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Sample records for activation-induced cytidine deaminase

  1. Biological function of activation-induced cytidine deaminase (AID

    Ritu Kumar

    2014-10-01

    Full Text Available Activation-induced Cytidine Deaminase (AID is an essential regulator of B cell diversification, but its full range of action has until recently been an enigma. Based on homology, it was originally proposed to be an RNA-editing enzyme, but so far, no RNA substrates are known. Rather, it functions by deaminating cytidine, and in this manner, coupled with base-excision repair or mismatch repair machinery, it is a natural mutator. This allows it to play a central role in adaptive immunity, whereby it initiates the processes of class switch recombination and somatic hypermutation to help generate a diverse and high-affinity repertoire of immunoglobulin isotypes. More recently, it has been appreciated that methylated cytidine, already known as a key epigenetic mark on DNA controlling gene expression, can also be a target for AID modification. Coupled with repair machinery, this can facilitate the active removal of methylated DNA. This activity can impact the process of cellular reprogramming, including transition of a somatic cell to pluripotency, which requires major reshuffling of epigenetic memory. Thus, seemingly disparate roles for AID in controlling immune diversity and epigenetic memory have a common mechanistic basis. However, the very activity that is so useful for B cell diversity and cellular reprogramming is dangerous for the integrity of the genome. Thus, AID expression and activity is tightly regulated, and deregulation is associated with diseases including cancer. Here, we review the range of AID functions with a focus on its mechanisms of action and regulation. Major questions remain to be answered concerning how and when AID is targeted to specific loci and how this impacts development and disease.

  2. Role of Activation-Induced Cytidine Deaminase in the Development of Oral Squamous Cell Carcinoma

    Yosuke Nakanishi; Satoru Kondo; Naohiro Wakisaka; Akira Tsuji; Kazuhira Endo; Shigeyuki Murono; Makoto Ito; Kouichi Kitamura; Masamichi Muramatsu; Tomokazu Yoshizaki

    2013-01-01

    PURPOSE: In humans, activation-induced cytidine deaminase (AID) expression results due to inflammation and this deaminase activity is also involved in carcinogenesis. The aim of this study is to investigate the correlation between AID expression and the clinical classification of oral cancer tissues. EXPERIMENTAL DESIGN: The current study investigated the correlation between AID expression and the clinical classification of oral cancer tissues from 27 patients who underwent surgical resection...

  3. A coming-of-age story: activation-induced cytidine deaminase turns 10

    Delker, Rebecca K; Fugmann, Sebastian D.; Papavasiliou, F. Nina

    2009-01-01

    The discovery and characterization of activation-induced cytidine deaminase (AID) 10 years ago provided the basis for a mechanistic understanding of secondary antibody diversification and the subsequent generation and maintenance of cellular memory in B lymphocytes, which signified a major advance in the field of B cell immunology. Here we celebrate and review the triumphs in the mission to understand the mechanisms through which AID influences antibody diversification, as well as the implica...

  4. Activation-induced cytidine deaminase acts on double-strand breaks in vitro.

    Shen, Hong Ming

    2007-02-01

    Activation-induced cytidine deaminase (AID) is likely responsible for DNA cytidine deamination, although it may also act as an RNA deaminase. It functions on single-stranded DNA, the non-template strand in double-stranded DNA during transcription, or both strands in supercoiled DNA. To ask whether AID is able to deaminate cytidine at DNA breaks, plasmids, containing a SnaBI site (TAC downward arrowGTA) that forms blunt ends after digestion with SnaBI, were generated. If AID deaminates cytidine at the upstream blunt end, the ATG start codon in either of two drug resistance genes will be regenerated after ligation and replication in UDG-null E. coli cells. This study shows that AID targets cytidine at the break. The extent of deamination activity beyond the break is correlated with the base composition in the break region. If the break region is A, T-rich, C > T transitions are extensive. However, when the break region is not A, T-rich, mutations are mainly restricted to the break, similar to findings in vivo. The results indicate that AID has activity on double strand breaks (DSBs). Based on previous and current findings, a somatic hypermutation (SHM) model is proposed, in which collision between the transcription apparatus and the replication fork generates DSBs. After AID acts on break ends, the error-prone DNA repair machinery fixes and creates mutations. PMID:16697045

  5. A coming-of-age story: activation-induced cytidine deaminase turns 10.

    Delker, Rebecca K; Fugmann, Sebastian D; Papavasiliou, F Nina

    2009-11-01

    The discovery and characterization of activation-induced cytidine deaminase (AID) 10 years ago provided the basis for a mechanistic understanding of secondary antibody diversification and the subsequent generation and maintenance of cellular memory in B lymphocytes, which signified a major advance in the field of B cell immunology. Here we celebrate and review the triumphs in the mission to understand the mechanisms through which AID influences antibody diversification, as well as the implications of AID function on human physiology. We also take time to point out important ongoing controversies and outstanding questions in the field and highlight key experiments and techniques that hold the potential to elucidate the remaining mysteries surrounding this vital protein. PMID:19841648

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

    Stav Sapoznik

    2016-02-01

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

  7. Acquisition of Genetic Aberrations by Activation-Induced Cytidine Deaminase (AID) during Inflammation-Associated Carcinogenesis

    Genetic abnormalities such as nucleotide alterations and chromosomal disorders that accumulate in various tumor-related genes have an important role in cancer development. The precise mechanism of the acquisition of genetic aberrations, however, remains unclear. Activation-induced cytidine deaminase (AID), a nucleotide editing enzyme, is essential for the diversification of antibody production. AID is expressed only in activated B lymphocytes under physiologic conditions and induces somatic hypermutation and class switch recombination in immunoglobulin genes. Inflammation leads to aberrant AID expression in various gastrointestinal organs and increased AID expression contributes to cancer development by inducing genetic alterations in epithelial cells. Studies of how AID induces genetic disorders are expected to elucidate the mechanism of inflammation-associated carcinogenesis

  8. Identification of DNA cleavage- and recombination-specific hnRNP cofactors for activation-induced cytidine deaminase

    Hu, Wenjun; Begum, Nasim A.; Mondal, Samiran; Stanlie, Andre; Honjo, Tasuku

    2015-01-01

    The B-cell–specific antibody gene-diversifying enzyme activation-induced cytidine deaminase (AID) shows high homology with apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC1), which edits apolipoprotein B-100 mRNA in the presence of cofactor APOBEC1 complementation factor/APOBEC complementation factor (A1CF/ACF). Here we show that the DNA cleavage and recombination functions of AID depend critically on its RNA-dependent interaction with distinct heterogeneous nuclear ribonu...

  9. Activation-induced cytidine deaminase deficiency causes organ-specific autoimmune disease.

    Koji Hase

    Full Text Available 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 organs (TLOs in non-lymphoid tissues including the stomach at around 6 months of age. At a later stage, AID-/- mice develop a severe gastritis characterized by loss of gastric glands and epithelial hyperplasia. The disease development was not attenuated even under germ-free (GF conditions. Gastric autoantigen -specific serum IgM was elevated in AID-/- mice, and the serum levels correlated with the gastritis pathological score. Adoptive transfer experiments suggest that autoimmune CD4+ T cells mediate gastritis development as terminal effector cells. These results suggest that abnormal B-cell expansion due to AID deficiency can drive B-cell autoimmunity, and in turn promote TLO formation, which ultimately leads to the propagation of organ-specific autoimmune effector CD4+ T cells. Thus, AID plays an important role in the containment of autoimmune diseases by negative regulation of autoreactive B cells.

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

    Hu, Yi, E-mail: yihooyi@gmail.com; Ericsson, Ida, E-mail: ida.ericsson@ntnu.no; Doseth, Berit, E-mail: berit.doseth@ntnu.no; Liabakk, Nina B., E-mail: nina.beate.liabakk@ntnu.no; Krokan, Hans E., E-mail: hans.krokan@ntnu.no; Kavli, Bodil, E-mail: bodil.kavli@ntnu.no

    2014-03-10

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

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

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

  12. Activation-induced cytidine deaminase-mediated sequence diversification is transiently targeted to newly integrated DNA substrates.

    Yang, Shu Yuan; Fugmann, Sebastian D; Gramlich, Hillary S; Schatz, David G

    2007-08-31

    The molecular features that allow activation-induced cytidine deaminase (AID) to target Ig and certain non-Ig genes are not understood, although transcription has been implicated as one important parameter. We explored this issue by testing the mutability of a non-Ig transcription cassette in Ig and non-Ig loci of the chicken B cell line DT40. The cassette did not act as a stable long term mutation target but was able to be mutated in an AID-dependent manner for a limited time post-integration. This indicates that newly integrated DNA has molecular characteristics that render it susceptible to modification by AID, with implications for how targeting and mis-targeting of AID occurs. PMID:17613522

  13. T-Independent Activation-Induced Cytidine Deaminase Expression, Class-Switch Recombination, and Antibody Production by Immature/Transitional 1 B Cells1

    Ueda, Yoshihiro; Liao, Dongmei; Yang, Kaiyong; Patel, Anjali; Kelsoe, Garnett

    2007-01-01

    Inflammation elicits a splenic lymphopoiesis of unknown physiologic significance but one that juxtaposes developing B cells and exogenous Ag. We show that immature and transitional 1 (immature/T1) B cells constitutively express activation-induced cytidine deaminase and B lymphocyte-induced maturation protein 1 in amounts that support accelerated plasmacytic differentiation and limited class-switch recombination. In vivo, activation of immature/T1 B cells by TLR ligands or bacterial vaccine ra...

  14. Isotype-switched follicular lymphoma displays dissociation between activation-induced cytidine deaminase expression and somatic hypermutation.

    Scherer, Florian; Navarrete, Marcelo A; Bertinetti-Lapatki, Cristina; Boehm, Joachim; Schmitt-Graeff, Annette; Veelken, Hendrik

    2016-01-01

    In B-cells, activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes. AID introduces mutations in immunoglobulin variable regions (IGV) during B-cell receptor affinity maturation, but may also introduce aberrant mutations into non-immunoglobulin genes, most commonly BCL6. Follicular lymphoma (FL) B-cells constitutively express AID and undergo CSR, SHM and aberrant SHM. We have studied AID expression, the presence of SHM mutations, CSR, and aberrant SHM in BCL6 in a cohort of 75 FL patients. Whereas IgM-expressing (non-switched) FL were characterized by an expected positive correlation between AID and IGV and BCL6 mutations, isotype-switched FL showed dissociation between AID expression and aberrant SHM, and inverse correlation between SHM and AID expression. Our results unveil two manifest biological subgroups of FL and indicate that the specific dissociation between AID and SHM after isotype switch may correlate with the clinical outcome of this heterogeneous disease. PMID:25860234

  15. SU-C-303-01: Activation-Induced Cytidine Deaminase Confers Cancer Resistance to Radiation Therapy

    Purpose: To study the role of activation-induced cytidine deaminase (AID) in malignant cell resistance to radiation therapy. Methods: We first developed several small devices that could be used to adopt radiation beams from clinical high dose rate brachy therapy (HDR) or linac-based megavoltage machines to perform pre-clinical cell and mouse experiments. Then we used these devices to deliver radiation to AID-positive and AID-silenced cancer cells or tumors formed by these cells in mice. Cells and mice bearing tumors received the same dose under the same experimental conditions. For cells, we observed the apoptosis and the cell survival rate over time. For mice bearing tumors, we measured and recorded the tumor sizes every other day for 4 weeks. Results: For cell experiments, we found that the AID-positive cells underwent much less apoptosis compared with AID-silenced cells upon radiation. And for mouse experiments, we found that AID-positive tumors grew significantly faster than the AID-silenced tumors despite of receiving the same doses of radiation. Conclusion: Our study suggests that AID may confer cancer resistance to radiation therapy, and AID may be a significant biomarker predicting cancer resistance to radiation therapy for certain cancer types

  16. ATM increases activation-induced cytidine deaminase activity at downstream S regions during class-switch recombination.

    Khair, Lyne; Guikema, Jeroen E J; Linehan, Erin K; Ucher, Anna J; Leus, Niek G J; Ogilvie, Colin; Lou, Zhenkun; Schrader, Carol E; Stavnezer, Janet

    2014-05-15

    Activation-induced cytidine deaminase (AID) initiates Ab class-switch recombination (CSR) in activated B cells resulting in exchanging the IgH C region and improved Ab effector function. During CSR, AID instigates DNA double-strand break (DSB) formation in switch (S) regions located upstream of C region genes. DSBs are necessary for CSR, but improper regulation of DSBs can lead to chromosomal translocations that can result in B cell lymphoma. The protein kinase ataxia telangiectasia mutated (ATM) is an important proximal regulator of the DNA damage response (DDR), and translocations involving S regions are increased in its absence. ATM phosphorylates H2AX, which recruits other DNA damage response (DDR) proteins, including mediator of DNA damage checkpoint 1 (Mdc1) and p53 binding protein 1 (53BP1), to sites of DNA damage. As these DDR proteins all function to promote repair and recombination of DSBs during CSR, we examined whether mouse splenic B cells deficient in these proteins would show alterations in S region DSBs when undergoing CSR. We find that in atm(-/-) cells Sμ DSBs are increased, whereas DSBs in downstream Sγ regions are decreased. We also find that mutations in the unrearranged Sγ3 segment are reduced in atm(-/-) cells. Our data suggest that ATM increases AID targeting and activity at downstream acceptor S regions during CSR and that in atm(-/-) cells Sμ DSBs accumulate as they lack a recombination partner. PMID:24729610

  17. Repression of human activation induced cytidine deaminase by miR-93 and miR-155

    Activation Induced cytidine Deaminase (AID) targets the immunoglobulin genes of activated B cells, where it converts cytidine to uracil to induce mutagenesis and recombination. While essential for immunoglobulin gene diversification, AID misregulation can result in genomic instability and oncogenic transformation. This is classically illustrated in Burkitt's lymphoma, which is characterized by AID-induced mutation and reciprocal translocation of the c-MYC oncogene with the IgH loci. Originally thought to be B cell-specific, AID now appears to be misexpressed in several epithelial cancers, raising the specter that AID may also participate in non-B cell carcinogenesis. The mutagenic potential of AID argues for the existence of cellular regulators capable of repressing inappropriate AID expression. MicroRNAs (miRs) have this capacity, and we have examined the publically available human AID EST dataset for miR complementarities to the human AID 3'UTR. In this work, we have evaluated the capacity of two candidate miRs to repress human AID expression in MCF-7 breast carcinoma cells. We have discovered moderate miR-155 and pronounced miR-93 complementary target sites encoded within the human AID mRNA. Luciferase reporter assays indicate that both miR-93 and miR-155 can interact with the 3'UTR of AID to block expression. In addition, over-expression of either miR in MCF-7 cells reduces endogenous AID protein, but not mRNA, levels. Similarly indicative of AID translational regulation, depletion of either miR in MCF-7 cells increases AID protein levels without concurrent increases in AID mRNA. Together, our findings demonstrate that miR-93 and miR-155 constitutively suppress AID translation in MCF-7 cells, suggesting widespread roles for these miRs in preventing genome cytidine deaminations, mutagenesis, and oncogenic transformation. In addition, our characterization of an obscured miR-93 target site located within the AID 3'UTR supports the recent

  18. Repression of human activation induced cytidine deaminase by miR-93 and miR-155

    Holton Nathaniel W

    2011-08-01

    Full Text Available Abstract Background Activation Induced cytidine Deaminase (AID targets the immunoglobulin genes of activated B cells, where it converts cytidine to uracil to induce mutagenesis and recombination. While essential for immunoglobulin gene diversification, AID misregulation can result in genomic instability and oncogenic transformation. This is classically illustrated in Burkitt's lymphoma, which is characterized by AID-induced mutation and reciprocal translocation of the c-MYC oncogene with the IgH loci. Originally thought to be B cell-specific, AID now appears to be misexpressed in several epithelial cancers, raising the specter that AID may also participate in non-B cell carcinogenesis. Methods The mutagenic potential of AID argues for the existence of cellular regulators capable of repressing inappropriate AID expression. MicroRNAs (miRs have this capacity, and we have examined the publically available human AID EST dataset for miR complementarities to the human AID 3'UTR. In this work, we have evaluated the capacity of two candidate miRs to repress human AID expression in MCF-7 breast carcinoma cells. Results We have discovered moderate miR-155 and pronounced miR-93 complementary target sites encoded within the human AID mRNA. Luciferase reporter assays indicate that both miR-93 and miR-155 can interact with the 3'UTR of AID to block expression. In addition, over-expression of either miR in MCF-7 cells reduces endogenous AID protein, but not mRNA, levels. Similarly indicative of AID translational regulation, depletion of either miR in MCF-7 cells increases AID protein levels without concurrent increases in AID mRNA. Conclusions Together, our findings demonstrate that miR-93 and miR-155 constitutively suppress AID translation in MCF-7 cells, suggesting widespread roles for these miRs in preventing genome cytidine deaminations, mutagenesis, and oncogenic transformation. In addition, our characterization of an obscured miR-93 target site located

  19. Activation-induced cytidine deaminase expression in CD4+ T cells is associated with a unique IL-10-producing subset that increases with age.

    Hongyan Qin

    Full Text Available Activation-induced cytidine deaminase (AID, produced by the Aicda gene, is essential for the immunoglobulin gene (Ig alterations that form immune memory. Using a Cre-mediated genetic system, we unexpectedly found CD4(+ T cells that had expressed Aicda (exAID cells as well as B cells. ExAID cells increased with age, reaching up to 25% of the CD4(+ and B220(+ cell populations. ExAID B cells remained IgM(+, suggesting that class-switched memory B cells do not accumulate in the spleen. In T cells, AID was expressed in a subset that produced IFN-γ and IL-10 but little IL-4 or IL-17, and showed no evidence of genetic mutation. Interestingly, the endogenous Aicda expression in T cells was enhanced in the absence of B cells, indicating that the process is independent from the germinal center reaction. These results suggest that in addition to its roles in B cells, AID may have previously unappreciated roles in T-cell function or tumorigenesis.

  20. Expression of activation-induced cytidine deaminase enhances the clearance of pneumococcal pneumonia: evidence of a subpopulation of protective anti-pneumococcal B1a cells.

    Yamamoto, Natsuo; Kerfoot, Steven M; Hutchinson, Andrew T; Dela Cruz, Charles S; Nakazawa, Naomi; Szczepanik, Marian; Majewska-Szczepanik, Monika; Nazimek, Katarzyna; Ohana, Noboru; Bryniarski, Krzysztof; Mori, Tsutomu; Muramatsu, Masamichi; Kanemitsu, Keiji; Askenase, Philip W

    2016-01-01

    We describe a protective early acquired immune response to pneumococcal pneumonia that is mediated by a subset of B1a cells. Mice deficient in B1 cells (xid), or activation-induced cytidine deaminase (AID(-/-) ), or invariant natural killer T (iNKT) cells (Jα18(-/-) ), or interleukin-13 (IL-13(-/-) ) had impaired early clearance of pneumococci in the lung, compared with wild-type mice. In contrast, AID(-/-) mice adoptively transferred with AID(+/+) B1a cells, significantly cleared bacteria from the lungs as early as 3 days post infection. We show that this early bacterial clearance corresponds to an allergic contact sensitivity-like cutaneous response, probably due to a subpopulation of initiating B1a cells. In the pneumonia model, these B1a cells were found to secrete higher affinity antigen-specific IgM. In addition, as in contact sensitivity, iNKT cells were required for the anti-pneumococcal B1a cell initiating response, probably through early production of IL-13, given that IL-13(-/-) mice also failed to clear infection. Our study is the first to demonstrate the importance of AID in generating an appropriate B1a cell response to pathogenic bacteria. Given the antibody affinity and pneumonia resistance data, natural IgM produced by conventional B1a cells are not responsible for pneumonia clearance compared with the AID-dependent subset. PMID:26456931

  1. Interleukin-21 stimulates B-cell immunoglobulin E synthesis in human beings concomitantly with activation-induced cytidine deaminase expression and differentiation into plasma cells.

    Kobayashi, Shinji; Haruo, Nagumo; Sugane, Kazuo; Ochs, Hans D; Agematsu, Kazunaga

    2009-01-01

    Interleukin (IL)-21 downregulates immunoglobulin E (IgE) production in murine systems by inhibiting germline epsilon transcription in IL-4-stimulated B cells. We here sought to clarify the function of IL-21 in human B-cell IgE synthesis. IL-21 dramatically enhanced IgE production by human mononuclear cells, or purified total, naive, or memory B cells in the presence of IL-4 plus anti-CD40 mAb cross-linked with CD32-transfectants, and the production was strengthened with further addition of IL-10. It was concomitant to the enhancement of activation-induced cytidine deaminase (AID) mRNA expression, but no increase of germline epsilon transcription. We also observed that IL-21 promoted B-cell differentiation into plasma cells with increase of B-lymphocyte-induced maturation protein-1 (Blimp-1), but not X-box binding protein 1 (XBP-1), which was further accentuated by co-stimulation with IL-4 plus CD40 signaling. Thus, IL-21 is a strong inducer of IgE production in human beings concomitantly with AID expression and the differentiation into plasma cells. Our data suggest that IL-21 plays an important role in occurrence and the treatment of allergic disorders. PMID:19026702

  2. Non-redundancy of cytidine deaminases in class switch recombination.

    Fugmann, Sebastian D; Rush, James S; Schatz, David G

    2004-03-01

    Class switch recombination (CSR), somatic hypermutation, and gene conversion are immunoglobulin diversification mechanisms that are strictly dependent on the activity of the activation-induced cytidine deaminase (AID). The precise role and substrate(s) of AID in these processes remain to be well defined. The closest homologue of AID is APOBEC-1, a bona fide mRNA-editing enzyme, which shares with AID the ability to deaminate cytidines within single-stranded DNA in vitro and in prokaryotic cells. To determine whether APOBEC-1 can therefore substitute for AID in activated B cells, we expressed human AID, a catalytic mutant thereof, and rat APOBEC-1 in AID-deficient murine B cells. Whereas AID rescued CSR, neither the inactive mutant nor APOBEC-1 could complement AID deficiency. This indicates that cytidine deaminase activity is necessary but not sufficient to initiate CSR, and suggests that AID is specifically targeted to its cognate substrate, the immunoglobulin genes or a distinct mRNA, by an as-yet-unknown mechanism. PMID:14991614

  3. Overexpression of Activation-Induced Cytidine Deaminase in MTX- and Age-Related Epstein-Barr Virus-Associated B-Cell Lymphoproliferative Disorders of the Head and Neck

    Kentaro Kikuchi

    2015-01-01

    Full Text Available Recent research has shown that activation-induced cytidine deaminase (AID triggers somatic hypermutation and recombination, in turn contributing to lymphomagenesis. Such aberrant AID expression is seen in B-cell leukemia/lymphomas, including Burkitt lymphoma which is associated with c-myc translocation. Moreover, Epstein-Barr virus (EBV latent membrane protein-1 (LMP-1 increases genomic instability through early growth transcription response-1 (Egr-1 mediated upregulation of AID in B-cell lymphoma. However, few clinicopathological studies have focused on AID expression in lymphoproliferative disorders (LPDs. Therefore, we conducted an immunohistochemical study to investigate the relationship between AID and LMP-1 expression in LPDs (MTX-/Age-related EBV-associated, including diffuse large B-cell lymphomas (DLBCLs. More intense AID expression was detected in LPDs (89.5% than in DLBCLs (20.0%, and the expression of LMP-1 and EBER was more intense in LPDs (68.4% and 94.7% than in DLBCLs (10.0% and 20.0%. Furthermore, stronger Egr-1 expression was found in MTX/Age-EBV-LPDs (83.3% than in DLBCLs (30.0%. AID expression was significantly constitutively overexpressed in LPDs as compared with DLBCLs. These results suggest that increased AID expression in LPDs may be one of the processes involved in lymphomagenesis, thereby further increasing the survival of genetically destabilized B-cells. AID expression may be a useful indicator for differentiation between LPDs and DLBCLs.

  4. Enzymatic conformational fluctuations along the reaction coordinate of cytidine deaminase

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

    2002-01-01

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

  5. Intrinsic immunity against retrotransposons by APOBEC cytidine deaminases

    AtsushiKoito

    2013-02-01

    Full Text Available 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 nonautonomous 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 mRNA, a protein involved in lipid transport, also plays a role in controlling mobile elements. These mammalian 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

  6. Myeloprotection by Cytidine Deaminase Gene Transfer in Antileukemic Therapy

    Nico Lachmann

    2013-03-01

    Full Text Available Gene transfer of drug resistance (CTX-R genes can be used to protect the hematopoietic system from the toxicity of anticancer chemotherapy and this concept recently has been proven by overexpression of a mutant O6-methylguaninemethyltransferase in the hematopoietic system of glioblastoma patients treated with temozolomide. Given its protection capacity against such relevant drugs as cytosine arabinoside (ara-C, gemcitabine, decitabine, or azacytidine and the highly hematopoiesis-specific toxicity profile of several of these agents, cytidine deaminase (CDD represents another interesting candidate CTX-R gene and our group recently has established the myeloprotective capacity of CDD gene transfer in a number of murine transplant studies. Clinically, CDD overexpression appears particularly suited to optimize treatment strategies for acute leukemias and myelodysplasias given the efficacy of ara-C (and to a lesser degree decitabine and azacytidine in these disease entities. This article will review the current state of the art with regard to CDD gene transfer and point out potential scenarios for a clinical application of this strategy. In addition, risks and potential side effects associated with this approach as well as strategies to overcome these problems will be highlighted.

  7. Activation induced deaminase: how much and where?

    Orthwein, Alexandre; Di Noia, Javier M

    2012-08-01

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

  8. Structural and biological function of NYD-SP15 as a new member of cytidine deaminases.

    Xu, Yidan; Li, Lei; Li, Jianmin; Liu, Qinghuai

    2016-05-25

    Recent studies were mainly focus on the cytidine deaminase family genes, which contained a lot of members that varied on the function of catalytic deamination in RNA or DNA and were involved in the process of growth maintenance, host immunity, retroviral infection, tumorigenesis, and drug resistance with a feature of C-U deamination. In this study, we identified a new member of cytidine deaminase family, NYD-SP15. Previous work showed that the deduced structure of the protein contained two dCMP_cyt_deam domains, which were involved in zinc ion binding. NYD-SP15 was expressed variably in a wide range of tissues, indicating its worthy biological function and creative significances. Sequence analysis, RT-PCR, western blot, flow cytometry, direct-site mutation and GST pull-down assay were performed to analyze the construction and function of NYD-SP15. The results in our studies showed that NYD-SP15 was closely related to deoxycytidylate deaminase and cytidine deaminase, with authentic cytidine deaminase activity in vivo and vitro as well as homo dimerization effects. NYD-SP15 contained nuclear localization sequence (NLS) and nuclear export-signal (NES) and could dynamically shuttle between the nucleus and cytoplasm. Furthermore, NYD-SP15 gene over-expression reduced the cells growth and blocked G1 to S phase, which implied a potential inhibition effect on cell growth. PMID:26945630

  9. Macrophages increase the resistance of pancreatic adenocarcinoma cells to gemcitabine by upregulating cytidine deaminase

    Amit, Moran; Gil, Ziv

    2013-01-01

    Tumor-associated macrophages play a central role in tumor progression and metastasis. Macrophages can also promote the resistance of malignant cells to chemotherapy by stimulating the upregulation of cytidine deaminase, an intracellular enzyme that catabolizes the active form of gemcitabine. Targeting macrophage-dependent chemoresistance may reduce tumor-associated morbidity and mortality. PMID:24498570

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

    Nowarski, Roni; Kotler, Moshe

    2013-06-15

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

  11. Complexes between nuclear factor-κB p65 and signal transducer and activator of transcription 3 are key actors in inducing activation-induced cytidine deaminase expression and immunoglobulin A production in CD40L plus interleukin-10-treated human blood B cells.

