Sample records for yeast cytosine deaminase

  1. Catalytic Mechanism of Yeast Cytosine Deaminase:  An ONIOM Computational Study

    Microsoft Academic Search

    Stepan Sklenak; Lishan Yao; Robert I. Cukier; Honggao Yan

    2004-01-01

    The complete path for the deamination reaction catalyzed by yeast cytosine deaminase (yCD), a zinc metalloenzyme of significant biomedical interest, has been investigated using the ONIOM method. Cytosine deamination proceeds via a sequential mechanism involving the protonation of N 3 , the nucleophilic attack of C 4 by the zinc-coordinated hydroxide, and the cleavage of the C 4 -N 4

  2. Yeast Cytosine Deaminase Mutants with Increased Thermostability Impart Sensitivity to 5-Fluorocytosine

    PubMed Central

    Stolworthy, Tiffany S.; Korkegian, Aaron M.; Willmon, Candice L.; Ardiani, Andressa; Cundiff, Jennifer; Stoddard, Barry L.; Black, Margaret E.

    2008-01-01

    SUMMARY Prodrug gene therapy (PGT) is a treatment strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme capable of converting a nontoxic prodrug into a potent cytotoxin. One of the most promising PGT enzymes is cytosine deaminase (CD), a microbial salvage enzyme that converts cytosine to uracil. CD also converts 5-fluorocytosine (5FC) to 5-fluorouracil (5FU), an inhibitor of DNA synthesis and RNA function. Over 150 studies of cytosine deaminase-mediated PGT applications have been reported since 2000, all using wild-type enzymes. However, various forms of cytosine deaminase are limited by inefficient turnover of 5FC and/or limited thermostability. In a previous study we stabilized and extended the half-life of yeast cytosine deaminase (yCD) by repacking of its hydrophobic core at several positions distant from the active site. Here we report that random mutagenesis of residues selected based on alignment with similar enzymes, followed by selection for enhanced sensitization to 5FC, also produces an enzyme variant (yCD-D92E) with elevated Tm values and increased activity half-life. The new mutation is located at the enzyme's dimer interface, indicating that independent mutational pathways can lead to an increase in the temperature that induces protein unfolding and aggregation in thermal denaturation experiments measured by circular dichroism spectroscopy, and an increase in the half-life of enzyme activity at physiological temperature, as well as more subtle effect on enzyme kinetics. Each independently derived set of mutations significantly improves the enzyme's performance in PGT assays both in cell culture and in animal models. PMID:18291415

  3. A Molecular Dynamics Study of the Ligand Release Path in Yeast Cytosine Deaminase

    PubMed Central

    Yao, Lishan; Yan, Honggao; Cukier, Robert I.

    2007-01-01

    Yeast cytosine deaminase, a zinc metalloenzyme, catalyzes the deamination of cytosine to uracil. Experimental and computational evidence indicates that the rate-limiting step is product release, instead of the chemical reaction step. In this work, we use molecular dynamics to suggest ligand exit paths. Simulation at 300 K shows that the active site is well protected by the C-terminal helix (residues 150–158) and F-114 loop (residues 111–117) and that on the molecular dynamics timescale water does not flow in or out of the active site. In contrast, simulation at 320 K shows a significant increase in flexibility of the C-terminal helix and F-114 loop. The motions of these two regions at 320 K open the active site and permit water molecules to diffuse into and out of the active site through two paths with one much more favored than the other. Cytosine is pushed out of the active site by a restraint method in two directions specified by these two paths. In path 1 the required motion of the protein is local—involving only the C-terminal helix and F-114 loop—and two residues, F-114 and I-156, are identified that have to be moved away to let cytosine out; whereas in path 2, the protein has to rearrange itself much more extensively, and the changes are also much larger compared to the path 1 simulation. PMID:17218460

  4. Role of Glutamate 64 in the Activation of the Prodrug 5-fluorocytosine by Yeast Cytosine Deaminase

    PubMed Central

    Wang, Jifeng; Sklenak, Stepan; Liu, Aizhuo; Felczak, Krzysztof; Wu, Yan; Li, Yue; Yan, Honggao

    2012-01-01

    Yeast cytosine deaminase catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, NMR, and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in kcat and a dramatic increase in Km, indicating Glu64 is important for both binding and catalysis in the activation of 5FC. 19F-NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. 1H and 15N NMR analysis revealed trans-hydrogen-bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has higher activation energy as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by 1H and 15N NMR analysis. To explore the functional role of Glu64 in catalysis, deamination of cytosine catalyzed by the E64A mutant was investigated by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and after the formation of the tetrahedral reaction intermediate become partially rate-limiting steps. The results of the experimental and computational studies together indicate that Glu64 plays a critical role in both binding and chemical transformation in the conversion of the prodrug 5FC to the anticancer drug 5-fluorouracil. PMID:22208667

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

    E-print Network

    Sklenak, Stepan

    that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild of cytosine catalyzed by the E64A mutant by ONIOM calculations. The results showed that without the assistance

  6. Anti-Tumor Therapy Mediated by 5-Fluorocytosine and a Recombinant Fusion Protein Containing TSG-6 Hyaluronan Binding Domain and Yeast Cytosine Deaminase

    PubMed Central

    Park, Joshua I.; Cao, Limin; Platt, Virginia M.; Huang, Zhaohua; Stull, Robert A.; Dy, Edward E.; Sperinde, Jeffrey J.; Yokoyama, Jennifer S.; Szoka, Francis C.

    2009-01-01

    Matrix Attachment Therapy (MAT) is an enzyme prodrug strategy that targets hyaluronan in the tumor extracellular matrix to deliver a prodrug converting enzyme near the tumor cells. A recombinant fusion protein containing the hyaluronan binding domain of TSG-6 (Link) and yeast cytosine deaminase (CD) with an N-terminal His(×6) tag was constructed to test MAT on the C26 colon adenocarcinoma in Balb/c mice that were given 5-fluorocytosine (5-FC) in the drinking water. LinkCD was expressed in E.coli and purified by metal-chelation affinity chromatography. The purified LinkCD fusion protein exhibits a Km of 0.33 mM and Vmax of 15 ?M/min/?g for the conversion of 5-FC to 5-fluorouracil (5-FU). The duration of the enzyme activity for LinkCD was longer than that of CD enzyme at 37 °C: the fusion protein retained 20% of its initial enzyme activity after 24 hr, and 12% after 48 hr. The LinkCD fusion protein can bind to a hyaluronan oligomer (12-mer) at a KD of 55 ?M at pH 7.4 and a KD of 5.32 ?M at pH 6.0 measured using surface plasmon resonance (SPR). To evaluate the anti-tumor effect of LinkCD/5-FC combination therapy in vivo, mice received intratumoral injections of LinkCD on days 11 and 14 after C26 tumor implantation and the drinking water containing 10 mg/mL of 5-FC starting on day 11. To examine if the Link domain by itself was able to reduce tumor growth, we included treatment groups that received LinkCD without 5-FC and Link-mtCD (a functional mutant that lacks cytosine deaminase activity) with 5-FC. Animals that received LinkCD/5-FC treatment showed significant tumor size reduction and increased survival compared to the CD/5-FC treatment group. Treatment groups that were unable to produce 5-FU had no effect on the tumor growth despite receiving the fusion protein that contained the Link domain. The results indicate that a treatment regime consisting of a fusion protein containing the Link domain, the active CD enzyme, and the prodrug 5-FC are sufficient to produce an anti-tumor effect. Thus, the LinkCD fusion protein is an alternative to antibody-directed prodrug enzyme therapy (ADEPT) approaches for cancer treatment. PMID:19265397

  7. 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.

  8. Improved cytotoxic effects of Salmonella-producing cytosine deaminase in tumour cells

    PubMed Central

    Mesa-Pereira, Beatriz; Medina, Carlos; Camacho, Eva María; Flores, Amando; Santero, Eduardo

    2015-01-01

    In order to increase the cytotoxic activity of a Salmonella strain carrying a salicylate-inducible expression system that controls cytosine deaminase production, we have modified both, the vector and the producer bacterium. First, the translation rates of the expression module containing the Escherichia coli?codA gene cloned under the control of the Pm promoter have been improved by using the T7 phage gene 10 ribosome binding site sequence and replacing the original GUG start codon by AUG. Second, to increase the time span in which cytosine deaminase may be produced by the bacteria in the presence of 5-fluorocytosine, a 5-fluorouracyl resistant Salmonella strain has been constructed by deleting its upp gene sequence. This new Salmonella strain shows increased cytosine deaminase activity and, after infecting tumour cell cultures, increased cytotoxic and bystander effects under standard induction conditions. In addition, we have generated a purD mutation in the producer strain to control its intracellular proliferation by the presence of adenine and avoid the intrinsic Salmonella cell death induction. This strategy allows the analysis and comparison of the cytotoxic effects of cytosine deaminase produced by different Salmonella strains in tumour cell cultures. PMID:25227763

  9. Engineered herpes simplex virus expressing bacterial cytosine deaminase for experimental therapy of brain tumors

    Microsoft Academic Search

    M B Guffey; J N Parker; W S Luckett; G Y Gillespie; S Meleth; R J Whitley; J M Markert

    2007-01-01

    Lack of effective therapy of primary brain tumors has promoted the development of novel experimental approaches utilizing oncolytic viruses combined with gene therapy. Towards this end, we have assessed a conditionally replication-competent, ?134.5-deleted herpes simplex virus type 1 (HSV-1) expressing cytosine deaminase (CD) for treatment of malignant brain tumors. Our results are summarized as follows: (i) a recombinant HSV (M012)

  10. Cytosine deaminase-producing human mesenchymal stem cells mediate an antitumor effect in a mouse xenograft model

    Microsoft Academic Search

    Mi-Hyeon You; Wang-Joon Kim; Wooyoung Shim; Sang-Rim Lee; Gwang Lee; Sangdun Choi; Dae-Yong Kim; Yong Man Kim; Hyunsoo Kimand; Sang-Uk Han

    2009-01-01

    Background and Aim: Stem cell transplantation offers potential gene therapy for brain tumors. However, this approach has received little attention as a treatment for gastrointes- tinal tumors. In the present study, we explored the possibility of human bone marrow- derived mesenchymal stem cells (hMSC) producing cytosine deaminase (CD), followed by systemic 5-fluorocytosine (5-FC) administration, showing an antitumor effect on a

  11. Treatment of Tumor in Mice by Oral Administration of Cytosine Deaminase Gene Carried in Live Attenuated Salmonella.

    PubMed

    Li, Yu-Hua; Xie, Yong-Mei; Guo, Kun-Yuan; Chen, Hui; Wei, Yi; Huang, Jian-Sheng; Ren, Da-Ming

    2001-01-01

    To study the possibility of oral gene therapy using live attenuated Salmonella, eukaryotic expression vectors EGFPN1, pLCDSN were introduced into a live attenuated AraA(-) auxotrophic mutant of Salmonella typhimurium (SL3261) and were administered orally to BALB/c and C57BL/6 mice. After six weeks, these mice were challenged with 4T(1) and Lewis cancer cells. Until the tumors reached to about 10 mm in diameter, 5-fluorocytosine was given through intraperitoneal injection. Flow cytometry, confocal microscopy and PCR methods were used to detect the integration and expression of the genes. The inhibition of the tumor and the survival time of the mice were also investigated. Results showed that cytosine deaminase gene integration could be detected in almost all kinds of mice tissue. And the GFP expression was much stronger in spleen and tumor than in other tissues. Cytosine deaminase/5-fluorocytosine system had significant antitumoractivities in vivo. The anti-tumor activities of cytosine deaminase/5-fluorocytosine at 500 mg/kg on 4T(1) and Lewis carcinoma in BALB/c and C57BL/6 mice were more potent than the efficiency of 5-fluorouracil 10 mg/kg(P 0.05). Therefor, this experiment demonstrates the potential value of live attenuated Salmonella as carrier for oral gene therapy. PMID:12050817

  12. 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

  13. Expression of Cytosine Deaminase Gene in Human Colon Carcinoma Cells by Recombinant Retroviral Vector.

    PubMed

    Jiang, Qiong; Ge, Kai; Xu, De-Hua; Sun, Lan-Ying; Zheng, Zhong-Cheng; Liu, Xin-Yuan

    1997-01-01

    The recombinant retroviral vector pLCDSN containing E. coli cytosine deaminase gene was constructed. After packaging with PA317 cell line, the infectious particles were used to infect human colon carcinoma cell line LoVo. A single clone harbouring EC-CD gene was picked after G418 selection. There was no significant difference in cell growth curve or morphology between the LoVo/LCDSN and LoVo cells. Both of them were very sensitive to 5-FU in vitro (IC(50), approximately 0.5 &mgr;mol/L). However, the expression of the CD gene did increase the sensitivity of these cells to the nontoxic prodrug, 5-FC, decreasing the IC(50) for 5-FC from 22 000 &mgr;mol/L for parental LoVo cells to 13 &mgr;mol/L for LoVo/LCDSN cells. Obvious by side effect was also observed. When cells transduced with CD gene were mixed with wild type cells at a ratio of 30:70, above 80% of the cancer cells could be killed after treatment with a nontoxic concentration of 5-FC. PMID:12219218

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

    PubMed Central

    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.

    2012-01-01

    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. 20/34 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 substrate DNA cytosines. PMID:22181350

  15. Crystal structure of the DNA cytosine deaminase APOBEC3F: the catalytically active and HIV-1 Vif-binding domain

    PubMed Central

    Bohn, Markus-Frederik; Shandilya, Shivender M. D.; Albin, John S.; Kouno, Takahide; Anderson, Brett D.; McDougle, Rebecca M.; Carpenter, Michael A.; Rathore, Anurag; Evans, Leah; Davis, Ahkillah N.; Zhang, Jing Ying; Lu, Yongjian; Somasundaran, Mohan; Matsuo, Hiroshi; Harris, Reuben S.; Schiffer, Celia A.

    2013-01-01

    Human APOBEC3F is an anti-retroviral single strand DNA cytosine deaminase, susceptible to degradation by the HIV-1 protein Vif. In this study the crystal structure of the HIV Vif binding, catalytically active, C-terminal domain of APOBEC3F (A3F-CTD) was determined. The A3F-CTD shares structural motifs with portions of APOBEC3G-CTD, APOBEC3C and APOBEC2. Residues identified to be critical for Vif-dependent degradation of APOBEC3F all fit within a predominantly negatively charged contiguous region on the surface of A3F-CTD. Specific sequence motifs, previously shown to play a role in Vif susceptibility and virion encapsidation, are conserved across APOBEC3’s and between APOBEC3’s and HIV-1 Vif. In this structure these motifs pack against each other at intermolecular interfaces, providing potential insights both into APOBEC3 oligomerization and Vif interactions. PMID:23685212

  16. 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

  17. Bacterial cytosine deaminase mutants created by molecular engineering show improved 5-fluorocytosine-mediated cell killing in vitro and in vivo.

    PubMed

    Fuchita, Michi; Ardiani, Andressa; Zhao, Lei; Serve, Kinta; Stoddard, Barry L; Black, Margaret E

    2009-06-01

    Cytosine deaminase is used in combination with 5-fluorocytosine as an enzyme-prodrug combination for targeted genetic cancer treatment. This approach is limited by inefficient gene delivery and poor prodrug conversion activities. Previously, we reported individual point mutations within the substrate binding pocket of bacterial cytosine deaminase (bCD) that result in marginal improvements in the ability to sensitize cells to 5-fluorocytosine (5FC). Here, we describe an expanded random mutagenesis and selection experiment that yielded enzyme variants, which provide significant improvement in prodrug sensitization. Three of these mutants were evaluated using enzyme kinetic analyses and then assayed in three cancer cell lines for 5FC sensitization, bystander effects, and formation of 5-fluorouracil metabolites. All variants displayed 18- to 19-fold shifts in substrate preference toward 5FC, a significant reduction in IC(50) values and improved bystander effect compared with wild-type bCD. In a xenograft tumor model, the best enzyme mutant was shown to prevent tumor growth at much lower doses of 5FC than is observed when tumor cells express wild-type bCD. Crystallographic analyses of this construct show the basis for improved activity toward 5FC, and also how two different mutagenesis strategies yield closely related but mutually exclusive mutations that each result in a significant alteration of enzyme specificity. PMID:19487291

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

    Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

    2008-02-01

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

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

    PubMed Central

    2014-01-01

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

  1. Cytosine 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

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

    PubMed

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

    2014-05-01

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

  3. 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

  4. The Role of Cytidine Deaminase and GATA1 Mutations in the Increased Cytosine Arabinoside Sensitivity of Down Syndrome Myeloblasts and Leukemia Cell Lines

    Microsoft Academic Search

    Yubin Ge; Tanya L. Jensen; Mark L. Stout; Robin M. Flatley; Patrick J. Grohar; Yaddanapudi Ravindranath; Larry H. Matherly; Jeffrey W. Taub

    2004-01-01

    Myeloblasts from Down syndrome (DS) children with acute myeloid leukemia (AML) are significantly more sensitive in vitro to 1--D-arabino- furanosylcytosine (ara-C) and generate higher 1--D-arabinofuranosylcy- tosine 5-triphosphate (ara-CTP) than non-DS AML myeloblasts. Semi- quantitative reverse transcription-PCR analyses demonstrated that transcripts for cytidine deaminase (CDA) were 2.7-fold lower in DS than for non-DS myeloblasts. In contrast, transcripts of cystathionine--syn- thase and

  5. Systemic administration of a recombinant vaccinia virus expressing the cytosine deaminase gene and subsequent treatment with 5-fluorocytosine leads to tumor-specific gene expression and prolongation of survival in mice.

    PubMed

    Gnant, M F; Puhlmann, M; Alexander, H R; Bartlett, D L

    1999-07-15

    Suicide gene therapy using the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) has shown promising results for the treatment of colon carcinoma cells in vitro. Efficient viral infection and tumor-specific gene delivery is crucial for clinically measurable treatment effects. After proving efficient gene transfer in vitro, we demonstrate here that genes can be delivered to metastatic liver tumors in vivo in a highly selective manner using systemic delivery of a thymidine kinase-deleted (TK-) recombinant vaccinia virus (Western Reserve strain). When the vector was administered systemically in C57BL/6 mice or nude/athymic mice with established disseminated MC38 liver metastases, transgene expression in tumors was usually 1,000 to 10,000-fold higher compared with other organs (n = 160; P < 0.0001). This tumor-specific gene transfer leads to significant tumor responses and subsequent survival benefits after the transfer of the CD gene to liver metastases and subsequent systemic treatment with the prodrug 5-FC (P < 0.0001). We describe reporter gene and survival experiments both in immunocompetent and athymic nude mice, establishing a gene expression pattern over time and characterizing the treatment effects of the virus delivery/prodrug system. Cure rates of up to 30% in animals with established liver metastases show that suicide gene therapy using TK- vaccinia virus as a vector may be a promising system for the clinical application of tumor-directed gene therapy. PMID:10416601

  6. The DNA Deaminase Activity of Human APOBEC3G Is Required for Ty1, MusD, and Human Immunodeficiency Virus Type 1 Restriction

    Microsoft Academic Search

    April J. Schumacher; Guylaine Hache; Donna A. MacDuff; William L. Brown; Reuben S. Harris

    2008-01-01

    Human APOBEC3G and several other APOBEC3 proteins have been shown to inhibit the replication of a variety of retrotransposons and retroviruses. All of these enzymes can deaminate cytosines within single-strand DNA, but the overall importance of this conserved activity in retroelement restriction has been questioned by reports of deaminase-independent mechanisms. Here, three distinct retroelements, a yeast retrotransposon, Ty1, a murine

  7. 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.

  8. Molecular Structure of Cytosine

    NSDL National Science Digital Library

    2004-10-12

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

  9. Processive DNA Demethylation via DNA Deaminase-Induced Lesion Resolution

    PubMed Central

    Morgan, Hugh; Incorvaia, Elisabetta; Rangam, Gopinath; Dean, Wendy; Santos, Fatima; Reik, Wolf; Petersen-Mahrt, Svend K.

    2014-01-01

    Base modifications of cytosine are an important aspect of chromatin biology, as they can directly regulate gene expression, while DNA repair ensures that those modifications retain genome integrity. Here we characterize how cytosine DNA deaminase AID can initiate DNA demethylation. In vitro, AID initiated targeted DNA demethylation of methyl CpGs when in combination with DNA repair competent extracts. Mechanistically, this is achieved by inducing base alterations at or near methyl-cytosine, with the lesion being resolved either via single base substitution or a more efficient processive polymerase dependent repair. The biochemical findings are recapitulated in an in vivo transgenic targeting assay, and provide the genetic support of the molecular insight into DNA demethylation. This targeting approach supports the hypothesis that mCpG DNA demethylation can proceed via various pathways and mCpGs do not have to be targeted to be demethylated. PMID:25025377

  10. Cytosine modifications in myeloid malignancies.

    PubMed

    Meldi, Kristen M; Figueroa, Maria E

    2015-08-01

    Aberrant DNA methylation is a hallmark of many cancers, including the myeloid malignancies acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). The discovery of TET-mediated demethylation of 5-methylcytosine (5mC) and technological advancements in next-generation sequencing have permitted the examination of other cytosine modifications, namely 5-hydroxymethylcytosine (5hmC), in these myeloid malignancies on a genome-wide scale. Due to the prominence of mutations in epigenetic modifiers that can influence cytosine modifications in these disorders, including IDH1/2, TET2, and DNMT3A, many recent studies have evaluated the relative levels, distribution, and functional consequences of cytosine modifications in leukemic cells. Furthermore, several therapies are being used to treat AML and MDS that target various proteins within the cytosine modification pathway in an effort to revert the abnormal epigenetic patterns that contribute to the diseases. In this review, we provide an overview of cytosine modifications and selected technologies currently used to distinguish and analyze these epigenetic marks in the genome. Then, we discuss the role of mutant enzymes, including DNMT3A, TET2, IDH1/2, and the transcription factor, WT1, in disrupting normal patterns of 5mC and 5hmC in AML and MDS. Finally, we describe several therapies, both standard, front-line treatments and new drugs in clinical trials, aimed at inhibiting the proteins that ultimately lead to aberrant cytosine modifications in these diseases. PMID:25956466

  11. Human cytidine deaminase: A biochemical characterization of its naturally occurring variants

    PubMed Central

    Micozzi, Daniela; Carpi, Francesco Martino; Pucciarelli, Stefania; Polzonetti, Valeria; Polidori, Paolo; Vilar, Santiago; Williams, Brian; Costanzi, Stefano; Vincenzetti, Silvia

    2014-01-01

    Human cytidine deaminase is an enzyme of the pyrimidine salvage pathways that metabolizes several cytosine nucleoside analogs used as prodrugs in chemotherapy. We carried out a characterization of the cytidine deaminase 79A>C and 208G>A Single Nucleotide Polymorphisms, in order to highlight their functional role and provide data that could help fine-tune the chemotherapic use of cytosine nucleosides in patients carrying the above mentioned SNPs. The 79A>C SNP results in a K27Q change in a protein region not involved in the catalytic event. The 208G>A SNP produces an alanine to threonine substitution (A70T) within the conserved catalytic domain. Q27 variant is endowed with a greater catalytic efficiency toward the natural substrates and the antileukemic agent cytarabine (Ara-C), when compared to K27 variant. Molecular modeling, protein stability experiments and site-directed mutagenesis suggest that K27 variant may have an increased stability with respect to Q27 due to an ionic interaction between a lysine residue at position 27 and a glutamate residue at position 24. The T70 variant has a lower catalytic efficiency toward the analyzed substrates when compared to the A70 variant, suggesting that patients carrying the 208G>A SNP may have a greater exposure to cytosine based pro drugs, with possible toxicity consequences. PMID:24183806

  12. Discovery of Deaminase Activities in COG1816 

    E-print Network

    Goble, Alissa M

    2013-04-24

    site in the human guanine deaminase, a model for isoxanthopterin binding has been proposed and can be seen in Scheme 1.31. Although isoxanthopterin deaminase activity has been reported previously in extracts of Alcaligenes faecalis, this enzyme does...

  13. RESEARCH Open Access The Plasmodium falciparum, Nima-related kinase

    E-print Network

    Paris-Sud XI, Université de

    markers, yeast cytosine deaminase-uridyl phosphoribosyl transferase, or human dihydrofolate reductase of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow

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

    PubMed Central

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

    2014-01-01

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

  15. AID and APOBEC deaminases: balancing DNA damage in epigenetics and immunity.

    PubMed

    Franchini, Don-Marc; Petersen-Mahrt, Svend K

    2014-01-01

    DNA mutations and genomic recombinations are the origin of oncogenesis, yet parts of developmental programs as well as immunity are intimately linked to, or even depend on, such DNA damages. Therefore, the balance between deleterious DNA damages and organismal survival utilizing DNA editing (modification and repair) is in continuous flux. The cytosine deaminases AID/APOBEC are a DNA editing family and actively participate in various biological processes. In conjunction with altered DNA repair, the mutagenic potential of the family allows for APOBEC3 proteins to restrict viral infection and transposons propagation, while AID can induce somatic hypermutation and class switch recombination in antibody genes. On the other hand, the synergy between effective DNA repair and the nonmutagenic potential of the DNA deaminases can induce local DNA demethylation to support epigenetic cellular identity. Here, we review the current state of knowledge on the mechanisms of action of the AID/APOBEC family in immunity and epigenetics. PMID:25333851

  16. Molecular biology of AMP deaminase deficiency

    Microsoft Academic Search

    Manfred Gross

    1994-01-01

    In man, there are at least four isoforms of adenosine monophosphate deaminase (AMPD): myoadenylate deaminase in skeletal muscle, the L isoform in liver, and the E1 and E2 isoforms in erythrocytes. Myoadenylafe deaminase is encoded by the AMPD1 gene located on chromosome 1 p13-p21, the L isoform by the AMPD2 gene, and both isoforms in erythrocytes by the AMPD3 gene.

  17. 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 ...

  18. 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

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

    PubMed

    Oehlenschlæger, Christian Berg; Løvgreen, Monika Nøhr; Reinauer, Eva; Lehtinen, Emilia; Pind, Marie-Louise Lindberg; Harris, Pernille; Martinussen, Jan; Willemoës, 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

  20. Nucleic acid determinants for selective deamination of DNA over RNA by activation-induced deaminase

    PubMed Central

    Nabel, Christopher S.; Lee, Jae W.; Wang, Laura C.; Kohli, Rahul M.

    2013-01-01

    Activation-induced deaminase (AID), a member of the larger AID/APOBEC family, is the key catalyst in initiating antibody somatic hypermutation and class-switch recombination. The DNA deamination model accounting for AID’s functional role posits that AID deaminates genomic deoxycytosine bases within the immunoglobulin locus, activating downstream repair pathways that result in antibody maturation. Although this model is well supported, the molecular basis for AID’s selectivity for DNA over RNA remains an open and pressing question, reflecting a broader need to elucidate how AID/APOBEC enzymes engage their substrates. To address these questions, we have synthesized a series of chimeric nucleic acid substrates and characterized their reactivity with AID. These chimeric substrates feature targeted variations at the 2?-position of nucleotide sugars, allowing us to interrogate the steric and conformational basis for nucleic acid selectivity. We demonstrate that modifications to the target nucleotide can significantly alter AID’s reactivity. Strikingly, within a substrate that is otherwise DNA, a single RNA-like 2?-hydroxyl substitution at the target cytosine is sufficient to compromise deamination. Alternatively, modifications that favor a DNA-like conformation (or sugar pucker) are compatible with deamination. AID’s closely related homolog APOBEC1 is similarly sensitive to RNA-like substitutions at the target cytosine. Inversely, with unreactive 2?-fluoro-RNA substrates, AID’s deaminase activity was rescued by introducing a trinucleotide DNA patch spanning the target cytosine and two nucleotides upstream. These data suggest a role for nucleotide sugar pucker in explaining the molecular basis for AID’s DNA selectivity and, more generally, suggest how other nucleic acid-modifying enzymes may distinguish DNA from RNA. PMID:23942124

  1. First principles potential for the cytosine dimer.

    PubMed

    Manukyan, Artür; Tekin, Adem

    2015-06-14

    We developed a new first principles potential for the cytosine dimer. The ab initio calculations were performed with a DFT-SAPT combination of the symmetry-adapted perturbation method and density functional theory, and fitted to a model site-site functional form. The model potential was used to predict cluster structures up to cytosine hexamers. The global cluster structure optimizations showed that the new potential is able to reproduce some of the 2D filament structures. Moreover many new non-planar cytosine cluster structures were also discovered. Interaction energies of these clusters were compared with B3LYP-D, MP2, SCS-MP2, SCS-MI-MP2 and AMBER. It has been shown that the model agrees well with all ab initio methods, especially for the cytosine hexamer. The model potential outperforms the AMBER force field and therefore it can be exploited to study more challenging larger systems. PMID:25971940

  2. Genetics Home Reference: Adenosine monophosphate deaminase deficiency

    MedlinePLUS

    ... it plays a role in producing energy within muscle cells. Mutations in the AMPD1 gene disrupt the function of AMP deaminase and impair the muscle cells' ability to produce energy. This lack of energy ...

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

    PubMed

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

    2014-02-26

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

  4. Molecular biology of AMP deaminase deficiency.

    PubMed

    Gross, M

    1994-04-15

    In man, there are at least four isoforms of adenosine monophosphate deaminase (AMPD): myoadenylate deaminase in skeletal muscle, the L isoform in liver, and the E1 and E2 isoforms in erythrocytes. Myoadenylate deaminase is encoded by the AMPD1 gene located on chromosome 1 p13-p21, the L isoform by the AMPD2 gene, and both isoforms in erythrocytes by the AMPD3 gene. Myoadenylate deaminase deficiency is found in 2-3% of all muscle biopsies. The inborn type of myoadenylate deaminase deficiency is caused by a single mutant allele harbouring two mutations: C34-->T (Gln-->Stop) and C143-->T (Pro-48-->Leu). Population studies revealed a frequency of the mutant allele of 0.12 in Caucasian Americans and Germans. The C34-->T mutation is located in exon 2, which is alternatively spliced in part of the AMPD1 transcript in human muscle. Since the second mutation does not affect enzyme function, alternatively spliced mRNA encodes a catalytically active enzyme. Only one patient with a disorder linked to liver AMPD has been described so far. In this patient the decreased inhibition of this enzyme by GTP resulted in uric acid overproduction and gout. A complete lack of erythroyte AMPD activity is found in asymptomatic subjects. The molecular basis of both disorders is not yet known. PMID:8032342

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

    PubMed Central

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

    2014-01-01

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

  6. How to distinguish methyl-cytosine from cytosine with high fidelity.

    PubMed

    Bianchi, Caterina; Zangi, Ronen

    2012-12-01

    Methylation of cytosines in the DNA is central to the epigenetic code. The patterns along the DNA formed by these chemical marks instruct the cell which proteins to express and their faithful maintenance after replication are vital to the organism's life. Although Dnmt1 is the enzyme catalyzing the methylation reaction, it was found that UHRF1 (ubiquitin-like, containing PHD and RING finger domain 1) is the protein that actually recognizes hemi-methylated CpG sites. Nevertheless, the physical mechanism driving the strikingly robust distinction between hemi-methylated and unmethylated sites is not known. In this paper, we show that the large difference in the binding affinities of UHRF1 to these sites is possible not due to the presence of the methyl group itself but is a result of the accompanying changes in the distribution of the electrons around the cytosine ring. In particular, methylation reduces the dipole moment of cytosine and, as a consequence, unmethylated DNA in its unbound state in water is more stable than hemi-methylated DNA. Furthermore, the interaction energy of hemi-methylated DNA bound to UHRF1 with its surrounding is stronger than that of unmethylated DNA. Thus, the change in the electronic structure of cytosine upon methylation destabilizes the unbound state and stabilizes the bound state rendering discrimination with high fidelity possible. PMID:23041422

  7. 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.

  8. Evolution and diversification of lamprey antigen receptors: evidence for involvement of an AID-APOBEC family cytosine deaminase

    Microsoft Academic Search

    Igor B Rogozin; Lakshminarayan M Iyer; Lizhi Liang; Galina V Glazko; Victoria G Liston; Youri I Pavlov; L Aravind; Zeev Pancer

    2007-01-01

    The variable lymphocyte receptors (VLRs) of jawless vertebrates such as lamprey and hagfish are composed of highly diverse modular leucine-rich repeats. Each lymphocyte assembles a unique VLR by rearrangement of the germline gene. In the lamprey genome, we identify here about 850 distinct cassettes encoding leucine-rich repeat modules that serve as sequence templates for the hypervariable VLR repertoires. The data

  9. 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.

  10. Adenine deaminase and adenine utilization in Saccharomyces cerevisiae.

    PubMed Central

    Deeley, M C

    1992-01-01

    Compared with other purine salvage and nitrogen catabolism enzymatic activities, adenine deaminase (adenine aminohydrolase [AAH]; EC 3.5.4.2) activity in Saccharomyces cerevisiae is uniquely regulated. AAH specific activity is not induced by adenine and is reduced sevenfold when cells are cultivated in medium containing proline in place of ammonium as the sole nitrogen source. Exogenous adenine enters metabolic pathways primarily via the function of either AAH or adenine phosphoribosyltransferase (APRT; EC 2.4.2.7). Exogenous adenosine cannot normally be utilized as a purine source. Strains efficiently utilized adenosine or inosine when grown in pH 4.5 medium containing Triton X-100. A recessive mutation permitting utilization of adenosine or inosine in standard media was isolated. In both situations, growth of purine auxotrophs required either AAH or APRT activity. With medium containing either ammonium or proline as a nitrogen source, minimum doubling times of purine auxotrophs deficient in either APRT or AAH were measured. In proline-based medium, AAH and APRT permitted equal utilization of exogenous adenine. In ammonium-based medium, the absence of APRT increased the minimum doubling time by 50%. Similar experiments using sufficient exogenous histidine to feedback inhibit histidine biosynthesis failed to affect the growth rates of adenine auxotrophs blocked in AAH or APRT, indicating that the histidine-biosynthetic pathway does not play a significant role in adenine utilization. The gene that encodes AAH in S. cerevisiae was isolated by complementation using yeast strain XD1-1, which is deficient in AAH, APRT, and purine synthesis. A 1.36-kb EcoRI-SphI fragment was demonstrated to contain the structural gene for AAH by expressing this DNA in Escherichia coli under control of the trp promoter-operator. Northern (RNA) studies using the AAH-, APRT-, and CDC3-coding regions indicated that AAH regulation was not mediated at the level of transcription or mRNA degradation. Images PMID:1577682

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

    PubMed

    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. PMID:24434356

  12. 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

  13. Tritium labelling of nucleic acids accompanied by conversion of cytosine to uracil.

    PubMed Central

    Scheinker, V S

    1976-01-01

    A new method of incorporation of tritium into nucleic acids with an accompanying conversion of cytosine to uracil is proposed. The method is based on the reaction of nucleic acids with bisulfite in the presence of 3H2O. Under certain conditions poly(C) is quantitatively converted to a radioactive poly(U), whereas similar bisulfite treatment of poly(U) does not result in any tritium incorporation. Specificity of the reaction is confirmed by the results of analysis of modified tRNA and rRNA. Incubation of tRNA with bisulfite and 3H2O does not lead to cleavage of the polynucleotide chain. Similar treatment of the denatured DNA results in tritium incorporation into DNA which is accompanied by a conversion of cytosine to uracil. There is virtually no reaction between native DNA and bisulfite. Only certain cytosone residues in yeast tRNAVal/2a interact with bisulfate providing that reaction is carried out under sufficiently mild conditions. PMID:1272798

  14. Cytosine methylation levels in the genome of Stellaria longipes

    Microsoft Academic Search

    Q. Cai; C. C. Chinnappa

    1999-01-01

    Environment-induced alteration of DNA methylation levels was investigated inStellaria longipes (Caryophyllaceae). Total cytosine methylation levels were measured using HPLC in 6 genets representing two ecotypes (alpine\\u000a and prairie) grown in short day photoperiod and cold temperature (SDC) and long day photoperiod and warm temperature (LDW)\\u000a conditions. The levels of methylated cytosine were 16.54-22.20% among the three genets from the alpine

  15. Guanine-Plus-Cytosine Content of Rothia dentocaviosa

    Microsoft Academic Search

    KATHLEEN B. GUSTAFSON; ANN SEDGWICK; ALAN L. COYKENDALL

    1985-01-01

    The guanine-plus-cytosine contents of Rothia dentocariosa strains ATCC 17931T (T = type strain) and ATCC 19426 were determined by thermal denaturation and found to be 54 and 57 mol%, respectively. These values are lower than the values given in many publications, but are consistent with data cited in some other references. The guanine-plus-cytosine (G + C) content of Rothia dentocariosa

  16. Serum adenosine deaminase activity in cutaneous anthrax

    PubMed Central

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

    2014-01-01

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

  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. Regulation of AMP deaminase by phosphoinositides.

    PubMed

    Sims, B; Mahnke-Zizelman, D K; Profit, A A; Prestwich, G D; Sabina, R L; Theibert, A B

    1999-09-01

    AMP deaminase (AMPD) converts AMP to IMP and is a diverse and highly regulated enzyme that is a key component of the adenylate catabolic pathway. In this report, we identify the high affinity interaction between AMPD and phosphoinositides as a mechanism for regulation of this enzyme. We demonstrate that endogenous rat brain AMPD and the human AMPD3 recombinant enzymes specifically bind inositide-based affinity probes and to mixed lipid micelles that contain phosphatidylinositol 4,5-bisphosphate. Moreover, we show that phosphoinositides specifically inhibit AMPD catalytic activity. Phosphatidylinositol 4,5-bisphosphate is the most potent inhibitor, effecting pure noncompetitive inhibition of the wild type human AMPD3 recombinant enzyme with a K(i) of 110 nM. AMPD activity can be released from membrane fractions by in vitro treatment with neomycin, a phosphoinositide-binding drug. In addition, in vivo modulation of phosphoinositide levels leads to a change in the soluble and membrane-associated pools of AMPD activity. The predicted human AMPD3 sequence contains pleckstrin homology domains and (R/K)X(n)(R/K)XKK sequences, both of which are characterized phosphoinositide-binding motifs. The interaction between AMPD and phosphoinositides may mediate membrane localization of the enzyme and function to modulate catalytic activity in vivo. PMID:10464307

  19. Activation induced deaminase: how much and where?

    PubMed

    Orthwein, Alexandre; Di Noia, Javier M

    2012-08-01

    Activation induced deaminase (AID) plays a central role in adaptive immunity by initiating the processes of somatic hypermutation (SHM) and class switch recombination (CSR). On the other hand, AID also predisposes to lymphoma and plays a role in some autoimmune diseases, for which reasons AID expression and activity are regulated at various levels. Post-translational mechanisms regulating the amount and subcellular localization of AID are prominent in balancing AID physiological and pathological functions in B cells. Mechanisms regulating AID protein levels include stabilizing chaperones in the cytoplasm and proteins efficiently targeting AID to the proteasome within the nucleus. Nuclear export and cytoplasmic retention contribute to limit the amount of AID accessing the genome. Additionally, a number of factors have been implicated in AID active nuclear import. We review these intertwined mechanisms proposing two scenarios in which they could interact as a network or as a cycle for defining the optimal amount of AID protein. We also comparatively review the expression levels of AID necessary for its function during the immune response, present in different cancers as well as in those tissues in which AID has been implicated in epigenetic remodeling of the genome by demethylating DNA. PMID:22687198

  20. 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

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

    NASA Astrophysics Data System (ADS)

    Das, Babulal; Baruah, Jubaraj B.

    2011-08-01

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

  2. 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

  3. Quantitative changes in adenosine deaminase isoenzymes in human colorectal adenocarcinomas.

    PubMed

    ten Kate, J; Wijnen, J T; van der Goes, R G; Quadt, R; Griffioen, G; Bosman, F T; Khan, P M

    1984-10-01

    Several reports have suggested that a decrease or absence of adenosine deaminase complexing protein (ADCP) is consistently associated with cancer. However, in other studies, decreased as well as increased ADCP levels were found. In the present study, we investigated ADCP levels in 37 colorectal adenocarcinomas and correlated the results with clinicopathological characteristics in individual carcinomas. The levels of adenosine deaminase (EC 3.5.4.4) and soluble ADCP were determined in tissue samples by, respectively, a spectrophotometric assay and an ADCP specific radioimmunoassay. The values in the individual tumors were compared with their histological characteristics, such as degree of differentiation, nuclear grading, and the preoperative plasma carcinoembryonic antigen levels in the patients. It was found that ADCP was decreased in about a third of the tumors but unaltered or even increased in others. However, there was an overall 40% increase of the adenosine deaminase activity in the tumors compared to normal tissue. There seems to be no simple correlation between any of the clinicopathological parameters and the ADCP or adenosine deaminase levels. Methods detecting ADCP at single cell level might be helpful in exploring its potential use as a cancer-associated marker. PMID:6147190

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

    PubMed

    Smith, Alexander E; Ford, Kevin G

    2007-01-01

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

  5. 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.

  6. 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.

  7. 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.

  8. Dry yeast

    NSDL National Science Digital Library

    Ranveig Thattai (None; )

    2005-09-27

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

  9. Yeast virology

    Microsoft Academic Search

    REED B. WICKNER

    The three families of double-stranded RNA (dsRNA) viruses and two families of retroviruses (retrotranspo- sons) of the yeast Sacc\\/zaromyces cerevisiaeare all trans- mitted between cells only by cell fusion, probably re- flecting the high frequency of mating of yeast cells in nature. One dsRNA virus and two retroviruses appar- ently use ribosomal \\

  10. Primary Adenosine Monophosphate (AMP) Deaminase Deficiency in a Hypotonic Infant

    Microsoft Academic Search

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

    2011-01-01

    The spectrum of the adenosine monophosphate (AMP) deaminase deficiency ranges from asymptomatic carriers to patients who manifest exercise-induced muscle pain, occasionally rhabdomyolysis, and idiopathic hyperCKemia. However, previous to the introduction of molecular techniques, rare cases with congenital weakness and hypotonia have also been reported. We report a 6-month-old girl with the association of congenital muscle weakness and hypotonia, muscle deficiency

  11. 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)

  12. Yeast Infections

    MedlinePLUS

    Candida is the scientific name for yeast. It is a fungus that lives almost everywhere, including in ... infection that causes white patches in your mouth Candida esophagitis is thrush that spreads to your esophagus, ...

  13. 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...

  14. Low cytosine triphosphate synthase 2 expression renders resistance to 5-fluorouracil in colorectal cancer

    E-print Network

    Wong, Limsoon

    in colorectal cancer Wen Lee Tana *, Bhaskar Bhattacharyaa *, Marie Loha, b , Indirikumar Balasubramaniana, Floxuridine; CRC, colorectal cancer; CTPS, cytosine triphosphate synthase 2; CTPS2, cytosine triphosphate-derived colorectal cancer xenografts characterised for 5FU resistance. Candidate gene function was tested by si

  15. Triplex-mediated analysis of cytosine methylation at CpA sites in DNA.

    PubMed

    Johannsen, Marie W; Gerrard, Simon R; Melvin, Tracy; Brown, Tom

    2014-01-18

    Modified triplex-forming oligonucleotides distinguish 5-methyl cytosine from unmethylated cytosine in DNA duplexes by differences in triplex melting temperatures. The discrimination is sequence-specific; dramatic differences in stabilisation are seen for CpA methylation, whereas CpG methylation is not detected. This direct detection of DNA methylation constitutes a new approach for epigenetic analysis. PMID:24276836

  16. Probing Guanine and Cytosine Tautomers in the Gas Phase

    NASA Astrophysics Data System (ADS)

    Pena, I.; Vaquero, V.; López, J. C.; Alonso, J. L.

    2009-06-01

    Using laser ablation molecular beam Fourier transform microwave spectroscopy (LA-MB-FTMW) we have recently studied the nucleic acid bases uracil and thymine. We have now successfully probed in isolation conditions in the gas phase cytosine and guanine which are solids with high melting points (m.p.>300°C) and a low vapour pressure, and consequently, they are elusive to gas-phase rotational studies. Five rotational species have been detected in the supersonic expansion of cytosine. The unambiguous assignment of the observed species to the various tautomer/conformer structures is based on the markedly different values of the quadrupole coupling constants of the three ^{14}N nuclei, which act as fingerprints for the identification of the various species. Four species have been observed in the rotational spectra of Guanine. The comparison between the experimental rotational constants and those calculated ab initio provide a definitive test for the identification of the four lowest energy forms. The planarity of the tautomers is discussed on the basis of the inertial defect values (?=I_c-I_a-I_b). V. Vaquero, M.E. Sanz, J.C. López and J.L. Alonso, J. Phys. Chem. A 111, 3443 (2007) J.C. López, M.I. Peña, M.E. Sanz and J.L. Alonso, J. Chem. Phys. 126, 191103 (2007)

  17. 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 Å, 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.

  18. Adenosine deaminase in rodent median eminence: detection by antibody to the mouse enzyme and co-localization with adenosine deaminase-complexing protein (CD26).

