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1

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

PubMed Central

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.

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

2008-01-01

2

Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast  

Microsoft Academic Search

BACKGROUND: The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT\\/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection

Alessandra Mallano; Silvia Zamboni; Giulia Carpinelli; Filippo Santoro; Michela Flego; Alessandro Ascione; Mara Gellini; Marina Tombesi; Franca Podo; Maurizio Cianfriglia

2008-01-01

3

Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast  

PubMed Central

Background The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies. Results An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU. Conclusion The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.

Mallano, Alessandra; Zamboni, Silvia; Carpinelli, Giulia; Santoro, Filippo; Flego, Michela; Ascione, Alessandro; Gellini, Mara; Tombesi, Marina; Podo, Franca; Cianfriglia, Maurizio

2008-01-01

4

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

PubMed

Detection of a therapeutic response early in the course of cancer treatment, before tumor growth delay or regression, is not currently possible in experimental models or clinical medicine. New interim measures of therapeutic response would be particularly useful in the development of cancer chemosensitization gene therapy by facilitating optimization of gene transfer protocols and prodrug dosing schedules. Diffusion MRI is a sensitive technique producing quantitative and noninvasive images of the apparent mobility of water within a tissue. We investigated the utility of diffusion MRI for detecting early changes associated with a refined cytosine deaminase (CD)/5-fluorocytosine (5FC) chemosensitization gene therapy paradigm in orthotopic 9L gliomas stably expressing the recently cloned S. cerevisiae CD gene. Mean tumor diffusion increased 31% within 8 days of initiating 5-FC treatment, preceding tumor growth arrest and regression. Complete regression of the intracranial tumor was observed in four of five treated animals, and recurrent tumor in the remaining animal exhibited water diffusion behavior similar to primary, untreated tumors. These results demonstrate the efficacy of the yCD/5FC strategy for glioma and suggest that increased tumor water diffusion is an indicator of active therapeutic intervention. PMID:10871748

Stegman, L D; Rehemtulla, A; Hamstra, D A; Rice, D J; Jonas, S J; Stout, K L; Chenevert, T L; Ross, B D

2000-06-01

5

AID/APOBEC cytosine deaminase induces genome-wide kataegis  

PubMed Central

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

2012-01-01

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

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.

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

7

Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase  

SciTech Connect

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.

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

2011-12-31

8

Alanine-Scanning Mutagenesis Reveals a Cytosine Deaminase Mutant with Altered Substrate Preference †  

Microsoft Academic Search

ABSTRACT: Suicide gene therapy of cancer is a method,whereby,cancerous,tumors,can be selectively eradicated while sparing damage to normal tissue. This is accomplished by delivering a gene, encoding an enzyme capable of specifically converting a nontoxic prodrug into a cytotoxin, to cancer cells followed by prodrug administration. The Escherichia coli gene, codA, encodes cytosine deaminase and is introduced into cancer cells followed

Sheri D. Mahan; Greg C. Ireton; Barry L. Stoddard; Margaret E. Black

2004-01-01

9

Efficacy of gene therapy-delivered cytosine deaminase is determined by enzymatic activity but not expression  

PubMed Central

The potential utility of tumour-selective 5-fluorouracil treatment using attenuated Salmonella serovar typhimurium recombinant for cytosine deaminase (TAPET-CD) has been documented in experimental settings. The present data demonstrate that in vivo 19F-magnetic resonance spectroscopy measurements allow the outcome prediction of this prokaryotic-based therapy, demonstrating the necessity of non-invasive real-time imaging techniques for treatment monitoring.

Dubois, L; Dresselaers, T; Landuyt, W; Paesmans, K; Mengesha, A; Wouters, B G; Hecke, P Van; Theys, J; Lambin, P

2007-01-01

10

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

Microsoft Academic Search

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)

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

2007-01-01

11

Combined cytosine deaminase expression, 5-fluorocytosine exposure, and radiotherapy increases cytotoxicity to cholangiocarcinoma cells  

Microsoft Academic Search

Cholangiocarcinoma is a malignancy that is resistant to current therapy. We applied the toxin gene therapy strategy of cytosine deaminase conversion of the nontoxic prodrug 5-fluorocytosine to 5-fluorouracil combined with radiotherapy to cholangiocarcinoma. The transduction efficiency of SK-ChA-1 cholangiocarcinoma cells was determined by fluorescence-activated cell-sorting analysis following infection with recombinant adenovirus AdCMVLacZ, which encodes the gene for ?-galactosidase. To evaluate

Lee C. Pederson; Selwyn M. Vickers; Donald J. Buchsbaum; Sreekanth R. Kancharla; Matthew S. Mayo; David T. Curiel; Murray A. Stackhouse

1998-01-01

12

Adenovirus-mediated hypoxia-targeting cytosine deaminase gene therapy enhances radiotherapy in tumour xenografts  

Microsoft Academic Search

Hypoxia is closely associated with the radioresistance of tumours; therefore, targeting hypoxic areas is very important for cancer therapy. The aim of this study is to establish such a targeting strategy by applying a bacterial cytosine deaminase (BCD)\\/5-fluorocytosine (5-FC) gene therapy system and to examine whether the strategy enhances the efficacy of radiotherapy in a tumour xenograft. The hypoxia-responsive promoter

J Liu; H Harada; M Ogura; T Shibata; M Hiraoka

2007-01-01

13

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

SciTech Connect

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

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

2007-04-01

14

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

Microsoft Academic Search

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

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

15

Combined radiation and gene therapy for brain tumors with adenovirus-mediated transfer of cytosine deaminase and uracil phosphoribosyltransferase genes  

Microsoft Academic Search

Radiation therapy is an established modality for the treatment of malignant gliomas. Several reports have shown the advantage of additional radiation in combination with gene therapy. In this study, we investigated the ability of radiation therapy to enhance 5-fluorocytosine (5-FC)\\/cytosine deaminase (CD) plus uracil phosphoribosyltransferase (UPRT) gene therapy in malignant gliomas. In vitro study suggested evidence of a significant cytotoxic

Hirokazu Kambara; Takashi Tamiya; Yasuhiro Ono; Shinji Ohtsuka; Kinya Terada; Yoshiaki Adachi; Tomotsugu Ichikawa; Hirofumi Hamada; Takashi Ohmoto

2002-01-01

16

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

Microsoft Academic Search

Herpes simplex virus type-1 thymidine kinase (HSV-1TK) and Escherichia coli cytosine deaminase (CD) fusion protein was designed using InsightII software. The structural rationality of the fusion proteins incorporating a series of flexible linker peptide was analyzed, and a suitable linker peptide was chosen for further investigated. The recombinant plasmid containing the coding regions of HSV-1TK and CD cDNA connected by

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

2007-01-01

17

In vivo efficacy and toxicity of 5-fluorocytosine\\/cytosine deaminase gene therapy for malignant gliomas mediated by adenovirus  

Microsoft Academic Search

We evaluated the therapeutic efficacy and neurotoxicity of adenovirus-mediated transduction of the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) for experimental malignant brain tumors. The 5-FC sensitivity in 9 L cells infected by an adenovirus vector expressing CD (AdexCACD) was increased 1700-fold compared with control cells. Rats bearing 9 L brain tumors were treated with an intratumoral injection of AdexCACD

Tomotsugu Ichikawa; Takashi Tamiya; Yoshiaki Adachi; Yasuhiro Ono; Kengo Matsumoto; Tomohisa Furuta; Yoko Yoshida; Hirofumi Hamada; Takashi Ohmoto

2000-01-01

18

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

PubMed Central

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.

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

19

Therapeutic effect of genetically modified human neural stem cells encoding cytosine deaminase on experimental glioma.  

PubMed

The aim of this study was to determine the efficacy of neural stem cell-based suicidal gene therapy in rats bearing human glioma. F3 human neural stem cells (NSCs) were transduced to encode cytosine deaminase (CD) which converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). Intratumoral or intravenous transplantation of F3.CD human NSCs led to marked reduction in tumor burden and significantly prolonged the survival of brain tumor-bearing rats. The systemic administration of 5-FC with direct intratumoral/intravenous transplantation of F3.CD cells had remarkable therapeutic effect in rats with human glioma cells as compared with transplantation of parental F3 cells. There was 74% reduction in tumor volume in rats receiving direct transplantation of F3.CD cells into tumor site, and 67% reduction in tumor volume in rats receiving intravenous injection of F3.CD cells as compared to control animals transplanted with human glioma U373 cells alone. The combination of F3.CD and 5-FC was a highly effective in the glioma rat model. Our observations suggest that genetically engineered NSCs encoding suicide gene CD could provide clinical application of suicide gene therapy for patients with glioma. PMID:22177952

Kim, Jae Ho; Kim, Jin Young; Kim, Seung U; Cho, Kyung Gi

2011-12-08

20

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

PubMed

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

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

2013-02-22

21

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

PubMed

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, gamma(1)34.5-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) was constructed in which both copies of the gamma(1)34.5 gene were replaced with the bacterial CD gene, under the control of the cellular promoter Egr-1; (ii) M012-infected cells in vitro efficiently convert 5-fluorocytosine (5-FC) to 5-fluorouracil, thereby enhancing cytotoxicity of neighboring, uninfected cells; (iii) both direct and bystander cytotoxicity of murine neuroblastoma and human glioma cell lines after infection with M012 were demonstrated; (iv) direct intracerebral inoculation of A/J mice demonstrated lack of neurotoxicity at doses similar to G207, a gamma(1)34.5-deleted HSV with demonstrated safety in human patient trials and (v) intratumoral injection of M012 into Neuro-2a flank tumors in combination with 5-FC administration significantly reduced tumor growth versus tumors treated with R3659 combined with 5-FC, or treated with M012 alone. Thus, M012 is a promising new oncolytic HSV vector with an enhanced prodrug-mediated, antineoplastic effect that is safe for intracranial administration. PMID:16990846

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

2006-09-22

22

Cytidine Deaminase Genotype and Toxicity of Cytosine Arabinoside Therapy in Children with Acute Myeloid Leukemia  

PubMed Central

Cytosine arabinoside (ara-C) is irreversibly deaminated by cytidine deaminase (CDD) to a nontoxic metabolite. A common polymorphism, A79C, in CDD changes a lysine residue to glutamine resulting in decreased enzyme activity. We determined CDD A79C genotypes for 457 children with AML treated on CCG 2941 and 2961 and analyzed the impact of CDD genotype on therapy outcomes. Post-Induction treatment related mortality (TRM) was significantly elevated in children with the CC genotype (5 year TRM 17 ± 13% CC vs 7 ± 4% AA, 5 ± 4% AC, p= 0.05). This was more notable in children who received IDA-FLAG (ara-C= 7590 mg/m2) (5 year TRM 24 ± 21% CC vs 6 ± 6% AA, 6 ± 7% AC, p=0.07) as consolidation therapy compared to IDA-DCTER (ara-C= 800 mg/m2) (5 year TRM 15 ± 20% CC vs 8 ± 6% AA, 4 ± 6% AC; p=0.29). Relapse-free survival was non-significantly increased in children with the CC genotype treated with IDA-FLAG (76 ± 20% CC vs 59 ± 12% AA and 55 ± 14% AC; p= 0.40). These data indicate that children with a low activity CDD genotype are at increased risk of treatment-related mortality with Ara-C based therapy for AML.

Bhatla, Deepika; Gerbing, Robert B; Alonzo, Todd A.; Conner, Heather; Ross, Julie A; Meshinchi, Soheil; Zhai, Xiaowen; Zamzow, Tiffany; Mehta, Parinda A; Geiger, Hartmut; Perentesis, John; Davies, Stella M

2010-01-01

23

CYTOTOXICITY OF ADENOVIRAL-MEDIATED CYTOSINE DEAMINASE PLUS 5-FLUOROCYTOSINE GENE THERAPY IS SUPERIOR TO THYMIDINE KINASE PLUS ACYCLOVIR IN A HUMAN RENAL CELL CARCINOMA MODEL  

Microsoft Academic Search

PurposeAn estimated 11,600 Americans will die of renal cell carcinoma in 1998. The lack of effective chemotherapy or radiotherapy requires the investigation of novel treatment modalities. We compared two forms of toxic gene therapy, cytosine deaminase (CD) plus 5-fluorocytosine (5-FC) and thymidine kinase (TK) plus acyclovir (ACV), in pre-clinical models of human renal cell carcinoma.

TOSHIRO SHIRAKAWA; THOMAS A. GARDNER; SONG-CHU KO; NEIL BANDER; SAVIO WOO; AKINOBU GOTOH; SADAO KAMIDONO; LELAND W. K. CHUNG; CHINGHAI KAO

1999-01-01

24

Engineering conditionally replication-competent adenoviral vectors carrying the cytosine deaminase gene increases the infectivity and therapeutic effect for breast cancer gene therapy  

Microsoft Academic Search

We constructed a conditionally replication-competent adenoviral vector Ad.Lp-CD-IRES-E1A(control) in which the expression of both the prodrug-activating cytosine deaminase gene and the viral replication E1A gene were driven by the L-plastin tumor-specific promoter. In order to overcome the low infectivity of the adenoviral vectors for breast cancer cells, and to increase the safety and efficacy for cancer gene therapy, this vector

Y Liu; T Ye; J Maynard; H Akbulut; A Deisseroth

2006-01-01

25

Cellular and molecular events associated with the antitumor response induced by the cytosine deaminase\\/5-fluorocytosine suicide gene therapy system in a rat liver metastasis model  

Microsoft Academic Search

The bacterial cytosine deaminase (CD) gene converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil. We have previously shown, in a rat liver metastasis model from colon carcinoma, that intratumoral injection of a CD-expressing plasmid into the animals followed by 5-FC treatment results in the regression of the treated tumor as well as distant uninjected tumors. The aim of this study

S Bertin; S Neves; A Gavelli; P Baqué; N Brossette; S Simões; M C Pedroso de Lima; V Pierrefite-Carle

2007-01-01

26

Gene amplification of human cytidine deaminase proviral cDNA and increased levels of its mRNA produces enhanced drug resistance to cytosine arabinoside in retroviral-transduced murine fibroblasts  

Microsoft Academic Search

Hematopoietic toxicity is one of the major problems that limits the effectiveness of many antineoplastic drugs. One approach to overcome this problem is to confer chemoresistance to the hematopoietic cells by gene transfer of drug resistance genes. Human cytidine deaminase (CD) inactivates the cytosine nucleoside analogues, such as cytosine arabinoside (ARA-C), by deamination. We have reported previously that retroviral-mediated gene

Christian M. Beauséjour; Richard L. Momparler

1998-01-01

27

A cyclooxygenase-2/prostaglandin E2 pathway augments activation-induced cytosine deaminase expression within replicating human B cells.  

PubMed

Within inflammatory environments, B cells encountering foreign or self-Ag can develop tertiary lymphoid tissue expressing activation-induced cytosine deaminase (AID). Recently, this DNA-modifying enzyme was detected in nonlymphoid cells within several inflamed tissues and strongly implicated in malignant transformation. This study examines whether a cyclooxygenase 2 (COX-2) pathway, often linked to inflammation, influences AID expression in activated B lymphocytes. In this paper, we report that dividing human B cells responding to surrogate C3d-coated Ag, IL-4, and BAFF express AID, as well as COX-2. A progressive increase in AID with each division was paralleled by a division-related increase in a COX-2-linked enzyme, microsomal PGE(2) synthase-1, and the PGE(2)R, EP2. Cells with the greatest expression of AID expressed the highest levels of EP2. Although COX-2 inhibitors diminished both AID expression and IgG class switching, exogenous PGE(2) and butaprost, a selective EP2 agonist, augmented AID mRNA/protein and increased the numbers of IgG(+) progeny. Despite the latter, the proportion of IgG(+) cells within viable progeny generally declined with PGE(2) supplementation. This was not due to PGE(2)-promoted differentiation to plasma cells or to greater downstream switching. Rather, because phosphorylated ataxia telangiectasia mutated levels were increased in progeny of PGE(2)-supplemented cultures, it appears more likely that PGE(2) facilitates AID-dependent DNA double-strand breaks that block B cell cycle progression or promote activation-induced cell death, or both. Taken together, the results suggest that a PGE(2) feed-forward mechanism for augmenting COX-2 pathway proteins promotes progressively increased levels of AID mRNA, protein, and function. PMID:20921530

Lee, Hyunjoo; Trott, Joshua S; Haque, Shabirul; McCormick, Steven; Chiorazzi, Nicholas; Mongini, Patricia K A

2010-10-04

28

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.  

PubMed

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. PMID:17663441

Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

2008-02-01

29

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

PubMed

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

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

2013-09-05

30

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

PubMed Central

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.

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

31

In vivo replication-deficient adenovirus vector-mediated transduction of the cytosine deaminase gene sensitizes glioma cells to 5-fluorocytosine.  

PubMed

Viral vector-mediated transfer of chemosensitization genes represents a promising new approach to the treatment of cancer. Previous reports have demonstrated that transfection of the bacterial cytosine deaminase (cd) gene into mammalian cells can sensitize them to the otherwise nontoxic nucleoside, 5-fluorocytosine (5-FC). We now report that a replication-deficient adenovirus vector that transduces the cd gene (Ad.CMV-cd) highly sensitizes 9L gliosarcoma cells to 5-FC, and that gene transduction is associated with a potent bystander effect that is not dependent on direct cell-to-cell contact. Stereotactic injection of Ad.CMV-cd into established rat gliomas, followed by systemic administration of 5-FC in vivo, results in prolongation of survival. PMID:8919593

Dong, Y; Wen, P; Manome, Y; Parr, M; Hirshowitz, A; Chen, L; Hirschowitz, E A; Crystal, R; Weichselbaum, R; Kufe, D W; Fine, H A

1996-04-10

32

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

PubMed

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

Gopinath, P; Ghosh, Siddhartha Sankar

2007-12-19

33

Oncolysis by an HSV-1 engineered to express cytosine deaminase and a fusogenic glycoprotein for head and neck squamous cell carcinoma  

PubMed Central

Background A replication-competent, attenuated, oncolytic herpes simplex virus-1, OncoVEXGALV/CD, has previously been engineered to express a fusogenic protein from the gibbon ape leukemia virus and cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT) which converts fluorocytosine (5-FC) to 5-fluorouracil (5-FU). OncoVEXGFP is an analogous vector that expresses enhanced green fluorescent protein. Methods We assessed the ability of OncoVEXGALV/CD and OncoVEXGFP to infect, replicate within, and lyse four head and neck squamous carcinoma (HNSCC) cell lines in vitro. The effects of adding 5-FC with OncoVEXGALV/CD were evaluated. Results HNSCC was permissive to GFP expression in100% of cells by OncoVEXGFP at a multiplicity of infection (MOI) of 1 after 48 hours, and supported logarithmic viral replication. Virus caused >60% cell death six days after exposure to virus at MOI 0.1 in three of the four cell lines. 5-FC failed to enhance cytotoxicity induced by OncoVEXGALV/CD at MOI 0.1. However, for the least sensitive SCC25 cell line, virus at MOI 0.01 was cytotoxic to only 4% of cells after six days, but was cytotoxic to 35% of cells with 5-FC. Conclusions OncoVEXGALV/CD efficiently infects, replicates within and lyses HNSCC at relatively low viral doses. Prodrug conversion by CD did not enhance therapy at viral doses which cause efficient cytotoxicity, but may have beneficial effects in less sensitive cell lines at low viral doses.

Price, Daniel L.; Lin, Shu-Fu; Han, Ziqun; Simpson, Guy; Coffin, Robert S.; Wong, Joyce; Li, Sen; Fong, Yuman; Wong, Richard J.

2009-01-01

34

Suppression of the growth of human colorectal cancer cells by therapeutic stem cells expressing cytosine deaminase and interferon-? via their tumor-tropic effect in cellular and xenograft mouse models.  

PubMed

Genetically engineered stem cells (GESTECs) exhibit a potent therapeutic efficacy via their strong tumor tropism toward cancer cells. In this study, we introduced the human parental neural stem cells, HB1.F3, with the human interferon beta (IFN-?) gene which is a typical cytokine gene that has an antitumor effect and the cytosine deaminase (CD) gene from Escherichia coli (E. coli) that could convert the non-toxic prodrug, 5-fluorocytosine (5-FC), to a toxic metabolite, 5-fluorouracil (5-FU). Two types of stem cells expressing the CD gene (HB1.F3.CD cells) and both the CD and human IFN-? genes (HB1.F3.CD.IFN-?) were generated. The present study was performed to examine the migratory and therapeutic effects of these GESTECs against the colorectal cancer cell line, HT-29. When co-cultured with colorectal cancer cells in the presence of 5-FC, HB1.F3.CD and HB1.F3.CD.IFN-? cells exhibited the cytotoxicity on HT-29 cells via the bystander effect. In particular, HB1.F3.CD.IFN-? cells showed the synergistic cytotoxic activity of 5-FU and IFN-?. We also confirmed the migration ability of HB1.F3.CD and HB1.F3.CD.IFN-? cells toward HT-29 cells by a modified migration assay in vitro, where chemoattractant factors secreted by HT-29 cells attracted the GESTECs. In a xenograft mouse model, the volume of tumor mass was decreased up to 56% in HB1.F3.CD injected mice while the tumor mass was greatly inhibited about 76% in HB1.F3.CD.IFN-? injected mice. The therapeutic treatment by these GESTECs is a novel strategy where the combination of the migration capacity of stem cells as a vector for therapeutic genes towards colorectal cancer and a synergistic antitumor effect of CD and IFN-? genes can selectively target this type of cancer. PMID:23403306

Yi, Bo-Rim; Park, Min-Ah; Lee, Hye-Rim; Kang, Nam-Hee; Choi, Kelvin J; Kim, Seung U; Choi, Kyung-Chul

2013-01-19

35

Low-dose etoposide enhances telomerase-dependent adenovirus-mediated cytosine deaminase gene therapy through augmentation of adenoviral infection and transgene expression in a syngeneic bladder tumor model.  

PubMed

The human telomerase reverse transcriptase (hTERT) promoter can selectively drive transgene expression in many telomerase-positive human cancer cells. Here we evaluated combination therapy of adenoviral vector Ad-hTERT-CD encoding E. coli cytosine deaminase (CD) driven by the hTERT promoter and low-dose etoposide (0.1 microg/mL) for treating bladder cancer. Ad-hTERT-CD conferred sensitivity to 5-fluorocytosine (5-FC) in bladder cancer cells, which could be enhanced by etoposide treatment, but not in normal cells. Such effect was correlated with up-regulation of hypoxia-inducible factor (HIF)-1alpha expression. By contrast, etoposide activated p53 and down-regulated hTERT promoter activity in normal cells. Etoposide also increased adenoviral infection via enhancement of coxsackie-adenovirus receptor expression on bladder cancer and normal cells. Combination index analysis revealed that combined therapy of Ad-hTERT-CD (10(9) plaque-forming units)/5-FC (200 mg/kg) with etoposide (2 mg/kg) synergistically suppressed tumor growth and prolonged survival in mice bearing syngeneic MBT-2 bladder tumors. This combination therapy regimen induced complete tumor regression and generated antitumor immunity in 75% of tumor-bearing mice. Furthermore, increased infiltrating CD4(+) and CD8(+) T cells and necrosis within tumors were found in mice receiving combination therapy of Ad-hTERT-CD and etoposide compared with those treated with either treatment alone. Thus, the potential high therapeutic index of the combination therapy may be an appealing therapeutic intervention for bladder cancer. Furthermore, because a majority of human tumors exhibit high telomerase activity, adenovirus-mediated CD gene therapy driven by the hTERT promoter in combination with low-dose etoposide may be applicable to a broad spectrum of cancers. PMID:17047058

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

2006-10-15

36

Rescue of the Orphan Enzyme Isoguanine Deaminase  

SciTech Connect

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.

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

2011-12-31

37

Statistical modelling and phylogenetic analysis of a deaminase domain.  

PubMed

Deamination reactions are catalyzed by a variety of enzymes including those involved in nucleoside/nucleotide metabolism and cytosine to uracil (C-->U) and adenosine to inosine (A-->I) mRNA editing. The active site of the deaminase (DM) domain in these enzymes contains a conserved histidine (or rarely cysteine), two cysteines and a glutamate proposed to act as a proton shuttle during deamination. Here, a statistical model, a hidden Markov model (HMM), of the DM domain has been created which identifies currently known DM domains and suggests new DM domains in viral, bacterial and eucaryotic proteins. However, no DM domains were identified in the currently predicted proteins from the archaeon Methanococcus jannaschii and possible causes for, and a potential means to ameliorate this situation are discussed. In some of the newly identified DM domains, the glutamate is changed to a residue that could not function as a proton shuttle and in one instance (Mus musculus spermatid protein TENR) the cysteines are also changed to lysine and serine. These may be non-competent DM domains able to bind but not act upon their substrate. Phylogenetic analysis using an HMM-generated alignment of DM domains reveals three branches with clear substructure in each branch. The results suggest DM domains that are candidates for yeast, platyhelminth, plant and mammalian C-->U and A-->I mRNA editing enzymes. Some bacterial and eucaryotic DM domains form distinct branches in the phylogenetic tree suggesting the existence of common, novel substrates. PMID:9541871

Mian, I S; Moser, M J; Holley, W R; Chatterjee, A

1998-01-01

38

Myoadenylate deaminase deficiency  

Microsoft Academic Search

Summary Myoadenylate deaminase (MAD) is the rate-limiting enzyme in the purine nucleotide cycle which is biochemically linked to glycolysis and the citric cycle and thereby providing energy during intense muscular activity. In muscle fibers, myoadenylate deaminase operates at considerably higher activity levels than in other organs. First detected using enzyme-histochemical methods, it now appears that deficiency of myoadenylate deaminase is

H. H. Goebel; A. Bardosi

1987-01-01

39

Site-selective in vivo targeting of cytosine-5 DNA methylation by zinc-finger proteins  

Microsoft Academic Search

Cytosine-5 DNA methylation is a critical signal defining heritable epigenetic states of transcription. As aberrant methylation patterns often accompany disease states, the ability to target cytosine methyl- ation to preselected regions could prove valuable in re-establishing proper gene regulation. We employ the strategy of targeted gene methylation in yeast, which has a naturally unmethylated genome, select- ively directing de novo

Christopher D. Carvin; Rebecca D. Parr; Michael P. Kladde

2003-01-01

40

Tad1p, a yeast tRNA-specific adenosine deaminase, is related to the mammalian pre-mRNA editing enzymes ADAR1 and ADAR2.  

PubMed Central

We have identified an RNA-specific adenosine deaminase (termed Tad1p/scADAT1) from Saccharomyces cerevisiae that selectively converts adenosine at position 37 of eukaryotic tRNAAla to inosine. The activity of purified recombinant Tad1p depends on the conformation of its tRNA substrate and the enzyme was found to be inactive on all other types of RNA tested. Mutant strains in which the TAD1 gene is disrupted are viable but lack Tad1p enzyme activity and their tRNAAla is not modified at position A37. Transformation of the mutant cells with the TAD1 gene restored enzyme activity. Tad1p has significant sequence similarity with the mammalian editing enzymes which act on specific precursor-mRNAs and on long double-stranded RNA. These findings suggest an evolutionary link between pre-mRNA editing and tRNA modification.

Gerber, A; Grosjean, H; Melcher, T; Keller, W

1998-01-01

41

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

Microsoft Academic Search

Naturally occurring drug resistance genes of human origin can be exploited for selection of genetically engineered cells co-expressing a desired therapeutic transgene. Their non-immunogenicity in clinical applications would be a major asset. Human cytidine deaminase (hCD) is a chemoresistance gene that inactivates cytotoxic cytosine nucleoside analogs, such as cytosine arabinoside (Ara-C). The aim of this study was to establish if

N Eliopoulos; A Al-Khaldi; CM Beauséjour; RL Momparler; LF Momparler; J Galipeau

2002-01-01

42

The Cytosine Water Complex  

NASA Astrophysics Data System (ADS)

A multi FID system has been adapted into the operation sequence of the LA-MB-FTMW spectrometer. Thanks to the reached sensitivity, one monohydrate of cytosine (A= 3725.61 (26) MHz, B=980.385 (76) MHz, C=777.231 (46) MHz) has been detected in the supersonic expansion. J. --U. Grabow, W. Stahl, H. Dreizler, Rev. Sci. Instrum. 1996, 67, 4072 -- 4084. J. L. Alonso, C. Pérez, M. E. Sanz, J. C. López, S. Blanco, Phys. Chem. Chem. Phys. 2009, 11, 617 -- 627.

Daly, A. M.; Mata, S.; Bermudez, C.; Berdakin, M.; Pena, I.; Cabezas, C.; Alonso, J. L.

2013-06-01

43

The Crystal Structure of the Bifunctional Deaminase\\/Reductase RibD of the Riboflavin Biosynthetic Pathway in Escherichia coli: Implications for the Reductive Mechanism  

Microsoft Academic Search

We have determined the crystal structure of the bi-functional deaminase\\/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1–145) is similar to cytosine deaminase and the C-terminal domain (146–367) is similar to dihydrofolate reductase. We showed that

Pål Stenmark; Martin Moche; Daniel Gurmu; Pär Nordlund

2007-01-01

44

A 43.5 kb segment of yeast chromosome XIV, which contains MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1, predicts an adenosine deaminase gene and 14 new open reading frames.  

PubMed

A 43,481 bp fragment from the left arm of chromosome XIV of Saccharomyces cerevisiae was sequenced. A gene for tRNA(phe) and 23 non-overlapping open reading frames (ORFs) were identified, seven of which correspond to known yeast genes: MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1. One ORF may correspond to the yet unidentified yeast adenosine deaminase gene. Among the 15 other ORFs, four exhibit known signatures, which include a protein tyrosine phosphatase, a cytoskeleton-associated protein and two ATP-binding proteins, four have similarities with putative proteins of yeast or proteins from other organisms and seven exibit no significant similarity with amino acid sequences described in data banks. One ORF is identical to yeast expressed sequence tags (EST) and therefore corresponds to an expressed gene. Six ORFs present similarities to human dbESTs, thus identifying motifs conserved during evolution. Nine ORFs are putative transmembrane proteins. In addition, one overlapping and three antisense ORFs, which are not likely to be functional, were detected. PMID:8619318

Mallet, L; Bussereau, F; Jacquet, M

1995-09-30

45

Inhibition of cytidine deaminase by zebularine enhances the antineoplastic action of 5-aza-2'-deoxycytidine.  

PubMed

Cytidine (CR) deaminase is a key enzyme in the catabolism of cytosine nucleoside analogues, since their deamination results in a loss of their pharmacological activity. In this report we have investigated the importance of CR deaminase with respect to the antineoplastic action of inhibitors of DNA methylation, 5-aza-2'-deoxycytidine (5-AZA-CdR) and zebularine. Zebularine has a dual mechanism of action, since it can also inhibit CR deaminase. The objective of our study was to investigate the importance of zebularine as an inhibitor of CR deaminase with respect to the antineoplastic action of 5-AZA-CdR. Using an in vitro clonogenic assay, we investigated the antineoplastic action of 5-AZA-CdR and zebularine, alone and in combination on wild type 3T3 murine fibroblasts and corresponding V5 cells transduced with CR deaminase gene to express a very high level of CR deaminase activity. The V5 cells were much less sensitive to 5-AZA-CdR than the wild type 3T3 cells. The addition of zebularine significantly enhanced the antineoplastic action of 5-AZA-CdR on V5 cells, but not 3T3 cells. Enzymatic analysis on CR deaminase purified from the V5 cells showed that zebularine is a competitive inhibitor of the deamination of 5-AZA-CdR. These in vitro observations are in accord with our in vivo study in mice with L1210 leukemia, which showed that zebularine increased the antileukemic activity of 5-AZA-CdR. Pharmacokinetic analysis also showed that zebularine increased the plasma level of 5-AZA-CdR during an i.v. infusion in mice. Our results indicate that the major mechanism by which zebularine enhances the antineoplastic action of 5-AZA-CdR is by inhibition of CR deaminase. These findings provide a rationale to investigate 5-AZA-CdR in combination with zebularine in patients with advanced leukemia. PMID:18398609

Lemaire, Maryse; Momparler, Louise F; Raynal, Noël J-M; Bernstein, Mark L; Momparler, Richard L

2008-04-09

46

DNA cytosine methylation in plant development.  

PubMed

Cytosine bases of the nuclear genome in higher plants are often extensively methylated. Cytosine methylation has been implicated in the silencing of both transposable elements (TEs) and endogenous genes, and loss of methylation may have severe functional consequences. The recent methylation profiling of the entire Arabidopsis genome has provided novel insights into the extent and pattern of cytosine methylation and its relationships with gene activity. In addition, the fresh studies also revealed the more dynamic nature of this epigenetic modification across plant development than previously believed. Cytosine methylation of gene promoter regions usually inhibits transcription, but methylation in coding regions (gene-body methylation) does not generally affect gene expression. Active demethylation (though probably act synergistically with passive loss of methylation) of promoters by the 5-methyl cytosine DNA glycosylase or DEMETER (DME) is required for the uni-parental expression of imprinting genes in endosperm, which is essential for seed viability. The opinion that cytosine methylation is indispensible for normal plant development has been reinforced by using single or combinations of diverse loss-of-function mutants for DNA methyltransferases, DNA glycosylases, components involved in siRNA biogenesis and chromatin remodeling factors. Patterns of cytosine methylation in plants are usually faithfully maintained across organismal generations by the concerted action of epigenetic inheritance and progressive correction of strayed patterns. However, some variant methylation patterns may escape from being corrected and hence produce novel epialleles in the affected somatic cells. This, coupled with the unique property of plants to produce germline cells late during development, may enable the newly acquired epialleles to be inherited to future generations, which if visible to selection may contribute to adaptation and evolution. PMID:20171573

Zhang, Meishan; Kimatu, Josphert N; Xu, Kezhang; Liu, Bao

2010-01-01

47

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

SciTech Connect

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

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

2011-10-28

48

Rat kidney porphobilinogen deaminase kinetics  

Microsoft Academic Search

Background and aims: Acute intermittent porphyria (AIP) is an inherited disease resulting from a reduced activity of the enzyme porphobilinogen deaminase (PBG-D). The kidney is an important target for numerous porphyrinogenic drugs and it may contribute to the clinical manifestations of porphyric attacks. An evaluation of kidney PBG-D role in the AIP pathophysiology requires detailed information on kidney PBG-D properties,

Guillermo Noriega; Guillermo Mattei; Alcira Batlle; Adela Ana Juknat

2002-01-01

49

Bacterial Ammeline Metabolism via Guanine Deaminase ?  

PubMed Central

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.

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

2010-01-01

50

Experimental Thermochemistry of Gas Phase Cytosine Tautomers  

NASA Astrophysics Data System (ADS)

Enthalpies of interconversion are measured for the three lowest energy tautomers of isolated cytosine. The equilibrium distribution of tautomers near 600 K is frozen upon the capture of the gas phase species by low temperature helium nanodroplets. The temperature dependence of the gas phase cytosine tautomer populations is determined with infrared laser spectroscopy of the helium solvated species. The interconverison enthalpies obtained from the van't Hoff relation are 1.14 ± 0.21 and 1.63 ± 0.12 for the C31 rightleftharpoons C32 and C31 rightleftharpoons C1 equilibria, respectively. C31 and C32 are rotamers of an enol tautomer, and C1 is a keto tautomer. The interconversion enthalpies are compared to recent CCSD(T) thermochemistry calculations of cytosine tautomers.