    Lafarge, S; Hamzeh-Cognasse, H; Richard, Y; Pozzetto, B; Cogné, M; Cognasse, F; Garraud, O

    2011-11-01

    The signal transducer and activator of transcription 3 (STAT3) transcription factor pathway plays an important role in many biological phenomena. STAT3 transcription is triggered by cytokine-associated signals. Here, we use isolated human B cells to analyse the role of STAT3 in interleukin (IL)-10 induced terminal B cell differentiation and in immunoglobulin (Ig)A production as a characteristic readout of IL-10 signalling. We identified optimal conditions for inducing in-vitro IgA production by purified blood naive B cells using IL-10 and soluble CD40L. We show that soluble CD40L consistently induces the phosphorylation of nuclear factor (NF)-κB p65 but not of STAT3, while IL-10 induces the phosphorylation of STAT3 but not of NF-κB p65. Interestingly, while soluble CD40L and IL-10 were synergistic in driving the terminal maturation of B cells into IgA-producing plasma cells, they did not co-operate earlier in the pathway with regard to the transcription factors NF-κB p65 or STAT3. Blocking either NF-κB p65 or STAT3 profoundly altered the production of IgA and mRNA for activation-induced cytidine deaminase (AID), an enzyme strictly necessary for Ig heavy chain recombination. Finally, the STAT3 pathway was directly activated by IL-10, while IL-6, the main cytokine otherwise known for activating the STAT3 pathway, did not appear to be involved in IL-10-induced-STAT3 activation. Our results suggest that STAT3 and NF-κB pathways co-operate in IgA production, with soluble CD40L rapidly activating the NF-κB pathway, probably rendering STAT3 probably more reactive to IL-10 signalling. This novel role for STAT3 in B cell development reveals a potential therapeutic or vaccine target for eliciting IgA humoral responses at mucosal interfaces. PMID:21985363

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

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

    2011-01-01

    APOBEC3G (A3G) is a cytidine deaminase that catalyzes deamination of deoxycytidine (dC) on single-stranded DNA (ssDNA). The oligomeric state of A3G required to support deaminase activity remains unknown. We show under defined in vitro conditions that full-length and native A3G formed complexes with ssDNA in an A3G concentration-dependent but temperature-independent manner. Complexes assembled and maintained at 4 °C did not have significant deaminase activity, but their enzymatic function coul...

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

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

    2012-07-12

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

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

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

    2011-01-01

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

  15. Synthesis of a bifunctional cytidine derivative and its conjugation to RNA for in vitro selection of a cytidine deaminase ribozyme

    Nico Rublack

    2014-08-01

    Full Text Available Over the past 20 years, the generation of functional RNAs by in vitro selection has become a standard technique. Apart from aptamers for simple binding of defined ligands, also RNAs for catalysis of chemical reactions have been selected. In the latter case, a key step often is the conjugation of one of the two reactants to the library, requiring suitable strategies for terminal or internal RNA functionalization. With the aim of selecting a ribozyme for deamination of cytidine, we have set up a selection scheme involving the attachment of the cytidine acting as deamination substrate to the 3'-terminus of the RNAs in the library, and library immobilization. Here, we report the synthesis of a bifunctional cytidine derivative suitable for conjugation to RNA and linkage of the conjugated library to a streptavidine-coated surface. Successful conjugation of the cytidine derivative to the 3'-terminus of a model RNA is demonstrated.

  16. Significance of determination of serum cytidine deaminase (CD) levels for diagnosis of active rheumatoid arthritis (RA)

    Objective: To determine the clinical value of measurement of serum cytidine deaminase (CD) levels in patients with active rheumatoid arthritis (RA). Methods: Serum levels of CD were detected with spectrophotometry, in 33 patients with active RA and 60 controls. The erythrocyte sedimentation rate (ESR) and CRP content were also determined in both groups. Results: The ser- um CD contents in patients with active RA(14.80 ± 2.11U/ml) were significantly higher than those in controls(4.86±1.86 U/ml,P<0.01). The CRP contents (51.46 ± 20.43mg/L) and ESR readings(85.03 ± 27.6mm/h) in the patients were also significantly higher than those in the controls(3.40 ± 2.21mg/L and 13.04 ± 4.89mm/h respectively, all P<0.01). In the patients, the serum CD contents were linearly positively correlated with the ESR contents and CRP readings (r=0.6324 and 0.8013 respectively, P <0.01). Conclusion: Serum CD is an early biochemical marker for diagnosis of active rheumatoid arthritis and is also of prognostic value. (authors)

  17. Assignment of the human cytidine deaminase (CDA) gene to chromosome 1 band p35-p36.2

    Saccone, S.; Andreozzi, L.; Della Valle, G. [Universita di Pavia (Italy)] [and others

    1994-08-01

    The enzyme cytidine deaminase (EC 3.5.4.12; CDA) catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. It can also catalyze the deamination of cytosine nucleoside analogues such as cytosine arabinoside and 5-azacytidine, which results in a loss of their cytotoxic and antitumor activity. Cytosine arabinoside is used in the treatment of acute myeloid leukemia, and the antileukemic activity of the drug is dependent on phosphorylation by deoxycytidine kinase. The occurrence of clinical cytosine arabinoside resistance is one of the main problems in the successful treatment of acute myeloid leukemia. Resistance to the drug has been ascribed to functional deoxycytidine kinase deficiency and to increased expression of the CDA gene. In this study, we report on the isolation of a CDA genomic fragment and its use as a probe for the chromosomal localization of the human CDA gene by in situ hybridization. 9 refs., 1 fig.

  18. HIV-1 Vif versus the APOBEC3 cytidine deaminases: an intracellular duel between pathogen and host restriction factors.

    Wissing, Silke; Galloway, Nicole L K; Greene, Warner C

    2010-10-01

    The Vif protein of HIV is essential for the effective propagation of this pathogenic retrovirus in vivo. Vif acts by preventing virion encapsidation of two potent antiviral factors, the APOBEC3G and APOBEC3F cytidine deaminases. Decreased encapsidation in part involves Vif-mediated recruitment of a ubiquitin E3 ligase complex that promotes polyubiquitylation and proteasome-mediated degradation of APOBEC3G/F. The resultant decline in intracellular levels of these enzymes leads to decreased encapsidation of APOBECG/F into budding virions. This review discusses recent advances in our understanding of the dynamic interplay of Vif with the antiviral APOBEC3 enzymes. PMID:20538015

  19. NF-IL6 (C/EBPβ) induces HIV-1 replication by inhibiting cytidine deaminase APOBEC3G

    Shigemi M Kinoshita; Taguchi, Shizuka

    2008-01-01

    T cell activation is crucial for the productive HIV-1 infection of primary T cells; however, little is known about the host molecules involved in this process. We show that the host transcription factor NF-IL6 (also called C/EBPβ) renders primary CD4+ T cells highly permissive for HIV-1 replication. NF-IL6 facilitates reverse transcription of the virus by binding to and inhibiting the antiviral cytidine deaminase APOBEC3G. A mutation in NF-IL6 at Ser-288 weakened its binding to APOBEC3G and s...

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

    McDougall, William M.; Smith, Harold C.

    2011-01-01

    APOBEC3G (A3G) is a deoxycytidine deaminase active on ssDNA substrates. In HIV infected cells A3G interacted with reverse transcription complexes where its activity as a deoxycytidine deaminase led to mutation of the viral genome. A3G not only bound ssDNA, but it also had an intrinsic ability to bind RNA. In many cell types that can support HIV replication, A3G ssDNA deaminase activity was suppressed and the enzyme resided in high molecular mass, ribonucleoprotein complexes associated with cy...

  1. Haploinsufficiency of activation-induced deaminase for antibody diversification and chromosome translocations both in vitro and in vivo.

    Isora V Sernández

    Full Text Available 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.

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

    McDougall, William M.; Smith, Harold C.

    2011-01-01

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

  3. Cloning, expression, and purification of cytidine deaminase from Arabidopsis thaliana

    Vincenzetti, Silvia; Cambi, Alessandra; Neuhard, Jan;

    1999-01-01

    plasma-optical emission spectroscopy analysis indicated that the enzyme contains 1 mol of zinc atom per mole of subunit. The kinetic properties of AT-CDA1 both toward the natural substrates and with analogs indicated that the catalytic mechanism of the plant enzyme is probably very similar to that of the......The complementary DNA (cDNA) coding forArabidopsis thalianacytidine deaminase 1 (AT-CDA1) was obtained from the amplifiedA. thalianacDNA expression library, provided by R. W. Davis (Stanford University, CA). AT-CDA1 cDNA was subcloned into the expression vector pTrc99-A and the protein, expressed...

  4. Of the Nine Cytidine Deaminase-Like Genes in Arabidopsis, Eight Are Pseudogenes and Only One Is Required to Maintain Pyrimidine Homeostasis in Vivo.

    Chen, Mingjia; Herde, Marco; Witte, Claus-Peter

    2016-06-01

    CYTIDINE DEAMINASE (CDA) catalyzes the deamination of cytidine to uridine and ammonia in the catabolic route of C nucleotides. The Arabidopsis (Arabidopsis thaliana) CDA gene family comprises nine members, one of which (AtCDA) was shown previously in vitro to encode an active CDA. A possible role in C-to-U RNA editing or in antiviral defense has been discussed for other members. A comprehensive bioinformatic analysis of plant CDA sequences, combined with biochemical functionality tests, strongly suggests that all Arabidopsis CDA family members except AtCDA are pseudogenes and that most plants only require a single CDA gene. Soybean (Glycine max) possesses three CDA genes, but only two encode functional enzymes and just one has very high catalytic efficiency. AtCDA and soybean CDAs are located in the cytosol. The functionality of AtCDA in vivo was demonstrated with loss-of-function mutants accumulating high amounts of cytidine but also CMP, cytosine, and some uridine in seeds. Cytidine hydrolysis in cda mutants is likely caused by NUCLEOSIDE HYDROLASE1 (NSH1) because cytosine accumulation is strongly reduced in a cda nsh1 double mutant. Altered responses of the cda mutants to fluorocytidine and fluorouridine indicate that a dual specific nucleoside kinase is involved in cytidine as well as uridine salvage. CDA mutants display a reduction in rosette size and have fewer leaves compared with the wild type, which is probably not caused by defective pyrimidine catabolism but by the accumulation of pyrimidine catabolism intermediates reaching toxic concentrations. PMID:27208239

  5. Molecular Analysis of Activation-Induced Cytidine Deaminase Gene in Immunoglobulin-E Deficient Patients

    Sergio Roa

    2008-01-01

    Full Text Available Understanding how class switch recombination (CSR is regulated to produce immunoglobulin E (IgE has become fundamental because of the dramatic increase in the prevalence of IgE-mediated hypersensitivity reactions. CSR requires the induction of the enzyme AICDA in B cells. Mutations in AICDA have been linked to Hyper-IgM syndrome (HIGM2, which shows absence of switching to IgE as well as to IgG and IgA. Although isolated IgE deficiency is a rare entity, here we show some individuals with normal serum IgM, IgG, and IgA levels that had undetectable total serum IgE levels. We have analyzed the AICDA gene in these individuals to determine if there are mutations in AICDA that could lead to selective IgE deficiency. Conformational sensitive gel electrophoresis (CSGE and sequencing analysis of AICDA coding sequences demonstrated sequence heterogeneity due to 5923A/G and 7888C/T polymorphisms, but did not reveal any novel mutation that might explain the selective IgE deficit.

  6. APOBEC1 and APOBEC3 cytidine deaminases as restriction factors for hepadnaviral genomes in non-humans in vivo.

    Renard, Myrtille; Henry, Michel; Guétard, Denise; Vartanian, Jean-Pierre; Wain-Hobson, Simon

    2010-07-16

    Reverse transcription of the hepadnavirus RNA pre-genome means that nascent cDNA may be vulnerable to genetic editing by host cell APOBEC cytidine deaminases that have specificity single-stranded DNA as substrate. Hepatitis B virus (HBV) is particularly vulnerable to editing by APOBEC3G (hA3G) in late-stage disease where up to 35% of genomes can be edited. Yet, the organization of the A3 locus varies considerably among mammals with a single gene for the mouse and seven genes for Old and New World monkeys, which suggests that the outcome may be very variable for other natural hepadnavirus infections. In addition, there is the powerful mouse transgenic model of HBV replication (mHBV) that has proved to be immensely useful in understanding HBV immunopathogenesis. Here, we show that mHBV is edited in vivo by mAPOBEC1 (mA1) and not mAPOBEC3 (mA3), which follows from the fact that unlike humans, the mA1 gene is highly expressed in the liver. For woodchuck hepatitis virus, an mA3 ortholog is probably operative. For HBV-infected tree shrew primary liver cultures, the editing profile more resembles that observed in humans in keeping with fact that this species belongs to the order closest to Primates. There seems to be more genetic editing in liver or cell-associated genomes than serum or culture supernatants, suggesting that too much editing of virion cDNA might impede completion of DNA synthesis. PMID:20546753

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

    Simon Gemble

    2015-07-01

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

  8. Cytidine deaminase motifs within the DYW domain of two pentatricopeptide repeat-containing proteins are required for site-specific chloroplast RNA editing.

    Wagoner, Jessica A; Sun, Tao; Lin, Lin; Hanson, Maureen R

    2015-01-30

    In angiosperm organelles, cytidines are converted to uridines by a deamination reaction in the process termed RNA editing. The C targets of editing are recognized by members of the pentatricopeptide repeat (PPR) protein family. Although other members of the editosome have begun to be identified, the enzyme that catalyzes the C-U conversion is still unknown. The DYW motif at the C terminus of many PPR editing factors contains residues conserved with known cytidine deaminase active sites; however, some PPR editing factors lack a DYW motif. Furthermore, in many PPR-DYW editing factors, the truncation of the DYW motif does not affect editing efficiency, so the role of the DYW motif in RNA editing is unclear. Here, a chloroplast PPR-DYW editing factor, quintuple editing factor 1 (QED1), was shown to affect five different plastid editing sites, the greatest number of chloroplast C targets known to be affected by a single PPR protein. Loss of editing at the five sites resulted in stunted growth and accumulation of apparent photodamage. Adding a C-terminal protein tag to QED1 was found to severely inhibit editing function. QED1 and RARE1, another plastid PPR-DYW editing factor, were discovered to require their DYW motifs for efficient editing. To identify specific residues critical for editing, conserved deaminase residues in each PPR protein were mutagenized. The mutant PPR proteins, when expressed in qed1 or rare1 mutant protoplasts, could not complement the editing defect. Therefore, the DYW motif, and specifically, the deaminase residues, of QED1 and RARE1 are required for editing efficiency. PMID:25512379

  9. Structural analysis of the activation-induced deoxycytidine deaminase required in immunoglobulin diversification.

    Pham, Phuong; Afif, Samir A; Shimoda, Mayuko; Maeda, Kazuhiko; Sakaguchi, Nobuo; Pedersen, Lars C; Goodman, Myron F

    2016-07-01

    Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) and class-switch recombination (CSR) by deaminating C→U during transcription of Ig-variable (V) and Ig-switch (S) region DNA, which is essential to produce high-affinity antibodies. Here we report the crystal structure of a soluble human AID variant at 2.8Å resolution that favors targeting WRC motifs (W=A/T, R=A/G) in vitro, and executes Ig V SHM in Ramos B-cells. A specificity loop extending away from the active site to accommodate two purine bases next to C, differs significantly in sequence, length, and conformation from APOBEC proteins Apo3A and Apo3G, which strongly favor pyrimidines at -1 and -2 positions. Individual amino acid contributions to specificity and processivity were measured in relation to a proposed ssDNA binding cleft. This study provides a structural basis for residue contributions to DNA scanning properties unique to AID, and for disease mutations in human HIGM-2 syndrome. PMID:27258794

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

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

    2015-12-01

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

  11. Synthesis and Conformational Analysis of Locked Carbocyclic Analogues of 1,3-Diazepinone Riboside, a High-Affinity Cytidine Deaminase Inhibitor

    2009-01-01

    Cytidine deaminase (CDA) catalyzes the deamination of cytidine via a hydrated transition-state intermediate that results from the nucleophilic attack of zinc-bound water at the active site. Nucleoside analogues where the leaving NH3 group is replaced by a proton and prevent conversion of the transition state to product are very potent inhibitors of the enzyme. However, stable carbocyclic versions of these analogues are less effective as the role of the ribose in facilitating formation of hydrated species is abolished. The discovery that a 1,3-diazepinone riboside (4) operated as a tight-binding inhibitor of CDA independent of hydration provided the opportunity to study novel inhibitors built as conformationally locked, carbocyclic 1,3-diazepinone nucleosides to determine the enzyme’s conformational preference for a specific form of sugar pucker. This work describes the synthesis of two target bicyclo[3.1.0]hexane nucleosides, locked as north (5) and south (6) conformers, as well as a flexible analogue (7) built with a cyclopentane ring. The seven-membered 1,3-diazepinone ring in all the three targets was built from the corresponding benzoyl-protected carbocyclic bis-allyl ureas by ring-closing metathesis. The results demonstrate CDA’s binding preference for a south sugar pucker in agreement with the high-resolution crystal structures of other CDA inhibitors bound at the active site. PMID:19618900

  12. 胞苷脱氨酶基因对小鼠大剂量化疗的保护作用%Protection of a cytidine deaminase gene gainst toxicity of high dose chemotherapy in mice

    Bo Chen; Caigang Liu; Yang Lu; Ping Lu; Feng Jin; Fan Yao; Ruishan Zhang; Jinlong Liu; Shicheng Zhao

    2008-01-01

    Objective:To explore the feasibility of transfecting cytidine deaminase(CD)gene into mouse bone marrow cells in order to observe the drug resistance of high dose Ara-C and improve the tolerance of myelosuppression following combination chemotherapy.Methods:Human cytidine deaminase gene was transfected into mice bone marrow cells by retroviral vector.Resistant colony-forming unit granulocyte-macrophage(CFU-GM)assay was performed after the transfected mice bone marrow cells treated by the Ara-C.DNA was extracted from mice bone marrow cells.The drug resistant gene in mice bone marrow cells after transfection was detected by PCR.Results:Bone marrow cells of lhe donor mice cultured with lhe retroviral producer cells showed the drug resistant colonies and resistance to Ara-C,so did accept mice transplanted with the CD gene(CFU-GM of donor mice was 52%,X2=124.62,P<0.01:accept mice was 54%,X2=126.26.P<0.01,both compared with the contrast group).The animal survival rate was significantly higher in gene transfected group than that of the control(X2=7.42.P<0.01).CD gene of transfected bone marrow cells was confirmed by PCR.Conclusion:CD gene can be transfected into bone marrow cells of mice efficiently and increase the drug resistance to Ara-C.

  13. Paclitaxel alters the expression and specific activity of deoxycytidine kinase and cytidine deaminase in non-small cell lung cancer cell lines

    Patel Shitalben R

    2009-06-01

    Full Text Available Abstract Background We observed that paclitaxel altered the pharmacokinetic properties of gemcitabine in patients with non-small cell lung cancer (NSCLC and limited the accumulation of gemcitabine and its metabolites in various primary and immortalized human cells. Therefore, we classified the drug-drug interaction and the effects of paclitaxel on deoxycytidine kinase (dCK and cytidine deaminase (CDA in three NSCLC cell lines. These enzymes are responsible for the metabolism of gemcitabine to its deaminated metabolite dFdU (80% of the parent drug and the phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP. These metabolites appear to relate to sensitivity and tolerability of gemcitabine based on previous animal and laboratory studies. Methods Three immortalized human cells representative of the most common histological subtypes identified in patients with advanced NSCLC were exposed to the individual drugs or combinations to complete a multiple drug effect analysis. These same cell lines were exposed to vehicle-control or paclitaxel and the mRNA levels, protein expression and specific activity of dCK and CDA were compared. Comparisons were made using a two-tailed paired t-test or analysis of variance with a P value of Results The multiple drug effect analysis indicated synergy for H460, H520 and H838 cells independent of sequence. As anticipated, paclitaxel-gemcitabine increased the number of G2/M cells, whereas gemcitabine-paclitaxel increased the number of G0/G1 or S cells. Paclitaxel significantly decreased dCK and CDA mRNA levels in H460 and H520 cells (40% to 60%, P Conclusion In summary, paclitaxel altered the mRNA levels and specific activity of dCK and CDA and these effects could be dependent on histological subtype. More cell and animal studies are needed to further characterize the relationship between mRNA levels and the overall drug-drug interaction and the potential to use histological subtype as a predictive factor in the

  14. APOBEC deaminases-mutases with defensive roles for immunity

    Courtney; PROCHNOW; Ronda; BRANSTEITTER

    2009-01-01

    In recent years, tremendous progress has been made in the elucidation of the biological roles and molecular mechanisms of the apolioprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family of enzymes. The APOBEC family of cytidine deaminases has important functional roles within the adaptive and innate immune system. Activation induced cytidine deaminase (AID) plays a central role in the biochemical steps of somatic hypermutation and class switch recombination during antibody maturation, and the APOBEC 3 enzymes are able to inhibit the mobility of retroelements and the replication of retroviruses and DNA viruses, such as the human immunodeficiency virus type-1 and hepatitis B virus. Recent advances in structural and functional studies of the APOBEC enzymes provide new biochemical insights for how these enzymes carry out their biological roles. In this review, we provide an overview of these recent advances in the APOBEC field with a special emphasis on AID and APOBEC3G.

  15. Analysis of 6912 unselected somatic hypermutations in human VDJ rearrangements reveals lack of strand specificity and correlation between phase II substitution rates and distance to the nearest 3' activation-induced cytidine deaminase target

    Ohm-Laursen, Line; Barington, Torben

    2007-01-01

    -23*01) from blood B lymphocytes enriched for CD27-positive memory cells. Analyses of 6,912 unique, unselected substitutions showed that in vivo hot and cold spots for the SHM of C and G residues corresponded closely to the target preferences reported for AID in vitro. A detailed analysis of all possible four......-nucleotide motifs present on both strands of the V(H) gene showed significant correlations between the substitution frequencies in reverse complementary motifs, suggesting that the SHM machinery targets both strands equally well. An analysis of individual J(H) and D gene segments showed that the substitution...... rates in G and T residues correlated inversely with the distance to the nearest 3' WRC AID hot spot motif on both the nontranscribed and transcribed strands. This suggests that phase II SHM takes place 5' of the initial AID deamination target and primarily targets T and G residues or, alternatively, the...

  16. 着色性干皮病基因组D和胞苷脱氨酶基因单核苷酸多态性与肺癌易感性的关系%Relationship between single nucleotide polymorphisms of xeroderma pigmentosum complementary group D and cytidine deaminase gene and susceptibility to lung cancer

    周敏; 戎霞君; 高蓓莉; 丁永杰; 万欢英

    2011-01-01

    Objective To investigate the relationship between single nucleotide polymorphisms of xeroderma pigmentosum complementary group D ( XPD) and cytidine deaminase ( CDA) gene and susceptibility to lung cancer, and explore the influence of the interaction between smoking and gene polymorphisms on the risk of development of lung cancer. Methods Case-control study was performed on 103 patients with lung cancer and 103 healthy controls. Polymerase chain reaction-restriction fragment length polymorphism ( PCR-RFLP) was employed to determine the genotype distribution of XPD exon 10 G→A (Asp312Asn) and 23A→C (.Lys751Gln) and that of CDA exon 79A→ C ( Lys27Gln) and 208G→ A (Ala70 Thr). Results There was no significant difference in genotype distribution of XPD312 and XPD751 between two groups (P > 0.05). However, smoking in combination with mutation at locus 751 of XPD increased the risk of development of lung cancer (P =0.044), and the risk of development of lung cancer increased 6. 13 times with mutations at both loci 312 and 751 of XPD (P =0. 047). There was no significant difference in genotype distribution of CDA Lys27Gln and CDA Ala70 Thr between two groups (P > 0. 05). There was no significant difference in genotype distribution of XPD and CDA among different pathological types. Conclusion Smoking in combination with mutation at locus 751 of XPD may increase the risk of development of lung cancer, and mutation' at both loci 312 and locus 751 of XPD may increase the risk of development of lung cancer.%目的 探讨着色性干皮病基因组D( XPD)和胞苷脱氨酶(CDA)基因单核苷酸多态性与肺癌易感性及其病理类型的关系,并探讨吸烟与基因多态性的交互作用对肺癌发病风险的影响.方法 采用病例-对照研究方法纳入肺癌患者和健康对照者各103人,采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)法分析两组人群XPD基因外显子10G→A (Asp312Asn)、23 A→C( Lys751 Gln

  17. A novel zinc-binding motif found in two ubiquitous deaminase families.

    Reizer, J.; Buskirk, S.; Bairoch, A.; Reizer, A.; Saier, M. H.

    1994-01-01

    Two families of deaminases, one specific for cytidine, the other for deoxycytidylate, are shown to possess a novel zinc-binding motif, here designated ZBS. We have (1) identified the protein members of these 2 families, (2) carried out sequence analyses that allow specification of this zinc-binding motif, and (3) determined signature sequences that will allow identification of additional members of these families as their sequences become available. PMID:8061614

  18. Genetics Home Reference: adenosine monophosphate deaminase deficiency

    ... links) CLIMB: Children Living with Inherited Metabolic Diseases Muscular Dystrophy Association: Myoadenylate Deaminase Deficiency Genetic Testing Registry (1 link) Muscle AMP deaminase deficiency ...

  19. Perspectives of bacterial ACC deaminase in phytoremediation.

    Arshad, Muhammad; Saleem, Muhammad; Hussain, Sarfraz

    2007-08-01

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

  20. The Frequency of Cytidine Editing of Viral DNA Is Differentially Influenced by Vpx and Nucleosides during HIV-1 or SIVMAC Infection of Dendritic Cells.

    Xuan-Nhi Nguyen

    Full Text Available Two cellular factors are currently known to modulate lentiviral infection specifically in myeloid cells: SAMHD1 and APOBEC3A (A3A. SAMHD1 is a deoxynucleoside triphosphohydrolase that interferes with viral infection mostly by limiting the intracellular concentrations of dNTPs, while A3A is a cytidine deaminase that has been described to edit incoming vDNA. The restrictive phenotype of myeloid cells can be alleviated through the direct degradation of SAMHD1 by the HIV-2/SIVSM Vpx protein or else, at least in the case of HIV-1, by the exogenous supplementation of nucleosides that artificially overcome the catabolic activity of SAMHD1 on dNTPs. Here, we have used Vpx and dNs to explore the relationship existing between vDNA cytidine deamination and SAMHD1 during HIV-1 or SIVMAC infection of primary dendritic cells. Our results reveal an interesting inverse correlation between conditions that promote efficient infection of DCs and the extent of vDNA editing that may reflect the different susceptibility of vDNA to cytoplasmic effectors during the infection of myeloid cells.