    PubMed

    Nagy, J I; Yamamoto, T; Uemura, H; Schrader, W P

    1996-07-01

    Adenosine deaminase in the hypothalamic tuberomammillary nucleus and median eminence of rat and mouse brains was investigated with two different antibodies generated against the enzyme derived from either calf or mouse. Both antibodies labelled neurons in the tuberomammillary nucleus and, as determined in rat, they immunolabelled the same neurons. In the median eminence, immunopositive fibres and terminals were detected with anti-mouse adenosine deaminase in both rat and mouse, while no such staining was seen in either species with antibody against the calf enzyme. These fibres were most concentrated in the external median eminence, had a more restricted distribution than those containing either galanin or tyrosine hydroxylase and only partially overlapped with oxytocin-positive fibres. By electron microscopy, adenosine deaminase was found in terminals containing both small, clear vesicles with diameters of 35 to 45 nm and large dense-core vesicles with diameters of 100 to 140 nm. Preadsorption of antibodies with purified enzyme derived from the species against which they were directed eliminated all staining in rat, while antibody adsorptions across species were less effective. Preadsorption of anti-mouse adenosine deaminase antibody with the mouse deaminase led to increased labelling in mouse median eminence, suggesting an interaction between tissue components and antibody-linked enzyme. Tests for the presence of adenosine deaminase-complexing protein (CD26) with an antibody against this protein gave positive labelling in the median eminence of both species and this labelling was co-distributed with that seen for adenosine deaminase. These results confirm the expression of adenosine deaminase in restricted populations of neurons in rodent brain as revealed with a novel antibody, suggest the presence of a distinct form or localization of the enzyme in the median eminence, and raise the possibility that it contributes, perhaps along with CD26, to purinergic regulation of hormone secretion in this structure. PMID:8783262

  19. 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

  20. 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/?L 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

  1. Detection of Cytosine methylation in ancient DNA from five native american populations using bisulfite sequencing.

    PubMed

    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/?L 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. Deaminase activity on single-stranded DNA (ssDNA) occurs in vitro when APOBEC3G cytidine deaminase forms homotetramers and higher-order complexes.

    PubMed

    McDougall, William M; Okany, Chinelo; Smith, Harold C

    2011-09-01

    APOBEC3G (A3G) is a cytidine deaminase that catalyzes deamination of deoxycytidine (dC) on single-stranded DNA (ssDNA). The oligomeric state of A3G required to support deaminase activity remains unknown. We show under defined in vitro conditions that full-length and native A3G formed complexes with ssDNA in an A3G concentration-dependent but temperature-independent manner. Complexes assembled and maintained at 4 °C did not have significant deaminase activity, but their enzymatic function could be restored by subsequent incubation at 37 °C. This approach enabled complexes of a defined size range to be isolated and subsequently evaluated for their contribution to enzymatic activity. The composition of A3G bound to ssDNA was determined by protein-protein chemical cross-linking. A3G-ssDNA complexes of 16 S were necessary for deaminase activity and consisted of cross-linked A3G homotetramers and homodimers. At lower concentrations, A3G only formed 5.8 S homodimers on ssDNA with low deaminase activity. Monomeric A3G was not identified in 5.8 S or 16 S complexes. We propose that deaminase-dependent antiviral activity of A3G in vivo may require a critical concentration of A3G in viral particles that will promote oligomerization on ssDNA during reverse transcription. PMID:21737457

  3. Intrinsic immunity against retrotransposons by APOBEC cytidine deaminases.

    PubMed

    Koito, Atsushi; Ikeda, Terumasa

    2013-01-01

    Over 40% of the human genome is recognizable as having been derived from ancient retroelements, transported by an intracellular copy-and-paste process involving an RNA intermediate, with an additional few percent classified as DNA transposable elements. Endogenous retroviruses are long terminal repeat (LTR)-type retroelements that account for ~8% of human genomic DNA. Non-LTR members are present at extremely high copy numbers, with ~17% of the human genome consisting of long interspersed nuclear elements (LINEs). These LINEs modify vertebrate genomes not only through insertions, but also by the indirect replication of non-autonomous retrotransposons, such as short interspersed nuclear elements. As expected, vertebrate intrinsic immunity has evolved to support a balance between retroelement insertions that confer beneficial genetic diversity and those that cause deleterious gene disruptions. The mammalian cytidine deaminases encoded by the APOBEC3 genes can restrict a broad number of exogenous pathogens, such as exogenous retroviruses, and the mobility of endogenous retroelements. Furthermore, APOBEC1 from a variety of mammalian species, which mediates the cytidine (C) to uridine (U) deamination of apolipoprotein B (apoB) mRNA, a protein involved in lipid transport, also plays a role in controlling mobile elements. These mammalian apoB mRNA-editing, catalytic polypeptide (APOBEC) cytidine deaminases, which can bind to single-stranded DNA (ssDNA) as well as RNA, are able to insert mutations into ssDNA and/or RNA as a result of their ability to deaminate C to U. While these APOBEC cytidine deaminases with DNA mutagenic activity can be deleterious to cells, their biological modifications, such as protein-protein interactions and subcellular localization, in addition to their ability to bind to RNA, appear to have conferred a role for APOBECs as a cellular defense system against retroviruses and retroelements. In support of this notion, the expansion of the single APOBEC3 gene in mice to the seven APOBEC3 genes found in primates apparently correlates with the significant enhancement of the restriction of endogenous retroelements seen in primates, including humans. This review discusses the current understanding of the mechanism of action of APOBEC cytidine deaminases and attempts to summarize their roles in controlling retrotransposons. PMID:23431045

  4. The ONIOM molecular dynamics method for biochemical applications: Cytidine deaminase

    NASA Astrophysics Data System (ADS)

    Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

    2007-03-01

    We developed 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.

  5. 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

  6. 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

  7. Ag Nanocluster Formation Using a Cytosine Oligonucleotide Template

    PubMed Central

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

    2008-01-01

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

  8. 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

  9. Cytosine deamination plays a primary role in the evolution of mammalian isochores.

    PubMed

    Fryxell, K J; Zuckerkandl, E

    2000-09-01

    DNA melting is rate-limiting for cytosine deamination, from which we infer that the rate of cytosine deamination should decline twofold for each 10% increase in GC content. Analysis of human DNA sequence data confirms that this is the case for 5-methylcytosine. Several lines of evidence further confirm that it is also the case for unmethylated cytosine and that cytosine deamination causes the majority of all C-->T and G-->A transitions in mammals. Thus, cytosine deamination and DNA base composition each affect the other, forming a positive feedback loop that facilitates divergent genetic drift to high or low GC content. Because a 10 degrees C increase in temperature in vitro increases the rate of cytosine deamination 5. 7-fold, cytosine deamination must be highly dependent on body temperature, which is consistent with the dramatic differences between the isochores of warm-blooded versus cold-blooded vertebrates. Because this process involves both DNA melting and positive feedback, it would be expected to spread progressively (in evolutionary time) down the length of the chromosome, which is consistent with the large size of isochores in modern mammals. PMID:10958853

  10. 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

  11. 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

  12. Characterization of a gene coding for a putative adenosine deaminase-related growth factor by RNA interference in the basidiomycete Flammulina velutipes.

    PubMed

    Sekiya, Shuichi; Yamada, Masato; Shibata, Kou; Okuhara, Toru; Yoshida, Masumi; Inatomi, Satoshi; Taguchi, Goro; Shimosaka, Makoto

    2013-04-01

    A full-length cDNA coding for a putative adenosine deaminase (Fv-ada) was isolated from the basidiomycete Flammulina velutipes. Fv-ada encodes a polypeptide consisting of 537 amino acid residues, which has a consensus sequence conserved among adenosine deaminase-related growth factors (ADGF) found in several metazoa, including chordates and insects. Fv-ada transcript was detected at all stages of growth in dikaryotic F. velutipes cells, with a peak at the primordial stage. Heterologous expression of Fv-ada in the yeast Pichia pastoris produced recombinant Fv-ADA that catalyzed the conversion of adenosine to inosine. Dikaryotic mycelia from F. velutipes were transformed with the binary plasmid pFungiway-Fv-ada, which was designed to suppress the expression of Fv-ada through RNA interference. The growth rates of the resulting transformants were retarded in response to the degree of suppression, indicating that Fv-ada plays an important role in the mycelial growth of F. velutipes. These results suggested that ADGF could function as growth factors in fungi, as is seen in other eukaryotes. PMID:23177216

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

    PubMed Central

    Geyer, Kathrin K.; Rodríguez López, 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

  14. DNA duplex stability of the thio-iso-guanine•methyl-iso-Cytosine base pair.

    PubMed

    Lee, Dongkye; Switzer, Christopher

    2015-06-01

    We report the synthesis, incorporation into oligonucleotides, and base-pairing properties of the 2-thio-variant of iso-guanine. Iso-guanine is the purine component of a nonstandard base pair with 5-methyl-iso-cytosine. The 2-thio-iso-guanine•5-methyl-iso-cytosine base pair is found to have similar stability to an adenine•thymine pair. PMID:25965331

  15. Cytosine methylation and the fate of CpG dinucleotides in vertebrate genomes

    Microsoft Academic Search

    David N. Cooper; Michael Krawczak

    1989-01-01

    The dinucleotide CpG is a “hotspot” for mutation in the human genome as a result of (1) the modification of the 5' cytosine by cellular DNA methyltransferases and (2) the consequent high frequency of spontaneous deamination of 5-methyl cytosine (5mC) to thymidine. DNA methylation thus contributes significantly, albeit indirectly, to the incidence of human genetic disease. We have attempted to

  16. IMP and AMP deaminase in reperfusion injury down-regulates neutrophil recruitment

    Microsoft Academic Search

    Fei-Hua Qiu; Koichiro Wada; Gregory L. Stahl; Charles N. Serhan

    2000-01-01

    We examined gene regulation in murine lungs after hind-limb vessel occlusion and reperfusion. A rapid increase of transcript for the AMP deaminase 3 gene (AMPD3) and its enzymatic activity (EC 3.5.4.6) generating inosine monophosphate (IMP) were identified with transcripts located in bronchial and alveolar epithelium. AMP deaminase inhibitor decreased IMP levels and significantly enhanced neutrophil recruitment within lung tissue during

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

    Microsoft Academic Search

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

    2010-01-01

    AMP-deaminase (AMPD, EC 3.5.4.6), which catalyzes the irreversible hydrolytic deamination of AMP to IMP and ammonia, is an\\u000a important energy-related enzyme. The partial genomic sequence of the gene encoding myoadenylate deaminase (AMPD1) from the\\u000a teleost fish Platichthys flesus was determined. The amino acid sequence of P. flesus AMPD1 shows 82% homology with that of the teleost fish Danio rerio. Comparison

  18. Phylogenetic Analysis Reveals a Novel Protein Family Closely Related to Adenosine Deaminase

    Microsoft Academic Search

    Stephanie A. Maier; Julia R. Galellis; Heather E. McDermid

    2005-01-01

    Adenosine deaminase (ADA) is a well-characterized enzyme involved in the depletion of adenosine levels. A group of proteins\\u000a with similarity to ADA, the adenosine deaminase-related growth factors (ADGF; known as CECR1 in vertebrates), has been described\\u000a recently in various organisms. We have determined the phylogenetic relationships of various gene products with significant\\u000a amino acid similarity to ADA using parsimony and

  19. Expression patterns of AMP-deaminase isozymes in human hepatocellular carcinoma (HCC)

    Microsoft Academic Search

    Magdalena Szyd?owska; Anna Roszkowska

    2008-01-01

    Background AMP-deaminase (EC 3.5.4.6) is an enzyme of nucleotide breakdown involved in regulation of energetic metabolism in mammalian\\u000a cells. The enzyme is coded by a family of three independent genes (AMPD1, AMPD2 and AMPD3), synthesizing three different isozymes. In mammalian liver, the reaction catalyzed by AMP-deaminase constitutes a rate-limiting\\u000a step in adenine nucleotide catabolism. In neoplastic liver, adenine nucleotide catabolism

  20. Cloning and Characterization of a Plasmid Encoded ACC Deaminase from an Indigenous Pseudomonas fluorescens FY32

    Microsoft Academic Search

    Davoud Farajzadeh; Naser Aliasgharzad; Nemat Sokhandan Bashir; Bagher Yakhchali

    2010-01-01

    In addition to the characterized mechanisms responsible for many direct effects of plant growth promoting bacteria (PGPB)\\u000a on plants, it has been suggested that a number of PGPB contain the enzyme ACC deaminase that catalyzes degradation of 1-aminocyclopropane-1-carboxylic\\u000a acid (ACC), the immediate precursor of ethylene, into ?-ketobutyrate and ammonia. As part of an effort to obtain an ACC deaminase\\u000a encoding

  1. Serum adenosine deaminase activity in bovine liver diseases.

    PubMed

    Abd Ellah, Mahmoud Rushdi; Nishimori, Kazuhiro; Goryo, Masanobu; Okada, Keiji; Yasuda, Jun

    2004-11-01

    A total of 60 cattle were examined for the presence of pathological liver lesions. The liver lesions were classified as glycogen degeneration, liver abscess, sawdust liver and fatty degeneration. The value of serum adenosine deaminase (ADA) activity was investigated as a pilot study for diagnosing liver diseases in cattle. Serum ADA activity was significantly higher in cases with glycogen degeneration (9.8 +/- 3.8 U/l) , liver abscess (10.4 +/- 3.2 U/l), sawdust liver (11.5 +/- 7.3 U/l) and fatty degeneration (20.8 +/- 7.7 U/l) than in the controls. The results indicate that ADA activity increases with the degree of hepatocellular damage. We concluded that serum ADA activity may be of value in bovine liver disease diagnosis. PMID:15585959

  2. Orthologous mammalian APOBEC3A cytidine deaminases hypermutate nuclear DNA.

    PubMed

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

    2014-02-01

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

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

    PubMed

    Zakrzewski, Falk; Schubert, Veit; Viehoever, Prisca; Minoche, André 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

  4. 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 ?-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 ?-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

  5. Metformin activates AMP kinase through inhibition of AMP deaminase.

    PubMed

    Ouyang, Jiangyong; Parakhia, Rahulkumar A; Ochs, Raymond S

    2011-01-01

    The mechanism for how metformin activates AMPK (AMP-activated kinase) was investigated in isolated skeletal muscle L6 cells. A widely held notion is that inhibition of the mitochondrial respiratory chain is central to the mechanism. We also considered other proposals for metformin action. As metabolic pathway markers, we focused on glucose transport and fatty acid oxidation. We also confirmed metformin actions on other metabolic processes in L6 cells. Metformin stimulated both glucose transport and fatty acid oxidation. The mitochondrial Complex I inhibitor rotenone also stimulated glucose transport but it inhibited fatty acid oxidation, independently of metformin. The peroxynitrite generator 3-morpholinosydnonimine stimulated glucose transport, but inhibited fatty acid oxidation. Addition of the nitric oxide precursor arginine to cells did not affect glucose transport. These studies differentiate metformin from inhibition of mitochondrial respiration and from active nitrogen species. Knockdown of adenylate kinase also failed to affect metformin stimulation of glucose transport. Hence, any means of increase in ADP appears not to be involved in the metformin mechanism. Knockdown of LKB1, an upstream kinase and AMPK activator, did not affect metformin action. Having ruled out existing proposals, we suggest a new one: metformin might increase AMP through inhibition of AMP deaminase (AMPD). We found that metformin inhibited purified AMP deaminase activity. Furthermore, a known inhibitor of AMPD stimulated glucose uptake and fatty acid oxidation. Both metformin and the AMPD inhibitor suppressed ammonia accumulation by the cells. Knockdown of AMPD obviated metformin stimulation of glucose transport. We conclude that AMPD inhibition is the mechanism of metformin action. PMID:21059655

  6. 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

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

    PubMed

    Bolaños-Villegas, Pablo; Jauh, Guang-Yuh

    2015-05-01

    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

  8. Real-time study of interactions between cytosine-cytosine pairs in DNA oligonucleotides and silver ions using dual polarization interferometry.

    PubMed

    Zheng, Yu; Yang, Cheng; Yang, Fan; Yang, Xiurong

    2014-04-15

    The real-time conformational changes of cytosine (C)-rich ssDNA oligonucleotides upon binding with silver ions (Ag(+)) were studied using dual polarization interferometry (DPI). Upon the addition of Ag(+), Ag(+) selectively bound to cytosine-cytosine mismatches and formed C-Ag(+)-C complexes, inducing change of the structure of the C-rich ssDNA from random coil conformation to duplex conformation, whereas the control ssDNA without cytosine-cytosine mismatches had no such signal, which was consistent with circular dichroism (CD) characterization. The conformational change of DNA was reflected on the changes of the mass, thickness, and density values resolved by DPI. The calibration curves showed that as the concentration of Ag(+) increased from 10 nM to 8 ?M, the thickness and mass values increased linearly while the density values decreased linearly. Other metal ions such as K(+), Ca(2+), Na(+), Mg(2+), Zn(2+), Mn(2+), Ni(2+), and Pb(2+) did not interfere with the interaction between Ag(+) and C-rich ssDNA, indicating that this method had a good selectivity. The practical application of this biosensor was also investigated in real samples such as drinking water. Besides, cysteine could specifically capture Ag(+) from C-Ag(+)-C complexes and transformed the structure of the C-rich DNA back from rigid double-stranded conformation to random coil conformation, which allowed cysteine to be detected selectively as well. It is expected that this biosensing strategy may be utilized to study the interaction of DNA with other molecules. PMID:24611666

  9. YEAST GENETICS Fred Winston

    E-print Network

    Winston, Fred

    YEAST GENETICS Fred Winston 7.1 Introduction Key Concepts · Genetic studies of the yeast. The yeast Saccharomyces cerevisiae is an ideal experimental organism. It is a microorganism that has a fast biology. Yeast has been the focus of extensive studies in many aspects of molecular biology. These areas

  10. Genital Yeast Infections

    Microsoft Academic Search

    J. D. Oriel; Betty M. Partridge; Maire J. Denny; J. C. Coleman

    1972-01-01

    Genital yeast infection was studied in 533 women seen in a department of venereology. Yeasts were recovered in culture from 138 patients (26% of the total). Candida albicans accounted for 112 (81%) of the isolates and Torulopsis glabrata for 22 (16%); other yeasts were uncommon. There was no evidence that the presence of yeasts was related to age. 32% of

  11. Lsh, chromatin remodeling family member, modulates genome-wide cytosine methylation patterns at nonrepeat sequences.

    PubMed

    Tao, Yongguang; Xi, Sichuan; Shan, Jigui; Maunakea, Alika; Che, Anney; Briones, Victorino; Lee, Eunice Y; Geiman, Theresa; Huang, Jiaqiang; Stephens, Robert; Leighty, Robert M; Zhao, Keji; Muegge, Kathrin

    2011-04-01

    DNA methylation is critical for normal development and plays important roles in genome organization and transcriptional regulation. Although DNA methyltransferases have been identified, the factors that establish and contribute to genome-wide methylation patterns remain elusive. Here, we report a high-resolution cytosine methylation map of the murine genome modulated by Lsh, a chromatin remodeling family member that has previously been shown to regulate CpG methylation at repetitive sequences. We provide evidence that Lsh also controls genome-wide cytosine methylation at nonrepeat sequences and relate those changes to alterations in H4K4me3 modification and gene expression. Deletion of Lsh alters the allocation of cytosine methylation in chromosomal regions of 50 kb to 2 Mb and, in addition, leads to changes in the methylation profile at the 5' end of genes. Furthermore, we demonstrate that loss of Lsh promotes--as well as prevents--cytosine methylation. Our data indicate that Lsh is an epigenetic modulator that is critical for normal distribution of cytosine methylation throughout the murine genome. PMID:21427231

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

    PubMed

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

    1997-06-01

    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. PMID:9199558

  13. AMP Deaminase 3 Deficiency Enhanced 5?-AMP Induction of Hypometabolism

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

  15. 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.

  16. 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.

  17. Oxidized, deaminated cytosines are a source of C ? T transitions in vivo

    PubMed Central

    Kreutzer, Deborah A.; Essigmann, John M.

    1998-01-01

    The most common base substitution arising from oxidative damage of DNA is a GC ? AT transition. In an effort to determine the oxidized lesion(s) that gives rise to this mutation, the mutagenicity of three oxidized cytosines, 5-hydroxycytosine, 5-hydroxyuracil, and uracil glycol, were investigated in Escherichia coli. An M13 viral genome was constructed to contain a single oxidized cytosine at a specific site. Replication in vivo of the single-stranded genomes yielded mutation frequencies of 0.05%, 83%, and 80% for 5-hydroxycytosine, 5-hydroxyuracil, and uracil glycol, respectively. The predominant mutation observed was C ? T. A model for C ? T oxidative mutagenesis is suggested in which initial cytosine oxidation is followed by deamination to a poorly repaired uracil derivative that is strongly miscoding during replication. PMID:9520408

  18. 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.

  19. Cytosine Arabinoside Influx and Nucleoside Transport Sites in Acute Leukemia

    PubMed Central

    Wiley, J. S.; Jones, S. P.; Sawyer, W. H.; Paterson, A. R. P.

    1982-01-01

    Although cytosine arabinoside (araC) can induce a remission in a majority of patients presenting with acute myeloblastic leukemia (AML), a minority fail to respond and moreover the drug has less effect in acute lymphoblastic leukemia (ALL). The carrier-mediated influx of araC into purified blasts from patients with AML, ALL, and acute undifferentiated leukemia (AUL) has been compared to that of normal lymphocytes and polymorphs. Blasts showed a larger mediated influx of araC than mature cells, since mean influxes for myeloblasts and lymphoblasts were 6- and 2.3-fold greater than polymorphs and lymphocytes, respectively. Also, the mean influx for myeloblasts was fourfold greater than the mean for lymphoblasts. The number of nucleoside transport sites was estimated for each cell type by measuring the equilibrium binding of [3H]nitrobenzylthioinosine (NBMPR), which inhibits nucleoside fluxes by binding with high affinity to specific sites on the transport mechanism. The mean binding site numbers for myeloblasts and lymphoblasts were 5- and 2.8-fold greater, respectively, than for the mature cells of the same maturation series. The mean number of NBMPR binding sites for myeloblasts was fourfold greater than for lymphoblasts. Patients with AUL were heterogeneous since blasts from some gave values within the myeloblastic range and others within the lymphoblastic range. The araC influx correlated closely with the number of NBMPR binding sites measured in the same cells on the same day. Transport parameters were measured on blasts from 15 patients with AML or AUL who were then treated with standard induction therapy containing araC. Eight patients entered complete remission, while seven failed therapy, among whom were the three patients with the lowest araC influx (<0.4 pmol/107 cells per min) and NBMPR binding (<3,000 sites/cell) for the treated group. In summary, myeloblasts have both higher araC transport rates and more nucleoside transport sites than lymphoblasts and this factor may contribute to the greater sensitivity of AML to this drug. AraC transport varied >10-fold between leukemic blasts and normal leukocytes, but transport capacity related directly to the number of nucleoside transport sites on the cell. Finally, low araC transport rates or few NBMPR binding sites on blasts were observed in a subset of patients with acute leukemia who failed to achieve remission with drug combinations containing araC. PMID:6948829

  20. RNA-editing cytidine deaminase Apobec-1 is unable to induce somatic hypermutation in mammalian cells

    PubMed Central

    Eto, Tomonori; Kinoshita, Kazuo; Yoshikawa, Kiyotsugu; Muramatsu, Masamichi; Honjo, Tasuku

    2003-01-01

    Antibody diversification by somatic hypermutation, gene conversion, and class switch recombination is completely dependent on activation-induced cytidine deaminase (AID). A recent report showing induction of DNA mutations in Escherichia coli by overexpression of AID, Apobec-1, and related members of the RNA-editing cytidine deaminase family suggested that they may directly modify deoxycytidine in DNA in mammalian cells (DNA-editing model). We therefore examined whether Apobec-1 bona fide RNA-editing enzyme could show somatic hypermutation and class switching activities in murine B lymphocytes and fibroblasts. Unlike AID, Apobec-1 was unable to induce somatic hypermutation or class switching. The results force a reevaluation of the physiological significance of the DNA deaminase activities of AID and Apobec-1 in E. coli and in vitro. PMID:14559972

  1. Infrared multiple photon dissociation action spectroscopy of proton-bound dimers of cytosine and modified cytosines: effects of modifications on gas-phase conformations.

    PubMed

    Yang, Bo; Wu, R R; Berden, G; Oomens, J; Rodgers, M T

    2013-11-21

    The gas-phase structures of proton-bound dimers of cytosine and modified cytosines and their d6-analogues generated by electrospray ionization are probed via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical electronic structure calculations. The modified cytosines examined include the 5-methyl-, 5-fluoro-, and 5-bromo-substituted species. IRMPD action spectra of seven proton-bound dimers exhibit both similar and distinctive spectral features over the range of ?2600-3700 cm(-1). The IRMPD spectra of all of these proton-bound dimers are relatively simple, but exhibit obvious shifts in the positions of several bands that correlate with the properties of the substituent. The measured IRMPD spectra are compared to linear IR spectra calculated for the stable low-energy tautomeric conformations, determined at the B3LYP/6-31G* level of theory, to identify the conformations accessed in the experiments. Comparison of the measured IRMPD and calculated IR spectra indicates that only a single conformation, the ground-state structure, is accessed for all proton-bound homodimers, whereas the ground-state and a small population of the first-excited tautomeric conformations are accessed for all proton-bound heterodimers. In all cases, three hydrogen-bonding interactions in which the nucleobases are aligned in an antiparallel fashion analogous to that of the DNA i-motif are responsible for stabilizing the base pairing. Thus, base modifications such as 5-methyl- and 5-halo-substitution of cytosine should not alter the structure of the DNA i-motif. PMID:24151932

  2. IRMPD action spectroscopy of alkali metal cation-cytosine complexes: effects of alkali metal cation size on gas phase conformation.

    PubMed

    Yang, Bo; Wu, R R; Polfer, N C; Berden, G; Oomens, J; Rodgers, M T

    2013-10-01

    The gas-phase structures of alkali metal cation-cytosine complexes generated by electrospray ionization are probed via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical calculations. IRMPD action spectra of five alkali metal cation-cytosine complexes exhibit both similar and distinctive spectral features over the range of ~1000-1900 cm(-1). The IRMPD spectra of the Li(+)(cytosine), Na(+)(cytosine), and K(+)(cytosine) complexes are relatively simple but exhibit changes in the shape and shifts in the positions of several bands that correlate with the size of the alkali metal cation. The IRMPD spectra of the Rb(+)(cytosine) and Cs(+)(cytosine) complexes are much richer as distinctive new IR bands are observed, and the positions of several bands continue to shift in relation to the size of the metal cation. The measured IRMPD spectra are compared to linear IR spectra of stable low-energy tautomeric conformations calculated at the B3LYP/def2-TZVPPD level of theory to identify the conformations accessed in the experiments. These comparisons suggest that the evolution in the features in the IRMPD action spectra with the size of the metal cation, and the appearance of new bands for the larger metal cations, are the result of the variations in the intensities at which these complexes can be generated and the strength of the alkali metal cation-cytosine binding interaction, not the presence of multiple tautomeric conformations. Only a single tautomeric conformation is accessed for all five alkali metal cation-cytosine complexes, where the alkali metal cation binds to the O2 and N3 atoms of the canonical amino-oxo tautomer of cytosine, M(+)(C1). PMID:23893433

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

    Microsoft Academic Search

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

    1988-01-01

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

  4. 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.

  5. Structural and metabolic specificity of methylthiocoformycin for malarial adenosine deaminases

    PubMed Central

    Ho, Meng-Chiao; Cassera, María B.; Madrid, Dennis C.; Ting, Li-Min; Tyler, Peter C.; Kim, Kami; Almo, Steven C.; Schramm, Vern L.

    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 (Tyler, P. C., Taylor, E. A., Fröhlich, R. G. G. and Schramm, V. L. (2007) J. Am. Chem. Soc. 129, 6872–6879). The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation (Larson, E. T. et al. (2008) J. Mol. Biol. 381, 975–988). Here, the crystal structure of ADA from Plasmodium vivax in 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°. 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 plasmodial ADAs establishes 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. PMID:19728741

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

    NASA Astrophysics Data System (ADS)

    Shi, Jinming

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

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

    E-print Network

    Xia, Xuhua

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

  8. 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

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

    PubMed Central

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

    2014-01-01

    The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is known to play a role in sensitivity to temozolomide. Promoter hypermethylation of MGMT is commonly used to predict low expression levels of MGMT in gliomas, despite observed discordance between promoter methylation and protein levels. Here, we investigated the functional role of gene body cytosine modification in regulating levels of MGMT gene expression and sensitivity to temozolomide. In 91 human glioblastoma samples, we observed significant variation in MGMT expression levels in patients with an unmethylated promoter, with higher levels of gene body cytosine modification correlating with higher gene expression levels. Furthermore, inducing hypomethylation across the MGMT gene body with decitabine corresponded with decreased levels of MGMT gene expression in lymphoblastoid and glioblastoma cell lines, indicating an important functional role for gene body cytosine modifications in maintaining gene expression. We reasoned that the decrease in MGMT expression induced by decitabine may render resistant glioblastoma cell lines more sensitive to temozolomide. Consistent with this reasoning, we found that the MGMT-expressing glioblastoma cell lines exhibiting an unmethylated MGMT promoter that were pre-treated with decitabine became significantly more sensitive to temozolomide. Overall, our results suggest a functional role for gene body cytosine modification in regulating gene expression of MGMT and indicate that pre-treating patients whose tumors have an unmethylated MGMT promoter with decitabine prior to temozolomide treatment may increase their response to therapy. PMID:24568970

  10. Yeast Education Network

    NSDL National Science Digital Library

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

  11. Vaginal Yeast Infections

    MedlinePLUS

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

  12. Vaginal Yeast Infection

    MedlinePLUS

    ... on. Read more information on enabling JavaScript. Vaginal Yeast Infection Skip Content Marketing Share this: Main Content Area Vaginal yeast infection, or vulvovaginal candidiasis, is a common cause ...

  13. Immunohistochemical analysis of human skeletal muscle AMP deaminase deficiency. Evidence of a correlation between the muscle HPRG content and the level of the residual AMP deaminase activity

    Microsoft Academic Search

    Antonietta R. M. Sabbatini; Antonio Toscano; Mohammed Aguennouz; Daniela Martini; Enza Polizzi; Maria Ranieri-Raggi; Arthur J. G. Moir; Alba Migliorato; Olimpia Musumeci; Giuseppe Vita; Antonio Raggi

    2006-01-01

    We have previously described that, in healthy human skeletal muscle, an anti-histidine-proline-rich-glycoprotein (HPRG) antibody\\u000a selectively binds to type IIB fibers that are well known to contain the highest level of AMP deaminase (AMPD) activity, suggesting\\u000a an association of the HPRG-like protein to the enzyme isoform M. The present paper reports an immunohistochemical study performed\\u000a on human skeletal muscle biopsies from

  14. CLONTECHInnovative Yeast Protocols Handbook

    E-print Network

    Erickson, F. Les

    CLONTECHInnovative Tools to Accelerate Discovery Yeast Protocols Handbook PT3024-1 (PR13103 FOR RESEARCH USE ONLY #12;Yeast Protocols Handbook CLONTECH Laboratories, Inc. www.clontech.com Protocol # PT3024-1 2 Version # PR13103 I. Introduction 4 II. Introduction to Yeast Promoters 5 III. Culturing

  15. Expression patterns of AMP-deaminase and cytosolic 5'-nucleotidase genes in human term placenta.

    PubMed

    Roszkowska, Anna; Klimek, Jerzy; Kaletha, Krystian

    2008-04-01

    Background AMP-deaminase (EC 3.5.4.6) and 5'-nucleotidase (EC 3.1.3.5) are enzymes responsible for the maintenance of cellular adenine nucleotides pool. Both exist in several isoforms that differ in kinetic properties and tissue distribution. Profile of isoforms of these enzymes in human placenta has not been analyzed so far while this could be important for understanding of pathology of placental ischemia such as in preeclampsia. Our aim was therefore to analyze expression of AMPD and CN-I genes in human term placenta. Methods RT-PCR analysis was used for determine expression of AMPD1, AMPD2, AMPD3 and CN-I. Results and conclusion The experimental results presented here indicate that genes coding "AMP-preferring", cytosolic isozyme of 5'-nucleotidase (cN-I) as well as "muscle-type" isozyme of AMP-deaminase (AMPD1) are not expressed in human term placenta. Among other AMPD family genes, only these coding "liver-type" isozyme (AMPD2) and, in lesser degree, "erythrocyte-type" isozyme (AMPD3) of AMP-deaminase are expressed in this organ. The expression level of AMPD3 was a half of that presented by AMPD2. We conclude that high abundance of AMP-deaminase 2 transcript suggest that this particular isoform is a predominant pathway of adenine nucleotides degradation in human term placenta that follows liver-type regulation of this process. PMID:18165923

  16. Genetic and other determinants of AMP deaminase activity in healthy adult skeletal muscle.

    PubMed

    Norman, B; Mahnke-Zizelman, D K; Vallis, A; Sabina, R L

    1998-10-01

    AMPD1 genotype, relative fiber type composition, training status, and gender were evaluated as contributing factors to the reported variation in AMP deaminase enzyme activity in healthy skeletal muscle. Multifactorial correlative analyses demonstrate that AMPD1 genotype has the greatest effect on enzyme activity. An AMPD1 mutant allele frequency of 13.7 and a 1.7% incidence of enzyme deficiency was found across 175 healthy subjects. Homozygotes for the AMPD1 normal allele have high enzyme activities, and heterozygotes display intermediate activities. When examined according to genotype, other factors were found to affect variability as follows: AMP deaminase activity in homozygotes for the normal allele exhibits a negative correlation with the relative percentage of type I fibers and training status. Conversely, residual AMP deaminase activity in homozygotes for the mutant allele displays a positive correlation with the relative percentage of type I fibers. Opposing correlations in different homozygous AMPD1 genotypes are likely due to relative fiber-type differences in the expression of AMPD1 and AMPD3 isoforms. Gender also contributes to variation in total skeletal muscle AMP deaminase activity, with normal homozygous and heterozygous women showing only 85-88% of the levels observed in genotype-matched men. PMID:9760316

  17. 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 ...

  18. Catalytic Mechanism of Guanine Deaminase:  An ONIOM and Molecular Dynamics Study

    Microsoft Academic Search

    Lishan Yao; Robert I. Cukier; Honggao Yan

    2007-01-01

    The catalytic mechanism of Bacillus subtilis guanine deaminase (bGD), a Zn metalloenzyme, has been investigated by a combination of quantum mechanical calculations using the multilayered ONIOM method and molecular dynamics simulations. In contrast to a previously proposed catalytic mechanism, which requires the bound guanine to assume a rare tautomeric state, the ONIOM calculations showed that the active-site residues of the

  19. MicroRNA-155 Is a Negative Regulator of Activation-Induced Cytidine Deaminase

    E-print Network

    Papavasiliou, F. Nina

    Immunity Article MicroRNA-155 Is a Negative Regulator of Activation-Induced Cytidine Deaminase was regulated post- transcriptionally by a lymphocyte-specific microRNA, miR-155. We found that miR-155 maturation. Thus, miR- 155, which has recently been shown to play important roles in regulating the germinal

  20. Direct Evidence that RNA Inhibits APOBEC3G ssDNA Cytidine Deaminase Activity

    PubMed Central

    McDougall, William M.; Smith, Harold C.

    2011-01-01

    APOBEC3G (A3G) is a deoxycytidine deaminase active on ssDNA substrates. In HIV infected cells A3G interacted with reverse transcription complexes where its activity as a deoxycytidine deaminase led to mutation of the viral genome. A3G not only bound ssDNA, but it also had an intrinsic ability to bind RNA. In many cell types that can support HIV replication, A3G ssDNA deaminase activity was suppressed and the enzyme resided in high molecular mass, ribonucleoprotein complexes associated with cytoplasmic P-bodies and stress granules. Using a defined in vitro system, we show that RNA alone was sufficient to suppress A3G deaminase activity and did so in an RNA concentration-dependent manner. RNAs of diverse sequences and as short as 25 nucleotides were effective inhibitors. Native PAGE analyses showed that RNA formed ribonucleoprotein complexes with A3G and in so doing prevented ssDNA substrates from binding to A3G. The data provided direct evidence that A3G binding to cellular RNAs constituted a substantial impediment to the enzyme’s ability to interact with ssDNA. PMID:21856286

  1. 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.

  2. Quantum-mechanical calculation of the intensity distribution in resonance Raman spectra of guanine-cytosine pair

    NASA Astrophysics Data System (ADS)

    Burova, T. G.; Ten, G. N.; Anashkin, A. A.

    2008-05-01

    The method of quantum-mechanical calculation of the relative line intensities in the resonance Raman (RR) spectra of polyatomic molecules, which was previously applied to the analysis of the spectra of individual cyclic molecules and makes it possible to take into account the Herzberg-Teller and Duschinsky effects, as well as the frequency effect, is applied for the first time to the calculation of the spectra of a pair guanine-cytosine. Satisfactory agreement between the calculated results and the available experimental data is obtained. The particular features of the intensity distribution in the RR spectra of the guanine-cytosine pair excited by laser radiation at 266, 240, 218, and 200 nm are analyzed. The RR spectra of the guanine-cytosine pair are compared with the spectra of the individual guanine and cytosine molecules excited by the laser radiation at the same wavelengths.

  3. Arabidopsis Methionine  Lyase Is Regulated According to Isoleucine Biosynthesis Needs But Plays a Subordinate Role to Threonine Deaminase

    Microsoft Academic Search

    Vijay Joshi; Georg Jander

    2009-01-01

    The canonical pathway for isoleucine biosynthesis in plants begins with the conversion of threonine to 2-ketobutyrate by threonine deaminase (OMR1). However, demonstration of methionine g-lyase (MGL) activity in Arabidopsis (Arabidopsis thaliana) suggested that production of 2-ketobutyrate from methionine can also lead to isoleucine biosynthesis. Rescue of the isoleucine deficit in a threonine deaminase mutant by MGL overexpression, as well as

  4. Investigating the target recognition of DNA cytosine-5 methyltransferase HhaI by library selection using in vitro compartmentalisation

    Microsoft Academic Search

    Yin-Fai Lee; Dan S. Tawfik; Andrew D. Griffiths

    2002-01-01

    In vitro compartmentalisation (IVC), a technique for selecting genes encoding enzymes based on com- partmentalising gene translation and enzymatic reactions in emulsions, was used to investigate the interaction of the DNA cytosine-5 methyltransferase M.HhaI with its target DNA (5¢-GCGC-3¢). Crystallog- raphy shows that the active site loop from the large domain of M.HhaI interacts with a flipped-out cytosine (the target

  5. Quantum-mechanical calculation of the intensity distribution in resonance Raman spectra of cytosine

    NASA Astrophysics Data System (ADS)

    Burova, T. G.; Ten, G. N.; Kucherova, V. V.

    2004-07-01

    A quantum-mechanical calculation of the relative intensities of lines in resonance Raman (RR) spectra of cytosine excited by laser radiation at 266, 218, and 200 nm was performed in different approximations of the vibronic theory. Both the Herzberg-Teller effect and the contribution from electronic states located close to the resonance state are shown to play a significant role in determining the relative intensities of lines. A satisfactory agreement between the calculated results and experimental data is obtained. The specific features of the intensity distribution in the RR spectra of cytosine are compared with those in the spectra of the previously studied thymine and uracil, which have a similar structure and also belong to the simplest nucleic acid bases.

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

    PubMed Central

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

    2012-01-01

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

  7. Transformation of Yeast

    Microsoft Academic Search

    Albert Hinnen; James B. Hicks; Gerald R. Fink

    1978-01-01

    A stable leu2- yeast strain has been transformed to LEU2+ by using a chimeric ColE1 plasmid carrying the yeast leu2 gene. We have used recently developed hybridization and restriction endonuclease mapping techniques to demonstrate directly the presence of the transforming DNA in the yeast genome and also to determine the arrangement of the sequences that were introduced. These studies show

  8. Vaginal Yeast Infections (For Parents)

    MedlinePLUS

    ... treatment is simple and painless. What Is a Yeast Infection? A yeast infection, also known as candidiasis ( ... you can be treated appropriately. Do Guys Get Yeast Infections? Guys can get an infection of the ...

  9. Red Yeast Rice: An Introduction

    MedlinePLUS

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

  10. IMP and AMP deaminase in reperfusion injury down-regulates neutrophil recruitment.

    PubMed

    Qiu, F H; Wada, K; Stahl, G L; Serhan, C N

    2000-04-11

    We examined gene regulation in murine lungs after hind-limb vessel occlusion and reperfusion. A rapid increase of transcript for the AMP deaminase 3 gene (AMPD3) and its enzymatic activity (EC) generating inosine monophosphate (IMP) were identified with transcripts located in bronchial and alveolar epithelium. AMP deaminase inhibitor decreased IMP levels and significantly enhanced neutrophil recruitment within lung tissue during reperfusion. In addition, IMP inhibited cytokine-initiated neutrophil infiltration in vivo and selectively attenuated neutrophil rolling by 90% in microvessels. We prepared labeled IMP and demonstrated that IMP specifically binds to neutrophils. IMP also stimulated binding of gamma-[(35)S]thio-GTP, suggesting that IMP is a potent regulator of neutrophils. Taken together, these results elucidate a previously unrecognized mechanism that protects tissues from the potentially deleterious consequences of aberrant neutrophil accumulation. Moreover, they are relevant for new therapeutic approaches to regulate neutrophil responses in inflammation and vascular disease. PMID:10760293

  11. Oxidation reactions of cytosine DNA components by hydroxyl radical and one-electron oxidants in aerated aqueous solutions.

    PubMed

    Wagner, J Richard; Cadet, Jean

    2010-04-20

    Indirect evidence strongly suggests that oxidation reactions of cytosine and its minor derivative 5-methylcytosine play a major role in mutagenesis and cancer. Therefore, there is an emerging necessity to identify the final oxidation products of these reactions, to search for their formation in cellular DNA, and to assess their mutagenic features. In this Account, we report and discuss the main *OH and one-electron-mediated oxidation reactions, two of the most potent sources of DNA damage, of cytosine and 5-methylcytosine nucleosides that have been recently characterized. The addition of *OH to the 5,6-unsaturated double bond of cytosine and 5-methylcytosine generates final degradation products that resemble those observed for uracil and thymine. The main product from the oxidation of cytosine, cytosine glycol, has been shown to undergo dehydration at a much faster rate as a free nucleoside than when inserted into double-stranded DNA. On the other hand, the predominant *OH addition at C5 of cytosine or 5-methylcytosine leads to the formation of 5-hydroxy-5,6-dihydro radicals that give rise to novel products with an imidazolidine structure. The mechanism of the formation of imidazolidine products is accounted for by rearrangement reactions that in the presence of molecular oxygen likely involve an intermediate pyrimidine endoperoxide. The reactions of the radical cations of cytosine and 5-methylcytosine are governed by competitive hydration, mainly at C6 of the pyrimidine ring, and deprotonation from the exocyclic amino and methyl group, leading in most cases to products similar to those generated by *OH. 5-Hydroxypyrimidines, the dehydration products of cytosine and uracil glycols, have a low oxidation potential, and their one-electron oxidation results in a cascade of decomposition reactions involving the formation of isodialuric acid, dialuric acid, 5-hydroxyhydantoin, and its hydroxyketone isomer. In biology, GC --> AT transitions are the most common mutations in the genome of aerobic organisms, including the lacI gene in bacteria, lacI transgenes in rodents, and the HPRT gene in rodents and humans, so a more complete understanding of cytosine oxidation is an essential research goal. The data and insights presented here shed new light on oxidation reactions of cytosine and 5-methylcytosine and should facilitate their validation in cellular DNA. PMID:20078112

  12. Expression patterns of AMP-deaminase and cytosolic 5?-nucleotidase genes in human term placenta

    Microsoft Academic Search

    Anna Roszkowska; Jerzy Klimek; Krystian Kaletha

    2008-01-01

    Background AMP-deaminase (EC 3.5.4.6) and 5?-nucleotidase (EC 3.1.3.5) are enzymes responsible for the maintenance of cellular adenine\\u000a nucleotides pool. Both exist in several isoforms that differ in kinetic properties and tissue distribution. Profile of isoforms\\u000a of these enzymes in human placenta has not been analyzed so far while this could be important for understanding of pathology\\u000a of placental ischemia such

  13. Cloning and expression of cDNA encoding heart-type isoform of AMP deaminase

    Microsoft Academic Search

    Xudong Wang; Hiroko Morisaki; Kannika Sermsuvitayawong; Ikuo Mineo; Keiko Toyama; Nobuaki Ogasawara; Tsunehiro Mukai; Takayuki Morisaki

    1997-01-01

    Mouse cDNA for the heart-type (H) isoform of AMP deaminase (EC 3.5.4.6., AMPD) has been isolated and characterized. The cDNA for the Ampd gene expressed in heart is predicted to encode a peptide of 766 amino acids with a molecular mass of 88 kDa. The coding region of this cDNA was quite homologous to the human AMPD3 gene which encodes

  14. Localization of N-Terminal Sequences in Human AMP Deaminase Isoforms That Influence Contractile Protein Binding

    Microsoft Academic Search

    Donna K. Mahnke-Zizelman; Richard L. Sabina

    2001-01-01

    The reversible association of AMP deaminase (AMPD, EC 3.5.4.6) with elements of the contractile apparatus is an identified mechanism of enzyme regulation in mammalian skeletal muscle. All three members of the human AMPD multigene family contain coding information for polypeptides with divergent N-terminal and conserved C-terminal domains. In this study, serial N-terminal deletion mutants of up to 111 (AMPD1), 214

  15. Structural studies of isolated native thick filaments from rabbit psoas muscle with AMP deaminase decoration.

    PubMed Central

    Koretz, J F

    1982-01-01

    AMP deaminase (adenylate deaminase; AMP aminohydrolase, EC 3.5.4.6), a large flat tetrameric enzyme found in skeletal muscle, binds strongly and specifically to the subfragment-2 region of rabbit skeletal muscle myosin. This allows its use as a structural probe in myosin and myosin rod aggregation studies. When mixed with a preparation of isolated native thick filaments, AMP deaminase decorates the entire filament backbone except for the central bare zone. Binding is particularly ordered in the banded region, where 11 stripes of about 43-nm spacing on either side of the bare zone have been observed in studies of isolated A-bands. No systematic absence of deaminase is seen here, indicating that the presence of the C-protein and H-protein bands does not make the binding site inaccessible to the tetramer. Optical diffraction patterns of the decorated filaments show the expected 42.9-nm periodicities and a reflection indexing at 28.6 nm. Because of the bulkiness of the tetramer relative to the number of available binding sites at each 14.3-nm interval along the filament shaft, the helix net is being sampled at a lower frequency than is the underlying myosin organization. As a result, reflections on layer lines other than orders of 42.9 nm are also observed (e.g., 57.2); these reflections strongly indicate a structure based on a 12/1 primitive helix. The results appear to eliminate the symmetric double two-fold and three-fold models of thick filament structure but are consistent with an asymmetric four-fold structure. Images PMID:6959110

  16. Evaluation of the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation

    Microsoft Academic Search

    A. Ebmeier; L. Allison; H. Cerutti; T. Clemente

    2004-01-01

    The initial step in the synthesis of isoleucine (Ile) is the conversion of threonine to a-ketobutyrate. This reaction is carried out by threonine deaminase (TD), which is feedback-regulated by Ile. Mutations in TD that manifest insensitivity to Ile feedback inhibition result in intracellular accumulation of Ile. Previous reports have shown that in planta expression of the wild-type Escherichia coli TD,

  17. The multifaceted roles of RNA binding in APOBEC cytidine deaminase functions.

    PubMed

    Prohaska, Kimberly M; Bennett, Ryan P; Salter, Jason D; Smith, Harold C

    2014-01-01

    Cytidine deaminases have important roles in the regulation of nucleoside/deoxynucleoside pools for DNA and RNA synthesis. The APOBEC family of cytidine deaminases (named after the first member of the family that was described, Apolipoprotein B mRNA Editing Catalytic Subunit 1, also known as APOBEC1 or A1) is a fascinating group of mutagenic proteins that use RNA and single-stranded DNA (ssDNA) as substrates for their cytidine or deoxycytidine deaminase activities. APOBEC proteins and base-modification nucleic acid editing have been the subject of numerous publications, reviews, and speculation. These proteins play diverse roles in host cell defense, protecting cells from invading genetic material, enabling the acquired immune response to antigens and changing protein expression at the level of the genetic code in mRNA or DNA. The amazing power these proteins have for interphase cell functions relies on structural and biochemical properties that are beginning to be understood. At the same time, the substrate selectivity of each member in the family and their regulation remains to be elucidated. This review of the APOBEC family will focus on an open question in regulation, namely what role the interactions of these proteins with RNA have in editing substrate recognition or allosteric regulation of DNA mutagenic and host-defense activities. PMID:24664896

  18. Bacteria with ACC deaminase can promote plant growth and help to feed the world.

    PubMed

    Glick, Bernard R

    2014-01-20

    To feed all of the world's people, it is necessary to sustainably increase agricultural productivity. One way to do this is through the increased use of plant growth-promoting bacteria; recently, scientists have developed a more profound understanding of the mechanisms employed by these bacteria to facilitate plant growth. Here, it is argued that the ability of plant growth-promoting bacteria that produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase to lower plant ethylene levels, often a result of various stresses, is a key component in the efficacious functioning of these bacteria. The optimal functioning of these bacteria includes the synergistic interaction between ACC deaminase and both plant and bacterial auxin, indole-3-acetic acid (IAA). These bacteria not only directly promote plant growth, they also protect plants against flooding, drought, salt, flower wilting, metals, organic contaminants, and both bacterial and fungal pathogens. While a considerable amount of both basic and applied work remains to be done before ACC deaminase-producing plant growth-promoting bacteria become a mainstay of plant agriculture, the evidence indicates that with the expected shift from chemicals to soil bacteria, the world is on the verge of a major paradigm shift in plant agriculture. PMID:24095256

  19. Mitochondrial assembly in yeast

    Microsoft Academic Search

    Les A Grivell; Marta Artal-Sanz; Gertjan Hakkaart; Liesbeth de Jong; Leo G. J Nijtmans; Katinka van Oosterum; Michel Siep; Hans van der Spek

    1999-01-01

    The yeast Saccharomyces cerevisiae is likely to be the first organism for which a complete inventory of mitochondrial proteins and their functions can be drawn up. A survey of the 340 or so proteins currently known to be localised in yeast mitochondria reveals the considerable investment required to maintain the organelle’s own genetic system, which itself contributes seven key components

  20. Alcoholic Fermentation in Yeast

    NSDL National Science Digital Library

    Ingrid Waldron

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

  1. Yeasts: Neglected Pathogens

    Microsoft Academic Search

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

    2009-01-01

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

  2. Strikingly different effects of hydrogen bonding on the photodynamics of individual nucleobases in DNA: comparison of guanine and cytosine.