Morrison, A. M.; Douberly, G. E.

2011-06-01

51

Photophysical pathways of cytosine in aqueous solution  

SciTech Connect

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

Kistler, Kurt A.; Matsika, Spiridoula

2010-02-28

52

Discovery and Structure Determination of the Orphan Enzyme Isoxanthopterin Deaminase  

PubMed Central

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

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

2010-01-01

53

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

PubMed Central

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.

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

2013-01-01

54

Confounded cytosine! Tinkering and the evolution of DNA  

Microsoft Academic Search

Early in the history of DNA, thymine replaced uracil, thus solving a short-term problem for storing genetic information — mutation of cytosine to uracil through deamination. Any engineer would have replaced cytosine, but evolution is a tinkerer not an engineer. By keeping cytosine and replacing uracil the problem was never eliminated, returning once again with the advent of DNA methylation.

Anthony Poole; David Penny; Britt-Marie Sjöberg

2001-01-01

55

Replication Fork Collapse and Genome Instability in a Deoxycytidylate Deaminase Mutant  

PubMed Central

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

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

2012-01-01

56

Genetics Home Reference: Adenosine monophosphate deaminase deficiency  

MedlinePLUS

... deaminase deficiency? asymptomatic ; autosomal ; autosomal recessive ; cell ; deficiency ; enzyme ; gene ; joint ; mutation ; myalgia ; nucleotide ; population ; prevalence ; recessive ; skeletal muscle You may find definitions for these and many other terms in the ...

57

An APOBEC Cytidine Deaminase Mutagenesis Pattern is Widespread in Human Cancers  

PubMed Central

Recent studies indicate that a subclass of APOBEC cytidine deaminases, which convert cytosine to uracil during RNA editing and retrovirus or retrotransposon restriction, may induce mutation clusters in human tumors. We show here that throughout cancer genomes APOBEC mutagenesis is pervasive and correlates with APOBEC mRNA levels. Mutation clusters in whole-genome and exome datasets conformed to stringent criteria indicative of an APOBEC mutation pattern. Applying these criteria to 954,247 mutations in 2,680 exomes of 14 cancer types, mostly from TCGA, revealed significant presence of the APOBEC mutation pattern in bladder, cervical, breast, head and neck and lung cancers, reaching 68% of all mutations in some samples. Within breast cancer, the HER2E subtype was clearly enriched with tumors displaying the APOBEC mutation pattern, suggesting this type of mutagenesis is functionally linked with cancer development. The APOBEC mutation pattern also extended to cancer-associated genes, implying that ubiquitous APOBEC mutagenesis is carcinogenic.

Roberts, Steven A.; Lawrence, Michael S.; Klimczak, Leszek J.; Grimm, Sara A.; Fargo, David; Stojanov, Petar; Kiezun, Adam; Kryukov, Gregory V.; Carter, Scott L.; Saksena, Gordon; Harris, Shawn; Shah, Ruchir R.; Resnick, Michael A.; Getz, Gad; Gordenin, Dmitry A.

2013-01-01

58

Evolution of vitamin B2 biosynthesis: eubacterial RibG and fungal Rib2 deaminases.  

PubMed

Eubacterial RibG and yeast Rib2 possess a deaminase domain for pyrimidine deamination in the second and third steps, respectively, of riboflavin biosynthesis. These enzymes are specific for ribose and ribitol, respectively. Here, the crystal structure of Bacillus subtilis RibG in complex with a deaminase product is reported at 2.56 Å resolution. Two loops move towards the product on substrate binding, resulting in interactions with the ribosyl and phosphate groups and significant conformational changes. The product carbonyl moiety is bent out of the pyrimidine ring to coordinate to the catalytic zinc ion. Such distortions in the bound substrate and product may play an essential role in enzyme catalysis. The yeast Rib2 structure was modelled and a mutational analysis was carried out in order to understand the mechanism of substrate recognition in these two enzymes. Detailed structural comparisons revealed that the two consecutive carbonyl backbones that occur prior to the PCXXC signature constitute a binding hole for the target amino group of the substrate. This amino-binding hole is essential in B. subtilis RibG and is also conserved in the RNA/DNA-editing deaminases. PMID:23385458

Chen, Sheng Chia; Shen, Chieh Yi; Yen, Te Ming; Yu, Hui Chia; Chang, Ting Hao; Lai, Wen Lin; Liaw, Shwu Huey

2013-01-19

59

Discovery and Structure Determination of the Orphan Enzyme Isoxanthopterin Deaminase  

SciTech Connect

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

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

2010-05-25

60

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

PubMed

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

Bianchi, Caterina; Zangi, Ronen

2012-10-04

61

Transcriptional pausing and stalling causes multiple clustered mutations by human activation-induced deaminase  

PubMed Central

Transcription of the rearranged immunoglobulin gene and expression of the enzyme activation-induced deaminase (AID) are essential for somatic hypermutations of this gene during antibody maturation. While AID acts as a single-strand DNA-cytosine deaminase creating U · G mispairs that lead to mutations, the role played by transcription in this process is less clear. We have used in vitro transcription of the kan gene by the T7 RNA polymerase (RNAP) in the presence of AID and a genetic reversion assay for kanamycin-resistance to investigate the causes of multiple clustered mutations (MCMs) during somatic hypermutations. We find that, depending on transcription conditions, AID can cause single-base substitutions or MCMs. When wild-type RNAP is used for transcription at physiologically relevant concentrations of ribonucleoside triphosphates (NTPs), few MCMs are found. In contrast, slowing the rate of elongation by reducing the NTP concentration or using a mutant RNAP increases several-fold the percent of revertants containing MCMs. Arresting the elongation complexes by a quick removal of NTPs leads to formation of RNA-DNA hybrids (R-loops). Treatment of these structures with AID results in a high percentage of KanR revertants with MCMs. Furthermore, selecting for transcription elongation complexes stalled near the codon that suffers mutations during acquisition of kanamycin-resistance results in an overwhelming majority of revertants with MCMs. These results show that if RNAP II pauses or stalls during transcription of immunoglobulin gene, AID is likely to promote MCMs. As changes in physiological conditions such as occurrence of certain DNA primary or secondary structures or DNA adducts are known to cause transcriptional pausing and stalling in mammalian cells, this process may cause MCMs during somatic hypermutation.—Canugovi, C., Samaranayake, M., Bhagwat, A. S. Transcriptional pausing and stalling causes multiple clustered mutations by human activation-induced deaminase.

Canugovi, Chandrika; Samaranayake, Mala; Bhagwat, Ashok S.

2009-01-01

62

Developmental changes of chicken liver AMP deaminase.  

PubMed Central

The AMP deaminase activity measured in crude chicken liver extract did not change significantly during development. The livers of 10- and 14-day chick embryos, 1-day, 5-, 10- and 16-week-old chickens and adult hens were examined for the existence of multiple forms of AMP deaminase. Phosphocellulose column chromatography revealed the existence of two peaks of enzyme activity in the liver of 10- and 16-week-old chickens and adult hens. Kinetic studies with the preparations of AMP deaminase revealed sigmoid-shaped substrate-saturation curves at all developmental stages and hyperbolic-shaped saturation curves for the enzyme form appearing in 10-week-old chickens. All AMP deaminases investigated were susceptible to activation by ATP and inhibition by Pi. Kinetic and regulatory properties as well as pH optima of all the enzyme preparations tested indicate that AMP deaminase isolated from the embryos and from 1-day-old chicks was similar to the form I isolated from adult hens and differed significantly from the form II of this enzyme.

Spychala, J; Kaletha, K; Makarewicz, W

1985-01-01

63

Perspectives of bacterial ACC deaminase in phytoremediation.  

PubMed

Phytoremediation of contaminated soil and water environments is regulated and coordinated by the plant root system, yet root growth is often inhibited by pollutant-induced stress. Prolific root growth could maximize rates of hyperaccumulation of inorganic contaminants or rhizodegradation of organic pollutants, and thus accelerate phytoremediation. Accelerated ethylene production in response to stress induced by contaminants is known to inhibit root growth and is considered as a major limitation in improving phytoremediation efficiency. Recent work shows that bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase regulates ethylene levels in plants by metabolizing its precursor ACC into alpha-ketobutyric acid and ammonia. Plants inoculated with ACC deaminase bacteria or transgenic plants that express bacterial ACC deaminase genes can regulate their ethylene levels and consequently contribute to a more extensive root system. Such proliferation of roots in contaminated soil can lead to enhanced uptake of heavy metals or rhizodegradation of xenobiotics. PMID:17573137

Arshad, Muhammad; Saleem, Muhammad; Hussain, Sarfraz

2007-06-18

64

Concerted action of activation-induced cytidine deaminase and uracil-DNA glycosylase reduces covalently closed circular DNA of duck hepatitis B virus.  

PubMed

Covalently closed circular DNA (cccDNA) forms a template for the replication of hepatitis B virus (HBV) and duck HBV (DHBV). Recent studies suggest that activation-induced cytidine deaminase (AID) functions in innate immunity, although its molecular mechanism of action remains unclear, particularly regarding HBV restriction. Here we demonstrated that overexpression of chicken AID caused hypermutation and reduction of DHBV cccDNA levels. Inhibition of uracil-DNA glycosylase (UNG) by UNG inhibitor protein (UGI) abolished AID-induced cccDNA reduction, suggesting that the AID/UNG pathway triggers the degradation of cccDNA via cytosine deamination and uracil excision. PMID:23954625

Chowdhury, Sajeda; Kitamura, Kouichi; Simadu, Miyuki; Koura, Miki; Muramatsu, Masamichi

2013-08-15

65

The interferon-inducible, double-stranded RNA-specific adenosine deaminase gene (DSRAD) maps to human chromosome 1q21.1-21.2  

SciTech Connect

The interferon-inducible double-stranded RNA-specific adenosine deaminase is an RNA-modifying enzyme implicated in the generation of biased hypermutations of viral RNAs and the site-selective editing of mammalian mRNAs of neural origin. The gene for the dsRNA-specific adenosine deaminase has been mapped by fluorescence in situ hybridization (FISH) of genomic clones to a single locus on human chromosome 1 bands q21.1-21.2. Simultaneous multicolor FISH including X clones and yeast artificial chromosomes showed a localization of the gene in band 1q21 centromeric of D1S1705. 22 refs., 1 fig.

Weier, H.U.G.; Greulich, K.M. [Lawrence Berkeley National Lab., CA (United States); George, C.X.; Samuel, C.E. [Univ. of California, Santa Barbara, CA (United States)

1995-11-20

66

Prenatal diagnosis for adenosine deaminase deficiency  

Microsoft Academic Search

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

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

1981-01-01

67

Adenosine deaminase deficiency with mosaicism for a \\  

Microsoft Academic Search

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

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

2002-01-01

68

Dipeptidyl peptidase IV and adenosine deaminase activity  

Microsoft Academic Search

Dipeptidyl peptidase IV (DPPIV) and adenosine deaminase (ADA), two T cell associated enzymes, are known to have a possible interaction and play essential roles in immune system functioning. On the other hand, depression has been shown to be accompanied with some immune-inflammatory alterations. In this regard, in order to make a contribution to the understanding of the ongoing immune disturbances

Serenay Elgün; Aytaç Keskinege; Hakan Kumbasar

1999-01-01

69

Isolation of a Saccharomyces cerevisiae mutant strain deficient in deoxycytidylate deaminase activity and partial characterization of the enzyme.  

PubMed Central

Deoxycytidylate deaminase activity in Saccharomyces cerevisiae has been partially characterized. The yeast enzyme was found to exhibit properties similar to those of dCMP deaminases isolated from higher eucaryotes. A mutant strain completely deficient in dCMP deaminase activity was isolated by selection for resistance to 5-fluoro-2'-deoxycytidylate followed by screening for cross sensitivity to 5-fluoro-2'-deoxyuridylate, a potent inhibitor of the yeast thymidylate synthetase. We have designated this new allele dcd1 . A strain exhibiting an auxotrophic requirement for dUMP was isolated after mutagenesis of a dcd1 tup7 haploid. Genetic analysis revealed that this auxotrophic phenotype resulted from a combination of the dcd1 allele and a second, unlinked, nuclear mutation that we designated dmp1 . This allele, which by itself conveys no readily discernible phenotype, presumably impairs efficient synthesis of dUMP from UDP. The auxotrophic requirement of dcd1 dmp1 tup7 strains also can be satisfied by exogenous dTMP but not deoxyuridine.

McIntosh, E M; Haynes, R H

1984-01-01

70

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

PubMed

The spectrum of the adenosine monophosphate (AMP) deaminase deficiency ranges from asymptomatic carriers to patients who manifest exercise-induced muscle pain, occasionally rhabdomyolysis, and idiopathic hyperCKemia. However, previous to the introduction of molecular techniques, rare cases with congenital weakness and hypotonia have also been reported. We report a 6-month-old girl with the association of congenital muscle weakness and hypotonia, muscle deficiency of adenosine monophosphate deaminase, and the homozygous C to T mutation at nucleotide 34 of the adenosine monophosphate deaminase-1 gene. This observation indicates the possible existence of a primary adenosine monophosphate deaminase deficiency manifested by congenital muscle weakness and hypotonia. PMID:21343608

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

2011-02-22

71

Purine Metabolism in Adenosine Deaminase Deficiency  

Microsoft Academic Search

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

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

1976-01-01

72

Hot Spot Mutations in Adenosine Deaminase Deficiency  

Microsoft Academic Search

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

Rochelle Hirschhorn; Stephanie Tzall; Amy Ellenbogen

1990-01-01

73

A New Nuclear Function of the Entamoeba histolytica Glycolytic Enzyme Enolase: The Metabolic Regulation of Cytosine5 Methyltransferase 2 (Dnmt2) Activity  

Microsoft Academic Search

Cytosine-5 methyltransferases of the Dnmt2 family function as DNA and tRNA methyltransferases. Insight into the role and biological significance of Dnmt2 is greatly hampered by a lack of knowledge about its protein interactions. In this report, we address the subject of protein interaction by identifying enolase through a yeast two-hybrid screen as a Dnmt2-binding protein. Enolase, which is known to

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

2010-01-01

74

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

SciTech Connect

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.

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

2006-01-01

75

Myoadenylate deaminase deficiency in twins with recessive olivopontocerebellar atrophy  

Microsoft Academic Search

Two adult non-identical twins with autosomal recessive olivopontocerebellar degeneration (OPCA) had markedly deficient adenylate deaminase in skeletal muscle homogenates. Ischemic exercise failed to increase the blood ammonia, while lactate increased normally. Glutamate dehydrogenase and NADP-dependent malic enzyme activities in muscle mitochondria of both patients were normal. The significance of adenylate deaminase deficiency in these twins with OPCA is discussed.

G. Uziel; F. Cornelio; C. Gellera; G. Perego; M. Rimoldi

1986-01-01

76

Adenosine deaminase in disorders of purine metabolism and in immune deficiency  

SciTech Connect

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

Tritsch, G.L.

1985-01-01

77

An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers.  

PubMed

Recent studies indicate that a subclass of APOBEC cytidine deaminases, which convert cytosine to uracil during RNA editing and retrovirus or retrotransposon restriction, may induce mutation clusters in human tumors. We show here that throughout cancer genomes APOBEC-mediated mutagenesis is pervasive and correlates with APOBEC mRNA levels. Mutation clusters in whole-genome and exome data sets conformed to the stringent criteria indicative of an APOBEC mutation pattern. Applying these criteria to 954,247 mutations in 2,680 exomes from 14 cancer types, mostly from The Cancer Genome Atlas (TCGA), showed a significant presence of the APOBEC mutation pattern in bladder, cervical, breast, head and neck, and lung cancers, reaching 68% of all mutations in some samples. Within breast cancer, the HER2-enriched subtype was clearly enriched for tumors with the APOBEC mutation pattern, suggesting that this type of mutagenesis is functionally linked with cancer development. The APOBEC mutation pattern also extended to cancer-associated genes, implying that ubiquitous APOBEC-mediated mutagenesis is carcinogenic. PMID:23852170

Roberts, Steven A; Lawrence, Michael S; Klimczak, Leszek J; Grimm, Sara A; Fargo, David; Stojanov, Petar; Kiezun, Adam; Kryukov, Gregory V; Carter, Scott L; Saksena, Gordon; Harris, Shawn; Shah, Ruchir R; Resnick, Michael A; Getz, Gad; Gordenin, Dmitry A

2013-07-14

78

Repeated Intravenous Administration of Cytosine Arabinoside Triacetate to Beagle Dogs.  

National Technical Information Service (NTIS)

Two beagle dogs, one male and one female, received daily intravenous doses of 50 mg/kg of cytosine arabinoside triacetate for 15 consecutive days. The surviving dog was observed for a period of 30 days following the 15-day dose regime. The criteria of eff...

H. Feinman T. W. Tusing E. R. Homan D. P. Rall

1967-01-01

79

Specific targeting of cytosine methylation to DNA sequences in vivo  

Microsoft Academic Search

Development of methods that will allow exogenous imposition of inheritable gene-specific methylation patterns has potential application in both therapeu- tics 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

Alexander E. Smith; Kevin G. Ford

2007-01-01

80

Cellular Pharmacology of Cyclopentenyl Cytosine in Molt4 Lymphoblasts  

Microsoft Academic Search

The toxicity, uptake, and metabolism of the oncolytic nucleoside cyclopentenyl cytosine (CPEC) have been examined in the Molt-4 line of human lymphoblasts. This compound is known to be converted to its 5'- triphosphate, which inhibits CTP synthetase and depletes the pools of cytidine nucleotides. In the Molt-4 system, the concentration of drug reducing proliferation by 50% in a 24-h incubation

Harry Ford; David A. Cooney; Gurpreet S. Ahluwalia; Michael E. Rommel; LeRoi Hicks; Kathryn A. Dobyns; Joseph E. Tomaszewski; David G. Johns

81

Transcriptional regulation of ACC deaminase gene expression in Pseudomonas putida UW4.  

PubMed

One of the major mechanisms that plant growth-promoting bacteria use to facilitate plant growth is through the lowering of plant ethylene levels by the bacterial enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Many of the bacterial ACC deaminase genes (acdS) that have been examined to date are under the transcriptional control of a leucine-responsive regulatory protein, Lrp, encoded by acdR and referred to here as AcdR. The work presented here is focused on how AcdR and the newly discovered AcdB protein from Pseudomonas putida UW4 are involved in the regulation of acdS expression. First, the results of gel retardation experiments showed that AcdR binds to the acdS regulatory region, and this binding activity in vitro is not affected by the addition of 2 mmol x L-1 ACC but can be eliminated by addition of 20 microg x mL-1 leucine. Second, a potential regulatory protein, AcdB, involved in the regulation of acdS expression, was identified through both yeast 2-hybrid screen and coimmunoprecipitation based on its ability to bind to AcdR; subsequently, its binding to the acdS regulatory region in the presence of ACC was shown by gel retardation experiments. The data are interpreted in terms of a model in which AcdR and AcdB co-regulate the expression of the acdS gene. PMID:18388982

Cheng, Zhenyu; Duncker, Bernard P; McConkey, Brendan J; Glick, Bernard R

2008-02-01

82

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

Microsoft Academic Search

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

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

1978-01-01

83

Renal Deoxyadenosine Transport and Immunodeficiency Associated with Adenosine Deaminase Deficiency  

Microsoft Academic Search

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

J. Arly Nelson

1989-01-01

84

Maintaining Genome Stability: The Role of Helicases and Deaminases.  

National Technical Information Service (NTIS)

Both helicases and deaminases are enzymes that play an important role in maintaining genomic stability and immune responses. Errors in duplicating DNA can result in genomic instability, leading to various human diseases, such as cancer, immune system diso...

X. Chen

2008-01-01

85

Pharmacological inhibition of AMP-deaminase in rat cardiac myocytes.  

PubMed

Because mutation of AMP deaminase 1 gene leading to reduced AMP deaminase activity may result in protection of cardiac function in patients with heart disease, inhibitors of AMP deaminase (AMPD) may have therapeutic applications. This study evaluated the effect of a specific inhibitor of AMP deaminase 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol (AMPDI) on the isolated human enzyme and on nucleotide catabolism in rat cardiomyocytes. AMPDI effectively inhibited isolated human AMPD with an IC(50) = 0.5 micro M. AMPDI was much less effective with isolated cardiomyocytes (IC(50) = 0.5 mM). AMPDI is a very effective inhibitor of AMPD that despite lower efficiency in the cell system examined could be useful for in vivo studies. PMID:18600554

Borkowski, T; Orlewska, C; Slominska, E M; Yuen, A; Lipinski, M; Rybakowska, I; Foks, H; Kaletha, K K; Yacoub, M H; Smolenski, R T

2008-06-01

86

Yeast Dihydroorotate Dehydrogenase as a New Selectable Marker for Plasmodium falciparum Transfection  

PubMed Central

Genetic manipulation of Plasmodium falciparum in culture through transfection has provided numerous insights into the molecular and cell biology of this parasite. The procedure is rather cumbersome, and is limited by the number of drug-resistant markers that can be used for selecting transfected parasites. Here we report a new selectable marker that could allow multiple transfections. We have taken advantage of our finding that a critical function of the mitochondrial electron transport chain (mtETC) in the erythrocytic stages of P. falciparum is the regeneration of ubiquinone as co-substrate of dihydroorotate dehydrogenase (DHODH), and that transgenic P. falciparum expressing ubiquinone-independent DHODH from yeast (yDHODH) are resistant to all mtETC inhibitors. We assessed the possibility of using yDHODH as a positive selectable marker for transfections of P. falciparum, including its use in gene disruption strategies. We constructed a transfection vector designed for gene disruption, termed pUF-1, containing the yDHODH gene as the positive selection marker in combination with a previously described fused yeast cytosine deaminase-uracil phosphoribosyl transferase gene as a negative selection marker. Transfection of the D10 strain followed by selection with atovaquone yielded positively selected parasites containing the plasmid, demonstrating that yDHODH can be used as a selective marker. Atovaquone, however, could not be used for such selection with the Dd2 strain of P. falciparum. On the other hand, we demonstrated that yDHODH transgenic parasites could be selected in both strains by Plasmodium DHODH-specific triazolopyrimidine-based inhibitors. Thus, selection with DHODH inhibitors was superior in that it successfully selected transgenic Dd2 parasites, as well as yielded transgenic parasites after a shorter period of selection. As a proof of concept, we have successfully disrupted the type II vacuolar proton-pumping pyrophosphatase gene (PfVP2) in P. falciparum by double crossover recombination, showing that this gene is not essential for the survival of blood stage parasites.

Ganesan, Suresh M.; Morrisey, Joanne M.; Ke, Hangjun; Painter, Heather J.; Laroiya, Kamal; Phillips, Margaret A.; Rathod, Pradipsinh K.; Mather, Michael W.; Vaidya, Akhil B.

2011-01-01

87

Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection.  

PubMed

Genetic manipulation of Plasmodium falciparum in culture through transfection has provided numerous insights into the molecular and cell biology of this parasite. The procedure is rather cumbersome, and is limited by the number of drug-resistant markers that can be used for selecting transfected parasites. Here we report a new selectable marker that could allow multiple transfections. We have taken advantage of our finding that a critical function of the mitochondrial electron transport chain (mtETC) in the erythrocytic stages of P. falciparum is the regeneration of ubiquinone as co-substrate of dihydroorotate dehydrogenase (DHODH), and that transgenic P. falciparum expressing ubiquinone-independent DHODH from yeast (yDHODH) are resistant to all mtETC inhibitors. We assessed the possibility of using yDHODH as a positive selectable marker for transfections of P. falciparum, including its use in gene disruption strategies. We constructed a transfection vector designed for gene disruption, termed pUF-1, containing the yDHODH gene as the positive selection marker in combination with a previously described fused yeast cytosine deaminase-uracil phosphoribosyl transferase gene as a negative selection marker. Transfection of the D10 strain followed by selection with atovaquone yielded positively selected parasites containing the plasmid, demonstrating that yDHODH can be used as a selective marker. Atovaquone, however, could not be used for such selection with the Dd2 strain of P. falciparum. On the other hand, we demonstrated that yDHODH transgenic parasites could be selected in both strains by Plasmodium DHODH-specific triazolopyrimidine-based inhibitors. Thus, selection with DHODH inhibitors was superior in that it successfully selected transgenic Dd2 parasites, as well as yielded transgenic parasites after a shorter period of selection. As a proof of concept, we have successfully disrupted the type II vacuolar proton-pumping pyrophosphatase gene (PfVP2) in P. falciparum by double crossover recombination, showing that this gene is not essential for the survival of blood stage parasites. PMID:21251930

Ganesan, Suresh M; Morrisey, Joanne M; Ke, Hangjun; Painter, Heather J; Laroiya, Kamal; Phillips, Margaret A; Rathod, Pradipsinh K; Mather, Michael W; Vaidya, Akhil B

2011-01-18

88

A screening protocol for identification of functional mutants of RNA editing adenosine deaminases.  

PubMed

Genetic screens can be used to evaluate a spectrum of mutations and thereby infer the function of particular residues within a protein. The Adenosine Deaminase Acting on RNA (ADAR) family of RNA-editing enzymes selectively deaminate adenosines (A) in double-helical RNA, generating inosine (I). The protocol described here exploits the editing activity of ADAR2 in a yeast-based screen by inserting an editing substrate sequence with a stop codon incorporated at the editing site upstream from the sequence encoding the reporter ?-galactosidase. A-to-I editing changes the stop codon to a tryptophan codon, allowing normal expression of the reporter. This technique is particularly well-suited for screening ADAR and ADAR substrate mutant libraries for editing activity. Curr. Protoc. Chem. Biol. 4:357-369 © 2012 by John Wiley & Sons, Inc. PMID:23788559

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

2012-12-01

89

ACC Deaminase Containing PGPR for Potential Exploitation in Agriculture  

Microsoft Academic Search

\\u000a The beneficial free-living bacteria present in the plant rhizosphere are usually referred to as plant growth promoting rhizobacteria\\u000a (PGPR). Among the various mechanisms of plant growth promotion, certain PGPR possess the enzyme 1-aminocyclopropane-1-carboxylic\\u000a acid (ACC) deaminase that cleaves plant-produced ACC, the immediate precursor of the stress hormone ethylene. ACC deaminase\\u000a containing PGPR act as a sink for ACC and protects

Venkadasamy Govindasamy; Murugesan Senthilkumar; Pranita Bose; Lakkineni Vithal Kumar; D. Ramadoss; Kannepalli Annapurna

90

[Adenosine deaminase in severe combined immunodeficiency syndrome].  

PubMed

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

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

2012-09-01

91

l-Serine Deaminase of Escherichia coli  

PubMed Central

The native l-serine deaminase (l-serine hydrolyase, deaminating, EC 4.2.1.13) of Escherichia coli K-12, which seems to be a very labile protein, is rather stable in concentrated solution. Dilution rapidly inactivates it, but in the presence of a saturating concentration of l-serine the molecule is protected from inactivation. It is a very specific enzyme; l-serine is the sole substrate with a Km value of 6.60 × 10?3m. d-Serine and l-cysteine are competitive inhibitors. Substrate saturation curves of the native enzyme show sigmoid shape, whereas the enzyme liberated from the bacteria in the presence of l-serine exhibits normal Michaelis-Menten kinetics.

Alfoldi, Lajos; Rasko, Istvan; Kerekes, Erzsebet

1968-01-01

92

The Curious Chemical Biology of Cytosine: Deamination, Methylation and Oxidation as Modulators of Genomic Potential  

PubMed Central

A multitude of functions have evolved around cytosine within DNA, endowing the base with physiological significance beyond simple information storage. This versatility arises from enzymes that chemically modify cytosine to expand the potential of the genome. Some modifications alter coding sequences, such as deamination of cytosine by AID/APOBEC enzymes to generate immunologic or virologic diversity. Other modifications are critical to epigenetic control, altering gene expression or cellular identity. Of these, cytosine methylation is well understood, in contrast to recently discovered modifications, such as oxidation by TET enzymes to 5-hydroxymethylcytosine. Further complexity results from cytosine demethylation, an enigmatic process that impacts cellular pluripotency. Recent insights help us to propose an integrated DNA demethylation model, accounting for contributions from cytosine oxidation, deamination and base excision repair. Taken together, this rich medley of alterations renders cytosine a genomic “wild card”, whose context-dependent functions make the base far more than a static letter in the code of life.

Nabel, Christopher S.; Manning, Sara A.; Kohli, Rahul M.

2011-01-01

93

The curious chemical biology of cytosine: deamination, methylation, and oxidation as modulators of genomic potential.  

PubMed

A multitude of functions have evolved around cytosine within DNA, endowing the base with physiological significance beyond simple information storage. This versatility arises from enzymes that chemically modify cytosine to expand the potential of the genome. Some modifications alter coding sequences, such as deamination of cytosine by AID/APOBEC enzymes to generate immunologic or virologic diversity. Other modifications are critical to epigenetic control, altering gene expression or cellular identity. Of these, cytosine methylation is well understood, in contrast to recently discovered modifications, such as oxidation by TET enzymes to 5-hydroxymethylcytosine. Further complexity results from cytosine demethylation, an enigmatic process that impacts cellular pluripotency. Recent insights help us to propose an integrated DNA demethylation model, accounting for contributions from cytosine oxidation, deamination, and base excision repair. Taken together, this rich medley of alterations renders cytosine a genomic "wild card", whose context-dependent functions make the base far more than a static letter in the code of life. PMID:22004246

Nabel, Christopher S; Manning, Sara A; Kohli, Rahul M

2011-10-31

94

Structural features of antiviral DNA cytidine deaminases.  

PubMed

Abstract The APOBEC3 (A3) family of cytidine deaminases plays a vital role for innate defense against retroviruses. Lentiviruses such as HIV-1 evolved the Vif protein that triggers A3 protein degradation. There are seven A3 proteins, A3A-A3H, found in humans. All A3 proteins can deaminate cytidines to uridines in single-stranded DNA (ssDNA), generated during viral reverse transcription. A3 proteins have either one or two cytidine deaminase domains (CD). The CDs coordinate a zinc ion, and their amino acid specificity classifies the A3s into A3Z1, A3Z2, and A3Z3. A3 proteins occur as monomers, dimers, and large oligomeric complexes. Studies on the nature of A3 oligomerization, as well as the mode of interaction of A3s with RNA and ssDNA are partially controversial. High-resolution structures of the catalytic CD2 of A3G and A3F as well as of the single CD proteins A3A and A3C have been published recently. The NMR and X-ray crystal structures show globular proteins with six ?-helices and five ? sheets arranged in a characteristic motif (?1-?1-?2/2'-?2-?3-?3-?4-?4-?5-?5-?6). However, the detailed arrangement and extension of individual structure elements and their relevance for A3 complex formation and activity remains a matter of debate and will be highlighted in this review. PMID:23787464

Vasudevan, Ananda Ayyappan Jaguva; Smits, Sander H J; Höppner, Astrid; Häussinger, Dieter; Koenig, Bernd W; Münk, Carsten

2013-11-01

95

The catalase activity of diiron adenine deaminase.  

PubMed

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS. PMID:21998098

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

2011-11-09

96

The control of natural variation in cytosine methylation in Arabidopsis.  

PubMed Central

We explore the extent and sources of epigenetic variation in cytosine methylation in natural accessions of the flowering plant, Arabidopsis thaliana, by focusing on the methylation of the major rRNA gene repeats at the two nucleolus organizer regions (NOR). Our findings indicate that natural variation in NOR methylation results from a combination of genetic and epigenetic mechanisms. Genetic variation in rRNA gene copy number and trans-acting modifier loci account for some of the natural variation in NOR methylation. Our results also suggest that divergence and inheritance of epigenetic information, independent of changes in underlying nucleotide sequence, may play an important role in maintaining natural variation in cytosine methylation.

Riddle, Nicole C; Richards, Eric J

2002-01-01

97

Specific targeting of cytosine methylation to DNA sequences in vivo.  

PubMed

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

Smith, Alexander E; Ford, Kevin G

2006-12-20

98

High-throughput sequencing of cytosine methylation in plant DNA  

PubMed Central

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

2013-01-01

99

Rhizobium leguminosarum Biovar viciae 1-Aminocyclopropane-1Carboxylate Deaminase Promotes Nodulation of Pea Plants  

Microsoft Academic Search

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and charac- terized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria

Wenbo Ma; Frederique C. Guinel; Bernard R. Glick

2003-01-01

100

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

PubMed Central

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.

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

2013-01-01

101

The catalase activity of diiron adenine deaminase  

SciTech Connect

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

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

2011-12-01

102

Effects of bacterial ACC deaminase on Brassica napus gene expression.  

PubMed

Plants in association with plant growth-promoting rhizobacteria can benefit from lower plant ethylene levels through the action of the bacterial enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. This enzyme cleaves the immediate biosynthetic precursor of ethylene, ACC. Ethylene is responsible for many aspects of plant growth and development but, under stressful conditions, it exacerbates stress symptoms. The ACC deaminase-containing bacterium Pseudomonas putida UW4 is a potent plant growth-promoting strain and, as such, was used to elaborate the detailed role of bacterial ACC deaminase in Brassica napus (canola) plant growth promotion. Transcriptional changes in bacterially treated canola plants were investigated with the use of an Arabidopsis thaliana oligonucleotide microarray. A heterologous approach was necessary because there are few tools available at present to measure global expression changes in nonmodel organisms, specifically with the sensitivity of microarrays. The results indicate that the transcription of genes involved in plant hormone regulation, secondary metabolism, and stress response was altered in plants by the presence of the bacterium, whereas the upregulation of genes for auxin response factors and the downregulation of stress response genes was observed only in the presence of bacterial ACC deaminase. These results support the suggestion that there is a direct link between ethylene and the auxin response, which has been suggested from physiological studies, and provide more evidence for the stress-reducing benefits of ACC deaminase-expressing plant growth-promoting bacteria. PMID:22352713

Stearns, Jennifer C; Woody, Owen Z; McConkey, Brendan J; Glick, Bernard R

2012-05-01

103

Hot spot mutations in adenosine deaminase deficiency  

SciTech Connect

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

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

1990-08-01

104

The preparation, characterisation and solubility characteristics of a hydrogen-bonded complex between acyclovir and cytosine  

Microsoft Academic Search

Acyclovir and cytosine form a Crick-Watson hydrogen-bonded complex in dimethylsulphoxide (DMSO). For the complex formation Acyclovir + cytosine ? complex the equilibrium constant, K, was determined using NMR spectroscopy to be K = 1.00 ± 0.07 mol?1 dm3 at 21°C in DMSO.The acyclovir-cytosine complex was formed in DMSO, then diluted with octan-1-ol to leave a 95.3% v\\/v octan-1-ol\\/47% v\\/v DMSO

C. S. Newby; M. Coke

1996-01-01

105

Delayed onset adenosine deaminase deficiency associated with acute disseminated encephalomyelitis.  

PubMed

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

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

2012-03-24

106

Adenosine deaminase activity in fertile and infertile men.  