  1. Increased CDA expression/activity in males contributes to decreased cytidine analogue half-life and likely contributes to worse outcomes with 5-azacytidine or decitabine therapy

    Mahfouz, Reda Z; Jankowska, Ania; Ebrahem, Quteba; Gu, Xiaorong; Visconte, Valeria; Tabarroki, Ali; Terse, Pramod; Covey, Joseph; Chan, Kenneth; Ling, Yonghua; Engelke, Kory J.; Sekeres, Mikkael A.; Tiu, Ramon; Maciejewski, Jaroslaw; Radivoyevitch, Tomas; Saunthararajah, Yogen

    2013-01-01

    Purpose The cytidine analogues 5-azacytidine and decitabine, used to treat myelodysplastic syndromes (MDS), produce a molecular epigenetic effect, depletion of DNA-methyltransferase (DNMT1). This action is S-phase dependent. Hence, genetic factors that decrease the half-lives of these drugs could impact efficacy. Documentation of such impact, and elucidation of underlying mechanisms, could lead to improved clinical application. Design Cytidine deaminase (CDA) rapidly inactivates 5-azacytidine/decitabine. The effect of CDA SNP A79C and gender on CDA expression, enzyme activity and drug pharmacokinetics/pharmacodynamics was examined in mice and humans, and the impact on overall survival (OS) was evaluated in 5-azacytidine/decitabine-treated MDS patients (n=90) and cytarabine-treated acute myeloid leukemia (AML) patients (n=76). Results By HPLC, plasma CDA activity was decreased as expected in individuals with the SNP A79C. Interestingly and significantly, there was an even larger decrease in females compared to males. Explaining this decrease, liver CDA expression was significantly lower in female versus male mice. As expected, decitabine plasma levels, measured by mass-spectrometry, were significantly higher in females. In mathematical modeling, the detrimental impact of shorter drug half-life (e.g., in males) was greater in low compared to high S-phase fraction disease (e.g., MDS versus AML), since in high S-phase fraction disease, even a short exposure treats a major portion of cells. Accordingly, in multivariate analysis, OS was significantly worse in male versus female MDS patients treated with 5-azacytidine/decitabine. Conclusions Increased CDA expression/activity in males contributes to decreased cytidine analogue half-life and likely contributes to worse outcomes with 5-azacytidine or decitabine therapy. PMID:23287564

  2. Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

    Zhang, Zheng; Jakkaraju, Sriram; Blain, Joy; Gogol, Kenneth; Zhao, Lei; Hartley, Robert C.; Karlsson, Courtney A.; Staker, Bart L.; Lance J Stewart; Myler, Peter J.; Clare, Michael; Begley, Darren W.; Horn, James R.; Hagen, Timothy J.

    2013-01-01

    Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fr...

  3. Influence of x-rays on the deamination of cytidine compounds in yeasts

    The investigation of deamination of cytidine, cytosine, and cytidine nucleotides (CMP, CDP and CTP) was carried out on yeast Candida guilliermondii. It has been shown, that after exposure to X-ray the value of deamination of (CTD) was decreased, and the value of deamination of cytosine was increased. The increase of deamination level was detected in presence of all substrates besides cytidine after the post radiation repair of cells

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

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

    2011-01-01

    The deaminase-like fold includes, in addition to nucleic acid/nucleotide deaminases, several catalytic domains such as the JAB domain, and others involved in nucleotide and ADP-ribose metabolism. Using sensitive sequence and structural comparison methods, we develop a comprehensive natural classification of the deaminase-like fold and show that its ancestral version was likely to operate on nucleotides or nucleic acids. Consequently, we present evidence that a specific group of JAB domains ar...

  5. Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei.

    Zhang, Zheng; Jakkaraju, Sriram; Blain, Joy; Gogol, Kenneth; Zhao, Lei; Hartley, Robert C; Karlsson, Courtney A; Staker, Bart L; Edwards, Thomas E; Stewart, Lance J; Myler, Peter J; Clare, Michael; Begley, Darren W; Horn, James R; Hagen, Timothy J

    2013-12-15

    Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series. PMID:24157367

  6. The Effect of Cytidine on the Structure and Function of an RNA Ligase Ribozyme

    Rogers, Jeff; Joyce, Gerald F.

    2001-01-01

    A cytidine-free ribozyme with RNA ligase activity was obtained by in vitro evolution, starting from a pool of random- sequence RNAs that contained only guanosine, adenosine, and uridine. This ribozyme contains 74 nt and catalyzes formation of a 3',5' -phosphodiester linkage with a catalytic rate of 0.016/min. The RNA adopts a simple secondary structure based on a three-way junction motif, with ligation occurring at the end of a stem region located several nucleotides away from the junction. Cytidine was introduced to the cytidine-free ribozyme in a combinatorial fashion and additional rounds of in vitro evolution were carried out to allow the molecule to adapt to this added component. The resulting cytidine-containing ribozyme formed a 3',5' linkage with a catalytic rate of 0.32/min. The improved rate of the cytidine-containing ribozyme was the result of 12 mutations, including seven added cytidines, that remodeled the internal bulge loops located adjacent to the three-way junction and stabilized the peripheral stem regions.

  7. [NIR-SERS Spectra Detection of Cytidine on Nano-Silver Films].

    Zhang, De-qing; Liu, Ren-ming; Zhang, Guo-qiang; Zhang, Yan; Xiong, Yang; Zhang, Chuan-yun; Li, Lun; Si, Min-zhen

    2016-03-01

    The polyvinyl alcohol (PVA) protected silver glass-like nanostructure (PVA-Ag-GNS) with high surface-enhanced Raman scattering (SERS) activity was prepared and employed to detect the near-infrared surface enhanced Raman scattering (NIR-SERS) spectra of cytidine aqueous solution (10(-2)-10(-8) mol x L(-1)). In the work, the near-infrared laser beam (785 nm) was used as the excitation light source. The experiment results show that high-quality NIR-SERS spectra were obtained in the ranges of 300 to 2 000 cm(-1) and the detection limit of cytidine aqueous solution was down to 10(-7) mol x L(-1). Meanwhile, the PVA-Ag-GNS shows a high enhancement factor (EF) of -10(8). In order to test the optical reproducibility of PVA-Ag-GNS, ten samples of cytidine aqueous solution (10(-2)-10(-5) mol x L(-1)) had been dropped onto the surface of PVA-Ag-GNS respectively. Meanwhile, these samples were measured by the portable Raman spectrometer. As a result, the PVA-Ag-GNS demonstrated good optical reproducibility in the detection of cytidine aqueous solution. In addition, to explain the reason of enhancement effect, the ultraviolet-visible (UV-Vis) extinction spectrum and scanning electron microscope (SEM) of cytidine molecules adsorbed on the surface of PVA-Ag-GNS were measured. There is plasmon resonance band at 800 nm in the UV-Vis extinction Spectrum of the compound system. Therefore, when the near-infrared laser beam (785 nm) was used as excitation light source, the compound system may produce strongly surface plasmon resonance (SPR). According to the SEM of PVA-Ag-GNS, there are much interstitial between the silver nanoparticles. So NIR-SERS is mainly attributed to electromagnetic (EM) fields associated with strong surface plasmon resonance. At last, the geometry optimization and pre-Raman spectrum of cytidine for the ground states were performed with DFT, B3LYP functional and the 6-311G basis set, and the near-infrared laser with wavelength of 785 nm was employed in the pre

  8. Study of Ectonucleotidases and Adenosine Deaminases in Drosophila

    PREUER, Kristina

    2013-01-01

    Extracellular adenosine triphosphate and extracellular adenosine are important regulatory molecules in the human immune system. The concentrations of these molecules are in turn regulated by ectonucleotidases and adenosine deaminases. In this thesis I attempt to test the gene silencing efficiency of RNA interference for three different genes coding for such enzymes in the model organism Drosophila melanogaster.

  9. Preparation of alpha-5-aza-2'-deoxy-[6-3H]cytidine

    Elbert, Tomáš; Černý, B.

    2008-01-01

    Roč. 73, č. 5 (2008), s. 701-704. ISSN 0010-0765 Institutional research plan: CEZ:AV0Z40550506 Keywords : alfa-5aza-2'-deoxy-cytidine Subject RIV: CC - Organic Chemistry Impact factor: 0.784, year: 2008

  10. Enzymatic production of 5'-inosinic acid by AMP deaminase from a newly isolated Aspergillus oryzae.

    Li, Shubo; Chen, Leitao; Hu, Yangjun; Fang, Guohui; Zhao, Mouming; Guo, Yuan; Pang, Zongwen

    2017-02-01

    5'-adenylic acid deaminase (AMP deaminase), an important enzyme for the food industry, can catalyze the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, a new strain was screened that efficiently produces 3191.6U/g of AMP deaminase at 32°C. After purification, the optimal temperature and pH of the AMP deaminase were found to be 40°C and 6.0, respectively, but it was partially inhibited by Fe(3+), Cu(2+), Al(3+), and Zn(2+). With amplification of the AMP deaminase production system, 6mL of crude enzyme could produce 2.00mg/g of IMP from 2.04mg/g of dried yeast with an 84.8% molar yield after 40min. These results provide a new insight into AMP deaminase production and offer a potential platform for producing 5'-IMP. PMID:27596420

  11. Characterization of unexplored amidohydrolase enzyme-pterin deaminase.

    Jayaraman, Angayarkanni; Thandeeswaran, Murugesan; Priyadarsini, Ulaganathan; Sabarathinam, Shanmugam; Nawaz, K A Ayub; Palaniswamy, Muthusamy

    2016-06-01

    Pterin deaminase is an amidohydrolase enzyme hydrolyzing pteridines to form lumazine derivatives and ammonia. The enzyme captured the attention of scientists as early as 1959 and had been patented for its application as an anticancer agent. It is ubiquitously present in prokaryotes and has been reported in some eukaryotes such as honey bee, silkworm and rats. The enzyme has been observed to have a spectrum of substrates with the formation of respective lumazines. The role of the substrates of the enzyme in various metabolic pathways warrants a significant role in the biological activity of both prokaryotes and eukaryotes. Even though the functions of the enzyme have been explored in prokaryotes, their niche in the eukaryotic system is not clear. There is very few information on the structural and functional properties of the enzyme. This review has been congregated to emphasize the significance of pterin deaminase and analyzes the lacunae in understanding the biological characters of the enzyme. PMID:27094187

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

    Lada Artem G

    2012-12-01

    Full Text Available Abstract 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.

  13. The emerging role of adenosine deaminases in insects

    Doleželová, Eva; Žurovec, Michal; Doležal, T.; Šimek, Petr; Bryant, P. J.

    2005-01-01

    Roč. 35, č. 5 (2005), s. 381-389. ISSN 0965-1748 R&D Projects: GA ČR(CZ) GA204/04/1205; GA AV ČR(CZ) IAA5007107 Grant ostatní: United States National Science Foundation(US) 440860-21565 Institutional research plan: CEZ:AV0Z50070508 Keywords : adenosine deaminase * ADA * growth factor Subject RIV: ED - Physiology Impact factor: 2.733, year: 2005

  14. Acute intermittent porphyria in two patients on anticonvulsant therapy and with normal erythrocyte porphobilinogen deaminase activity.

    Herrick, A. L.; McColl, K E; Moore, M R; Brodie, M J; Adamson, A R; Goldberg, A

    1989-01-01

    1. Acute intermittent porphyria (AIP) is sometimes termed a 'pharmacogenetic' disease. patients with genetic deficiency of the enzyme porphobilinogen deaminase are liable to develop acute attacks of porphyria if exposed to a variety of drugs. 2. Two patients are reported who had no evidence of deficiency of erythrocyte porphobilinogen deaminase yet developed typical attacks of AIP while on anticonvulsant therapy. 3. Normal activity of erythrocyte porphobilinogen deaminase does not completely ...

  15. Potentiation of cytotoxicity and radiosensitization of (E)-2'-deoxy-2' (fluoromethylene) cytidine by pentoxifylline in vitro.

    Li, Y-X; Sun, L-Q; Paschoud, N; Weber-Johnson, K.; Coucke, Philippe

    1999-01-01

    (E)-28-deoxy-28-(fluoromethylene) cytidine (FMdC), a novel inhibitor of ribonucleotide-diphosphate reductase, has been shown to have anti-tumor activity against solid tumors and sensitize tumor cells to ionizing radiation. Pentoxifylline (PTX) can potentiate the cell killing induced by DNAdamaging agents through abrogation of DNA-damagedependent G2 checkpoint. We investigated the cytotoxic, radiosensitizing and cell-cycle effects of FMdC and PTX in a human colon-cancer...

  16. Class switch recombination in selective IgA-deficient subjects

    Jensen, Lone Hummelshøj; Ryder, L P; Nielsen, L K;

    2006-01-01

    control donors, all Caucasians. Stimulation of B cells from IgA-deficient donors with the cytokines transforming growth factor (TGF)-beta, interferon (IFN)-gamma or interleukin (IL)-10 in the presence of anti-CD40 antibodies showed reduced expression of both activation-induced cytidine deaminase (AID) and...

  17. Transcriptional stalling in B-lymphocytes: a mechanism for antibody diversification and maintenance of genomic integrity.

    Sun, Jianbo; Rothschild, Gerson; Pefanis, Evangelos; Basu, Uttiya

    2013-01-01

    B cells utilize three DNA alteration strategies-V(D)J recombination, somatic hypermutation (SHM) and class switch recombination (CSR)-to somatically mutate their genome, thereby expressing a plethora of antibodies tailor-made against the innumerable antigens they encounter while in circulation. Of these three events, the single-strand DNA cytidine deaminase, Activation Induced cytidine Deaminase (AID), is responsible for SHM and CSR. Recent advances, discussed in this review article, point toward various components of RNA polymerase II "stalling" machinery as regulators of AID activity during antibody diversification and maintenance of B cell genome integrity. PMID:23584095

  18. Characterization of the Escherichia coli codBA operon encoding cytosine permease and cytosine deaminase

    Danielsen, S; Kilstrup, M; Barilla, K;

    1992-01-01

    The nucleotide sequence of a 3.1 kb segment carrying the cytosine deaminase gene (codA) from Escherichia coli was determined. The sequence revealed the presence of two open reading frames, the first (codB) specifying a highly hydrophobic polypeptide and the second specifying cytosine deaminase. A...

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

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

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

    Frydman, R B; Frydman, B

    1979-06-01

    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

  1. Identification, Expression, and Characterization of Escherichia coli Guanine Deaminase

    Maynes, Jason T.; Yuan, Richard G.; Snyder, Floyd F.

    2000-01-01

    Using the human cDNA sequence corresponding to guanine deaminase, the Escherichia coli genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown function was identified as having 36% identity to the human protein. The putative gene was amplified, subcloned into the pMAL-c2 vector, expressed, purified, and characterized enzymatically. The 50.2-kDa protein catalyzed the conversion of guanine to xanthine, having a Km of ...

  2. Study the effect of bacterial 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase on resistance to salt stress in tomato plant

    Maryam SADRNIA

    2011-11-01

    Full Text Available 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase produced by rhizobacteria could be remove theethylene precursor and stimulate plant growth. Aim of the work was investigation on effect of rhizosphere bacteria Pseudomonasmendocina containing plasmid carrying gene encoding ACC deaminase on resistance of tomato plant to salinity. Amplification ofacds gene in selected Pseudomonas was performed; the g e n e w a s c l o n e d i n Escherichia coli and was cloned subsequently in P.mendocina. Enzyme activity was determined in cloned Escherichia coli and cloned P. mendocina for confirmation of geneexpression. Effect of bacterial ACC deaminase on resistance of tomato plants to NaCl was studied in Pot and Greenhouse. In potexperiment, tomato plant treated by cloned P. mendocina was compared with plants treated by P. mendocina (without plasmid andcontrol group. Salinity were established by adding 172 and 207 mM of NaCl to irrigated water. Greenhouse experiments wereconducted in similar groups of bacteria in 207 mM of NaCl. Results obtained from pot experiment revealed that plants treated bycloned P. mendocina in 172 mM of NaCl was showed increasing content of growth than ones treated by P. mendocina and controlas 11%, 18.4% growth for the shoot, 16.6%, 3.7% for roots and 9.6%, 27.5% for wet weight after five weeks, respectively. In 207mM of NaCl, the results were as 14.9 %, 9.7% for shoot, 94.3%, 15.7% for roots and 96.4%, 50.6% for wet weight, respectively. Ingreenhouse experiment, results in same parameter in 207 mM of NaCl were revealed as 63.7%, 7 times for shoot, 2.8, 14 times forroots and 66.1%, 154 times for wet weight, respectively. We concluded that recombinant P. mendocina producing ACC deaminaseby reduction of ethylene content of tomato plant in high salt concentrations could result in improvement of plant resistance tosalinity.

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

    Hanisch, Frank; Hellsten, Ylva; Zierz, Stephan

    2006-01-01

    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...

  4. Alternative transcription and splicing of the human porphobilinogen deaminase gene

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

  5. ESR study of irradiated single crystals of the cocrystalline complex of cytidine: Salicylic acid

    Irradiation at 77 K of single crystals of the 1:1 complex of cytidine and salicylic acid produces a phenoxyl radical formed by oxidation of the salicylic acid. Anisotropic hyperfine coupling tensors have been determined for this radical which are associated with the para and ortho hydrogens. No cytidine oxidation products (alkoxy or hydroxyalkyl radicals) were observed at 77 K. Following the decay of the phenoxyl radical at room temperature, four radicals were detected. These include the cytosine 5--yl and 6--yl radicals, formed by H addition to the cytosine ring, and an anisotropic doublet. By UV irradiation at room temperature, it is possible to convert a significant fraction of 6-yl radicals into 5-yl radicals. Hyperfine coupling and g tensors determined for the anisotropic doublet indicate that this radical is formed in the C/sub 1'/-C/sub 2'/ region of the sugar moiety. These results indicate a shift in radiation damage away from the salicylic acid upon warming, and show that the radiation chemistry of the cocrystalline complex is different from that of the isolated bases

  6. Serum adenosine deaminase as oxidative stress marker in type 2 diabetes mellitus

    Shashikala Magadi Dasegowda

    2015-05-01

    Results: The study observed an increased level of serum adenosine deaminase, malondialdehyde and decreased levels of total antioxidant capacity in type 2 diabetes mellitus compared to controls. Serum adenosine deaminase levels in type 2 diabetics were 50.77 +/- 6.95 and in controls was 17.86 +/- 4.04. Serum Malondialdehyde levels in type 2 diabetics was 512.13 +/- 70.15 and in controls was 239.32 +/- 23.97. Serum total antioxidant levels in type 2 diabetics was 0.39+/-0.15 and in controls was 1.66+/-0.25. Positive correlation was seen between serum adenosine deaminase and malondialdehyde and it was statistically significant. Statistically significant negative correlation was seen between serum adenosine deaminase and total antioxidant capacity. Conclusion: Adenosine deaminase can be used as oxidative stress marker. Their increased levels indicate oxidative stress in type 2 diabetes mellitus. Therefore, estimation of serum adenosine deaminase levels help in early prediction and prevention of long term complications occurring due to oxidative stress in diabetics, thereby decreasing the mortality and morbidity in them. [Int J Res Med Sci 2015; 3(5.000: 1195-1198

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

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

    2010-05-25

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

  8. APOBEC3B-Mediated Cytidine Deamination Is Required for Estrogen Receptor Action in Breast Cancer

    Manikandan Periyasamy

    2015-10-01

    Full Text Available Estrogen receptor α (ERα is the key transcriptional driver in a large proportion of breast cancers. We report that APOBEC3B (A3B is required for regulation of gene expression by ER and acts by causing C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER and to repair by non-homologous end-joining (NHEJ pathways. We provide evidence that transient cytidine deamination by A3B aids chromatin modification and remodelling at the regulatory regions of ER target genes that promotes their expression. A3B expression is associated with poor patient survival in ER+ breast cancer, reinforcing the physiological significance of A3B for ER action.

  9. Regulation of Adenosine Deaminase on Induced Mouse Experimental Autoimmune Uveitis.

    Liang, Dongchun; Zuo, Aijun; Zhao, Ronglan; Shao, Hui; Kaplan, Henry J; Sun, Deming

    2016-03-15

    Adenosine is an important regulator of the immune response, and adenosine deaminase (ADA) inhibits this regulatory effect by converting adenosine into functionally inactive molecules. Studies showed that adenosine receptor agonists can be anti- or proinflammatory. Clarification of the mechanisms that cause these opposing effects should provide a better guide for therapeutic intervention. In this study, we investigated the effect of ADA on the development of experimental autoimmune uveitis (EAU) induced by immunizing EAU-prone mice with a known uveitogenic peptide, IRBP1-20. Our results showed that the effective time to administer a single dose of ADA to suppress induction of EAU was 8-14 d postimmunization, shortly before EAU expression; however, ADA treatment at other time points exacerbated disease. ADA preferentially inhibited Th17 responses, and this effect was γδ T cell dependent. Our results demonstrated that the existing immune status strongly influences the anti- or proinflammatory effects of ADA. Our observations should help to improve the design of ADA- and adenosine receptor-targeted therapies. PMID:26856700

  10. Mechanish of dTTP Inhibition of the Bifunctional dCTP Deaminase:dUTPase Encoded by Mycobacterium tuberculosis

    Helt, Signe Smedegaard; Thymark, Majbritt; Harris, Pernille;

    2008-01-01

    Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme to be cha...

  11. Cytidine 5’-diphosphocholine administration prevents peripheral neuropathic pain after sciatic nerve crush injury in rats

    Emril, Dessy

    2016-01-01

    Dessy R Emril,1 Samekto Wibowo,2 Lucas Meliala,2 Rina Susilowati3 1Department of Neurology, Faculty of Medicine, Syiah Kuala University, Banda Aceh, 2Department of Neurology, 3Department of Histology and Cell Biology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, IndonesiaBackground: Cytidine 5’-diphosphocholine (citicoline) has been shown to have beneficial effects in central nervous system injury as well as in motoric functional recovery after peripheral nerve injury. ...

  12. Cytidine 5’-diphosphocholine administration prevents peripheral neuropathic pain after sciatic nerve crush injury in rats

    Emril DR; Wibowo S; Meliala L; Susilowati R

    2016-01-01

    Dessy R Emril,1 Samekto Wibowo,2 Lucas Meliala,2 Rina Susilowati3 1Department of Neurology, Faculty of Medicine, Syiah Kuala University, Banda Aceh, 2Department of Neurology, 3Department of Histology and Cell Biology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, IndonesiaBackground: Cytidine 5’-diphosphocholine (citicoline) has been shown to have beneficial effects in central nervous system injury as well as in motoric functional recovery after peripheral nerve injury. This...

  13. Isolation and characterization of human liver guanine deaminase.

    Gupta, N K; Glantz, M D

    1985-01-01

    Guanine deaminase (EC 3.5.4.3, guanine aminohydrolase [GAH]) was purified 3248-fold from human liver to homogeneity with a specific activity of 21.5. A combination of ammonium sulfate fractionation, and DEAE-cellulose, hydroxylapatite, and affinity chromatography with guanine triphosphate ligand were used to purify the enzyme. The enzyme was a dimer protein of a molecular weight of 120,000 with each subunit of 59,000 as determined by gel filtration and sodium dodecyl sulfate-gel electrophoresis. Isoelectric focusing gave a pI of 4.76. It was found to be an acidic protein, as evidenced by the amino acid analysis, enriched with glutamate, aspartate, alanine and glycine. It showed a sharp pH optimum of 8.0. The apparent Km for guanine was determined to be 1.53 X 10(-5) M at pH 6.0 and 2 X 10(-4) M for 8-azaguanine as a substrate at pH 6.0. The enzyme was found to be sensitive to p-hydroxymercuribenzoate inhibition with a Ki of 1.53 X 10(-5) M and a Ki of 5 X 10(-5) M with 5-aminoimidazole-4-carboxamide as an inhibitor. The inhibition with iodoacetic acid showed only a 7% loss in the activity at 1 X 10(-4) M and a 24% loss at 1 X 10(-3) M after 30 min of incubation, whereas p-hydroxymercuribenzoate incubation for 30 min resulted in a 91% loss of activity at a concentration of 1 X 10(-4) M. Guanine was the substrate for all of the inhibition studies. The enzyme was observed to be stable up to 40 degrees C, with a loss of almost all activity at 65 degrees C with 30 min incubation. Two pKa values were obtained at 5.85 and 8.0. Analysis of the N-terminal amino acid proved to be valine while the C-terminal residue was identified as alanine. PMID:3966794

  14. SIGNIFICANCE OF ADENOSINE DEAMINASE SERUM CONCENTRATIONS IN THE DIAGNOSIS OF EXTRA-PULMONARY TUBERCULOSIS

    Stevanovic G,

    2011-06-01

    Full Text Available Extra pulmonary tuberculosis (EPTB is a growing problem worldwide. Due to the nature of the disease, the diversity of clinical pictures as well as its minor epidemiological importance, the diagnosis is difficult and often late.In addition to standard TB diagnostic techniques use of new biochemical (surrogate markers are increased. With this work we wanted to examine the usefulness of serum adenosine deaminase levels as a diagnostic parameter for EPTB.The work included 116 patients with fever of unknown origin in which tuberculosis or infectious mononucleosis was not proven and 51 person who had proven EPTB. Correlated adenosine deaminase levels between these two groups we obtained significantly higher values ​​in patients with EPTB. The calculated sensitivity was 0.56, specificity 0.89, positive predictive value 0.80 and negative predictive value 0.72. Certain reducing of the values observed during anti TB therapy. In previous studies the diagnostic importance of adenosine deaminase in the diagnosis of tuberculosis serosityes was demonstrated. The significance of serum levels in diagnosis is rarely evaluated during EPTB. Our findings are similar to the results of authors who have conducted such testing in the pediatric population.Increased concentrations of serum adenosine deaminase have shown the potential of usable screening test and can be used as an indicative EPTB parameter. To fully assess its diagnostic significance require future clinical research.

  15. Outcome of hematopoietic stem cell transplantation for adenosine deaminase-deficient severe combined immunodeficiency

    Hassan, Amel; Booth, Claire; Brightwell, Alex; Allwood, Zoe; Veys, Paul; Rao, Kanchan; Hoenig, Manfred; Friedrich, Wilhelm; Gennery, Andrew; Slatter, Mary; Bredius, Robbert; Finocchi, Andrea; Cancrini, Caterina; Aiuti, Alessandro; Porta, Fulvio; Lanfranchi, Arnalda; Ridella, Michela; Steward, Colin; Filipovich, Alexandra; Marsh, Rebecca; Bordon, Victoria; Al-Muhsen, Saleh; Al-Mousa, Hamoud; Alsum, Zobaida; Al-Dhekri, Hasan; Al Ghonaium, Abdulaziz; Speckmann, Carsten; Fischer, Alain; Mahlaoui, Nizar; Nichols, Kim E.; Grunebaum, Eyal; Al Zahrani, Daifulah; Roifman, Chaim M.; Boelens, Jaap; Davies, E. Graham; Cavazzana-Calvo, Marina; Notarangelo, Luigi; Gaspar, H. Bobby

    2012-01-01

    Deficiency of the purine salvage enzyme adenosine deaminase leads to SCID (ADA-SCID). Hematopoietic cell transplantation (HCT) can lead to a permanent cure of SCID; however, little data are available on outcome of HCT for ADA-SCID in particular. In this multicenter retrospective study, we analyzed o

  16. Cutting edge: a cis-acting DNA element targets AID-mediated sequence diversification to the chicken Ig light chain gene locus.

