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1

Yeast Cytosine Deaminase Improves Radiosensitization and Bystander Effect by 5-Fluorocytosine of Human Colorectal Cancer Xenografts1  

Microsoft Academic Search

The efficacy of cancer gene therapy using bacterial cytosine deaminase (bCD)\\/5-fluorocytosine (5-FC) enzyme\\/prodrug strategy is limited by the inefficiency of cytosine deaminase (CD)-catalyzed conversion of 5-FC into 5-fluorouracil (5-FU). We have shown previously that yeast CD (yCD) is more efficient at the conversion of 5-FC than bCD. In the current study, we hypothesized that the increased production of 5-FU by

Els Kievit; Mukesh K. Nyati; Emily Ng; Lauren D. Stegman; Josh Parsels; Brian D. Ross; Alnawaz Rehemtulla; Theodore S. Lawrence

2

Oncolytic Herpes simplex virus expressing yeast cytosine deaminase: relationship between viral replication, transgene expression, prodrug bioactivation  

PubMed Central

Yeast cytosine deaminase (yCD) is a well-characterized prodrug/enzyme system that converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), and has been combined with oncolytic viruses. However, in vivo studies of the interactions between 5-FC bioactivation and viral replication have not been previously reported, nor have the kinetics of transgene expression and the pharmacokinetics of 5-FC and 5-FU. We constructed a replication-conditional HSV-1 expressing yCD and examined cytotoxicity when 5-FC was initiated at different times after viral infection, and observed that earlier 5-FC administration led to greater cytotoxicity than later 5-FC administration in vitro and in vivo. Twelve days of 5-FC administration was superior to 6 days in animal models, but dosing beyond 12 days did not further enhance efficacy. Consistent with the dosing schedule results, both viral genomic DNA copy number and viral titers were observed to peak on Day 3 after viral injection and gradually decrease thereafter. The virus is replication-conditional and was detected in tumors for as long as 2 weeks after viral injection. The maximum relative extent of yCD conversion of 5-FC to 5-FU in tumors was observed on Day 6 after viral injection and it decreased progressively thereafter. The observation that 5-FU generation within tumors did not lead to appreciable levels of systemic 5-FU (<10 ng/ml) is important and has not been previously reported. The approaches used in these studies of the relationship between the viral replication kinetics, transgene expression, prodrug administration and anti-tumor efficacy are useful in the design of clinical trials of armed, oncolytic viruses.

Yamada, Suguru; Kuroda, Toshihiko; Fuchs, Bryan C.; He, Xiaoying; Supko, Jeffrey G.; Schmitt, Anthony; McGinn, Christopher M.; Lanuti, Michael; Tanabe, Kenneth K.

2011-01-01

3

Targeted tumor therapy with a fusion protein of an antiangiogenic human recombinant scFv and yeast cytosine deaminase.  

PubMed

In adults, endothelial cell division occurs only in wound healing, during menstruation, or in diseases such as wet age-related macular degeneration or development of benign or malignant tissues. Angiogenesis is one of the major requirements to supply the fast developing tumor tissue with oxygen and nutrients, and enables it to spread into other tissues far from its origin. We selected the extradomain B (ED-B), a splice variant of fibronectin, which is exclusively expressed in ovaries, uterus, during wound healing, and in tumor tissues, as a target for the development of an innovative antiangiogenic, prodrug-based targeted tumor therapy approach. We designed a fusion protein termed L19CDy-His, consisting of the antibody single chain fragment L19 for targeting ED-B and yeast cytosine deaminase for the conversion of 5-fluorocytosine into cytotoxic 5-fluorouracil. We purified high amounts of the fusion protein from Pichia pastoris that is stable, enzymatically active, and retains 75% of its activity after incubation with human plasma for up to 72 hours. The binding of L19CDy-His to ED-B was confirmed by an enzyme-linked immunosorbent assay and quantified by surface plasmon resonance spectroscopy determining a KD value of 81±7 nM. L19CDy-His successfully decreased cell survival of the murine ED-B-expressing teratocarcinoma cell line F9 upon addition of the prodrug 5-fluorocytosine. Our data demonstrate the suitability of targeting ED-B by L19CDy-His for effective prodrug-based tumor therapy. PMID:22892453

Schellmann, Nicole; Panjideh, Hossein; Fasold, Patricia; Bachran, Diana; Bachran, Christopher; Deckert, Peter M; Fuchs, Hendrik

2012-09-01

4

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

5

Antitumor effects of genetically engineered stem cells expressing yeast cytosine deaminase in lung cancer brain metastases via their tumor-tropic properties.  

PubMed

Although mortality related with primary tumors is approximately 10%, metastasis leads to 90% of cancer-associated death. The majority of brain metastases result from lung cancer, but the metastatic mechanism remains unclear. In general, chemotherapy for treating brain diseases is disrupted by the brain blood barrier (BBB). As an approach to improve treatment of lung cancer metastasis to the brain, we employed genetically engineered stem cells (GESTECs), consisting of neural stem cells (NSCs) expressing a suicide gene. Cytosine deaminase (CD), one of the suicide genes, originating from bacterial (bCD) or yeast (yCD), which can convert the non-toxic prodrug, 5-fluorocytosine (5-FC), into 5-fluorouracil (5-FU), can inhibit cancer cell growth. We examined the therapeutic efficacy and migratory properties of GESTECs expressing yCD, designated as HB1.F3.yCD, in a xenograft mouse model of lung cancer metastasis to the brain. In this model, A549 lung cancer cells were implanted in the right hemisphere of the mouse brain, while CM-DiI pre-stained HB1.F3.yCD cells were implanted in the contralateral brain. Two days after the injection of stem cells, 5-FC was administered via intraperitoneal injection. The tumor-tropic effect of HB1.F3.yCD was evident by fluorescent analysis, in which red-colored stem cells migrated to the lung tumor mass of the contralateral brain. By histological analysis of extracted brain, the therapeutic efficacy of HB1.F3.yCD in the presence of 5-FC was confirmed by the reduction in density and aggressive tendency of lung cancer cells following treatment with 5-FC, compared to a negative control or HB1.F3.yCD injection without 5-FC. Taken together, these results indicate that HB1.F3.yCD expressing a suicide gene may be a new therapeutic strategy for lung cancer metastases to the brain in the presence of a prodrug. PMID:22426744

Yi, Bo-Rim; Kim, Seung U; Kim, Yun-Bae; Lee, Hong Jun; Cho, Myung-Haing; Choi, Kyung-Chul

2012-06-01

6

Cytosine deaminase as a negative selective marker for Arabidopsis  

Microsoft Academic Search

Cytosine deaminase (CD), produced by prokaryotes but not by higher eukaryotes including plants, deaminates cytosine to uracil. The enzyme likewise converts 5-fluorocytosine (5FC), which by itself is not toxic, to 5-fluorouracil (5FU), which is toxic. The Escherichia coli codA-coding sequence encoding CD, together with appropriate regulatory elements, was introduced into Arabidopsis. Neither untransformed controls, nor transgenic plants expressing no CD

Ranian J. Perera; Christian G. Linard; Ethan R. Signer

1993-01-01

7

Emerging implications of nonmammalian cytosine deaminases on cancer therapeutics.  

PubMed

Nonmammalian cytosine deaminases (CDs) have been investigated for last 30 years in the context of cancer therapy. The therapeutic effect of CD is based on its ability to catalyze the conversion of nontoxic prodrug 5-fluorocytosine (5FC) into the anticancer drug 5-fluorouracil (5FU) by deamination of the number 4 carbon of 5FC. This deamination property of CD has been explored to develop innovative therapeutic approach for treatment of cancer. A general overview is needed for the identification of efficient cytosine deaminases for potential use in cancer therapy. In this review, we have discussed about nonmammalian CDs for a variety of prodrug gene/enzyme therapy applications with several recent examples. Finally, we have provided a prospective on the future aspects of CDs and their applications in cancer therapy. PMID:22673971

Yata, Vinod Kumar; Gopinath, P; Ghosh, Siddhartha Sankar

2012-08-01

8

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

9

Replication Protein A (RPA) Hampers the Processive Action of APOBEC3G Cytosine Deaminase on Single-Stranded DNA  

PubMed Central

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

Lada, Artem G.; Prakash, Aishwarya; Borgstahl, Gloria E. O.; Rogozin, Igor B.; Pavlov, Youri I.

2011-01-01

10

Targeted endostatin-cytosine deaminase fusion gene therapy plus 5-fluorocytosine suppresses ovarian tumor growth.  

PubMed

There are currently no effective therapies for cancer patients with advanced ovarian cancer, therefore developing an efficient and safe strategy is urgent. To ensure cancer-specific targeting, efficient delivery, and efficacy, we developed an ovarian cancer-specific construct (Survivin-VISA-hEndoyCD) composed of the cancer specific promoter survivin in a transgene amplification vector (VISA; VP16-GAL4-WPRE integrated systemic amplifier) to express a secreted human endostatin-yeast cytosine deaminase fusion protein (hEndoyCD) for advanced ovarian cancer treatment. hEndoyCD contains an endostatin domain that has tumor-targeting ability for anti-angiogenesis and a cytosine deaminase domain that converts the prodrug 5-fluorocytosine (5-FC) into the chemotherapeutic drug, 5-fluorouracil. Survivin-VISA-hEndoyCD was found to be highly specific, selectively express secreted hEndoyCD from ovarian cancer cells, and induce cancer-cell killing in vitro and in vivo in the presence of 5-FC without affecting normal cells. In addition, Survivin-VISA-hEndoyCD plus 5-FC showed strong synergistic effects in combination with cisplatin in ovarian cancer cell lines. Intraperitoneal (i.p.) treatment with Survivin-VISA-hEndoyCD coupled with liposome attenuated tumor growth and prolonged survival in mice bearing advanced ovarian tumors. Importantly, there was virtually no severe toxicity when hEndoyCD is expressed by Survivin-VISA plus 5-FC compared with CMV plus 5-FC. Thus, the current study demonstrates an effective cancer-targeted gene therapy that is worthy of development in clinical trials for treating advanced ovarian cancer. PMID:22562248

Sher, Y-P; Chang, C-M; Juo, C-G; Chen, C-T; Hsu, J L; Lin, C-Y; Han, Z; Shiah, S-G; Hung, M-C

2013-02-28

11

Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene  

Microsoft Academic Search

AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined

Li-Zong Shen; Wen-Xi Wu; De-Hua Xu; Zhong-Cheng Zheng; Xin-Yuan Liu; Qiang Ding; Yi-Bing Hua; Kun Yao

12

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

13

Stabilization of Aspergillus parasiticus cytosine deaminase by immobilization on calcium alginate beads improved enzyme operational stability.  

PubMed

Cytosine deaminase (CD) from Aspergillus parasiticus, which has half-life of 1.10 h at 37°C, was stabilized by immobilization on calcium alginate beads. The immobilized CD had pH and temperature optimum of 5 and 50°C respectively. The immobilized enzyme also stoichiometrically deaminated Cytosine and 5-fluorocytosine (5-FC) with the apparent K(M) values of 0.60 mM and 0.65 mM respectively, displaying activation energy of 10.72 KJ/mol. The immobilization of native CD on calcium alginate beads gave the highest yield of apparent enzymatic activity of 51.60% of the original activity and the enzymatic activity was lost exponentially at 37°C over 12 h with a half-life of 5.80 h. Hence, the operational stability of native CD can be improved by immobilization on calcium alginate beads. PMID:23030840

Zanna, H; Nok, A J; Ibrahim, S; Inuwa, H M

2013-12-01

14

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

PubMed

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

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

2014-05-29

15

The Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase  

PubMed Central

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

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

2011-01-01

16

Human neural stem cells transduced with IFN-? and cytosine deaminase genes intensify bystander effect in experimental glioma  

Microsoft Academic Search

Previously, we have shown that the genetically modified human neural stem cells (NSCs) show remarkable migratory and tumor-tropic capability to track down brain tumor cells and deliver therapeutic agents with significant therapeutic benefit. Human NSCs that were retrovirally transduced with cytosine deaminase (CD) gene showed remarkable ‘bystander killer effect’ on the glioma cells after application of the prodrug, 5-fluorocytosine (5-FC).

S Ito; A Natsume; S Shimato; M Ohno; T Kato; P Chansakul; T Wakabayashi; S U Kim

2010-01-01

17

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

PubMed

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

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

2014-02-01

18

Nitrogen Regulation of the codBA (Cytosine Deaminase) Operon from Escherichia coli by the Nitrogen Assimilation Control Protein, NAC  

PubMed Central

Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. ?-Galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position ?59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions ?120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions ?83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.

Muse, Wilson B.; Rosario, Christopher J.; Bender, Robert A.

2003-01-01

19

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

20

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

2007-01-01

21

Endogenous APOBEC3A DNA cytosine deaminase is cytoplasmic and nongenotoxic.  

PubMed

APOBEC3A (A3A) is a myeloid lineage-specific DNA cytosine deaminase with a role in innate immunity to foreign DNA. Previous studies have shown that heterologously expressed A3A is genotoxic, suggesting that monocytes may have a mechanism to regulate this enzyme. Indeed, we observed no significant cytotoxicity when interferon was used to induce the expression of endogenous A3A in CD14(+)-enriched primary cells or the monocytic cell line THP-1. In contrast, doxycycline-induced A3A in HEK293 cells caused major cytotoxicity at protein levels lower than those observed when CD14(+) cells were stimulated with interferon. Immunofluorescent microscopy of interferon-stimulated CD14(+) and THP-1 cells revealed that endogenous A3A is cytoplasmic, in stark contrast to stably or transiently transfected A3A, which has a cell-wide localization. A3A constructs engineered to be cytoplasmic are also nontoxic in HEK293 cells. These data combine to suggest that monocytic cells use a cytoplasmic retention mechanism to control A3A and avert genotoxicity during innate immune responses. PMID:23640892

Land, Allison M; Law, Emily K; Carpenter, Michael A; Lackey, Lela; Brown, William L; Harris, Reuben S

2013-06-14

22

Molecular chemotherapy of pancreatic cancer using novel mutant bacterial cytosine deaminase gene.  

PubMed

The combination of molecular chemotherapy with radiation therapy has the potential to become a powerful approach for treatment of pancreatic cancer. We have developed an adenoviral vector (AdbCD-D314A) encoding a mutant bacterial cytosine deaminase (bCD) gene, which converts the prodrug 5-fluorocytosine (5-FC) into the active drug 5-fluorouracil. The aim of this study was to investigate AdbCD-D314A/5-FC-mediated cytotoxicity in vitro and therapeutic efficacy in vivo alone and in combination with radiation against human pancreatic cancer cells and xenografts. AdbCD-D314A/5-FC-mediated cytotoxicity alone and in combination with radiation was analyzed using crystal violet inclusion and clonogenic survival assays. CD enzyme activity was determined by measuring conversion of [3H]5-FC to [3H]5-fluorouracil after adenoviral infection of pancreatic cancer cells in vitro and pancreatic tumor xenografts by TLC. S.c. pancreatic tumor xenografts were used to evaluate the therapeutic efficacy of AdbCD-D314A/5-FC molecular chemotherapy in combination with radiation therapy. AdbCD-D314A infection resulted in increased 5-FC-mediated pancreatic cancer cell killing that correlated with significantly enhanced CD enzyme activity compared with AdbCDwt encoding wild-type of bCD. Animal studies showed significant inhibition of growth of human pancreatic tumors treated with AdbCD-D314A/5-FC in comparison with AdbCDwt/5-FC. Also, a significantly greater inhibition of growth of Panc2.03 and MIA PaCA-2 tumor xenografts was produced by the combination of AdbCD-D314A/5-FC with radiation compared with either agent alone. The results indicate that the combination of AdbCD-D314A/5-FC molecular chemotherapy with radiation therapy significantly enhanced cytotoxicity of pancreatic cancer cells in vitro and increased therapeutic efficacy against human pancreatic tumor xenografts. PMID:18790765

Kaliberova, Lyudmila N; Della Manna, Debbie L; Krendelchtchikova, Valentina; Black, Margaret E; Buchsbaum, Donald J; Kaliberov, Sergey A

2008-09-01

23

Adenosine potentiates the therapeutic effects of neural stem cells expressing cytosine deaminase against metastatic brain tumors.  

PubMed

Tumor-tropic properties of neural stem cells (NSCs) provide a novel approach with which to deliver targeting therapeutic genes to brain tumors. Previously, we developed a therapeutic strategy against metastatic brain tumors using a human NSC line (F3) expressing cytosine deaminase (F3.CD). F3.CD converts systemically administered 5-fluorocytosine (5-FC), a blood-brain barrier permeable nontoxic prodrug, into the anticancer agent 5-fluorouracil (5-FU). In this study, we potentiated a therapeutic strategy of treatment with nucleosides in order to chemically facilitate the endogenous conversion of 5-FU to its toxic metabolite 5-FU ribonucleoside (5-FUR). In vitro, 5-FUR showed superior cytotoxic activity against MDA-MB-435 cancer cells when compared to 5-FU. Although adenosine had little cytotoxic activity, the addition of adenosine significantly potentiated the in vitro cytotoxicity of 5-FU. When MDA-MB?435 cells were co-cultured with F3.CD cells, F3.CD cells and 5-FC inhibited the growth of MDA-MB-435 cells more significantly in the presence of adenosine. Facilitated 5-FUR production by F3.CD was confirmed by an HPLC analysis of the conditioned media derived from F3.CD cells treated with 5-FC and adenosine. In vivo systemic adenosine treatment also significantly potentiated the therapeutic effects of F3.CD cells and 5-FC in an MDA-MB-435 metastatic brain tumor model. Simple adenosine addition improved the antitumor activity of the NSCs carrying the therapeutic gene. Our results demonstrated an increased therapeutic potential, and thereby, clinical applicability of NSC-based gene therapy. PMID:23828015

Kang, Wonyoung; Seol, Ho Jun; Seong, Dong-Ho; Kim, Jandi; Kim, Yonghyun; Kim, Seung U; Nam, Do-Hyun; Joo, Kyeung Min

2013-09-01

24

Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients.  

PubMed

We performed a pilot trial in refractory cancer patients to investigate the feasibility of intratumoral injection of TAPET-CD, an attenuated Salmonella bacterium expressing the E. coli cytosine deaminase gene. A total of three patients received three dose levels of TAPET-CD (3 x 10(6)-3 x 10(7) CFU/m(2)) via intratumoral injection once every 28 days as long as progression of disease or intolerable toxicity was not observed. From days 4 to 14 of each 28 day cycle, patients also received 5-fluorocytosine (5-FC) at a dose of 100 mg/kg/day p.o. divided three times daily. Six cycles of treatment were administered. No significant adverse events clearly attributable to TAPET-CD were demonstrated. Two patients had intratumor evidence of bacterial colonization with TAPET-CD, which persisted for at least 15 days after initial injection. Conversion of 5-FC to 5-fluorouracil (5-FU) as a result of cytosine deaminase expression was demonstrated in these two patients. The tumor to plasma ratio of 5-FU for these two colonized patients was 3.0, demonstrating significantly increased levels of 5-FU at the site of TAPET-CD colonization and insignificant systemic spread of the bacteria. In contrast, the tumor to plasma ratio of 5-FU of the patient who did not show colonization of TAPET-CD was less than 1.0. These results support the principle that a Salmonella bacterium can be utilized as a delivery vehicle of the cytosine deaminase gene to malignant tissue and that the delivered gene is functional (i.e. able to convert 5-FC to 5-FU) at doses at or below 3 x 10(7) CFU/m(2). PMID:14502226

Nemunaitis, John; Cunningham, Casey; Senzer, Neil; Kuhn, Joseph; Cramm, Jennifer; Litz, Craig; Cavagnolo, Robert; Cahill, Ann; Clairmont, Caroline; Sznol, Mario

2003-10-01

25

Antitumor activity of mutant bacterial cytosine deaminase gene for colon cancer  

PubMed Central

AIM: To evaluate bacterial cytosine deaminase (bCD) mutant D314A and 5-fluorocytosine (5-FC) for treatment of colon cancer in a mouse model. METHODS: Recombinant lentivirus vectors that contained wild-type bCD gene (bCDwt), and bCD mutant D314A gene (bCD-D314A) with green fluorescence protein gene were constructed and used to infect human colon carcinoma LoVo cells, to generate stable transfected cells, LoVo/null, LoVo/bCDwt or LoVo/bCD-D314A. These were injected subcutaneously into Balb/c nude mice to establish xenograft models. Two weeks post-LoVo cell inoculation, PBS or 5-FC (500 mg/kg) was administered by intraperitoneal (i.p.) injection once daily for 14 d. On the day after LoVo cell injection, mice were monitored daily for tumor volume and survival. RESULTS: Sequence analyses confirmed the construction of recombinant lentiviral plasmids that contained bCDwt or bCD-D314A. The lentiviral vector had high efficacy for gene delivery, and RT-PCR showed that bCDwt or bCD-D314A gene was transferred to LoVo cells. Among these treatment groups, gene delivery or 5-FC administration alone had no effect on tumor growth. However, bCDwt/5-FC or bCD-D314A/5-FC treatment inhibited tumor growth and prolonged survival of mice significantly (P < 0.05). Importantly, the tumor volume in the bCD-D314A/5-FC-treated group was lower than that in the bCDwt/5-FC group (P < 0.05), and bCD-D314A plus 5-FC significantly prolonged survival of mice in comparison with bCDwt plus 5-FC (P < 0.05). CONCLUSION: The bCD mutant D314A enhanced significantly antitumor activity in human colon cancer xenograft models, which provides a promising approach for human colon carcinoma therapy.

Deng, Long-Ying; Wang, Jian-Ping; Gui, Zhi-Fu; Shen, Li-Zong

2011-01-01

26

Pyrimidine, purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K12. Involvement of the glnLG and purR genes in the regulation of codA expression  

Microsoft Academic Search

Cytosine deaminase, encoded by the codA gene in Escherichia coli catalyzes the deamination of cytosine to uracil and ammonia. Regulation of codA expression was studied by determining the level of cytosine deaminase in E. coli K12 grown in various defined media. Addition of either pyrimidine or purine nucleobases to the growth medium caused repressed enzyme levels, whereas growth on a

Lennart Andersen; Mogens Kilstrup; Jan Neuhard

1989-01-01

27

Bystander effect from cytosine deaminase and uracil phosphoribosyl transferase genes in vitro: a partial contribution of gap junctions.  

PubMed

Among gene therapy strategies elaborated to kill cancer cells, one uses the CodA gene, coding for cytosine deaminase (CD) that converts 5-fluorocytosine (5-FC) into toxic 5-fluorouracil (5-FU). To enhance 5-FC metabolic activation, we prepared a vector carrying CodA and upp (uracil phosphoribosyl transferase) genes which rendered HeLa cells sensitive to 5-FC and enhanced a bystander effect not mediated by gap junctions. However, 1% CD(+)-UPP(+) cells were able to kill 40% of the cell population if the cells were communicating. This suggests that, at very low percentages of CD(+)-UPP(+) cells, CodA and upp induce a bystander effect through gap junction-dependent mechanisms. PMID:19342154

Tanaka, Toshiaki; Duflot-Dancer, Agnès; Tiraby, Michèle; Piccoli, Colette; Tiraby, Gérard; Yamasaki, Hiroshi; Mesnil, Marc

2009-09-01

28

Quantitation of Cytosine Deaminase mRNA by Real-Time Reverse Transcription Polymerase Chain Reaction: A Sensitive Method for Assessing 5-Fluorocytosine Toxicity in Vitro  

Microsoft Academic Search

Cytosine deaminase\\/5-fluorocytosine (CD\\/5-FC) is a promising strategy for local cancer gene therapy. We hypothesized that CD expression within tumor cells would be directly related to efficacy and that quantitation of markers of CD expression such as mRNA, protein, and enzyme activity would therefore facilitate prediction of 5-FC toxicity. These three markers were thus quantitated by real-time quantitative reverse transcription polymerase

C. Ryan Miller; Allen N. Gustin; Donald J. Buchsbaum; Selwyn M. Vickers; Upender Manne; William E. Grizzle; Gretchen A. Cloud; Robert B. Diasio; Martin R. Johnson

2002-01-01

29

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

30

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

Microsoft Academic Search

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

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

31

Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase.  

PubMed Central

The gene encoding cytosine deaminase (CD) has been expressed in the human colorectal carcinoma cell line WiDr. Metabolism studies confirm that tumor cells expressing CD convert the very nontoxic prodrug 5-fluorocytosine (5FCyt) to 5-fluorouracil (5FUra) and 5FUra metabolites. Tumor xenografts composed of CD-expressing cells can selectively generate tumor levels of > 400 microM 5FUra when the host mouse is dosed with nontoxic levels of 5FCyt. The selective metabolic conversion of 5FCyt to 5FUra in CD-expressing tumor cells results in the inhibition of thymidylate synthase and incorporation of 5FUra into RNA. 5FUra is also liberated into the surrounding environment when CD-expressing tumor cells are treated with 5FCyt. The liberated 5FUra is able to kill neighboring, non-CD-expressing tumor cells in vitro and in vivo. Most importantly, when only 2% of the tumor mass contains CD-expressing cells (98% non-CD-expressing cells), significant regressions in all tumors are observed when the host mouse is dosed with nontoxic levels of 5FCyt.

Huber, B E; Austin, E A; Richards, C A; Davis, S T; Good, S S

1994-01-01

32

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

PubMed Central

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

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

2014-01-01

33

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

SciTech Connect

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

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

2007-08-17

34

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

PubMed

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

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

2013-07-10

35

Thymidine kinase/ganciclovir and cytosine deaminase/5-fluorocytosine suicide gene therapy-induced cell apoptosis in breast cancer cells.  

PubMed

The present study was conducted to explore the efficacy of suicide gene therapy with thymidine kinase (TK) in combination with cytosine deaminase (CD) for breast cancer. The expression of CD/TK was detected in the infected cells by RT-PCR. The killing effect on MCF-7 cells following treatment was analyzed by MTT assay. The morphological characteristics of the cells were observed by electron microscopy, and the distribution of the cell cycle was analyzed by flow cytometry. Caspase?3 and -8 activities were detected by absorption spectrometry. Cytotoxic assays showed that cells transfected with CD/TK became more sensitive to the prodrugs. Morphological features characteristic of apoptosis were noted in the MCF?7 cells via electron microscopy. The experimental data showed that the proportion of MCF-7 cells during the different phases of the cell cycle varied significantly following treatment with the prodrugs. The activity of caspase?3 gradually increased following treatment with increasing concentrations of the prodrugs. We conclude that the TK/ganciclovir and CD/5-fluorocytosine suicide gene system used here induces apoptosis in breast cancer cells, and provides a promising treatment modality for breast cancer. PMID:23799574

Kong, H; Tao, L; Qi, K; Wang, Y; Li, Q; Du, J; Huang, Z

2013-09-01

36

Hypoxia imaging predicts success of hypoxia-induced cytosine deaminase/5-fluorocytosine gene therapy in a murine lung tumor model.  

PubMed

Tc-99m-HL91 is a hypoxia imaging biomarker. The aim of this study was to investigate the value of Tc-99m-HL91 imaging for hypoxia-induced cytosine deaminase (CD)/5-fluorocytosine (5-FC) gene therapy in a murine lung tumor model. C57BL/6 mice were implanted with Lewis lung carcinoma cells transduced with the hypoxia-inducible promoter-driven CD gene (LL2/CD) or luciferase gene (LL2/Luc) serving as the control. When tumor volumes reached 100?mm(3), pretreatment images were acquired after injection of Tc-99m-HL91. The mice were divided into low and high hypoxic groups based on the tumor-to-non-tumor ratio of Tc-99m-HL91. They were injected daily with 5-FC (500?mg?kg(-1)) or the vehicle for 1 week. When tumor volumes reached 1000?mm(3), autoradiography and histological examinations were performed. Treatment with 5-FC delayed tumor growth and enhanced the survival of mice bearing high hypoxic LL2/CD tumors. The therapeutic effect of hypoxia-induced CD/5-FC gene therapy was more pronounced in high hypoxic tumors than in low hypoxic tumors. This study provides the first evidence that Tc-99m-HL91 can serve as an imaging biomarker for predicting the treatment responses of hypoxia-regulated CD/5-FC gene therapy in animal tumor models. Our results suggest that hypoxia imaging using Tc-99m-HL91 has the predictive value for the success of hypoxia-directed treatment regimens. PMID:22281757

Lee, B-F; Lee, C-H; Chiu, N-T; Hsia, C-C; Shen, L-H; Shiau, A-L

2012-04-01

37

Potent inhibitors for the deamination of cytosine arabinoside and 5-aza-2?-deoxycytidine by human cytidine deaminase  

Microsoft Academic Search

Deamination of the nucleoside analogues ARA-C and 5-AZA-CdR by CR deaminase results in a loss of antileukemic activity. To prevent the inactivation of these analogues, inhibitors of CR deaminase may prove to be useful agents. In the present study we investigated the effects of the deaminase inhibitors Zebularine, 5-F-Zebularine, and diazepinone riboside on the deamination of CR, ARA-C, and 5-AZA-CdR

Josée Laliberté; Victor E. Marquez; Richard L. Momparler

1992-01-01

38

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

2013-01-01

39

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

40

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

PubMed

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

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

1995-07-01

41

Systemic Administration of a Recombinant Vaccinia Virus Expressing the Cytosine Deaminase Gene and Subsequent Treatment with 5-Fluorocytosine Leads to Tumor-specific Gene Expression and Prolongation of Survival in Mice1  

Microsoft Academic Search

Suicide gene therapy using the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) has shown promising results for the treatment of colon carcinoma cells in vitro. Efficient viral infection and tumor-specific gene delivery is crucial for clinically measurable treatment effects. After proving efficient gene transfer in vitro, we demonstrate here that genes can be delivered to metastatic liver tumors in vivo

Michael F. X. Gnant; Markus Puhlmann; H. Richard Alexander; David L. Bartlett

1999-01-01

42

Cytotoxic effect of replication-competent adenoviral vectors carrying L-plastin promoter regulated E1A and cytosine deaminase genes in cancers of the breast, ovary and colon  

Microsoft Academic Search

Prodrug activating transcription unit gene therapy is one of several promising approaches to cancer gene therapy. Combining that approach with conditionally replication-competent viral vectors that are truly tumor specific has been an important objective of recent work. In this study, we report the construction of a new conditionally replication-competent bicistronic adenoviral vector in which the cytosine deaminase (CD) gene and

Hakan Akbulut; Lixin Zhang; Yucheng Tang; Albert Deisseroth

2003-01-01

43

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

SciTech Connect

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

Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

2008-02-01

44

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

PubMed

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

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

2014-04-15

45

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

PubMed Central

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

2014-01-01

46

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

PubMed

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

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

2004-01-01

47

Effects of Genetically Engineered Stem Cells Expressing Cytosine Deaminase and Interferon-Beta or Carboxyl Esterase on the Growth of LNCaP Prostate Cancer Cells.  

PubMed

The risk of prostate cancer has been increasing in men by degrees. To develop a new prostate cancer therapy, we used a stem cell-derived gene directed prodrug enzyme system using human neural stem cells (hNSCs) that have a tumor-tropic effect. These hNSCs were transduced with the therapeutic genes for bacterial cytosine deaminase (CD), alone or in combination with the one encoding human interferon-beta (IFN-?) or rabbit carboxyl esterase (CE) to generate HB1.F3.CD, HB1.F3.CD.IFN-?, and HB1.F3.CE cells, respectively. CD enzyme can convert the prodrug 5-fluorocytosine (5-FC) into the activated form 5-fluorouracil (5-FU). In addition, CE enzyme can convert the prodrug CPT-11 into a toxic agent, SN-38. In our study, the human stem cells were found to migrate toward LNCaP human prostate cancer cells rather than primary cells. This phenomenon may be due to interactions between chemoattractant ligands and receptors, such as VEGF/VEGFR2 and SCF/c-Kit, expressed as cancer and stem cells, respectively. The HB1.F3.CE, HB.F3.CD, or HB1.F3.CD.IFN-? cells significantly reduced the LNCaP cell viability in the presence of the prodrugs 5-FC or CPT-11. These results indicate that stem cells expressing therapeutic genes can be used to develop a new strategy for selectively treating human prostate cancer. PMID:23202910

Yi, Bo-Rim; Hwang, Kyung-A; Kim, Yun-Bae; Kim, Seung U; Choi, Kyung-Chul

2012-01-01

48

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

PubMed

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

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

2014-05-01

49

Synergistic effects of genetically engineered stem cells expressing cytosine deaminase and interferon-? via their tumor tropism to selectively target human hepatocarcinoma cells.  