PubMed

Adenosine deaminase (ADA; E.C.3.5.4.4) catalyses the deamination of adenosine to inosine. In the human reproductive system, the importance of enzymes that affect metabolism of adenosine, particularly adenosine deaminase, has been noticed. The purpose of this study was to determine the plasma activities of total adenosine deaminase (ADAT), and its isoenzymes, ADA1 and ADA2, in fertile and infertile men. Plasma activities of ADA and its isoenzymes were measured in 55 fertile men and 70 infertile men. There was a significant difference in the ADA1 and ADA2 activities between fertile and infertile individuals (P < 0.01). The activity of ADAT, ADA2 and ADA1 in infertile men was higher than that in fertile individuals. This alteration in ADA activity can lead to reduced adenosine levels, which may be involved in disturbing the fertility process. PMID:21919946

Rostampour, F; Biglari, M; Vaisi-Raygani, A; Salimi, S; Tavilani, H

2011-09-15

107

Identification, expression, and characterization of Escherichia coli guanine deaminase.  

PubMed

Using the human cDNA sequence corresponding to guanine deaminase, the Escherichia coli genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown function was identified as having 36% identity to the human protein. The putative gene was amplified, subcloned into the pMAL-c2 vector, expressed, purified, and characterized enzymatically. The 50.2-kDa protein catalyzed the conversion of guanine to xanthine, having a K(m) of 15 microM with guanine and a k(cat) of 3.2 s(-1). The bacterial enzyme shares a nine-residue heavy metal binding site with human guanine deaminase, PG[FL]VDTHIH, and was found to contain approximately 1 mol of zinc per mol of subunit of protein. The E. coli guanine deaminase locus is 3' from an open reading frame which shows homology to a bacterial purine base permease. PMID:10913105

Maynes, J T; Yuan, R G; Snyder, F F

2000-08-01

108

Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. Evidence for a dinuclear zinc site.  

PubMed

XAS of Zn-peptide binary and ternary complexes prepared using peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMP deaminase (AMPD) strongly suggest that the region 48-61 of the enzyme contains a zinc binding site, whilst the region 360-372 of the enzyme is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD. XAS performed on fresh preparations of rabbit skeletal muscle AMPD provides evidence for a dinuclear zinc site in the enzyme compatible with a (mu-aqua)(mu-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn-Zn distance of about 3.3 Angstrom. The data indicate that zinc is not required for HPRG/AMPD interaction, both zinc ions being bound to the catalytic subunit of the enzyme, one to the three conserved amino acid residues among those four assumed to be in contact with zinc in yeast AMPD, and the other at the N-terminal region, probably to His-52, Glu-53 and His-57. Tryptic digests of different enzyme preparations demonstrate the existence of two different protein conformations and of a zinc ion connecting the N-terminal and C-terminal regions of AMPD. PMID:17254852

Mangani, Stefano; Benvenuti, Manuela; Moir, Arthur J G; Ranieri-Raggi, Maria; Martini, Daniela; Sabbatini, Antonietta R M; Raggi, Antonio

2006-12-23

109

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

PubMed

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

Pornbanlualap, Somchai; Chalopagorn, Pornchanok

2011-04-12

110

Fluorocyclopentenyl-cytosine with broad spectrum and potent antitumor activity.  

PubMed

On the basis of the potent biological activity of cyclopentenyl-pyrimidines, fluorocyclopentenyl-pyrimidines were designed and synthesized from D-ribose. Among these, the cytosine derivative 5a showed highly potent antigrowth effects in a broad range of tumor cell lines and very potent antitumor activity in a nude mouse tumor xenograft model implanted with A549 human lung cancer cells. However, its 2'-deoxycytidine derivative 5b did not show any antigrowth effects, indicating that 2'-hydroxyl group is essential for the biological activity. PMID:22524616

Choi, Won Jun; Chung, Hwa-Jin; Chandra, Girish; Alexander, Varughese; Zhao, Long Xuan; Lee, Hyuk Woo; Nayak, Akshata; Majik, Mahesh S; Kim, Hea Ok; Kim, Jin-Hee; Lee, Young B; Ahn, Chang H; Lee, Sang Kook; Jeong, Lak Shin

2012-05-01

111

Elastic electron scattering from the DNA bases cytosine and thymine  

SciTech Connect

Cross-section data for electron scattering from biologically relevant molecules are important for the modeling of energy deposition in living tissue. Relative elastic differential cross sections have been measured for cytosine and thymine using the crossed-beam method. These measurements have been performed for six discrete electron energies between 60 and 500 eV and for detection angles between 15 deg. and 130 deg. Calculations have been performed via the screen-corrected additivity rule method and are in good agreement with the present experiment.

Colyer, C. J.; Bellm, S. M.; Lohmann, B. [ARC Centre of Excellence for Antimatter-Matter Studies, University of Adelaide, Adelaide, South Australia 5005 (Australia); Blanco, F. [Departamento de Fisica Atomica Molecular y Nuclear, Facultad de Ciencias Fisicas. Universidad Complutense, Avenida Complutense s/n, E-28040 Madrid (Spain); Garcia, G. [Instituto de Fisica Fundamental, Consejo Superior de Investigaciones Cientificas, Serrano 113-bis, E-28006 Madrid (Spain)

2011-10-15

112

A New Family of High-Affinity Transporters for Adenine, Cytosine, and Purine Derivatives in Arabidopsis  

PubMed Central

In many organisms, including plants, nucleic acid bases and derivatives such as caffeine are transported across the plasma membrane. Cytokinins, important hormones structurally related to adenine, are produced mainly in root apices, from where they are translocated to shoots to control a multitude of physiological processes. Complementation of a yeast mutant deficient in adenine uptake (fcy2) with an Arabidopsis cDNA expression library enabled the identification of a gene, AtPUP1 (for Arabidopsis thaliana purine permease1), belonging to a large gene family (AtPUP1 to AtPUP15) encoding a new class of small, integral membrane proteins. AtPUP1 transports adenine and cytosine with high affinity. Uptake is energy dependent, occurs against a concentration gradient, and is sensitive to protonophores, potentially indicating secondary active transport. Competition studies show that purine derivatives (e.g., hypoxanthine), phytohormones (e.g., zeatin and kinetin), and alkaloids (e.g., caffeine) are potent inhibitors of adenine and cytosine uptake. Inhibition by cytokinins is competitive (competitive inhibition constant \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}K\\end{equation*}\\end{document}i = \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}20\\;{\\mathrm{to}}\\;35\\end{equation*}\\end{document} ?\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{M}}\\end{equation*}\\end{document}), indicating that cytokinins are transported by this system. AtPUP1 is expressed in all organs except roots, indicating that the gene encodes an uptake system for root-derived nucleic acid base derivatives in shoots or that it exports nucleic acid base analogs from shoots by way of the phloem. The other family members may have different affinities for nucleic acid bases, perhaps functioning as transporters for nucleosides, nucleotides, and their derivatives.

Gillissen, Bernd; Burkle, Lukas; Andre, Bruno; Kuhn, Christina; Rentsch, Doris; Brandl, Birgit; Frommer, Wolf B.

2000-01-01

113

Metal-modified nucleobase pairs and triplets as cytosine receptors.  

PubMed

A preorganized cationic receptor 2 for cytosine (C) is described which is composed of trans-a2PtII (a= NH3 or CH3NH2) cross-linked modules with adenine (A), guanine (G), and uracil (U) or thymine (T) model nucleobases. The functions of these three modules are as follows: i) Adenine orientates the two other bases at right angles, thus producing the L-shape of the receptor. ii) Guanine is the primary receptor. iii) Uracil or thymine act as coreceptors. Compared with the normal Watson-Crick pair between G and C, the association constant between 2 and C increases by a factor of 3 (in DMSO). As deduced from 1H NMR spectroscopy and confirmed by the X-ray crystal structure of the C adduct 4b, cytosine is fixed through five hydrogen bonds to the receptor, one of which involves the aromatic H(5) of C. A comparison of C binding is made with a structurally related linkage isomer receptor as well as the precursor molecule trans[alpha2PtAG]2+. The potential of modular, cationic receptors is illustrated. PMID:11411983

Lüth, M S; Freisinger, E; Lippert, B

2001-05-18

114

Arabidopsis PAI gene arrangements, cytosine methylation and expression.  

PubMed Central

Previous analysis of the PAI tryptophan biosynthetic gene family in Arabidopsis thaliana revealed that the Wassilewskija (WS) ecotype has four PAI genes at three unlinked sites: a tail-to-tail inverted repeat at one locus (PAI1-PAI4) plus singlet genes at two other loci (PAI2 and PAI3). The four WS PAI genes are densely cytosine methylated over their regions of DNA identity. In contrast, the Columbia (Col) ecotype has three singlet PAI genes at the analogous loci (PAI1, PAI2, and PAI3) and no cytosine methylation. To understand the mechanism of PAI gene duplication at the polymorphic PAI1 locus, and to investigate the relationship between PAI gene arrangement and PAI gene methylation, we analyzed 39 additional ecotypes of Arabidopsis. Six ecotypes had PAI arrangements similar to WS, with an inverted repeat and dense PAI methylation. All other ecotypes had PAI arrangements similar to Col, with no PAI methylation. The novel PAI-methylated ecotypes provide insights into the mechanisms underlying PAI gene duplication and methylation, as well as the relationship between methylation and gene expression.

Melquist, S; Luff, B; Bender, J

1999-01-01

115

Chemical carcinogens in non-enzymatic cytosine deamination: 3-isocyanatoacrylonitrile  

PubMed Central

Uracil has long been known as the main product of nitrosative cytosine deamination in aqueous solution. Recent mechanistic studies of cytosinediazonium ion suggest that the cation formed by its dediazoniation can ring-open to N-protonated (Z,s-cis)-3-isocyanatoacrylonitrile 7. Stereochemical preferences are discussed of the 3-isocyanatoacrylonitriles (Z,s-cis)-10, (E,s-cis)-11, (Z,s-trans)-12, and (E,s-trans)-13. The electronic structures of 7 and 10–13 have been analyzed and a rationale is provided for the thermodynamic preference for (Z,s-cis)-10. It is shown that s-cis/s-trans-interconversion occurs via C?N rotation–inversion paths with barriers below 3 kcal mol?1. The proton affinities of 3-isocyanatoacrylonitrile 10 and water are nearly identical and, thus, 3-isocyanatoacrylonitriles can and should be formed in aqueous media from 7 along with 3-aminoacrylonitriles 9. The results highlight the relevance of the chemistry of 3-isocyanatoacrylonitriles for the understanding of the chemical toxicology of nitrosation of the nucleobase cytosine. Electronic Supplementary Material Supplementary Material is available for this article at http://dx.doi.org/10.1007/s00894-005-0048-0

Wu, Hong; von Saint Paul, Francisca

2006-01-01

116

Effects of cytosine methylation on DNA charge transport  

NASA Astrophysics Data System (ADS)

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.

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

2012-04-01

117

Haploinsufficiency of Activation-Induced Deaminase for Antibody Diversification and Chromosome Translocations both In Vitro and In Vivo  

PubMed Central

The humoral immune response critically relies on the secondary diversification of antibodies. This diversification takes places through somatic remodelling of the antibody genes by two molecular mechanisms, Class Switch Recombination (CSR) and Somatic Hypermutation (SHM). The enzyme Activation Induced Cytidine Deaminase (AID) initiates both SHM and CSR by deaminating cytosine residues on the DNA of immunoglobulin genes. While crucial for immunity, AID-catalysed deamination is also the triggering event for the generation of lymphomagenic chromosome translocations. To address whether restricting the levels of AID expression in vivo contributes to the regulation of its function, we analysed mice harbouring a single copy of the AID gene (AID+/?). AID+/? mice express roughly 50% of normal AID levels, and display a mild hyperplasia, reminiscent of AID deficient mice and humans. Moreover, we found that AID+/? cells have an impaired competence for CSR and SHM, which indicates that AID gene dose is limiting for its physiologic function. We next evaluated the impact of AID reduction in AID+/? mice on the generation of chromosome translocations. Our results show that the frequency of AID-promoted c-myc/IgH translocations is reduced in AID+/? mice, both in vivo and in vitro. Therefore, AID is haploinsufficient for antibody diversification and chromosome translocations. These findings suggest that limiting the physiologic levels of AID expression can be a regulatory mechanism that ensures an optimal balance between immune proficiency and genome integrity.

Sernandez, Isora V.; de Yebenes, Virginia G.; Dorsett, Yair; Ramiro, Almudena R.

2008-01-01

118

Activation-induced cytidine deaminase (AID) is necessary for the epithelial-mesenchymal transition in mammary epithelial cells.  

PubMed

Activation-induced cytidine deaminase (AID), which functions in antibody diversification, is also expressed in a variety of germ and somatic cells. Evidence that AID promotes DNA demethylation in epigenetic reprogramming phenomena, and that it is induced by inflammatory signals, led us to investigate its role in the epithelial-mesenchymal transition (EMT), a critical process in normal morphogenesis and tumor metastasis. We find that expression of AID is induced by inflammatory signals that induce the EMT in nontransformed mammary epithelial cells and in ZR75.1 breast cancer cells. shRNA-mediated knockdown of AID blocks induction of the EMT and prevents cells from acquiring invasive properties. Knockdown of AID suppresses expression of several key EMT transcriptional regulators and is associated with increased methylation of CpG islands proximal to the promoters of these genes; furthermore, the DNA demethylating agent 5 aza-2'deoxycytidine (5-Aza-dC) antagonizes the effects of AID knockdown on the expression of EMT factors. We conclude that AID is necessary for the EMT in this breast cancer cell model and in nontransformed mammary epithelial cells. Our results suggest that AID may act near the apex of a hierarchy of regulatory steps that drive the EMT, and are consistent with this effect being mediated by cytosine demethylation. This evidence links our findings to other reports of a role for AID in epigenetic reprogramming and control of gene expression. PMID:23882083

Muñoz, Denise P; Lee, Elbert L; Takayama, Sachiko; Coppé, Jean-Philippe; Heo, Seok-Jin; Boffelli, Dario; Di Noia, Javier M; Martin, David I K

2013-07-23

119

Activation-Induced Cytidine Deaminase Expression in Gastric Cancer  

Microsoft Academic Search

Helicobacter pylori increases the risk of gastric cancer development and triggers aberrant expression of activation-induced cytidine deaminase (AID). The goal of the present study was to investigate whether AID expression is involved in the development or progression of gastric cancer and the nuclear expression of p53 protein in cancer cells. We examined the expression pattern of the AID and p53

Chang Jae Kim; Jae Hwi Song; Yong Gu Cho; Zhang Cao; Su Young Kim; Suk Woo Nam; Jung Young Lee; Won Sang Park

2007-01-01

120

Gene Therapy for Immunodeficiency Due to Adenosine Deaminase Deficiency  

Microsoft Academic Search

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

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

2009-01-01

121

Sensorineural deafness in siblings with adenosine deaminase deficiency  

Microsoft Academic Search

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

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

1996-01-01

122

ACC deaminase activity in avirulent Agrobacterium tumefaciens D3.  

PubMed

Some plant-growth-promoting bacteria encode the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which breaks down ACC, the direct precursor of ethylene biosynthesis in all higher plants, into ammonia and ?-ketobutyrate and, as a result, reduces stress ethylene levels in plants caused by a wide range of biotic and abiotic stresses. It was previously shown that ACC deaminase can inhibit crown gall development induced by Agrobacterium tumefaciens and can partially protect plants from this disease. Agrobacterium tumefaciens D3 has been previously reported to contain a putative ACC deaminase structural gene (acdS) and a regulatory gene (acdR = lrpL). In the present study, it was found that A. tumefaciens D3 is an avirulent strain. ACC deaminase activity and its regulation were also characterized. Under gnotobiotic conditions, wild-type A. tumefaciens D3 was shown to be able to promote plant root elongation, while the acdS and lrpL double mutant strain A. tumefaciens D3-1 lost that ability. When co-inoculated with the virulent strain, A. tumefaciens C58, in wounded castor bean plants, both the wild-type A. tumefaciens D3 and the mutant A. tumefaciens D3-1 were found to be able to significantly inhibit crown gall development induced by A. tumefaciens C58. PMID:21491979

Hao, Youai; Charles, Trevor C; Glick, Bernard R

2011-04-01

123

Clinical expression, genetics and therapy of adenosine deaminase (ADA) deficiency  

Microsoft Academic Search

Adenosine deaminase (ADA) deficiency was the first known cause of primary immunodeficiency. Over the past 25 years the basis for immune deficiency has largely been established. Now it appears that ADA deficiency may also cause hepatic toxicity, raising new questions about its pathogenesis. The ADA gene has been sequenced and the ADA three-dimensional structure solved. The relationship between genotype and

M. S. Hershfield; F. X. Arredondo-Vega; I. Santisteban

1997-01-01

124

A Role for Adenosine Deaminase in Drosophila Larval Development  

Microsoft Academic Search

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of

Tomas Dolezal; Eva Dolezelova; Michal Zurovec; Peter J. Bryant

2005-01-01

125

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

PubMed Central

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

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

1997-01-01

126

Dry yeast  

NSDL National Science Digital Library

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.

Ranveig Thattai (None;)

2005-09-27

127

Chemical mapping of cytosines enzymatically flipped out of the DNA helix  

PubMed Central

Haloacetaldehydes can be employed for probing unpaired DNA structures involving cytosine and adenine residues. Using an enzyme that was structurally proven to flip its target cytosine out of the DNA helix, the HhaI DNA methyltransferase (M.HhaI), we demonstrate the suitability of the chloroacetaldehyde modification for mapping extrahelical (flipped-out) cytosine bases in protein–DNA complexes. The generality of this method was verified with two other DNA cytosine-5 methyltransferases, M.AluI and M.SssI, as well as with two restriction endonucleases, R.Ecl18kI and R.PspGI, which represent a novel class of base-flipping enzymes. Our results thus offer a simple and convenient laboratory tool for detection and mapping of flipped-out cytosines in protein–DNA complexes.

Liutkeviciute, Zita; Tamulaitis, Gintautas; Klimasauskas, Saulius

2008-01-01

128

Vaginal Yeast Infections  

MedlinePLUS

... HIV/AIDS Sexually transmitted infections fact sheet Vaginal yeast infections fact sheet What is a vaginal yeast ... on vaginal yeast infections What is a vaginal yeast infection? A vaginal yeast infection is irritation of ...

129

Hot spot mutations in adenosine deaminase deficiency.  

PubMed Central

We have previously characterized mutant adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) enzymes in seven children with partial ADA deficiency. Six children shared common origins, suggesting a common progenitor. However, we found evidence for multiple phenotypically different mutant enzymes. We hypothesized that many of the mutations would be at CpG dinucleotides, hot spots at which spontaneous deamination of 5-methylcytosine results in C to T or G to A transitions. Digestion of DNA from these children with Msp I and Taq I, enzymes recognizing CpG dinucleotides, identified three different mutations, each correlating with expression of a different mutant enzyme. Sequencing of cDNA clones and genomic DNA amplified by polymerase chain reaction confirmed the presence of C to T or G to A transitions at CpG dinucleotides (C226 to T, G446 to A, and C821 to T, resulting in Arg76 to Trp, Arg149 to Gln, and Pro274 to Leu). A "null" mutation, also found in two ADA-deficient severe combined immunodeficient children, was serendipitously detected as gain of a site for Msp I. Simultaneous loss of a site for Bal I defined the precise base substitution (T320 to C, Leu107 to Pro), confirmed by sequence analysis. To determine the true frequency of hot spot mutation in these children, consecutively ascertained through a newborn screening program, we sequenced cDNA from the remaining alleles. Two others were hot spot mutations (C631 to T and G643 to A, resulting in Arg211 to Cys and Ala215 to Thr), each again resulting in expression of a phenotypically different mutant enzyme. Only one additional mutation (previously identified by us) is not in a hot spot. These seven mutations account for all 14 chromosomes in these children. There is thus a very high frequency of hot spot mutations in partial ADA deficiency. Most of these children carry two different mutant alleles. We were able to correlate genotype and phenotype and to dissect the activity of individual mutant alleles. Images

Hirschhorn, R; Tzall, S; Ellenbogen, A

1990-01-01

130

Photochemical selectivity in guanine-cytosine base-pair structures  

NASA Astrophysics Data System (ADS)

Prebiotic chemistry presumably took place before formation of an oxygen-rich atmosphere and thus under conditions of intense short wavelength UV irradiation. Therefore, the UV photochemical stability of the molecular building blocks of life may have been an important selective factor in determining the eventual chemical makeup of critical biomolecules. To investigate the role of UV irradiation in base-pairing we have studied guanine (G) and cytosine (C) base pairs in the absence of the RNA backbone. We distinguished base-pair structures by IR-UV hole-burning spectroscopy as well as by high-level correlated ab initio calculations. The Watson-Crick structure exhibits broad UV absorption, in stark contrast to other GC structures and other base-pair structures. This broad absorption may be explained by a rapid internal conversion that makes this specific base pair arrangement uniquely photochemically stable. ab initio computation | DNA base pairs | IR-UV spectroscopy | jet cooling | photochemistry

Abo-Riziq, Ali; Grace, Louis; Nir, Eyal; Kabelac, Martin; Hobza, Pavel; de Vries, Mattanjah S.

2005-01-01

131

Chemical synthesis of DNA oligomers containing cytosine arabinoside.  

PubMed Central

The solid phase phospite triester synthesis of oligodeoxynucleotides containing cytosine arabinoside (araC) is described. A protected araC phosphoramadite was prepared for the introduction of araC residues at 5'termini and internucleotide positions in DNA oligomers. These oligomers were utilized to demonstrate the formation of correct 3'-5' linkages, to test for alkaline lability at the araC site, and to study the stability of duplexes containing araC-G base pairs. For the introduction of araC residues at 3' terminal positions, a protected derivative of araC was coupled to functionalized silica. This material was used to prepare a test oligomer which was characterized enzymatically. Images

Beardsley, G P; Mikita, T; Klaus, M M; Nussbaum, A L

1988-01-01

132

The Triplex-Hairpin Transition in Cytosine-Rich DNA  

PubMed Central

We present a theoretical study of the self-complementary single-stranded 30-mer d(TC*TTC*C*TTTTCCTTCTC*CCGAGAAGGTTTT) (PDB ID: 1b4y) that was designed to form an intramolecular triplex by folding back twice on itself. At neutral pH the molecule exists in a duplex hairpin conformation, whereas at acidic pH the cytosines labeled by an asterisk (*) are protonated, forming Hoogsteen hydrogen bonds with guanine of a GC Watson-Crick basepair to generate a triplex. As a first step in an investigation of the energetics of the triplex-hairpin transition, we applied the Bashford-Karplus multiple site model of protonation to calculate the titration curves for the two conformations. Based on these data, a two-state model is used to study the equilibrium properties of transition. Although this model properly describes the thermodynamics of the protonation-deprotonation steps that drive the folding-unfolding of the oligomer, it cannot provide insight into the time-dependent mechanism of the process. A series of molecular dynamics simulations using the ff94 force field of the AMBER 6.0 package was therefore run to explore the dynamics of the folding/unfolding pathway. The molecular dynamics method was combined with Poisson-Boltzmann calculations to determine when a change in protonation state was warranted during a trajectory. This revealed a sequence of elementary protonation steps during the folding/unfolding transition and suggests a strong coupling between ionization and folding in cytosine-rich triple-helical triplexes.

Petrov, Anton S.; Lamm, Gene; Pack, George R.

2004-01-01

133

Catalytic mechanism and three-dimensional structure of adenine deaminase.  

PubMed

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(cat) = 2.0 s(-1); k(cat)/K(m) = 2.5 × 10(3) M(-1) s(-1)). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn(2+) prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k(cat) and k(cat)/K(m) values of 200 s(-1) and 5 × 10(5) M(-1) s(-1), respectively. The apoenzyme was prepared and reconstituted with Fe(2+), Zn(2+), or Mn(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(II)/Fe(II)]-ADE was oxidized to [Fe(III)/Fe(III)]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe(III)/Fe(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 Mo?ssbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 Å 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. PMID:21247091

Kamat, Siddhesh S; Bagaria, Ashima; Kumaran, Desigan; Holmes-Hampton, Gregory P; Fan, Hao; Sali, Andrej; Sauder, J Michael; Burley, Stephen K; Lindahl, Paul A; Swaminathan, Subramanyam; Raushel, Frank M

2011-02-04

134

Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase  

SciTech Connect

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.

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

135

Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase  

SciTech Connect

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.

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

2011-12-31

136

Arabidopsis thaliana cytidine deaminase 1 shows more similarity to prokaryotic enzymes than to eukaryotic enzymes  

Microsoft Academic Search

Two Expressed Sequence Tagged (EST) clones were identified from the Arabidopsis database as encoding putative cytidine deaminases.\\u000a Sequence analysis determined that the two clones overlapped and encoded a single cDNA. This cytidine deaminase corresponds\\u000a to theArabidopsis thaliana gene,cda1. The deduced amino acid sequence was more closely related to prokaryotic cytidine deaminases than to eukaryotic enzymes.\\u000a The cDNA shares 44% amino

Chris Kafer; Robert W. Thornburg

2000-01-01

137

Counting Yeast.  

ERIC Educational Resources Information Center

|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)|

Bealer, Jonathan; Welton, Briana

1998-01-01

138

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, ...

139

Hydroxyl radical induced cross-linking of cytosine and tyrosine in nucleohistone  

SciTech Connect

Hydroxyl radical induced formation of a DNA-protein cross-link involving cytosine and tyrosine in nucleohistone in buffered aqueous solution is reported. The technique of gas chromatography-mass spectrometry was used for this investigation. A {gamma}-irradiated aqueous mixture of cytosine and tyrosine was first investigated in order to obtain gas chromatographic-mass spectrometric properties of possible cytosine-tyrosine cross-links. One cross-link was observed, and its structure was identified as the product from the formation of a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. With the use of gas chromatography-mass spectrometry with selected-ion monitoring, this cytosine-tyrosine cross-link was identified in acidic hydrolysates of calf thymus nucleohistone {gamma}-irradiated in N{sub 2}O-saturated aqueous solution. The yield of this DNA-protein cross-link in nucleohistone was found to be a linear function of the radiation dose in the range of 100-500 Gy (J{center dot}kg{sup {minus}1}). This yield amounted to 0.05 nmol{center dot}J{sup {minus}1}. Mechanisms underlying the formation of the cytosine-tyrosine cross-link in nucleohistone were proposed to involve radical-radical and/or radical addition reactions of hydroxyl adduct radicals of cytosine and tyrosine moieties, forming a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. When oxygen was present in irradiated solutions, no cytosine-tyrosine cross-links were observed.

Gajewski, E.; Dizdaroglu, M. (National Institute of Standards and Technology, Gaithersburg, MD (USA))

1990-01-30

140

ADA2 isoform of adenosine deaminase from pleural fluid.  

PubMed

Adenosine deaminase isoenzyme 2 (ADA2) was isolated from human pleural fluid for the first time. Molecular and kinetic properties were characterized. It was shown that the inhibitors of adenosine deaminase isoenzyme 1 (ADA1), adenosine, and erithro-9-(2-hydroxy-3-nonyl)adenine (EHNA) derivatives are poor inhibitors of ADA2. Comparison of the interaction of ADA2 and ADA1 with adenosine and its derivative, 1-deazaadenosine, indicates that the isoenzymes have similar active centers. The absence of ADA2 inhibition by EHNA is evidence of a difference of these active centers in a close environment. The possible role of Zn2+ ions and the participation of acidic amino acids Glu and Asp in adenosine deamination catalyzed by ADA2 were shown. PMID:15670822

Andreasyan, Nune A; Hairapetyan, Hripsime L; Sargisova, Yelizaveta G; Mardanyan, Sona S

2005-01-31

141

Structure and control of pyridoxal phosphate dependent allosteric threonine deaminase  

Microsoft Academic Search

Background: Feedback inhibition of biosynthetic threonine deaminase (TD) from Escherichia coli provided one of the earliest examples of protein-based metabolic regulation. Isoleucine, the pathway end-product, and valine, the product of a parallel pathway, serve as allosteric inhibitor and activator, respectively. This enzyme is thus a useful model system for studying the structural basis of allosteric control mechanisms.Results: We report the

D Travis Gallagher; Gary L Gilliland; Gaoyi Xiao; James Zondlo; Kathryn E Fisher; Diana Chinchilla; Edward Eisenstein

1998-01-01

142

Severe combined immunodeficiency due to adenosine deaminase deficiency.  

PubMed

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

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

2012-03-01

143

Capillary Electrophoresis in Diagnosis and Monitoring of Adenosine Deaminase Deficiency  

Microsoft Academic Search

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

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

144

Gene therapy for adenosine-deaminase-deficient severe combined immunodeficiency  

Microsoft Academic Search

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

Alessandro Aiuti

2004-01-01

145

Enzymatic conformational fluctuations along the reaction coordinate of cytidine deaminase  

Microsoft Academic Search

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

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

2002-01-01

146

Regulation of adenosine receptor engagement by ecto-adenosine deaminase  

Microsoft Academic Search

Adenosine deaminase (ADA) can localize to the cell surface through its interaction with CD26. Using CD26-transfected cells, we demonstrate that cell surface ADA (ecto-ADA) can regulate adenosine receptor engagement by degrading extracellular adenosine (Ado) to inosine. This ability was dependent upon CD26 expression, the extent of CD26 saturation with ecto-ADA, and the kinetics of the cAMP response. Thus, the cAMP

Tomoko Hashikawa; Scott W. Hooker; Jerzy G. Maj; Christopher J. Knott-Craig; Masahide Takedachi; Shinya Murakami; Linda F. Thompson

2003-01-01

147

Variegate porphyria with coexistent decrease in porphobilinogen deaminase activity.  

PubMed

Variegate porphyria is a rare disease caused by a deficiency of protoporphyrinogen oxidase. In most cases, the clinical findings are a combination of systemic symptoms similar to those occurring in acute intermittent porphyria and cutaneous lesions indistinguishable from those of porphyria cutanea tarda. We report on a 24-year-old woman with variegate porphyria who, after intake of lynestrenol, developed typical cutaneous lesions but no viscero-neurological symptoms. The diagnosis was based on the characteristic urinary coproporphyrin and faecal protoporphyrin excretion patterns, and the specific peak of plasma fluorescence at 626 nm in spectrofluorometry. Biochemical analysis revealed that most of the family members, though free of clinical symptoms, excrete porphyrin metabolites in urine and stool similar to variegate porphyria, accompanied by a significant decrease of porphobilinogen deaminase activity of a range which is ordinarily found in patients with acute intermittent porphyria only (approximately 50%). These data first led to the assumption of two separate and independently inherited genetic defects, similar to the dual porphyria of Chester. Molecular analysis, however, revealed only a missense mutation of the protoporphyrinogen oxidase gene, but not of the porphobilinogen deaminase gene. Thus, in the family presented, porphobilinogen deaminase deficiency is likely to be a phenomenon secondary to the genetic defect of protoporphyrinogen oxidase. PMID:11800145

Weinlich, G; Doss, M O; Sepp, N; Fritsch, P

148

Sequence motifs characteristic of DNA[cytosine-N4]methyltransferases: similarity to adenine and cytosine-C5 DNA-methylases.  

PubMed

The sequences coding for DNA[cytosine-N4]methyltransferases MvaI (from Micrococcus varians RFL19) and Cfr9I (from Citrobacter freundii RFL9) have been determined. The predicted methylases are proteins of 454 and 300 amino acids, respectively. Primary structure comparison of M.Cfr9I and another m4C-forming methylase, M.Pvu II, revealed extended regions of homology. The sequence comparison of the three DNA[cytosine-N4]-methylases using originally developed software revealed two conserved patterns, DPF-GSGT and TSPPY, which were found similar also to those of adenine and DNA[cytosine-C5]-methylases. These data provided a basis for global alignment and classification of DNA-methylase sequences. Structural considerations led us to suggest that the first region could be the binding site of AdoMet, while the second is thought to be directly involved in the modification of the exocyclic amino group. PMID:2690010

Klimasauskas, S; Timinskas, A; Menkevicius, S; Butkienè, D; Butkus, V; Janulaitis, A

1989-12-11

149

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

PubMed

The fluctuation of surface-enhanced Raman scattering (SERS) spectra has been an obstacle to the analysis of the adsorbate on the metal surface. In this paper, we aim at using the density functional theory (DFT) to study the fluctuant Raman spectra of the cytosine molecule which interacts with a coinage metal atom or cation via N1 and N3 sites. The results show that the adsorption site strongly influences the Raman spectral property of cytosine molecule, especially the relative intensity of some bands. In addition, the SERS spectra of cytosine which is adsorbed on the gold, silver, and copper electrodes are measured, and the possible orientation and adsorption site of the cytosine molecule adsorbed on metal electrodes surface are proposed with the help of DFT simulations. PMID:21676649

Liu, Shuanjiang; Zheng, Guimei; Li, Jianxin

2011-05-24

150

Yeast Infection (Candidiasis)  

MedlinePLUS

newsletter | contact Share | Yeast Infection (Candidiasis) Information for adults A A A This is a candida (yeast) infection of the skin folds of the abdomen. Overview Candidiasis, commonly known as a yeast infection, is an infection with the common yeast ( ...

151

New Plasmid System To Select for Saccharomyces cerevisiae Purine-Cytosine Permease Affinity Mutants  

PubMed Central

The FCY2 gene of Saccharomyces cerevisiae encodes a purine-cytosine permease (PCP) that mediates the active transport of purines and cytosine. A structure-function model for this PCP has been recently proposed. In this study, we developed a plasmid-based system that generated a number of affinity-mutated alleles, enabling us to define new amino acids critical for permease function.

Wagner, Renaud; Straub, Marie-Laure; Souciet, Jean-Luc; Potier, Serge; de Montigny, Jacky

2001-01-01

152

Cytosine methylation and the fate of CpG dinucleotides in vertebrate genomes  

Microsoft Academic Search

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

David N. Cooper; Michael Krawczak

1989-01-01

153

The role of cytosine arabinoside maintenance in acute nonlymphoblastic leukemia.  

PubMed

A series of 30 unselected patients with acute nonlymphoblastic leukemia (ANLL) was treated with combination chemotherapy, including three courses of cytosine arabinoside (Ara-C) by 5-day continuous i.v. infusion, vincristine i.v. weekly, and prednisone daily to complete remission. Ara-C was administered alone as a 5-day continuous i.v. infusion monthly for maintenance. Ten (33%) achieved a complete remission (CR). The remaining 30 (67%), including temporary partial remissions, hematologic improvements, inadequate trials, and early deaths, were all considered failures. The CR rate was 57% in those 17 cases receiving an adequate trial. After After 5 1/2 years' followup, the overall median survival, including cases failing to achieve CR, was 3.1 months. For those having adequate trials the median survival was 16.6 months, and for those achieving a CR, 36.6 months. Two patients are still alive, one at 55.2 months on maintenance therapy, and the other at 62.8 months, currently unmaintained. PMID:1182675

Grozea, P N; Bottonley, R H; Shaw, M T; Tu, H; Chanes, R E; Condit, P T

1975-09-01

154

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

PubMed

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

Delatte, Benjamin; Fuks, François

2013-04-26

155

TET proteins: on the frenetic hunt for new cytosine modifications  

PubMed Central

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

Delatte, Benjamin

2013-01-01

156

Yeast Droplets  

NASA Astrophysics Data System (ADS)

It is well known that the Young's law and surface tension govern the shape of liquid droplets on solid surfaces. Here we address through experiments and theory the shape of growing aggregates of yeast on agar substrates, and assess whether these ideas still hold. Experiments are carried out on Baker's yeast, with different levels of expressions of an adhesive protein governing cell-cell and cell-substrate adhesion. Changing either the agar concentration or the expression of this protein modifies the local contact angle of a yeast droplet. When the colony is small, the shape is a spherical cap with the contact angle obeying Young's law. However, above a critical volume this structure is unstable, and the droplet becomes nonspherical. We present a theoretical model where this instability is caused by bulk elastic effects. The model predicts that the transition depends on both volume and contact angle, in a manner quantitatively consistent with our experiments.