    Kothapalli, Nagarama; Norton, Darrell D; Fugmann, Sebastian D

    2008-02-15

    Somatic hypermutation and gene conversion are two closely related processes that increase the diversity of the primary Ig repertoire. Both processes are initiated by the activation-induced cytidine deaminase that converts cytosine residues to uracils in a transcription-dependent manner; these lesions are subsequently fixed in the genome by direct replication and error-prone DNA repair. Two alternative mechanisms were proposed to explain why this mutagenic activity is targeted almost exclusively to Ig loci: 1) specific cis-acting DNA sequences; or 2) very high levels of Ig gene transcription. In this study we now identify a novel 3' regulatory region in the chicken Ig light chain gene containing not only a classical transcriptional enhancer but also cis-acting DNA elements essential for targeting activation-induced cytidine deaminase-mediated sequence diversification to this locus. PMID:18250404

  17. Stability of RNA and DNA in Bone Marrow Cells, Demonstrated with Tritiated Cytidine and Thymidine

    DNA and RNA metabolism was studied using tritiated thymidine (H3Th), a specific precursor for DNA, and tritiated cytidine (H3C), a common precursor for both RNA and DNA. With H3C, differential incorporation into RNA, DNA or the soluble pool was determined autoradiographically in the single cell, and/or chemically for cell populations by means of differential extraction using appropriate treatment with perchloric acid. Initial turnover studies in the Hela cell with H3C indicated the precursor role of nuclear RNA for cytoplasmic RNA. Conservation and distribution of label in the RNA fraction was consistent with major macromolecular RNA stability, and continued incorporation of label into the DNA fraction was consistent with the presence of a late precursor for DNA. Similar findings were observed in the immature bone marrow cells of the rat studied over a period of several days after intravenous administration of H3C. The amount of tritium activity in the acid-soluble' RNA and DNA fractions was followed chemically and/or autoradiographically. The three curves were found to be parallel from the first day after injection and parallel to curves for tritium label in DNA following H3Th administration. The expected rate of fall off in label, calculated from kinetics of the rat bone marrow cell populations studied separately by H3Th and autoradiography, assuming no turnover of RNA or DNA and loss of label only by loss of marrow cells by division and maturation, was in agreement with the slopes obtained. The results indicate that, once synthesized, soluble and macromolecular RNA is retained by the bone marrow cell in a manner similar to DNA. Newly formed RNA and DNA are diluted in the cells only through cell division. (author)

  18. p53 Represses Class Switch Recombination to IgG2a through its Antioxidant Function1

    Guikema, Jeroen E. J.; Schrader, Carol E.; Brodsky, Michael H.; Linehan, Erin K.; Richards, Adam; El Falaky, Nahla; Li, Daniel H.; Sluss, Hayla K.; Szomolanyi-Tsuda, Eva; Stavnezer, Janet

    2010-01-01

    Immunoglobulin class switch recombination (CSR) occurs in activated mature B cells, and causes an exchange of the IgM isotype for IgG, IgE or IgA isotypes, which increases the effectiveness of the humoral immune response. DNA double-stranded breaks (DSBs) in recombining switch (S) regions, where CSR occurs, are required for recombination. Activation-induced cytidine deaminase (AID) initiates DSB formation by deamination of cytosines in S regions. This reaction requires reactive oxygen species...

  19. Separation of mutational and transcriptional enhancers in immunoglobulin genes

    Kothapalli, Naga Rama; Collura, Kaitlin M.; Norton, Darrell D.; Fugmann, Sebastian D.

    2011-01-01

    Secondary immunoglobulin (Ig) gene diversification relies on activation-induced cytidine deaminase (AID) to create U:G mismatches that are subsequently fixed by mutagenic repair pathways. AID activity is focused to Ig loci by cis-regulatory DNA sequences named targeting elements. Here we show that in contrast to prevailing thought in the field, the targeting elements in the chicken IGL locus are distinct from classical transcriptional enhancers. These mutational enhancer elements (MEEs) are r...

  20. AID-mediated diversification within the IgL locus of chicken DT40 cells is restricted to the transcribed IgL gene

    Gopal, Anjali R.; Fugmann, Sebastian D.

    2007-01-01

    Somatic hypermutation (SHM) and gene conversion (GCV) are closely related processes that increase the diversity the primary immunoglobulin repertoire. In both processes the activation-induced cytidine deaminase (AID) converts cytosine residues to uracils within the DNA of the immunoglobulin (Ig) genes in a transcription-dependent manner, and subsequent error-prone repair processes lead to changes in the antigen recognition site of the encoded receptors. This activity is specifically recruited...

  1. Control of gene conversion and somatic hypermutation by immunoglobulin promoter and enhancer sequences

    Yang, Shu Yuan; Fugmann, Sebastian D.; Schatz, David G.

    2006-01-01

    It is thought that gene conversion (GCV) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes occur in two steps: the generation of uracils in DNA by activation-induced cytidine deaminase, followed by their subsequent repair by various DNA repair pathways to generate sequence-diversified products. It is not known how either of the two steps is targeted specifically to Ig loci. Because of the tight link between transcription and SHM, we have investigated the role of endogenous Ig light...

  2. Targeting of AID-mediated sequence diversification to immunoglobulin genes

    Kothapalli, Naga Rama; Fugmann, Sebastian D.

    2011-01-01

    Activation-induced cytidine deaminase (AID) is a key enzyme for antibody-mediated immune responses. Antibodies are encoded by the immunoglobulin genes and AID acts as a transcription-dependent DNA mutator on these genes to improve antibody affinity and effector functions. An emerging theme in field is that many transcribed genes are potential targets of AID, presenting an obvious danger to genomic integrity. Thus there are mechanisms in place to ensure that mutagenic outcomes of AID activity ...

  3. The roles of APE1, APE2, DNA polymerase β and mismatch repair in creating S region DNA breaks during antibody class switch

    Schrader, Carol E.; Guikema, Jeroen E.J.; Wu, Xiaoming; Stavnezer, Janet

    2008-01-01

    Immunoglobulin class switch recombination (CSR) occurs by an intrachromosomal deletion requiring generation of double-stranded DNA breaks (DSBs) in immunoglobulin switch region DNA. The initial steps of DSB formation have been elucidated: cytosine deamination by activation-induced cytidine deaminase (AID) and the generation of abasic sites by uracil-DNA glycosylase (UNG). We show that abasic sites are converted into single-strand breaks (SSBs) by apurinic/apyrimidinic endonucleases (APE1 and ...

  4. APRIL stimulates NF-κB-mediated HoxC4 induction for AID expression in mouse B cells

    Park, Seok-Rae; Kim, Pyeung-Hyeun; Lee, Kyu-Seon; Lee, Sang-Hoon; Seo, Goo-Young; Yoo, Yung-Choon; Lee, Junglim; Casali, Paolo

    2012-01-01

    Activation-induced cytidine deaminase (AID) plays a key role in B cell immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM). We have previously reported that the highly conserved homeodomain HoxC4 transcription factor binds to the Aicda (AID gene) promoter to induce AID expression. Here, we investigated the regulation of HoxC4 transcription by a proliferation-inducing ligand (APRIL) and B cell-activating factor belonging to the TNF family (BAFF) in mouse B cell...

  5. Covalent modification of Lys19 in the CTP binding site of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase.

    Tullius, M. V.; Vann, W.F.; Gibson, B W

    1999-01-01

    Periodate oxidized CTP (oCTP) was used to investigate the importance of lysine residues in the CTP binding site of the cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) from Haemophilus ducreyi. The reaction of oCTP with the enzyme follows pseudo-first-order saturation kinetics, giving a maximum rate of inactivation of 0.6 min(-1) and a K(I) of 6.0 mM at pH 7.1. Mass spectrometric analysis of the modified enzyme provided data that was consistent with beta-...

  6. Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) mutations associated with the domestic cat AB blood group

    Millon Lee V; Pedersen Niels C; Grahn Robert A; Niini Tirri; Bighignoli Barbara; Polli Michele; Longeri Maria; Lyons Leslie A

    2007-01-01

    Abstract Background The cat has one common blood group with two major serotypes, blood type A that is dominant to type B. A rare type AB may also be allelic and is suspected to be recessive to A and dominant to B. Cat blood type antigens are defined, N-glycolylneuraminic acid (NeuGc) is associated with type A and N-acetylneuraminic acid (NeuAc) with type B. The enzyme cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) determines the sugar bound to the red cell by converting NeuAc...

  7. Characterization of the Porphobilinogen Deaminase Deficiency in Acute Intermittent Porphyria: IMMUNOLOGIC EVIDENCE FOR HETEROGENEITY OF THE GENETIC DEFECT

    Anderson, Peter M.; Reddy, Raman M.; Anderson, Karl E.; Desnick, Robert J

    1981-01-01

    The molecular pathology of the porphobilinogen (PBG)-deaminase deficiency in heterozygotes for acute intermittent porphyria (AIP) was investigated by means of biochemical and immunologic techniques. The stable enzyme-substrate intermediates (A, B, C, D, and E) of PBG-deaminase were separated by anion-exchange chromatography of erythrocyte lysates from heterozygotes for AIP and normal individuals. In normal lysates, the intermediates eluted in a characteristic pattern with decreasing amounts o...

  8. Comparative study of adenosine deaminase activity, insulin resistance and lipoprotein(a) among smokers and healthy non-smokers

    Ramesh Ramasamy; Sathish Babu Murugaiyan; Arulkumaran U.; Sathiya R.; Kuzhandai Velu V.; Niranjan Gopal

    2016-01-01

    Background: Adenosine deaminase also known as adenosine aminohydrolase involved in purine metabolism. Its primary function is development and maintenance of immune system. The main objective of the study was to estimate adenosine deaminase (ADA) enzyme and find its correlation with lipoprotein(a) and insulin resistance among smokers and healthy non-smokers. Methods: Fifty smokers and fifty healthy non-smokers were selected based on WHO definition. ADA, lipid profile and glucose was estimat...

  9. Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae.

    Medin, J A; Hunt, L; Gathy, K; Evans, R K; Coleman, M S

    1990-01-01

    Human adenosine deaminase (EC 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in Spodoptera frugiperda cells and in Trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. The coding sequence of human adenosine deaminase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. Approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellul...

  10. Assessing the effects of heavy metals in ACC deaminase and IAA production on plant growth-promoting bacteria.

    Carlos, Mendoza-Hernández José; Stefani, Perea-Vélez Yazmin; Janette, Arriola-Morales; Melani, Martínez-Simón Sara; Gabriela, Pérez-Osorio

    2016-01-01

    This study poses a methodology in order to simultaneously quantify ACC deaminase and IAA levels in the same culture medium. Ten bacterial strains isolated from plant rhizosphere naturally settled in mining residues were chosen. These bacterial strains were characterized as PGPB, and all of them showed at least three characteristics (indole-3 acetic acid and siderophore production, ACC deaminase enzyme activity, and inorganic phosphate solubilization). Taxonomic identification showed that the strains belong to Enterobacter, Serratia, Klebsiella, and Escherichia genera. Similarly, both the ACC deaminase enzyme activity and the IAA synthesis in the presence of Cu, As, Pb, Ni, Cd, and Mn were measured. The results showed that both the ACC deaminase enzyme activity and the IAA synthesis were higher with the Pb, As, and Cu treatments than with the Escherichia N16, Enterobacter K131, Enterobacter N9, and Serratia K120 control treatments. On the other hand, Ni, Cd, and Mn negatively affected both the ACC deaminase enzyme activity and the IAA production on every bacterium except on the Klebsiella Mc173 strain. Serratia K120 bacterium got a positive correlation between ACC deaminase and IAA in the presence of every heavy metal, and it also promoted Helianthus annuus plant growth, showing a potential use in phytoremediation systems. PMID:27296962

  11. Lymphomagenesis-related gene expression in B cells from sustained virological responders with occult hepatitis C virus infection.

    Roque Cuéllar, M C; García-Lozano, J R; Sánchez, B; Praena-Fernández, J M; Martínez Sierra, C; Núñez-Roldán, A; Aguilar-Reina, J

    2016-08-01

    The expression of activation-induced cytidine deaminase, B-aggressive lymphoma, cyclin D1 and serine/threonine kinase 15 genes, among others, is increased in B cells from patients with chronic hepatitis C virus (HCV) infection. It is unknown whether the level of expression of these genes in B cells is increased in patients with hepatitis C who have achieved a sustained virological response (SVR) but who have persistent, detectable HCV RNA, so-called occult infection. Eighty-three patients who achieved and SVR, 27 with detectable HCV and 56 without detectable HCV RNA, 28 chronic hepatitis C patients and 32 healthy controls were studied. RNA was extracted from B cells, and gene expression levels were measured by RT-PCR. Patients with chronic HCV and those who achieved an SVR (with and without persistent low-level HCV RNA) showed a statistically significant higher expression compared to healthy controls, of activation-induced cytidine deaminase (P = 0.004, P occult infection' had a statistically significantly higher expression level than patients with and SVR without 'occult infection' (P = 0.014). The higher expression levels found for activation-induced cytidine deaminase, together with other genes indicates that these B lymphomagenesis-related genes are upregulated following HCV therapy and this is more marked when HCV can be detected in PBMCs. PMID:26946048

  12. Studies of ferredoxins and dCTP deaminase:dUTPase

    Helt, Signe Smedegaard

    2008-01-01

    være mulige. En anden del af arbejdet omhandler krystallisation og strukturbestemmelse af enzymet dCTP deaminase:dUTPase fra tuberkulosebakterien. Dette enzym er involveret i dannelsen af nukleotidet, dTTP, der er nødvendig for at danne DNA Idet enzymet ikke findes i mennesker, kan det betragtes som et...... muligt medicinsk mål i behandlingen af f.eks. tuberkulose. Ved sammenligning af strukturerne for apo-enzymet og enzym-dTTP komplekset var store forskelle synlige. dTTPs strukturelle hæmning af enzymet har givet ny indsigt i reaktionsmekanismen for de bifunktionelle dCTP deaminase:dUTPaser såvel som for...

  13. Autoimmune dysregulation and purine metabolism in adenosine deaminase (ADA)-deficiency

    Aisha Vanessa Sauer; Immacolata eBrigida; Nicola eCarriglio; Alessandro eAiuti

    2012-01-01

    Genetic defects in the adenosine deaminase (ADA) gene are among the most common causes for severe combined immunodeficiency (SCID). ADA-SCID patients suffer from lymphopenia, severely impaired cellular and humoral immunity, failure to thrive and recurrent infections. Currently available therapeutic options for this otherwise fatal disorder include bone marrow transplantation (BMT), enzyme replacement therapy with bovine ADA (PEG-ADA) or hematopoietic stem cell gene therapy (HSC-GT). Although ...

  14. Serum adenosine deaminase as oxidative stress marker in type 2 diabetes mellitus

    Shashikala Magadi Dasegowda; Ashok Kumar Jeppu; Sushith; Kavitha Ashok Kumar

    2015-01-01

    Background: Oxidative stress markers are increased in type 2 diabetes mellitus and its estimation helps in predicting the long term complications. In present study comparison and correlation of the levels of serum adenosine deaminase, serum malondialdehyde, and serum total antioxidant capacity in type 2 diabetes mellitus and in age and sex matched healthy controls. Methods: Study group consisted of 100 individuals between the age group of 35-65 years of age. Of which 50 individuals with t...

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

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

    2009-01-01

    Claudia A Montiel-Equihua, Adrian J Thrasher, H Bobby GasparCentre for Immunodeficiency, Molecular Immunology Unit, UCL Institute of Child Health, London, UKAbstract: The history of stem cell gene therapy is strongly linked to the development of gene therapy for severe combined immunodeficiencies (SCID) and especially adenosine deaminase (ADA)-deficient SCID. Here we discuss the developments achieved in over two decades of clinical and laboratory research that led to the establishment of a pr...

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

    Thrasher, Adrian

    2009-01-01

    Claudia A Montiel-Equihua, Adrian J Thrasher, H Bobby GasparCentre for Immunodeficiency, Molecular Immunology Unit, UCL Institute of Child Health, London, UKAbstract: The history of stem cell gene therapy is strongly linked to the development of gene therapy for severe combined immunodeficiencies (SCID) and especially adenosine deaminase (ADA)-deficient SCID. Here we discuss the developments achieved in over two decades of clinical and laboratory research that led to the establishment of a pr...

  17. SIGNIFICANCE OF ADENOSINE DEAMINASE SERUM CONCENTRATIONS IN THE DIAGNOSIS OF EXTRA-PULMONARY TUBERCULOSIS

    Stevanovic G.; Pelemis M.; Pavlovic M.; Lavadinovic L,; Dakic Z,; Milosevic I,; Milosevic B.

    2011-01-01

    Extra pulmonary tuberculosis (EPTB) is a growing problem worldwide. Due to the nature of the disease, the diversity of clinical pictures as well as its minor epidemiological importance, the diagnosis is difficult and often late.In addition to standard TB diagnostic techniques use of new biochemical (surrogate markers) are increased. With this work we wanted to examine the usefulness of serum adenosine deaminase levels as a diagnostic parameter for EPTB.The work included 116 patients with feve...

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

    Fernández, José R.; Sweet, Eric S; Welsh, William J.; Firestein, Bonnie L

    2010-01-01

    Guanine deaminase (GDA; cypin) is an important metalloenzyme that processes the first step in purine catabolism, converting guanine to xanthine by hydrolytic deamination. In higher eukaryotes, GDA also plays an important role in the development of neuronal morphology by regulating dendritic arborization. In addition to its role in the maturing brain, GDA is thought to be involved in proper liver function since increased levels of GDA activity have been correlated with liver disease and transp...

  19. Radio-chromatographic determination of plasmatic adenosine deaminase (A.D.)

    We were able, by using a radio-chromatographic method, to measure an adenosine deaminase activity in normal human heparinized platelet-poor plasma, which can degrade 0.016 μM adenosine. This activity suppressed by heating 56 C for 30 minutes is inhibited by high concentrations of urea and is proportional to the amount of plasma, source of enzyme, in the systems. (authors)

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

    Magnucka, Elżbieta G; Pietr, Stanisław J

    2015-12-01

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

  1. Inducing salt tolerance in maize through ACC-deaminase biotechnology (abstract)

    Ethylene is one of the five established classes of phytohormones. Its involvement in evoking physiological responses in plants exposed to any kind of environmental stresses (such as salinity, drought and metal toxicity stresses) is well established, thus it has also been designated as 'stress' hormone. It is generally believed that stress induces accelerated synthesis of ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), which subsequently results in the out burst of ethylene production and plant responds to this higher level of ethylene. Thus any check on this accelerated ethylene production in plants exposed to salinity stress could help in minimizing the negative impact of this stress and plants might survive better. There are some soil bacteria which carry ACC-deaminase enzyme and their presence on root surface results in lowering the accelerated production of ethylene. Trials were conducted under axenic condition to study the effect of inoculation with ACC-deaminase containing rhizobacteria to ameliorate the effect of salinity on seedling growth. Maize seedlings were exposed to different salinity levels and results indicated that inoculation with ACC-deaminase containing rhizobacteria significantly increased root elongation, shoot length, fresh and dry weight of seedlings at all the salinity levels. (author)

  2. In Silico Discovery of Potential Uridine-Cytidine Kinase 2 Inhibitors from the Rhizome of Alpinia mutica.

    Malami, Ibrahim; Abdul, Ahmad Bustamam; Abdullah, Rasedee; Bt Kassim, Nur Kartinee; Waziri, Peter; Christopher Etti, Imaobong

    2016-01-01

    Uridine-cytidine kinase 2 is implicated in uncontrolled proliferation of abnormal cells and it is a hallmark of cancer, therefore, there is need for effective inhibitors of this key enzyme. In this study, we employed the used of in silico studies to find effective UCK2 inhibitors of natural origin using bioinformatics tools. An in vitro kinase assay was established by measuring the amount of ADP production in the presence of ATP and 5-fluorouridine as a substrate. Molecular docking studies revealed an interesting ligand interaction with the UCK2 protein for both flavokawain B and alpinetin. Both compounds were found to reduce ADP production, possibly by inhibiting UCK2 activity in vitro. In conclusion, we have identified flavokawain B and alpinetin as potential natural UCK2 inhibitors as determined by their interactions with UCK2 protein using in silico molecular docking studies. This can provide information to identify lead candidates for further drug design and development. PMID:27070566

  3. Ag(I)-mediated homo and hetero pairs of guanosine and cytidine: Monitoring by circular dichroism spectroscopy

    Goncharova, Iryna

    2014-01-01

    Ag(I)-containing compounds are attractive as antibacterial and antifungal agents. The renewed interest in the application of silver(I) compounds has led to the need for detailed knowledge of the mechanism of their action. One of the possible ways is the coordination of Ag(I) to G-C pairs of DNA, where Ag+ ions form Ag(I)-mediated base pairs and inhibit the transcription. Herein, a systematic chiroptical study on silver(I)-mediated homo and mixed pairs of the C-G complementary-base derivatives cytidine(C) and 5‧-guanosine monophosphate(G) in water is presented. Ag(I)-mediated homo and hetero pairs of G and C and their self-assembled species were studied under two pH levels (7.0 and 10.0) by vibrational (VCD) and electronic circular dichroism(ECD). VCD was used for the first time in this field and showed itself to be a powerful method for obtaining specific structural information in solution. Based on results of the VCD experiments, the different geometries of the homo pairs were proposed under pH 7.0 and 10.0. ECD was used as a diagnostic tool to characterize the studied systems and as a contact point between the previously defined structures of the metal or proton mediated pairs of nucleobases and the systems studied here. On the basis of the obtained data, the formation of the self-assembled species of cytidine with a structure similar to the i-motif structure in DNA was proposed at pH 10.0.

  4. Regulation of Expression of the prb-1b / ACC Deaminase gene by UV-B in Transgenic tomatoes

    Transgenic tomato plants with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UWA4 under the control of a pathogenesis-related promoter (prb-1b) from tobacco were challenged by abiotic stresses to determine the expression patterns of the transgene. No ACC deaminase RNA or protein was detected bu RT-PCR and in western blots prepared from leaf proteins of transgenic plants after wounding or treatment with alpha-amino butyric acid, xylanase, ethephon, salicylic acid, jasmonic acid , ethylene, or ethylene plus jasmonic acid. However, expression of the ACC deaminase transgene was observed in leaves and roots of transformed tomato lines exposed to UV light. The UV response required a minimum of 48 h of exposure and was specific to UV-B light

  5. Magnetically assisted fluorescence ratiometric assays for adenosine deaminase using water-soluble conjusated polymers

    HE Fang; YU MingHui; WANG Shu

    2009-01-01

    A magnetically assisted fluorescence ratiometric technique has been developed for adenosine deami-nase assays with high sensitivity using water-soluble cationic conjugated polymers (CCPs).The assay contains three elements:a biotin-labeled aptamer of adenosine (biotin-aptamer),a signaling probe single-stranded DNA-tagged fiuorescein at terminus (ssDNA-FI) and a CCP.The specific binding of adenosine to biotin-aptamer makes biotin-aptamer and ssDNA-FI unhybridized,and the ssDNA-FI is washed out after streptavidin-coated magnetic beads are added and separated from the assay solution under magnetic field.In this case,after the addition of CCP to the magnetic beads solution,the fluo-rescence resonance energy transfer (FRET) from CCP to fluorescein is inefficient.Upon adding adenosine deaminase,the adenosine is converted into inosine,and the biotin-aptamer is hybridized with ssDNA-FI to form doubled stranded DNA (biotin-dsDNA-FI).The ssONA-FI is attached to the mag-netic beads at the separation step,and the addition of CCP to the magnetic beads solution leads to efficient FRET from CCP to fluorescein.Thus the adenosine deaminase activity can be monitored by fluorescence spectra in view of the intensity decrease of CCP emission or the increase of fluorescein emission in aqueous solutions.The assay integrates surface-functionalized magnetic particles with significant amplification of detection signal of water-soluble cationic conjugated polymers.

  6. Biochemistry and Genetics of ACC deaminase: A weapon to 'stress ethylene' produced in plants

    Rajnish Prakash Singh

    2015-09-01

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

  7. In vitro and in vivo activation of L-serine deaminase in Escherichia coli K-12.

    Newman, E. B.; D. Dumont; Walker, C.

    1985-01-01

    Escherichia coli L-serine deaminase (L-SD) in crude extracts made in glycylglycine could be activated by incubation with iron sulfate and dithiothreitol. This activation could also be demonstrated in vitro in two mutants which were physiologically deficient in L-SD activity in vivo. This suggests that these mutants were deficient not in L-SD but in an enzyme(s) activating L-SD. The suggestion is made that production of a functional L-SD in vivo requires activation of the structural gene produ...

  8. A novel L-serine deaminase activity in Escherichia coli K-12.

    Su, H. (Hsin-Ning); Newman, E B

    1991-01-01

    We demonstrate here that Escherichia coli K-12 synthesizes two different L-serine deaminases (L-SD) catalyzing the nonoxidative deamination of L-serine to pyruvate, one coded for by the previously described sdaA gene and a second, hitherto undescribed enzyme which we call L-SD2. A strain carrying a null mutation in sdaA made no detectable L-SD in minimal medium, but had activity in Luria broth. We describe a mutation, sdaX, which affects the regulation of L-SD2 and permits its expression in m...

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

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

    2016-01-01

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

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

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

    2007-01-01

    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...... 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 d......CTP 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...

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

    Claudia A Montiel-Equihua

    2009-12-01

    Full Text Available Claudia A Montiel-Equihua, Adrian J Thrasher, H Bobby GasparCentre for Immunodeficiency, Molecular Immunology Unit, UCL Institute of Child Health, London, UKAbstract: The history of stem cell gene therapy is strongly linked to the development of gene therapy for severe combined immunodeficiencies (SCID and especially adenosine deaminase (ADA-deficient SCID. Here we discuss the developments achieved in over two decades of clinical and laboratory research that led to the establishment of a protocol for the autologous transplant of retroviral vector-mediated gene-modified hematopoietic stem cells, which has proved to be both successful and, to date, safe. Patients in trials in three different countries have shown long-term immunological and metabolic correction. Nevertheless, improvements to the safety profile of viral vectors are underway and will undoubtedly reinforce the position of stem cell gene therapy as a treatment option for ADA-SCID.Keywords: adenosine deaminase, severe combined immunodeficiency, gene therapy, hematopoietic stem cell, retrovirus, clinical trial

  12. Optimization of catalyst-solvent system for preparation of alpha-5,6-dihydro-5-aza-2'-deoxy-[6-3H]-cytidine

    Elbert, Tomáš

    2011-01-01

    Roč. 54, č. 5 (2011), s. 285-285. ISSN 0362-4803. [Workshop of the International Isotope Society - Central European Division. The Synthesis and Applications of Isotopes and Isotopically Labelled Compounds /17./. 23.09.2010-24.09.2010, Bad Soden] Institutional research plan: CEZ:AV0Z40550506 Keywords : tritium * labelled compounds * alfa-5,6-dihydro-5-aza-2'-deoxy-cytidine Subject RIV: CC - Organic Chemistry

  13. Morphological effects of cytidin-diphosphate-choline on rats with lesions of the substantia nigra: study using horse radish peroxidase method.

    Stanzani, S

    1981-09-15

    Morphological effects of Cytidin-diphosphate-Choline (CDP-choline) (Ni-cholin) on rat brain with Substantia nigra lesions were studied by using the horse radish peroxidase method (HRP). Three groups of animals were studied. Post-lesion axonal and cellular regeneration was detected only in the group of rats treated with CDP-choline q.d. i.m. for 15 days. PMID:7306424

  14. Conformational studies of the smallest structural motifs of DNA detectable via vibrational circular dichroism: cytidylyl-(3'-5')-guanosine and guanylyl-(3'-5')-cytidine.

    Birke, S S; Diem, M.

    1995-01-01

    The infrared absorption and vibrational circular dichroism (VCD) spectra of buffered aqueous solutions of cytidylyl-(3'-5')-guanosine (5'(CG)3') and guanylyl-(3'-5')-cytidine (5'(GC)3') are reported. Under low ionic strength conditions, these dinucleotides exhibit VCD features that can be predicted qualitatively from structural data of (CG)2 and (GC)2 sequences of poly(dG-dC).poly(dG-dC), using the exciton model for infrared VCD intensities.

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

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

    2015-07-01

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

  16. Isolation and properties of AMP deaminase from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California, Mexico.