PubMed

Stem cells have received a great deal of attention for their clinical and therapeutic potential for treating human diseases and disorders. Recent studies have shown that it is possible to genetically engineered stem cells (GESTECs) to produce suicide enzymes that convert non-toxic prodrugs to toxic metabolites, selectively migrate toward tumor sites and reduce tumor growth. In this study, we evaluated whether these GESTECs are capable of migrating to hepatocarcinoma cells and examined the potential therapeutic efficacy of gene-directed enzyme prodrug therapy against liver cancer cells in cellular and animal models. A modified transwell migration assay was performed to determine the migratory capacity of GESTECs to Hep3B hepatocarcinoma cells. GESTECs, that is, HB1.F3.CD or HB1.F3.CD.interferon-? (IFN-?) cells, engineered to express a suicide gene, cytosine deaminase (CD), selectively migrated toward liver cancer cells. Treatment of Hep3B, human liver cancer cells, with the prodrug 5-fluorocytosine (5-FC) in the presence of HB1.F3.CD or HB1.F3.CD.IFN-? cells resulted in the inhibition of Hep3B cell growth. In a xenografted mouse model injected with hepatocarcinoma, we investigated the therapeutic effect of these stem cells. For 9 weeks, the xenografted mice were treated with HB1.F3.CD or HB1.F3.CD.IFN-? in the presence of 5-FC. A growth of tumor mass was inhibited about 40-50% in the mice treated with GESTECs and a prodrug. In addition, we further confirmed the cytotoxic effect on tumor cells by histological analysis and migratory effect of therapeutic stem cells. Taken together, GESTECs expressing a fusion gene encoding CD and IFN-? may exert a synergistic antitumor effect on this type of tumor. PMID:22790964

Yi, B-R; Hwang, K-A; Kang, N-H; Kim, S U; Jeung, E-B; Kim, H-C; Choi, K-C

2012-09-01

50

Interaction of endothelial progenitor cells expressing cytosine deaminase in tumor tissues and 5-fluorocytosine administration suppresses growth of 5-fluorouracil-sensitive liver cancer in mice.  

PubMed

The drug delivery system to tumors is a critical factor in upregulating the effect of anticancer drugs and reducing adverse events. Recent studies indicated selective migration of bone marrow-derived endothelial progenitor cells (EPC) into tumor tissues. Cytosine deaminase (CD) transforms nontoxic 5-fluorocytosine (5-FC) into the highly toxic 5-fluorouracil (5-FU). We investigated the antitumor effect of a new CD/5-FC system with CD cDNA transfected EPC for hepatocellular carcinoma (HCC) in mice. We used human hepatoma cell lines (HuH-7, HLF, HAK1-B, KYN-2, KIM-1) and a rat EPC cell line (TR-BME-2). Escherichia coli CD cDNA was transfected into TR-BME-2 (CD-TR-BME). The inhibitory effect of 5-FU on the proliferation of hepatoma cell lines and the inhibitory effect of 5-FU secreted by CD-TR-BME and 5-FC on the proliferation of co-cultured hepatoma cells were evaluated by a tetrazolium-based assay. In mouse subcutaneous xenograft models of KYN-2 and HuH-7, CD-TR-BME was transplanted intravenously followed by 5-FC injection intraperitoneally. HuH-7 cells were the most sensitive to 5-FU and KYN-2 cells were the most resistant. CD-TR-BME secreted 5-FU and inhibited HuH-7 proliferation in a 5-FC dose-dependent manner. CD-TR-BME were recruited into the tumor tissues and some were incorporated into tumor vessels. Tumor growth of HuH-7 was significantly suppressed during 5-FC administration. No bodyweight loss, ALT abnormality or bone marrow suppression was observed. These findings suggest that our new CD/5-FC system with CD cDNA transfected EPC could be an effective and safe treatment for suppression of 5-FU-sensitive HCC growth. PMID:22151662

Torimura, Takuji; Ueno, Takato; Taniguchi, Eitaro; Masuda, Hiroshi; Iwamoto, Hideki; Nakamura, Toru; Inoue, Kinya; Hashimoto, Osamu; Abe, Mitsuhiko; Koga, Hironori; Barresi, Vincenza; Nakashima, Emi; Yano, Hirohisa; Sata, Michio

2012-03-01

51

Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo.  

PubMed

Suicide genes such as cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) encode products that convert nontoxic substances (prodrugs) into toxic metabolites. Suicide gene transfer is currently being used in cancer therapy or can be used as a safety modality. To analyze the reliability of suicide genes as a safety modality for a vaccination study with viable cytokine/B7 gene-modified tumor cells, the individual and combined efficacy of the two suicide genes was compared for in vitro and in vivo cell killing of a murine mammary adenocarcinoma cell line (TS/A). To adapt the system to an in vivo gene delivery situation, bulk cultures cotransfected with the CD and TK gene were used instead of selected clones. In vitro, both CD and TK conferred sensitivity to the respective prodrug but the combined cytotoxic effects of both gene products were always superior. For in vivo analysis BALB/c mice were injected subcutaneously with CD- and TK-modified TS/A cells, treated with prodrugs, and tumor size was evaluated for a period of 100 days. In the in vivo situation the combination of both enzyme/prodrug systems was again most effective. The highest single concentration of 5-FC (500 mg/kg) or GCV (100 mg/kg) was not able to fully protect the animals from developing tumors, whereas a combination of 5-FC (250 mg/kg) and GCV (50 mg/kg) resulted in complete tumor eradication. In nude mice treated in the same way, most CD/TK tumors could not be eliminated. Furthermore, BALB/c mice cured of TS/A-CD/TK tumors developed a systemic tumor immunity against challenge with parental TS/A cells. These findings indicate that reliable tumor elimination by the suicide genes depends on T cells. The cooperative effect of both suicide genes was confirmed in vitro with the human renal cell carcinoma line RCC26. We conclude that TK and CD together, but neither gene alone, act as a safety mechanism for the elimination of tumor cells in a reliable fashion and suggest that a rapid and quantitative antigen release by effective TK- and CD-mediated tumor destruction is necessary for T cell immunity to develop. PMID:9581908

Uckert, W; Kammertöns, T; Haack, K; Qin, Z; Gebert, J; Schendel, D J; Blankenstein, T

1998-04-10

52

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

PubMed

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

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

2014-01-01

53

Human amniotic fluid-derived stem cells expressing cytosine deaminase and thymidine kinase inhibits the growth of breast cancer cells in cellular and xenograft mouse models.  

PubMed

As human amniotic fluid-derived stem cells (hAFSCs) are capable of multiple lineage differentiation, extensive self-renewal and tumor targeting, they may be valuable for clinical anticancer therapies. In this study, we used hAFSCs as vehicles for targeted delivery of therapeutic suicide genes to breast cancer cells. hAFSCs were engineered to produce AF2.CD-TK cells in order to express two suicide genes encoding bacterial cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK) that convert non-toxic prodrugs, 5-fluorocytosine (5-FC) and mono-phosphorylate ganciclovir (GCV-MP), into cytotoxic metabolites, 5-fluorouracil (5-FU) and triphosphate ganciclovir (GCV-TP), respectively. In cell viability test in vitro, AF2.CD-TK cells inhibited the growth of MDA-MB-231 human breast cancer cells in the presence of the 5-FC or GCV prodrugs, or a combination of these two reagents. When the mixture of 5-FC and GCV was treated together, an additive cytotoxic effect was observed in the cell viability. In animal experiments using female BALB/c nude mouse xenografts, which developed by injecting MDA-MB-231 cells, treatment with AF2.CD-TK cells in the presence of 5-FC and GCV significantly reduced tumor volume and weight to the same extent seen in the mice treated with 5-FU. Histopathological and fluorescent staining assays further showed that AF2.CD-TK cells were located exactly at the site of tumor formation. Furthermore, breast tissues treated with AF2.CD-TK cells and two prodrugs maintained their normal structures (for example, the epidermis and reticular layers) while breast tissue structures in 5-FU-treated mice were almost destroyed by the potent cytotoxicity of the drug. Taken together, these results indicate that AF2.CD-TK cells can serve as excellent vehicles in a novel therapeutic cell-based gene-directed prodrug system to selectively target breast malignancies. PMID:22498724

Kang, N-H; Hwang, K-A; Yi, B-R; Lee, H J; Jeung, E-B; Kim, S U; Choi, K-C

2012-06-01

54

Rescue of the Orphan Enzyme Isoguanine Deaminase  

Microsoft Academic Search

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

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

2011-01-01

55

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

2013-06-01

56

The role of protein kinase A in the action of activation-induced deaminase  

Microsoft Academic Search

DNA cytosine deaminases are a newly discovered class of enzymes that convert cytosines in DNA to uracil. Human activation-induced cytidine deaminase (AID) plays a key role in antibody maturation in vertebrates. There are three major genetic events by which antibodies acquire their specificity. They are somatic hypermutation (SHM), Class switch recombination (CSR) and gene conversion (GC). Approximately 10 to 15%

He Huang

2008-01-01

57

Cytosine accumulation as a measure of the proton electrochemical gradient acting on the overexpressed cytosine permease of Saccharomyces cerevisiae.  

PubMed

The magnitude of the proton gradient (delta mu H+) driving solute accumulation in Saccharomyces cerevisiae has long been in doubt, principally because of the lack of an agreed method for assaying its electrical component, the membrane potential (delta psi). In the present work, the size of the cytosine gradient (delta mu cyt) that the yeast generated was used as a measure of the driving gradient (delta mu H+). The selected yeast lacked cytosine deaminase and overexpressed cytosine permease, a 1 H+/cytosine system. delta mu cyt, assayed in washed cell suspensions fermenting glucose and containing 0.5 or 50 mM KCl, was about 260 mV at pH 4 or 5, falling to about 194 mV at pH 7. As a first estimate, -delta mu H+ was thus at least as large at the respective pH value. A 20 mM solution of the lipophilic cation tetraphenylphosphonium lowered delta mu cyt to a value roughly equal to the magnitude of the pH gradient (delta pH). A mathematical model was used to correct the first estimates of delta mu H+ for the effect of cytosine leakage outside the symport. In such a system, delta mu cyt cannot exceed the equivalent ratio Vmax/KmL, where Vmax and Km are kinetic parameters of the symport and L is the rate coefficient for leakage. The feasibility of assaying delta mu H+ depends on it not being much larger than that ratio. The model was tested successfully against observations made with yeast preparations depleted of ATP. After correction, -delta mu H+ during fermentation was estimated to be up to 25 mV larger than delta mu cyt and at least 70 mV larger than previous estimates in the literature involving lipophilic cations. From a knowledge of delta pH, delta psi was in turn deduced and compared with the maximum methylamine gradient (delta mu M) the yeast formed. The results supported the claim in the literature that, at acid pH, delta mu M is a measure of delta psi. PMID:8868419

Eddy, A A; Hopkins, P

1996-03-01

58

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

59

Molecular Structure of Cytosine  

NSDL National Science Digital Library

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

2004-10-12

60

APOBEC3G cytosine deamination hotspots are defined by both sequence context and single-stranded DNA secondary structure.  

PubMed

Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (i.e., APOBEC3G or A3G) is an evolutionarily conserved cytosine deaminase that potently restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons and other viruses. A3G has a nucleotide target site specificity for cytosine dinucleotides, though only certain cytosine dinucleotides are 'hotspots' for cytosine deamination, and others experience little or no editing by A3G. The factors that define these critical A3G hotspots are not fully understood. To investigate how A3G hotspots are defined, we used an in vitro fluorescence resonance energy transfer-based oligonucleotide assay to probe the site specificity of A3G. Our findings strongly suggest that the target single-stranded DNA (ssDNA) secondary structure as well as the bases directly 3' and 5' of the cytosine dinucleotide are critically important A3G recognition. For instance, A3G cannot readily deaminate a cytosine dinucleotide in ssDNA stem structures or in nucleotide base loops composed of three bases. Single-stranded nucleotide loops up to seven bases in length were poor targets for A3G activity unless cytosine residues flanked the cytosine dinucleotide. Furthermore, we observed that A3G favors adenines, cytosines and thymines flanking the cytosine dinucleotide target in unstructured regions of ssDNA. Low cytosine deaminase activity was detected when guanines flanked the cytosine dinucleotide. Taken together, our findings provide the first demonstration that A3G cytosine deamination hotspots are defined by both the sequence context of the cytosine dinucleotide target as well as the ssDNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on processes such as HIV-1 genetic variation. PMID:23620282

Holtz, Colleen M; Sadler, Holly A; Mansky, Louis M

2013-07-01

61

APOBEC3G cytosine deamination hotspots are defined by both sequence context and single-stranded DNA secondary structure  

PubMed Central

Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (i.e., APOBEC3G or A3G) is an evolutionarily conserved cytosine deaminase that potently restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons and other viruses. A3G has a nucleotide target site specificity for cytosine dinucleotides, though only certain cytosine dinucleotides are ‘hotspots’ for cytosine deamination, and others experience little or no editing by A3G. The factors that define these critical A3G hotspots are not fully understood. To investigate how A3G hotspots are defined, we used an in vitro fluorescence resonance energy transfer-based oligonucleotide assay to probe the site specificity of A3G. Our findings strongly suggest that the target single-stranded DNA (ssDNA) secondary structure as well as the bases directly 3? and 5? of the cytosine dinucleotide are critically important A3G recognition. For instance, A3G cannot readily deaminate a cytosine dinucleotide in ssDNA stem structures or in nucleotide base loops composed of three bases. Single-stranded nucleotide loops up to seven bases in length were poor targets for A3G activity unless cytosine residues flanked the cytosine dinucleotide. Furthermore, we observed that A3G favors adenines, cytosines and thymines flanking the cytosine dinucleotide target in unstructured regions of ssDNA. Low cytosine deaminase activity was detected when guanines flanked the cytosine dinucleotide. Taken together, our findings provide the first demonstration that A3G cytosine deamination hotspots are defined by both the sequence context of the cytosine dinucleotide target as well as the ssDNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on processes such as HIV-1 genetic variation.

Holtz, Colleen M.; Sadler, Holly A.; Mansky, Louis M.

2013-01-01

62

Processive DNA Demethylation via DNA Deaminase-Induced Lesion Resolution  

PubMed Central

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

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

2014-01-01

63

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

64

Arxula adeninivorans Recombinant Guanine Deaminase and Its Application in the Production of Food with Low Purine Content.  

PubMed

Purines of exogenous and endogenous sources are degraded to uric acid in human beings. Concentrations >6.8 mg uric acid/dl serum cause hyperuricemia and its symptoms. Pharmaceuticals and the reduction of the intake of purine-rich food are used to control uric acid levels. A novel approach to the latter proposition is the enzymatic reduction of the purine content of food by purine-degrading enzymes. Here we describe the production of recombinant guanine deaminase by the yeast Arxula adeninivorans LS3 and its application in food. In media supplemented with nitrogen sources hypoxanthine or adenine, guanine deaminase (AGDA) gene expression is induced and intracellular accumulation of guanine deaminase (Agdap) protein occurs. The characteristics of the guanine deaminase isolated from wild-type strain LS3 and a transgenic strain expressing the AGDA gene under control of the strong constitutive TEF1 promoter were determined and compared. Both enzymes were dimeric and had temperature optima of 55°C with high substrate specificity for guanine and localisation in both the cytoplasm and vacuole of yeast. The enzyme was demonstrated to reduce levels of guanine in food. A mixture of guanine deaminase and other purine degradation enzymes will allow the reduction of purines in purine-rich foods. © 2014 S. Karger AG, Basel. PMID:24481069

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

2014-01-01

65

Arabidopsis MET1 Cytosine Methyltransferase Mutants  

Microsoft Academic Search

We describe the isolation and characterization of two missense mutations in the cytosine-DNA-methyl- transferase gene, MET1, from the flowering plant Arabidopsis thaliana. Both missense mutations, which affect the catalytic domain of the protein, led to a global reduction of cytosine methylation throughout the genome. Surprisingly, the met1-2 allele, with the weaker DNA hypomethylation phenotype, alters a well-conserved residue in methyltransferase

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

2003-01-01

66

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

67

Replication fork collapse and genome instability in a deoxycytidylate deaminase mutant.  

PubMed

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

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

2012-11-01

68

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

69

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

70

Active demethylation in mouse zygotes involves cytosine deamination and base excision repair  

PubMed Central

Background DNA methylation in mammals is an epigenetic mark necessary for normal embryogenesis. During development active loss of methylation occurs in the male pronucleus during the first cell cycle after fertilisation. This is accompanied by major chromatin remodelling and generates a marked asymmetry between the paternal and maternal genomes. The mechanism(s) by which this is achieved implicate, among others, base excision repair (BER) components and more recently a major role for TET3 hydroxylase. To investigate these methylation dynamics further we have analysed DNA methylation and hydroxymethylation in fertilised mouse oocytes by indirect immunofluorescence (IF) and evaluated the relative contribution of different candidate factors for active demethylation in knock-out zygotes by three-dimensional imaging and IF semi-quantification. Results We find two distinct phases of loss of paternal methylation in the zygote, one prior to and another coincident with, but not dependent on, DNA replication. TET3-mediated hydroxymethylation is limited to the replication associated second phase of demethylation. Analysis of cytosine deaminase (AID) null fertilised oocytes revealed a role for this enzyme in the second phase of loss of paternal methylation, which is independent from hydroxymethylation. Investigation into the possible repair pathways involved supports a role for AID-mediated cytosine deamination with subsequent U-G mismatch long-patch BER by UNG2 while no evidence could be found for an involvement of TDG. Conclusions There are two observable phases of DNA demethylation in the mouse zygote, before and coincident with DNA replication. TET3 is only involved in the second phase of loss of methylation. Cytosine deamination and long-patch BER mediated by UNG2 appear to independently contribute to this second phase of active demethylation. Further work will be necessary to elucidate the mechanism(s) involved in the first phase of active demethylation that will potentially involve activities required for early sperm chromatin remodelling.

2013-01-01

71

Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae.  

PubMed Central

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1). Images

McIntosh, E M; Haynes, R H

1986-01-01

72

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

73

Discovery and structure determination of the orphan enzyme isoxanthopterin deaminase .  

PubMed

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

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

2010-05-25

74

De novo synthesis of thymidylate via deoxycytidine in dcd (dCTP deaminase) mutants of Escherichia coli.  

PubMed Central

dcd (dCTP deaminase) mutants of Escherichia coli were reported not to require thymidine for growth even though most of the thymidylate that is synthesized de novo arises from cytosine nucleotides through a pathway involving dCTP deaminase. We found, however, that the fresh introduction of dcd mutations into many strains of E. coli produced a requirement for thymidine for optimum aerobic growth, but the mutants readily reverted to prototrophy via mutations in other genes. One such mutation was in deoA, the gene for deoxyuridine phosphorylase. However, a dcd deo mutant became thymidine dependent once again if a cdd mutation (affecting deoxycytidine deaminase) were introduced. The results indicate that dcd mutants utilize an alternative pathway of TMP synthesis in which deoxycytidine and deoxyuridine are intermediates. A cdd mutation blocks the pathway by preventing the conversion of deoxycytidine to deoxyuridine, whereas a deoA mutation enhances it by sparing deoxyuridine from catabolism. The deoxycytidine must arise from dCTP or dCDP via unknown steps. It is not known to what extent this pathway is utilized in wild-type cells, which, unlike the dcd mutants, do not accumulate dCTP.

Weiss, B; Wang, L

1994-01-01

75

Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways  

PubMed Central

During immunoglobulin (Ig) diversification, activation-induced deaminase (AID) initiates somatic hypermutation and class switch recombination by catalysing the conversion of cytosine to uracil. The synergy between AID and DNA repair pathways is fundamental for the introduction of mutations, however the molecular and biochemical mechanisms underlying this process are not fully elucidated. We describe a novel method to efficiently decipher the composition and activity of DNA repair pathways that are activated by AID-induced lesions. The in vitro resolution (IVR) assay combines AID based deamination and DNA repair activities from a cellular milieu in a single assay, thus avoiding synthetically created DNA-lesions or genetic-based readouts. Recombinant GAL4-AID fusion protein is targeted to a plasmid containing GAL4 binding sites, allowing for controlled cytosine deamination within a substrate plasmid. Subsequently, the Xenopus laevis egg extract provides a source of DNA repair proteins and functional repair pathways. Our results demonstrated that DNA repair pathways which are in vitro activated by AID-induced lesions are reminiscent of those found during AID-induced in vivo Ig diversification. The comparative ease of manipulation of this in vitro systems provides a new approach to dissect the complex DNA repair pathways acting on defined physiologically lesions, can be adapted to use with other DNA damaging proteins (e.g. APOBECs), and provide a means to develop and characterise pharmacological agents to inhibit these potentially oncogenic processes.

Franchini, Don-Marc; Incorvaia, Elisabetta; Rangam, Gopinath; Coker, Heather A.; Petersen-Mahrt, Svend K.

2013-01-01

76

Arabidopsis MET1 cytosine methyltransferase mutants.  

PubMed Central

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

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

2003-01-01

77

Product activation of human erythrocyte AMP deaminase.  

PubMed

IMP was found to activate AMP deaminase in crude glucose-depleted human erythrocyte lysates. Activation of the enzyme by IMP is due to prevention of the inhibitory effect of inorganic phosphate. At 1 mM AMP and 2-3 mM phosphate the addition of 2-5 mM IMP accelerates the AMP deamination two to three times. PMID:9862426

Mosharov, E V; Vitvitsky, V M; Ataullakhanov, F I

1998-11-27

78

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

PubMed

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

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

2013-10-01

79

Crystal and Molecular Structure of N-Methyl Cytosine  

Microsoft Academic Search

IN the nucleic acids, cytosine is connected to a sugar residue through N1 of the pyrimidine ring. Here we describe the results of an X-ray diffraction analysis of crystals of N1-methyl cytosine. The presence of the methyl group makes it likely that the molecule has the same tautomeric form as in the nucleic acids. This molecule crystallizes in hydrogen bonded

F. Scott Mathews; Alexander Rich

1964-01-01

80

Comparative isoschizomer profiling of cytosine methylation: The HELP assay  

Microsoft Academic Search

The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. The intragenomic profile shows most of the genome to be contiguous

Batbayar Khulan; Reid F. Thompson; Kenny Ye; Melissa J. Fazzari; Masako Suzuki; Edyta Stasiek; Maria E. Figueroa; Jacob L. Glass; Quan Chen; Cristina Montagna; Eli Hatchwell; Rebecca R. Selzer; Todd A. Richmond; Roland D. Green; Ari Melnick; John M. Greally

2006-01-01

81

Trace derivatization of cytosine with pentafluorobenzoyl chloride and dimethyl sulfate.  

PubMed Central

Methods are being developed for derivatizing trace amounts of DNA adducts for ultimate determination by gas chromatographic techniques. High-pressure liquid chromatography is used to optimize appropriate derivatization reactions for the determination of cytosine. A single vial reaction scheme involves acylation with electrophoric pentafluorobenzoylchloride followed by alkylation with dimethyl sulfate. Currently, the overall yield for this reaction starting with 50 nmole of cytosine is 59 +/- 4.6%.

Fisher, D H; Adams, J; Giese, R W

1985-01-01

82

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

PubMed Central

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

Scheinker, V S

1976-01-01

83

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

PubMed Central

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 methylation 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, selectively directing de novo DNA methylation via the fusion of C5 DNA methyltransferases to heterologous DNA-binding proteins. The zinc-finger proteins Zif268 and Zip53 can target DNA methylation by M.CviPI or M.SssI 5–52 nt from single zinc-factor binding sites. Modification at specific GC (M.CviPI) or CG (M.SssI) sites is enhanced as much as 20-fold compared with strains expressing either the free enzyme or a fusion protein with the zinc-finger protein moiety unable to bind to DNA. Interestingly, methylation is also selectively targeted as far as 353 nt from the zinc-finger protein binding sites, possibly indicative of looping, nucleosomes or higher-order chromatin structure. These data demonstrate that methylation can be targeted in vivo to a potentially broad range of sequences using specifically engineered zinc-finger proteins. Further more, the selective targeting of methylation by zinc-finger proteins demonstrates that binding of distinct classes of factors can be monitored in living cells.

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

2003-01-01

84

Intraspecies diversity of the industrial yeast strains Saccharomyces cerevisiae and Saccharomyces pastorianus based on analysis of the sequences of the internal transcribed spacer (ITS) regions and the D1/D2 region of 26S rDNA.  

PubMed

We divided industrial yeast strains of Saccharomyces cerevisiae into three groups based on the sequences of their internal transcribed spacer (ITS) regions. One group contained sake yeasts, shochu yeasts, and one bakery yeast, another group contained wine yeasts, and the third group contained beer and whisky yeasts, including seven bakery yeasts. The three groups were distinguished by polymorphisms at two positions, designated positions B and C, corresponding to nucleotide numbers 279 and 301 respectively in the S288C strain. The yeasts in the Japanese group had one thymine at position B and one thymine at position C. The wine yeasts had one thymine at position B and one cytosine at position C. And the beer and whisky yeasts had two thymines at position B and one cytosine at position C. Strains of S. pastorianus were divided into three groups based on the sequences of their 26S rDNA D1/D2 and ITS regions. PMID:17617725

Kawahata, Miho; Fujii, Tsutomu; Iefuji, Haruyuki

2007-07-01

85

The clinical pharmacology of the adenosine deaminase inhibitor 2?-deoxycoformycin  

Microsoft Academic Search

2'-deoxycoformycin (2'-dCF; Pentostatin), a stoichiometric inhibitor of mammalian adenosine deaminase (ado deaminase), exhibits immunosuppressive and antilymphocytic activity in animal test systems. A clinical pharmacology\\/phase I study of 2'-dCF administered as a single agent has been completed (18 patients). Dose levels ranged from 0.1 mg\\/kgx1 to 0.25 mg\\/kg\\/dayx5; ado deaminase and 2'-dCF were measured spectrophotometrically. Plasma decay curves were bi-exponential (a

John F. Smyth; Rosanne M. Paine; Ann L. Jackman; Kenneth R. Harrap; Marvin M. Chassin; Richard H. Adamson; David G. Johns

1980-01-01

86

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

PubMed

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

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

2012-09-17

87

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

88

Serum adenosine deaminase activity in cutaneous anthrax  

PubMed Central

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

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

2014-01-01

89

Serum adenosine deaminase activity in cutaneous anthrax.  

PubMed

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

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

2014-01-01

90

The catalase activity of diiron adenine deaminase  

PubMed Central

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn2+ before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO4. Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [FeII/FeII]-ADE catalyzed the conversion of H2O2 to O2 and H2O. The values of kcat and kcat/Km for the catalase activity are 200 s?1 and 2.4 × 104 M?1 s?1, respectively. [FeII/FeII]-ADE underwent more than 100 turnovers with H2O2 before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with gave = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H2O2 by [FeII/FeII]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.

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

2011-01-01

91

Ionization-induced tautomerization in Cytosine and effect of solvation.  

PubMed

The recent observation of excitation-induced tautomerization in gas-phase cytosine motivated us to investigate the possibility of facile tautomerization in ionized cytosine and the effect of solvation on the tautomerization barriers. The tautomerization mechanisms were characterized at the density functional theory (DFT)/?B97X-D and coupled-cluster singles and doubles (CCSD) levels of theory. Vertical and adiabatic ionization energies (VIEs and AIEs, respectively) of the tautomers of cytosine and the microsolvated species were calculated with the equation-of-motion ionization-potential coupled-cluster (EOM-IP-CCSD) method. We observed that, in microsolvated cytosine, the solvatochromic shifts of the VIEs can be both blue- and red-shifted depending on the tautomers. This is explained by the analysis of the charge-dipole interactions between the cytosine and water molecules. We noticed that, upon ionization, gas-phase tautomerization barriers are reduced by 0-4 kcal/mol, whereas microsolvated (with one water) tautomerization barriers are reduced by 4-5 kcal/mol. We also investigated the tautomerization process in solvation using a continuum model with one active water molecule in the quantum mechanical region. We noticed that, even though bulk solvation has a significant effect on ionization energies, its effect on the ionization-induced tautomerization barrier is minimal. PMID:24955479

Das, Tamal; Ghosh, Debashree

2014-07-17

92

Generation of discrete yeast DNA fragments by endonuclease RI  

Microsoft Academic Search

FINE structure genetic analysis has shown that the ilv1 gene of yeast, Saccharomyces cerevisiae, is multifunctional1-3. The ilv1 gene product, threonine deaminase, shows catalytic activity, as well as participating in multivalent repression of other enzymes involved in isoleucine-valine biosynthetic pathways1-3. A better understanding of the regulatory role of the ilv1 gene product and other proteins, such as isoleucyl-tRNA synthetase, which

Kamalendu Nath; Arthur P. Bollon

1975-01-01

93

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

94

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

95

APOBEC-Mediated Cytosine Deamination Links PIK3CA Helical Domain Mutations to Human Papillomavirus-Driven Tumor Development.  

PubMed

APOBEC3B cytosine deaminase activity has recently emerged as a significant mutagenic factor in human cancer. APOBEC activity is induced in virally infected cells, and APOBEC signature mutations occur at high frequency in cervical cancers (CESC), over 99% of which are caused by human papillomavirus (HPV). We tested whether APOBEC-mediated mutagenesis is particularly important in HPV-associated tumors by comparing the exomes of HPV+ and HPV- head and neck squamous cell carcinomas (HNSCCs) sequenced by The Cancer Genome Atlas project. As expected, HPV- HNSCC displays a smoking-associated mutational signature, whereas our data suggest that reduced exposure to exogenous carcinogens in HPV+ HNSCC creates a selective pressure that favors emergence of tumors with APOBEC-mediated driver mutations. Finally, we provide evidence that APOBEC activity is responsible for the generation of helical domain hot spot mutations in the PIK3CA gene across multiple cancers. Our findings implicate APOBEC activity as a key driver of PIK3CA mutagenesis and HPV-induced transformation. PMID:24910434

Henderson, Stephen; Chakravarthy, Ankur; Su, Xiaoping; Boshoff, Chris; Fenton, Tim Robert

2014-06-26

96

Compositional dynamics of guanine and cytosine content in prokaryotic genomes  

Microsoft Academic Search

Nucleotide compositional analyses of disparities in genomic guanine and cytosine (gGC) content directly relate to the amino acid composition, through the union of the genetic code. Here we analyzed 229 prokaryotic genomes to address the intricate relationships between gGC, amino acids and their codons in the context of genes. First, we not only confirmed the universal rule that the average

Jianfei Hu; Xiaoqian Zhao; Zhang Zhang; Jun Yu

2007-01-01

97

Genetic and biochemical consequences of adenosine deaminase deficiency in humans.  

PubMed

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

Bose, Rahul; Nandagopal, Krishnadas

2013-10-01

98

[Serum adenosine deaminase activity in pulmonary tuberculosis].  

PubMed

Adenosine deaminase (ADA) activity has been helpful for the diagnosis of tuberculous pleurisy. However, there are few studies about the role of ADA in the diagnosis and follow-up of pulmonary tuberculosis. In our study, serum ADA activity was determined in order to investigate the role of the enzyme in the diagnosis of pulmonary tuberculosis and monitoring the efficiency of therapy. The ADA activity was (mean +/- SD) 21.77 +/- 8.51 U/L in pulmonary tuberculosis patients (n= 44), 6.24 +/- 3.25 U/L in old tuberculosis patients (n= 24), 8.58 +/- 4.38 U/L in healthy control subjects (n= 20), whereas the mean for the patients with bronchial cancer (n= 20) was 18.51 +/- 7.85 U/L. There was no statistical difference between the results of pulmonary tuberculosis patients and the patients with bronchial cancer. On the contrary, the result of these two group were significantly different from both old tuberculosis patients and healthy control subjects (p< 0.001 for both). In 10 pulmonary tuberculosis patients, ADA activities were determined both before and after treatment and a significant decrease was observed in ADA activities after treatment (p< 0.001). In conclusion, serum ADA activity is increased in pulmonary tuberculosis patients, therefore it may be a helpful parameter for monitoring therapy. PMID:15143406

Alata?, Füsun; Uslu, Sema; Moral, Hale; Alata?, Ozkan; Metinta?, Muzaffer; Erginel, Sinan; Uçgun, Irfan

2003-01-01

99

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

100

Characterization of cytosine methylated regions and 5-cytosine DNA methyltransferase (Ehmeth) in the protozoan parasite Entamoeba histolytica  

Microsoft Academic Search

The DNA methylation status of the protozoan para- site Entamoeba histolytica was heretofore unknown. In the present study, we developed a new technique, based on the affinity of methylated DNA to 5-methyl- cytosine antibodies, to identify methylated DNA in this parasite. Ribosomal DNA and ribosomal DNA circles were isolated by this method and we con- firmed the validity of our

Ohad Fisher; Rama Siman-Tov; Serge Ankri

2004-01-01

101

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

2011-08-01

102

Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase  

PubMed Central

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 (kcat = 2.0 s?1; kcat/Km = 2.5 × 103 M?1 s?1). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn2+ prior to induction, the purified enzyme was substantially more active for the deamination of adenine with values of kcat and kcat/Km of 200 s?1 and 5 × 105 M?1s?1, respectively. The apo-enzyme was prepared and reconstituted with Fe2+, Zn2+, or Mn2+. 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 within the deaminase sub-family of the amidohydrolase superfamily (AHS) to utilize a binuclear metal center for the catalysis of a deamination reaction. [FeII/FeII]-ADE was oxidized to [FeIII/FeIII]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [FeIII/FeIII]-ADE with dithionite restored the deaminase activity and thus the di-ferrous form of the enzyme is essential for catalytic activity. No evidence for spin-coupling between metal ions was evident by EPR or Mössbauer spectroscopies. 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 based on homology to other members of the amidohydrolase superfamily. Based on 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, 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.