Nguyen, Baochi; Upadhyaya, Arpita; van Oudenaarden, Alexander; Brenner, Michael

2002-11-01

157

Cytotoxic effect of a replication-incompetent adenoviral vector with cytosine deaminase gene driven by L-plastin promoter in hepatocellular carcinoma cells  

Microsoft Academic Search

Great expectations are set on gene therapy for the treatment of malignant hepatocellular carcinomas (HCC) in East Asia. Recombinant\\u000a adenoviral vectors (AV) have been developed in which the L-plastin promoter (LP) regulates the expression of transgenes, in\\u000a a tumor cell specific manner, resulting in an increase in the therapeutic index. The development of the AdLPCD vector, a replication-incompetent\\u000a AV, containing

Kihwa Jung; Sunja Kim; Kyumhyang Lee; Changmin Kim; Injae Chung

2007-01-01

158

Acute Intermittent Porphyria: Expression of Mutant and Wild-Type Porphobilinogen Deaminase in COS1 Cells  

Microsoft Academic Search

Background: Acute intermittent porphyria (AIP) is an autosomal dominant disorder that results from the partial deficiency of porphobilinogen deaminase (PBGD) in the heme biosynthetic pathway. Patients with AIP can experience acute attacks consisting of abdominal pain and various neuropsychiatric symp- toms. Although molecular biological studies on the porphobilinogen deaminase (PBGD) gene have re- vealed several mutations responsible for AIP, the

Sami Mustajoki; Minna Laine; Maija Lahtela; Pertti Mustajoki; Leena Peltonen; Raili Kauppinen

2000-01-01

159

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

Microsoft Academic Search

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

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

1986-01-01

160

Rhizobium leguminosarum Biovar viciae 1-Aminocyclopropane-1-Carboxylate Deaminase Promotes Nodulation of Pea Plants  

PubMed Central

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.

Ma, Wenbo; Guinel, Frederique C.; Glick, Bernard R.

2003-01-01

161

Serum adenosine deaminase, catalase and carbonic anhydrase activities in patients with bladder cancer  

PubMed Central

OBJECTIVES: The relationship between adenosine deaminase and various cancers has been investigated in several studies. However, serum adenosine deaminase activity and carbonic anhydrase and catalase activities in patients with bladder cancer have not previously been reported. Therefore, the aim of this study was to measure serum adenosine deaminase, carbonic anhydrase and catalase activities in patients with bladder cancer. MATERIALS AND METHODS: Forty patients with bladder cancer and 30 healthy controls were enrolled in the study. Serum adenosine deaminase, carbonic anhydrase and catalase activities were measured spectrophotometrically. RESULTS: Serum adenosine deaminase, carbonic anhydrase and catalase activities were significantly higher in patients with bladder cancer than controls (all significant, p<0.001). CONCLUSIONS: These markers might be a potentially important finding as an additional diagnostic biochemical tool for bladder cancer.

Pirincci, Necip; Gecit, Ilhan; Gunes, Mustafa; Bilgehan Y?ksel, Mehmet; Kaba, Mehmet; Tan?k, Serhat; Demir, Halit; Aslan, Mehmet

2012-01-01

162

Genetic heterogeneity in acute intermittent porphyria: characterisation and frequency of porphobilinogen deaminase mutations in Finland.  

PubMed Central

The occurrence of different porphobilinogen deaminase mutant types in 68 patients with acute intermittent porphyria from 33 unrelated families in Finland was studied with biochemical and immunological techniques. In this fairly homogenous population four different porphobilinogen deaminase mutant types were identified and their frequencies determined. Most (about 80%) of the mutations were cross reacting immunological material (CRIM) negative, including a large kindred with normal erythrocyte porphobilinogen deaminase activities. The remainder of the families had CRIM positive mutations, including an unusual type (type 2) that had an immunoreactive, non-catalytic porphobilinogen deaminase level considerably greater than the maximal theoretical ratio of CRIM to activity of 2.0 for a single mutant allele. Correlations of the amount of residual porphobilinogen deaminase activity and the occurrence of acute clinical manifestations in each mutant type suggested that CRIM positive type 2 patients may have fewer acute symptoms. Images FIG 2

Mustajoki, P; Desnick, R J

1985-01-01

163

Structure of the cytosine-cytosine mismatch in the thymidylate synthase mRNA binding site and analysis of its interaction with the aminoglycoside paromomycin  

PubMed Central

The structure of a cytosine–cytosine (CC) mismatch-containing RNA molecule derived from a hairpin structure in the thymidylate synthase mRNA that binds the aminoglycoside paromomycin with high affinity was determined using nuclear magnetic resonance (NMR) spectroscopy. The cytosines in the mismatch form a noncanonical base pair where both cytosines are uncharged and stack within the stem of the RNA structure. Binding to paromomycin was analyzed using isothermal titration calorimetry (ITC) to demonstrate the necessity of the CC mismatch and to determine the affinity dissociation constant of this RNA to paromomycin to be 0.5 ± 0.3 ?M. The CC mismatch, and the neighboring GC base pairs experienced the highest degree of chemical shift changes in their H6 and H5 resonances indicating that paromomycin binds in the major groove at the CC mismatch site. In comparing the structure of CC mismatch RNA with a fully Watson–Crick GC base paired stem, the CC mismatch is shown to confer a widening of the major groove. This widening, combined with the dynamic nature of the CC mismatch, enables binding of paromomycin to this RNA molecule.

Tavares, Tony J.; Beribisky, Alexander V.; Johnson, Philip E.

2009-01-01

164

Mutagenesis dependent upon the combination of activation-induced deaminase expression and a double-strand break  

PubMed Central

We explored DNA metabolic events potentially relevant to somatic hypermutation (SHM) of immunoglobulin genes using a yeast model system. Double-strand break (DSB) formation has been discussed as a possible component of the SHM process during immunoglobulin gene maturation. Yet, possible mechanisms linking DSB formation with mutagenesis have not been well understood. In the present study, a linkage between mutagenesis in a reporter gene and a double-strand break at a distal site was examined as a function of activation-induced deaminase (AID) expression. Induction of the DSB was found to be associated with mutagenesis in a genomic marker gene located 7kb upstream of the break site: mutagenesis was strongest with the combination of AID expression and DSB induction. The mutation spectrum of this DSB and AID-mediated mutagenesis was characteristic of replicative bypass of uracil in one strand and was dependent on expression of DNA polymerase delta (Pol ?). These results in a yeast model system illustrate that the combination of DSB induction and AID expression could be associated with mutagenesis observed in SHM. Implications of these findings for SHM of immunoglobulin genes in human B cells are discussed.

Poltoratsky, Vladimir; Heacock, Michelle; Kissling, Grace E.; Prasad, Rajendra; Wilson, Samuel H.

2010-01-01

165

EMBRYONIC FACTOR 1 encodes an AMP deaminase and is essential for the zygote to embryo transition in Arabidopsis.  

PubMed

Fusion of the egg and the sperm cells in plants produces a zygote that develops into an embryo. Screening of ethyl methanesulfonate-mutagenized populations of Arabidopsis led to the identification of EMBRYONIC FACTOR 1 (FAC1), a locus that gives a zygote-lethal phenotype when mutated. The FAC1 gene was identified by positional cloning and confirmed by a genetic complementation test against a T-DNA insertion allele. It encodes an AMP deaminase (AMPD) that is known in human and yeast to convert AMP to IMP to maintain the energy potential. Expression of FAC1 in a yeast AMPD mutant after removal of its N-terminal putative transmembrane domain complemented the mutant phenotype, suggesting a functional conservancy but a structural divergence through evolution. Although a low level of FAC1 expression was observed in all organs tested, using a reporter construct we observed a significantly increased FAC1 expression in the zygote, early embryo and endosperm. Furthermore, during somatic embryogenesis, a high level of FAC1 expression was observed in developing embryos including putative embryogenic cells. FAC1, therefore, represents one of the earliest expressed genes known in plants. It may act through AMP depletion to provide sufficient energy for the zygote to proceed through development. PMID:15918887

Xu, Jun; Zhang, Hai-Ying; Xie, Cong-Hua; Xue, Hong-Wei; Dijkhuis, Paul; Liu, Chun-Ming

2005-06-01

166

A Gene of Bacteriophage T4 whose Product Prevents True Late Transcription on Cytosine-Containing T4 DNA  

Microsoft Academic Search

T-even coliphages have 5-hydroxymethylcytosine in their DNA instead of cytosine. In some T4 mutants, the replicated DNA contains cytosine, but then no late gene products are made. We show that the inability to make late gene products with cytosine-containing T4 DNA is due to a T4 gene product. This gene product, while probably nonessential under normal conditions, interacts with an

Larry Snyder; Larry Gold; Elizabeth Kutter

1976-01-01

167

The Role of ACC Deaminase Producing PGPR in Sustainable Agriculture  

Microsoft Academic Search

\\u000a The plant rhizosphere is a multidimensional and dynamic ecological environment of complicated microbe–plant interactions for\\u000a harnessing essential macro and micronutrients from a limited nutrient pool. Certain plant growth promoting rhizobacteria (PGPR)\\u000a contain a vital enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (EC 4.1.99.4), which regulates ethylene production\\u000a by metabolizing ACC (an intermediate precursor of ethylene biosynthesis in higher plants) into ?-ketobutyrate and

Meenu Saraf; Chaitanya Kumar Jha; Dhara Patel

168

Human plasma adenosine deaminase 2 is secreted by activated monocytes.  

PubMed

Adenosine deaminase (ADA) plays an important role in the immune system, and its activity is composed of two kinetically distinct isozymes, ADA1 and ADA2. ADA2 is a major component of human plasma total ADA activity and ADA2 activity is significantly elevated in patients with various diseases such as HIV infection and chronic hepatitis. However, relatively little is known about ADA2 because of difficulties in purifying this enzyme. In this study we succeeded in purifying human plasma ADA2 and demonstrate the extracellular secretion of ADA2 from human peripheral blood monocytes stimulated with phorbol 12-myristate 13-acetate and calcium ionophore. PMID:16542154

Iwaki-Egawa, Sachiko; Yamamoto, Takaya; Watanabe, Yasuhiro

2006-03-01

169

Cytosine methylation and hydroxymethylation mark DNA for elimination in Oxytricha trifallax  

PubMed Central

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

2012-01-01

170

Identification of direct targets and modified bases of RNA cytosine methyltransferases.  

PubMed

The extent and biological impact of RNA cytosine methylation are poorly understood, in part owing to limitations of current techniques for determining the targets of RNA methyltransferases. Here we describe 5-azacytidine-mediated RNA immunoprecipitation (Aza-IP), a technique that exploits the covalent bond formed between an RNA methyltransferase and the cytidine analog 5-azacytidine to recover RNA targets by immunoprecipitation. Targets are subsequently identified by high-throughput sequencing. When applied in a human cell line to the RNA methyltransferases DNMT2 and NSUN2, Aza-IP enabled >200-fold enrichment of tRNAs that are known targets of the enzymes. In addition, it revealed many tRNA and noncoding RNA targets not previously associated with NSUN2. Notably, we observed a high frequency of C?G transversions at the cytosine residues targeted by both enzymes, allowing identification of the specific methylated cytosine(s) in target RNAs. Given the mechanistic similarity of RNA cytosine methyltransferases, Aza-IP may be generally applicable for target identification. PMID:23604283

Khoddami, Vahid; Cairns, Bradley R

2013-04-21

171

Baker's yeast cytidine deaminase: Substrate and inhibitor specificity, and a hypothesis on metabolic and regulatory significance  

Microsoft Academic Search

Riassunio La citidina deaminasi, parzialmente purificata da lievito di pane, è capace di deaminare sia la citidina che la deossicitidina. I valori delleKm per ambedue i 2 substrati sono 25×10?5M e 9.1×10?5M rispettivamente. Inoltre l'enzima è inibito da numerosi nucleosidi monofosfati, difosfati e trifosfati. È molto significativa il tipo di inibizione allosterica esercitata dal dTTP. Si riporta una ipotesi sul

G. Magni; P. Ipata; P. Natalini; R. A. Felicioli; G. Cercignani

1974-01-01

172

HPLC-UV, MALDI-TOF-MS and ESI-MS/MS Analysis of the Mechlorethamine DNA Crosslink at a Cytosine-Cytosine Mismatch Pair  

PubMed Central

Background Mechlorethamine [ClCH2CH2N(CH3)CH2CH2Cl], a nitrogen mustard alkylating agent, has been proven to form a DNA interstrand crosslink at a cytosine-cytosine (C-C) mismatch pair using gel electrophoresis. However, the atomic connectivity of this unusual crosslink is unknown. Methodology/Principal Findings HPLC-UV, MALDI-TOF-MS, and ESI-MS/MS were used to determine the atomic connectivity of the DNA C-C crosslink formed by mechlorethamine, MALDI-TOF-MS of the HPLC-purified reaction product of mechlorethamine with the DNA duplex d[CTCACACCGTGGTTC]•d[GAACCACCGTGTGAG] (underlined bases are a C-C mismatch pair) indicated formation of an interstrand crosslink at m/z 9222.088 [M?2H+Na]+. Following enzymatic digestion of the crosslinked duplex by snake venom phosphodiesterase and calf intestinal phosphatase, ESI-MS/MS indicated the presence of dC-mech-dC [mech?=?CH2CH2N(CH3)CH2CH2] at m/z 269.2 [M]2+ (expected m/z 269.6, exact mass 539.27) and its hydrolytic product dC-mech-OH at m/z 329.6 [M]+ (expected m/z 329.2). Fragmentation of dC-mech-dC gave product ions at m/z 294.3 and 236.9 [M]+, which are both due to loss of the 4-amino group of cytosine (as ammonia), in addition to dC and dC+HN(CH3)CH?=?CH2, respectively. The presence of m/z 269.2 [M]2+ and loss of ammonia exclude crosslink formation at cytosine N4 or O2 and indicate crosslinking through cytosine N3 with formation of two quaternary ammonium ions. Conclusions Our results provide an important addition to the literature, as the first example of the use of HPLC and MS for analysis of a DNA adduct at the N3 position of cytosine.

Rojsitthisak, Pornchai; Jongaroonngamsang, Nutthapon; Romero, Rebecca M.; Haworth, Ian S.

2011-01-01

173

Laser photobleaching leads to a fluorescence grade adenosine deaminase  

SciTech Connect

The enzyme adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) from calf intestinal mucosa is commercially available at high purity grade yet, at the sensitivity at which fluorescence studies may be undertaken, a nonpeptidic fluorescence is detectable at lambda exmax = 350 nm and lambda emmax = 420 nm. A sevenfold decrease of this nonpeptidic fluorescence was obtained upon irradiation by the third harmonic (355 nm) of a Nd:YAG laser for 16 min, at 5 mJ/pulse, with a pulse width of 6 ns at a repetition rate of 10 Hz. The decline of fluorescence was accompanied by a negligible loss of enzymatic activity. Moreover, the integrity of the protein was ascertained by (i) its fluorescence (lambda exmax = 305 nm, lambda emmax = 335 nm) and lifetime distribution and (ii) its kinetics in the presence of the substrate adenosine and two inhibitors, all of which remained essentially unaltered. Laser photobleaching is a simple way to achieve a fluorescence grade adenosine deaminase.

Parola, A.H.; Caiolfa, V.R.; Bar, I.; Rosenwaks, S. (Ben Gurion Univ. of the Negev, Beer Sheva (Israel))

1989-09-01

174

Restriction of Foamy Viruses by APOBEC Cytidine Deaminases  

PubMed Central

Foamy viruses (FVs) are nonpathogenic retroviruses infecting many species of mammals, notably primates, cattle, and cats. We have examined whether members of the apolipoprotein B-editing catalytic polypeptide-like subunit (APOBEC) family of antiviral cytidine deaminases restrict replication of simian FV. We show that human APOBEC3G is a potent inhibitor of FV infectivity in cell culture experiments. This antiviral activity is associated with cytidine editing of the viral genome. Both molecular FV clones and primary uncloned viruses were susceptible to APOBEC3G, and viral infectivity was also inhibited by murine and simian APOBEC3G homologues, as well as by human APOBEC3F. Wild-type and bet-deleted viruses were similarly sensitive to this antiviral activity, suggesting that Bet does not significantly counteract APOBEC proteins. Moreover, we did not detect FV sequences that may have been targeted by APOBEC in naturally infected macaques, but we observed a few G-to-A substitutions in humans that have been accidentally contaminated by simian FV. In infected hosts, the persistence strategy employed by FV might be based on low levels of replication, as well as avoidance of cells expressing large amounts of active cytidine deaminases.

Delebecque, Frederic; Suspene, Rodolphe; Calattini, Sara; Casartelli, Nicoletta; Saib, Ali; Froment, Alain; Wain-Hobson, Simon; Gessain, Antoine; Vartanian, Jean-Pierre; Schwartz, Olivier

2006-01-01

175

A Linear-Scaling Quantum Mechanical Investigation of Cytidine Deaminase  

NASA Astrophysics Data System (ADS)

We describe the divide-and-conquer technique for linear-scaling semiempirical quantum mechanical calculations. This method has been successfully applied to study cytidine deaminase. Large-scale simulations were performed for optimizing geometries surrounding the active site of the enzyme and obtaining related energetics. The results of the minimizations provide a significant complement to experimental efforts and aid in the understanding of the enzymatic profile of cytidine deaminase. More specifically, we present our predictions about the structure of the active species and the structure of the active site for low pH. Finally, we present our results for the structure of the zinc ion coordination for different substrates which represent points along the reaction profile. In particular, we find that our results for the Zn-S?132 and the Zn-S?129 bondlengths yield similar trends compared to x-ray crystallography data as the enzyme structure changes from the ground-state to the transition-state analog and from the transition-state analog to the product.

Lewis, James P.; Liu, Shubin; Lee, Tai-Sung; Yang, Weitao

1999-05-01

176

Electrochemical and fluorescence detection of cytosine-related SNPs using a ferrocenyl naphthyridine derivative.  

PubMed

A novel hydrogen bond-forming ligand for cytosine-related single nucleotide polymorphism, which contains both a fluorescent naphthyridine moiety and a ferrocene group as an electrochemical indicator, is described. Hydrogen bond-mediated recognition for a target nucleobase within an abasic site-containing DNA duplex was confirmed by both fluorescence and electrochemical measurements. The analysis by fluorescence titration reveals that the ligand shows significant fluorescent quenching upon formation of a 1: 1 complex with the target nucleobase opposite an AP site, and the selectivity was in the order of cytosine > thymine > adenine, guanine, reflecting the stability of hydrogen bond formation. PMID:18029703

Morita, Kotaro; Sato, Yusuke; Seino, Takehiro; Nishizawa, Seiichi; Teramae, Norio

2007-01-01

177

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

PubMed

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

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

2013-10-15

178

Myoadenylate deaminase deficiency in a patient with facial and limb girdle myopathy  

Microsoft Academic Search

Absence of AMP-deaminase was demonstrated by histochemical and biochemical methods in a muscle biopsy of a 25-year-old woman with facial and limb girdle myopathy. Venous ammonia failed to rise after ischaemic exercise.

R. Mercelis; J. J. Martin; I. Dehaene; Th. Barsy; G. Berghe

1981-01-01

179

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

Microsoft Academic Search

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

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

1992-01-01

180

IRMPD Action Spectroscopy of Alkali Metal Cation-Cytosine Complexes: Effects of Alkali Metal Cation Size on Gas Phase Conformation.  

PubMed

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

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

2013-07-27

181

IRMPD Action Spectroscopy of Alkali Metal Cation-Cytosine Complexes: Effects of Alkali Metal Cation Size on Gas Phase Conformation  

NASA Astrophysics Data System (ADS)

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).

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

2013-10-01

182

Deoxyadenosine Triphosphate as a Potentially Toxic Metabolite in Adenosine Deaminase Deficiency  

Microsoft Academic Search

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

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

1978-01-01

183

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

Microsoft Academic Search

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

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

1997-01-01

184

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

PubMed

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

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

2010-12-01

185

Binding properties of adenosine deaminase interacted with theophylline.  

PubMed

Thermodynamic studies were carried out to evaluate the binding of theophylline on adenosine deaminase (ADA) in 50 mM sodium phosphate buffer pH 7.5, at 300 K, using isothermal titration calorimetry (ITC). A simple method for determination of binding isotherm in the drug--ADA interaction was applied using ITC data. ADA has two binding sites for theophylline, which show positive cooperativity in its sites. The intrinsic association equilibrium constants are 6 and 52 mM(-1) in the first and second binding sites, respectively. Hence, occupation of the first site has produced an appreciable enhancement by 8.7 of the binding affinity of the second site. The molar enthalpies of binding are -12.2 and -14.9 kJ/mol in the first and second binding sites, respectively. PMID:15467230

Saboury, Ali Akbar; Bagheri, Soghra; Ataie, Ghasem; Amanlou, Masoud; Moosavi-Movahedi, Ali Akbar; Hakimelahi, Gholam Hossein; Cristalli, Gloria; Namaki, Saeid

2004-10-01

186

Alternative transcription and splicing of the human porphobilinogen deaminase gene  

SciTech Connect

Porphobilinogen deaminase is a cytosolic enzyme involved in the heme biosynthetic pathway. Two isoforms of PBGD, encoded by two mRNAs differing solely in their 5' end, are known: one is found in all cells and the other is present only in erythroid cells. The authors have previously shown that the human PBGD is encoded by a single gene and have now cloned and characterized this gene, which is split into 15 exons spread over 10 kilobases of DNA. They demonstrate that the two mRNAs arise from two overlapping transcription units. The first one (upstream) is active in all tissues and its promoter has some of the structural features of a housekeeping promoter; the second, located 3 kilobases downstream, is active only in erythroid cells and its promoter displays structural homologies with the ..beta..-globin gene promoters.

Chretien, S.; Dubart, A.; Beaupain, D.; Raich, N.; Grandchamp, B.; Rosa, J.; Goossens, M.; Romeo, P.H.

1988-01-01

187

Adenosine deaminase activity in normal pregnancy and pregnancy associated disorders.  

PubMed

Adenosine deaminase (ADA) is an enzyme of purine salvage pathway and has two important isoenzymes ADA1 and ADA2. The activity of ADA has been changed in diseases characterized by altered cell-mediated immunity. It was observed that total serum ADA activity was decreased during normal pregnancy compared with non-pregnant women. However, total serum ADA activity and serum ADA2 activity was increased in hyperemesis gravidarum and pre-eclampsia in pregnant women. Less information is available regarding role of ADA in abortions (recurrent and missed) and anembryonic pregnancies. Here, we review the activity of ADA and its isoenzymes. Despite these findings, it will be interesting to know whether activity of ADA will be same if ADA is estimated throughout the pregnancy and in pregnancy related complications from early first trimester to third trimester, as all studies until now were carried out at a particular stage of pregnancy. PMID:23527577

Jadhav, Ashish Anantrao; Jain, Anuradha

2013-03-25

188

ACC deaminase from plant growth-promoting bacteria affects crown gall development.  

PubMed

In addition to the well-known roles of indoleacetic acid and cytokinin in crown gall formation, the plant hormone ethylene also plays an important role in this process. Many plant growth-promoting bacteria (PGPB) encode the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which can degrade ACC, the immediate precursor of ethylene in plants, to alpha-ketobutyrate and ammonia and thereby lower plant ethylene levels. To study the effect of ACC deaminase on crown gall development, an ACC deaminase gene from the PGPB Pseudomonas putida UW4 was introduced into Agrobacterium tumefaciens C58, so that the effect of ACC deaminase activity on tumour formation in tomato and castor bean plants could be assessed. Plants were also coinoculated with A. tumefaciens C58 and P. putida UW4 or P. putida UW4-acdS- (an ACC deaminase minus mutant strain). In both types of experiments, it was observed that the presence of ACC deaminase generally inhibited tumour development on both tomato and castor bean plants. PMID:18059561

Hao, Youai; Charles, Trevor C; Glick, Bernard R

2007-12-01

189

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

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

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

1988-01-01

190

Ultrafast IR spectroscopy of the short-lived transients formed by UV excitation of cytosine derivatives.  

PubMed

A strong infrared band at 1574 cm(-1) is observed following 267 nm excitation of 2'-deoxycytidine (tau = 37 +/- 4 ps) or 2'-deoxycytidine 5'-monophosphate (tau = 33 +/- 4 ps); this band is provisionally attributed to an 1n(N)pi* state and is absent for cytosine. PMID:17520112

Quinn, Susan; Doorley, Gerard W; Watson, Graeme W; Cowan, Alexander J; George, Michael W; Parker, Anthony W; Ronayne, Kate L; Towrie, Michael; Kelly, John M

2007-05-03

191

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

NASA Astrophysics Data System (ADS)

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.

Shi, Jinming

192

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

Microsoft Academic Search

The fluctuation of surface-enhanced Raman scattering (SERS) spectra has been an obstacle to the analysis of the adsorbate on the metal surface. In this paper, we aim at using the density functional theory (DFT) to study the fluctuant Raman spectra of the cytosine molecule which interacts with a coinage metal atom or cation via N1 and N3 sites. The results

Shuanjiang Liu; Guimei Zheng; Jianxin Li

2011-01-01

193

5'-Methyl-cytosine in the macronuclear DNA of Blepharisma japonicum.  

PubMed

Brief report on the presence of 5'-methyl-cytosine as a minor base (0.56%) in the macronuclear DNA of the ciliate protozoan Blepharisma japonicum. The evidence comes from electrophoresis of macronuclear DNA digested by appropriate restriction endonucleases and high-performance liquid chromatography. PMID:6210211

Salvini, M; Durante, M; Citti, L; Nobili, R

1984-12-15

194

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

PubMed Central

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

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

2003-01-01

195

High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells  

Microsoft Academic Search

Mammalian genes are highly heterogeneous with respect to their nucleotide composition, but the functional consequences of this heterogeneity are not clear. In the previous studies, weak positive or negative correlations have been found between the silent-site guanine and cytosine (GC) content and expression of mammalian genes. However, previous studies disregarded differences in the genomic context of genes, which could potentially

Grzegorz Kudla; Leszek Lipinski; Fanny Caffin; Aleksandra Helwak; Maciej Zylicz

2006-01-01

196

Cytosine Derivatives Form Hemiprotonated Dimers in Solution and the Gas Phase.  

PubMed

Hemiprotonated dimers of cytosine derivatives, implicated in the formation of the i-motif of DNA, have been created in solution and the gas phase. The mechanism of dimerization has been analyzed by mass spectrometry and multidimensional NMR spectroscopy. PMID:22571841

Moehlig, Aaron R; Djernes, Katherine E; Krishnan, V Mahesh; Hooley, Richard J

2012-05-01

197

Expression systems for industrial Gram-positive bacteria with low guanine and cytosine content  

Microsoft Academic Search

Recent years have seen an increase in the development of gene expression systems for industrial Gram-positive bacteria with low guanine and cytosine content that belong to the genera Bacillus, Clostridium, Lactococcus, Lactobacillus, Staphylococcus and Streptococcus. In particular, considerable advances have been made in the construction of inducible gene expression systems based on the capacity of these bacteria to utilize specific

Willem M. de Vos; Michiel Kleerebezem; Oscar P. Kuipers

1997-01-01

198

Orientation of Ordered Structures of Cytosine and Cytidine 5'-Monophosphate Adsorbed at Au(110)/Liquid Interfaces  

NASA Astrophysics Data System (ADS)

It is demonstrated using reflection anisotropy spectroscopy that the adsorption of cytosine and cytidine 5'-monophosphate at the Au(110) 1×2/electrolyte interface gives rise to ordered structures in which the base is oriented vertical to the surface and parallel to the [11¯0] axis of the Au(110) plane.

Weightman, P.; Dolan, G. J.; Smith, C. I.; Cuquerella, M. C.; Almond, N. J.; Farrell, T.; Fernig, D. G.; Edwards, C.; Martin, D. S.

2006-03-01

199

Androgen Receptor Cytosine, Adenine, Guanine Repeats, and Haplotypes in Relation to Ovarian Cancer Risk  

Microsoft Academic Search

Biological and epidemiologic evidence suggest that androgen or its receptor may play a role in ovarian cancer pathogenesis. The most notable genetic factor influencing androgen receptor (AR) activity is the functional cytosine, adenine, guanine (CAG) repeat in which length is inversely propor- tional to its transactivational activity. Additional genetic variation due to single nucleotide polymorphisms in the AR gene may

Kathryn L. Terry; Linda Titus-Ernstoff; Daniel W. Cramer

200

Failure of Cytosine Arabinoside in Treating Smallpox: A Double-Blind Study.  

National Technical Information Service (NTIS)

The effects of cytosine arabinoside (cytarabine) in the treatment of smallpox have been assessed in a controlled double-blind study of 18 Ethiopian patients. The drug, given in single intravenous doses of 100 mg. per sq. m. body-surface on each of four co...

D. T. Dennis E. B. Doberstyn S. Awoke

1974-01-01

201

Metformin Activates AMP Kinase through Inhibition of AMP Deaminase  

PubMed Central

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.

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

2011-01-01

202

Metformin activates AMP kinase through inhibition of AMP deaminase.  

PubMed

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

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

2010-11-08

203

Comparative enzymology of AMP deaminase, adenylate kinase, and creatine kinase in vertebrate heart and skeletal muscle: the characteristic AMP deaminase levels of skeletal versus cardiac muscle are reversed in the North American toad.  

PubMed

The specific activity of three characteristic enzymes, adenylate deaminase, adenylate kinase, and creatine kinase, in the skeletal muscles and heart of a variety of vertebrate land animals, including the human, are surveyed. Data from this study and available studies in the literature suggest that adenosine monophosphate deaminase in land vertebrates is quite high in white skeletal muscle, usually somewhat lower in red muscle, and 15- to 500-fold lower in cardiac muscle. Adenosine monophosphate deaminase is active primarily under ischemic or hypoxic conditions which occur frequently in white muscle, only occasionally in red muscle, and ought never occur in heart muscle, and this may therefore account for observed enzyme levels. The common North American toad, Bufo americanus, provides a striking exception to the rule with cardiac adenosine monophosphate deaminase as high as in mammalian skeletal muscle, whereas its skeletal muscle level of adenosine monophosphate deaminase is several times lower. The exceptional levels in the toad are not due to a change in substrate binding and are not accompanied by comparable change in the level of adenylate or creatine kinase. Nor do they signal any major change in isozyme composition, since a human muscle adenosine monophosphate deaminase-specific antiserum reacts with toad muscle adenosine monophosphate deaminase, but not with toad heart adenosine monophosphate deaminase. They do not represent any general anuran evolutionary strategy, since the bullfrog (Rana catesbeiana) and the giant tropic toad (Bufo marinus) have the usual vertebrate pattern of adenosine monophosphate deaminase distribution.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8394393

Fishbein, W N; Davis, J I; Foellmer, J W

1993-01-01

204

Yeast-Air Balloons  

NSDL National Science Digital Library

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

Exploratorium, The

2012-03-10

205

A Feast for Yeast  

NSDL National Science Digital Library

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

Society, American C.

2000-01-01

206

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

PubMed Central

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

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

2013-01-01

207

Conformational Variants of Duplex DNA Correlated with Cytosine-rich Chromosomal Fragile Sites*S?  

PubMed Central

We found that several major chromosomal fragile sites in human lymphomas, including the bcl-2 major breakpoint region, bcl-1 major translocation cluster, and c-Myc exon 1-intron 1 boundary, contain distinctive sequences of consecutive cytosines exhibiting a high degree of reactivity with the structure-specific chemical probe bisulfite. To assess the inherent structural variability of duplex DNA in these regions and to determine the range of structures reactive to bisulfite, we have performed bisulfite probing on genomic DNA in vitro and in situ; on duplex DNA in supercoiled and linearized plasmids; and on oligonucleotide DNA/DNA and DNA/2?-O-methyl RNA duplexes. Bisulfite is significantly more reactive at the frayed ends of DNA duplexes, which is expected given that bisulfite is an established probe of single-stranded DNA. We observed that bisulfite also distinguishes between more subtle sequence/structural differences in duplex DNA. Supercoiled plasmids are more reactive than linear DNA; and sequences containing consecutive cytosines, namely GGGCCC, are more reactive than those with alternating guanine and cytosine, namely GCGCGC. Circular dichroism and x-ray crystallography show that the GGGCCC sequence forms an intermediate B/A structure. Molecular dynamics simulations also predict an intermediate B/A structure for this sequence, and probe calculations suggest greater bisulfite accessibility of cytosine bases in the intermediate B/A structure over canonical B- or A-form DNA. Electrostatic calculations reveal that consecutive cytosine bases create electropositive patches in the major groove, predicting enhanced localization of the bisulfite anion at homo-C tracts over alternating G/C sequences. These characteristics of homo-C tracts in duplex DNA may be associated with DNA-protein interactions in vivo that predispose certain genomic regions to chromosomal fragility.

Tsai, Albert G.; Engelhart, Aaron E.; Hatmal, Ma'mon M.; Houston, Sabrina I.; Hud, Nicholas V.; Haworth, Ian S.; Lieber, Michael R.

2009-01-01

208

Conformational variants of duplex DNA correlated with cytosine-rich chromosomal fragile sites.  

