rafael; McCormick, Peter J; Climent, Nria

    2016-02-01

    Regulatory T cells have an important role in immune suppression during HIV-1 infection. As regulatory T cells produce the immunomodulatory molecule adenosine, our aim here was to assess the potential of adenosine removal to revert the suppression of anti-HIV responses exerted by regulatory T cells. The experimental setup consisted of ex vivo cocultures of T and dendritic cells, to which adenosine deaminase, an enzyme that hydrolyzes adenosine, was added. In cells from healthy individuals, adenosine hydrolysis decreased CD4(+)CD25(hi) regulatory T cells. Addition of 5'-N-ethylcarboxamidoadenosine, an adenosine receptor agonist, significantly decreased CD4(+)CD25(lo) cells, confirming a modulatory role of adenosine acting via adenosine receptors. In autologous cocultures of T cells with HIV-1-pulsed dendritic cells, addition of adenosine deaminase led to a significant decrease of HIV-1-induced CD4(+)CD25(hi) forkhead box p3(+) cells and to a significant enhancement of the HIV-1-specific CD4(+) responder T cells. An increase in the effector response was confirmed by the enhanced production of CD4(+) and CD8(+) CD25(-)CD45RO(+) memory cell generation and secretion of Th1 cytokines, including IFN-? and IL-15 and chemokines MIP-1?/CCL3, MIP-1?/CCL4, and RANTES/CCL5. These ex vivo results show, in a physiologically relevant model, that adenosine deaminase is able to enhance HIV-1 effector responses markedly. The possibility to revert regulatory T cell-mediated inhibition of immune responses by use of adenosine deaminase, an enzyme that hydrolyzes adenosine, merits attention for restoring T lymphocyte function in HIV-1 infection. PMID:26310829

  5. 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.

  6. Water Transport in Yeasts.

    PubMed

    Sabir, Farzana; Prista, Catarina; Madeira, Ana; Moura, Teresa; Loureiro-Dias, Maria C; Soveral, Graça

    2016-01-01

    Water moves across membranes through the lipid bilayer and through aquaporins, in this case in a regulated manner. Aquaporins belong to the MIP superfamily and two subfamilies are represented in yeasts: orthodox aquaporins considered to be specific water channels and aquaglyceroporins (heterodox aquaporins). In Saccharomyces cerevisiae genome, four aquaporin isoforms were identified, two of which are genetically close to orthodox aquaporins (ScAqy1 and ScAqy2) and the other two are more closely related to the aquaglyceroporins (ScFps1 and ScAqy3). Advances in the establishment of water channels structure are reviewed in this chapter in relation with the mechanisms of selectivity, conductance and gating. Aquaporins are important for key aspects of yeast physiology. They have been shown to be involved in sporulation, rapid freeze-thaw tolerance, osmo-sensitivity, and modulation of cell surface properties and colony morphology, although the underlying exact mechanisms are still unknown. PMID:26721272

  7. Genetic editing of HBV DNA by monodomain human APOBEC3 cytidine deaminases and the recombinant nature of APOBEC3G.

    PubMed

    Henry, Michel; Gutard, Denise; Suspne, Rodolphe; Rusniok, Christophe; Wain-Hobson, Simon; Vartanian, Jean-Pierre

    2009-01-01

    Hepatitis B virus (HBV) DNA is vulnerable to editing by human cytidine deaminases of the APOBEC3 (A3A-H) family albeit to much lower levels than HIV cDNA. We have analyzed and compared HBV editing by all seven enzymes in a quail cell line that does not produce any endogenous DNA cytidine deaminase activity. Using 3DPCR it was possible to show that all but A3DE were able to deaminate HBV DNA at levels from 10(-2) to 10(-5)in vitro, with A3A proving to be the most efficient editor. The amino terminal domain of A3G alone was completely devoid of deaminase activity to within the sensitivity of 3DPCR ( approximately 10(-4) to 10(-5)). Detailed analysis of the dinucleotide editing context showed that only A3G and A3H have strong preferences, notably CpC and TpC. A phylogenic analysis of A3 exons revealed that A3G is in fact a chimera with the first two exons being derived from the A3F gene. This might allow co-expression of the two genes that are able to restrict HIV-1Deltavif efficiently. PMID:19169351

  8. Solid-phase molecular recognition of cytosine based on proton-transfer reaction. Part II. supramolecular architecture in the cocrystals of cytosine and its 5-Fluoroderivative with 5-Nitrouracil

    PubMed Central

    2011-01-01

    Background Cytosine is a biologically important compound owing to its natural occurrence as a component of nucleic acids. Cytosine plays a crucial role in DNA/RNA base pairing, through several hydrogen-bonding patterns, and controls the essential features of life as it is involved in genetic codon of 17 amino acids. The molecular recognition among cytosines, and the molecular heterosynthons of molecular salts fabricated through proton-transfer reactions, might be used to investigate the theoretical sites of cytosine-specific DNA-binding proteins and the design for molecular imprint. Results Reaction of cytosine (Cyt) and 5-fluorocytosine (5Fcyt) with 5-nitrouracil (Nit) in aqueous solution yielded two new products, which have been characterized by single-crystal X-ray diffraction. The products include a dihydrated molecular salt (CytNit) having both ionic and neutral hydrogen-bonded species, and a dihydrated cocrystal of neutral species (5FcytNit). In CytNit a protonated and an unprotonated cytosine form a triply hydrogen-bonded aggregate in a self-recognition ion-pair complex, and this dimer is then hydrogen bonded to one neutral and one anionic 5-nitrouracil molecule. In 5FcytNit the two neutral nucleobase derivatives are hydrogen bonded in pairs. In both structures conventional N-H...O, O-H...O, N-H+...N and N-H...N- intermolecular interactions are most significant in the structural assembly. Conclusion The supramolecular structure of the molecular adducts formed by cytosine and 5-fluorocytosine with 5-nitrouracil, CytNit and 5FcytNit, respectively, have been investigated in detail. CytNit and 5FcytNit exhibit widely differing hydrogen-bonding patterns, though both possess layered structures. The crystal structures of CytNit (Dpka = -0.7, molecular salt) and 5FcytNit (Dpka = -2.0, cocrystal) confirm that, at the present level of knowledge about the nature of proton-transfer process, there is not a strict correlation between the Dpka values and the proton transfer, in that the acid/base pka strength is not a definite guide to predict the location of H atoms in the solid state. Eventually, the absence in 5FcytNit of hydrogen bonds involving fluorine is in agreement with findings that covalently bound fluorine hardly ever acts as acceptor for available Brnsted acidic sites in the presence of competing heteroatom acceptors. PMID:21888640

  9. Tapping into yeast diversity.

    PubMed

    Fay, Justin C

    2012-11-01

    Domesticated organisms demonstrate our capacity to influence wild species but also provide us with the opportunity to understand rapid evolution in the context of substantially altered environments and novel selective pressures. Recent advances in genetics and genomics have brought unprecedented insights into the domestication of many organisms and have opened new avenues for further improvements to be made. Yet, our ability to engineer biological systems is not without limits; genetic manipulation is often quite difficult. The budding yeast, Saccharomyces cerevisiae, is not only one of the most powerful model organisms, but is also the premier producer of fermented foods and beverages around the globe. As a model system, it entertains a hefty workforce dedicated to deciphering its genome and the function it encodes at a rich mechanistic level. As a producer, it is used to make leavened bread, and dozens of different alcoholic beverages, such as beer and wine. Yet, applying the awesome power of yeast genetics to understanding its origins and evolution requires some knowledge of its wild ancestors and the environments from which they were derived. A number of surprisingly diverse lineages of S. cerevisiae from both primeval and secondary forests in China have been discovered by Wang and his colleagues. These lineages substantially expand our knowledge of wild yeast diversity and will be a boon to elucidating the ecology, evolution and domestication of this academic and industrial workhorse. PMID:23281494

  10. 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

  11. Conformational switch-mediated accelerated release of drug from cytosine-rich nucleic acid-capped magnetic nanovehicles.

    PubMed

    Liu, Zhen; Huang, Yanyan; Pu, Fang; Ren, Jinsong; Qu, Xiaogang

    2016-02-16

    A novel concept that the conformational switch of cytosine-rich DNA can accelerate the release of drug from DNA-capped nanovehicles is rationally devised. Our present strategy can greatly extend the potential usages of DNA molecules with specific sequences as conformational switch-controlled devices. PMID:26879607

  12. 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.

  13. A study of the excited states in cytosine and guanine stacks in the Hartree-Fock and exciton approximations.

    PubMed

    Grobelsek-Vracko, M; Zaider, M

    1994-04-01

    We report calculated exciton energies for the cytosine and guanine stacks obtained in the ab initio Hartree-Fock crystal orbital and exciton approximation, which includes the excited electron-hole interaction. This interaction plays an important role in the description of excited electron spectra in the low-energy region. The stacks were chosen as examples of polymers with helical symmetry. PMID:8146296

  14. Metal Ion Induced Pairing of Cytosine Bases: Formation of I-Motif Structures Identified by IR Ion Spectroscopy

    NASA Astrophysics Data System (ADS)

    Gao, Juehan; Berden, Giel; Oomens, J.

    2015-06-01

    While the Watson-Crick structure of DNA is among the most well-known molecular structures of our time, alternative base-pairing motifs are also known to occur, often depending on base sequence, pH, or presence of cations. Pairing of two cytosine (C) bases induced by the sharing of a single proton (C-H^+-C) gives rise to the so-called i-motif, occurring particularly in the telomeric region of DNA, and particularly at low pH. At physiological pH, silver cations were recently suggested to form cytosine dimers in a C-Ag^+-C structure analogous to the hemiprotonated cytosine dimer, which was later confirmed by IR spectroscopy.^1 Here we investigate whether Ag^+ is unique in this behavior. Using infrared action spectroscopy employing the free-electron laser FELIX and a tandem mass spectrometer in combination with quantum-chemical computations, we investigate a series of C-M^+-C complexes, where M is Cu, Li and Na. The complexes are formed by electrospray ionization (ESI) from a solution of cytosine and the metal chloride salt in acetonitrile/water. The complexes of interest are mass-isolated in the cell of a FT ion cyclotron resonance mass spectrometer, where they are irradiated with the tunable IR radiation from FELIX in the 600 - 1800 wn range. Spectra in the H-stretching range are obtained with a LaserVision OPO. Both experimental spectra as well as theoretical calculations indicate that while Cu behaves as Ag, the alkali metal ions induce a clearly different dimer structure, in which the two cytosine units are parallelly displaced. In addition to coordination to the ring nitrogen atom, the alkali metal ions coordinate to the carbonyl oxygen atoms of both cytosine bases, indicating that the alkali metal ion coordination favorably competes with hydrogen bonding between the two cytosine sub-units of the i-motif like structure. 1. Berdakin, Steinmetz, Maitre, Pino, J. Phys. Chem. A 2014, 118, 3804

  15. Ultrafast repair of irradiated DNA: Nonadiabatic ab initio simulations of the guanine-cytosine photocycle

    NASA Astrophysics Data System (ADS)

    Markwick, Phineus R. L.; Doltsinis, Nikos L.

    2007-05-01

    Nonadiabatic first-principles molecular dynamics simulations have been performed of the photoexcited Watson-Crick guanine-cytosine (GC) DNA base pair in the gas phase and in aqueous solution. An excited state coupled proton-electron transfer (CPET) from G to C along the central hydrogen bond is observed upon excitation of the ??* state initially localized on G. In the resulting charge transfer state a conical intersection between the excited state and the ground state is easily accessible. Therefore radiationless decay is fast, of the order of 100fs, followed by a rapid CPET back reaction retrieving the initial Watson-Crick structure. A detailed analysis of the mechanism of nonradiative decay suggests a biexponential behavior in which out-of-plane motion plays a special role for the longer decay component.

  16. Absolute cross sections for vibrational excitations of cytosine by low energy electron impact

    PubMed Central

    Michaud, M.; Bazin, M.; Sanche, L.

    2013-01-01

    The absolute cross sections (CSs) for vibrational excitations of cytosine by electron impact between 0.5 and 18 eV were measured by electron-energy loss (EEL) spectroscopy of the molecule deposited at monolayer coverage on an inert Ar substrate. The vibrational energies compare to those that have been reported from IR spectroscopy of cytosine isolated in Ar matrix, IR and Raman spectra of poly-crystalline cytosine, and ab initio calculation. The CSs for the various H bending modes at 142 and 160 meV are both rising from their energy threshold up to 1.7 and 2.1 × 10−17 cm2 at about 4 eV, respectively, and then decrease moderately while maintaining some intensity at 18 eV. The latter trend is displayed as well for the CS assigned to the NH2 scissor along with bending of all H at 179 meV. This overall behavior in electron-molecule collision is attributed to direct processes such as the dipole, quadrupole, and polarization contributions, etc. of the interaction of the incident electron with a molecule. The CSs for the ring deformation at 61 meV, the ring deformation with N-H symmetric wag at 77 meV, and the ring deformations with symmetric bending of all H at 119 meV exhibit common enhancement maxima at 1.5, 3.5, and 5.5 eV followed by a broad hump at about 12 eV, which are superimposed on the contribution due to the direct processes. At 3.5 eV, the CS values for the 61-, 77-, and 119-meV modes reach 4.0, 3.0, and 4.5 × 10−17 cm2, respectively. The CS for the C-C and C-O stretches at 202 meV, which dominates in the intermediate EEL region, rises sharply until 1.5 eV, reaches its maximum of 5.7 × 10−17 cm2 at 3.5 eV and then decreases toward 18 eV. The present vibrational enhancements, correspond to the features found around 1.5 and 4.5 eV in electron transmission spectroscopy (ETS) and those lying within 1.5–2.1 eV, 5.2–6.8 eV, and 9.5–10.9 eV range in dissociative electron attachment (DEA) experiments with cytosine in gas phase. While the ETS features are ascribed to shape resonances associated with the electron occupation of the second and third antibonding π-orbitals of the molecule in its ground state, the correspondence with DEA features suggests the existence of common precursor anion states decaying with certain probabilities into the vibrationally excited ground state. PMID:22998289

  17. Systematic detection of hidden complexities in the unfolding mechanism of a cytosine-rich DNA strand

    NASA Astrophysics Data System (ADS)

    Smiatek, Jens; Janssen-Mller, Daniel; Friedrich, Rudolf; Heuer, Andreas

    2014-01-01

    We investigate the unfolding pathway of a cytosine-rich DNA structure via molecular dynamics simulations. By the study of the essential dynamics, we are able to identify a hidden complexity in the description of the dynamics in terms of the first two eigenvectors which are used as collective variables. This complexity can be mainly explained by non-Gaussian fluctuations due to contributions arising from the disregarded set of eigenvectors. We introduce the local non-Gaussian parameter as a tool for the detection of hidden complexities. The usage of this parameter allows a fast and reliable investigation for the determination of the important minimal number of eigenvectors which is needed for a sufficient description of molecular unfolding motion.

  18. Semiclassical dynamics of electron attachment to guanine-cytosine base pair

    NASA Astrophysics Data System (ADS)

    Honda, Tomohiro; Minoshima, Yusuke; Yokoi, Yuki; Takayanagi, Toshiyuki; Shiga, Motoyuki

    2015-04-01

    Electron attachment dynamics to the guanine-cytosine (G-C) base pair in the gas phase is studied using DFT and molecular dynamics. The potential energy surface of the G-C anion is constructed with the empirical-valence-bond method using force-field information obtained from long-range corrected DFT calculations. Ring-polymer molecular dynamics simulations predict that the initial dipole-bound anion readily converts into the valence-bound anion within 0.1 ps and proton-transfer occurs subsequently within 10 ps. The same process was found in classical simulations, but on a much slower time scale. This result suggests that nuclear quantum effects are important in understanding DNA damage by low-energy electrons.

  19. Mapping global changes in nuclear cytosine base modifications in the early mouse embryo

    PubMed Central

    Li, Y; Seah, Michelle K Y; O'Neill, C

    2016-01-01

    Reprogramming epigenetic modifications to cytosine is required for normal embryo development. We used improved immunolocalization techniques to simultaneously map global changes in the levels of 5′-methylcytosine (5meC) and 5′-hydroxymethylcytosine (5hmC) in each cell of the embryo from fertilization through the first rounds of cellular differentiation. The male and female pronuclei of the zygote showed similar staining levels, and these remained elevated over the next three cell cycles. The inner cells of the morula showed a progressive reduction in global levels of both 5meC and 5hmC and further losses occurred in the pluripotent inner cell mass (ICM) of the blastocyst. This was accompanied by undetectable levels of DNA methyltransferase of each class in the nuclei of the ICM, while DNA methyltransferase 3B was elevated in the hypermethylated nuclei of the trophectoderm (TE). Segregation of the ICM into hypoblast and epiblast was accompanied by increased levels in the hypoblast compared with the epiblast. Blastocyst outgrowth in vitro is a model for implantation and showed that a demethylated state persisted in the epiblast while the hypoblast had higher levels of both 5meC and 5hmC staining. The high levels of 5meC and 5hmC evident in the TE persisted in trophoblast and trophoblast giant cells after attachment of the blastocyst to the substratum in vitro. This study shows that global cytosine hypomethylation and hypohydroxymethylation accompanied the formation of the pluripotent ICM and this persisted into the epiblast after blastocyst outgrowth, and each differentiated lineage formed in the early embryo showed higher global levels of 5meC and 5hmC. PMID:26660107

  20. Singlet excited-state dynamics of 5-fluorocytosine and cytosine: An experimental and computational study

    PubMed Central

    Blancafort, Llus; Cohen, Boiko; Hare, Patrick M.; Kohler, Bern; Robb, Michael A.

    2005-01-01

    The photophysics of singlet excited 5-fluorocytosine (5FC) was studied in steady-state and time-resolved experiments and theoretically by quantum chemical calculations. Femtosecond transient absorption measurements show that replacement of the C5 hydrogen of cytosine by fluorine increases the excited-state lifetime by two orders of magnitude from 720 fs to 73 4 ps. Experimental evidence indicates that emission in both compounds originates from a single tautomeric form. The lifetime of 5FC is the same within experimental uncertainty in the solvents ethanol and dimethylsulfoxide. The insensitivity of the S1 lifetime to the protic nature of the solvent suggests that proton transfer is not the principal quenching mechanism for the excited state. Excited state calculations were carried out for the amino-keto tautomer of 5FC, the dominant species in polar environments, in order to understand its longer excited-state lifetime. CASSCF and CAS-PT2 calculations of the excited states show that the minimum energy path connecting the minimum of the 1?, ?* state with the conical intersection responsible for internal conversion has essentially the same energetics for cytosine and 5FC, suggesting that both bases decay nonradiatively by the same mechanism. The dramatic difference in lifetimes may be due to subtle changes along the decay coordinate. A possible reason may be differences in the intramolecular vibrational redistribution rate from the Franck-Condon active, in-plane modes to the out-of-plane modes that must be activated to reach the conical intersection region. PMID:16833777

  1. Mapping global changes in nuclear cytosine base modifications in the early mouse embryo.

    PubMed

    Li, Y; Seah, Michelle K Y; O'Neill, C

    2016-02-01

    Reprogramming epigenetic modifications to cytosine is required for normal embryo development. We used improved immunolocalization techniques to simultaneously map global changes in the levels of 5'-methylcytosine (5meC) and 5'-hydroxymethylcytosine (5hmC) in each cell of the embryo from fertilization through the first rounds of cellular differentiation. The male and female pronuclei of the zygote showed similar staining levels, and these remained elevated over the next three cell cycles. The inner cells of the morula showed a progressive reduction in global levels of both 5meC and 5hmC and further losses occurred in the pluripotent inner cell mass (ICM) of the blastocyst. This was accompanied by undetectable levels of DNA methyltransferase of each class in the nuclei of the ICM, while DNA methyltransferase 3B was elevated in the hypermethylated nuclei of the trophectoderm (TE). Segregation of the ICM into hypoblast and epiblast was accompanied by increased levels in the hypoblast compared with the epiblast. Blastocyst outgrowth in vitro is a model for implantation and showed that a demethylated state persisted in the epiblast while the hypoblast had higher levels of both 5meC and 5hmC staining. The high levels of 5meC and 5hmC evident in the TE persisted in trophoblast and trophoblast giant cells after attachment of the blastocyst to the substratum in vitro. This study shows that global cytosine hypomethylation and hypohydroxymethylation accompanied the formation of the pluripotent ICM and this persisted into the epiblast after blastocyst outgrowth, and each differentiated lineage formed in the early embryo showed higher global levels of 5meC and 5hmC. PMID:26660107

  2. High Dose Cytosine Arabinoside in the consolidation of adult acute myeloid leukemia.

    PubMed

    Rahman, M H; Khan, M A; Islam, M S; Afrose, S; Ara, T

    2012-04-01

    This interventional study was done to evaluate the duration of remission with High Dose Cytosine Arabinoside (Ara-C) as post-remission chemotherapy in the consolidation of adult acute myeloid leukemia. A total of 32 patients were included in this study. Among them, 19 were male and 13 were female and the age of the patients ranges from 15-60 years. We use High Dose Cytosine Arabinoside 1.5-2.5 g/m2 i.v, 12 hourly, over 2-3 hours on day 1, 3 and 5 in a 28 days cycle. This study was done during the period of April 2007 to March 2009 in the department of hematology, Dhaka Medical College & Hospital. History, clinical features and laboratory investigations were included. Among 32 patients, 5 patients (15.6%) received one cycle, 20 patients (62.5%) received two cycles and 7 patients (21.9%) received three cycles. The mean SD duration of remission (disease free survival) was 5.20 3.83 months who received one cycle, 9.55 3.30 months and 10.71 1.70 months who received two cycles and three cycles respectively. The adverse effects of the therapy were neutropenia and neutropenic fever, purpuric rash, gum bleeding, mucositis and peripheral neuropathy. The supportive materials needed were antibiotics (both prophylactic and treatment) 86.13%, blood and blood products 51.7% and G-CSF 14.9% patients of all cycles. High Dose Ara-C (HiDAC) is a safe and cost effective consolidation treatment for AML patients in complete remission. This therapy merits multi-center control study to define its efficacy and cost-effectiveness in contrast to our socio-economic condition. PMID:22561761

  3. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...) may be safely used in food provided the total folic acid content of the yeast does not exceed 0.04 milligram per gram of yeast (approximately 0.008 milligram of pteroyglutamic acid per gram of yeast)....

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

    PubMed

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

    2015-11-01

    Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalisisolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival. PMID:26517498

  5. Sequence requirements for transcriptional arrest in exon 1 of the human adenosine deaminase gene

    SciTech Connect

    Zhi Chen; Kellems, R.E.; Innis, J.W. ); Sun, Minghua; Wright, D.A. )

    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.

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

    PubMed Central

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

    2015-01-01

    Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalis isolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival. PMID:26517498

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

    PubMed

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

    2016-04-01

    Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria, AIP) is characterized by neurovisceral attacks when hepatic heme synthesis is activated by endogenous or environmental factors including fasting. While the molecular mechanisms underlying the nutritional regulation of hepatic heme synthesis have been described, glucose homeostasis during fasting is poorly understood in porphyria. Our study aimed to analyse glucose homeostasis and hepatic carbohydrate metabolism during fasting in PBGD-deficient mice. To determine the contribution of hepatic PBGD deficiency to carbohydrate metabolism, AIP mice injected with a PBGD-liver gene delivery vector were included. After a 14 h fasting period, serum and liver metabolomics analyses showed that wild-type mice stimulated hepatic glycogen degradation to maintain glucose homeostasis while AIP livers activated gluconeogenesis and ketogenesis due to their inability to use stored glycogen. The serum of fasted AIP mice showed increased concentrations of insulin and reduced glucagon levels. Specific over-expression of the PBGD protein in the liver tended to normalize circulating insulin and glucagon levels, stimulated hepatic glycogen catabolism and blocked ketone body production. Reduced glucose uptake was observed in the primary somatosensorial brain cortex of fasted AIP mice, which could be reversed by PBGD-liver gene delivery. In conclusion, AIP mice showed a different response to fasting as measured by altered carbohydrate metabolism in the liver and modified glucose consumption in the brain cortex. Glucose homeostasis in fasted AIP mice was efficiently normalized after restoration of PBGD gene expression in the liver. PMID:26908609

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

    PubMed Central

    BRAVO-TOBAR, Ivn Daro; NELLO-PREZ, Carlota; FERNNDEZ, Al; MOGOLLN, Nora; PREZ, Mary Carmen; VERDE, Juan; CONCEPCIN, Juan Luis; RODRIGUEZ-BONFANTE, Claudina; BONFANTE-CABARCAS, Rafael

    2015-01-01

    SUMMARY Chagas disease is a public health problem worldwide. The availability of diagnostic tools to predict the development of chronic Chagas cardiomyopathy is crucial to reduce morbidity and mortality. Here we analyze the prognostic value of adenosine deaminase serum activity (ADA) and C-reactive protein serum levels (CRP) in chagasic individuals. One hundred and ten individuals, 28 healthy and 82 chagasic patients were divided according to disease severity in phase I (n = 35), II (n = 29), and III (n = 18). A complete medical history, 12-lead electrocardiogram, chest X-ray, and M-mode echocardiogram were performed on each individual. Diagnosis of Chagas disease was confirmed by ELISA and MABA using recombinant antigens; ADA was determined spectrophotometrically and CRP by ELISA. The results have shown that CRP and ADA increased linearly in relation to disease phase, CRP being significantly higher in phase III and ADA at all phases. Also, CRP and ADA were positively correlated with echocardiographic parameters of cardiac remodeling and with electrocardiographic abnormalities, and negatively with ejection fraction. CRP and ADA were higher in patients with cardiothoracic index ? 50%, while ADA was higher in patients with ventricular repolarization disturbances. Finally, CRP was positively correlated with ADA. In conclusion, ADA and CRP are prognostic markers of cardiac dysfunction and remodeling in Chagas disease. PMID:26603224

  9. Heme-Biosynthetic Porphobilinogen Deaminase Protects Aspergillus nidulans from Nitrosative Stress

    PubMed Central

    Zhou, Shengmin; Narukami, Toshiaki; Nameki, Misuzu; Ozawa, Tomoko; Kamimura, Yosuke; Hoshino, Takayuki

    2012-01-01

    Microorganisms have developed mechanisms to combat reactive nitrogen species (RNS); however, only a few of the fungal genes involved have been characterized. Here we screened RNS-resistant Aspergillus nidulans strains from fungal transformants obtained by introducing a genomic DNA library constructed in a multicopy vector. We found that the AN0121.3 gene (hemC) encodes a protein similar to the heme biosynthesis enzyme porphobilinogen deaminase (PBG-D) and facilitates RNS-tolerant fungal growth. The overproduction of PBG-D in A. nidulans promoted RNS tolerance, whereas PBG-D repression caused growth that was hypersensitive to RNS. PBG-D levels were comparable to those of cellular protoheme synthesis as well as flavohemoglobin (FHb; encoded by fhbA and fhbB) and nitrite reductase (NiR; encoded by niiA) activities. Both FHb and NiR are hemoproteins that consume nitric oxide and nitrite, respectively, and we found that they are required for maximal growth in the presence of RNS. The transcription of hemC was upregulated by RNS. These results demonstrated that PBG-D is a novel NO-tolerant protein that modulates the reduction of environmental NO and nitrite levels by FHb and NiR. PMID:22038601