2011-01-01

103

Phosphorylation directly regulates the intrinsic DNA cytidine deaminase activity of activation-induced deaminase and APOBEC3G protein.  

PubMed

The beneficial effects of DNA cytidine deamination by activation-induced deaminase (AID; antibody gene diversification) and APOBEC3G (retrovirus restriction) are tempered by probable contributions to carcinogenesis. Multiple regulatory mechanisms serve to minimize this detrimental outcome. Here, we show that phosphorylation of a conserved threonine attenuates the intrinsic activity of activation-induced deaminase (Thr-27) and APOBEC3G (Thr-218). Phospho-null alanine mutants maintain intrinsic DNA deaminase activity, whereas phospho-mimetic glutamate mutants are inactive. The phospho-mimetic variants fail to mediate isotype switching in activated mouse splenic B lymphocytes or suppress HIV-1 replication in human T cells. Our data combine to suggest a model in which this critical threonine acts as a phospho-switch that fine-tunes the adaptive and innate immune responses and helps protect mammalian genomic DNA from procarcinogenic lesions. PMID:21659520

Demorest, Zachary L; Li, Ming; Harris, Reuben S

2011-07-29

104

High-throughput sequencing of cytosine methylation in plant DNA.  

PubMed

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

Hardcastle, Thomas J

2013-01-01

105

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

PubMed

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

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

1997-05-01

106

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

107

Effects of cytosine methylation on transcription factor binding sites  

PubMed Central

Background DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect the affinity of transcription factors (TFs) for their binding sites (TFBSs). If it is a consequence, then gene repression caused by chromatin modification may be stabilized by DNA methylation. Until now, these two possibilities have been supported only by non-systematic evidence and they have not been tested on a wide range of TFs. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important. Results We found that the methylation profiles of 16.6% of cytosines and the expression profiles of neighboring transcriptional start sites (TSSs) were significantly negatively correlated. We called the CpGs corresponding to such cytosines “traffic lights”. We observed a strong selection against CpG “traffic lights” within TFBSs. The negative selection was stronger for transcriptional repressors as compared with transcriptional activators or multifunctional TFs as well as for core TFBS positions as compared with flanking TFBS positions. Conclusions Our results indicate that direct and selective methylation of certain TFBS that prevents TF binding is restricted to special cases and cannot be considered as a general regulatory mechanism of transcription.

2014-01-01

108

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

109

DNA-templated silver nanoclusters based label-free fluorescent molecular beacon for the detection of adenosine deaminase.  

PubMed

A general and reliable fluorescent molecular beacon is proposed in this work utilizing DNA-templated silver nanoclusters (AgNCs). The fluorescent molecular beacon has been employed for sensitive determination of the concentration of adenosine deaminase (ADA) and its inhibition. A well-designed oligonucleotide containing three functional regions (an aptamer region for adenosine assembly, a sequence complementary to the region of the adenosine aptamer, and an inserted six bases cytosine-loop) is adopted as the core element in the strategy. The enzymatic reaction of adenosine catalyzed by ADA plays a key role as well in the regulation of the synthesis of the DNA-templated AgNCs, i.e. the signal indicator. The intensity of the fluorescence signal may thereby determine the concentration of the enzyme and its inhibitor. The detection limit of the ADA can be lowered to 0.05 UL(-1). Also, 100 fM of a known inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) is enough to present distinguishable fluorescence emission. Moreover, since the fluorescent signal indicator is not required to be bound with the oligonucleotide, this fluorescent molecular beacon may integrate the advantages of both the label-free and signal-on strategies. PMID:24035856

Zhang, Kai; Wang, Ke; Xie, Minhao; Zhu, Xue; Xu, Lan; Yang, Runlin; Huang, Biao; Zhu, Xiaoli

2014-02-15

110

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

PubMed Central

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.

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

2013-01-01

111

Cytosine photoproduct-DNA glycosylase in Escherichia coli and cultured human cells  

SciTech Connect

Ultraviolet irradiation of DNA produces a variety of pyrimidine base damages. The activities of Escherichia coli endonuclease III and a human lymphoblast endonuclease that incises ultraviolet-irradiated DNA at modified cytosine moieties were compared. Both the bacterial and human enzymes release this cytosine photoproduct as a free base. These glycosylase activities are linear with times of reaction, quantities of enzyme, and irradiation dosages of the substrates. Both enzyme activities are similarly inhibited by the addition of monovalent and divalent cations. Analysis by DNA sequencing identified loci of endonucleolytic incision as cytosines. These are neither cyclobutane pyrimidine dimers, 6-(1,2-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4(1H,3H)-pyrimidinediones, nor apyrimidinic sites. This cytosine photoproduct is separable from unmodified cytosine by high-performance liquid chromatography. This separation should facilitate identification of this modified cytosine and elucidation of its biological significance.

Weiss, R.B.; Gallagher, P.E.; Brent, T.P.; Duker, N.J. (Temple Univ. School of Medicine, Philadelphia, PA (USA))

1989-02-21

112

Red yeast  

MedlinePLUS

... with this combination.Talk with your health provider.Cyclosporine (Neoral, Sandimmune)Red yeast might affect the muscles. Cyclosporine (Neoral, Sandimmune) might also affect the muscles. Taking ...

113

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, ...

114

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

115

Relationship between adenosine deaminase activity and cytokine-secreting T cells in normal pregnancy  

Microsoft Academic Search

OBJECTIVE:To evaluate the relationship between plasma adenosine deaminase activity and the proportion of cytokine-secreting T cells as causes of changes in adenosine deaminase activity in normal pregnancy.METHODS:Plasma adenosine deaminase activity and the proportions of cytokine-secreting T cells were measured in the peripheral blood of 26 nonpregnant and normal pregnant women in the third trimester. The proportion of CD4-positive T cells

Yoshio Yoneyama; Rintaro Sawa; Shunji Suzuki; Koichi Yoneyama; Daisuke Doi; Tsutomu Araki

2002-01-01

116

Biosynthetic threonine deaminase gene of tomato: isolation, structure, and upregulation in floral organs.  

PubMed

The gene encoding the plant biosynthetic threonine deaminase (Td; EC 4.2.1.16) has been cloned as a result of its unusual upregulation in tomato flowers. The Td gene of tomato encodes a polypeptide of 595 residues, the first 80 of which comprise a putative two-domain transit peptide cleaved at position 51. Comparison of the amino acid sequence with the corresponding enzymes from yeast and bacteria reveals a near identity of the important catalytic regions and greater than 40% overall similarity. The Td gene is unique in the tomato genome and its coding region is interrupted by eight introns. Its expression is greater than 50-fold higher in sepals and greater than 500-fold higher in the rest of the flower than in leaves or roots. Its overexpression, however, is strictly confined to the parenchymal cells of the floral organs. In young tomato leaves, the chloroplast-bound enzyme is found almost exclusively in the subepidermal spongy mesophyll cells. PMID:2011578

Samach, A; Hareven, D; Gutfinger, T; Ken-Dror, S; Lifschitz, E

1991-04-01

117

DNA deaminases induce break-associated mutation showers with implication of APOBEC3B and 3A in breast cancer kataegis  

PubMed Central

Breast cancer genomes have revealed a novel form of mutation showers (kataegis) in which multiple same-strand substitutions at C:G pairs spaced one to several hundred nucleotides apart are clustered over kilobase-sized regions, often associated with sites of DNA rearrangement. We show kataegis can result from AID/APOBEC-catalysed cytidine deamination in the vicinity of DNA breaks, likely through action on single-stranded DNA exposed during resection. Cancer-like kataegis can be recapitulated by expression of AID/APOBEC family deaminases in yeast where it largely depends on uracil excision, which generates an abasic site for strand breakage. Localized kataegis can also be nucleated by an I-SceI-induced break. Genome-wide patterns of APOBEC3-catalyzed deamination in yeast reveal APOBEC3B and 3A as the deaminases whose mutational signatures are most similar to those of breast cancer kataegic mutations. Together with expression and functional assays, the results implicate APOBEC3B/A in breast cancer hypermutation and give insight into the mechanism of kataegis. DOI: http://dx.doi.org/10.7554/eLife.00534.001

Taylor, Benjamin JM; Nik-Zainal, Serena; Wu, Yee Ling; Stebbings, Lucy A; Raine, Keiran; Campbell, Peter J; Rada, Cristina; Stratton, Michael R; Neuberger, Michael S

2013-01-01

118

Alkylation of cytosine and 5-hydroxymethyl-cytosine by methyl methanesulphonate and N-methyl-N-nitrosourea: its relevance to mutagenesis.  

PubMed

The reaction of cytosine and 5-hydroxymethyl-cytosine (OHMeCyt) with a variety of monofunctional alkylating agents has been investigated to evaluate further the possible role of cytosine alkylation in mutagenesis and the possibility that the immunity of T-even phages to mutation by methyl methanesulphonate (MMS) was due to the unreactivity of OHMeCyt towards this agent. Both cytosine and OHMeCyt reacted equally well with the methylating agents MMS and N-methyl-N-nitrosourea (MNU) affording 6% and less than 1% respectively of the 3-substituted derivative. No product was isolated following subjection of the bases to reaction with ethyl methane-sulphonate (EMS), N-ethyl-N-nitrosourea (ENU) or iso-propyl methane-sulphonate (iPMS). PMID:193650

Smith, B J

1977-03-01

119

Enzymic excision of ultraviolet-induced cytosine hydrates from left-handed DNA  

SciTech Connect

Ultraviolet irradiation of DNA produces a variety of pyrimidine modifications. These include cytosine hydrate (5,6-dihydro-6-hydroxycytosine), released as a free base by E. coli endonuclease III. The enzymic excision of cytosine hydrate by this purified enzyme was investigated by assaying release of labeled photoproduct from DNA into the ethanol-soluble fraction. Ultraviolet-irradiated poly(dG-dC):poly(dG-dC), radio-labeled in cytosines, was used as substrate. Shifts between the right-handed B-conformation and the left-handed Z-conformation were effected by heating the polymer in the presence of either Ni(II) or Co(II). Conformational states were determined by ultraviolet circular dichroism. Rates of enzymic cytosine hydrate release did not differ between the different substrate conformations, B-DNA and Z-DNA. Irradiation of left-handed poly(dG-dC):poly(dG-dC) resulted in cytosine hydrate formation. Therefore, neither formation nor enzymic excision of ultraviolet-induced cytosine hydrates are substantially affected by the DNA conformational state. Cytosine hydrates are most likely to occur in alternating purine:pyrimidine sequences. Such segments can adopt the Z-conformation as a result of reactions with chemical carcinogens, the presence of metal ions, or negative superhelicity. These results indicate repair of cytosine hydrates to be likely, regardless of the DNA conformational state.

Duker, N.J. (Temple Univ., Philadelphia, PA (United States))

1990-02-26

120

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

PubMed

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

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

2014-01-18

121

Base-pairing energies of proton-bound homodimers determined by guided ion beam tandem mass spectrometry: application to cytosine and 5-substituted cytosines.  

PubMed

Base-pairing interactions in proton-bound dimers of cytosine (C(+)·C) are the major forces responsible for stabilization of DNA i-motif conformations. Permethylation of cytosine in extended (CCG)·(CGG)n trinucleotide repeats has been shown to cause fragile-X syndrome, the most widespread inherited cause of mental retardation in humans. Oligonucleotides containing 5-bromo- or 5-fluorocytosine can bind to proteins that selectively bind methylated DNA, suggesting that halogenated cytosine damage products can potentially mimic methylation signals. However, the influence of methylation or halogenation on the base-pairing energies (BPEs) of proton-bound dimers of cytosine and their impact on the stability of DNA i-motif conformations is presently unknown. To address this, proton-bound homodimers of cytosine and 5-methyl-, 5-fluoro-, 5-bromo-, and 5-iodocytosine are investigated in detail both experimentally and theoretically. The BPEs of proton-bound homodimers of cytosine and the modified cytosines are measured by threshold collision-induced dissociation (TCID) techniques. 5-Methylation of cytosine is found to increase the BPE and would therefore tend to stabilize DNA i-motif conformations. In contrast, 5-halogenation lowers the BPE. However, the BPEs of the proton-bound 5-halocytosine homodimers examined here still significantly exceed that of Watson-Crick G·C base pairs, such that DNA i-motif conformations should be preserved in the presence of these modifications. Excellent agreement between TCID measured and B3LYP calculated BPEs is found, suggesting that B3LYP calculations can be used to provide reliable energetic predictions for related systems. PMID:24117448

Yang, Bo; Wu, R R; Rodgers, M T

2013-11-19

122

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

EPA Science Inventory

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

123

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

Microsoft Academic Search

A new selective medium has been developed for cells containing the enzyme deoxycytidine deaminase. This medium contains hypoxanthine, aminopterin, and 5-methyldeoxycytidine (HAM medium). To survive in the presence of the aminopterin, the cells must utilize deoxycytidine deaminase to convert the 5-methyldeoxycytidine to thymidine. The cells must also have thymidine kinase and hypoxanthine phosphoribosyltransferase. A mouse cell line deficient in deoxycytidine

Teh-sheng Chan; Cedric Long; Howard Green

1975-01-01

124

Disappearance of porphobilinogen deaminase activity in leaves before the onset of senescence.  

PubMed

The activity of porphobilinogen deaminase was measured in young and senescent or mature leaves of pepper (Capsicum annuum), and poinsettia (Euphorbia pulcherrima). Whereas high activity was found in the crude extracts of the young leaves, almost no activity was found in the extracts of senescent or mature leaves. The decrease in deaminase activity was not due to the presence of an isolatable inhibitor. By purifying the crude enzyme extracts from leaves of different ages on DEAE-cellulose columns it was shown that the decrease in deaminase activity was due to a real decrease in the amount of enzyme. Fruiting also decreased porphobilinogen deaminase activity. Several kinetic constants of the C. annuum deaminase were determined. PMID:16660874

Frydman, R B; Frydman, B

1979-06-01

125

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

126

Base-pairing energies of proton-bound heterodimers of cytosine and modified cytosines: implications for the stability of DNA i-motif conformations.  

PubMed

The DNA i-motif conformation was discovered in (CCG)•(CGG)n trinucleotide repeats, which are associated with fragile X syndrome, the most widespread inherited cause of mental retardation in humans. The DNA i-motif is a four-stranded structure whose strands are held together by proton-bound dimers of cytosine (C(+)•C). The stronger base-pairing interactions in C(+)•C proton-bound dimers as compared to Watson-Crick G•C base pairs are the major forces responsible for stabilization of i-motif conformations. Methylation of cytosine results in silencing of the FMR1 gene and causes fragile X syndrome. However, the influence of methylation or other modifications such as halogenation of cytosine on the base-pairing energies (BPEs) in the i-motif remains elusive. To address this, proton-bound heterodimers of cytosine and 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, and 5-iodocytosine are probed in detail. Experimentally, the BPEs of proton-bound heterodimers of cytosine and modified cytosines are determined using threshold collision-induced dissociation (TCID) techniques. All modifications at the 5-position of cytosine are found to lower the BPE and therefore would tend to destabilize DNA i-motif conformations. However, the BPEs in these proton-bound heterodimers still significantly exceed those of the Watson-Crick G•C and neutral C•C base pairs, suggesting that C(+)•C mismatches are still energetically favored such that i-motif conformations are preserved. Excellent agreement between TCID measured BPEs and B3LYP calculated values is found with the def2-TZVPPD and 6-311+G(2d,2p) basis sets, suggesting that calculations at these levels of theory can be employed to provide reliable energetic predictions for related systems. PMID:24320604

Yang, Bo; Rodgers, M T

2014-01-01

127

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

PubMed Central

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

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

2014-01-01

128

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

129

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

PubMed Central

A number of theories propose that RNA, or an RNA-like substance, played a role in the origin of life. Usually, such hypotheses presume that the Watson–Crick bases were readily available on prebiotic Earth, for spontaneous incorporation into a replicator. Cytosine, however, has not been reported in analyses of meteorites nor is it among the products of electric spark discharge experiments. The reported prebiotic syntheses of cytosine involve the reaction of cyanoacetylene (or its hydrolysis product, cyanoacetaldehyde), with cyanate, cyanogen, or urea. These substances undergo side reactions with common nucleophiles that appear to proceed more rapidly than cytosine formation. To favor cytosine formation, reactant concentrations are required that are implausible in a natural setting. Furthermore, cytosine is consumed by deamination (the half-life for deamination at 25°C is ?340 yr) and other reactions. No reactions have been described thus far that would produce cytosine, even in a specialized local setting, at a rate sufficient to compensate for its decomposition. On the basis of this evidence, it appears quite unlikely that cytosine played a role in the origin of life. Theories that involve replicators that function without the Watson–Crick pairs, or no replicator at all, remain as viable alternatives.

Shapiro, Robert

1999-01-01

130

Deaminase Activity on Single-stranded DNA (ssDNA) Occurs in Vitro when APOBEC3G Cytidine Deaminase Forms Homotetramers and Higher-order Complexes*  

PubMed Central

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

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

2011-01-01

131

Dual base-flipping of cytosines in a CpG dinucleotide sequence.  

PubMed

Simultaneous flipped-out conformation of two neighboring bases on opposite strands of DNAs has been observed in several X-ray structures. It has also been detected for two cytosines on opposite strands in different contexts of CpG sites. In this paper, we study by MD simulations the dual base flipping of the two cytosines in hemi-methylated CpG site. We calculate the potential of mean force of flipping-out the unmethylated cytosine in three model systems. The first is for DNA bound to the regulatory protein UHRF1. In this case, the methyl-cytosine on the complementary strand is flipped-out into the binding pocket of the SRA domain of the protein. The other two systems are for unbound DNAs in which the methyl-cytosine is either intra-helical or extra-helical. We find that when the methyl-cytosine is flipped-out it is easier to flip-out the other (unmethylated) cytosine on the opposite strand by about 14-16kJ/mol. This lower penalty for dual-base flipping is observed for both the bound and unbound states of the DNA. Analyses of the hydrogen bond network and stacking interactions within the CpG site indicate that the lower penalty is due to stabilization of the dual-base flipped-out conformation via interactions involving the orphan guanines. The results presented in this paper suggest that the extra-helical conformation of the methyl-cytosine recognized by UHRF1 can facilitate the base-flipping process of the target cytosine to be methylated by Dnmt1. PMID:24469333

Bianchi, Caterina; Zangi, Ronen

2014-01-01

132

Intrinsic immunity against retrotransposons by APOBEC cytidine deaminases  

PubMed Central

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

Koito, Atsushi; Ikeda, Terumasa

2013-01-01

133

Threonine deaminase from Salmonella typhimurium. Relationship between regulatory sites.  

PubMed

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

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

1975-02-25

134

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

135

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

ClinicalTrials.gov

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

2014-05-06

136

Detection of single methylated cytosine using junction-forming DNA probes.  

PubMed

DNA methylation is an epigenetic mechanism for transcriptional regulation. The methylation process controls cellular differentiation and is defective in many diseases including cancer. Therefore, the development of a simple method for analysing cytosine methylation in a target gene is required. Here we report a conceptually new method for sequence-selective chemical modification of a single cytosine in single-stranded DNA (ssDNA) using two DNA probes to form a DNA three-way junction with the ssDNA. The method was successfully used in a simple quantitative polymerase-chain-reaction-based assay for discrimination of a single methylated cytosine. PMID:24614975

Takanashi, Kenta; Kato, Teru

2014-03-31

137

Electrophoretic mobility of duplex DNA cross-linked by mechlorethamine at a cytosine-cytosine mismatch pair.  

PubMed

The common nitrogen mustard, mechlorethamine, can form a covalent cross-link between the two bases of a cytosine-cytosine mismatch pair within a DNA duplex. The cross-linked species can be readily separated from DNA monoadducts and unreacted strands using denaturing polyacrylamide gel electrophoresis. Here, using DNA 19 mer duplexes that are mechlorethamine cross-linked at a C(4)-C(35), C(7)-C(32), C(10)-C(29), or C(13)-C(26) mismatch pair, we show that the denaturing polyacrylamide gel electrophoresis mobility of the cross-linked species is particularly sensitive to the proximity of the C-C cross-link to the duplex end. Species that are cross-linked at a C(4)-C(35) mismatch have greater mobilities than those cross-linked at C(7)-C(32) or C(13)-C(26), and the species with a central C(10)-C(29) cross-link have the lowest mobility. The mobility is also dependent on the proximity of the cross-link to a 5'-(32)P-phosphate or a 5'-fluorescein label. We interpret these results in terms of the conformational properties of the cross-linked species in the denaturing gel. The results are consistent with the retention of partial duplex character at the end proximal to the cross-link, with an influence on the mobility of the GC/AT ratio proximal to the cross-link and at the duplex end, and a small but discernible effect of the label. PMID:23334930

Romero, Rebecca M; Rojsittisak, Pornchai; Haworth, Ian S

2013-03-01

138

Essential role for diacylglycerol in protein transport from the yeast Golgi complex  

Microsoft Academic Search

Yeast phosphatidylinositol transfer protein (Secl4p) is required for the production of secretory vesicles from the Golgi. This requirement can be relieved by inactivation of the cytosine 5'diphosphate (CDP)-choline pathway for phosphatidy1choline biosynthesis, indicating that Secl4p is an essential component of a regulatory pathway linking phospholipid metabolism with vesicle trafficking (the Secl4p pathway1-6). Sac1p (refs 7 and 8) is an integral

Brian G. Kearns; Todd P. McGee; Peter Mayinger; Alma Gedvilaite; Scott E. Phillips; Satoshi Kagiwada; Vytas A. Bankaitis

1997-01-01

139

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

PubMed Central

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

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

2001-01-01

140

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

PubMed Central

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

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

1995-01-01

141

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

PubMed Central

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.

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

1989-01-01

142

A Mutant of Uracil DNA Glycosylase That Distinguishes between Cytosine and 5-Methylcytosine.  

PubMed

We demonstrate that a mutant of uracil DNA glycosylase (N123D:L191A) distinguishes between cytosine and methylcytosine. Uracil DNA glycosylase (UDG) efficiently removes uracil from DNA in a reaction in which the base is flipped into the enzyme's active site. Uracil is selected over cytosine by a pattern of specific hydrogen bonds, and thymine is excluded by steric clash of its 5-methyl group with Y66. The N123D mutation generates an enzyme that excises cytosine. This N123D:L191A mutant excises C when it is mispaired with A or opposite an abasic site, but not when it is paired with G. In contrast no cleavage is observed with any substrates that contain 5-methylcytosine. This enzyme may offer a new approach for discriminating between cytosine and 5-methylcytosine. PMID:24740413

Kimber, Scott T; Brown, Tom; Fox, Keith R

2014-01-01

143

A Mutant of Uracil DNA Glycosylase That Distinguishes between Cytosine and 5-Methylcytosine  

PubMed Central

We demonstrate that a mutant of uracil DNA glycosylase (N123D:L191A) distinguishes between cytosine and methylcytosine. Uracil DNA glycosylase (UDG) efficiently removes uracil from DNA in a reaction in which the base is flipped into the enzyme’s active site. Uracil is selected over cytosine by a pattern of specific hydrogen bonds, and thymine is excluded by steric clash of its 5-methyl group with Y66. The N123D mutation generates an enzyme that excises cytosine. This N123D:L191A mutant excises C when it is mispaired with A or opposite an abasic site, but not when it is paired with G. In contrast no cleavage is observed with any substrates that contain 5-methylcytosine. This enzyme may offer a new approach for discriminating between cytosine and 5-methylcytosine.

Kimber, Scott T.; Brown, Tom; Fox, Keith R.

2014-01-01

144

Analysis of activation-induced cytidine deaminase mRNA levels in patients with chronic lymphocytic leukemia with different cytogenetic status.  

PubMed

Activation induced cytidine deaminase (AID) enzyme, which converts cytosine into uracil and is expressed only by activated B lymphocytes, plays a role in B cells in both the mechanisms of somatic hypermutation (SHM) and class switch recombination (CSR). There are studies showing that AID can cause numerous translocations in different lymphoproliferative diseases. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B cells in bone marrow and peripheral blood. The predictability and clinical status of B-CLL are difficult to determine. About 30-50% of patients have chromosomal abnormalities. AID, which is thought to create fraction segments for translocations, might also cause deletions in DNA regions of 17p13, 11q22.3, 13q14 and 13q34 that are associated with prognostic implications in patients with CLL. In this study, the AID gene expression in patients with CLL with and without deletions was investigated. When compared to healthy subjects and patients without deletions, increased levels of AID expression in patients with deletions of 17p13, 11q22.3 or 13q14 were found, but not for the 13q34 region. Our results show that AID expression may be associated with deletions in patients with CLL. PMID:23662991

Gelmez, Metin Y; Teker, Aysegul B A; Aday, Aynur D; Yavuz, Akif S; Soysal, Teoman; Deniz, Gunnur; Aktan, Melih

2014-02-01

145

Safety and efficacy of suicide gene therapy with adenosine deaminase 5-fluorocytosine silmutaneously in in vitro cultures of melanoma and retinal cell lines.  

PubMed

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

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

2014-01-01

146

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

PubMed Central

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

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

2014-01-01

147

Roles, and establishment, maintenance and erasing of the epigenetic cytosine methylation marks in plants.  

PubMed

Heritable information in plants consists of genomic information in DNA sequence and epigenetic information superimposed on DNA sequence. The latter is in the form of cytosine methylation at CG, CHG and CHH elements (where H = A, T orC) and a variety of histone modifications in nucleosomes. The epialleles arising from cytosine methylation marks on the nuclear genomic loci have better heritability than the epiallelic variation due to chromatin marks. Phenotypic variation is increased manifold by epiallele comprised methylomes. Plants (angiosperms) have highly conserved genetic mechanisms to establish, maintain or erase cytosine methylation from epialleles. The methylation marks in plants fluctuate according to the cell/tissue/organ in the vegetative and reproductive phases of plant life cycle. They also change according to environment. Epialleles arise by gain or loss of cytosine methylation marks on genes. The changes occur due to the imperfection of the processes that establish and maintain the marks and on account of spontaneous and stress imposed removal of marks. Cytosine methylation pattern acquired in response to abiotic or biotic stress is often inherited over one to several subsequent generations.Cytosine methylation marks affect physiological functions of plants via their effect(s) on gene expression levels. They also repress transposable elements that are abundantly present in plant genomes. The density of their distribution along chromosome lengths affects meiotic recombination rate, while their removal increases mutation rate. Transposon activation due to loss of methylation causes rearrangements such that new gene regulatory networks arise and genes for microRNAs may originate. Cytosine methylation dynamics contribute to evolutionary changes. This review presents and discusses the available evidence on origin, removal and roles of cytosine methylation and on related processes, such as RNA directed DNA methylation, imprinting, paramutation and transgenerational memory in plants. PMID:24371187

Kumar, Sushil; Kumari, Renu; Sharma, Vishakha; Sharma, Vinay

2013-12-01

148

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

149

Electron attachment to the cytosine-centered DNA single strands: does base stacking matter?  

PubMed

Electron attachment to the trimer of nucleotide, dGpdCpdG, has been investigated by a quantum mechanical approach at a reliable level of theory. The study of the electron attached dGpdCpdG species demonstrates that cytosine contained DNA single strands have a strong tendency to capture low-energy electrons and to form electronically stable cytosine-centered radical anions. The comparative study of the model molecules pdCpdG and dGpdCp reveals that base stacking has little contribution to the adiabatic electron affinity (AEA) of cytosine in DNA single strands. Additionally, the base-base stacking does not affect the vertical detachment energy (VDE) of the cytosine-centered radicals. Intrastrand H-bonding is found to be critical in increasing the values of the AEA and VDE. However, base-base stacking is revealed to be important in enlarging the vertical electron affinity (VEA) of cytosine. The electron attachment to the cytosine moiety intensifies the intrastrand H-bonding between the neighboring G and C bases. This process disrupts the base-base stacking interaction in the radical anion of dGpdCpdG. PMID:22225006

Gu, Jiande; Wang, Jing; Leszczynski, Jerzy

2012-02-01

150

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

PubMed

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

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

2014-06-01

151

High-speed conversion of cytosine to uracil in bisulfite genomic sequencing analysis of DNA methylation.  

PubMed

Bisulfite genomic sequencing is a widely used technique for analyzing cytosine-methylation of DNA. By treating DNA with bisulfite, cytosine residues are deaminated to uracil, while leaving 5-methylcytosine largely intact. Subsequent PCR and nucleotide sequence analysis permit unequivocal determination of the methylation status at cytosine residues. A major caveat associated with the currently practiced procedure is that it takes 16-20 hr for completion of the conversion of cytosine to uracil. Here we report that a complete deamination of cytosine to uracil can be achieved in shorter periods by using a highly concentrated bisulfite solution at an elevated temperature. Time course experiments demonstrated that treating DNA with 9 M bisulfite for 20 min at 90 degrees C or 40 min at 70 degrees C all cytosine residues in the DNA were converted to uracil. Under these conditions, the majority of 5-methylcytosines remained intact. When a high molecular weight DNA derived from a cell line (containing a number of genes whose methylation status was known) was treated with bisulfite under the above conditions and amplified and sequenced, the results obtained were consistent with those reported in the literature. Although some degradation of DNA occurred during this process, the amount of treated DNA required for the amplification was nearly equal to that required for the conventional bisulfite genomic sequencing procedure. The increased speed of DNA methylation analysis with this novel procedure is expected to advance various aspects of DNA sciences. PMID:15871463

Shiraishi, Masahiko; Hayatsu, Hikoya

2004-12-31

152

Ras Signaling in Yeast  

PubMed Central

Since the study of yeast RAS and adenylate cyclase in the early 1980s, yeasts including budding and fission yeasts contributed significantly to the study of Ras signaling. First, yeast studies provided insights into how Ras activates downstream signaling pathways. Second, yeast studies contributed to the identification and characterization of GAP and GEF proteins, key regulators of Ras. Finally, the study of yeast provided many important insights into the understanding of C-terminal processing and membrane association of Ras proteins.

Tamanoi, Fuyuhiko

2011-01-01

153

Pharmacokinetics of cytosine arabinoside in patients with acute myeloid leukaemia.  

PubMed Central

1 The pharmacokinetics of cytosine arabinoside were studied after a single i.v. bolus of 2 mg/kg ara-C in patients with newly diagnosed untreated AML, using a bioassay and GC-MS method to measure the plasma concentrations. 2 Most patients showed a bi- or tri-phasic decline in plasma concentrations with time. Plasma clearance was 3.9 to 18.1 l/min as measured by the GC-MS method, and terminal half-lives varied from 7--107 min. 3 There was poor correlation of the GC-MS assay with the bioassay, probably because the latter was interfered with by the release of endogenous nucleosides from blasts after after ara-C. 4 Plasma concentrations were measured by GC-MS during continuous infusions in 14 patients. Plasma clearances were much lower than after a bolus, 0.39 to 5.25 l/min. 5 There was no correlation of response (remission or fall in peripheral blast count) with exposure to ara-C calculated from infusion dose, clearance and duration of infusion. 6 This study shows that ara-C pharmacokinetics varies markedly from patient to patient and that there is a wide range in the plasma concentrations associated with therapeutic response.

Harris, A L; Potter, C; Bunch, C; Boutagy, J; Harvey, D J; Grahame-Smith, D G

1979-01-01

154

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

155

Prophylactic treatment for cytosine arabinoside-induced keratoconjunctivitis.  

