RNA Editing, ADAR1, and the Innate Immune Response.

PubMed

Wang, Qingde; Li, Xiaoni; Qi, Ruofan; Billiar, Timothy

2017-01-18

RNA editing, particularly A-to-I RNA editing, has been shown to play an essential role in mammalian embryonic development and tissue homeostasis, and is implicated in the pathogenesis of many diseases including skin pigmentation disorder, autoimmune and inflammatory tissue injury, neuron degeneration, and various malignancies. A-to-I RNA editing is carried out by a small group of enzymes, the adenosine deaminase acting on RNAs (ADARs). Only three members of this protein family, ADAR1-3, exist in mammalian cells. ADAR3 is a catalytically null enzyme and the most significant function of ADAR2 was found to be in editing on the neuron receptor GluR-B mRNA. ADAR1, however, has been shown to play more significant roles in biological and pathological conditions. Although there remains much that is not known about how ADAR1 regulates cellular function, recent findings point to regulation of the innate immune response as an important function of ADAR1. Without appropriate RNA editing by ADAR1, endogenous RNA transcripts stimulate cytosolic RNA sensing receptors and therefore activate the IFN-inducing signaling pathways. Overactivation of innate immune pathways can lead to tissue injury and dysfunction. However, obvious gaps in our knowledge persist as to how ADAR1 regulates innate immune responses through RNA editing. Here, we review critical findings from ADAR1 mechanistic studies focusing on its regulatory function in innate immune responses and identify some of the important unanswered questions in the field.

A-to-I RNA editing independent of ADARs in filamentous fungi

PubMed Central

Wang, Chenfang; Xu, Jin-Rong; Liu, Huiquan

2016-01-01

ABSTRACT ADAR mediated A-to-I RNA editing is thought to be unique to animals and occurs mainly in the non-coding regions. Recently filamentous fungi such as Fusarium graminearum were found to lack orthologs of animal ADARs but have stage-specific A-to-I editing during sexual reproduction. Unlike animals, majority of editing sites are in the coding regions and often result in missense and stop loss changes in fungi. Furthermore, whereas As in RNA stems are targeted by animal ADARs, RNA editing in fungi preferentially targets As in hairpin loops, implying that fungal RNA editing involves mechanisms related to editing of the anticodon loop by ADATs. Identification and characterization of fungal adenosine deaminases and their stage-specific co-factors may be helpful to understand the evolution of human ADARs. Fungi also can be used to study biological functions of missense and stop loss RNA editing events in eukaryotic organisms. PMID:27533598

The Role of ADAR1 and ADAR2 in the Regulation of miRNA-21 in Idiopathic Pulmonary Fibrosis.

PubMed

Díaz-Piña, Gabriela; Ordoñez-Razo, Rosa Ma; Montes, Eduardo; Páramo, Ignacio; Becerril, Carina; Salgado, Alfonso; Santibañez-Salgado, J Alfredo; Maldonado, Mariel; Ruiz, Victor

2018-04-10

microRNAs (miRNAs) are small non-coding 1RNAs that post-transcriptionally regulate gene expression. Recent evidence shows that adenosine deaminases that act on RNA (ADAR) can edit miRNAs. miRNAs are involved in the development of different diseases, such as idiopathic pulmonary fibrosis (IPF). In IPF, about 40% of the miRNAs are differentially expressed with respect to controls. Among these miRNAs, miRNA-21 has been found over-expressed in IPF and its targets are anti-fibrosing molecules such as PELI1 and SPRY2. The objective of this study is to determine the role of ADAR1 and 2 on the expression of miRNA-21 in human lung fibroblasts trough quantification of gene expression, protein levels, and overexpression of ADAR1 and 2. Six control and six fibrotic primary fibroblast cell cultures were used for RNA extraction, ADAR1, ADAR2, PELI1, SPRY2, miRNA-21, and pri-miRNA-21 expression was measured. Subsequently, two fibrotic fibroblast cultures were used for overexpression of ADAR1 and ADAR2, and they were stimulated with TGFβ1. Real-time PCR and Western blot were performed. ADAR1 is significantly downregulated in IPF fibroblasts; the overexpression of ADAR1 and ADAR2 reestablishes the expression levels of miRNA-21, PELI1, and SPRY2 in fibroblasts of patients with IPF. These changes in the processing of miRNAs have great value in pathology diagnosis, including lung diseases, and play an important role in the understanding of molecular mechanisms involved in the development of different pathologies, as well as representing new therapeutic targets.