    PubMed

    Zelený, Tomáš; Ruckenbauer, Matthias; Aquino, Adelia J A; Müller, Thomas; Lankaš, Filip; Dršata, Tomáš; Hase, William L; Nachtigallova, Dana; Lischka, Hans

    2012-08-22

    Ab initio surface hopping dynamics calculations were performed to study the photophysical behavior of cytosine and guanine embedded in DNA using a quantum mechanical/molecular mechanics (QM/MM) approach. It was found that the decay rates of photo excited cytosine and guanine were affected in a completely different way by the hydrogen bonding to the DNA environment. In case of cytosine, the geometrical restrictions exerted by the hydrogen bonds did not influence the relaxation time of cytosine significantly due to the generally small cytosine ring puckering required to access the crossing region between excited and ground state. On the contrary, the presence of hydrogen bonds significantly altered the photodynamics of guanine. The analysis of the dynamics indicates that the major contribution to the lifetime changes comes from the interstrand hydrogen bonds. These bonds considerably restricted the out-of-plane motions of the NH(2) group of guanine which are necessary for the ultrafast decay to the ground state. As a result, only a negligible amount of trajectories decayed into the ground state for guanine embedded in DNA within the simulation time of 0.5 ps, while for comparison, the isolated guanine relaxed to the ground state with a lifetime of about 0.22 ps. These examples show that, in addition to phenomena related to electronic interactions between nucleobases, there also exist relatively simple mechanisms in DNA by which the lifetime of a nucleobase is significantly enhanced as compared to the gas phase. PMID:22845192

  3. Genome-Wide Variation of Cytosine Modifications Between European and African Populations and the Implications for Complex Traits

    PubMed Central

    Moen, Erika L.; Zhang, Xu; Mu, Wenbo; Delaney, Shannon M.; Wing, Claudia; McQuade, Jennifer; Myers, Jamie; Godley, Lucy A.

    2013-01-01

    Elucidating cytosine modification differences between human populations can enhance our understanding of ethnic specificity in complex traits. In this study, cytosine modification levels in 133 HapMap lymphoblastoid cell lines derived from individuals of European or African ancestry were profiled using the Illumina HumanMethylation450 BeadChip. Approximately 13% of the analyzed CpG sites showed differential modification between the two populations at a false discovery rate of 1%. The CpG sites with greater modification levels in European descent were enriched in the proximal regulatory regions, while those greater in African descent were biased toward gene bodies. More than half of the detected population-specific cytosine modifications could be explained primarily by local genetic variation. In addition, a substantial proportion of local modification quantitative trait loci exhibited population-specific effects, suggesting that genetic epistasis and/or genotype × environment interactions could be common. Distinct correlations were observed between gene expression levels and cytosine modifications in proximal regions and gene bodies, suggesting epigenetic regulation of interindividual expression variation. Furthermore, quantitative trait loci associated with population-specific modifications can be colocalized with expression quantitative trait loci and single nucleotide polymorphisms previously identified for complex traits with known racial disparities. Our findings revealed abundant population-specific cytosine modifications and the underlying genetic basis, as well as the relatively independent contribution of genetic and epigenetic variations to population differences in gene expression. PMID:23792949

  4. Determination of Serum Adenosine Deaminase and Xanthine Oxidase Levels in Patients with Crimean–Congo Hemorrhagic Fever

    PubMed Central

    Celik, V. Kenan; Sari, Ismail; Engin, Aynur; Yildiz, Gürsel; Aydin, Hüseyin; Bakir, Sevtap

    2010-01-01

    OBJECTIVE: Crimean–Congo hemorrhagic fever is an acute viral hemorrhagic fever with a high mortality rate. Despite increasing knowledge about hemorrhagic fever viruses, little is known about the pathogenesis of Crimean–Congo hemorrhagic fever. In this study, we measured serum adenosine deaminase and xanthine oxidase levels in Crimean–Congo hemorrhagic fever patients. METHODS: Serum adenosine deaminase levels were measured with a sensitive colorimetric method described by Giusti and xanthine oxidase levels by the method of Worthington in 30 consecutive hospitalized patients (mean age 42.6 ± 21.0). Laboratory tests confirmed their diagnoses of Crimean–Congo hemorrhagic fever. Thirty-five subjects (mean age 42.9 ± 19.1) served as the control group. RESULTS: There was a significant difference in adenosine deaminase and xanthine oxidase levels between cases and controls (p<0.05). However, neither adenosine deaminase nor xanthine oxidase levels varied with the severity of disease in the cases assessed (p>0.05). CONCLUSION: Adenosine deaminase and xanthine oxidase levels were increased in patients with Crimean–Congo hemorrhagic fever. Elevated serum xanthine oxidase activity in patients with Crimean–Congo hemorrhagic fever may be associated with reactive oxygen species generated by the xanthine/xanthine oxidase system during inflammatory responses. In addition, elevated lipid peroxidation may contribute to cell damage and hemorrhage. The association of cell damage and hemorrhage with xanthine oxidase activity should be further investigated in large-scale studies. PMID:20668627

  5. Yeast transcription factors Kevin Struhl

    E-print Network

    Yeast transcription factors Kevin Struhl Harvard Medical School, Boston, USA Studies of yeast Transcriptional regulatory mechanisms are fundamentally similar in eukaryotic organisms from yeasts to humans (for reviews of yeast transcription, see [1,2]). Compo- nents of the chromatin template and the basic RNA

  6. Yeast ecology of Kombucha fermentation

    Microsoft Academic Search

    Ai Leng Teoh; Gillian Heard; Julian Cox

    2004-01-01

    Kombucha is a traditional fermentation of sweetened tea, involving a symbiosis of yeast species and acetic acid bacteria. Despite reports of different yeast species being associated with the fermentation, little is known of the quantitative ecology of yeasts in Kombucha. Using oxytetracycline-supplemented malt extract agar, yeasts were isolated from four commercially available Kombucha products and identified using conventional biochemical and

  7. 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

  8. Photo protection of RNA building blocks: Adenosine 5?-monophosphate, cytidine 5?-monophosphate and cytosine

    NASA Astrophysics Data System (ADS)

    Nielsen, Jakob Brun; Thøgersen, Jan; Jensen, Svend Knak; Keiding, Søren Rud

    2013-04-01

    Photoprotection of the RNA nucleotides adenosine 5'-monophosphate and cytidine 5'-monophosphate, and the nucleobase cytosine was studied using UV pump, IR probe femtosecond transient absorption spectroscopy. The excitation energy is contained in the aromatic ring system, protecting the RNA backbone. All three molecules dissipate the excitation energy by internal conversion and subsequent vibrational relaxation to the electronic ground state in less than 10 ps. In addition, a second deactivation channel is found in cytidine 5'-monophosphate, illustrated by a signal at 1563 cm-1 with a lifetime of 33 ps assigned to an n?? state in agreement with observations in the UV region.

  9. Methylation of DNA in early development: 5-methyl cytosine content of DNA in sea urchin sperm and embryos

    PubMed Central

    Pollock, James M.; Swihart, Marcia; Taylor, J.Herbert

    1978-01-01

    By separating formic acid hydrolysates with high pressure chromatography on an Aminex-10 column, we determined the ratio of 5-methyl cytosine to cytosine and other bases of DNA from sea urchin sperm and nuclei of embryos from early cleavage through pluteus stages. Contrary to several previous reports, we could not find any measurable changes in the methylation levels of embryonic nuclear DNAs at different stages of development. We also found no consistent differences between the methylation levels of sea urchin sperm and embryonic nuclei or the 5-methyl cytosine content of fish (Mugilcephalus) sperm and liver nuclei. While these measurements would not have detected subtle variations associated with differentiation, they would have indicated the gross changes previously reported for embryos or between sperm and somatic nuclei had those changes been present. PMID:745996

  10. Dynamic modes of the flipped-out cytosine during HhaI methyltransferase-DNA interactions in solution.

    PubMed Central

    Klimasauskas, S; Szyperski, T; Serva, S; Wüthrich, K

    1998-01-01

    Flipping of a nucleotide out of a B-DNA helix into the active site of an enzyme has been observed for the HhaI and HaeIII cytosine-5 methyltransferases (M.HhaI and M.HaeIII) and for numerous DNA repair enzymes. Here we studied the base flipping motions in the binary M. HhaI-DNA and the ternary M.HhaI-DNA-cofactor systems in solution. Two 5-fluorocytosines were introduced into the DNA in the places of the target cytosine and, as an internal control, a cytosine positioned two nucleotides upstream of the recognition sequence 5'-GCGC-3'. The 19F NMR spectra combined with gel mobility data show that interaction with the enzyme induces partition of the target base among three states, i.e. stacked in the B-DNA, an ensemble of flipped-out forms and the flipped-out form locked in the enzyme active site. Addition of the cofactor analogue S-adenosyl-L-homocysteine greatly enhances the trapping of the target cytosine in the catalytic site. Distinct dynamic modes of the target cytosine have thus been identified along the reaction pathway, which includes novel base-flipping intermediates that were not observed in previous X-ray structures. The new data indicate that flipping of the target base out of the DNA helix is not dependent on binding of the cytosine in the catalytic pocket of M.HhaI, and suggest an active role of the enzyme in the opening of the DNA duplex. PMID:9427765

  11. Yeast expression platforms

    Microsoft Academic Search

    Erik Böer; Gerhard Steinborn; Gotthard Kunze; Gerd Gellissen

    2007-01-01

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

  12. 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.

  13. 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.

  14. 3-Ureidoacrylonitriles: Novel products from the photoisomerization of cytosine, 5-methylcytosine, and related compounds

    SciTech Connect

    Shaw, A.A.; Shetlar, M.D. (Univ. of California, San Francisco (USA))

    1990-10-10

    During studies of the effects of far-ultraviolet light on DNA and its components, the authors have discovered that cytosine and 5-methylcytosine, as well as their nucleosides and N1-methyl derivatives, undergo photoisomerization reactions to yield the corresponding cis- and trans-3-ureidoacrylonitriles. For example, N1-methylcytosine reacts to give cis- and trans-3-(N3-methylureido)acrylonitrile. These products have been characterized through use of one- and two-dimensional high-resolution {sup 1}H and {sup 13}C NMR spectroscopy, ultraviolet and infrared spectroscopy, electron impact and liquid secondary ion mass spectrometry, and, in some cases, synthesis by an alternate route. Detailed {sup 13}C NMR data for the parent compounds are presented for comparison purposes. These photoisomerization reactions take place cleanly in acetonitrile; the corresponding reactions occur in aqueous solutions as well. For cytosine and 5-methylcytosine the photoreaction in acetonitrile is slow; however, the rate of reaction is greatly enhanced by N1 substitution.

  15. The experimental and theoretical gas phase acidities of adenine, guanine, cytosine, uracil, thymine and halouracils

    NASA Astrophysics Data System (ADS)

    Chen, Edward C. M.; Herder, Charles; Chen, Edward S.

    2006-10-01

    The gas phase acidities GPA (? H (298) for deprotonation) of the most stable tautomers of adenine, guanine, cytosine, uracil and thymine are evaluated. New GPA are obtained from electron impact spectra and acid dissociation constants measured in dimethylsulfoxide for A, U and 5-FU. The average experimental GPA are: [N1 sbnd H] C 340(2); T 333(2); U 333(2); 5-FU 329(4); [N9 sbnd H] A 333(1); G 332(4); all in kcal/mol. Only cytosine is a weaker acid than HCl in the gas phase. The most acidic hydrogens in the nucleotides are replaced by the sugar in DNA and RNA. The experimental N3 sbnd H GPA are G 334(4); U 347(2), T 347(4), while the predicted N3 sbnd H 5-FU GPA is 343 kcal/mol. The NH sbnd H GPA are: C 346(4); A 352(2); G 336(4) (all in kcal/mol). These are supported by semi-empirical multiconfiguration configuration interaction calculations. The predicted C8 sbnd H acidities of G and A and the C6 sbnd H of T are about the same, 360(2) kcal/mol. The remaining CH acidities are 370-380 kcal/mol. The 5-halouracils are predicted to be more acidic than HCl.

  16. Cytosine methylation does not affect binding of transcription factor Sp1

    SciTech Connect

    Harrington, M.A.; Jones, P.A. (Univ. of Southern California School of Medicine, Los Angeles (USA)); Imagawa, M.; Karin, M. (Univ. of California, San Diego, La Jolla (USA))

    1988-04-01

    DNA methylation may be a component of a multilevel control mechanism that regulates eukaryotic gene expression. The authors used synthetic oligonucleotides to investigate the effect of cytosine methylation on the binding of the transcription factor Sp1 to its target sequence (a G+C-rich sequence known as a GC box). Concatemers of double-stranded 14-mers containing a GC box successfully competed with the human metallothionein IIA promoter for binding to Sp1 in DNase I protection experiments. The presence of 5-methylcytosine in the CpG sequence of the GC box did not influence Sp1 binding. The result was confirmed using double-stranded 20-mers containing 16 base pairs of complementary sequence. Electrophoretic gel retardation analysis of annealed 28-mers containing a GC box incubated with an Sp1-containing HeLa cell nuclear extract demonstrated the formation of DNA-protein complexes; formation of these complexes was not inhibited when an oligomer without a GC box was used as a competitor. Once again, the presence of a 5-methylcytosine residue in the GC box did not influence the binding of the protein to DNA. The results therefore preclude a direct effect of cytosine methylation on Sp1-DNA interactions.

  17. Insulin Mimetic Peroxo Complexes of Vanadium Containing Uracil or Cytosine as Ligand

    PubMed Central

    Mandal, Shipra

    2000-01-01

    Mixed ligand oxo peroxo complexes of vanadium (V), M[VO(O2)L2].nH2O where M = K or NH4, HL = uracil or cytosine and n = 1 or 2, have been isolated from aqueous methanolic medium. The complexes were characterised by elemental analysis, conductance, TGA, UV-Visible, IR and NMR spectral studies. Both the peroxide and the other ligands acts as chelates coordinating through their oxygen at C (2) and nitrogen at N (3) and the presence of monomeric oxoperoxovanadium (V) species, have been established by IR, 1H and 51V NMR studies. The complexes appeared to possess pentagonal bipyramidal geometry. The terminal oxo ligand and the oxygen of the uracil or cytosine at C (2) occupy the axial position while the peroxide and the other donor ligands are in the equatorial position. The UV spectral studies confirm the presence of Vanadium in its +5 oxidation state. The administration of the potassium salts of the complexes reduces the blood glucose level in Swiss Albino mice compared to that of KVO3. The complexes also readily oxidise cysteine to Cystine in aqueous solution. The possible mechanism for the insulin-mimic activity of the complexes is discussed. PMID:18475940

  18. Inflammation-Mediated Cytosine Damage: A Mechanistic Link between Inflammation and the Epigenetic Alterations in Human Cancers

    Microsoft Academic Search

    Victoria Valinluck; Lawrence C. Sowers

    Aberrant methylation patterns have long been known to exist in the promoter regions of key regulatory genes in the DNA of tumor cells. However, the mechanisms by which these methylation patterns become altered during the transforma- tion of normal cells to tumor cells have remained elusive. We have recently shown in in vitro studies that inflammation- mediated halogenated cytosine damage

  19. REMPI spectroscopy of cytosine E. Nir, M. Muuller, L.I. Grace, M.S. de Vries *

    E-print Network

    de Vries, Mattanjah S.

    lasers, as well as from an excimer laser, used for two-photon ionization and for spectral hole burning hole burning spectroscopy to distinguish and identify different tautomers and different cluster bases, cytosine exhibits vibronic spectra with sharp features in two spectral regions, separated

  20. The mid-IR absorption spectrum of gas-phase clusters of the nucleobases guanine and cytosine

    E-print Network

    de Vries, Mattanjah S.

    The mid-IR absorption spectrum of gas-phase clusters of the nucleobases guanine and cytosine Joost pairs that cross-link the two strands, (ii) the interaction between the nucleobases on one strand the factors that play a role, which can be achieved by studying isolated molecules in the gas phase

  1. The role of cytidine deaminases on innate immune responses against human viral infections.

    PubMed

    Vieira, Valdimara C; Soares, Marcelo A

    2013-01-01

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

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

    PubMed

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

    2014-06-01

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

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

    PubMed Central

    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

    2012-01-01

    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

  4. 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.

  5. An RNA Editor, Adenosine Deaminase Acting on Double-Stranded RNA (ADAR1)

    PubMed Central

    George, Cyril X.; John, Lijo

    2014-01-01

    Adenosine deaminase acting on RNA1 (ADAR1) catalyzes the C6 deamination of adenosine (A) to produce inosine (I) in regions of RNA with double-stranded (ds) character. This process is known as A-to-I RNA editing. Alternative promoters drive the expression of the Adar1 gene and alternative splicing gives rise to transcripts that encode 2 ADAR1 protein size isoforms. ADAR1 p150 is an interferon (IFN)-inducible dsRNA adenosine deaminase found in the cytoplasm and nucleus, whereas ADAR1 p110 is constitutively expressed and nuclear in localization. Dependent on the duplex structure of the dsRNA substrate, deamination of adenosine by ADAR can be either highly site-selective or nonspecific. A-to-I editing can alter the stability of RNA structures and the coding of RNA as I is read as G instead of A by ribosomes during mRNA translation and by polymerases during RNA replication. A-to-I editing is of broad physiologic significance. Both the production and the action of IFNs, and hence the subsequent interaction of viruses with their hosts, are among the processes affected by A-to-I editing. PMID:24905200

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

    PubMed Central

    Vieira, Valdimara C.; Soares, Marcelo A.

    2013-01-01

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

  7. Rhizobacteria containing ACC-deaminase confer salt tolerance in maize grown on salt-affected fields.

    PubMed

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

    2009-11-01

    Salt stress is one of the major constraints hampering agricultural production owing to its impact on ethylene production and nutritional imbalance. A check on the accelerated ethylene production in plants could be helpful in minimizing the negative effect of salt stress on plant growth and development. Four Pseudomonas, 1 Flavobacterium, and 1 Enterobacter strain of plant growth promoting rhizobacteria containing 1-aminocyclopropane-1-carboxylate (ACC)-deaminase were selected and their effects on growth and yield of maize were investigated to improve the salt tolerance of maize grown on salt-affected fields. The selected rhizobacterial isolates reduced or eliminated the classical "triple" response, indicating their ability to reduce stress-induced ethylene levels. Results showed that rhizobacterial strains, particularly Pseudomonas and Enterobacter spp., significantly promoted the growth and yield of maize compared with the non-inoculated control. Pseudomonas fluorescens increased plant height, biomass, cob yield, grain yield, 1000 grain mass, and straw yield of maize up to 29%, 127%, 67%, 60%, 17%, and 166%, respectively, over the control. Under stress conditions, more N, P, and K uptake and high K+-Na+ ratios were recorded in inoculated plants compared with the control. The results imply that inoculation with plant growth promoting rhizobacteria containing ACC-deaminase could be a useful approach for improving growth and yield of maize under salt-stressed conditions. PMID:19940939

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

    PubMed Central

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

    2015-01-01

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

  9. 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...

  10. 1-Aminocyclopropane-1-carboxylate (ACC) deaminases from Methylobacterium radiotolerans and Methylobacterium nodulans with higher specificity for ACC.

    PubMed

    Fedorov, Dmitry N; Ekimova, Galina A; Doronina, Nina V; Trotsenko, Yuri A

    2013-06-01

    The 1-aminocyclopropane-1-carboxylate (ACC) deaminases (EC 3.4.99.7), the key enzymes of degradation of the precursor of the phytohormone ethylene, have not been well studied despite their great importance for plant-bacterial interactions. Using blast, the open reading frames encoding ACC deaminases were found in the genomes of epiphytic methylotroph Methylobacterium radiotolerans JCM2831 and nodule-forming endosymbiont Methylobacterium nodulans ORS2060. These genes were named acdS and cloned; recombinant proteins were expressed and purified from Escherichia coli. The enzyme from M. nodulans displayed the highest substrate specificity among all of the characterized ACC deaminases (Km 0.80 ± 0.04 mM), whereas the enzyme from M. radiotolerans had Km 1.8 ± 0.3 mM. The kcat values were 111.8 ± 0.2 and 65.8 ± 2.8 min(-1) for the enzymes of M. nodulans and M. radiotolerans, respectively. Both enzymes are homotetramers with a molecular mass of 144 kDa, as was demonstrated by size exclusion chromatography and native PAGE. The purified enzymes displayed the maximum activity at 45-50 °C and pH 8.0. Thus, the priority data have been obtained, extending the knowledge of biochemical properties of bacterial ACC deaminases. PMID:23517598

  11. Identification of the Most Common Mutation within the Porphobilinogen Deaminase Gene in Swedish Patients with Acute Intermittent Porphyria

    Microsoft Academic Search

    Jin-Sung Lee; Maria Anvret

    1991-01-01

    Acute intermittent porphyria (AIP) is a metabolic disorder characterized by a partial deficiency of the porphobilinogen deaminase (PBGD, EC 4.3.1.8) activity. Previous haplotype analysis combined with genealogical data suggested a common origin of the PBGD gene mutation in the AIP families originating from northern Sweden (Lappland), where the highest prevalence of the disease (1 in 1500) is observed. An AIP

  12. High prevalence of a point mutation in the porphobilinogen deaminase gene in Dutch patients with acute intermittent porphyria

    Microsoft Academic Search

    Xue-Fan Gu; Felix de Rooij; J. S. Lee; Kor Te Velde; Jean Charles Deybach; Yves Nordmann; Bernard Grandchamp

    1993-01-01

    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

  13. Virtual Yeast Cell

    NSDL National Science Digital Library

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

  14. 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

  15. 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 ?-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

  16. 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.

  17. Yeast Metabolism Lab Mrs. Zimmerman

    E-print Network

    Rose, Michael R.

    Yeast Metabolism Lab Mrs. Zimmerman 10/22/10 #12;Photosynthesis 6 CO2 + 6 H2O C6H12O6 + 6 O2 Oxygen Glucose Carbon Dioxide Water Energy #12;Yeast · Unicellular · Eukaryotic (like us) · Kingdom Fungi" Saccharomyces cerevisiae #12;Alcoholic Fermentation · Some organisms, including yeast, can create energy without

  18. Genetics of Yeasts

    NASA Astrophysics Data System (ADS)

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

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

  19. Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer12

    PubMed Central

    Wielscher, Matthias; Liou, Willy; Pulverer, Walter; Singer, Christian F; Rappaport-Fuerhauser, Christine; Kandioler, Daniela; Egger, Gerda; Weinhäusel, Andreas

    2013-01-01

    Change of DNA cytosine methylation (5mC) is an early event in the development of cancer, and the recent discovery of a 5-hydroxymethylated form (5hmC) of cytosine suggests a regulatory epigenetic role that might be different from 5-methylcytosine. Here, we aimed at elucidating the role of 5hmC in breast cancer. To interrogate the 5hmC levels of the leucine zipper, putative tumor suppressor 1 (LZTS1) gene in detail, we analyzed 75 primary breast cancer tissue samples from initial diagnosis and 12 normal breast tissue samples derived from healthy persons. Samples were subjected to 5hmC glucosyltransferase treatment followed by restriction digestion and segment-specific amplification of 11 polymerase chain reaction products. Nine of the 11 5?LZTS1 fragments showed significantly lower (fold change of 1.61–6.01, P < .05) 5hmC content in primary breast cancer tissue compared to normal breast tissue samples. No significant differences were observed for 5mC DNA methylation. Furthermore, both LZTS1 and TET1 mRNA expressions were significantly reduced in tumor samples (n = 75, P < .001, Student's t test), which correlated significantly with 5hmC levels in samples. 5hmC levels in breast cancer tissues were associated with unfavorable histopathologic parameters such as lymph node involvement (P < .05, Student's t test). A decrease of 5hmC levels of LZTS1, a classic tumor suppressor gene known to influence metastasis in breast cancer progression, is correlated to down-regulation of LZTS1 mRNA expression in breast cancer and might epigenetically enhance carcinogenesis. The study provides support for the novel hypothesis that suggests a strong influence of 5hmC on mRNA expression. Finally, one may also consider 5hmC as a new biomarker. PMID:24466374

  20. Genome evolution in yeasts

    Microsoft Academic Search

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

    2004-01-01

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

  1. METHODS TO IDENTIFY YEAST

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yeasts are commonly identified from either phenotype or, more recently, from diagnostic gene sequences. Methods based on phenotype include fermentation reactions on a select set of sugars and growth responses on various carbon and nitrogen sources or on other diagnostic compounds. Isolates are fur...

  2. 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

  3. Role of ascites adenosine deaminase in differentiating between tuberculous peritonitis and peritoneal carcinomatosis

    PubMed Central

    Kang, Seung Joo; Kim, Ji Won; Baek, Jee Hyun; Kim, Se Hyung; Kim, Byeong Gwan; Lee, Kook Lae; Jeong, Ji Bong; Jung, Yong Jin; Kim, Joo Sung; Jung, Hyun Chae; Song, In Sung

    2012-01-01

    AIM: To investigate the usefulness of tumor markers and adenosine deaminase in differentiating between tuberculous peritonitis (TBP) and peritoneal carcinomatosis (PC). METHODS: A retrospective analysis of data was performed on consecutive patients who underwent peritoneoscopic and abdominal computed tomography (CT) evaluations. Among 75 patients at the Seoul National University Hospital from January 2000 to June 2010 who underwent both tests, 27 patients (36.0%) and 25 patients (33.3%) were diagnosed with TBP and PC, respectively. Diagnosis was confirmed by peritoneoscopic biopsy. RESULTS: Serum c-reactive protein (7.88 ± 6.62 mg/dL vs 3.12 ± 2.69 mg/dL, P = 0.01), ascites adenosine deaminase (66.76 ± 32.09 IU/L vs 13.89 ± 8.95 IU/L, P < 0.01), ascites lymphocyte proportion (67.77 ± 23.41% vs 48.36 ± 18.78%, P < 0.01), and serum-ascites albumin gradient (0.72 ± 0.49 g/dL vs 1.05 ± 0.50 g/dL, P = 0.03) were significantly different between the two groups. Among tumor markers, serum and ascites carcinoembryonic antigen, serum carbohydrate antigen 19-9 showed significant difference between two groups. Abdominal CT examinations showed that smooth involvement of the parietal peritoneum was more common in the TBP group (77.8% vs 40.7%) whereas nodular involvement was more common in the PC group (14.8% vs 40.7%, P = 0.04). From receiver operating characteristic (ROC) curves ascites adenosines deaminase (ADA) showed better discriminative capability than tumor markers. An ADA cut-off level of 21 IU/L was found to yield the best results of differential diagnosis; sensitivity, specificity, positive predictive value, and negative predictive value were 92.0%, 85.0%, 88.5% and 89.5%, respectively. CONCLUSION: Besides clinical and radiologic findings, ascitic fluid ADA measurement is helpful in the differential diagnosis of TBP and PC. PMID:22719194

  4. Flavour-active wine yeasts.

    PubMed

    Cordente, Antonio G; Curtin, Christopher D; Varela, Cristian; Pretorius, Isak S

    2012-11-01

    The flavour of fermented beverages such as beer, cider, saké and wine owe much to the primary fermentation yeast used in their production, Saccharomyces cerevisiae. Where once the role of yeast in fermented beverage flavour was thought to be limited to a small number of volatile esters and higher alcohols, the discovery that wine yeast release highly potent sulfur compounds from non-volatile precursors found in grapes has driven researchers to look more closely at how choice of yeast can influence wine style. This review explores recent progress towards understanding the range of 'flavour phenotypes' that wine yeast exhibit, and how this knowledge has been used to develop novel flavour-active yeasts. In addition, emerging opportunities to augment these phenotypes by engineering yeast to produce so-called grape varietal compounds, such as monoterpenoids, will be discussed. PMID:22940803

  5. Yeasts associated with Manteca.

    PubMed

    Suzzi, Giovanna; Schirone, Maria; Martuscelli, Maria; Gatti, Monica; Fornasari, Maria Emanuela; Neviani, Erasmo

    2003-04-01

    Manteca is a traditional milk product of southern Italy produced from whey deriving from Caciocavallo Podolico cheese-making. This study was undertaken to obtain more information about the microbiological properties of this product and particularly about the presence, metabolic activities, and technological significance of the different yeast species naturally occurring in Manteca. High numbers of yeasts were counted after 7 days ripening (10(4)-10(5) cfu g(-1)) and then decreased to 10(2) at the end. A total of 179 isolates were identified and studied for their phenotypic and genotypic characteristics. The most frequently encountered species were Trichosporon asahii (45), Candida parapsilosis (33), Rhodotorula mucilaginosa (32), Candida inconspicua (29). Some of these yeasts showed lipolytic activity (32 strains) and proteolytic activity (29 strains), NaCl resistance up to 10% and growth up to 45 degrees C (42 strains). Biogenic amines were formed by proteolytic strains, in particular phenylethylamine, putrescine and spermidine. Spermidine was produced by all the yeasts tested in this work, but only Trichosporon produced a great quantity of this compound. Histamine was not detectable. Caseinolytic activity was common to almost all strains, corresponding to the ability to efficiently split off amino-terminal amino acids. The highest and most constant activity expressed by all species was X-prolyl-dipeptidyl aminopeptidase. The findings suggest that the presence of yeasts may play a significant role in justifying interactions with lactic acid bacteria, and consequently with their metabolic activity in the definition of the peculiar characteristics of Manteca cheese. PMID:12702448

  6. Engineered ACC deaminase-expressing free-living cells of Mesorhizobium loti show increased nodulation efficiency and competitiveness on Lotus spp.

    PubMed

    Conforte, Valeria P; Echeverria, Mariela; Sánchez, Cintia; Ugalde, Rodolfo A; Menéndez, Ana B; Lepek, Viviana C

    2010-08-01

    Ethylene inhibits the establishment of symbiosis between rhizobia and legumes. Several rhizobia species express the enzyme ACC deaminase, which degrades the ethylene precursor 1-cyclopropane-1-carboxilate (ACC), leading to reductions in the amount of ethylene evolved by the plant. M. loti has a gene encoding ACC deaminase, but this gene is under the activity of the NifA-RpoN-dependent promoter; thus, it is only expressed inside the nodule. The M. loti structural gene ACC deaminase (acdS) was integrated into the M. loti chromosome under a constitutive promoter activity. The resulting strain induced the formation of a higher number of nodules and was more competitive than the wild-type strain on Lotus japonicus and L. tenuis. These results suggest that the introduction of the ACC deaminase activity within M. loti in a constitutive way could be a novel strategy to increase nodulation competitiveness of the bacteria, which could be useful for the forage inoculants industry. PMID:20953097

  7. 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

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

    Microsoft Academic Search

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

    2000-01-01

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

  9. [Adenosine deaminase 1 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 2months 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

  10. 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

  11. Catalytic pocket inaccessibility of activation-induced cytidine deaminase is a safeguard against excessive mutagenic activity.

    PubMed

    King, Justin J; Manuel, Courtney A; Barrett, Crystal V; Raber, Susanne; Lucas, Heather; Sutter, Patricia; Larijani, Mani

    2015-04-01

    Activation-induced cytidine deaminase (AID) mutates cytidine to uridine at immunoglobulin loci to initiate secondary antibody diversification but also causes genome-wide damage. We previously demonstrated that AID has a relatively low catalytic rate. The structure of AID has not been solved. Thus, to probe the basis for its catalytic lethargy we generated a panel of free or DNA-bound AID models based on eight recently resolved APOBEC structures. Docking revealed that the majority of AID:DNA complexes would be inactive due to substrate binding such that a cytidine is not positioned for deamination. Furthermore, we found that most AID conformations exhibit fully or partially occluded catalytic pockets. We constructed mutant and chimeric AID variants predicted to have altered catalytic pocket accessibility dynamics and observed significant correlation with catalytic rate. Data from modeling simulations and functional tests of AID variants support the notion that catalytic pocket accessibility is an inherent bottleneck for AID activity. PMID:25728927

  12. 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

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

    PubMed Central

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

    2013-01-01

    The absolute cross sections (CS) 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 3A?(???*) and 2 3A?(???*) valence states of the molecule. Their energy dependent CS exhibit essentially a common maximum at about 6 eV with a value of 1.84 × 10?17 cm2 for the former and 4.94 × 10?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 1A?(???*) valence state, shows only a steep rise to about 1.04 × 10?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 3, 1A?(???*), 4 1A?(???*), 5 1A?(???*), and 6 1A?(???*) valence states, respectively. The CS for the 3 3, 1A? and 4 1A? 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 1.79 × 10?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 3.8 × 10?18 cm2 near its excitation threshold is attributed to transitions from the ground state to the 1 3, 1A?(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 8.1 × 10?16 cm2 at the incident energy of 13 eV. PMID:20969430

  14. 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.

  15. Strand displacement recognition of mixed adenine–cytosine sequences in double stranded DNA by thymine–Guanine PNA (Peptide nucleic acid)

    Microsoft Academic Search

    Peter E Nielsen; Michael Egholm

    2001-01-01

    Mixed pyrimidine–purine peptide nucleic acids (PNAs) composed of thymines and guanines are shown to form a PNA2–DNA triplex with Watson–Crick complementary adenine–cytosine oligonucleotides and to bind complementary adenine–cytosine targets in double stranded DNA by helix invasion. These results for the first time demonstrate binding of an unmodified PNA oligomer to a mixed pyrimidine–purine target in double stranded DNA and illustrate

  16. 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).

  17. Serum glucose and insulin response in rats administered with sucrose or starch containing adenosine, inosine or cytosine.

    PubMed

    Fukumori, Y; Maeda, N; Takeda, H; Onodera, S; Shiomi, N

    2000-02-01

    Blood glucose and insulin responses and gastric emptying were examined in rats intubated with sucrose or soluble starch that contained adenosine, inosine and cytosine. The increase in serum glucose and insulin levels in the rats following loading with sucrose (2.5 g/kg of body weight) or soluble starch (1.875 g/kg of body weight) was significantly reduced by the administration of adenosine, inosine and cytosine (0.0625-0.125 g/kg of body weight). The gastric emptying rates were only marginally affected by the nucleoside administration. The activities of sucrase, maltase, isomaltase and glucoamylase in a crude preparation from the small intestinal mucosa of rats were mildly inhibited by the nucleosides. The decrease in blood glucose and insulin levels may have been in response to a decrease in glucose absorption caused by the inhibiting effect of the nucleosides on the mucosal enzymes that digest sucrose, maltose, and malto- and isomalto-oligosaccharides. PMID:10737175

  18. Potential derived point charge model study of electrostatic interaction energies in some complexes of water with uracil, thymine, and cytosine.

    PubMed

    Ray, N K; Bolis, G; Shibata, M; Rein, R

    1984-01-01

    Potential derived (PD) point charges and segmental multipole moments are calculated for water, uracil, thymine, and cytosine using STO-3G quality wave functions. The PD point charges are used to estimate the electrostatic interaction energies for a series of complexes of water with these nucleic acid bases. It is shown here that the results obtained using simple PD charge model is very similar to those obtained from more elaborate segmental multipole moment analysis. PMID:11540815

  19. The Drosophila Cytosine5 Methyltransferase Dnmt2 Is Associated with the Nuclear Matrix and Can Access DNA during Mitosis

    Microsoft Academic Search

    Matthias Schaefer; Julia P. Steringer; Frank Lyko; Neil Hotchin

    2008-01-01

    Cytosine-5 methyltransferases of the Dnmt2 family are highly conserved in evolution and their biological function is being studied in several organisms. Although all structural DNA methyltransferase motifs are present in Dnmt2, these enzymes show a strong tRNA methyltransferase activity. In line with an enzymatic activity towards substrates other than DNA, Dnmt2 has been described to localize to the cytoplasm. Using

  20. Thermodynamic Potential for the Abiotic Synthesis of Adenine, Cytosine, Guanine, Thymine, Uracil, Ribose, and Deoxyribose in Hydrothermal Systems

    Microsoft Academic Search

    Douglas E. LaRowe; Pierre Regnier

    2008-01-01

    The thermodynamic potential for the abiotic synthesis of the five common nucleobases (adenine, cytosine, guanine, thymine,\\u000a and uracil) and two monosaccharides (ribose and deoxyribose) from formaldehyde and hydrogen cyanide has been quantified under\\u000a temperature, pressure, and bulk composition conditions that are representative of hydrothermal systems. The activities of\\u000a the precursor molecules (formaldehyde and hydrogen cyanide) required to evaluate the thermodynamics

  1. ''Is Yeast Alive?''

    NSDL National Science Digital Library

    Ms. Katrenia Hosea-Flanigan (Frank Cody High School)

    2006-04-01

    In this inquiry activity students explore the characteristics of living organisms to determine whether yeast meets the criteria of a living thing. This inquiry activity was developed by a K-12 science teacher in the American Physiological SocietyÂ?s 2006 Frontiers in Physiology Program. The NSES Standards addressed by this activity are current as of the year of development. For more information on the Frontiers in Physiology Program, please visit www.frontiersinphys.org.

  2. Emerging opportunistic yeast infections.

    PubMed

    Miceli, Marisa H; Díaz, José A; Lee, Samuel A

    2011-02-01

    A growing population of immunosuppressed patients has resulted in increasingly frequent diagnoses of invasive fungal infections, including those caused by unusual yeasts. The incidence of non-albicans species of Candida is increasing compared with that of Candida albicans, and several species, such as Candida glabrata and Candida krusei, may be resistant to azole antifungal therapy. Trichosporon species are the second most common cause of fungaemia in patients with haematological malignant disease and are characterised by resistance to amphotericin and echinocandins and poor prognosis. Rhodotorula species belong to the family Cryptococcaceae, and are a cause of catheter-related fungaemia, sepsis, and invasive disease in severely immunosuppressed patients. An increasing number of sporadic cases of invasive fungal infections by non-neoformans cryptococci have been reported in immunocompromised hosts, especially for patients with advanced HIV infection or cancer who are undergoing transplant. Other uncommon yeasts that can cause invasive disease in severely immunosuppressed patients include Geotrichum, Hansenula, Malassezia, and Saccharomyces. Host immune status is a crucial determinant of the type of invasive fungal infection a patient is at risk for. Diagnosis can be challenging and relies heavily on traditional cultures of blood and other sterile sites, although serum (1,3)-?-D-glucan testing might have an adjunctive role. Although rare yeasts are emerging as opportunistic human pathogens, diagnosis remains challenging and treatment suboptimal. PMID:21272794

  3. Sequence-selective binding of phenazinium dyes phenosafranin and safranin O to guanine-cytosine deoxyribopolynucleotides: spectroscopic and thermodynamic studies.