PubMed

We found that several major chromosomal fragile sites in human lymphomas, including the bcl-2 major breakpoint region, bcl-1 major translocation cluster, and c-Myc exon 1-intron 1 boundary, contain distinctive sequences of consecutive cytosines exhibiting a high degree of reactivity with the structure-specific chemical probe bisulfite. To assess the inherent structural variability of duplex DNA in these regions and to determine the range of structures reactive to bisulfite, we have performed bisulfite probing on genomic DNA in vitro and in situ; on duplex DNA in supercoiled and linearized plasmids; and on oligonucleotide DNA/DNA and DNA/2'-O-methyl RNA duplexes. Bisulfite is significantly more reactive at the frayed ends of DNA duplexes, which is expected given that bisulfite is an established probe of single-stranded DNA. We observed that bisulfite also distinguishes between more subtle sequence/structural differences in duplex DNA. Supercoiled plasmids are more reactive than linear DNA; and sequences containing consecutive cytosines, namely GGGCCC, are more reactive than those with alternating guanine and cytosine, namely GCGCGC. Circular dichroism and x-ray crystallography show that the GGGCCC sequence forms an intermediate B/A structure. Molecular dynamics simulations also predict an intermediate B/A structure for this sequence, and probe calculations suggest greater bisulfite accessibility of cytosine bases in the intermediate B/A structure over canonical B- or A-form DNA. Electrostatic calculations reveal that consecutive cytosine bases create electropositive patches in the major groove, predicting enhanced localization of the bisulfite anion at homo-C tracts over alternating G/C sequences. These characteristics of homo-C tracts in duplex DNA may be associated with DNA-protein interactions in vivo that predispose certain genomic regions to chromosomal fragility. PMID:19106104

Tsai, Albert G; Engelhart, Aaron E; Hatmal, Ma'mon M; Houston, Sabrina I; Hud, Nicholas V; Haworth, Ian S; Lieber, Michael R

2008-12-23

209

Decrease in topoisomerase I is responsible for activation-induced cytidine deaminase (AID)-dependent somatic hypermutation  

PubMed Central

Somatic hypermutation (SHM) and class-switch recombination (CSR) of the Ig gene require both the transcription of the locus and the expression of activation-induced cytidine deaminase (AID). During CSR, AID decreases the amount of topoisomerase I (Top1); this decrease alters the DNA structure and induces cleavage in the S region. Similarly, Top1 is involved in transcription-associated mutation at dinucleotide repeats in yeast and in triplet-repeat contraction in mammals. Here, we report that the AID-induced decrease in Top1 is critical for SHM. Top1 knockdown or haploinsufficiency enhanced SHM, whereas Top1 overexpression down-regulated it. A specific Top1 inhibitor, camptothecin, suppressed SHM, indicating that Top1's activity is required for DNA cleavage. Nonetheless, suppression of transcription abolished SHM, even in cells with Top1 knockdown, suggesting that transcription is critical. These results are consistent with a model proposed for CSR and triplet instability, in which transcription-induced non-B structure formation is enhanced by Top1 reduction and provides the target for irreversible cleavage by Top1. We speculate that the mechanism for transcription-coupled genome instability was adopted to generate immune diversity when AID evolved.

Kobayashi, Maki; Sabouri, Zahra; Sabouri, Somayeh; Kitawaki, Yoko; Pommier, Yves; Abe, Takaya; Kiyonari, Hiroshi; Honjo, Tasuku

2011-01-01

210

Cytosine ribose flexibility in DNA: a combined NMR 13C spin relaxation and molecular dynamics simulation study  

PubMed Central

Using 13C spin relaxation NMR in combination with molecular dynamic (MD) simulations, we characterized internal motions within double-stranded DNA on the pico- to nano-second time scale. We found that the C–H vectors in all cytosine ribose moieties within the Dickerson–Drew dodecamer (5?-CGCGAATTCGCG-3?) are subject to high amplitude motions, while the other nucleotides are essentially rigid. MD simulations showed that repuckering is a likely motional model for the cytosine ribose moiety. Repuckering occurs with a time constant of around 100 ps. Knowledge of DNA dynamics will contribute to our understanding of the recognition specificity of DNA-binding proteins such as cytosine methyltransferase.

Duchardt, Elke; Nilsson, Lennart

2008-01-01

211

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

NASA Astrophysics Data System (ADS)

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

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

2010-12-01

212

Cytosine neutral molecules and cation-radicals in the gas-phase  

NASA Astrophysics Data System (ADS)

Gas-phase cytosine molecules and cation-radicals represent a complex system of several nearly isoenergetic tautomers within each group. Computational methods differ in ordering the relative enthalpies of neutral cytosine tautomers. At our highest level of theory, CCSD(T)/aug-cc-pVTZ calculations find an enol form, anti-2-hydroxy-4-aminopyrimidine (2), to be the most stable neutral tautomer in the gas-phase, followed by its rotamer, syn-2-hydroxy-4-aminopyrimidine (3), the canonical oxo-form, 4-amino-1,2-dihydropyrimidin-2(1H)-one (1), imino-forms, 2-oxo-4-iminodihydro(1H,3H)pyrimidine (4 and 5), and another oxo-form, 4-amino-dihydropyrimidin-2(3H)-one (6). Other tautomers, such as anti-anti, syn-syn and syn-anti-2-hydroxy-4-iminodihydro(3H,4H)pyrimidines (7-9), are less stable. The adiabatic ionization energies of the major cytosine tautomers have been calculated to be 8.71, 8.64, 8.62, 8.58, 8.64, and 8.31 eV for 1, 2, 3, 4, 5, and 6, respectively. Cytosine cation-radicals show very close relative energies that increase in the order of 6+ (most stable) <2+ [approximate] 3+ < 4+ [approximate] 7+ [approximate] 1+ < 5+. In addition, distonic ions having radical centers at C-5 (10+) and C-6 (11+ are found as low-energy isomers of 1+-7+. Metastable cytosine cation-radicals undergo ring-cleavage dissociations by eliminations of CO (major) and HNCO (minor). The energetics of these and other higher-energy dissociations, including the pertinent transition states, have been established by high-level ab initio and density functional theory calculations and plausible mechanisms have been proposed. Collisional neutralization of cytosine cation-radicals with trimethylamine and dimethyldisulfide as electron donors forms stable molecules that are detected as cation-radicals following collisional reionization. The dissociations observed upon neutralization-reionization mainly include ring-cleavages followed by loss of NCO, HNCO, and formation of C2H3N, C2H2N, and CO neutral fragments that are assigned to ion dissociations following reionization.

Wolken, Jill K.; Yao, Chunxiang; Turecek, Frantisek; Polce, Michael J.; Wesdemiotis, Chrys

2007-11-01

213

Specific solvation effects on the structures and properties of Watson–Crick and reverse Watson–Crick isocytosine–cytosine and guanine–cytosine base pairs: a theoretical ab initio study  

Microsoft Academic Search

Ab initio quantum chemical studies were performed for the standard and most favorable reverse Watson–Crick isocytosine–cytosine (iCC) and guanine–cytosine (GC) complexes in the gas phase and in a water solution. Full geometry optimizations at the Hartree–Fock (HF) level with the 6-31G(d) basis set without any constraints on the planarity of these complexes were carried out. The water solution was modeled

N. U Zhanpeisov; J Leszczynski

1999-01-01

214

Molecular evolution of minisatellites in hemiascomycetous yeasts.  

PubMed

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

Richard, Guy-Franck; Dujon, Bernard

2005-09-21

215

Perspective of plant growth promoting rhizobacteria (PGPR) containing ACC deaminase in stress agriculture.  

PubMed

Ethylene is a gaseous plant growth hormone produced endogenously by almost all plants. It is also produced in soil through a variety of biotic and abiotic mechanisms, and plays a key role in inducing multifarious physiological changes in plants at molecular level. Apart from being a plant growth regulator, ethylene has also been established as a stress hormone. Under stress conditions like those generated by salinity, drought, waterlogging, heavy metals and pathogenicity, the endogenous production of ethylene is accelerated substantially which adversely affects the root growth and consequently the growth of the plant as a whole. Certain plant growth promoting rhizobacteria (PGPR) contain a vital enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which regulates ethylene production by metabolizing ACC (an immediate precursor of ethylene biosynthesis in higher plants) into alpha-ketobutyrate and ammonia. Inoculation with PGPR containing ACC deaminase activity could be helpful in sustaining plant growth and development under stress conditions by reducing stress-induced ethylene production. Lately, efforts have been made to introduce ACC deaminase genes into plants to regulate ethylene level in the plants for optimum growth, particularly under stressed conditions. In this review, the primary focus is on giving account of all aspects of PGPR containing ACC deaminase regarding alleviation of impact of both biotic and abiotic stresses onto plants and of recent trends in terms of introduction of ACC deaminase genes into plant and microbial species. PMID:17665234

Saleem, Muhammad; Arshad, Muhammad; Hussain, Sarfraz; Bhatti, Ahmad Saeed

2007-07-31

216

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

NASA Astrophysics Data System (ADS)

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.

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

2004-07-01

217

Active cytosine demethylation triggered by a nuclear receptor involves DNA strand breaks  

PubMed Central

Cytosine methylation at CpG dinucleotides contributes to the epigenetic maintenance of gene silencing. Dynamic reprogramming of DNA methylation patterns is believed to play a key role during development and differentiation in vertebrates. The mechanisms of DNA demethylation remain unclear and controversial. Here, we present a detailed characterization of the demethylation of an endogenous gene in cultured cells. This demethylation is triggered in a regulatory region by a transcriptional activator, the glucocorticoid receptor. We show that DNA demethylation is an active process, occurring independently of DNA replication, and in a distributive manner without concerted demethylation of cytosines on both strands. We demonstrate that the DNA backbone is cleaved 3? to the methyl cytidine during demethylation, and we suggest that a DNA repair pathway may therefore be involved in this demethylation.

Kress, Clemence; Thomassin, Helene; Grange, Thierry

2006-01-01

218

Neurodevelopmental Disabilities in Children With Intermediate and Premutation Range Fragile X Cytosine-Guanine-Guanine Expansions.  

PubMed

To determine the range of neurodevelopmental diagnoses associated with intermediate (45-54 repeats) and premutation (55-200 repeats) range cytosine-guanine-guanine fragile X expansions, the medical records of children with intermediate or premutation range expansions were retrospectively reviewed, and all neurodevelopmental diagnoses were abstracted. Twenty-nine children (9 female, 20 male; age, 13 months to 17 years) with intermediate (n = 25) or premutation (n = 4) range expansions were identified with neurodevelopmental diagnoses, including global developmental delay/intellectual disability (n = 15), language and learning disorders (n = 9), attention-deficit hyperactivity disorder (n = 5), epilepsy (n = 5), and motor disorders (n = 12), including 2 boys younger than 4 years of age with tremor and ataxia. Thus, children with intermediate or premutation range fragile X cytosine-guanine-guanine expansions may be more susceptible than children without such expansions to other processes, both genetic and environmental, that contribute to neurodevelopmental disability. PMID:23266944

Renda, Meredith M; Voigt, Robert G; Babovic-Vuksanovic, Dusica; Highsmith, W Edward; Vinson, Sherry S; Sadowski, Christine M; Hagerman, Randi J

2012-12-23

219

SCREENING AND EVALUATION OF RHIZOBACTERIA CONTAINING ACC-DEAMINASE FOR GROWTH PROMOTION OF WHEAT (TRITICUM AESTIVUM L.) UNDER SALINITY STRESS  

Microsoft Academic Search

Effect of rhizobacteria containing ACC-deaminase (1-aminocyclopropane-1- carboxylate deaminase) activity was studied in the Department of Soil and Environmental Sciences, University of Agriculture, Faisalabad, Pakistan during 2005 for promoting wheat growth under salt stressed conditions. Minimal salt medium containing ACC as sole nitrogen source was used for growth of rhizobacteria. Disinfected wheat seeds were germinated in petri plates. Uniformly germinated seeds

Rizwana Kausar; Sher Muhammad Shahzad; Muhammad Arshad; Muhammad Ashfaq Anjum

220

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

Microsoft Academic Search

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

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

1991-01-01

221

Cytidine Deaminases from B. subtilis and E. coli :  Compensating Effects of Changing Zinc Coordination and Quaternary Structure †  

Microsoft Academic Search

Cytidine deaminase from E. coli is a dimer of identical subunits (Mr ) 31 540), each containing a single zinc atom. Cytidine deaminase from B. subtilis is a tetramer of identical subunits ( Mr ) 14 800). After purification from an overexpressing strain, the enzyme from B. subtilis is found to contain a single atom of zinc per enzyme subunit

Dean C. Carlow; Nina Mejlhede; Jan Neuhard; Richard Wolfenden

1999-01-01

222

Vaginal Yeast Infections (For Parents)  

MedlinePLUS

... infection 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 don't get vaginal yeast infections, ...

223

A Potent Cell-active Allosteric Inhibitor of Murine DNA Cytosine C5 Methyltransferase  

Microsoft Academic Search

The major DNA cytosine methyltransferase isoform in mouse erythroleukemia cells, Dnmt1, exhibits potent dead-end inhibition with a single-stranded nucleic acid by binding to an allosteric site on the enzyme. The pre- viously reported substrate inhibition with double- stranded substrates also involves binding to an allo- steric site. Thus, both forms of inhibition involve ternary enzyme-DNA-DNA complexes. The inhibition potency of

James Flynn; Jing-Yuan Fang; Judy A. Mikovits; Norbert O. Reich

2003-01-01

224

Structural Effects of Cytosine Methylation on DNA Sugar Pucker Studied by FTIR  

Microsoft Academic Search

This FTIR investigation concerns structural consequences of 5-methylation of cytosine in a DNA decamer in solution. Methylation of DNA is an important functional signal in transcription, but its effect on DNA structure is variable and not fully understood. Here, single and multiple 5-methylcytosine substitutions are introduced into the self-complementary sequence d(CCGGCGCCGG)2. No major structural effect of methylation on the DNA

Martina Banyay; Astrid Gräslund

2002-01-01

225

Nonplanarity and solvent effects on structural and polarizability properties of cytosine tautomers  

Microsoft Academic Search

We report equilibrium geometries, relative stabilities, electronic and vibrational polarizabilities of cytosine tautomers and of reference compounds phenol, aniline and pyrimidine, in the gas phase and in solution, calculated by conventional correlated ab initio (CCSD, CCSD(T) and MP2) and density functional (B97-1) methods, using a variety of basis sets. Gas phase ionization potentials, electron affinities and torsional potentials for the

A. Alparone; A. Millefiori; S. Millefiori

2005-01-01

226

Dependence on temperature and guanine-cytosine content of bubble length distributions in DNA  

Microsoft Academic Search

We present numerical results on the temperature dependence of the distribution of bubble lengths in DNA segments of various guanine-cytosine (GC) concentrations. Base-pair openings are described by the Peyrard-Bishop-Dauxois model and the corresponding thermal equilibrium distributions of bubbles are obtained through Monte Carlo calculations for bubble sizes up to the order of a hundred base pairs. The dependence of the

G. Kalosakas; S. Ares

2009-01-01

227

5?Cytosine DNA-methyltransferase mRNA levels in hereditary colon carcinoma  

Microsoft Academic Search

DNA methylation plays an important part in the regulation of gene expression. Alterations in DNA methylation in tumours have\\u000a been reported and have been used to generate hypotheses about mutagenesis and silencing of tumour suppressor genes. However,\\u000a the underlying mechanism is still poorly understood, and conflicting data on the levels of overexpression of 5?-cytosine DNA\\u000a methyltransferase in sporadic colon carcinoma

Claude A. Jakob; Irene Guldenschuh; Rainer Hürlimann; Beat Müllhaupt; Andreas Müller; Rudolf Ammann; Michael Fried; J. Roth

1999-01-01

228

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

PubMed

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

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

2007-07-07

229

Reversal by cytidine of cyclopentenyl cytosine-induced toxicity in mice without compromise of antitumor activity  

Microsoft Academic Search

Among nine compounds surveyed, cytidine was found to be the most effective in reversing the antiproliferative effects of cyclopentenyl cytosine (CPEC) on human T-lymphoblasts (MOLT-4) in culture. Cytidine, at concentrations of 1–25 ?M, enabled cells to maintain normal logarithmic growth when added up to 12 hr after exposure to a 200 nM concentration of the oncolytic nucleoside, CPEC. The most

Harry Ford; John S. Driscoll; Zhang Hao; Katherine A. Dobyns; Michael E. Rommel; Emily Stowe; Joseph O. Anderson; Jacqueline Plowman; William R. Waud; David G. Johns; David A. Cooney

1995-01-01

230

Reverse Banding on Chromosomes Produced by a Guanosine-Cytosine Specific DNA Binding Antibiotic: Olivomycin  

Microsoft Academic Search

Characteristic reverse fluorescent banding patterns (R bands) on human, bovine, and mouse metaphase chromosomes are produced by treating chromosome preparations directly with olivomycin. With the DNA in solution, the repeating polymer poly[d(G-C)] \\\\cdot poly[d(G-C)] (where G is guanine and C is cytosine) enhanced the fluorescence of olivomycin, while the antibiotic fluorescence was not affected by the alternating polynucleotide poly[d(A-T)] \\\\cdot

J. H. van de Sande; C. C. Lin; K. F. Jorgenson

1977-01-01

231

Persistence of Cytosine Methylation of DNA following Fertilisation in the Mouse  

Microsoft Academic Search

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

Yan Li; Chris ONeill

2012-01-01

232

Photoaffinity Labeling and Characterization of the Cloned Purine-Cytosine Transport System in Saccharomyces cerevisiae  

Microsoft Academic Search

8-Azido[2-3H]adenine was used as a photoaffinity label for the purine-cytosine transport system. After irradiation in the presence of the photoaffinity label, the cells were converted into protoplasts, their plasma membranes were purified, and the membrane proteins were extracted and separated by NaDodSO4\\/PAGE. The radioactivity was specifically incorporated into a protein with a molecular weight of 120,000. Photoaffinity labeling of this

Rainer Schmidt; Morris F. Manolson; Marie-Renee Chevallier

1984-01-01

233

AMP Deaminase 3 Deficiency Enhanced 5'-AMP Induction of Hypometabolism.  

PubMed

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

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

2013-09-16

234

Erythrocyte adenosine deaminase: diagnostic value for Diamond-Blackfan anaemia.  

PubMed

Diamond-Blackfan anaemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterized by red cell aplasia. Mutations in ribosomal genes are found in more than 50% of cases. Elevated erythrocyte adenosine deaminase (eADA) was first noted in DBA in 1983. In this study we determined the value of eADA for the diagnosis of DBA compared with other IBMFS; the association of eADA in DBA with age, gender or other haematological parameters; and the association with known DBA-related gene mutations. For the diagnosis of DBA compared with non-DBA patients with other bone marrow failure syndromes, eADA had a sensitivity of 84%, specificity 95%, and positive and negative predictive values of 91%. In patients with DBA there was no association between eADA and gender, age, or other haematological parameters. Erythrocyte ADA segregated with, as well as independent of, known DBA gene mutations. While eADA was an excellent confirmatory test for DBA, 16% of patients with classical clinical DBA had a normal eADA. PMID:23252420

Fargo, John H; Kratz, Christian P; Giri, Neelam; Savage, Sharon A; Wong, Carolyn; Backer, Karen; Alter, Blanche P; Glader, Bertil

2012-12-17

235

Dipeptidyl peptidase IV and adenosine deaminase activity. Decrease in depression.  

PubMed

Dipeptidyl peptidase IV (DPPIV) and adenosine deaminase (ADA), two T cell associated enzymes, are known to have a possible interaction and play essential roles in immune system functioning. On the other hand, depression has been shown to be accompanied with some immune-inflammatory alterations. In this regard, in order to make a contribution to the understanding of the ongoing immune disturbances in depression, serum DPPIV and ADA activities were determined in minor and major depressives and compared with healthy controls. Both enzyme activities were found to be decreased in major depressives compared to controls while only DPPIV activity was significantly lower in major depressives than the minor depressives. There were significant inverse relationships between enzyme activities and the severity of depression. Moreover, a positive intracorrelation was found between decreased DPPIV and ADA levels. The correlated decrease in DPPIV and ADA, might be a further support for their possible association. Results also suggest that decreased enzyme activities might reflect the impaired immune state in depression while major depressed patients might have a greater tendency to immune dysfunction than the minor depressed ones. PMID:10581653

Elgün, S; Keskinege, A; Kumbasar, H

1999-11-01

236

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

PubMed Central

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.

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

2013-01-01

237

Diagnostic value of serum adenosine deaminase level in pulmonary tuberculosis  

PubMed Central

Background: In some studies, the level of adenosine deaminase (ADA) in sputum and effusion liquids was used for the diagnosis of tuberculosis (TB). But it is not always possible to access these materials. The goal of this study is to assess the diagnostic value of serum ADA levels in pulmonary TB patients. Materials and Methods: In this study, 40 sputum smear-positive TB patients who were hospitalized and 40 non-TB patients who referred for surgeries were selected. A serum sample was collected and serum ADA level was measured by ADA kit. Results: The average (SD) of serum ADA in TB and non-TB patients were 20.88 (±5.97) and 10.69 (±2.98) U/L, respectively (P value < 0.05). The best cut-off point was 14 U/L. The calculated area under the receiver operating characteristic (ROC) curve was 0.955 (95% CI, 0.914-0.995); sensitivity was 92.7% (95% CI, 84.7-100) and specificity was 88.1% (95% CI, 78.3-97.8) (P < 0.001). Conclusion: Serum ADA level may be proposed as a proper index for TB diagnosis.

Afrasiabian, Shahla; Mohsenpour, Behzad; Bagheri, Katayoun Haji; Sigari, Naseh; Aftabi, Kaveh

2013-01-01

238

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

NASA Astrophysics Data System (ADS)

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

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

2012-02-01

239

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

SciTech Connect

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

Reva, Igor; Fausto, Rui [Department of Chemistry, University of Coimbra, 3004-535 Coimbra (Portugal); Nowak, Maciej J.; Lapinski, Leszek [Institute of Physics, Polish Academy of Sciences, Al. Lotnikow 32/46, 02-668 Warsaw (Poland)

2012-02-14

240

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

PubMed

We studied the single photon ionization of gas phase adenine and cytosine by means of vacuum ultraviolet synchrotron radiation coupled to a velocity map imaging electron?ion coincidence spectrometer. Both in-vacuum temperature-controlled oven and aerosol thermodesorption were successfully applied to promote the intact neutral biological species into the gas phase. The photoion yields are consistent with previous measurements. In addition, we deduced the threshold photoelectron spectra and the slow photoelectron spectra for both species, where the close to zero kinetic energy photoelectrons and the corresponding photoions are measured in coincidence. The photoionization close and above the ionization energies are found to occur mainly via direct processes. Both vaporization techniques lead to similar electronic spectra for the two molecules, which consist of broadbands due to the complex electronic structure of the cationic species and to the possible contribution of several neutral tautomers for cytosine prior to ionization. Accurate ionization energies are measured for adenine and cytosine at, respectively, 8.267 ± 0.005 eV and 8.66 ± 0.01 eV, and we deduce precise thermochemical data for the adenine radical cation. Finally, we performed an evaluation and a comparison of the two vaporization techniques addressing the following criteria: measurement precision, thermal fragmentation, sensitivity, and sample consumption. The aerosol thermodesorption technique appears as a promising alternative to vaporize large thermolabile biological compounds, where extended thermal decomposition or low sensitivity could be encountered when using a simple oven vaporization technique. PMID:23485287

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

2013-03-01

241

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

PubMed

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

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

2012-02-14

242

Yeast Infection during Pregnancy  

MedlinePLUS

... may be reprinted for personal, noncommercial use only. Yeast infection during pregnancy: Are over-the-counter treatments ... share your e-mail address Sign up Question Yeast infection during pregnancy: Are over-the-counter treatments ...

243

Yeast Infection (Vaginal)  

MedlinePLUS

... may be reprinted for personal, noncommercial use only. Yeast infection (vaginal) By Mayo Clinic staff Original Article: http://www.mayoclinic.com/health/yeast-infection/DS01182 Definition Symptoms Causes Risk factors Preparing ...

244

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.

245

Yeast Based Sensors  

NASA Astrophysics Data System (ADS)

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

Shimomura-Shimizu, Mifumi; Karube, Isao

246

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

NASA Astrophysics Data System (ADS)

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

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

1999-10-01

247

Lager brewing yeast  

Microsoft Academic Search

Lager brewing yeast is a group of closely related strains of Saccharomyces pastorianus\\/S. carlsbergensis used for lager beer production all over the world, making it one of the most important industrial yeasts. The pure cultivation\\u000a of yeast was established in the early 1880’s with immediate practical success for lager brewing yeast. However, almost a century\\u000a would elapse before its genetics

Yukiko Kodama; Morten C. Kielland-Brandt; Jørgen Hansen

248

Enthalpy and enzyme activity of modified histidine residues of adenosine deaminase and diethyl pyrocarbonate complexes.  

PubMed

Kinetic and thermodynamic studies have been made on the effect of diethyl pyrocarbonate as a histidine modifier on the active site of adenosine deaminase in 50 mM sodium phosphate buffer pH 6.8, at 27 degrees C using UV spectrophotometry and isothermal titration calorimetry (ITC). Inactivation of adenosine deaminase by diethyl pyrocarbonate is correlated with modification of histidyl residues. The number of modified histidine residues complexed to active site of adenosine deaminase are equivalent to 4. The number and energy of histidine binding sets are determined by enthalpy curve, which represents triple stages. These stages are composed of 3,1 and 1 sites of histidyl modified residues at diethyl pyrocarbonate concentrations, 0.63, 1.8, 3.3 mM. The heat contents corresponding to the first, second and third sets are found to be 18000, 22000 and 21900 kJ mol(-1) respectively. PMID:10704983

Ataie, G; Moosavi-Movahedi, A A; Saboury, A A; Hakimelahi, G H; Hwu, J R; Tsay, S C

2000-03-16

249

[The influence of nonsteroidal anti-inflammatory drugs on deaminase adenosine activity].  

PubMed

The aim of the study was to evaluate the influence ofnonsteroidal anti-inflammatory drugs on deaminase adenosine activity using animal model. 70 rats of Wistar breed was divided into equal groups of ten. Animals were given intragastrically for three weeks: acetylsalicylic acid (ASA) at doses of 2 and 10 mg/kg bw/day, diclofenac at doses of 1 and 5 mg/kg bw/day, nimesulide at doses of 2.5 mg and 12.5 mg/kg bw/day. Control group received only water. After 21 days rats were decapitated and blood was collected to assess deaminase adenosine activity in plasma and erythrocytes. It was found that all investigated drugs inhibit ADA activity in plasma and ADA2 isoenzyme is responsible for this inhibition. Deaminase adenosine activity in erythrocytes is inhibited by ASA and diclofenac but increased by nimesulide. PMID:18652275

Kopff, Anna; Kopff, Maria; Kowalczyk, Edward

2006-09-01

250

Maintaining Genome Stability: The Role of Helicases and Deaminases.  

National Technical Information Service (NTIS)

Study the in vitro functions of MCM proteins from archaea and yeast cells using the genetically engineered protein constructs. In this aim, we will also extend our prior success in the X-ray structural studies of an N-terminal fragment of an archaea MCM b...

X. J. Chen

2007-01-01

251

Maintaining Genome Stability: The Role of Helicases and Deaminases.  

National Technical Information Service (NTIS)

The Brief Description of The Four Aims For The Grant: (Aim 1). Study the in vitro functions of MCM proteins from archaea and yeast cells using the genetically engineered protein constructs. In this aim, we will also extend our prior success in the X-ray s...

X. Chen

2006-01-01

252

[Yeasts contaminating salmon roe].  

PubMed

Quantitative and species compositions of yeast contaminating eggs, fry and fingerlings of Salmo gairdneri Rich under artificial breeding have been studied. Prevalence of species of genera Candida, Rhodotorula, Cryptococcus and Debaryomyces is noted. Yeast isolated from perished eggs and sick fry do not possess pathogenic properties. Certain strains of yeast make stimulating effect on the studied microorganisms. PMID:8983527

Nagornaia, S S; Ignatova, E A; Isaeva, N M; Davydov, O N; Podgorski?, V S

253

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

SciTech Connect

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

Daddona, P.E.

1981-12-10

254

AMPD1 C34T mutation selectively affects AMP-deaminase activity in the human heart.  

PubMed

Possession of the nonsense mutation in AMPD 1 C34T gene has been linked to improved survival in patients with heart failure, possibly by promoting the formation of adenosine. This mutation is known to decrease the activity of AMP-deaminase in skeletal muscle. We have found that the AMPD1 mutation decreases the activity of AMP-deaminase in the heart without changing the activity of any other enzymes of adenine nucleotide metabolism. Protective mechanism of this mutation may be thus induced by local cardiac metabolic changes. PMID:16021918

Kalsi, K K; Yuen, A H Y; Johnson, P H; Birks, E J; Yacoub, M H; Smolenski, R T

2005-01-01

255

Prions in Yeast  

PubMed Central

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

Liebman, Susan W.; Chernoff, Yury O.

2012-01-01

256

Autoimmune dysregulation and purine metabolism in adenosine deaminase deficiency.  

PubMed

Genetic defects in the adenosine deaminase (ADA) gene are among the most common causes for severe combined immunodeficiency (SCID). ADA-SCID patients suffer from lymphopenia, severely impaired cellular and humoral immunity, failure to thrive, and recurrent infections. Currently available therapeutic options for this otherwise fatal disorder include bone marrow transplantation (BMT), enzyme replacement therapy with bovine ADA (PEG-ADA), or hematopoietic stem cell gene therapy (HSC-GT). Although varying degrees of immune reconstitution can be achieved by these treatments, breakdown of tolerance is a major concern in ADA-SCID. Immune dysregulation such as autoimmune hypothyroidism, diabetes mellitus, hemolytic anemia, and immune thrombocytopenia are frequently observed in milder forms of the disease. However, several reports document similar complications also in patients on long-term PEG-ADA and after BMT or GT treatment. A skewed repertoire and decreased immune functions have been implicated in autoimmunity observed in certain B-cell and/or T-cell immunodeficiencies, but it remains unclear to what extent specific mechanisms of tolerance are affected in ADA deficiency. Herein we provide an overview about ADA-SCID and the autoimmune manifestations reported in these patients before and after treatment. We also assess the value of the ADA-deficient mouse model as a useful tool to study both immune and metabolic disease mechanisms. With focus on regulatory T- and B-cells we discuss the lymphocyte subpopulations particularly prone to contribute to the loss of self-tolerance and onset of autoimmunity in ADA deficiency. Moreover we address which aspects of immune dysregulation are specifically related to alterations in purine metabolism caused by the lack of ADA and the subsequent accumulation of metabolites with immunomodulatory properties. PMID:22969765

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

2012-08-27

257

Autoimmune Dysregulation and Purine Metabolism in Adenosine Deaminase Deficiency  

PubMed Central

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

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

2012-01-01

258

Adenosine deaminase activity in COPD patients and healthy subjects.  

PubMed

Chronic obstructive pulmonary disease (COPD) has been defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD), as a disease state characterized by airflow limitation which is not fully reversible. COPD consists of emphysema which is the destruction and inflammation of the lung alveoli. Adenosine deaminase (ADA, E.C.3.5.4.4) converts adenosine to inosine. There are two isoenzymes of ADA in serum; ADA1 and ADA2. It has been established that in COPD patients the adenosine levels increase, which can contribute to decrease of ADA activity. In this research we studied the ADA and its isoenzyme activity in COPD patients. This descriptive analytical case-control study was performed on thirty patients who were hospitalized in the pulmonary wards with an acute exacerbation of COPD. ADA activity was determined in 30 COPD patients, 30 nonsmokers and 30 smokers controls. All subjects were male. We used colorimetric (Giusti) method for measuring of ADA activity. The data were analyzed using SPSS 13 software and Kruskall-Wallis and two-way ANOVA tests. Total ADA activity in the COPD and smoker control groups was significantly lower than in non smoker group (18.99 -/+ 7, 19.03 -/+ 9.1 and 22.95 -/+ 6.7 U/L, respectively). There was a significant difference for ADA2 between the three groups. Whereas the ADA1 activity in the three groups had no significant difference. Based on the obtained data, decrease of ADA activity may play an important role in the formation of pulmonary injury in COPD patients. PMID:20548128

Goodarzi, Mohammad Taghi; Abdi, Mohammad; Tavilani, Heidar; Nadi, Ebrahim; Rashidi, Mojtaba

2010-03-01

259

[Adenosine deaminase activity in tuberculous and malignant pleural effusions].  

PubMed

Measurement of pleural adenosine deaminase activity (ADA) is a useful diagnostic tool for tuberculous pleurisy, but false-positive findings from non-tuberculous effusions have been reported. In order to improve diagnostic value of ADA it is recommended to estimate activity of both ADA1 and ADA2 izoenzymes or 2'-deoxyadenosine/adenosine activity ratio. In order to evaluate ADA as a diagnostic parameter total ADA, with adenosine as a substrate, and 2'-deoxyadenosine/adenosine activity ratio were measured in tuberculous and malignant pleural effusions. Altogether, 26 pleural exudates (11 tuberculous and 15 malignant) were selected. ADA either with adenosine or 2'-deoxyadenosine was determined by colorimetric method of Giusti. Each pleural fluid sample was diluted prior to the assay (1:8) to avoid enzyme inhibition which was observed in nondiluted pleural effusions. The ADA level reached the diagnostic cut-off set for tuberculous effusions (40 U/L) in every 11 tuberculous exudates with the mean value of 85,3+/-47,1 U/L; in 9 of these the 2'-deoxyadenosine/adenosine ratio was less than 0,45. In the malignant group of patients, no one ADA level exceed 40 U/L, being estimated at 10,6+/-7,7 U/L (p<0,001). In 10 of these 15 exudates the 2'-deoxyadenosine/adenosine ratio was undetectable, in four it was less than 0,45 and only in one it was over 0,45. We concluded that ADA measured by the Giusti method proceeded by the dilution 1:8 of the pleural effusion samples very good differentiates tuberculous from malignant pleurisy, without the necessity to determine the 2'-deoxyadenosine/adenosine ratio. The investigation needs to be continued on the more numerous groups of patients. PMID:17175968

Safianowska, Aleksandra; Krenke, Rafa?; Dmowska-Sobstyl, Barbara; Bogacka-Zatorska, Elzbieta; Domaga?a-Kulawik, Joanna; Chazan, Ryszarda

2006-01-01

260

Potential roles of adenosine deaminase-2 in diabetic retinopathy.  