PubMed

High-dose cytosine arabinoside (Ara-C) is known to cause keratoconjunctivitis in a large proportion of patients. Topical steroids are a popular choice of prophylactic treatment. The objective of this survey was to evaluate the type, dose, frequency and duration of drops used in all centres (n = 117) participating in the acute myeloid leukaemia trial 15 (AML 15), and to assess adherence to the trial protocol prescribed guidelines. All centres used prophylactic treatment; however, the dose of Ara-C at which it was initiated varied from 100 to 6000 mg/m(2)/day. All centres used some form of steroid prophylaxis with prednisolone 0.5% being the most commonly used (98/117), but only 6 of the 117 centres initiated treatment at doses recommended in the trial protocol. Ten centres used lubricants in addition to steroids. No centre reported the use of non-steroidal anti-inflammatory drops. The duration of treatment ranged from 2 days prior to commencing Ara-C to 28 days post-treatment. Twenty-two centres reported complications of which preservative allergy was the commonest. There is a large variation in prophylactic treatment practice in the UK. Clear guidance on the use of prophylaxis and further studies are required to ascertain the most effective prophylactic treatment and regimen. PMID:21424238

Patel, A K; Sheehan, W; Jenkins, A; Lane, C; Kell, J

2011-06-01

156

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

2013-05-01

157

Basic defect in the expression of adenosine deaminase in ADA - SCID disease  

Microsoft Academic Search

A specific competitive radioimmunoassay (RIA) was employed to quantify human adenosine deaminase molecules produced in human-Chinese hamster somatic cell hybrids. Studies on a set of hybrids in which the normal and aberrant expressions of adenosine deaminase (assigned earlier to human chromosome 20) were segregating, have demonstrated that in the patient with ADA -SCID disease reported by Herbschleb-Voogt et al (1981a),

Elly Herbschleb-Voogt; Jan-Willem Scholten; P. Meera Khan

1983-01-01

158

Cloning of cDNAs Encoding Mammalian Double-Stranded RNA-Specific Adenosine Deaminase  

Microsoft Academic Search

Double-stranded RNA (dsRNA)-specific adenosine deaminase converts adenosine to inosine in dsRNA. The protein has been purified from calf thymus, and here we describe the cloning of cDNAs encoding both the human and rat proteins as well as a partial bovine clone. The human and rat clones are very similar at the aminoacidlevelexceptattheirNterminiandcontainthreedsRNAbindingmotifs,aputativenucleartargeting signal, and a possible deaminase motif. Antibodies raised

MARY A. O'CONNELL; SABINE KRAUSE; MIYOKO HIGUCHI; J. JUSTIN HSUAN; NICHOLAS F. TOTTY; ANDREAS JENNY; ANDWALTER KELLER

1995-01-01

159

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

Microsoft Academic Search

Recently, considerable interest has been directed to red-fluorescence photodiagnosis of brain and other tumours during surgery using the protoporphyrin IX natural precursor, 5-aminolaevulinic acid. In the present study we focused on the role of the rate-limiting enzyme porphobilinogen deaminase in glioma C6 cell activity, differentiation and sub-cellular distribution. Over-expression of the human housekeeping porphobilinogen deaminase in the glioma cells, using

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

2002-01-01

160

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

161

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

162

Peripheral neuropathy caused by high-dose cytosine arabinoside treatment in a patient with acute myeloid leukemia  

Microsoft Academic Search

The central nervous system toxicity of high-dose cytosine arabinoside is well recognized, but the toxicity of cytosine arabinoside\\u000a in the peripheral nervous system has been infrequently reported. A 49-year-old Japanese man was diagnosed with acute myeloid\\u000a leukemia. After he achieved complete remission, he received high-dose cytosine arabinoside treatment (2?g\\/m2 twice a day for 5 days; total, 20?g\\/m2) as consolidation therapy.

Takeshi Saito; Osamu Asai; Nobuaki Dobashi; Shingo Yano; Hiroshi Osawa; Yutaka Takei; Shinobu Takahara; Yoji Ogasawara; Yuko Yamaguchi; Jiro Minami; Noriko Usui

2006-01-01

163

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

PubMed

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

Dahncke, Kathleen; Witte, Claus-Peter

2013-10-01

164

Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase.  

PubMed

Double-stranded RNA (dsRNA)-specific adenosine deaminase converts adenosine to inosine in dsRNA. The protein has been purified from calf thymus, and here we describe the cloning of cDNAs encoding both the human and rat proteins as well as a partial bovine clone. The human and rat clones are very similar at the amino acid level except at their N termini and contain three dsRNA binding motifs, a putative nuclear targeting signal, and a possible deaminase motif. Antibodies raised against the protein encoded by the partial bovine clone specifically recognize the calf thymus dsRNA adenosine deaminase. Furthermore, the antibodies can immunodeplete a calf thymus extract of dsRNA adenosine deaminase activity, and the activity can be restored by addition of pure bovine deaminase. Staining of HeLa cells confirms the nuclear localization of the dsRNA-specific adenosine deaminase. In situ hybridization in rat brain slices indicates a widespread distribution of the enzyme in the brain. PMID:7862132

O'Connell, M A; Krause, S; Higuchi, M; Hsuan, J J; Totty, N F; Jenny, A; Keller, W

1995-03-01

165

Anti-inflammatory activity of non-nucleoside adenosine deaminase inhibitor FR234938.  

PubMed

Adenosine has anti-inflammatory activity. Adenosine deaminase (EC 3.5.4.4) metabolizes extracellular adenosine, resulting in an exacerbation of inflammation. Consequently, it was hypothesized that adenosine deaminase inhibitors produce anti-inflammatory activity by increasing extracellular adenosine concentration. This group recently developed a non-nucleoside adenosine deaminase inhibitor, FR234938, by using rational structure-based drug design. FR234938 inhibits recombinant human adenosine deaminase enzyme competitively. FR234938 inhibits interleukin (IL)-6-dependent immunoglobulin (Ig) M production by SKW6.4 cells, in the presence of adenosine. Inhibitory effect of FR234938/adenosine combination is blocked by an A2a adenosine receptor antagonist. FR234938 also inhibits anti-type II collagen delayed type hypersensitivity (DTH) in a dose-dependent manner, both in the presence and absence of recombinant human adenosine deaminase. Moreover, FR234938 inhibits tumor necrosis factor (TNF)-alpha and IL-10 production in a lipopolysaccharide (LPS)-induced cytokine production model in mice. These results indicate that FR234938 has potential anti-inflammatory activity. Non-nucleoside adenosine deaminase inhibitor FR234938 has good potential as a new type of anti-rheumatic and anti-inflammatory drug, by modulating host-defense concentrations of adenosine. PMID:16515782

Kuno, Masako; Seki, Nobuo; Tsujimoto, Susumu; Nakanishi, Isao; Kinoshita, Takayoshi; Nakamura, Katsuya; Terasaka, Tadashi; Nishio, Nobuya; Sato, Akihiro; Fujii, Takashi

2006-03-18

166

Real-Time Study of Interactions between Cytosine-Cytosine Pairs in DNA Oligonucleotides and Silver Ions Using Dual Polarization Interferometry.  

PubMed

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

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

2014-04-15

167

Transcriptome-wide target profiling of RNA cytosine methyltransferases using the mechanism-based enrichment procedure Aza-IP.  

PubMed

Cytosine methylation within RNA is common, but its full scope and functions are poorly understood, as the RNA targets of most mammalian cytosine RNA methyltransferases (m(5)C-RMTs) remain uncharacterized. To enable their characterization, we developed a mechanism-based method for transcriptome-wide m(5)C-RMT target profiling. All characterized mammalian m(5)C-RMTs form a reversible covalent intermediate with their cytosine substrate-a covalent linkage that is trapped when conducted on the cytosine analog 5-azacytidine (5-aza-C). We used this property to develop Aza-immunoprecipitation (Aza-IP), a methodology to form stable m(5)C-RMT-RNA linkages in cell culture, followed by IP and high-throughput sequencing, to identify direct RNA substrates of m(5)C-RMTs. Remarkably, a cytosine-to-guanine (C?G) transversion occurs specifically at target cytosines, allowing the simultaneous identification of the precise target cytosine within each RNA. Thus, Aza-IP reports only direct RNA substrates and the C?G transversion provides an important criterion for target cytosine identification, which is not available in alternative approaches. Here we present a step-by-step protocol for Aza-IP and downstream analysis, designed to reveal identification of substrate RNAs and precise cytosine targets of m(5)C-RMTs. The entire protocol takes 40-50 d to complete. PMID:24434802

Khoddami, Vahid; Cairns, Bradley R

2014-02-01

168

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

NASA Astrophysics Data System (ADS)

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

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

169

Identification of direct targets and modified bases of RNA cytosine methyltransferases  

PubMed Central

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 mechanism-based 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 non-coding 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 cytosine RNA methyltransferases, Aza-IP may be generally applicable for target identification.

Khoddami, Vahid; Cairns, Bradley R.

2013-01-01

170

A functional single-nucleotide polymorphism in the human cytidine deaminase gene contributing to ara-C sensitivity.  

PubMed

To test the hypothesis that analyses of drug targets for polymorphism will help to establish gene-based information for the treatment of cancer patients, we investigated the functional single-nucleotide polymorphisms in the human cytidine deaminase (HDCA) gene. The cDNAs from 52 leukaemia/lymphoma samples and 169 control blood samples were direct-sequenced and analysed for the polymorphisms. Three different polymorphisms (A79C, G208A and T435C) were identified in the coding region of the HDCA gene and displayed allelic frequencies of 20.1%, 4.3% and 70.1%, respectively. No association with susceptibility to disease was observed. A novel polymorphism, G208A produced an alanine to threonine substitution (A70T) within the conserved catalytic domain. By introduction of the polymorphic HCDA genes into the yeast CDA-null mutants, the HCDA-70T showed 40% and 32% activity of prototype for cytidine and ara-C substrates, respectively (P < 0.01). The ara-C IC50 value of the yeast transformants carrying HCDA-70T was 757 +/- 33 micromol and was significantly lower (P < 0.01) than that of prototype (941 +/- 58 micromol). This study demonstrated a population characterized with 208A genotype for, which potentially leads one more sensitive to ara-C treatment than prototype. Accumulation of polymorphisms in the genes responsible for drug metabolism and determination of polymorphism-induced biological variations could provide the additional therapeutic strategies in risk-stratified protocols for the treatment of childhood malignancies. PMID:12544510

Yue, Lijie; Saikawa, Yutaka; Ota, Kazuhisa; Tanaka, Motohiro; Nishimura, Ryosei; Uehara, Takahiro; Maeba, Hideaki; Ito, Takashi; Sasaki, Takuma; Koizumi, Shoichi

2003-01-01

171

Ionic and tautomeric composition of cytosine in aqueous solution: resonance and non-resonance Raman spectroscopy study.  

PubMed

A complex experimental and theoretical study of the structural composition of cytosine in water was performed. Raman and resonance Raman spectra of cytosine in acidic, neutral, and alkaline water solutions (pH = 3, 7, and 10, respectively) were recorded at excitation wavelengths of 514, 266, 218, and 200 nm. The temperature dependence of the frequencies and intensities of the resonance Raman bands was obtained in the temperature interval of 4-80 °C. To interpret the experimental Raman and resonance Raman spectra and to determine the structural composition of the water solution of cytosine, the spectra of cytosine and its cation, anion, oxoimine, and hydroxoamine forms were calculated at the B3LYP/6-311++G(d,p) level using the SCRF method. The electronic spectra and geometric parameters of the cytosine and its molecular forms in the excited electronic states close to the excitation wavelengths of the resonance Raman spectra were calculated using the DFT method. The cation exists in the acidic solution together with cytosine and its oxoimine and hydroxoamine tautomeric forms. Cytosine with a small amount of anion dominates in the alkaline medium. The structural composition of the water solution of cytosine is confirmed by the results of quantum-mechanical calculations of the intensity distribution in the resonance Raman spectra. PMID:24215239

Burova, Tatiana G; Ermolenkov, Vladimir V; Ten, Galina N; Kadrov, Dmitriy M; Nurlygaianova, Marina N; Baranov, Victor I; Lednev, Igor K

2013-12-01

172

Diagnostic value of ascites gamma interferon levels in tuberculous peritonitis. Comparison with adenosine deaminase activity.  

PubMed

The value of ascites gamma interferon concentration and ascites adenosine deaminase activity in distinguishing tuberculosis from other causes of ascites was examined in a prospective study of 86 patients with ascites, including 16 with tuberculous peritonitis. Gamma interferon concentration was higher in tuberculous peritonitis than in the other causes of ascites (p less than 0.0001), and a cut-off between 3 and 9 u/ml reached a sensitivity and a specificity of 100%. The mean (+/- SD) gamma interferon level in tuberculous ascites was 39.3 +/- 18.3 u/ml in patients seronegative for HIV and 14.2 +/- 4.7 u/ml in patients with AIDS (p = 0.01). Adenosine deaminase activity in tuberculous ascites was also higher than in the other causes of ascites (p less than 0.0001), and a cut-off of 40 u/l reached a sensitivity of 100% and a specificity of 97%. The two false positives for adenosine deaminase test were true negatives for the gamma interferon test. There was no significant correlation between gamma interferon concentration and adenosine deaminase activity either in tuberculous ascitis or in any other group. This study suggests that ascites gamma interferon determination may be very useful in the screening of tuberculous peritonitis, but its cost makes it advisable to use adenosine deaminase activity as a routine test, at least in areas where tuberculosis is endemic. PMID:1771679

Ribera, E; Martínez Vásquez, J M; Ocaña, I; Ruiz, I; Jimínez, J G; Encabo, G; Segura, R M; Pascual, C

1991-09-01

173

Structural phylogenetic analysis of activation-induced deaminase function.  

PubMed

In mammals, activation-induced deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of Ig genes. SHM and CSR activities require separate regions within AID. A chromosome region maintenance 1 (CRM1)-dependent nuclear export signal (NES) at the AID C terminus is necessary for CSR, and has been suggested to associate with CSR-specific cofactors. CSR appeared late in AID evolution, during the emergence of land vertebrates from bony fish, which only display SHM. Here, we show that AID from African clawed frog (Xenopus laevis), but not pufferfish (Takifugu rubripes), can induce CSR in AID-deficient mouse B cells, although both are catalytically active in bacteria and mammalian cell systems, albeit at decreased level. Like mammalian AID, Takifugu AID is actively exported from the cell nucleus by CRM1, and the Takifugu NES can substitute for the equivalent region in human AID, indicating that all the CSR-essential NES motif functions evolutionarily predated CSR activity. We also show that fusion of the Takifugu AID catalytic domain to the entire human noncatalytic domain restores activity in mammalian cells, suggesting that AID features mapping within the noncatalytic domain, but outside the NES, influence its function. PMID:16785531

Ichikawa, H Travis; Sowden, Mark P; Torelli, Andrew T; Bachl, Jürgen; Huang, Pinwei; Dance, Geoffrey S C; Marr, Shauna H; Robert, Jacques; Wedekind, Joseph E; Smith, Harold C; Bottaro, Andrea

2006-07-01

174

Changes in Serum Adenosine Deaminase Activity during Normal Pregnancy  

PubMed Central

Adenosine deaminase (ADA), an enzyme essential for the differentiation of lymphoid cells, has been used for monitoring diseases with altered immunity. The purpose of this study was to investigate the changes in serum ADA activity throughout normal pregnancy. We measured the catalytic values of serum ADA from 202 normal pregnant women using a commercial kit. Subjects were divided into four groups according to the gestational age in weeks (Gwks) (Group I: 5-9 Gwks [n=58]; Group II: 15-20 Gwks [n= 63]; Group III: 24-30 Gwks [n=34]; Group IV: 30-39 Gwks [n=47]). The serum ADA levels for the Groups I, II, III, and IV were as follows: 20.1±6.9 IU/L, 20.0±7.6 IU/L, 37.9±19.9 IU/L, and 24.5±8.6 IU/L, respectively. The serum ADA activity of group III was significantly higher than the other groups (p<0.05). However, there was no significant correlation between the Gwks and the serum ADA activity. Furthermore, other parameters, such as maternal age (p=0.29), gestational age at delivery (p=0.07), delivery mode (p=0.39), and birth weight (p=0.59) had no correlation with ADA activity. Reference values of serum ADA in normal pregnancy may provide important database for making clinical decisions in pregnancies complicated by conditions where cellular immunity has been altered.

Lee, Soo Jin; Hwang, Han Sung; Kim, Bit Na Rae; Kim, Min A; Lee, Jae Wook; Park, Yong Won

2007-01-01

175

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

176

Functions and Regulation of RNA Editing by ADAR Deaminases  

PubMed Central

One type of RNA editing converts adenosines to inosines (A?I editing) in double-stranded RNA (dsRNA) substrates. A?I RNA editing is mediated by adenosine deaminase acting on RNA (ADAR) enzymes. A?I RNA editing of protein-coding sequences of a limited number of mammalian genes results in recoding and subsequent alterations of their functions. However, A?I RNA editing most frequently targets repetitive RNA sequences located within introns and 5? and 3? untranslated regions (UTRs). Although the biological significance of noncoding RNA editing remains largely unknown, several possibilities, including its role in the control of endogenous short interfering RNAs (esiRNAs), have been proposed. Furthermore, recent studies have revealed that the biogenesis and functions of certain microRNAs (miRNAs) are regulated by the editing of their precursors. Here, I review the recent findings that indicate new functions for A?I editing in the regulation of noncoding RNAs and for interactions between RNA editing and RNA interference mechanisms.

Nishikura, Kazuko

2010-01-01

177

Validity of serum adenosine deaminase in diagnosis of tuberculosis  

PubMed Central

Introduction Tuberculosis is one of the most important infectious causes of death worldwide. Ziehl-Neelsen staining of sputum has high specificity in tuberculosis endemic countries, but modest sensitivity which varies among laboratories. This study was set up to investigate the diagnostic value of serum Adenosine deaminase in diagnosis of tuberculosis. Methods In a cross sectional and prospective study Serums of 200 patients of positive sputum smear, negative sputum smear, extra-pulmonary tuberculosis and bacterial community acquired pneumonia collected from March 2011 to May 2012 were evaluated. The data were analyzed using SPSS software and P-value of <0.05 was considered significant. Results A total of 200 subjects were included in the study designed in four groups. In cut-off value of ?24 U/l for ADA in smear positive patients defined the sensitivity, specificity and positive predictive value 12%, 98% and 86% respectively. In smear negative patients defined the 6%, 98% and 75%, and in extra-pulmonary tuberculosis patients defined the sensitivity 14%, 98% and 88% respectively. Conclusion This study indicated that measurement of serum ADA level do not have enough sensitivity to assist in the diagnoses of tuberculosis patients from other respiratory diseases and not evaluated perform well enough to replace sputum smear microscopy. Thus, this tests have little role in the diagnosis of pulmonary tuberculosis.

Farazi, Aliasghar; Moharamkhani, Ayda; Sofian, Masoome

2013-01-01

178

ADA (adenosine deaminase) gene therapy enters the competition  

SciTech Connect

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

Culliton, B.J.

1990-08-31

179

Purification and characterization of Plasmodium yoelii adenosine deaminase.  

PubMed

Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed K(m) values of 41 ?M and 34 ?M for adenosine and 2'-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with K(i) value of 0.5 ?M. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional -SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA. PMID:21945268

Yadav, Sarika; Saxena, Jitendra Kumar; Dwivedi, U N

2011-12-01

180

Regulation of hypermutation by activation-induced cytidine deaminase phosphorylation  

PubMed Central

Activation-induced cytidine deaminase (AID) initiates Ig class switch recombination and somatic hypermutation by producing U:G mismatches in DNA. These mismatches also have the potential to induce DNA damage including double-stranded breaks and chromosome translocations; therefore, strict regulation of AID is important for maintaining genomic stability. In addition to transcriptional regulation, it has been proposed that phosphorylation can also modulate AID activity. Using a combination of MS and immunochemical approaches we found that 5–15% of the AID expressed in activated B cells was phosphorylated at serine-38 (p38AID). This form of AID was enriched in the chromatin fraction in activated B cells, suggesting a role for phosphorylation in targeting AID to DNA. Consistent with this idea, serine-38 to alanine mutant AID (AIDS38A) showed diminished somatic hypermutation activity on artificial and physiological DNA targets. We conclude that a small fraction of AID is phosphorylated in activated B cells and that the modified form contributes disproportionately to hypermutation.

McBride, Kevin M.; Gazumyan, Anna; Woo, Eileen M.; Barreto, Vasco M.; Robbiani, Davide F.; Chait, Brian T.; Nussenzweig, Michel C.

2006-01-01

181

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

182

Phase I clinical trial of continuous infusion cyclopentenyl cytosine.  

PubMed

Cyclopentenyl cytosine (CPE-C) is an investigational drug that is active against human solid tumor xenografts. The 5'-triphosphate of CPE-C inhibits CTP synthase, and depletes CTP and dCTP pools. We conducted a phase I clinical trial of CPE-C given as a 24-h continuous i.v. infusion every 3 weeks in 26 adults with solid tumors. The starting dose rate, 1 mg/m2 per h, was selected on the basis of both preclinical studies and pharmacokinetic data from two patients obtained after a test dose of 24 mg/m2 CPE-C as an i.v. bolus. Dose escalation was guided by clinical toxicity. A total of 87 cycles were given, and ten patients received four or more cycles. The mean CPE-C steady-state plasma levels (Cpss) increased linearly from 0.4 microM to 3.1 microM at dose levels ranging from 1 to 5.9 mg/m2 per h (actual body weight); the mean total body clearance was 146 +/- 38 ml/min per m2. CPE-C was eliminated by both renal excretion of intact drug and deamination to cyclopentenyl uracil in an apparent 2:1 ratio. CTP synthase activity in intact bone marrow mononuclear cells was inhibited by 58% to 100% at 22 h compared to matched pretreatment samples at all CPE-C dose levels. When all data were combined, flux through CTP synthase was decreased by 89.6% +/- 3.1% at 22 h (mean +/- SE, n = 16), and remained inhibited by 67.6% +/- 7.7% (n = 10) for at least 24 h post-CPE-C infusion. Granulocyte and platelet toxicities were dose-dependent, and dose-limiting myelosuppression occurred during the initial cycle in two of three patients treated with 5.9 mg/m2 per h. Four of 11 patients (4 of 20 cycles) who received 4.7 mg/m2 per h CPE-C experienced hypotension 24-48 h after completion of the CPE-C infusion during their first (n = 2), third (n = 1) and sixth cycles (n = 1), respectively. Two of these patients died with refractory hypotension despite aggressive hydration and cardiopulmonary resuscitation. One of 12 patients (28 total cycles) treated with 3.5 mg/m2 per h CPE-C experienced orthostatic hypotension during cycle 1, and this patient had a second episode of orthostatic hypotension at a lower dose (3.0 mg/m2 per h). Hypotension was not seen in patients receiving < or = 2.5 mg/m2 per h CPE-C.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7554044

Politi, P M; Xie, F; Dahut, W; Ford, H; Kelley, J A; Bastian, A; Setser, A; Allegra, C J; Chen, A P; Hamilton, J M

1995-01-01

183

Yeast Infection (Candidiasis)  

MedlinePLUS

... for adults A A A This is a candida (yeast) infection of the skin folds of the ... infection with the common yeast (or fungus) organism, Candida albicans, which is commonly found in the environment. ...

184

Phenoptosis in yeasts.  

PubMed

The current view on phenoptosis and apoptosis as genetic programs aimed at eliminating potentially dangerous organisms and cells, respectively, is given. Special emphasis is placed on apoptosis (phenoptosis) in yeasts: intracellular defects and a plethora of external stimuli inducing apoptosis in yeasts; distinctive morphological and biochemical hallmarks accompanying apoptosis in yeasts; pro- and antiapoptotic factors involved in yeast apoptosis signaling; consecutive stages of apoptosis from external stimulus to the cell death; a prominent role of mitochondria and other organelles in yeast apoptosis; possible pathways for release of apoptotic factors from the intermembrane mitochondrial space into the cytosol are described. Using some concrete examples, the obvious physiological importance and expediency of altruistic death of yeast cells is shown. Poorly known aspects of yeast apoptosis and prospects for yeast apoptosis study are defined. PMID:22817540

Sukhanova, E I; Rogov, A G; Severin, F F; Zvyagilskaya, R A

2012-07-01

185

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

186

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

187

Identification of the reactive sulfhydryl group of 1-aminocyclopropane-1-carboxylate deaminase.  

PubMed

1-Aminocyclopropane-1-carboxylate (ACC) deaminase, a pyridoxal phosphate enzyme that catalyzes cyclopropane ring-opening and deamination of ACC, formed a quinoid intermediate with D-alanine, as shown by the appearance of a 510-nm absorption band. The presence of D-alanine also stimulated the inactivation of ACC deaminase with iodoacetamide. The increase of absorbance at 510 nm and the stimulation of the enzyme inactivation were temperature-dependent with a critical point at around 20 degrees C, indicating a conformational change of the enzyme. To identify a reactive thiol group, this stimulated inactivation and an iodoacetamide derivative, N-(iodoacetamidoethyl)-1-aminonaphthalene-5- sulfonic acid were used. The residue that was modified by the specific reagent was monitored by absorbance at 350 nm through the digestion by lysylendopeptidase and the fractionation of peptides, and it was located at Cys-162 near the midpoint of the whole peptide chain of the ACC deaminase. PMID:7764364

Honma, M; Kawai, J; Yamada, M

1993-12-01

188

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

PubMed

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

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

2011-05-28

189

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

Microsoft Academic Search

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

Robert Shapiro

1999-01-01

190

pH-controlled reversible drug binding and release using a cytosine-rich hairpin DNA.  

PubMed

Here we report that a cytosine-rich DNA carrier, that oscillates between a hairpin and an i-motif structure in its response to pH variation, can be used as a drug binding and release device. PMID:21677978

Xu, Can; Zhao, Chuanqi; Ren, Jinsong; Qu, Xiaogang

2011-07-28

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

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

Microsoft Academic Search

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

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

1992-01-01

193

The Role of Gene Body Cytosine Modifications in MGMT Expression and Sensitivity to Temozolomide.  

PubMed

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is known to play a role in sensitivity to temozolomide. Promoter hypermethylation of MGMT is commonly used to predict low expression levels of MGMT in gliomas, despite observed discordance between promoter methylation and protein levels. Here, we investigated the functional role of gene body cytosine modification in regulating levels of MGMT gene expression and sensitivity to temozolomide. In 91 human glioblastoma samples, we observed significant variation in MGMT expression levels in patients with an unmethylated promoter, with higher levels of gene body cytosine modification correlating with higher gene expression levels. Furthermore, inducing hypomethylation across the MGMT gene body with decitabine corresponded with decreased levels of MGMT gene expression in lymphoblastoid and glioblastoma cell lines, indicating an important functional role for gene body cytosine modifications in maintaining gene expression. We reasoned that the decrease in MGMT expression induced by decitabine may render resistant glioblastoma cell lines more sensitive to temozolomide. Consistent with this reasoning, we found that the MGMT-expressing glioblastoma cell lines exhibiting an unmethylated MGMT promoter that were pretreated with decitabine became significantly more sensitive to temozolomide. Overall, our results suggest a functional role for gene body cytosine modification in regulating gene expression of MGMT and indicate that pretreating patients whose tumors have an unmethylated MGMT promoter with decitabine before temozolomide treatment may increase their response to therapy. Mol Cancer Ther; 13(5); 1334-44. ©2014 AACR. PMID:24568970

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

2014-05-01

194

Xylose fermentation by yeasts  

Microsoft Academic Search

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

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

1984-01-01

195

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

PubMed

In skeletal muscle, adenosine monophosphate (AMP) is mainly deaminated by AMP deaminase. However, the C34T mutation in the AMPD1 gene severely reduces AMP deaminase activity. Alternatively, intracellular AMP is dephosphorylated to adenosine via cytosolic AMP 5'-nucleotidase (cN-I). In individuals with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with different AMPD1 genotypes [homozygotes for C34T mutation (TT); heterozygotes for C34T mutation (CT); and homozygotes for wild type (CC): diseased controls CC; and normal controls CC]. AMP deaminase activity showed genotype-dependent differences. Total cN activity in normal controls accounted for 57+/-22% of whole homogenate 5'-nucleotidase activity and was not significantly different from the other groups. A weak inverse correlation was found between AMP deaminase and cN-I activities (r2=0.18, p<0.01). There were no significant differences between different groups in the activities of cN-I, whole homogenate 5'-nucleotidase and ectoN, or in cN-I expression on Western blots. No correlation for age, fibre type distribution and AMPD1 genotype was found for whole homogenate nucleotidase, total cN and cN-I using multiple linear regression analysis. There was no gender-specific difference in the activities of whole homogenate nucleotidase, total cN and cN-I. The results indicate no changes in the relative expression or catalytic behaviour of cN-I in AMP deaminase-deficient human skeletal muscle, but suggest that increased turnover of AMP by cN-I in working skeletal muscle is due to higher substrate availability of AMP. PMID:16497164

Hanisch, Frank; Hellsten, Ylva; Zierz, Stephan

2006-01-01

196

Role of adenosine monophosphate deaminase-1 gene polymorphism in patients with congestive heart failure (influence on tumor necrosis factor-alpha level and outcome).  

PubMed

The Cytosin-->thymidin transition at codon 12 of the adenosine monophosphate deaminase-1 (AMPD1) gene results in a complete loss of its catalytic activity. The increased conversion of adenosine monophosphate to adenosine, which in turn attenuates the expression of tumor necrosis factor-alpha (TNF-alpha) expression, has been suggested as a putative mechanism for prolonged survival in patients with congestive heart failure (CHF) carrying the mutant AMPD1 allele. Therefore, the impact of this polymorphism on circulatory TNF-alpha concentrations and outcome in patients with CHF should be studied. The AMPD1 genotype of each patient with CHF (n = 90; idiopathic dilated cardiomyopathy n = 53; coronary artery disease n = 20; other n = 17) was determined by direct sequencing. Serum TNF-alpha concentrations were measured by enzyme-linked immunosorbent assay. We found 66 patients (75.6%) to be homozygous for the wild-type allele (AMPD1 +/+), and 20 patients (22.2%) were heterozygous and 2 were homozygous (2.2%) for the mutant AMPD1 allele (AMPD1 +/- or -/-). TNF-alpha serum concentrations were 4.2 +/- 2.0 pg/ml for the AMPD1 +/+ genotype and 5.3 +/- 2.9 pg/ml for the AMPD1 +/- and -/- genotypes (p = 0.045). A downregulation of TNF-alpha in patients carrying the mutant allele could therefore be not detected. However, Kaplan-Meier analysis demonstrated a significantly prolonged survival without heart transplantation or revival from sudden death in the AMPD1 +/- & -/- group (p = 0.020). Multivariate analysis identified the AMPD1 wild-type genotype as an independent risk factor (odds ratio 9.34, 95% confidence interval 1.78 to 48.96). The mutant AMPD1 allele, in the context of CHF, is associated with a prognostic benefit. The underlying mechanism of TNF-alpha is unrelated. PMID:15135700

Gastmann, Anja; Sigusch, Holger H; Henke, Andreas; Reinhardt, Dirk; Surber, Ralf; Gastmann, Oliver; Figulla, Hans R

2004-05-15

197

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

198

Serum and synovial fluid adenosine deaminase activity in patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis.  

PubMed Central

Adenosine deaminase activity was determined in paired samples of serum and synovial fluid taken from patients with rheumatoid arthritis (n = 12), reactive arthritis (n = 13), and osteoarthritis (n = 7), and the value of this investigation in the diagnosis of synovial swellings was assessed. Increased activity was found in the synovial fluid taken from patients with rheumatoid disease and reactive arthritis, though values were less raised in the latter. Synovial fluid taken from patients with osteoarthritis did not show significantly raised adenosine deaminase activity as compared with that of normal controls (n = 3).

Yuksel, H; Akoglu, T F

1988-01-01

199

Cloning and overproduction of biodegradative threonine deaminase from Escherichia coli W strain.  

PubMed

We have cloned the structural gene (tdcB) of biodegradative threonine deaminase from Escherichia coli W strain by utilizing the polymerase chain reaction. The JM109/pUCTDA strain, which was obtained by transforming E. coli JM109 with a vector plasmid (pUCTDA) containing the cloned tdcB gene, produced a large amount of the enzyme corresponding to more than 5% of the total soluble protein. Amino acid sequence analysis of this recombinant enzyme showed that the amino acid sequence is identical to the nucleotide-deduced sequence of biodegradative threonine deaminase from E. coli K-12. PMID:1586456

Hirose, K; Fujita, M; Takeuchi, M; Yumoto, N; Tokushige, M; Kawata, Y

1992-04-01

200

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

201

Structural and metabolic specificity of methylthiocoformycin for malarial adenosine deaminases  

PubMed Central

Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation. Methylthioadenosine (MTA) is a substrate for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA (Tyler, P. C., Taylor, E. A., Fröhlich, R. G. G. and Schramm, V. L. (2007) J. Am. Chem. Soc. 129, 6872–6879). The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation (Larson, E. T. et al. (2008) J. Mol. Biol. 381, 975–988). Here, the crystal structure of ADA from Plasmodium vivax in complex with MT-coformycin reveals an unprecedented binding geometry for 5’-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5’-methylthioribosyl groups are rotated 130°. A hydrogen bonding network between Asp172 and the 3'-hydroxyl of MT-coformycin is essential for recognition of the 5’-methylthioribosyl group. Water occupies the 5'-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic and structural analyses of PvADA and kinetic analysis of five other plasmodial ADAs establishes the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin and is missing Asp172. Treatment of P. falciparum cultures with coformycin or MT-coformycin in the presence of MTA is effective in inhibiting parasite growth.