Fmrp Interacts with Adar and Regulates RNA Editing, Synaptic Density and Locomotor Activity in Zebrafish

PubMed Central

Porath, Hagit T.; Barak, Michal; Pinto, Yishay; Wachtel, Chaim; Zilberberg, Alona; Lerer-Goldshtein, Tali; Efroni, Sol; Levanon, Erez Y.; Appelbaum, Lior

2015-01-01

Fragile X syndrome (FXS) is the most frequent inherited form of mental retardation. The cause for this X-linked disorder is the silencing of the fragile X mental retardation 1 (fmr1) gene and the absence of the fragile X mental retardation protein (Fmrp). The RNA-binding protein Fmrp represses protein translation, particularly in synapses. In Drosophila, Fmrp interacts with the adenosine deaminase acting on RNA (Adar) enzymes. Adar enzymes convert adenosine to inosine (A-to-I) and modify the sequence of RNA transcripts. Utilizing the fmr1 zebrafish mutant (fmr1-/-), we studied Fmrp-dependent neuronal circuit formation, behavior, and Adar-mediated RNA editing. By combining behavior analyses and live imaging of single axons and synapses, we showed hyperlocomotor activity, as well as increased axonal branching and synaptic density, in fmr1-/- larvae. We identified thousands of clustered RNA editing sites in the zebrafish transcriptome and showed that Fmrp biochemically interacts with the Adar2a protein. The expression levels of the adar genes and Adar2 protein increased in fmr1-/- zebrafish. Microfluidic-based multiplex PCR coupled with deep sequencing showed a mild increase in A-to-I RNA editing levels in evolutionarily conserved neuronal and synaptic Adar-targets in fmr1-/- larvae. These findings suggest that loss of Fmrp results in increased Adar-mediated RNA editing activity on target-specific RNAs, which, in turn, might alter neuronal circuit formation and behavior in FXS. PMID:26637167

Genetics Home Reference: adenosine deaminase deficiency

MedlinePlus

... to eliminate a molecule called deoxyadenosine, which is generated when DNA is broken down. Adenosine deaminase converts ... a substitute for professional medical care or advice. Users with questions about a personal health condition should ...

An RNA editing/dsRNA binding-independent gene regulatory mechanism of ADARs and its clinical implication in cancer.

PubMed

Qi, Lihua; Song, Yangyang; Chan, Tim Hon Man; Yang, Henry; Lin, Chi Ho; Tay, Daryl Jin Tai; Hong, HuiQi; Tang, Sze Jing; Tan, Kar Tong; Huang, Xi Xiao; Lin, Jaymie Siqi; Ng, Vanessa Hui En; Maury, Julien Jean Pierre; Tenen, Daniel G; Chen, Leilei

2017-10-13

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by Adenosine DeAminases acting on double-stranded RNA(dsRNA) (ADAR), occurs predominantly in the 3' untranslated regions (3'UTRs) of spliced mRNA. Here we uncover an unanticipated link between ADARs (ADAR1 and ADAR2) and the expression of target genes undergoing extensive 3'UTR editing. Using METTL7A (Methyltransferase Like 7A), a novel tumor suppressor gene with multiple editing sites at its 3'UTR, we demonstrate that its expression could be repressed by ADARs beyond their RNA editing and double-stranded RNA (dsRNA) binding functions. ADARs interact with Dicer to augment the processing of pre-miR-27a to mature miR-27a. Consequently, mature miR-27a targets the METTL7A 3'UTR to repress its expression level. In sum, our study unveils that the extensive 3'UTR editing of METTL7A is merely a footprint of ADAR binding, and there are a subset of target genes that are equivalently regulated by ADAR1 and ADAR2 through their non-canonical RNA editing and dsRNA binding-independent functions, albeit maybe less common. The functional significance of ADARs is much more diverse than previously appreciated and this gene regulatory function of ADARs is most likely to be of high biological importance beyond the best-studied editing function. This non-editing side of ADARs opens another door to target cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

An RNA editing/dsRNA binding-independent gene regulatory mechanism of ADARs and its clinical implication in cancer

PubMed Central

Qi, Lihua; Song, Yangyang; Chan, Tim Hon Man; Yang, Henry; Lin, Chi Ho; Tay, Daryl Jin Tai; Hong, HuiQi; Tang, Sze Jing; Tan, Kar Tong; Huang, Xi Xiao; Lin, Jaymie Siqi; Ng, Vanessa Hui En; Maury, Julien Jean Pierre