    PubMed

    Saha, Ishita; Hossain, Maidul; Suresh Kumar, Gopinatha

    2010-11-25

    The sequence selectivity of the DNA binding of the phenazinium dyes phenosafranin and safranin O have been investigated with four sequence-specific deoxyribopolynucleotides from spectroscopic and calorimetric studies. The alternating guanine-cytosine sequence selectivity of the dyes has been revealed from binding affinity values, circular dichroism, thermal melting, competition dialysis, and calorimetric results. The binding affinities of both the dyes to the polynucleotides were of the order of 10(5) M(-1), but the values were higher for the guanine-cytosine polynucleotides over adenine-thymine ones. Phenosafranin had a higher binding affinity compared to safranin O. Isothermal titration calorimetric studies revealed that the binding reactions were exothermic and favored by negative enthalpy and predominantly large positive entropy contributions in all cases except poly(dA)·poly(dT) where the profile was anomalous. Although charged, nonpolyelectrolytic contribution was revealed to be dominant to the free energy of binding. The negative heat capacity values obtained from the temperature dependence of enthalpy changes, which were higher for phenosafranin compared to safranin O, suggested significant hydrophobic contribution to the binding process. In aggregate, the data presents evidence for the alternating guanine-cytosine base pair selectivity of these phenazinium dyes and a stronger binding of phenosafranin over safranin O. PMID:20979425

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

    PubMed

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

    2014-01-01

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

  5. Yeast interactions and wine flavour.

    PubMed

    Fleet, Graham H

    2003-09-01

    Wine is the product of complex interactions between fungi, yeasts and bacteria that commence in the vineyard and continue throughout the fermentation process until packaging. Although grape cultivar and cultivation provide the foundations of wine flavour, microorganisms, especially yeasts, impact on the subtlety and individuality of the flavour response. Consequently, it is important to identify and understand the ecological interactions that occur between the different microbial groups, species and strains. These interactions encompass yeast-yeast, yeast-filamentous fungi and yeast-bacteria responses. The surface of healthy grapes has a predominance of Aureobasidium pullulans, Metschnikowia, Hanseniaspora (Kloeckera), Cryptococcus and Rhodotorula species depending on stage of maturity. This microflora moderates the growth of spoilage and mycotoxigenic fungi on grapes, the species and strains of yeasts that contribute to alcoholic fermentation, and the bacteria that contribute to malolactic fermentation. Damaged grapes have increased populations of lactic and acetic acid bacteria that impact on yeasts during alcoholic fermentation. Alcoholic fermentation is characterised by the successional growth of various yeast species and strains, where yeast-yeast interactions determine the ecology. Through yeast-bacterial interactions, this ecology can determine progression of the malolactic fermentation, and potential growth of spoilage bacteria in the final product. The mechanisms by which one species/strain impacts on another in grape-wine ecosystems include: production of lytic enzymes, ethanol, sulphur dioxide and killer toxin/bacteriocin like peptides; nutrient depletion including removal of oxygen, and production of carbon dioxide; and release of cell autolytic components. Cell-cell communication through quorum sensing molecules needs investigation. PMID:12892919

  6. Conservation of yeasts by dehydration

    Microsoft Academic Search

    Martin Beker; Alexander Rapoport

    The presented material concerns the theoretical basis for obtaining high-quality active dry biopreparations. It deals with the present understanding of anabiosis, contains data on yeast resistance against dehydration and the limits for preserving the viability of microorganisms in anabiosis. The process of water transport in yeast biomass during dehydration is discussed.\\u000a The changes and transformations in yeast cells occuring after

  7. Persistence of Cytosine Methylation of DNA following Fertilisation in the Mouse

    PubMed Central

    Li, Yan; O'Neill, Chris

    2012-01-01

    Normal development of the mammalian embryo requires epigenetic reprogramming of the genome. The level of cytosine methylation of CpG-rich (5meC) regions of the genome is a major epigenetic regulator and active global demethylation of 5meC throughout the genome is reported to occur within the first cell-cycle following fertilization. An enzyme or mechanism capable of catalysing such rapid global demethylation has not been identified. The mouse is a widely used model for studying developmental epigenetics. We have reassessed the evidence for this phenomenon of genome-wide demethylation following fertilisation in the mouse. We found when using conventional methods of immunolocalization that 5meC showed a progressive acid-resistant antigenic masking during zygotic maturation which gave the appearance of demethylation. Changing the unmasking strategy by also performing tryptic digestion revealed a persistence of a methylated state. Analysis of methyl binding domain 1 protein (MBD1) binding confirmed that the genome remained methylated following fertilisation. The maintenance of this methylated state over the first several cell-cycles required the actions of DNA methyltransferase activity. The study shows that any 5meC remodelling that occurs during early development is not explained by a global active loss of 5meC staining during the cleavage stage of development and global loss of methylation following fertilization is not a major component of epigenetic reprogramming in the mouse zygote. PMID:22292019

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

    PubMed Central

    Pedersen, Jakob Skou; Valen, Eivind; Velazquez, Amhed M. Vargas; Parker, Brian J.; Rasmussen, Morten; Lindgreen, Stinus; Lilje, Berit; Tobin, Desmond J.; Kelly, Theresa K.; Vang, Søren; Andersson, Robin; Jones, Peter A.; Hoover, Cindi A.; Tikhonov, Alexei; Prokhortchouk, Egor; Rubin, Edward M.; Sandelin, Albin; Gilbert, M. Thomas P.; Krogh, Anders; Willerslev, Eske; Orlando, Ludovic

    2014-01-01

    Epigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence data generated from hair shafts of a 4000-yr-old Paleo-Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery of the expected signals at promoter regions, exon/intron boundaries, and CTCF sites. The top-scoring nucleosome calls revealed distinct DNA positioning biases, attesting to nucleotide-level accuracy. The ancient methylation levels exhibited high conservation over time, clustering closely with modern hair tissues. Using ancient methylation information, we estimated the age at death of the Saqqaq individual and illustrate how epigenetic information can be used to infer ancient gene expression. Similar epigenetic signatures were found in other fossil material, such as 110,000- to 130,000-yr-old bones, supporting the contention that ancient epigenomic information can be reconstructed from a deep past. Our findings lay the foundation for extracting epigenomic information from ancient samples, allowing shifts in epialleles to be tracked through evolutionary time, as well as providing an original window into modern epigenomics. PMID:24299735

  9. Triple-helix formation by oligonucleotides containing the three bases thymine, cytosine, and guanine.

    PubMed Central

    Giovannangéli, C; Rougée, M; Garestier, T; Thuong, N T; Hélène, C

    1992-01-01

    A homopurine-homopyrimidine sequence of human immunodeficiency virus (HIV) proviral DNA was chosen as a target for triple-helix-forming oligonucleotides. An oligonucleotide containing three bases (thymine, cytosine, and guanine) was shown to bind to its target sequence under physiological conditions. This oligonucleotide is bound in a parallel orientation with respect to the homopurine sequence. Thymines recognize A.T base pairs to form T.A.T base triplets and guanines recognize a run of G.C base pairs to form G.G.C base triplets. A single 5-methylcytosine was shown to stabilize the triple helix when incorporated in a stretch of thymines; it recognizes a single G.C base pair in a run of A.T base pairs. These results provide some of the rules required for choosing the more appropriate oligonucleotide sequence to form a triple helix at a homopurine-homopyrimidine sequence of duplex DNA. A psoralen derivative attached to the oligonucleotide containing thymine, 5-methylcytosine, and guanine was shown to photoinduce cross-linking of the two DNA strands at the target sequence in a plasmid containing part of the HIV proviral DNA sequence. Triplex formation and cross-linking were monitored by inhibition of Dra I restriction enzyme cleavage. The present results provide a rational basis for the development of triplex-forming oligonucleotides targeted to specific sequences of the HIV provirus integrated in its host genome. Images PMID:1528873

  10. Maturation of Purkinje cells in mouse cerebellum after neonatal administrations of cytosine arabinoside.

    PubMed

    Yamano, T; Shimada, M; Nakao, K; Nakamura, T; Wakaizumi, S; Kusunoki, T

    1978-10-13

    ICR-JCL strain mice were injected subcutaneously with 30 mg/kg body weight of cytosine arabinoside at 2, 3, and 4 days of age. This chemical prevented the production of the basket cells, stellate cells, and granule cells in the external granular layer of the cerebellum. Decrease in number of these microneutrons affected the noraml synaptic connections between the Purkinje cells and the microneurons, thus causing the disarrangement and abnormal arborization of the Purkinje cells. Of the three types of microneurons, the basket and a few stellate cells played a more important role in the disarrangement of the Purkinje cells and abnormal arborization of their primary dendrites than the granule cells did. Abnormal outgrowing directions of other smooth dendrites of the Purkinje cells were caused mainly by the diminution of stellate cells. Although parallel fibers were grossly decreased in number in the treated cerebellums, spines of the spiny dendrites of the Purkinje cells sprouted considerably in the 15-day-old mice, and then their morphological features remained even after 100 days of age. PMID:707044

  11. Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA.

    PubMed

    Lukinavicius, Grazvydas; Lapinaite, Audrone; Urbanaviciute, Giedre; Gerasimaite, Ruta; Klimasauskas, Saulius

    2012-12-01

    DNA methyltransferases catalyse the transfer of a methyl group from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) onto specific target sites on DNA and play important roles in organisms from bacteria to humans. AdoMet analogs with extended propargylic side chains have been chemically produced for methyltransferase-directed transfer of activated groups (mTAG) onto DNA, although the efficiency of reactions with synthetic analogs remained low. We performed steric engineering of the cofactor pocket in a model DNA cytosine-5 methyltransferase (C5-MTase), M.HhaI, by systematic replacement of three non-essential positions, located in two conserved sequence motifs and in a variable region, with smaller residues. We found that double and triple replacements lead to a substantial improvement of the transalkylation activity, which manifests itself in a mild increase of cofactor binding affinity and a larger increase of the rate of alkyl transfer. These effects are accompanied with reduction of both the stability of the product DNA-M.HhaI-AdoHcy complex and the rate of methylation, permitting competitive mTAG labeling in the presence of AdoMet. Analogous replacements of two conserved residues in M.HpaII and M2.Eco31I also resulted in improved transalkylation activity attesting a general applicability of the homology-guided engineering to the C5-MTase family and expanding the repertoire of sequence-specific tools for covalent in vitro and ex vivo labeling of DNA. PMID:23042683

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

    PubMed Central

    Tsuboi, Hidenori; Sutoh, Keita; Wada, Masamitsu

    2012-01-01

    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

  13. 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

  14. 5?-Cytosine-Phosphoguanine (CpG) Methylation Impacts the Activity of Natural and Engineered Meganucleases

    PubMed Central

    Valton, Julien; Daboussi, Fayza; Leduc, Sophie; Molina, Rafael; Redondo, Pilar; Macmaster, Rachel; Montoya, Guillermo; Duchateau, Philippe

    2012-01-01

    In this study, we asked whether CpG methylation could influence the DNA binding affinity and activity of meganucleases used for genome engineering applications. A combination of biochemical and structural approaches enabled us to demonstrate that CpG methylation decreases I-CreI DNA binding affinity and inhibits its endonuclease activity in vitro. This inhibition depends on the position of the methylated cytosine within the DNA target and was almost total when it is located inside the central tetrabase. Crystal structures of I-CreI bound to methylated cognate target DNA suggested a molecular basis for such inhibition, although the precise mechanism still has to be specified. Finally, we demonstrated that the efficacy of engineered meganucleases can be diminished by CpG methylation of the targeted endogenous site, and we proposed a rational design of the meganuclease DNA binding domain to alleviate such an effect. We conclude that although activity and sequence specificity of engineered meganucleases are crucial parameters, target DNA epigenetic modifications need to be considered for successful gene editions. PMID:22740697

  15. Growth promotion of Vigna mungo (L.) by Pseudomonas spp. exhibiting auxin production and ACC-deaminase activity

    Microsoft Academic Search

    Shahzadi Noreen; Basharat Ali; Shahida Hasnain

    Auxin production and 1-aminocyclopropane-1-carboxylate (ACC) deaminase of rhizobacteria are very important plant growth promoting\\u000a attributes. In the present study, Pseudomonas strains exhibiting these traits were evaluated for their growth promoting effects on Vigna mungo (L.). Colorimetric analysis revealed that Pseudomonas alcaliphila AvR-2, Pseudomonas sp. AvH-4 and Pseudomonas aeruginosa As-17, respectively, produced 40.30, 32.90 and 36.50 ?g auxin ml?1 in the presence

  16. Effects of alcohol–kola nut interactions on brain glucose oxidase, glutamine synthase, and adenylate deaminase activities in Wistar rats

    Microsoft Academic Search

    G. O. Obochi; S. P. Malu; A. E. Abara

    2009-01-01

    The effects of alcohol–kola nut interactions on activites of whole brain glucose oxidase, glutamine synthetase, and adenylate deaminase were examined in Wistar rats. Thirty Wistar albino rats were divided into six groups. Control group (1) received a placebo (4 mL of distilled water). Groups 2–6 were treated for a 21-day period with either 10% (v\\/v) alcohol, kola nut, caffeine, alcohol

  17. Expression of cytidine deaminase in human solid tumors and its regulation by 1 ?,25-dihydroxyvitamin D 3

    Microsoft Academic Search

    Shin-Ichi Watanabe; Takafumi Uchida

    1996-01-01

    We found that vitamin D3 up-regulates the expression of cytidine deaminase (CDD) gene in some human solid tumor cell lines as well as the monocytic leukemia cell lines. Two kinds of full length CDD cDNA were identified from human placenta: one has glutamine and the other one has lysine at codon 27. The expression was tested in various normal tissues

  18. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 2013-04-01 2013-04-01 false Dried yeasts. 172.896 Section 172.896 Food and Drugs...CONSUMPTION Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces...

  19. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 2011-04-01 2011-04-01 false Dried yeasts. 172.896 Section 172.896 Food and Drugs...CONSUMPTION Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces...

  20. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 2012-04-01 2012-04-01 false Dried yeasts. 172.896 Section 172.896 Food and Drugs...CONSUMPTION Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces...

  1. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 2014-04-01 2014-04-01 false Dried yeasts. 172.896 Section 172.896 Food and Drugs...CONSUMPTION Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces...

  2. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...3 2010-04-01 2009-04-01 true Dried yeasts. 172.896 Section 172.896 Food and Drugs...CONSUMPTION Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces...

  3. Molecular analysis of porphobilinogen (PBG) deaminase gene mutations in acute intermittent porphyria: first study in patients of Slavic origin.

    PubMed

    Rosipal, R; Puy, H; Lamoril, J; Martasek, P; Nordmann, Y; Deybach, J C

    1997-05-01

    Acute intermittent porphyria (AIP) is an autosomally dominant inherited metabolic disorders caused by decreased activity of porphobilinogen deaminase, the third enzyme in the human heme biosynthetic pathway. We report here the first mutations in the human porphobilinogen deaminase gene in seven unrelated patients from the Czech and Slovak Republics with acute intermittent porphyria. We used denaturing gradient gel electrophoresis to screen all 15 exons and exon/intron boundaries of the porphobilinogen deaminase gene. Polymerase chain reaction products of abnormal migration patterns were subjected to direct sequencing to identify the causative mutations. Thus we revealed four novel mutations and three which have been previously described. Of the four novel mutations, two were mis-sense (G24S, V267M), one was a single base insertion (158insA) that produced a stop codon 12 codons downstream, and one was a single base substitution in intron 12 (771 + 1) resulting in a splicing defect. The three previously detected mutations were mis-sense mutations (R26C, R26H, G111R). These results suggest a high allelic heterogeneity in Czech and Slovak patients. PMID:9238757

  4. STRIPE2 encodes a putative dCMP deaminase that plays an important role in chloroplast development in rice.

    PubMed

    Xu, Jing; Deng, Yiwen; Li, Qun; Zhu, Xudong; He, Zuhua

    2014-10-20

    Mutants with abnormal leaf coloration are good genetic materials for understanding the mechanism of chloroplast development and chlorophyll biosynthesis. In this study, a rice mutant st2 (stripe2) with stripe leaves was identified from the ?-ray irradiated mutant pool. The st2 mutant exhibited decreased accumulation of chlorophyll and aberrant chloroplasts. Genetic analysis indicated that the st2 mutant was controlled by a single recessive locus. The ST2 gene was finely confined to a 27-kb region on chromosome 1 by the map-based cloning strategy and a 5-bp deletion in Os01g0765000 was identified by sequence analysis. The deletion happened in the joint of exon 3 and intron 3 and led to new spliced products of mRNA. Genetic complementation confirmed that Os01g0765000 is the ST2 gene. We found that the ST2 gene was expressed ubiquitously. Subcellular localization assay showed that the ST2 protein was located in mitochondria. ST2 belongs to the cytidine deaminase-like family and possibly functions as the dCMP deaminase, which catalyzes the formation of dUMP from dCMP by deamination. Additionally, exogenous application of dUMP could partially rescue the st2 phenotype. Therefore, our study identified a putative dCMP deaminase as a novel regulator in chloroplast development for the first time. PMID:25438698

  5. Production of Food Grade Yeasts

    Microsoft Academic Search

    Argyro Bekatorou; Costas Psarianos; Athanasios A. Koutinas

    2006-01-01

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

  6. Yeast Infections in Immunocompromised Hosts

    Microsoft Academic Search

    Emmanuel Rollides; Thomas J. Walsh

    A number of yeast fungi are pathogenic, but the two genera that contain the most important animal and human pathogens are\\u000a Candida and Cryptococcus. In addition, there are a number of other yeasts that have been, more rarely, implicated in disease.

  7. Yeast interactions and wine flavour

    Microsoft Academic Search

    Graham H. Fleet

    2003-01-01

    Wine is the product of complex interactions between fungi, yeasts and bacteria that commence in the vineyard and continue throughout the fermentation process until packaging. Although grape cultivar and cultivation provide the foundations of wine flavour, microorganisms, especially yeasts, impact on the subtlety and individuality of the flavour response. Consequently, it is important to identify and understand the ecological interactions

  8. Preservation of manipulated yeast diet

    Microsoft Academic Search

    Joong Kyun Kim; Hae-Yoon Chung

    2002-01-01

    Manipulated yeast diet can be usedfor seed production of aquacultural organisms.Various methods for preserving the yeast dietduring the periods of circulation in marketwere tested, and the preservation of the yeastdiet by freeze-drying was the best. With thispreservation method, the manipulated yeastswere maintained fairly well (up to 71%) whenstored for three weeks under refrigeratedcondition (4 °C), while more than 80% ofthe

  9. Epigenetic Variation, Inheritance, and Parent-of-Origin Effects of Cytosine Methylation in Maize (Zea mays)

    PubMed Central

    Lauria, Massimiliano; Piccinini, Sara; Pirona, Raul; Lund, Gertrud; Viotti, Angelo; Motto, Mario

    2014-01-01

    Pure epigenetic variation, or epigenetic variation that is independent of genetic context, may provide a mechanism for phenotypic variation in the absence of DNA mutations. To estimate the extent of pure epigenetic variation within and across generations and to identify the DNA regions targeted, a group of eight plants derived from a highly inbred line of maize (Zea mays) was analyzed by the methylation-sensitive amplified polymorphism (MSAP) technique. We found that cytosine methylation (mC) differences among individuals accounted for up to 7.4% of CCGG sites investigated by MSAP. Of the differentially methylated fragments (DMFs) identified in the S0 generation, ?12% were meiotically inherited for at least six generations. We show that meiotically heritable mC variation was consistently generated for an average of 0.5% CCGG sites per generation and that it largely occurred somatically. We provide evidence that mC variation can be established and inherited in a parent-of-origin manner, given that the paternal lineage is more prone to both forward and reverse mC changes. The molecular characterization of selected DMFs revealed that the variation was largely determined by CG methylation changes that map within gene regions. The expression analysis of genes overlapping with DMFs did not reveal an obvious correlation between mC variation and transcription, reinforcing the idea that the primary function of gene-body methylation is not to control gene expression. Because this study focuses on epigenetic variation in field-grown plants, the data presented herein pertain to spontaneous epigenetic changes of the maize genome in a natural context. PMID:24374354

  10. Formation and genotoxicity of a guanine–cytosine intrastrand cross-link lesion in vivo

    PubMed Central

    Hong, Haizheng; Cao, Huachuan; Wang, Yinsheng

    2007-01-01

    Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes, and they can damage biological molecules including nucleic acids. Exposure of isolated DNA to X/?-rays and Fenton reagents was shown to lead to the formation of intrastrand cross-link lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. By employing HPLC coupled with tandem mass spectrometry (LC-MS/MS) with the isotope dilution method, we assessed quantitatively the formation of a guanine–cytosine (G[8-5]C) intrastrand cross-link lesion in HeLa-S3 cells upon exposure to ?-rays. The yield of the G[8-5]C cross-link was 0.037 lesions per 109 nucleosides per Gy, which was ?300 times lower than that of 5-formyl-2?-deoxyuridine (0.011 lesions per 106 nucleosides per Gy) under identical exposure conditions. We further constructed a single-stranded M13 genome harboring a site-specifically incorporated G[8-5]C lesion and developed a novel mass spectrometry-based method for interrogating the products emanating from the replication of the genome in Escherichia coli cells. The results demonstrated that G[8-5]C blocked considerably DNA replication as represented by a 20% bypass efficiency, and the lesion was significantly mutagenic in vivo, which included a 8.7% G?T and a 1.2% G?C transversion mutations. DNA replication in E. coli hosts deficient in SOS-induced polymerases revealed that polymerase V was responsible for the error-prone translesion synthesis in vivo. PMID:17942427

  11. Cytosine arabinoside induces ectoderm and inhibits mesoderm expression in human embryonic stem cells during multilineage differentiation

    PubMed Central

    Jagtap, S; Meganathan, K; Gaspar, J; Wagh, V; Winkler, J; Hescheler, J; Sachinidis, A

    2011-01-01

    BACKGROUND AND PURPOSE Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here, we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals. This study was undertaken to explore the adverse effects of cytosine arabinoside (Ara-C) on randomly differentiated hESCs. EXPERIMENTAL APPROACH Human embryonic stem cells were used to investigate the effects of a developmental toxicant Ara-C. Sublethal concentrations of Ara-C were given for two time points, day 7 and day 14 during the differentiation. Gene expression was assessed with microarrays to determine the dysregulated transcripts in presence of Ara-C. KEY RESULTS Randomly differentiated hESCs were able to generate the multilineage markers. The low concentration of Ara-C (1 nM) induced the ectoderm and inhibited the mesoderm at day 14. The induction of ectodermal markers such as MAP2, TUBB III, PAX6, TH and NESTIN was observed with an inhibition of mesodermal markers such as HAND2, PITX2, GATA5, MYL4, TNNT2, COL1A1 and COL1A2. In addition, no induction of apoptosis was observed. Gene ontology revealed unique dysregulated biological process related to neuronal differentiation and mesoderm development. Pathway analysis showed the axon guidance pathway to be dysregulated. CONCLUSIONS AND IMPLICATIONS Our results suggest that hESCs in combination with toxicogenomics offer a sensitive in vitro developmental toxicity model as an alternative to traditional animal experiments. PMID:21198554

  12. 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...

  13. Transformation of Azospirillum brasilense Cd with an ACC Deaminase Gene from Enterobacter cloacae UW4 Fused to the Tet r Gene Promoter Improves Its Fitness and Plant Growth Promoting Ability

    Microsoft Academic Search

    G. Holguin; B. R. Glick

    2003-01-01

    It has been reported that PGPB, containing ACC deaminase, can cleave the plant ethylene precursor ACC and thereby lower ethylene concentration in a developing or stressed plant, protecting it against the deleterious effects of stress ethylene and facilitating the formation of longer roots. In a previous work we have demonstrated expression of the ACC deaminase gene (acdS) from Enterobacter cloacae

  14. Cloning and expression of cDNA encoding heart-type isoform of AMP deaminase.

    PubMed

    Wang, X; Morisaki, H; Sermsuvitayawong, K; Mineo, I; Toyama, K; Ogasawara, N; Mukai, T; Morisaki, T

    1997-04-01

    Mouse cDNA for the heart-type (H) isoform of AMP deaminase (EC 3.5.4.6., AMPD) has been isolated and characterized. The cDNA for the Ampd gene expressed in heart is predicted to encode a peptide of 766 amino acids with a molecular mass of 88 kDa. The coding region of this cDNA was quite homologous to the human AMPD3 gene which encodes the erythrocyte-type (E) isoform of AMPD, although the H-isoform of rodent AMPD was reported to be immunologically distinct from the E-isoform of human AMPD. The non-coding region of the isolated cDNA is not homologous to human AMPD3, while rodent Ampd1 has similarity to human AMPD1 in the non-coding region as well as in the coding region. The transcripts for the cloned cDNA were expressed in heart, slow-twitch skeletal muscle and non-muscle tissues. Prokaryotic expression showed that this cDNA encodes a catalytically active enzyme which reacts to the specific antibody raised to the H-isoform of AMPD. PMID:9133604

  15. AMP deaminase 3 plays a critical role in remote reperfusion lung injury.

    PubMed

    Li, Peili; Ogino, Kazuhide; Hoshikawa, Yoshiko; Morisaki, Hiroko; Toyama, Keiko; Morisaki, Takayuki; Morikawa, Kumi; Ninomiya, Haruaki; Yoshida, Akio; Hashimoto, Kiyoshi; Shirayoshi, Yasuaki; Hisatome, Ichiro

    2013-04-26

    Remote reperfusion lung injury following skeletal muscle ischemia and reperfusion accounts for high morbidity and mortality. AMP deaminase (AMPD), a key enzyme for nucleotide cycle, has been implicated in the regulation of this phenomenon. However, the function of Ampd2 and Ampd3 subtype has not been elucidated in remote reperfusion rodent lung injury. We utilized AMPD3 and AMPD2-deficient mice. The two types of AMPD-deficient mice and wild-type (WT) littermates were subjected to ischemia-reperfusion injury. After 3h bilateral hind-limb ischemia and reperfusion, AMPD3 mRNA, AMPD activity and inosine monophosphate (IMP) increased significantly in WT and AMPD2-deficient mice lungs, while they did not show significant alterations in AMPD3-deficient mice lungs. Genetic inactivation of Ampd3 resulted in markedly accelerated myeloperoxidase (MPO) activity along with exaggerated neutrophils infiltration and hemorrhage in the lungs compared to WT and AMPD2-deficient mice, furthermore, IMP treatment significantly attenuated MPO activity and neutrophils infiltration in WT and the two types of AMPD-deficient mice lungs after 3h reperfusion. These findings demonstrate for the first time in AMP-deficient mice models that AMPD3 plays a critical role in remote reperfusion lung injury via generation of IMP and validate the potential to use IMP into the clinical arena to attenuate remote ischemia-reperfusion lung injury. PMID:23542464

  16. Localization of N-terminal sequences in human AMP deaminase isoforms that influence contractile protein binding.

    PubMed

    Mahnke-Zizelman, D K; Sabina, R L

    2001-07-13

    The reversible association of AMP deaminase (AMPD, EC 3.5.4.6) with elements of the contractile apparatus is an identified mechanism of enzyme regulation in mammalian skeletal muscle. All three members of the human AMPD multigene family contain coding information for polypeptides with divergent N-terminal and conserved C-terminal domains. In this study, serial N-terminal deletion mutants of up to 111 (AMPD1), 214 (AMPD2), and 126 (AMPD3) residues have been constructed without significant alteration of catalytic function or protein solubility. The entire sets of active enzymes are used to extend our understanding of the contractile protein binding of AMPD. Analysis of the most truncated active enzymes demonstrates that all three isoforms can associate with skeletal muscle actomyosin and suggests that a primary binding domain is located within the C-terminal 635-640 residues of each polypeptide. However, discrete stretches of N-terminal sequence alter this behavior. Residues 54-83 in the AMPD1 polypeptide contribute to a high actomyosin binding capacity of both isoform M spliceoforms, although the exon 2- enzyme exhibits significantly greater association compared to its exon 2+ counterpart. Conversely, residues 129-183 in the AMPD2 polypeptide reduce actomyosin binding of isoform L. In addition, residues 1-48 in the AMPD3 polypeptide dramatically suppress contractile protein binding of isoform E, thus allowing this enzyme to participate in other intracellular interactions. PMID:11444869

  17. LINE-1 Retroelements Complexed and Inhibited by Activation Induced Cytidine Deaminase

    PubMed Central

    Metzner, Mirjam; Jäck, Hans-Martin; Wabl, Matthias

    2012-01-01

    LINE-1 (abbreviated L1) is a major class of retroelements in humans and mice. If unrestricted, retroelements accumulate in the cytoplasm and insert their DNA into the host genome, with the potential to cause autoimmune disease and cancer. Retroviruses and other retroelements are inhibited by proteins of the APOBEC family, of which activation-induced cytidine deaminase (AID) is a member. Although AID is mainly known for being a DNA mutator shaping the antibody repertoire in B lymphocytes, we found that AID also restricts de novo L1 integrations in B- and non-B-cell lines. It does so by decreasing the protein level of open reading frame 1 (ORF1) of both exogenous and endogenous L1. In activated B lymphocytes, AID deficiency increased L1 mRNA 1.6-fold and murine leukemia virus (MLV) mRNA 2.7-fold. In cell lines and activated B lymphocytes, AID forms cytoplasmic high-molecular-mass complexes with L1 mRNA, which may contribute to L1 restriction. Because AID-deficient activated B lymphocytes do not express ORF1 protein, we suggest that ORF1 protein expression is inhibited by additional restriction factors in these cells. The greater increase in MLV compared to L1 mRNA in AID-deficient activated B lymphocytes may indicate less strict surveillance of retrovirus. PMID:23133680

  18. 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. (MSU); (UW)

    2011-11-07

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

  19. Syzygium cumini inhibits adenosine deaminase activity and reduces glucose levels in hyperglycemic patients.

    PubMed

    Bopp, A; De Bona, K S; Bellé, L P; Moresco, R N; Moretto, M B

    2009-08-01

    Syzigium cumini (L.) Skeels from the Myrtaceae family is among the most common medicinal plants used to treat diabetes in Brazil. Leaves, fruits, and barks of S. cumini have been used for their hypoglycemic activity. Adenosine deaminase (ADA) is an important enzyme that plays a relevant role in purine and DNA metabolism, immune responses, and peptidase activity. ADA is suggested to be an important enzyme for modulating the bioactivity of insulin, but its clinical significance in diabetes mellitus (DM) has not yet been proven. In this study, we examined the effect of aqueous leaf extracts of S. cumini (L.) (ASC) on ADA activity of hyperglycemic subjects and the activity of total ADA, and its isoenzymes in serum and erythrocytes. The present study indicates that: (i) the ADA activity in hyperglycemic serum was higher than normoglycemic serum and ADA activity was higher when the blood glucose level was more elevated; (ii) ASC (60-1000 microg/mL) in vitro caused a concentration-dependent inhibition of total ADA activity and a decrease in the blood glucose level in serum; (iii) ADA1 and 2 were reduced both in erythrocytes and in hyperglycemic serum. These results suggest that the decrease of ADA activity provoked by ASC may contribute to control adenosine levels and the antioxidant defense system of red cells and could be related to the complex ADA/DPP-IV-CD26 and the properties of dipeptidyl peptidase IV (DPP-IV) inhibitors which serve as important regulators of blood glucose. PMID:19709327

  20. Sensitivity and specificity of adenosine deaminase in diagnosis of juvenile idiopathic arthritis

    PubMed Central

    Doudkani-Fard, Mina; Ziaee, Vahid; Moradinejad, Mohamad-Hassan; Sedaghat, Mojtaba; Haghi-Ashtiani, Mohammad-Taghi; Ahmadinejad, Zahra

    2014-01-01

    Background: Juvenile Idiopathic Arthritis (JIA) is one of the most common chronic rheumatic diseases inchildren with unknown etiology and pathogenesis. It also has no diagnostic test and its clinical diagnosis ismade through ruling out other types of arthritis. The aim of this study was to evaluate the level of ADA (AdenosineDeaminase) in the serum of JIA patients and to compare it with that of patients with Reactive Arthritis(RA). Evaluation of sensitivity and specificity of serum ADA level in JIA was another objective. Methods: The study included 120 children with JIA (mean age= 7.6 ± 4.3 years) and 40 children with RA(mean age= 5.5 ± 3.1 years). The ADA was measured in the active phase of both diseases. Results: The mean ADA serum level was obtained as 15.8 ± 11.8 U/l in JIA patients and 14.3 ± 7.5 U/l in RApatients. The difference was statistically insignificant (p= 0.4). Another finding of this study was the significantspecificity (77.5%) of this laboratory parameter for JIA in comparison with its low sensitivity (36.7%). Positivepredictive value was 83% and negative predictive value 29%. Conclusion: Determination of ADA serum levels is a noninvasive reliable and easy biomarker for diagnosis ofJIA and it can be used as alternative parameters representing disease activity. PMID:25678992

  1. Combined detections of interleukin-33 and adenosine deaminase for diagnosis of tuberculous pleural effusion

    PubMed Central

    Li, Diandian; Shen, Yongchun; Fu, Xiaomin; Li, Min; Wang, Tao; Wen, Fuqiang

    2015-01-01

    Objective: To investigate the diagnostic accuracy of the combination of interleukin-33 (IL-33) and adenosine deaminase (ADA) for differentiating TPE from pleural effusions with the other etiologies. Methods: Pleural effusion samples were collected from 32 TPE patients and 55 non-TPE patients. Pleural levels of IL-33 and ADA were measured by ELISA. The corresponding biochemical indexes were also simultaneously determined. Results: The pleural levels of IL-33 and ADA in the TPE group were significantly higher than those in the non-TPE group. With a cut-off value of 68.3 pg/ml, the sensitivity and specificity for IL-33 were 83.9% and 70.9%, respectively. While for ADA, the sensitivity and specificity were 87.5% and 87.3%, respectively at a cut-off value of 10.25 U/L. Combined use of IL-33 and ADA measurements further increased the sensitivity or specificity. Conclusion: Our study suggests that the applications of new biomarker IL-33, along with ADA, may serve as efficient diagnosis strategies in the management of pleural TB. Further studies at a large scale should be performed to validate our findings. PMID:25755791

  2. Hereditary overexpression of adenosine deaminase in erythrocytes: evidence for a cis-acting mutation.

    PubMed Central

    Chen, E H; Tartaglia, A P; Mitchell, B S

    1993-01-01

    Overexpression of adenosine deaminase (ADA) in red blood cells is inherited as an autosomal dominant trait and causes hemolytic anemia. The increased ADA activity in erythrocytes is due to an increase in steady-state levels of ADA mRNA of normal sequence. Increased ADA mRNA may be due to a cis-acting mutation which results in increased transcription or a loss of down-regulation during erythroid differentiation. Alternatively, it is possible that the mutation is in a trans-acting factor which interacts with normal ADA transcriptional elements to cause overexpression in red blood cells. To discriminate between a cis-acting and a trans-acting mutation, we took advantage of a highly polymorphic TAAA repeat located at the tail end of an Alu repeat approximately 1.1 kb upstream of the ADA gene. Using PCR to amplify this region, we identified five different alleles in 19 members of the family. All 11 affected individuals had an ADA allele with 12 TAAA repeats, whereas none of the 8 normal individuals did. We conclude that this disorder results from a cis-acting mutation in the vicinity of the ADA gene. Images Figure 2 Figure 3 PMID:8213817

  3. Control of transcription arrest in intron 1 of the murine adenosine deaminase gene.

    PubMed Central

    Kash, S F; Kellems, R E

    1994-01-01

    Transcription arrest plays a key role in the regulation of the murine adenosine deaminase (ADA) gene, as well as a number of other cellular and viral genes. We have previously characterized the ADA intron 1 arrest site, located 145 nucleotides downstream of the transcription start site, with respect to sequence and elongation factor requirements. Here, we show that the optimal conditions for both intron 1 arrest and overall ADA transcription involve the addition of high concentrations of KCl soon after initiation. As we have further delineated the sequence requirements for intron 1 arrest, we have found that sequences downstream of the arrest site are unnecessary for arrest. Also, a 24-bp fragment containing sequences upstream of the arrest site is sufficient to generate arrest downstream of the adenovirus major late promoter only in the native orientation. Surprisingly, we found that deletion of sequences encompassing the ADA transcription start site substantially reduced intron 1 arrest, with no effect on overall levels of transcription. At the same time, deletion of sequences upstream of the TATA box had no significant effect on either process. We believe the start site mutations have disrupted either the assembly or the composition of the transcription complex such that intron 1 site read-through is now favored. This finding, coupled with the increase in overall transcription after high-concentration KCl treatment, allows us to further refine our model of ADA gene regulation. Images PMID:8065352

  4. Activation-induced Cytidine Deaminase in B Cell Immunity and Cancers.

    PubMed

    Park, Seok-Rae

    2012-12-01

    Activation-induced cytidine deaminase (AID) is an enzyme that is predominantly expressed in germinal center B cells and plays a pivotal role in immunoglobulin class switch recombination and somatic hypermutation for antibody (Ab) maturation. These two genetic processes endow Abs with protective functions against a multitude of antigens (pathogens) during humoral immune responses. In B cells, AID expression is regulated at the level of either transcriptional activation on AID gene loci or post-transcriptional suppression of AID mRNA. Furthermore, AID stabilization and targeting are determined by post-translational modifications and interactions with other cellular/nuclear factors. On the other hand, aberrant expression of AID causes B cell leukemias and lymphomas, including Burkitt's lymphoma caused by c-myc/IgH translocation. AID is also ectopically expressed in T cells and non-immune cells, and triggers point mutations in relevant DNA loci, resulting in tumorigenesis. Here, I review the recent literatures on the function of AID, regulation of AID expression, stability and targeting in B cells, and AID-related tumor formation. PMID:23396757

  5. A Role for Host Activation-Induced Cytidine Deaminase in Innate Immune Defense against KSHV

    PubMed Central

    Bekerman, Elena; Jeon, Diana; Ardolino, Michele; Coscoy, Laurent

    2013-01-01

    Activation-induced cytidine deaminase (AID) is specifically induced in germinal center B cells to carry out somatic hypermutation and class-switch recombination, two processes responsible for antibody diversification. Because of its mutagenic potential, AID expression and activity are tightly regulated to minimize unwanted DNA damage. Surprisingly, AID expression has been observed ectopically during pathogenic infections. However, the function of AID outside of the germinal centers remains largely uncharacterized. In this study, we demonstrate that infection of human primary naïve B cells with Kaposi's sarcoma-associated herpesvirus (KSHV) rapidly induces AID expression in a cell intrinsic manner. We find that infected cells are marked for elimination by Natural Killer cells through upregulation of NKG2D ligands via the DNA damage pathway, a pathway triggered by AID. Moreover, without having a measurable effect on KSHV latency, AID impinges directly on the viral fitness by inhibiting lytic reactivation and reducing infectivity of KSHV virions. Importantly, we uncover two KSHV-encoded microRNAs that directly regulate AID abundance, further reinforcing the role for AID in the antiviral response. Together our findings reveal additional functions for AID in innate immune defense against KSHV with implications for a broader involvement in innate immunity to other pathogens. PMID:24244169

  6. The Adenosine Deaminase Gene Polymorphism Is Associated with Chronic Heart Failure Risk in Chinese

    PubMed Central

    He, Hai-Rong; Li, Yuan-Jie; He, Gong-Hao; Wang, Ya-Jun; Zhai, Ya-Jing; Xie, Jiao; Zhang, Wei-Peng; Dong, Ya-Lin; Lu, Jun

    2014-01-01

    Adenosine (Ado) is an important cardioprotective agent. Since endogenous Ado levels are affected by the enzyme Ado deaminase (ADA), polymorphisms within the ADA gene may exert some effect on chronic heart failure (CHF). This study applied a case-control investigation to 300 northern Chinese Han CHF patients and 400 ethnicity-matched healthy controls in which nine single-nucleotide polymorphisms (SNPs) of ADA were genotyped and association analyses were performed. Odds ratios (ORs) with 95% confidence intervals (CI) were used to assess the association. Overall, rs452159 polymorphism in ADA gene was significantly associated with susceptibility to CHF under the dominant model (p = 0.013, OR = 1.537, 95% CI = 1.10–2.16), after adjustment for age, sex, and traditional cardiovascular risk factors. No difference in genotype distribution and allele frequency for the rs452159 according to the functional New York Heart Association class was found. Furthermore, the values of left ventricular ejection fraction, left-ventricle end-diastolic diameter or left-ventricle end-systolic diameter did not differ significantly among the different rs452159 genotype CHF patients. Although further studies with larger cohorts and other ethnicities are required to validate the conclusions, the findings of this study potentially provide novel insight into the pathogenesis of CHF. PMID:25170811

  7. Adenosine Deaminase Acting on RNA-1 (ADAR1) Inhibits HIV-1 Replication in Human Alveolar Macrophages

    PubMed Central

    Levy, David N.; Li, Yonghua; Kumar, Rajnish; Burke, Sean A.; Dawson, Rodney; Hioe, Catarina E.; Borkowsky, William; Rom, William N.; Hoshino, Yoshihiko

    2014-01-01

    While exploring the effects of aerosol IFN-? treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL) of aerosol IFN-?-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1) in the BAL cells. IFN-? induced ADAR1 expression in monocyte-derived macrophages (MDM) but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro. Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-? suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages. PMID:25272020

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

    PubMed

    Garca, Mehmet Fatih; Demir, Halit; Turan, Mahfuz; Bozan, Naz?m; Kozan, Ahmet; Belli, ?eyda Bayel; Arslan, Ay?e; Cankaya, Hakan

    2014-06-01

    To emphasize the effectiveness of adenosine deaminase (ADA) enzyme, which has important roles in the differentiation of lymphoid cells, and oxidative stress in patients with chronic tonsillitis. Serum and tissue samples were obtained from 25 patients who underwent tonsillectomy due to recurrent episodes of acute tonsillitis. In the control group, which also had 25 subjects, only serum samples were taken as obtaining tissue samples would not have been ethically appropriate. ADA enzyme activity, catalase (CAT), carbonic anhydrase (CA), nitric oxide (NO) and malondialdehyde (MDA) were measured in the serum and tissue samples of patients and control group subjects. The serum values of both groups were compared. In addition, the tissue and serum values of patients were compared. Serum ADA activity and the oxidant enzymes MDA and NO values of the patient group were significantly higher than those of the control group (p < 0.001), the antioxidant enzymes CA and CAT values of the patient group were significantly lower than those of the control group (p < 0.001). In addition, while CA, CAT and NO enzyme levels were found to be significantly higher in the tonsil tissue of the patient group when compared to serum levels (p < 0.05), there was no difference between tissue and serum MDA and ADA activity (p > 0.05). Elevated ADA activity may be effective in the pathogenesis of chronic tonsillitis both by impairing tissue structure and contributing to SOR formation. PMID:24305782

  9. Inflammation-mediated genomic instability: roles of activation-induced cytidine deaminase in carcinogenesis.

    PubMed

    Shimizu, Takahiro; Marusawa, Hiroyuki; Endo, Yoko; Chiba, Tsutomu

    2012-07-01

    Chronic inflammation is a strong risk factor for the development of cancer. Many previous studies have demonstrated that a transcriptional factor, nuclear factor (NF)-?B, plays an important role in the association between inflammation and cancer development, particularly tumor promotion and tumor progression. Although it is well recognized that cancer develops via stepwise accumulation of genetic aberrations, the mechanisms underlying the generation of these genetic alterations in normal epithelial cells under inflammatory conditions are not known. We recently demonstrated that pathogenic bacterial or viral factors and the subsequent inflammatory reactions lead to the aberrant expression of a DNA mutator enzyme, activation-induced cytidine deaminase (AID), in various epithelial cells via NF-?B activation, which causes the accumulation of genetic alterations in tumor-related genes. AID activation is widely observed in gastrointestinal tissues with cancer-associated inflammation, such as chronic viral hepatitis, Helicobacter pylori-related gastritis, Barrett's esophagus and inflammatory bowel disease. Furthermore, a deficiency of endogenous AID expression reduces both accumulation of somatic mutations in tumor-related genes and tumor incidence in a mouse model of inflammation-associated cancer development. These findings strongly suggest that AID plays an integral role in inflammation-associated carcinogenesis and is therefore a potential target molecule for the prevention and treatment of cancers. PMID:22469133

  10. Yeasts: From genetics to biotechnology

    SciTech Connect

    Russo, S.; Poli, G. [Univ. of Milan (Italy); Siman-Tov, R.B. [Univ. of Jerusalem, Rehovot (Israel)

    1995-12-31

    Yeasts have been known and used in food and alcoholic fermentations ever since the Neolithic Age. In more recent times, on the basis of their peculiar features and history, yeasts have become very important experimental models in both microbiological and genetic research, as well as the main characters in many fermentative production processes. In the last 40 years, advances in molecular biology and genetic engineering have made possible not only the genetic selection of organisms, but also the genetic modification of some of them, especially the simplest of them, such as bacteria and yeasts. These discoveries have led to the availability of new yeast strains fit to fulfill requests of industrial production and fermentation. Moreover, genetically modified and transformed yeasts have been constructed that are able to produce large amounts of biologically active proteins and enzymes. Thus, recombinant yeasts make it easier to produce drugs, biologically active products, diagnostics, and vaccines, by inexpensive and relatively simple techniques. Yeasts are going to become more and more important in the {open_quotes}biotechnological revolution{close_quotes} by virtue of both their features and their very long and safe use in human nutrition and industry. 175 refs., 4 figs., 6 tabs.

  11. Yeast ecology of Kombucha fermentation.

    PubMed

    Teoh, Ai Leng; Heard, Gillian; Cox, Julian

    2004-09-01

    Kombucha is a traditional fermentation of sweetened tea, involving a symbiosis of yeast species and acetic acid bacteria. Despite reports of different yeast species being associated with the fermentation, little is known of the quantitative ecology of yeasts in Kombucha. Using oxytetracycline-supplemented malt extract agar, yeasts were isolated from four commercially available Kombucha products and identified using conventional biochemical and physiological tests. During the fermentation of each of the four products, yeasts were enumerated from both the cellulosic pellicle and liquor of the Kombucha. The number and diversity of species varied between products, but included Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii and Zygosaccharomyces bailii. While these yeast species are known to occur in Kombucha, the enumeration of each species present throughout fermentation of each of the four Kombucha cultures demonstrated for the first time the dynamic nature of the yeast ecology. Kombucha fermentation is, in general, initiated by osmotolerant species, succeeded and ultimately dominated by acid-tolerant species. PMID:15282124

  12. A new and efficient synthetic method for 15N3-labeled cytosine nucleosides: Dimroth rearrangement of cytidine N3-oxides.

    PubMed

    Sako, Magoichi; Kawada, Hiroyoshi

    2004-11-12

    The treatment of (15)N(4)-labeled cytidine N(3)-oxide and (15)N(4)-labeled 2'-deoxycytidine N(3)-oxide, prepared from the appropriate unprotected uridines in three reaction steps, with benzyl bromide in the presence of excess lithium methoxide allowed the smooth occurrence of their Dimroth rearrangement even under mild conditions leading to the corresponding (15)N(3)-labeled uridine 4-O-benzyloximes which can easily undergo the reductive N-O bond cleavage to give the desirable (15)N(3)-labeled cytosine nucleosides in high total yields. PMID:15527310

  13. Yeast Breads: Made at Home. 

    E-print Network

    Cox, Maeona; Harris, Jimmie Nell; Reasonover, Frances; Mason, Lousie

    1957-01-01

    tablespoons sugar ll/z teaspoons salt cup shortening 1/4 CUP lukewarm water 2 packages yeast or 2 yeast cakes 39'4 CUPS flour Apple Coffee Cake. Scald milk and stir in sugar, salt and shorten~r , Cool to lukewarm. Sprinkle or crumble yeast tnto nit1.... Honey twist. Streusel coffee cake. Butterscotch pecan rolls. STREUSEL COFFEE CAKE 2 tablespoons butter or margarine 2 tablespoons sugar cup flour CUP fine bread crumbs 1/2 teaspoon cinnamon Cream fat and sugar. Add flour, bread crumbs...

  14. Polyglutamine misfolding in yeast

    PubMed Central

    2011-01-01

    Protein misfolding is associated with many human diseases, including neurodegenerative diseases, such as Alzheimer disease, Parkinson disease and Huntington disease. Protein misfolding often results in the formation of intracellular or extracellular inclusions or aggregates. Even though deciphering the role of these aggregates has been the object of intense research activity, their role in protein misfolding diseases is unclear. Here, I discuss the implications of studies on polyglutamine aggregation and toxicity in yeast and other model organisms. These studies provide an excellent experimental and conceptual paradigm that contributes to understanding the differences between toxic and protective trajectories of protein misfolding. Future studies like the ones discussed here have the potential to transform basic concepts of protein misfolding in human diseases and may thus help to identify new therapeutic strategies for their treatment. PMID:22052348

  15. Activation-Induced Cytidine Deaminase Alters the Subcellular Localization of Tet Family Proteins

    PubMed Central

    Arioka, Yuko; Watanabe, Akira; Saito, Kuniaki; Yamada, Yasuhiro

    2012-01-01

    Activation-induced cytidine deminase (Aid), a unique enzyme that deaminates cytosine in DNA, shuttles between the nucleus and the cytoplasm. A recent study proposed a novel function of Aid in active DNA demethylation via deamination of 5-hydroxymethylcytosine, which is converted from 5-methylcytosine by the Ten-eleven translocation (Tet) family of enzymes. In this study, we examined the effect of simultaneous expression of Aid and Tet family proteins on the subcellular localization of each protein. We found that overexpressed Aid is mainly localized in the cytoplasm, whereas Tet1 and Tet2 are localized in the nucleus, and Tet3 is localized in both the cytoplasm and the nucleus. However, nuclear Tet proteins were gradually translocated to the cytoplasm when co-expressed with Aid. We also show that Aid-mediated translocation of Tet proteins is associated with Aid shuttling. Here we propose a possible role for Aid as a regulator of the subcellular localization of Tet family proteins. PMID:23028748

  16. Electron transfer in amino acid.nucleic acid base complexes: EPR, ENDOR, and DFT study of X-irradiated N-formylglycine.cytosine complex crystals.