PubMed

The early activation of microglia that induces retinal inflammation in DR may serve as a target for therapeutic intervention of DR. Our demonstration that retinal inflammation is attenuated via adenosine receptor A(2A)AR supports the hypothesis that a mechanism to maintain extracellular concentrations of adenosine important in normal physiology is impaired in DR. Extracellular concentrations of adenosine are regulated by the interplay of equiliberative nucleoside transporter (ENT)s with enzymes of adenosine metabolism including adenosine deaminase-1 (ADA1), adenosine kinase (AK) and CD73. In the vertebrates but not rodents, a macrophage-associated ADA2 is identified. The role of ADA2 is, therefore, understudied as the sequencing probes or antibodies to mouse ADA2 are not available. We identified increased ADA2 expression and activity in human and porcine retinas with diabetes, and in Amadori glycated albumin (AGA)- or hyperglycemia-treated porcine and human microglia. In rodent as well as porcine cells, modulation of TNF-? release is mediated by A(2A)AR. Quantitative analysis of normal and diabetic porcine retinas reveals that while the expression levels of ADA2, A2AAR, ENT1, TNF-? and MMP9 are increased, the levels of AK are reduced during inflammation as an endogenous protective mechanism. To determine the role of ADA2, we found that AGA induces ADA2 expression, ADA2 activity and TNF-? release, and that TNF-? release is blocked by ADA2-neutralizing antibody or ADA2 siRNA, but not by scrambled siRNA. These results suggest that retinal inflammation in DR is mediated by ADA2, and that the anti-inflammatory activity of A(2A)AR signaling is impaired in diabetes due to increased ADA2 activity. PMID:23685153

Elsherbiny, Nehal M; Naime, Mohammad; Ahmad, Saif; Elsherbini, Ahmed M; Mohammad, Shuaib; Fulzele, Sadanand; El-Remessy, Azza B; Al-Gayyar, Mohammed M; Eissa, Laila A; El-Shishtawy, Mamdouh M; Han, Guichun; White, Richard; Haroldo, Toque Flores; Liou, Gregory I

2013-05-16

261

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

PubMed Central

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

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

2013-01-01

262

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

PubMed Central

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

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

2010-01-01

263

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

PubMed

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

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

2013-08-07

264

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

Microsoft Academic Search

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

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

1999-01-01

265

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

PubMed

Yeast 25S rRNA was reported to contain a single cytosine methylation (m(5)C). In the present study using a combination of RP-HPLC, mung bean nuclease assay and rRNA mutagenesis, we discovered that instead of one, yeast contains two m(5)C residues at position 2278 and 2870. Furthermore, we identified and characterized two putative methyltransferases, Rcm1 and Nop2 to be responsible for these two cytosine methylations, respectively. Both proteins are highly conserved, which correlates with the presence of two m(5)C residues at identical positions in higher eukaryotes, including humans. The human homolog of yeast Nop2, p120 has been discovered to be upregulated in various cancer tissues, whereas the human homolog of Rcm1, NSUN5 is completely deleted in the William's-Beuren Syndrome. The substrates and function of both human homologs remained unknown. In the present study, we also provide insights into the significance of these two m(5)C residues. The loss of m(5)C2278 results in anisomycin hypersensitivity, whereas the loss of m(5)C2870 affects ribosome synthesis and processing. Establishing the locations and enzymes in yeast will not only help identifying the function of their homologs in higher organisms, but will also enable understanding the role of these modifications in ribosome function and architecture. PMID:23913415

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

2013-08-02

266

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

PubMed Central

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

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

2013-01-01

267

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

PubMed

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

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

1998-08-01

268

Urinary excretion of adenosine deaminase binding protein in neonates treated with tobramycin  

Microsoft Academic Search

The potential tubulotoxicity of tobramycin and cefotaxim were assessed in neonates by measuring the urinary level of adenosine deaminase binding protein (ABP) and urinary a1-microglobulin and ß2-microglobulin. In a prospective study, 33 neonates who received tobramycin and cefotaxim for suspected neonatal sepsis were compared with 48 untreated newborns during the first 10 days of life. The urinary concentrations of ABP

Nader Gordjani; Rainer Burghard; Dirk Miiller; Helga Mathiii; Gunther Mergehenn; Jekabs U. Leititis; Matthias Brandis

1995-01-01

269

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

Microsoft Academic Search

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

Michael R. Blackburn; Rodney E. Kellems

2005-01-01

270

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

Microsoft Academic Search

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

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

2007-01-01

271

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

Microsoft Academic Search

Inherited deficiency of the enzyme adenosine deaminase (ADA) has been found in a significant proportion of patients with severe combined immunodeficiency disease and inherited defect generally characterized by a deficiency of both B and T cells. Two questions are central to understanding the pathophysiology of this disease: (1) at what stage or stages in lymphocyte development are the effects of

R BARTON

1985-01-01

272

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

Microsoft Academic Search

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

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

1991-01-01

273

Genotype is an important determinant of phenotype in adenosine deaminase deficiency  

Microsoft Academic Search

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

Michael S Hershfield

2003-01-01

274

Bilateral sensorineural deafness in adenosine deaminase-deficient severe combined immunodeficiency  

Microsoft Academic Search

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

Wendy Albuquerque; Hubert B. Gaspar

2004-01-01

275

Adenosine deaminase activity in serum and placenta of patients with anembryonic pregnancies and missed abortions  

Microsoft Academic Search

Aim: The aim of this study is to assess adenosine deaminase (ADA) activity in the serum and placenta of patients with missed abortions, anembryonic pregnan- cies, and voluntary abortions. Methods: Nine cases of anembryonic pregnancies and 21 cases of missed abor- tions (group I, n=30), and voluntary dilatation and curettage cases (group II, n=30) were included in this prospective study.

Irfan Kutlar; Fuat Aksoy; Oya Koyluoglu; Mete Gurol Ugur; Ozcan Balat; Mehmet Tarakcioglu

2005-01-01

276

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 ...

277

Targeting activation-induced cytidine deaminase prevents colon cancer development despite persistent colonic inflammation  

Microsoft Academic Search

Inflammatory bowel disease (IBD) is an important etiologic factor in the development of colorectal cancer. However, the mechanism underlying carcinogenesis through chronic inflammation is still unknown. Activation-induced cytidine deaminase (AID) is induced by the inflammation and involved in various human carcinogenesis via its mutagenic activity. In the current study, we investigated whether the inflammation\\/AID axis plays an integral role in

A Takai; H Marusawa; Y Minaki; T Watanabe; H Nakase; K Kinoshita; G Tsujimoto; T Chiba

2012-01-01

278

Immune Function in Mice Exposed to the Adenosine Deaminase Inhibitor 2'-Deoxycoformycin During Immune System Development.  

National Technical Information Service (NTIS)

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...

R. W. Luebke L. D. Lawson R. R. Rogers M. M. Riddle R. J. Smialowicz

1987-01-01

279

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

ERIC Educational Resources Information Center

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

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

2012-01-01

280

Expression of activation-induced cytidine deaminase in human hepatocytes via NF-?B signaling  

Microsoft Academic Search

Activation-induced cytidine deaminase (AID) is involved in somatic DNA alterations of the immunoglobulin gene for amplification of immune diversity. The fact that constitutive expression of AID in mice causes tumors in various organs, including lymphoid tissues and lungs, suggests the important role of the aberrant editing activity of AID on various tumor-related genes for carcinogenesis. AID expression, however, is restricted

Y Endo; H Marusawa; K Kinoshita; T Morisawa; T Sakurai; I-M Okazaki; K Watashi; K Shimotohno; T Honjo; T Chiba; T Chiba

2007-01-01

281

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

PubMed

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

Roszkowska, Anna; Klimek, Jerzy; Kaletha, Krystian

2007-12-30

282

Modification by liposomes of the adenosine triphosphate-activating effect on adenylate deaminase from pig heart.  

PubMed

Adenylate deaminase (AMP deaminase, EC 3.5.4.6) of a high substrate specificity was purified from pig heart by chromatography on cellulose phosphate. The enzyme shows a co-operative binding of AMP [h (Hill coefficient) 2.35, with SO.5 (half-saturating substrate concentration) 5mM]. ATP and ADP act as positive effectors, lowering h to 1.55 and SO.5 to 1 mM. The addition of liposomes (phospholipid bilayers) to ATP-activated or ADP-activated enzyme causes a further shift of the h value to 1.04 and SO.5 to 0.5 mM. For ATP-activated enzyme the addition of liposomes increases Vmax. by about 100%, and for ADP-activated enzyme by 50%. Liposomes have no effect on the kinetics of AMP deaminase in the absence of ATP and ADP, and neither do they influence the inhibitory effect of orthophosphate on heart muscle AMP deaminase. Metabolic implications of these findings are discussed. PMID:743213

Purzycka-Preis, J; Prus, E; Wo?niak, M; Zydowo, M

1978-11-01

283

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

EPA Science Inventory

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

284

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

NASA Astrophysics Data System (ADS)

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

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

2013-03-01

285

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

PubMed

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

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

2012-02-22

286

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

NASA Astrophysics Data System (ADS)

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

Bende, A.; Muntean, C. M.

2012-02-01

287

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

NASA Astrophysics Data System (ADS)

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

Sridhar, B.; Ravikumar, K.

2010-03-01

288

Nuclear quantum effect on the hydrogen-bonded structure of guanine–cytosine pair  

Microsoft Academic Search

The structure of Watson–Crick type guanine–cytosine (G–C) base pair has been studied by classical hybrid Monte Carlo (HMC)\\u000a and quantum path integral hybrid Monte Carlo (PIHMC) simulations on the semiempirical PM6 potential energy surface. For the\\u000a three NH?X hydrogen-bonded moieties, the intramolecular NH bonds are found systematically longer while the H?X distance shorter\\u000a in the PIHMC simulation than in the

Masashi Daido; Akihito Koizumi; Motoyuki Shiga; Masanori Tachikawa

289

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

NASA Astrophysics Data System (ADS)

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.

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

2013-04-01

290

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

PubMed

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

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

2009-12-22

291

Cloning and characterization of a plasmid encoded ACC deaminase from an indigenous Pseudomonas fluorescens FY32.  

PubMed

In addition to the characterized mechanisms responsible for many direct effects of plant growth promoting bacteria (PGPB) on plants, it has been suggested that a number of PGPB contain the enzyme ACC deaminase that catalyzes degradation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, into alpha-ketobutyrate and ammonia. As part of an effort to obtain an ACC deaminase encoding gene from a collection of soil samples, only one bacterial isolate, Pseudomonas fluorescens FY32 was capable of growing on ACC as a sole source of nitrogen. The ACC deaminase gene was amplified from the above isolate by polymerase chain reaction (PCR) giving an expected DNA fragment, 1017 bp. Sequence analysis of the fragment showed that it was highly homologous (94% and 98% identities at nucleotide and amino acid levels, respectively) to the previously characterized acdS gene from Pseudomonas sp. 6G5. Furthermore, fusion of the ACC deaminase ORF with lacZ gene resulted in the expression of active enzyme in Escherichia coli. In addition, further analyses revealed that the acdS gene was plasmid-encoded so that a large plasmid (pFY32) with almost 50 kb in size was identified from this bacterium. Furthermore, transfer of pFY32 into E. coli DH5alpha proved its ACC deaminase activity. This result was in accordance with previous reports suggesting horizontal transfer of the acdS gene. However, it needs more investigation to identify whether this pFY32 plasmid has undergone lateral gene transfer during the evolutionary process. PMID:20049599

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

2010-01-05

292

Phylogenetic analysis reveals a novel protein family closely related to adenosine deaminase.  

PubMed

Adenosine deaminase (ADA) is a well-characterized enzyme involved in the depletion of adenosine levels. A group of proteins with similarity to ADA, the adenosine deaminase-related growth factors (ADGF; known as CECR1 in vertebrates), has been described recently in various organisms. We have determined the phylogenetic relationships of various gene products with significant amino acid similarity to ADA using parsimony and Bayesian methods, and discovered a novel paralogue, termed ADA-like (ADAL). The ADGF proteins share a novel amino acid motif, "MPKG," within which the proline and lysine residues are also conserved in the ADAL and ADA subfamilies. The significance of this new domain is unknown, but it is located just upstream of two ADA catalytic residues, of which all eight are conserved among the ADGF and ADAL proteins. This conservation suggests that ADGF and ADAL may share the same catalytic function as ADA, which has been proven for some ADGF members. These analyses also revealed that some genes previously thought to be classic ADAs are instead ADAL or ADGFs. We here define the ADGF, ADAL, ADA, adenine deaminase (ADE), and AMP deaminase (AMPD) groups as subfamilies of the adenyl-deaminase family. The availability of genomic data for the members of this family allowed us to reconstruct the intron evolution within the phylogeny and strengthen the introns-late hypothesis of the synthetic introns theory. This study shows that ADA activity is clearly more complex than once thought, perhaps involving a delicately balanced pattern of temporal and spatial expression of a number of paralogous proteins. PMID:16245011

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

2005-10-20

293

Transformation of Yeast  

Microsoft Academic Search

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

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

1978-01-01

294

Population Growth in Yeasts  

NSDL National Science Digital Library

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

Engineering K-Ph.d. Program

295

Cytosine ribose flexibility in DNA: a combined NMR 13C spin relaxation and molecular dynamics simulation study.  

PubMed

Using (13)C spin relaxation NMR in combination with molecular dynamic (MD) simulations, we characterized internal motions within double-stranded DNA on the pico- to nano-second time scale. We found that the C-H vectors in all cytosine ribose moieties within the Dickerson-Drew dodecamer (5'-CGCGAATTCGCG-3') are subject to high amplitude motions, while the other nucleotides are essentially rigid. MD simulations showed that repuckering is a likely motional model for the cytosine ribose moiety. Repuckering occurs with a time constant of around 100 ps. Knowledge of DNA dynamics will contribute to our understanding of the recognition specificity of DNA-binding proteins such as cytosine methyltransferase. PMID:18579564

Duchardt, Elke; Nilsson, Lennart; Schleucher, Jürgen

2008-06-25

296

Nucleic Acid Amplification in Yeast.  

National Technical Information Service (NTIS)

Plasmid DNA from single yeast colonies was efficiently amplified using rolling circle amplification (RCA). The amplified DNA was directly used for restriction digestion, DNA sequencing, and yeast transformation. The RCA of plasmid DNA from single yeast co...

W. Farmerie W. Y. Song X. Ding

2004-01-01

297

The Role of a Common Adenosine Monophosphate Deaminase (AMPD)-1 Polymorphism in Outcomes of Ischemic and Nonischemic Heart Failure  

Microsoft Academic Search

BackgroundA common variant of the adenosine monophosphate deaminase (AMPD)-1 gene (C34T) results in enzymatic inactivity and may increase adenosine in cardiac muscle and confer cardioprotection through ischemic preconditioning.

Matthew J. Kolek; John F. Carlquist; Surai Thaneemit-Chen; Laura C. Lazzeroni; Bryant M. Whiting; Benjamin D. Horne; Joseph B. Muhlestein; Philip Lavori; Jeffrey L. Anderson

2005-01-01

298

Comparative immunologic and kinetic evaluation of AMP-deaminase isolated from normal human liver and hepatocellular carcinoma (HCC).  

PubMed

In the present paper physico-chemical properties of AMP-deaminase purified from human liver neoplasm-hepatocellular carcinoma (HCC) were investigated and compared with these obtained for the enzyme from normal, unaffected tissue. PMID:15571293

Szydlowska, M; Sledzinski, Z; Krzyzanowski, M; Nagel-Starczynowska, G; Kaletha, K

2004-10-01

299

L-Serine deaminase activity is induced by exposure of Escherichia coli K-12 to DNA-damaging agents.  

PubMed

The synthesis of L-serine deaminase in Escherichia coli K-12 was induced after exposure of cells to a variety of DNA-damaging agents, including UV irradiation, nalidixic acid, and mitomycin C. Synthesis was also induced during growth at high temperature. A mutant constitutive for SOS functions showed an elevated level of L-serine deaminase activity. The response to DNA-damaging agents thus may be mediated via the SOS system. PMID:6813312

Newman, E B; Ahmad, D; Walker, C

1982-11-01

300

Moonlighting Proteins in Yeasts  

PubMed Central

Proteins able to participate in unrelated biological processes have been grouped under the generic name of moonlighting proteins. Work with different yeast species has uncovered a great number of moonlighting proteins and shown their importance for adequate functioning of the yeast cell. Moonlighting activities in yeasts include such diverse functions as control of gene expression, organelle assembly, and modification of the activity of metabolic pathways. In this review, we consider several well-studied moonlighting proteins in different yeast species, paying attention to the experimental approaches used to identify them and the evidence that supports their participation in the unexpected function. Usually, moonlighting activities have been uncovered unexpectedly, and up to now, no satisfactory way to predict moonlighting activities has been found. Among the well-characterized moonlighting proteins in yeasts, enzymes from the glycolytic pathway appear to be prominent. For some cases, it is shown that despite close phylogenetic relationships, moonlighting activities are not necessarily conserved among yeast species. Organisms may utilize moonlighting to add a new layer of regulation to conventional regulatory networks. The existence of this type of proteins in yeasts should be taken into account when designing mutant screens or in attempts to model or modify yeast metabolism.

Gancedo, Carlos; Flores, Carmen-Lisset

2008-01-01

301

Expression and characterization of a second L-amino acid deaminase isolated from Proteus mirabilis in Escherichia coli.  

PubMed

L-amino acid deaminases catalyze the deamination of natural L-amino acids. Two types of L-amino acid deaminase have been identified in Proteus species. One exhibits high levels of activity toward a wide range of aliphatic and aromatic L-amino acids, typically L-phenylalanine, whereas the other acts on a relatively narrow range of basic L-amino acids, typically L-histidine. In this study, we cloned, expressed, and characterized a second amino acid deaminase, termed Pm1, from P. mirabilis KCTC 2566. Homology alignment of the deduced amino acid sequence of Pm1 demonstrated that the greatest similarity (96%) was with the L-amino acid deaminase (LAD) of P. vulgaris, and that homology with Pma was relatively low (72%). Also, similar to LAD, Pm1 was most active on L-histidine, indicating that Pm1 belongs to the second type of amino acid deaminase. In agreement with this conclusion, the V(max) and K(m) values of Pm1 were 119.7 (?g phenylpyruvic acid/mg/min) and 31.55 mM phenylalanine, respectively, values lower than those of Pma. The Pml deaminase will be very useful industrially in the preparation of commercially valuable materials including urocanic acid and ?-oxoglutarate. PMID:21298676

Baek, Jin-Oh; Seo, Jeong-Woo; Kwon, Ohsuk; Seong, Su-Il; Kim, Ik-Hwan; Kim, Chul Ho

2011-02-07

302

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

Microsoft Academic Search

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

Rochelle Hirschhorn; Nicholas Beratis; Fred S. Rosen

1976-01-01

303

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  

Microsoft Academic Search

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,

Yan-feng Zhang; Lin-yan He; Zhao-jin Chen; Qing-ya Wang; Meng Qian; Xia-fang Sheng

2011-01-01

304

Characterization of lead-resistant and ACC deaminase-producing endophytic bacteria and their potential in promoting lead accumulation of rape  

Microsoft Academic Search

Forty-nine lead (Pb)-resistant endophytic bacteria were isolated from metal-tolerant Commelina communis plants grown on lead and zinc mine tailing, of which, seven 1-aminocyclopropane-1-carboxylate (ACC) deaminase-producing endophytic bacteria were initially obtained and characterized with respect to heavy metal resistance and production of ACC deaminase, indole-3-acetic acid (IAA) as well as siderophores. Two isolates (Q2BJ2 and Q2BG1) showing higher ACC deaminase activity

Yan-feng Zhang; Lin-yan He; Zhao-jin Chen; Wen-hui Zhang; Qing-ya Wang; Meng Qian; Xia-fang Sheng

2011-01-01

305

Significant strength of charged DNA-protein ?-? interactions: a preliminary study of cytosine.  

PubMed

The present work characterized the preferred gas-phase structure and optimum interaction energy of both parallel stacked and perpendicular T-shaped dimers between cytosine (C), as a representative nucleobase, and aspartic/glutamic acid (DE), aspartate/glutamate (DE(-)) or arginine (R(+)), using detailed M06-2X/6-31+G(d,p) potential energy surface scans as a function of the relative monomer orientation. Through comparison to previous literature on the ?-? interactions between the DNA nucleobases and the aromatic amino acid residues, this work will allow for comparisons between DNA-protein interactions involving aromatic and acyclic R-side chains, as well as comparisons of the relative geometric dependence and magnitude of ?-? (C:DE), ?cation-? (C:R(+)), and ?anion-? (C:DE(-)) interactions. Our results show that the preferred relative monomer orientation is highly dependent on the monomer composition and charge, and is dictated by electrostatic-driven interactions. More importantly, for the first time, we report that the ?-? interactions between cytosine and (neutral) aspartic/glutamic acid are up to approximately -40 kJ mol(-1), while the ?cation-? or ?anion-? interactions between cytosine and arginine or aspartate/glutamate are up to approximately -90 and -99 kJ mol(-1), respectively. An extensive investigation of the effects of the computational methodology implemented, including comparisons to detailed CCSD(T)/CBS potential energy surfaces and interaction energies, supports the use of M06-2X, as well as ?B97X-D, to study DNA-protein ?-? interactions of varying composition and charge. Most importantly, the CCSD(T)/CBS results verify the strong nature of these DNA-protein ?-? interactions, as well as the unique nature of the ?cation-? and ?anion-? counterparts. Therefore, our results emphasize that a wide variety of different types of noncovalent interactions between both cyclic and acyclic ?-containing components can significantly contribute to the stability of DNA-protein complexes and likely play a larger role in biology than currently accepted. PMID:23991905

Wells, Rachael A; Kellie, Jennifer L; Wetmore, Stacey D

2013-08-30

306

Tissue culture-induced transpositional activity of mPing is correlated with cytosine methylation in rice  

Microsoft Academic Search

BACKGROUND: mPing is an endogenous MITE in the rice genome, which is quiescent under normal conditions but can be induced towards mobilization under various stresses. The cellular mechanism responsible for modulating the activity of mPing remains unknown. Cytosine methylation is a major epigenetic modification in most eukaryotes, and the primary function of which is to serve as a genome defense

Frédéric Ngezahayo; Chunming Xu; Hongyan Wang; Lily Jiang; Jinsong Pang; Bao Liu

2009-01-01

307

Post-injury stress signals alter epigenetic profile of cytosine methylation in the proviral genome of endogenous retroviruses  

Microsoft Academic Search

The majority of epigenetic methylation events at cytosine residues of the genome are reported to occur in transposable elements, as a result, it contributes to genome stability by repressing their transposition activity. Our recent studies demonstrated that the expression of certain murine endogenous retroviruses (MuERVs), a family of retrotransposons, is modulated in the liver after burn injury and sepsis. In

Sophia Chiu; Karen Hsu; David G. Greenhalgh; Kiho Cho

2010-01-01

308

Highly selective binding of naphthyridine with a trifluoromethyl group to cytosine opposite an abasic site in DNA duplexes.  

PubMed

We report on highly selective binding of a naphthyridine derivative with a trifluoromethyl group to cytosine opposite an abasic site in DNA duplexes; the binding-induced fluorescence quenching is applicable to the analysis of a C-related single-base mutation in DNAs amplified by PCR. PMID:22526917

Sato, Yusuke; Zhang, Yushuang; Seino, Takehiro; Sugimoto, Takashi; Nishizawa, Seiichi; Teramae, Norio

2012-04-23

309

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

Microsoft Academic Search

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

Michael C. Golding; Mark E. Westhusin

2003-01-01

310

Electron transport through 5-substituted pyrimidines in DNA: electron affinities of uracil and cytosine derivatives differently affect the apparent efficiencies.  

PubMed

We investigated excess electron transport (EET) in DNA containing cytosine derivatives. By arranging the derivatives according to their electron affinities, the apparent EET efficiency was successfully regulated. Unexpectedly, however, providing gradients of electron affinity by inserting 5-fluorocytosine did not always enhance EET. PMID:24061333

Ito, Takeo; Kurihara, Ryohsuke; Utsumi, Nihiro; Hamaguchi, Yuta; Tanabe, Kazuhito; Nishimoto, Sei-Ichi

2013-10-01

311

The biochemistry of activation-induced deaminase and its physiological functions.  

PubMed

Activation-induced deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) by inducing mutations and double-strand breaks at the immunoglobulin (Ig) locus in B cells. AID converts deoxycytidine (dC) to deoxyuridine (dU) in single-stranded DNA (ssDNA). This deamination reaction is enzymatically straightforward, but ultimately results in diverse biological consequences. Here, we review the enzymatic features of AID, such as the parameters that govern substrate binding and catalysis. We discuss how these properties of AID relate to secondary antibody diversification processes and the manners in which they may regulate the targeting of AID to various loci. Based on the current data on AID and other related deaminases, we propose a 3-dimensional structure for AID and how this model provides clues into AID's catalytic mechanism. PMID:22695318

Larijani, Mani; Martin, Alberto

2012-06-12

312

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

PubMed

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

Nowarski, Roni; Kotler, Moshe

2013-04-18

313

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

PubMed

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

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

2009-10-11

314

[Inhibition of adenosine deaminase activity by drugs influencing the cardiovascular system].  

PubMed

Adenosine deaminase (ADA) is an unique enzyme which catalyzes conversion of adenosine and 2'-deoxyadenosine to inosine and 2'-deoxyinosine respectively. One of physiological roles of this enzyme is modulation of its substrate--adenosine concentration (both intracellular and extraectocellular). In presented work the influence of acetylsalicylic acid, metoprolol, simvastatin, isosorbide mononitrate and molsidomine on total activity of adenosine deaminase and its isoenzymes--ADA1 and ADA2 in vivo was studied. We have affirmed that simvastatin decreased of tADA activity by 50%, acetylsalicylic acid by 34%, metoprolol by 29.1% and isosorbide mononitrate by 19.3%. Only after molsidomine administration were no significant changes in ADA activity observed. The result showed that the decline of ADA activity was mainly due to marked decrease in ADA2 isoenzyme. PMID:15508788

Kopff, Maria; Kowalczyk, Edward; Kopff, Anna

2004-06-01

315

Formulation, quality control and shelf life of the experimental cytostatic drug cyclopentenyl cytosine.  

PubMed

This paper describes the formulation and quality control of an aqueous sterilized formulation of the experimental cytostatic drug cyclopentenyl cytosine (CPEC) to be used in Phase I/II clinical trials. The raw drug substance was extensively tested. A High Pressure Liquid Chromotography (HPLC) method was validated for the quality control of the formulated product. The aqueous formulation was found to be stable for at least 2 years at 2-8 degrees C. Sterilization (15 min at 121 degrees C) showed no influence on drug stability. The results show that CPEC can be formulated in an aqueous solution. The described HPLC method is a useful tool in the pharmaceutical quality control. PMID:16638688

Schimmel, Kirsten; Guchelaar, Henk-Jan; van Kan, Erik

2006-04-01

316

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

NASA Astrophysics Data System (ADS)

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

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

2007-05-01

317

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

PubMed

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

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

2013-06-05

318

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

PubMed Central

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.

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

2013-01-01

319

Dependence on temperature and guanine-cytosine content of bubble length distributions in DNA.  

PubMed

We present numerical results on the temperature dependence of the distribution of bubble lengths in DNA segments of various guanine-cytosine (GC) concentrations. Base-pair openings are described by the Peyrard-Bishop-Dauxois model and the corresponding thermal equilibrium distributions of bubbles are obtained through Monte Carlo calculations for bubble sizes up to the order of a hundred base pairs. The dependence of the parameters of bubble length distribution on temperature and the GC content is investigated. We provide simple expressions which approximately describe these relations. The variation of the average bubble length is also presented. We find a temperature dependence of the exponent c that appears in the distribution of bubble lengths. If an analogous dependence exists in the loop entropy exponent of real DNA, it may be relevant to understand overstretching in force-extension experiments. PMID:19548765

Kalosakas, G; Ares, S

2009-06-21

320

Hypomethylation of cytosine residues in cold-sensitive regions of Cestrum strigilatum (Solanaceae)  

PubMed Central

Heterochromatin comprises a fraction of the genome usually with highly repeated DNA sequences and lacks of functional genes. This region can be revealed by using Giemsa C-banding, fluorochrome staining and cytomolecular tools. Some plant species are of particular interest through having a special type of heterochromatin denominated the cold-sensitive region (CSR). Independent of other chromosomal regions, when biological materials are subjected to low temperatures (about 0 °C), CSRs appear slightly stained and decondensed. In this study, we used Cestrum strigilatum (Solanaceae) to understand some aspects of CSR condensation associated with cytosine methylation levels, and to compare the behavior of different heterochromatin types of this species, when subjected to low temperatures.

Guarido, Paula Carolina Paes; de Paula, Adriano Alves; da Silva, Carlos Roberto Maximiano; Rodriguez, Carmen; Vanzela, Andre Luis Laforga

2012-01-01

321

Cytosine methylation inhibits replication of African cassava mosaic virus by two distinct mechanisms.  

PubMed Central

Extrachromosomally replicating viral DNA is usually free of cytosine methylation and viral templates methylated in vitro are poor substrates when used in replication assays. We have investigated the mechanism of inhibition of viral replication by DNA methylation using as a model the DNA A of African cassava mosaic virus. We have constructed two component helper systems which allow for separation of the transcriptional inhibition of viral genes necessary for replication from replication inhibition due to altered interaction between the replication complex and methylated viral DNA. Our results suggest that methylation-mediated reduction of viral replication is due to both repression mechanisms and that this provides two independent selection pressures for the maintenance of methylation-free replicons in infected cells. Images

Ermak, G; Paszkowski, U; Wohlmuth, M; Scheid, O M; Paszkowski, J

1993-01-01

322

Specific Activity of Phenylalanine Deaminase in Extracts of the Proteus-Providence Group  

Microsoft Academic Search

THE possession of a phenylalanine deaminase which converts phenylalanine to phenylpyruvic acid is one of a combination of five properties unique for the Proteus-Providence group of organisms1. Of a total of 185 P. hauseri (P. mirabilis + P. vulgaris), 155 P. morganii, 29 P. rettgeri and 239 Providence strains qualitatively examined for the presence of this enzyme2-4, all but two

J. A. Smit

1966-01-01

323

Purification of Adenosine Deaminase from Chicken-Egg Yolk by Affinity Column Chromatography  

Microsoft Academic Search

Adenosine deaminase (adenosine aminohydrolase; E.C. 3.5.4.4) has been purified 4686-fold from egg yolk. The procedure developed was used to isolate the enzyme from eight chicken eggs. An easily prepared affinity column employing purine riboside was used as the final step in the purification. The method developed permits the rapid isolation and a high recovery of the protein. The specific activity

R. Lopez; F. Cabre; R. Franco; H. Cascante; E. I. Canela

1990-01-01

324

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

Microsoft Academic Search

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

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

325

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

Microsoft Academic Search

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

Patricia Benveniste; Amos Cohen

1995-01-01

326

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

Microsoft Academic Search

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

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

1992-01-01

327

Cognitive and behavioral abnormalities in adenosine deaminase deficient severe combined immunodeficiency  

Microsoft Academic Search

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

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

2001-01-01

328

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

PubMed

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

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

2012-02-15

329

Hematopoietic stem cell gene therapy for adenosine deaminase deficient-SCID  

Microsoft Academic Search

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

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

2009-01-01

330

Discovery of Activation?Induced Cytidine Deaminase, the Engraver of Antibody Memory  

Microsoft Academic Search

Discovery of activation?induced cytidine deaminase (AID) paved a new path to unite two genetic alterations induced by antigen stimulation; class switch recombination (CSR) and somatic hypermutation (SHM). AID is now established to cleave specific target DNA and to serve as engraver of these genetic alterations. AID of a 198?residue protein has four important domains: nuclear localization signal and SHM?specific region

Masamichi Muramatsu; Hitoshi Nagaoka; Reiko Shinkura; Nasim A. Begum; Tasuku Honjo

2007-01-01

331

Molecularly cloned mammalian glucosamine-6- phosphate deaminase localizes to transporting epithelium and lacks oscillin activity  

Microsoft Academic Search

Glucosamine-6-phosphate deaminase (GNPDA) catalyzes the conversion of glucosamine-6- phosphate to fructose-6-phosphate, a reaction that under physiological conditions proceeds to the for- mation of fructose-6-phosphate. Though first identi- fied in mammalian tissues in 1956, the enzyme has not previously been molecularly characterized in mammalian tissues, although a bacterial GNPDA has been cloned. Recently, a protein displaying similarity to bacterial GNPDA was

HERMAN WOLOSKER; DOUGLAS KLINE; YING BIAN; Å SETH BLACKSHAW; ANDREW M. CAMERON; THOMAS J. FRALICH; RONALD L. SCHNAAR; SOLOMON H. SNYDER

332

Expression of activation-induced cytidine deaminase in human B-cell non-Hodgkin lymphomas  

Microsoft Academic Search

Activation-induced cytidine deaminase (AID) induces somatic hypermutation (SHM), class switch recombination (CSR), and immunoglobulin gene conversion in B-lymphocytes. Here we report for the first time the expression of AID in healthy human B-lymphocytes and in B-cell non- Hodgkin lymphomas (B-NHL). AID mRNA expression in humans is restricted to the CD19CD38IgD germinal center cells, namely the CD19CD38CD44 centro- blasts. After in

Jobst Greeve; Antje Philipsen; Kristina Krause; Wolfram Klapper; Klaus Heidorn; Brian E. Castle; Joe Janda; Kenneth B. Marcu; Reza Parwaresch

2003-01-01

333

Promotion of plant growth by ACC deaminase-producing soil bacteria  

Microsoft Academic Search

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

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

2007-01-01

334

Promotion of plant growth by ACC deaminase-producing soil bacteria  

Microsoft Academic Search

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

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

335

Deaminase-independent inhibition of HIV1 reverse transcription by APOBEC3G  

Microsoft Academic Search

APOBEC3G (A3G), a host protein that inhibits HIV-1 reverse transcription and replication in the absence of Vif, displays cytidine deaminase and single- stranded (ss) nucleic acid binding activities. HIV-1 nucleocapsid protein (NC) also binds nucleic acids and has a unique property, nucleic acid chaperone activity, which is crucial for efficient reverse tran- scription. Here we report the interplay between A3G,

Yasumasa Iwatani; Denise S. B. Chan; F. Wang; Kristen Stewart Maynard; Wataru Sugiura; Angela M. Gronenborn; Ioulia Rouzina; Mark C. Williams; Karin Musier-Forsyth; Judith G. Levin

2007-01-01

336

Activation-induced deaminase, AID, is catalytically active as a monomer on single-stranded DNA  

Microsoft Academic Search

Hypermutation and class switch recombination of immunoglobulin genes are antigen-activated mechanisms triggered by AID, a cytidine deaminase. AID deaminates cytidine residues in the DNA of the variable and the switch regions of the immunoglobulin locus. The resulting uracil induces error-prone DNA synthesis in the case of hypermutation or DNA breaks that activate non-homologous recombination in the case of class switch

Sukhdev S. Brar; Elizabeth J. Sacho; Ingrid Tessmer; Deborah L. Croteau; Dorothy A. Erie; Marilyn Diaz

2008-01-01

337

Myoadenylate deaminase deficiency: Successful symptomatic therapy by high dose oral administration of ribose  

Microsoft Academic Search

Summary A 55 years old patient suffering from exercise-induced muscle pain and stiffness due to primary myoadenylate deaminase deficiency has been successfully treated with D-ribose since 1984: single doses of 4 grams administered at the beginning of exercise prevented the symptoms completely; on continuation of exercise this dose had to be repeated all 10–30 min. Total doses of 50–60 g

N. Zöllner; S. Reiter; M. Gross; D. Pongratz; C. D. Reimers; K. Gerbitz; I. Paetzke; T. Deufel; G. Hübner

1986-01-01

338

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

Microsoft Academic Search

We discovered a d-phenylserine deaminase that catalyzed the pyridoxal 5?-phosphate (PLP)-dependent deamination reaction from d-threo-phenylserine to phenylpyruvate in newly isolated Arthrobacter sp. TKS1. The enzyme was partially purified, and its N-terminal amino acid sequence was analyzed. Based on the sequence information,\\u000a the gene encoding the enzyme was identified and expressed in Escherichia coli. The expressed protein was purified to homogeneity

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

2011-01-01

339

Direct Association of Adenosine Deaminase with a T Cell Activation Antigen, CD26  

Microsoft Academic Search

CD26, the T cell activation molecule dipeptidyl peptidase IV (DPPIV), associates with a 43-kilodalton protein. Amino acid sequence analysis and immunoprecipitation studies demonstrated that this 43-kilodalton protein was adenosine deaminase (ADA). ADA was coexpressed with CD26 on the Jurkat T cell lines, and an in vitro binding assay showed that the binding was through the extracellular domain of CD26. ADA

Junichi Kameoka; Toshiaki Tanaka; Yoshihisa Nojima; Stuart F. Schlossman; Chikao Morimoto

1993-01-01

340

Two tissue-specific factors bind the erythroid promoter of the human porphobilinogen deaminase gene  

Microsoft Academic Search

We have studied the erythroid-specific promoter of the human gene coding for Porphobilinogen Deaminase (PBGD) by DNaseI footprinting, gel retardation and methylation interference assays. We show that this promoter, which is inducible during MEL cell differentiation, contains three binding sites for the erythroid-specific factor NF-E1 and one site for a second erythroid-specific factor, which we name NF-E2. NF-E1 is a

Vincent Mignotte; Lee Wall; Boer de E; Frank Grosveld; Paul-Henri Romeo

1989-01-01

341

Mutations in Activation-Induced Cytidine Deaminase in Patients with Hyper IgM Syndrome  

Microsoft Academic Search

Recent studies have shown that mutations in a newly described RNA editing enzyme, activation-induced cytidine deaminase (AID), can cause an autosomal recessive form of hyper IgM syndrome. To determine the relative frequency of mutations in AID, we evaluated a group of 27 patients with hyper IgM syndrome who did not have defects in CD40 ligand and 23 patients with common

Yoshiyuki Minegishi; Aubert Lavoie; Charlotte Cunningham-Rundles; Pierre-Michel Bédard; Jacques Hébert; Louise Côté; Kazuo Dan; Debra Sedlak; Rebecca H. Buckley; Alain Fischer; Anne Durandy; Mary Ellen Conley

2000-01-01

342

Myoadenylate deaminase deficiency: absence of correlation with exercise intolerance in 452 muscle biopsies  

Microsoft Academic Search

A histochemical assay was routinely performed of myoadenylate deaminase (MAD) in muscle biopsy specimens. MAD was absent in 13 cases, i.e. 2.9% of the specimens. In 10 cases the deficiency was confirmed biochemically. The diagnoses in the 13 patients were: polyneuropathy (n=5), infantile spinal muscular atrophy (n=3), congenital myopathy with type 2 fibre atrophy, facioscapulohumeral myopathy, polymyositis, myotonic dystrophy and

Rudolf Mercelis; Jean-Jacques Martin; Thierry de Barsy; Georges Van den Berghe

1987-01-01

343

Rat Harderian Gland Porphobilinogen Deaminase: Characterization Studies and Regulatory Action of Protoporphyrin IX  

Microsoft Academic Search

Properties of purified porphobilinogen deaminase (PBG-D; EC 4.3.1.8) from rat harderian gland are here presented. The enzyme behaves as a monomer ofMr38 ± 2 kDa and is optimally active at pH 8.0–8.2. Its activation energy, determined by an Arrhenius plot, is 76.1 kJ\\/mol. Initial velocity studies showed a linear progress curve for uroporphyringen I formation and a hyperbolic dependence of

Carina A. Cardalda; Adela Ana Juknat; Fernando G. Princ; Alcira Batlle

1997-01-01

344

Kinetic behaviour and allosteric regulation of human deoxycytidylate deaminase derived from leukemic cells  

Microsoft Academic Search

Deoxycytidylate deaminase has been highly purified (1232-fold) from human leukemia CCRF-CEM cells. The native molecular weight of the enzyme is 108 000 and subunit molecular weight 50 500, suggesting that the native enzyme exists as a dimer. The enzyme exhibits a sigmoidal initial velocity vs substrate concentration curve and is regulated by allosteric effectors, dCTP and TTP. The curve relating

Peter H. Ellims; Ann Y. Kao; Bruce A. Chabner

1983-01-01

345

Solubility-based genetic screen identifies RING finger protein 126 as an E3 ligase for activation-induced cytidine deaminase.  