2017-01-01

Abstract Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by Adenosine DeAminases acting on double-stranded RNA(dsRNA) (ADAR), occurs predominantly in the 3′ untranslated regions (3′UTRs) of spliced mRNA. Here we uncover an unanticipated link between ADARs (ADAR1 and ADAR2) and the expression of target genes undergoing extensive 3′UTR editing. Using METTL7A (Methyltransferase Like 7A), a novel tumor suppressor gene with multiple editing sites at its 3′UTR, we demonstrate that its expression could be repressed by ADARs beyond their RNA editing and double-stranded RNA (dsRNA) binding functions. ADARs interact with Dicer to augment the processing of pre-miR-27a to mature miR-27a. Consequently, mature miR-27a targets the METTL7A 3′UTR to repress its expression level. In sum, our study unveils that the extensive 3′UTR editing of METTL7A is merely a footprint of ADAR binding, and there are a subset of target genes that are equivalently regulated by ADAR1 and ADAR2 through their non-canonical RNA editing and dsRNA binding-independent functions, albeit maybe less common. The functional significance of ADARs is much more diverse than previously appreciated and this gene regulatory function of ADARs is most likely to be of high biological importance beyond the best-studied editing function. This non-editing side of ADARs opens another door to target cancer. PMID:28985428

ADAR2 induces reproducible changes in sequence and abundance of mature microRNAs in the mouse brain

PubMed Central

Vesely, Cornelia; Tauber, Stefanie; Sedlazeck, Fritz J.; Tajaddod, Mansoureh; von Haeseler, Arndt; Jantsch, Michael F.

2014-01-01

Adenosine deaminases that act on RNA (ADARs) deaminate adenosines to inosines in double-stranded RNAs including miRNA precursors. A to I editing is widespread and required for normal life. By comparing deep sequencing data of brain miRNAs from wild-type and ADAR2 deficient mouse strains, we detect editing sites and altered miRNA processing at high sensitivity. We detect 48 novel editing events in miRNAs. Some editing events reach frequencies of up to 80%. About half of all editing events depend on ADAR2 while some miRNAs are preferentially edited by ADAR1. Sixty-four percent of all editing events are located within the seed region of mature miRNAs. For the highly edited miR-3099, we experimentally prove retargeting of the edited miRNA to novel 3′ UTRs. We show further that an abundant editing event in miR-497 promotes processing by Drosha of the corresponding pri-miRNA. We also detect reproducible changes in the abundance of specific miRNAs in ADAR2-deficient mice that occur independent of adjacent A to I editing events. This indicates that ADAR2 binding but not editing of miRNA precursors may influence their processing. Correlating with changes in miRNA abundance we find misregulation of putative targets of these miRNAs in the presence or absence of ADAR2. PMID:25260591

Adenosine deaminase deficiency: a review.

PubMed

Flinn, Aisling M; Gennery, Andrew R

2018-04-24

Adenosine deaminase (ADA) deficiency leads to an accumulation of toxic purine degradation by-products, most potently affecting lymphocytes, leading to adenosine deaminase-deficient severe combined immunodeficiency. Whilst most notable affects are on lymphocytes, other manifestations include skeletal abnormalities, neurodevelopmental affects and pulmonary manifestations associated with pulmonary-alveolar proteinosis. Affected patients present in early infancy, usually with persistent infection, or with pulmonary insufficiency. Three treatment options are currently available. Initial treatment with enzyme replacement therapy may alleviate acute symptoms and enable partial immunological reconstitution, but treatment is life-long, immune reconstitution is incomplete, and the reconstituted immune system may nullify the effects of the enzyme replacement. Hematopoietic stem cell transplant has long been established as the treatment of choice, particularly where a matched sibling or well matched unrelated donor is available. More recently, the use of gene addition techniques to correct the genetic defect in autologous haematopoietic stem cells treatment has demonstrated immunological and clinical efficacy. This article reviews the biology, clinical presentation, diagnosis and treatment of ADA-deficiency.

Genome-wide A-to-I RNA editing in fungi independent of ADAR enzymes

PubMed Central

Liu, Huiquan; Wang, Qinhu; He, Yi; Chen, Lingfeng; Hao, Chaofeng; Jiang, Cong; Li, Yang; Dai, Yafeng; Kang, Zhensheng; Xu, Jin-Rong