    PubMed

    Sagstuen, Einar; Close, David M; Vågane, Randi; Hole, Eli O; Nelson, William H

    2006-07-20

    Single crystals of the 1:1 complex of the nucleic acid base cytosine and the dipeptide N-formylglycine (C.NFG) have been irradiated at 10 and 273 K to doses of about 70 kGy and studied at temperatures between 10 and 293 K using 24 GHz (K-band) and 9.5 GHz (X-band) electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and ENDOR-induced EPR (EIE) spectroscopy. In this complex, the cytosine base is hydrogen bonded at positions N3 and N4 to the carboxylic group of the dipeptide, and the N3 position of cytosine has become protonated by the carboxylic group. At 10 K, two major radicals were characterized and identified. One of these (R1) is ascribed to the decarboxylated N-formylglycine one-electron oxidized species. The other (R2) is the N3-protonated cytosine one-electron reduced species. A third minority species (R3) appears to be a different conformation or protonation state of the one-electron reduced cytosine radical. Upon warming, the R2 and R3 radicals decay at about 100 K, and at 295 K, the only cytosine-centered radicals present are the C5 and C6 H-addition radicals (R5, R6). The R1 radical decays at about 150 K, and a glycine backbone radical (R4) grows in slowly. Thus, in the complex, a complete separation of initial oxidation and reduction events occurs, with oxidation localized at the dipeptide moiety, whereas reduction occurs at the nucleic acid base moiety. DFT calculations indicate that this separation is driven by large differences in electron affinities and ionization potentials between the two constituents of the complex. Once the initial oxidation and reduction products are trapped, no further electron transfer between the two constituents of the complex takes place. PMID:16836426

  17. Red Yeast Rice: An Introduction

    MedlinePLUS

    ... drugs, and some may contain a potentially harmful contaminant. This fact sheet provides basic information about red ... supplements. Some red yeast rice products contain a contaminant called citrinin, which can cause kidney failure. Tell ...

  18. A novel mutation in the porphobilinogen deaminase gene in an extended Chinese family with acute intermittent porphyria.

    PubMed

    Yang, Jing; Wang, Honglian; Yin, Kunlun; Hua, Baolai; Zhu, Tienan; Zhao, Yongqiang; Guo, Shubin; Yu, Xuezhong; Wu, Wei; Zhou, Zhou

    2015-07-10

    Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by a partial deficiency of porphobilinogen deaminase (PBGD), the third enzyme of the heme biosynthetic pathway. Establishing accurate diagnoses of the patient and asymptomatic family members with AIP involves identifying the PBGD enzyme mutations directly. Genetic testing provides a precise diagnosis for the patient and other asymptomatic family members, and thereby proper treatments can be initiated to prevent the disease from progressing. In this study, we report a novel PBGD missense mutation, A G-to-C, at the position 988 resulting in Alanine to Proline (Ala330Pro), in a Chinese family. PMID:25870942

  19. Mössbauer studies on yeast metallothionein

    Microsoft Academic Search

    X.-Q. Ding; E. Bill; A. X. Trautwein; H. J. Hartmann; U. Weser

    1994-01-01

    Iron-substituted yeast metallothionein, Fe(II)-yeast-MT, has been studied by Mössbauer spectroscopy. The iron in the protein is in the high-spin ferrous state. As maximum metal content, 4 Fe(II)\\/molecule has been determined, with the 4 metal ions forming a diamagnetic cluster due to the antiferromagnetic exchange interaction between the Fe(II) ions via bridging thiolates. In case the iron titration is less than

  20. Sociobiology of the budding yeast.

    PubMed

    Wloch-Salamon, Dominika M

    2014-04-01

    Social theory has provided a useful framework for research with microorganisms. Here I describe the advantages and possible risks of using a well-known model organism, the unicellular yeast Saccharomyces cerevisiae, for sociobiological research. I discuss the problems connected with clear classification of yeast behaviour based on the fitnessbased Hamilton paradigm. Relevant traits include different types of communities, production of flocculins, invertase and toxins, and the presence of apoptosis. PMID:24736156

  1. Mutagenic effects induced by the attack of NO2 radical to the guanine-cytosine base pair

    PubMed Central

    Cerón-Carrasco, José P.; Requena, Alberto; Zúñiga, José; Jacquemin, Denis

    2015-01-01

    We investigate the attack of the nitrogen dioxide radical (NO•2) to the guanine—cytosine (GC) base pair and the subsequent tautomeric reactions able to induce mutations, by means of density functional theory (DFT) calculations. The conducted simulations allow us to identify the most reactive sites of the GC base pair. Indeed, the computed relative energies demonstrate that the addition of the NO•2 radical to the C8 position of the guanine base forms to the most stable adduct. Although the initial adducts might evolve to non-canonical structures via inter-base hydrogen bonds rearrangements, the probability for the proton exchange to occur lies in the same range as that observed for undamaged DNA. As a result, tautomeric errors in NO2-attacked DNA arises at the same rate as in canonical DNA, with no macroscopic impact on the overall stability of DNA. The potential mutagenic effects of the GC–NO•2 radical adducts likely involve side reactions, e.g., the GC deprotonation to the solvent, rather than proton exchange between guanine and cytosine basis. PMID:25798437

  2. Synthesis and spectral properties of cytosine nucleosides of 2-amino-2-deoxy-alpha-D-arabino-furanose and -pyranose.

    PubMed

    Wolfrom, M L; Inouye, S

    1975-07-01

    Ethyl 2-deoxy-3,5-di-O-p-nitrobenzoyl-1-thio-2-(trifluoroacetamido)-beta-D-arabinofuranoside (3) was converted into the glycosyl chloride. Condensation of the latter with 2,4-dimethoxypyrimidine, followed by amination, gave 1-(2-amino-2-deoxy-alpha-D-arabinofuranosyl)cytosine (6), which was also obtained from the alpha-D anomer (4) of 3. Similarly, 1-(2-amino-2-deoxy-alpha-D-arabinopyranosyl)cytosine (12) was synthesized from ethyl 2-deoxy-3,4-di-O-p-nitrobenzoyl-1-thio-2-(trifluoroacetamido)-alpha-D-arabinopyranoside (9). The p.m.r. spectra of these nucleosides, as well as those of the 1-thioglycosides, are discussed in terms of the conformation of the sugar portion. In particular, a large change of the J1,2 coupling constants of the alpha-D-furanosides, according to the substituents at C-1 and C-2, was interpreted on the basis of conformational mobility. PMID:1139561

  3. 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

  4. B-cell development and functions and therapeutic options in adenosine deaminase–deficient patients

    PubMed Central

    Brigida, Immacolata; Sauer, Aisha V.; Ferrua, Francesca; Giannelli, Stefania; Scaramuzza, Samantha; Pistoia, Valentina; Castiello, Maria Carmina; Barendregt, Barbara H.; Cicalese, Maria Pia; Casiraghi, Miriam; Brombin, Chiara; Puck, Jennifer; Müller, Klaus; Notarangelo, Lucia Dora; Montin, Davide; van Montfrans, Joris M.; Roncarolo, Maria Grazia; Traggiai, Elisabetta; van Dongen, Jacques J. M.; van der Burg, Mirjam; Aiuti, Alessandro

    2015-01-01

    Background Adenosine deaminase (ADA) deficiency causes severe cellular and humoral immune defects and dysregulation because of metabolic toxicity. Alterations in B-cell development and function have been poorly studied. Enzyme replacement therapy (ERT) and hematopoietic stem cell (HSC) gene therapy (GT) are therapeutic options for patients lacking a suitable bone marrow (BM) transplant donor. Objective We sought to study alterations in B-cell development in ADA-deficient patients and investigate the ability of ERT and HSC-GT to restore normal B-cell differentiation and function. Methods Flow cytometry was used to characterize B-cell development in BM and the periphery. The percentage of gene-corrected B cells was measured by using quantitative PCR. B cells were assessed for their capacity to proliferate and release IgM after stimulation. Results Despite the severe peripheral B-cell lymphopenia, patients with ADA-deficient severe combined immunodeficiency showed a partial block in central BM development. Treatment with ERT or HSC-GT reverted most BM alterations, but ERT led to immature B-cell expansion. In the periphery transitional B cells accumulated under ERT, and the defect in maturation persisted long-term. HSC-GT led to a progressive improvement in B-cell numbers and development, along with increased levels of gene correction. The strongest selective advantage for ADA-transduced cells occurred at the transition from immature to naive cells. B-cell proliferative responses and differentiation to immunoglobulin secreting IgM after B-cell receptor and Toll-like receptor triggering were severely impaired after ERT and improved significantly after HSC-GT. Conclusions ADA-deficient patients show specific defects in B-cell development and functions that are differently corrected after ERT and HSC-GT. PMID:24506932

  5. Pleural Fluid Adenosine Deaminase (Pfada) in the Diagnosis of Tuberculous Effusions in a Low Incidence Population

    PubMed Central

    Arnold, David T.; Bhatnagar, Rahul; Fairbanks, Lynette D.; Zahan-Evans, Natalie; Clive, Amelia O.; Morley, Anna J.; Medford, Andrew R. L.; Maskell, Nicholas A.

    2015-01-01

    Introduction Previous studies have assessed the diagnostic ability of pleural fluid adenosine deaminase (pfADA) in detecting tuberculous pleural effusions, with good specificity and sensitivity reported. However, in North Western Europe pfADA is not routinely used in the investigation of a patient with an undiagnosed pleural effusion, mainly due to a lack of evidence as to its utility in populations with low mycobacterium tuberculosis (mTB) incidence. Methods Patients presenting with an undiagnosed pleural effusion to a tertiary pleural centre in South-West England over a 3 year period, were prospectively recruited to a pleural biomarker study. Pleural fluid from consecutive patients with robust 12-month follow up data and confirmed diagnosis were sent for pfADA analysis. Results Of 338 patients enrolled, 7 had confirmed tuberculous pleural effusion (2%). All mTB effusions were lymphocyte predominant with a median pfADA of 72.0 IU/L (range- 26.7 to 91.5) compared to a population median of 12.0 IU/L (range- 0.3 to 568.4). The optimal pfADA cut off was 35 IU/L, which had a negative predictive value (NPV) of 99.7% (95% CI; 98.2-99.9%) for the exclusion of mTB, and sensitivity of 85.7% (95% CI; 42.2-97.6%) with an area under the curve of 0.88 (95% CI; 0.732–1.000). Discussion This is the first study examining the diagnostic utility of pfADA in a low mTB incidence area. The chance of an effusion with a pfADA under 35 IU/L being of tuberculous aetiology was negligible. A pfADA of over 35 IU/L in lymphocyte-predominant pleural fluid gives a strong suspicion of mTB. PMID:25647479

  6. 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.

  7. 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

  8. Two Arabidopsis Threonine Aldolases Are Nonredundant and Compete with Threonine Deaminase for a Common Substrate Pool[W

    PubMed Central

    Joshi, Vijay; Laubengayer, Karen M.; Schauer, Nicolas; Fernie, Alisdair R.; Jander, Georg

    2006-01-01

    Amino acids are not only fundamental protein constituents but also serve as precursors for many essential plant metabolites. Although amino acid biosynthetic pathways in plants have been identified, pathway regulation, catabolism, and downstream metabolite partitioning remain relatively uninvestigated. Conversion of Thr to Gly and acetaldehyde by Thr aldolase (EC 4.1.2.5) was only recently shown to play a role in plant amino acid metabolism. Whereas one Arabidopsis thaliana Thr aldolase (THA1) is expressed primarily in seeds and seedlings, the other (THA2) is expressed in vascular tissue throughout the plant. Metabolite profiling of tha1 mutants identified a >50-fold increase in the seed Thr content, a 50% decrease in seedling Gly content, and few other significant metabolic changes. By contrast, homozygous tha2 mutations cause a lethal albino phenotype. Rescue of tha2 mutants and tha1 tha2 double mutants by overproduction of feedback-insensitive Thr deaminase (OMR1) shows that Gly formation by THA1 and THA2 is not essential in Arabidopsis. Seed-specific expression of feedback-insensitive Thr deaminase in both tha1 and tha2 Thr aldolase mutants greatly increases seed Ile content, suggesting that these two Thr catabolic enzymes compete for a common substrate pool. PMID:17172352

  9. Cloning of human AMP deaminase isoform E cDNAs. Evidence for a third AMPD gene exhibiting alternatively spliced 5'-exons.

    PubMed

    Mahnke-Zizelman, D K; Sabina, R L

    1992-10-15

    Higher eukaryotes express multiple isoforms of AMP deaminase (EC 3.5.4.6). In humans, four AMP deaminase variants, termed M (muscle), L (liver), E1, and E2 (erythrocyte) can be distinguished by a variety of biochemical and immunological criteria. Previous molecular studies have reported two genes, AMPD1 and AMPD2, that produce isoform M and L transcripts, respectively. This study identifies a third human AMP deaminase gene, AMPD3. Nucleotide sequence alignments between AMPD3 cDNAs isolated from several human libraries indicate three different extreme 5'-ends. Alternate forms of the AMPD3 cDNAs contain a common 2301-bp open reading frame (ORF) and 3'-untranslated region of 1245 bp. Two of the three forms, however, exhibit additional 5'-end nucleotide sequences that would extend their respective ORFs by 21 and 27 nucleotides. RNase protection analyses and the partial characterization of human AMPD3 genomic clones demonstrate alternative splicing of three different 5'-terminal exons. Western blot analyses detect anti-E-specific immunoreactivity in affinity-purified extracts derived from the bacterial expression of a truncated AMPD3 cDNA. These results are discussed in relation to AMP deaminase isoform diversity. PMID:1400401

  10. Structures of Substrate and Inhibitor-Bound Adenosine Deaminase from a Human Malaria Parasite Show a Dramatic Conformational Change and Shed Light on Drug Selectivity

    Microsoft Academic Search

    Eric T. Larson; Wei Deng; Brian E. Krumm; Alberto Napuli; Natascha Mueller; Wesley C. Van Voorhis; Frederick S. Buckner; Erkang Fan; Angela Lauricella; George DeTitta; Joseph Luft; Frank Zucker; Wim G. J. Hol; Christophe L. M. J. Verlinde; Ethan A. Merritt

    2008-01-01

    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

  11. Structures of Substrate-And Inhibitor-Bound Adenosine Deaminase From a Human Malaria Parasite Show a Dramatic Conformational Change And Shed Light on Drug Selectivity

    Microsoft Academic Search

    E. T. Larson; W. Deng; B. E. Krumm; A. Napuli; N. Mueller; W. C. Van Voorhis; F. S. Buckner; E. Fan; A. Lauricella; G. DeTitta; J. Luft; F. Zucker; W. G. J. Hol; C. L. M. J. Verlinde; E. A. Merritt

    2009-01-01

    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

  12. Detection of seven point mutations in the porphobilinogen deaminase gene in patients with acute intermittent porphyria, by direct sequencing of in vitro amplified cDNA

    Microsoft Academic Search

    C. S. Mgone; W. G. Lanyon; M. R. Moore; J. M. Connor

    1992-01-01

    Direct cDNA sequencing has been performed on asymmetrically amplified transcripts from the human porphobilinogen deaminase gene. Lymphocytes from 30 patients with acute intermittent porphyria were the source of mRNA; of the seven separate point mutations detected, three were silent, whereas four resulted in amino acid changes. Three of these changes involved highly conserved amino acids, and the remaining one a

  13. Arabidopsis Methionine ?-Lyase Is Regulated According to Isoleucine Biosynthesis Needs But Plays a Subordinate Role to Threonine Deaminase1[W][OA

    PubMed Central

    Joshi, Vijay; Jander, Georg

    2009-01-01

    The canonical pathway for isoleucine biosynthesis in plants begins with the conversion of threonine to 2-ketobutyrate by threonine deaminase (OMR1). However, demonstration of methionine ?-lyase (MGL) activity in Arabidopsis (Arabidopsis thaliana) suggested that production of 2-ketobutyrate from methionine can also lead to isoleucine biosynthesis. Rescue of the isoleucine deficit in a threonine deaminase mutant by MGL overexpression, as well as decreased transcription of endogenous Arabidopsis MGL in a feedback-insensitive threonine deaminase mutant background, shows that these two enzymes have overlapping functions in amino acid biosynthesis. In mgl mutant flowers and seeds, methionine levels are significantly increased and incorporation of [13C]Met into isoleucine is decreased, but isoleucine levels are unaffected. Accumulation of free isoleucine and other branched-chain amino acids is greatly elevated in response to drought stress in Arabidopsis. Gene expression analyses, amino acid phenotypes, and labeled precursor feeding experiments demonstrate that MGL activity is up-regulated by osmotic stress but likely plays a less prominent role in isoleucine biosynthesis than threonine deaminase. The observation that MGL makes a significant contribution to methionine degradation, particularly in reproductive tissue, suggests practical applications for silencing the expression of MGL in crop plants and thereby increasing the abundance of methionine, a limiting essential amino acid. PMID:19571310

  14. Coherent regulation in yeast’s cell-cycle network

    NASA Astrophysics Data System (ADS)

    Aral, Ne?e; Kabakç?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 yeast’s 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.

  15. The Plant Cell, Vol. 13, 17491759, August 2001, www.plantcell.org 2001 American Society of Plant Biologists Sequence Elimination and Cytosine Methylation Are Rapid and

    E-print Network

    Bhattacharyya, Madan Kumar

    Biologists Sequence Elimination and Cytosine Methylation Are Rapid and Reproducible Responses of the Genome with such genomic stress is a significant, al- beit poorly understood, question. In plants, it is estimated that polyploidy occurred in the history of 70% of flowering species (Masterson, 1994). Genome sequencing data may

  16. Pairing of the nucleobases guanine and cytosine in the gas phase studied by IRUV double-resonance spectroscopy and ab initio

    E-print Network

    de Vries, Mattanjah S.

    Pairing of the nucleobases guanine and cytosine in the gas phase studied by IR­UV double wavelength region. Introduction It is well known that the two strands of DNA are held together by guanine the inherent properties of the base pair interaction from those external effects, we study isolated gas

  17. Nuclear Transport of Yeast Proteasomes

    PubMed Central

    Enenkel, Cordula

    2014-01-01

    Proteasomes are conserved protease complexes enriched in the nuclei of dividing yeast cells, a major site for protein degradation. If yeast cells do not proliferate and transit to quiescence, metabolic changes result in the dissociation of proteasomes into proteolytic core and regulatory complexes and their sequestration into motile cytosolic proteasome storage granuli. These granuli rapidly clear with the resumption of growth, releasing the stored proteasomes, which relocalize back to the nucleus to promote cell cycle progression. Here, I report on three models of how proteasomes are transported from the cytoplasm into the nucleus of yeast cells. The first model applies for dividing yeast and is based on the canonical pathway using classical nuclear localization sequences of proteasomal subcomplexes and the classical import receptor importin/karyopherin ??. The second model applies for quiescent yeast cells, which resume growth and use Blm10, a HEAT-like repeat protein structurally related to karyopherin ?, for nuclear import of proteasome core particles. In the third model, the fully-assembled proteasome is imported into the nucleus. Our still marginal knowledge about proteasome dynamics will inspire the discussion on how protein degradation by proteasomes may be regulated in different cellular compartments of dividing and quiescent eukaryotic cells. PMID:25333764

  18. 280 EXPRESSION IN YEAST [23] [23] Manipulating Yeast Genome Using Plasmid Vectors

    E-print Network

    Botstein, David

    280 EXPRESSION IN YEAST [23] [23] Manipulating Yeast Genome Using Plasmid Vectors By TIM STEARNS, HONG MA, and DAVID BOTSTEIN The yeast Saccharomyces cerevisiae has proved to be a popular high status of yeast as an experimental system is in large part due to the work of the many geneticists

  19. Pre-Absorbing Antibody with Yeast Cells Preparation of Fixed Yeast

    E-print Network

    Aris, John P.

    106 Pre-Absorbing Antibody with Yeast Cells Preparation of Fixed Yeast 1. Plan to do steps 1-10 in the yeast immunofluorescence method. But, start with 100 mls of cells at OD600=0.2. Then, do all steps in quadruplicate. Do pretreatment, and digest cells for 10 minutes. 2. Pool all yeast in SPC + Pics in one

  20. Yeast through the ages: A statistical analysis of genetic changes in aging yeast

    E-print Network

    Hardin, Jo

    Yeast through the ages: A statistical analysis of genetic changes in aging yeast A. Wise J. Hardin focuses on the analysis of data from a yeast DNA microarray experiment. The biological question that motivates our research is "What genetic changes in yeast happen over time?" In order to explore the research

  1. APPENDIX 4LGrowth and Manipulation of Yeast PREPARATION OF SELECTED YEAST MEDIA

    E-print Network

    Winston, Fred

    containers in which 2.5 kg of dextrose is packaged. Throughout this chapter, YNB -AA/AS refers to yeastAPPENDIX 4LGrowth and Manipulation of Yeast PREPARATION OF SELECTED YEAST MEDIA Like Escherichia coli, yeast can be grown in either liquid media or on the surface of (or embedded in) solid agar plates

  2. Regulation of rat AMP deaminase 3 (isoform C) by development and skeletal muscle fibre type.

    PubMed

    Mahnke-Zizelman, D K; D'cunha, J; Wojnar, J M; Brogley, M A; Sabina, R L

    1997-09-01

    AMP deaminase (AMPD) is characterized by a multigene family in rodents and man. Highly conserved rat and human AMPD1 and AMPD2 genes produce protein products that exhibit cross-species immunoreactivities (AMPD1, rat isoform A and human isoform M; AMPD2, rat isoform B and human isoform L). A third gene, AMPD3, has been described in humans, but antisera raised against its purified protein product (isoform E) reportedly does not cross-react with a third activity purified from rat tissues (isoform C). This study was designed to address this latter issue by cloning, sequencing and expressing rat AMPD3 cDNA species. Similarly to the human AMPD3 gene, the rat AMPD3 gene produces multiple transcripts that differ at or near their 5' ends. The boundary at which these alternative sequences diverge is precisely conserved in both species. Across the region that is common to all rat and human AMPD3 cDNA species, nucleotide and predicted amino acid sequences are 89% and 93% identical respectively, although the rat open reading frame is lacking two separate in-frame codons in the 5' end. Extreme 5' regions between the two species are entirely divergent, and one alternative rat sequence is predicted to confer at least 36 additional N-terminal residues to its encoded AMPD3 polypeptide. A comparison of 3' untranslated regions indicates that the rat sequence is 250 bp longer and contains multiple consensus polyadenylation signals. Examination of relative rat AMPD3 gene expression shows (1) variable patterns of alternative mRNA abundance across adult tissues, (2) developmental regulation in skeletal muscle and liver, and (3) greater mRNA abundance in adult red (soleus) than in mixed (plantaris) and white (outer gastrocnemius) skeletal muscle. Finally, baculoviral expression of rat and human AMPD3 proteins produces enzymes that are chromatographically and kinetically similar. Moreover, both recombinant activities immunoreact with anti-C and anti-E serum. These combined results demonstrate that rat isoform C and human isoform E are homologous cross-species AMPD3 proteins. PMID:9291127

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

    PubMed Central

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

    2002-01-01

    The extra-enzymic function of cell-surface adenosine deaminase (ADA), an enzyme mainly localized in the cytosol but also found on the cell surface of monocytes, B cells and T cells, has lately been the subject of numerous studies. Cell-surface ADA is able to transduce co-stimulatory signals in T cells via its interaction with CD26, an integral membrane protein that acts as ADA-binding protein. The aim of the present study was to explore whether ADA-CD26 interaction plays a role in the adhesion of lymphocyte cells to human epithelial cells. To meet this aim, different lymphocyte cell lines (Jurkat and CEM T) expressing endogenous, or overexpressing human, CD26 protein were tested in adhesion assays to monolayers of colon adenocarcinoma human epithelial cells, Caco-2, which express high levels of cell-surface ADA. Interestingly, the adhesion of Jurkat and CEM T cells to a monolayer of Caco-2 cells was greatly dependent on CD26. An increase by 50% in the cell-to-cell adhesion was found in cells containing higher levels of CD26. Incubation with an anti-CD26 antibody raised against the ADA-binding site or with exogenous ADA resulted in a significant reduction (50-70%) of T-cell adhesion to monolayers of epithelial cells. The role of ADA-CD26 interaction in the lymphocyte-epithelial cell adhesion appears to be mediated by CD26 molecules that are not interacting with endogenous ADA (ADA-free CD26), since SKW6.4 (B cells) that express more cell-surface ADA showed lower adhesion than T cells. Adhesion stimulated by CD26 and ADA is mediated by T cell lymphocyte function-associated antigen. A role for ADA-CD26 interaction in cell-to-cell adhesion was confirmed further in integrin activation assays. FACS analysis revealed a higher expression of activated integrins on T cell lines in the presence of increasing amounts of exogenous ADA. Taken together, these results suggest that the ADA-CD26 interaction on the cell surface has a role in lymphocyte-epithelial cell adhesion. PMID:11772392

  4. Bacteria, Yeast and Chemicals on Human Skin

    MedlinePLUS

    ... gov/medlineplus/videos/news/Microbes_040115-1.html Bacteria, Yeast and Chemicals on Human Skin HealthDay News ... on this page, please enable JavaScript. Play video: Bacteria, Yeast and Chemicals on Human Skin For closed ...

  5. 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)

  6. 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

  7. Microhydration of guanine...cytosine base pairs, a theoretical Study on the role of water in stability, structure and tautomeric equilibrium.

    PubMed

    Zelený, Tomás; Hobza, Pavel; Kabelác, Martin

    2009-05-14

    The potential energy surfaces of guanine...cytosine complexes and microhydrated guanine...cytosine (one and two water molecules) were investigated by the molecular dynamics/quenching method (MD/Q), using the empirical potential Parm94 force field, implemented in the Amber program package. The calculations were conducted for all the possible combinations of the four most stable tautomers of guanine and three of cytosine (covering the canonical forms in both cases). The obtained structures were sorted by their structural motifs into three main groups: planar hydrogen-bonded; stacked; and T-shaped structures. The most stable structures found at the empirical potential energy surfaces were fully reoptimised at the second-order Møller-Plesset perturbation theory as well as using the density functional method with an empirical dispersion term (DFT-D). A combination of the canonical form of guanine and cytosine and canonical cytosine with a guanine tautomer where the hydrogen is switched from position N9 to N7 are energetically preferred in microsolvated systems as well as those without the presence of a solvent. The rising number of water molecules leads to smaller differences between the stability of the various combinations of the tautomers of bases in the base pairs. For some of the tautomer combinations (mainly the enol-enol combination), two water molecules are sufficient for the preference of stacked structures over the H-bonded ones. The interaction energies and geometries obtained by the second-order Møller-Plesset perturbation theory method and the much less computationally demanding DFT-D method are comparable, except for stacked complexes, where the interaction energies are overestimated on average by 3 kcal mol(-1) at the MP2 level. PMID:19421545

  8. Cdc42 Oscillations in Yeasts

    NSDL National Science Digital Library

    Felipe O. Bendezu (Switzerland; University of Lausanne REV)

    2012-12-04

    A fundamental problem in cell biology is how cells define one or several discrete sites of polarity. Through mechanisms involving positive and negative feedback, the small Rho-family guanosine triphosphatase Cdc42 breaks symmetry in round budding yeast cells to define a single site of polarized cell growth. However, it is not clear how cells can define multiple sites of polarization concurrently. We discuss a study in which rod-shaped fission yeast cells, which naturally polarize growth at their two cell ends, exhibited oscillations of Cdc42 activity between these sites. We compare these findings with similar oscillatory behavior of Cdc42 detected in budding yeast cells and discuss the possible mechanism and functional outputs of these oscillations.

  9. Yeast proteome map (update 2006).

    PubMed

    Perrot, Michel; Guieysse-Peugeot, Anne-Laure; Massoni, Aurélie; Espagne, Christelle; Claverol, Stéphane; Silva, Raquel Monteiro; Jenö, Paul; Santos, Manuel; Bonneu, Marc; Boucherie, Hélian

    2007-04-01

    To improve the potential of two-dimensional gel electrophoresis for proteomic investigations in yeast we have undertaken the systematic identification of Saccharomyces cerevisiae proteins separated on 2-D gels. We report here the identification of 187 novel protein spots. They were identified by two methods, mass spectrometry and gene inactivation. These identifications extend the number of protein spots identified on our yeast 2-D proteome map to 602, i.e. nearly half the detectable spots of the proteome map. These spots correspond to 417 different proteins. The reference map and the list of identified proteins can be accessed on the Yeast Protein Map server (www.ibgc.u-bordeaux2.fr/YPM). PMID:17351888

  10. Excited state proton transfer is not involved in the ultrafast deactivation of Guanine-Cytosine pair in solution.

    PubMed

    Biemann, Lars; Kovalenko, Sergey A; Kleinermanns, Karl; Mahrwald, Rainer; Markert, Morris; Improta, Roberto

    2011-12-14

    Different derivatives of Guanine (G) and Cytosine (C), which sterically enforce the Watson-Crick (WC) conformer, have been studied in CHCl(3) by means of broad-band transient absorption spectroscopy. Our experiments rule out the involvement of an Excited State Proton Transfer (ESPT), which dominates the excited state decay of GC in the gas phase. Instead, the ultrafast dynamics via internal conversion occurs in a polar environment mainly by relaxation in the monomer moieties. Time-dependent density functional theory (TD-DFT) calculations in solution indeed indicate that population transfer from the bright excited states toward the charge transfer state is not effective in CHCl(3) and a noticeable energy barrier is associated with the ESPT reaction. ESPT is therefore not expected to be a main deactivation route for GC pairs within DNA. PMID:22074113

  11. 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

  12. Functionalized Tricyclic Cytosine Analogues Provide Nucleoside Fluorophores with Improved Photophysical Properties and a Range of Solvent Sensitivities

    PubMed Central

    Rodgers, Brittney J.; Elsharif, Nada A.; Vashisht, Nisha; Mingus, Macy M.; Mulvahill, Mark A.; Stengel, Gudrun; Kuchta, Robert D.

    2014-01-01

    Tricyclic cytosines (tC and tCO frameworks) have emerged as a unique class of fluorescent nucleobase analogues that minimally perturb the structure of B-form DNA and that are not quenched in duplex nucleic acids. Systematic derivatization of these frameworks is a likely approach to improve on and diversify photophysical properties, but has not so far been examined. Synthetic methods were refined to improve on tolerance for electron donating and electron withdrawing groups, resulting in a series of eight new, fluorescent cytidine analogues. Photophysical studies show that substitution of the framework results in a pattern of effects largely consistent across tC and tCO and provides nucleoside fluorophores that are brighter than either parent. Moreover, a range of solvent sensitivities is observed, offering promise that this family of probes can be extended to new applications that require reporting on the local environment. PMID:24311229

  13. Understanding the structural and dynamic consequences of DNA epigenetic modifications: computational insights into cytosine methylation and hydroxymethylation.

    PubMed

    Carvalho, Alexandra T P; Gouveia, Leonor; Kanna, Charan Raju; Wärmländer, Sebastian K T S; Platts, Jamie A; Kamerlin, Shina Caroline Lynn

    2014-12-01

    We report a series of molecular dynamics (MD) simulations of up to a microsecond combined simulation time designed to probe epigenetically modified DNA sequences. More specifically, by monitoring the effects of methylation and hydroxymethylation of cytosine in different DNA sequences, we show, for the first time, that DNA epigenetic modifications change the molecule's dynamical landscape, increasing the propensity of DNA toward different values of twist and/or roll/tilt angles (in relation to the unmodified DNA) at the modification sites. Moreover, both the extent and position of different modifications have significant effects on the amount of structural variation observed. We propose that these conformational differences, which are dependent on the sequence environment, can provide specificity for protein binding. PMID:25625845

  14. 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.; (NIEHS); (UW)

    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.

  15. Inhibition of transcription of cytosine-containing DNA in vitro by the alc gene product of bacteriophage T4

    SciTech Connect

    Drivdahl, R.H.; Kutter, E.M. (Evergreen State College, Olympia, WA (USA))

    1990-05-01

    The alc gene product (gpalc) of bacteriophage T4 inhibits the transcription of cytosine-containing DNA in vivo. The authors examined its effect on transcription in vitro by comparing RNA polymerase isolated from Escherichia coli infected with either wild-type T4D{sup +} or alc mutants. A 50 to 60% decline in RNA polymerase activity, measured on phage T7 DNA, was observed by 1 min after infection with either T4D{sup +} or alc mutants; this did not occur when the infecting phage lacked gpalt. In the case of the T4D{sup +} strain but not alc mutants, this was followed by a further decrease. By 5 min after infection the activity of alc mutants was 1.5 to 2.5 times greater than that of the wild type on various cytosine-containing DNA templates, whereas there was little or no difference in activity on T4 HMdC-DNA, in agreement with the in vivo specificity. Effects on transcript initiation and elongation were distinguished by using a T7 phage DNA template. Rifampin challenge, end-labeling with ({gamma}-{sup 32}P)ATP, and selective initiation with a dinucleotide all indicate that the decreased in vitro activity of the wild-type polymerase relative to that of the alc mutants was due to inhibition of elongation, not to any difference in initiation rates. Wild-type (but not mutated) gpalc copurified with RNA polymerase on heparin agarose but not in subsequent steps. Immunoprecipitation of modified RNA polymerase also indicated that gpalc was not tightly bound to RNA polymerase intracellularly.

  16. Red yeast rice: a new hypolipidemic drug

    Microsoft Academic Search

    Mélanie Journoud; Peter J. H Jones

    2004-01-01

    Red yeast rice is a source of fermented pigment with possible bioactive effect. Evidence shows that fermented red yeast rice lowers cholesterol levels moderately compared to other statin drugs, but with the added advantage of causing less adverse effects. A review of the body of evidence surrounding the properties of red yeast rice underscores its potential as a new alternative

  17. Enological functions of parietal yeast mannoproteins

    Microsoft Academic Search

    Andrea Caridi

    2006-01-01

    Parietal yeast mannoproteins play a very important role in the overall vinification process. Their production and release, both during winemaking and aging on lees, depends on the specific yeast strain and the nutritional conditions. The following enological functions of parietal yeast mannoproteins have been described: (a) adsorption of ochratoxin A; (b) combination with phenolic compounds; (c) increased growth of malolactic

  18. YEASTBOOK PERSPECTIVES Yeast: An Experimental Organism

    E-print Network

    Botstein, David

    YEASTBOOK PERSPECTIVES Yeast: An Experimental Organism for 21st Century Biology David Botstein*,1, Cambridge, Massachusetts 02139 ABSTRACT In this essay, we revisit the status of yeast as a model system for biology. We first summarize important contributions of yeast to eukaryotic biology that we anticipated

  19. 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)

  20. Mössbauer studies on yeast metallothionein

    NASA Astrophysics Data System (ADS)

    Ding, X.-Q.; Bill, E.; Trautwein, A. X.; Hartmann, H. J.; Weser, U.

    1994-12-01

    Iron-substituted yeast metallothionein, Fe(II)-yeast-MT, has been studied by Mössbauer spectroscopy. The iron in the protein is in the high-spin ferrous state. As maximum metal content, 4 Fe(II)/molecule has been determined, with the 4 metal ions forming a diamagnetic cluster due to the antiferromagnetic exchange interaction between the Fe(II) ions via bridging thiolates. In case the iron titration is less than 4 Fe(II)/apoprotein, the ions are magnetically noninteracting, with each individual Fe(II) behaving similar to Fe(II) in reduced rubredoxin.

  1. Combined nuclear measurements of yeast

    NASA Astrophysics Data System (ADS)

    Saleh, N. S.; Al-Saleh, K. A.; Arafah, D.-E.; Halim, N. A.

    1987-05-01

    Combined Rutherford backscattering (RBS), X-ray fluorescence (XRF) and proton induced X-ray emission (PIXE) techniques were used to determine the elemental composition of yeast. Results reveal no toxic elements (e.g. Ag, Pb, etc) in yeast. Yet results display some similarities in concentrations of some elements (e.g. Ti, Mn, Ni, Cu and Sr), large differences are observed for others (e.g. S, Cl, K, Ca, Fe and Zn). Variations are accounted due to different growing media or contamination during processing.

  2. Beer brewing using a fusant between a sake yeast and a brewer's yeast

    Microsoft Academic Search

    Nobuhiko Mukai; Chiharu Nishimori; Ikuko Wilson Fujishige; Akihiro Mizuno; Toshiro Takahashi; Kazuo Sato

    2001-01-01

    Beer brewing using a fusant between a sake yeast (a lysine auxotrophic mutant of sake yeast K-14) and a brewer's yeast (a respiratory-deficient mutant of the top fermentation yeast NCYC1333) was performed to take advantage of the beneficial characteristics of sake yeasts, i.e., the high productivity of esters, high tolerance to ethanol, and high osmotolerance. The fusant (F-32) obtained was

  3. Control of ATP homeostasis during the respiro-fermentative transition in yeast

    PubMed Central

    Walther, Thomas; Novo, Maite; Rössger, Katrin; Létisse, Fabien; Loret, Marie-Odile; Portais, Jean-Charles; François, Jean-Marie

    2010-01-01

    Respiring Saccharomyces cerevisiae cells respond to a sudden increase in glucose concentration by a pronounced drop of their adenine nucleotide content ([ATP]+[ADP]+[AMP]=[AXP]). The unknown fate of ‘lost' AXP nucleotides represented a long-standing problem for the understanding of the yeast's physiological response to changing growth conditions. Transient accumulation of the purine salvage pathway intermediate, inosine, accounted for the apparent loss of adenine nucleotides. Conversion of AXPs into inosine was facilitated by AMP deaminase, Amd1, and IMP-specific 5?-nucleotidase, Isn1. Inosine recycling into the AXP pool was facilitated by purine nucleoside phosphorylase, Pnp1, and joint action of the phosphoribosyltransferases, Hpt1 and Xpt1. Analysis of changes in 24 intracellular metabolite pools during the respiro-fermentative growth transition in wild-type, amd1, isn1, and pnp1 strains revealed that only the amd1 mutant exhibited significant deviations from the wild-type behavior. Moreover, mutants that were blocked in inosine production exhibited delayed growth acceleration after glucose addition. It is proposed that interconversion of adenine nucleotides and inosine facilitates rapid and energy-cost efficient adaptation of the AXP pool size to changing environmental conditions. PMID:20087341

  4. Yeast Cultures in Ruminant Nutrition

    Microsoft Academic Search

    S. A. DENEV; Tz. PEEVA; P. RADULOVA; N. STANCHEVA; G. STAYKOVA; G. BEEV; P. TODOROVA; S. TCHOBANOVA

    2007-01-01

    Abstract DENEV,, S. A., Tz. PEEVA, P. RADULOVA, P. STANCHEVA, G. STAYKOVA, G. BEEV, P. TODOROVA and S. TCHOBANOVA, 2007. Yeast cultures in ruminant nutrition. Bulg. J. Agric. Sci.13: 357-374 Interest in the use of fungal direct-fed microbials in ruminant nutrition is considerable.The

  5. Malassezia Baillon, emerging clinical yeasts

    Microsoft Academic Search

    Roma Batra; Teun Boekhout; Eveline Guého; F. Javier Cabañes; Thomas L. Dawson; Aditya K. Gupta

    2005-01-01

    The human and animal pathogenic yeast genus Malassezia has received considerable attention in recent years from dermatologists, other clinicians, veterinarians and mycologists. Some points highlighted in this review include recent advances in the technological developments related to detection, identification, and classification of Malassezia species. The clinical association of Malassezia species with a number of mammalian dermatological diseases including dandruff, seborrhoeic

  6. Mutational comparison of the single-domained APOBEC3C and double-domained APOBEC3F\\/G anti-retroviral cytidine deaminases provides insight into their DNA target site specificities

    Microsoft Academic Search

    Rupert C. L. Beale; Silvestro G. Conticello; Michael S. Neuberger

    Human APOBEC3F and APOBEC3G are double- domained deaminases that can catalyze dC!dU deamination in HIV-1 and MLV retroviral DNA replica- tion intermediates, targeting T-C or C-C dinucleo- tides, respectively. HIV-1 antagonizes their action through its vif gene product, which has been shown (at least in the case of APOBEC3G) to interact with the N-terminal domain of the deaminase, triggering its

  7. Detection of four novel mutations in the porphobilinogen deaminase gene in French Caucasian patients with acute intermittent porphyria.

    PubMed

    Puy, H; Deybach, J C; Lamoril, J; Robreau, A M; Nordmann, Y

    1996-01-01

    Acute intermittent porphyria (AIP) is an autosomal dominant disorder characterized by alterations of the gene encoding porphobilinogen deaminase (PBGD: EC 4.3.1.8), the third enzyme of the heme biosynthetic pathway. The molecular heterogeneity of the mutations causing AlP has been demonstrated with a reported predominance of single base substitutions resulting in amino acid changes. The molecular basis of AIP in four French patients was investigated using denaturing gradient gel electrophoresis followed by direct sequencing. We describe four different novel mutations that affected exon 12 (a frameshift and an exon skipping), exon 4 (a stop codon) and exon 15 (a frameshift inducing a stop codon). This study further documents the molecular heterogeneity of mutations in the PBGD gene in the French Caucasian population and reports types of mutations relatively uncommon in AIP. PMID:8860014

  8. AID upmutants isolated using a high-throughput screen highlight the immunity/cancer balance limiting DNA deaminase activity.

    PubMed

    Wang, Meng; Yang, Zizhen; Rada, Cristina; Neuberger, Michael S

    2009-07-01

    DNA deaminases underpin pathways in antibody diversification (AID) and anti-viral immunity (APOBEC3s). Here we show how a high-throughput bacterial papillation assay can be used to screen for AID mutants with increased catalytic activity. The upmutations focus on a small number of residues, some highlighting regions implicated in AID's substrate interaction. Many of the upmutations bring the sequence of AID closer to that of APOBEC3s. AID upmutants can yield increased antibody diversification, raising the possibility that modification of AID's specific activity might be used to regulate antibody diversification in vivo. However, upmutation of AID also led to an increased frequency of chromosomal translocations, suggesting that AID's specific activity may have been limited by the risk of genomic instability. PMID:19543289

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

    SciTech Connect

    Sadat Hayatshahi, Sayyed Hamed [Department of Biophysics, Faculty of Science, Tarbiat Modares University, P.O. Box: 14115/175, Tehran (Iran, Islamic Republic of) ; Abdolmaleki, Parviz [Department of Biophysics, Faculty of Science, Tarbiat Modares University, P.O. Box: 14115/175, Tehran (Iran, Islamic Republic of) ]. E-mail: parviz@modares.ac.ir; Safarian, Shahrokh [Department of Biology, Faculty of Science, Tehran University, P.O. Box: 13155-6455, Tehran (Iran, Islamic Republic of) ; Khajeh, Khosro [Department of Biochemistry, Faculty of Science, Tarbiat Modares University, P.O. Box: 14115/175, Tehran (Iran, Islamic Republic of)

    2005-12-16

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

  10. Endoplasmic reticulum involvement in yeast cell death

    PubMed Central

    Austriaco, O. P., Nicanor

    2012-01-01

    Yeast cells undergo programed cell death (PCD) with characteristic markers associated with apoptosis in mammalian cells including chromatin breakage, nuclear fragmentation, reactive oxygen species generation, and metacaspase activation. Though significant research has focused on mitochondrial involvement in this phenomenon, more recent work with both Saccharomyces cerevisiae and Schizosaccharomyces pombe has also implicated the endoplasmic reticulum (ER) in yeast PCD. This minireview provides an overview of ER stress-associated cell death (ER-SAD) in yeast. It begins with a description of ER structure and function in yeast before moving to a discussion of ER-SAD in both mammalian and yeast cells. Three examples of yeast cell death associated with the ER will be highlighted here including inositol starvation, lipid toxicity, and the inhibition of N-glycosylation. It closes by suggesting ways to further examine the involvement of the ER in yeast cell death. PMID:22876361

  11. Occurrence and Growth of Yeasts in Yogurts

    PubMed Central

    Suriyarachchi, V. R.; Fleet, G. H.

    1981-01-01

    Yogurts purchased from retail outlets were examined for the presence of yeasts by being plated onto oxytetracycline malt extract agar. Of the 128 samples examined, 45% exhibited yeast counts above 103 cells per g. A total of 73 yeast strains were isolated and identified as belonging to the genera Torulopsis, Kluyveromyces, Saccharomyces, Candida, Rhodotorula, Pichia, Debaryomyces, and Sporobolomyces. Torulopsis candida and Kluyveromyces fragilis were the most frequently isolated species, followed by Saccharomyces cerevisiae, Rhodotorula rubra, Kluyveromyces lactis, and Torulopsis versatilis. The growth of yeasts in yogurts was related to the ability of the yeasts to grow at refrigeration temperatures, to ferment lactose and sucrose, and to hydrolyze milk casein. Most yeast isolates grew in the presence of 100 ?g of sorbate and benzoate preservatives per ml. Higher yeast counts from yogurts were obtained when the yogurts were plated onto oxytetracycline malt extract agar than when they were plated onto acidified malt extract agar. PMID:16345853

  12. Electron paramagnetic resonance and electron nuclear double resonance studies of X-irradiated crystals of cytosine hydrochloride. Part I: free radical formation at 10 K after high radiation doses.