  10. Trypanosoma evansi: adenosine deaminase activity in the brain of infected rats.

    PubMed

    Da Silva, Aleksandro S; Bell, Luziane P; Bitencourt, Paula E R; Perez, Herakles A Garcia; Thom, 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

  11. Activity of ectonucleotidases and adenosine deaminase in rats exposed to cigarette smoke.

    PubMed

    Thom, G R; Mazzanti, C M; Ahmed, M; Corra, M; Spanevello, R M; Maldonado, P A; Luchese, C; Cargnelutti, D; Morsch, V M; Duarte, M M M F; Fiorenza, A M; Nogueira, C W; De Bona, K S; Moretto, M B; Da Luz, S C A; Mazzanti, A; Schetinger, M R C

    2009-09-01

    Cigarette smoke is a complex mixture of various toxic substances that are capable of initiating oxidative damage and promoting blood platelet alterations. In this study, we investigated the activities of the ectoenzymes NTPDase (ectonucleoside triphosphate diphosphohydrolase, CD39) and 5'-nucleotidase (CD73) in platelets as well as adenosine deaminase (ADA) in the plasma of rats exposed to aged and diluted sidestream smoke during 4 weeks. The rats were divided into two groups: I (control) and II (exposed to smoke). After the exposure period, blood was collected and the platelets and plasma were separated for enzymatic assay. The results demonstrated that NTPDase (with ATP as substrate) and 5'-nucleotidase (AMP as substrate) activities were significantly higher in group II (p < 0.05) as compared to group I, while no significant difference was observed for NTPDase with ADP as substrate. The ADA activity was significantly reduced in group II (p < 0.05) as compared with group I. Platelet aggregation was significantly increased in group II (p < 0.05) as compared with group I. We suggest that these alterations in the activity of enzymes from the purinergic system are associated with an increase in platelet aggregation. However, our study has demonstrated that the organism tries to compensate for this enhanced aggregation by increasing hydrolysis of AMP and reducing hydrolysis of adenosine, a potent inhibitor of aggregation and an important modulator of vascular tone. PMID:19459774

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  13. Human ADA2 belongs to a new family of growth factors with adenosine deaminase activity

    PubMed Central

    2005-01-01

    Two distinct isoenzymes of ADA (adenosine deaminase), ADA1 and ADA2, have been found in humans. Inherited mutations in ADA1 result in SCID (severe combined immunodeficiency). This observation has led to extensive studies of the structure and function of this enzyme that have revealed an important role for it in lymphocyte activation. In contrast, the physiological role of ADA2 is unknown. ADA2 is found in negligible quantities in serum and may be produced by monocytes/macrophages. ADA2 activity in the serum is increased in various diseases in which monocyte/macrophage cells are activated. In the present study, we report that ADA2 is a heparin-binding protein. This allowed us to obtain a highly purified enzyme and to study its biochemistry. ADA2 was identified as a member of a new class of ADGFs (ADA-related growth factors), which is present in almost all organisms from flies to humans. Our results suggest that ADA2 may be active in sites of inflammation during hypoxia and in areas of tumour growth where the adenosine concentration is significantly elevated and the extracellular pH is acidic. Our finding that ADA2 co-purified and concentrated together with IgG in commercially available preparations offers an intriguing explanation for the observation that treatment with such preparations leads to non-specific immune-system stimulation. PMID:15926889

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

    PubMed

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

    2015-01-01

    Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ∼ 5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer. PMID:26681117

  15. 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

  16. PKA-mediated phosphorylation regulates the function of activation-induced deaminase (AID) in B cells.

    PubMed

    Pasqualucci, Laura; Kitaura, Yasuyuki; Gu, Hua; Dalla-Favera, Riccardo

    2006-01-10

    During humoral immune responses, two distinct genetic modification events diversify the Ig genes in germinal center (GC) B cells: somatic hypermutation and class switch recombination (CSR). Both processes require the activity of activation-induced cytidine deaminase (AID), an enzyme expressed specifically in GC B cells. However, the mechanisms that regulate AID activity are largely unknown. Here we report that protein kinase A (PKA) phosphorylates AID and regulates its activity in GC B cells. AID physically interacts with the PKA holoenzyme in the cytoplasm and is phosphorylated by the PKA catalytic subunit at specific residues. AID phosphorylation is required for CSR, because substitution of the two phosphorylation targets impairs its ability to rescue CSR in AID-deficient B cells. Pharmacologic inhibition of PKA prevents isotype class switching in a murine B-cell lymphoma cell line; conversely, B cells from mice where PKA activity is made constitutive by conditional deletion of the PKA regulatory subunit gene display enhanced CSR. These findings implicate PKA in the regulation of AID function and suggest that the control of T cell-dependent immune responses may be modulated, via AID, by signals that activate PKA. PMID:16387847

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

    SciTech Connect

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

    2011-11-07

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

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

    PubMed Central

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

    2015-01-01

    Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ∼5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer. PMID:26681117

  19. Myoadenylate deaminase deficiency does not affect muscle anaplerosis during exhaustive exercise in humans

    PubMed Central

    Tarnopolsky, Mark A; Parise, Gianni; Gibala, Martin J; Graham, Terry E; Rush, James W E

    2001-01-01

    Myoadenylate deaminase (AMPD) deficiency is present in 1–2 % of the population. In theory, this deficiency may alter exercise energy metabolism by impairing the purine nucleotide cycle (PNC) and reducing tricarboxylic acid (TCA) cycle anaplerosis. The role of the PNC in TCA cycle anaplerosis is still a debated issue in physiology. Using patients with the AMPD1 mutation will allow a human ‘knockout’ approach to answering this question. Muscle AMPD activity and genotype (whole blood AMPD1 analysis) was used to classify participants into three groups: n = 3 with absence of AMPD activity and -/- AMPD1 genotype (homozygous); n = 4 with less than 50 % normal AMPD activity and +/- genotype (heterozygous) and n = 12 with normal AMPD activity and +/+ genotype (control). Biopsies were taken from the vastus lateralis muscle before and after incremental cycle ergometry exercise to exhaustion. The muscle biopsies were analysed for AMPD activity, purine nucleotides/nucleosides and bases, creatine, phosphocreatine, amino acids, and the TCA cycle intermediates malate, citrate and fumarate. Time to exhaustion on the cycle ergometer was not different between groups. Muscle adenosine monophosphate increased significantly with exercise for homozygous subjects as compared with the other groups (P < 0.05). Inosine monophosphate increased significantly after exercise for control (P < 0.05) but not for the homozygous subjects. There were no other between-group differences for any other measured variables. In summary, complete and partial muscle AMPD deficiency did not affect TCA cycle anaplerosis, phosphocreatine hydrolysis, energy charge or exercise performance. PMID:11410643

  20. Abrogation of lupus nephritis in Activation-Induced Deaminase-deficient MRL/lpr Mice1

    PubMed Central

    Jiang, Chuancang; Foley, Julie; Clayton, Natasha; Kissling, Grace; Jokinen, Micheal; Herbert, Ronald; Diaz, Marilyn

    2007-01-01

    We generated MRL/lpr mice deficient in the Activation Induced Deaminase (AID). Because AID is required for immunoglobulin hypermutation and class switch recombination, these mice lack hypermutated IgG antibodies. Unlike their AID wild-type littermates, AID-deficient MRL/lpr mice not only lacked autoreactive IgG antibodies, but also experienced a dramatic increase in the levels of autoreactive IgM. This phenotype in AID-deficient mice translated into a dramatic reduction in glomerulonephritis, minimal mononuclear cell infiltration in the kidney, and a dramatic increase in survival to levels comparable to previously reported for MRL/lpr mice completely lacking B cells and levels well below those of mice lacking secreted antibodies. Therefore, this study, wherein littermates with either high levels of autoreactive IgM or autorective IgG are directly examined, proves that autoreactive IgM antibodies alone are not sufficient to promote kidney disease in MRL/lpr mice. In addition, the substantial decrease in mortality combined with a dramatic increase in autoreactive IgM antibodies in AID-deficient MRL/lpr mice, suggest that autoreactive IgM antibodies might not only fail to promote nephritis, but may also provide a protective role in MRL/lpr mice. This novel mouse model containing high levels of autoreactive, unmutated IgM antibodies will help delineate the contribution of autoreactive IgM to autoimmunity. PMID:17513793

  1. 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 (μ-aqua)(μ-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

  2. A source of the single stranded DNA substrate for activation-induced deaminase during somatic hypermutation

    PubMed Central

    Wang, Xiaohua; Fan, Manxia; Kalis, Susan; Wei, Lirong; Scharff, Matthew D.

    2014-01-01

    During somatic hypermutation (SHM), activation-induced deaminase (AID) mutates deoxycytidine on single-stranded DNA (ssDNA) generated by the transcription machinery, but the detailed mechanism remains unclear. Here we report a higher abundance of RNA polymerase II (Pol II) at the immunoglobulin heavy chain variable (Igh-V) region compared to the constant region and partially transcribed Igh RNAs, suggesting a slower Pol II progression at Igh-V that could result in some early/premature transcription termination after prolonged pausing/stalling of Pol II. Knocking down RNA exosome complexes, which could decrease premature transcription termination, leads to decreased SHM. Knocking down Spt5, which can augment premature transcription termination, leads to increases in both SHM and the abundance of ssDNA substrates. Collectively, our data support the model that, following the reduction of Pol II progression (pausing or stalling) at the Igh-V, additional steps such as premature transcription termination are involved in providing ssDNA substrates for AID during SHM. PMID:24923561

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

    PubMed

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

    2008-12-01

    Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture to study ADA-deficient human thymocyte development in an "in vivo-like" environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular dATP. Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients. PMID:19018008

  4. 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

  5. Adenosine deaminase deficiency as the first target disorder in gene therapy.

    PubMed

    Onodera, M; Sakiyama, Y

    2000-03-01

    In the past decade, the advent of gene therapy has been acclaimed as a revolutionary medical intervention, embraced with great enthusiasm. However, recent disappointing results of the considerable clinical trials have also clearly demonstrated that such an initial expectation was an overestimation of gene therapy. There are only a few successful cases despite the 3000 patients who have been treated with various forms of gene therapy. Gene therapy for severe combined immunodeficiency (SCID) caused by adenosine deaminase (ADA) deficiency is one of the few such cases where results have been promising. In particular, peripheral T-lymphocytes-directed gene therapy provides further immunological improvements for patients with ADA-SCID receiving the PEG-ADA treatment whereas gene therapy targeting haematopoietic stem cell has so far proved insufficient for clinical benefits. This report will review crucial problems elucidated in the past five clinical trials for ADA-SCID and gives an outline of the next generation of stem cell gene therapy in Japan. PMID:11060694

  6. Visible integration of the adenosine deaminase (ADA) gene into the recipient genome after gene therapy.

    PubMed

    Egashira, M; Ariga, T; Kawamura, N; Miyoshi, O; Niikawa, N; Sakiyama, Y

    1998-01-23

    Gene therapy for patients with adenosine deaminase (ADA) deficiency has become practical in the 1990s, and the exogenous gene has been reported to survive for several years in the recipient genome. To evaluate the integration efficiency of the ADA gene (ADA) into peripheral blood lymphocytes (PBL) of a patient with ADA deficiency who is receiving gene therapy, we performed two-color interphase fluorescence in situ hybridization (FISH) analysis by using digoxigenin-labeled ADA-cDNA and the biotin-labeled lambda-genomic ADA clone as probes. After each of 9 sequential series of gene therapy, interphase nuclei of 100 mononuclear cells from the patient were analyzed, and those of a LASN-producing cell line were used as a control. FISH signals were detected with rhodamine and FITC for the cDNA and the genomic DNA, respectively. The number of PBL giving a transgene signal grew after the sequential gene therapies, and the proportion of signal-positive cells reached about 10%. Our results indicate that the two-color FISH system can be used as a potential aid to monitor the efficiency of the ADA gene therapy. PMID:9475605

  7. 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

  8. Expression of activation-induced cytidine deaminase in oral epithelial dysplasia and oral squamous cell carcinoma.

    PubMed

    Miyazaki, Yuji; Fujinami, Masahiro; Inoue, Harumi; Kikuchi, Kentaro; Ide, Fumio; Kusama, Kaoru

    2013-01-01

    Oral epithelial dysplasia is thought to be a precursor state of carcinogenesis and may harbor gene alterations. Recently, it was reported that gene editing enzyme, activation-induced cytidine deaminase (AID), is expressed in precursor and cancer epithelial cells during carcinogenesis associated with chronic inflammation/infection and that this enzyme induces mutation of tumor-suppressor genes. Thus, AID may have a role in carcinogenesis via oral epithelial dysplasia. In this study, we classified oral mucosal epithelium exhibiting epithelial dysplasia as squamous intraepithelial neoplasia (SIN) grades 1-3, according to the 2005 World Health Organization classification, and used immunohistochemical techniques to examine AID expression in oral mucosal epithelium exhibiting SIN and oral cancer tissues. AID was observed in prickle cells in oral mucosal epithelium with epithelial dysplasia and in oral cancer cells. Additionally, to investigate the mechanism of AID expression and its role in cancer progression, we incubated the oral cancer cell line HSC-2 with inflammatory cytokines. In the HSC-2 cell line, AID expression was enhanced by TNF-? via NF-?B activation and promoted expression of N-cadherin by regulating Snail expression. These findings suggest that AID has a role in the development of oral epithelial dysplasia and promotes progression of oral cancer. PMID:24351917

  9. Integrase-defective lentiviral vectors as a delivery platform for targeted modification of adenosine deaminase locus.

    PubMed

    Joglekar, Alok V; Hollis, Roger P; Kuftinec, Gabriela; Senadheera, Shantha; Chan, Rebecca; Kohn, Donald B

    2013-09-01

    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

  10. Expression of activation-induced cytidine deaminase in oral epithelial dysplasia and oral squamous cell carcinoma.

    TOXLINE Toxicology Bibliographic Information

    Miyazaki Y; Fujinami M; Inoue H; Kikuchi K; Ide F; Kusama K

    2013-01-01

    Oral epithelial dysplasia is thought to be a precursor state of carcinogenesis and may harbor gene alterations. Recently, it was reported that gene editing enzyme, activation-induced cytidine deaminase (AID), is expressed in precursor and cancer epithelial cells during carcinogenesis associated with chronic inflammation/infection and that this enzyme induces mutation of tumor-suppressor genes. Thus, AID may have a role in carcinogenesis via oral epithelial dysplasia. In this study, we classified oral mucosal epithelium exhibiting epithelial dysplasia as squamous intraepithelial neoplasia (SIN) grades 1-3, according to the 2005 World Health Organization classification, and used immunohistochemical techniques to examine AID expression in oral mucosal epithelium exhibiting SIN and oral cancer tissues. AID was observed in prickle cells in oral mucosal epithelium with epithelial dysplasia and in oral cancer cells. Additionally, to investigate the mechanism of AID expression and its role in cancer progression, we incubated the oral cancer cell line HSC-2 with inflammatory cytokines. In the HSC-2 cell line, AID expression was enhanced by TNF-? via NF-?B activation and promoted expression of N-cadherin by regulating Snail expression. These findings suggest that AID has a role in the development of oral epithelial dysplasia and promotes progression of oral cancer.

  11. 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.

  12. 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

  13. Adenosine deaminase is a useful biomarker to diagnose pleural tuberculosis in low to medium prevalence settings.

    PubMed

    Michot, Jean-Marie; Madec, Yoann; Bulifon, Sophie; Thorette-Tcherniak, Ccile; Fortineau, Nicolas; Nol, Nicolas; Lambotte, Olivier; El Jahiri, Younes; Delacour, Herv; Delfraissy, Jean-Franois; Blanc, Franois-Xavier

    2016-03-01

    Adenosine deaminase (ADA) activity measurement in pleural fluid is a relevant test to diagnose pleural tuberculosis (pTB) in high tuberculosis prevalence settings. We investigated the diagnostic utility of pleural ADA using a retrospective analysis of patients admitted with newly diagnosed pleural effusion without identified etiology between 2001 and 2008 in Paris suburb, a low to medium tuberculosis prevalence area. 104 adults (mean age 55 years; 34 with pTB, 70 with other diagnoses) were analyzed. Median follow-up was 15.6 months. Mean [interquartile range] pleural ADA was 119 U/L [IQR: 83-143] in pTB and 24 U/L [IQR: 15-31] in non-tuberculous effusions (P<0.001). With an optimal pleural ADA cut-off value of 41.5 U/L for pTB diagnosis, sensitivity and specificity were 97.1% and 92.9%, while positive and negative predictive values were 86.8% and 98.5%, respectively. We conclude that pleural ADA activity could be integrated in the diagnostic procedures of pTB in low to medium tuberculosis prevalence settings. PMID:26707067

  14. 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. 88:16881694. 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

  15. Ethanol tolerance in yeasts.

    PubMed

    Casey, G P; Ingledew, W M

    1986-01-01

    It is now certain that the inherent ethanol tolerance of the Saccharomyces strain used is not the prime factor regulating the level of ethanol that can be produced in a high sugar brewing, wine, sake, or distillery fermentation. In fact, in terms of the maximum concentration that these yeasts can produce under batch (16 to 17% [v/v]) or fed-batch conditions, there is clearly no difference in ethanol tolerance. This is not to say, however, that under defined conditions there is no difference in ethanol tolerance among different Saccharomyces yeasts. This property, although a genetic determinant, is clearly influenced by many factors (carbohydrate level, wort nutrition, temperature, osmotic pressure/water activity, and substrate concentration), and each yeast strain reacts to each factor differently. This will indeed lead to differences in measured tolerance. Thus, it is extremely important that each of these be taken into consideration when determining "tolerance" for a particular set of fermentation conditions. The manner in which each alcohol-related industry has evolved is now known to have played a major role in determining traditional thinking on ethanol tolerance in Saccharomyces yeasts. It is interesting to speculate on how different our thinking on ethanol tolerance would be today if sake fermentations had not evolved with successive mashing and simultaneous saccharification and fermentation of rice carbohydrate, if distillers' worts were clarified prior to fermentation but brewers' wort were not, and if grape skins with their associated unsaturated lipids had not been an integral part of red wine musts. The time is now ripe for ethanol-related industries to take advantage of these findings to improve the economies of production. In the authors' opinion, breweries could produce higher alcohol beers if oxygenation (leading to unsaturated lipids) and "usable" nitrogen source levels were increased in high gravity worts. White wine fermentations could also, if desired, match the higher ethanol levels in red wines if oxygenation (to provide the unsaturated lipids deleted in part by the removal of the grape skins) were practiced and if care were given to assimilable nitrogen concentrations. This would hold true even at 10 to 14 degrees C, and the more rapid fermentations would maximize utilization of winery tankage.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:3533426

  16. The Budding Yeast Nucleus

    PubMed Central

    Taddei, Angela; Schober, Heiko; Gasser, Susan M.

    2010-01-01

    The budding yeast nucleus, like those of other eukaryotic species, is highly organized with respect to both chromosomal sequences and enzymatic activities. At the nuclear periphery interactions of nuclear pores with chromatin, mRNA, and transport factors promote efficient gene expression, whereas centromeres, telomeres, and silent chromatin are clustered and anchored away from pores. Internal nuclear organization appears to be function-dependent, reflecting localized sites for tRNA transcription, rDNA transcription, ribosome assembly, and DNA repair. Recent advances have identified new proteins involved in the positioning of chromatin and have allowed testing of the functional role of higher-order chromatin organization. The unequal distribution of silent information regulatory factors and histone modifying enzymes, which arises in part from the juxtaposition of telomeric repeats, has been shown to influence chromatin-mediated transcriptional repression. Other localization events suppress unwanted recombination. These findings highlight the contribution budding yeast genetics and cytology have made to dissecting the functional role of nuclear structure. PMID:20554704

  17. Red yeast rice for dysipidemia.

    PubMed

    Shamim, Shariq; Al Badarin, Firas J; DiNicolantonio, James J; Lavie, Carl J; O'Keefe, James H

    2013-01-01

    Red yeast rice is an ancient Chinese food product that contains monacolins, chemical substances that are similar to statins in their mechanisms of action and lipid lowering properties. Several studies have found red yeast rice to be moderately effective at improving the lipid profile, particularly for lowering the low-density lipoprotein cholesterol levels. One large randomized controlled study from China found that red yeast rice significantly improved risk of major adverse cardiovascular events and overall survival in patients following myocardial infarction. Thus, red yeast rice is a potentially useful over-the-counter cholesterol-lowering agent. However, many red yeast rice formulations are non-standardized and unregulated food supplements, and there is a need for further research and regulation of production. PMID:24003656

  18. Brewer's yeast and sugarcane yeast as protein sources for dogs.

    PubMed

    Martins, M S; Sakomura, N K; Souza, D F; Filho, F O R; Gomes, M O S; Vasconcellos, R S; Carciofi, A C

    2014-10-01

    Brewer's yeast (BY), autolysed sugarcane yeast (ASCY) and integral sugar cane yeast (ISCY) were studied in two experiments as ingredients for dog diets. In the first experiment, 28 dogs were randomly assigned to four diets; one reference diet and three test diets containing 15% of BY, ASCY or ISCY and 85% of the reference diet (as-fed basis). The digestibilities of the yeasts were calculated by the substitution method. In the second experiment, 35 dogs were randomized to five diets with similar chemical composition but different levels of sugarcane yeast inclusion (0%, 7.5% ASCY, 15% ASCY, 7.5% ISCY and 15% ISCY). In both experiments, the coefficient of total tract apparent digestibility (CTTAD) of nutrients was determined through total collection of faeces. During experiment, two additional analyses of food palatability, nitrogen balance and urea postprandial responses were performed. The data were submitted to analysis of variance, and the means were compared by orthogonal or polynomial contrasts or Tukey's test (p<0.05). In experiment 1, CTTAD of protein was lower for both sugarcane yeasts than for BY (p=0.012), as was metabolizable energy content (p=0.025). In experiment 2, a linear reduction in energy digestibility with ASCY inclusion (p=0.05) was verified. Furthermore, faecal score and DM content were reduced with ISCY inclusion (p<0.003). No effect of yeast inclusion on nitrogen balance or postprandial urea response was found. Also, the inclusion of 7.5% of ASCY or ISCY increased diet palatability (p<0.01). Yeasts present adequate digestibility by dogs, but its effect on faecal formation needs to be considered. No clear advantage for the use of ASCY over ISCY was found. In conclusion, we find that sugarcane yeast is suitable for inclusion in dog food and can enhance the overall palatability of the diet. PMID:24304448

  19. Agriculturally important yeasts: Biological control of field and postharvest diseases using yeast antagonists, and yeasts as pathogens of plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two important agricultural aspects of yeasts, control of plant diseases through application of yeasts as the control agent, and yeasts that are plant pathogens are reviewed. Yeasts as biocontrol organisms are presented first, followed by a discussion of some of the more common plant pathogenic yeas...

  20. Absolute cross sections for electronic excitations of cytosine by low energy electron impact

    NASA Astrophysics Data System (ADS)

    Bazin, M.; Michaud, M.; Sanche, L.

    2010-10-01

    The absolute cross sections (CSs) for electronic excitations of cytosine by electron impact between 5 and 18 eV were measured by electron-energy-loss (EEL) spectroscopy of the molecule deposited at low coverage on an inert Ar substrate. The lowest EEL features found at 3.55 and 4.02 eV are ascribed to transitions from the ground state to the two lowest triplet 1 A3'(? ???) and 2 A3'(? ???) valence states of the molecule. Their energy dependent CSs exhibit essentially a common maximum at about 6 eV with a value of 1.8410-17 cm2 for the former and 4.9410-17 cm2 for the latter. In contrast, the CS for the next EEL feature at 4.65 eV, which is ascribed to the optically allowed transition to the 2 A1'(? ???) valence state, shows only a steep rise to about 1.0410-16 cm2 followed by a monotonous decrease with the incident electron energy. The higher EEL features at 5.39, 6.18, 6.83, and 7.55 eV are assigned to the excitations of the 3 A3,1'(? ???), 4 A1'(? ???), 5 A1'(? ???), and 6 A1'(? ???) valence states, respectively. The CSs for the 3 A3,1' and 4 A1' states exhibit a common enhancement at about 10 eV superimposed on a more or less a steep rise, reaching, respectively, a maximum of 1.27 and 1.7910-16 cm2, followed by a monotonous decrease. This latter enhancement and the maximum seen at about 6 eV in the lowest triplet states correspond to the core-excited electron resonances that have been found by dissociative electron attachment experiments with cytosine in the gas phase. The weak EEL feature found at 5.01 eV with a maximum CS of 3.810-18 cm2 near its excitation threshold is attributed to transitions from the ground state to the 1 A3,1?(n ???) states. The monotonous rise of the EEL signal above 8 eV is attributed to the ionization of the molecule. It is partitioned into four excitation energy regions at about 8.55, 9.21, 9.83, and 11.53 eV, which correspond closely to the ionization energies of the four highest occupied molecular orbitals of cytosine. The sum of the ionization CS for these four excitation regions reaches a maximum of 8.110-16 cm2 at the incident energy of 13 eV.

  1. Crystallization and preliminary X-ray crystallographic analysis of biodegradative threonine deaminase (TdcB) from Salmonella typhimurium

    SciTech Connect

    Simanshu, Dhirendra K.; Chittori, Sagar; Savithri, H. S.; Murthy, M. R. N.

    2006-03-01

    S. typhimurium biodegradative threonine deaminase (TdcB), a member of the β-family of PLP-dependent enzymes, has been overexpressed, purified and crystallized in three different crystal forms using the hanging-drop vapour-diffusion method. Biodegradative threonine deaminase (TdcB) catalyzes the deamination of l-threonine to α-ketobutyrate, the first reaction in the anaerobic breakdown of l-threonine to propionate. Unlike the biosynthetic threonine deaminase, TdcB is insensitive to l-isoleucine and is activated by AMP. Here, the cloning of TdcB (molecular weight 36 kDa) from Salmonella typhimurium with an N-terminal hexahistidine affinity tag and its overexpression in Escherichia coli is reported. TdcB was purified to homogeneity using Ni–NTA affinity column chromatography and crystallized using the hanging-drop vapour-diffusion technique in three different crystal forms. Crystal forms I (unit-cell parameters a = 46.32, b = 55.30, c = 67.24 Å, α = 103.09, β = 94.70, γ = 112.94°) and II (a = 56.68, b = 76.83, c = 78.50 Å, α = 66.12, β = 89.16, γ = 77.08°) belong to space group P1 and contain two and four molecules of TdcB, respectively, in the asymmetric unit. Poorly diffracting form III crystals were obtained in space group C2 and based on the unit-cell volume are most likely to contain one molecule per asymmetric unit. Two complete data sets of resolutions 2.2 Å (crystal form I) and 1.7 Å (crystal form II) were collected at 100 K using an in-house X-ray source.

  2. 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

  3. Studies on Plant Growth Promoting Properties of Fruit-Associated Bacteria from Elettaria cardamomum and Molecular Analysis of ACC Deaminase Gene.