    Marquez-Rios, E; Pacheco-Aguilar, R; Castillo-Yañez, F J; Figueroa-Soto, C G; Ezquerra-Brauer, J M; Gollas-Galvan, T

    2008-09-01

    Adenosine monophosphate (AMP) deaminase was purified from jumbo squid mantle muscle by chromatography in cellulose phosphate, Q-Fast and 5'-AMP sepharose. Specific activity of 2.5U/mg protein, 4.5% recovery and 133.68 purification fold were obtained at the end of the experiment. SDS-PAGE showed a single band with 87kDa molecular mass, native PAGE proved a band of 178kDa, whereas gel filtration detected a 180kDa protein, suggesting the homodimeric nature of this enzyme, in which subunits are not linked by covalent forces. Isoelectric focusing of this enzyme showed a pI of 5.76, which agrees with pI values of AMP deaminase from other invertebrate organisms. AMP deaminase presented a kinetic sigmoidal plot with Vmax of 1.16μM/min/mg, Km of 13mM, Kcat of 3.48μM.s(-1) and a Kcat/Km of 267 (mol/L)(-1).s(-1). The apparent relative low catalytic activity of jumbo squid muscle AMP deaminase in the absence of positive effectors is similar to that reported for homologous enzymes in other invertebrate organisms. PMID:26050167

  17. Isolation and characterization of novel bacteria containing acc deaminase from the rhizosphere resource on dry-farming lands

    Soil-microbe-plant interactions are known to be intricate and they can greatly influence the crop vigor and yield. Plant growth promoting rhizobacteria (PGPR) containing ACC deaminase can markedly affect plant metabolic processes under stress conditions. In the present study, we isolated 300 bacterial strains from the rhizosphere of maize or apple grown in drought-hit soil including four different locations of the Loess Plateau, China. Of all isolated strains, four with ACC deaminase activity (ranging from 28.88 to 155.12 nmol alpha-ketobutyrate mg-1 h-1) were further studied by determining their biological characters and sequencing the 16S rRNA gene. All four strains showed positive performance in terms of arabinose, citrate utilization, urease, indol, glucose and melibiose. In connection with the results of biochemical characters and phylogenetic analysis, these strains commonly belong to three different genera: Klebsiella, Pseudomonas and Raoultella and four different species: Klebsiella oxytoca, Klebsiella variicola, Pseudomonas fluorescens and Raoultella planticola. Although some researchers have reported their performance under stress conditions, we are the first to report Klebsiella oxytoca, Klebsiella variicola and Raoultella planticola containing ACC deaminase under drought stress. These findings are a reasonable explanation to their superb ability of causing stress-resistance in maize (Zea mays) or apple (Malus domestica) plants. The presence of diverse PGPR possessing potential ACC deaminase activity may be beneficial for enhancing crop production under different stress conditions. (author)

  18. Comparison of interferon-gamma release assays and adenosine deaminase of pleural fluid for the diagnosis of pleural tuberculosis

    刘菲

    2014-01-01

    Objective To compare the diagnostic performance of interferon gamma releasing assays(T-SPOT.TB)and adenosine deaminase(ADA)in pleural tuberculosis,and therefore to evaluate the value of T-SPOT.TB in a high tuberculosis burden country.Methods From June 2011to November 2012,111 patients with pleural fluid in Beijing Chest Hospital,Capital Medical University were

  19. Short Communication: Rhizosphere bacteria containing ACC-deaminase conferred drought tolerance in wheat grown under semi-arid climate

    Masood Ahmad Shakir

    2012-05-01

    Full Text Available Certain rhizobacteria have the potential of lowering endogenous ethylene levels in plants because of their 1- aminocyclopropane-1-carboxylate (ACC-deaminase activity and promoting root growth. This mechanism is of great agronomic significance under stress environments, which are known to induce accelerated production of ethylene. Thirty rhizobacteria were isolated from rhizosphere of wheat (Triticum aestivum L. growing in the Southern Punjab, a semi-arid region of Pakistan. Rhizobacteria were screened for ACC-deaminase activity and their potential to confer drought tolerance in wheat crop. Results of laboratory study revealed that selected rhizobacteria lowered endogenous ethylene levels in the rhizosphere as measured by Gas Chromatograph. Axenic studies showed that inoculation increased root-shoot length, root-shoot mass and lateral root number of the inoculated plants by 141, 44, 196, 52 and 30%, respectively, over control. Better-developed roots because of inoculation with plant growth promoting rhizobacteria (PGPR helped plants, a better crop stand that enhanced moisture and nutrient feeding volume resulting-in improved growth and yields of wheat crop. Two-year multi-location field trials inferred optimum yields with low delta water in semi-arid climate by PGPR containing ACC-deaminase. The enzyme ACC-deaminase probably lowered harmful ethylene levels which partially eliminated drought stress consequently utilizing soil moisture from lower profiles through proliferated roots.

  20. Concerted bifunctionality of the dCTP deaminase-dUTPase from Methanocaldococcus jannaschii: A structural and pre-steady state kinetic analysis

    Siggaard, Julie; Johansson, Eva; Vognsen, Tina Reinholdt; Helt, Signe Smedegaard; Harris, Pernille; Larsen, Sine; Willemoës, Martin

    2009-01-01

    Two mutant dCTP deaminase-dUTPases from Methanocaldococcus jannaschii were crystallised and the crystal structures were solved: E145A in complex with the substrate analogue alpha,beta-imido-dUTP and E145Q in complex with diphosphate. Both mutant enzymes were defect in the deaminase reaction and had...... nucleotidyl diphosphorylase reaction was observed in the E145A:alpha,beta-imido-dUTP complex and positioned similarly as in the monofunctional trimeric dUTPase. A comparison of the active sites of the bifunctional enzyme and the monofunctional family members, dCTP deaminase and dUTPase, suggests similar...

  1. Radio-chromatographic determination of plasmatic adenosine deaminase (A.D.); Determination radiochromatographique de l'adenosine deaminase (A.D.)

    Chivot, J.J.; Depernet, D.; Caen, J. [Commissariat a l' Energie Atomique, Bruyeres-le-Chatel (France). Centre d' Etudes

    1970-07-01

    We were able, by using a radio-chromatographic method, to measure an adenosine deaminase activity in normal human heparinized platelet-poor plasma, which can degrade 0.016 {mu}M adenosine. This activity suppressed by heating 56 C for 30 minutes is inhibited by high concentrations of urea and is proportional to the amount of plasma, source of enzyme, in the systems. (authors) [French] Nous avons pu, en utilisant une methode radiochromatographique, mesurer une activite adenosine deaminasique dans le plasma humain pauvre en plaquettes heparine qui peut degrader 0,016 {mu}M d'adenosine. Cette activite qui est supprimee par chauffage a 56 degres pendant 30 minutes, est reduite par conservation a -20 C pendant une semaine, est inhibee par d'importantes concentrations d'uree et ne l'est pas, ni par le dipyridamol, ni par le pHMB. Cette activite est proportionnelle a la quantite de plasma, source d'enzyme, mise dans les differents systemes reactifs. (auteur)

  2. Cytidine 5′-diphosphocholine administration prevents peripheral neuropathic pain after sciatic nerve crush injury in rats

    Emril, Dessy R; Wibowo, Samekto; Meliala, Lucas; Susilowati, Rina

    2016-01-01

    Background Cytidine 5′-diphosphocholine (citicoline) has been shown to have beneficial effects in central nervous system injury as well as in motoric functional recovery after peripheral nerve injury. This study aimed to examine the effect of citicoline on prevention of neuropathic pain in a rat model of sciatic nerve crush injury. Methods Forty experimental rats were divided into four groups. In three groups, the right sciatic nerves were crushed in the mid-thigh region, and a gelatin sponge moistened with 0.4 or 0.8 mL of 100 µmol/L citicoline, or saline 0.4 mL in the control group, was applied. The fourth group of rats was sham-operated, ie the sciatic nerve was exposed with no crush. Functional assessments were performed 4 weeks after crush injury. von Frey filaments (100 g threshold) were used to assess neuropathic pain. In addition, the sciatic functional index and extensor postural thrust (EPT) tests were used to assess motoric function. Results The crush/citicoline 0.4 mL group had a lower percentage of pain (23.53%, n=17) compared with the crush/saline group (53.33%, n=15, Pnerve injury due to compression with a larger administered volume. Conclusion In situ administration of 0.4 mL of 100 µmol/L citicoline prevents the occurrence of neuropathic pain and induces motoric recovery, evaluated by EPT test, 4 weeks after sciatic nerve injury. PMID:27284264

  3. Platelet aggregation and serum adenosine deaminase (ADA) activity in pregnancy associated with diabetes, hypertension and HIV.

    Leal, Claudio A M; Leal, Daniela B R; Adefegha, Stephen A; Morsch, Vera M; da Silva, José E P; Rezer, João F P; Schrekker, Clarissa M L; Abdalla, Faida H; Schetinger, Maria R C

    2016-07-01

    Platelet aggregation and adenosine deaminase (ADA) activity were evaluated in pregnant women living with some disease conditions including hypertension, diabetes mellitus and human immunodeficiency virus infection. The subject population is consisted of 15 non-pregnant healthy women [control group (CG)], 15 women with normal pregnancy (NP), 7 women with hypertensive pregnancy (HP), 10 women with gestational diabetes mellitus (GDM) and 12 women with human immunodeficiency virus-infected pregnancy (HIP) groups. The aggregation of platelets was checked using an optical aggregometer, and serum ADA activity was determined using the colorimetric method. After the addition of 5 µM of agonist adenosine diphosphate, the percentage of platelet aggregation was significantly (p pregnancy and pregnancy-associated diseases suggest that platelet aggregation and ADA activity could serve as peripheral markers for the development of effective therapy in the maintenance of homeostasis and some inflammatory process in these pathophysiological conditions. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27273565

  4. Serum adenosine deaminase activity and its isoenzyme in patients treated for tuberculosis

    Objective: Increased serum adenosine deaminase (ADA) activity, mainly associated with tuberculosis can also occur in a number of other diseases thus negatively affecting the diagnostic utility of ADA measurements in tuberculosis. The aim of the study was to determine whether or not the combined use of the activity of ADA, its isoenzymes and differential cell counts would provide a more efficient means of diagnosing tuberculosis than the use of ADA levels alone. Results: Data suggested significant (p0.75) of ADA/sub 2/ADA was found to be better indicator of tuberculosis. Lymphocyte neutrophil ratio (L/N)> 0.69 gave additional benefit to increase the sensitivity and specificity for the use of ADA as marker in diagnosing tuberculosis. Conclusion: The combined use of activity of ADA, its isoenzymes and total and differential cell counts is a better indicator and gives better understanding to diagnose and evaluate tuberculosis and response to therapy. (author)

  5. Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells

    Recombinant retroviruses encoding human adenosine deaminase have been used to infect murine hematopoietic stem cells. In bone marrow transplant recipients reconstituted with the genetically modified cells, human ADA was detected in peripheral blood mononuclear cells of the recipients for at least 6 months after transplantation. In animals analyzed in detail 4 months after transplantation, human ADA and proviral sequences were detected in all hematopoietic lineages; in several cases, human ADA activity exceeded the endogenous activity. These studies demonstrate the feasibility of introducing a functional human ADA gene into hematopoietic stem cells and obtaining expression in multiple hematopoietic lineages long after transplantation. This approach should be helpful in designing effective gene therapies for severe combined immunodeficiency syndromes in humans

  6. Serum Adenosine deaminase activity and C-reactive protein levels in unstable angina

    Rani Surekha

    2003-01-01

    Full Text Available In unstable angina (USA patients, immunological responses contributing to inflammation play a vital role in plaque rupture and thrombosis causing stroke. In the present study an attempt is made to estimate the levels of adenosine deaminase activity, an immunoenzyme marker and C-reactive protein, a marker of inflammation in USA patients. 45 patients presenting USA and 50 age and sex matched healthy controls were included in the study. Serum ADA activity was measured spectrophotometrically at 630nm and serum C-reactive protein was detected using Avitex CRP kit, which is a rapid latex agglutination test. The Mean ADA levels were 41.15 ± 11.04 in patients and 20.71±5.63 in controls and 66.6% of patients and none of the controls were positive to CRP. The present study observed the importance of ADA as a serum marker in addition to CRP for assessing the immune response in USA patients.

  7. ACC (1-Aminocyclopropane-1-Carboxylate) Deaminase Activity, a Widespread Trait in Burkholderia Species, and Its Growth-Promoting Effect on Tomato Plants▿

    Onofre-Lemus, Janette; Hernández-Lucas, Ismael; Girard, Lourdes; Caballero-Mellado, Jesús

    2009-01-01

    The genus Burkholderia includes pathogens of plants and animals and some human opportunistic pathogens, such as the Burkholderia cepacia complex (Bcc), but most species are nonpathogenic, plant associated, and rhizospheric or endophytic. Since rhizobacteria expressing ACC (1-aminocyclopropane-1-carboxylate) deaminase may enhance plant growth by lowering plant ethylene levels, in this work we investigated the presence of ACC deaminase activity and the acdS gene in 45 strains, most of which are...

  8. Mixed Inhibition of Adenosine Deaminase Activity by 1,3-Dinitrobenzene: A Model for Understanding Cell-Selective Neurotoxicity in Chemically-Induced Energy Deprivation Syndromes in Brain

    Wang, Yipei; Liu, Xin; Schneider, Brandon; Zverina, Elaina A.; Russ, Kristen; Wijeyesakere, Sanjeeva J.; Fierke, Carol A.; Richardson., Rudy J.; Philbert, Martin A.

    2011-01-01

    Astrocytes are acutely sensitive to 1,3-dinitrobenzene (1,3-DNB) while adjacent neurons are relatively unaffected, consistent with other chemically-induced energy deprivation syndromes. Previous studies have investigated the role of astrocytes in protecting neurons from hypoxia and chemical injury via adenosine release. Adenosine is considered neuroprotective, but it is rapidly removed by extracellular deaminases such as adenosine deaminase (ADA). The present study tested the hypothesis that ...

  9. Characterization and optimization of 1-Aminocyclopropane-1-Carboxylate Deaminase (ACCD activity in different rhizospheric PGPR along with Microbacterium sp. strain ECI-12A

    Ashok Kumar

    2013-03-01

    Full Text Available A total of nine strains of plant growth promoting rhizobacteria were analyzed for ACC deaminase activity, where highest ACC deaminase activity was found in Klebsiella sp strain ECI-10A (539.1 nmol α-keto butyrate/ mg protein/ h and lowest in Microbacterium sp strain ECI-12A (122.0 nmol α-keto butyrate/ mg protein/ h. Although Microbacterium sp strain ECI-12A showed lowest level of ACC deaminase activity, but, the species of Microbacterium isolated from rhizosphere is the first report. Microbacterium sp strain ECI-12A was also analyzed under varying conditions of time, amount of 1-Aminocyclopropane-1- carboxylate (ACC, and temperature for optimization of the ACC deaminase activity. The optimum activity was recorded with the supplementation of 5mM ACC at 30oC temperature after 24h of culture growth. All the nine strains showed acdS gene in the PCR amplification of that gene. No any rhizospheric Microbacterium species showing ACC deaminase activity have been reported earlier, therefore, we report here ACC deaminase activity in Microbacterium sp ECI-12A isolated from rice rhizosphere is a novel finding.

  10. Residual activity induced by ion bombardment on insulating samples

    In this work we investigate some properties of the residual activity induced by protons impinging on quartz, mylar and other insulating materials. In particular, we discuss the time constant related to the decay of the emitted radiation after the primary ion beam is turned off. This radiation includes a continuum of bremsstrahlung and, in some cases, characteristic X-rays induced in the process as well. In general, the results indicate the presence of two time constants in the decaying process. Moreover, it appears that the residual activity has a strong dependence on the material specifications and on the conditions of the surface under bombardment. A simple mechanism for this process is suggested

  11. Activation-Induced Cell Death in T Cells and Autoimmunity

    JianZhang; XuemeiXu; YongLiu

    2004-01-01

    Activation-induced cell death (AICD), which results from the interaction between Fas and Fas ligand, is responsible for maintaining tolerance to self-antigen. A defect in AICD may lead to development of autoimmunity. During the last several years, much progress has been made in understanding the mechanism(s) of AICD and its potential role in the pathogenesis of autoimmune diseases. In this review, we summarize the most recent progress on the regulation of the susceptibility of T cells to AICD and its possible involvement in autoimmune diseases.

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

    The tRNA-specific adenosine deaminase from the pathogenic bacteria S. pyogenes has been overexpressed and crystallized. The tRNA-specific adenosine deaminase from the pathogenic bacteria Streptococcus pyogenes (spTAD) has been overexpressed in Escherichia coli and crystallized in the presence of Zn2+ ion at 295 K using ammonium sulfate as a precipitant. Flash-cooled crystals of spTAD diffracted to 2.0 Å using 30%(v/v) glycerol as a cryoprotectant. X-ray diffraction data have been collected to 2.0 Å using synchrotron radiation. The crystal belongs to the tetragonal space group P42212, with unit-cell parameters a = b = 81.042, c = 81.270 Å. The asymmetric unit contains one subunit of spTAD, with a corresponding crystal volume per protein weight (VM) of 3.3 Å3 Da−1 and a solvent content of 62.7%

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

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P212121, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å

  14. Retrovirus-mediated gene transfer of human adenosine deaminase: expression of functional enzyme in murine hematopoietic stem cells in vivo.

    Lim, B; Williams, D A; Orkin, S H

    1987-01-01

    Simplified Moloney murine leukemia virus-based recombinant retrovirus vectors have been constructed which transduce human adenosine deaminase (ADA) cDNA. ADA transcription is under the control of the constitutive promoter for the human X chromosome phosphoglycerate kinase (pgk) gene. In these simplified vectors, dominant selectable markers are not included and selection is dependent on overproduction of functional ADA enzyme. Primary murine hematopoietic cells were infected with helper-free r...

  15. Purine metabolic enzymes in lymphocytes. III. Effects of immunosuppressants on adenosine deaminase and purine nucleoside phosphorylase activities

    Kurashige, Satonori; Akuzawa, Yuki; Yoshida, Toshiharu; Kodama, Kazue

    1983-01-01

    Mice were treated with a single injection of 6-mercaptopurine riboside (6MP-R), predonine or cyclophosphamide (CY), and the effects of these immunosuppressants on blastogenic responses to phytohemagglutinin P (PHA-P) or bacterial lipopoly saccharide (LPS) and on adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP) activities were studied with spleen lymphocytes. The retardation of blastogenic responses to both PHA-P and LPS were associated with the retardation of both intracellu...

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

    Naval-Macabuhay, Isaac; Casanova, Víctor; Navarro, Gemma; García, Felipe; León, Agathe; Miralles, Laia; Rovira, Cristina; Martinez-Navio, José M; Gallart, Teresa; Mallol, Josefa; Gatell, José M; Lluís, Carme; Franco, Rafael; McCormick, Peter J; Climent, Núria

    2016-02-01

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

  17. Short Communication: Rhizosphere bacteria containing ACC-deaminase conferred drought tolerance in wheat grown under semi-arid climate

    Masood Ahmad Shakir; Asghari Bano; and Muhammad Arshad

    2012-01-01

    Certain rhizobacteria have the potential of lowering endogenous ethylene levels in plants because of their 1- aminocyclopropane-1-carboxylate (ACC)-deaminase activity and promoting root growth. This mechanism is of great agronomic significance under stress environments, which are known to induce accelerated production of ethylene. Thirty rhizobacteria were isolated from rhizosphere of wheat (Triticum aestivum L.) growing in the Southern Punjab, a semi-arid region of Pakistan. Rhizobacteria we...

  18. Antitumor and radiosensitizing effects of (E)-2'-Deoxy-2'-(Fluoromethylene) cytidine, a novel inhibior of ribonucleotide diphosphate reductase on human colon carcinoma xenografts in nude mice.

    Sun, Lin-Quan; Li, Ye-Xiong; Guillou, Louis; Mirimanoff, René-Olivier; Coucke, Philippe

    1997-01-01

    Antitumor and radiosensitizing effects of (E).2'-deoxy.2'-(fluromethyl ene) cytidine (FMdC), a novel inhibitor of ribonucleotide reductase, were evaluated on nude mice bearing s.c. xenografts and liver metastases of a human colon carcinoma. FMdC given once daily or twice weekly has a dose-dependent antitumor effect. The maximum tolerated dose In the mice was reached with 10 mgi'kg applied daily over 12 days. Twice weekly administration of FMdC reduced its toxicity but lowere...

  19. The ribonucleoside diphosphate reductase inhibitor (E)-2'-Deoxy-(fluoromethylene) cytidine, acts as a cytotoxic radiosensitizer on human cancer cell lines in vitro.

    Coucke, Philippe; Decosterd, L-A; Li, Y-X; Cottin, E.; Chen, X; Sun, L-Q; Stern, S.; Paschoud, N; Denekamp, J.

    1999-01-01

    ABSTRACT (E)-2*-Deoxy-(fluoromethylene)cytidine (FMdC) is known as an inhibitor of ribonucleoside diphosphate reductase, a key enzyme in the de novo pathway of DNA synthesis. FMdC was tested as a modifier of radiation response in vitro on a human colon carcinoma cell line (WiDr), and the observed radiosensitization was confirmed on two human cervix cancer cell lines (C33-A and SiHa). Using the clonogenic assay, the effect ratio (ER) at a clinically relevant dose level of ...

  20. Positive interactive radiosensitization observed in vitro with the combination of two nucleoside analogs (E)-2'-deoxy-2'-(fluoromethylene) cytidine (FMdC) and iododeoxyuridine (IdUrd).

    Coucke, Philippe; Cottin, E.; Azria, D; Martineau, P.; Adamer, F; Decosterd, L-A; Buchegger, F; Do, H-P

    2004-01-01

    (E)-20-Deoxy-20-(fluoromethylene) cytidine (FMdC), an inhibitor of ribonucleotide diphosphate reductase (RR), is a potent radiation-sensitiser acting through alterations in the deoxyribonucleoside triphosphate (dNTP) pool in the de novo pathway to DNA synthesis. The activity of thymidine kinase (TK), a key enzyme in the ‘salvage pathway’, is known to increase in response to a lowering of dATP induced by FMdC. Nucleoside analogues such as iododeoxyuridine (IdUrd) are incorporated into DNA a...

  1. Cytidine 5’-diphosphocholine administration prevents peripheral neuropathic pain after sciatic nerve crush injury in rats

    Emril DR

    2016-05-01

    Full Text Available Dessy R Emril,1 Samekto Wibowo,2 Lucas Meliala,2 Rina Susilowati3 1Department of Neurology, Faculty of Medicine, Syiah Kuala University, Banda Aceh, 2Department of Neurology, 3Department of Histology and Cell Biology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, IndonesiaBackground: Cytidine 5’-diphosphocholine (citicoline has been shown to have beneficial effects in central nervous system injury as well as in motoric functional recovery after peripheral nerve injury. This study aimed to examine the effect of citicoline on prevention of neuropathic pain in a rat model of sciatic nerve crush injury.Methods: Forty experimental rats were divided into four groups. In three groups, the right sciatic nerves were crushed in the mid-thigh region, and a gelatin sponge moistened with 0.4 or 0.8 mL of 100 µmol/L citicoline, or saline 0.4 mL in the control group, was applied. The fourth group of rats was sham-operated, ie the sciatic nerve was exposed with no crush. Functional assessments were performed 4 weeks after crush injury. von Frey filaments (100 g threshold were used to assess neuropathic pain. In addition, the sciatic functional index and extensor postural thrust (EPT tests were used to assess motoric function.Results: The crush/citicoline 0.4 mL group had a lower percentage of pain (23.53%, n=17 compared with the crush/saline group (53.33%, n=15, P<0.005. The crush/citicoline 0.4 mL group also showed better motoric recovery, as seen in stronger EPT results (P<0.001. However, the sciatic functional index analysis did not show significant differences between groups (P=0.35. The crush/citicoline 0.8 mL group showed a higher percentage of pain (66.67%, n=18 and less EPT recovery. These results may be explained by more severe nerve injury due to compression with a larger administered volume.Conclusion: In situ administration of 0.4 mL of 100 μmol/L citicoline prevents the occurrence of neuropathic pain and induces motoric recovery

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

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

    2011-11-07

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

  3. The Role of Histidine-Proline-Rich Glycoprotein as Zinc Chaperone for Skeletal Muscle AMP Deaminase

    Maria Ranieri-Raggi

    2014-05-01

    Full Text Available Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP deaminase (AMPD and the metal binding protein histidine-proline-rich glycoprotein (HPRG acts in this manner. HPRG is a major plasma protein. Recent investigations have reported that skeletal muscle cells do not synthesize HPRG but instead actively internalize plasma HPRG. X-ray absorption spectroscopy (XAS performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (μ-aqua(μ-carboxylatodizinc(II core with two histidine residues at each metal site. XAS on HPRG isolated from the AMPD complex showed that zinc is bound to the protein in a dinuclear cluster where each Zn2+ ion is coordinated by three histidine and one heavier ligand, likely sulfur from cysteine. We describe the existence in mammalian HPRG of a specific zinc binding site distinct from the His-Pro-rich region. The participation of HPRG in the assembly and maintenance of skeletal muscle AMPD by acting as a zinc chaperone is also demonstrated.

  4. Mitochondrial Damage and Apoptosis Induced by Adenosine Deaminase Inhibition and Deoxyadenosine in Human Neuroblastoma Cell Lines.

    Garcia-Gil, Mercedes; Tozzi, Maria Grazia; Balestri, Francesco; Colombaioni, Laura; Camici, Marcella

    2016-07-01

    The treatment with deoxycoformycin, a strong adenosine deaminase inhibitor, in combination with deoxyadenosine, causes apoptotic cell death of two human neuroblastoma cell lines, SH-SY5Y and LAN5. Herein we demonstrate that, in SH-SY5Y cells, this combination rapidly decreases mitochondrial reactive oxygen species and, in parallel, increases mitochondrial mass, while, later, induces nuclear fragmentation, and activation of caspase-8, -9, and -3. In previous papers we have shown that a human astrocytoma cell line, subjected to the same treatment, undergoes apoptotic death as well. Therefore, both astrocytoma and neuroblastoma cell lines undergo apoptotic death following the combined treatment with deoxycoformycin and deoxyadenosine, but several differences have been found in the mode of action, possibly reflecting a different functional and metabolic profile of the two cell lines. Overall this work indicates that the neuroblastoma cell lines, like the line of astrocytic origin, are very sensitive to purine metabolism perturbation thus suggesting new therapeutic approaches to nervous system tumors. J. Cell. Biochem. 117: 1671-1679, 2016. © 2015 Wiley Periodicals, Inc. PMID:26659614

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

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

    2016-04-01

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

  6. Hereditary overexpression of adenosine deaminase in erythrocytes: Evidence for a cis-acting mutation

    Chen, E.H. (Univ. of Michigan, Ann Arbor, MI (United States)); Tartaglia, A.P. (Albany Medical College, Albany, MI (United States)); Mitchell, B.S. (Univ. of North Carolina, Chapel Hill, NC (United States))

    1993-10-01

    Overexpression of adenosine deaminase (ADA) in red blood cells is inherited as an autosomal dominant trait and causes hemolytic anemia. The increased ADA activity in erythrocytes is due to an increase in steady-state levels of ADA mRNA of normal sequence. Increased ADA mRNA may be due to a cis-acting mutation which results in increased transcription or a loss of down-regulation during erythroid differentiation. Alternatively, it is possible that the mutation is in a trans-acting factor which interacts with normal ADA transcriptional elements to cause overexpression in red blood cells. To discriminate between a cis-acting and a trans-acting mutation, the authors took advantage of a highly polymorphic TAAA repeat located at the tail end of an Alu repeat approximately 1.1 kb upstream of the ADA gene. Using PCR to amplify this region, the authors identified five different alleles in 19 members of the family. All 11 affected individuals had an ADA allele with 12 TAAA repeats, whereas none of the 8 normal individuals did. The authors conclude that this disorder results from a cis-acting mutation in the vicinity of the ADA gene. 24 refs., 3 figs.