    PubMed

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

    1998-02-01

    Anhydrous single crystals of cytosine hydrochloride (protonated at N3) have been X-irradiated at 10 K and studied using K-band EPR, ENDOR and FSE spectroscopy. At least seven radicals were present at 10 K after X irradiation with a dose of about 150 kGy. Two different protonation states of the one-electron reduced cytosine cation were observed: an amino-protonated species (R1) and the pristine one-electron reduced species (R2) with zero net charge. Apparently three deprotonated versions of the one-electron oxidized cytosine cation were formed: the amino-deprotonated cation (R3), an N3-deprotonated cation (R4) and an N1-deprotonated cation (R5). Finally, two products formed by net hydrogen addition to the cytosine base were observed: a C5 hydrogen-addition radical (R6) and a C6 hydrogen-addition radical (R7). The crystalline lattice of cytosine hydrochloride is characterized in part by a cytosine base initially protonated at the N3-position, thus forming a cytosine base cation, and in part by an extended network of hydrogen bonding involving the chlorine anions. Proton transfer properties of pristine one-electron oxidation and reduction base products in this lattice are discussed and are suggested as explanations of the unusual multitude of positions for deprotonation of the one-electron oxidized species as well as for the two protonation states of the reduction product observed. The magnetic parameters for the amino-protonated species R1 agree well with those extracted from previous studies of cytosine derivatives in frozen solutions and in various glasses. PMID:9457889

  13. Mycotoxins - prevention and decontamination by yeasts.

    PubMed

    Pfliegler, Walter P; Pusztahelyi, Tünde; Pócsi, István

    2015-07-01

    The application of yeasts has great potential in reducing the economic damage caused by toxigenic fungi in the agriculture. Some yeasts may act as biocontrol agents inhibiting the growth of filamentous fungi. These species may also gain importance in the preservation of agricultural products and in the reduction of their mycotoxin contamination, yet the extent of mycotoxin production in the presence of biocontrol agents is relatively less understood. The application of yeasts in various technological processes may have a direct inhibitory effect on the toxin production of certain molds, which is independent of their growth suppressing effect. Furthermore, several yeast species are capable of accumulating mycotoxins from agricultural products, thereby effectively decontaminating them. Probiotic yeasts or products containing yeast cell wall are also applied to counteract mycotoxicosis in livestock. Several yeast strains are also able to degrade toxins to less-toxic or even non-toxic substances. This intensively researched field would greatly benefit from a deeper knowledge on the genetic and molecular basis of toxin degradation. Moreover, yeasts and their biotechnologically important enzymes may exhibit sensitivity to certain mycotoxins, thereby mounting a considerable problem for the biotechnological industry. It is noted that yeasts are generally regarded as safe; however, there are reports of toxin degrading species that may cause human fungal infections. The aspects of yeast-mycotoxin relations with a brief consideration of strain improvement strategies and genetic modification for improved detoxifying properties and/or mycotoxin resistance are reviewed here. PMID:25682759

  14. Does cytosine-thymine-guanine (CTG) expansion size predict cardiac events and electrocardiographic progression in myotonic dystrophy?

    PubMed Central

    Clarke, N; Kelion, A; Nixon, J; Hilton-Jones, D; Forfar, J

    2001-01-01

    OBJECTIVE—To assess whether the size of the cytosine-thymine-guanine (CTG) expansion mutation in myotonic dystrophy predicts progression of conduction system disease and cardiac events.?DESIGN—Longitudinal study involving ECG and clinical follow up over (mean (SD)) 4.8 (1.8) and 6.2 (1.9) years, respectively, of patients stratified by CTG expansion size (E0 to E4).?PATIENTS—73 adult patients under annual review in a regional myotonic dystrophy clinic. Patients were grouped into E0/E1 (n = 25), E2 (n = 34), and E3/E4 (n = 14).?RESULTS—The proportion of patients with a QRS complex > 100 ms at baseline increased with the size of the CTG expansion (EO/E1, 4%; E2, 12%; E3/E4, 36%; p = 0.02). This trend was more pronounced at follow up (E0/E1, 4%; E2, 21%; E3/E4, 57%; p = 0.0004). The rate of widening of the QRS complex (ms/year) was similarly related to the size of the mutation (EO/E1, 0.4 (1.3); E2, 1.4 (2.5); E3/E4, 1.5 (1.6); p = 0.04). First degree atrioventricular block was present in 23% of patients at baseline and 34% at follow up, with no significant relation to expansion size. Seven patients suffered a cardiac event during follow up (sudden death in two, permanent pacemaker insertion in three, chronic atrial arrhythmia in two), of whom six were in CTG expansion group E2 or greater. Patients who experienced a cardiac event during follow up had more rapid rates of PR interval increase (9.9 (11.1) v 1.6 (2.9) ms/year; p = 0.008) and a trend to greater QRS complex widening (3.6 (4.5) v 0.9 (1.5) ms/year; p = 0.06) than those who did not.?CONCLUSIONS—Larger CTG expansions are associated with a higher rate of conduction disease progression and a trend to increased risk of cardiac events in myotonic dystrophy.???Keywords: myotonic dystrophy; atrioventricular block; delayed intraventricular conduction; cytosine-thymine-guanine expansion PMID:11559681

  15. Yeast Metabolism Lab Purpose: To determine the effects of different

    E-print Network

    Rose, Michael R.

    1 Yeast Metabolism Lab Purpose: To determine the effects of different carbohydrates on the metabolism of live yeast. Background: Some organisms are capable of photosynthesis- using energy captured treatment (yeast+water, yeast+glucose, or yeast+sweetener) will produce the most carbon dioxide (CO2) from

  16. Morphology of a human-derived YAC in yeast meiosis

    Microsoft Academic Search

    Josef Loidl; Harry Scherthan; Johan T. Den Dunnen; Franz Klein

    1995-01-01

    In meiosis of human males DNA is packaged along pachytene chromosomes about 20 time more compactly than in meiosis of yeast. Nevertheless, a human-derived yeast artificial chromosome (YAC) shows the same degree of compaction of DNA as endogenous chromosomes in meiotic prophase nuclei of yeast. This suggests that in yeast meiosis, human and yeast DNA adopt a similar organization of

  17. Morphology of a human-derived YAC in yeast meiosis

    Microsoft Academic Search

    Josef Loidl; Harry Scherthan; Johan T. Den Dunnen; Franz Klein

    1995-01-01

    In meiosis of human males DNA is packaged along pachytene chromosomes about 20 times more compactly than in meiosis of yeast. Nevertheless, a human-derived yeast artificial chromosome (YAC) shows the same degree of compaction of DNA as endogenous chromosomes in meiotic prophase nuclei of yeast. This suggests that in yeast meiosis, human and yeast DNA adopt a similar organization of

  18. Synthesis and Biochemical Testing of 3-(Carboxyphenylethyl)imidazo[2,1-f][1,2,4]triazines as Inhibitors of AMP Deaminase.

    PubMed

    Lindell, Stephen D; Maechling, Simon; Sabina, Richard L

    2010-09-01

    C-Ribosyl imidazo[2,1-f][1,2,4]triazines and 3-[2-(3-carboxyphenyl)ethyl]-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ols represent two classes of known AMP deaminase inhibitors. A combination of the aglycone from the former class with the ribose phosphate mimic from the latter led to the 3-[2-(3-carboxyphenyl)ethyl]imidazo[2,1-f][1,2,4]triazines, which represent a new class of AMP deaminase inhibitors. The best compound, 3-[2-(3-carboxy-5,6,7,8-tetrahydronaphthyl)ethyl]imidazo[2,1-f][1,2,4]triazine (8), was a good inhibitor of all three human AMPD recombinant isozymes (AMPD1, AMPD2, and AMPD3; IC50 = 0.9-5.7 ?M) but a poor inhibitor of the plant recombinant enzyme (Arabidopsis FAC1; IC50 = 200 ?M). PMID:24900209

  19. Synthesis and Biochemical Testing of 3-(Carboxyphenylethyl)imidazo[2,1-f][1,2,4]triazines as Inhibitors of AMP Deaminase

    PubMed Central

    2010-01-01

    C-Ribosyl imidazo[2,1-f][1,2,4]triazines and 3-[2-(3-carboxyphenyl)ethyl]-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ols represent two classes of known AMP deaminase inhibitors. A combination of the aglycone from the former class with the ribose phosphate mimic from the latter led to the 3-[2-(3-carboxyphenyl)ethyl]imidazo[2,1-f][1,2,4]triazines, which represent a new class of AMP deaminase inhibitors. The best compound, 3-[2-(3-carboxy-5,6,7,8-tetrahydronaphthyl)ethyl]imidazo[2,1-f][1,2,4]triazine (8), was a good inhibitor of all three human AMPD recombinant isozymes (AMPD1, AMPD2, and AMPD3; IC50 = 0.9?5.7 ?M) but a poor inhibitor of the plant recombinant enzyme (Arabidopsis FAC1; IC50 = 200 ?M). PMID:24900209

  20. LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition

    Microsoft Academic Search

    Alexei A. Koshkin; Sanjay K. Singh; Poul Nielsen; Vivek K. Rajwanshi; Ravindra Kumar; Michael Meldgaard; Carl Erik Olsen; Jesper Wengel

    1998-01-01

    LNA (Locked Nucleic Acids), consisting of 2?-O,4?-C-methylene bicyclonucleoside monomers, is efficiently synthesized and its nucleic acid recognition potential evaluated for six different nucleobases, namely adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil. Unprecedented increases (+3 to +8 °C per modification) in the thermal stability of duplexes towards both DNA and RNA were obtained when evaluating mixed sequences of partly or fully

  1. Transgene silencing in grapevines transformed with GFLV resistance genes: analysis of variable expression of transgene, siRNAs production and cytosine methylation

    Microsoft Academic Search

    Giorgio Gambino; Irene Perrone; Andrea Carra; Walter Chitarra; Paolo Boccacci; Daniela Torello Marinoni; Marco Barberis; Fatemeh Maghuly; Margit Laimer; Ivana Gribaudo

    2010-01-01

    Eight transgenic grapevine lines transformed with the coat protein gene of Grapevine fanleaf virus (GFLV-CP) were analyzed for a correlation between transgene expression, siRNAs production and DNA methylation. Bisulphite\\u000a genome sequencing was used for a comprehensive analysis of DNA methylation. Methylated cytosine residues of CpG and CpNpG\\u000a sites were detected in the GFLV-CP transgene, in the T7 terminator and in

  2. Adsorption of adenine, cytosine, thymine, and uracil on sulfide-modified montmorillonite: FT-IR, Mössbauer and EPR spectroscopy and X-ray diffractometry studies.

    PubMed

    Carneiro, Cristine E A; Berndt, Graciele; de Souza Junior, Ivan G; de Souza, Cláudio M D; Paesano, Andrea; da Costa, Antonio C S; di Mauro, Eduardo; de Santana, Henrique; Zaia, Cássia T B V; Zaia, Dimas A M

    2011-10-01

    In the present work the interactions of nucleic acid bases with and adsorption on clays were studied at two pHs (2.00, 7.00) using different techniques. As shown by Mössbauer and EPR spectroscopies and X-ray diffractometry, the most important finding of this work is that nucleic acid bases penetrate into the interlayer of the clays and oxidize Fe(2+) to Fe(3+), thus, this interaction cannot be regarded as a simple physical adsorption. For the two pHs the order of the adsorption of nucleic acid bases on the clays was: adenine???cytosine?>?thymine?>?uracil. The adsorption of adenine and cytosine on clays increased with decreasing of the pH. For unaltered montmorillonite this result could be explained by electrostatic forces between adenine/cytosine positively charged and clay negatively charged. However for montmorillonite modified with Na(2)S, probably van der Waals forces also play an important role since both adenine/cytosine and clay were positively charged. FT-IR spectra showed that the interaction between nucleic acid bases and clays was through NH(+) or NH (2) (+) groups. X-ray diffractograms showed that nucleic acid bases adsorbed on clays were distributed into the interlayer surface, edge sites and external surface functional groups (aluminol, silanol) EPR spectra showed that the intensity of the line g???2 increased probably because the oxidation of Fe(2+) to Fe(3+) by nucleic acid bases and intensity of the line g?=?4.1 increased due to the interaction of Fe(3+) with nucleic acid bases. Mössbauer spectra showed a large decreased on the Fe(2+) doublet area of the clays due to the reaction of nucleic acid bases with Fe(2+). PMID:21717172

  3. Comparative transcriptomics of H. pylori strains AM5, SS1 and their hpyAVIBM deletion mutants: possible roles of cytosine methylation.

    PubMed

    Kumar, Ritesh; Mukhopadhyay, Asish K; Ghosh, Prachetash; Rao, Desirazu N

    2012-01-01

    Helicobacter pylori is an important human pathogen and one of the most successful chronic colonizers of the human body. H. pylori uses diverse mechanisms to modulate its interaction with the host in order to promote chronic infection and overcome host immune response. Restriction-modification genes are a major part of strain-specific genes present in H. pylori. The role of N(6)--adenine methylation in bacterial gene regulation and virulence is well established but not much is known about the effect of C(5) -cytosine methylation on gene expression in prokaryotes. In this study, it was observed by microarray analysis and RT-PCR, that deletion of an orphan C(5) -cytosine methyltransferase, hpyAVIBM in H. pylori strains AM5and SS1 has a significant effect on the expression of number of genes belonging to motility, adhesion and virulence. AM5?hpyAVIBM mutant strain has a different LPS profile and is able to induce high IL-8 production compared to wild-type. hpyAVIBM from strain 26695 is able to complement mutant SS1 and AM5 strains. This study highlights a possible significance of cytosine methylation in the physiology of H. pylori. PMID:22879937

  4. Low energy electron induced cytosine base release in 2?-deoxycytidine-3?-monophosphate via glycosidic bond cleavage: A time-dependent wavepacket study

    SciTech Connect

    Bhaskaran, Renjith; Sarma, Manabendra, E-mail: msarma@iitg.ernet.in [Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati 781 039 (India)

    2014-09-14

    Low energy electron (LEE) induced cytosine base release in a selected pyrimidine nucleotide, viz., 2?-deoxycytidine-3?-monophosphate is investigated using ab initio electronic structure methods and time dependent quantum mechanical calculations. It has been noted that the cytosine base scission is comparatively difficult process than the 3? C–O bond cleavage from the lowest ?{sup *} shape resonance in energy region <1 eV. This is mainly due to the high activation energy barrier associated with the electron transfer from the ?{sup *} orbital of the base to the ?{sup *} orbital of the glycosidic N–C bond. In addition, the metastable state formed after impinging LEE (0–1 eV) has very short lifetime (10 fs) which may decay in either of the two competing auto-detachment or dissociation process simultaneously. On the other hand, the selected N–C mode may cleave to form the cytosine base anion at higher energy regions (>2 eV) via tunneling of the glycosidic bond. Resonance states generated within this energy regime will exist for a duration of ?35–55 fs. Comparison of salient features of the two dissociation events, i.e., 3? C–O single strand break and glycosidic N–C bond cleavage in 3?-dCMPH molecule are also provided.

  5. IL-6 enhances the nuclear translocation of DNA cytosine-5-methyltransferase 1 (DNMT1) via phosphorylation of the nuclear localization sequence by the AKT kinase.

    PubMed

    Hodge, David R; Cho, Edward; Copeland, Terry D; Guszczynski, Tad; Yang, Eric; Seth, Arun K; Farrar, William L

    2007-01-01

    The epigenetic programming of genomic DNA is accomplished, in part, by several DNA cytosine-5-methyltransferases that act by covalently modifying cytosines with the addition of a methyl group. This covalent modification is maintained by the DNA cytosine-5-methyltransferase-1 enzyme (DNMT1), which is capable of acting in concert with other similar enzymes to silence important tumor suppressor genes. IL-6 is a multifunctional mediator of inflammation, acting through several major signaling cascades, including the phosphatidylinositol-3-kinase pathway (PI-3-K), which activates protein kinase B (AKT/PKB) downstream. Here, we show that the subcellular localization of DNMT1 can be altered by the addition of IL-6, increasing the rate of nuclear translocation of the enzyme from the cytosolic compartment. The mechanism of nuclear translocation of DNMT1 is greatly enhanced by phosphorylation of the DNMT1 nuclear localization signal (NLS) by PKB/AKT kinase. Mutagenic alteration of the two AKT target amino acids within the NLS results in a major loss of DNMT1 nuclear translocation, while the creation of a "phospho-mimic" amino acid (mutation to acidic residues) restores this compartmentation ability. These observations suggest an interesting hypothesis regarding how mediators of chronic inflammation may disturb the delicate balance of cellular compartmentalization of important proteins, and reveals a potential mechanism for the induction or enhancement of tumor growth via alteration of the components involved in the epigenetic programming of a cell. PMID:18204201

  6. Molecular Characterization of Adenosine 5?-monophosphate Deaminase—The Key Enzyme Responsible for the Umami Taste of Nori ( Porphyra yezoensis Ueda, Rhodophyta)

    Microsoft Academic Search

    Seiko Minami; Minoru Sato; Yoshihiro Shiraiwa; Koji Iwamoto

    The enzyme adenosine 5?-monophosphate deaminase (AMPD, EC 3.5.4.6) catalyzes the conversion of adenosine 5?-monophosphate\\u000a to inosine 5?-mononucleotide (IMP). IMP is generally known as the compound responsible for the umami taste of the edible red alga Porphyra yezoensis Ueda that is known in Japan as nori. Therefore, we suspect that AMPD plays a key role in providing a favorable nori taste.

  7. Different isoforms of the B-cell mutator activation-induced cytidine deaminase are aberrantly expressed in BCR–ABL1-positive acute lymphoblastic leukemia patients

    Microsoft Academic Search

    I Iacobucci; A Lonetti; F Messa; A Ferrari; D Cilloni; S Soverini; F Paoloni; F Arruga; E Ottaviani; S Chiaretti; M Messina; M Vignetti; C Papayannidis; A Vitale; F Pane; P P Piccaluga; S Paolini; G Berton; A Baruzzi; G Saglio; M Baccarani; R Foà; G Martinelli

    2010-01-01

    The main reason for the unfavorable clinical outcome of BCR–ABL1-positive acute lymphoblastic leukemia (ALL) is genetic instability. However, how normal B-cell precursors acquire the genetic changes that lead to transformation has not yet been completely defined. We investigated the expression of the activation-induced cytidine deaminase (AID) and its role in clinical outcome in 61 adult BCR–ABL1-positive ALL patients. AID expression

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

    Microsoft Academic Search

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

    2004-01-01

    The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) converts ACC, the precursor of the plant hormone ethylene, to ?-ketobutyrate and ammonium. This enzyme has been identified in soil bacteria and has been proposed to play a key role in microbe-plant association. A soluble recombinant ACCD from Pseudomonas putida UW4 of molecular weight 41 kDa has been cloned, expressed, and purified. It showed selectivity

  9. Sedative and electroencephalographic actions of erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA): Relationship to inhibition of brain adenosine deaminase

    Microsoft Academic Search

    Wallace B. Mendelson; Alice Kuruvilla; Thomas Watlington; Kevin Goehl; Steven M. Paul; Phil Skolnick

    1983-01-01

    Parenteral administration of the adenosine deaminase (ADA) inhibitor erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) results in a profound decrease in spontaneous motor activity in mice and rats. The inhibition of cortical ADA activity measured ex vivo parallels the decrease in spontaneous motor activity in a time-dependent manner. Nonetheless, a marked reduction in electroencephalographically defined sleep was observed in rats during a period when both

  10. Metagenomic analysis of the 1-aminocyclopropane-1-carboxylate deaminase gene ( acdS ) operon of an uncultured bacterial endophyte colonizing Solanum tuberosum L

    Microsoft Academic Search

    Branislav Nikolic; Helmut Schwab; Angela Sessitsch

    Deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is a key plant-beneficial trait found in\\u000a many plant growth-promoting bacteria. In this study, we analysed ACC deaminase genes (acdS) of bacterial endophytes colonizing field-grown potato plants. PCR analysis revealed the presence of two types of acdS genes, the dominant one showing high homology to an acdS gene derived from Pseudomonas fluorescens.

  11. Characterization of the Yeast Transcriptome

    Microsoft Academic Search

    Victor E. Velculescu; Lin Zhang; Wei Zhou; Jacob Vogelstein; Munira A. Basrai; Douglas E Bassett; Phil Hieter; Bert Vogelstein; Kenneth W. Kinzler

    1997-01-01

    We have analyzed the set of genes expressed from the yeast genome, herein called the transcriptome, using serial analysis of gene expression. Analysis of 60,633 transcripts revealed 4,665 genes, with expression levels ranging from 0.3 to over 200 transcripts per cell. Of these genes, 1981 had known functions, while 2684 were previously uncharacterized. The integration of positional information with gene

  12. Malassezia Baillon, emerging clinical yeasts.

    PubMed

    Batra, Roma; Boekhout, Teun; Guého, Eveline; Cabañes, F Javier; Dawson, Thomas L; Gupta, Aditya K

    2005-12-01

    The human and animal pathogenic yeast genus Malassezia has received considerable attention in recent years from dermatologists, other clinicians, veterinarians and mycologists. Some points highlighted in this review include recent advances in the technological developments related to detection, identification, and classification of Malassezia species. The clinical association of Malassezia species with a number of mammalian dermatological diseases including dandruff, seborrhoeic dermatitis, pityriasis versicolor, psoriasis, folliculitis and otitis is also discussed. PMID:16084129

  13. Yeast diversity in hypersaline habitats.

    PubMed

    Butinar, L; Santos, S; Spencer-Martins, I; Oren, A; Gunde-Cimerman, N

    2005-03-15

    Thus far it has been considered that hypersaline natural brines which are subjected to extreme solar heating, do not contain non-melanized yeast populations. Nevertheless we have isolated yeasts in eight different salterns worldwide, as well as from the Dead Sea, Enriquillo Lake (Dominican Republic) and the Great Salt Lake (Utah). Among the isolates obtained from hypersaline waters, Pichia guilliermondii, Debaryomyces hansenii, Yarrowia lipolytica and Candida parapsilosis are known contaminants of low water activity food, whereas Rhodosporidium sphaerocarpum, R. babjevae, Rhodotorula laryngis, Trichosporon mucoides, and a new species resembling C. glabrata were not known for their halotolerance and were identified for the first time in hypersaline habitats. Moreover, the ascomycetous yeast Metschnikowia bicuspidata, known to be a parasite of the brine shrimp, was isolated as a free-living form from the Great Salt Lake brine. In water rich in magnesium chloride (bitterns) from the La Trinitat salterns (Spain), two new species provisionally named C. atmosphaerica - like and P. philogaea - like were discovered. PMID:15766773

  14. 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

  15. Improved negative selection protocol for Plasmodium berghei in the rodent malarial model

    PubMed Central

    2012-01-01

    An improved methodology is presented here for transgenic Plasmodium berghei lines that express the negative selectable marker yFCU (a bifunctional protein that combines yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT)) and substitutes delivery of selection drug 5-fluorocytosine (5FC) by intraperitoneal injection for administration via the drinking water of the mice. The improved methodology is shown to be as effective, less labour-intensive, reduces animal handling and animal numbers required for successful selection thereby contributing to two of the "three Rs" of animal experimentation, namely refinement and reduction. PMID:22463060

  16. 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

  17. Genomic Change, Retrotransposon Mobilization and Extensive Cytosine Methylation Alteration in Brassica napus Introgressions from Two Intertribal Hybridizations

    PubMed Central

    Zhang, Xueli; Ge, Xianhong; Shao, Yujiao; Sun, Genlou; Li, Zaiyun

    2013-01-01

    Hybridization and introgression represent important means for the transfer and/or de novo origination of traits and play an important role in facilitating speciation and plant breeding. Two sets of introgression lines in Brassica napus L. were previously established by its intertribal hybridizations with two wild species and long-term selection. In this study, the methods of amplified fragment length polymorphisms (AFLP), sequence-specific amplification polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) were used to determine their genomic change, retrotransposon mobilization and cytosine methylation alteration in these lines. The genomic change revealed by the loss or gain of AFLP bands occurred for ?10% of the total bands amplified in the two sets of introgressions, while no bands specific for wild species were detected. The new and absent SSAP bands appeared for 9 out of 11 retrotransposons analyzed, with low frequency of new bands and their total percentage of about 5% in both sets. MSAP analysis indicated that methylation changes were common in these lines (33.4–39.8%) and the hypermethylation was more frequent than hypomethylation. Our results suggested that certain extents of genetic and epigenetic alterations were induced by hybridization and alien DNA introgression. The cryptic mechanism of these changes and potential application of these lines in breeding were also discussed. PMID:23468861

  18. The Drosophila Cytosine-5 Methyltransferase Dnmt2 Is Associated with the Nuclear Matrix and Can Access DNA during Mitosis

    PubMed Central

    Schaefer, Matthias; Steringer, Julia P.; Lyko, Frank

    2008-01-01

    Cytosine-5 methyltransferases of the Dnmt2 family are highly conserved in evolution and their biological function is being studied in several organisms. Although all structural DNA methyltransferase motifs are present in Dnmt2, these enzymes show a strong tRNA methyltransferase activity. In line with an enzymatic activity towards substrates other than DNA, Dnmt2 has been described to localize to the cytoplasm. Using molecular and biochemical approaches we show here that Dnmt2 is both a cytoplasmic and a nuclear protein. Sub-cellular fractionation shows that a significant amount of Dnmt2 is bound to the nuclear matrix. Sub-cellular localization analysis reveals that Dnmt2 proteins are enriched in actively dividing cells. Dnmt2 localization is highly dynamic during the cell cycle. Using live imaging we observed that Dnmt2-EGFP enters prophase nuclei and shows a spindle-like localization pattern during mitotic divisions. Additional experiments suggest that this localization is microtubule dependent and that Dnmt2 can access DNA during mitotic cell divisions. Our results represent the first comprehensive characterization of Dnmt2 proteins on the cellular level and have important implications for our understanding of the molecular activities of Dnmt2. PMID:18183295

  19. Methylation by a Unique ?-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725

    PubMed Central

    Chung, Daehwan; Farkas, Joel; Huddleston, Jennifer R.; Olivar, Estefania; Westpheling, Janet

    2012-01-01

    Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the Gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute) and that methylation with an endogenous unique ?-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation. PMID:22928042

  20. Methylation by a unique ?-class N4-cytosine methyltransferase is required for DNA transformation of Caldicellulosiruptor bescii DSM6725.

    PubMed

    Chung, Daehwan; Farkas, Joel; Huddleston, Jennifer R; Olivar, Estefania; Westpheling, Janet

    2012-01-01

    Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute) and that methylation with an endogenous unique ?-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation. PMID:22928042

  1. Biochemical behavior of N-oxidized cytosine and adenine bases in DNA polymerase-mediated primer extension reactions

    PubMed Central

    Tsunoda, Hirosuke; Kudo, Tomomi; Masaki, Yoshiaki; Ohkubo, Akihiro; Seio, Kohji; Sekine, Mitsuo

    2011-01-01

    To clarify the biochemical behavior of 2?-deoxyribonucleoside 5?-triphosphates and oligodeoxyribonucleotides (ODNs) containing cytosine N-oxide (Co) and adenine N-oxide (Ao), we examined their base recognition ability in DNA duplex formation using melting temperature (Tm) experiments and their substrate specificity in DNA polymerase-mediated replication. As the result, it was found that the Tm values of modified DNA–DNA duplexes incorporating 2?-deoxyribonucleoside N-oxide derivatives significantly decreased compared with those of the unmodified duplexes. However, single insertion reactions by DNA polymerases of Klenow fragment (KF) (exo?) and Vent (exo?) suggested that Co and Ao selectively recognized G and T, respectively. Meanwhile, the kinetic study showed that the incorporation efficiencies of the modified bases were lower than those of natural bases. Ab initio calculations suggest that these modified bases can form the stable base pairs with the original complementary bases. These results indicate that the modified bases usually recognize the original bases as partners for base pairing, except for misrecognition of dATP by the action of KF (exo?) toward Ao on the template, and the primers could be extended on the template DNA. When they misrecognized wrong bases, the chain could not be elongated so that the modified base served as the chain terminator. PMID:21300642

  2. Assembly of eukaryotic algal chromosomes in yeast

    PubMed Central

    2013-01-01

    Background Synthetic genomic approaches offer unique opportunities to use powerful yeast and Escherichia coli genetic systems to assemble and modify chromosome-sized molecules before returning the modified DNA to the target host. For example, the entire 1 Mb Mycoplasma mycoides chromosome can be stably maintained and manipulated in yeast before being transplanted back into recipient cells. We have previously demonstrated that cloning in yeast of large (>?~?150 kb), high G?+?C (55%) prokaryotic DNA fragments was improved by addition of yeast replication origins every ~100 kb. Conversely, low G?+?C DNA is stable (up to at least 1.8 Mb) without adding supplemental yeast origins. It has not been previously tested whether addition of yeast replication origins similarly improves the yeast-based cloning of large (>150 kb) eukaryotic DNA with moderate G?+?C content. The model diatom Phaeodactylum tricornutum has an average G?+?C content of 48% and a 27.4 Mb genome sequence that has been assembled into chromosome-sized scaffolds making it an ideal test case for assembly and maintenance of eukaryotic chromosomes in yeast. Results We present a modified chromosome assembly technique in which eukaryotic chromosomes as large as ~500 kb can be assembled from cloned ~100 kb fragments. We used this technique to clone fragments spanning P. tricornutum chromosomes 25 and 26 and to assemble these fragments into single, chromosome-sized molecules. We found that addition of yeast replication origins improved the cloning, assembly, and maintenance of the large chromosomes in yeast. Furthermore, purification of the fragments to be assembled by electroelution greatly increased assembly efficiency. Conclusions Entire eukaryotic chromosomes can be successfully cloned, maintained, and manipulated in yeast. These results highlight the improvement in assembly and maintenance afforded by including yeast replication origins in eukaryotic DNA with moderate G?+?C content (48%). They also highlight the increased efficiency of assembly that can be achieved by purifying fragments before assembly. PMID:24325901

  3. 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

  4. Efficacy of marine yeasts and baker's yeast as immunostimulants in Fenneropenaeus indicus: A comparative study

    Microsoft Academic Search

    P. J. Sarlin; Rosamma Philip

    2011-01-01

    Efficacy of marine yeasts Debaryomyces hansenii (S8) and Candida tropicalis (S186) as immunostimulants to Indian white prawn Fenneropenaeus indicus was estimated in comparison with Saccharomyces cerevisiae S36. Biomass of yeast strains was prepared using Malt Extract Agar and incorporated into a standard diet to prepare yeast diets of varying concentrations. F. indicus were fed these diets for a period of

  5. Role of glucose signaling in yeast metabolism

    SciTech Connect

    Dam, K. van [Univ. of Amsterdam (Netherlands). E.C. Slater Inst.

    1996-10-05

    The conversion of glucose to ethanol and carbon dioxide by yeast was the first biochemical pathway to be studied in detail. The initial observation that this process is catalyzed by an extract of yeast led to the discovery of enzymes and coenzymes and laid the foundation for modern biochemistry. In this article, knowledge concerning the relation between uptake of and signaling by glucose in the yeast Saccharomyces cerevisiae is reviewed and compared to the analogous process in prokaryotes. It is concluded that (much) more fundamental knowledge concerning these processes is required before rational redesign of metabolic fluxes from glucose in yeast can be achieved.

  6. 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.

  7. 21 CFR 184.1983 - Bakers yeast extract.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...2011-04-01 2011-04-01 false Bakers yeast extract. 184.1983 Section 184.1983 Food...Substances Affirmed as GRAS § 184.1983 Bakers yeast extract. (a) Bakers yeast extract is the food ingredient resulting...

  8. 21 CFR 184.1983 - Bakers yeast extract.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 2010-04-01 2009-04-01 true Bakers yeast extract. 184.1983 Section 184.1983 Food...Substances Affirmed as GRAS § 184.1983 Bakers yeast extract. (a) Bakers yeast extract is the food ingredient resulting...

  9. 21 CFR 184.1983 - Bakers yeast extract.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...2014-04-01 2014-04-01 false Bakers yeast extract. 184.1983 Section 184.1983 Food...Substances Affirmed as GRAS § 184.1983 Bakers yeast extract. (a) Bakers yeast extract is the food ingredient resulting...

  10. 21 CFR 184.1983 - Bakers yeast extract.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...2013-04-01 2013-04-01 false Bakers yeast extract. 184.1983 Section 184.1983 Food...Substances Affirmed as GRAS § 184.1983 Bakers yeast extract. (a) Bakers yeast extract is the food ingredient resulting...

  11. Drosophila Regulate Yeast Density and Increase Yeast Community Similarity in a Natural Substrate

    PubMed Central

    Stamps, Judy A.; Yang, Louie H.; Morales, Vanessa M.; Boundy-Mills, Kyria L.

    2012-01-01

    Drosophila melanogaster adults and larvae, but especially larvae, had profound effects on the densities and community structure of yeasts that developed in banana fruits. Pieces of fruit exposed to adult female flies previously fed fly-conditioned bananas developed higher yeast densities than pieces of the same fruits that were not exposed to flies, supporting previous suggestions that adult Drosophila vector yeasts to new substrates. However, larvae alone had dramatic effects on yeast density and species composition. When yeast densities were compared in pieces of the same fruits assigned to different treatments, fruits that developed low yeast densities in the absence of flies developed significantly higher yeast densities when exposed to larvae. Across all of the fruits, larvae regulated yeast densities within narrow limits, as compared to a much wider range of yeast densities that developed in pieces of the same fruits not exposed to flies. Larvae also affected yeast species composition, dramatically reducing species diversity across fruits, reducing variation in yeast communities from one fruit to the next (beta diversity), and encouraging the consistent development of a yeast community composed of three species of yeast (Candida californica, C. zemplinina, and Pichia kluvyeri), all of which were palatable to larvae. Larvae excreted viable cells of these three yeast species in their fecal pools, and discouraged the growth of filamentous fungi, processes which may have contributed to their effects on the yeast communities in banana fruits. These and other findings suggest that D. melanogaster adults and their larval offspring together engage in ‘niche construction’, facilitating a predictable microbial environment in the fruit substrates in which the larvae live and develop. PMID:22860093

  12. Prevention of Yeast Spoilage in Feed and Food by the Yeast Mycocin HMK

    PubMed Central

    Lowes, K. F.; Shearman, C. A.; Payne, J.; MacKenzie, D.; Archer, D. B.; Merry, R. J.; Gasson, M. J.

    2000-01-01

    The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts. PMID:10698773

  13. Anhydrobiosis in yeast: influence of calcium and magnesium ions on yeast resistance to dehydration-rehydration.

    PubMed

    Trofimova, Yuliya; Walker, Graeme; Rapoport, Alexander

    2010-07-01

    The influence of calcium and magnesium ions on resistance to dehydration in the yeast, Saccharomyces cerevisiae, was investigated. Magnesium ion availability directly influenced yeast cells' resistance to dehydration and, when additionally supplemented with calcium ions, this provided further significant increase of yeast resistance to dehydration. Gradual rehydration of dry yeast cells in water vapour indicated that both magnesium and calcium may be important for the stabilization of yeast cell membranes. In particular, calcium ions were shown for the first time to increase the resistance of yeast cells to dehydration in stress-sensitive cultures from exponential growth phases. It is concluded that magnesium and calcium ion supplementations in nutrient media may increase the dehydration stress tolerance of S. cerevisiae cells significantly, and this finding is important for the production of active dry yeast preparations for food and fermentation industries. PMID:20487021

  14. Yeast Breads: Made at Home. 

    E-print Network

    Reasonover, Frances

    1971-01-01

    and salt. Cool to lukewarm. Add softened yeast. Blend well. Gradually add flour to form a soft dough. Cover. Let rise in warm place until light and doubled in bulk from 1% to 2 hours. Roll out dough on well-floured surface or pastry cloth to a 15... Turn dough onto surface well dusted with . Knead until smooth, about 20 times. :sired, into crescents, rolls, etc. Place on jed baking sheet. Cover with damp cloth. warm place, free from draft, about 1 hour. F. 10 to 15 minutes, depending on size...

  15. YEAST MEIOSIS Sister kinetochores are mechanically

    E-print Network

    Asbury, Chip

    YEAST MEIOSIS Sister kinetochores are mechanically fused during meiosis I in yeast Krishna K Production of healthy gametes requires a reductional meiosis I division in which replicated sister chromatids comigrate, rather than separate as in mitosis or meiosis II. Fusion of sister kinetochores during meiosis I

  16. Chronological aging leads to apoptosis in yeast

    Microsoft Academic Search

    Eva Herker; Helmut Jungwirth; Katharina A. Lehmann; Corinna Maldener; Kai-Uwe Fröhlich; Silke Wissing; Sabrina Büttner; Markus Fehr; Stephan Sigrist; Frank Madeo

    2004-01-01

    uring the past years, yeast has been successfully established as a model to study mechanisms of apoptotic regulation. However, the beneficial effects of such a cell suicide program for a unicellular organism remained obscure. Here, we demonstrate that chronologi- cally aged yeast cultures die exhibiting typical markers of apoptosis, accumulate oxygen radicals, and show caspase activation. Age-induced cell death is

  17. Yeast flora of grape berries during ripening

    Microsoft Academic Search

    Gianfranco Rosini; Federico Federici; Alessandro Martini

    1982-01-01

    The yeast flora associated with the surface of grapes during ripening was studied with regard to different sectors of the grape skin and the position in the bunch by means of traditional as well as more vigorous preisolation and precounting treatments. The yeast number per square centimeter of skin increases with ripening and is highest in the area immediately surrounding

  18. Yeast: An Experimental Organism for Modern Biology.

    ERIC Educational Resources Information Center

    Botstein, David; Fink, Gerald R.

    1988-01-01

    Discusses the applicability and advantages of using yeasts as popular and ideal model systems for studying and understanding eukaryotic biology at the cellular and molecular levels. Cites experimental tractability and the cooperative tradition of the research community of yeast biologists as reasons for this success. (RT)

  19. Phosphoinositides in yeast: genetically tractable signalling

    Microsoft Academic Search

    Stefaan Wera; Jan C. T. Bergsma; Johan M. Thevelein

    2001-01-01

    Research on signalling through phosphoinositides has made tremendous advances over the last few years. Studies with budding yeast (Saccharomyces cerevisiae) combine the advantage of a eukaryotic system with those of a rapidly growing, genetically modifiable and tractable organism of which the genome is fully sequenced. Hence, despite some differences in phosphoinositide signalling between mammals and yeast (e.g. the absence of

  20. The oenological characteristics of commercial dry yeasts

    Microsoft Academic Search

    Giuseppe Comi; Isabella Croattini; Marilena Marino; Michela Maifreni; Roberto Zironi

    1997-01-01

    Twenty preparations of dry active yeast (18 Saccharomyces cerevisiae and two Saccharomyces bayanus) available in Italy were tested in white and red musts (Moscato, Albana and Sangiovese) to study their oenological characteristics (i.e. fermentation rate, total alcohol and acetic acid production). After the application of chemiometric techniques for descriptive analyses to the results of the oenological assessment, the yeasts were

  1. PERSPECTIVES Chromatin Conformation of Yeast Centromeres

    E-print Network

    PERSPECTIVES Chromatin Conformation of Yeast Centromeres KERRY S. BLOOM, ENRIQUE AMAYA, JOHN CARBON The centromere region of Saccharomyces cerevisiae chromosome III has been replaced by various DNA fragments from the centromere regions of yeast chromosomes III and Xl. A 289-base pair centromere (CEN3) sequence can stabilize

  2. Definition, classification and nomenclature of the yeasts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This submission includes sections for the Preface, Use of this Book, Table of Contents and a chapter entitled Definition, classification and nomenclature of the yeasts, which are to be published in The Yeasts, A Taxonomic Study, 5th edition. This book has been prepared by a team of international ex...

  3. Topical Therapy for Mucosal Yeast Infections

    Microsoft Academic Search

    Paul R. Summers

    2011-01-01

    Mucosal yeast infection is best understood as a consequence of compromised mucosal cell-mediated and innate immunity. Defense against oral candidiasis is dominantly cell mediated. The innate immune system may play the main role in regulating vulvovaginal yeast infection. Conditions that compromise cell-mediated immunity such as leukemia, severe illness and HIV infection must be considered as predisposing factors for recurrent oral

  4. Life Stress and Chronic Yeast Infections

    Microsoft Academic Search

    Nancy A. Williams; Jerry L. Deffenbacher

    1983-01-01

    This study investigated the relationships of positive and negative life change to yeast infections in women having a gynecological examination at a university health center. Subjects completed the Life Experiences Survey and a questionnaire about experiences with yeast infections and received, as a routine part of their visitation of the gynecology service, a standard gynecological examination, including a laboratory test

  5. Can yeast transcriptomics help improve wine fermentation?

    Microsoft Academic Search

    C. Varela; J. Cárdenas; E. Agosin

    Wine fermentation is a dynamic and complex process in which the yeast cell is subjected to multiple stress conditions. A successful adaptation involves changes in gene expression profiles where a large number of genes are up- or down-regulated. Functional genomic approaches are com- monly used to obtain global gene expression profiles, providing a comprehensive view of yeast physiology. We used

  6. Fermentation studies using Saccharomyces diastaticus yeast strains

    SciTech Connect

    Erratt, J.A.; Stewart, G.G.

    1981-01-01

    The yeast species, Saccharomyces diastaticus, has the ability to ferment starch and dextrin, because of the extracellular enzyme, glucoamylase, which hydrolyzes the starch/dextrin to glucose. A number of nonallelic genes--DEX 1, DEX 2, and dextrinase B which is allelic to STA 3--have been isolated, which impart to the yeast the ability to ferment dextrin. Various diploid yeast strains were constructed, each being either heterozygous or homozygous for the individual dextrinase genes. Using 12 (sup 0) plato hopped wort (30% corn adjunct) under agitated conditions, the fermentation rates of the various diploid yeast strains were monitored. A gene-dosage effect was exhibited by yeast strains containing DEX 1 or DEX 2, however, not with yeast strains containing dextrinase B (STA 3). The fermentation and growth rates and extents were determined under static conditions at 14.4 C and 21 C. With all yeast strains containing the dextrinase genes, both fermentation and growth were increased at the higher incubation temperature. Using 30-liter fermentors, beer was produced with the various yeast strains containing the dextrinase genes and the physical and organoleptic characteristics of the products were determined. The concentration of glucose in the beer was found to increase during a 3-mo storage period at 21 C, indicating that the glucoamylase from Saccharomyces diastaticus is not inactivated by pasteurization. (Refs. 36).

  7. Computational study of substituent effects on the interaction energies of hydrogen-bonded Watson-Crick cytosine: guanine base pairs.

    PubMed

    Xue, Chunxia; Popelier, Paul L A

    2008-04-24

    The substituent effects on interaction energies of hydrogen-bonded DNA Watson-Crick base pairs in the gas phase were captured in a model using ab initio descriptors (at the B3LYP/6-311+G(2d,p) level). While forming a noncovalently bonded complex with unsubstituted guanine (G), cytosine (C) carried 42 possible substituents both at the C6 position (C6X:G) and at the C5 position (C5X:G). We rationalize why complexes possessing a more strongly electron-withdrawing group in CX form less stable base pairs. Multivariate linear regression constructed the quantitative relationships between the interaction energies of the complexes and the descriptors, which were drawn from quantum chemical topology (QCT). For the C6X dataset, the best model yielded r2 = 0.93 and a root-mean-square (rms) energy of 0.53 kJ/mol for the 28 complexes in the training set. This model was evaluated by an external test set (14 complexes), yielding an r2 value of 0.96 and an rms error of 0.42 kJ/mol. For the C5X dataset, the QCT descriptors generated a linear model, with r2 values of 0.92 and 0.97 and rms values of 1.69 and 1.24 kJ/mol for the training set (31 compounds) and the external test set (11 compounds), respectively. The models built here could therefore be useful for the assessment of the interaction energy of C6X:G and C5X:G purely from monomeric data. PMID:18373374

  8. 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

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

    PubMed

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

    2014-11-14

    Abstract Poliovirus, the causative agent of poliomyelitis, is a human enterovirus and member of the Picornaviridae family. An effective live-attenuated poliovirus vaccine strain (Sabin 1) has been developed and has protected humans from polio. However, a few cases of vaccine virulence reversion have been documented in several countries. For instance, circulating type 1 vaccine-derived poliovirus is a highly pathogenic poliovirus that evolved from an avirulent strain, but the mechanism by which vaccine strains undergo reversion remains unclear. In this study, vaccine strains exhibited A to G/U to C and G to A/C to U hypermutations in the reversed evolution of Sabin 1. Furthermore, the mutation ratios of U to C and C to U were higher than those of other mutation types. Dinucleotide editing context was then analyzed. Results showed that A to G and U to C mutations exhibited preferences similar to adenosine deaminases acting on RNA (ADAR). Hence, ADARs may participate in poliovirus vaccine evolution. PMID:25330844

  10. A rare case of complete human erythrocyte AMP deaminase deficiency due to two novel missense mutations in AMPD3.

    PubMed

    Yamada, Y; Goto, H; Wakamatsu, N; Ogasawara, N

    2001-01-01

    Human erythrocyte AMP deaminase (AMPD3) deficiency is a clinically asymptomatic condition characterized by a 50% increase in steady-state levels of ATP in affected cells. The deficiency in Japanese is associated 75% of the time with the same mutation of R573C, and 25% of the time with other heterogeneous mutations of the AMPD3 gene. The heterozygote frequency was estimated at about 1/30. We previously reported five Japanese individuals who had a complete deficiency of AMPD3. Four were homozygotes for the major mutation of R573C; however, one female did not have the R573C allele. To clarify the mutations in her AMPD3 gene, we analyzed the AMPD3 gene and detected a minor mutation, W450R, derived from the mother and a novel mutation,Q712P, derived from the father. The expression study using AMPD3 cDNA containing both mutations showed that each mutation completely reduced the enzyme activity of AMPD3. As the frequency of carriers heterozygous for these mutations seems to be very low, identifying them may lead to a better understanding of the genetic background of populations in Japan. PMID:11139257

  11. Efficiency of purine utilization by Helicobacter pylori: roles for adenosine deaminase and a NupC homolog.

    PubMed

    Miller, Erica F; Vaish, Soumya; Maier, Robert J

    2012-01-01

    The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase. PMID:22701700

  12. Effects of aqueous extract from Silybum marianum on adenosine deaminase activity in cancerous and noncancerous human gastric and colon tissues

    PubMed Central

    Öztürk, 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

  13. Fate mapping for activation-induced cytidine deaminase (AID) marks non-lymphoid cells during mouse development.

    PubMed

    Rommel, Philipp C; Bosque, David; Gitlin, Alexander D; Croft, Gist F; Heintz, Nathaniel; Casellas, Rafael; Nussenzweig, Michel C; Kriaucionis, Skirmantas; Robbiani, Davide F

    2013-01-01

    The Aicda gene encodes Activation-Induced cytidine Deaminase (AID), an enzyme essential for remodeling antibody genes in mature B lymphocytes. AID is also responsible for DNA damage at oncogenes, leading to their mutation and cancer-associated chromosome translocation in lymphoma. We used fate mapping and AID(GFP) reporter mice to determine if AID expression in the mouse extends beyond lymphocytes. We discovered that AID(cre) tags a small fraction of non-lymphoid cells starting at 10.5 days post conception (dpc), and that AID(GFP+) cells are detectable at dpc 11.5 and 12.5. Embryonic cells are tagged by AID(cre) in the submandibular region, where conditional deletion of the tumor suppressor PTEN causes squamous papillomas. AID(cre) also tags non-lymphoid cells in the embryonic central nervous system. Finally, in the adult mouse brain, AID(cre) marks a small fraction of diverse neurons and distinct neuronal populations, including pyramidal cells in cortical layer IV. PMID:23861962

  14. 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

  15. A carbon nanotubes based fluorescent aptasensor for highly sensitive detection of adenosine deaminase activity and inhibitor screening in natural extracts.