    PubMed

    Jasim, B; Anish, Mathew Chacko; Shimil, Vellakudiyan; Jyothis, Mathew; Radhakrishnan, E K

    2015-09-01

    Endophytic microorganisms have been reported to have diverse plant growth promoting mechanisms including phosphate solubilization, N2 fixation, production of phyto-hormones and ACC (1-aminocyclopropane-1-carboxylate) deaminase and antiphyto-pathogenic properties. Among these, ACC deaminase production is very important because of its regulatory effect on ethylene which is a stress hormone with precise role in the control of fruit development and ripening. However, distribution of these properties among various endophytic bacteria associated with fruit tissue and its genetic basis is least investigated. In the current study, 11 endophytic bacteria were isolated and identified from the fruit tissue of Elettaria cardamomum and were studied in detail for various plant growth promoting properties especially ACC deaminase activity using both culture-based and PCR-based methods. PCR-based screening identified the isolates EcB 2 (Pantoea sp.), EcB 7 (Polaromonas sp.), EcB 9 (Pseudomonas sp.), EcB 10 (Pseudomonas sp.) and EcB 11 (Ralstonia sp.) as positive for ACC deaminase. The PCR products were further subjected to sequence analysis which proved the similarity of the sequences identified in the study with ACC deaminase sequences reported from other sources. The detailed bioinformatic analysis of the sequence including homology-based modelling and molecular docking confirmed the sequences to have ACC deaminase activity. The docking of the modelled proteins was done using patch dock, and the detailed scrutiny of the protein ligand interaction revealed conservation of key amino acids like Lys51, Ser78, Tyr268 and Tyr294 which play important role in the enzyme activity. These suggest the possible regulatory effect of these isolates on fruit physiology. PMID:26164855

  4. Adenosine deaminase activity in serum, erythrocytes and lymphocytes of rats infected with Leptospira icterohaemorrhagiae.

    PubMed

    Tonin, Alexandre A; Pimentel, Victor C; da Silva, Aleksandro S; de Azevedo, Maria Isabel; Souza, Viviane C G; Wolkmer, Patrcia; Rezer, Joo F P; Badke, Manoel R T; Leal, Daniela B R; Schetinger, Maria Rosa C; Monteiro, Silvia G; Lopes, Sonia T A

    2012-04-01

    Leptospirosis is a systemic disease of humans and domestic animals, mainly dogs, cattle and swine. The course of human leptospirosis varies from mild to severe fatal forms and the most severe form of human leptospirosis is principally caused by Leptospira interrogans serovar icterohaemorrhagiae (L. icterohaemorrhagiae). The enzyme adenosine deaminase (ADA) plays an important role in the production and differentiation of blood cells. The aim of this study was to evaluate the activity of ADA in serum, erythrocytes and lymphocytes of rats infected with L. icterohaemorrhagiae, as compared with non-infected rats. Twenty-four adult rats, divided into two uniform groups (A and B) were used for the enzymatic assays. The animals in Group B were inoculated intraperitoneally with 210(8) leptospires/rat, and the rodents in Group A (control) were not-inoculated. Blood collection was performed on days 5 and 15 post-infection (PI) and the blood used to assess the ADA activity. The infection by L.icterohaemorrhagiae altered erythrocyte count, hemoglobin concentration and hematocrit, causing a decrease in all these parameters on day 15 PI. Lymphocytes decreased significantly on day 15 PI, and ADA activity in serum was inhibited in infected rats on days 5 and 15 PI and its activity in erythrocytes were increased on day 5 PI. On day 5 PI, we found an increase in ADA activity in erythrocytes of infected rats. No correlation was observed between hematocrit and erythrocyte ADA activity on days 5 and 15 PI. The ADA activity was inhibited in rats infected on day 15 PI. A positive correlation (r(2)=60) was also observed between the number of lymphocytes and ADA activity in lymphocytes on day 15 PI (P<0.05). In conclusion, our results showed that the ADA activity is altered in serum, lymphocytes and erythrocytes in experimental infection by L.icterohaemorrhagiae in rats, concomitantly with hematological parameters. PMID:21320715

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

    PubMed Central

    Gui, Xuwei; Xiao, Heping

    2014-01-01

    This systematic review and meta-analysis was performed to determine accuracy and usefulness of adenosine deaminase (ADA) in diagnosis of tuberculosis pleurisy. Medline, Google scholar and Web of Science databases were searched to identify related studies until 2014. Two reviewers independently assessed quality of studies included according to standard Quality Assessment of Diagnosis Accuracy Studies (QUADAS) criteria. The sensitivity, specificity, diagnostic odds ratio and other parameters of ADA in diagnosis of tuberculosis pleurisy were analyzed with Meta-DiSC1.4 software, and pooled using the random effects model. Twelve studies including 865 tuberculosis pleurisy patients and 1379 non-tuberculosis pleurisy subjects were identified from 110 studies for this meta-analysis. The sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnosis odds ratio (DOR) of ADA in the diagnosis of tuberculosis pleurisy were 45.25 (95% CI 27.63-74.08), 0.86 (95% CI 0.84-0.88), 0.88 (95% CI 0.86-0.90), 6.32 (95% CI 4.83-8.26) and 0.15 (95% 0.11-0.22), respectively. The area under the summary receiver operating characteristic curve (SROC) was 0.9340. Our results demonstrate that the sensitivity and specificity of ADA are high in the diagnosis of tuberculosis pleurisy especially when ADA?50 (U/L). Thus, ADA is a relatively sensitive and specific marker for tuberculosis pleurisy diagnosis. However, it is cautious to apply these results due to the heterogeneity in study design of these studies. Further studies are required to confirm the optimal cut-off value of ADA. PMID:25419343

  6. Graphene oxide based fluorescent aptasensor for adenosine deaminase detection using adenosine as the substrate.

    PubMed

    Xing, Xiao-Jing; Liu, Xue-Guo; Yue-He; Luo, Qing-Ying; Tang, Hong-Wu; Pang, Dai-Wen

    2012-01-01

    We present a novel fluorescent aptasensor for simple and accurate detection of adenosine deaminase (ADA) activity and inhibition on the basis of graphene oxide (GO) using adenosine (AD) as the substrate. This aptasensor consists of a dye-labeled single-stranded AD specific aptamer, GO and AD. The fluorescence intensity of the dye-labeled AD specific aptamer is quenched very efficiently by GO as a result of strong ?-? stacking interaction and excellent electronic transference of GO. In the presence of AD, the fluorescence of the GO-based probe is recovered since the competitive binding of AD and GO with the dye-labeled aptamer prevents the adsorption of dye-labeled aptamer on GO. When ADA was introduced to this GO-based probe solution, the fluorescence of the probe was quenched owing to ADA can convert AD into inosine which has no affinity to the dye-labeled aptamer, thus allowing quantitative investigation of ADA activity. The as-proposed sensor is highly selective and sensitive for the assay of ADA activity with a detection limit of 0.0129U/mL in clean buffer, which is more than one order of magnitude lower than the previous reports. Meanwhile, a good linear relationship with the correlation coefficient of R=0.9922 was obtained by testing 5% human serum containing a series of concentrations of ADA. Additionally, the inhibition effect of erythro-9-(2-hydroxy-3-nonyl) adenine on ADA activity was investigated in this design. The GO-based fluorescence aptasensor not only provides a simple, cost-effective and sensitive platform for the detection of ADA and its inhibitor but also shows great potential in the diagnosis of ADA-relevant diseases and drug development. PMID:22613226

  7. Activities of ectonucleotidases and adenosine deaminase in platelets of dogs experimentally infected with Rangelia vitalii.

    PubMed

    Paim, Carlos Breno V; Da Silva, Aleksandro S; Paim, Francine C; Frana, Raqueli T; Costa, Marcio M; Souza, Viviane C G; Pimentel, Victor C; Jaques, Jeandre A; Mazzanti, Cinthia M; Leal, Daniela B R; Monteiro, Silvia G; Schetinger, Maria Rosa C; Lopes, Sonia T A

    2012-06-01

    Rangeliosis is a disease which affects dogs in Brazil, caused by a piroplasm known as Rangelia vitalii. This disease causes a lot of clinico-pathological features, including the coagulation disorders associated with bleeding. The cause of these changes has not yet been determined. Considering the association of purinergic system and hemostasis this study aimed to evaluate the activity of enzymes that hydrolyze ATP, ADP and AMP; and deamination of adenosine in platelets from dogs experimentally infected with R. vitalii. For this study, 12 healthy young dogs (females) were used, separated in two groups. Group A (n=5) were uninfected controls, and group B were experimentally infected with R. vitalii (n=7). After being inoculated with R. vitalii-infected blood, animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite after five days post-inoculation (PI). Blood samples were collected to quantitate and separate platelets (Day 0, 12 and 21 PI) and to measure the enzymatic activities (Day 12 and 21 PI). The activity of NTPDase, 5'-nucleotidase and adenosine deaminase (ADA) was measured in platelets. A reduction (P<0.01) in the number of platelets was observed in R. vitalii-infected blood at Days 12 and 21 PI. At Day 12 PI, a reduction (P<0.01) in the hydrolysis of ATP, ADP and AMP, and deamination of adenosine was observed in dogs infected with R. vitalii. At Day 21 PI the ADA activity remained decreased, unlike the activity of NTPDase which increased (P<0.05). Based on these results we can conclude that ATP, ADP and AMP hydrolysis and adenosine deamination were altered in platelets of R. vitalii-infected dogs. Considering the importance of the purinergic system in hemostasis, it is believed that those changes contribute to the coagulation disorders and bleeding observed in R. vitalii-infected dogs and discussed in this manuscript. PMID:22475775

  8. Non-viral mediated gene transfer of porphobilinogen deaminase into mammalian cells.

    PubMed

    Johansson, A; Mller, C; Gellerfors, P; Harper, P

    2002-01-01

    Acute intermittent porphyria (AIP) is an inborn error of heme synthesis in which the third enzyme, porphobilinogen deaminase (PBGD), is deficient. The disease is characterized by recurrent attacks of acute abdominal pain often accompanied by neuropsychiatric symptoms. Current therapeutic treatment with heme is only palliative and no curative alternative exists. The present report describes the first step towards a gene therapy treatment for AIP. Mouse cDNA encoding the PBGD enzyme was cloned and four vectors containing the full-length mouse and human cDNA of the housekeeping and erythroid PBGD isoforms under the control of a cytomegalovirus promoter were constructed. The vectors, condensed to polyethylenimine, were successfully transfected to NIH 3T3 and HeLa cells as determined by enzymatic activity measurements. Thus, the PBGD activity was increased 3-10 times in NIH 3T3 cells and 95-240 times in HeLa cells. The expression was shown to be dose and time dependent, with the highest level of activity observed in HeLa cells after 72 h posttransfection. Non-viral gene transfer was also undertaken in PBGD-deficient fibroblasts established from an AIP patient. Complete normalization of the PBGD activity was accomplished after the addition of 2.5 microg DNA per well. Further addition of DNA increased the PBGD activity up to threefold the normal value. The study documents a successful gene transfer and a high degree of PBGD expression in different cell-lines, indicating a potential for future gene therapy in AIP. PMID:12004925

  9. Discovery and characterization of D-phenylserine deaminase from Arthrobacter sp. TKS1.

    PubMed

    Muramatsu, Hisashi; Suzuki, Yuri; Imai, Takeshi; Ueshima, Sakuko; Ozaki, Jun; Matsui, Yuji; Kato, Shin-ichiro; Ohnishi, Kouhei; Kimoto, Norihiro; Yamamoto, Hiroaki; Nagata, Shinji

    2011-04-01

    We discovered a D-phenylserine deaminase that catalyzed the pyridoxal 5'-phosphate (PLP)-dependent deamination reaction from D-threo-phenylserine to phenylpyruvate in newly isolated Arthrobacter sp. TKS1. The enzyme was partially purified, and its N-terminal amino acid sequence was analyzed. Based on the sequence information, the gene encoding the enzyme was identified and expressed in Escherichia coli. The expressed protein was purified to homogeneity and characterized. The enzyme consisted of two identical 46-kDa subunits and showed maximum activity at pH 8.5 and 55C. The enzyme was stable in the range of pH 7.5 to pH 8.5 and up to 50C. The enzyme acted on the D-forms of ?-hydroxy-?-amino acids, such as D-threo-phenylserine (K(m), 19 mM), D-serine (K(m), 5.8 mM), and D-threonine (K(m), 102 mM). As L-threonine, D-allo-threonine, L-allo-threonine, and DL-erythro-phenylserine were inert, the enzyme could distinguish D-threo-form from among the four stereoisomers of phenylserine or threonine. The enzyme was activated by ZnSO(4), CuSO(4), BaCl(2), and CoCl(2) and strongly inhibited by phenylhydrazine, sodium borohydride, hydroxylamine, and DL-penicillamine. The enzyme exhibited absorption maxima at 280 and around 415 nm. The enzyme has an N-terminal domain similar to that of alanine racemase, which belongs to the fold type III group of pyridoxal enzymes. PMID:21190106

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

    PubMed Central

    Rannou, Fabrice; Uguen, Arnaud; Scotet, Virginie; Le Maréchal, Cédric; Rigal, Odile; Marcorelles, Pascale; Gobin, Eric; Carré, Jean-Luc; Zagnoli, Fabien; Giroux-Metges, Marie-Agnès

    2015-01-01

    Aim Our aim was to evaluate the accuracy of aerobic exercise testing to diagnose metabolic myopathies. Methods From December 2008 to September 2012, all the consecutive patients that underwent both metabolic exercise testing and a muscle biopsy were prospectively enrolled. Subjects performed an incremental and maximal exercise testing on a cycle ergometer. Lactate, pyruvate, and ammonia concentrations were determined from venous blood samples drawn at rest, during exercise (50% predicted maximal power, peak exercise), and recovery (2, 5, 10, and 15 min). Biopsies from vastus lateralis or deltoid muscles were analysed using standard techniques (reference test). Myoadenylate deaminase (MAD) activity was determined using p-nitro blue tetrazolium staining in muscle cryostat sections. Glycogen storage was assessed using periodic acid-Schiff staining. The diagnostic accuracy of plasma metabolite levels to identify absent and decreased MAD activity was assessed using Receiver Operating Characteristic (ROC) curve analysis. Results The study involved 51 patients. Omitting patients with glycogenoses (n = 3), MAD staining was absent in 5, decreased in 6, and normal in 37 subjects. Lactate/pyruvate at the 10th minute of recovery provided the greatest area under the ROC curves (AUC, 0.893 ± 0.067) to differentiate Abnormal from Normal MAD activity. The lactate/rest ratio at the 10th minute of recovery from exercise displayed the best AUC (1.0) for discriminating between Decreased and Absent MAD activities. The resulting decision tree achieved a diagnostic accuracy of 86.3%. Conclusion The present algorithm provides a non-invasive test to accurately predict absent and decreased MAD activity, facilitating the selection of patients for muscle biopsy and target appropriate histochemical analysis. PMID:26207760

  11. Preliminary investigations on inducing salt tolerance in maize through inoculation with rhizobacteria containing ACC deaminase activity.

    PubMed

    Nadeem, Sajid Mahmood; Zahir, Zahir Ahmad; Naveed, Muhammad; Arshad, Muhammad

    2007-10-01

    Twenty rhizobacterial strains containing 1-aminocyclopropane-1-carboxylate deaminase were isolated from the rhizosphere of salt-affected maize fields. They were screened for their growth-promoting activities under axenic conditions at 1, 4, 8, and 12 dS x m-1 salinity levels. Based upon the data of the axenic study, the 6 most effective strains were selected to conduct pot trials in the wire house. Besides one original salinity level (1.6 dS x m-1), 3 other salinity levels (4, 8, and 12 dS x m-1) were maintained in pots and maize seeds inoculated with selected strains of plant growth-promoting rhizobacteria, as well as uninoculated controls were sown. Results showed that the increase in salinity level decreased the growth of maize seedlings. However, inoculation with rhizobacterial strains reduced this depression effect and improved the growth and yield at all the salinity levels tested. Selected strains significantly increased plant height, root length, total biomass, cob mass, and grain yield up to 82%, 93%, 51%, 40%, and 50%, respectively, over respective uninoculated controls at the electrical conductivity of 12 dS x m-1. Among various plant growth-promoting rhizobacterial strains, S5 (Pseudomonas syringae), S14 (Enterobacter aerogenes), and S20 (Pseudomonas fluorescens) were the most effective strains for promoting the growth and yield of maize, even at high salt stress. The relatively better salt tolerance of inoculated plants was associated with a high K+/Na+ ratio as well as high relative water and chlorophyll and low proline contents. PMID:18026206

  12. Investigation into effects of antipsychotics on ectonucleotidase and adenosine deaminase in zebrafish brain.

    PubMed

    Seibt, Kelly Juliana; Oliveira, Renata da Luz; Bogo, Mauricio Reis; Senger, Mario Roberto; Bonan, Carla Denise

    2015-12-01

    Antipsychotic agents are used for the treatment of psychotic symptoms in patients with several brain disorders, such as schizophrenia. Atypical and typical antipsychotics differ regarding their clinical and side-effects profile. Haloperidol is a representative typical antipsychotic drug and has potent dopamine receptor antagonistic functions; however, atypical antipsychotics have been developed and characterized an important advance in the treatment of schizophrenia and other psychotic disorders. Purine nucleotides and nucleosides, such as ATP and adenosine, constitute a ubiquitous class of extracellular signaling molecules crucial for normal functioning of the nervous system. Indirect findings suggest that changes in the purinergic system, more specifically in adenosinergic activity, could be involved in the pathophysiology of schizophrenia. We investigated the effects of typical and atypical antipsychotics on ectonucleotidase and adenosine deaminase (ADA) activities, followed by an analysis of gene expression patterns in zebrafish brain. Haloperidol treatment (9 µM) was able to decrease ATP hydrolysis (35%), whereas there were no changes in hydrolysis of ADP and AMP in brain membranes after antipsychotic exposure. Adenosine deamination in membrane fractions was inhibited (38%) after haloperidol treatment when compared to the control; however, no changes were observed in ADA soluble fractions after haloperidol exposure. Sulpiride (250 µM) and olanzapine (100 µM) did not alter ectonucleotidase and ADA activities. Haloperidol also led to a decrease in entpd2_mq, entpd3 and adal mRNA transcripts. These findings demonstrate that haloperidol is an inhibitor of NTPDase and ADA activities in zebrafish brain, suggesting that purinergic signaling may also be a target of pharmacological effects promoted by this drug. PMID:26156500

  13. 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

  14. Delivery of a secretable adenosine deaminase through microcapsules--a novel approach to somatic gene therapy.

    PubMed

    Hughes, M; Vassilakos, A; Andrews, D W; Hortelano, G; Belmont, J W; Chang, P L

    1994-12-01

    Many current gene therapy protocols require genetic modification of autologous cells. An alternate approach is to use universal recombinant cell lines engineered to secrete in vivo the desired gene products. Enclosing these cells within immunoprotective devices before implantation would prevent rejection of the nonautologous donor cells. To overcome the limitation that not all therapeutic gene products are secreted, we now propose to fuse a signal sequence to the amino terminus of a nonsecreted protein such as human adenosine deaminase (ADA), thus directing the product into a secretory pathway for release from the cells. A fusion gene constructed between the cDNA of the beta-lactamase signal sequence and human ADA expressed a product after in vitro transcription and translation that was immunologically similar to the human protein. Mouse fibroblasts transfected with the fusion gene demonstrated secreted ADA activity that resembled the human cytosolic enzyme in its heat stability, pH optimum, KM, electrophoretic mobility, and immunologic reactivity. Hence, the secreted enzyme expressed from the fusion gene is antigenically and enzymatically similar to the authentic human form. When transfected mouse fibroblasts or myoblasts were enclosed in permselective alginate-poly-L-lysine alginate microcapsules, ADA activity was secreted from the microcapsules and the cells remained viable for over 5 months. Hence, a secretable and functional human ADA has been constructed that can be delivered from recombinant cells within immunoprotective capsules. The success of this strategy provides the prototype for engineering nonsecreted gene products for therapy via this novel method of somatic gene therapy. PMID:7711137

  15. Serum cytidine deaminase levels after withdrawal of non-steroidal anti-inflammatory treatment in rheumatoid arthritis.

    PubMed Central

    Thompson, P W; Kirwan, J R; Jones, D D; Currey, H L

    1988-01-01

    Increases in joint inflammation in nine patients with rheumatoid arthritis were provoked by withdrawal of their non-steroidal anti-inflammatory drugs. Pain score, duration of morning stiffness, Ritchie articular index score, and the number of analgesic tablets consumed reached peaks after five, three, five, and five days respectively compared with values during six days of normal treatment. Changes in serum cytidine deaminase (believed to reflect polymorph turnover in inflamed joints) showed a different pattern, with a sharp peak after two days and a subsequent trough. Possible mechanisms for these differences are discussed. PMID:3365029

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

    PubMed

    Khl, J S; Schwarz, K; Mnch, 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

  17. Lager yeast comes of age.

    PubMed

    Wendland, Jrgen

    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

  18. 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

  19. 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

  20. Yeasts: From genetics to biotechnology

    SciTech Connect

    Russo, S.; Poli, G.; Siman-Tov, R.B.

    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.

  1. Revisiting yeast trehalose metabolism.

    PubMed

    Eleutherio, Elis; Panek, Anita; De Mesquita, Joelma Freire; Trevisol, Eduardo; Magalhes, Rayne

    2015-08-01

    Establishing the function of trehalose in yeast cells has led us, over the years, through a long path-from simple energy storage carbohydrate, then a stabilizer and protector of membranes and proteins, through a safety valve against damage caused by oxygen radicals, up to regulator of the glycolytic path. In addition, trehalose biosynthesis has been proposed as a target for novel drugs against several pathogens. Since this pathway is entirely absent in mammalian cells and makes use of highly specific enzymes, trehalose metabolism might be an interesting target for the development of novel therapies. In this review, we want to address some recent points investigated about trehalose metabolism in Saccharomyces cerevisiae, focusing mainly on the mechanism by which this simple disaccharide protects against stress and on the enzymes involved in its synthesis and breakdown. We believe that these concepts are of great importance for medical and biotechnological applications. PMID:25209979

  2. BIOSYNTHESIS OF YEAST CAROTENOIDS.

    PubMed

    SIMPSON, K L; NAKAYAMA, T O; CHICHESTER, C O

    1964-12-01

    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, zeta-carotene, neurosporene, beta-zeacarotene, gamma-carotene, torulin, a C(40) aldehyde, and torularhodin. Torulin and torularhodin were established as the main carotenoids of 62-506. PMID:14240958

  3. Assignment of cytosine N3 resonances in nucleic acids via intrabase three-bond coupling to amino protons.

    PubMed

    Rdisser, S; Pelton, J G; Tinoco, I

    1999-10-01

    Coherences were observed between 15N3 of cytosine and its trans amino proton (H42) using a modified gradient-based heteronuclear single quantum coherence (HSQC) pulse sequence optimized for three-bond proton-nitrogen couplings. The method is demonstrated with a 22-nucleotide RNA fragment of the P5abc region of a group I intron uniformly labeled with 15N. Use of intraresidue 15N3-amino proton couplings to assign cytosine 15N3 signals complements the recently proposed JNN HNN COSY [Dingley, A.J. and Grzesiek, S. (1998) J. Am. Chem. Soc., 120, 8293-8297] method of identifying hydrogen-bonded base pairs in RNA. PMID:10605090

  4. 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).

  5. Quantum chemical MP2 results on some hydrates of cytosine: binding sites, energies and the first hydration shell.

    PubMed

    Fogarasi, Gza; Szalay, Pter G

    2015-11-28

    A detailed quantum chemical investigation was undertaken to obtain the structure and energetics of cytosine hydrates CytnH2O, with n = 1 to 7. The MP2(fc)/aug-cc-pVDZ level was used as the standard, with some DFT (B3LYP) and coupled cluster calculations, as well as calculations with the aug-cc-pVTZ basis set added for comparison. In a systematic search for microhydrated forms of cytosine, we have found that several structures have not yet been reported in the literature. The energies of different isomers, as well as binding energies are compared. When predicting the stability of a complex, we suggest using a scheme where the water molecules are extracted from a finite model of bulk water. Finally, based on energetic data, we suggest a rational definition of the first hydration shell; with this definition, it contains just six water molecules. PMID:26487481

  6. A study of the excited states in cytosine and guanine stacks in the Hartree-Fock and exciton approximations

    SciTech Connect

    Grobelsek-Vracko, M.; Zaider, M. )

    1994-04-01

    We report calculated exciton energies for the cytosine and guanine stacks obtained in the ab initio Hartree-Fock crystal orbital and exciton approximation, which includes the excited electron-hole interaction. This interaction plays an important role in the description of excited electron spectra in the low-energy region. The stacks were chosen as examples of polymers with helical symmetry. 21 refs., 3 figs., 3 tabs.

  7. Genome-Wide Identification and Comparative Analysis of Cytosine-5 DNA Methyltransferase and Demethylase Families in Wild and Cultivated Peanut

    PubMed Central

    Wang, Pengfei; Gao, Chao; Bian, Xiaotong; Zhao, Shuzhen; Zhao, Chuanzhi; Xia, Han; Song, Hui; Hou, Lei; Wan, Shubo; Wang, Xingjun

    2016-01-01

    DNA methylation plays important roles in genome protection, regulation of gene expression and is associated with plants development. Plant DNA methylation pattern was mediated by cytosine-5 DNA methyltransferase and demethylase. Although the genomes of AA and BB wild peanuts have been fully sequenced, these two gene families have not been studied. In this study we report the identification and analysis of putative cytosine-5 DNA methyltransferases (C5-MTases) and demethylases in AA and BB wild peanuts. Cytosine-5 DNA methyltransferases in AA and BB wild peanuts could be classified in MET, CMT, and DRM2 groups based on their domain organization. This result was supported by the gene and protein structural characteristics and phylogenetic analysis. We found that some wild peanut DRM2 members didn't contain UBA domain which was different from other plants such as Arabidopsis, maize and soybean. Five DNA demethylase encoding genes were found in AA genome and five in BB genome. The selective pressure analysis showed that wild peanut C5-MTase genes mainly underwent purifying selection but many positive selection sites can be detected. Conversely, DNA demethylase genes mainly underwent positive selection during evolution. Additionally, the expression dynamic of cytosine-5 DNA methyltransferase and demethylase genes in different cultivated peanut tissues were analyzed. Expression result showed that cold, heat or PEG stress could influence the expression level of C5-MTase and DNA demethylase genes in cultivated peanut. These results are useful for better understanding the complexity of these two gene families, and will facilitate epigenetic studies in peanut in the future. PMID:26870046

  8. Genome-Wide Identification and Comparative Analysis of Cytosine-5 DNA Methyltransferase and Demethylase Families in Wild and Cultivated Peanut.