PubMed

Protein-protein interactions are typically identified by either biochemical purification coupled to mass spectrometry or genetic approaches exemplified by the yeast two-hybrid assay; however, neither assay works well for the identification of cofactors for poorly soluble proteins. Solubility of a poorly soluble protein is thought to increase upon cofactor binding, possibly by masking otherwise exposed hydrophobic domains. We have exploited this notion to develop a high-throughput genetic screen to identify interacting partners of an insoluble protein fused to chloramphenicol acetyltransferase by monitoring the survival of bacteria in the presence of a drug. In addition to presenting proof-of-principle experiments, we apply this screen to activation-induced cytidine deaminase (AID), a poorly soluble protein that is essential for antibody diversification. We identify a unique cofactor, RING finger protein 126 (RNF126), verify its interaction by traditional techniques, and show that it has functional consequences as RNF126 is able to ubiquitylate AID. Our results underpin the value of this screening technique and suggest a unique form of AID regulation involving RNF126 and ubiquitylation. PMID:23277564

Delker, Rebecca K; Zhou, Yanjiao; Strikoudis, Alexandros; Stebbins, C Erec; Papavasiliou, F Nina

2012-12-31

346

Genetic Improvement of Baker's Yeasts  

Microsoft Academic Search

Yeasts have been used for many thousands of years to produce leavened bread. Nowadays the production of baker's yeast biomass represents a highly competitive multi-billion dollar global industry. The environmental conditions that prevail during manufacture and application of baker's yeasts, coupled with the sheer variety of bread making processes and recipes used around the world, place considerable demands on yeasts.

Paul V. Attfield; Philip J. L. Bell

2003-01-01

347

TRP channels in yeast.  

PubMed

Microbes have made numerous contributions to the study of biology and medicine. Those contributions also include many original discovery's in the study of ion channels often thought as the province of neuroscientists or cardiophysiologists. Yeast have long been used as a model organism and TRP channel genes and their transmembrane products touted as the "vanguards of the sensory system" can be identified in the genomes of many yeasts. This article aims to review the study of these TRP channels in yeast their discovery, electrophysiological properties and physiological function. PMID:21290303

Kaleta, Marta; Palmer, Christopher

2011-01-01

348

Proton-coupled hole transfer in X-irradiated doped crystalline cytosine.H2O.  

PubMed

Following exposure to X-irradiation at low temperatures, the main reactions taking place in single crystals of cytosine monohydrate doped with minute amounts of 2-thiocytosine are hole transfer (HT) from the electron-loss centers to the dopant and recombination of oxidation and reduction products, assumedly by electron transfer. A huge deuterium kinetic isotope effect (KIE; >102-103) at 100 K, together with the kinetic curves obtained and density functional theory (DFT) calculations of equilibrium energy changes, indicates that these reactions proceed through a concerted proton-coupled electron/hole transfer where the proton transfer occurs between hydrogen-bonded cytosine molecules. The temperature dependence of these reaction rates between 10 and 150 K in normal and partially deuterated samples was investigated by monitoring the growth and decay of the various radical species over time using electron paramagnetic resonance (EPR) spectroscopy. By assuming a random distribution of the hole donors and acceptors in the crystals, the data are consistent with an exponential distance-dependent rate, giving a distance decay constant (beta) around 1 A-1 for the HT, which indicates that a long-range single-step superexchange mechanism mediates the charge transfer. The reactions undergo a transition from a slow, weakly temperature-dependent rate to an Arrhenius-type rate at 40-50 K, presumably being activated by excitation of low-frequency intermolecular vibrations that couple to the process. Below this transition temperature, the transfer probability might be dominated by temperature-independent nuclear tunneling. A similar beta value in both temperature regions suggests that hopping is not activated. PMID:18341308

Krivokapi?, André; Herak, Janko N; Sagstuen, Einar

2008-03-15

349

Structural basis for the growth factor activity of human adenosine deaminase ADA2.  

PubMed

Two distinct adenosine deaminases, ADA1 and ADA2, are found in humans. ADA1 has an important role in lymphocyte function and inherited mutations in ADA1 result in severe combined immunodeficiency. The recently isolated ADA2 belongs to the novel family of adenosine deaminase growth factors (ADGFs), which play an important role in tissue development. The crystal structures of ADA2 and ADA2 bound to a transition state analogue presented here reveal the structural basis of the catalytic/signaling activity of ADGF/ADA2 proteins. In addition to the catalytic domain, the structures discovered two ADGF/ADA2-specific domains of novel folds that mediate the protein dimerization and binding to the cell surface receptors. This complex architecture is in sharp contrast with that of monomeric single domain ADA1. An extensive glycosylation and the presence of a conserved disulfide bond and a signal peptide in ADA2 strongly suggest that ADA2, in contrast to ADA1, is specifically designed to act in the extracellular environment. The comparison of catalytic sites of ADA2 and ADA1 demonstrates large differences in the arrangement of the substrate-binding pockets. These structural differences explain the substrate and inhibitor specificity of adenosine deaminases and provide the basis for a rational design of ADA2-targeting drugs to modulate the immune system responses in pathophysiological conditions. PMID:20147294

Zavialov, Anton V; Yu, Xiaodi; Spillmann, Dorothe; Lauvau, Grégoire; Zavialov, Andrey V

2010-02-09

350

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

PubMed Central

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

Richter, G; Fischer, M; Krieger, C; Eberhardt, S; Luttgen, H; Gerstenschlager, I; Bacher, A

1997-01-01

351

Structural Basis for the Growth Factor Activity of Human Adenosine Deaminase ADA2*?  

PubMed Central

Two distinct adenosine deaminases, ADA1 and ADA2, are found in humans. ADA1 has an important role in lymphocyte function and inherited mutations in ADA1 result in severe combined immunodeficiency. The recently isolated ADA2 belongs to the novel family of adenosine deaminase growth factors (ADGFs), which play an important role in tissue development. The crystal structures of ADA2 and ADA2 bound to a transition state analogue presented here reveal the structural basis of the catalytic/signaling activity of ADGF/ADA2 proteins. In addition to the catalytic domain, the structures discovered two ADGF/ADA2-specific domains of novel folds that mediate the protein dimerization and binding to the cell surface receptors. This complex architecture is in sharp contrast with that of monomeric single domain ADA1. An extensive glycosylation and the presence of a conserved disulfide bond and a signal peptide in ADA2 strongly suggest that ADA2, in contrast to ADA1, is specifically designed to act in the extracellular environment. The comparison of catalytic sites of ADA2 and ADA1 demonstrates large differences in the arrangement of the substrate-binding pockets. These structural differences explain the substrate and inhibitor specificity of adenosine deaminases and provide the basis for a rational design of ADA2-targeting drugs to modulate the immune system responses in pathophysiological conditions.

Zavialov, Anton V.; Yu, Xiaodi; Spillmann, Dorothe; Lauvau, Gregoire; Zavialov, Andrey V.

2010-01-01

352

Yeast infections (image)  

MedlinePLUS

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

353

Mutant yeast on drugs  

Microsoft Academic Search

Analyzing drug-treated and mutant yeast cells with the new tools of genomics enables the identification of drug targets and should improve the odds of developing useful therapeutics (pages 1293–1301).

David J. Lockhart

1998-01-01

354

[Aspects of yeast biodiversity].  

PubMed

Yeast biodiversity represents a dynamic scientific domain characterized by permanent emerging theories and accumulation of new data. Identification of genome structure for a number of yeast species and elucidation of regulatory pathways for species-specific metabolic networks, lead to development of numerous applications of yeasts in industry, biotechnology, therapeutics and bioremediation. The studies of the scientific community were long time focused on Saccharomyces cerevisae due mainly to its use in food production. Therefore, the species belonging to Saccharomyces genus became reference points for genomics and biodiversity studies. During last decades there is a growing interest for yeast species able to produce biomass by assimilating or degrading various compounds such as methanol, hydrocarbons, wood hydrolisates and other residues or by-products from different industries. PMID:23745219

Csutak, Ortansa; Vassu, Tatiana

355

Yeast expression platforms  

Microsoft Academic Search

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

Erik Böer; Gerhard Steinborn; Gotthard Kunze; Gerd Gellissen

2007-01-01

356

Nitrile Metabolizing Yeasts  

NASA Astrophysics Data System (ADS)

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.

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

357

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

PubMed

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

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

2008-09-30

358

Expression of an Exogenous 1-Aminocyclopropane-1-Carboxylate Deaminase Gene in Sinorhizobium meliloti Increases Its Ability To Nodulate Alfalfa  

PubMed Central

1-Aminocyclopropane-1-carboxylate (ACC) deaminase has been found in various plant growth-promoting rhizobacteria, including rhizobia. This enzyme degrades ACC, the immediate precursor of ethylene, and thus decreases the biosynthesis of ethylene in higher plants. The ACC deaminase of Rhizobium leguminosarum bv. viciae 128C53K was previously reported to be able to enhance nodulation of peas. The ACC deaminase structural gene (acdS) and its upstream regulatory gene, a leucine-responsive regulatory protein (LRP)-like gene (lrpL), from R. leguminosarum bv. viciae 128C53K were introduced into Sinorhizobium meliloti, which does not produce this enzyme, in two different ways: through a plasmid vector and by in situ transposon replacement. The resulting ACC deaminase-producing S. meliloti strains showed 35 to 40% greater efficiency in nodulating Medicago sativa (alfalfa), likely by reducing ethylene production in the host plants. Furthermore, the ACC deaminase-producing S. meliloti strain was more competitive in nodulation than the wild-type strain. We postulate that the increased competitiveness might be related to utilization of ACC as a nutrient within the infection threads.

Ma, Wenbo; Charles, Trevor C.; Glick, Bernard R.

2004-01-01

359

Cloning of the blasticidin S deaminase gene ( BSD ) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae  

Microsoft Academic Search

Aspergillus terreus produces a unique enzyme, blasticidin S deaminase, which catalyzes the deamination of blasticidin S (BS), and in consequence confers high resistance to the antibiotic. A cDNA clone derived from the structural gene for BS deaminase (BSD) was isolated by transforming Escherichia coli with an Aspergillus cDNA expression library and directly selecting for the ability to grow in the

Makoto Kimura; Takashi Kamakura; Quan Zhou Tao; Isao Kaneko; Isamu Yamaguchi

1994-01-01

360

Oxygen requirements of yeasts.  

PubMed

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

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

1990-12-01

361

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.

362

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.

2008-02-28

363

Yeast killer systems.  

PubMed Central

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.

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

1997-01-01

364

Cytosine arabinoside affects the heat and capsaicin receptor TRPV1 localisation and sensitivity in human sensory neurons  

Microsoft Academic Search

Background Cytosine arabinoside (Ara C) is a useful chemotherapy agent, used for treating acute myeloid leukaemia, although it may be\\u000a associated with side effects including painful neuropathy. It is also used for in vitro neuronal studies to limit the proliferation\\u000a of non-neuronal cells and thereby select nondividing neuronal cells. We studied the effects of Ara C on human dorsal root

Uma Anand; William R. Otto; Chas Bountra; Iain Chessell; Marco Sinisi; Rolfe Birch; Praveen Anand

2008-01-01

365

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

Microsoft Academic Search

The mid-infrared (IR) absorption spectrum of jet-cooled clusters of the nucleobases guanine and cytosine has been recorded in the 500-1800 cm? 1 range by ion-dip spectroscopy. Some 30 clearly separated and sharp resonances are observed. The combination of the experimental data with new high-level ab initio calculations is consistent with a previous structural assignment and a tentative assignment is made

Joost M. Bakker; Isabelle Compagnon; Gerard Meijer; Gert von Helden; Martin Kabelác; Pavel Hobza; Mattanjah S. de Vries

2004-01-01

366

Alterations in inheritance pattern and level of cytosine DNA methylation, and their relationship with heterosis in rice  

Microsoft Academic Search

Novel hybrid-specific\\/heterotic gene expression patterns observed from expression studies suggest the need to characterize\\u000a the underlying regulatory mechanism(s) to reveal the biological basis of heterosis in crop plants. To gain an insight into\\u000a the molecular basis of heterosis in rice, we investigated the inheritance pattern and level of cytosine methylation, a major\\u000a epigenetic regulatory mechanism, in the leaf tissue of

K. Sakthivel; K. Girishkumar; G. Ramkumar; V. V. Shenoy; S. T. Kajjidoni; P. M. Salimath

2010-01-01

367

Cytosine-phosphate-guanine (CpG) motifs are sensitizing agents for lipopolysaccharide in toxic shock model  

Microsoft Academic Search

Objective. Unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides are highly frequent motifs in bacterial DNA and rare in the mammalian genome. They are potent inducers of inflammatory cytokines and act synergistically with lipopolysaccharide (LPS) for the induction of tumor necrosis factor ! (TNF-!) production in vivo. It has therefore been suggested that innate immune reaction to bacterial unmethylated CpG motifs might contribute to

Sylvie Cornélie; Eric Wiel; Niels Lund; Gilles Lebuffe; Catherine Vendeville; Gilles Riveau; Benoît Vallet; Elisabeth Ban

2002-01-01

368

5-hydroxymethyl-cytosine enrichment of non-committed cells is not a universal feature of vertebrate development.  

PubMed

5-hydroxymethyl-cytosine (5-hmC) is a cytosine modification that is relatively abundant in mammalian pre-implantation embryos and embryonic stem cells (ESC) derived from mammalian blastocysts. Recent observations imply that both 5-hmC and Tet1/2/3 proteins, catalyzing the conversion of 5-methyl-cytosine to 5-hmC, may play an important role in self renewal and differentiation of ESCs. Here we assessed the distribution of 5-hmC in zebrafish and chick embryos and found that, unlike in mammals, 5-hmC is immunochemically undetectable in these systems before the onset of organogenesis. In addition, Tet1/2/3 transcripts are either low or undetectable at corresponding stages of zebrafish development. However, 5-hmC is enriched in later zebrafish and chick embryos and exhibits tissue-specific distribution in adult zebrafish. Our findings show that 5-hmC enrichment of non-committed cells is not a universal feature of vertebrate development and give insights both into evolution of embryonic pluripotency and the potential role of 5-hmC in its regulation. PMID:22419071

Almeida, Rimple D; Loose, Matthew; Sottile, Virginie; Matsa, Elena; Denning, Chris; Young, Lorraine; Johnson, Andrew D; Gering, Martin; Ruzov, Alexey

2012-04-01

369

Semi-quantitative immunohistochemical detection of 5-hydroxymethyl-cytosine reveals conservation of its tissue distribution between amphibians and mammals.  

PubMed

5-Hydroxymethyl-cytosine (5-hmC) is a form of modified cytosine, which has recently attracted a considerable attention due to its potential role in transcriptional regulation. According to several reports 5-hydroxymethyl-cytosine distribution is tissue-specific in mammals. Thus, 5-hmC is enriched in embryonic cell populations and in adult neuronal tissue. Here, we describe a novel method of semi-quantitative immunohistochemical detection of 5-hmC and utilize it to assess the levels of this modification in amphibian tissues. We show that, similar to mammalian embryos, 5-hmC is enriched in axolotl tadpoles compared with adult tissues. Our data demonstrate that 5-hmC distribution is tissue-specific in amphibians, and that strong 5-hmC enrichment in neuronal cells is conserved between amphibians and mammals. In addition, we identify 5-hmC-enriched cell populations that are distributed in amphibian skin and connective tissue in a mosaic manner. Our results illustrate that immunochemistry can be successfully used not only for spatial identification of cells enriched with 5-hmC, but also for the semi-quantitative assessment of the levels of this epigenetic modification in single cells of different tissues. PMID:22395462

Almeida, Rimple D; Sottile, Virginie; Loose, Matthew; De Sousa, Paul A; Johnson, Andrew D; Ruzov, Alexey

2012-02-01

370

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

PubMed Central

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.

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

2012-01-01

371

Singlet and triplet excited-state dynamics study of the keto and enol tautomers of cytosine.  

PubMed

The photoinduced excited-state dynamics of the keto and enol forms of cytosine have been investigated by using ab initio surface-hopping to gain an understanding of the outcome of molecular beam femtosecond pump-probe photoionisation spectroscopy experiments. Both singlet and triplet states were included in the dynamics. The results show that triplet states play a significant role in the relaxation of the keto tautomer, whereas they are less important in the enol tautomer. In both forms, the T1 state minimum was found to be too low in energy to be detected in standard photoionisation spectroscopy experiments and therefore experimental decay times should arise from simultaneous relaxation to the ground state and additional intersystem crossing followed by internal conversion to the T1 state. In agreement with available experimental lifetimes, we observed three decay constants of 7, 270 and 1900 fs, the first two coming from the keto tautomer and the third from the enol form. Deactivation of the enol tautomer is due to internal conversion to the ground state through two ethylenic-type S1/S0 conical intersections. PMID:23893944

Mai, Sebastian; Marquetand, Philipp; Richter, Martin; González-Vázquez, Jesús; González, Leticia

2013-07-25

372

Theoretical investigation of hydrogen transfer mechanism in the guanine cytosine base pair  

NASA Astrophysics Data System (ADS)

We have studied the quantum-dynamics of the hydrogen bonds in the guanine cytosine base pair. Due to the position of hydrogen atoms, different tautomers are possible: the stable Watson Crick G C, the imino-enol G* C*, the imino-enol imino-enol G# C# and some zwitterionic structures. The common idea in the literature is that only the G C and the G* C* tautomers are stable with an estimate of G C ? G* C* transition probability of 10-6 10-9 by the help of Boltzmann statistics. Here we show a detailed quantum theoretical study that suggests the following conclusion: G C is the stablest tautomer, some partially charged systems (due to the movement of only one hydrogen atom) are important and a large amount of the imino-enol G* C* (and less of the imino-enol imino-enol G# C# structure) tautomer is present at any time. The corresponding transition probabilities from different tautomers are not due to thermal passage, but they are a pure quantum phenomenon. These large probabilities definitively disprove the idea of these tautomers as mutation points. The mechanisms of passage from the G C tautomer to the others have also been investigated.

Villani, Giovanni

2006-05-01

373

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

PubMed Central

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.

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

2012-01-01

374

Photoalkylated DNA and ultraviolet-irradiated DNA are incised at cytosines by endonuclease III.  

PubMed Central

Photoalkylation, the ultraviolet irradiation of DNA with isopropanol and di-tert-butylperoxide, causes a variety of base alterations. These include 8-(2-hydroxy-2-propyl)guanines, 8-(2-hydroxy-2-propyl)adenines and thymine dimers. An E. coli endonuclease against photoalkylated DNA was assayed by conversion of superhelical PM2 phage DNA to the nicked form. Enzyme activities were compared between extracts of strain BW9109 (xth-), lacking exonuclease III activity, and strain BW434 (xth-,nth-), deficient in both exonuclease III and endonuclease III. The endonuclease level in the double mutant against substrate photoalkylated DNA was under 20% of the activity in the mutant lacking only exonuclease III. Irradiation of the DNA substrate in the absence of isopropanol did not affect the activity in either strain. Analysis by polyacrylamide gel electrophoresis identified the sites of DNA cleavage by purified E. coli endonuclease III as cytosines, both in DNA irradiated at biologically significant wavelengths and in photoalkylated DNA. Neither 8-(2-hydroxy-2-propyl)purines, pyrimidine dimers, uracils nor 6-4'-(pyrimidin-2'-one)pyrimidines were substrates for the enzyme. Images

Weiss, R B; Duker, N J

1986-01-01

375

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

PubMed Central

Gibbon species have accumulated an unusually high number of chromosomal changes since diverging from the common hominoid ancestor 15–18 million years ago. The cause of this increased rate of chromosomal rearrangements is not known, nor is it known if genome architecture has a role. To address this question, we analyzed sequences spanning 57 breaks of synteny between northern white-cheeked gibbons (Nomascus l. leucogenys) and humans. We find that the breakpoint regions are enriched in segmental duplications and repeats, with Alu elements being the most abundant. Alus located near the gibbon breakpoints (<150 bp) have a higher CpG content than other Alus. Bisulphite allelic sequencing reveals that these gibbon Alus have a lower average density of methylated cytosine that their human orthologues. The finding of higher CpG content and lower average CpG methylation suggests that the gibbon Alu elements are epigenetically distinct from their human orthologues. The association between undermethylation and chromosomal rearrangement in gibbons suggests a correlation between epigenetic state and structural genome variation in evolution.

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

2009-01-01

376

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

PubMed Central

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.

Tsuboi, Hidenori; Sutoh, Keita; Wada, Masamitsu

2012-01-01

377

RNA-Mediated Epigenetic Heredity Requires the Cytosine Methyltransferase Dnmt2  

PubMed Central

RNA–mediated transmission of phenotypes is an important way to explain non-Mendelian heredity. We have previously shown that small non-coding RNAs can induce hereditary epigenetic variations in mice and act as the transgenerational signalling molecules. Two prominent examples for these paramutations include the epigenetic modulation of the Kit gene, resulting in altered fur coloration, and the modulation of the Sox9 gene, resulting in an overgrowth phenotype. We now report that expression of the Dnmt2 RNA methyltransferase is required for the establishment and hereditary maintenance of both paramutations. Our data show that the Kit paramutant phenotype was not transmitted to the progeny of Dnmt2?/? mice and that the Sox9 paramutation was also not established in Dnmt2?/? embryos. Similarly, RNA from Dnmt2-negative Kit heterozygotes did not induce the paramutant phenotype when microinjected into Dnmt2-deficient fertilized eggs and microinjection of the miR-124 microRNA failed to induce the characteristic giant phenotype. In agreement with an RNA–mediated mechanism of inheritance, no change was observed in the DNA methylation profiles of the Kit locus between the wild-type and paramutant mice. RNA bisulfite sequencing confirmed Dnmt2-dependent tRNA methylation in mouse sperm and also indicated Dnmt2-dependent cytosine methylation in Kit RNA in paramutant embryos. Together, these findings uncover a novel function of Dnmt2 in RNA–mediated epigenetic heredity.

Liebers, Reinhard; Tuorto, Francesca; Ghanbarian, Hossein; Lyko, Frank; Cuzin, Francois; Rassoulzadegan, Minoo

2013-01-01

378

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

PubMed Central

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

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

2009-01-01

379

Stability of the guanine-cytosine radical cation in DNA base pairs triplets  

NASA Astrophysics Data System (ADS)

To investigate the influence of flanking nucleic acid base pairs on the stability of the guanine-cytosine radical cation (GC+), ab initio Hartree Fock calculations on 16 DNA base pair triplets were carried out using cc-pVDZ and cc-pVTZ basis sets. Furthermore, second order Møller Plesset (MP2) correlation energy correction were computed using the cc-pVDZ basis set. The results from homodesmotic reactions suggest that GC+ is energetically most favored when being embedded by two neighboring GC base pairs, while the strongest destabilization of around 39 kJ mol-1 (9.2 kcal mol-1) occurs in the TGC triplet. The destabilization was also found to be dependent on the actual sequence where the nucleotide in 5? position has a weaker influence than that in 3? position. As effects from the DNA backbone and further environment are excluded, the results indicates that the strong localization of the positive charge on the middle GC base pair is dominated by electrostatic interactions. Consequences for the DNA in terms of susceptibility to mutations by oxidative damage and charge hopping are discussed.

Hutter, Michael C.

2006-07-01

380

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

PubMed Central

During sexual development, Neurospora crassa inactivates genes in duplicated DNA segments by a hypermutation process, repeat-induced point mutation (RIP). RIP introduces C:G to T:A transition mutations and creates targets for subsequent DNA methylation in vegetative tissue. The mechanism of RIP and its relationship to DNA methylation are not fully understood. Mutations in DIM-2, a DNA methyltransferase (DMT) responsible for all known cytosine methylation in Neurospora, does not prevent RIP. We used RIP to disrupt a second putative DMT gene in the Neurospora genome and tested mutants for defects in DNA methylation and RIP. No effect on DNA methylation was detected in the tissues that could be assayed, but the mutants showed recessive defects in RIP. Duplications of the am and mtr genes were completely stable in crosses homozygous for the mutated potential DMT gene, which we call rid (RIP defective). The same duplications were inactivated normally in heterozygous crosses. Disruption of the rid gene did not noticeably affect fertility, growth, or development. In contrast, crosses homozygous for a mutation in a related gene in Ascobolus immersus, masc1, reportedly fail to develop and heterozygous crosses reduce methylation induced premeiotically [Malagnac, F., Wendel, B., Goyon, C., Faugeron, G., Zickler, D., et al. (1997) Cell 91, 281–290]. We isolated homologues of rid from Neurospora tetrasperma and Neurospora intermedia to identify conserved regions. Homologues possess all motifs characteristic of eukaryotic DMTs and have large distinctive C- and N-terminal domains.

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

2002-01-01

381

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

PubMed

During sexual development, Neurospora crassa inactivates genes in duplicated DNA segments by a hypermutation process, repeat-induced point mutation (RIP). RIP introduces C:G to T:A transition mutations and creates targets for subsequent DNA methylation in vegetative tissue. The mechanism of RIP and its relationship to DNA methylation are not fully understood. Mutations in DIM-2, a DNA methyltransferase (DMT) responsible for all known cytosine methylation in Neurospora, does not prevent RIP. We used RIP to disrupt a second putative DMT gene in the Neurospora genome and tested mutants for defects in DNA methylation and RIP. No effect on DNA methylation was detected in the tissues that could be assayed, but the mutants showed recessive defects in RIP. Duplications of the am and mtr genes were completely stable in crosses homozygous for the mutated potential DMT gene, which we call rid (RIP defective). The same duplications were inactivated normally in heterozygous crosses. Disruption of the rid gene did not noticeably affect fertility, growth, or development. In contrast, crosses homozygous for a mutation in a related gene in Ascobolus immersus, masc1, reportedly fail to develop and heterozygous crosses reduce methylation induced premeiotically [Malagnac, F., Wendel, B., Goyon, C., Faugeron, G., Zickler, D., et al. (1997) Cell 91, 281-290]. We isolated homologues of rid from Neurospora tetrasperma and Neurospora intermedia to identify conserved regions. Homologues possess all motifs characteristic of eukaryotic DMTs and have large distinctive C- and N-terminal domains. PMID:12072568

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

2002-06-18

382

P170 (Pgp) expression in leukemic cells after therapeutic exposure to arabinosyl cytosine.  

PubMed

The expression of the mdr-1 gene coding for a transmembrane 170 KD glycoprotein (P170 or PGP) is an important cause of multidrug resistance (MDR). In tumor cells the expression of the gene may vary and there is experimental evidence that it can be induced by exposure to MDR-unrelated agents. We investigated if the therapeutic exposure to Arabinosyl Cytosine (AC) could affect the level of P170 expression in the blast cells of acute non-lymphocytic leukemia (ANLL). The reactivity to the P170-directed MRK 16 monoclonal antibody of the marrow blast cells from 27 patients with ANLL prior and after treatment with standard dose AC was evaluated by flow cytometry. After treatment with AC the MRK 16 mean fluorescence index (MFI) was increased in 5/18 cases of primary and previously untreated ANLL and in 7/9 cases of relapsed or secondary leukemia. Overall, the mean value of the MFI was 6.8+/-3.6 before and 9.0+/-3.8 after AC (P=0.001, Wilkoxon matched pairs test). Therapeutic exposure to AC in vivo may increase P170 expression in leukemic cells. This may influence the definition and the quantitation of resistance and may have therapeutic implications, concerning the association with other cytotoxics and the use of MDR modifiers. PMID:10358333

Grimaz; Damiani; Michieli; Sperotto; Masolini; Baccarani

1998-01-01

383

Aplidin synergizes with cytosine arabinoside: functional relevance of mitochondria in Aplidin-induced cytotoxicity.  

PubMed

Aplidin (plitidepsin) is a novel marine-derived antitumor agent presently undergoing phase II clinical trials in hematological malignancies and solid tumors. Lack of bone marrow toxicity has encouraged further development of this drug for treatment of leukemia and lymphoma. Multiple signaling pathways have been shown to be involved in Aplidin-induced apoptosis and cell cycle arrest in G1 and G2 phase. However, the exact mechanism(s) of Aplidin action remains to be elucidated. Here we demonstrate that mitochondria-associated or -localized processes are the potential cellular targets of Aplidin. Whole genome gene-expression profiling (GEP) revealed that fatty acid metabolism, sterol biosynthesis and energy metabolism, including the tricarboxylic acid cycle and ATP synthesis are affected by Aplidin treatment. Moreover, mutant MOLT-4, human leukemia cells lacking functional mitochondria, were found to be resistant to Aplidin. Cytosine arabinoside (araC), which also generates oxidative stress but does not affect the ATP pool, showed synergism with Aplidin in our leukemia and lymphoma models in vitro and in vivo. These studies provide new insights into the mechanism of action of Aplidin. The efficacy of the combination of Aplidin and araC is currently being evaluated in clinical phase I/II program for the treatment of patients with relapsed leukemia and high-grade lymphoma. PMID:17713546

Humeniuk, R; Menon, L G; Mishra, P J; Saydam, G; Longo-Sorbello, G S A; Elisseyeff, Y; Lewis, L D; Aracil, M; Jimeno, J; Bertino, J R; Banerjee, D

2007-08-23

384

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

NASA Astrophysics Data System (ADS)

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

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

1992-02-01

385

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

SciTech Connect

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

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

1992-02-01

386

Thermodynamic and kinetic studies of competitive inhibition of adenosine deaminase by ring opened analogues of adenine nucleoside.  

PubMed

Ring opened analogues of adenine nucleosides substituted at the ninth adenine positions, with and without esterification (compounds I and II), have been studied kinetically and thermodynamically at various temperatures in order to determine the sites of ring opened analogues. These are deemed to be important for binding to the adenosine deaminase. Adenosine deaminase is found to bind more strongly to compound I than to compound II, therefore compound I is a stronger inhibitor than II, because the position of (5') OH on the ribose moiety decreases the inhibitory strength on the ring opened analogue. PMID:8485104

Moosavi-Movahedi, A A; Rahmani, Y; Hakimelahi, G H

1993-04-01

387

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

PubMed Central

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

Burrows, R B; Brown, G M

1978-01-01

388

Methods for yeast characterization from industrial products  

Microsoft Academic Search

This work compared the efficiency of four methods for the identification of industrial yeast strains and the establishment of a pattern for yeast characterization to be used during industrial fermentation processes, allowing the detection of yeast contaminants. Five strains of yeast currently used in the Brazilian fuel alcohol industry (about 99% of the yeast used for this purpose), and yeast

Luiz H Gomes; Keila M. R Duarte; Juan L Argueso; Sergio Echeverrigaray; Flavio C. A Tavares

2000-01-01

389

L-arabinose fermenting yeast  

DOEpatents

An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. Methods of producing ethanol include utilizing these modified yeast strains. ##STR00001##

Zhang, Min (Lakewood, CO); Singh, Arjun (Lakewood, CO); Knoshaug, Eric (Golden, CO); Franden, Mary Ann (Centennial, CO); Jarvis, Eric (Boulder, CO); Suominen, Pirkko (Maple Grove, MN)

2010-12-07

390

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

PubMed

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

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

2012-02-01

391

The complex regulation and function of activation-induced cytidine deaminase (AID)  

PubMed Central

Activation-induced cytidine deaminase (AID) instigates mutations and DNA breaks in Ig genes undergoing somatic hypermutation (SHM) and class switch recombination (CSR) during B cell activation in response to immunization and infection. This review discusses how AID expression and activity are regulated, including recent discoveries of AID interacting proteins that might recruit AID to immunoglobulin (Ig) genes and also allow it to target both DNA strands. Also discussed is the accumulating evidence that AID binds to, mutates, and creates breaks at numerous non-Ig sites in the genome, initiating cell transformation and malignancies.