  7. Camouflage Patterning in Maize Leaves Results from a Defect in Porphobilinogen Deaminase

    Mingshu Huang; Thomas L.Slewinski; R.Frank Baker; Diane Janick-Buckner; Brent Buckner; Gurmukh S.Johal; David M.Braun

    2009-01-01

    Maize leaves are produced from polarized cell divisions that result in clonal cell lineages arrayed along the long axis of the leaf.We utilized this stereotypical division pattern to identify a collection of mutants that form chloroplast pigmentation sectors that violate the clonal cell lineages.Here,we describe the camouflage1 (cf1) mutant,which develops nonclonal,yellow-green sectors in its leaves.We cloned the cf1 gene by transposon tagging and determined that it encodes porphobilinogen deaminase (PBGD),an enzyme that functions early in chlorophyll and heme biosynthesis.While PBGD has been characterized biochemically,no viable mutations in this gene have been reported in plants.To investigate the in vivo function of PBGD,we characterized the cf1 mutant.Histological analyses revealed that of 1 yellow sectors display the novel phenotype of bundle sheath cell-specific death.Light-shift experiments determined that constant light suppressed cf1 sector formation,a dark/light transition is required to induce yellow sectors,and that sectors form only during a limited time of leaf development.Biochemical experiments determined that cf1 mutant leaves have decreased PBGD activity and increased levels of the enzyme substrate in both green and yellow regions.Furthermore,the cf1 yellow regions displayed a reduction in catalase activity.A threshold model is hypothesized to explain the cf1 variegation and incorporates photosynthetic cell differentiation,reactive oxygen species scavenging,and PBGD function.

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

    Iván Darío BRAVO-TOBAR

    2015-10-01

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

  9. Outcome of hematopoietic stem cell transplantation for adenosine deaminase-deficient severe combined immunodeficiency.

    Hassan, Amel; Booth, Claire; Brightwell, Alex; Allwood, Zoe; Veys, Paul; Rao, Kanchan; Hönig, Manfred; Friedrich, Wilhelm; Gennery, Andrew; Slatter, Mary; Bredius, Robbert; Finocchi, Andrea; Cancrini, Caterina; Aiuti, Alessandro; Porta, Fulvio; Lanfranchi, Arnalda; Ridella, Michela; Steward, Colin; Filipovich, Alexandra; Marsh, Rebecca; Bordon, Victoria; Al-Muhsen, Saleh; Al-Mousa, Hamoud; Alsum, Zobaida; Al-Dhekri, Hasan; Al Ghonaium, Abdulaziz; Speckmann, Carsten; Fischer, Alain; Mahlaoui, Nizar; Nichols, Kim E; Grunebaum, Eyal; Al Zahrani, Daifulah; Roifman, Chaim M; Boelens, Jaap; Davies, E Graham; Cavazzana-Calvo, Marina; Notarangelo, Luigi; Gaspar, H Bobby

    2012-10-25

    Deficiency of the purine salvage enzyme adenosine deaminase leads to SCID (ADA-SCID). Hematopoietic cell transplantation (HCT) can lead to a permanent cure of SCID; however, little data are available on outcome of HCT for ADA-SCID in particular. In this multicenter retrospective study, we analyzed outcome of HCT in 106 patients with ADA-SCID who received a total of 119 transplants. HCT from matched sibling and family donors (MSDs, MFDs) had significantly better overall survival (86% and 81%) in comparison with HCT from matched unrelated (66%; P HCT, nonengraftment was a major problem. Long-term immune recovery showed that regardless of transplant type, overall T-cell numbers were similar, although a faster rate of T-cell recovery was observed after MSD/MFD HCT. Humoral immunity and donor B-cell engraftment was achieved in nearly all evaluable surviving patients and was seen even after unconditioned HCT. These data detail for the first time the outcomes of HCT for ADA-SCID and show that, if patients survive HCT, long-term cellular and humoral immune recovery is achieved. PMID:22791287

  10. A novel pathway for the synthesis of inositol phospholipids uses cytidine diphosphate (CDP)-inositol as donor of the polar head group.

    Jorge, Carla D; Borges, Nuno; Santos, Helena

    2015-07-01

    We describe a novel biosynthetic pathway for glycerophosphoinositides in Rhodothermus marinus in which inositol is activated by cytidine triphosphate (CTP); this is unlike all known pathways that involve activation of the lipid group instead. This work was motivated by the detection in the R. marinus genome of a gene with high similarity to CTP:L-myo-inositol-1-phosphate cytidylyltransferase, the enzyme that synthesizes cytidine diphosphate (CDP)-inositol, a metabolite only known in the synthesis of di-myo-inositol phosphate. However, this solute is absent in R. marinus. The fate of radiolabelled CDP-inositol was investigated in cell extracts to reveal that radioactive inositol was incorporated into the chloroform-soluble fraction. Mass spectrometry showed that the major lipid product has a molecular mass of 810 Da and contains inositol phosphate and alkyl chains attached to glycerol by ether bonds. The occurrence of ether-linked lipids is rare in bacteria and has not been described previously in R. marinus. The relevant synthase was identified by functional expression of the candidate gene in Escherichia coli. The enzyme catalyses the transfer of L-myo-inositol-1-phosphate from CDP-inositol to dialkylether glycerol yielding dialkylether glycerophosphoinositol. Database searching showed homologous proteins in two bacterial classes, Sphingobacteria and Alphaproteobacteria. This is the first report of the involvement of CDP-inositol in phospholipid synthesis. PMID:25472423

  11. Volumetric properties of the nucleosides adenosine, cytidine, and uridine in aqueous solution at T = 298.15 K and p = (10 to 120) MPa

    Highlights: ► Sound speeds were measured for aqueous solutions of nucleosides at high pressures. ► Volumetric properties were derived for T = 298.15 K and p = (10 to 120) MPa. ► The isothermal compression of adenosine is positive and increases as p increases. ► The isothermal compressions for cytidine and uridine change sign as p increases. -- Abstract: Sound speeds have been measured for aqueous solutions of the nucleosides adenosine, cytidine, and uridine at T = 298.15 K and at the pressures p = (10, 20, 40, 60, 80, 100, and 120) MPa. Using methods described in previous work, these sound speeds were used to derive the partial molar volumes at infinite dilution, V2o, the partial molar isentropic compressions at infinite dilution, KS,2o, and the partial molar isothermal compressions at infinite dilution, KT,2o{KT,2o=-(∂V2o/∂p)T}, for the nucleosides in aqueous solution at elevated pressures. The V2o and KT,2o results were rationalized in terms of the likely solute–water interactions. The V2o results were also critically compared with those calculated using the revised Helgeson–Kirkham–Flowers (HKF) equation of state

  12. Dosagem da atividade da adenosina deaminase no líquido pleural para o diagnóstico da tuberculose pleural Pleural fluid adenosine deaminase detection for the diagnosis of pleural tuberculosis

    Morrys Casagrande Kaisemann; Afrânio Lineu Kritski; Maria de Fátima C Pereira; Anete Trajman

    2004-01-01

    INTRODUÇÃO: O diagnóstico da tuberculose pleural permanece um desafio, pois a sensibilidade dos testes tradicionais é baixa. O exame histopatológico da pleura é o método mais preciso, com até 80% de sensibilidade. A dosagem da adenosina deaminase foi introduzida mais recentemente, mas sua utilidade no diagnóstico da tuberculose pleural no Brasil não foi suficientemente esclarecida. OBJETIVO: Verificar a sensibilidade e a especificidade de um método experimental de dosagem da atividade da aden...

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

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

    2015-12-01

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

  14. Oxidative Stress Biomarkers and Adenosine Deaminase over the Alopecic Area of the Patients with Alopecia Areata

    Öztürk, Perihan; Arıcan, Özer; Kurutaş, Ergül Belge; Mülayim, Kamil

    2016-01-01

    Background: Alopecia areata (AA) is an autoimmune, T-cell mediated, and chronic inflammatory disorder. The pathological mechanisms of disease are unclear, but oxidative stress may be involved. To our knowledge, no studies have examined the oxidative stress levels or biomarkers within the lesional area and skin surface in patients with AA. Similarly, adenosine deaminase (ADA) has not been characterized in AA. Aims: Therefore, we aimed to define ADA levels and the factors involved in oxidative stress from scalp-scrapes of patients with AA. Study Design: Case-control study. Method: A total of 60 patients (30 diagnosed AA patients and 30 healthy controls) were included in the study. ADA as well as oxidative stress factors, including malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) were analyzed from scalp-scrapes in both groups and quantified by spectrophotometry. Results: Activities of SOD (p=0.000), CAT (p=0.033), and ADA (p=0.004) as well as levels of GSH (p=0.000) and MDA (p=0.032) in patients with AA were higher than the controls statistically significant. Conclusion: Based on these results, factors associated with oxidative stress were elevated in AA patient scalp-scrapes compared to controls and may have a defined role the disease pathogenesis. Alterations in the activities of antioxidant enzymes from AA patient scraping samples may be a local effect of elevated oxidative stress levels. In this disease, oxidative stress may affect not only hair follicle but also any layers of the skin.

  15. Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase.

    Miller, Danielle V; Brown, Anne M; Xu, Huimin; Bevan, David R; White, Robert H

    2016-06-01

    Adenine deaminases (Ade) and hypoxanthine/guanine phosphoribosyltransferases (Hpt) are widely distributed enzymes involved in purine salvage. Characterization of the previously uncharacterized Ade (MJ1459 gene product) and Hpt (MJ1655 gene product) are discussed here and provide insight into purine salvage in Methanocaldococcus jannaschii. Ade was demonstrated to use either Fe(II) and/or Mn(II) as the catalytic metal. Hpt demonstrated no detectable activity with adenine, but was equally specific for hypoxanthine and guanine with a kcat /KM of 3.2 × 10(7) and 3.0 × 10(7) s(- 1) M(- 1) , respectively. These results demonstrate that hypoxanthine and IMP are the central metabolites in purine salvage in M. jannaschii for AMP and GMP production. A conserved cysteine (C127, M. jannaschii numbering) was examined due to its high conservation in bacterial and archaeal homologues. To assess the role of this highly conserved cysteine in M. jannaschii Ade, site-directed mutagenesis was performed. It was determined that mutation to serine (C127S) completely abolished Ade activity and mutation to alanine (C127A) exhibited 10-fold decrease in kcat over the wild type Ade. To further investigate the role of C127, detailed molecular docking and dynamics studies were performed and revealed adenine was unable to properly orient in the active site in the C127A and C127S Ade model structures due to distinct differences in active site conformation and rotation of D261. Together this work illuminates purine salvage in M. jannaschii and the critical role of a cysteine residue in maintaining active site conformation of Ade. Proteins 2016; 84:828-840. © 2016 Wiley Periodicals, Inc. PMID:26990095

  16. Cerebrospinal fluid adenosine deaminase activity: A complimentary tool in the early diagnosis of tuberculous meningitis

    Taori Girdhar M

    2006-03-01

    Full Text Available Abstract Background Tuberculous meningitis (TBM is the commonest form of neurotuberculosis caused by Mycobacterium tuberculosis bacilli (MTB. The diagnosis of TBM is often difficult. A reliable, cost-effective and rapid diagnostic test, which can be performed in any standard pathology laboratory, could be of help in the diagnosis of TBM. In the present study we measured the adenosine deaminase (ADA activity in cerebrospinal fluid (CSF of TBM and non-TBM patients. Method ADA activity in CSF was determined according to a method based on the Berthlot reaction, which is the formation of a colored indophenol complex from ammonia liberated from adenosine, and quantified spectrophotometrically. Results The CSF ADA activity from TBM patients was compared with CSF ADA from non-TBM infectious meningitis patients, and from patients with non-infectious neurological disorders. The mean CSF ADA activity was found to be significantly higher in CSF of TBM patients, 14.31 ± 3.87 (2.99–26.94, mean ± SD with range, than in the CSF from non-TBM infectious meningitis, 9.25 ± 2.14 (4.99–13.96 and from the non-infectious neurological disorders group, 2.71 ± 1.96 (0.00–7.68, P Conclusion This study demonstrated that ADA activity in the CSF of TBM patients, using a cut-off value 11.39 U/L/min, can be useful for the early differential diagnosis of TBM. This test can be performed in any pathology laboratory where more sophisticated methods are not available.

  17. Autoimmune dysregulation and purine metabolism in adenosine deaminase (ADA-deficiency

    Aisha Vanessa Sauer

    2012-08-01

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

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

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

  19. Evaluation of adenosine deaminase seric activity in the diagnosis of bovine tuberculosis

    Márcio Roberto Silva

    2006-06-01

    Full Text Available Determination of seric levels of adenosine deaminase (ADA, an enzyme produced by monocytes/macrophages and lymphocytes, has been used in the diagnosis of human tuberculosis (TB. In the present study, ADA seric activity was evaluated comparatively to the comparative tuberculin test in the diagnosis of bovine tuberculosis. Two hundred fifty-six cattle were classified by origin and by the comparative tuberculin test as TB-positive animals (n = 52, from herds where the Mycobacterium bovis had previously been isolated, and TB-negative animals (n = 204, TB-free herds. The mean ADA seric value from the TB-positive group (4.45 ± 2.33 U/L was significantly lower (p = 0.008 than that observed in sera from the TB-negative group (6.12 ± 4.47 U/L. When animals from a herd with clinical cases of enzootic bovine leukosis of TB-negative group were withdrawn from analysis, the mean ADA seric values of TB-negative group (5.12 ± 3.75 U/L was not significantly different anymore from that of the TB-positive group (p = 0.28. There was no agreement in the diagnosis of bovine TB between comparative tuberculin test and determination of ADA seric values, using two different cutoff points, being 6.12 U/L and 15.0 U/L, (kappa = -0.086 and kappa = -0.082, respectively. In conclusion, the determination of ADA seric activity was not a good auxiliary test for bovine TB, because it was not able to distinguish between TB-positive and TB-negative animals.

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

    Adenosine deaminase deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, the authors synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNA showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These change do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency

  1. Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

    Monica K Akre

    Full Text Available Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

  2. Classical Mus musculus Igκ Enhancers Support Transcription but not High Level Somatic Hypermutation from a V-Lambda Promoter in Chicken DT40 Cells

    Kothapalli, Naga Rama; Norton, Darrell D.; Fugmann, Sebastian D.

    2011-01-01

    Somatic hypermutation (SHM) of immunoglobulin genes is initiated by activation-induced cytidine deaminase (AID) in activated B cells. This process is strictly dependent on transcription. Hence, cis-acting transcriptional control elements have been proposed to target SHM to immunoglobulin loci. The Mus musculus Igκ locus is regulated by the intronic enhancer (iE/MAR) and the 3′ enhancer (3′E), and multiple studies using transgenic and knock-out approaches in mice and cell lines have reported s...

  3. APE1 is dispensable for S-region cleavage but required for its repair in class switch recombination

    Xu, Jianliang; Husain, Afzal; Hu, Wenjun; Honjo, Tasuku; Kobayashi, Maki

    2014-01-01

    Apurinic/apyrimidinic endonuclease 1 (APE1) has been shown to be a critical endonuclease required for class switch recombination (CSR). Here we show that APE1’s endonuclease activity, but not its redox regulation or transcriptional regulation activity, is important for CSR. Conversely, APE1 is dispensable for activation-induced cytidine deaminase (AID)-induced somatic hypermutation (SHM) as well as IgH/c-myc translocation. Moreover, during CSR, APE1 is not required for AID-induced S-region br...

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

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

  5. Southern blight disease of tomato control by 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing Paenibacillus lentimorbus B-30488.

    Dixit, Ritu; Agrawal, Lalit; Gupta, Swati; Kumar, Manoj; Yadav, Sumit; Chauhan, Puneet Singh; Nautiyal, Chandra Shekhar

    2016-02-01

    Tomato cultivation is highly susceptible for soil born diseases and among them southern blight disease caused by Scelerotium rolfsii is very common. For its management use of chemical fungicides is not very successful as their spores are able to survive for many years in the soil. As an alternative eco-friendly approach to control the disease antagonistic microbes are being characterized.Among them plant growth promoting rhizobacteria Paenibacillus lentimorbus B-30488 (B-30488) with antagonistic properties, multiple PGP attributes stress tolerance and ACC deaminase enzyme activity is characterized to decipher its mode of action against S. rolfsii under in vitro and in vivo conditions. In vitro results obtained from this study clearly demonstrate that B-30488 has ability to show antagonistic properties under different abiotic stresses against S. rolfsii. Similar results were also obtained from in vivo experiments where B-30488 inoculation has efficiently controlled the disease caused by S. rolfsii and improve the plant growth. Deleterious enhanced ethylene level in S. rolfsii infected plants was also ameliorated by inoculation of ACC deaminase producing B-30488. The ACC accumulation, ACO and ACS activities were also modulated in S. rolfsii infected plants. Results from defense enzymes and other biochemical attributes were also support the role of B-30488 inoculation in ameliorating the biotic stress caused by S. rolfsii in tomato plants. These results were further validated by pathogen related gene expression analysis by real time PCR. Overall results from the present study may be concluded that ACC deaminase producing B-30488 has ability to control the southern blight disease caused by S. rolfsii and commercial bioinoculant package may be developed. PMID:26825539

  6. RNA binding to APOBEC3G induces the disassembly of functional deaminase complexes by displacing single-stranded DNA substrates

    Polevoda, Bogdan; McDougall, William M.; Tun, Bradley N.; Cheung, Michael; Salter, Jason D.; Friedman, Alan E.; Smith, Harold C.

    2015-01-01

    APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G d...

  7. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent ...

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

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

    2015-07-10

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

  9. PCR detection of a C/T polymorphism in exon 1 of the porphobilinogen deaminase gene (PBGD)

    Picat, C.; Bourgeois, F.; Grandchamp, B. (Faculte de Medicine, Paris (France))

    1991-09-25

    Sequencing of exon 1 revealed a C/T polymorphism in exon 1 of human porphobilinogen deaminase gene (PBGD) at position {minus}64 relatively to the initiation translational codon. Genetic defects of PBGD are responsible for acute intermittent porphyria. The use of a 5{prime} primer with a mutated sequence to amplify the region containing this polymorphism allows its restriction analysis. After a ApaI digest of the amplified fragment, two alleles can be identified: F1: 164 bp, F2: 145 bp + 19 bp. Codominant inheritance was demonstrated in two large families with AIP.

  10. A robust quantitative solid phase immunoassay for the acute phase protein C-reactive protein (CRP) based on cytidine 5 '-diphosphocholine coupled dendrimers

    Heegaard, Peter M. H.; Pedersen, H. G.; Jensen, A. L.; Boas, Ulrik

    2009-01-01

    C-reactive protein (CRP) is an important acute phase protein, being used as a sensitive indicator of inflammation and infection and is also associated with the risk of cardiovascular problems. The present paper describes a robust and sensitive ELISA for CRP, based on the affinity of CRP for...... phosphocholine. In this design synthetic globular polymers (dendrimers) are used as scaffolds for the multivalent display of phosphocholine molecules. CRP present in a sample binds to the phosphocholine moiety presented at high density in the coating layer and is detectable by specific antibodies. The ELISA was...... applied to determination of pig and human CRP using commercially available antibodies against human CRP. The assay was shown to be more sensitive than previously published immunoassays employing albumin-coupled cytidine diphosphocholine. The coating was stable for at least 30 days at room temperature and...

  11. Kinetic Analysis of Oligo(C) Formation from the 5‧-Monophosphorimidazolide of Cytidine with Pb(II) Ion Catalyst at 10 75°C

    Kawamura, Kunio; Maeda, Jun

    2007-04-01

    The temperature dependence of the rate constants for the formation of oligocytidylate (oligo(C)) from the 5‧-monophosphorimidazolide of cytidine (ImpC) in the presence of Pb(II) ion catalyst has been investigated at 10 75°C. The rate constants for the formation of oligo(C) increased in the order of the formation of 2-mer 51.8 kJ mol-1 for 4-mer, and 66.8 ± 4.5 kJ mol-1 for the hydrolysis of ImpC. The significance of the temperature dependence of the formation rate constants of the model prebiotic formation of RNA is discussed.

  12. Mannosylated liposomal cytidine 5' diphosphocholine prevent age related global moderate cerebral ischemia reperfusion induced mitochondrial cytochrome c release in aged rat brain.

    Ghosh, S; Das, N; Mandal, A K; Dungdung, S R; Sarkar, S

    2010-12-29

    Mitochondrial dysfunctions generating from cerebral ischemia-reperfusion exert a potential threat on neuronal cell survival and hence, accelerate the aging process and age dependent neuropathology. Thirty min moderate cerebral ischemia induced by bilateral common carotid artery occlusion (BCCAO) followed by 30 min reperfusion caused an increased diene production, depleted glutathione (GSH) content, reduced superoxide dismutase (SOD) and catalase activities and pyramidal neuronal loss in young (2 months old) and aged (20 months old) rat brain compared to sham operated controls. Cytidine 5' diphosphocholine (CDP-Choline) is a known neuroprotective drug. CDP-Choline after metabolism in the liver suffers hydrolysis and splits into cytidine and choline before entering systemic circulation and hardly circumvents blood brain barrier (BBB) as such. Previous reports show CDP-Choline liposomes significantly increased in vivo uptake compared to "free drug" administration in cerebral ischemia. To enhance the therapeutic concentration build up in brain we sought to formulate mannosylated liposomal CDP-Choline (MLCDP) utilizing the mannose receptors. We tested the therapeutic supremacy of MLCDP over liposomal CDP-Choline (LCDP) in global moderate cerebral ischemia reperfusion induced neuronal damage. CDP-Choline in MLCDP delivery system was found potent to exert substantial protection against global moderate cerebral ischemia reperfusion induced mitochondrial damage in aged rat brain. Membrane lipid peroxidation, GSSG/GSH ratio and reactive oxygen species (ROS) generation in cerebral tissue were found to be higher in aged, compared to young rat. Further decline of those parameters was observed in aged rat brain by the induction of global moderate cerebral ischemia and reperfusion. MLCDP treatment when compared to free or LCDP treatment prevented global moderate cerebral ischemia-reperfusion induced mitochondrial damage as evident ultra structurally and release of cytochrome c

  13. Crystallization and preliminary X-ray characterization of the tetrapyrrole-biosynthetic enzyme porphobilinogen deaminase from Arabidopsis thaliana

    The enzyme porphobilinogen deaminase (PBGD) catalyses a key early step in the biosynthesis of haem in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. PBGD from the model plant organism A. thaliana has been expressed and the enzyme was crystallized in a form that diffracted synchrotron radiation to high resolution. The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step of the haem-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Since PBGD catalyses a reaction which is common to the biosynthesis of both haem and chlorophyll, structural studies of a plant PBGD enzyme offer great potential for the discovery of novel herbicides. Until recently, structural data have only been available for the Escherichia coli and human forms of the enzyme. Expression in E. coli of a codon-optimized gene for Arabidopsis thaliana PBGD has permitted for the first time the crystallization and preliminary X-ray analysis of the enzyme from a plant species at high resolution

  14. Selection of efficient salt-tolerant bacteria containing ACC deaminase for promotion of tomato growth under salinity stress

    Kannika Chookietwattana* and Kedsukon Maneewan

    2012-05-01

    Full Text Available For successful application of plant growth promoting bacteria (PGPB in salt-affected soil, bioinoculant with salt-tolerant property is required in order to provide better survival and perform well in the field. The present study aimed to select the most efficient salt-tolerant bacterium containing 1-aminocyclopropane-1-carboxylic acid (ACC deaminase from eighty four bacterial strains and to investigate the effects of the selected bacterium on the germination and growth of tomato (Licopersicon esculentum Mill. cv. Seeda under saline conditions. The Bacillus licheniformis B2r was selected for its ability to utilize ACC as a sole nitrogen source under salinity stress. It also showed a high ACC deaminase activity at 0.6 M NaCl salinity. Tomato plants inoculated with the selected bacterium under various saline conditions (0, 30, 60, 90 and 120 mM NaCl revealed a significant increase in the germination percentage, germination index, root length, and seedling dry weight especially at salinity levels ranging from 30-90 mM NaCl. The work described in this report is an important step in developing an efficient salt-tolerant bioinoculant to facilitate plant growth in saline soil.

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

    Ku, Min-Je [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Won-Ho [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biotechnology and Genetic Engineering, Korea University, Seoul 136-701 (Korea, Republic of); Nam, Ki-hyun; Rhee, Kyeong-hee [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Ki-Seog [Biotechnology and Genetic Engineering, Korea University, Seoul 136-701 (Korea, Republic of); Kim, Eunice EunKyung [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Yu, Myung-Hee [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Hwang, Kwang Yeon, E-mail: hwangky@kist.re.kr [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of)

    2005-04-01

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

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

    Storey Kenneth B

    2008-04-01

    Full Text Available Abstract Background The wood frog, Rana sylvatica, is one of a few vertebrate species that have developed natural freeze tolerance, surviving days or weeks with 65–70% of its total body water frozen in extracellular ice masses. Frozen frogs exhibit no vital signs and their organs must endure multiple stresses, particularly long term anoxia and ischemia. Maintenance of cellular energy supply is critical to viability in the frozen state and in skeletal muscle, AMP deaminase (AMPD plays a key role in stabilizing cellular energetics. The present study investigated AMPD control in wood frog muscle. Results Wood frog AMPD was subject to multiple regulatory controls: binding to subcellular structures, protein phosphorylation, and effects of allosteric effectors, cryoprotectants and temperature. The percentage of bound AMPD activity increased from 20 to 35% with the transition to the frozen state. Bound AMPD showed altered kinetic parameters compared with the free enzyme (S0.5 AMP was reduced, Hill coefficient fell to ~1.0 and the transition to the frozen state led to a 3-fold increase in S0.5 AMP of the bound enzyme. AMPD was a target of protein phosphorylation. Bound AMPD from control frogs proved to be a low phosphate form with a low S0.5 AMP and was phosphorylated in incubations that stimulated PKA, PKC, CaMK, or AMPK. Bound AMPD from frozen frogs was a high phosphate form with a high S0.5 AMP that was reduced under incubation conditions that stimulated protein phosphatases. Frog muscle AMPD was activated by Mg·ATP and Mg·ADP and inhibited by Mg·GTP, KCl, NaCl and NH4Cl. The enzyme product, IMP, uniquely inhibited only the bound (phosphorylated enzyme from muscle of frozen frogs. Activators and inhibitors differentially affected the free versus bound enzyme. S0.5 AMP of bound AMPD was also differentially affected by high versus low assay temperature (25 vs 5°C and by the presence/absence of the natural cryoprotectant (250 mM glucose that

  17. Structures of dCTP deaminase from Escherichia coli with bound substrate and product: reaction mechanism and determinants of mono- and bifunctionality for a family of enzymes

    Johansson, Eva; Fanø, Mathias; Bynck, Julie H;

    2005-01-01

    complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely...

  18. Targeted cytosine deaminase-uracil phosphoribosyl transferase suicide gene therapy induces small cell lung cancer-specific cytotoxicity and tumor growth delay

    Christensen, Camilla L; Gjetting, Torben; Poulsen, Thomas Tuxen;

    2010-01-01

    deaminase (YCD) gene alone or fused with the yeast uracil phosphoribosyl transferase (YUPRT) gene followed by administration of 5-fluorocytosine (5-FC) prodrug. Experimental design: The YCD gene or the YCD-YUPRT gene was placed under regulation of the SCLC-specific promoter insulinoma-associated 1 (INSM1...

  19. Prospective Use of 1-Aminocyclopropane-1-Carboxylate Deaminase-Producing Bacteria for Plant Growth Promotion and Defense against Biotic and Abiotic Stresses in Peat-Soil-Agriculture

    EDI HUSEN

    2008-12-01

    Full Text Available The 1-aminocyclopropane-1-carboxylate (ACC deaminase (EC4.1.99.4 is an enzyme produced by some soil bacteria to degrade ACC (the immediate precursor of ethylene to reduce ethylene biosynthesis in higher plants. Increased concentrations of ethylene in plant tissues, which are triggered by various biotic and abiotic stresses, inhibits plant growth and weakens the plant defense against the stressors. Various findings on the successful use of ACC deaminase producing bacteria for plant growth under unfavorable soil conditions are inspiring their use in tropical peat-soil-agriculture, which possesses bio-physical constraints. It has been proven that inoculation of plants with ACC deaminase producing bacteria decreased ethylene inhibition generated by unfavorable environmental conditions, such as nutrient shortage, flooding, drought, high salts, and the presence of heavy metals and organic pollutants. Understanding the mechanisms by which ACC deaminase-producing bacteria act to reduce plant stress and the fitness of bacterial traits with the properties and constraints of peat-soils becomes a key to utilize these bacteria in improving crop productivity. The bacteria may ameliorate plant stress as well as promote plant growth under seasonal bio-physical changes of peat-soils that are usually encountered in the field.