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

2009-01-01

202

Diagnostic utility of adenosine deaminase in exudative pleural effusions  

PubMed Central

Objective: To determine the diagnostic utility of adenosine deaminase (ADA) in exudative pleural effusions of different etiologies. Setting and Design: It was an observational study conducted at a tertiary care teaching institute. Materials and Methods: Of a total of 171 pleural fluid samples, 122 were found to be exudates and were included in the study. Pleural fluid ADA was done for all included patients. Pleural fluid ADA ?40 U/l was taken as diagnostic cut off for TB effusion. Statistical Analysis: Sensitivity, specificity positive and negative predictive value of pleural fluid ADA for diagnosing TB was calculated by using clinical calculator – 1, Richard Lowry 2001-2013. Results: There were 171 patients with pleural effusion, out of which 122 (71.8%) were found to be exudative and were studied further. There were 49 (40.1%), 36 (29.5%) and 33 (27%) cases of TB, malignancy and para pneumonic effusion respectively, whereas 4 (3.3%) cases remained undiagnosed. Median ADA values for TB, malignancy and para pneumonic effusion were 55.8 U/l (range 9.7-756 U/l), 18 U/l (6.5-81 U/l) and 25 U/l (3.4-172 U/l) respectively. Pleural fluid ADA >40U/l yielded 85.7% sensitivity, 80.8% specificity, 75% positive predictive value and 89.5% negative predictive value. Conclusion: Pleural fluid ADA remains useful in diagnosing tuberculosis pleural effusion. The median ADA for TB effusion in present cohort was 51.8 IU/ml. Pleural fluid ADA of 40 U/L yielded 89.5% negative predictive value and 75% positive predictive value. Pleural fluid ADA is cost effective and good screening test for diagnosis of TB.

Mehta, Asmita A.; Gupta, Amit Satish; Ahmed, Subin; Rajesh, V.

2014-01-01

203

Activity and immunohistochemical localization of porphobilinogen deaminase in rat tissues.  

PubMed

Porphobilinogen deaminase (PBGD) is an enzyme involved in the synthesis of heme. Acute intermittent porphyria (AIP) is an inherited disease resulting from a reduced activity of PBGD. The symptoms seem to be due to a neurological dysfunction. Attacks of AIP are often provoked by conditions where the PBGD activity becomes insufficient as a result of an increased synthesis of heme in the liver. How this affects the nervous tissue is still unknown. It may well be that a reduced activity of PBGD in other tissues than the liver is of importance too. The aim of the present study was to examine the activity and the immunohistochemical localization of PBGD in the following tissues of wistar female rats: brain, heart, submandibular gland, liver, kidney, pancreas, ovary, stomach, duodenum, jejunum, ileum, colon and musculature. The PBGD activity varied considerably among the tissues. It was highest in the liver, 14 pkat/g, and lowest in the jejunum, 0.7 pkat/g. The immunohistochemical localization of PBGD was studied by antibodies raised against a 40 amino acid synthetic peptide that corresponds to a segment in the C-terminal part of PBGD. The study demonstrated that the PBGD immunoreactivity was not evenly distributed among the various cell types in a given tissue. Immunohistochemical reactions were pronounced in Kupffer cells in the liver, in smooth muscle cells of arteries and arterioles, in distal and collecting tubules in the kidney, in nerve axons in the brain and in ganglionic cells in the intestine. Especially, the immunohistochemical reaction in nerve cells is notable considering the nervous dysfunction in AIP. PMID:11202056

Jørgensen, P E; Erlandsen, E J; Poulsen, S S; Markussen, S; Koch, C; Brock, A

2000-11-01

204

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

2013-07-01

205

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

Microsoft Academic Search

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

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

2006-01-01

206

Adenosine deaminase messenger RNAs in lymphoblast cell lines derived from leukemic patients and patients with hereditary adenosine deaminase deficiency.  

PubMed Central

Hereditary deficiency of adenosine deaminase (ADA) usually causes profound lymphopenia with severe combined immunodeficiency disease. Cells from patients with ADA deficiency contain less than normal, and sometimes undetectable, amounts of ADA catalytic activity and ADA protein. The molecular defects responsible for hereditary ADA deficiency are poorly understood. ADA messenger RNAs and their translation products have been characterized in seven human lymphoblast cell lines derived as follows: GM-130, GM-131, and GM-2184 from normal adults; GM-3043 from a partially ADA deficient, immunocompetent !Kung tribesman; GM-2606 from an ADA deficient, immunodeficient child; CCRF-CEM and HPB-ALL from leukemic children. ADA messenger (m)RNA was present in all lines and was polyadenylated. The ADA synthesized by in vitro translation of mRNA from each line reacted with antisera to normal human ADA and was of normal molecular size. There was no evidence that posttranslational processing of ADA occurred in normal, leukemic, or mutant lymphoblast lines. Relative levels of specific translatable mRNA paralleled levels of ADA protein in extracts of the three normal and two leukemic lines. However, unexpectedly high levels of ADA specific, translatable mRNA were found in the mutant GM-2606 and GM-3043 lines, amounting to three to four times those of the three normal lines. Differences in the amounts of ADA mRNA and rates of ADA synthesis appear to be of primary importance in maintaining the differences in ADA levels among lymphoblast lines with structurally normal ADA. ADA deficiency in at least two mutant cell lines is not caused by deficient levels of translatable mRNA, and unless there is some translational control of this mRNA, the characteristic cellular ADA deficiency is most likely secondary to synthesis and rapid degradation of a defective ADA protein. Images

Adrian, G S; Hutton, J J

1983-01-01

207

Threonine overproduction in yeast strains carrying the HOM3-R2 mutant allele under the control of different inducible promoters.  

PubMed

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

Farfán, M J; Aparicio, L; Calderón, I L

1999-01-01

208

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

PubMed Central

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

Farfan, Maria-Jose; Aparicio, Luis; Calderon, Isabel L.

1999-01-01

209

Active nuclear import and cytoplasmic retention of activation-induced deaminase  

Microsoft Academic Search

The enzyme activation-induced deaminase (AID) triggers antibody diversification in B cells by catalyzing deamination and consequently mutation of immunoglobulin genes. To minimize off-target deamination, AID is restrained by several regulatory mechanisms including nuclear exclusion, thought to be mediated exclusively by active nuclear export. Here we identify two other mechanisms involved in controlling AID subcellular localization. AID is unable to passively

Anne-Marie Patenaude; Alexandre Orthwein; Yi Hu; Vanina A Campo; Bodil Kavli; Alejandro Buschiazzo; Javier M Di Noia

2009-01-01

210

The Activities of Paraoxonase, Xanthine Oxidase, Adenosine Deaminase and the Level of Nitrite in Pseudoexfoliation Syndrome  

Microsoft Academic Search

Aim: Our purpose was to investigate the possible roles of nitrite levels and the activity of paraoxonase (PON), xanthine oxidase (XO) and adenosine deaminase (ADA) in the pathogenesis of pseudoexfoliation (PEX) syndrome. Methods: Serum samples were taken from 43 patients with PEX and 41 control subjects. The serum PON, XO and ADA activities and nitrite levels were assayed by spectrophotometric

Ahmet Gürel; Sunay Duman

2009-01-01

211

Identification of a selective nuclear import signal in adenosine deaminases acting on RNA  

Microsoft Academic Search

The adenosine deaminases acting on RNA (ADARs) comprise a family of RNA editing enzymes that selectively modify single codons within RNA primary transcripts with often profound impact on protein function. Little is known about the mechanisms that regulate nuclear RNA editing activity. Editing levels show cell-type specific and developmental modulation that does not strictly coincide with observed expression levels of

Stefan Maas; Willemijn M. Gommans

2009-01-01

212

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

213

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...

214

Mapping of the gene for cytidine deaminase (cdd) in Escherichia coli K-12.  

PubMed

The structural gene encoding cytidine deaminase (cdd) has been mapped in Escherichia coli K-12. It is located counterclockwise to ptsF between 46 and 47 min. The gene order in this region of the E. coli chromosome was found to be his-udk-gat-dld-cdd-ptsF. PMID:6339482

Josephsen, J; Hammer-Jespersen, K; Hansen, T D

1983-04-01

215

Isolation and characterization of the threonine deaminase promoter in Nicotiana attenuata  

Microsoft Academic Search

The enzyme encoded by the threonine deaminase (TD) gene catalyzes the conversion of threonine to ?-keto butyrate in the biosynthesis of isoleucine (Ile). In Nicotiana attenuata, TD transcripts accumulate constitutively in cotyledons and flowers and are elicited in leaves by wounding, herbivore attack, and methyl jasmonic acid (MeJA) treatment. To understand TD's unique pattern of expression, we isolated a genomic

Jin-Ho Kang; Ian T. Baldwin

2006-01-01

216

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

2008-04-01

217

Direct Evidence that RNA Inhibits APOBEC3G ssDNA Cytidine Deaminase Activity  

PubMed Central

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

McDougall, William M.; Smith, Harold C.

2011-01-01

218

Immunoenzymological Evidence Suggesting a Change in Conformation of Adenylic Acid Deaminase and Creatine Kinase during Substrate Combination  

PubMed Central

The kinetics of inhibition of 5?-adenylic acid deaminase and creatine-ATP transphosphorylase by their respective antibodies are studied and rate constants of combination are ascertained. It is shown that the single substrate 5?-adenylic acid (AMP) of deaminase “protects” the enzyme against antibody inhibition. However, phosphate, a competitive inhibitor of the highly specific deaminase, enhances combination with antibody. With creatine kinase, however, addition of either of the substrates, alone or in combination with the required magnesium, each of which separately bind to the enzyme, does not prevent inhibition of the enzyme by its antibody. However, the “working” enzyme combined with all substrates is “protected” against antibody inhibition.

Samuels, Arthur J.

1961-01-01

219

Heterologous Protein Secretion from Yeast  

Microsoft Academic Search

Secretion of calf prochymosin from yeast yields fully activable zymogen while production in the yeast cytoplasm yields insoluble, unactivable enzyme with aberrant disulfide bonding. Factors that increase the efficiency of secretion of prochymosin from yeast are use of a yeast secretion signal sequence, integration of the transcriptional unit into the yeast genome, and specific mutations in a number of host

Robert A. Smith; Margaret J. Duncan; Donald T. Moir

1985-01-01

220

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

221

Precise estimates of mutation rate and spectrum in yeast  

PubMed Central

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

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

2014-01-01

222

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

NASA Technical Reports Server (NTRS)

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

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

1995-01-01

223

Cloning, expression, purification, and characterization of biosynthetic threonine deaminase from Escherichia coli.  

PubMed

Feedback inhibition of the regulatory enzyme threonine deaminase by isoleucine provides an important level of enzymic control over branched chain amino acid biosynthesis in Escherichia coli. Cloning ilvA, the structural gene for threonine deaminase, under control of the trc promoter results in expression of active enzyme upon induction by isopropyl 1-thio-beta-D-galactoside to levels of approximately 20% of the soluble protein in cell extracts. High level expression of threonine deaminase has facilitated the development of a rapid and efficient protocol for the purification of gram quantities of enzyme with a specific activity 3-fold greater than previous preparations. The catalytic activity of threonine deaminase is absolutely dependent on the presence of pyridoxal phosphate, and the tetrameric molecule is isolated containing 1 mol of cofactor/56,000-Da chain. Wild-type threonine deaminase demonstrates a sigmoidal dependence of initial velocity on threonine concentration in the absence of isoleucine, consistent with a substrate-promoted conversion of the enzyme from a low activity to a high activity conformation. The enzymic dehydration of threonine to alpha-ketobutyrate measured by steady-state kinetics, performed at 20 degrees C in 0.05 M potassium phosphate, pH 7.5, is described by a Hill coefficient, nH, of 2.3 and a K0.5 of 8.0 mM. The negative allosteric effector L-isoleucine strongly inhibits the enzyme, yielding a value for nH of 3.9 and K0.5 of 74 mM whereas enzyme activity is greatly increased by L-valine, which yields nearly hyperbolic kinetics characterized by a value for nH of 1.0 and a K0.5 of 5.7 mM. Thus, these effectors promote dramatic and opposing effects on the transition from the low activity to the high activity conformation of the tetrameric enzyme. PMID:2005118

Eisenstein, E

1991-03-25

224

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

225

Cytosylglucuronic acid synthase (cytosine: UDP-glucuronosyltransferase) from Streptomyces griseochromogenes, the first prokaryotic UDP-glucuronosyltransferase.  

PubMed Central

Cytosylglucuronic acid synthase (cytosine: UDP-glucuronosyltransferase), the first prokaryotic UDP-GT and a key enzyme in the biosynthesis of the antibiotic blasticidin S, was purified 870-fold. It has optimum activity at a pH of 8.4 to 8.6, Kms of 6.0 (UDP-glucuronic acid) and 243 (cytosine) microM, and a maximum rate of metabolism of 14.6 mumol/min/mg. The apparent M(r) is 43,000. Activity was slightly enhanced by Mg2+ or Ca2+ but was not inhibited by EDTA. Activity was strongly inhibited by UDP. Cytosylglucuronic acid differs from eukaryotic UDP-glucuronosyltransferases in being a soluble protein with no apparent phospholipid requirement. Images

Gould, S J; Guo, J

1994-01-01

226

Chemical modification of cytosine residues of U6 snRNA with hydrogen sulfide (nucleosides and nucleotides. Part 49 [1]).  

PubMed Central

Sulfhydrolysis of cytosine residues to 4-thiouracil residues in mouse U6 snRNA was carried out to examine the secondary structure of U6 snRNA. The cytosine residues at positions 6, 42 and 68 were modified significantly, and at positions 11, 19 (or/and 25), 61 and 66 in moderate extent. Based on the result, the plausible secondary structure of U6 snRNA is discussed. Images

Miura, K; Tsuda, S; Harada, F; Ueda, T

1983-01-01

227

Alterations in cytosine methylation and species-specific transcription induced by interspecific hybridization between Oryza sativa and O. officinalis  

Microsoft Academic Search

Interspecific hybridization and polyploidization may involve programmed genetic and epigenetic changes. In this study, we\\u000a used the methylation-sensitive amplified polymorphism (MSAP) method to survey cytosine methylation alterations that occurred\\u000a in F1 hybrid and BC1 progeny following interspecific hybridization between Oryza sativa and O. officinalis. Across all 316 parental methylated sites, 25 (7.9%) cytosine methylation alterations were detected in the F1

Huajun Jin; Wei Hu; Zhe Wei; Linglin Wan; Gang Li; Guangxuan Tan; Lili Zhu; Guangcun He

2008-01-01

228

Concerted bis-alkylating reactivity of clerocidin towards unpaired cytosine residues in DNA  

Microsoft Academic Search

Clerocidin (CL) is a topoisomerase II poison, which cleaves DNA irreversibly at guanines (G) and revers- ibly at cytosines (C). Furthermore, the drug can induce enzyme-independent strand breaks at the G and C level. It has been previously shown that G-damage is induced by alkylation of the guanine N7, followed by spontaneous depurination and nucleic acid cleav- age, whereas scission

Sara N. Richter; Ileana Menegazzo; Daniele Fabris; Manlio Palumbo

2004-01-01

229

NONMEM Population Models of Cytosine Arabinoside and Fludarabine Phosphate in Pediatric Patients With Leukemia  

Microsoft Academic Search

Computer software NONMEM for nonlinear mixed-effects modeling was used to determine the population pharmacokinetics (PPK) and pharmacodynamics (PPD)\\u000a of intravenously administered nucleoside analogs cytosine arabinoside (ara-C) and fludarabine phosphate (F-ara-A) in pediatric\\u000a patients with leukemias. A description of the major NONMEM tasks as well as the statistical models has been given for these\\u000a drugs. Two pharmacokineticpharmacodynamic (PK-PD) models have been

Vassilios I. Avramis

230

Spectral Analysis of Guanine and Cytosine Fluctuations of Mouse Genomic DNA  

Microsoft Academic Search

We study global fluctuations of the guanine and cytosine base content (GC%) in mouse genomic DNA using spectral analyses. Power spectra S(f) of GC% fluctuations in all nineteen autosomal and two sex chromosomes are observed to have the universal functional form S(f) \\\\sim 1\\/f^alpha (alpha \\\\approx 1) over several orders of magnitude in the frequency range 10^-7< f 10^-5 cycle\\/base)

Wentian Li; Dirk Holste

2004-01-01

231

Is the cytosine DNA methyltransferase gene MET1 regulated by DNA methylation in Arabidopsis thaliana plants?  

Microsoft Academic Search

The methylation patterns of the MET1 gene in organs of Arabidopsis thaliana were studied by Southern blot hybridization of DNA samples hydrolyzed with differentially methylation-sensitive restriction\\u000a endonucleases. A highly methylated on internal cytosine residue CCGG site was found 1.5 kb upstream of the gene, whereas CCGG\\u000a sites located in more proximal parts of the 5?-flanking region and the gene itself

V. V. Ashapkin; L. I. Kutueva; B. F. Vanyushin

2011-01-01

232

The Guanine and Cytosine Content of Genomic DNA and Bacterial Evolution  

Microsoft Academic Search

The genomic guanine and cytosine (G+C) content of eubacteria is related to their phylogeny. The G+C content of various parts of the genome (protein genes, stable RNA genes, and spacers) reveals a positive linear correlation with the G+C content of their genomic DNA. However, the plotted correlation slopes differ among various parts of the genome or among the first, second,

Akira Muto; Syozo Osawa

1987-01-01

233

Cytosine methylation regulates oviposition in the pathogenic blood fluke Schistosoma mansoni  

Microsoft Academic Search

Similar to other metazoan pathogens, Schistosoma mansoni undergoes transcriptional and developmental regulation during its complex lifecycle and host interactions. DNA methylation as a mechanism to control these processes has, to date, been discounted in this parasite. Here we show the first evidence for cytosine methylation in the S. mansoni genome. Transcriptional coregulation of novel DNA methyltransferase (SmDnmt2) and methyl-CpG-binding domain

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

2011-01-01

234

Covalent Bond Formation between a DNA-Cytosine Methyltransferase and DNA Containing 5-azacytosine  

Microsoft Academic Search

DNA containing 5-azacytosine (azaC) has previously been shown to be a potent inhibitor of DNA-cytosine methyltransferases. In this report, we describe experiments which demonstrate that azaC-DNA forms a covalent complex with Hpa II methylase, a bacterial enzyme that methylates the internal C of C-C-G-G sequences. The complex does not undergo detectable dissociation over at least 3 days and is stable

Daniel V. Santi; Anne Norment; Charles E. Garrett

1984-01-01

235

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

NASA Astrophysics Data System (ADS)

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.

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

2013-03-01

236

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

237

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

PubMed Central

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

Finnegan, E J; Dennis, E S

1993-01-01

238

Error rates for nanopore discrimination among cytosine, methylcytosine, and hydroxymethylcytosine along individual DNA strands.  

PubMed

Cytosine, 5-methylcytosine, and 5-hydroxymethylcytosine were identified during translocation of single DNA template strands through a modified Mycobacterium smegmatis porin A (M2MspA) nanopore under control of phi29 DNA polymerase. This identification was based on three consecutive ionic current states that correspond to passage of modified or unmodified CG dinucleotides and their immediate neighbors through the nanopore limiting aperture. To establish quality scores for these calls, we examined ~3,300 translocation events for 48 distinct DNA constructs. Each experiment analyzed a mixture of cytosine-, 5-methylcytosine-, and 5-hydroxymethylcytosine-bearing DNA strands that contained a marker that independently established the correct cytosine methylation status at the target CG of each molecule tested. To calculate error rates for these calls, we established decision boundaries using a variety of machine-learning methods. These error rates depended upon the identity of the bases immediately 5' and 3' of the targeted CG dinucleotide, and ranged from 1.7% to 12.2% for a single-pass read. We estimate that Q40 values (0.01% error rates) for methylation status calls could be achieved by reading single molecules 5-19 times depending upon sequence context. PMID:24167260

Schreiber, Jacob; Wescoe, Zachary L; Abu-Shumays, Robin; Vivian, John T; Baatar, Baldandorj; Karplus, Kevin; Akeson, Mark

2013-11-19

239

Error rates for nanopore discrimination among cytosine, methylcytosine, and hydroxymethylcytosine along individual DNA strands  

PubMed Central

Cytosine, 5-methylcytosine, and 5-hydroxymethylcytosine were identified during translocation of single DNA template strands through a modified Mycobacterium smegmatis porin A (M2MspA) nanopore under control of phi29 DNA polymerase. This identification was based on three consecutive ionic current states that correspond to passage of modified or unmodified CG dinucleotides and their immediate neighbors through the nanopore limiting aperture. To establish quality scores for these calls, we examined ?3,300 translocation events for 48 distinct DNA constructs. Each experiment analyzed a mixture of cytosine-, 5-methylcytosine–, and 5-hydroxymethylcytosine–bearing DNA strands that contained a marker that independently established the correct cytosine methylation status at the target CG of each molecule tested. To calculate error rates for these calls, we established decision boundaries using a variety of machine-learning methods. These error rates depended upon the identity of the bases immediately 5? and 3? of the targeted CG dinucleotide, and ranged from 1.7% to 12.2% for a single-pass read. We estimate that Q40 values (0.01% error rates) for methylation status calls could be achieved by reading single molecules 5–19 times depending upon sequence context.

Schreiber, Jacob; Wescoe, Zachary L.; Abu-Shumays, Robin; Vivian, John T.; Baatar, Baldandorj; Karplus, Kevin; Akeson, Mark

2013-01-01

240

Accelerated bisulfite-deamination of cytosine in the genomic sequencing procedure for DNA methylation analysis.  

PubMed

Understanding the biological consequences of DNA methylation is a current focus of intensive studies. A standard method for analyzing the methylation at position 5 of cytosines in genomic DNA involves chemical modification of the DNA with bisulfite, followed by PCR amplification and sequencing. Bisulfite deaminates cytosine, but it deaminates 5-methylcytosine only very slowly, thereby allowing determination of the methylated sites. The determination is usually performed using sodium bisulfite solutions of 3-5 M concentration with an incubation period of 12-16 hr at 50 degrees C. We demonstrate here that this deamination can be speeded up significantly by increasing the bisulfite concentration and the temperature with which the reaction is performed. In an experiment, in which denatured DNA was treated with 9 M bisulfite for 10 min at pH 5.4 and 90 degrees C, deamination of cytosines occurred to an extent of 99.6%, while 5-methylcytosine residues in the DNA were deaminated at less than 10%. Using a plasmid DNA fragment, we observed that the DNA can serve as a template for PCR amplification after the bisulfite treatment. This new procedure is expected to offer an improved genomic sequencing method, leading to the promotion of research on understanding the biological and medical significance of DNA methylation. PMID:17150578

Hayatsu, Hikoya; Negishi, Kazuo; Shiraishi, Masahiko

2004-01-01

241

Chromone-fused cytosine analogues: synthesis, biological activity, and structure-activity relationship.  

PubMed

The preparation of a series of novel chromone-fused cytosine analogues, i.e., chromeno[2,3-d]pyrimidines has been carried out from substituted 2-amino-4-oxo-4H-chromene-3-carbonitriles with urea, thiourea, and guanidine under different reaction conditions. These chromone-fused cytosine analogues were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv strain and different microbial pathogenic strains in cell culture for their structure-activity relationships, respectively. Among the synthesized compounds, 2d, 3a, and 4e showed better results against Mycobacterium tuberculosis H37Rv. The compounds 2a, 2b, and 3a showed potential antibacterial activity against E. coli and P. aeruginosa, while the majority of compounds were found to be active against S. aureus as compared to ampicillin. The synthesized cytosine analogues having an imine (-C&dbnd;NH) have been less sensitive to the bacterial and fungal strains but have a more beneficial effect on Mycobacterium tuberculosis H37Rv. PMID:24660882

Haveliwala, Dhaval D; Kamdar, Nimesh R; Mistry, Prashant T; Patel, Saurabh K

2014-01-01

242

Amelioration of high salinity stress damage by plant growth-promoting bacterial endophytes that contain ACC deaminase.  

PubMed

Plant growth and productivity is negatively affected by soil salinity. However, it is predicted that plant growth-promoting bacterial (PGPB) endophytes that contain 1-aminocyclopropane-1-carboxylate (ACC) deaminase (E.C. 4.1.99.4) can facilitate plant growth and development in the presence of a number of different stresses. In present study, the ability of ACC deaminase containing PGPB endophytes Pseudomonas fluorescens YsS6, Pseudomonas migulae 8R6, and their ACC deaminase deficient mutants to promote tomato plant growth in the absence of salt and under two different levels of salt stress (165 mM and 185 mM) was assessed. It was evidence that wild-type bacterial endophytes (P. fluorescens YsS6 and P. migulae 8R6) promoted tomato plant growth significantly even in the absence of stress (salinity). Plants pretreated with wild-type ACC deaminase containing endophytic strains were healthier and grew to a much larger size under high salinity stress compared to plants pretreated with the ACC deaminase deficient mutants or no bacterial treatment (control). The plants pretreated with ACC deaminase containing bacterial endophytes exhibit higher fresh and dry biomass, higher chlorophyll contents, and a greater number of flowers and buds than the other treatments. Since the only difference between wild-type and mutant bacterial endophytes was ACC deaminase activity, it is concluded that this enzyme is directly responsible for the different behavior of tomato plants in response to salt stress. The use of PGPB endophytes with ACC deaminase activity has the potential to facilitate plant growth on land that is not normally suitable for the majority of crops due to their high salt contents. PMID:24769617

Ali, Shimaila; Charles, Trevor C; Glick, Bernard R

2014-07-01

243

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

244

Relationship Between Escherichia coli B Titer and the Level of Deoxycytidylate Deaminase Activity Induced on Bacteriophage T2r+ Infection  

PubMed Central

The activities of six bacteriophage T2r+-induced enzymes (thymidylate synthetase, deoxycytidylate deaminase, thymidylate kinase, deoxycytidylate hydroxymethylase, deoxycytidine pyrophosphatase, and dihydrofolate reductase) were measured after dilution of phage-infected Escherichia coli B from 8 × 108 to 2 × 108 cells per ml. The only enzyme activity altered was that of deoxycytidylate deaminase, which increased three- to fourfold. Conversely, the rapid concentration of cells from 2 × 108 to 8 × 108 per ml did not result in a reduction in deaminase activity. Although an enhancement in aeration reduced the response of deoxycytidylate deaminase to cellular dilution, the influence of potential metabolic inhibitors or activators could not be shown. The change in deoxycytidylate deaminase activity appeared to be associated with an altered translational event, since the increase could not be prevented by rifampin but was blocked effectively by chloramphenicol and hydroxylamine. In addition, antibody to the T2 phage-induced deoxycytidylate deaminase demonstrated that the increase in enzyme activity was associated with a corresponding increase in radioactive leucine incorporated into the enzyme antigen.

Trimble, Robert B.; Maley, Gladys F.; Maley, Frank

1972-01-01

245

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

246

Yeast cystathionine beta-synthase is a pyridoxal phosphate enzyme but, unlike the human enzyme, is not a heme protein.  

PubMed

Our studies of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are aimed at clarifying the cofactor dependence and catalytic mechanism and obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease). We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine beta-synthase. The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine beta-synthase. The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism. The reaction of the enzyme with L-serine in the absence of L-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm. The formation of this intermediate from the product, L-cystathionine, demonstrates the partial reversibility of the reaction. Our results establish the overall catalytic mechanism of yeast cystathionine beta-synthase and provide a useful system for future studies of structure and function. The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymes O-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase. PMID:10766767

Jhee, K H; McPhie, P; Miles, E W

2000-04-21

247

The De Novo Cytosine Methyltransferase DRM2 Requires Intact UBA Domains and a Catalytically Mutated Paralog DRM3 during RNA–Directed DNA Methylation in Arabidopsis thaliana  

Microsoft Academic Search

Eukaryotic DNA cytosine methylation can be used to transcriptionally silence repetitive sequences, including transposons and retroviruses. This silencing is stable between cell generations as cytosine methylation is maintained epigenetically through DNA replication. The Arabidopsis thaliana Dnmt3 cytosine methyltransferase ortholog DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) is required for establishment of small interfering RNA (siRNA) directed DNA methylation. In mammals PIWI proteins and

Ian R. Henderson; Angelique Deleris; William Wong; Xuehua Zhong; Hang Gyeong Chin; Gregory A. Horwitz; Krystyna A. Kelly; Sriharsa Pradhan; Steven E. Jacobsen

2010-01-01

248

Cytosine hypomethylation at CHG and CHH sites in the pleiotropic mutants of Mendelian inheritance in Catharanthus roseus.  

PubMed

The 5S and 18S rDNA sequences of Catharanthus roseus cv 'Nirmal' (wild type) and its leafless inflorescence (lli), evergreen dwarf (egd) and irregular leaf lamina (ill) single mutants and lli egd, lli ill and egd ill double mutants were characterized. The lli, egd and ill mutants of Mendelian inheritance bore the names after their most conspicuous morphological feature(s). They had been chemically induced and isolated for their salt tolerance. The double mutants were isolated as morphological segregants from crosses between single mutants. The morphological features of the two parents accompanied salt tolerance in the double mutants. All the six mutants were hypomethylated at repeat sequences, upregulated and downregulated for many genes and carried pleiotropic alterations for several traits. Here the 5S and 18S rDNAs of C. roseus were found to be relatively low in cytosine content. Cytosines were preponderantly in CG context (53%) and almost all of them were methylated (97%). The cytosines in CHH and CHG (where H = A, T or C) contexts were largely demethylated (92%) in mutants. The demethylation was attributable to reduced expression of RDR2 and DRM2 led RNA dependant DNA methylation and CMT3 led maintenance methylation pathways. Mutants had gained some cytosines by substitution of C at T sites. These perhaps arose on account of errors in DNA replication, mediated by widespread cytosine demethylation at CHG and CHH sites. It was concluded that the regulation of cytosine ethylation mechanisms was disturbed in the mutants. ILL, EGD and LLI genes were identified as the positive regulators of other genes mediating the RdDM and CMT3 pathways, for establishment and maintenance of cytosine methylation in C. roseus. PMID:24371171

Kumari, Renu; Yadav, Gitanjali; Sharma, Vishakha; Sharma, Vinay; Kumar, Sushil

2013-12-01

249

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

2013-06-15

250

Validity of the continuous spectrophotometric assay of Kalckar for adenosine deaminase activity.  

PubMed

Both adenosine and inosine obey Beer's law to 1.0 mM at 265 nm and pH 7.4 at 25 degrees C. Murphy et al. (1) claimed serious deviation from Beer's law above 200 microM for both substances, and concluded that the assay of adenosine deaminase activity based on recording spectrophotometric change at 265 nm as originally suggested by Kalckar produces anomalous results. The data herein presented show that this is not so, and that the large number of published studies of adenosine deaminase activity assayed by this method are indeed valid and should not be dismissed as artifactual as suggested by Murphy et al. PMID:6859524

Tritsch, G L

1983-02-15

251

Chicken double-stranded RNA adenosine deaminase has apparent specificity for Z-DNA.  

PubMed Central

A M(r) 140,000 protein has been purified from chicken lungs to apparent homogeneity. The protein binds with high affinity to a non-BNA conformation, which is most likely to the Z-DNA. The protein also has a binding site for double-stranded RNA (dsRNA). Peptide sequences from this protein show similarity to dsRNA adenosine deaminase, an enzyme that deaminates adenosine in dsRNA to form inosine. Assays for this enzyme confirm that dsRNA adenosine deaminase activity and Z-DNA binding are properties of the same molecule. The coupling of these two activities in a single molecule may indicate a distinctive mechanism of gene regulation that is, in part, dependent on DNA topology. As such, DNA topology, through its effects on the efficiency and extent of RNA editing may be important in the generation of new phenotypes during evolution. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

Herbert, A; Lowenhaupt, K; Spitzner, J; Rich, A

1995-01-01

252

Overproduction of adenine deoxynucleosides and deoxynucletides in adenosine deaminase deficiency with severe combined immunodeficiency disease.  