    PubMed

    Wang, Pengfei; Gao, Chao; Bian, Xiaotong; Zhao, Shuzhen; Zhao, Chuanzhi; Xia, Han; Song, Hui; Hou, Lei; Wan, Shubo; Wang, Xingjun

    2016-01-01

    DNA methylation plays important roles in genome protection, regulation of gene expression and is associated with plants development. Plant DNA methylation pattern was mediated by cytosine-5 DNA methyltransferase and demethylase. Although the genomes of AA and BB wild peanuts have been fully sequenced, these two gene families have not been studied. In this study we report the identification and analysis of putative cytosine-5 DNA methyltransferases (C5-MTases) and demethylases in AA and BB wild peanuts. Cytosine-5 DNA methyltransferases in AA and BB wild peanuts could be classified in MET, CMT, and DRM2 groups based on their domain organization. This result was supported by the gene and protein structural characteristics and phylogenetic analysis. We found that some wild peanut DRM2 members didn't contain UBA domain which was different from other plants such as Arabidopsis, maize and soybean. Five DNA demethylase encoding genes were found in AA genome and five in BB genome. The selective pressure analysis showed that wild peanut C5-MTase genes mainly underwent purifying selection but many positive selection sites can be detected. Conversely, DNA demethylase genes mainly underwent positive selection during evolution. Additionally, the expression dynamic of cytosine-5 DNA methyltransferase and demethylase genes in different cultivated peanut tissues were analyzed. Expression result showed that cold, heat or PEG stress could influence the expression level of C5-MTase and DNA demethylase genes in cultivated peanut. These results are useful for better understanding the complexity of these two gene families, and will facilitate epigenetic studies in peanut in the future. PMID:26870046

  9. Single Site Discrimination of Cytosine, 5-Methylcytosine, and 5-Hydroxymethylcytosine in Target DNA Using Anthracene-Tagged Fluorescent Probes.

    PubMed

    Duprey, Jean-Louis H A; Bullen, Gemma A; Zhao, Zheng-Yun; Bassani, Dario M; Peacock, Anna F A; Wilkie, John; Tucker, James H R

    2016-03-18

    The ability to discriminate between epigenetic variants in DNA is a necessary tool if we are to increase our understanding of the roles that they play in various biological processes and medical conditions. Herein, it is demonstrated how a simple two-step fluorescent probe assay can be used to differentiate all three major epigenetic variants of cytosine at a single locus site in a target strand of DNA. PMID:26580817

  10. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... 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 provided the total folic...

  11. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... 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 provided the total folic...

  12. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... 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 provided the total folic...

  13. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Dried yeasts. 172.896 Section 172.896 Food and... 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 provided the total folic...

  14. Biosorption of copper by yeasts.

    PubMed

    Junghans, K; Straube, G

    1991-01-01

    The ability to accumulate copper from aqueous solutions was determined with different yeast species. Yeast cells did not show any significant differences in process kinetics. The uptake was very fast and was influenced by environmental factors. The metal-accumulating capacity differed among the tested strains. The yeast Candida tropicalis and Pichia guilliermondii were chosen for extensive research. Cells of the stationary growth phase were able to adsorb a high amount of copper. The uptake capacity decreased with increasing biomass concentration. Copper adsorption obeyed the Freundlich isotherm. Optimal pH range was between 5 and 7. The biomass could be used repeatedly for biosorption after desorption by mineral acids. PMID:1777357

  15. Molybdate induces thermotolerance in yeast.

    PubMed

    Tiligada, E; Miligkos, V; Ypsilantis, E; Papamichael, K; Delitheos, A

    1999-08-01

    Application of a mild heat pretreatment, performed by shifting cells from 27 degrees C to 37 degrees C led to the protection of yeast cells from death due to a subsequent extreme heat shock at 53 degrees C. The presence of cycloheximide inhibited this induction of thermotolerance, indicating the involvement of de novo protein. The phosphatase inhibitor sodium molybdate induced thermotolerance to the non-pretreated yeast cells. This induction of thermotolerance did not seem to depend upon de novo protein synthesis. Thus, acquisition of thermotolerance in yeast may involve a number of cellular mechanisms depending on the conditions the organism encounters at any particular time. PMID:10499293

  16. 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

  17. Discovery of bisulfite-mediated cytosine conversion to uracil, the key reaction for DNA methylation analysis — A personal account

    PubMed Central

    Hayatsu, Hikoya

    2008-01-01

    Methylation at position 5 of cytosine in DNA is being intensively studied in many areas of biological sciences, as the methylation is intimately associated with the control of gene functions. The principal analytical method for determining the sites of 5-methylcytosine in genome at the sequence level involves bisulfite modification of DNA. The utility of this chemical treatment is based on the property of bisulfite to selectively deaminate cytosine residues. The bisulfite-mediated cytosine deamination was discovered in 1970 by us in the University of Tokyo. At the same time, Shapiro and his coworkers in New York University found the same reaction independently. We also reported that 5-methylcytosine was deaminated by bisulfite only very slowly. These findings were later utilized by a group of Australian scientists to devise a means to analyze 5-methylcytosine in DNA; thus, a method called ‘bisulfite genomic sequencing’ was invented by these researchers in 1992. This review describes the author’s reflection of the discovery of bisulfite reactions with pyrimidine bases. The author’s recent work that has resulted in an improvement of the procedure of analysis by use of a newly devised high concentration bisulfite solution is also described. PMID:18941305

  18. Intramolecular folding of a fragment of the cytosine-rich strand of telomeric DNA into an i-motif.

    PubMed

    Leroy, J L; Guéron, M; Mergny, J L; Hélène, C

    1994-05-11

    In the recently discovered i-motif, four stretches of cytosine form two parallel-stranded duplexes whose C.C+ base pairs are fully intercalated. The i-motif may be recognized by characteristic Overhauser cross-peaks of the proton NMR spectrum, reflecting short H1'-H1' distances across the minor groove, and short internucleotide amino-proton-H2'/H2" across the major groove. We report the observation of such cross-peaks in the spectra of a fragment of the C-rich telomeric strand of vertebrates, d[CCCTAA]3CCC. The spectra also demonstrate that the cytosines are base-paired and that proton exchange is very slow, as reported previously for the i-motif. From UV absorbance and gel chromatography measurements, we assign these properties to an i-motif which includes all or nearly all the cytosines, and which is formed by intramolecular folding at slightly acid or neutral pH. A fragment of telomeric DNA of Tetrahymena, d[CCCCAA]3CCCC, has the same properties. Hence four consecutive C stretches of a C-rich telomeric strand can fold into an i-motif. Hypothetically, this could occur in vivo. PMID:8202359

  19. A cytosine methyltransferase homologue is essential for repeat-induced point mutation in Neurospora crassa

    PubMed Central

    Freitag, Michael; Williams, Rebecca L.; Kothe, Gregory O.; Selker, Eric U.

    2002-01-01

    During sexual development, Neurospora crassa inactivates genes in duplicated DNA segments by a hypermutation process, repeat-induced point mutation (RIP). RIP introduces C:G to T:A transition mutations and creates targets for subsequent DNA methylation in vegetative tissue. The mechanism of RIP and its relationship to DNA methylation are not fully understood. Mutations in DIM-2, a DNA methyltransferase (DMT) responsible for all known cytosine methylation in Neurospora, does not prevent RIP. We used RIP to disrupt a second putative DMT gene in the Neurospora genome and tested mutants for defects in DNA methylation and RIP. No effect on DNA methylation was detected in the tissues that could be assayed, but the mutants showed recessive defects in RIP. Duplications of the am and mtr genes were completely stable in crosses homozygous for the mutated potential DMT gene, which we call rid (RIP defective). The same duplications were inactivated normally in heterozygous crosses. Disruption of the rid gene did not noticeably affect fertility, growth, or development. In contrast, crosses homozygous for a mutation in a related gene in Ascobolus immersus, masc1, reportedly fail to develop and heterozygous crosses reduce methylation induced premeiotically [Malagnac, F., Wendel, B., Goyon, C., Faugeron, G., Zickler, D., et al. (1997) Cell 91, 281290]. We isolated homologues of rid from Neurospora tetrasperma and Neurospora intermedia to identify conserved regions. Homologues possess all motifs characteristic of eukaryotic DMTs and have large distinctive C- and N-terminal domains. PMID:12072568

  20. 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

  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, Sren; 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. Effects of cytosine methylation on DNA morphology: An atomic force microscopy study.

    PubMed

    Cassina, V; Manghi, M; Salerno, D; Tempestini, A; Iadarola, V; Nardo, L; Brioschi, S; Mantegazza, F

    2016-01-01

    Methylation is one of the most important epigenetic mechanisms in eukaryotes. As a consequence of cytosine methylation, the binding of proteins that are implicated in transcription to gene promoters is severely hindered, which results in gene regulation and, eventually, gene silencing. To date, the mechanisms by which methylation biases the binding affinities of proteins to DNA are not fully understood; however, it has been proposed that changes in double-strand conformations, such as stretching, bending, and over-twisting, as well as local variations in DNA stiffness/flexibility may play a role. The present work investigates, at the single molecule level, the morphological consequences of DNA methylation in vitro. By tracking the atomic force microscopy images of single DNA molecules, we characterize DNA conformations pertaining to two different degrees of methylation. In particular, we observe that methylation induces no relevant variations in DNA contour lengths, but produces measurable incremental changes in persistence lengths. Furthermore, we observe that for the methylated chains, the statistical distribution of angles along the DNA coordinate length is characterized by a double exponential decay, in agreement with what is predicted for polyelectrolytes. The results reported herein support the claim that the biological consequences of the methylation process, specifically difficulties in protein-DNA binding, are at least partially due to DNA conformation modifications. PMID:26475643

  3. Ultrafast two-dimensional infrared spectroscopy of guanine-cytosine base pairs in DNA oligomers.

    PubMed

    Greve, Christian; Elsaesser, Thomas

    2013-11-14

    NH and OH stretching excitations of hydrated double-stranded DNA oligomers containing guanine-cytosine (GC) base pairs in a Watson-Crick geometry are studied by two-dimensional (2D) infrared spectroscopy. The 2D spectra measured at a low hydration level (?4 water molecules/base pair) are dominated by NH stretch contributions from the NH2 groups of G and C and the NH group of G. Partially hydrated NH2 groups display red-shifted NH stretch frequencies and a mixing of the wave functions of the two local NH oscillators via the mechanical vibrational coupling. The NH stretch lifetimes are of the order of 200-300 fs. Weak couplings exist between NH stretch oscillators within a base pair, while interactions between neighboring GC pairs in the double helix are negligible. The absence of spectral diffusion on a 1 ps time scale suggests a relatively rigid structure of the hydrogen bonds between DNA and residual water molecules. 2D spectra recorded with fully hydrated DNA oligomers exhibit NH and OH stretch contributions with a weak influence of water fluctuations on the NH stretch lineshapes. The femtosecond spectral diffusion of OH stretch excitations is slower than that in bulk H2O and originates from structural fluctuations of the water shell and the formation of a vibrationally hot ground state by vibrational relaxation. We compare our findings with measurements on hydrated adenine-thymine DNA oligomers and anhydrous GC base pairs in solution. PMID:24127664

  4. Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome.

    PubMed

    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, Sren; 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

    2014-03-01

    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

  5. Oxidative stress mediates neuronal DNA damage and apoptosis in response to cytosine arabinoside.

    PubMed

    Geller, H M; Cheng, K Y; Goldsmith, N K; Romero, A A; Zhang, A L; Morris, E J; Grandison, L

    2001-07-01

    Cytosine arabinoside (AraC) is a nucleoside analog that produces significant neurotoxicity in cancer patients. The mechanism by which AraC causes neuronal death is a matter of some debate because the conventional understanding of AraC toxicity requires incorporation into newly synthesized DNA. Here we demonstrate that AraC-induced apoptosis of cultured cerebral cortical neurons is mediated by oxidative stress. AraC-induced cell death was reduced by treatment with several different free-radical scavengers (N-acetyl-L-cysteine, dipyridamole, uric acid, and vitamin E) and was increased following depletion of cellular glutathione stores. AraC induced the formation of reactive oxygen species in neurons as measured by an increase in the fluorescence of the dye 5-(6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate. AraC produced DNA single-strand breaks as measured by single-cell gel electrophoresis and the level of DNA strand breakage was reduced by treatment with the free radical scavengers. These data support a model in which AraC induces neuronal apoptosis by provoking the generation of reactive oxygen species, causing oxidative DNA damage and initiating the p53-dependent apoptotic program. These observations suggest the use of antioxidant therapies to reduce neurotoxicity in AraC chemotherapeutic regimens. PMID:11461962

  6. Evolutionary Breakpoints in the Gibbon Suggest Association between Cytosine Methylation and Karyotype Evolution

    PubMed Central

    Carbone, Lucia; Harris, R. Alan; Vessere, Gery M.; Mootnick, Alan R.; Humphray, Sean; Rogers, Jane; Kim, Sung K.; Wall, Jeffrey D.; Martin, David; Jurka, Jerzy; Milosavljevic, Aleksandar; de Jong, Pieter J.

    2009-01-01

    Gibbon species have accumulated an unusually high number of chromosomal changes since diverging from the common hominoid ancestor 1518 million years ago. The cause of this increased rate of chromosomal rearrangements is not known, nor is it known if genome architecture has a role. To address this question, we analyzed sequences spanning 57 breaks of synteny between northern white-cheeked gibbons (Nomascus l. leucogenys) and humans. We find that the breakpoint regions are enriched in segmental duplications and repeats, with Alu elements being the most abundant. Alus located near the gibbon breakpoints (<150 bp) have a higher CpG content than other Alus. Bisulphite allelic sequencing reveals that these gibbon Alus have a lower average density of methylated cytosine that their human orthologues. The finding of higher CpG content and lower average CpG methylation suggests that the gibbon Alu elements are epigenetically distinct from their human orthologues. The association between undermethylation and chromosomal rearrangement in gibbons suggests a correlation between epigenetic state and structural genome variation in evolution. PMID:19557196

  7. Epigenetic memory of DNAi associated with cytosine methylation and histone modification in fern.

    PubMed

    Tsuboi, Hidenori; Sutoh, Keita; Wada, Masamitsu

    2012-11-01

    Gene silencing technology, such as RNA interference (RNAi), is commonly used to reduce gene expression in plant cells, and exogenous double-stranded RNA (dsRNA) can induce gene silencing in higher plants. Previously, we showed that the delivery of double-stranded DNA (dsDNA) fragments, such as PCR products of an endogenous gene sequence, into fern (Adiantum capillus-veneris) gametophytic cells induces a sequence-specific gene silencing that we termed DNAi. In this study, we used a neochrome 1 gene (NEO1) that mediates both red light-induced chloroplast movement and phototropism as a model of DNAi and confirmed that the NEO1 function was suppressed by the repression of the NEO1 gene. Interestingly, the gene silencing effect by DNAi was found in the progeny. Cytosine methylation was detected in the NEO1-silenced lines. The DNA modifications was present in the transcriptional region of NEO1, but no differences between wild type and the silenced lines were found in the downstream region of NEO1. Our data suggest that the DNAi gene silencing effect that was inherited throughout the next generation is regulated by epigenetic modification. Furthermore, the histone deacetylase inhibitor, trichostatin A (TSA), recovered the expression and function of NEO1 in the silenced lines, suggesting that histone deacetylation is essential for the direct suppression of target genes by DNAi. PMID:22990449

  8. Transcription-dependent cytosine deamination: a novel mechanism in ultraviolet light-induced mutagenesis

    PubMed Central

    Hendriks, Giel; Callja, Fabienne; Besaratinia, Ahmad; Vrieling, Harry; Pfeifer, Gerd P.; Mullenders, Leon H.F.; Jansen, Jacob G.; de Wind, Niels

    2009-01-01

    Summary Skin cancer is the most ubiquitous cancer type in the Caucasian population and its incidence is increasing rapidly [1]. Transcribed, proliferation-related, genes in dermal stem cells are targets for the induction of ultraviolet light (UV)-induced mutations that drive carcinogenesis. We have recently found that gene transcription increases UV-induced mutagenesis in mammalian stem cells, suggesting a role of transcription in skin carcinogenesis [2]. Here we show that transcription-associated, UV-induced, nucleotide substitutions are caused by increased deamination of cytosines to uracil within photolesions at the transcribed strand, presumably at sites of stalled transcription complexes. Additionally, via an independent mechanism, transcription of UV-damaged DNA induces the generation of intragenic deletions. We provide evidence that transcription-coupled nucleotide excision repair (TC-NER) provides protection against both classes of transcription-associated mutagenesis. Combined, these results unveil the existence of two mutagenic pathways, operating specifically at the transcribed DNA strand of active genes. Moreover, these results uncover a novel role for TC-NER in the suppression of UV light-induced genome aberrations and provide a rationale for the efficient induction of apoptosis by stalled transcription complexes. PMID:20045328

  9. Isoschizomers and amplified fragment length polymorphism for the detection of specific cytosine methylation changes.

    PubMed

    Ruiz-Garca, Leonor; Cabezas, Jose Antonio; de Mara, Nuria; Cervera, Mara-Teresa

    2010-01-01

    Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is a modification of the Amplified Fragment Length Polymorphism (AFLP) technique that has been used to study methylation of anonymous CCGG sequences in different fungi, plant and animal species. The main variation of this technique is based on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent cutter restriction enzyme. For each sample, AFLP analysis is performed using both EcoRI/HpaII and EcoRI/MspI digested samples. Comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) "Methylation-insensitive polymorphisms" that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples; and (2) "Methylation-sensitive polymorphisms" that are associated with amplified fragments differing in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses modifications that can be applied to adjust the technology to different species of interest. PMID:20204869

  10. Hydroxymethyl cytosine marks in the human mitochondrial genome are dynamic in nature.

    PubMed

    Ghosh, Sourav; Sengupta, Shantanu; Scaria, Vinod

    2016-03-01

    Apart from DNA methylation, hydroxymethylation has increasingly been studied as an important epigenetic mark. 5- hydroxymethylcytosines, though initially were thought to be an intermediary product of demethylation, recent studies suggest this to be a highly regulated process and modulated by the TET family of enzymes. Recent genome wide studies have shown that hydroxymethylcytosine marks are closely associated with the regulation of important biological processes like transcription and embryonic development. It is also known that aberrant hydroxymethylation marks have been associated with diseases like cancer. The presence of hydroxymethylcytosines in the mitochondrial genome has been earlier suggested, though the genome-scale map has not been laid out. In this present study, we have mapped and analyzed the hydroxymethylcytosine marks in the mitochondrial genome using 23 different publicly available datasets. We cross validated our data by checking for consistency across a subset of genomic regions previously annotated to hydroxymethylcytosines and show good consistency. We observe a dynamic distribution of hydroxymethylation marks in the mitochondrial genome. Unlike the methylcytosine marks, hydroxymethylcytosine marks are characterized by the lack of conservation across the samples considered, though similar cell types shared the pattern. We additionally observed that the hydroxymethylation marks are enriched in the upstream of GSS (gene start site) regions and in gene body as similar as nuclear genes. To the best of our knowledge, this is the first genome-scale map of hydroxymethyl cytosines in the human mitochondrial genome. PMID:26826294

  11. Susceptibility to cytosine arabinoside (Ara-C)-induced cytotoxicity in human leukemia cell lines.

    PubMed

    Kanno, Syu-Ichi; Higurashi, Ayako; Watanabe, Yurie; Shouji, Ai; Asou, Keiko; Ishikawa, Masaaki

    2004-09-10

    Cytosine arabinoside (1-beta-d-arabinofuranosylcytosine; Ara-C) is the most important antimetabolite chemotherapeutic drug used for acute leukemia. We examined the difference in susceptibility to Ara-C-induced cell death among a number of typical human leukemia cell lines, NALM-6, MOLT-4, Jurkat, U937 and HL-60. NALM-6, which had a high expression level of p53, a tumor suppressor gene, was most susceptible to Ara-C. U937 and HL-60, with p53-null human leukemia cell lines were little affected by Ara-C. There was not always a correlation between susceptibility and the uptake of Ara-C. The production of reactive oxygen species (ROS) was increased in all leukemia cells. Pifithrin-alpha, a chemical inhibitor of wild-type p53, ameliorated the cytotoxicity of Ara-C in NALM-6 and MOLT-4, but not Jurkat, U937 or HL-60. Our data suggest that the mechanism of Ara-C-induced cell death is a common one, involving an increase in the production of ROS and p53-dependent cell death. PMID:15302096

  12. Electronic excited states of guanine-cytosine hairpins and duplexes studied by fluorescence spectroscopy.

    PubMed

    Brazard, Johanna; Thazhathveetil, Arun K; Vayá, Ignacio; Lewis, Frederick D; Gustavsson, Thomas; Markovitsi, Dimitra

    2013-08-01

    Guanine-cytosine hairpins, containing a hexaethylene glycol bridge, are studied by steady-state fluorescence spectroscopy and time-correlated single photon counting; their properties are compared to those of duplexes with the same sequence. It is shown that, both in hairpins and in duplexes, base pairing induces quenching of the ππ* fluorescence, the quantum yield decreasing by at least two orders of magnitude. When the size of the systems increases from two to ten base pairs, a fluorescent component decaying on the nanosecond time-scale appears at energy higher than that stemming from the bright states of non-interacting mono-nucleotides (ca. 330 nm). For ten base pairs, this new fluorescence forms a well-defined band peaking at 305 nm. Its intensity is about 20% higher for the hairpin compared to the duplex. Its position (red-shifted by 1600 cm(-1)) and width (broader by 1800 cm(-1) FWHM) differ from those observed for large duplexes containing 1000 base pairs, suggesting the involvement of electronic coupling. Fluorescence anisotropy reveals that the excited states responsible for high energy emission are not populated directly upon photon absorption but are reached during a relaxation process. They are assigned to charge transfer states. According to the emerging picture, the amplitude of conformational motions determines whether instantaneous deactivation to the ground state or emission from charge transfer states will take place, while ππ* fluorescence is associated to imperfect base-pairing. PMID:23736116

  13. Photosensitized [2 + 2] cycloaddition of N-acetylated cytosine affords stereoselective formation of cyclobutane pyrimidine dimer

    PubMed Central

    Yamamoto, Junpei; Nishiguchi, Kosuke; Manabe, Koichiro; Masutani, Chikahide; Hanaoka, Fumio; Iwai, Shigenori

    2011-01-01

    Photocycloaddition between two adjacent bases in DNA produces a cyclobutane pyrimidine dimer (CPD), which is one of the major UV-induced DNA lesions, with either the cis-syn or trans-syn structure. In this study, we investigated the photosensitized intramolecular cycloaddition of partially-protected thymidylyl-(3??5?)-N4-acetyl-2?-deoxy-5-methylcytidine, to clarify the effect of the base modification on the cycloaddition reaction. The reaction resulted in the stereoselective formation of the trans-syn CPD, followed by hydrolysis of the acetylamino group. The same result was obtained for the photocycloaddition of thymidylyl-(3??5?)-N4-acetyl-2?-deoxycytidine, whereas both the cis-syn and trans-syn CPDs were formed from thymidylyl-(3??5?)-thymidine. Kinetic analyses revealed that the activation energy of the acid-catalyzed hydrolysis is comparable to that reported for the thymine-cytosine CPD. These findings provided a new strategy for the synthesis of oligonucleotides containing the trans-syn CPD. Using the synthesized oligonucleotide, translesion synthesis by human DNA polymerase ? was analyzed. PMID:20880992

  14. Red Yeast Rice: An Introduction

    MedlinePLUS

    ... as certain cholesterol-lowering drugs, and some may contain a potentially harmful contaminant. This fact sheet provides ... information. Key Facts Some red yeast rice products contain substantial amounts of monacolin K, which is chemically ...

  15. The Yeast Sphingolipid Signaling Landscape

    PubMed Central

    Montefusco, David J.; Matmati, Nabil

    2014-01-01

    Sphingolipids are recognized as signaling mediators in a growing number of pathways, and represent potential targets to address many diseases. The study of sphingolipid signaling in yeast has created a number of breakthroughs in the field, and has the potential to lead future advances. The aim of this article is to provide an inclusive view of two major frontiers in yeast sphingolipid signaling. In the first section, several key studies in the field of sphingolipidomics are consolidated to create a yeast sphingolipidome that ranks nearly all known sphingolipid species by their level in a resting yeast cell. The second section presents an overview of most known phenotypes identified for sphingolipid gene mutants, presented with the intention of illuminating not yet discovered connections outside and inside of the field. PMID:24220500

  16. A quantum chemical insight to intermolecular hydrogen bonding interaction between cytosine and nitrosamine: Structural and energetic investigations

    NASA Astrophysics Data System (ADS)

    Khalili, Behzad

    2016-03-01

    Hydrogen bond interactions which are formed during complex formation between cytosine and nitrosamine have been fully investigated using B3LYP, B3PW91 and MP2 methods in conjunction with various basis sets including 6-311++G (d,p), 6-311++G (2d,2p), 6-311++G (df,pd) and AUG-cc-pVDZ. Three regions around the most stable conformer of cytosine in the gas phase with six possible double H-bonded interactions were considered. Two intermolecular hydrogen bonds of type NC-N-HNA and O-H(N-H)C-ONA were found on the potential energy surface in a cyclic system with 8-member in CN1, CN3, CN5 and 7-member in CN2, CN4, CN6 systems. Results of binding energy calculation at all applied methods reveal that the CN1 structure is the most stable one which is formed by interaction of nitrosamine with cytosine in S1 region. The BSSE-corrected binding energy for six complex system is ranging from -23.8 to -43.6 kJ/mol at MP2/6-311++G (df,pd) level and the stability order is as CN1 > CN2 > CN3 > CN4 > CN5 > CN6 in all studied levels of theories. The NBO results reveal that the charge transfer occurred from cytosine to nitrosamine in CN1, CN3, CN5 and CN6 whereas this matter in the case of CN2 and CN4 was reversed. The relationship between BEs with red shift of H-bond involved bonds vibrational frequencies, charge transfer energies during complex formation and electron densities at H-bond BCPs were discussed. In addition activation energetic properties related to the proton transfer process between cytosine and nitrosamine have been calculated at MP2/6-311++G (df,pd) level. AIM results imply that H-bond interactions are electrostatic with partially covalent characteristic in nature.

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

    PubMed

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

    2013-08-01

    Piracetam improves cognitive function in animals and in human beings, but its mechanism of action is still not completely known. In the present study, we investigated whether enzymes involved in extracellular adenine nucleotide metabolism, adenosine triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase and adenosine deaminase (ADA) are affected by piracetam in the hippocampus and cerebral cortex of animals subjected to scopolamine-induced memory impairment. Piracetam (0.02?mol/5?L, intracerebroventricular, 60min pre-training) prevented memory impairment induced by scopolamine (1mg/kg, intraperitoneal, immediately post-training) in the inhibitory avoidance learning and in the object recognition task. Scopolamine reduced the activity of NTPDase in hippocampus (53% for ATP and 53% for ADP hydrolysis) and cerebral cortex (28% for ATP hydrolysis). Scopolamine also decreased the activity of 5'-nucleotidase (43%) and ADA (91%) in hippocampus. The same effect was observed in the cerebral cortex for 5'-nucleotidase (38%) and ADA (68%) activities. Piracetam fully prevented scopolamine-induced memory impairment and decrease of NTPDase, 5'-nucleotidase and adenosine deaminase activities in synaptosomes from cerebral cortex and hippocampus. In vitro experiments show that piracetam and scopolamine did not alter enzymatic activity in cerebral cortex synaptosomes. Moreover, piracetam prevented scopolamine-induced increase of TBARS levels in hippocampus and cerebral cortex. These results suggest that piracetam-induced improvement of memory is associated with protection against oxidative stress and maintenance of NTPDase, 5'-nucleotidase and ADA activities, and suggest the purinergic system as a putative target of piracetam. PMID:23677777

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

    SciTech Connect

    Ku, Min-Je; Lee, Won-Ho; Nam, Ki-hyun; Rhee, Kyeong-hee; Lee, Ki-Seog; Kim, Eunice EunKyung; Yu, Myung-Hee; Hwang, Kwang Yeon

    2005-04-01

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

  19. Investigation of Radical-Mediated Catalysis in Coenzyme B12-Dependent Ethanolamine Deaminase by using Transient Optical Absorption Spectroscopy.