    PubMed

    Hu, Kun; Huang, Yong; Wang, Sheng'e; Zhao, Shulin

    2014-07-01

    A carbon nanotubes (CNTs) based fluorescent aptasensor was developed for adenosine deaminase (ADA) activity detection and inhibitor screening by using adenosine (AD) as the substrate. This sensing system consists of CNTs, AD, split anti-AD aptamer fragment and dye-labeled aptamer fragment. In the absence of ADA, two aptamer fragments bind simultaneously with AD to form an AD-aptamer complex. This AD-aptamer complex cannot adsorb onto CNTs, and has high fluorescence intensity. When ADA is introduced into this system, ADA can convert AD into inosine, which has not affinity to the split anti-AD aptamer fragment. Thus, the split anti-AD aptamer fragments were adsorbed onto CNTs via strong ?-? stacking interactions, resulting in the quenching of the fluorescence of the dye-labeled aptamer fragment. The proposed aptasensor can detect ADA activity from 0.005 to 0.2U/mL with a low detection limit of 0.002 U/mL. Moreover, it has been also demonstrated that this CNTs-based fluorescence aptasensor is suitable for ADA inhibitor screening from traditional Chinese medicine (TCM). Considering the superior sensitivity and specificity, the proposed CNTs-based fluorescent aptasensor can be expected to provide a simple, cost-effective and sensitive platform for the detection of ADA activity and screening of potential drugs. PMID:24682016

  16. The A2B adenosine receptor colocalizes with adenosine deaminase in resting parietal cells from gastric mucosa.

    PubMed

    Arin, R M; Vallejo, A I; Rueda, Y; Fresnedo, O; Ochoa, B

    2015-01-01

    The A2B adenosine receptor (A2BR) mediates biological responses to extracellular adenosine in a wide variety of cell types. Adenosine deaminase (ADA) can degrade adenosine and bind extracellularly to adenosine receptors. Adenosine modulates chloride secretion in gastric glands and gastric mucosa parietal cells. A close functional link between surface A2BR and ADA has been found on cells of the immune system, but whether this occurs in the gastrointestinal tract is unknown. The goal of this study was to determine whether A2BR and ADA are coexpressed at the plasma membrane of the acid-secreting gastric mucosa parietal cells. We used isolated gastric parietal cells after purification by centrifugal elutriation. The membrane fraction was obtained by sucrose gradient centrifugation. A2BR mRNA expression was analyzed by RT-PCR. The surface expression of A2BR and ADA proteins was evaluated by Western blotting, flow cytometry and confocal microscopy. Our findings demonstrate that A2BR and ADA are expressed in cell membranes isolated from gastric parietal cells. They show a high degree of colocalization that is particularly evident in the surface of contact between parietal cells. The confocal microscopy data together with flow cytometry analysis suggest a tight association between A2BR and ADA that might be specifically linked to glandular secretory function. PMID:25754047

  17. Anaerobic digestion of food waste using yeast.

    PubMed

    Suwannarat, Jutarat; Ritchie, Raymond J

    2015-08-01

    Fermentative breakdown of food waste seems a plausible alternative to feeding food waste to pigs, incineration or garbage disposal in tourist areas. We determined the optimal conditions for the fermentative breakdown of food waste using yeast (Saccharomyces cerevisiae) in incubations up to 30days. Yeast efficiently broke down food waste with food waste loadings as high as 700g FW/l. The optimum inoculation was ?46×10(6)cells/l of culture with a 40°C optimum (25-40°C). COD and BOD were reduced by ?30-50%. Yeast used practically all the available sugars and reduced proteins and lipids by ?50%. Yeast was able to metabolize lipids much better than expected. Starch was mobilized after very long term incubations (>20days). Yeast was effective in breaking down the organic components of food waste but CO2 gas and ethanol production (?1.5%) were only significant during the first 7days of incubations. PMID:25987287

  18. Encapsulation of yeast cells in colloidosomes.

    PubMed

    Keen, Polly H R; Slater, Nigel K H; Routh, Alexander F

    2012-01-17

    Polymeric colloidosomes encapsulating viable Baker's yeast cells were prepared. To make the capsules, an aqueous suspension of 153 nm poly(methyl methacrylate-co-butyl acrylate) latex particles plus yeast cells is emulsified in a continuous phase of sunflower oil. By adding a small amount of ethanol to the oil phase, the latex particles at the surface of the emulsion droplets aggregate, forming the colloidosome shells. The microcapsules have been examined using optical, confocal, and scanning electron microscopies. The viability of the yeast cells was tested using fluorescent molecular probes. The encapsulated Baker's yeast cells were able to metabolize glucose from solution, although at a slower rate compared to nonencapsulated yeast. This demonstrates diffusion limitation through the colloidosome shell. The diffusive resistance could be increased by manufacturing colloidosomes with a double latex shell. PMID:22149136

  19. Ultrastructural organization of yeast chromatin

    PubMed Central

    1982-01-01

    The ultrastructural organization of yeast chromatin was examined in Miller spread preparations of samples prepared from spheroplasts or isolated nuclei of Saccharomyces cerevisiae. Micrographs from preparations dispersed in 1 mM Tris (pH 7.2) illustrate that the basic chromatin fiber in yeast exists in two ultrastructurally distinct conformations. The majority (up to 95%) of the chromatin displays a beaded nucleosomal organization, although adjacent nucleosomes are separated by internucleosomal linkers of variable lengths. Ribonucleoprotein (RNP) fibrils are only occasionally associated with chromatin displaying the conformation. The remaining 5-10% of the chromatin appears to be devoid of discrete nucleosomes and has a smooth contour with a fiber diameter of 30-40 A. Transcriptional units, including putative ribosomal precursor RNA genes, defined by the presence of nascent RNP fibrils are restricted to chromatin displaying this smooth morphology. Chromatin released from nuclei in the presence of 5 mM Mg++ displays higher-order chromatin fibers, 200-300 A in diameter, these fibers appear to be arranged in a manner than reflects the two forms of the basic chromatin fiber. PMID:7040415

  20. New search for pectolytic yeasts.

    PubMed

    Biely, P; Sláviková, E

    1994-01-01

    A new screening method for pectin-depolymerizing microorganisms is described. The method is based on precipitation of non-hydrolyzed citrus pectin with hexadecyltrimethylammonium bromide in a medium solidified with a bacterial gelling gum. A substrate depolymerized by the secreted enzymes does not precipitate, and the positive strains thus show transparent areas around the colonies. The method was used to screen 300 yeast and yeast-like microorganisms belonging to 52 different genera. The secretion of pectin-depolymerizing enzymes occurred with different frequencies in 13 genera (69 positive strains of 207 tested), the lowest frequency being found in the genus Candida (13 positive out of 125 strains tested) and the highest frequency in the genera Aureobasidium (4 of 6) Cryptococcus (29 of 38), Geotrichum (4 of 9), Kluyveromyces (5 of 5), Rhodosporidium (2 of 2), Leucosporidium (2 of 2), Trichosporon (3 of 6) and Ustilago (2 of 2). Strains giving the highest number of harvested cells after growth on pectin in a liquid medium have been identified. PMID:8549997

  1. Photoinduced electron detachment and proton transfer: the proposal for alternative path of formation of triplet states of guanine (G) and cytosine (C) pair.

    PubMed

    Gu, Jiande; Wang, Jing; Leszczynski, Jerzy

    2015-02-12

    A viable pathway is proposed for the formation of the triplet state of the GC Watson-Crick base pair. It includes the following steps: (a) a low-energy electron is captured by cytosine in the GC pair, forming the cytosine base-centered radical anion GC(-•); and (b) photoradiation with energy around 5 eV initiates the electron detachment from either cytosine (in the gas phase) or guanine (in aqueous solutions). This triggers interbase proton transfer from G to C, creating the triplet state of the GC pair. Double proton transfer involving the triplet state of GC pair leads to the formation of less stable tautomer G(N2-H)(•)C(O2H)(•). Tautomerization is accomplished through a double proton transfer process in which one proton at the N3 of C(H)(•) migrates to the N1 of G(-H)(•); meanwhile, the proton at the N2 of G transfers to the O2 of C. This process is energetically viable; the corresponding activation energy is around 12-13 kcal/mol. The base-pairing energy of the triplet is found to be ?3-5 kcal/mol smaller than that of the singlet state. Thus, the formation of the triplet state GC pair in DNA double strand only slightly weakens its stability. The obtained highly reactive radicals are expected to cause serious damage in the DNA involved in biochemical processes, such as DNA replication where radicals are exposed in the single strands. PMID:25340559

  2. A Caspase-Related Protease Regulates Apoptosis in Yeast

    Microsoft Academic Search

    Frank Madeo; Eva Herker; Corinna Maldener; Silke Wissing; Stephan Lächelt; Mark Herlan; Markus Fehr; Kirsten Lauber; Stephan J Sigrist; Sebastian Wesselborg; Kai-Uwe Fröhlich

    2002-01-01

    Yeast can undergo cell death accompanied by cellular markers of apoptosis. However, orthologs of classical mammalian apoptosis regulators appeared to be missing from the yeast genome, challenging a common mechanism of yeast and mammalian apoptosis. Here we investigate Yor197w, a yeast protein with structural homology to mammalian caspases, and demonstrate caspase-like processing of the protein. Hydrogen peroxide treatment induces apoptosis

  3. Original article Screening for the potential probiotic yeast strains

    E-print Network

    Paris-Sud XI, Université de

    Original article Screening for the potential probiotic yeast strains from raw milk to assimilate yeast strains, isolated from raw milk, were tested to obtain potential probiotic yeasts for assimilating cholesterol. During in vitro tests, 17 yeast strains were capable of growth in bile salt solutions, and most

  4. Genome Analysis The pattern and evolution of yeast promoter

    E-print Network

    Barkai, Naama

    Genome Analysis The pattern and evolution of yeast promoter bendability Itay Tirosh1 , Judith-less promoters from 11 yeast species, whereas the position of the rigid DNA varies substantially among species. cerevisiae promoters We examined the bendability pattern of yeast promoters. The promoters of many yeast

  5. Computational Predictions of Structures of Multichromosomes of Budding Yeast

    E-print Network

    Liang, Jie

    Computational Predictions of Structures of Multichromosomes of Budding Yeast (Accepted, Conf Proc of budding yeast nucleus. We successfully generated a large number of model genomes of yeast with appropriate yeast genome realistically. The model developed here provides a general computational framework

  6. Yeast (in press) Published online in Wiley InterScience

    E-print Network

    Lycan, Deborah E.

    Yeast Yeast (in press) Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/yea.1502 Yeast Functional Analysis Report A suite of Gateway cloning vectors for high of overexpression plasmids containing the entire complement of yeast open reading frames (ORFs) have recently been

  7. The wine and beer yeast Dekkera bruxellensis.

    PubMed

    Schifferdecker, Anna Judith; Dashko, Sofia; Ishchuk, Olena P; Piškur, Jure

    2014-09-01

    Recently, the non-conventional yeast Dekkera bruxellensis has been gaining more and more attention in the food industry and academic research. This yeast species is a distant relative of Saccharomyces cerevisiae and is especially known for two important characteristics: on the one hand, it is considered to be one of the main spoilage organisms in the wine and bioethanol industry; on the other hand, it is 'indispensable' as a contributor to the flavour profile of Belgium lambic and gueuze beers. Additionally, it adds to the characteristic aromatic properties of some red wines. Recently this yeast has also become a model for the study of yeast evolution. In this review we focus on the recently developed molecular and genetic tools, such as complete genome sequencing and transformation, to study and manipulate this yeast. We also focus on the areas that are particularly well explored in this yeast, such as the synthesis of off-flavours, yeast detection methods, carbon metabolism and evolutionary history. PMID:24932634

  8. Novel aspects of tetramer assembly and N-terminal domain structure and function are revealed by recombinant expression of human AMP deaminase isoforms.

    PubMed

    Mahnke-Zizelman, D K; Tullson, P C; Sabina, R L

    1998-12-25

    AMP deaminase isoforms purified from endogenous sources display smaller than predicted subunit molecular masses, whereas baculoviral expression of human AMPD1 (isoform M) and AMPD3 (isoform E) cDNAs produces full-sized recombinant enzymes. However, nearly 100 N-terminal amino acid residues are cleaved from each recombinant polypeptide during storage at 4 degreesC. Expression of N-truncated cDNAs (DeltaL96AMPD1 and DeltaM90AMPD3) produces stable recombinant enzymes exhibiting subunit molecular masses and kinetic properties that are similar to those reported for purified isoforms M and E. Conversely, wild type recombinant isoforms display significantly higher Km(app) values in the absence of ATP. Gel filtration analysis demonstrates native tetrameric structures for all recombinant proteins, except the wild type AMPD1 enzyme, which forms aggregates of tetramers that disperse upon cleavage of N-terminal residues at 4 degreesC. These data: 1) confirm that available literature on AMP deaminase is likely derived from N-truncated enzymes and 2) are inconsistent with a new model proposing native trimeric structure of an N-truncated rabbit skeletal muscle AMP deaminase (Ranieri-Raggi, M., Montali, U., Ronca, F., Sabbatini, A., Brown, P. E., Moir, A. J. G., and Raggi, A. (1997) Biochem. J. 326, 641-648). N-terminal residues also influence actomyosin-binding properties of the enzyme, which are enhanced and suppressed by AMPD1 and AMPD3 sequences, respectively. Finally, co-expression of AMPD1 and AMPD3 recombinant polypeptides produces tetrameric enzymes with either isoform-specific or mixed subunits, and also reveals that tetramer assembly is driven by relative polypeptide abundance with no apparent preference for like subunits. PMID:9857047

  9. Alpha-keto acids are novel siderophores in the genera Proteus, Providencia, and Morganella and are produced by amino acid deaminases.

    PubMed

    Drechsel, H; Thieken, A; Reissbrodt, R; Jung, G; Winkelmann, G

    1993-05-01

    Growth promotion and iron transport studies revealed that certain alpha-keto acids generated by amino acid deaminases, by enterobacteria of the Proteus-Providencia-Morganella group (of the tribe Proteeae), show significant siderophore activity. Their iron-binding properties were confirmed by the chrome azurol S assay and UV spectra. These compounds form ligand-to-metal charge transfer bands in the range of 400 to 500 nm. Additional absorption bands of the enolized ligands at 500 to 700 nm are responsible for color formation. Siderophore activity was most pronounced with alpha-keto acids possessing an aromatic or heteroaromatic side chain, like phenylpyruvic acid and indolylpyruvic acid, resulting from deamination of phenylalanine and tryptophan, respectively. In addition, alpha-keto acids possessing longer nonpolar side chains, like alpha-ketoisocaproic acid or alpha-ketoisovaleric acid and even alpha-ketoadipic acid, also showed siderophore activity which was absent or negligible with smaller alpha-keto acids or those possessing polar functional groups, like pyruvic acid, alpha-ketobutyric acid, or alpha-ketoglutaric acid. The fact that deaminase-negative enterobacteria, like Escherichia coli and Salmonella spp., could not utilize alpha-keto acids supports the view that specific iron-carboxylate transport systems have evolved in members of the tribe Proteeae and are designed to recognize ferric complexes of both alpha-hydroxy acids and alpha-keto acids, of which the latter can easily be generated by L-amino acid deaminases in an amino acid-rich medium. Exogenous siderophores, like ferric hydroxamates (ferrichromes) and ferric polycarboxylates (rhizoferrin and citrate), were also utilized by members of the tribe Proteeae. PMID:8478334

  10. Quality assessment of lager brewery yeast samples and strains using barley malt extracts with anti-yeast activity.

    PubMed

    van Nierop, Sandra N E; Axcell, Barry C; Cantrell, Ian C; Rautenbach, Marina

    2009-04-01

    Membrane active anti-yeast compounds, such as antimicrobial peptides and proteins, cause yeast membrane damage which is likely to affect yeast vitality and fermentation performance, parameters which are notoriously difficult to analyse. In this work the sensitivity of lager brewery yeast strains towards barley malt extracts with anti-yeast activity was assessed with an optimised assay. It was found that yeast, obtained directly from a brewery, was much more sensitive towards the malt extracts than the same yeast strain propagated in the laboratory. Sensitivity to the malt extracts increased during the course of a laboratory scale fermentation when inoculated with brewery yeast. As the assay was able to differentiate yeast samples with different histories, it shows promise as a yeast quality assay measuring the yeast's ability to withstand stress which can be equated to vitality. The assay was also able to differentiate between different lager yeast strains of Saccharomyces cerevisiae propagated in the laboratory when challenged with a number of malt extracts of varying anti-yeast activity. The assessment of yeast strains in the presence of malt extracts will lead to the identification of yeast strains with improved quality/vitality that can withstand malt-associated anti-yeast activity during brewery fermentations. PMID:19171262

  11. Yeast is one of the most common causes of vaginal infections. Yeast infections occur when a fungus called

    E-print Network

    Virginia Tech

    Yeast is one of the most common causes of vaginal infections. Yeast infections occur when a fungus bacteria in the vagina, which creates an imbalance and promotes yeast proliferation. Excessive fatigue, stress, or illness may lower the body's ability to control excessive yeast growth. Tight clothing

  12. Yeast Transformation (introducing plasmid vector into a yeast strain): This protocol is a modification (shortened version) of "The BEST

    E-print Network

    Yeast Transformation (introducing plasmid vector into a yeast strain): This protocol://www.umanitoba.ca/medicine/biochem/gietz/Trafo.html) 1. Inoculate 5 ml of YPD with a yeast colony from plate. 2. Grow culture overnight at 300 C. 3 and centrifuging at 1750xg (high speed in clinical centrifuge) for 2 minutes. 6. Carefully pour media off of yeast

  13. MAP kinase dynamics in yeast.

    PubMed

    van Drogen, F; Peter, M

    2001-09-01

    MAP kinase pathways play key roles in cellular responses towards extracellular signals. In several cases, the three core kinases interact with a scaffold molecule, but the function of these scaffolds is poorly understood. They have been proposed to contribute to signal specificity, signal amplification, or subcellular localization of MAP kinases. Several MAP kinases translocate to the nucleus in response to their activation, suggesting that nuclear transport may provide a regulatory mechanism. Here we describe new applications for Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP), to study dynamic translocations of MAPKs between different subcellular compartments. We have used these methods to measure the nuclear/cytoplasmic dynamics of several yeast MAP kinases, and in particular to address the role of scaffold proteins for MAP-kinase signaling. PMID:11730324

  14. Carboxylation of cytosine (5caC) in the CG dinucleotide in the E-box motif (CGCAG|GTG) increases binding of the Tcf3|Ascl1 helix-loop-helix heterodimer 10-fold.

    PubMed

    Golla, Jaya Prakash; Zhao, Jianfei; Mann, Ishminder K; Sayeed, Syed K; Mandal, Ajeet; Rose, Robert B; Vinson, Charles

    2014-06-27

    Three oxidative products of 5-methylcytosine (5mC) occur in mammalian genomes. We evaluated if these cytosine modifications in a CG dinucleotide altered DNA binding of four B-HLH homodimers and three heterodimers to the E-Box motif CGCAG|GTG. We examined 25 DNA probes containing all combinations of cytosine in a CG dinucleotide and none changed binding except for carboxylation of cytosine (5caC) in the strand CGCAG|GTG. 5caC enhanced binding of all examined B-HLH homodimers and heterodimers, particularly the Tcf3|Ascl1 heterodimer which increased binding ~10-fold. These results highlight a potential function of the oxidative products of 5mC, changing the DNA binding of sequence-specific transcription factors. PMID:24835951

  15. Pityrosporum yeasts--what's new?

    PubMed

    Faergemann, J

    1997-01-01

    The lipophilic yeast Pityrosporum ovale is a member of the normal human cutaneous flora in adults but also associated with several skin diseases. In pityriasis versicolor, under the influence of predisposing factors, P. ovale changes from the round blastospore form to the mycelial form. A great problem in pityriasis versicolor is the high rate of recurrence and to avoid this a prophylactic treatment is mandatory. Pityrosporum folliculitis is a chronic disease characterized by pruritic follicular papules and pustules located primarily on the upper trunk, neck and upper arms. In direct microscopy clusters of round budding yeast cells are found. The disease responds rapidly to antimycotic therapy. There are now many studies indicating that P. ovale plays an important role in seborrhoeic dermatitis. Many of these are treatment studies showing a good effect of antimycotics paralleled by a reduction in number of organisms. Severe seborrhoeic dermatitis often difficult to treat is associated with AIDS. In peripheral blood from a high number of patients with seborrhoeic dermatitis we found an increase in number of natural killer T-cells and decreased PHA and Con-A stimulation. Secondary we found low serum IgG antibody titres in patients compared to controls. Other studies have found a reduced lymphocyte stimulation reaction when lymphocytes from patients with seborrhoeic dermatitis were stimulated with a P. ovale extract. Additionally, IL-2 and IFN gamma production by lymphocytes from patients was markedly depressed and IL-10 synthesis were increased after stimulation with P. ovale extract. The majority of adult patients with atopic dermatitis localized to the head, neck and scalp are prick-test positive to a protein P. ovale extract. One study showed that p. ovale extracts increased IL-4, IL-10 and IgE synthesis in patients with atopic dermatitis. There are also treatment studies indicating that antifungal treatment may be beneficial in these patients. PMID:9370147

  16. Assimilation spectrum of the yeast Candida utilis 49 used for producing fodder yeast from synthetic ethanol

    Microsoft Academic Search

    M. Šestáková

    1976-01-01

    Oxidizing and assimilating ability of the yeastCandida utilis 49 was tested with 21 different low-boiling organic compounds which come as components of raw synthetic ethanol. The highest\\u000a yields of yeast dry weight were obtained with ethanol (72.0%), propanol (48.2%), ethyl acetate (43.4%) and acetic acid (34.2%).\\u000a To a minor extent, the yeast was capable of utilizing also 2-propanol, butanol and

  17. Signature-Based Small Molecule Screening Identifies Cytosine Arabinoside as an EWS/FLI Modulator in Ewing Sarcoma

    PubMed Central

    Stegmaier, Kimberly; Wong, Jenny S; Ross, Kenneth N; Chow, Kwan T; Peck, David; Wright, Renee D; Lessnick, Stephen L; Kung, Andrew L; Golub, Todd R

    2007-01-01

    Background The presence of tumor-specific mutations in the cancer genome represents a potential opportunity for pharmacologic intervention to therapeutic benefit. Unfortunately, many classes of oncoproteins (e.g., transcription factors) are not amenable to conventional small-molecule screening. Despite the identification of tumor-specific somatic mutations, most cancer therapy still utilizes nonspecific, cytotoxic drugs. One illustrative example is the treatment of Ewing sarcoma. Although the EWS/FLI oncoprotein, present in the vast majority of Ewing tumors, was characterized over ten years ago, it has never been exploited as a target of therapy. Previously, this target has been intractable to modulation with traditional small-molecule library screening approaches. Here we describe a gene expression–based approach to identify compounds that induce a signature of EWS/FLI attenuation. We hypothesize that screening small-molecule libraries highly enriched for FDA-approved drugs will provide a more rapid path to clinical application. Methods and Findings A gene expression signature for the EWS/FLI off state was determined with microarray expression profiling of Ewing sarcoma cell lines with EWS/FLI-directed RNA interference. A small-molecule library enriched for FDA-approved drugs was screened with a high-throughput, ligation-mediated amplification assay with a fluorescent, bead-based detection. Screening identified cytosine arabinoside (ARA-C) as a modulator of EWS/FLI. ARA-C reduced EWS/FLI protein abundance and accordingly diminished cell viability and transformation and abrogated tumor growth in a xenograft model. Given the poor outcomes of many patients with Ewing sarcoma and the well-established ARA-C safety profile, clinical trials testing ARA-C are warranted. Conclusions We demonstrate that a gene expression–based approach to small-molecule library screening can identify, for rapid clinical testing, candidate drugs that modulate previously intractable targets. Furthermore, this is a generic approach that can, in principle, be applied to the identification of modulators of any tumor-associated oncoprotein in the rare pediatric malignancies, but also in the more common adult cancers. PMID:17425403

  18. Targeting the Immune System to Fight Cancer Using Chemical Receptor Homing Vectors Carrying Polyinosine/Cytosine (PolyIC)

    PubMed Central

    Levitzki, Alexander

    2012-01-01

    Cancer researchers have been looking for ways to harness the immune system and to reinstate immune surveillance, to kill cancer cells without collateral damage. Here we scan current approaches to targeting the immune system against cancer, and emphasize our own approach. We are using chemical vectors attached to a specific ligand, to introduce synthetic dsRNA, polyinosine/cytosine (polyIC), into tumors. The ligand binds to a receptor protein that is overexpressed on the surface of the tumor cells. Upon ligand binding, the receptor complex is internalized, introducing the polyIC into the cell. In this fashion a large amount of synthetic dsRNA can be internalized, leading to the activation of dsRNA-binding proteins, such as dsRNA dependent protein kinase (PKR), Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-1), and melanoma differentiation-associated gene 5 (MDA5). The simultaneous activation of these signaling proteins leads to the rapid demise of the targeted cell and to cytokine secretion. The cytokines lead to a strong bystander effect and to the recruitment of immune cells that converge upon the targeted cells. The bystander effects lead to the destruction of neighboring tumor cells not targeted themselves by the vector. Normal cells, being more robust than tumor cells, survive. This strategy has several advantages: (1) recruitment of the immune system is localized to the tumor. (2) The response is rapid, leading to fast tumor eradication. (3) The bystander effects lead to the eradication of tumor cells not harboring the target. (4) The multiplicity of pro-death signaling pathways elicited by PolyIC minimizes the likelihood of the emergence of resistance. In this chapter we focus on EGFR as the targeted receptor, which is overexpressed in many tumors. In principle, the strategy can be extended to other tumors that overexpress a protein that can be internalized by a ligand, which can be a small molecule, a single chain antibody, or an affibody. PMID:22649773

  19. Mechanistic Studies of 1-Aminocyclopropane-1-carboxylate Deaminase (ACCD): Characterization of an Unusual PLP-dependent Reaction

    PubMed Central

    Thibodeaux, Christopher J.; Liu, Hung-wen

    2011-01-01

    1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5?-phosphate (PLP) dependent enzyme that cleaves the cyclopropane ring of ACC, to give ?-ketobutyric acid and ammonia as products. The cleavage of the C?-C? bond of an amino acid substrate is a rare event in PLP-dependent enzyme catalysis. Potential chemical mechanisms involving nucleophile- or acid-catalyzed cyclopropane ring opening have been proposed for the unusual transformation catalyzed by ACCD, but the actual mode of cyclopropane ring cleavage remains obscure. In this report, we aim to elucidate the mechanistic features of ACCD catalysis by investigating the kinetic properties of ACCD from Pseudomonas sp. ACP and several of its mutant enzymes. Our studies suggest that the pKa of the conserved active site residue, Tyr294, is lowered by a hydrogen bonding interaction with a second conserved residue, Tyr268. This allows Tyr294 to deprotonate the incoming amino group of ACC in order to initiate the aldimine exchange reaction between ACC and the PLP coenzyme, and also likely helps to activate Tyr294 for a role as nucleophile to attack and cleave the cyclopropane ring of the substrate. In addition, solvent kinetic isotope effect, proton inventory, and 13C-KIE studies of the wild type enzyme suggest that the C?-C? bond cleavage step in the chemical mechanism is at least partially rate limiting under kcat/Km conditions, and is likely preceded in the mechanism by a partially rate limiting step involving the conversion of a stable gem-diamine intermediate into a reactive external aldimine intermediate that is poised for cyclopropane ring cleavage. When viewed within the context of previous mechanistic and structural studies of ACCD enzymes, our studies are most consistent with a mode of cyclopropane ring cleavage involving nucleophilic catalysis by Tyr294. PMID:21244019

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

    PubMed Central

    Gemble, Simon; Ahuja, Akshay; Buhagiar-Labarchède, Géraldine; Onclercq-Delic, Rosine; Dairou, Julien; Biard, Denis S. F.; Lambert, Sarah; Lopes, Massimo; Amor-Guéret, Mounira

    2015-01-01

    Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at “difficult-to-replicate” sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3’-5’ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects. PMID:26181065

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

    PubMed Central

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

    2014-01-01

    Objective: Tuberculosis kills 3.70 lakh patients in India every year,out of which 7-12 % are meningeal involvement. Delay in its diagnosis and initiation of treatment results in poor prognosis and squeal in up to 25% of cases. The aim of the present study is to look for a simple, rapid, cost effective, and fairly specific test in differentiating tubercular aetiology from other causes of meningitis. In the present study we measured the adenosine deaminase activity (ADA) in Cerebrospinal Fluid (CSF) of Tubercular Meningitis (TBM) and non-TBM patients. Methods: Fifty six patients attending hospital with symptoms and signs of meningitis were selected and divided into three groups: tubercular, pyogenic, and aseptic meningitis, depending upon the accepted criteria. CSF was drawn and ADA estimated. Results: Out of 32 tubercular patients, 28 had CSF-ADA at or above the cut-off value while four had below. Out of 24 non-tuberculous patients (pyogenic and aseptic meningitis), two aseptic meningitis (AM) patient had ADA levels at or above the cut-off value while 22 had below this value. Results of our study indicate that ADA level estimation in CSF is not only of considerable value in the diagnosis of TBM, CSF, and ADA level 10 U/L as a cut-off value with sensitivity 87.5% and specificity 83.33% and positive predictive value of the test was 87.5%.and 83.3% negative predictive value. Conclusion: It can be concluded that ADA estimation in CSF is not only simple, inexpensive and rapid but also fairly specific method for making a diagnosis of tuberculous aetiology in TBM, especially when there is a dilemma of differentiating the tuberculous aetiology from non-tuberculous ones. For this reason ADA estimation in TBM may find a place as a routine investigation. PMID:24701487

  2. Characterization of rhizobacterial strain Rs-2 with ACC deaminase activity and its performance in promoting cotton growth under salinity stress.

    PubMed

    Wu, Zhansheng; Yue, Haitao; Lu, Jianjiang; Li, Chun

    2012-06-01

    A plant growth-promoting rhizobacterial strain Rs-2 with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity was isolated from salinized soils using ACC as the sole nitrogen source. Based on its physiological and biochemical properties and 16S rDNA sequence analysis, this strain was identified as Raoultella planticola. The maximum value of nitrogen fixation, dissolved phosphorus and dissolved potassium of Rs-2 were 148.8 ?g/ml, 205.0 and 4.31 mg/l, respectively within 192 h liquid culture. The germination rate of cotton seeds (Gossypium hirsutum L.) inoculated with Rs-2 (Rs-2-S) was enhanced by 29.5 % in pot experiments compared with that of the control (CK-S). Subsequently, individual plant height, fresh weight and dry weight of cotton seedlings in Rs-2-S treatment increased by 15.0, 33.7 and 33.3 %, respectively, compared with those in CK-S treatment. Statistical analysis showed that the inoculums of Rs-2 promoted significantly (P < 0.05) cotton growth. Further analysis showed that Rs-2 reduced the quantities of ethylene and abscisic acid in cotton seedlings, and increased indole acetic acid content in cotton seedlings under salinity stress. The accumulation of N, P, K(+), Ca(2+) and Fe(2+) in the cotton plants was increased significantly (P < 0.05) in Rs-2-S treatment, whereas the uptake of Na(+) in cotton seedlings decreased (P < 0.05). Hence, the present observations suggested that R. planticola Rs-2 could have a promising potential for promoting cotton growth and alleviating salinity stress. PMID:22806112

  3. Kinetochore Structure: Pulling Answers from Yeast

    E-print Network

    Cheeseman, Iain M.

    Despite the identification of multiple kinetochore proteins, their structure and organization has remained unclear. New work uses electron microscopy to visualize isolated budding yeast kinetochore particles and reveal the ...

  4. [Malassezia yeasts and their significance in dermatology].

    PubMed

    Hort, W; Nilles, M; Mayser, P

    2006-07-01

    Yeasts of the genus Malassezia belong to the normal microflora of the human skin. In addition they are known to cause a variety of skin diseases; the most frequent of which is pityriasis versicolor. Malassezia yeasts are also thought to be associated with seborrheic dermatitis, dandruff and Malassezia folliculitis. Recently the significance of Malassezia yeasts as a trigger factor for atopic dermatitis of the head and neck region has been pointed out. The role of the Malassezia yeasts in these different diseases has been controversial in the past and remains an issue because of difficulties in isolation, culture and differentiation of the organism. Thanks to molecular techniques, 10 species can actually be differentiated. The article presents the different Malassezia-associated diseases, their clinical picture, diagnosis and appropriate therapy. In addition the speciation of Malassezia is reviewed. PMID:16758222

  5. Monitoring Air Quality with Leaf Yeasts.

    ERIC Educational Resources Information Center

    Richardson, D. H. S.; And Others

    1985-01-01

    Proposes that leaf yeast serve as quick, inexpensive, and effective techniques for monitoring air quality. Outlines procedures and provides suggestions for data analysis. Includes results from sample school groups who employed this technique. (ML)

  6. Nanodosimetry of Auger electrons: A case study from the decay of 125I and 0–18-eV electron stopping cross sections of cytosine

    PubMed Central

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

    2013-01-01

    Radiopharmaceuticals emitting Auger electrons are often injected into patients undergoing cancer treatment with targeted radionuclide therapy (TRT). In this type of radiotherapy, the radiation source is radial and most of the emitted primary particles are low-energy electrons (LEEs) having kinetic energies distributed mostly from zero to a few hundred electron volts with very short ranges in biological media. These LEEs generate a high density of energy deposits and clustered damage, thus offering a relative biological effectiveness comparable to that of alpha particles. In this paper, we present a simple model and corresponding measurements to assess the energy deposited near the site of the radiopharmaceuticals in TRT. As an example, a calculation is performed for the decay of a single 125I radionuclide surrounded by a 1-nm-radius spherical shell of cytosine molecules using the energy spectrum of LEEs emitted by 125I along with their stopping cross sections between 0 and 18 eV. The dose absorbed by the cytosine shell, which occupies a volume of 4 nm3, is extremely high. It amounts to 79 kGy per decay of which 3%, 39%, and 58% is attributed to vibrational excitations, electronic excitations, and ionization processes, respectively. PMID:24976798

  7. Structure of the 2-Aminopurine-Cytosine Base Pair Formed in the Polymerase Active Site of the RB69 Y567A-DNA Polymerase

    SciTech Connect

    Reha-Krantz, Linda J.; Hariharan, Chithra; Subuddhi, Usharani; Xia, Shuangluo; Zhao, Chao; Beckman, Jeff; Christian, Thomas; Konigsberg, William (Yale); (Alberta)

    2011-11-21

    The adenine base analogue 2-aminopurine (2AP) is a potent base substitution mutagen in prokaryotes because of its enhanceed ability to form a mutagenic base pair with an incoming dCTP. Despite more than 50 years of research, the structure of the 2AP-C base pair remains unclear. We report the structure of the 2AP-dCTP base pair formed within the polymerase active site of the RB69 Y567A-DNA polymerase. A modified wobble 2AP-C base pair was detected with one H-bond between N1 of 2AP and a proton from the C4 amino group of cytosine and an apparent bifurcated H-bond between a proton on the 2-amino group of 2-aminopurine and the ring N3 and O2 atoms of cytosine. Interestingly, a primer-terminal region rich in AT base pairs, compared to GC base pairs, facilitated dCTP binding opposite template 2AP. We propose that the increased flexibility of the nucleotide binding pocket formed in the Y567A-DNA polymerase and increased 'breathing' at the primer-terminal junction of A+T-rich DNA facilitate dCTP binding opposite template 2AP. Thus, interactions between DNA polymerase residues with a dynamic primer-terminal junction play a role in determining base selectivity within the polymerase active site of RB69 DNA polymerase.

  8. Transgene silencing in grapevines transformed with GFLV resistance genes: analysis of variable expression of transgene, siRNAs production and cytosine methylation.

    PubMed

    Gambino, Giorgio; Perrone, Irene; Carra, Andrea; Chitarra, Walter; Boccacci, Paolo; Torello Marinoni, Daniela; Barberis, Marco; Maghuly, Fatemeh; Laimer, Margit; Gribaudo, Ivana

    2010-02-01

    Eight transgenic grapevine lines transformed with the coat protein gene of Grapevine fanleaf virus (GFLV-CP) were analyzed for a correlation between transgene expression, siRNAs production and DNA methylation. Bisulphite genome sequencing was used for a comprehensive analysis of DNA methylation. Methylated cytosine residues of CpG and CpNpG sites were detected in the GFLV-CP transgene, in the T7 terminator and in the 35S promoter of three grapevines without transgene expression, but no detectable level of siRNAs was recorded in these lines. The detailed analysis of 8 lines revealed the complex arrangements of T-DNA and integrated binary vector sequences as crucial factors that influence transgene expression. After inoculation with GFLV, no change in the levels of cytosine methylation was observed, but transgenic and untransformed plants produced short siRNAs (21-22 nt) indicating that the grapevine plants responded to GFLV infection by activating a post-transcriptional gene silencing mechanism. PMID:19507046

  9. Universal 1/f noise, crossovers of scaling exponents, and chromosome-specific patterns of guanine-cytosine content in DNA sequences of the human genome

    NASA Astrophysics Data System (ADS)

    Li, Wentian; Holste, Dirk

    2005-04-01

    Spatial fluctuations of guanine and cytosine base content (GC%) are studied by spectral analysis for the complete set of human genomic DNA sequences. We find that (i) 1/f? decay is universally observed in the power spectra of all 24 chromosomes, and (ii) the exponent ??1 extends to about 107 bases, one order of magnitude longer than has previously been observed. We further find that (iii) almost all human chromosomes exhibit a crossover from ?1?1 (1/f?1) at lower frequency to ?2<1 (1/f?2) at higher frequency, typically occurring at around 30 000-100 000 bases, while (iv) the crossover in this frequency range is virtually absent in human chromosome 22. In addition to the universal 1/f? noise in power spectra, we find (v) several lines of evidence for chromosome-specific correlation structures, including a 500 000 base long oscillation in human chromosome 21. The universal 1/f? spectrum in the human genome is further substantiated by a resistance to reduction in variance of guanine and cytosine content when the window size is increased.

  10. A novel radiochemical approach to 1-(2'-deoxy-2'-[(18) F]fluoro-?-d-arabinofuranosyl)cytosine ((18) F-FAC).

    PubMed

    Meyer, Jan-Philip; Probst, Katrin C; Trist, Iuni M L; McGuigan, Christopher; Westwell, Andrew D

    2014-09-01

    (18) F-FAC (1-(2'-deoxy-2'-[(18) F]fluoro-?-D-arabinofuranosyl)-cytosine) is an important 2'-fluoro-nucleoside-based positron emission tomography (PET) tracer that has been used for in vivo prediction of response to the widely used cancer chemotherapy drug gemcitabine. Previously reported synthetic routes to (18) F-FAC have relied on early introduction of the (18) F radiolabel prior to attachment to protected cytosine base. Considering the (18) F radiochemical half-life (110?min) and the technical challenges of multi-step syntheses on PET radiochemistry modular systems, late-stage radiofluorination is preferred for reproducible and reliable radiosynthesis with in vivo applications. Herein, we report the first late-stage radiosynthesis of (18) F-FAC. Cytidine derivatives with leaving groups at the 2'-position are particularly prone to undergo anhydro side-product formation upon heating because of their electron density at the 2-carbonyl pyrimidone oxygen. Our rationally developed fluorination precursor showed an improved reactivity-to-stability ratio at elevated temperatures. (18) F-FAC was obtained in radiochemical yields of 4.3-5.5% (n?=?8, decay-corrected from end of bombardment), with purities ?98% and specific activities ?63?GBq/µmol. The synthesis time was 168?min. PMID:25257474

  11. DNA (Cytosine-C5) Methyltransferase Inhibition by Oligodeoxyribonucleotides Containing 2-(1H)-Pyrimidinone (Zebularine Aglycon) at the Enzymatic Target Site

    PubMed Central

    van Bemmel, Dana M.; Brank, Adam S.; Eritja, Ramon; Marquez, Victor E.; Christman, Judith K.

    2009-01-01

    Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors. PMID:19467223

  12. Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation*

    PubMed Central

    Estève, Pierre-Olivier; Terragni, Jolyon; Deepti, Kanneganti; Chin, Hang Gyeong; Dai, Nan; Espejo, Alexsandra; Corrêa, Ivan R.; Bedford, Mark T.; Pradhan, Sriharsa

    2014-01-01

    Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation. PMID:24492612

  13. The growth of solar radiated yeast

    SciTech Connect

    Kraft, T.

    1995-09-01

    This researcher plans to determine if solar radiation affects the growth of yeast. The irradiated yeast was obtained from a sample exposed in space during a Space Shuttle flight of September 9-20, 1994. Further, the control groups were held at: (1) Goddard Space Flight Center (GSFC) in Greenbelt, Maryland; and (2) South Dakota School of Mines and Technology. The procedure used was based on the fact that yeast is most often used in consumable baked goods. Therefore, the yeast was incorporated into a basic Betty Crocker bread recipe. Data was collected by placing measured amounts of dough into sample containers with fifteen minute growth in height measurements collected and recorded. This researcher assumed the viability of yeast to be relative to its ability to produce carbon dioxide gas and cause the dough to rise. As all ingredients and surroundings were equal, this researcher assumed the yeast will produce the only significant difference in data collected. This researcher noted the approximate use date on all sample packages to be prior to arrival and experiment date. All dates equal, it was then assumed each would act in a similar manner of response. This assumption will allow for equally correct data collection.

  14. The growth of solar radiated yeast

    NASA Technical Reports Server (NTRS)

    Kraft, Tyrone

    1995-01-01

    This researcher plans to determine if solar radiation affects the growth of yeast. The irradiated yeast was obtained from a sample exposed in space during a Space Shuttle flight of September 9-20, 1994. Further, the control groups were held at: (1) Goddard Space Flight Center (GSFC) in Greenbelt, Maryland; and (2) South Dakota School of Mines and Technology. The procedure used was based on the fact that yeast is most often used in consumable baked goods. Therefore, the yeast was incorporated into a basic Betty Crocker bread recipe. Data was collected by placing measured amounts of dough into sample containers with fifteen minute growth in height measurements collected and recorded. This researcher assumed the viability of yeast to be relative to its ability to produce carbon dioxide gas and cause the dough to rise. As all ingredients and surroundings were equal, this researcher assumed the yeast will produce the only significant difference in data collected. This researcher noted the approximate use date on all sample packages to be prior to arrival and experiment date. All dates equal, it was then assumed each would act in a similar manner of response. This assumption will allow for equally correct data collection.

  15. Physiological and environmental control of yeast prions

    PubMed Central

    Chernova, Tatiana A.; Wilkinson, Keith D.; Chernoff, Yury O.

    2014-01-01

    Prions are self-perpetuating protein isoforms that cause fatal and incurable neurodegenerative disease in mammals. Recent evidence indicates that a majority of human proteins involved in amyloid and neural inclusion disorders possess at least some prion properties. In lower eukaryotes, such as yeast, prions act as epigenetic elements, which increase phenotypic diversity by altering a range of cellular processes. While some yeast prions are clearly pathogenic, it is also postulated that prion formation could be beneficial in variable environmental conditions. Yeast and mammalian prions have similar molecular properties. Crucial cellular factors and conditions influencing prion formation and propagation were uncovered in the yeast models. Stress-related chaperones, protein quality control deposits, degradation pathways and cytoskeletal networks control prion formation and propagation in yeast. Environmental stresses trigger prion formation and loss, supposedly acting via influencing intracellular concentrations of the prion-inducing proteins, and/or by localizing prionogenic proteins to the prion induction sites via heterologous ancillary helpers. Physiological and environmental modulation of yeast prions points to new opportunities for pharmacological intervention and/or prophylactic measures targeting general cellular systems rather than the properties of individual amyloids and prions. PMID:24236638

  16. Functional adaptation between yeast actin and its cognate myosin motors.

    PubMed

    Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S; Rubenstein, Peter A; Lord, Matthew

    2011-09-01

    We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins. PMID:21757693

  17. Cloning of the cDNA encoding adenosine 5'-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

    NASA Astrophysics Data System (ADS)

    Jiang, Keyong; Sun, Shujuan; Liu, Mei; Wang, Baojie; Meng, Xiaolin; Wang, Lei

    2013-01-01

    AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) ( P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) ( P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.