Stavnezer, Janet

2011-01-01

392

"REVERSE" CARBOCYCLIC FLEXIMERS: SYNTHESIS OF A NEW CLASS OF ADENOSINE DEAMINASE INHIBITORS  

PubMed Central

A series of flexible carbocyclic pyrimidine nucleosides has been designed and synthesized. In contrast to previously reported “fleximers” from our laboratory, these analogues have the connectivity of the heterocyclic base system “reversed”, where the pyrimidine ring is attached to the sugar moiety, rather than the five membered imidazole ring. As was previously seen with the ribose fleximers, their inherent flexibility should allow them to adjust to enzyme binding site mutations, as well as increase the affinity for atypical enzymes. Preliminary biological screening has revealed surprising inhibition of adenosine deaminase, despite their lack of resemblance to adenosine.

Zimmermann, Sarah C.; Sadler, Joshua M.; O'Daniel, Peter I.; Kim, Nathaniel T.; Seley-Radtke, Katherine L.

2013-01-01

393

Modification of retroviral RNA by double-stranded RNA adenosine deaminase.  

PubMed Central

In this report, we describe a recombinant provirus generated during in vitro passage that contains a short region of adenosine-to-guanosine hypermutation. The hypermutated region is restricted to complementary sequences present in the recombinant provirus. We propose that a duplex was formed in the recombinant RNA prior to reverse transcription. This duplex was a substrate for double-stranded RNA adenosine deaminase, an activity found in all cells examined that deaminates A in double-stranded RNA, converting it to inosine, which is further converted to a guanosine by reverse transcription. It appears that cis viral sequences facilitated the A-->G transitions.

Hajjar, A M; Linial, M L

1995-01-01

394

[Adenosine deaminase in the blood cells and serum of patients with tuberculous pleurisy].  

PubMed

The common complication of pulmonary tuberculosis is pleurisy that requires not only special treatments, but also gives rise to difficulties in the differential diagnosis of tuberculosis. The investigation has studied the possibility of determining the activity of adenosine deaminase (ADA) as a marker of pleural effusion of tuberculous etiology. The patients with tuberculous pleurisy have been found to have higher lymphocytic and red blood ADA, and ADA, activities and a significantly increased serum ADA2 activity. Thus, the changes in the activity of ADA and its isozymes have been shown to be specific to pleurisy of tuberculous etiology and to be of diagnostic value. PMID:18318225

Alinezhad, S M; Gurevich, G L; Zakharevski?, F I; Kamali, F; Taganovich, A D

2008-01-01

395

[Diagnostic characteristics of adenosine deaminase test in Byelorussian patients with tuberculous pleurisy].  

PubMed

The results of studying the diagnostic value of changes in the pleural fluid and serum activities of adenosine deaminase (ADA) and its isozymes are analyzed in Byelorussian patients with tuberculous pleurisy. There is an increased serum and pleural fluid ADA activity in the patients with tuberculous pleurisy caused mainly by a rise in ADA2 activity. The test determining the activity of ADA, and ADA2 in particular, has shown high sensitivity and specificity as compared with the results obtained in other countries. The diagnostic efficacy of determination of these indices in the serum is also high, but it is lower than that in the pleural fluid. PMID:18822475

Taganovich, A D; Alinezhad, Said M

2008-01-01

396

Maize haplotype with a helitron-amplified cytidine deaminase gene copy  

PubMed Central

Background Genetic maps are based on recombination of orthologous gene sequences between different strains of the same species. Therefore, it was unexpected to find extensive non-collinearity of genes between different inbred strains of maize. Interestingly, disruption of gene collinearity can be caused among others by a rolling circle-type copy and paste mechanism facilitated by Helitrons. However, understanding the role of this type of gene amplification has been hampered by the lack of finding intact gene sequences within Helitrons. Results By aligning two haplotypes of the z1C1 locus of maize we found a Helitron that contains two genes, one encoding a putative cytidine deaminase and one a hypothetical protein with part of a 40S ribosomal protein. The cytidine deaminase gene, called ZmCDA3, has been copied from the ZmCDA1 gene on maize chromosome 7 about 4.5 million years ago (mya) after maize was formed by whole-genome duplication from two progenitors. Inbred lines contain gene copies of both progenitors, the ZmCDA1 and ZmCDA2 genes. Both genes diverged when the progenitors of maize split and are derived from the same progenitor as the rice OsCDA1 gene. The ZmCDA1 and ZmCDA2 genes are both transcribed in leaf and seed tissue, but transcripts of the paralogous ZmCDA3 gene have not been found yet. Based on their protein structure the maize CDA genes encode a nucleoside deaminase that is found in bacterial systems and is distinct from the mammalian RNA and/or DNA modifying enzymes. Conclusion The conservation of a paralogous gene sequence encoding a cytidine deaminase gene over 4.5 million years suggests that Helitrons could add functional gene sequences to new chromosomal positions and thereby create new haplotypes. However, the function of such paralogous gene copies cannot be essential because they are not present in all maize strains. However, it is interesting to note that maize hybrids can outperform their inbred parents. Therefore, certain haplotypes may function only in combination with other haplotypes or under specialized environmental conditions.

Xu, Jian-Hong; Messing, Joachim

2006-01-01

397

Conversion of pentoses by yeasts  

SciTech Connect

The utilization and conversion of D-xylose, D-xyulose, L-arabinose, and xylitol by yeast strains have been investigated with the following results: 1) The majority of yeasts tested utilize D-xylose and produce polyols, ethanol, and organic acids. The type and amount of products formed varies with the yeast strains used. The most commonly detected product is xylitol. 2) The majority of yeasts tested utilize D-xylulose aerobically and fermentatively to produce ethanol, xylitol D-arabitol, and organic acids. The type and amount of products varies depending upon the yeast strains used. 3) Xylitol is a poor carbon and energy source for most yeasts tested. Some yeast strains produce small amounts of ethanol from xylitol. 4) Most yeast strains utilize L-arabinose, and L-arabitol is the common product. Small amounts of ethanol are also produced by some yeast strains. 5) Of the four substrates examined, D-xylulose was the preferred substrate, followed by D-xylose, L-arabinose, and xylitol. 6) Mutant yeast strains that exhibit different metabolic product patterns can be induced and isolated from Candida sp. Saccharomyces cerevisiae, and other yeasts. These mutant strains can be used for ethanol production from D-xylose as well as for the study of metabolic regulation of pentose utilization in yeasts.

Gong, C.S.; Claypool, T.A.; Maun, C.M.; Mccracken, L.D.; Tsao, G.T.; Ueng, P.P.

1983-01-01

398

Yeasts from the North Sea  

Microsoft Academic Search

Yeasts were isolated from twelve established sites in the North Sea from 1964 to 1966. A percentage frequency of 99% with populations varying from 3000 viable cells\\/L was observed. This mycota was characterized by considerable spatial and temporal fluctuation, with the dominant yeast present being the ascosporogenous species, Debaryomyces hansenii. This taxon, as well as other common North Sea yeasts,

S. P. Meyers; D. G. Ahearn; W. Gunkel; F. J. Roth

1967-01-01

399

Evaluation of YeastIdent and Uni-Yeast-Tek yeast identification systems.  

PubMed Central

The accuracy of the new API YeastIdent system and the Flow Laboratories Uni-Yeast-Tek identification kit with an expanded data base was evaluated in comparison to the API 20C yeast identification system by three laboratories. A total of 489 test isolates were used, biased toward yeasts commonly encountered in clinical specimens. Isolates not in a system's data base were not counted in the evaluation of that system. For isolates in their data base, YeastIdent was 55% accurate and Uni-Yeast-Tek was 40% accurate. By the manufacturer's criteria of reliable identification without additional tests, both systems failed to identify many common and uncommon species. The limited number of substrates and difficulties in assessing results obtained with 11 of the API YeastIdent substrates and apparent errors in the expanded Uni-Yeast-Tek data base appeared to be major factors limiting the accuracy of these systems.

Salkin, I F; Land, G A; Hurd, N J; Goldson, P R; McGinnis, M R

1987-01-01

400

Tissue culture-induced transpositional activity of mPing is correlated with cytosine methylation in rice  

PubMed Central

Background mPing is an endogenous MITE in the rice genome, which is quiescent under normal conditions but can be induced towards mobilization under various stresses. The cellular mechanism responsible for modulating the activity of mPing remains unknown. Cytosine methylation is a major epigenetic modification in most eukaryotes, and the primary function of which is to serve as a genome defense system including taming activity of transposable elements (TEs). Given that tissue-culture is capable of inducing both methylation alteration and mPing transposition in certain rice genotypes, it provides a tractable system to investigate the possible relationship between the two phenomena. Results mPing transposition and cytosine methylation alteration were measured in callus and regenerated plants in three rice (ssp. indica) genotypes, V14, V27 and R09. All three genotypes showed transposition of mPing, though at various frequencies. Cytosine methylation alteration occurred both at the mPing-flanks and at random loci sampled globally in callus and regenerated plants of all three genotypes. However, a sharp difference in the changing patterns was noted between the mPing-flanks and random genomic loci, with a particular type of methylation modification, i.e., CNG hypermethylation, occurred predominantly at the mPing-flanks. Pearson's test on pairwise correlations indicated that mPing activity is positively correlated with specific patterns of methylation alteration at random genomic loci, while the element's immobility is positively correlated with methylation levels of the mPing's 5'-flanks. Bisulfite sequencing of two mPing-containing loci showed that whereas for the immobile locus loss of CG methylation in the 5'-flank was accompanied by an increase in CHG methylation, together with an overall increase in methylation of all three types (CG, CHG and CHH) in the mPing-body region, for the active locus erasure of CG methylation in the 5'-flank was not followed by such a change. Conclusion Our results documented that tissue culture-induced mPing activity in rice ssp. indica is correlated with alteration in cytosine methylation patterns at both random genomic loci and the elements' flanks, while the stability of mPing positively correlates with enhanced methylation levels of both the flanks and probably the elements per se. Thus, our results implicate a possible role of cytosine methylation in maintaining mPing stability under normal conditions, and in releasing the element's activity as a consequence of epigenetic perturbation in a locus-specific manner under certain stress conditions.

Ngezahayo, Frederic; Xu, Chunming; Wang, Hongyan; Jiang, Lily; Pang, Jinsong; Liu, Bao

2009-01-01

401

Genome evolution in yeasts  

Microsoft Academic Search

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

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

402

Biosynthesis of yeast mitochondria  

Microsoft Academic Search

Ethidium bromide selectively inhibits growth of the petite negative yeast Kluyveromyces fragilis on a non-fermentable carbon source. In short term experiments, when growth in ethidium is continued for about 11 generations, this inhibition is accompanied by a loss of cyanide sensitive respiration and particulate cytochromes, an initial phase of microcolony production, and an inhibition of mitochondrial DNA synthesis. The loss

A. A. Luha; P. A. Whittaker; R. C. Hammond

1974-01-01

403

Yeast DNA Extraction  

NSDL National Science Digital Library

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

Hays, Lana

2009-01-01

404

Stratification of Nucleoside Analog Chemotherapy Using 1-(2?-Deoxy-2?-18F-Fluoro-?-D-Arabinofuranosyl)Cytosine and 1-(2?-Deoxy-2?-18F-Fluoro-?-L-Arabinofuranosyl)-5-Methylcytosine PET  

PubMed Central

The ability to measure tumor determinants of response to nucleoside analog (NA) chemotherapy agents such as gemcitabine and related compounds could significantly affect the management of several types of cancer. Previously we showed that the accumulation in tumors of the new PET tracer 1-(2?-deoxy-2?-18F-fluoro-?-D-arabinofuranosyl)cytosine (18F-FAC) is predictive of responses to gemcitabine. 18F-FAC retention in cells requires deoxycytidine kinase (dCK), a rate-limiting enzyme in the deoxyribonucleoside salvage metabolism and in gemcitabine conversion from an inactive prodrug to a cytotoxic compound. The objectives of the current study were to determine whether 18F-FAC tumor uptake is also influenced by cytidine deaminase (CDA), a determinant of resistance to gemcitabine; to develop a new PET assay using 18F-FAC and the related probe 1-(2?-deoxy-2?-18F-fluoro-?-L-arabinofuranosyl)-5-methylcytosine (L-18F-FMAC) to profile tumor lesions for both dCK and CDA enzymatic activities; and to determine whether this PET assay can identify the most effective NA chemotherapy against tumors with differential expression of dCK and CDA. Methods Isogenic murine leukemic cell lines with defined dCK and CDA activities were generated by retroviral transduction. A cell viability assay was used to determine the sensitivity of the isogenic cell lines to the dCK-dependent NA prodrugs gemcitabine and clofarabine. In vitro enzymatic and cell-based tracer uptake assays and in vivo PET with 18F-FAC and L-18F-FMAC were used to predict tumor responses to gemcitabine and clofarabine. Results dCK and CDA activities measured by kinase and tracer uptake assays correlated with the sensitivity of isogenic cell lines to gemcitabine and clofarabine. Coexpression of CDA decreased the sensitivity of dCK-positive cells to gemcitabine treatment in vitro by 15-fold but did not affect responses to clofarabine. Coexpression of CDA decreased 18F-FAC but not L-18F-FMAC, phosphorylation, and uptake by dCK-positive cells. 18F-FAC and L-18F-FMAC PET estimates of the enzymatic activities of dCK and CDA in tumor implants in mice were predictive of responses to gemcitabine and clofarabine treatment in vivo. Conclusion These findings support the utility of PET-based phenotyping of tumor nucleoside metabolism for guiding the selection of NA prodrugs.

Lee, Jason T.; Campbell, Dean O.; Satyamurthy, Nagichettiar; Czernin, Johannes; Radu, Caius G.

2012-01-01

405

L-arabinose fermenting yeast  

DOEpatents

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

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

2013-02-12

406

Innate Immune Signaling Induces High Levels of TC-specific Deaminase Activity in Primary Monocyte-derived Cells through Expression of APOBEC3A Isoforms*  

PubMed Central

In HIV-1-infected individuals, G-to-A hypermutation is found in HIV-1 DNA isolated from peripheral blood mononuclear cells (PBMCs). These mutations are thought to result from editing by one or more host enzymes in the APOBEC3 (A3) family of cytidine deaminases, which act on CC (APOBEC3G) and TC (other A3 proteins) dinucleotide motifs in DNA (edited cytidine underlined). Although many A3 proteins display high levels of deaminase activity in model systems, only low levels of A3 deaminase activity have been found in primary cells examined to date. In contrast, here we report high levels of deaminase activity at TC motifs when whole PBMCs or isolated primary monocyte-derived cells were treated with interferon-? (IFN?) or IFN?-inducing toll-like receptor ligands. Induction of TC-specific deaminase activity required new transcription and translation and correlated with the appearance of two APOBEC3A (A3A) isoforms. Knockdown of A3A in monocytes with siRNA abolished TC-specific deaminase activity, confirming that A3A isoforms are responsible for all TC-specific deaminase activity observed. Both A3A isoforms appear to be enzymatically active; moreover, our mutational studies raise the possibility that the smaller isoform results from internal translational initiation. In contrast to the high levels of TC-specific activity observed in IFN?-treated monocytes, CC-specific activity remained low in PBMCs, suggesting that A3G deaminase activity is relatively inhibited, unlike that of A3A. Together, these findings suggest that deaminase activity of A3A isoforms in monocytes and macrophages may play an important role in host defense against viruses.

Thielen, Beth K.; McNevin, John P.; McElrath, M. Juliana; Hunt, Brook Vander Stoep; Klein, Kevin C.; Lingappa, Jaisri R.

2010-01-01

407

Clustered Adenine/Thymine Stretches Are Essential for Function of a Fission Yeast Replication Origin  

PubMed Central

We have determined functional elements required for autonomous replication of the Schizosaccharomyces pombe ars2004 that acts as an intrinsic chromosomal replication origin. Internal deletion analysis of a 940-bp fragment (ars2004M) showed three regions, I to III, to be required for autonomously replicating sequence (ARS) activity. Eight-base-pair substitutions in the 40-bp region I, composed of arrays of adenines on a DNA strand, resulted in a great reduction of ARS activity. Substitutions of region I with synthetic sequences showed that no specific sequence but rather repeats of three or more consecutive adenines or thymines, without interruption by guanine or cytosine, are required for the ARS activity. The 65-bp region III contains 11 repeats of the AAAAT sequence, while the 165-bp region II has short adenine or thymine stretches and a guanine- and cytosine-rich region which enhances ARS activity. All three regions in ars2004M can be replaced with 40-bp poly(dA/dT) fragments without reduction of ARS activity. Although spacer regions in the ars2004M enhance ARS activity, all could be deleted when an 40-bp poly(dA/dT) fragment was added in place of region I. Our results suggest that the origin activity of fission yeast replicators depends on the number of adenine/thymine stretches, the extent of their clustering, and presence of certain replication-enhancing elements.

Okuno, Yukiko; Satoh, Hiroyasu; Sekiguchi, Mariko; Masukata, Hisao

1999-01-01

408

DNA sequence of the D-serine deaminase activator gene dsdC.  

PubMed Central

We have determined the DNA sequence of dsdC, the gene that encodes the D-serine deaminase activator protein of Escherichia coli K-12. The sequence contains a single open reading frame that terminates in a UGA codon. One the basis of the size of the protein, 33 kilodaltons, and the amino acid sequence encoded by the open reading frame, we identified a likely translation initiation codon 731 base pairs upstream of the translation initiation codon for the divergently transcribed D-serine deaminase gene. There is a broad range of codon usage, not surprising in view of the weak expression of the gene. The N-terminal two-thirds of the activator is arginine-lysine rich and quite polar; the remainder is more neutral. The segment of the protein that seems most likely to have potential to form the helix-turn-helix structure characteristic of DNA-regulatory proteins is located near the end of the polar region. The protein contains a region with significant homology to lambda attB.

Palchaudhuri, S; Patel, V; McFall, E

1988-01-01

409

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

PubMed

Beneficial plant-associated bacteria play a key role in supporting and/or promoting plant growth and health. Plant growth promoting bacteria present in the rhizosphere of crop plants can directly affect plant metabolism or modulate phytohormone production or degradation. We isolated 355 bacteria from the rhizosphere of rice plants grown in the farmers' fields in the coastal rice field soil from five different locations of the Ganjam district of Odisha, India. Six bacteria producing both ACC deaminase (ranging from 603.94 to 1350.02?nmol ?-ketobutyrate mg(-1) ?h(-1) ) and indole acetic acid (IAA; ranging from 10.54 to 37.65??M?ml(-1) ) in pure cultures were further identified using polyphasic taxonomy including BIOLOG((R)) , FAME analysis and the 16S rRNA gene sequencing. Phylogenetic analyses of the isolates resulted into five major clusters to include members of the genera Bacillus, Microbacterium, Methylophaga, Agromyces, and Paenibacillus. Seed inoculation of rice (cv. Naveen) by the six individual PGPR isolates had a considerable impact on different growth parameters including root elongation that was positively correlated with ACC deaminase activity and IAA production. The cultures also had other plant growth attributes including ammonia production and at least two isolates produced siderophores. Study indicates that presence of diverse rhizobacteria with effective growth-promoting traits, in the rice rhizosphere, may be exploited for a sustainable crop management under field conditions. PMID:23681643

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

2013-05-17

410

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

PubMed

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

Fernández, José R; Sweet, Eric S; Welsh, William J; Firestein, Bonnie L

2010-07-27

411

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

PubMed Central

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.

Vieira, Valdimara C.; Soares, Marcelo A.

2013-01-01

412

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

PubMed Central

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

Fernandez, Jose R.; Sweet, Eric S.; Welsh, William J.; Firestein, Bonnie L.

2010-01-01

413

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

PubMed

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

Vieira, Valdimara C; Soares, Marcelo A

2013-06-25

414

Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA  

PubMed Central

CONTEXT: Amplification of Guanine-Cytosine (GC) -rich sequences becomes important in screening and diagnosis of certain genetic diseases such as diseases arising due to expansion of GC-rich trinucleotide repeat regions. However, GC-rich sequences in the genome are refractory to standard polymerase chain reaction (PCR) amplification and require a special reaction conditions and/or modified PCR cycle parameters. AIM: Optimize a cost effective PCR assay to amplify the GC-rich DNA templates. SETTINGS AND DESIGN: Fragile X mental retardation gene (FMR 1) is an ideal candidate for PCR optimization as its GC content is more than 80%. Primers designed to amplify the GC rich 5’ untranslated region of the FMR 1 gene, was selected for the optimization of amplification using DNA extracted from buccal mucosal cells. MATERIALS AND METHODS: A simple and rapid protocol was used to extract DNA from buccal cells. PCR optimization was carried out using three methods, (a) substituting a substrate analog 7-deaza-dGTP to dGTP (b) in the presence of a single PCR additive and (c) using a combination of PCR additives. All PCR amplifications were carried out using a low-cost thermostable polymerase. RESULTS: Optimum PCR conditions were achieved when a combination of 1M betaine and 5% dimethyl sulfoxide (DMSO) was used. CONCLUSIONS: It was possible to amplify the GC rich region of FMR 1 gene with reproducibility in the presence of betaine and DMSO as additives without the use of commercially available kits for DNA extraction and the expensive thermostable polymerases.

Bhagya, C. H. W. M. R. Chandrasekara; Wijesundera Sulochana, W. S.; Hemamali, N. Perera

2013-01-01

415

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

PubMed Central

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.

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

2011-01-01

416

Uptake of [18F]1-(2'-deoxy-2'-arabinofuranosyl) Cytosine Indicates Intestinal Inflammation in Mice  

PubMed Central

Background & Aims Uptake of [18F]1-(2’-deoxy-2’-arabinofuranosyl)cytosine (D-FAC) is a distinctive trait of activated lymphocytes; its biodistribution predominates in the spleen, thymus, and bone marrow. In addition to these immune compartments, D-FAC is taken up at high levels by the intestine. We analyzed the regional specificity of uptake and the cell types that mediate it. Methods In mice, 3-dimensional isocontour regions of interest (ROIs) were drawn based on computed tomographic images to quantify intestinal signals from micro positron emission tomography (PET) scans. To ascertain the cell type responsible, intestinal epithelium and immune cells were isolated and D-FAC uptake was analyzed, in vitro. Mice deficient in mucosal homing (?7 integrin-/-), enteric microbiota (germ-free), or active for immune colitis (G?i2-/- CD3+ transferred into Rag-/- recipients) were studied. Results Strong uptake of D-FAC was detected throughout the intestine, with greatest signal per ROI in the duodenum. Fractionation of intestinal cell types after in vivo uptake revealed that the signal was almost entirely from epithelial cells. Among resident immune cell types, CD4+ T cells showed the greatest per-cell and total uptake. D-FAC uptake increased in both the intestine and systemic lymphoid sites during colitis. Compared to fluorodeoxyglucose, increased uptake of D-FAC in the small and large intestine occurred at an earlier stage of disease development. Conclusions Uptake of D-FAC is a prominent trait of normal mouse intestinal epithelial cells, which is useful for their non-invasive visualization by PET. Increased uptake of D-FAC reflects the activity of the epithelium and lymphocytes, providing a unique early marker of intestinal inflammation.

Brewer, Sarah; Nair-Gill, Evan; Wei, Bo; Chen, Ling; Li, Xiaoxiao; Riedinger, Mireille; Campbell, Dean O.; Wiltzius, Stephanie; Satyamurthy, Nagichettiar; Phelps, Michael E.; Radu, Caius; Witte, Owen N.; Braun, Jonathan

2010-01-01

417

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

Microsoft Academic Search

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

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

2003-01-01

418

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

Microsoft Academic Search

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

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

1999-01-01

419

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

Microsoft Academic Search

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

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

2007-01-01

420

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

PubMed

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

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

2013-09-02

421

Comparative investigation of adenosine deaminase activity in spleen, thymus, and bone marrow of AKR and C57BL mice  

Microsoft Academic Search

In C57BL mice, with a low incidence of spontaneous leukemia, there is a progressive decrease with age in adenosine deaminase activity in the thymus and an increase in its activity in the spleen, whereas in AKR mice, with a high incidence of spontaneous leukemia, the changes in activity of this emzyme follow aparallel course and activity in the thymus is

A. I. Svirnovskii; V. I. Levin

1971-01-01

422

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

Microsoft Academic Search

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

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

423

Mammalian Homology to Yeast  

NSDL National Science Digital Library

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

1997-01-01

424

''Is Yeast Alive?''  

NSDL National Science Digital Library

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 SocietyÃÂs 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.

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

2006-04-01

425

Chemical transformation of yeast.  

PubMed

Transformation of chemically competent yeast cells is a method for introducing exogenous DNA into living cells. Typically, the DNA is either a plasmid carrying an autonomous replication sequence that allows for propagation or a linear piece of DNA to be integrated into the genome. The DNA usually also carries a marker that allows for selection of successfully transformed cells by plating on the appropriate selective media. PMID:24011057

Bergkessel, Megan; Guthrie, Christine

2013-01-01

426

Glutathione Production in Yeast  

NASA Astrophysics Data System (ADS)

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

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

427

Incorporation of 5-chlorocytosine into mammalian DNA results in heritable gene silencing and altered cytosine methylation patterns  

PubMed Central

Cytosine methylation patterns are essential for the proper control of gene expression in higher vertebrates. Although alterations in methylation patterns are frequently observed in human tumors, neither the mechanisms for establishing methylation patterns during normal development nor the mechanisms leading to pathological alterations of methylation patterns are currently known. While epidemiological studies have implicated inflammation in cancer etiology, a mechanistic link has yet to be established. Investigations of inflammation-mediated DNA damage may have provided important new insights. Our in vitro studies revealed that the inflammation-mediated DNA damage product, 5-chlorocytosine, could direct fraudulent methylation of previously unmethylated CpG sites. The purpose of this study was to recapitulate our in vitro findings by introducing 5-chlorocytosine residues into the DNA of replicating mammalian cells and to examine its impact on gene expression and cytosine methylation patterns. CHO-K1 cells hemizygous for the hprt gene were electroporated with the triphosphates of cytosine [2?-deoxycytidine-5?-triphosphate (dCTP)], 5-methylcytosine [5-methyl-2?-deoxycytidine-5?-triphosphate (MedCTP)] and 5?-chloro-2?-deoxycytidine-5?-triphosphate (CldCTP), and then selected with 6-thioguanine for silencing the hprt gene. Both modified nucleotides, MedCTP and CldCTP, but not unmodified dCTP, silenced hprt gene expression. Subsequent bisulfite pyrosequencing of CpG sites within the hprt promoter region of the selected cells confirmed hypermethylation, although global methylation levels as measured by gas chromatography–mass spectrometry did not change. Modified nucleotide-induced gene silencing could be reversed with 5-aza-2?-deoxycytidine indicating an epigenetic rather than mutagenic alteration. These results provide further evidence that the inflammation damage product 5-chlorocytosine could be a link between inflammation and cancer development.

Lao, Victoria Valinluck; Herring, Jason L.; Kim, Cherine H.; Darwanto, Agus; Soto, Ubaldo; Sowers, Lawrence C.

2009-01-01

428

Characterizing the protonation state of cytosine in transient G·C Hoogsteen base pairs in duplex DNA.  

PubMed

G·C Hoogsteen base pairs can form transiently in duplex DNA and play important roles in DNA recognition, replication, and repair. G·C Hoogsteen base pairs are thought to be stabilized by protonation of cytosine N3, which affords a second key hydrogen bond, but experimental evidence for this is sparse because the proton cannot be directly visualized by X-ray crystallography and nuclear magnetic resonance spectroscopy. Here, we combine NMR and constant pH molecular dynamics simulations to directly investigate the pKa of cytosine N3 in a chemically trapped N1-methyl-G·C Hoogsteen base pair within duplex DNA. Analysis of NMR chemical shift perturbations and NOESY data as a function of pH revealed that cytosine deprotonation is coupled to a syn-to-anti transition in N1-methyl-G, which results in a distorted Watson-Crick geometry at pH >9. A four-state analysis of the pH titration profiles yields a lower bound pKa estimate of 7.2 ± 0.1 for the G·C Hoogsteen base pair, which is in good agreement with the pKa value (7.1 ± 0.1) calculated independently using constant pH MD simulations. Based on these results and pH-dependent NMR relaxation dispersion measurements, we estimate that under physiological pH (pH 7-8), G·C Hoogsteen base pairs in naked DNA have a population of 0.02-0.002%, as compared to 0.4% for A·T Hoogsteen base pairs, and likely exist primarily as protonated species. PMID:23506098

Nikolova, Evgenia N; Goh, Garrett B; Brooks, Charles L; Al-Hashimi, Hashim M

2013-04-29

429

Spatial and Functional Relationships Among Pol V-Associated loci, Pol IV-Dependent siRNAs, and Cytosine Methylation in the Arabidopsis Epigenome  

SciTech Connect

Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%-60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites.

Wierzbicki, A. T.; Cocklin, Ross; Mayampurath, Anoop; Lister, Ryan; Rowley, M. J.; Gregory, Brian D.; Ecker, Joseph R.; Tang, Haixu; Pikaard, Craig S.

2012-08-15

430

Spatial and functional relationships among Pol V-associated loci, Pol IV-dependent siRNAs, and cytosine methylation in the Arabidopsis epigenome  

PubMed Central

Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%–60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites.

Wierzbicki, Andrzej T.; Cocklin, Ross; Mayampurath, Anoop; Lister, Ryan; Rowley, M. Jordan; Gregory, Brian D.; Ecker, Joseph R.; Tang, Haixu; Pikaard, Craig S.

2012-01-01

431

N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.  

PubMed

When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

1982-10-25

432

N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.  

PubMed Central

When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides.

Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

1982-01-01

433

Uptake of cytosine arabinoside (Ara-C) by LSA lymphoma after irradiation in the stationary phase of growth.  

PubMed

An in vitro system was used to assess uptake of cytosine arabinoside by LSA ascites tumor cells growing in vivo. Changes in tumor uptake with aging after depopulation and perturbation of the cell population with 1,000 rad were studied. Both DNA synthesis and drug uptake were highest during early rapid tumor growth and decreased with aging and the stationary phase of growth. However, irradiation caused a second peak of cellular activity and drug uptake. These results indicate that irradiation stimulates regrowth of tumor. PMID:7063699

Young, J A; Maruyama, Y

1982-03-01

434

Effect of Granulocyte Colony-Stimulating Factor on Chemotherapeutic Activity of Cytosine Arabinoside in Acute Leukemic Cell Lines  

Microsoft Academic Search

Recent studies have shown the presence of receptors for granulocyte colony-stimulating factor (G-CSF) on lymphoid leukemic\\u000a cells. To determine the effect of G-CSF on chemotherapeutic activity of cytosine arabinoside (Ara-C) on lymphoid as well as\\u000a myeloid leukemic cells, we evaluated cell counts, apoptosis, and growth inhibition in HL-60, KG-1, Molt-4, Jijoye, and CCRF-CEM\\u000a cell lines after incubation with Ara-C (0.1

Kwang Eun Cha; Soo Young Yoon; Kap No Lee

2001-01-01

435

Transition state structure of E. coli tRNA-specific adenosine deaminase.  

PubMed

Bacterial tRNA-specific adenosine deaminase (TadA) catalyzes the essential deamination of adenosine to inosine at the wobble position of tRNAs and is necessary to permit a single tRNA species to recognize multiple codons. The transition state structure of Escherichia coli TadA was characterized by kinetic isotope effects (KIEs) and quantum chemical calculations. A stem loop of E. coli tRNA(Arg2) was used as a minimized TadA substrate, and its adenylate editing site was isotopically labeled as [1'-(3)H], [5'-(3)H2], [1'-(14)C], [6-(13)C], [6-(15)N], [6-(13)C, 6-(15)N] and [1-(15)N]. The intrinsic KIEs of 1.014, 1.022, 0.994, 1.014 and 0.963 were obtained for [6-(13)C]-, [6-(15)N]-, [1-(15)N]-, [1'-(3)H]-, [5'-(3)H2]-labeled substrates, respectively. The suite of KIEs are consistent with a late SNAr transition state with a complete, pro-S-face hydroxyl attack and nearly complete N1 protonation. A significant N6-C6 dissociation at the transition state of TadA is indicated by the large [6-(15)N] KIE of 1.022 and corresponds to an N6-C6 distance of 2.0 A in the transition state structure. Another remarkable feature of the E. coli TadA transition state structure is the Glu70-mediated, partial proton transfer from the hydroxyl nucleophile to the N6 leaving group. KIEs correspond to H-O and H-N distances of 2.02 and 1.60 A, respectively. The large inverse [5'-(3)H] KIE of -3.7% and modest normal [1'-(3)H] KIE of 1.4% indicate that significant ribosyl 5'-reconfiguration and purine rotation occur on the path to the transition state. The late SNAr transition-state established here for E. coli TadA is similar to the late transition state reported for cytidine deaminase. It differs from the early SNAr transition states described recently for the adenosine deaminases from human, bovine, and Plasmodium falciparum sources. The ecTadA transition state structure reveals the detailed architecture for enzymatic catalysis. This approach should be readily transferable for transition state characterization of other RNA editing enzymes. PMID:18251477

Luo, Minkui; Schramm, Vern L

2008-02-02

436

A label-free fluorescent molecular beacon based on DNA-templated silver nanoclusters for detection of adenosine and adenosine deaminase.  

PubMed

A simple and reliable fluorescent molecular beacon is developed utilizing DNA-templated silver nanoclusters as a signal indicator and adenosine triphosphate (ATP) and adenosine deaminase as mechanical activators. PMID:22543727

Zhang, Min; Guo, Su-Miao; Li, Ying-Ru; Zuo, Peng; Ye, Bang-Ce

2012-04-27

437

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

SciTech Connect

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

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

1987-02-01

438

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.  

PubMed

We have previously described that, in healthy human skeletal muscle, an anti-histidine-proline-rich-glycoprotein (HPRG) antibody selectively binds to type IIB fibers that are well known to contain the highest level of AMP deaminase (AMPD) activity, suggesting an association of the HPRG-like protein to the enzyme isoform M. The present paper reports an immunohistochemical study performed on human skeletal muscle biopsies from patients with AMPD deficiency and carried out utilizing both the anti-HPRG antibody and an anti-AMPD antibody specific for the isoform M. A correlation between the muscle content of the HPRG-like protein and the level of AMPD activity was demonstrated. In the specimens from patients with Acquired AMPD deficiency the HPRG-immunoreactivity was less intense than that shown by the control subjects and was related to the residual AMPD activity. The patients affected by Primary and Coincidental AMPD deficiency, which were characterized by an absence of enzyme activity and AMPD immunoreactivity, showed the lowest HPRG immunoreactivity that was clearly detectable by Western blot analysis, but not by immunohistochemistry. The interpretation of the significance of these observations suggests a physiological mutual dependence between skeletal muscle HPRG and AMPD polypeptides with regard to their stability. PMID:16570231

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

2006-03-29

439

Conservation of yeasts by dehydration  

Microsoft Academic Search

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

Martin Beker; Alexander Rapoport

440

Yeast interactions and wine flavour.  

PubMed

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; nutrien