    NASA Astrophysics Data System (ADS)

    Gadbaw, Michael C.; Warncke, Kurt

    2002-10-01

    Radical-mediated catalysis in the B12-dependent enzymes is initiated by the thermal cleavage of the cobalt-carbon bond of coenzyme B12 (adenosylcob(III)alamin), which produces a geminate CoII-deoxyadenosyl radical pair. Migration of the deoxyadenosyl radical center over 6 promotes radical pair stabilization, so that hydrogen atom abstraction from substrate by the deoxyadenosyl radical can occur to initiate the radical rearrangement to the product radical. Cob(II)alamin is present during these reactions, which occur on the sub-ms timescale. We are investigating the mechanism of radical pair stabilization by using pulsed-laser excitation (Nd-YAG at 355, 532 nm; 10 ns fwhm) to photolyze the cobalt-carbon bond in adenosylcob(III)alamin bound to purified ethanolamine deaminase isolated from Salmonella typhimurium. Absorbance changes are monitored on timescales of >100 ns at approximately 480 nm [cob(II)alamin] or 524 nm [cob(III)alamin] by using a home-constructed transient UV-visible spectrophotometer. The lifetime of the radical pair state is measured in free holoenzyme, and in the presence of substrate analogs and inhibitors. The results provide insights into the mechanism of high-yield radical pair separation in ethanolamine deaminase. [1] Canfield & Warncke, J. Phys. Chem. B 2002, 106, 8831.

  20. 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.

  1. Yeasts preservation: alternatives for lyophilisation.

    PubMed

    Nyanga, Loveness K; Nout, Martinus J R; Smid, Eddy J; Boekhout, Teun; Zwietering, Marcel H

    2012-11-01

    The aim of the study was to compare the effect of two low-cost, low technology traditional methods for drying starter cultures with standard lyophilisation. Lyophilised yeast cultures and yeast cultures preserved in dry rice cakes and dry plant fibre strands were examined for viable cell counts during 6months storage at 4 and 25C. None of the yeast cultures showed a significant loss in viable cell count during 6months of storage at 4C upon lyophilisation and preservation in dry rice cakes. During storage at 25C in the dark, yeast cultures preserved in dry rice cakes, and lyophilised cultures of Saccharomyces cerevisiae and Issatchenkia orientalis showed no significant loss of viable cells up to 4months of storage. Yeast cultures preserved in dry plant fibre strands had the greatest loss of viable count during the 6months of storage at 25C. Preservation of yeasts cultures in dry rice cakes provided better survival during storage at 4C than lyophilisation. The current study demonstrated that traditional methods can be useful and effective for starter culture preservation in small-scale, low-tech applications. PMID:22806747

  2. 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

  3. 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.

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

    PubMed Central

    Dieni, Christopher A; Storey, Kenneth B

    2008-01-01

    Background The wood frog, Rana sylvatica, is one of a few vertebrate species that have developed natural freeze tolerance, surviving days or weeks with 6570% of its total body water frozen in extracellular ice masses. Frozen frogs exhibit no vital signs and their organs must endure multiple stresses, particularly long term anoxia and ischemia. Maintenance of cellular energy supply is critical to viability in the frozen state and in skeletal muscle, AMP deaminase (AMPD) plays a key role in stabilizing cellular energetics. The present study investigated AMPD control in wood frog muscle. Results Wood frog AMPD was subject to multiple regulatory controls: binding to subcellular structures, protein phosphorylation, and effects of allosteric effectors, cryoprotectants and temperature. The percentage of bound AMPD activity increased from 20 to 35% with the transition to the frozen state. Bound AMPD showed altered kinetic parameters compared with the free enzyme (S0.5 AMP was reduced, Hill coefficient fell to ~1.0) and the transition to the frozen state led to a 3-fold increase in S0.5 AMP of the bound enzyme. AMPD was a target of protein phosphorylation. Bound AMPD from control frogs proved to be a low phosphate form with a low S0.5 AMP and was phosphorylated in incubations that stimulated PKA, PKC, CaMK, or AMPK. Bound AMPD from frozen frogs was a high phosphate form with a high S0.5 AMP that was reduced under incubation conditions that stimulated protein phosphatases. Frog muscle AMPD was activated by MgATP and MgADP and inhibited by MgGTP, KCl, NaCl and NH4Cl. The enzyme product, IMP, uniquely inhibited only the bound (phosphorylated) enzyme from muscle of frozen frogs. Activators and inhibitors differentially affected the free versus bound enzyme. S0.5 AMP of bound AMPD was also differentially affected by high versus low assay temperature (25 vs 5C) and by the presence/absence of the natural cryoprotectant (250 mM glucose) that accumulates during freezing. Conclusion Maintenance of long term viability under the ischemic conditions in frozen muscle requires attention to the control of cellular energetics. Differential regulatory controls on AMPD by mechanisms including binding to muscle proteins, actions allosteric effectors, glucose and temperature effects and reversible phosphorylation adjust enzyme function for an optimal role in controlling cellular adenylate levels in ischemic frozen muscle. Stable modification of AMPD properties via freeze-responsive phosphorylation may contribute both to AMPD control and to coordinating AMPD function with other enzymes of energy metabolism in cold ischemic muscle. PMID:18430211

  5. 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.

  6. Yeast mitochondrial transcriptomics.

    PubMed

    Garcia, Mathilde; Darzacq, Xavier; Devaux, Frederic; Singer, Robert H; Jacq, Claude

    2007-01-01

    Although 30 years ago it was strongly suggested that some cytoplasmic ribosomes are bound to the surface of yeast mitochondria, the mechanisms and the raison d'être of this process are not understood. For instance, it is not perfectly known which of the several hundred nuclearly encoded genes have to be translated to the mitochondrial vicinity to guide the import of the corresponding proteins. One can take advantage of several modern methods to address a number of aspects of the site-specific translation process of messenger ribonucleic acid (mRNA) coding for proteins imported into mitochondria. Three complementary approaches are presented to analyze the spatial distribution of mRNAs coding for proteins imported into mitochondria. Starting from biochemical purifications of mitochondria-bound polysomes, we describe a genomewide approach to classify all the cellular mRNAs according to their physical proximity with mitochondria; we also present real-time quantitative reverse transcription polymerase chain reaction monitoring of mRNA distribution to provide a quantified description of this localization. Finally, a fluorescence microscopy approach on a single living cell is described to visualize the in vivo localization of mRNAs involved in mitochondria biogenesis. PMID:18314748

  7. 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing rhizobacteria protect Ocimum sanctum plants during waterlogging stress via reduced ethylene generation.

    PubMed

    Barnawal, Deepti; Bharti, Nidhi; Maji, Deepamala; Chanotiya, Chandan Singh; Kalra, Alok

    2012-09-01

    Ocimum sanctum grown as rain-fed crop, is known to be poorly adapted to waterlogged conditions. Many a times the crop suffers extreme damages because of anoxia and excessive ethylene generation due to waterlogging conditions present under heavy rain. The usefulness of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth promoting rhizobacteria was investigated under waterlogging stress. The comparison of herb yield and stress induced biochemical changes of waterlogged and non-waterlogged plants with and without ACC deaminase-containing microbiological treatments were monitored in this study. Ten plant growth promoting rhizobacteria strains containing ACC-deaminase were isolated and characterized. Four selected isolates Fd2 (Achromobacter xylosoxidans), Bac5 (Serratia ureilytica), Oci9 (Herbaspirillum seropedicae) and Oci13 (Ochrobactrum rhizosphaerae) had the potential to protect Ocimum plants from flood induced damage under waterlogged glass house conditions. Pot experiments were conducted to evaluate the potential of these ACC deaminase-containing selected strains for reducing the yield losses caused by waterlogging conditions. Bacterial treatments protected plants from waterlogging induced detrimental changes like stress ethylene production, reduced chlorophyll concentration, higher lipid peroxidation, proline concentration and reduced foliar nutrient uptake. Fd2 (A. xylosoxidans) induced maximum waterlogging tolerance as treated waterlogged plants recorded maximum growth and herb yield (46.5% higher than uninoculated waterlogged plants) with minimum stress ethylene levels (53% lower ACC concentration as compared to waterlogged plants without bacterial inoculation) whereas under normal non-waterlogged conditions O. rhizosphaerae was most effective in plant growth promotion. PMID:22846334

  8. The cloned 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Sinorhizobium sp. strain BL3 in Rhizobium sp. strain TAL1145 promotes nodulation and growth of Leucaena leucocephala.

    PubMed

    Tittabutr, Panlada; Awaya, Jonathan D; Li, Qing X; Borthakur, Dulal

    2008-06-01

    The objective of this study was to determine the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase of symbionts in nodulation and growth of Leucaena leucocephala. The acdS genes encoding ACC deaminase were cloned from Rhizobium sp. strain TAL1145 and Sinorhizobium sp. BL3 in multicopy plasmids, and transferred to TAL1145. The BL3-acdS gene greatly enhanced ACC deaminase activity in TAL1145 compared to the native acdS gene. The transconjugants of TAL1145 containing the native or BL3 acdS gene could grow in minimal media containing 1.5mM ACC, whereas BL3 could tolerate up to 3mM ACC. The TAL1145 acdS gene was inducible by mimosine and not by ACC, while the BL3 acdS gene was highly inducible by ACC and not by mimosine. The transconjugants of TAL1145 containing the native- and BL3-acdS genes formed nodules with greater number and sizes, and produced higher root mass on L. leucocephala than by TAL1145. This study shows that the introduction of multiple copies of the acdS gene increased ACC deaminase activities of TAL1145 and enhanced its symbiotic efficiency on L. leucocephala. PMID:18406559

  9. Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation.

    PubMed Central

    Smith, S S; Kaplan, B E; Sowers, L C; Newman, E M

    1992-01-01

    The properties of the methyl-directed DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) suggest that it is the enzyme that maintains patterns of methylation in the human genome. Proposals for the enzyme's mechanism of action suggest that 5-methyldeoxycytidine is produced from deoxycytidine via a dihydrocytosine intermediate. We have used an oligodeoxynucleotide containing 5-fluorodeoxycytidine as a suicide substrate to capture the enzyme and the dihydrocytosine intermediate. Gel retardation experiments demonstrate the formation of the expected covalent complex between duplex DNA containing 5-fluorodeoxycytidine and the human enzyme. Formation of the complex was dependent upon the presence of the methyl donor S-adenosylmethionine, suggesting that it comprises an enzyme-linked 5-substituted dihydrocytosine moiety in DNA. Dihydrocytosine derivatives are extremely labile toward hydrolytic deamination in aqueous solution. Because C-to-T transition mutations are especially prevalent at CG sites in human DNA, we have used high-performance liquid chromatography to search for thymidine that might be generated by hydrolysis during the methyl transfer reaction. Despite the potential for deamination inherent in the formation of the intermediate, the methyltransferase did not produce detectable amounts of thymidine. The data suggest that the ability of the human methyltransferase to preserve genetic information when copying a methylation pattern (i.e., its fidelity) is comparable to the ability of a mammalian DNA polymerase to preserve genetic information when copying a DNA sequence. Thus the high frequency of C-to-T transitions at CG sites in human DNA does not appear to be due to the normal enzymatic maintenance of methylation patterns. Images PMID:1584813

  10. Neurodegenerative Central Nervous System Langerhans Cell Histiocytosis and Coincident Hydrocephalus: Treated with Vincristine/Cytosine Arabinoside

    PubMed Central

    Allen, Carl E.; Flores, Ricardo; Rauch, Ronald; Dauser, Robert; Murray, Jeffrey C.; Puccetti, Diane; Hsu, David A.; Sondel, Paul; Hetherington, Maxine; Goldman, Stan; McClain, Kenneth L.

    2012-01-01

    Background Central nervous system (CNS) complications of Langerhans cell histiocytosis (LCH) include mass lesions and a neurodegenerative (ND) syndrome with ataxia, dysarthria, dysmetria, learning and behavior difficulties and/or characteristic changes on brain MRIs. Hydrocephalus has rarely been reported in LCH. LCH lesions of the orbit, mastoid and temporal bones (CNS-Risk lesions) and diabetes insipidus predispose patients to ND-CNS-LCH. Treatment options have been limited and only a case series using trans-retinoic acid (ATRA) and intravenous immunoglobulin (IVIG) have been published. Methods We have used cytosine arabinoside (ARA-C) with or without vincristine to treat 8 patients with ND-CNS LCH. Patients:7 male children and one young adult male with clinical and radiologic ND- CNS-LCH were treated with a regimen of vincristine 1.5 mg/m2 on day 1 and ARA-C 100 mg/m2 daily for 5 days or ARA-C alone monthly for 419 months. Seven patients were evaluated with an ataxia rating scale (ARS) and all with serial MRIs of the brain. Results Five of 7 patients had decreases in their ARS scores and/or decreased T2 hyperintense lesions on MRI images. Grade 2 neutropenia was the most frequent adverse event. Vincristine-associated neuropathy occurred in two patients. Hydrocephalus caused symptoms and signs that confounded the diagnosis and management of ND-CNS-LCH in all 4 patients affected with both. Conclusions Subtle changes in neurologic function may be complicated by hydrocephalus. Vcr/ARA-C or ARA-C were an effective therapies for some ND-CNS LCH patients. A clinical trial using this and possibly other modalities such as IVIG or ATRA should be done. PMID:19908293

  11. 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

  12. Vacuum-Ultraviolet photoionization studies of the microhydrationof DNA bases (Guanine, Cytosine, Adenine and Thymine)

    SciTech Connect

    Belau, L.; Wilson, K.R.; Leone, S.R.; Musahid, Ahmed

    2007-01-22

    In this work, we report on a photoionization study of the microhydration of the four DNA bases. Gas-phase clusters of water with DNA bases [guanine (G), cytosine (C), adenine (A), and thymine (T)] are generated via thermal vaporization of the bases and expansion of the resultant vapor in a continuous supersonic jet expansion of water seeded in Ar. The resulting clusters are investigated by single-photon ionization with tunable vacuum-ultraviolet synchrotron radiation and mass analyzed using reflectron mass spectrometry. Photoionization efficiency (PIE) curves are recorded for the DNA bases and the following water (W) clusters: G, GW{sub n} (n = 1-3); C, CW{sub n} (n = 1-3); A, AW{sub n} (n = 1,2); and T, TW{sub n} (n = 1-3). Appearance energies (AE) are derived from the onset of these PIE curves (all energies in eV): G (8.1 {+-} 0.1), GW (8.0 {+-} 0.1), GW{sub 2} (8.0 {+-} 0.1), and GW{sub 3} (8.0); C (8.65 {+-} 0.05), CW (8.45 {+-} 0.05), CW{sub 2} (8.4 {+-} 0.1), and CW{sub 3} (8.3 {+-} 0.1); A (8.30 {+-} 0.05), AW (8.20 {+-} 0.05), and AW{sub 2} (8.1 {+-} 0.1); T (8.90 {+-} 0.05); and TW (8.75 {+-} 0.05), TW{sub 2} (8.6 {+-} 0.1), and TW{sub 3} (8.6 {+-} 0.1). The AEs of the DNA bases decrease slightly with the addition of water molecules (up to three) but do not converge to values found for photoinduced electron removal from DNA bases in solution.

  13. Vacuum-ultraviolet photoionization studies of the microhydration of DNA bases (guanine, cytosine, adenine, and thymine).

    PubMed

    Belau, Leonid; Wilson, Kevin R; Leone, Stephen R; Ahmed, Musahid

    2007-08-01

    In this work, we report on a photoionization study of the microhydration of the four DNA bases. Gas-phase clusters of water with DNA bases [guanine (G), cytosine (C), adenine (A), and thymine (T)] are generated via thermal vaporization of the bases and expansion of the resultant vapor in a continuous supersonic jet expansion of water seeded in Ar. The resulting clusters are investigated by single-photon ionization with tunable vacuum-ultraviolet synchrotron radiation and mass analyzed using reflectron mass spectrometry. Photoionization efficiency (PIE) curves are recorded for the DNA bases and the following water (W) clusters: G, GWn (n = 1-3); C, CWn (n = 1-3); A, AWn (n = 1,2); and T, TWn (n = 1-3). Appearance energies (AE) are derived from the onset of these PIE curves (all energies in eV): G (8.1 +/- 0.1), GW (8.0 +/- 0.1), GW2 (8.0 +/- 0.1), and GW3 (8.0); C (8.65 +/- 0.05), CW (8.45 +/- 0.05), CW2 (8.4 +/- 0.1), and CW3 (8.3 +/- 0.1); A (8.30 +/- 0.05), AW (8.20 +/- 0.05), and AW2 (8.1 +/- 0.1); T (8.90 +/- 0.05); and TW (8.75 +/- 0.05), TW2 (8.6 +/- 0.1), and TW3 (8.6 +/- 0.1). The AEs of the DNA bases decrease slightly with the addition of water molecules (up to three) but do not converge to values found for photoinduced electron removal from DNA bases in solution. PMID:17419600

  14. McrB: a prokaryotic protein specifically recognizing DNA containing modified cytosine residues.

    PubMed Central

    Krger, 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

  15. Characterization of ACC deaminase-producing endophytic bacteria isolated from copper-tolerant plants and their potential in promoting the growth and copper accumulation of Brassica napus.

    PubMed

    Zhang, Yan-Feng; He, Lin-Yan; Chen, Zhao-Jin; Wang, Qing-Ya; Qian, Meng; Sheng, Xia-Fang

    2011-03-01

    One hundred Cu-resistant-endophytic bacteria were isolated from Cu-tolerant plants grown on Cu mine wasteland, of which, eight Cu-resistant and 1-aminocyclopropane-1-carboxylate (ACC) deaminase-producing endophytic bacteria were obtained based on the ACC deaminase activity of the bacteria and characterized with respect to metal resistance, production of ACC deaminase, indole-3-acetic acid (IAA) as well as siderophores and mineral phosphate solubilization. Ralstonia sp. J1-22-2, Pantoea agglomerans Jp3-3, and Pseudomonas thivervalensis Y1-3-9 with higher ACC deaminase activity (ranging from 213 to 370 ?M ?-ketobutyrate mg(-1)h(-1)) were evaluated for promoting plant growth and Cu uptake of rape grown in quartz sand containing 0, 2.5, and 5 mg kg(-1) of Cu in pot experiments. The eight bacteria were found to exhibit different multiple heavy metal resistance characteristics, to show different levels of ACC deaminase activity and to produce indole acetic acid. Seven bacteria produced siderophores and solubilized inorganic phosphate. Pot experiments showed that inoculation with the strains (J1-22-2, Jp3-3, and Y1-3-9) was found to increase the biomass of rape. Increases in above-ground tissue Cu contents of rape cultivated in 2.5 and 5 mg kg(-1) of Cu-contaminated substrates varied from 9% to 31% and from 3 to 4-fold respectively in inoculated-rape plants compared to the uninoculated control. The maximum Cu uptake of rape was observed after inoculation with P. agglomerans Jp3-3. The results show that metal-resistant and plant growth promoting endophytic bacteria play an important role in plant growth and Cu uptake which may provide a new endophytic bacterial-assisted phytoremediation of Cu-contaminated environment. PMID:21315404

  16. Coherent regulation in yeasts cell-cycle network

    NASA Astrophysics Data System (ADS)

    Aral, Ne?e; Kabak?o?lu, Alkan

    2015-05-01

    We define a measure of coherent activity for gene regulatory networks, a property that reflects the unity of purpose between the regulatory agents with a common target. We propose that such harmonious regulatory action is desirable under a demand for energy efficiency and may be selected for under evolutionary pressures. We consider two recent models of the cell-cycle regulatory network of the yeast, Saccharomyces cerevisiae as a case study and calculate their degree of coherence. A comparison with random networks of similar size and composition reveals that the yeasts cell-cycle regulation is wired to yield an exceptionally high level of coherent regulatory activity. We also investigate the mean degree of coherence as a function of the network size, connectivity and the fraction of repressory/activatory interactions.

  17. 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.

  18. Progress in Yeast Glycosylation Engineering.

    PubMed

    Hamilton, Stephen R; Zha, Dongxing

    2015-01-01

    While yeast are lower eukaryotic organisms, they share many common features and biological processes with higher eukaryotes. As such, yeasts have been used as model organisms to facilitate our understanding of such features and processes. To this end, a large number of powerful genetic tools have been developed to investigate and manipulate these organisms. Going hand-in-hand with these genetic tools is the ability to efficiently scale up the fermentation of these organisms, thus making them attractive hosts for the production of recombinant proteins. A key feature of producing recombinant proteins in yeast is that these proteins can be readily secreted into the culture supernatant, simplifying any downstream processing. A consequence of this secretion is that the proteins typically pass through the secretory pathway, during which they may be exposed to various posttranslational modifications. The addition of glycans is one such modification. Unfortunately, while certain aspects of glycosylation are shared between lower and higher eukaryotes, significant differences exist. Over the last two decades much research has focused on engineering the glycosylation pathways of yeast to more closely resemble those of higher eukaryotes, particularly those of humans for the production of therapeutic proteins. In the current review we shall highlight some of the key achievements in yeast glyco-engineering which have led to humanization of both the N- and O-linked glycosylation pathways. PMID:26082216

  19. 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.

  20. Decreased expression of manganese superoxide dismutase in transformed cells is associated with increased cytosine methylation of the SOD2 gene.

    PubMed

    Huang, Y; He, T; Domann, F E

    1999-08-01

    Tumor cells express lower levels of manganese superoxide dismutase (MnSOD) than their normal counterparts. Enforced expression of MnSOD reverses the malignant phenotype of many transformed cells, suggesting that SOD2 is a tumor suppressor. The SOD2 gene contains a large CpG island spanning > 3.5 kb that starts near the 5' edge of the promoter and extends into intron 2. We hypothesized that the difference in SOD2 expression between tumor cells and their normal cell counterparts might be secondary to differences in their cytosine methylation patterns in this CpG island. To test this hypothesis, we analyzed the methylation status of the SOD2 gene in two cell line models that show differential MnSOD expression between normal and SV40-transformed cells: WI38 and MRC5 and their SV40-transformed variants, WI38-VA and MRC5-VA. We subdivided the SOD2 gene CpG island into 10 individual regions for analysis by bisulfite genomic sequencing. A region located in intron 2 displayed a significant increase in cytosine methylation in both transformed cell lines that expressed low levels of MnSOD mRNA compared with their normal cell counterparts. Recent studies by others have shown that SOD2 intron 2 is a potent transcriptional enhancer. The association between increased cytosine methylation of the SOD2 intron 2 region and decreased MnSOD expression in transformed cells compared with their normal counterparts suggests that an epigenetic mechanism contributes to the differential SOD2 gene expression between these normal and SV40-transformed cells. PMID:10463060

  1. Toxicogenomics using yeast DNA microarrays.

    PubMed

    Yasokawa, Daisuke; Iwahashi, Hitoshi

    2010-11-01

    Development of genomics and bioinformatics enable us to analyze the global gene expression profiles of cells by DNA microarray. Changes in gene expression patterns indicate changes in its physiological conditions. Following the exposure of an organism or cell to toxic chemicals or other environmental stresses, the global genetic responses can be expeditiously and easily analyzed. Baker's yeast, Saccharomyces cerevisiae, is one of the most studied and useful model eukaryotes. The biggest advantage of yeast genomics is the available functional information for each gene and a considerable number of data are accumulating in the field of toxicity assessment using yeast DNA microarray. In this review, we discuss the toxicogenomics of metal ions, alcohols and aldehydes, and other chemicals. PMID:20624688

  2. [Alkalitolerant yeasts from natural biotopes].

    PubMed

    Lisichkina, G A; Bab'eva, I P; Sorokin, D Iu

    2003-01-01

    Using a solid nutrient medium containing alkaline buffer (pH 10) and an antibiotic, alkalitolerant yeasts were isolated from samples of soda-rich saline soils (solonchaks) of Armenia (Arazdayan) and the Transbaikal Region (the Kungur Steppe). The species diversity of the yeast populations of the tested soda-rich soils was relatively insignificant. They only contained alkalitolerant representatives of asporogenic capsulated yeasts belonging to the species Cryptococcus laurentii, C. albidus, Rhodotorula glutinis, R. mucilaginosa, and Sporobolomyces roseus. C. laurentii representatives clearly dominated the isolates obtained, their number exceeding that of the other species by 2-3 orders of magnitude. All of the isolates grew on acidic wort agar, suggesting that they did not include obligate alkaliphiles. PMID:14679910

  3. The effect of breathing vibration on the charge carrier mobility of a guanine cytosine base pair stack

    NASA Astrophysics Data System (ADS)

    Beleznay, F. B.; Szekeres, Zs.; Bogr, F.; Ladik, J.

    2006-06-01

    Ab initio HF crystal orbital calculations were performed for a guanine-cytosine base pair stack at its equilibrium position and at 0.05 dilatation and compression, respectively. The shifts of the conduction and valence band edges were used to determine the deformation potentials of the electrons and the holes. After performing an approximate calculation for the elastic constant, the mobilities of the electrons and holes were determined. They were combined with the mobilities belonging to the longitudinal and torsional motions at a stack in DNA.

  4. Ultrafast IR spectroscopy of polymeric cytosine nucleic acids reveal the long-lived species is due to a localised state.

    PubMed

    Keane, Praic M; Wojdyla, Michal; Doorley, Gerard W; Kelly, John M; Clark, Ian P; Parker, Anthony W; Greetham, Gregory M; Towrie, Michael; Magno, Lus M; Quinn, Susan J

    2012-05-14

    The decay pathways of UV-excited cytosine polymers are investigated using picosecond time-resolved infrared spectroscopy. Similar yields of a non-emissive (1)n?* state are found in the single-stranded dC(30) polymer as in the dCMP monomer, but with a longer lifetime in the polymer (80 ps vs. 39 ps). A longer lifetime is also found in the d(CpC) dinucleotide. No evidence of excimer states is observed, suggesting that localised (1)n?* excited states are the most significant intermediates present on the picosecond timescale. PMID:22358255

  5. 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

  6. 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

  7. 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...

  8. Yeast Can Affect Behavior and Learning.

    ERIC Educational Resources Information Center

    Crook, William G.