PubMed Central

The deoxynucleotide, dATP, is elevated 50- to 1,000-fold above normal in erythrocytes, lymphocytes, and bone marrow from a child with adenosine deaminase deficiency and severe combined immunodeficiency disease. The child, when 17 mo of age, was also excreting approximately 30 mg of deoxyadenosine per day in urine (normal is less than 0.1 mg/day). Urinary excretion of uric acid was decreased. Elevated dATP levels in lymphocytes and bone marrow, and increased urinary excretion of deoxyadenosine, persisted despite hypertransfusion of the child with irradiated erythrocytes from a donor with normal adenosine deaminase. Overproduction of deoxynucleotides by increased salvage of adenosine appears to be the primary metabolic abnormality in patients with adenosine de aminase deficiency.

Donofrio, J; Coleman, M S; Hutton, J J; Daoud, A; Lampkin, B; Dyminski, J

1978-01-01

253

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

2010-12-01

254

Late-onset adenosine deaminase deficiency presenting with Heck’s disease  

Microsoft Academic Search

Focal epithelial hyperplasia, also known as Heck’s disease, is a rare but distinctive entity of viral etiology with characteristic\\u000a clinical and histopathological features. It is a benign, asymptomatic disease of the oral mucosa caused by human papilloma\\u000a viruses (HPV). Previous studies postulated an association between these lesions and immunodeficiency. Genetic deficiency of\\u000a adenosine deaminase (ADA) results in varying degrees of

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

2010-01-01

255

Activation-Induced Cytidine Deaminase Deficiency Causes Organ-Specific Autoimmune Disease  

Microsoft Academic Search

Activation-induced cytidine deaminase (AID) expressed by germinal center B cells is a central regulator of somatic hypermutation (SHM) and class switch recombination (CSR). Humans with AID mutations develop not only the autosomal recessive form of hyper-IgM syndrome (HIGM2) associated with B cell hyperplasia, but also autoimmune disorders by unknown mechanisms. We report here that AID?\\/? mice spontaneously develop tertiary lymphoid

Koji Hase; Daisuke Takahashi; Masashi Ebisawa; Sayaka Kawano; Kikuji Itoh; Hiroshi Ohno; Lena Alexopoulou

2008-01-01

256

Novel adenosine deaminase 2 mutations in a child with a fatal vasculopathy.  

PubMed

Adenosine deaminase 2 (ADA2) deficiency due to CECR1 mutations is a recently defined disorder that involves systemic inflammation and vasculopathy often associated with polyarteritis nodosa. We report on a 5-year-old girl with a severe vasculopathy who carried two novel mutations in CECR1. Conclusion: Identification of CECR1 mutations in patients with vasculopathy may lead to earlier diagnosis of ADA2 deficiency. PMID:24737293

Garg, Nisha; Kasapcopur, Ozgur; Foster, Joseph; Barut, Kenan; Tekin, Ay?e; K?z?lk?l?ç, Osman; Tekin, Mustafa

2014-06-01

257

Vif and the Role of Antiviral Cytidine Deaminases in HIV1 Replication  

Microsoft Academic Search

Vif is required for HIV-1 replication in primary cells and other nonpermissive cell lines. The function of Vif was unknown until recent papers revealed that it prevents the encapsidation of APOBEC3G and APOBEC3F, two potent antiretroviral cytidine deaminases. APOBEC3G and APOBEC3F inhibit the replication of ?vif HIV-1 by deaminating the minus-strand of the viral reverse transcripts, introduc- ing numerous G?A

Qin Yu; Nathaniel R. Landau; Renate König

258

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

SciTech Connect

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

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

1988-12-13

259

Methylthioadenosine Deaminase in an Alternative Quorum Sensing Pathway in Pseudomonas aeruginosa  

PubMed Central

Pseudomonas aeruginosa possesses an unusual pathway for 5?-methylthioadenosine (MTA) metabolism involving deamination to 5?-methylthioinosine (MTI) followed by N-ribosyl phosphorolysis to hypoxanthine and 5-methylthio-?-D-ribose 1-phosphate. The specific MTI phosphorylase of P. aeruginosa has been reported (Guan, R., Ho, M. C., Almo, S. C. and Schramm, V. L. (2011) Biochemistry 50, 1247–1254) and here we characterize MTA deaminase from P. aeruginosa (PaMTADA). Genomic analysis indicated the PA3170 locus to be a candidate for MTA deaminase (MTADA). Protein encoded by PA3170 was expressed and shown to deaminate MTA with 40-fold greater catalytic efficiency for MTA than for adenosine. The kcat/Km value of 1.6 × 107 M?1s?1 for MTA is the highest catalytic efficiency known for an MTA deaminase. 5?-Methylthiocoformycin (MTCF) is a 4.8 pM transition state analogue for PaMTADA but causes no significant inhibition of human adenosine deaminase or MTA phosphorylase. MTCF is permeable to P. aeruginosa and exhibits an IC50 of 3 nM on cellular PaMTADA activity. PaMTADA is the only activity in P. aeruginosa extracts to act on MTA. MTA and 5-methylthio-?-D-ribose are involved in quorum sensing pathways, thus PaMTADA is a potential target for quorum sensing. The crystal structure of PaMTADA in complex with MTCF shows the transition state mimic 8-R-hydroxyl group in contact with a catalytic site Zn2+, the 5?-methylthio group in a hydrophobic pocket and the transition state mimic of the diazepine ring in contact with a catalytic site Glu.

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

2012-01-01

260

Partial Characterization of AMP Deaminase from Jumbo Squid (Dosidicus gigas) Mantle  

Microsoft Academic Search

AMP deaminase enzyme showed an optimum temperature at 35°C. It was relatively stable between 30 and 40°C but extremely unstable at higher temperatures. The optimum pH was 6.0 and the enzyme presented an excellent stability with pH between 5.5 and 6.5. The K ion acted as a negative effector when used in concentrations higher than 50 mM. The nucleotide ATP

R. Pacheco-Aguilar; J. M. Ezquerra-Brauer; J. Castillo-Yañez; M. E. Lugo-Sanchez; E. Marquez-Rios

2009-01-01

261

Serum adenosine deaminase, 5? nucleotidase and malondialdehyde in acute infective hepatitis  

Microsoft Academic Search

Serum adenosine deaminase (ADA), 5? nucleotidase (5?NT) and malondialdehyde (MDA) were estimated in patients with acute infective\\u000a hepatitis (AIH) along with the routine parameters of liver disease. Present study is done to evaluate these special parameters\\u000a in patients with clinical history of AIH and to assess the utility of these parameters as diagnostic\\/ prognostic indices of\\u000a liver function and to

K. Pratibha; Usha Anand; Rajni Agarwal

2004-01-01

262

1-Aminocyclopropane-1Carboxylate (ACC) Deaminase Genes in Rhizobia from Southern Saskatchewan  

Microsoft Academic Search

A collection of 233 rhizobia strains from 30 different sites across Saskatchewan, Canada was assayed for 1-aminocyclopropane-1-carboxylate\\u000a (ACC) deaminase activity, with 27 of the strains displaying activity. When all 27 strains were characterized based on 16S\\u000a rRNA gene sequences, it was noted that 26 strains are close to Rhizobium leguminosarum and one strain is close to Rhizobium gallicum. Polymerase chain

Jin Duan; Kirsten M. Müller; Trevor C. Charles; Susanne Vesely; Bernard R. Glick

2009-01-01

263

Synthetic substrate analogs for the RNA-editing adenosine deaminase ADAR2  

Microsoft Academic Search

We have synthesized structural analogs of a natural RNA editing substrate and compared editing reactions of these substrates by recombinant ADAR-2, an RNA-editing adenosine deaminase. Deamination rates were shown to be sensitive to structural changes at the 2'-carbon of the edited adenosine. Methylation of the 2'-OH caused a large decrease in deamination rate, whereas 2'-deoxyadenosine and 2'- deoxy-2'-fluoroadenosine were deaminated

Hye Young Yi-Brunozzi; LaHoma M. Easterwood; Gregg M. Kamilar; Peter A. Beal

1999-01-01

264

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

Microsoft Academic Search

Adenosine deaminase deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, the authors synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine

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

1987-01-01

265

Identification of a New Class of Adenosine Deaminase from Helicobacter pylori with Homologs among Diverse Taxa  

PubMed Central

Early studies of Helicobacter pylori's nutritional requirements alluded to a complete purine salvage network in this organism. Recently, this hypothesis was confirmed in two strains of H. pylori, whose purine requirements were satisfied by any single purine base or nucleoside. Most of the purine conversion enzymes in H. pylori have been studied using mutant analysis; however, the gene encoding adenosine deaminase (ADD) in H. pylori remained unidentified. Through stepwise protein purification followed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF), we discovered that H. pylori ADD is encoded by hp0267, an apparently essential gene. Hp0267 shares no sequence homology with previously characterized ADDs, yet both are members of the amidohydrolase superfamily. Hp0267 is grouped within cog0402, while other ADDs studied to date are found in cog1816. The hp0267 locus was previously misannotated as encoding a chlorohydrolase. Using purified recombinant Hp0267, we determined the enzyme's pH optimum, temperature optimum, substrate specificity, and estimated kinetic constants. In contrast to other known ADDs, Hp0267 contains Fe(II) as the relevant metal ligand. Furthermore, Hp0267 exhibits very low deaminase activity on 2?-deoxyadenosine, a substrate that is readily hydrolyzed by cog1816 ADDs. Our preliminary comparative genomic analysis suggests that Hp0267 represents a second enzyme class of adenosine deaminase whose phyletic distribution among prokaryotes is broad.

Miller, Erica F.

2013-01-01

266

Identification of a new class of adenosine deaminase from Helicobacter pylori with homologs among diverse taxa.  

PubMed

Early studies of Helicobacter pylori's nutritional requirements alluded to a complete purine salvage network in this organism. Recently, this hypothesis was confirmed in two strains of H. pylori, whose purine requirements were satisfied by any single purine base or nucleoside. Most of the purine conversion enzymes in H. pylori have been studied using mutant analysis; however, the gene encoding adenosine deaminase (ADD) in H. pylori remained unidentified. Through stepwise protein purification followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), we discovered that H. pylori ADD is encoded by hp0267, an apparently essential gene. Hp0267 shares no sequence homology with previously characterized ADDs, yet both are members of the amidohydrolase superfamily. Hp0267 is grouped within cog0402, while other ADDs studied to date are found in cog1816. The hp0267 locus was previously misannotated as encoding a chlorohydrolase. Using purified recombinant Hp0267, we determined the enzyme's pH optimum, temperature optimum, substrate specificity, and estimated kinetic constants. In contrast to other known ADDs, Hp0267 contains Fe(II) as the relevant metal ligand. Furthermore, Hp0267 exhibits very low deaminase activity on 2'-deoxyadenosine, a substrate that is readily hydrolyzed by cog1816 ADDs. Our preliminary comparative genomic analysis suggests that Hp0267 represents a second enzyme class of adenosine deaminase whose phyletic distribution among prokaryotes is broad. PMID:23852874

Miller, Erica F; Maier, Robert J

2013-09-01

267

The mechanism of M.HhaI DNA C5 cytosine methyltransferase enzyme: A quantum mechanics/molecular mechanics approach  

PubMed Central

The mechanism of DNA cytosine-5-methylation catalyzed by the bacterial M.HhaI enzyme has been considered as a stepwise nucleophilic addition of Cys-81-S? to cytosine C6 followed by C5 nucleophilic replacement of the methyl of S-adenosyl-l-methionine to produce 5-methyl-6-Cys-81-S-5,6-dihydrocytosine. In this study, we show that the reaction is concerted from a series of energy calculations by using the quantum mechanical/molecular mechanical hybrid method. Deprotonation of 5-methyl-6-Cys-81-S-5,6-dihydrocytosine and expulsion of Cys-81-S? provides the product DNA 5-methylcytosine. A required base catalyst for this deprotonation is not available as a member of the active site structure. A water channel between the active site and bulk water allows entrance of solvent to the active site. Hydroxide at 10?7 mole fraction (pH = 7) is shown to be sufficient for the required catalysis. We also show that Glu-119-CO2H can divert the reaction by protonating cytosine N3 when Cys-81-S? attacks cytosine, to form the 6-Cys-81-S-3-hydrocytosine. The reactants and 6-Cys-81-S-3-hydrocytosine product are in rapid equilibrium, and this explains the observed hydrogen exchange of cytosine with solvent.

Zhang, Xiaodong; Bruice, Thomas C.

2006-01-01

268

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

269

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

270

Strong changes in the dielectric functions of cytosine upon molecular modification  

NASA Astrophysics Data System (ADS)

The DNA base cytosine and its variant 1-allylcytosine were deposited as thick layers onto silicon substrates using organic molecular beam deposition. The dielectric functions of these materials were measured in the vacuum ultraviolet range from 4.0 to 9.5 eV using synchrotron radiation. In the experiments, the slight modification of the molecules results in very pronounced differences in the optical response. This was unexpected in the light of time dependent density functional theory calculation predicting a very similar optical response for both molecules. We attribute the substantial change in the optical response to stronger intermolecular interaction upon the molecular modification.

Suzuki, Yu; Gordan, Ovidiu D.; Silaghi, Simona D.; Zahn, Dietrich R. T.; Schubert, Anett; Thiel, Werner R.; Cobet, Christoph; Esser, Nobert; Braun, Walter

2005-11-01

271

Finding the fifth base: Genome-wide sequencing of cytosine methylation  

PubMed Central

Complete sequences of myriad eukaryotic genomes, including several human genomes, are now available, and recent dramatic developments in DNA sequencing technology are opening the floodgates to vast volumes of sequence data. Yet, despite knowing for several decades that a significant proportion of cytosines in the genomes of plants and animals are present in the form of methylcytosine, until very recently the precise locations of these modified bases have never been accurately mapped throughout a eukaryotic genome. Advanced “next-generation” DNA sequencing technologies are now enabling the global mapping of this epigenetic modification at single-base resolution, providing new insights into the regulation and dynamics of DNA methylation in genomes.

Lister, Ryan; Ecker, Joseph R.

2009-01-01

272

RNAi in budding yeast  

PubMed Central

RNAi, a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast, Saccharomyces cerevisiae. Here, we show that RNAi is present in other budding-yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate siRNAs, which mostly correspond to transposable elements and Y’ subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y’ mRNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a novel class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi.

Mower, Jeffrey P.; Wolfe, Kenneth H.; Fink, Gerald R.; Bartel, David P.

2013-01-01

273

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

274

Viral induced yeast apoptosis.  

PubMed

In an analogous system to mammals, induction of an apoptotic cell death programme (PCD) in yeast is not only restricted to various exogenous factors and stimuli, but can also be triggered by viral killer toxins and viral pathogens. In yeast, toxin secreting killer strains are frequently infected with double-stranded (ds)RNA viruses that are responsible for killer phenotype expression and toxin secretion in the infected host. In most cases, the viral toxins are either pore-forming proteins (such as K1, K2, and zygocin) that kill non-infected and sensitive yeast cells by disrupting cytoplasmic membrane function, or protein toxins (such as K28) that act in the nucleus by blocking DNA synthesis and subsequently causing a G1/S cell cycle arrest. Interestingly, while all these virus toxins cause necrotic cell death at high concentration, they trigger caspase- and ROS-mediated apoptosis at low-to-moderate concentration, indicating that even low toxin doses are deadly by triggering PCD in enemy cells. Remarkably, viral toxins are not solely responsible for cell death induction in vivo, as killer viruses themselves were shown to trigger apoptosis in non-infected yeast. Thus, as killer virus-infected and toxin secreting yeasts are effectively protected and immune to their own toxin, killer yeasts bear the intrinsic potential to dominate over time in their natural habitat. PMID:18291112

Schmitt, Manfred J; Reiter, Jochen

2008-07-01

275

Tautomerism in cytosine and uracil: a theoretical and experimental X-ray absorption and resonant auger study.  

PubMed

The core level photoabsorption spectra of the nucleobases cytosine and uracil in the gas phase have been measured and the results interpreted with theoretical calculations using an ab initio Green’s function approach. A single tautomer of uracil is populated, in agreement with previous work, while three tautomers of cytosine are clearly identified, whose identity and relative populations at the temperature of the experiment were reported previously. The second-order ADC approach to polarization propagator was employed in calculations of X-ray photoabsorption energies and intensities. The theoretical spectra have been constructed as Boltzmann-factor-weighted sums of individual tautomer spectra. These theoretical spectra are in good agreement with the experimental photoabsorption results at the oxygen, nitrogen, and carbon edges. In addition we report resonant Auger spectra of the valence band of cytosine, which support previous assignments of the character of the valence band states. PMID:20795686

Feyer, Vitaliy; Plekan, Oksana; Richter, Robert; Coreno, Marcello; de Simone, Monica; Prince, Kevin C; Trofimov, Alexander B; Zaytseva, Irina L; Schirmer, Jochen

2010-09-23

276

Contribution of cytosine-containing cyclobutane dimers to DNA damage produced by photosensitized triplet-triplet energy transfer.  

PubMed

Mutagenic cyclobutane pyrimidine dimers (CPDs) can be induced in DNA through either direct excitation or photosensitized triplet-triplet energy transfer (TTET). In the latter pathway, thymines are expected to receive the excitation energy from the photosensitizer and react with adjacent pyrimidines. By using state-of-the art analytical tools, we provide herein additional information on the formation of cytosine-containing CPDs. We thus determined the yield of all possible CPDs upon TTET in a series of natural DNAs with various base compositions. We show that the distribution of CPDs cannot be explained only by excitation of individual thymines. We propose that the mechanism for TTET involves at least dinucleotides as the minimal targets. The observation of the formation of cytosine-cytosine CPDs also suggests that additional pathways are involved in this photosensitized reaction. PMID:24668918

Douki, Thierry; Bérard, Izabel; Wack, Aude; Andrä, Sigrid

2014-05-01

277

Formation of cytosine glycol and 5,6-dihydroxycytosine in deoxyribonucleic acid on treatment with osmium tetroxide.  

PubMed Central

OsO4 selectively forms thymine glycol lesions in DNA. In the past, OsO4-treated DNA has been used as a substrate in studies of DNA repair utilizing base-excision repair enzymes such as DNA glycosylases. There is, however, no information available on the chemical identity of other OsO4-induced base lesions in DNA. A complete knowledge of such DNA lesions may be of importance for repair studies. Using a methodology developed recently for characterization of oxidative base damage in DNA, we provide evidence for the formation of cytosine glycol and 5,6-dihydroxycytosine moieties, in addition to thymine glycol, in DNA on treatment with OsO4. For this purpose, samples of OsO4-treated DNA were hydrolysed with formic acid, then trimethylsilylated and analysed by capillary gas chromatography-mass spectrometry. In addition to thymine glycol, 5-hydroxyuracil (isobarbituric acid), 5-hydroxycytosine and 5,6-dihydroxyuracil (isodialuric acid or dialuric acid) were identified in OsO4-treated DNA. It is suggested that 5-hydroxyuracil was formed by formic acid-induced deamination and dehydration of cytosine glycol, which was the actual oxidation product of the cytosine moiety in DNA. 5-Hydroxycytosine obviously resulted from dehydration of cytosine glycol, and 5,6-dihydroxyuracil from deamination of 5,6-dihydroxycytosine. This scheme was supported by the presence of 5-hydroxyuracil, uracil glycol and 5,6-dihydroxyuracil in OsO4-treated cytosine. Treatment of OsO4-treated cytosine with formic acid caused the complete conversion of uracil glycol into 5-hydroxyuracil. The implications of these findings relative to studies of DNA repair are discussed.

Dizdaroglu, M; Holwitt, E; Hagan, M P; Blakely, W F

1986-01-01

278

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

PubMed Central

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

2013-01-01

279

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

NASA Astrophysics Data System (ADS)

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

van der Burgt, Peter J. M.

2014-05-01

280

Insulin mimetic peroxo complexes of vanadium containing uracil or Cytosine as ligand.  

PubMed

Mixed ligand oxo peroxo complexes of vanadium (V), M[VO(O(2))L(2)].nH(2)O where M = K or NH(4), HL = uracil or cytosine and n = 1 or 2, have been isolated from aqueous methanolic medium. The complexes were characterised by elemental analysis, conductance, TGA, UV-Visible, IR and NMR spectral studies. Both the peroxide and the other ligands acts as chelates coordinating through their oxygen at C (2) and nitrogen at N (3) and the presence of monomeric oxoperoxovanadium (V) species, have been established by IR, (1)H and (51)V NMR studies. The complexes appeared to possess pentagonal bipyramidal geometry. The terminal oxo ligand and the oxygen of the uracil or cytosine at C (2) occupy the axial position while the peroxide and the other donor ligands are in the equatorial position. The UV spectral studies confirm the presence of Vanadium in its +5 oxidation state. The administration of the potassium salts of the complexes reduces the blood glucose level in Swiss Albino mice compared to that of KVO(3). The complexes also readily oxidise cysteine to Cystine in aqueous solution. The possible mechanism for the insulin-mimic activity of the complexes is discussed. PMID:18475940

Sarkar, A R; Mandal, S

2000-01-01

281

Cytosine methylation does not affect binding of transcription factor Sp1  

SciTech Connect

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

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

1988-04-01

282

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

283

Activation and Deactivation of a Broad-Spectrum Antiviral Drug by a Single Enzyme: Adenosine Deaminase Catalyzes Two Consecutive Deamination Reactions  

Microsoft Academic Search

Ribavirin is an approved broad-spectrum antiviral drug. A liver-targeting prodrug of ribavirin, viramidine, is in clinical trial in an attempt to provide a better therapeutic index. The conversion of viramidine to ribavirin, and of ribavirin to an inactive metabolite through adenosine deaminase, is reported. Kinetic analysis indicates that adenosine deaminase is likely involved in activation of viramidine in vivo, and

Jim Zhen Wu; Heli Walker; Johnson Y. N. Lau; Zhi Hong

2003-01-01

284

Identification of two pentatricopeptide repeat genes required for RNA editing and zinc binding by C-terminal cytidine deaminase-like domains.  

PubMed

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

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

2013-12-20

285

Oxygen requirements of yeasts.  

PubMed Central

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

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

1990-01-01

286

Mapping Yeast Transcriptional Networks  

PubMed Central

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

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

2013-01-01

287

21 CFR 172.896 - Dried yeasts.  

Code of Federal Regulations, 2013 CFR

...172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis ) and dried torula yeast (Candida utilis ) may be safely used in food provided the total folic acid content of the yeast does not exceed 0.04 milligram...

2013-04-01

288

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

PubMed Central

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

Suspene, Rodolphe; Aynaud, Marie-Ming; Guetard, Denise; Henry, Michel; Eckhoff, Grace; Marchio, Agnes; Pineau, Pascal; Dejean, Anne; Vartanian, Jean-Pierre; Wain-Hobson, Simon

2011-01-01

289

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

290

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

291

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.

292

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

PubMed Central

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

Sakinc, Turkan; Kline, Kimberly; Nielsen, Hailyn V.; Hultgren, Scott; Gatermann, Soren G.

2013-01-01

293

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

PubMed

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

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

2013-12-01

294

Vaginal Yeast Infections (For Parents)  

MedlinePLUS

... a common infection caused by a yeast called candida albicans (a type of fungus). Yeast infections usually ... the vagina, it is known as vulvovaginal candidiasis . Candida can overgrow for many reasons. Stress, pregnancy, and ...

295

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

296

Mechanistic studies on the pyridoxal phosphate enzyme 1-aminocyclopropane-1-carboxylate deaminase from Pseudomonas sp.  

PubMed

The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACPC deaminase) from a pseudomonad is a pyridoxal phosphate (PLP) linked catalyst which fragments the cyclopropane substrate to alpha-ketobutyrate and ammonia [Honma, M., & Shimomura, T. (1978) Agric. Biol. Chem. 42, 1825]. Enzymatic incubations in D2O yield alpha-ketobutyrate with one deuterium at the C-4 methyl group and one deuterium at one of the C-3 prochiral methylene hydrogens. Stereochemical analysis of the location of the C-3 deuteron was accomplished by in situ enzymatic reduction to (2S)-2-hydroxybutyrate with L-lactate dehydrogenase and conversion to the phenacyl ester. The C-3 hydrogens of the (2S)-2-hydroxybutyryl moiety are fully resolved in a 250-MHz NMR spectrum. Absolute assignment of 3S and 3R loci was obtained with phenacyl (2S,3S)-2-hydroxy[3-2H]butyrate generated enzymatically by D-serine dehydratase action on D-threonine. ACPC deaminase shows a stereoselective outcome with a 3R:3S deuterated product ratio of 72:28. 2-Vinyl-ACPC is also a fragmentation substrate with exclusive regiospecific cleavage to yield the straight-chain keto acid product 2-keto-5-hexenoate. The D isomer of vinylglycine is processed to alpha-ketobutyrate and ammonia at 8% the Vmax of ACPC, while L-vinylglycine is not a substrate. It is likely that ACPC and D-vinylglycine yield a common intermediate--the vinylglycine-PLP-p-quinoid adduct--which is then protonated sequentially at C-4 and then C-3 to account for the observed deuterium incorporation. The D isomers of beta-substituted alanines (fluoroalanine, chloroalanine, and O-acetyl-D-serine) partition between catalytic elimination and enzyme inactivation. Each shows a different partition ratio, arguing against the common aminoacrylyl-PLP as the inactivating species. PMID:7326243

Walsh, C; Pascal, R A; Johnston, M; Raines, R; Dikshit, D; Krantz, A; Honma, M

1981-12-22

297

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

PubMed

Cytidine deaminases have important roles in the regulation of nucleoside/deoxynucleoside pools for DNA and RNA synthesis. The APOBEC family of cytidine deaminases (named after the first member of the family that was described, Apolipoprotein B mRNA Editing Catalytic Subunit 1, also known as APOBEC1 or A1) is a fascinating group of mutagenic proteins that use RNA and single-stranded DNA (ssDNA) as substrates for their cytidine or deoxycytidine deaminase activities. APOBEC proteins and base-modification nucleic acid editing have been the subject of numerous publications, reviews, and speculation. These proteins play diverse roles in host cell defense, protecting cells from invading genetic material, enabling the acquired immune response to antigens and changing protein expression at the level of the genetic code in mRNA or DNA. The amazing power these proteins have for interphase cell functions relies on structural and biochemical properties that are beginning to be understood. At the same time, the substrate selectivity of each member in the family and their regulation remains to be elucidated. This review of the APOBEC family will focus on an open question in regulation, namely what role the interactions of these proteins with RNA have in editing substrate recognition or allosteric regulation of DNA mutagenic and host-defense activities. WIREs RNA 2014, 5:493-508. doi: 10.1002/wrna.1226 For further resources related to this article, please visit the WIREs website. Conflict of interest: KMP, RPB, and JDS are employees of OyaGen Inc, which is a drug development biotechnology company engaged in the therapeutic targeting of editing enzymes. Dr. Smith is the founder of OyaGen and, as a consultant, serves as CEO and CSO for OyaGen. Dr. Smith is a tenured full professor in the Department of Biochemistry and Biophysics, Center for RNA Biology and Cancer Center at the University of Rochester, School of Medicine and Dentistry in Rochester, NY. PMID:24664896

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

2014-07-01

298

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

NASA Technical Reports Server (NTRS)

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

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

1997-01-01

299

Cytosine unstacking and strand slippage at an insertion-deletion mutation sequence in an overhang-containing DNA duplex.  

PubMed

Base unstacking in template strands, when accompanied by strand slippage, can result in deletion mutations during strand extension by nucleic acid polymerases. In a GCCC mutation hot-spot sequence, which was previously identified to have a 50% probability of causing such mutations during DNA replication by a Y-family polymerase, a single-base deletion mutation could result from such unstacking of any one of its three template cytosines. In this study, the intrinsic energetic differences in unstacking among these three cytosines in a solvated DNA duplex overhang model were examined using umbrella sampling molecular dynamics simulations. The free energy profiles obtained show that cytosine unstacking grows progressively more unfavorable as one moves inside the duplex from the 5'-end of the overhang template strand. Spontaneous strand slippage occurs in response to such base unstacking in the direction of both the major and minor grooves for all three cytosines. Unrestrained simulations run from three distinct strand-slipped states and one non-strand-slipped state suggest that a more duplexlike environment can help stabilize strand slippage. The possible underlying reasons and biological implications of these observations are discussed in the context of nucleic acid replication active site dynamics. PMID:24854722

Manjari, Swati R; Pata, Janice D; Banavali, Nilesh K

2014-06-17

300

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

301

Cytosine arabinoside in the treatment of acute myeloid leukemia: The role and place of high-dose regimens  

Microsoft Academic Search

Summary Cytosine arabinoside (AraC) is one of the most active drugs in the treatment of acute leukemias and is widely applied at all phases of therapy. In spite of extensive clinical and experimental investigations, its intracellular metabolism and especially its mechanism of action are still not fully elucidated. Controversy also continues about the most appropriate way and dose of administration;

W. Hiddemann

1991-01-01

302

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

2013-11-11

303

NSun2-Mediated Cytosine-5 Methylation of Vault Noncoding RNA Determines Its Processing into Regulatory Small RNAs  

PubMed Central

Summary Autosomal-recessive loss of the NSUN2 gene has been identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNAs), yet the identification of cytosine methylation in other RNA species has been hampered by the lack of sensitive and reliable molecular techniques. Here, we describe miCLIP as an additional approach for identifying RNA methylation sites in transcriptomes. miCLIP is a customized version of the individual-nucleotide-resolution crosslinking and immunoprecipitation (iCLIP) method. We confirm site-specific methylation in tRNAs and additional messenger and noncoding RNAs (ncRNAs). Among these, vault ncRNAs contained six NSun2-methylated cytosines, three of which were confirmed by RNA bisulfite sequencing. Using patient cells lacking the NSun2 protein, we further show that loss of cytosine-5 methylation in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Thus, impaired processing of vault ncRNA may contribute to the etiology of NSun2-deficiency human disorders.

Hussain, Shobbir; Sajini, Abdulrahim A.; Blanco, Sandra; Dietmann, Sabine; Lombard, Patrick; Sugimoto, Yoichiro; Paramor, Maike; Gleeson, Joseph G.; Odom, Duncan T.; Ule, Jernej; Frye, Michaela

2013-01-01

304

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

305

"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

306

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

307

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

PubMed Central

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

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

1995-01-01

308

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

309

[Multiple forms of adenosine deaminase in the thymus of the bovine fetus and calf].  

PubMed

We present data on values of adenosine deaminase (ADA) activity in bovine fetus and calf thymus. Between 13 and 17 weeks of fetal life enzyme activity in thymus increases up to levels observed in older fetuses and calf. ADA activity of thymus is characterized by the presence of three enzymic form ("A", "B", "C") which show molecular weights of greater than 1 x 10(6), 310,000 and 55,000, respectively. Forms "B" and "C" were compared. The relative amounts of the three forms in 17 weeks old fetus were quite similar to those observed in calf thymus. PMID:7337749

Rotondaro, L

1981-12-15

310

Mechanism, specificity and general properties of the yeast enzyme catalysing the formation of inosine 34 in the anticodon of transfer RNA.  

PubMed

In yeast, inosine is found at the first position of the anticodon (position 34) of seven different isoacceptor tRNA species, while in Escherichia coli it is present only in tRNAArg. The corresponding tRNA genes all have adenosine at position 34. Using as substrates in vitro T7-runoff transcripts of 31 plasmids carrying each natural of synthetic tRNA gene harbouring an anticodon with adenosine 34, we have characterised a yeast enzyme that catalyses the conversion of adenosine 34 to inosine 34. The homologous E. coli enzyme modifies adenosine 34 only in tRNAs with an arginine anticodon ACG. The base conversion occurs by a hydrolytic deamination-type reaction. This was determined by reversed phase high-pressure liquid chromatography/electrospray mass spectrometry analysis of the reaction product after in vitro modification in [18O]water. This newly characterised tRNA:adenosine 34 deaminase was partially purified from yeast. It has a molecular mass of approximately 75 kDa, and it does not require any cofactor, except magnesium ions, to deaminate adenosine 34 efficiently in tRNA. The observed dependence of the enzymatic reaction on magnesium ions probably reflects the need for a correct tRNA architecture. Enzymatic recognition of tRNA does not depend on the presence of any "identify" nucleoside other than adenosine 34. Likewise, the presence of pseudouridine 32 or 1-methyl-guanosine 37 in the anticodon loop does not interfere with inosine 34 biosynthesis. However, the efficacy of adenosine 34 to inosine 34 conversion depends on the nucleotide sequence of the anticodon loop and its proximal stem, the best tRNA substrates being those with a purine at position 35. Mutations that affect the size of the anticodon loop or one of several three-dimensional base-pairs abolish the capacity of the tRNA to be substrate for the yeast tRNA:adenosine 34 deaminase. Evidently, the activity of yeast tRNA:adenosine 34 deaminase depends more on the global structural feature (conformational stability/flexibility) of the L-shaped tRNA substrates than on the identity of any particular nucleotide other than adenosine 34. An apparent K(m) of 2.3 nM for its natural substrate tRNASer (anticodon AGA) was measured. Altogether, these results suggest that a single enzyme can account for the presence of inosine 34 in all seven cytoplasmic A34-containing precursor tRNAs in yeast. PMID:8893855

Auxilien, S; Crain, P F; Trewyn, R W; Grosjean, H

1996-10-01

311

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

312

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

PubMed

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 polycrystalline 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) cm(2) 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 NH(2) 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) cm(2), 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) cm(2) at 3.5 eV and then decreases toward 18 eV. The present vibrational enhancements, correspond to the features found around 1.5 and 4.5 eV in electron transmission spectroscopy (ETS) and those lying within 1.5-2.1 eV, 5.2-6.8 eV, and 9.5-10.9 eV range in dissociative electron attachment (DEA) experiments with cytosine in gas phase. While the ETS features are ascribed to shape resonances associated with the electron occupation of the second and third antibonding ?-orbitals of the molecule in its ground state, the correspondence with DEA features suggests the existence of common precursor anion states decaying with certain probabilities into the vibrationally excited ground state. PMID:22998289

Michaud, M; Bazin, M; Sanche, L

2012-09-21

313

Paramutation of the r1 locus of maize is associated with increased cytosine methylation.  