2016-01-01

Yeasts and filamentous fungi do not have adenosine deaminase acting on RNA (ADAR) orthologs and are believed to lack A-to-I RNA editing, which is the most prevalent editing of mRNA in animals. However, during this study with the PUK1 (FGRRES_01058) pseudokinase gene important for sexual reproduction in Fusarium graminearum, we found that two tandem stop codons, UA1831GUA1834G, in its kinase domain were changed to UG1831GUG1834G by RNA editing in perithecia. To confirm A-to-I editing of PUK1 transcripts, strand-specific RNA-seq data were generated with RNA isolated from conidia, hyphae, and perithecia. PUK1 was almost specifically expressed in perithecia, and 90% of transcripts were edited to UG1831GUG1834G. Genome-wide analysis identified 26,056 perithecium-specific A-to-I editing sites. Unlike those in animals, 70.5% of A-to-I editing sites in F. graminearum occur in coding regions, and more than two-thirds of them result in amino acid changes, including editing of 69 PUK1-like pseudogenes with stop codons in ORFs. PUK1 orthologs and other pseudogenes also displayed stage-specific expression and editing in Neurospora crassa and F. verticillioides. Furthermore, F. graminearum differs from animals in the sequence preference and structure selectivity of A-to-I editing sites. Whereas A's embedded in RNA stems are targeted by ADARs, RNA editing in F. graminearum preferentially targets A's in hairpin loops, which is similar to the anticodon loop of tRNA targeted by adenosine deaminases acting on tRNA (ADATs). Overall, our results showed that A-to-I RNA editing occurs specifically during sexual reproduction and mainly in the coding regions in filamentous ascomycetes, involving adenosine deamination mechanisms distinct from metazoan ADARs. PMID:26934920

A Role for Adenosine Deaminase in Drosophila Larval Development

PubMed Central

Dolezal, Tomas; Dolezelova, Eva; Zurovec, Michal

2005-01-01

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organ. Here we show that the hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as was speculated previously for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals. PMID:15907156

Mutations in the ADAR1 gene in Chinese families with dyschromatosis symmetrica hereditaria.

PubMed

Zhang, G L; Shi, H J; Shao, M H; Li, M; Mu, H J; Gu, Y; Du, X F; Xie, P

2013-01-04

We investigated 2 Chinese families with dyschromatosis symmetrica hereditaria (DSH) and search for mutations in the adenosine deaminase acting on RNA1 (ADAR1) gene in these 2 pedigrees. We performed a mutation analysis of the ADAR1 gene in 2 Chinese families with DSH and reviewed all articles published regarding ADAR1 mutations reported since 2003 by using PubMed. By direct sequencing, a 2-nucleotide AG deletion, 2099-2100delAG, was found in family 1, and a C→T mutation was identified at nucleotide 1420 that changed codon 474 from arginine to a translational termination codon in family 2. Two different pathogenic mutations were identified, c.2099-2100delAG and c.1420C>T, the former being a novel mutation, and the latter previously reported in 3 other families with DSH. To date, a total of 110 mutations in the ADAR1 gene have been reported, and 10 of them were recurrent; the mutations R474X, R1083C, R1096X, and R1155W might be the DSH-related hotspots.

Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations

PubMed Central

Pfaller, Christian K.; Mastorakos, George M.; Matchett, William E.; Ma, Xiao; Samuel, Charles E.

2015-01-01

ABSTRACT Defective interfering RNAs (DI-RNAs) of the viral genome can form during infections of negative-strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document the frequent de novo generation of copy-back DI-RNAs from independent rescue events both for a vaccine measles virus (vac2) and for a wild-type measles virus (IC323) as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C-protein-deficient (C-protein-knockout [CKO]) measles viruses generated about 10 times more DI-RNAs than parental virus, suggesting that C enhances the processivity of the viral polymerase. We obtained the nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and reinitiation sites, and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA-1 (ADAR1), which catalyzes in double-stranded RNA the C-6 deamination of adenosine to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that form double-strand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced the infectivity of virus stocks. The CKO phenotype was dominant: DI-RNAs derived from vac2 with a CKO suppressed the replication of vac2, as shown by coinfections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, coinfection with a C-protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs. IMPORTANCE Recombinant measles viruses (MVs) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and

Combinatory RNA-Sequencing Analyses Reveal a Dual Mode of Gene Regulation by ADAR1 in Gastric Cancer.

PubMed

Cho, Charles J; Jung, Jaeeun; Jiang, Lushang; Lee, Eun Ji; Kim, Dae-Soo; Kim, Byung Sik; Kim, Hee Sung; Jung, Hwoon-Yong; Song, Ho-June; Hwang, Sung Wook; Park, Yangsoon; Jung, Min Kyo; Pack, Chan Gi; Myung, Seung-Jae; Chang, Suhwan