  20. Sensitization of prostate cancer cell lines to 5-fluorocytosine induced by a replication incompetent adenoviral vector carrying a cytosine deaminase transcription unit

    2001-01-01

    AIM: To investigate the efficiency of cytosine deaminase adenoviral/5-fluorocytosine system on prostate cancer cell lines. METHODS: Cell culture, infectivity test and sensitivity test, observing the bystander effect and animal model experiment were carried out. RESULTS: All the established prostate cancer cell lines were eventually infectable, but ratio of vector/cell and time of exposed at which infection occurs was dependent on the cell lines. The expression of transfered cytosine deaminase gene peaked at different days, but persisted beyond 11 days. The prostate cell lines were sensitized to the 5-fluorocytosine by infection with the cytosine deaminase gene adenoviral vector, and only 5% of the LNCap and 10% of the RM-1 cells infected were required for 100% cell death. In the animal model, there was significant eradiation of tumor growth at the ratio of 400 vector particles/cell and with the systematic treatment of 5-fluorocytosine. CONCLUSION: The adenoviral vector carrying a cytosine deaminase transcription unit can sensitize the prostate cancer cell lines to 5-fluorocytosine, and the system can significantly inhibit the growth of prostatic tumor in mice.

  1. Killing effect of coexpressing cytosine deaminase and thymidine kinase on rat vascular smooth muscle cells

    曹慧青; 孟宪敏; 刘冬青; 赵秀文; 丁金凤

    2004-01-01

    Background Vascular smooth muscle cell (VSMC) proliferation following arterial injury plays a critical role in a variety of vascular proliferative disorders, such as atherosclerosis and restenosis after balloon angioplasty. Herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and E.coli cytosine deaminase (CD)/5-fluorocytosine (5-Fc) suicide gene systems have been successfully employed in cardiovascular gene therapy, respectively. We reasoned that coexpression of both HSV-TK with CD suicide genes would lead to increased cell killing. To test this imagine, the adenoviral vectors expressing TK and/or CD genes were developed and tested on vascular smooth muscle cells. Methods Adenoviral vectors, including Ad-EF1α-CD-cytomegolovirus (CMV)-TK coexpressing both CD and TK double suicide genes, Ad-EF1α-CD and Ad-CMV-TK expressing CD and TK respectively, and control vector Ad-CMV-LacZ, were constructed and prepared with homologous recombination in RecA+E.coli cells. Integration and expression of CD and/or TK gene were identified by PCR and Western blot. Primary cultured VSMCs were infected at a multiplicity of infection (MOI) of 20 with exposure to their matching prodrugs 5-Fc and GCV. Cell mortality was measured by methyl thiazolyl tetrazolium (MTT) assays. Flow cytometry analysis was used to detect cell death. Apoptotic cells were analyzed using Hoechst 33342 fluorescence dye as a DNA probe. Genomic DNA cleavage of apoptotic VSMCs was tested by agarose gel electrophoresis. Results Recombinant adenovirus expressing CD and/or TK suicide genes were successfully constructed. Both single and double suicide genes could be integrated into adenoviral genome and expressed. Cytotoxic effects of Ad-EF1α-CD-CMV-TK double suicide genes combined with 5-Fc and GCV were higher than those of Ad-CMV-TK and Ad-EF1α-CD single gene groups. The rate of cell survival was only (9±3)% in the Ad-EF1α-CD-CMV-TK group, but (37±3)% in the Ad-CMV-TK and (46±4)% in the Ad-EF1

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

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

  3. Increased level of soluble adenosine deaminase in bone marrow of visceral leishmaniasis patients: an inverse relation with parasite load.

    Rai, Ambak K; Kumar, Prabin; Saini, Sheetal; Thakur, Chandreshwar P; Seth, Tulika; Mitra, Dipendra K

    2016-09-01

    Adenosine deaminase (ADA) which degrades adenosine to inosine, is known to be pro-inflammatory molecule in many diseases. Adenosine suppresses the functioning of the immune system and thus promotes dissemination of the parasite. In our previous finding, the level of soluble ADA in serum of visceral leishmaniasis (VL) was found to be increased as compared to healthy controls. However, it cannot be fairly interpreted unless their level is demonstrated at the disease site, where the parasite resides. We designed this study to correlate the level of soluble ADA (sADA) with parasitic load at the disease site i.e. bone marrow (BM). We found increased levels of sADA in BM as compared to the unaffected BM. Furthermore, a significant inverse correlation is observed between the parasite load and level of sADA at the disease site. PMID:27447233

  4. The genetics of feto-placental development: A study of acid phosphatase locus 1 and adenosine deaminase polymorphisms in a consecutive series of newborn infants

    Bergamaschi Antonio

    2008-09-01

    Full Text Available Abstract Background Acid phosphatase locus 1 and adenosine deaminase locus 1 polymorphisms show cooperative effects on glucose metabolism and immunological functions. The recent observation of cooperation between the two systems on susceptibility to repeated spontaneous miscarriage prompted us to search for possible interactional effects between these genes and the correlation between birth weight and placental weight. Deviation from a balanced development of the feto-placental unit has been found to be associated with perinatal morbidity and mortality and with cardiovascular diseases in adulthood. Methods We examined 400 consecutive newborns from the Caucasian population of Rome. Birth weight, placental weight, and gestational length were registered. Acid phosphatase locus 1 and adenosine deaminase locus 1 phenotypes were determined by starch gel electrophoresis and correlation analysis was performed by SPSS programs. Informed verbal consent to participate in the study was obtained from the mothers. Results Highly significant differences in birth weight-placental weight correlations were observed among acid phosphatase locus 1 phenotypes (p = 0.005. The correlation between birth weight and placental weight was markedly elevated in subjects carrying acid phosphatase locus 1 phenotypes with medium-low F isoform concentration (A, CA and CB phenotypes compared to those carrying acid phosphatase locus 1 phenotypes with medium-high F isoform concentration (BA and B phenotypes (p = 0.002. Environmental and developmental variables were found to exert a significant effect on birth weight-placental weight correlation in subjects with medium-high F isoform concentrations, but only a marginal effect was observed in those with medium-low F isoform concentrations. The correlation between birth weight and placental weight is higher among carriers of the adenosine deaminase locus 1 allele*2, which is associated with low activity, than in homozygous adenosine

  5. The rhizosphere bacterium Variovorax paradoxus 5C-2 containing ACC deaminase does not increase systemic ABA signaling in maize (Zea mays L.)

    Dodd, Ian C.; Jiang, Fan; Teijeiro, Rosalia Garcia; Belimov, Andrey A.; Hartung, Wolfram

    2009-01-01

    Soil inoculation with the ACC deaminase-containing rhizobacterium Variovorax paradoxus 5C-2 increased pea (Pisum sativum) growth and yield in both well watered and drying soil, with an attenuation of systemic ACC signaling likely key in the latter case.1 However, inoculated plants also had increased xylem ABA concentrations (which may also promote growth) in drying soil. Possible mediation of ABA levels by V. paradoxus 5C-2 was investigated in two experiments in which maize (Zea mays) growth ...

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

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

    2015-01-01

    Abstract Aim: To investigate the enhancing effect of radionuclide therapy by the therapeutic gene placed under the control of radio-responsive promoter. Methods: The recombinant lentivirus E8-codA-GFP, including a synthetic radiation-sensitive promoter E8, cytosine deaminase (CD) gene, and green fluorescent protein gene, was constructed. The gene expression activated by 125I radiation was assessed by observation of green fluorescence. The ability of converting 5-fluorocytosine (5-FC) to 5-flu...

  7. Conversion of a stem cell leukemia from a T-lymphoid to a myeloid phenotype induced by the adenosine deaminase inhibitor 2'-deoxycoformycin.

    Hershfield, M S; Kurtzberg, J; Harden, E; Moore, J O; Whang-Peng, J; Haynes, B. F.

    1984-01-01

    Selective failure of lymphoid development occurs in genetic deficiency of adenosine deaminase (ADA). We examined the in vivo effects of a potent inhibitor of ADA, 2'-deoxycoformycin, which was used to treat a patient with refractory acute leukemia. Unexpectedly, within 7 days of starting treatment, the leukemic phenotype underwent complete conversion from T lymphoblastic to promyelocytic, with kinetics that suggested a precursor-product relationship between the two cell populations. Pretreatm...

  8. A CORRELATIVE STUDY OF ADENOSINE DEAMINASE ACTIVITY & T.B. IgG IN SERUM IN CASES OF TUBERCULOSIS

    Ajay

    2012-11-01

    Full Text Available ABSTRACT: INTRODUCTION: Tuberculosis is major cause of morbidity and mort ality in India as well in other parts of world. It is caused by myc obacterium tuberculosis which primarily affects lung and cause pulmonary tuberculosis. Diag nosis of tuberculosis rests upon a positive history of contact, clinical symptoms, x-ray chest, sputum positivity and AFB culture. Adenosine deaminase (ADA is an enzyme which catalyzes the de amination of adenosine into inosine and ammonia. ADA level is found to be elevated in tuber culosis and typhoid fever where cell mediated immunity is elevated. The ADA level is sig nificantly elevated in tuberculosis and helps to differentiate between tubercular and non tubercu lar diseases. The ADA level is also found to be elevated in serum and pleural fluid in patients of tubercular pleural effusion, tubercular ascitis and tubercular pericardial effusion. METHODS : Routine hemogram, Montoux test, X-ray chest, FNAC of lymph nodes, biopsy of lymph node whene ver required, estimation of serum ADA level and T.B.IgG studies were performed in each cas e. RESULTS: In the present study a total of 45 cases were selected for the study. There are 30 cases of pulmonary tuberculosis and 15 controls. The values of serum ADA and tubercular Ig G in pulmonary tubercular group are significantly higher as compared to those of control s. None of the control for ADA showed significant ratio of positivity (≥1.7. One of the 1 5 cases showed remarkable ratio of positivity (>1.2-1.6 and 14 (93.3% cases showed insignifican t ratio of positivity. Only 2 (13.33% of the 15 cases showed positivity for TB IgG and rest 13 (8 6.66% were regarded negative. CONCLUSIONS: Thus it can be concluded that determination of ser um adenosine deaminase levels can effectively diagnose tuberculosis with s ensitivity of 96.66% and specificity of 93.33% as compared to TBIgG showing sensitivity of 90% and specificity of 86.6%. Also cost of ADA estimation is remarkably

  9. The use of pyr-mutations to modify pyrimidine pools in Lactococci

    Hansen, Steen Lyders Lerche; Martinussen, Jan; Hammer, Karin

    1999-01-01

    The specific engineering of organisms used for startercultures has become an effective way of improving and developing new products in the dairy industry. In order to obtain strains with specific characteristics, it is imperative to have a good understanding of the central biochemical pathways...... pyrimidines are only available through salvage of uracil and/or cytidine, the pools of CTP and UTP can be varied independently by controlling the activities of cytidine-deaminase (cdd) and CTP-synthetase (pyrG). Cytidine-deaminase catalyses the deamination of cytidine leading to uridine and CTP...

  10. The use of Pyr-mutants to modify pyrimidine metabolism in lactococci

    Hansen, Steen Lyders Lerche; Martinussen, Jan; Hammer, Karin

    1998-01-01

    The specific engineering of organisms used for startercultures has become an effective way of improving and developing new products in the dairy industry. In order to obtain strains with specific characteristics, it is imperative to have a good understanding of the central biochemical pathways...... pyrimidines are only available through salvage of uracil and/or cytidine, the pools of CTP and UTP can be varied independently by controlling the activities of cytidine-deaminase (cdd) and CTP-synthetase (pyrG). Cytidine-deaminase catalyses the deamination of cytidine leading to uridine and CTP...

  11. Isolation, characterization and colonization of 1-aminocyclopropane-1-carboxylate deaminase-producing bacteria XG32 and DP24.

    Wang, Mei-Xia; Liu, Jia; Chen, Shuang-Lin; Yan, Shu-Zhen

    2012-03-01

    Two 1-aminocyclopropane-1-carboxylate deaminase-producing bacterial strains (DP24 and XG32) were isolated from surface-sterilized tomato roots and rizhospere soil. The strains were identified as Pseudomonas fluorescens biovar. IV (XG2) and Erwinia herbicola (DP24) by physiological and biochemical tests, and 16S rRNA gene analysis. Both strains showed positive plant growth-promoting activity when inoculated into cucumber (Cucumis sativus), tomato (Lycopersicon esculentum), pepper (Capsicum annuum) and rapeseed (Brassica napus L.). Colonization ability and behavior of these two strains were determined by treating mutant strains with rifampicin and fluorescence in situ hybridization (FISH) assay with rRNA targeted probes, respectively. Both strains were endophytic colonizers of pepper plants. The behavior of the two strains was not identical. Strain XG32 only colonized the root and reached the max level of 27.7 × 10(7) c.f.u./g (fresh weight), after 12 days postinoculation, while strain DP24 was able to colonize the roots, stems and leaves. The max level was reached at 40.87 × 10(7) c.f.u./g (fresh weight) in the roots, 17 × 10(7) c.f.u./g in the stems after 7 days postinoculation and 44.84 × 10(7) c.f.u./g in the leaves after 12 days postinoculation. PMID:22805836

  12. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1. PMID:19651874

  13. Long-term expression of human adenosine deaminase in mice transplanted with retrovirus-infected hematopoietic stem cells

    Long-term stable expression of foreign genetic sequences transferred into hematopoietic stem cells by using retroviral vectors constitutes a relevant model for somatic gene therapy. Such stability of expression may depend on vector design, including the presence or absence of specific sequences within the vector, in combination with the nature and efficiency of infection of the hematopoietic target cells. The authors have previously reported successful transfer of human DNA encoding adenosine deaminase (ADA) into CFU-S (colony-forming unit-spleen) stem cells using simplified recombinant retroviral vectors. Human ADA was expressed in CFU-S-derived spleen colonies at levels near to endogenous enzyme. However, because of the lack of an efficient dominant selectable marker and low recombinant viral titers, stability of long-term expression of human ADA was not examined. They report here the development of an efficient method of infection of hematopoietic stem cells (HSC) without reliance on in vitro selection. Peripheral blood samples of 100% of mice transplanted with HSC infected by this protocol exhibit expression of human ADA 30 days after transplantation. Some mice (6 of 13) continue to express human ADA in all lineages after complete hematopoietic reconstitution (4 months). The use of recombinant retroviral vectors that efficiently transfer human ADA cDNA into HSC leading to stable expression of functional ADA in reconstituted mice, provides an experimental framework for future development of approaches to somatic gene therapy

  14. Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening.

    Qi, Yanfei; Li, Youxin; Bao, James J

    2016-08-01

    A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237-0.833 U/ml and 9.013-10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity. PMID:27173606

  15. Dietary Supplementation of Ginger and Turmeric Rhizomes Modulates Platelets Ectonucleotidase and Adenosine Deaminase Activities in Normotensive and Hypertensive Rats.

    Akinyemi, Ayodele Jacob; Thomé, Gustavo Roberto; Morsch, Vera Maria; Bottari, Nathieli B; Baldissarelli, Jucimara; de Oliveira, Lizielle Souza; Goularte, Jeferson Ferraz; Belló-Klein, Adriane; Oboh, Ganiyu; Schetinger, Maria Rosa Chitolina

    2016-07-01

    Hypertension is associated with platelet alterations that could contribute to the development of cardiovascular complications. Several studies have reported antiplatelet aggregation properties of ginger (Zingiber officinale) and turmeric (Curcuma longa) with limited scientific basis. Hence, this study assessed the effect of dietary supplementation of these rhizomes on platelet ectonucleotidase and adenosine deaminase (ADA) activities in Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) induced hypertensive rats. Animals were divided into seven groups (n = 10): normotensive control rats; induced (l-NAME hypertensive) rats; hypertensive rats treated with atenolol (10 mg/kg/day); normotensive and hypertensive rats treated with 4% supplementation of turmeric or ginger, respectively. After 14 days of pre-treatment, the animals were induced with hypertension by oral administration of l-NAME (40 mg/kg/day). The results revealed a significant (p < 0.05) increase in platelet ADA activity and ATP hydrolysis with a concomitant decrease in ADP and AMP hydrolysis of l-NAME hypertensive rats when compared with the control. However, dietary supplementation with turmeric or ginger efficiently prevented these alterations by modulating the hydrolysis of ATP, ADP and AMP with a concomitant decrease in ADA activity. Thus, these activities could suggest some possible mechanism of the rhizomes against hypertension-derived complications associated to platelet hyperactivity. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27151061

  16. STUDY OF ADENOSINE DEAMINASE AND LYMPHOCYTE/NEUTROPHIL RATIO IN COMBINATION AS DIAGNOSTIC TOOL FOR TUBERCULAR PLEURAL EFFUSION

    Md. Faizur

    2015-10-01

    Full Text Available Adenosine deaminase, considered one of the key enzyme of purine metabolism, has been used in work up of lymphocytic pleural effusion. Low level of ADA 50IU/L specially when combined with Lymphocytic/neutrophil ratio >0.75 in pleural fluid is useful test in the diagnosis of tubercular pleurisy. AIM AND OBJECTIVE: To suggest a better diagnostic tool in the diagnosis of pleural effusion of tubercular origin by estimating the activity of ADA along with L/N ratio in pleural effusion. METHOD: Biochemical, cytological and microbiology studies were done by obtaining pleural fluid by thoracocentesis in 100 patients after excluding pleural effusion cases of malignancy, transudative effusion. RESULT: 84 cases were tubercular and had high level of ADA in comparison to rest of 16 non-tubercular cases. At level of 50 IU/L of ADA activity test had sensitivity of 97.6%, specificity 87.5%, positive predictive value 97.6%, negative predictive value 87.5%which increased to 100% and 92.8%, 98.6%, and 100% respectively in combination with test of Lymphocytic/Neutrophilic ratio >0.75. CONCLUSION: ADA level with L/N ratio can be important investigation in diagnosis of tubercular pleural effusion cases.

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

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

    2014-02-15

    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

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

    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

  19. Combined QM(DFT)/MM molecular dynamics simulations of the deamination of cytosine by yeast cytosine deaminase (yCD).

    Zhang, Xin; Zhao, Yuan; Yan, Honggao; Cao, Zexing; Mo, Yirong

    2016-05-15

    Extensive combined quantum mechanical (B3LYP/6-31G*) and molecular mechanical (QM/MM) molecular dynamics simulations have been performed to elucidate the hydrolytic deamination mechanism of cytosine to uracil catalyzed by the yeast cytosine deaminase (yCD). Though cytosine has no direct binding to the zinc center, it reacts with the water molecule coordinated to zinc, and the adjacent conserved Glu64 serves as a general acid/base to shuttle protons from water to cytosine. The overall reaction consists of several proton-transfer processes and nucleophilic attacks. A tetrahedral intermediate adduct of cytosine and water binding to zinc is identified and similar to the crystal structure of yCD with the inhibitor 2-pyrimidinone. The rate-determining step with the barrier of 18.0 kcal/mol in the whole catalytic cycle occurs in the process of uracil departure where the proton transfer from water to Glu64 and nucleophilic attack of the resulting hydroxide anion to C2 of the uracil ring occurs synchronously. © 2016 Wiley Periodicals, Inc. PMID:26813441

  20. Aqueous extract from taxus baccata inhibits adenosine deaminase activity significantly in cancerous and noncancerous human gastric and colon tissues

    Zahide Esra Durak

    2014-01-01

    Full Text Available Objective: To investigate possible effects of aqueous taxus baccata extract on adenosine deaminase (ADA activity in cancerous and noncancerous human tissues and to clarify mechanism(s of its anticancer potential. Materials and Methods: Cancerous and noncancerous human gastric and colon tissues were used in the study. The extracts were prepared in distilled water. Before and after treatment with the extracts, ADA activities in the tissue homogenates were measured. Results: ADA activity was found to be higher in gastric tissue compared with colon tissue, but no differences were found between ADA activities of cancerous and noncancerous tissues for both as well. In the plant extract studies, it was found that taxus extract significantly inhibited ADA activity both in cancerous and noncancerous gastric and colon tissues. Conclusion: Our results suggest that aqueous extract from taxus baccata inhibits ADA activities in both gastric and colon tissues significantly. It is suggested that in addition to other proposed mechanisms, accumulated adenosine due to the inhibition of ADA enzyme might also play part in the anticancer properties of taxus species.

  1. Antitumor effects and radiosensitization of cytosine deaminase and thymidine kinase fusion suicide gene on colorectal carcinoma cells

    De-Hua Wu; Li Liu; Long-Hua Chen

    2005-01-01

    AIM: To investigate the killing effect and radiosensitization of double suicide gene mediated by adenovirus on colorectal carcinoma cells.METHODS: Colorectal carcinoma cell line SW480 was transfected with adenovirus expression vector containing cytosine deaminase (CD) and thymidine kinase (Tk) fusion gene. The expression of CD-TK fusion gene was detected by reverse transcriptase-polymerase chain reaction. The toxic effect of ganciclovir (GCV) and 5-fiuorocytosine (5FC) on infected cells was determined by MTT assay. The radiosensitization of double suicide gene was evaluated by clonogenic assay.RESULTS: After prodrugs were used, the survival rate of colorectal carcinoma cells was markedly decreased. When GCV and 5-FC were used in combination, the cytotoxicity and bystandereffect were markedly superior to a single prodrug (x2 = 30.371, P<0.01). Both GCV and 5-FC could sensitize colorectal carcinoma cells to the toxic effect of radiation, and greater radiosensitization was achieved when both prodrug were used in combination. CONCLUSION: CD-TK double suicide gene can kill and radiosensitize colorectal carcinoma cells.

  2. Escherichia coli ASKA Clone Library Harboring tRNA-Specific Adenosine Deaminase (tadA Reveals Resistance towards Xanthorrhizol

    Yogiara

    2015-09-01

    Full Text Available Xanthorrhizol is a potent antimicrobial compound isolated from the rhizome of Curcuma xanthorrhiza. However, the mechanism of xanthorrhizol action is unknown. To screen for probable target(s, we introduced the ASKA pooled-plasmid library into Escherichia coli W3110 imp4213 and enriched the library for resistant clones with increasing concentrations of xanthorrhizol. After three rounds of enrichment, we found nine genes that increased xanthorrhizol resistance. The resistant clones were able to grow in LB medium containing 256 µg/mL xanthorrhizol, representing a 16-fold increase in the minimum inhibitory concentration. Subsequent DNA sequence analysis revealed that overexpression of tadA, galU, fucU, ydeA, ydaC, soxS, nrdH, yiiD, and mltF genes conferred increased resistance towards xanthorrhizol. Among these nine genes, tadA is the only essential gene. tadA encodes a tRNA-specific adenosine deaminase. Overexpression of E. coli W3110 imp4213 (pCA24N-tadA conferred resistance to xanthorrhizol up to 128 µg/mL. Moreover, overexpression of two tadA mutant enzymes (A143V and F149G led to a twofold increase in the MIC. These results suggest that the targets of xanthorrhizol may include tadA, which has never before been explored as an antibiotic target.

  3. Simultaneous alteration of de novo and salvage pathway to the deoxynucleoside thriphosphate pool by (E)-2'-deoxy-(fluoromethylene)cytidine (FMDC) and zidovudine (AZT) results in increased radiosensitivity in vitro.

    Coucke, Philippe; Cottin, Eliane; Laurent, A.; Decosterd, A.

    2007-01-01

    Abstract To test whether a thymidine analog zidovudine (=AZT), is able to modify the radiosensitizing effects of (E)-2'-Deoxy-(fluoromethylene)cytidine (FMdC). A human colon cancer cell line Widr was exposed for 48 hours prior to irradiation to FMdC. Zidovudine was added at various concentrations immediately before irradiation. We measured cell survival and the effect of FMdC, AZT and FMdC + AZT on deoxynucleotide triphosphate pool. FMdC results in a significant increase of radiosensitivit...

  4. Signaling Mechanism of Cannabinoid Receptor-2 Activation-Induced β-Endorphin Release.

    Gao, Fang; Zhang, Ling-Hong; Su, Tang-Feng; Li, Lin; Zhou, Rui; Peng, Miao; Wu, Cai-Hua; Yuan, Xiao-Cui; Sun, Ning; Meng, Xian-Fang; Tian, Bo; Shi, Jing; Pan, Hui-Lin; Li, Man

    2016-08-01

    Activation of cannabinoid receptor-2 (CB2) results in β-endorphin release from keratinocytes, which then acts on primary afferent neurons to inhibit nociception. However, the underlying mechanism is still unknown. The CB2 receptor is generally thought to couple to Gi/o to inhibit cAMP production, which cannot explain the peripheral stimulatory effects of CB2 receptor activation. In this study, we found that in a keratinocyte cell line, the Gβγ subunits from Gi/o, but not Gαs, were involved in CB2 receptor activation-induced β-endorphin release. Inhibition of MAPK kinase, but not PLC, abolished CB2 receptor activation-induced β-endorphin release. Also, CB2 receptor activation significantly increased intracellular Ca(2+). Treatment with BAPTA-AM or thapsigargin blocked CB2 receptor activation-induced β-endorphin release. Using a rat model of inflammatory pain, we showed that the MAPK kinase inhibitor PD98059 abolished the peripheral effect of the CB2 receptor agonist on nociception. We thus present a novel mechanism of CB2 receptor activation-induced β-endorphin release through Gi/o-Gβγ-MAPK-Ca(2+) signaling pathway. Our data also suggest that stimulation of MAPK contributes to the peripheral analgesic effect of CB2 receptor agonists. PMID:26108183

  5. Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C*

    Lum, Michelle A.; Balaburski, Gregor M; Murphy, Maureen E.; Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    Background: Mechanisms of activation-induced PKC down-regulation are poorly understood. A characterized pathway involves priming site dephosphorylation and degradation of the dephosphorylated species.

  6. MicroRNA-146b-3p Regulates Retinal Inflammation by Suppressing Adenosine Deaminase-2 in Diabetes

    Sadanand Fulzele

    2015-01-01

    Full Text Available Hyperglycemia- (HG- Amadori-glycated albumin- (AGA- induced activation of microglia and monocytes and their adherence to retinal vascular endothelial cells contribute to retinal inflammation leading to diabetic retinopathy (DR. There is a great need for early detection of DR before demonstrable tissue damages become irreversible. Extracellular adenosine, required for endogenous anti-inflammation, is regulated by the interplay of equilibrative nucleoside transporter with adenosine deaminase (ADA and adenosine kinase. ADA, including ADA1 and ADA2, exists in all organisms. However, because ADA2 gene has not been identified in mouse genome, how diabetes alters adenosine-dependent anti-inflammation remains unclear. Studies of pig retinal microglia and human macrophages revealed a causal role of ADA2 in inflammation. Database search suggested miR-146b-3p recognition sites in the 3′-UTR of ADA2 mRNA. Coexpression of miR-146b-3p, but not miR-146-5p or nontargeting miRNA, with 3′-UTR of the ADA2 gene was necessary to suppress a linked reporter gene. In the vitreous of diabetic patients, decreased miR-146b-3p is associated with increased ADA2 activity. Ectopic expression of miR-146b-3p suppressed ADA2 expression, activity, and TNF-α release in the AGA-treated human macrophages. These results suggest a regulatory role of miR-146b-3p in diabetes related retinal inflammation by suppressing ADA2.