    1984-01-01

    A pediatrician recounts his experiences in diagnosing and treating allergies to common yeast germs that may result in behavior and learning problems. He lists characteristics that may predispose children to yeast-connected health problems. (CL)

  9. C-Reactive Protein, Sialic Acid and Adenosine Deaminase Levels in Serum and Pleural Fluid from Patients with Pleural Effusion

    PubMed Central

    Kim, Ji Woon; Yang, In Ae; Oh, Eun A; Rhyoo, Young Gun; Jang, Young Ho; Ryang, Dong Wook; Yoo, JooYong

    1988-01-01

    Laboratory analysis of pleural fluids is essential to determine underlying diseases. The authors evaluated the clinical significance of C-reactive protein (C-RP), sialic acid (SA), and adenosine deaminase (ADA) determinations in sera and pleural fluids from 37 patients with pleural effusion. (FP12)C-RP and sialic acid levels and ADA activities were higher in exudates than in transudates of pleural fluids. Serum and pleural fluid C-RP levels were high in patients with pyothorax. Determinations of serum sialic acid and the pleural fluid to serum ratio were useful for the differential diagnosis of pulmonary tuberculosis and malignancy. ADA activities of pleural fluid and serum are useful for the differentiation of malignancy from tuberculosis and nonspecific pyothorax. C-RP concentrations of pleural fluid correlated to serum levels. However, concentrations of sialic acid and ADA activities were not correlated to serum levels and only correlated to protein concentrations of pleural fluids. PMID:3154188

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

    SciTech Connect

    Sadat Hayatshahi, Sayyed Hamed; 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 {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%.

  11. Activation-Induced Cytidine Deaminase Expression in Human B Cell Precursors Is Essential for Central B Cell Tolerance.

    PubMed

    Cantaert, Tineke; Schickel, Jean-Nicolas; Bannock, Jason M; Ng, Yen-Shing; Massad, Christopher; Oe, Tyler; Wu, Renee; Lavoie, Aubert; Walter, Jolan E; Notarangelo, Luigi D; Al-Herz, Waleed; Kilic, Sara Sebnem; Ochs, Hans D; Nonoyama, Shigeaki; Durandy, Anne; Meffre, Eric

    2015-11-17

    Activation-induced cytidine deaminase (AID), the enzyme-mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells. PMID:26546282

  12. High prevalence of a point mutation in the porphobilinogen deaminase gene in Dutch patients with acute intermittent porphyria.

    PubMed

    Gu, X F; de Rooij, F; Lee, J S; Te Velde, K; Deybach, J C; Nordmann, Y; Grandchamp, B

    1993-03-01

    Acute intermittent porphyria (AIP) is an autosomal dominant disease characterized by a deficiency of porphobilinogen deaminase (PBGD). Up to now 14 different mutations have been described. In an effort to investigate the molecular epidemiology of AIP we have undertaken a systematic study of different exons of the PBGD gene from a large number of unrelated patients. Here, exon 8 from 82 unrelated Dutch and French AIP patients was examined using single strand confirmation polymorphism analysis (SSCP) after polymerase chain reaction (PCR) amplification. A single base mutation, C to T, at position 346 of the sequence coding for PBGD was observed in 15 Dutch families but in only 1 French family. A simple PCR assay is described to facilitate the diagnosis of this common mutation at the DNA level. PMID:8096492

  13. Inhibition of AMP deaminase activity does not improve glucose control in rodent models of insulin resistance or diabetes.

    PubMed

    Admyre, Therese; Amrot-Fors, Lena; Andersson, Maria; Bauer, Martin; Bjursell, Mikael; Drmota, Tomas; Hallen, Stefan; Hartleib-Geschwindner, Judith; Lindmark, Bo; Liu, Jianming; Lfgren, Lars; Rohman, Mattias; Selmi, Nidhal; Wallenius, Kristina

    2014-11-20

    Inhibition of AMP deaminase (AMPD) holds the potential to elevate intracellular adenosine and AMP levels and, therefore, to augment adenosine signaling and activation of AMP-activated protein kinase (AMPK). To test the latter hypothesis, novel AMPD pan inhibitors were synthesized and explored using a panel of in vitro, ex vivo, and in vivo models focusing on confirming AMPD inhibitory potency and the potential of AMPD inhibition to improve glucose control in vivo. Repeated dosing of selected inhibitors did not improve glucose control in insulin-resistant or diabetic rodent disease models. Mice with genetic deletion of the muscle-specific isoform Ampd1 did not showany favorable metabolic phenotype despite being challenged with high-fat diet feeding. Therefore, these results do not support the development of AMPD inhibitors for the treatment of type 2 diabetes. PMID:25459661

  14. Chromatin and Transcription in Yeast

    PubMed Central

    Rando, Oliver J.; Winston, Fred

    2012-01-01

    Understanding the mechanisms by which chromatin structure controls eukaryotic transcription has been an intense area of investigation for the past 25 years. Many of the key discoveries that created the foundation for this field came from studies of Saccharomyces cerevisiae, including the discovery of the role of chromatin in transcriptional silencing, as well as the discovery of chromatin-remodeling factors and histone modification activities. Since that time, studies in yeast have continued to contribute in leading ways. This review article summarizes the large body of yeast studies in this field. PMID:22345607

  15. Mitochondrial inheritance in budding yeast.

    PubMed

    Boldogh, I R; Yang, H C; Pon, L A

    2001-06-01

    During the past decade significant advances were made toward understanding the mechanism of mitochondrial inheritance in the yeast Saccharomyces cerevisiae. A combination of genetics, cell-free assays and microscopy has led to the discovery of a great number of components. These fall into three major categories: cytoskeletal elements, mitochondrial membrane components and regulatory proteins. These proteins mediate activities, including movement of mitochondria from mother cells to buds, segregation of mitochondria and mitochondrial DNA, and equal distribution of the organelle between mother cells and buds during yeast cell division. PMID:11389764

  16. Altered multidrug resistance phenotype caused by anthracycline analogues and cytosine arabinoside in myeloid leukemia.

    PubMed

    Hu, X F; Slater, A; Kantharidis, P; Rischin, D; Juneja, S; Rossi, R; Lee, G; Parkin, J D; Zalcberg, J R

    1999-06-15

    The expression of P-glycoprotein (Pgp) is often increased in acute myeloid leukemia (AML). However, little is known of the regulation of Pgp expression by cytotoxics in AML. We examined whether Pgp expression and function in leukemic blasts was altered after a short exposure to cytotoxics. Blasts were isolated from 19 patients with AML (15 patients) or chronic myeloid leukemia in blastic transformation (BT-CML, 4 patients). Pgp expression and function were analyzed by flow cytometric analysis of MRK 16 binding and Rhodamine 123 retention, respectively. At equitoxic concentrations, ex vivo exposure for 16 hours to the anthracyclines epirubicin (EPI), daunomycin (DAU), idarubicin (IDA), or MX2 or the nucleoside analogue cytosine arabinoside (AraC) differentially upregulated MDR1/Pgp expression in Pgp-negative and Pgp-positive blast cells. In Pgp-negative blasts, all four anthracyclines and AraC significantly increased Pgp expression (P =.01) and Pgp function (P =.03). In contrast, MX2, DAU, and AraC were the most potent in inducing Pgp expression and function in Pgp positive blasts (P <.05). A good correlation between increased Pgp expression and function was observed in Pgp-negative (r =.90, P =.0001) and Pgp-positive blasts (r =.77, P =.0002). This increase in Pgp expression and function was inhibited by the addition of 1 micromol/L PSC 833 to blast cells at the time of their exposure to these cytotoxics. In 1 patient with AML, an increase in Pgp levels was observed in vivo at 4 and 16 hours after the administration of standard chemotherapy with DAU/AraC. Upregulation of Pgp expression was also demonstrated ex vivo in blasts harvested from this patient before the commencement of treatment. In 3 other cases (1 patient with AML and 2 with BT-CML) in which blasts were Pgp negative at the time of initial clinical presentation, serial samples at 1 to 5 months after chemotherapy showed the presence of Pgp-positive blasts. All 3 patients had refractory disease. Interestingly, in all 3 cases, upregulation of Pgp by cytotoxics was demonstrated ex vivo in blasts harvested at the time of presentation. These data suggest that upregulation of the MDR1 gene may represent a normal response of leukemic cells to cytotoxic stress and may contribute to clinical drug resistance. PMID:10361105

  17. Pulsed magnetic field from video display terminals enhances teratogenic effects of cytosine arabinoside in mice

    SciTech Connect

    Chiang, H.; Wu, R.Y.; Shao, B.J.; Fu, Y.D.; Yao, G.D.; Lu, D.J.

    1995-05-01

    Eighty-nine Swiss Webster mice were randomly divided into four groups: a control group, a pulsed magnetic field (PMF) group, a cytosine arabinoside (ara-C, a teratogen) group, and a combined PMF + ara-C group. Mice in the PMF and PMF + ara-C groups were irradiated with a PMF (a sawtooth waveform with 52 {mu}s rise time, 12{mu}s decay time, and 15.6 kHz frequency) at a peak magnetic flux density of 40 {mu}T for 4 hours daily on days 6-17 of gestation. The mice in the ara-C and the PMF + ara-C groups were injected intraperitoneally on day 9 of gestation with 10 mg/kg of ara-C. The incidence of resorption and dead fetuses was not affected by PMF but was increased by ara-C injection. The malformation incidence of cleft palate (CP) and/or cleft lip (CL) was significantly higher in all three of the treated groups than in the control group (P < 0.05). If, however, statistical analyses had been done on litters rather than on individual fetuses, they would show that the incidence of CP and/or CL in the PMF group is not significantly greater than that in the control group. A significantly higher incidence of CP and/or CL was found in the PMF + ara-C group (49%) than the ara-C alone group (26.1%). These data suggest that PMF might enhance the development of ara-C-induced CP and/or CL. The incidence of minor variations in skeletal development, including reduction of skeletal calcification and loss of skeleton, was not statistically significant in the PMF group. However, it was higher in the two ara-C-treated groups, and there was no significant difference between the ara-C alone group and the ara-C + PMF group. From these results it is concluded that the very weak embryotoxic effects of PMF exposure may be revealed and enhanced in combination with a teratogenic agent.

  18. Cytosine arabinoside promotes cytotoxic effect of T cells on leukemia cells mediated by bispecific antibody.

    PubMed

    Li, Wei; Fan, DongMei; Yang, Ming; Yan, Yan; Shi, RuiZan; Cheng, JunPing; Li, ZhenZhen; Zhang, MengNan; Wang, JianXiang; Xiong, Dongsheng

    2013-08-01

    Chemotherapeutic drugs can enhance an immune response of the host against the tumor in addition to killing cancer cells by direct cytotoxicity. Therefore, the combination of chemotherapy and immunotherapy is a promising approach for eliminating tumors, particularly in advanced stages. A strategic medication is to use a bispecific antibody format that is capable of recruiting polyclonal T cells around antibody-target-expressing tumor cells. Recently, we have constructed a bispecific antibody, anti-CD3anti-CD19, in a diabody configuration. In this study, we measured B7 family members B7.1 (CD80) and B7.2 (CD86) expressed on a CD19(+) human leukemia cell line, Nalm-6, stimulated by cytosine arabinoside (Ara-C). We found that a low concentration of Ara-C could upregulate CD80 expressed on CD19(+) Nalm-6 cells. The cytotoxicity of T lymphocytes against Nalm-6 cells in vitro and in vivo mediated by the anti-CD3anti-CD19 diabody with or without a low dose of Ara-C was compared. The combination of the anti-CD3anti-CD19 diabody and Ara-C showed the greatest effectiveness in enhancing the cytotoxicity of T cells against the tumor cells in vitro and in vivo. Activated T cells expressed higher levels of CD25 and CD69 and released more interleukin 2. Both perforin/granzyme B system and Fas/FasL pathway were involved in the diabody-induced T-cell cytotoxicity. Moreover, the activated T cells could upregulate ICAM-3 expression on Nalm-6 cells, and inhibition of LFA-1-ICAM-3 interaction impaired cytotoxicity of T cells. It was noted that Ara-C could upregulate CD80 expressed on two of five specimens of acute B lymphoblastic leukemia patient-derived cells. Cytotoxicity of T cells against these two patient-derived cells was enhanced in the presence of the anti-CD3anti-CD19 diabody. These findings indicate that treatment strategy using both cytotoxic lymphocyte-based immunotherapy and chemotherapy may have synergistic effects. PMID:23879717

  19. Intersystem crossing rates of S1 state keto-amino cytosine at low excess energy.

    PubMed

    Lobsiger, Simon; Etinski, Mihajlo; Blaser, Susan; Frey, Hans-Martin; Marian, Christel; Leutwyler, Samuel

    2015-12-21

    The amino-keto tautomer of supersonic jet-cooled cytosine undergoes intersystem crossing (ISC) from the v = 0 and low-lying vibronic levels of its S1((1)??(?)) state. We investigate these ISC rates experimentally and theoretically as a function of S1 state vibrational excess energy Eexc. The S1 vibronic levels are pumped with a ?5 ns UV laser, the S1 and triplet state ion signals are separated by prompt or delayed ionization with a second UV laser pulse. After correcting the raw ISC yields for the relative S1 and T1 ionization cross sections, we obtain energy dependent ISC quantum yields QISC (corr)=1%-5%. These are combined with previously measured vibronic state-specific decay rates, giving ISC rates kISC = 0.4-1.5 ? 10(9) s(-1), the corresponding S1?S0 internal conversion (IC) rates are 30-100 times larger. Theoretical ISC rates are computed using SCS-CC2 methods, which predict rapid ISC from the S1; v = 0 state with kISC = 3 ? 10(9) s(-1) to the T1((3)??(?)) triplet state. The surprisingly high rate of this El Sayed-forbidden transition is caused by a substantial admixture of (1)nO?(?) character into the S1((1)??(?)) wave function at its non-planar minimum geometry. The combination of experiment and theory implies that (1) below Eexc = 550 cm(-1) in the S1 state, S1?S0 internal conversion dominates the nonradiative decay with kIC ? 2 ? 10(10) s(-1), (2) the calculated S1?T1 ((1)??(?)?(3)??(?)) ISC rate is in good agreement with experiment, (3) being El-Sayed forbidden, the S1?T1 ISC is moderately fast (kISC = 3 ? 10(9) s(-1)), and not ultrafast, as claimed by other calculations, and (4) at Eexc ? 550 cm(-1) the IC rate increases by ?50 times, probably by accessing the lowest conical intersection (the C5-twist CI) and thereby effectively switching off the ISC decay channels. PMID:26696056

  20. Intersystem crossing rates of S1 state keto-amino cytosine at low excess energy

    NASA Astrophysics Data System (ADS)

    Lobsiger, Simon; Etinski, Mihajlo; Blaser, Susan; Frey, Hans-Martin; Marian, Christel; Leutwyler, Samuel

    2015-12-01

    The amino-keto tautomer of supersonic jet-cooled cytosine undergoes intersystem crossing (ISC) from the v = 0 and low-lying vibronic levels of its S1(1ππ∗) state. We investigate these ISC rates experimentally and theoretically as a function of S1 state vibrational excess energy Eexc. The S1 vibronic levels are pumped with a ˜5 ns UV laser, the S1 and triplet state ion signals are separated by prompt or delayed ionization with a second UV laser pulse. After correcting the raw ISC yields for the relative S1 and T1 ionization cross sections, we obtain energy dependent ISC quantum yields QISC corr = 1 % -5%. These are combined with previously measured vibronic state-specific decay rates, giving ISC rates kISC = 0.4-1.5 ṡ 109 s-1, the corresponding S1⇝S0 internal conversion (IC) rates are 30-100 times larger. Theoretical ISC rates are computed using SCS-CC2 methods, which predict rapid ISC from the S1; v = 0 state with kISC = 3 ṡ 109 s-1 to the T1(3ππ∗) triplet state. The surprisingly high rate of this El Sayed-forbidden transition is caused by a substantial admixture of 1nOπ∗ character into the S1(1ππ∗) wave function at its non-planar minimum geometry. The combination of experiment and theory implies that (1) below Eexc = 550 cm-1 in the S1 state, S1⇝S0 internal conversion dominates the nonradiative decay with kIC ≥ 2 ṡ 1010 s-1, (2) the calculated S1⇝T1 (1ππ∗⇝3ππ∗) ISC rate is in good agreement with experiment, (3) being El-Sayed forbidden, the S1⇝T1 ISC is moderately fast (kISC = 3 ṡ 109 s-1), and not ultrafast, as claimed by other calculations, and (4) at Eexc ˜ 550 cm-1 the IC rate increases by ˜50 times, probably by accessing the lowest conical intersection (the C5-twist CI) and thereby effectively switching off the ISC decay channels.

  1. 21 CFR 73.355 - Phaffia yeast.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 1 2014-04-01 2014-04-01 false Phaffia yeast. 73.355 Section 73.355 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATION Foods § 73.355 Phaffia yeast. (a) Identity. (1) The color additive phaffia yeast consists of the killed, dried...

  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. Targeted mutation at cytosine-containing pyrimidine dimers: studies of Escherichia coli B/r with acetophenone and 313-nm light.

    PubMed Central

    Fix, D; Bockrath, R

    1983-01-01

    We have tested the two-event model for UV mutagenesis producing class 2 suppressor mutations at glutamine tRNA genes in Escherichia coli. In the model used, the induction/indexing lesion is any type of pyrimidine dimer and the premutational photoproduct at the target site is a cytosine-containing dimer. Specific mutation-frequency responses were analyzed under conditions in which the ratio of thymine-thymine dimers to cytosine-containing dimers was modified by using 313-nm light and 0.0%, 0.1%, or 0.2% acetophenone. Changes observed in the production of class 2 suppressor mutations were consistent with the model and suggested that the G X C leads to A X T transitions responsible for class 2 suppressor mutations are targeted by cytosine-containing pyrimidine dimers at the mutational sites. PMID:6348769

  4. A 7-Deazaadenosylaziridine Cofactor for Sequence-Specific Labeling of DNA by the DNA Cytosine-C5 Methyltransferase M.HhaI.

    PubMed

    Kunkel, Falk; Lurz, Rudi; Weinhold, Elmar

    2015-01-01

    DNA methyltransferases (MTases) catalyze the transfer of the activated methyl group of the cofactor S-adenosyl-l-methionine (AdoMet or SAM) to the exocyclic amino groups of adenine or cytosine or the C5 ring atom of cytosine within specific DNA sequences. The DNA adenine-N6 MTase from Thermus aquaticus (M.TaqI) is also capable of coupling synthetic N-adenosylaziridine cofactor analogues to its target adenine within the double-stranded 5'-TCGA-3' sequence. This M.TaqI-mediated coupling reaction was exploited to sequence-specifically deliver fluorophores and biotin to DNA using N-adenosylaziridine derivatives carrying reporter groups at the 8-position of the adenine ring. However, these 8-modified aziridine cofactors were poor substrates for the DNA cytosine-C5 MTase from Haemophilus haemolyticus (M.HhaI). Based on the crystal structure of M.HhaI in complex with a duplex oligodeoxynucleotide and the cofactor product, we synthesized a stable 7-deazaadenosylaziridine derivative with a biotin group attached to the 7-position via a flexible linker. This 7-modified aziridine cofactor can be efficiently used by M.HhaI for the direct, quantitative and sequence-specific delivery of biotin to the second cytosine within 5'-GCGC-3' sequences in short duplex oligodeoxynucleotides and plasmid DNA. In addition, we demonstrate that biotinylation by M.HhaI depends on the methylation status of the target cytosine and, thus, could provide a method for cytosine-C5 DNA methylation detection in mammalian DNA. PMID:26610450

  5. 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

  6. Yeast as factory and factotum.

    PubMed

    Dixon, B

    2000-02-01

    After centuries of vigorous activity in making fine wines, beers and breads, Saccharomyces cerevisiae is now acquiring a rich new portfolio of skills, bestowed by genetic manipulation. As shown in a recent shop-window of research supported by the European Commission, yeasts will soon be benefiting industries as diverse as fish farming, pharmaceuticals and laundering. PMID:11190211

  7. Mutagenic Effects Induced by the Attack of NO2 Radical to the Guanine-Cytosine Base Pair

    NASA Astrophysics Data System (ADS)

    Cern-Carrasco, Jos Pedro; Requena, Alberto; Ziga, Jos; 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.

  8. Adsorption of cytosine, thymine, guanine and adenine on Cu(1 1 0) studied by infrared reflection absorption spectroscopy

    NASA Astrophysics Data System (ADS)

    Yamada, Taro; Shirasaka, Kaoru; Takano, Akira; Kawai, Maki

    2004-07-01

    The molecular internal vibration modes and orientation of the DNA base molecules (cytosine, thymine, guanine and adenine) adsorbed on copper(1 1 0) clean single crystal surface were investigated by infrared reflection adsorption spectroscopy in ultrahigh vacuum. The adsorption was performed by thermal evaporation of powder of each species onto Cu(1 1 0) at room temperature. The surface uptakes of the molecules were determined by Auger electron spectroscopy. Cytosine exhibited characteristic asymmetric and symmetric NH 2 stretching signal. The asymmetric stretching signal was missing in the submonolayer region, indicating that the whole molecule was standing upright, with the NH 2 group sticking out of the surface. Thymine was also proved to stand upright at surface. Guanine exhibited weakened infrared signals, indicating that the core purine ring was tilted on the surface, with an interaction of NH?C?O side of the molecule. Adenine at coverages below one monolayer gave no absorption signals, evidencing the molecules laid flat along the surface. The vibrational information obtained will be helpful in developing the future methods of surface DNA sequencing.

  9. Medium-dependent interactions of quinones with cytosine and cytidine: a laser flash photolysis study with magnetic field effect.

    PubMed

    Bose, Adity; Basu, Samita

    2009-03-01

    Laser flash photolysis and an external magnetic field have been used for the study of the interaction of two quinone molecules, namely, 9,10-anthraquinone (AQ) and 2-methyl 1,4-naphthoquinone (or menadione, MQ) with a DNA base, cytosine (C) and its nucleoside cytidine (dC) in two media, a homogeneous one composed of acetonitrile/water (ACN/H(2)O, 9:1, v/v) and a SDS micellar heterogeneous one. We have applied an external magnetic field for the proper identification of the transients formed during the interactions in micellar media. Cytosine exhibits electron transfer (ET) followed by hydrogen abstraction (HA) while dC reveals a reduced ET compared to C, with both quinones in organic homogeneous medium (ACN/H(2)O). Due to a higher electron affinity, AQ supports more faciler ET than MQ with dC in ACN/H(2)O but observations in SDS have been just the reverse. In SDS, ET from dC is completely quenched and a dominant HA is all that could be discerned. This work reveals two main findings: first, a drop in ET on addition of a ribose unit to C, which has been attributed to a role of keto-enol tautomerism in inducing ET from electron-rich nucleus and second, the effect of medium in controlling reaction mechanism by favoring HA with AQ although it is intrinsically more prone towards ET. PMID:19121557

  10. DPPA3 prevents cytosine hydroxymethylation of the maternal pronucleus and is required for normal development in bovine embryos

    PubMed Central

    Bakhtari, Azizollah; Ross, Pablo J

    2014-01-01

    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

  11. Mutagenic effects induced by the attack of NO2 radical to the guanine-cytosine base pair

    PubMed Central

    Cern-Carrasco, Jos P.; Requena, Alberto; Ziga, Jos; Jacquemin, Denis

    2015-01-01

    We investigate the attack of the nitrogen dioxide radical (NO2) to the guaninecytosine (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 GCNO2 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

  12. Photoreaction channels of the guanine-cytosine base pair explored by long-range corrected TDDFT calculations.

    PubMed

    Yamazaki, Shohei; Taketsugu, Tetsuya

    2012-07-01

    Photoinduced processes in the Watson-Crick guanine-cytosine base pair are comprehensively studied by means of long-range corrected (LC) TDDFT calculations of potential energy profiles using the LC-BLYP and CAM-B3LYP functionals. The ab initio CC2 method and the conventional TDDFT method with the B3LYP functional are also employed to assess the reliability of the LC-TDDFT method. The present approach allows us to compare the potential energy profiles at the same computational level for excited-state reactions of the base pair, including single and double proton transfer between the bases and nonradiative decay via ring puckering in each base. In particular, long-range correction to the TDDFT method is critical for a qualitatively correct description of the proton transfer reactions. The calculated energy profiles exhibit low barriers for out-of-plane deformation of the guanine moiety in the locally-excited state, which is expected to lead to a conical intersection with the ground state, as well as for single proton transfer from guanine to cytosine with the well-known electron-driven proton transfer mechanism. Thus the present results suggest that both processes can compete in hydrogen-bonded base pairs and play a significant role in the mechanism of photostability. PMID:22596076

  13. Bacteroides induce higher IgA production than Lactobacillus by increasing activation-induced cytidine deaminase expression in B cells in murine Peyer's patches.

    PubMed

    Yanagibashi, Tsutomu; Hosono, Akira; Oyama, Akihito; Tsuda, Masato; Hachimura, Satoshi; Takahashi, Yoshimasa; Itoh, Kikuji; Hirayama, Kazuhiro; Takahashi, Kyoko; Kaminogawa, Shuichi

    2009-02-01

    The gut mucosal immune system is crucial in host defense against infection by pathogenic microbacteria and viruses via the production of IgA. Previous studies have shown that intestinal commensal bacteria enhance mucosal IgA production. However, it is poorly understood how these bacteria induce IgA production and which genera of intestinal commensal bacteria induce IgA production effectively. In this study, we compared the immunomodulatory effects of Bacteroides and Lactobacillus on IgA production by Peyer's patches lymphocytes. IgA production by Peyer's patches lymphocytes co-cultured with Bacteroides was higher than with Lactobacillus. In addition, the expression of activation-induced cytidine deaminase increased in co-culture with Bacteroides but not with Lactobacillus. We found that intestinal commensal bacteria elicited IgA production. In particular, Bacteroides induced the differentiation of Peyer's patches B cell into IgA(+) B cells by increasing activation-induced cytidine deaminase expression. PMID:19202287

  14. Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay

    PubMed Central

    Jin, Zhao; Di Rienzi, Sara C.; Janzon, Anders; Werner, Jeff J.; Angenent, Largus T.; Dangl, Jeffrey L.; Fowler, Douglas M.

    2015-01-01

    Metagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen to Escherichia coli in vitro. The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate. PMID:26637602

  15. Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay.