2018-04-25

Adenosine deaminase acting on RNA 1 (ADAR1) is known to mediate deamination of adenosine-to-inosine through binding to double-stranded RNA, the phenomenon known as RNA editing. Currently, the function of ADAR1 in gastric cancer is unclear. This study was aimed at investigating RNA editing-dependent and editing-independent functions of ADAR1 in gastric cancer, especially focusing on its influence on editing of 3' untranslated regions (UTRs) and subsequent changes in expression of messenger RNAs (mRNAs) as well as microRNAs (miRNAs). RNA-sequencing and small RNA-sequencing were performed on AGS and MKN-45 cells with a stable ADAR1 knockdown. Changed frequencies of editing and mRNA and miRNA expression were then identified by bioinformatic analyses. Targets of RNA editing were further validated in patients' samples. In the Alu region of both gastric cell lines, editing was most commonly of the A-to-I type in 3'-UTR or intron. mRNA and protein levels of PHACTR4 increased in ADAR1 knockdown cells, because of the loss of seed sequences in 3'-UTR of PHACTR4 mRNA that are required for miRNA-196a-3p binding. Immunohistochemical analyses of tumor and paired normal samples from 16 gastric cancer patients showed that ADAR1 expression was higher in tumors than in normal tissues and inversely correlated with PHACTR4 staining. On the other hand, decreased miRNA-148a-3p expression in ADAR1 knockdown cells led to increased mRNA and protein expression of NFYA, demonstrating ADAR1's editing-independent function. ADAR1 regulates post-transcriptional gene expression in gastric cancer through both RNA editing-dependent and editing-independent mechanisms.

Bidirectional regulation of adenosine-to-inosine (A-to-I) RNA editing by DEAH box helicase 9 (DHX9) in cancer.

PubMed

Hong, HuiQi; An, Omer; Chan, Tim H M; Ng, Vanessa H E; Kwok, Hui Si; Lin, Jaymie S; Qi, Lihua; Han, Jian; Tay, Daryl J T; Tang, Sze Jing; Yang, Henry; Song, Yangyang; Bellido Molias, Fernando; Tenen, Daniel G; Chen, Leilei

2018-05-18

Adenosine-to-inosine (A-to-I) RNA editing entails the enzymatic deamination of adenosines to inosines by adenosine deaminases acting on RNA (ADARs). Dysregulated A-to-I editing has been implicated in various diseases, including cancers. However, the precise factors governing the A-to-I editing and their physiopathological implications remain as a long-standing question. Herein, we unravel that DEAH box helicase 9 (DHX9), at least partially dependent of its helicase activity, functions as a bidirectional regulator of A-to-I editing in cancer cells. Intriguingly, the ADAR substrate specificity determines the opposing effects of DHX9 on editing as DHX9 silencing preferentially represses editing of ADAR1-specific substrates, whereas augments ADAR2-specific substrate editing. Analysis of 11 cancer types from The Cancer Genome Atlas (TCGA) reveals a striking overexpression of DHX9 in tumors. Further, tumorigenicity studies demonstrate a helicase-dependent oncogenic role of DHX9 in cancer development. In sum, DHX9 constitutes a bidirectional regulatory mode in A-to-I editing, which is in part responsible for the dysregulated editome profile in cancer.

Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations.

PubMed

Pfaller, Christian K; Mastorakos, George M; Matchett, William E; Ma, Xiao; Samuel, Charles E; Cattaneo, Roberto

2015-08-01

Defective interfering RNAs (DI-RNAs) of the viral genome can form during infections of negative-strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document the frequent de novo generation of copy-back DI-RNAs from independent rescue events both for a vaccine measles virus (vac2) and for a wild-type measles virus (IC323) as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C-protein-deficient (C-protein-knockout [CKO]) measles viruses generated about 10 times more DI-RNAs than parental virus, suggesting that C enhances the processivity of the viral polymerase. We obtained the nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and reinitiation sites, and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA-1 (ADAR1), which catalyzes in double-stranded RNA the C-6 deamination of adenosine to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that form double-strand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced the infectivity of virus stocks. The CKO phenotype was dominant: DI-RNAs derived from vac2 with a CKO suppressed the replication of vac2, as shown by coinfections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, coinfection with a C-protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs. Recombinant measles viruses (MVs) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and other infectious

A novel immune resistance mechanism of melanoma cells controlled by the ADAR1 enzyme

PubMed Central

Galore-Haskel, Gilli; Nemlich, Yael; Greenberg, Eyal; Ashkenazi, Shira; Hakim, Motti; Itzhaki, Orit; Shoshani, Noa; Shapira-Fromer, Ronnie; Ben-Ami, Eytan; Ofek, Efrat; Anafi, Liat; Besser, Michal J.