  7. RNA binding to APOBEC3G induces the disassembly of functional deaminase complexes by displacing single-stranded DNA substrates.

    Polevoda, Bogdan; McDougall, William M; Tun, Bradley N; Cheung, Michael; Salter, Jason D; Friedman, Alan E; Smith, Harold C

    2015-10-30

    APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G dimers and multimers thereof. Although, fluorescence anisotropy showed that A3G had similar nanomolar affinity for RNA and ssDNA, RNA stochastically dissociated A3G dimers and higher-order oligomers from ssDNA, suggesting a different modality for RNA binding. Mass spectrometry mapping of A3G peptides cross-linked to nucleic acid suggested ssDNA only bound to three peptides, amino acids (aa) 181-194 in the N-terminus and aa 314-320 and 345-374 in the C-terminus that were part of a continuous exposed surface. RNA bound to these peptides and uniquely associated with three additional peptides in the N- terminus, aa 15-29, 41-52 and 83-99, that formed a continuous surface area adjacent to the ssDNA binding surface. The data predict a mechanistic model of RNA inhibition of ssDNA binding to A3G in which competitive and allosteric interactions determine RNA-bound versus ssDNA-bound conformational states. PMID:26424853

  8. RNA binding to APOBEC3G induces the disassembly of functional deaminase complexes by displacing single-stranded DNA substrates

    Polevoda, Bogdan; McDougall, William M.; Tun, Bradley N.; Cheung, Michael; Salter, Jason D.; Friedman, Alan E.; Smith, Harold C.

    2015-01-01

    APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G dimers and multimers thereof. Although, fluorescence anisotropy showed that A3G had similar nanomolar affinity for RNA and ssDNA, RNA stochastically dissociated A3G dimers and higher-order oligomers from ssDNA, suggesting a different modality for RNA binding. Mass spectrometry mapping of A3G peptides cross-linked to nucleic acid suggested ssDNA only bound to three peptides, amino acids (aa) 181–194 in the N-terminus and aa 314–320 and 345–374 in the C-terminus that were part of a continuous exposed surface. RNA bound to these peptides and uniquely associated with three additional peptides in the N- terminus, aa 15–29, 41–52 and 83–99, that formed a continuous surface area adjacent to the ssDNA binding surface. The data predict a mechanistic model of RNA inhibition of ssDNA binding to A3G in which competitive and allosteric interactions determine RNA-bound versus ssDNA-bound conformational states. PMID:26424853

  9. Potential benefits of combining cytosine deaminase/5-fluorocytosine gene therapy and irradiation for prostate cancer. Experimental study

    The purpose of this study was to investigate the potential of combining cytosine deaminase/5-fluorocytosine (CD/5-FC) gene therapy and radiation therapy (either external beam radiation or radioimmunotherapy [RIT]), for the treatment of prostate cancer. Tumor xenografts of CD-transduced LNCaP cells grown in the testes of severe combined immunodeficiency (SCID) mice were used to evaluate antitumor effect. The mice were injected intraperitoneally with 500 mg/kg of 5-FC, or with 5, 15 or 30 mg/kg of 5-fluorouracil (5-FU), for 9 days. The tumors were treated with fractionated radiation at a dose of 1 or 3 Gy/day for 3 days, or I-131 labelled anti-prostate specific antigen (anti-PSA) monoclonal antibody (mAb) administration at a subtherapeutic dose of 20 or 80 μCi. Intratumoral and serum concentrations of 5-FU were measured using high performance liquid chromatography. Mice treated with CD/5-FC gene therapy presented a significant tumor growth inhibition comparable to that obtained with 15 mg/kg, 5-FU systemic administration without marked weight loss. Treatment with CD/5-FC gene therapy resulted in higher tumor but lower serum concentrations of 5-FU than treatment with systemic 5-FU chemotherapy. An additive antitumor effect was obtained when CD/5-FC therapy was combined with 1 Gy irradiation, which by itself did not produce a significant antitumor effect. However, the efficacy of CD/5-FC therapy was not enhanced when combined with RIT, probably due to poor accumulation of the mAb as the tumor/blood ratio never exceeded 1. These findings indicate that CD/5-FC gene therapy for prostate cancer may function with enhanced antitumor effect when combined with external beam radiation. However, combining CD/5-FC gene therapy and RIT using an anti-PSA mAb may not be effective because of insufficient accumulation of the mAb at the target tumors. (author)

  10. Antitumor effect of cytosine deaminase/5-fluorocytosine suicide gene therapy system mediated by Bifidobacterium infantis on melanoma

    Cheng YI; Ying HUANG; Zhi-ying GUO; Shu-ren WANG

    2005-01-01

    Aim: To construct a Bifidobacterium infantis/CD targeting gene therapy system and observe the antitumor effect of cytosine deaminase/5-fluorocytosine (CD/5-FC) suicide gene therapy system mediated by Bifidobacterium infantis on melanoma in vitro and in vivo. Methods: A recombinant CD/pGEX- 1LamdaT plasmid was transfected into Bifidobacterium infantis by electroporation. Bifidobac terium infantis transfected by recombinant CD/pGEX-1LamdaT plasmid was in cubated with 5-FC anaerobically. Then the supernatant fluid was collected and added to melanoma B16-F10 cells to observe the killing effect for B16-F10 cells.Mice were inoculated with melanoma B 16-F10 cells to establish animal models.The mice were then injected with 5-FC and Bifidobacterium infantis transfected by recombinant CD/pGEX-1LamdaT plasmid. Results:Two segments of approxi mate 4.9 kb and 1.3 kb were extracted from the 6.2 kb recombinant plasmid, which were equal to the size of the pGEX-1LamdaT plasmid and CD gene, respectively.Sequencing results showed that the full length and sequence of nucleotide acid of the inserted gene in extracted recombinant plasmid was completely identical to the CD gene. In vitro, B 16-F10 cells treated by supernatant fluid were remarkably damaged morphologically, and the cell growth was significantly inhibited. Experi ments on the mice melanoma model showed that after treatment with a combination of transfected Bifidobacterium infantis and 5-FC, the tumor volume was significantly inhibited compared with controls. Conclusion: The foreign gene,CD gene, was correctly inserted into pGEX-1LambdaT plasmid and transferred into Bifidobacterium infantis. CD/5-FC suicide gene therapy system mediated by Bifidobacterium infantis demonstrated a good antitumor effect on melanoma in vitro and in vivo.

  11. Diagnostic value of serum adenosine deaminase levels in sputum smear negative pulmonary tuberculosis patients in Nepalese population

    Anil Chander; Chandrika Devi Shrestha

    2012-01-01

    Objective: To assess the levels of adenosine deaminase (ADA) in serum in patients with sputum smear negative pulmonary tuberculosis (SNPTB) and to compare it with serum ADA levels in patients with non-tuberculous pulmonary disease - chronic obstructive pulmonary disease (COPD) and with healthy control group and to explore its validity as a diagnostic marker in serum in SNPTB patients.Methods:Three groups of study populations were made. Group I: SNPTB - 142 cases, Group II:non-tubercular pulmonary disease - COPD - 40 cases, Group III: healthy controls - 80 cases. Serum samples were collected and ADA assay was done by the method of Guisti and Galanti. Results: ADA levels (Mean±SD, U/L) in the three groups were as follows: Group I: 42.26±21.22, Group II: 23.31±8.22, Group III: 18.88±6.67. Difference between Group I and Group III was statistically significant (P < 0.0001). The test showed a high specificity 91.25% (95% confidence interval - CI 83.00 - 95.7) and a sensitivity of 83.10% (95% CI 76.08-88.37) in Group I. Positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio and accuracy in Group I were 94.00%, 69.52%, 9.49, 0.18 and 82.43% respectively.Conclusions: Overall assessment of the use of serum ADA levels as a diagnostic biochemical marker in smear-negative pulmonary tuberculosis patients showed promising results. Studies with a larger population group are required to validate its use as a routine diagnostic test in these cases.

  12. Opposing activity changes in AMP deaminase and AMP-activated protein kinase in the hibernating ground squirrel.

    Miguel A Lanaspa

    Full Text Available Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2 (summer and activation of AMP-activated protein kinase (AMPK (winter. Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and β-fatty acid oxidation were evaluated. Changes in fat metabolism were correlated with changes in AMPD2 activity and intrahepatic uric acid (downstream product of AMPD2, as well as changes in AMPK and intrahepatic β-hydroxybutyrate (a marker of fat oxidation. Hepatic fat accumulation occurred during the summer with relatively increased enzymes associated with fat synthesis (FAS, ACL and ACC and decreased enoyl CoA hydratase (ECH1 and carnitine palmitoyltransferase 1A (CPT1A, rate limiting enzymes of fat oxidation. In summer, AMPD2 activity and intrahepatic uric acid levels were high and hepatic AMPK activity was low. In contrast, the active phosphorylated form of AMPK and β-hydroxybutyrate both increased during winter hibernation. Therefore, changes in AMPD2 and AMPK activity were paralleled with changes in fat synthesis and fat oxidation rates during the summer-winter cycle. These data illuminate the opposing forces of metabolism of AMP by AMPD2 and its availability to activate AMPK as a switch that governs fat metabolism in the liver of hibernating ground squirrel.

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

    Tombesi Marina

    2008-09-01

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

  14. SUB-LETHAL INJECTION OF COBRA VENOM DECREASES ADENOSINE DEAMINASE, HISTAMINE, AND IgE IN ORGANS OF MICE

    B. V. LIPPS

    2002-01-01

    Full Text Available The pharmacological substances adenosine deaminase (ADA, histamine, and IgE are endogenously present in animals. They are implicated in allergy and asthma and detectable in blood serum. This research reports the presence of ADA, histamine, and IgE at varying levels in almost all major organs of mice. This research further reports that intramuscular injection of sub-lethal dose of Naja kaouthia venom disrupted homeostasis and lowered the levels of ADA, histamine, and IgE in the organs of mice. Adult Balb/C male mice were injected with a half lethal dose of the venom. The mice were sacrificed at 2, 8, and 24 hours post-injection and different organs were collected. Organs were homogenized, centrifuged, and the supernatants were assayed for ADA, histamine, and IgE using respective antisera by immunological test enzyme-linked immunosorbent assay (ELISA. Organs from mice injected with PBS served as controls. No major decrease in the levels of ADA, histamine, and IgE was observed after 2 hours of venom injection. However, tremendous decreases in the levels of ADA, histamine, and IgE was observed in organs 24 h post-injection. The highest decrease for ADA was observed in the brain, liver, lung, muscle, and testis; for histamine, in the heart, muscle, lung, and testis; and for IgE, in the bone, heart, lung, muscle, and testis. This is a first-hand investigation showing the effect of envenomation on the pharmacokinetics of ADA, histamine, and IgE in organs.

  15. Hyperglycemia alters E-NTPDases, ecto-5'-nucleotidase, and ectosolic and cytosolic adenosine deaminase activities and expression from encephala of adult zebrafish (Danio rerio).

    Capiotti, Katiucia Marques; Siebel, Anna Maria; Kist, Luiza Wilges; Bogo, Maurício Reis; Bonan, Carla Denise; Da Silva, Rosane Souza

    2016-06-01

    Hyperglycemia is the main feature for the diagnosis of diabetes mellitus (DM). Some studies have demonstrated the relationship between DM and dysfunction on neurotransmission systems, such as the purinergic system. In this study, we evaluated the extracellular nucleotide hydrolysis and adenosine deamination activities from encephalic membranes of hyperglycemic zebrafish. A significant decrease in ATP, ADP, and AMP hydrolyses was observed at 111-mM glucose-treated group, which returned to normal levels after 7 days of glucose withdrawal. A significant increase in ecto-adenosine deaminase activity was observed in 111-mM glucose group, which remain elevated after 7 days of glucose withdrawal. The soluble-adenosine deaminase activity was significantly increased just after 7 days of glucose withdrawal. We also evaluated the gene expressions of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-5'-nucleotidase, ADA, and adenosine receptors from encephala of adult zebrafish. The entpd 2a.1, entpd 2a.2, entpd 3, and entpd 8 mRNA levels from encephala of adult zebrafish were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expressions of adenosine receptors (adora 1 , adora 2aa , adora 2ab , and adora 2b ) were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expression of ADA (ada 2a.1) was decreased in glucose withdrawal group. Maltodextrin, used as a control, did not affect the expression of adenosine receptors, ADA and E-NTPDases 2, 3, and 8, while the expression of ecto-5'-nucleotidase was slightly increased and the E-NTPDases 1 decreased. These findings demonstrated that hyperglycemia might affect the ecto-nucleotidase and adenosine deaminase activities and gene expression in zebrafish, probably through a mechanism involving the osmotic effect, suggesting that the modifications caused on purinergic system may also contribute to the diabetes-induced progressive cognitive impairment. PMID:26769247

  16. Inoculation with Pseudomonas spp.containing ACC-deaminase partially eliminates the effects of drought stress on growth,yield,and ripening of pea (Pisum sativum L.)

    M.ARSHAD; B.SHAHAROONA; T.MAHMOOD

    2008-01-01

    Two preselected plant growth promoting rhizobacteria (PGPR) containing 1-aminocyelopropane-1-carboxylate (ACC)deaminase (EC 4.1.99.4) were used to investigate their potential to ameliorate the effects of drought stress on growth,yield,and ripening of pea (Pisum sativum L.).Inoculated and uninoculated (control) seeds of pea cultivar 2000 were sown in pots (four seeds pot-1) and placed in a wire house.The plants were exposed to drought stress at different stages of growth (vegetative,flowering,and pod formation) by skipping the respective irrigation.Results revealed that inoculation of peas with PGPR containing ACC-deaminase significantly decreased the "drought stress imposed effects" on the growth and yield of peas.Exposure of plants to drought stress at vegetative growth stage significantly decreased shoot growth by 41% in the ease of uninoculated plants,whereas,by only 18% in the case of inoculated plants compared to nonstressed unlnoculated controlGrain yield was decreased when plants were exposed to drought stress at the flowering and pod formation stage,but inoculation resulted in better grain yield (up to 62% and 40% higher,respectively) than the respective uninoculated nonstressed control.Ripening of pods was also delayed in plants inoculated with PGPR,which may imply decreased endogenous ethylene production in inoculated plants.This premise is further supported by the observation that inoculation with PGPR reduced the intensity of classical "triple" response in etiolated pea seedlings,caused by externally applied ACC.It is very probable that the drought stress induced inhibitory effects of ethylene could be partially or completely eliminated by inoculation with PGPR containing ACC-deaminase.

  17. Effect of repeated pesticide applications on soil properties in cotton fields: II. Insecticide residues and impact on dehydrogenase and arginine deaminase activities

    Insecticides were applied sequentially at recommended dosages post crop emergence in cotton fields and soil was sampled at regular intervals after each treatment. Soil was analysed for insecticide residues and activity of the enzymes dehydrogenase and arginine deaminase. Insecticide residues detected in the soil were in small quantities and they did not persist for long. Only endosulfan leached below 15 cm. Insecticides had only temporary effects on enzyme activities which disappeared either before the next insecticide treatment or by the end of the experimental period. (author)

  18. The value of adenosine deaminase, interferon-gamma, and interferon-gamma induced protein of 10kD in the diagnosis of tuberculous pleuritis

    Ya-kun DONG; Li, Ai-zhen; Li-heng ZHENG; Yue-peng CHI; Wang, Yu-Hong; Qiu-mei WANG; Zhu, Gui-Yun; Lan-pin XIE

    2015-01-01

    Objective To explore the value of adenosine deaminase (ADA) activity, interferon-gamma (IFN-γ) and IFN-γ induced protein of 10kD (IP-10) levels in pleural effusion for the diagnosis of tuberculous pleuritis. Methods ADA activity, IFN-γ and IP-10 levels in pleural effusion were determined in sixty-three patients with tuberculous pleuritis and 50 patients with malignant pleural effusion. Results The mean levels of ADA, IFN-γ and IP-10 in the tuberculous pleural effusion were significantly highe...

  19. Targeted cytosine deaminase-uracil phosphoribosyl transferase suicide gene therapy induces small cell lung cancer-specific cytotoxicity and tumor growth delay

    Christensen, Camilla L; Gjetting, Torben; Poulsen, Thomas Tuxen;

    2010-01-01

    Small cell lung cancer (SCLC) is a highly malignant cancer for which there is no curable treatment. Novel therapies are therefore in great demand. In the present study we investigated the therapeutic effect of transcriptionally targeted suicide gene therapy for SCLC based on the yeast cytosine...... deaminase (YCD) gene alone or fused with the yeast uracil phosphoribosyl transferase (YUPRT) gene followed by administration of 5-fluorocytosine (5-FC) prodrug. Experimental design: The YCD gene or the YCD-YUPRT gene was placed under regulation of the SCLC-specific promoter insulinoma-associated 1 (INSM1...

  20. A Novel Serine Protease Secreted by Medicinal Maggots Enhances Plasminogen Activator-Induced Fibrinolysis

    van der Plas, Mariena J. A.; Andersen, Anders S.; Nazir, Sheresma; van Tilburg, Nico H.; Oestergaard, Peter R.; Krogfelt, Karen A.; van Dissel, Jaap T.; Hensbergen, Paul J.; Rogier M Bertina; Nibbering, Peter H.

    2014-01-01

    Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. As haemostatic processes play an important role in wound healing, this study focused on the effects of maggot secretions on coagulation and fibrinolysis. The results showed that maggot secretions enhance plasminogen activator-induced formation of plasmin and fibrinolysis in a dose- and time-dependent manner. By contrast, coagulation was not affected by secretions. Biochemical studies indicated that a novel s...

  1. Stable transformation of Toxoplasma gondii based on a pyrimethamine resistant trifunctional dihydrofolate reductase-cytosine deaminase-thymidylate synthase gene that confers sensitivity to 5-fluorocytosine.

    Fox, B A; Belperron, A A; Bzik, D J

    1999-01-01

    To improve genetic models available for the analysis of apicomplexan protozoan parasites, bacterial sequences encoding the 427 amino acid cytosine deaminase (CD) gene were fused, in-frame, to an engineered linker domain of the high level pyrimethamine resistant form of the parasite bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. Toxoplasma gondii was transformed with the plasmid containing the fused pyrimethamine resistant dihydrofolate reductase-cytosine deaminase-thymidylate synthase (DHFRm2m3-CD-TS) gene and parasites were selected in a high level of pyrimethamine. Transfected parasites that acquired resistance to pyrimethamine were cloned and evaluated for expression of the CD genetic marker. CD transgenic parasites acquired a high sensitivity to 5-fluorocytosine due to the intraparasitic conversion of this non-toxic prodrug to the cytotoxic compound 5-fluorouracil. Exogenously supplied cytosine or uracil rescued the growth of CD transgenic T. gondii parasites that were cultured in the presence of cytotoxic concentrations of 5-fluorouracil or 5-fluorocytosine. Bacterial CD fused to the pyrimethamine resistant DHFR-TS marker provides a novel genetic tool for new positive and negative genetic selection strategies in several protozoan parasites. An advantage of the CD genetic marker is that it is derived from a bacterial gene and can therefore be used in nearly any parasite genetic background for negative selection. This novel system should facilitate new approaches for the development of improved model genetic systems for the biological investigation of apicomplexan parasites. PMID:10029312

  2. Cloning of the cDNA encoding adenosine 5'-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

    Jiang, Keyong; Sun, Shujuan; Liu, Mei; Wang, Baojie; Meng, Xiaolin; Wang, Lei

    2013-01-01

    AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) ( Panimals.

  3. Regulation of dCTP deaminase from Escherichia coli by nonallosteric dTTP binding to an inactive form of the enzyme

    Johansson, Eva; Thymark, Majbritt; Bynck, Julie H;

    2007-01-01

    The trimeric dCTP deaminase produces dUTP that is hydrolysed to dUMP by the structurally closely related dUTPase. This pathway provides 70-80% of the total dUMP as a precursor for dTTP. Accordingly, dCTP deaminase is regulated by dTTP, which increases the substrate concentration for half......-maximal activity and the cooperativity of dCTP saturation. Likewise, increasing concentrations of dCTP increase the cooperativity of dTTP inhibition. Previous structural studies showed that the complexes of inactive mutant protein, E138A, with dUTP or dCTP bound, and wild-type enzyme with dUTP bound were all...... highly similar and characterized by having an ordered C-terminal. When comparing with a new structure in which dTTP is bound to the active site of E138A, the region between Val120 and His125 was found to be in a new conformation. This and the previous conformation were mutually exclusive within the...

  4. High frequency of mutations in exon 10 of the porphobilinogen deaminase gene in patients with a CRIM-positive subtype of acute intermittent porphyria

    Gu, X.F.; Rooij, F. de; Voortman, G.; Velde, K.T.; Nordmann, Y.; Grandchamp, B.

    1992-09-01

    Acute intermittent porphyria (AIP) is an autosomal dominant disease characterized by a partial deficiency of porphobilinogen (PBG) deaminase. Different subtypes of the disease have been defined, and more than 10 different mutations have been described. The authors focused their study on exon 10, since they previously found that three different mutations were located in this exon and that two of them seemed to be relatively common. They used denaturing gradient gel electrophoresis (DGGE) after in vitro amplification to detect all possible mutations in exon 10 in 41 unrelated AIP patients. In about one-fourth of these patients they could distinguish three abnormal migration patterns, indicating the presence of various mutations. Additional sequencing demonstrated the presence of three different single-base substitutions. Two of these mutations had already been described. A third one consisted of a C-to-T transition located at position 499 of the PBG deaminase mRNA and resulted in an Arg-to-Trp substitution. All three mutations were found in patients with crossreacting immunological material (CRIM)-positive forms of AlP. The high frequency of these mutations make DGGE analysis of exon 10 a useful approach allowing the direct detection of the DNA abnormality in most of the families with the CRIM-positive subtype of AlP. 23 refs., 3 figs., 1 tab.

  5. EFFICACY OF PLEURAL FLUID ADENOSINE DEAMINASE AND C -REACTIVE PROTEIN LEVELS IN EARLY DIFFERENTIAL DIAGNOSIS OF P LEURAL EFFUSION.

    Aliya

    2013-05-01

    Full Text Available ABSTRACT: - CONTEXT: Pleural effusion occurs secondary to various disea ses. Common causes of exudative effusion are tuberculosis, bacterial pneu monia, and malignancy. Transudative effusion is due to systemic diseases like cardiac failure, cirr hosis of liver. Conventional methods of diagnosis may not be able to establish the cause of pleural e ffusion. Early diagnosis and management reduces the morbidity and mortality. AIM: The objective of the study is to estimate pleural fluid Adenosine Deaminase (ADA and C - reactive protein (CRP leve ls and to evaluate their efficacy in differential diagnosis of transudative and exudative, tuberculou s and non tuberculous and inflammatory and non inflammatory effusions. MATERIAL AND METHODS: Fifty two patients of pleural effusion were investigated and divided into four groups base d on diagnosis. Group I, II, III and IV had 24 cases of tuberculous effusion, 13 cases of transuda tive effusion, 08 cases of malignant effusion and 07 cases of parapneumonic effusion respectively. Pl eural fluid was analyzed for ADA (Guisti and Galanti’s method and CRP (turbidometric immunoassa y. STATISTICAL ANALYSIS: The statistical analysis was done using unpaired student‘t’ test an d p value < 0.05 was considered statistically significant. RESULTS: In the present study pleural fluid ADA revealed hig hly significant increase in tuberculous effusion than non tuberculous effusions (p <0.001 and also when compared with non tuberculous subgroups, transudative effusion (p < 0 .001, malignant effusion (p<0.001, and PPE (p<0.01. ADA levels at a cutoff value of 40U/L, sh owed sensitivity, specificity, positive predictive value and negative predictive value of 91.67%, 89.3 %, 88% & 92.6% respectively in tuberculous effusion. Pleural fluid CRP levels in parapneumonic effusion were significantly higher compared to other types of effusions (p<0.001. Significantly h igher levels of CRP were seen in exudative effusion compared to

  6. Combined detections of interleukin 27, interferon-γ, and adenosine deaminase in pleural effusion for diagnosis of tuberculous pleurisy

    WU Yan-bin; YE Zhi-jian; QIN Sou-ming; WU Cong; CHEN Yi-qiang; SHI Huan-zhong

    2013-01-01

    Background Previous studies reported interleukin-27 (IL-27),interferon-γ (IFN-γ),or adenosine deaminase (ADA) alone plays a helpful role in diagnosing tuberculous pleural effusion (TPE).The present study aims at comparing the diagnostic accuracy of pleural IL-27,IFN-γ,and ADA,and investigate the diagnostic accuracy of the combination of IL-27,IFN-γ,or/and ADA for differentiating TPE from pleural effusions with the other etiologies.Methods The concentrations of IL-27,IFN-γ and ADA were simultaneously determined in pleural fluids and sera from 40 patients with TPE; 26 with malignant pleural effusion,seven with infectious pleural effusion,and eight with transudative pleural effusion by enzyme linked immunosorbent assay and colorimetric method.The corresponding biochemical indexs were also simultaneously determined.Results The concentrations of pleural IL-27 and IFN-γ in the tuberculous group were significantly higher than those in the malignant,infectious,and transudative groups.The concentrations of ADA in TPE were significantly higher than those in MPE or transudative effusions,while much lower than those in infectious effusions.Among these three biomarkers,IL-27 was the most effective for TPE diagnosis,with the cut off value of 900.8 ng/L.IL-27 had a high sensitivity of 95% and specificity of 97.6% for differential diagnosis of TPE from non-TPEs.Combinations of IL-27,IFN-γ and ADA measurements further increased the sensitivity or specificity up to 100%.Conclusions Compared to non-TPEs,IL-27,IFN-γ and ADA all simultaneously increased in TPE; and among these three rapid detection methods,IL-27 appeared to be the best for distinguishing tuberculous from non-TPEs,especially from MPE.Combinations of the three markers (IL-27,IFN-γ and ADA) yielded the highest sensitivity and specificity.These findings suggest that the applications of a new biomarker,IL-27,alone or with IFN-γ and ADA,may contribute to more efficient diagnosis strategies in the management

  7. Bacillus subtilis guanine deaminase is encoded by the yknA gene and is induced during growth with purines as the nitrogen source

    Nygaard, Per; Bedsted, Søren; Andersen, Kasper A.K.;

    2000-01-01

    Bacillus subtilis can utilize the purine bases adenine, hypoxanthine and xanthine as nitrogen sources. The utilization of guanine as a nitrogen source is reported here. The first step is the deamination of guanine to xanthine catalysed by guanine deaminase (GDEase). To isolate mutants defective in...

  8. Neutrons formed by heavy ions and activation induced in different materials

    This work deals with the Spiral project and more particularly with the neutrons flux formed by heavy ions and the activation induced in different materials. Indeed, the beams power suggests the interest of different materials behaviour study for allowing a possible selection to optimize radioprotection. Moreover, it is important to establish the activation mechanisms in order to be able to extrapolate the measures realized at 400 W (actual GANIL) to those of the future running taking into account the radioisotopes real mixtures formed during the reaction and their daughter products. A best knowledge of energizing and angular neutrons distributions is searched too. (O.L.). 11 refs., 23 figs., 9 tabs

  9. AID up-mutants isolated using a high-throughput screen highlight the immunity/cancer balance limiting DNA deaminase activity

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

    2016-01-01

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

  10. AcEST: BP913604 [AcEST

    Full Text Available YMU001_000032_B02 433 Adiantum capillus-veneris mRNA. clone: YMU001_000032_B02. BP913604 - Show ... Q0E4W2|Q0E4W2_9POAL Cytidine deaminase OS=Paspalum simplex ... Align length 38 Score (bit) 49.7 E-value 7.0e-05 R ... Q0E4W2|Q0E4W2_9POAL Cytidine deaminase OS=Paspalum simplex ... GN... 50 7e-05 tr|B4FPE0|B4FPE0_MAIZE Cytidine/deo ...