    PubMed

    Jin, Zhao; Di Rienzi, Sara C; Janzon, Anders; Werner, Jeff J; Angenent, Largus T; Dangl, Jeffrey L; Fowler, Douglas M; Ley, Ruth E

    2015-01-01

    Metagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen to Escherichia coli in vitro. The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate. PMID:26637602

  16. Ecto-AMP deaminase blunts the ATP-derived adenosine A2A receptor facilitation of acetylcholine release at rat motor nerve endings

    PubMed Central

    Magalhes-Cardoso, M Teresa; Pereira, M Ftima; Oliveira, Laura; Ribeiro, J A; Cunha, Rodrigo A; Correia-de-S, Paulo

    2003-01-01

    At synapses, ATP is released and metabolised through ecto-nucleotidases forming adenosine, which modulates neurotransmitter release through inhibitory A1 or facilitatory A2A receptors, according to the amounts of extracellular adenosine. Neuromuscular junctions possess an ecto-AMP deaminase that can dissociate extracellular ATP catabolism from adenosine formation. In this study we have investigated the pattern of ATP release and its conversion into adenosine, to probe the role of ecto-AMP deaminase in controlling acetylcholine release from rat phrenic nerve terminals. Nerve-evoked ATP release was 28 12 pmol (mg tissue)?1 at 1 Hz, 54 3 pmol (mg tissue)?1 at 5 Hz and disproportionally higher at 50 Hz (324 23 pmol (mg tissue)?1). Extracellular ATP (30 ?m) was metabolised with a half time of 8 2 min, being converted into ADP then into AMP. AMP was either dephosphorylated into adenosine by ecto-5?-nucleotidase (inhibited by ATP and blocked by 200 ?m?,?-methylene ADP) or deaminated into IMP by ecto-AMP deaminase (inhibited by 200 ?m deoxycoformycin, which increased adenosine formation). Dephosphorylation and deamination pathways also catabolised endogenously released adenine nucleotides, since the nerve-evoked extracellular AMP accumulation was increased by either ?,?-methylene ADP (200 ?m) or deoxycoformycin (200 ?m). In the presence of nitrobenzylthioinosine (30 ?m) to inhibit adenosine transport, deoxycoformycin (200 ?m) facilitated nerve-evoked [3H]acetylcholine release by 77 9 %, an effect prevented by the A2A receptor antagonist, ZM 241385 (10 nm). It is concluded that, while ecto-5?-nucleotidase is inhibited by released ATP, ecto-AMP deaminase activity transiently blunts adenosine formation, which would otherwise reach levels high enough to activate facilitatory A2A receptors on motor nerve terminals. PMID:12679375

  17. Possible Role of 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Activity of Sinorhizobium sp. BL3 on Symbiosis with Mung Bean and Determinate Nodule Senescence

    PubMed Central

    Tittabutr, Panlada; Sripakdi, Sudarat; Boonkerd, Nantakorn; Tanthanuch, Waraporn; Minamisawa, Kiwamu; Teaumroong, Neung

    2015-01-01

    Sinorhizobium sp. BL3 forms symbiotic interactions with mung bean (Vigna radiata) and contains lrpL-acdS genes, which encode the 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme that cleaves ACC, a precursor of plant ethylene synthesis. Since ethylene interferes with nodule formation in some legumes and plays a role in senescence in plant cells, BL3-enhancing ACC deaminase activity (BL3+) and defective mutant (BL3−) strains were constructed in order to investigate the effects of this enzyme on symbiosis and nodule senescence. Nodulation competitiveness was weaker in BL3− than in the wild-type, but was stronger in BL3+. The inoculation of BL3− into mung bean resulted in less plant growth, a lower nodule dry weight, and smaller nodule number than those in the wild-type, whereas the inoculation of BL3+ had no marked effects. However, similar nitrogenase activity was observed with all treatments; it was strongly detected 3 weeks after the inoculation and gradually declined with time, indicating senescence. The rate of plant nodulation by BL3+ increased in a time-dependent manner. Nodules occupied by BL3− formed smaller symbiosomes, and bacteroid degradation was more prominent than that in the wild-type 7 weeks after the inoculation. Changes in biochemical molecules during nodulation were tracked by Fourier Transform Infrared (FT-IR) microspectroscopy, and the results obtained confirmed that aging processes differed in nodules occupied by BL3 and BL3−. This is the first study to show the possible role of ACC deaminase activity in senescence in determinate nodules. Our results suggest that an increase in ACC deaminase activity in this strain does not extend the lifespan of nodules, whereas the lack of this activity may accelerate nodule senescence. PMID:26657304

  18. Possible Role of 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Activity of Sinorhizobium sp. BL3 on Symbiosis with Mung Bean and Determinate Nodule Senescence.

    PubMed

    Tittabutr, Panlada; Sripakdi, Sudarat; Boonkerd, Nantakorn; Tanthanuch, Waraporn; Minamisawa, Kiwamu; Teaumroong, Neung

    2015-12-22

    Sinorhizobium sp. BL3 forms symbiotic interactions with mung bean (Vigna radiata) and contains lrpL-acdS genes, which encode the 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme that cleaves ACC, a precursor of plant ethylene synthesis. Since ethylene interferes with nodule formation in some legumes and plays a role in senescence in plant cells, BL3-enhancing ACC deaminase activity (BL3(+)) and defective mutant (BL3(-)) strains were constructed in order to investigate the effects of this enzyme on symbiosis and nodule senescence. Nodulation competitiveness was weaker in BL3(-) than in the wild-type, but was stronger in BL3(+). The inoculation of BL3(-) into mung bean resulted in less plant growth, a lower nodule dry weight, and smaller nodule number than those in the wild-type, whereas the inoculation of BL3(+) had no marked effects. However, similar nitrogenase activity was observed with all treatments; it was strongly detected 3 weeks after the inoculation and gradually declined with time, indicating senescence. The rate of plant nodulation by BL3(+) increased in a time-dependent manner. Nodules occupied by BL3(-) formed smaller symbiosomes, and bacteroid degradation was more prominent than that in the wild-type 7 weeks after the inoculation. Changes in biochemical molecules during nodulation were tracked by Fourier Transform Infrared (FT-IR) microspectroscopy, and the results obtained confirmed that aging processes differed in nodules occupied by BL3 and BL3(-). This is the first study to show the possible role of ACC deaminase activity in senescence in determinate nodules. Our results suggest that an increase in ACC deaminase activity in this strain does not extend the lifespan of nodules, whereas the lack of this activity may accelerate nodule senescence. PMID:26657304

  19. Structures of substrate- and inhibitor-bound adenosine deaminase from a human malaria parasite show a dramatic conformational change and shed light on drug selectivity

    PubMed Central

    Larson, Eric T.; Deng, Wei; Krumm, Brian E.; Napuli, Alberto; Mueller, Natascha; Van Voorhis, Wesley C.; Buckner, Frederick S.; Fan, Erkang; Lauricella, Angela; DeTitta, George; Luft, Joseph; Zucker, Frank; Hol, Wim G. J.; Verlinde, Christophe L. M. J.; Merritt, Ethan A.

    2008-01-01

    Summary Plasmodium and other apicomplexan parasites are deficient in purine biosynthesis, relying instead on the salvage of purines from their host environment. Therefore interference with the purine salvage pathway is an attractive therapeutic target. The plasmodial enzyme adenosine deaminase (ADA) plays a central role in purine salvage and, unlike mammalian ADA homologs, has a further secondary role in methylthiopurine recycling. For this reason, plasmodial adenosine deaminase accepts a wider range of substrates, as it is responsible for deamination of both adenosine and 5?-methylthioadenosine. The latter substrate is not accepted by mammalian ADA homologs. The structural basis for this natural difference in specificity between plasmodial and mammalian ADA has not been well understood. We now report crystal structures of Plasmodium vivax adenosine deaminase in complex with adenosine, guanosine, and the picomolar inhibitor 2?-deoxycoformycin. These structures highlight a drastic conformational change in plasmodial ADA upon substrate-binding that has not been observed for mammalian ADA enzymes. Further, these complexes illuminate the structural basis for the differential substrate specificity and potential drug selectivity between mammalian and parasite enzymes. PMID:18602399

  20. Accelerated deamination of cytosine residues in UV-induced cyclobutane pyrimidine dimers leads to CC-->TT transitions.

    PubMed

    Peng, W; Shaw, B R

    1996-08-01

    The rate of UV-induced deamination of cytosine to uracil at a specific site in double-stranded (ds) DNA was monitored using a genetic reversion assay. M13mp2C141 ds DNA was exposed to 160 J/m2 UV (254 nm), incubated at 37 degrees C, pH 7.4, for various time intervals to allow for deamination, and treated with Escherichia coli photolyase in the presence of 365 nm light to reverse cyclobutane-type pyrimidine dimers. Upon transfection into uracil-glycosylase deficient (ung-) E. coli cells, the mutation (i.e., reversion) frequencies in the CCCC target sequence increased greatly with post-UV time of incubation at 37 degrees C, nearly doubling every day that the DNA had been held at 37 degrees C. After 8 days, the reversion frequencies had increased by two orders of magnitude upon transfection into ung- cells, relative to isogenic ung+ cells, indicating that most of the mutations arising in UV/photolyase-treated ds DNA were C-->T mutations mediated by a uracil intermediate. Sequencing of the revertants revealed that all mutations were single C-->T or tandem double CC-->TT mutations. An increasing percentage of tandem double CC-->TT mutations was found with longer post-UV incubation times, yet none occurred if the post-UV delay time step was omitted before photoreversal. After a 4-day delay between UV and photoreversal at 37 degrees C, greater than 84% of the total revertants had tandem double CC-->TT mutations. Thus, the generation of a tandem double mutation is a time-dependent process that arises in DNA after the initial UV exposure. The rate of appearance (with a pseudo-first-order rate constant ca. 10(-6) s-1) of tandem double mutations during incubation of UV-irradiated DNA is inconsistent with two random, independently occurring mutational events and suggests a concerted deamination of both residues in a tandem cytosine pyrimidine (C < > C) dimer. Considering that deamination in a C < > C dimer occurred here with a half-life of ca. 5 days, in contrast to the measured half-life of ca. 20,000 years for spontaneous (non-UV-treated) cytosine deamination for the same target, these studies show that the formation of pyrimidine dimers in DNA increases the rate of deamination by six orders of magnitude, leading to the accelerated formation of single C-->T and tandem double CC-->TT mutations. PMID:8756482

  1. The birth of yeast peroxisomes.

    PubMed

    Yuan, Wei; Veenhuis, Marten; van der Klei, Ida J

    2016-05-01

    This contribution describes the phenotypic differences of yeast peroxisome-deficient mutants (pex mutants). In some cases different phenotypes were reported for yeast mutants deleted in the same PEX gene. These differences are most likely related to the marker proteins and methods used to detect peroxisomal remnants. This is especially evident for pex3 and pex19 mutants, where the localization of receptor docking proteins (Pex13, Pex14) resulted in the identification of peroxisomal membrane remnants, which do not contain other peroxisomal membrane proteins, such as the ring proteins Pex2, Pex10 and Pex12. These structures in pex3 and pex19 cells are the template for peroxisome formation upon introduction of the missing gene. Taken together, these data suggest that in all yeast pex mutants analyzed so far peroxisomes are not formed de novo but use membrane remnant structures as a template for peroxisome formation upon reintroduction of the missing gene. The relevance of this model for peroxisomal membrane protein and lipid sorting to peroxisomes is discussed. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann. PMID:26367802

  2. Nuclear Import of Yeast Proteasomes

    PubMed Central

    Burcoglu, Julianne; Zhao, Liang; Enenkel, Cordula

    2015-01-01

    Proteasomes are highly conserved protease complexes responsible for the degradation of aberrant and short-lived proteins. In highly proliferating yeast and mammalian cells, proteasomes are predominantly nuclear. During quiescence and cell cycle arrest, proteasomes accumulate in granules in close proximity to the nuclear envelope/ER. With prolonged quiescence in yeast, these proteasome granules pinch off as membraneless organelles, and migrate as stable entities through the cytoplasm. Upon exit from quiescence, the proteasome granules clear and the proteasomes are rapidly transported into the nucleus, a process reflecting the dynamic nature of these multisubunit complexes. Due to the scarcity of studies on the nuclear transport of mammalian proteasomes, we summarised the current knowledge on the nuclear import of yeast proteasomes. This pathway uses canonical nuclear localisation signals within proteasomal subunits and Srp1/Kap95, and the canonical import receptor, named importin/karyopherin αβ. Blm10, a conserved 240 kDa protein, which is structurally related to Kap95, provides an alternative import pathway. Two models exist upon which either inactive precursor complexes or active holo-enzymes serve as the import cargo. Here, we reconcile both models and suggest that the import of inactive precursor complexes predominates in dividing cells, while the import of mature enzymes mainly occurs upon exit from quiescence. PMID:26262643

  3. A 5? cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs

    SciTech Connect

    Zhu, Deyu; Stumpf, Craig R.; Krahn, Joseph M.; Wickens, Marvin; Tanaka Hall, Traci M.

    2010-11-03

    A single regulatory protein can control the fate of many mRNAs with related functions. The Puf3 protein of Saccharomyces cerevisiae is exemplary, as it binds and regulates more than 100 mRNAs that encode proteins with mitochondrial function. Here we elucidate the structural basis of that specificity. To do so, we explore the crystal structures of Puf3p complexes with 2 cognate RNAs. The key determinant of Puf3p specificity is an unusual interaction between a distinctive pocket of the protein with an RNA base outside the 'core' PUF-binding site. That interaction dramatically affects binding affinity in vitro and is required for regulation in vivo. The Puf3p structures, combined with those of Puf4p in the same organism, illuminate the structural basis of natural PUF-RNA networks. Yeast Puf3p binds its own RNAs because they possess a -2C and is excluded from those of Puf4p which contain an additional nucleotide in the core-binding site.

  4. 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

  5. Coupling between hydrogen atoms transfer and stacking interaction in adenine-thymine/guanine-cytosine complexes: a theoretical study.

    PubMed

    Villani, Giovanni

    2014-05-22

    Four different complexes of two base pairs, an adenine-thymine and a guanine-cytosine one, have been studied in order to understand the modifications induced by the staking interaction between the two base pairs on the hydrogen atoms transfers between the bases in either base pair. The inclusion of these two kinds of interactions allows us to clarify if some properties, as the mechanism of hydrogen transfer, is exclusively a local effect of a base pair or can be modified by a more long-range interaction between the base pairs. The results on these four complexes are compared with those of the monomeric systems, the A-T and G-C base pair, and with those of the A-T and G-C dimers. The specificity of each complex and of each hydrogen bond has been analyzed. PMID:24813562

  6. 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; Wrmlnder, 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

  7. Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field

    PubMed Central

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

    2016-01-01

    Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds’ vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field. PMID:26999445

  8. Ultrasensitive Direct Quantification of Nucleobase Modifications in DNA by Surface-Enhanced Raman Scattering: The Case of Cytosine.

    PubMed

    Morla-Folch, Judit; Xie, Hai-nan; Gisbert-Quilis, Patricia; Gmez-de Pedro, Sara; Pazos-Perez, Nicolas; Alvarez-Puebla, Ramon A; Guerrini, Luca

    2015-11-01

    Recognition of chemical modifications in canonical nucleobases of nucleic acids is of key importance since such modified variants act as different genetic encoders, introducing variability in the biological information contained in DNA. Herein, we demonstrate the feasibility of direct SERS in combination with chemometrics and microfluidics for the identification and relative quantification of 4 different cytosine modifications in both single- and double-stranded DNA. The minute amount of DNA required per measurement, in the sub-nanogram regime, removes the necessity of pre-amplification or enrichment steps (which are also potential sources of artificial DNA damages). These findings show great potentials for the development of fast, low-cost and high-throughput screening analytical devices capable of detecting known and unknown modifications in nucleic acids (DNA and RNA) opening new windows of activity in several fields such as biology, medicine and forensic sciences. PMID:26447808

  9. Hydrogen-bonded double-proton transfer in five guanine-cytosine base pairs after hydrogen atom addition.

    PubMed

    Lin, Yuexia; Wang, Hongyan; Gao, Simin; Li, Ruhu; Schaefer, Henry F

    2012-08-01

    The double-proton transfer reactions in Watson-Crick guanine-cytosine (GC) base pairs after hydrogen atom addition are studied theoretically. The structural changes and energy differences among the structures are compared to explore the double-proton transfer mechanisms, concerted and stepwise. The concerted mechanism is found in all five radicals (GC+H)() considered, while the stepwise mechanism is predicted only for structures G-H()C(C6) and H()G(N7)-C. The geometrical features have been found to change regularly in the concerted double-proton transfer. This is different from the single-proton transfer, for which the structural perturbations are dispersed throughout the GC base pair. The energy analyses demonstrate that the concerted double-proton transfer mechanism is more favorable in the gas phase, while the stepwise mechanism dominates in water. The structures of proton transfer products become less favored energetically. PMID:22774934

  10. Stability of the valence anion of cytosine is governed by nucleobases sequence in the double stranded DNA ?-stack: A computational study

    NASA Astrophysics Data System (ADS)

    Koby?ecka, Monika; Leszczynski, Jerzy; Rak, Janusz

    2009-08-01

    The stabilities of the valence anion of cytosine (C-) in model trimers of complementary base pairs that possess the B-DNA geometry but differ in base sequence are reported. In order to estimate the energetics of electron attachment to the middle cytosine incorporated in the trimer, a thermodynamic cycle employing all possible two-body interaction energies in the neutral and anionic duplex as well as the adiabatic electron affinity of isolated cytosine were developed. All calculations were carried out at the MP2 level of theory with the aug-cc-pVDZ basis set. We have demonstrated that contrary to the literature reports, concerning single stranded DNA, the sequence of nucleic bases has a profound effect on the stability of the cytosine valence anion. The anionic 3'-CCC-5' complex is the most stable configuration (EA=0.399 eV) and the 3'-GCG-5' trimer anion is the most unstable species (EA=-0.193 eV). Moreover, with the energetic correction for the presence of sugar-phosphate backbone all possible double stranded DNA sequences lead to the stable C-. The predicted electron affinities of the cytosine anion have been compared to the results of analogous studies on the thymine anion published recently [M. Koby?ecka et al., J. Am. Chem. Soc. 130, 15683 (2008)]. The consequences of low-energy barrier proton transfer in the GC anion have been discussed in the context of induced by electrons DNA single strand breaks. The DNA sequences that should dramatically differ in their vulnerability to be damaged by low energy electrons have been proposed.

  11. 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

  12. 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

  13. A periplasmic aldehyde oxidoreductase represents the first molybdopterin cytosine dinucleotide cofactor containing molybdo-flavoenzyme from Escherichia coli.

    PubMed

    Neumann, Meina; Mittelstdt, Gerd; Iobbi-Nivol, Chantal; Saggu, Miguel; Lendzian, Friedhelm; Hildebrandt, Peter; Leimkhler, Silke

    2009-05-01

    Three DNA regions carrying genes encoding putative homologs of xanthine dehydrogenases were identified in Escherichia coli, named xdhABC, xdhD, and yagTSRQ. Here, we describe the purification and characterization of gene products of the yagTSRQ operon, a molybdenum-containing iron-sulfur flavoprotein from E. coli, which is located in the periplasm. The 135 kDa enzyme comprised a noncovalent (alpha beta gamma) heterotrimer with a large (78.1 kDa) molybdenum cofactor (Moco)-containing YagR subunit, a medium (33.9 kDa) FAD-containing YagS subunit, and a small (21.0 kDa) 2 x [2Fe2S]-containing YagT subunit. YagQ is not a subunit of the mature enzyme, and the protein is expected to be involved in Moco modification and insertion into YagTSR. Analysis of the form of Moco present in YagTSR revealed the presence of the molybdopterin cytosine dinucleotide cofactor. Two different [2Fe2S] clusters, typical for this class of enzyme, were identified by EPR. YagTSR represents the first example of a molybdopterin cytosine dinucleotide-containing protein in E. coli. Kinetic characterization of the enzyme revealed that YagTSR converts a broad spectrum of aldehydes, with a preference for aromatic aldehydes. Ferredoxin instead of NAD(+) or molecular oxygen was used as terminal electron acceptor. Complete growth inhibition of E. coli cells devoid of genes from the yagTSRQ operon was observed by the addition of cinnamaldehyde to a low-pH medium. This finding shows that YagTSR might have a role in the detoxification of aromatic aldehydes for E. coli under certain growth conditions. PMID:19368556

  14. Kinetics of extension of O sup 6 -methylguanine paired with cytosine or thymine in defined oligonucleotide sequences

    SciTech Connect

    Dosanjh, M.K.; Galeros, G.; Singer, B. ); Goodman, M.F. )

    1991-12-10

    The frequency of extending m{sup 6}G{center dot}C or m{sup 6}G{center dot}T pairs, when the 3{prime} and 5{prime} flanking neighbors of m{sup 6}G are either cytosines or thymines, was investigated using primed 25-base-long oligonucleotides and the Klenow fragment of Escherichia coli DNA polymerase I (Kf). The efficiency, V{sub max}/K{sub m}, of extension to the following normal base pair was up to 40-fold greater than for the formation of the m{sup 6}G{center dot}T or m{sup 6}G{center dot}C pair. The frequencies of inserting either dCMP or dTMP opposite these m{sup 6}G bases did not appear to be different in the two sequences, C-m{sup 6}G-C and T-m{sup 6}G-T, but extension was favored in the C-m{sup 6}G-C sequence. The m{sup 6}G{center dot}T pair extended to a C{center dot}G pair most efficiently, indicating that it was not a strong block to continued replication past the template lesion. Thus, m{sup 6}G{center dot}T flanked by cytosines replicates more readily than when flanked by thymines, increasing G {yields} A transitions. These data lend further support to the importance of sequence context in mutagenesis.

  15. Novel activities of human uracil DNA N-glycosylase for cytosine-derived products of oxidative DNA damage.

    PubMed Central

    Dizdaroglu, M; Karakaya, A; Jaruga, P; Slupphaug, G; Krokan, H E

    1996-01-01

    Uracil DNA N-glycosylase is a repair enzyme that releases uracil from DNA. A major function of this enzyme is presumably to protect the genome from pre-mutagenic uracil resulting from deamination of cytosine in DNA. Here, we report that human uracil DNA N-glycosylase also recognizes three uracil derivatives that are generated as major products of cytosine in DNA by hydroxyl radical attack or other oxidative processes. DNA substrates were prepared by gamma-irradiation of DNA in aerated aqueous solution and incubated with human uracil DNA N-glycosylase, heat-inactivated enzyme or buffer. Ethanol-precipitated DNA and supernatant fractions were then separated. Supernatant fractions after derivatization, and pellets after hydrolysis and derivatization were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results demonstrated that human uracil DNA N-glycosylase excised isodialuric acid, 5-hydroxyuracil and alloxan from DNA with apparent K(m) values of approximately 530, 450 and 660 nM, respectively. The excision of these uracil analogues is consistent with the recently described mechanism for recognition of uracil by human uracil DNA N-glycosylase [Mol,C.D., Arval,A.S., Slupphaug,G., Kavil,B., Alseth,I., Krokan,H.E. and Tainer,J.A. (1995) Cell, 80, 869-878]. Nine other pyrimidine- and purine-derived products that were identified in DNA samples were not substrates for the enzyme. The results indicate that human uracil DNA N-glycosylase may have a function in the repair of oxidative DNA damage. PMID:8602352

  16. Yeasts in floral nectar: a quantitative survey

    PubMed Central

    Herrera, Carlos M.; de Vega, Clara; Canto, Azucena; Pozo, María I.

    2009-01-01

    Background and Aims One peculiarity of floral nectar that remains relatively unexplored from an ecological perspective is its role as a natural habitat for micro-organisms. This study assesses the frequency of occurrence and abundance of yeast cells in floral nectar of insect-pollinated plants from three contrasting plant communities on two continents. Possible correlations between interspecific differences in yeast incidence and pollinator composition are also explored. Methods The study was conducted at three widely separated areas, two in the Iberian Peninsula (Spain) and one in the Yucatán Peninsula (Mexico). Floral nectar samples from 130 species (37–63 species per region) in 44 families were examined microscopically for the presence of yeast cells. For one of the Spanish sites, the relationship across species between incidence of yeasts in nectar and the proportion of flowers visited by each of five major pollinator categories was also investigated. Key Results Yeasts occurred regularly in the floral nectar of many species, where they sometimes reached extraordinary densities (up to 4 × 105 cells mm−3). Depending on the region, between 32 and 44 % of all nectar samples contained yeasts. Yeast cell densities in the order of 104 cells mm−3 were commonplace, and densities >105 cells mm−3 were not rare. About one-fifth of species at each site had mean yeast cell densities >104 cells mm−3. Across species, yeast frequency and abundance were directly correlated with the proportion of floral visits by bumble-bees, and inversely with the proportion of visits by solitary bees. Conclusions Incorporating nectar yeasts into the scenario of plant–pollinator interactions opens up a number of intriguing avenues for research. In addition, with yeasts being as ubiquitous and abundant in floral nectars as revealed by this study, and given their astounding metabolic versatility, studies focusing on nectar chemical features should carefully control for the presence of yeasts in nectar samples. PMID:19208669

  17. The yeast Golgi apparatus: insights and mysteries

    PubMed Central

    Papanikou, Effrosyni; Glick, Benjamin S.

    2009-01-01

    The Golgi apparatus is known to modify and sort newly synthesized secretory proteins. However, fundamental mysteries remain about the structure, operation, and dynamics of this organelle. Important insights have emerged from studying the Golgi in yeasts. For example, yeasts have provided direct evidence for Golgi cisternal maturation, a mechanism that is likely to be broadly conserved. Here, we highlight features of the yeast Golgi as well as challenges that lie ahead. PMID:19879270

  18. Plant growth-promoting traits of epiphytic and endophytic yeasts isolated from rice and sugar cane leaves in Thailand.

    PubMed

    Nutaratat, Pumin; Srisuk, Nantana; Arunrattiyakorn, Panarat; Limtong, Savitree

    2014-08-01

    A total of 1035 yeast isolates, obtained from rice and sugar cane leaves, were screened primarily for indole-3-acetic acid (IAA) production. Thirteen isolates were selected, due to their IAA production ranging from 1.2 to 29.3 mg g(-)(1) DCW. These isolates were investigated for their capabilities of calcium phosphate and ZnO(3) solubilisation, and also for production of NH(3), polyamine, and siderophore. Their 1-aminocyclopropane-1-carboxylate (ACC) deaminase, catalase and fungal cell wall-degrading enzyme activities were assessed. Their antagonism against rice fungal pathogens was also evaluated. Strain identification, based on molecular taxonomy, of the thirteen yeast isolates revealed that four yeast species - i.e. Hannaella sinensis (DMKU-RP45), Cryptococcus flavus (DMKU-RE12, DMKU-RE19, DMKU-RE67, and DMKU-RP128), Rhodosporidium paludigenum (DMKU-RP301) and Torulaspora globosa (DMKU-RP31) - were capable of high IAA production. Catalase activity was detected in all yeast strains tested. The yeast R. paludigenum DMKU-RP301 was the best IAA producer, yielding 29.3 mg g(-)(1) DCW, and showed the ability to produce NH3 and siderophore. Different levels of IAA production (7.2-9.7 mg g(-)(1) DCW) were found in four strains of C. flavus DMKU-RE12, DMKU-RE19, and DMKU-RE67, which are rice leaf endophytes, and strain DMKU-RP128, which is a rice leaf epiphyte. NH(3) production and carboxymethyl cellulase (CMCase) activity was also detected in these four strains. Antagonism to fungal plant pathogens and production of antifungal volatile compounds were exhibited in T. globosa DMKU-RP31, as well as a moderate level of IAA production (4.9 mg g(-)(1) DCW). The overall results indicated that T. globosa DMKU-RP31 might be used in two ways: enhancing plant growth and acting as a biocontrol agent. In addition, four C. flavus were also found to be strains of interest for optimal IAA production. PMID:25110131

  19. Role of glucose signaling in yeast metabolism

    SciTech Connect

    Dam, K. van

    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.

  20. 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.

  1. Effects of aqueous soybean, mistletoe and red clover extracts on activities of adenosine deaminase and xanthine oxidase enzyme.