2015-01-01

The blossom of immunotherapy in melanoma highlights the need to delineate mechanisms of immune resistance. Recently, we have demonstrated that the RNA editing protein, adenosine deaminase acting on RNA-1 (ADAR1) is down-regulated during metastatic transition of melanoma, which enhances melanoma cell proliferation and tumorigenicity. Here we investigate the role of ADAR1 in melanoma immune resistance. Importantly, knockdown of ADAR1 in human melanoma cells induces resistance to tumor infiltrating lymphocytes in a cell contact-dependent mechanism. We show that ADAR1, in an editing-independent manner, regulates the biogenesis of miR-222 at the transcription level and thereby Intercellular Adhesion Molecule 1 (ICAM1) expression, which consequently affects melanoma immune resistance. ADAR1 thus has a novel, pivotal, role in cancer immune resistance. Corroborating with these results, the expression of miR-222 in melanoma tissue specimens was significantly higher in patients who had no clinical benefit from treatment with ipilimumab as compared to patients that responded clinically, suggesting that miR-222 could function as a biomarker for the prediction of response to ipilimumab. These results provide not only novel insights on melanoma immune resistance, but also pave the way to the development of innovative personalized tools to enable optimal drug selection and treatment. PMID:26338962

Hyperactive hypothalamus, motivated and non-distractible chronic overeating in ADAR2 transgenic mice

PubMed Central

Akubuiro, Ashley; Zimmerman, M. Bridget; Boles Ponto, Laura L.; Walsh, Susan A.; Sunderland, John; McCormick, Laurie; Singh, Minati

2013-01-01

ADAR2 transgenic mice misexpressing the RNA editing enzyme ADAR2 (Adenosine Deaminase that act on RNA) show characteristics of overeating and experience adult onset obesity. Behavioral patterns and brain changes related to a possible addictive overeating in these transgenic mice were explored as transgenic mice display chronic hyperphagia. ADAR2 transgenic mice were assessed in their food preference and motivation to overeat in a competing reward environment with ad lib access to a running wheel and food. Metabolic activity of brain and peripheral tissue were assessed with [18F] fluorodeoxyglucose positron emission tomography (FDG-PET) and RNA expression of feeding related genes, ADAR2, dopamine and opiate receptors from the hypothalamus and striatum were examined. The results indicate that ADAR2 transgenic mice exhibit, (1) a food preference for diets with higher fat content, (2) significantly increased food intake that is non-distractible in a competing reward environment, (3) significantly increased mRNA expressions of ADAR2, serotonin 2C receptor (5HT2CR), D1, D2, and mu opioid receptors and no change in CRH mRNAs and significantly reduced ADAR2 protein expression in the hypothalamus, (4) significantly increased D1 receptor and altered bioamines with no change in ADAR2, mu opioid and D2 receptor mRNA expression in the striatum, and (5) significantly greater glucose metabolism in the hypothalamus, brain stem, right hippocampus, left and right mid brain regions and suprascapular peripheral tissue than controls. These results suggest that highly motivated and goal-oriented overeating behaviors of ADAR2 transgenic mice are associated with altered feeding, reward-related mRNAs, and hyperactive brain mesolimbic region. PMID:23323881

ADAR1 deletion induces NFκB and interferon signaling dependent liver inflammation and fibrosis.

PubMed

Ben-Shoshan, Shirley Oren; Kagan, Polina; Sultan, Maya; Barabash, Zohar; Dor, Chen; Jacob-Hirsch, Jasmine; Harmelin, Alon; Pappo, Orit; Marcu-Malina, Victoria; Ben-Ari, Ziv; Amariglio, Ninette; Rechavi, Gideon; Goldstein, Itamar; Safran, Michal

2017-05-04

Adenosine deaminase acting on RNA (ADAR) 1 binds and edits double-stranded (ds) RNA secondary structures found mainly within untranslated regions of many transcripts. In the current research, our aim was to study the role of ADAR1 in liver homeostasis. As previous studies show a conserved immunoregulatory function for ADAR1 in mammalians, we focused on its role in preventing chronic hepatic inflammation and the associated activation of hepatic stellate cells to produce extracellular matrix and promote fibrosis. We show that hepatocytes specific ADAR1 knock out (KO) mice display massive liver damage with multifocal inflammation and fibrogenesis. The bioinformatics analysis of the microarray gene-expression datasets of ADAR1 KO livers reveled a type-I interferons signature and an enrichment for immune response genes compared to control littermate livers. Furthermore, we found that in vitro silencing of ADAR1 expression in HepG2 cells leads to enhanced transcription of NFκB target genes, foremost of the pro-inflammatory cytokines IL6 and IL8. We also discovered immune cell-independent paracrine signaling among ADAR1-depleted HepG2 cells and hepatic stellate cells, leading to the activation of the latter cell type to adopt a profibrogenic phenotype. This paracrine communication dependent mainly on the production and secretion of the cytokine IL6 induced by ADAR1 silencing in hepatocytes. Thus, our findings shed a new light on the vital regulatory role of ADAR1 in hepatic immune homeostasis, chiefly its inhibitory function on the crosstalk between the NFκB and type-I interferons signaling cascades, restraining the development of liver inflammation and fibrosis.