PubMed Central

In paramutation two alleles of a gene interact so that one of the alleles is epigenetically silenced. The silenced state is then genetically transmissible for many generations. The large (220 kbp) multigenic complex R-r is paramutable: its level of expression is changed during paramutation. R-r was found to exhibit increases in its level of cytosine methylation (C-methylation) following paramutation. These C-methylation changes are localized to the 5' portions of the two genes in the complex that are most sensitive to paramutation. These methylation changes flank a small region called sigma that is thought to have been derived from a transposon named doppia. A mutant derivative of R-r that has a deletion of the sigma region fails to become methylated under conditions in which R-r is heavily methylated. This suggests that the presence of sigma sequences at the locus is required for the methylation changes that are observed following paramutation.

Walker, E L

1998-01-01

314

I-motif nanospheres: unusual self-assembly of long cytosine strands.  

PubMed

The synthesis and characterization of novel DNA structures based on tetraplex cytosine (C) arrangements, known as i-motifs or i-tetraplexes, is reported. Atomic force microscopy (AFM) investigation shows that long C-strands in mild acidic conditions form compact spherically shaped nanostructures. The DNA nanospheres are characterized by a typical uniform shape and narrow height distribution. Electrostatic force microscopy (EFM) measurements performed on the i-motif spheres clearly show their electrical polarizability. Further investigations by scanning tunneling microscopy (STM) at ultrahigh vacuum reveals that the structures exhibit an average voltage gap of 1.9 eV, which is narrower than the voltage gap previously measured for poly(dG)-poly(dC) molecules in similar conditions. PMID:21381197

Zikich, Dragoslav; Liu, Ke; Sagiv, Lior; Porath, Danny; Kotlyar, Alexander

2011-04-18

315

DNA-directed assembly of polyanilines: modified cytosine nucleotides transfer sequence programmability to a conjoined polymer.  

PubMed

A series of polyaniline (PANI) oligomers was constructed from monomer units covalently linked to duplex DNA through N-(2-aminoethyl) groups bonded through cytosines. DNA oligomers containing the aniline monomers were treated with horseradish peroxidase (HRP) and H2O2 under conditions known to cause polymerization of aniline. No change in the absorption spectrum of the DNA was observed for samples containing fewer than four contiguous aniline groups. However, for oligomers containing four, five, or six aniline units, treatment with HRP and H2O2 led to the appearance of absorption features characteristic of the conducting "proton doped" emeraldine oxidation state of PANI. Molecular modeling shows that the DNA is distorted in the region of the PANI, but flanking regions of the DNA maintain their B-form structure. These findings provide a method to exploit the self-recognition, self-assembly, and sequence programmability of DNA for the formation of conducting polymers. PMID:17090004

Datta, Bhaskar; Schuster, Gary B; McCook, Amanda; Harvey, Stephen C; Zakrzewska, Krystyna

2006-11-15

316

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

PubMed Central

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.

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

2013-01-01

317

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

318

Tissue-Specific Differences in Cytosine Methylation and Their Association with Differential Gene Expression in Sorghum1[W  

PubMed Central

It has been well established that DNA cytosine methylation plays essential regulatory roles in imprinting gene expression in endosperm, and hence normal embryonic development, in the model plant Arabidopsis (Arabidopsis thaliana). Nonetheless, the developmental role of this epigenetic marker in cereal crops remains largely unexplored. Here, we report for sorghum (Sorghum bicolor) differences in relative cytosine methylation levels and patterns at 5?-CCGG sites in seven tissues (endosperm, embryo, leaf, root, young inflorescence, anther, and ovary), and characterize a set of tissue-specific differentially methylated regions (TDMRs). We found that the most enriched TDMRs in sorghum are specific for the endosperm and are generated concomitantly but imbalanced by decrease versus increase in cytosine methylation at multiple 5?-CCGG sites across the genome. This leads to more extensive demethylation in the endosperm than in other tissues, where TDMRs are mainly tissue nonspecific rather than specific to a particular tissue. Accordingly, relative to endosperm, the other six tissues showed grossly similar levels though distinct patterns of cytosine methylation, presumably as a result of a similar extent of concomitant decrease versus increase in cytosine methylation that occurred at variable genomic loci. All four tested TDMRs were validated by bisulfite genomic sequencing. Diverse sequences were found to underlie the TDMRs, including those encoding various known-function or predicted proteins, transposable elements, and those bearing homology to putative imprinted genes in maize (Zea mays). We further found that the expression pattern of at least some genic TDMRs was correlated with its tissue-specific methylation state, implicating a developmental role of DNA methylation in regulating tissue-specific or -preferential gene expression in sorghum.

Zhang, Meishan; Xu, Chunming; von Wettstein, Diter; Liu, Bao

2011-01-01

319

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

PubMed Central

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

Rayat, Sundeep; Qian, Ming; Glaser, Rainer

2008-01-01

320

Mutagen testing with yeast.  

PubMed

This article deals primarily with the practical aspects of mutagen testing with yeast. Equipment necessary for a laboratory where mutagen testing with yeast is performed, and the most commonly used media, are listed. Some general procedures are described and, finally, for those who have little experience with work of this kind, a precise protocol is given for an experiment with stationary phase cells of the strain D7 of Saccharomyces cerevisiae using the heteroallelic ade2 system as the genetic endpoint. Some experimental data were obtained by students following this protocol using the direct-acting mutagen ethyl methanesulfonate (EMS); these data are discussed and analyzed. More details on the various genetic endpoints available in numerous yeast strains and on the interpretation of dose-dependence data, as well as an extended list of yeast literature, can be found in an article by Eckardt and von Borstel in this volume. Further technical advice is provided in our references to Zimmermann (1975), von Borstel (1981), and Zimmermann et al. (1984). PMID:3904715

Eckardt, F; Siede, W

1985-01-01

321

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

322

Nucleoside analog studies indicate mechanistic differences between RNA-editing adenosine deaminases  

PubMed Central

Adenosine deaminases acting on RNA (ADAR1 and ADAR2) are human RNA-editing adenosine deaminases responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. Since inosine is recognized during translation as guanosine, this often results in the expression of protein sequences different from those encoded in the genome. While our knowledge of the ADAR2 structure and catalytic mechanism has grown over the years, our knowledge of ADAR1 has lagged. This is due, at least in part, to the lack of well defined, small RNA substrates useful for mechanistic studies of ADAR1. Here, we describe an ADAR1 substrate RNA that can be prepared by a combination of chemical synthesis and enzymatic ligation. Incorporation of adenosine analogs into this RNA and analysis of the rate of ADAR1 catalyzed deamination revealed similarities and differences in the way the ADARs recognize the edited nucleotide. Importantly, ADAR1 is more dependent than ADAR2 on the presence of N7 in the edited base. This difference between ADAR1 and ADAR2 appears to be dependent on the identity of a single amino acid residue near the active site. Thus, this work provides an important starting point in defining mechanistic differences between two functionally distinct human RNA editing ADARs.

Mizrahi, Rena A.; Phelps, Kelly J.; Ching, Andrea Y.; Beal, Peter A.

2012-01-01

323

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

PubMed

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

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

2014-06-01

324

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

PubMed

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

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

2012-07-12

325

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

PubMed Central

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

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

2012-01-01

326

On the validity of continuous spectrophotometric assays for adenosine deaminase activity: a critical reappraisal.  

PubMed

Kinetic investigations on adenosine deaminase from calf intestinal mucosa by spectrophotometric monitoring of the reaction at 264, 270, or 228 nm show that this method does not produce artifactual inhibition by substrate excess up to 0.7 mM concentration, when either adenosine or 2'-deoxyadenosine are employed with calf adenosine deaminase. The evaluation of kinetic parameters for this system was carried out both by initial rate measurements and by numerical differentiation of time progress curves according to a recently published method (S. C. Koerber and A. L. Fink, 1987, Anal. Biochem. 165, 75-87). The following results were obtained by the latter method at pH 7.0 and 30 degrees C: for the conversion of adenosine to inosine, kcat = 251 +/- 15 s-1, KMs = 29.7 +/- 2.8 microM, KMp = 613 +/- 62 microM; for the conversion of 2'-deoxyadenosine to 2'-deoxyinosine, kcat = 283 +/- 17 s-1, KMs = 22.4 +/- 2.2 microM, KMp = 331 +/- 35 microM. At 285 nm, a slight negative deviation from Beer's law was observed for adenosine at concentrations higher than 0.9 mM. No deviation was found for inosine up to 2.0 mM at the same wavelength. PMID:2035831

Cercignani, G; Allegrini, S

1991-02-01

327

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

SciTech Connect

We applied the ONIOM-molecular dynamics (MD) method to the hydrolytic deamination of cytidine by cytidine deaminase, which is an essential step of the activation process of the anticancer drug inside the human body. The direct MD simulations were performed for the realistic model of cytidine deaminase calculating the energy and its gradient by the ab initio ONIOM method on the fly. The ONIOM-MD calculations including the thermal motion show that the neighboring amino acid residue is an important factor of the environmental effects and significantly affects not only the geometry and energy of the substrate trapped in the pocket of the active site but also the elementary step of the catalytic reaction. We successfully simulate the second half of the catalytic cycle, which has been considered to involve the rate-determining step, and reveal that the rate-determing step is the release of the NH3 molecule. TM and MA were supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. MD was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, and by the Office of Biological and Environmental Research of the U.S. Department of Energy DOE. Battelle operates Pacific Northwest National Laboratory for DOE.

Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

2007-08-23

328

Two Arabidopsis threonine aldolases are nonredundant and compete with threonine deaminase for a common substrate pool.  

PubMed

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

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

2006-12-01

329

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

PubMed

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

George, Cyril X; John, Lijo; Samuel, Charles E

2014-06-01

330

Effects of Tet-induced oxidation products of 5-methylcytosine on Dnmt1- and DNMT3a-mediated cytosine methylation.  

PubMed

We investigated systematically the effects of Tet-induced oxidation products of 5-methylcytosine on Dnmt1- and DNMT3a-mediated cytosine methylation in synthetic duplex DNA. We found that the replacement of 5-methylcytosine at a CpG site with a 5-hydroxymethylcytosine, 5-formylcytosine, 5-carboxylcytosine or 5-hydroxymethyluracil resulted in altered methylation of cytosine at both the opposite and the neighboring CpG sites. Our results provided important new knowledge about the implications of the 5-methylcytosine oxidation products in maintenance cytosine methylation. PMID:24789765

Ji, Debin; Lin, Krystal; Song, Jikui; Wang, Yinsheng

2014-07-01

331

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

332

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

SciTech Connect

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

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

1988-10-18

333

Evidence for two distinct effector-binding sites in threonine deaminase by site-directed mutagenesis, kinetic, and binding experiments.  

PubMed

A three-dimensional structure comparison between the dimeric regulatory serine-binding domain of Escherichia coli D-3-phosphoglycerate dehydrogenase [Schuller, D. J., Grant, G. A., and Banaszak, L. J. (1995) Nat. Struct. Biol. 2, 69-76] and the regulatory domain of E. coli threonine deaminase [Gallagher, D. T., Gilliland, G. L., Xiao, G., Zondlo, J., Fisher, K. E., Chinchilla, D. , and Eisenstein, E. (1998) Structure 6, 465-475] led us to make the hypothesis that threonine deaminase could have two binding sites per monomer. To test this hypothesis about the corresponding plant enzyme, site-directed mutagenesis was carried out on the recombinant Arabidopsis thaliana threonine deaminase. Kinetic and binding experiments demonstrated for the first time that each regulatory domain of the monomers of A. thaliana threonine deaminase possesses two different effector-binding sites constituted in part by Y449 and Y543. Our results demonstrate that Y449 belongs to a high-affinity binding site whose interaction with a first isoleucine induces conformational modifications yielding a conformer displaying a higher activity and with enhanced ability to bind a second isoleucine on a lower-affinity binding site containing Y543. Isoleucine interaction with this latter binding site is responsible for conformational modifications leading to final inhibition of the enzyme. Y449 interacts with both regulators, isoleucine and valine. However, interaction of valine with the high-affinity binding site induces different conformational modifications leading to reversal of isoleucine binding and reversal of inhibition. PMID:11106492

Wessel, P M; Graciet, E; Douce, R; Dumas, R

2000-12-12

334

Arabidopsis methionine gamma-lyase is regulated according to isoleucine biosynthesis needs but plays a subordinate role to threonine deaminase.  

PubMed

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

Joshi, Vijay; Jander, Georg

2009-09-01

335

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

336

Extracellular Polysaccharides Produced by Yeasts and Yeast-Like Fungi  

NASA Astrophysics Data System (ADS)

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

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

337

5-methyl-cytosine and 5-hydroxy-methyl-cytosine in the genome of Biomphalaria glabrata, a snail intermediate host of Schistosoma mansoni  

PubMed Central

Background Biomphalaria glabrata is the mollusc intermediate host for Schistosoma mansoni, a digenean flatworm parasite that causes human intestinal schistosomiasis. An estimated 200 million people in 74 countries suffer from schistosomiasis, in terms of morbidity this is the most severe tropical disease after malaria. Epigenetic information informs on the status of gene activity that is heritable, for which changes are reversible and that is not based on the DNA sequence. Epigenetic mechanisms generate variability that provides a source for potentially heritable phenotypic variation and therefore could be involved in the adaptation to environmental constraint. Phenotypic variations are particularly important in host-parasite interactions in which both selective pressure and rate of evolution are high. In this context, epigenetic changes are expected to be major drivers of phenotypic plasticity and co-adaptation between host and parasite. Consequently, with characterization of the genomes of invertebrates that are parasite vectors or intermediate hosts, it is also essential to understand how the epigenetic machinery functions to better decipher the interplay between host and parasite. Methods The CpGo/e ratios were used as a proxy to investigate the occurrence of CpG methylation in B. glabrata coding regions. The presence of DNA methylation in B. glabrata was also confirmed by several experimental approaches: restriction enzymatic digestion with isoschizomers, bisulfite conversion based techniques and LC-MS/MS analysis. Results In this work, we report that DNA methylation, which is one of the carriers of epigenetic information, occurs in B. glabrata; approximately 2% of cytosine nucleotides are methylated. We describe the methylation machinery of B. glabrata. Methylation occurs predominantly at CpG sites, present at high ratios in coding regions of genes associated with housekeeping functions. We also demonstrate by bisulfite treatment that methylation occurs in multiple copies of Nimbus, a transposable element. Conclusions This study details DNA methylation for the first time, one of the carriers of epigenetic information in B. glabrata. The general characteristics of DNA methylation that we observed in the B. glabrata genome conform to what epigenetic studies have reported from other invertebrate species.

2013-01-01

338

Yeast-Hyphal Dimorphism  

Microsoft Academic Search

All fungi have some capacity to grow in two basic morphological forms — spheres and tubes — therefore it could be argued that\\u000a they are all, to some extent, dimorphic. For many filamentous fungi spherical growth may only be expressed during the formation\\u000a of spores and many yeast-like fungi have only the remnants of a true filamentous growth habit. However,

N. A. R. Gow

339

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

PubMed Central

The absolute cross sections (CS) for electronic excitations of cytosine by electron impact between 5 and 18 eV were measured by electron-energy loss (EEL) spectroscopy of the molecule deposited at low coverage on an inert Ar substrate. The lowest EEL features found at 3.55 and 4.02 eV are ascribed to transitions from the ground state to the two lowest triplet 1 3A?(???*) and 2 3A?(???*) valence states of the molecule. Their energy dependent CS exhibit essentially a common maximum at about 6 eV with a value of 1.84 × 10?17 cm2 for the former and 4.94 × 10?17 cm2 for the latter. In contrast, the CS for the next EEL feature at 4.65 eV, which is ascribed to the optically allowed transition to the 2 1A?(???*) valence state, shows only a steep rise to about 1.04 × 10?16 cm2 followed by a monotonous decrease with the incident electron energy. The higher EEL features at 5.39, 6.18, 6.83, and 7.55 eV are assigned to the excitations of the 3 3, 1A?(???*), 4 1A?(???*), 5 1A?(???*), and 6 1A?(???*) valence states, respectively. The CS for the 3 3, 1A? and 4 1A? states exhibit a common enhancement at about 10 eV superimposed on a more or less a steep rise, reaching respectively a maximum of 1.27 and 1.79 × 10?16 cm2, followed by a monotonous decrease. This latter enhancement and the maximum seen at about 6 eV in the lowest triplet states correspond to the core-excited electron resonances that have been found by dissociative electron attachment experiments with cytosine in the gas phase. The weak EEL feature found at 5.01 eV with a maximum CS of 3.8 × 10?18 cm2 near its excitation threshold is attributed to transitions from the ground state to the 1 3, 1A?(n??*) states. The monotonous rise of the EEL signal above 8 eV is attributed to the ionization of the molecule. It is partitioned into four excitation energy regions at about 8.55, 9.21, 9.83, and 11.53 eV, which correspond closely to the ionization energies of the four highest occupied molecular orbitals of cytosine. The sum of the ionization CS for these four excitation regions reaches a maximum of 8.1 × 10?16 cm2 at the incident energy of 13 eV.

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

2013-01-01

340

Yeast Colony Embedding Method  

PubMed Central

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood. One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 ?) useful for light microscopy and thin sections (0.1 ?) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM. The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

Piccirillo, Sarah; Honigberg, Saul M.

2011-01-01

341

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

2006-01-01

342

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

PubMed Central

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

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

2012-01-01

343

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

2008-02-27

344

Ab initio study of a biradical radiationless decay channel of the lowest excited electronic state of cytosine and its derivatives  

NASA Astrophysics Data System (ADS)

A theoretical model for the ultrafast S1-->S0 internal conversion of cytosine is presented, in which a state switch from the initially prepared 1??* state to the out-of-plane deformed excited state of biradical character controls the rate of the S1(1??*) decay. This mechanism successfully accounts for the dramatically longer S1 lifetimes of 5-fluorocytosine and N-acetylcytosine relative to cytosine. The replacement of the C5 hydrogen atom by a methyl group is predicted to lead to a substantial, but not dramatic, increase in the S1 lifetime, also consistent with experiment. It is this ability to correctly predict the substituent effects that distinguishes the present model from the previously proposed mechanisms.

Zgierski, Marek Z.; Patchkovskii, Serguei; Lim, Edward C.

2005-08-01

345

Synthesis of adenine, guanine, cytosine, and other nitrogen organic compounds by a Fischer-Tropsch-like process.  

NASA Technical Reports Server (NTRS)

Study of the formation of purines, pyrimidines, and other bases from CO, H2, and NH3 under conditions similar to those used in the Fischer-Tropsch process. It is found that industrial nickel/iron alloy catalyzes the synthesis of adenine, guanine, cytosine, and other nitrogenous compounds from mixtures of CO, H2, and NH3 at temperatures of about 600 C. Sufficient sample was accumulated to isolate as solid products adenine, guanine, and cytosine, which were identified by infrared spectrophotometry. In the absence of nickel/iron catalyst, at 650 C, or in the presence of this catalyst, at 450 C, no purines or pyrimidines were synthesized. These results confirm and extend some of the work reported by Kayatsu et al. (1968).

Yang, C. C.; Oro, J.

1971-01-01

346

Methylation-Dependent Fragment Separation: Novel Analysis Of 5Methyl Cytosine By Capillary Electrophoresis Of Amplified Dna Using Pcr Incorporation Of Chemically Modified Dctp  

Microsoft Academic Search

Methylation of the cytosine (C) ring to form 5-methyl cytosine (MeC) in normally unmethylated CpG-rich regions of promoters in genes is associated with transcriptional silencing. Quantification of MeC is of current interest in findining new biomarkers for cancer. To this end, and for basic research in epigenomics, we have investigated a new method for relatively simple measurement of MeC content

Victoria L. Boyd; Kristina I. Moody; Achim E. Karger; Kenneth J. Livak; Gerald Zon; John W. Burns

2007-01-01

347

The effect of 1-?-d-arabinofuranosyl-cytosine on the expression of the common fragile site at 3p14  

Microsoft Academic Search

The effect of the G2-treatment of 1-ß-d-arabino-furanosyl-cytosine (araC) on the expression of the common fragile site at 3p14 (FRA3B) was studied. A significantly increased frequency of FRA3B induced by G2 treatment of araC was found in the lymphocytes grown in folate-deficient medium (positive rate 100%). A relatively low frequency of FRA3B was also induced in the cultures with folate in

Xinzhi Li; Zu-an Yan; Xianting Zhou

1986-01-01

348

Synthetic unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides are potent stimulators of antileukemia responses in naive and bone marrow transplant recipients  

Microsoft Academic Search

Immunostimulatory cytosine-phophate- guanosine (CpG)-containing motifs in bacterial DNA are potent immune system activators. Depending on the bases flank- ing the CpG motif and on the DNA back- bone, CpG oligodeoxynucleotides (ODNs) can induce relatively more B-cell activa- tion or relatively more natural killer (NK)- cell activation. To evaluate their antitu- mor activities, an NK-optimized ODN (1585) and 2 B-cell-optimized ODNs

Bruce R. Blazar; Arthur M. Krieg; Patricia A. Taylor

349

Impact of androgen receptor cytosine-adenine-guanine polymorphisms on clinical outcome in BRCA mutation-associated epithelial ovarian cancers  

Microsoft Academic Search

PurposeAndrogen signaling may function in the pathobiology of epithelial ovarian cancers associated with mutations in the BRCA1\\/2 genes. Androgen receptor (AR) activity correlates inversely with length of a polymorphic cytosine-adenine-guanine (CAG) repeat in exon 1. We hypothesized that AR CAG allele length is a modifier of clinical outcome in BRCA1\\/2 mutation positive women with ovarian cancer.

Andrew J. Li; Paula McAllister; Beth Y. Karlan

2010-01-01

350

Cyclophosphamide, cytosine arabinoside and TBI as a conditioning regimen for allogeneic bone marrow transplantation in patients with leukemia  

Microsoft Academic Search

This is a prospective study designed to determine the toxicity, efficacy and antileukemic effect of high-dose cytosine arabinoside (ara-C), cyclophosphamide and total body irradiation (TBI) as a myeloablative regimen prior to allogeneic bone marrow transplantation for patients with hematologic malignancies. Fifty-eight patients with hematologic malignancies were treated with cyclophosphamide, high-dose ara-C and total body irradiation (TBI) followed by allogeneic bone

AP Jillella; R Doria; K Khan; D Zelterman; YH Ahmad; BR Smith; W Holmes; P Becker; KB Roberts; JM Rappeport

1999-01-01

351

Retrotransposon silencing and telomere integrity in somatic cells of Drosophila depends on the cytosine-5 methyltransferase DNMT2  

Microsoft Academic Search

Here we show that the cytosine-5 methyltransferase DNMT2 controls retrotransposon silencing in Drosophila somatic cells. In Drosophila, significant DNMT2-dependent DNA methylation occurs during early embryogenesis. Suppression of white gene silencing by Mt2 (Dnmt2) null mutations in variegated P[w+] element insertions identified functional targets of DNMT2. The enzyme controls DNA methylation at retrotransposons in early embryos and initiates histone H4K20 trimethylation

Sameer Phalke; Olaf Nickel; Diana Walluscheck; Frank Hortig; Maria Cristina Onorati; Gunter Reuter

2009-01-01

352

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

SciTech Connect

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

Grobelsek-Vracko, M.; Zaider, M. (College of Physicians and Surgeons of Columbia Univ., New York, NY (United States))

1994-04-01

353

[Determination of guanine plus cytosine content of Pseudomonas cocovenenans subsp farinofermantans with two methods and the comparative study].  

PubMed

The guanine plus cytosine content (G + C) mol% of Pseudomonas cocovenenans subsp farinofermantans, Burkholderia gladioli and Burkholderia cocovenenans, which were similar in biochemical characteristics, were determined with Tm values method and HPLC method, and the comparative study of these two methods was discussed. A negative conclusion could not be made from the results. The three bacteria were the same species. The HPLC method was more accurate than the Tm values method, but the repeatability of the latter was better. PMID:12520932

Jiao, Z; Liu, X; Yang, R; Guo, Z

2000-07-01

354

Human adipose tissue-derived mesenchymal stem cells expressing yeast cytosinedeaminase::uracil phosphoribosyltransferase inhibit intracerebral rat glioblastoma.  

PubMed

Prodrug cancer gene therapy by mesenchymal stem cells (MSCs) targeted to tumors represents an attractive tool to activate prodrugs directly within the tumor mass, thus avoiding systemic toxicity. In this study, we tested the feasibility and efficacy of human adipose tissue-derived MSCs, engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase to treat intracranial rat C6 glioblastoma. Experiments were designed to simulate conditions of future clinical application for high-grade glioblastoma therapy by direct injections of therapeutic stem cells into tumor. We demonstrated that genetically modified therapeutic stem cells still have the tumor tropism when injected to a distant intracranial site and effectively inhibited glioblastoma growth after 5-fluorocytosine (5-FC) therapy. Coadministration of C6 cells and therapeutic stem cells with delayed 5-FC therapy improved the survival in a therapeutic stem cell dose-dependent manner and induced complete tumor regression in a significant number of animals. Continuous intracerebroventricular delivery of 5-FC using osmotic pump reduced the dose of prodrug required for the same therapeutic effect, and along with repeated administration of therapeutic stem cells increased the survival time. Intracerebral injection of therapeutic stem cells and treatment with 5-FC did not show any detectable adverse effects. Results support the arguments to begin clinical studies for treatment of high-grade brain tumors. PMID:21732344

Altanerova, Veronika; Cihova, Marina; Babic, Michal; Rychly, Boris; Ondicova, Katarina; Mravec, Boris; Altaner, Cestmir

2012-05-15

355

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

PubMed

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

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

2010-11-25

356

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

PubMed

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

Hayatsu, Hikoya

2008-01-01

357

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

PubMed Central

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

Hayatsu, Hikoya

2008-01-01

358

The cysteine conserved among DNA cytosine methylases is required for methyl transfer, but not for specific DNA binding.  

PubMed Central

All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating the normally inert C5 position. We show here that substitutions of this cysteine in the E. coli methylase M. EcoRII with either serine or tryptophan results in a complete loss of ability to transfer methyl groups to DNA. Interestingly, mutants with either serine or glycine substitution bind tightly to substrate DNA. These mutants resemble the wild-type enzyme in that their binding to substrate is not eliminated by the presence of non-specific DNA in the reaction, it is sensitive to methylation status of the substrate and is stimulated by an analog of the methyl donor. Hence the conserved cysteine is not essential for the specific stable binding of the enzyme to its substrate. However, substitution of the cysteine with the bulkier tryptophan does reduce DNA binding. We also report here a novel procedure for the synthesis of DNA containing 5-fluorocytosine. Further, we show that a DNA substrate for M. EcoRII in which the target cytosine is replaced by 5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms an irreversible complex with the enzyme. As expected, this modified substrate does not form irreversible complexes with the mutants. Images

Wyszynski, M W; Gabbara, S; Kubareva, E A; Romanova, E A; Oretskaya, T S; Gromova, E S; Shabarova, Z A; Bhagwat, A S

1993-01-01

359

Mitochondrial genome of the basidiomycetous yeast Jaminaea angkorensis.  

PubMed

Jaminaea angkorensis is an anamorphic basidiomycetous yeast species originally isolated from decaying leaves in Cambodia. Taxonomically, J. angkorensis is affiliated with Microstromatales (Exobasidiomycetes, Ustilaginomycotina, Basidiomycota) and represents a basal phylogenetic lineage of this fungal order. To perform a comparative analysis of J. angkorensis with other basidiomycetes, we determined and analyzed its complete mitochondrial DNA sequence. The mitochondrial genome is represented by 29,999 base pairs long, circular DNA containing 32 % guanine and cytosine residues. Its genetic organization is relatively compact and comprises typical genes for 15 conserved proteins involved in oxidative phosphorylation (atp6, 8, and 9; cob; cox1, 2, and 3; and nad1, 2, 3, 4, 4L, 5, and 6) and translation (rps3), two ribosomal RNAs (rnl and rns) and twenty-two transfer RNAs (trnA-Y). Although the gene content is similar to other basidiomycetes, the gene orders in the examined species exhibit only a limited synteny, reflecting their phylogenetic distances and extensive genome rearrangements. In addition, a comparative analysis of basidiomycete mitochondrial genomes indicates that stop-to-tryptophan reassignment of the UGA codon was accompanied by structural alterations of tRNA-Trp(CCA). These results provide an insight into the evolution of the genetic code in fungal mitochondria. PMID:24071901

Hegedusova, Eva; Brejova, Brona; Tomaska, Lubomir; Sipiczki, Matthias; Nosek, Jozef

2014-02-01

360

Selection and amplification of heterologous genes encoding adenosine deaminase in mammalian cells.  

PubMed Central

We demonstrate that an adenosine deaminase (ADA) cDNA gene can function as a dominant selectable and amplifiable marker for gene transfer experiments in mammalian cells. Cells that incorporate the gene can be selected by growth in the presence of low concentrations of the ADA inhibitor 2'-deoxycoformycin with cytotoxic concentrations of adenosine or its analogue 9-beta-D-xylofuranosyl adenine. The DNA copy number of the transfected ADA minigene in the isolated transformants of Chinese hamster ovary cells can be amplified greater than 100-fold by growth in ADA selection media and increasing concentrations of 2'-deoxycoformycin. This selection scheme may allow for the introduction and subsequent amplification of heterologous DNA in a variety of mammalian cells. Images

Kaufman, R J; Murtha, P; Ingolia, D E; Yeung, C Y; Kellems, R E

1986-01-01

361

Potential roles of Activation-Induced cytidine Deaminase in promotion or prevention of autoimmunity in humans  

PubMed Central

Autoimmune manifestations are paradoxical and frequent complications of primary immunodeficiencies, including T and/or B cell defects. Among pure B cell defects, the Activation-induced cytidine Deaminase (AID)-deficiency, characterized by a complete lack of immunoglobulin class switch recombination and somatic hypermutation, is especially complicated by autoimmune disorders. We summarized in this review the different autoimmune and inflammatory manifestations present in twelve patients out of a cohort of 45 patients. Moreover, we also review the impact of AID mutations on B-cell tolerance and discuss hypotheses that may explain why central and peripheral B-cell tolerance was abnormal in the absence of functional AID. Hence, AID plays an essential role in controlling autoreactive B cells in humans and prevents the development of autoimmune syndromes.

Durandy, Anne; Cantaert, Tineke; Kracker, Sven; Meffre, Eric

2014-01-01

362

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

PubMed Central

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

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

2011-01-01

363

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

PubMed Central

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

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

2014-01-01

364

Leptomeningeal gliomatosis with high levels of adenosine deaminase in the cerebrospinal fluid.  

PubMed

A 61-year-old man developed disturbance of consciousness for 2 weeks. He showed neck stiffness and hyporeflexia. Analysis of his cerebrospinal fluid (CSF) revealed pleocytosis and markedly reduced glucose contents. Adenosine deaminase (ADA) levels in the CSF were elevated (28.8 IU/l). Brain magnetic resonance imagings showed enhancement of the leptomeninges. Tuberculous meningitis was considered, but antituberculous drug was not effective. Repeated cytological analysis of the CSF demonstrated atypical cells with enlarged unevenly distributed nuclei and immunoreactive with glial fibrillary acidic protein. We diagnosed him as leptomeningeal gliomatosis. CSF ADA may be elevated in this rare disorder, and here we emphasize that repeated cytological analysis with immunohistochemical staining was useful for diagnosis. PMID:24807273

Nishihara, Hideaki; Omoto, Masatoshi; Ogasawara, Jun-Ichi; Koga, Michiaki; Kawai, Motoharu; Kanda, Takashi

2014-01-01

365

Crystal structure determination and dynamic studies of Mycobacterium tuberculosis Cytidine deaminase in complex with products.  