    PubMed

    Namuslu, M; Kocaoglu, H; Celik, H T; Avci, A; Devrim, E; Genc, Y; Gocmen, E; Erguder, I B; Durak, I

    2014-01-01

    Soybean (Glycine max), mistletoe (Viscum album) and red clover (Trifolium pratence) have been argued to have anti-cancer effects. In the present study it was aimed to investigate possible effects of these plant extracts on the activities of DNA turn-over enzymes, namely adenosine deaminase (ADA) and xanthine oxidase (XO) in cancerous and non-cancerous gastric and colon tissues. For this aim, 6 cancerous and 6 non-cancerous adjacent human gastric tissues, and 7 cancerous and 7 non-cancerous adjacent colon tissues were obtained by surgical operations. Our results suggest that aqueous soybean, mistletoe and red clover extracts may exhibit anti-tumoral activity by depleting hypoxanthine concentration in the cancer cells through XO activation, which may lead to lowered salvage pathway activity necessary for the cancer cells to proliferate in the cancerous colon tissue. Some foods like soybean, mistletoe and red clover may provide nutritional support to medical cancer therapy through inhibiting and/or activating key enzymes in cancer metabolism (Tab. 4, Ref.?33). PMID:25023428

  2. Opposing activity changes in AMP deaminase and AMP-activated protein kinase in the hibernating ground squirrel.

    PubMed

    Lanaspa, Miguel A; Epperson, L Elaine; Li, Nanxing; Cicerchi, Christina; Garcia, Gabriela E; Roncal-Jimenez, Carlos A; Trostel, Jessica; Jain, Swati; Mant, Colin T; Rivard, Christopher J; Ishimoto, Takuji; Shimada, Michiko; Sanchez-Lozada, Laura Gabriela; Nakagawa, Takahiko; Jani, Alkesh; Stenvinkel, Peter; Martin, Sandra L; Johnson, Richard J

    2015-01-01

    Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2) (summer) and activation of AMP-activated protein kinase (AMPK) (winter). Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and ?-fatty acid oxidation were evaluated. Changes in fat metabolism were correlated with changes in AMPD2 activity and intrahepatic uric acid (downstream product of AMPD2), as well as changes in AMPK and intrahepatic ?-hydroxybutyrate (a marker of fat oxidation). Hepatic fat accumulation occurred during the summer with relatively increased enzymes associated with fat synthesis (FAS, ACL and ACC) and decreased enoyl CoA hydratase (ECH1) and carnitine palmitoyltransferase 1A (CPT1A), rate limiting enzymes of fat oxidation. In summer, AMPD2 activity and intrahepatic uric acid levels were high and hepatic AMPK activity was low. In contrast, the active phosphorylated form of AMPK and ?-hydroxybutyrate both increased during winter hibernation. Therefore, changes in AMPD2 and AMPK activity were paralleled with changes in fat synthesis and fat oxidation rates during the summer-winter cycle. These data illuminate the opposing forces of metabolism of AMP by AMPD2 and its availability to activate AMPK as a switch that governs fat metabolism in the liver of hibernating ground squirrel. PMID:25856396

  3. Selection of the best blood compartment to measure cytidine deaminase activity to stratify for optimal gemcitabine or cytarabine treatment.

    PubMed

    Peters, Godefridus J; Honeywell, Richard J; Maulandi, Marie; Giovannetti, Elisa; Losekoot, Nienke; Etienne-Grimaldi, Marie-Christine; Milano, Gerard; Serdjebi, Cindy; Ciccolini, Joseph

    2014-01-01

    Cytidine deaminase (CDA) plays a crucial role in the degradation of cytidine analogs, such as gemcitabine and cytarabine. Several studies showed that a low CDA activity is associated with more toxicity but a higher efficacy, while a high activity will lead to a lower efficacy but less toxicity. A stratified dosing strategy based on the relative CDA activity would increase efficiency. In order to predict these events, a reliable measurement of CDA with a validated method is crucial. We aimed to determine which phenotype assay would be most suitable; a spectrophotometric assay using cytidine as a substrate, or an HPLC assay using gemcitabine as a substrate. In serum and whole blood of 26 volunteers, both assays showed an excellent correlation (R>0.999), but not in plasma nor in red blood cells. Moreover, there was no difference between males and females. In conclusion, the spectrophotometric assay seems the most simple and cost-effective test. It should be performed in serum, while it should be normalized on protein content as measured by the Bicinchoninic Acid. PMID:24940698

  4. Structure of human porphobilinogen deaminase at 2.8 A: the molecular basis of acute intermittent porphyria.

    PubMed

    Gill, Raj; Kolstoe, Simon E; Mohammed, Fiyaz; Al D-Bass, Abeer; Mosely, Julie E; Sarwar, Mohammed; Cooper, Jonathan B; Wood, Stephen P; Shoolingin-Jordan, Peter M

    2009-05-15

    Mutations in the human PBGD (porphobilinogen deaminase) gene cause the inherited defect AIP (acute intermittent porphyria). In the present study we report the structure of the human uPBGD (ubiquitous PBGD) mutant, R167Q, that has been determined by X-ray crystallography and refined to 2.8 A (1 A=0.1 nm) resolution (Rfactor=0.26, Rfree=0.29). The protein crystallized in space group P2(1)2(1)2 with two molecules in the asymmetric unit (a=81.0 A, b=104.4 A and c=109.7 A). Phases were obtained by molecular replacement using the Escherichia coli PBGD structure as a search model. The human enzyme is composed of three domains each of approx. 110 amino acids and possesses a dipyrromethane cofactor at the active site, which is located between domains 1 and 2. An ordered sulfate ion is hydrogen-bonded to Arg26 and Ser28 at the proposed substrate-binding site in domain 1. An insert of 29 amino acid residues, present only in mammalian PBGD enzymes, has been modelled into domain 3 where it extends helix alpha2(3) and forms a beta-hairpin structure that contributes to a continuous hydrogen-bonding network spanning domains 1 and 3. The structural and functional implications of the R167Q mutation and other mutations that result in AIP are discussed. PMID:19207107

  5. Chronic lung injury by constitutive expression of activation-induced cytidine deaminase leads to focal mucous cell metaplasia and cancer.

    PubMed

    Kitamura, Jiro; Uemura, Munehiro; Kurozumi, Mafumi; Sonobe, Makoto; Manabe, Toshiaki; Hiai, Hiroshi; Date, Hiroshi; Kinoshita, Kazuo

    2015-01-01

    Activation-induced cytidine deaminase (AID) is an enzyme required for antibody diversification, and it causes DNA mutations and strand breaks. Constitutive AID expression in mice invariably caused lung lesions morphologically similar to human atypical adenomatous hyperplasia (AAH), which can be a precursor of bronchioloalveolar carcinoma. Similar to AAH, mouse AAH-like lesion (MALL) exhibited signs of alveolar differentiation, judging from the expression of alveolar type II (AT2) cell marker surfactant protein C (SP-C). However, electron microscopy indicated that MALL, which possessed certain features of a mucous cell, is distinct from an AAH or AT2 cell. Although MALL developed in all individuals within 30 weeks after birth, lung tumors occurred in only 10%; this suggests that the vast majority of MALLs fail to grow into visible tumors. MALL expressed several recently described markers of lung alveolar regeneration such as p63, keratin 5, keratin 14, leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), and Lgr6. Increased cell death was observed in the lungs of AID transgenic mice compared with wild-type mice. Based on these observations, we speculate that MALL is a regenerating tissue compensating for cellular loss caused by AID cytotoxicity. AID expression in such regenerating tissue should predispose cells to malignant transformation via its mutagenic activity. PMID:25659078

  6. Opposing Activity Changes in AMP Deaminase and AMP-Activated Protein Kinase in the Hibernating Ground Squirrel

    PubMed Central

    Cicerchi, Christina; Garcia, Gabriela E.; Roncal-Jimenez, Carlos A.; Trostel, Jessica; Jain, Swati; Mant, Colin T.; Rivard, Christopher J.; Ishimoto, Takuji; Shimada, Michiko; Sanchez-Lozada, Laura Gabriela; Nakagawa, Takahiko; Jani, Alkesh; Stenvinkel, Peter; Martin, Sandra L.; Johnson, Richard J.

    2015-01-01

    Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2) (summer) and activation of AMP-activated protein kinase (AMPK) (winter). Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and ?-fatty acid oxidation were evaluated. Changes in fat metabolism were correlated with changes in AMPD2 activity and intrahepatic uric acid (downstream product of AMPD2), as well as changes in AMPK and intrahepatic ?-hydroxybutyrate (a marker of fat oxidation). Hepatic fat accumulation occurred during the summer with relatively increased enzymes associated with fat synthesis (FAS, ACL and ACC) and decreased enoyl CoA hydratase (ECH1) and carnitine palmitoyltransferase 1A (CPT1A), rate limiting enzymes of fat oxidation. In summer, AMPD2 activity and intrahepatic uric acid levels were high and hepatic AMPK activity was low. In contrast, the active phosphorylated form of AMPK and ?-hydroxybutyrate both increased during winter hibernation. Therefore, changes in AMPD2 and AMPK activity were paralleled with changes in fat synthesis and fat oxidation rates during the summer-winter cycle. These data illuminate the opposing forces of metabolism of AMP by AMPD2 and its availability to activate AMPK as a switch that governs fat metabolism in the liver of hibernating ground squirrel. PMID:25856396

  7. MicroRNA-146b-3p Regulates Retinal Inflammation by Suppressing Adenosine Deaminase-2 in Diabetes

    PubMed Central

    Fulzele, Sadanand; El-Sherbini, Ahmed; Ahmad, Saif; Sangani, Rajnikumar; Matragoon, Suraporn; Radhakrishnan, Reshmitha; Liou, Gregory I.

    2015-01-01

    Hyperglycemia- (HG-) Amadori-glycated albumin- (AGA-) induced activation of microglia and monocytes and their adherence to retinal vascular endothelial cells contribute to retinal inflammation leading to diabetic retinopathy (DR). There is a great need for early detection of DR before demonstrable tissue damages become irreversible. Extracellular adenosine, required for endogenous anti-inflammation, is regulated by the interplay of equilibrative nucleoside transporter with adenosine deaminase (ADA) and adenosine kinase. ADA, including ADA1 and ADA2, exists in all organisms. However, because ADA2 gene has not been identified in mouse genome, how diabetes alters adenosine-dependent anti-inflammation remains unclear. Studies of pig retinal microglia and human macrophages revealed a causal role of ADA2 in inflammation. Database search suggested miR-146b-3p recognition sites in the 3?-UTR of ADA2 mRNA. Coexpression of miR-146b-3p, but not miR-146-5p or nontargeting miRNA, with 3?-UTR of the ADA2 gene was necessary to suppress a linked reporter gene. In the vitreous of diabetic patients, decreased miR-146b-3p is associated with increased ADA2 activity. Ectopic expression of miR-146b-3p suppressed ADA2 expression, activity, and TNF-? release in the AGA-treated human macrophages. These results suggest a regulatory role of miR-146b-3p in diabetes related retinal inflammation by suppressing ADA2. PMID:25815338

  8. Editing of the GLuR-B ion channel RNA in vitro by recombinant double-stranded RNA adenosine deaminase.

    PubMed Central

    Dabiri, G A; Lai, F; Drakas, R A; Nishikura, K

    1996-01-01

    Double-stranded RNA (dsRNA)-specific adenosine deaminase (DRADA) has been implicated as an enzyme responsible for the editing of RNA transcripts encoding glutamate-gated ion channel subunits (GLuR) in brain. In one case, the editing alters the gene-encoded glutamine (Q) to an arginine (R) located within the channel-forming domain of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor subunit GLuR-B. The result of editing at this site, called the 'Q/R' site, is a profound alteration of the Ca2+ permeability of the GLuR channel. Using recombinantly expressed DRADA proteins, we now demonstrate in vitro that DRADA is indeed involved in editing of the GLuR-B RNA. In addition to the formation of an RNA duplex structure involving exon and intron sequences, Q/R site-selective editing by DRADA also requires a cofactor protein(s) commonly present even in non-neuronal cells. The accuracy and efficiency of this RNA editing system appear to be determined by the quantitative balance between DRADA, cofactor and substrate GLuR-B RNA. Images PMID:8598204

  9. Syzygium cumini extract decrease adenosine deaminase, 5'nucleotidase activities and oxidative damage in platelets of diabetic patients.

    PubMed

    De Bona, Karine S; Bell, Luziane P; Sari, Marcel H; Thom, Gustavo; Schetinger, Maria R C; Morsch, Vera M; Boligon, Aline; Athayde, Margareth L; Pigatto, Aline S; Moretto, Maria B

    2010-01-01

    Diabetes mellitus, a chronic metabolic disorder, has assumed epidemic proportions and its long-term complications can have devastating consequences. The oxidative stress in diabetes was greatly increased due to prolonged exposure to hyperglycemia and impairment of oxidant/antioxidant equilibrium. Syzygium cumini is being widely used to treat diabetes by the traditional practitioners over many centuries. Adenosine deaminase (ADA) and 5'-Nucleotidase (5'NT) are enzymes of purine nucleoside metabolism that play an important role in the regulation of adenosine (Ado) levels. In this study, we investigated the effect of Syzygium cumini aqueous leaves extract (ASc) on ADA and 5'NT activities and on parameters of oxidative stress under in vitro conditions, using platelets of patients with Type 2 diabetes mellitus. Platelet-Rich Plasma (PRP) was assayed by ADA, 5'NT, Catalase (CAT), Superoxide Dismutase (SOD) activities and Thiobarbituric acid reactive substances (TBARS) levels. We observed that ADA, 5'NT activities and TBARS levels were significantly higher when compared to the control group, and ASc (100 and 200 ?g/mL) prevented these effects. Our study demonstrates that ASc was able to remove oxidant species generated in diabetic conditions and modulates in the Ado levels. Then, ASc may promote a compensatory response in platelet function, improving the susceptibility-induced by the diabetes mellitus. PMID:21063110

  10. Identification of DNA cleavage- and recombination-specific hnRNP cofactors for activation-induced cytidine deaminase

    PubMed Central

    Hu, Wenjun; Begum, Nasim A.; Mondal, Samiran; Stanlie, Andre; Honjo, Tasuku

    2015-01-01

    Activation-induced cytidine deaminase (AID) is essential for antibody class switch recombination (CSR) and somatic hypermutation (SHM). AID originally was postulated to function as an RNA-editing enzyme, based on its strong homology with apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC1), the enzyme that edits apolipoprotein B-100 mRNA in the presence of the APOBEC cofactor APOBEC1 complementation factor/APOBEC complementation factor (A1CF/ACF). Because A1CF is structurally similar to heterogeneous nuclear ribonucleoproteins (hnRNPs), we investigated the involvement of several well-known hnRNPs in AID function by using siRNA knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9mediated disruption. We found that hnRNP K deficiency inhibited DNA cleavage and thereby induced both CSR and SHM, whereas hnRNP L deficiency inhibited only CSR and somewhat enhanced SHM. Interestingly, both hnRNPs exhibited RNA-dependent interactions with AID, and mutant forms of these proteins containing deletions in the RNA-recognition motif failed to rescue CSR. Thus, our study suggests that hnRNP K and hnRNP L may serve as A1CF-like cofactors in AID-mediated CSR and SHM. PMID:25902538

  11. Whole-genome sequencing reveals activation-induced cytidine deaminase signatures during indolent chronic lymphocytic leukaemia evolution

    PubMed Central

    Kasar, S.; Kim, J.; Improgo, R.; Tiao, G.; Polak, P.; Haradhvala, N.; Lawrence, M. S.; Kiezun, A.; Fernandes, S. M.; Bahl, S.; Sougnez, C.; Gabriel, S.; Lander, E. S.; Kim, H. T.; Getz, G.; Brown, J. R.

    2015-01-01

    Patients with chromosome 13q deletion or normal cytogenetics represent the majority of chronic lymphocytic leukaemia (CLL) cases, yet have relatively few driver mutations. To better understand their genomic landscape, here we perform whole-genome sequencing on a cohort of patients enriched with these cytogenetic characteristics. Mutations in known CLL drivers are seen in only 33% of this cohort, and associated with normal cytogenetics and unmutated IGHV. The most commonly mutated gene in our cohort, IGLL5, shows a mutational pattern suggestive of activation-induced cytidine deaminase (AID) activity. Unsupervised analysis of mutational signatures demonstrates the activities of canonical AID (c-AID), leading to clustered mutations near active transcriptional start sites; non-canonical AID (nc-AID), leading to genome-wide non-clustered mutations, and an ageing signature responsible for most mutations. Using mutation clonality to infer time of onset, we find that while ageing and c-AID activities are ongoing, nc-AID-associated mutations likely occur earlier in tumour evolution. PMID:26638776

  12. A new strategy based on pharmacophore-based virtual screening in adenosine deaminase inhibitors detection and in-vitro study

    PubMed Central

    2012-01-01

    Background and the purpose of the study Adenosine deaminase (ADA) inhibition not only may be applied for the treatment of ischemic injury, hypertension, lymphomas and leukaemia, but also they have been considered as anti- inflammatory drugs. On the other hand according to literatures, ADA inhibitors without a nucleoside framework would improve pharmacokinetics and decrease toxicity. Hence we have carried out a rational pharmacophore design for non-nucleoside inhibitors filtration. Methods A merged pharmacophore model based on the most potent non-nucleoside inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and natural products were generated and applied for compounds filtration. The effects of filtrated compounds based on pharmacophore and docking studies investigated on ADA by UV and Fluorescence spectroscopy techniques. Results Extracted compounds were find efficiently inhibit ADA, and the most potent (2) shows an inhibition constant equal to 20 μM. Besides, Fluorescence spectroscopy studies revealed that enzyme 3D structure bear further change in lower concentrations of compound 2. Conclusion 3 non-nucleoside inhibitors for ADA are presented. According to obtained results from UV and fluorescence spectroscopy, such interesting pharmacophore template with multiple approaches will help us to extract or design compound with desired properties. PMID:23351306

  13. Activation induced deaminase C-terminal domain links DNA breaks to end protection and repair during class switch recombination

    PubMed Central

    Zahn, Astrid; Eranki, Anil K.; Patenaude, Anne-Marie; Methot, Stephen P.; Fifield, Heather; Cortizas, Elena M.; Foster, Paul; Imai, Kohsuke; Durandy, Anne; Larijani, Mani; Verdun, Ramiro E.; Di Noia, Javier M.

    2014-01-01

    Activation-induced deaminase (AID) triggers antibody class switch recombination (CSR) in B cells by initiating DNA double strand breaks that are repaired by nonhomologous end-joining pathways. A role for AID at the repair step is unclear. We show that specific inactivation of the C-terminal AID domain encoded by exon 5 (E5) allows very efficient deamination of the AID target regions but greatly impacts the efficiency and quality of subsequent DNA repair. Specifically eliminating E5 not only precludes CSR but also, causes an atypical, enzymatic activity-dependent dominant-negative effect on CSR. Moreover, the E5 domain is required for the formation of AID-dependent Igh-cMyc chromosomal translocations. DNA breaks at the Igh switch regions induced by AID lacking E5 display defective end joining, failing to recruit DNA damage response factors and undergoing extensive end resection. These defects lead to nonproductive resolutions, such as rearrangements and homologous recombination that can antagonize CSR. Our results can explain the autosomal dominant inheritance of AID variants with truncated E5 in patients with hyper-IgM syndrome 2 and establish that AID, through the E5 domain, provides a link between DNA damage and repair during CSR. PMID:24591601

  14. MicroRNA-146b-3p regulates retinal inflammation by suppressing adenosine deaminase-2 in diabetes.

    PubMed

    Fulzele, Sadanand; El-Sherbini, Ahmed; Ahmad, Saif; Sangani, Rajnikumar; Matragoon, Suraporn; El-Remessy, Azza; Radhakrishnan, Reshmitha; Liou, Gregory I

    2015-01-01

    Hyperglycemia- (HG-) Amadori-glycated albumin- (AGA-) induced activation of microglia and monocytes and their adherence to retinal vascular endothelial cells contribute to retinal inflammation leading to diabetic retinopathy (DR). There is a great need for early detection of DR before demonstrable tissue damages become irreversible. Extracellular adenosine, required for endogenous anti-inflammation, is regulated by the interplay of equilibrative nucleoside transporter with adenosine deaminase (ADA) and adenosine kinase. ADA, including ADA1 and ADA2, exists in all organisms. However, because ADA2 gene has not been identified in mouse genome, how diabetes alters adenosine-dependent anti-inflammation remains unclear. Studies of pig retinal microglia and human macrophages revealed a causal role of ADA2 in inflammation. Database search suggested miR-146b-3p recognition sites in the 3'-UTR of ADA2 mRNA. Coexpression of miR-146b-3p, but not miR-146-5p or nontargeting miRNA, with 3'-UTR of the ADA2 gene was necessary to suppress a linked reporter gene. In the vitreous of diabetic patients, decreased miR-146b-3p is associated with increased ADA2 activity. Ectopic expression of miR-146b-3p suppressed ADA2 expression, activity, and TNF-? release in the AGA-treated human macrophages. These results suggest a regulatory role of miR-146b-3p in diabetes related retinal inflammation by suppressing ADA2. PMID:25815338

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

    PubMed

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

    2012-11-01

    We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34(+) cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m(2)). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency. PMID:22968453

  16. High-yield production of apoplast-directed human adenosine deaminase in transgenic tobacco BY-2 cell suspensions.

    PubMed

    Singhabahu, Sanjeewa; George, John; Bringloe, David

    2015-01-01

    Adenosine deaminase (ADA) deficiency, where a deleterious mutation in the ADA gene of patients results in a dysfunctional immune system, is ultimately caused by an absence of ADA. Over the last 25 years the disease has been treated with PEG-ADA, made from purified bovine ADA coupled with polyethylene glycol (PEG). However, it is thought that an enzyme replacement therapy protocol based on recombinant human ADA would probably be a more effective treatment. With this end in mind, a human ADA cDNA was inserted into plant expression vectors used to transform tobacco plant cell suspensions. Transgenic calli expressing constructs containing apoplast-directing signals showed significantly higher levels of recombinant ADA expression than calli transformed with cytosolic constructs. The most significant ADA activities, however, were measured in the media of transgenic cell suspensions prepared from high expressing transformed calli: where incorporation of a signal for arabinogalactan addition to ADA led to a recombinant protein yield of approximately 16mgL(-1) , a 336-fold increase over ADA produced by cell suspensions transformed with a cytosolic construct. PMID:24825606

  17. PEG-ADA: an alternative to haploidentical bone marrow transplantation and an adjunct to gene therapy for adenosine deaminase deficiency.

    PubMed

    Hershfield, M S

    1995-01-01

    PEG-ADA is a long-circulating form of adenosine deaminase (ADA) that has been in use for > 8 years as replacement therapy for severe combined immunodeficiency disease due to ADA deficiency. Treatment with PEG-ADA almost completely corrects metabolic abnormalities, allowing the recovery of a variable degree of immune function. Although not normal, the level of function achieved has in most cases been sufficient to protect against opportunistic and life-threatening infections. PEG-ADA has been used as an alternative for patients who lack an HLA-identical bone marrow donor, but are judged to be at too high a risk for undergoing HLA-haploidentical marrow transplantation. To date, mortality and morbidity with PEG-ADA have been less than for the latter procedure. PEG-ADA has also been an important adjunct to attempts to develop somatic cell gene therapy for ADA deficiency, although its continued use poses a problem for evaluation of the benefit of gene therapy. As a true "orphan drug" developed to treat a very small patient population, the cost per patient of PEG-ADA is very high. PMID:7749407

  18. Effects of aqueous extract from Silybum marianum on adenosine deaminase activity in cancerous and noncancerous human gastric and colon tissues

    PubMed Central

    ztrk, Bahad?r; Kocao?lu, Ender Hilmi; Durak, Zahide Esra

    2015-01-01

    Objective: Investigation of possible effects of Silybum marianum extract (SME) on adenosine deaminase (ADA) activity in cancerous and noncancerous human gastric and colon tissues to obtain information about possible mechanism of anticancer action of S. marianum. Materials and Methods: Cancerous and noncancerous human gastric and colon tissues removed from patients by surgical operations were used in the studies. The extract was prepared in distilled water. Before and after treatment with the extract, ADA activities in the samples were measured. Results: ADA activity was found to be lowered significantly in cancerous gastric tissues but not in noncancerous gastric tissues after treatment with the SME. In the colon tissues, ADA activities were however found to increase after the treatment of SME. Conclusion: Our results suggest that the aqueous extract from S. marianum inhibits ADA activity in cancerous gastric tissues significantly. It is suggested that in addition to other proposed mechanisms, accumulated adenosine due to the inhibition of ADA might also play a part in the anticancer properties of the S. marianum. PMID:25709224

  19. A novel bipartite intronic splicing enhancer promotes the inclusion of a mini-exon in the AMP deaminase 1 gene.

    PubMed

    Genetta, T; Morisaki, H; Morisaki, T; Holmes, E W

    2001-07-01

    Alternative splicing of the 12-base exon 2 of the adenosine monophosphate deaminase (AMPD) gene is subject to regulation by both cis- and trans-regulatory signals. The extent of exon 2 inclusion is stage- and cell type-specific and is subject to the physiological state of the cell. In adult skeletal muscle, a cell type that regulates the activity of this allosteric enzyme at several levels, the exon 2-plus form of AMPD, predominates. We have performed a systematic analysis of the cis-acting regulatory sequences that reside in the intron immediately downstream of this mini-exon. A complex element comprising sequences that enhance exon 2 inclusion and sequences that counteract this effect resides in the middle of this intron. We demonstrate that the enhancing component is bipartite, with more than a kilobase of sequence separating the two functional sites. The presence of even minimal levels the mini-exon in the fully processed AMPD mRNA requires both of these sites, neither of which appears in any other published splicing enhancer. An RNA binding activity derived from a muscle cell line requires both of the enhancing sites. Mutations in either of the sites that eliminate exon 2 inclusion abrogate this binding activity. PMID:11331279

  20. A Study on the Serum Adenosine Deaminase Activity in Patients with Typhoid Fever and Other Febrile Illnesses

    PubMed Central

    Ketavarapu, Sameera; Ramani G., Uma; Modi, Prabhavathi

    2013-01-01

    Background: Adenosine Deaminase (ADA) has been suggested to be an important enzyme which is associated with the cell mediated immunity, but its clinical significance in typhoid fever has not yet been characterized. The present study was taken up to evaluate the serum ADA activity in patients of typhoid fever. The levels of ADA were also measured in the patients who were suffering from other febrile illnesses. Material and Method: This was a case control study. The subjects who were included in this study were divided into 3 groups. Group A consisted of 50 normal healthy individuals who served as the controls. Group B consisted of 50 patients, both males and females of all age groups, who were suffering from culture positive typhoid fever. Group C consisted of 50 patients who were suffering from febrile illnesses other than typhoid fever like viral fever, gastro enteritis, malaria, tonsillitis, upper respiratory tract infections, etc. The serum levels of ADA were estimated in all the subjects who were under study. Results: The serum ADA level was found to be increased in the patients of typhoid fever as compared to that in those with other febrile illnesses and in the controls. Conclusion: From the present study, it can be concluded that there was a statistically significant increase in the serum ADA levels in the patients with typhoid. PMID:23730630