ADAR1 deletion induces NFκB and interferon signaling dependent liver inflammation and fibrosis

PubMed Central

Ben-Shoshan, Shirley Oren; Kagan, Polina; Sultan, Maya; Barabash, Zohar; Dor, Chen; Jacob-Hirsch, Jasmine; Harmelin, Alon; Pappo, Orit; Marcu-Malina, Victoria; Ben-Ari, Ziv; Amariglio, Ninette; Rechavi, Gideon; Goldstein, Itamar; Safran, Michal

2017-01-01

ABSTRACT Adenosine deaminase acting on RNA (ADAR) 1 binds and edits double-stranded (ds) RNA secondary structures found mainly within untranslated regions of many transcripts. In the current research, our aim was to study the role of ADAR1 in liver homeostasis. As previous studies show a conserved immunoregulatory function for ADAR1 in mammalians, we focused on its role in preventing chronic hepatic inflammation and the associated activation of hepatic stellate cells to produce extracellular matrix and promote fibrosis. We show that hepatocytes specific ADAR1 knock out (KO) mice display massive liver damage with multifocal inflammation and fibrogenesis. The bioinformatics analysis of the microarray gene-expression datasets of ADAR1 KO livers reveled a type-I interferons signature and an enrichment for immune response genes compared to control littermate livers. Furthermore, we found that in vitro silencing of ADAR1 expression in HepG2 cells leads to enhanced transcription of NFκB target genes, foremost of the pro-inflammatory cytokines IL6 and IL8. We also discovered immune cell-independent paracrine signaling among ADAR1-depleted HepG2 cells and hepatic stellate cells, leading to the activation of the latter cell type to adopt a profibrogenic phenotype. This paracrine communication dependent mainly on the production and secretion of the cytokine IL6 induced by ADAR1 silencing in hepatocytes. Thus, our findings shed a new light on the vital regulatory role of ADAR1 in hepatic immune homeostasis, chiefly its inhibitory function on the crosstalk between the NFκB and type-I interferons signaling cascades, restraining the development of liver inflammation and fibrosis. PMID:27362366

Hyperactive hypothalamus, motivated and non-distractible chronic overeating in ADAR2 transgenic mice.

PubMed

Akubuiro, A; Bridget Zimmerman, M; Boles Ponto, L L; Walsh, S A; Sunderland, J; McCormick, L; Singh, M

2013-04-01

ADAR2 transgenic mice misexpressing the RNA editing enzyme ADAR2 (Adenosine Deaminase that act on RNA) show characteristics of overeating and experience adult onset obesity. Behavioral patterns and brain changes related to a possible addictive overeating in these transgenic mice were explored as transgenic mice display chronic hyperphagia. ADAR2 transgenic mice were assessed in their food preference and motivation to overeat in a competing reward environment with ad lib access to a running wheel and food. Metabolic activity of brain and peripheral tissue were assessed with [(18) F] fluorodeoxyglucose positron emission tomography (FDG-PET) and RNA expression of feeding related genes, ADAR2, dopamine and opiate receptors from the hypothalamus and striatum were examined. The results indicate that ADAR2 transgenic mice exhibit, (1) a food preference for diets with higher fat content, (2) significantly increased food intake that is non-distractible in a competing reward environment, (3) significantly increased messenger RNA (mRNA) expressions of ADAR2, serotonin 2C receptor (5HT2C R), D1, D2 and mu opioid receptors and no change in corticotropin-releasing hormone mRNAs and significantly reduced ADAR2 protein expression in the hypothalamus, (4) significantly increased D1 receptor and altered bioamines with no change in ADAR2, mu opioid and D2 receptor mRNA expression in the striatum and (5) significantly greater glucose metabolism in the hypothalamus, brain stem, right hippocampus, left and right mid brain regions and suprascapular peripheral tissue than controls. These results suggest that highly motivated and goal-oriented overeating behaviors of ADAR2 transgenic mice are associated with altered feeding, reward-related mRNAs and hyperactive brain mesolimbic region. Genes, Brain and Behavior © 2013 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.

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

PubMed Central

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

2011-01-01

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

Understanding the recognition mechanisms of Zα domain of human editing enzyme ADAR1 (hZα(ADAR1)) and various Z-DNAs from molecular dynamics simulation.