PubMed

Cytidine deaminase (CDA) is a key enzyme in the pyrimidine salvage pathway. It is involved in the hydrolytic deamination of cytidine or 2'-deoxycytidine to uridine or 2'-deoxyuridine, respectively. Here we report the crystal structures of Mycobacterium tuberculosis CDA (MtCDA) in complex with uridine (2.4 Å resolution) and deoxyuridine (1.9 Å resolution). Molecular dynamics (MD) simulation was performed to analyze the physically relevant motions involved in the protein-ligand recognition process, showing that structural flexibility of some protein regions are important to product binding. In addition, MD simulations allowed the analysis of the stability of tetrameric MtCDA structure. These findings open-up the possibility to use MtCDA as a target in future studies aiming to the rational design of new inhibitor of MtCDA-catalyzed chemical reaction with potential anti-proliferative activity on cell growth of M. tuberculosis, the major causative agent of tuberculosis. PMID:21295009

Sánchez-Quitian, Zilpa A; Timmers, Luís F S M; Caceres, Rafael A; Rehm, Jacqueline G; Thompson, Claudia E; Basso, Luiz A; de Azevedo, Walter F; Santos, Diógenes S

2011-05-01

366

Combining molecular dynamics and docking simulations of the cytidine deaminase from Mycobacterium tuberculosis H37Rv.  

PubMed

Cytidine Deaminase (CD) is an evolutionarily conserved enzyme that participates in the pyrimidine salvage pathway recycling cytidine and deoxycytidine into uridine and deoxyuridine, respectively. Here, our goal is to apply computational techniques in the pursuit of potential inhibitors of Mycobacterium tuberculosis CD (MtCDA) enzyme activity. Molecular docking simulation was applied to find the possible hit compounds. Molecular dynamics simulations were also carried out to investigate the physically relevant motions involved in the protein-ligand recognition process, aiming at providing estimates for free energy of binding. The proposed approach was capable of identifying a potential inhibitor, which was experimentally confirmed by IC(50) evaluation. Our findings open up the possibility to extend this protocol to different databases in order to find new potential inhibitors for promising targets based on a rational drug design process. PMID:21541749

Timmers, Luís Fernando Saraiva Macedo; Ducati, Rodrigo Gay; Sánchez-Quitian, Zilpa Adriana; Basso, Luiz Augusto; Santos, Diógenes Santiago; de Azevedo, Walter Filgueira

2012-02-01

367

Identification of a specific domain required for dimerization of activation-induced cytidine deaminase.  

PubMed

Activation-induced cytidine deaminase (AID) is essential to all three genetic alterations required for generation of antigen-specific immunoglobulin: class switch recombination, somatic hypermutation, and gene conversion. Here we demonstrate that AID molecules form a homodimer autonomously in the absence of RNA, DNA, other cofactors, or post-translational modifications. Studies on serial deletion mutants revealed the minimum region between Thr27 and His56 responsible for dimerization. Analyses of point mutations within this region revealed that the residues between Gly47 and Gly54 are most important for the dimer formation. Functional analyses of these mutations indicate that all mutations impairing the dimer formation are inefficient for class switching, suggesting that dimer formation is required for class switching activity. Dimer formation and its requirement for the function of AID are features that AID shares with APOBEC-1, an RNA editing enzyme of apolipoprotein B100 mRNA. PMID:16687409

Wang, Jishu; Shinkura, Reiko; Muramatsu, Masamichi; Nagaoka, Hitoshi; Kinoshita, Kazuo; Honjo, Tasuku

2006-07-14

368

Biochemical and mass spectrometric evidence for quaternary structure modifications of plant threonine deaminase induced by isoleucine.  

PubMed

Arabidopsis thaliana threonine deaminase (TD) is a tetramer composed of identical approximately 59600 Da subunits. TD activity has been shown to be inhibited by isoleucine. This effect is reversed by a large excess of valine. Nondenaturant gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry experiments demonstrated that binding of isoleucine on TD induces dimerization of the enzyme, whereas tetramerization is restored by addition of a high valine concentration. Nondenaturant gel filtration and electrospray ionization mass spectrometry of the enzyme in the presence of increasing amounts of isoleucine suggest a fast equilibrium between the tetramer and the dimer. Finally, study of TD mutants allowed us to focus on the specific role of each isoleucine-binding site. PMID:12427039

Halgand, Frédéric; Wessel, Peter M; Laprévote, Olivier; Dumas, Renaud

2002-11-19

369

Design, synthesis, and pharmacological evaluation of fluorinated tetrahydrouridine derivatives as inhibitors of cytidine deaminase.  

PubMed

Several 2'-fluorinated tetrahydrouridine derivatives were synthesized as inhibitors of cytidine deaminase (CDA). (4R)-2'-Deoxy-2',2'-difluoro-3,4,5,6-tetrahydrouridine (7a) showed enhanced acid stability over tetrahydrouridine (THU) 5 at its N-glycosyl bond. As a result, compound 7a showed an improved oral pharmacokinetic profile with a higher and more reproducible plasma exposure in rhesus monkeys compared to 5. Co-administration of 7a with decitabine, a CDA substrate, boosted the plasma levels of decitabine in rhesus monkeys. These results demonstrate that compound 7a can serve as an acid-stable alternative to 5 as a pharmacoenhancer of drugs subject to CDA-mediated metabolism. PMID:24520856

Ferraris, Dana; Duvall, Bridget; Delahanty, Greg; Mistry, Bipin; Alt, Jesse; Rojas, Camilo; Rowbottom, Christopher; Sanders, Kristen; Schuck, Edgar; Huang, Kuan-Chun; Redkar, Sanjeev; Slusher, Barbara B; Tsukamoto, Takashi

2014-03-27

370

Static magnetic field inhibits adenosine deaminase activity in cancerous and noncancerous human gastric tissues.  

PubMed

Abstract Aim: Investigation of possible effects of static magnetic field (SMF) on adenosine deaminase (ADA) activity in cancerous and noncancerous human gastric and colon tissues to obtain information about possible action mechanism of SMF. Materials and Methods: Cancerous and noncancerous human gastric and colon tissues removed from patients by surgical operations were used in the studies. SMF was created by using two static magnets. Before and after treatment with SMF, ADA activities in the tissue samples were measured. Results: The ADA activity was found to be lowered in gastric tissues treated with the SMF. However, no change was observed in the ADA activity of colon tissues. Conclusions: Our results suggest that SMF inhibits the ADA enzyme in gastric tissues significantly. It is supposed that, in addition to other proposed mechanisms, accumulated adenosine due to the inhibition of the ADA enzyme might also play a part in the anticancer activity of SMF. PMID:24784458

Durak, Zahide Esra; Kocao?lu, Ender Hilmi; Oztürk, Bahad?r

2014-05-01

371

Negative regulation of activation-induced cytidine deaminase in B cells  

PubMed Central

Both class switch recombination (CSR) and somatic hypermutation (SHM) of the Ig genes require the activity of activation-induced cytidine deaminase (AID). Expression of AID is restricted to B cells in the germinal centers of the lymphoid organs, where activated B cells undergo CSR and SHM. We previously showed that constitutive and systemic expression of AID leads to tumorigenesis in T cells and lung epithelium, but not in B cells. This finding led us to suspect that transgenic AID may be inactivated at least in part in B cells. To address this issue, we generated conditional AID-transgenic mice that constitutively express AID only in B cells. Studies on the cross between the AID-transgenic and AID-deficient mice showed that abundant AID protein accumulated by constitutive expression is inactivated in B cells, possibly providing an explanation for the absence of deregulation of CSR and SHM in AID-transgenic B cells.

Muto, Taro; Okazaki, Il-mi; Yamada, Shuichi; Tanaka, Yoshimasa; Kinoshita, Kazuo; Muramatsu, Masamichi; Nagaoka, Hitoshi; Honjo, Tasuku

2006-01-01

372

Simultaneous detection of guanine, adenine, thymine and cytosine at choline monolayer supported multiwalled carbon nanotubes film.  

PubMed

A rapid, convenient and accurate method for the simultaneous detection of guanine (G), adenine (A), thymine (T) and cytosine (C) was developed at a multiwalled carbon nanotube (MWCNT)/choline (Ch) monolayer-modified glassy carbon electrode (GCE). X-ray photoelectron spectroscopy data demonstrated that Ch was covalently immobilised on the surface of GCE through oxygen atom. The Ch monolayer provides a positively charged surface with -N(+)(CH(3))(3) polar groups, so that it can attract negatively charged MWCNTs to the surface. Consequently, the MWCNT/Ch film exhibited remarkable electrocatalytic activities towards the oxidation of G, A, T and C due to the advantages of high electrode activity, large surface area, prominent antifouling property, and high electron transfer kinetics. All purine and pyrimidine bases showed well-defined catalytic oxidation peaks at MWCNT/Ch/GCE. The peak separations between G and A, A and T, and T and C are 270, 200, and 190 mV, respectively, which are sufficiently large for their potential recognition and simultaneous detection in mixture. Under the optimum conditions, the designed MWCNT/Ch/GCE exhibited low detection limit, high sensitivity and wide linear range for simultaneous detection of G, A, T and C. Moreover, the proposed method was successfully applied to the assessment of G, A, T and C contents in a herring sperm DNA sample with satisfactory results. PMID:21296567

Wang, Po; Wu, Hai; Dai, Zong; Zou, Xiaoyong

2011-03-15

373

Molecular cloning, expression, and characterization of starfish DNA (cytosine-5)-methyltransferases.  

PubMed

To determine whether and if so how a DNA methylation-dependent epigenetic mechanism for transcriptional gene silencing functions in Echinoderms, we cloned and sequenced dnmt1 and dnmt3 cDNAs of the starfish Asterina pectinifera. Since the Strongylocentrotus purpuratus genome has only two loci of DNA (cytosine-5)-methyltransferase genes encoding Dnmt1 and Dnmt3, they might constitute a sufficient set of dnmt genes in Echinoderms. The starfish Dnmt3 whose cDNA we cloned showed highest homology to a mammalian Dnmt3a2 splicing variant. Essentially all the characteristic motifs and sequences of the mammalian counterparts were found in the starfish Dnmts as well, except that a typical PCNA binding domain motif was lacking in the starfish Dnmt1. RT-PCR analysis indicated that the dnmt1 mRNA exists in both ovary and oocytes, but its levels in other tissues were very low or almost negligible. In contrast, the dnmt3 mRNA was detected only in the ovary, and not at all in the oocytes. The size of a dnmt1 transcript was about 6.5 kb on Northern blot analysis. On heterologous expression, the starfish Dnmt1 protein was expressed in insect cells in catalytically active form. PMID:22972351

Fujihara, Yoshihito; Miyasako, Hiroshi; Kato, Kumiko; Hayashi, Tadahiro; Toraya, Tetsuo

2012-01-01

374

Morphologic and phenotypic changes of human neuroblastoma cells in culture induced by cytosine arabinoside  

SciTech Connect

The effects of cytosine-arabinoside (ARA-C) on the growth and phenotypic expression of a new human neuroblastoma (NB) cell line (GI-ME-N) have been extensively tested. Low doses of ARA-C allowing more than 90% cell viability induce morphological differentiation and growth inhibition. Differentiated cells were larger and flattened with elongated dendritic processes; such cells appeared within 48 hours after a dose of ARA-C as low as 0.1 {mu}g/ml. The new morphological aspect reached the maximum expression after 5-6 days of culture being independent from the addition of extra drug to the culture. A decrease in ({sup 3}H)thymidine incorporation was also observed within 24 hours and the cell growth was completely inhibited on the sixth day. Moreover, ARA-C strongly inhibited anchorage-independent growth in soft agar assay. Membrane immunofluorescence showed several dramatic changes in NB-specific antigen expression after 5 days of treatment with ARA-C. At the same time ARA-C also modulated cytoskeletal proteins and slightly increased catecholamine expression. These findings suggest that noncytotoxic doses of ARA-C do promote the differentiation of GI-ME-N neuroblastoma cells associated with reduced expression of the malignant phenotype.

Ponzoni, M.; Lanciotti, M.; Melodia, A.; Casalaro, A.; Cornaglia-Ferraris, P. (G. Gaslini Children's Hospital, Genoa (Italy))

1989-03-01

375

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

PubMed Central

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

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

2011-01-01

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

Cytosine Arabinoside Therapy for Herpes Simplex Encephalitis--Clinical Experience with Six Patients  

PubMed Central

Two neonates and four adults with herpes simplex virus (HSV) encephalitis were treated with cytosine arabinoside (Ara-C). A low dose of 40 to 160 mg per m2 per day was given for 4 to 6 days by continuous intravenous infusion and, except in two cases, by intrathecal administration. In one patient, idoxuridine (IUdR) at the dose of 1 g every 4 h was also administered after 4 days of Ara-C therapy. Both neonates and two of four adults survived. Their clinical improvement was closely related in time to the onset of therapy with Ara-C (cases 1, 2, 3) and with IUdR (case 4). In one adult who died on the 27th day of illness of a massive pulmonary embolus, postmortem examination of the brain did not disclose viral inclusions, and viral culture was negative. In the other patient who died, however, brain culture postmortem was still positive for HSV despite 4 days of Ara-C therapy. Ara-C, in addition to IUdR, may be effective in HSV encephalitis treatment, but double-blind, controlled studies appear to be necessary with these agents.

Chow, Anthony W.; Ronald, Allan; Fiala, Milan; Hryniuk, William; Weil, Marvin L.; Geme, Joseph St.; Guze, Lucien B.

1973-01-01

378

High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells  

PubMed Central

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 obscure any correlation between GC content and expression. In the present work, we directly compared the expression of GC-rich and GC-poor genes placed in the context of identical promoters and UTR sequences. We performed transient and stable transfections of mammalian cells with GC-rich and GC-poor versions of Hsp70, green fluorescent protein, and IL2 genes. The GC-rich genes were expressed several-fold to over a 100-fold more efficiently than their GC-poor counterparts. This effect was not due to different translation rates of GC-rich and GC-poor mRNA. On the contrary, the efficient expression of GC-rich genes resulted from their increased steady-state mRNA levels. mRNA degradation rates were not correlated with GC content, suggesting that efficient transcription or mRNA processing is responsible for the high expression of GC-rich genes. We conclude that silent-site GC content correlates with gene expression efficiency in mammalian cells.

Caffin, Fanny; Helwak, Aleksandra; Zylicz, Maciej

2006-01-01

379

The extended and eccentric E-DNA structure induced by cytosine methylation or bromination.  

PubMed

Cytosine methylation or bromination of the DNA sequence d(GGCGCC)2 is shown here to induce a novel extended and eccentric double helix, which we call E-DNA. Like B-DNA, E-DNA has a long helical rise and bases perpendicular to the helix axis. However, the 3'-endo sugar conformation gives the characteristic deep major groove and shallow minor groove of A-DNA. Also, if allowed to crystallize for a period of time longer than that yielding E-DNA, the methylated sequence forms standard A-DNA, suggesting that E-DNA is a kinetically trapped intermediate in the transition to A-DNA. Thus, the structures presented here chart a crystallographic pathway from B-DNA to A-DNA through the E-DNA intermediate in a single sequence. The E-DNA surface is highly accessible to solvent, with waters in the major groove sitting on exposed faces of the stacked nucleotides. We suggest that the geometry of the waters and the stacked base pairs would promote the spontaneous deamination of 5-methylcytosine in the transition mutation of dm5C-dG to dT-dA base pairs. PMID:10966645

Vargason, J M; Eichman, B F; Ho, P S

2000-09-01

380

Evolutionary breakpoints in the gibbon suggest association between cytosine methylation and karyotype evolution.  

PubMed

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. PMID:19557196

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

2009-06-01

381

Dyson norms in XUV and strong-field ionization of polyatomics: Cytosine and uracil  

NASA Astrophysics Data System (ADS)

The extreme-ultraviolet (XUV) and strong-field valence ionization of cytosine and uracil is considered. We examine some simple estimates of the relative yields of the cation states populated following ionization and compare these to the results of a recently developed ab initio-type numerical model designed to compute strong-field ionization of molecules, the so-called time-dependent resolution in ionic states (TD-RIS) method. In analogy with one-photon XUV ionization, where the photoionization matrix elements can be related to the Dyson orbitals, we construct estimates for the yield of strong-field ionization (SFI) to different cation states based on the Dyson orbital norms and the Keldysh tunneling ionization rate. In the case of XUV ionization, the Dyson norms are shown to be good predictors of the relative cation yields when compared with the TD-RIS yields. The Dyson- and Keldysh-based models underestimate the yield to excited cation states in the case of SFI. The increased yield to the excited cation states in the TD-RIS results is attributed to the inclusion of multielectron effects and continuum structure not present in the simple models. The molecular Ammosov-Delone-Krainov (MO-ADK) method of calculating SFI is also considered. This later method is seen to agree more closely with the Dyson- and Keldysh-based estimates as it also fails to capture the multielectron effects and continuum structure included in the TD-RIS approach.

Spanner, Michael; Patchkovskii, Serguei; Zhou, Congyi; Matsika, Spiridoula; Kotur, Marija; Weinacht, Thomas C.

2012-11-01

382

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

PubMed

Photocycloaddition between two adjacent bases in DNA produces a cyclobutane pyrimidine dimer (CPD), which is one of the major UV-induced DNA lesions, with either the cis-syn or trans-syn structure. In this study, we investigated the photosensitized intramolecular cycloaddition of partially-protected thymidylyl-(3'?5')-N(4)-acetyl-2'-deoxy-5-methylcytidine, to clarify the effect of the base modification on the cycloaddition reaction. The reaction resulted in the stereoselective formation of the trans-syn CPD, followed by hydrolysis of the acetylamino group. The same result was obtained for the photocycloaddition of thymidylyl-(3'?5')-N(4)-acetyl-2'-deoxycytidine, whereas both the cis-syn and trans-syn CPDs were formed from thymidylyl-(3'?5')-thymidine. Kinetic analyses revealed that the activation energy of the acid-catalyzed hydrolysis is comparable to that reported for the thymine-cytosine CPD. These findings provided a new strategy for the synthesis of oligonucleotides containing the trans-syn CPD. Using the synthesized oligonucleotide, translesion synthesis by human DNA polymerase ? was analyzed. PMID:20880992

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

2011-02-01

383

Cytosine Methylation Associated with Repeat-Induced Point Mutation Causes Epigenetic Gene Silencing in Neurospora Crassa  

PubMed Central

Repeated DNA sequences are frequently mutated during the sexual cycle in Neurospora crassa by a process named repeat-induced point mutation (RIP). RIP is often associated with methylation of cytosine residues in and around the mutated sequences. Here we demonstrate that this methylation can silence a gene located in nearby, unique sequences. A large proportion of strains that had undergone RIP of a linked duplication flanking a single-copy transgene, hph (hygromycin B phosphotransferase), showed partial silencing of hph. These strains were all heavily methylated throughout the single-copy hph sequences and the flanking sequences. Silencing was alleviated by preventing methylation, either by 5-azacytidine (5AC) treatment or by introduction of a mutation (eth-1) known to reduce intracellular levels of S-adenosylmethionine. Silenced strains exhibited spontaneous reactivation of hph at frequencies of 10(-4) to 0.5. Reactivated strains, as well as cells that were treated with 5AC, gave rise to cultures that were hypomethylated and partially hygromycin resistant, indicating that some of the original methylation was propagated by a maintenance mechanism. Gene expression levels were found to be variable within a population of clonally related cells, and this variation was correlated with epigenetically propagated differences in methylation patterns.

Irelan, J. T.; Selker, E. U.

1997-01-01

384

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

385

RNA-mediated epigenetic heredity requires the cytosine methyltransferase Dnmt2.  

PubMed

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. PMID:23717211

Kiani, Jafar; Grandjean, Valérie; Liebers, Reinhard; Tuorto, Francesca; Ghanbarian, Hossein; Lyko, Frank; Cuzin, François; Rassoulzadegan, Minoo

2013-05-01

386

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

Microsoft Academic Search

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

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

2000-01-01

387

Enhancement of heavy metal accumulation by tissue specific co-expression of iaaM and ACC deaminase genes in plants  

Microsoft Academic Search

1-Aminocyclopropane deaminase (ACC) and tryptophan monooxygenase are two enzymes involved in plant senescence-inhibiting and growth-promoting regulation, respectively. In this study, two binary vectors were constructed in which the AgrobacteriumiaaM gene was under the transcriptional control of a xylem-specific glycine-rich protein promoter alone, or co-expressed with the bacterial ACC deaminase gene, which was driven by the constitutive CaMV 35S promoter. Transgenic

Yong Zhang; Lihong Zhao; Yao Wang; Baoyu Yang; Shiyun Chen

2008-01-01

388

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

PubMed Central

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

Nunes, Daniela Prudente Teixeira; Spegiorin, Ligia Cosentino Junqueira Franco; de Mattos, Cinara Cassia Brandao; Oliani, Antonio Helio; Vaz-Oliani, Denise Cristina Mos; de Mattos, Luiz Carlos

2011-01-01

389

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

PubMed

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

Singhabahu, Sanjeewa; George, John; Bringloe, David

2013-06-01

390

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

PubMed Central

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

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

2008-01-01

391

Role of Adenosine Deaminase Estimation in Differentiation of Tuberculous and Non-tuberculous Exudative Pleural Effusions  

PubMed Central

Background Tuberculosis kills five lakh patients every year in India, commonest being pulmonary tuberculosis and is often associated with effusion. Delay in diagnosis and treatment results in poor prognosis. Several studies have suggested the role of adenosine deaminase (ADA) in the diagnosis of tuberculous pleural effusions, but false-positive results from lymphocytic effusions have also been reported. The purpose of this study is to find out the role of ADA levels in differentiation of tuberculous and non-tuberculous exudative pleural effusions of different etiologies. Methods Ninety-six lymphocytic pleural fluid samples were consecutively selected and divided into two groups: tuberculous (n = 56) and non-tuberculous (n = 40), depending upon the etiology [Malignancy (n = 16), Infectious diseases (n = 18), Pulmonary embolism (n = 1), Collagen vascular diseases (n = 3) and Sarcoidosis (n = 2)]. ADA was estimated in pleural fluid in all the cases. Results In all 56 samples, ADA level of tuberculous group was above diagnostic cut-off (40 U/L), while only one sample was above cut-off in non-tuberculous group (2.5%). The negative predictive value of ADA for the diagnosis of non-tuberculous etiology was 97.5% (39 of 40) lymphocytic pleural effusion patients. Conclusions In this study, ADA levels in nontuberculous exudative pleural effusions rarely exceeded the cut-off; set for tuberculous disease. The pleural fluid ADA levels were significantly higher in tuberculous exudative pleural effusions when compared with non-tuberculous exudative pleural effusions. Keywords Adenosine deaminase; Tuberculous effusion; Pleural fluid; Exudative pleural effusions

Gupta, Bharat Kumar; Bharat, Vinay; Bandyopadhyay, Debapriya

2010-01-01

392

Evolution of the primate APOBEC3A cytidine deaminase gene and identification of related coding regions.  

PubMed

The APOBEC3 gene cluster encodes six cytidine deaminases (A3A-C, A3DE, A3F-H) with single stranded DNA (ssDNA) substrate specificity. For the moment A3A is the only enzyme that can initiate catabolism of both mitochondrial and nuclear DNA. Human A3A expression is initiated from two different methionine codons M1 or M13, both of which are in adequate but sub-optimal Kozak environments. In the present study, we have analyzed the genetic diversity among A3A genes across a wide range of 12 primates including New World monkeys, Old World monkeys and Hominids. Sequence variation was observed in exons 1-4 in all primates with up to 31% overall amino acid variation. Importantly for 3 hominids codon M1 was mutated to a threonine codon or valine codon, while for 5/12 primates strong Kozak M1 or M13 codons were found. Positive selection was apparent along a few branches which differed compared to positive selection in the carboxy-terminal of A3G that clusters with A3A among human cytidine deaminases. In the course of analyses, two novel non-functional A3A-related fragments were identified on chromosome 4 and 8 kb upstream of the A3 locus. This qualitative and quantitative variation among primate A3A genes suggest that subtle differences in function might ensue as more light is shed on this increasingly important enzyme. PMID:22272271

Henry, Michel; Terzian, Christophe; Peeters, Martine; Wain-Hobson, Simon; Vartanian, Jean-Pierre

2012-01-01

393

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

PubMed

In Escherichia coli and Salmonella typhimurium about 80% of the dUMP used for dTMP synthesis is derived from deamination of dCTP. The dCTP deaminase produces dUTP that subsequently is hydrolyzed by dUTPase to dUMP and diphosphate. The dCTP deaminase is regulated by dTTP that inhibits the enzyme by binding to the active site and induces an inactive conformation of the trimeric enzyme. We have analyzed the role of residues previously suggested to play a role in catalysis. The mutant enzymes R115Q, S111C, S111T and E138D were all purified and analyzed for activity. Only S111T and E138D displayed detectable activity with a 30- and 140-fold reduction in k(cat), respectively. Furthermore, S111T and E138D both showed altered dTTP inhibition compared to wild-type enzyme. S111T was almost insensitive to the presence of dTTP. With the E138D enzyme the dTTP dependent increase in cooperativity of dCTP saturation was absent, although the dTTP inhibition itself was still cooperative. Modeling of the active site of the S111T enzyme indicated that this enzyme is restricted in forming the inactive dTTP binding conformer due to steric hindrance by the additional methyl group in threonine. The crystal structure of E138D in complex with dUTP showed a hydrogen bonding network in the active site similar to wild-type enzyme. However, changes in the hydrogen bond lengths between the carboxylate and a catalytic water molecule as well as a slightly different orientation of the pyrimidine ring of the bound nucleotide may provide an explanation for the reduced activity. PMID:17996716

Thymark, Majbritt; Johansson, Eva; Larsen, Sine; Willemoës, Martin

2008-02-01

394

Site-directed mutagenesis of active site glutamate-217 in mouse adenosine deaminase.  

PubMed

Mouse adenosine deaminase (ADA) contains an active site glutamate residue at position-217 that is highly conserved in other adenosine and AMP deaminases. Previous research has suggested that proton donation to N-1 of the adenosine ring occurs prior to catalysis and supports the mechanism as proceeding via formation of a tetrahedral intermediate at C-6 of adenosine. The proposed catalytic mechanism of ADA based on the recent elucidations of the crystal structure of this enzyme with transition- and ground-state analogs hypothesized that Glu217 was involved in this proton donation step [Wilson, D. K., Rudolph, F. B., & Quiocho, F. A. (1991) Science 252, 1278-1284; Wilson, D. K., & Quiocho, F. A. (1993) Biochemistry 32, 1689-1693]. Site-directed mutagenesis of the equivalent glutamate in human ADA resulted in a dramatic loss of enzyme activity [Bhaumik, D., Medin, J., Gathy, K., & Coleman, M. (1993) J. Biol. Chem. 268, 5464-5470]. To further study the importance of this residue, site-directed mutagenesis was used to create mouse ADA mutants. Glu217 was mutated to Asp, Gly, Gln, and Ser, and all mutants were successfully expressed and purified. Circular dichroism and zinc analysis showed no significant changes in secondary structure or zinc content, respectively, compared to the native protein. The mutants showed only a slight variation in Km but dramatically reduced kcat, less than 0.2% of wild-type activity. UV difference and 13C NMR spectra conclusively demonstrated the failure of any of these mutants to hydrate purine riboside, a reaction carried out by the wild-type enzyme that results in formation of an enzyme-inhibitor complex. Surprisingly, Ki values for binding of the inhibitor to the mutants and to wild-type protein are similar, irrespective of whether the inhibitor is hydrated upon binding. These data confirm the importance of Glu217 in catalysis as suggested by the crystal structure of mouse ADA. PMID:8634299

Mohamedali, K A; Kurz, L C; Rudolph, F B

1996-02-01

395

Yeast interactions and wine flavour  

Microsoft Academic Search

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

Graham H. Fleet

2003-01-01

396

Yeast as a screening tool  

Microsoft Academic Search

The versatile genetic malleability of yeast, and the high degree of conservation between its cellular processes and those of human cells, have made it the model of choice for pioneering research in molecular and cell biology over the past four decades. These character- istics of yeast, taken together with technical advan- tages such as simple growth conditions, rapid cell division

Alcide Barberis; Tea Gunde; Catherine Berset; Stephan Audetat; Urs Lüthi

2005-01-01

397

Monitoring polyglutamine toxicity in yeast.  

PubMed

Experiments in yeast have significantly contributed to our understanding of general aspects of biochemistry, genetics, and cell biology. Yeast models have also delivered deep insights in to the molecular mechanism underpinning human diseases, including neurodegenerative diseases. Many neurodegenerative diseases are associated with the conversion of a protein from a normal and benign conformation into a disease-associated and toxic conformation - a process called protein misfolding. The misfolding of proteins with abnormally expanded polyglutamine (polyQ) regions causes several neurodegenerative diseases, such as Huntington's disease and the Spinocerebellar Ataxias. Yeast cells expressing polyQ expansion proteins recapitulate polyQ length-dependent aggregation and toxicity, which are hallmarks of all polyQ-expansion diseases. The identification of modifiers of polyQ toxicity in yeast revealed molecular mechanisms and cellular pathways that contribute to polyQ toxicity. Notably, several of these findings in yeast were reproduced in other model organisms and in human patients, indicating the validity of the yeast polyQ model. Here, we describe different expression systems for polyQ-expansion proteins in yeast and we outline experimental protocols to reliably and quantitatively monitor polyQ toxicity in yeast. PMID:21144902

Duennwald, Martin L

2011-03-01

398

Modelling the yeast interactome.  

PubMed

The topology behind biological interaction networks has been studied for over a decade. Yet, there is no definite agreement on the theoretical models which best describe protein-protein interaction (PPI) networks. Such models are critical to quantifying the significance of any empirical observation regarding those networks. Here, we perform a comprehensive analysis of yeast PPI networks in order to gain insights into their topology and its dependency on interaction-screening technology. We find that: (1) interaction-detection technology has little effect on the topology of PPI networks; (2) topology of these interaction networks differs in organisms with different cellular complexity (human and yeast); (3) clear topological difference is present between PPI networks, their functional sub-modules, and their inter-functional "linkers"; (4) high confidence PPI networks have more "geometrical" topology compared to predicted, incomplete, or noisy PPI networks; and (5) inter-functional "linker" proteins serve as mediators in signal transduction, transport, regulation and organisational cellular processes. PMID:24589662

Janji?, Vuk; Sharan, Roded; Pržulj, Nataša

2014-01-01

399

Red yeast rice for dysipidemia.  

PubMed

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

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

2013-01-01

400

Programmed nuclear destruction in yeast  

PubMed Central

Studies of the budding yeast Saccharomyces cerevisiae have provided many of the most important insights into the mechanisms of autophagy, which are common to all eukaryotes. However, investigation of yeast self-destruction pathways, including autophagy and programmed cell death, has been almost exclusively restricted to cells undergoing vegetative growth, leaving very little exploration of their functions during developmental transitions in the yeast life cycle. We have recently discovered that whole nuclei are subject to programmed destruction during yeast gametogenesis. Programmed nuclear destruction (PND) possesses characteristics of apoptosis in the form of DNA cleavage by endonuclease G, and involves bulk protein turnover through an unusual autophagic pathway involving lysis of the vacuole rather than delivery of components to it through macroautophagy. We thus illuminate an example of developmentally programmed cellular “self-eating” in yeast, which is associated with the rupture of a lytic organelle, reminiscent of programmed cell death mechanisms in plants and animals.

Eastwood, Michael D.; Cheung, Sally W.T.; Meneghini, Marc D.

2013-01-01

401

Adenosine deaminase restores the ability of atrial natriuretic peptide to induce glomerular hyperfiltration in low-sodium rats.  

PubMed

Administration of a large dose of atrial natriuretic peptide is associated with an increase in glomerular filtration rate, diuresis and natriuresis in normal-sodium rats. However, glomerular hyperfiltration induced by atrial natriuretic peptide is markedly decreased in low-sodium rats. Glomerular insensitivity to atrial natriuretic peptide may be due to increased activity of the renin-angiotensin system in low-sodium rats and to an accompanying hypersensitivity to adenosine. The results indicate that attenuated glomerular responses to atrial natriuretic peptide are restored by the administration of adenosine deaminase in low-sodium rats. Moreover atrial natriuretic peptide markedly increases the urinary excretion of adenosine deaminase, which may be due to increased permeability of glomeruli to the enzyme. PMID:1493157

Bizon, D; Kalinowski, L; Angielski, S

1992-10-01