  18. Identification of a 5'-deoxyadenosine deaminase in Methanocaldococcus jannaschii and its possible role in recycling the radical S-adenosylmethionine enzyme reaction product 5'-deoxyadenosine.

    PubMed

    Miller, Danielle; O'Brien, Kaitlin; Xu, Huimin; White, Robert H

    2014-03-01

    We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5'-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5'-deoxyadenosine to 5'-deoxyinosine as its major product but will also deaminate 5'-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5'-deoxyadenosine deaminase (DadD). The Km for 5'-deoxyadenosine was found to be 14.0 ± 1.2 ?M with a kcat/Km of 9.1 × 10(9) M(-1) s(-1). Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5'-deoxyadenosine. Since 5'-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5'-deoxyadenosine, whereupon the 5'-deoxyribose moiety of 5'-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens. PMID:24375099

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

    PubMed Central

    Magalhães-Cardoso, Maria Teresa; Ferreirinha, Fátima; Dias, Ana Sofia; Pelletier, Julie

    2014-01-01

    Purinergic signalling is remarkably plastic during gastrointestinal inflammation. Thus, selective drugs targeting the “purinome” may be helpful for inflammatory gastrointestinal diseases. The myenteric neuromuscular transmission of healthy individuals is fine-tuned and controlled by adenosine acting on A2A excitatory receptors. Here, we investigated the neuromodulatory role of adenosine in TNBS-inflamed longitudinal muscle-myenteric plexus of the rat ileum. Seven-day postinflammation ileitis lacks adenosine neuromodulation, which may contribute to acceleration of gastrointestinal transit. The loss of adenosine neuromodulation results from deficient accumulation of the nucleoside at the myenteric synapse despite the fact that the increases in ATP release were observed. Disparity between ATP outflow and adenosine deficit in postinflammatory ileitis is ascribed to feed-forward inhibition of ecto-5?-nucleotidase/CD73 by high extracellular ATP and/or ADP. Redistribution of NTPDase2, but not of NTPDase3, from ganglion cell bodies to myenteric nerve terminals leads to preferential ADP accumulation from released ATP, thus contributing to the prolonged inhibition of muscle-bound ecto-5?-nucleotidase/CD73 and to the delay of adenosine formation at the inflamed neuromuscular synapse. On the other hand, depression of endogenous adenosine accumulation may also occur due to enhancement of adenosine deaminase activity. Both membrane-bound and soluble forms of ecto-5?-nucleotidase/CD73 and adenosine deaminase were detected in the inflamed myenteric plexus. These findings provide novel therapeutic targets for inflammatory gut motility disorders. PMID:25210228

  20. High frequency of mutations in exon 10 of the porphobilinogen deaminase gene in patients with a CRIM-positive subtype of acute intermittent porphyria

    SciTech Connect

    Gu, X.F.; Rooij, F. de; Voortman, G.; Velde, K.T.; Nordmann, Y.; Grandchamp, B.

    1992-09-01

    Acute intermittent porphyria (AIP) is an autosomal dominant disease characterized by a partial deficiency of porphobilinogen (PBG) deaminase. Different subtypes of the disease have been defined, and more than 10 different mutations have been described. The authors focused their study on exon 10, since they previously found that three different mutations were located in this exon and that two of them seemed to be relatively common. They used denaturing gradient gel electrophoresis (DGGE) after in vitro amplification to detect all possible mutations in exon 10 in 41 unrelated AIP patients. In about one-fourth of these patients they could distinguish three abnormal migration patterns, indicating the presence of various mutations. Additional sequencing demonstrated the presence of three different single-base substitutions. Two of these mutations had already been described. A third one consisted of a C-to-T transition located at position 499 of the PBG deaminase mRNA and resulted in an Arg-to-Trp substitution. All three mutations were found in patients with crossreacting immunological material (CRIM)-positive forms of AlP. The high frequency of these mutations make DGGE analysis of exon 10 a useful approach allowing the direct detection of the DNA abnormality in most of the families with the CRIM-positive subtype of AlP. 23 refs., 3 figs., 1 tab.

  1. Yeast and human mitochondrial helicases.

    PubMed

    Szczesny, Roman J; Wojcik, Magdalena A; Borowski, Lukasz S; Szewczyk, Maciej J; Skrok, Magda M; Golik, Pawel; Stepien, Piotr P

    2013-08-01

    Mitochondria are semiautonomous organelles which contain their own genome. Both maintenance and expression of mitochondrial DNA require activity of RNA and DNA helicases. In Saccharomyces cerevisiae the nuclear genome encodes four DExH/D superfamily members (MSS116, SUV3, MRH4, IRC3) that act as helicases and/or RNA chaperones. Their activity is necessary for mitochondrial RNA splicing, degradation, translation and genome maintenance. In humans the ortholog of SUV3 (hSUV3, SUPV3L1) so far is the best described mitochondrial RNA helicase. The enzyme, together with the matrix-localized pool of PNPase (PNPT1), forms an RNA-degrading complex called the mitochondrial degradosome, which localizes to distinct structures (D-foci). Global regulation of mitochondrially encoded genes can be achieved by changing mitochondrial DNA copy number. This way the proteins involved in its replication, like the Twinkle helicase (c10orf2), can indirectly regulate gene expression. Here, we describe yeast and human mitochondrial helicases that are directly involved in mitochondrial RNA metabolism, and present other helicases that participate in mitochondrial DNA replication and maintenance. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life. PMID:23454114

  2. Yeast prions assembly and propagation

    PubMed Central

    2011-01-01

    Yeast prions are self-perpetuating protein aggregates that are at the origin of heritable and transmissible non-Mendelian phenotypic traits. Among these, [PSI+], [URE3] and [PIN+] are the most well documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Fibril assembly depends on the presence of N- or C-terminal prion domains (PrDs) which are not homologous in sequence but share unusual amino-acid compositions, such as enrichment in polar residues (glutamines and asparagines) or the presence of oligopeptide repeats. Purified PrDs form amyloid fibrils that can convert prion-free cells to the prion state upon transformation. Nonetheless, isolated PrDs and full-length prion proteins have different aggregation, structural and infectious properties. In addition, mutations in the “non-prion” domains (non-PrDs) of Sup35p, Ure2p and Rnq1p were shown to affect their prion properties in vitro and in vivo. Despite these evidences, the implication of the functional non-PrDs in fibril assembly and prion propagation has been mostly overlooked. In this review, we discuss the contribution of non-PrDs to prion assemblies, and the structure-function relationship in prion infectivity in the light of recent findings on Sup35p and Ure2p assembly into infectious fibrils from our laboratory and others. PMID:22052349

  3. Ribonucleoprotein particle appearing during sporulation in yeast.

    PubMed Central

    Wejksnora, P J; Haber, J E

    1978-01-01

    During sporulation of Saccharomyces cerevisiae, most strains accumulate an unmethylated 20S RNA. Contrary to previous reports, this sporulation 20S RNA is distinct from the short-lived methylated 20S RNA precursor of 18S rRNA. This RNA species was found in a cytoplasmic 32S ribonucleoprotein particle consisting of one single-stranded 20S RNA molecule and 18 to 20 identical protein subunits of molecular weight 23,000. The ribonucleoprotein particle was resistant to ribonuclease digestion, although purified 20S RNA was ribonuclease sensitive. Both the RNA and the protein of the 32S ribonucleoprotein particle were only synthesized under conditions that induce sporulation. The accumulation of 20S RNA depended on continued protein synthesis but was actinomycin D insensitive, despite a high guanine-plus-cytosine content. Synthesis of 20S RNA stopped when cells were removed from sporulation conditions and placed in growth medium. Images PMID:348681

  4. Aggregated platelets enhance adherence of Candida yeasts to endothelium.

    PubMed

    Klotz, S A; Harrison, J L; Misra, R P

    1989-10-01

    The adherence of Candida albicans yeasts to human umbilical vein endothelium to subendothelial extracellular matrix (ECM) was investigated. Yeasts added to confluent endothelium in citrated platelet-poor plasma adhered on the average of 1 colony forming unit (cfu) per culture well. When platelets were added as platelet-rich plasma, a significant increase of yeast adherence was not seen. However, when endothelium was contracted by treatment with 2 mM EDTA, resulting in exposure of ECM, yeast adherence was increased to 10 cfu/well. When platelets were added with these yeasts, the number of adhering yeasts was further increased to 23 cfu/well (P less than .01). This represented an increase in adherence of yeasts of 230%. When the endothelial cells were completely removed and ECM exposed, platelets were found to likewise augment yeast adherence. Platelets, when added to the ECM, formed aggregates to which the yeasts firmly adhered. Likewise, when platelets were aggregated by adenosine diphosphate and mixed with yeasts, yeasts were shown to bind avidly to aggregated platelets, whereas yeasts did not adhere to unactivated, discoid platelets. Thus, exposed subendothelial ECM induces the aggregation of platelets and yeasts bind avidly to these platelet aggregates. PMID:2677163

  5. Production of alpha-amylase by yeast

    SciTech Connect

    Thomse, K.K.

    1987-01-01

    The enzyme alpha-amylase confers to an organism the enzymatic activity for the degradation of polyglucosides with alpha-1,4 glycosidic bonds such as starch and glycogen which are among the major storage compounds in plants and animals. Most alpha-amylases are single polypeptides of molecular weights around 50,000 dalton. They are generally found in the digestive tract of animals and in germinating seeds. Among the products released upon enzymatic degradation of polyglucosides maltose, a sugar that can be utilized as carbon source by yeast, is a major constituent. A cDNA segment complementary to mouse salivary amylase messenger RNA has been inserted into the yeast expression vector pMA56 behind the promoter of the gene encoding alcohol dehydrogenase I of yeast. Yeast transformants harboring plasmids with the normal orientation of the promoter and the mouse amylase cDNA gene produce amylase and release the enzyme in free form into the culture medium. Approximately 90% of the amylase activity is found in the medium. Yeast strains carrying MAL allele and transformed with a plasmid which directed the synthesis of mouse alpha-amylase were tested on plates containing starch and in batch fermentations using different high molecular weight sugars and oligosaccharides as carbon source. The results of these experiments will be discussed. (Refs. 21).

  6. Influence of pesticides on yeasts colonizing leaves.

    PubMed

    Vadkertiová, Renata; Sláviková, Elena

    2011-01-01

    The effect of nine different pesticides on the growth of yeasts isolated from the leaves of fruit and forest trees was investigated. Four insecticides (with the active ingredients: thiacloprid, deltamethrin, lambdacyhalothrin, and thiamethoxam) and five fungicides (with the effective substances: bitertanol, kresoxim-methyl, mancozeb, trifloxystrobin, and cupric oxychloride) were tested. The concentrations of chemicals were those recommended by the manufacturers for the spraying of trees. The yeast strains isolated from the leaves of fruit trees were not sensitive to any of the insecticides. The majority of yeast strains isolated from the leaves of forest trees were either not sensitive or only to a small extent. While Rhodotorula mucilaginosa and Pichia anomala were not affected by any insecticide, the strains of Cryptococcus laurentii and Rhodotorula glutinis showed the highest sensitivity. The effects of fungicides on the growth of isolated yeasts were more substantial. The fungicide Dithane DG (mancozeb) completely inhibited the growth of all yeasts. All strains isolated from fruit tree leaves were more resistant to the tested fungicides than those isolated from the leaves of forest trees. The most resistant strains from the leaves of fruit trees belonged to the species Metschnikowia pulcherrima, Pichia anomala, and Saccharomyces cerevisiae, whereas Cryptococcus albidus and C. laurentii, originating from the leaves of forest trees, showed the highest sensitivity to fungicides. PMID:22351984

  7. Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases

    E-print Network

    Burgers, Peter M.

    Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases Stephanie A. Nick Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, SE-901 87, Umeå, Sweden Edited in vitro using the physiological nucleoside triphosphate concentra- tions, yeast DNA polymerase , which

  8. Accumulation of polycyclic aromatic hydrocarbon-induced single strand breaks is attributed to slower rejoining processes by DNA polymerase inhibitor, cytosine arabinoside in CHO-K1 cells

    SciTech Connect

    Park, Jongkun (Wonkwang Univ. (Korea)); Lee, Jungsup; Lee, Hyungho; Choi, Insoon; Park, Sangdai (Seoul National Univ. (Korea))

    1991-01-01

    The authors demonstrate a successful induction of DNA single breaks in CHO-K1 cells by cocultivation with mouse embryonic fibroblasts (MEF) during exposure to benzo(a)pyrene (BP) or 3-methylcholanthrene (MC). When compared to those induced by methyl methanesulfonate (MMS), the DNA single strand breaks induced by BP and MC were markedly accumulated by post-incubation with cytosine arabinoside (araC) and were much more delayed in their rejoining. These results suggest that the active metabolites of BP or MC produced by cocultivation with MEF or microsomal fraction (S-15) result in the formation of large DNA adducts which require an active participation of DNA polymerase {alpha}({delta}) in the polymerization step of excision repair for their removal.

  9. Folding of the hammerhead ribozyme: Pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change

    PubMed Central

    Buskiewicz, Iwona A.; Burke, John M.

    2012-01-01

    The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme–substrate complex in real time. Here, we report the development and application of a new folding assay in which we use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. We conclude that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair. PMID:22274955

  10. Cytotoxic Mechanism of Selenomethionine in Yeast*

    PubMed Central

    Kitajima, Toshihiko; Jigami, Yoshifumi; Chiba, Yasunori

    2012-01-01

    Although selenium is an essential element, its excessive uptake is detrimental to living organisms. The significance of selenium for living organisms has been exploited for various purposes. However, the molecular basis of selenium toxicity is not completely understood. Here, we applied a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach to analysis of yeast cells treated with selenomethionine. The data indicated that intracellular thiol compounds are significantly decreased, and diselenide and selenosulfide compounds are increased in selenomethionine-treated cells. The growth defect induced by selenomethionine was recovered by extracellular addition of cysteine and by genetic modification of yeast cells that have an additional de novo synthetic pathway for cysteine. Because cysteine is an intermediate of thiol compounds, these results suggested that the loss of a reduced form of thiol compounds due to selenomethionine causes a growth defect of yeast cells. PMID:22311978

  11. New yeast recombineering tools for bacteria.

    PubMed

    Shanks, Robert M Q; Kadouri, Daniel E; MacEachran, Daniel P; O'Toole, George A

    2009-09-01

    Recombineering with Saccharomyces cerevisiae is a powerful methodology that can be used to clone multiple unmarked pieces of DNA to generate complex constructs with high efficiency. Here, we introduce two new tools that utilize the native recombination enzymes of S. cerevisiae to facilitate the manipulation of DNA. First, yeast recombineering was used to make directed nested deletions in a bacteria-yeast shuttle plasmid using only one or two single stranded oligomers, thus obviating the need for a PCR step. Second, we have generated several new shuttle vectors for yeast recombineering capable of replication in a wide variety of bacterial genera. As a demonstration of utility, some of the approaches and vectors generated in this study were used to make a pigP deletion mutation in the opportunistic pathogen Serratia marcescens. PMID:19477196

  12. The organization of oligonucleosomes in yeast.

    PubMed Central

    Szent-Gyorgyi, C; Isenberg, I

    1983-01-01

    We have developed a method of preparing yeast chromatin that facilitates the analysis of nucleoprotein organization. Yeast chromatin, isolated as an insoluble complex, is digested with micrococcal nuclease and fractionated into major insoluble and soluble fractions. No nucleosomal repeat is seen early in digestion for the insoluble fraction. Nucleosomal complexes of the soluble fraction are excised by nuclease in a distinctive non-random pattern; they are markedly depleted in mononucleosomes. When we analyze the soluble material by high resolution native electrophoresis, we find that the nucleoproteins resolve into two bands for each DNA multimer of the nucleosomal repeat. Our results suggest that there are structural similarities between bulk yeast chromatin and chromatin configurations found in transcribing genes of complex eukaryotes. Images PMID:6344013

  13. Yeast Oligo-mediated Genome Engineering (YOGE)

    PubMed Central

    DiCarlo, JE; Conley, AJ; Penttilä, M; Jäntti, J; Wang, HH; Church, GM

    2014-01-01

    High-frequency oligonucleotide-directed recombination engineering (recombineering) has enabled rapid modification of several prokaryotic genomes to date. Here, we present a method for oligonucleotide-mediated recombineering in the model eukaryote and industrial production host S. cerevisiae, which we call Yeast Oligo-mediated Genome Engineering (YOGE). Through a combination of overexpression and knockouts of relevant genes and optimization of transformation and oligonucleotide designs, we achieve high gene modification frequencies at levels that only require screening of dozens of cells. We demonstrate the robustness of our approach in three divergent yeast strains, including those involved in industrial production of bio-based chemicals. Furthermore, YOGE can be iteratively executed via cycling to generate genomic libraries up to 105 individuals at each round for diversity generation. YOGE cycling alone, or in combination with phenotypic selections or endonuclease-based negative genotypic selections, can be used to easily generate modified alleles in yeast populations with high frequencies. PMID:24160921

  14. RNA degradation in yeast and human mitochondria.

    PubMed

    Szczesny, Roman J; Borowski, Lukasz S; Malecki, Michal; Wojcik, Magdalena A; Stepien, Piotr P; Golik, Pawel

    2012-01-01

    Expression of mitochondrially encoded genes must be finely tuned according to the cell's requirements. Since yeast and human mitochondria have limited possibilities to regulate gene expression by altering the transcription initiation rate, posttranscriptional processes, including RNA degradation, are of great importance. In both organisms mitochondrial RNA degradation seems to be mostly depending on the RNA helicase Suv3. Yeast Suv3 functions in cooperation with Dss1 ribonuclease by forming a two-subunit complex called the mitochondrial degradosome. The human ortholog of Suv3 (hSuv3, hSuv3p, SUPV3L1) is also indispensable for mitochondrial RNA decay but its ribonucleolytic partner has so far escaped identification. In this review we summarize the current knowledge about RNA degradation in human and yeast mitochondria. This article is part of a Special Issue entitled: Mitochondrial Gene Expression. PMID:22178375

  15. DNA Cytosine Methylation in the Bovine Leukemia Virus Promoter Is Associated with Latency in a Lymphoma-derived B-cell Line

    PubMed Central

    Pierard, Valérie; Guiguen, Allan; Colin, Laurence; Wijmeersch, Gaëlle; Vanhulle, Caroline; Van Driessche, Benoît; Dekoninck, Ann; Blazkova, Jana; Cardona, Christelle; Merimi, Makram; Vierendeel, Valérie; Calomme, Claire; Nguyên, Thi Liên-Anh; Nuttinck, Michèle; Twizere, Jean-Claude; Kettmann, Richard; Portetelle, Daniel; Burny, Arsène; Hirsch, Ivan; Rohr, Olivier; Van Lint, Carine

    2010-01-01

    Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2?-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5?-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator TaxBLV decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267LTaxSN 5?-LTR compared with the L267 5?-LTR. Interestingly, DNA methylation inhibitors and TaxBLV synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the ?154 or ?129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at ?129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5?-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency. PMID:20413592

  16. Cytosine methylation at CG and CNG sites is not a prerequisite for the initiation of transcriptional gene silencing in plants, but it is required for its maintenance.

    PubMed

    Diéguez, M J; Vaucheret, H; Paszkowski, J; Mittelsten Scheid, O

    1998-08-01

    Transgenes integrated into plant chromosomes, and/or endogenous plant genes, may be subjected to epigenetic silencing at the transcriptional or post-transcriptional level. Transcriptional inactivation is correlated with hypermethylation of CG/CNG sites at the silent loci. It is not known whether local hypermethylation is part of the inactivation process, or just an outcome of the silent state. To address this issue, we generated transgenic tobacco lines containing a selectable marker gene controlled by a derivative of the 35S promoter of the cauliflower mosaic virus (CaMV) devoid of CG and CNG methylation acceptor sites. Silencing was triggered by crossing to the silencer locus of tobacco line 271. This line contains inactive and methylated copies of the 35S promoter and is able to silence homologous promoter copies at ectopic chromosomal positions. The mutated promoter lacking CG/CNG methylation acceptor sites was as susceptible to Trans-silencing as the unmodified 35S promoter control. Thus, methylation at CG and CNG sites is not a prerequisite for the initiation of epigenetic gene inactivation. Interestingly, while methylation of the remaining cytosines is usually only slightly affected by silencing, it was significantly increased in the absence of CG/CNG sequences. Since this sequence preference is the same as that of known methyltransferases, this may imply that silencing is accompanied or directly followed by recruitment of methyltransferase, which, in the absence of cytosines in the optimal sequence context, modifies other C residues in the affected area. However, silencing without CG/CNG methylation was immediately relieved in the absence of the silencer. Thus, CG/CNG methylation is probably essential for the maintenance of previously established epigenetic states. PMID:9747712

  17. Circular permutation of DNA cytosine-N4 methyltransferases: in vivo coexistence in the BcnI system and in vitro probing by hybrid formation.

    PubMed

    Vilkaitis, Giedrius; Lubys, Arvydas; Merkiene, Egle; Timinskas, Albertas; Janulaitis, Arvydas; Klimasauskas, Saulius

    2002-04-01

    Sequence analysis of the BcnI restriction-modification system from Bacillus centrosporus revealed four open reading frames (bcnIC, bcnIR, bcnIB and bcnIA) that are arranged as two converging collinear pairs. One pair encodes a putative small regulatory protein, C.BcnI, and the restriction endonuclease R.BcnI. The other two gene products are the DNA cytosine-N4 methyltransferases M.BcnIA and M.BcnIB, which differ by circular permutation of conserved sequence motifs. The BcnI methyltransferases are isospecific on double-stranded DNA [methylation specificity CC(C/G)GG], but M.BcnIA can also methylate the target sites in single-stranded DNA. Functional analysis shows that bcnIA is dispensable (bcnIB is capable of protecting the DNA against the in vivo activity of bcnIR); in contrast, no stable clones were obtained if bcnIB alone was deleted from the system. By analogy with the DpnII system, the second methylase M.BcnIA may play a role in the transformation proficiency of its gram-positive host. The interchangeability of homologous elements in the beta class of cytosine-N4 methylases was probed by hybrid formation between M.BcnIB and its closest homolog M.Cfr9I (CCCGGG) employing a novel semi-random strategy combined with selection for catalytic activity. The fusion points in the active hybrids mapped in a narrow region located between sequence motifs X and I. Our data illustrate that recombination of two related sequences by circular permutation may serve as an evolutionary mechanism for creating new specificities of amino MTases. PMID:11917015

  18. The long physiological reach of the yeast vacuolar H + ATPase

    Microsoft Academic Search

    Patricia M. Kane

    2007-01-01

    V-ATPases are structurally conserved and functionally versatile proton pumps found in all eukaryotes. The yeast V-ATPase has\\u000a emerged as a major model system, in part because yeast mutants lacking V-ATPase subunits (vma mutants) are viable and exhibit a distinctive Vma- phenotype. Yeast vma mutants are present in ordered collections of all non-essential yeast deletion mutants, and a number of additional

  19. Fermentation of maltotriose by brewer's and baker's yeasts

    Microsoft Academic Search

    John Londesborough

    2001-01-01

    Two brewer's yeasts and one baker's yeast grew with =95% (w\\/w) pure maltotriose as carbon source in the presence of antimycin A to block respiration. Biomass yields (0.15 and 0.24 g dry yeast g-1 sugar, respectively, with and without antimycin A) were similar for growth on maltose and maltotriose, and yields of ethanol were 80% of stoichiometric. Yeasts harvested during

  20. Biochemical Comparison of Commercial Selenium Yeast Preparations.

    PubMed

    Fagan, Sheena; Owens, Rebecca; Ward, Patrick; Connolly, Cathal; Doyle, Sean; Murphy, Richard

    2015-08-01

    The trace mineral selenium (Se) is an essential element for human and animal nutrition. The addition of Se to the diet through dietary supplements or fortified food/feed is increasingly common owing to the often sub-optimal content of standard diets of many countries. Se supplements commercially available include the inorganic mineral salts such as sodium selenite or selenate, and organic forms such as Se-enriched yeast. Today, Se yeast is produced by several manufacturers and has become the most widely used source of Se for human supplementation and is also widely employed in animal nutrition where approval in all species has been granted by regulatory bodies such as the European Food Safety Authority (EFSA). Characterisation and comparison of Se-enriched yeast products has traditionally been made by quantifying total selenomethionine (SeMet) content. A disadvantage of this approach, however, is that it does not consider the effects of Se deposition on subsequent digestive availability. In this study, an assessment was made of the water-soluble extracts of commercially available Se-enriched yeast samples for free, peptide-bound and total water-soluble SeMet. Using LC-MS/MS, a total of 62 Se-containing proteins were identified across four Se yeast products, displaying quantitative/qualitative changes in abundance relative to the certified reference material, SELM-1 (P value <0.05; fold change ?2). Overall, the study indicates that significant differences exist between Se yeast products in terms of SeMet content, Se-containing protein abundance and associated metabolic pathways. PMID:25855372

  1. [The yeast biofilm in human medicine].

    PubMed

    R?zicka, Filip; Holá, Veronika; Votava, Miroslav

    2007-08-01

    In recent years, the role of Candida yeasts as causative agents of nosocomial infections has increased. One of the important virulence factors contributing to the development of such infections is biofilm production. This virulence factor enables yeast to colonize both native surfaces and artificial implants. The most common sources of infection are patients themselves, in particular the gastrointestinal tract and skin. The vectors of exogenous yeast infections are predominantly the hands of the health personnel and contaminated medical instruments. The adhesion of yeasts to the implant surfaces is determined both by implant surface and yeast characteristics. This is followed by proliferation and production of microcolonies and extracellular matrix. The final biofilm structure is also influenced by the production of hyphae and pseudohyphae. The entire process of biofilm production is controlled by numerous regulatory systems, with the key role being played by the quorum sensing system. Like the adhered bacterial cultures, candidas growing in the form of a biofilm are highly resistant to antimicrobial therapy. Resistance of yeast biofilms to antifungals is a complex process with multiple contributing factors. These are especially increased gene expression (e.g. genes encoding the so called multidrug efflux pumps), limited penetration of substances through the extracellular matrix, inhibited cell growth and altered microenvironment in deeper biofilm layers. The concentrations of antifungals able to effectively affect the biofilm cells exceed, by several orders of magnitude, the values of conventionally determined MICs. High biofilm resistance results in ineffective antifungal therapy of biofilm infections. Therefore, if possible, the colonized implant should be removed. Conservative therapy should involve antifungals with a proven effect on the biofilm (e.g. caspofungin). The most effective measure in fighting biofilm infections is prevention, especially adhering to aseptic techniques when manipulating with implants and their correct maintenance. PMID:17929219

  2. Research Articles Yeast Ancestral Genome Reconstructions: The Possibilities

    E-print Network

    Chauve, Cedric

    Research Articles Yeast Ancestral Genome Reconstructions: The Possibilities of Computational the availability of assembled eukaryotic genomes, the first one being a budding yeast, many computational methods them to infer and analyse the architectures of two ancestral yeast genomes, based on the sequence

  3. Growth and survival of a probiotic yeast in dairy products

    Microsoft Academic Search

    A Lourens-Hattingh; B. C Viljoen

    2001-01-01

    Poor survival of probiotic bacteria in yogurt has been recorded. Growth of a probiotic yeast, Saccharomyces boulardii, in association with the bio-yogurt microflora, by incorporating the yeast into commercial bio-yogurt, has been suggested to stimulate the growth of the probiotic organisms and to assure their survival during shelflife. Therefore, the ability of growth and survival of the probiotic yeast itself

  4. The humanization of N-glycosylation pathways in yeast

    Microsoft Academic Search

    Stefan Wildt; Tilllman U. Gerngross

    2005-01-01

    Yeast and other fungal protein-expression hosts have been extensively used to produce industrial enzymes, and are often the expression system of choice when manufacturing costs are of primary concern. However, for the production of therapeutic glycoproteins intended for use in humans, yeast have been less useful owing to their inability to modify proteins with human glycosylation structures. Yeast N-glycosylation is

  5. Cycloheximide resistance as marker for monitoring yeasts in wine fermentations

    Microsoft Academic Search

    F Pérez; J. A Regodón; M. E Valdés; C De Miguel; M Ram??rez

    2000-01-01

    When selected yeast strains are used in wine-making, it is necessary to ensure that the fermentation process is really conducted by the inoculated yeast. Saccharomyces cerevisiae spontaneous mutants resistant to cycloheximide (cyhr) were isolated from industrial strains. The mutations did not affect the fermentation kinetics, the quality of the wines, or the viability of active dry yeast made with the

  6. Tripartite organization of centromeric chromatin in budding yeast

    E-print Network

    Henikoff, Steven

    Tripartite organization of centromeric chromatin in budding yeast Kristina Krassovskya,b , Jorja G by nucleosomes containing the CenH3 histone variant, whereas in budding yeast, a 120-bp centromere DNA element this is the case in the budding yeast, Saccharomyces cerevisiae, where each of the 16 centromeres consists of a 120

  7. Lovastatin Content of Commercially Available Red Yeast Rice Supplements

    Microsoft Academic Search

    Philip J. Gregory; Rebecca Pettit; Zara Risoldi Cochrane; Amy F. Wilson; Andrew M. Abe

    2012-01-01

    Red yeast rice is a commonly used supplement in North America, primarily promoted for lowering cholesterol. The fermentation process for producing red yeast rice naturally produces a small concentration of lovastatin and related compounds. The authors evaluated label information and contacted manufacturers to inquire about lovastatin content in 117 commercially available red yeast rice supplement products. Only 14% of the

  8. 21 CFR 573.750 - Pichia pastoris dried yeast.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...2014-04-01 false Pichia pastoris dried yeast. 573.750 Section 573.750 Food...Listing § 573.750 Pichia pastoris dried yeast. (a) Identity. The food additive Pichia pastoris dried yeast may be used in feed formulations...

  9. 21 CFR 573.750 - Pichia pastoris dried yeast.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...2011-04-01 false Pichia pastoris dried yeast. 573.750 Section 573.750 Food...Listing § 573.750 Pichia pastoris dried yeast. (a) Identity . The food additive Pichia pastoris dried yeast may be used in feed formulations...

  10. ORIGINAL PAPER Candida gelsemii sp. nov., a yeast

    E-print Network

    Thomson, James D.

    ORIGINAL PAPER Candida gelsemii sp. nov., a yeast of the Metschnikowiaceae clade isolated from+Business Media B.V. 2006 Abstract A new yeast species, Candida gelsemii, is described to accommodate three Metschnikowiaceae Á Gelsemium sempervirens Á Nectar alkaloids Á Gelsemine Á New yeast species Introduction Floral

  11. 21 CFR 172.590 - Yeast-malt sprout extract.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...Drugs 3 2010-04-01 2009-04-01 true Yeast-malt sprout extract. 172.590 Section 172...Flavoring Agents and Related Substances § 172.590 Yeast-malt sprout extract. Yeast-malt sprout extract, as described in this...

  12. 21 CFR 172.590 - Yeast-malt sprout extract.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 3 2013-04-01 2013-04-01 false Yeast-malt sprout extract. 172.590 Section 172...Flavoring Agents and Related Substances § 172.590 Yeast-malt sprout extract. Yeast-malt sprout extract, as described in this...

  13. Yeast Genes That Enhance the Toxicity of a Mutant Huntingtin

    E-print Network

    Lindquist, Susan

    Yeast Genes That Enhance the Toxicity of a Mutant Huntingtin Fragment or -Synuclein Stephen-wide screens were performed in yeast to identify genes that enhance the toxicity of a mutant huntingtin's yeast Sac- charomyces cerevisiae as a model eukaryotic organism to test the hypothesis that the down

  14. Glucose and sucrose: hazardous fast-food for industrial yeast?

    E-print Network

    Glucose and sucrose: hazardous fast-food for industrial yeast? Kevin J. Verstrepen1,2 , Dirk 197, Glen Osmond, Adelaide SA-5064, Australia Yeast cells often encounter a mixture of different resist- ance. In an industrial context, these effects lead to several yeast-related problems

  15. Clustering, Communication and Environmental Oscillations in Populations of Budding Yeast

    E-print Network

    Young, Todd

    Clustering, Communication and Environmental Oscillations in Populations of Budding Yeast Chris describe how simple models of communication, consistent with known yeast phys- iological mechanisms relevant variables during yeast growth and division have been reported and studied for over 40 years [8, 12

  16. 21 CFR 573.750 - Pichia pastoris dried yeast.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...2013-04-01 false Pichia pastoris dried yeast. 573.750 Section 573.750 Food...Listing § 573.750 Pichia pastoris dried yeast. (a) Identity . The food additive Pichia pastoris dried yeast may be used in feed formulations...

  17. GENE ENGINEERING OF YEASTS FOR THE DEGRADATION OF HAZARDOUS WASTE

    EPA Science Inventory

    The research examined the structure and function of cytochrome P-450 genes in yeast as a model for gene engineering such as eukaryotic P-450 enzymes for biodegradation of hazardous waste by yeasts. Saccharomyces cerevisiae and Candida tropicalis are two yeasts known to produce ma...

  18. 21 CFR 172.590 - Yeast-malt sprout extract.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 3 2012-04-01 2012-04-01 false Yeast-malt sprout extract. 172.590 Section 172...Flavoring Agents and Related Substances § 172.590 Yeast-malt sprout extract. Yeast-malt sprout extract, as described in this...

  19. Boolean Network Model Predicts Cell Cycle Sequence of Fission Yeast

    E-print Network

    Bornholdt, Stefan

    Boolean Network Model Predicts Cell Cycle Sequence of Fission Yeast Maria I. Davidich, Stefan network model of the cell-cycle regulatory network of fission yeast (Schizosaccharomyces Pombe sequence being a strongly attractive trajectory. Comparing the fission yeast cell-cycle model to a similar

  20. Robust Spatial Sensing of Mating Pheromone Gradients by Yeast Cells

    E-print Network

    Nie, Qing

    Robust Spatial Sensing of Mating Pheromone Gradients by Yeast Cells Travis I. Moore1,2 , Ching not degrade the pheromone input. The yeast cells exhibited good accuracy with the mating projection typically caused defects in both sensing and response. Interestingly, yeast cells employed adaptive mechanisms

  1. 21 CFR 172.590 - Yeast-malt sprout extract.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 3 2011-04-01 2011-04-01 false Yeast-malt sprout extract. 172.590 Section 172...Flavoring Agents and Related Substances § 172.590 Yeast-malt sprout extract. Yeast-malt sprout extract, as described in this...

  2. 21 CFR 172.590 - Yeast-malt sprout extract.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 3 2014-04-01 2014-04-01 false Yeast-malt sprout extract. 172.590 Section 172...Flavoring Agents and Related Substances § 172.590 Yeast-malt sprout extract. Yeast-malt sprout extract, as described in this...

  3. 21 CFR 573.750 - Pichia pastoris dried yeast.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...2010-04-01 false Pichia pastoris dried yeast. 573.750 Section 573.750 Food...Listing § 573.750 Pichia pastoris dried yeast. (a) Identity . The food additive Pichia pastoris dried yeast may be used in feed formulations...

  4. Research Focus A short history of recombination in yeast

    E-print Network

    Otto, Sarah

    Research Focus A short history of recombination in yeast Clifford W. Zeyl1* and Sarah P. Otto2* 1 of fungal genomics, we know little about either the ecology or reproductive biology of the budding yeast of a studyofhistoricalpoutcrossingeventsand inferthe genomic positions of previous recombination events in the yeast Saccharomyces cerevisiae

  5. Exploring the Yeast Genome with Generalized Singular Value

    E-print Network

    Fonseca, Rodrigo

    Exploring the Yeast Genome with Generalized Singular Value Decomposition Andrew Ferguson Advisor courses of the yeast Saccharomyces cerevisiae under two different experimental con- ditions. In the first analysis, a comparison is performed between the yeast stress response to hydrogen peroxide (H2O2

  6. 21 CFR 573.750 - Pichia pastoris dried yeast.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...2012-04-01 false Pichia pastoris dried yeast. 573.750 Section 573.750 Food...Listing § 573.750 Pichia pastoris dried yeast. (a) Identity . The food additive Pichia pastoris dried yeast may be used in feed formulations...

  7. Invited Review Functional expression of heterologous proteins in yeast: insights

    E-print Network

    Rao, Rajini

    Invited Review Functional expression of heterologous proteins in yeast: insights into Ca2 signaling of heterologous proteins in yeast: insights into Ca2 signaling and Ca2 -transporting ATPases. Am J Physiol Cell Physiol 287: C580­C589, 2004; 10.1152/ajpcell.00135.2004.-- The baker's yeast Saccharomyces cerevisiae

  8. Population genomic analysis of outcrossing and recombination in yeast

    Microsoft Academic Search

    Douglas M Ruderfer; Stephen C Pratt; Hannah S Seidel; Leonid Kruglyak

    2006-01-01

    The budding yeast Saccharomyces cerevisiae has been used by humans for millennia to make wine, beer and bread. More recently, it became a key model organism for studies of eukaryotic biology and for genomic analysis. However, relatively little is known about the natural lifestyle and population genetics of yeast. One major question is whether genetically diverse yeast strains mate and

  9. Triacetic acid lactone production in industrial Saccharomyces yeast strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Triacetic acid lactone (TAL) is a potential platform chemical that can be produced in yeast. To evaluate the potential for industrial yeast strains to produce TAL, the g2ps1 gene encoding 2-pyrone synthase was transformed into thirteen industrial yeast strains of varied genetic background. TAL produ...

  10. Principles of chromosomal organization: lessons from yeast

    PubMed Central

    Zimmer, Christophe

    2011-01-01

    The spatial organization of genes and chromosomes plays an important role in the regulation of several DNA processes. However, the principles and forces underlying this nonrandom organization are mostly unknown. Despite its small dimension, and thanks to new imaging and biochemical techniques, studies of the budding yeast nucleus have led to significant insights into chromosome arrangement and dynamics. The dynamic organization of the yeast genome during interphase argues for both the physical properties of the chromatin fiber and specific molecular interactions as drivers of nuclear order. PMID:21383075

  11. Hajjoul/Mathon et al. Chromatin dynamics in living yeasts High throughput chromatin motion tracking in living yeast reveals the

    E-print Network

    Boyer, Edmond

    Hajjoul/Mathon et al. Chromatin dynamics in living yeasts - 1 - High throughput chromatin motion tracking in living yeast reveals the flexibility of the fiber throughout the genome Houssam Hajjoul1.1101/gr.157008.113 #12;Hajjoul/Mathon et al. Chromatin dynamics in living yeasts - 2 - ABSTRACT (211 words

  12. Biodiversity of Saccharomyces yeast strains from grape berries of wine-producing areas using starter commercial yeasts

    Microsoft Academic Search

    Eva Valero; Brigitte Cambon; Dorit Schuller; Margarida Casal; Sylvie Dequin

    2007-01-01

    The use of commercial wine yeast strains as starters has grown extensively over the past two decades. In this study, a large-scale sampling plan was devised over a period of 3 years in three different vineyards in the south of France, to evaluate autochthonous wine yeast biodiversity in vineyards around wineries where active dry yeasts have been used as fermentation

  13. A point mutation G----A in exon 12 of the porphobilinogen deaminase gene results in exon skipping and is responsible for acute intermittent porphyria.

    PubMed Central

    Grandchamp, B; Picat, C; de Rooij, F; Beaumont, C; Wilson, P; Deybach, J C; Nordmann, Y

    1989-01-01

    We have determined the mutation in a patient with acute intermittent porphyria. The mRNA coding for porphobilinogen deaminase was reverse transcribed then the cDNA was enzymatically amplified in vitro. Upon sequencing of a polymerase chain reaction product of abnormal size we found that this fragment lacked exon 12 of the gene. We analysed a genomic fragment containing exon 12 and determined that the patient was heterozygous for a point mutation G A at the last position of exon 12. We propose that this base change is responsible for an abnormal processing of the mutant allele such that exon 12 is missing in the mature mRNA. The resulting aberrant mRNA encodes a truncated protein which is inactive but stable and can be detected using antibodies directed against the normal enzyme. Images PMID:2789372

  14. Splice variants of activation induced deaminase (AID) do not affect the efficiency of class switch recombination in murine CH12F3 cells.

    PubMed

    Sala, Cesare; Mattiuz, Giorgio; Pietrobono, Silvia; Chicca, Andrea; Conticello, Silvestro G

    2015-01-01

    Activation Induced Deaminase (AID) triggers the antigen-driven antibody diversification processes through its ability to edit DNA. AID dependent DNA damage is also the cause of genetic alterations often found in mature B cell tumors. A number of splice variants of AID have been identified, for which a role in the modulation of its activity has been hypothesized. We have thus tested two of these splice variants, which we find catalytically inactive, for their ability to modulate the activity of endogenous AID in CH12F3 cells, a murine lymphoma cell line in which Class Switch Recombination (CSR) can be induced. In contrast to full-length AID, neither these splice variants or a catalytically impaired AID mutant affect the efficiency of Class Switch Recombination. Thus, while a role for these splice variants at the RNA level remains possible, it is unlikely that they exert any regulatory effect on the function of AID. PMID:25803053

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

    PubMed

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

    2014-09-01

    Lead (Pb) pollution is appearing as an alarming threat nowadays. Excessive Pb concentrations in agricultural soils result in minimizing the soil fertility and health which affects the plant growth and leads to decrease in crop production. Plant growth promoting rhizobacteria (PGPR) are beneficial bacteria which can protect the plants against many abiotic stresses, and enhance the growth. The study aimed to identify important rhizobacterial strains by using the 1-aminocyclopropane-1-carboxylate (ACC) enrichment technique and examine their inoculation effects in the growth promotion of maize, under Pb pollution. A pot experiment was conducted and six rhizobacterial isolates were used. Pb was added to 2 kg soil in each pot (with 4 seeds/pot) using Pb(NO3)2 at the rate of 0, 100, 200, 300, and 400 mg kg(-1) Pb with three replications in completely randomized design. Rhizobacterial isolates performed significantly better under all Pb levels, i.e., 100 to 400 Pb mg kg(-1) soil, compared to control. Comparing the efficacy of the rhizobacterial isolates under different Pb levels, rhizobacterial isolates having both ACC-deaminase and nitrogen-fixing activities (AN8 and AN12) showed highest increase in terms of the physical, chemical and enzymatic growth parameters of maize, followed by the rhizobacterial isolates having ACC-deaminase activity only (ACC5 and ACC8), and then the nitrogen-fixing rhizobia (Azotobacter and RN5). However, the AN8 isolate showed maximum efficiency, and highest shoot and root length (14.2 and 6.1 cm), seedling fresh and dry weights (1.91 and 0.14 g), chlorophyll a, b, and carotenoids (24.1, 30.2 and 77.7 ?g/l), protein (0.82 mg/g), proline (3.42 ?mol/g), glutathione S-transferase, peroxidase and catalase (12.3, 4.2 and 7.2 units/mg protein), while the lowest Pb uptake in the shoot and root (0.83 and 0.48 mg/kg) were observed under this rhizobial isolate at the highest Pb level (i.e., 400 Pb mg kg(-1) soil). The results revealed that PGPR significantly decreases the deleterious effects of Pb pollution and increases the maize growth under all Pb concentrations, i.e., 100-400 Pb mg kg(-1) soil. PGPR chelate the Pb in the soil, and ultimately influence its bioavailability, release and uptake. The PGPR having both ACC-deaminase and nitrogen-fixing abilities are more effective and resistive against Pb pollution than PGPR having either ACC-deaminase or nitrogen-fixing activity alone. The ACC enrichment technique is an efficient approach to select promising PGPR. PMID:24888619

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

    SciTech Connect

    Azim, N. [University of Punjab, New Campus, Lahore-54590 (Pakistan); Deery, E.; Warren, M. J. [University of Kent, Stacey Building, Canterbury CT2 7NJ (United Kingdom); Wolfenden, B. A. A.; Erskine, P.; Cooper, J. B., E-mail: jon.cooper@ucl.ac.uk; Coker, A.; Wood, S. P. [UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW3 2PF (United Kingdom); Akhtar, M. [University of Punjab, New Campus, Lahore-54590 (Pakistan)

    2014-03-01

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

  17. The spindle pole body of yeast

    Microsoft Academic Search

    Michael Snyder

    1994-01-01

    Microtubule organizing centers play an essential cellular role in nucleating microtubule assembly and establishing the microtubule array. The microtubule organizing center of yeast, the spindle pole body (SPB), shares many functions and properties with those other organisms. In recent years considerable new information has been generated concerning components associated with the SPB, and the mechanism by which it duplicates. This

  18. Turning yeast sequence into protein function

    SciTech Connect

    Heijne, G. von

    1996-04-01

    The complete genome sequencing of the yeast Saccharomyces Cerevisiae leads us into a new era of potential use for such data base information. Protein engineering studies suggest that genetic selection of overproducing strains may aid the assignment of protein function. Data base management and sequencing software have been developed to scan entire genomes.

  19. Number of Cytoplasmic Factors in Yeast Cells

    Microsoft Academic Search

    Takashi Sugimura; Kazuko Okabe; Akira Imamura

    1966-01-01

    IT is well known that the self-reproducing genetic factors (rho), required for the development of mitochondria, are present in the cytoplasm of yeast cell. When the cells are allowed to grow in a medium containing acriflavine or related dyes, large numbers of mutant cells with deficient respiration are produced, which tend to dominate the entire cell population after several generations1.

  20. Actin and Endocytosis in Budding Yeast

    PubMed Central

    Goode, Bruce L.; Eskin, Julian A.; Wendland, Beverly

    2015-01-01

    Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed. PMID:25657349