PubMed

Wang, Qianqian; Li, Lanlan; Wang, Xiaoting; Liu, Huanxiang; Yao, Xiaojun

2014-11-01

The Z-DNA-binding domain of human double-stranded RNA adenosine deaminase I (hZαADAR1) can specifically recognize the left-handed Z-DNA which preferentially occurs at alternating purine-pyrimidine repeats, especially the CG-repeats. The interactions of hZαADAR1 and Z-DNAs in different sequence contexts can affect many important biological functions including gene regulation and chromatin remodeling. Therefore it is of great necessity to fully understand their recognition mechanisms. However, most existing studies are aimed at the standard CG-repeat Z-DNA rather than the non-CG-repeats, and whether the molecular basis of hZαADAR1 binding to various Z-DNAs are identical or not is still unclear on the atomic level. Here, based on the recently determined crystal structures of three representative non-CG-repeat Z-DNAs (d(CACGTG)2, d(CGTACG)2 and d(CGGCCG)2) in complex with hZαADAR1, 40 ns molecular dynamics simulation together with binding free energy calculation were performed for each system. For comparison, the standard CG-repeat Z-DNA (d(CGCGCG)2) complexed with hZαADAR1 was also simulated. The consistent results demonstrate that nonpolar interaction is the driving force during the protein-DNA binding process, and that polar interaction mainly from helix α3 also provides important contributions. Five common hot-spot residues were identified, namely Lys169, Lys170, Asn173, Arg174 and Tyr177. Hydrogen bond analysis coupled with surface charge distribution further reveal the interfacial information between hZαADAR1 and Z-DNA in detail. All of the analysis illustrate that four complexes share the common key features and the similar binding modes irrespective of Z-DNA sequences, suggesting that Z-DNA recognition by hZαADAR1 is conformation-specific rather than sequence-specific. Additionally, by analyzing the conformational changes of hZαADAR1, we found that the binding of Z-DNA could effectively stabilize hZαADAR1 protein. Our study can provide some valuable

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matthews, Melissa M.; Thomas, Justin M.; Zheng, Yuxuan

Adenosine deaminases acting on RNA (ADARs) are editing enzymes that convert adenosine to inosine in duplex RNA, a modification reaction with wide-ranging consequences in RNA function. Understanding of the ADAR reaction mechanism, the origin of editing-site selectivity, and the effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. In this paper, we describe four crystal structures of the human ADAR2 deaminase domain bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis of the ADARmore » deaminase domain's dsRNA specificity, its base-flipping mechanism, and its nearest-neighbor preferences. In addition, we identified an ADAR2-specific RNA-binding loop near the enzyme active site, thus rationalizing differences in selectivity observed between different ADARs. In conclusion, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease.« less

Deficient RNA-editing enzyme ADAR2 in an amyotrophic lateral sclerosis patient with a FUS(P525L) mutation.

PubMed

Aizawa, Hitoshi; Hideyama, Takuto; Yamashita, Takenari; Kimura, Takashi; Suzuki, Naoki; Aoki, Masashi; Kwak, Shin

2016-10-01

Mutations in the fused in sarcoma (FUS) gene can cause amyotrophic lateral sclerosis (ALS), and FUS gene mutations have been reported in sporadic ALS patients with basophilic cytoplasmic inclusions. Deficiency of adenosine deaminase acting on RNA 2 (ADAR2), an enzyme that specifically catalyzes GluA2 Q/R site-editing, has been reported in considerable proportions of spinal motor neurons of the majority of sporadic ALS patients. We describe the relationship between GluA2 Q/R site-editing efficiency and FUS-positive inclusions in a patient with FUS(P525L). A 24-year-old woman with ALS presented with basophilic cytoplasmic inclusions, significantly reduced GluA2 Q/R site-editing efficiency in the spinal motor neurons, and markedly decreased ADAR2 mRNA levels. Neuropathologic examination showed that not all spinal motor neurons expressed ADAR2 and revealed FUS-positive cytoplasmic inclusions in motor neurons irrespective of ADAR2 immunoreactivity. There were no phosphorylated transactive response (TAR) DNA-binding protein 43 kDa (TDP-43)-positive inclusions, indicating that there was no tight correlation between ADAR2 deficiency and TDP-43 deposition. ADAR2 deficiency can occur in ALS patients with a FUS(P525L) mutation and is unrelated to the presence of FUS-positive inclusions. FUS-associated ALS may share neurodegenerative characteristics with classical sporadic ALS. Copyright © 2016 Elsevier Ltd. All rights reserved.

RNA editing enzyme ADAR2 is a mediator of neuropathic pain after peripheral nerve injury.

PubMed

Uchida, Hitoshi; Matsumura, Shinji; Okada, Shunpei; Suzuki, Tsutomu; Minami, Toshiaki; Ito, Seiji

2017-05-01

Transcriptional and post-translational regulations are important in peripheral nerve injury-induced neuropathic pain, but little is known about the role of post-transcriptional modification. Our objective was to determine the possible effect of adenosine deaminase acting on RNA (ADAR) enzymes, which catalyze post-transcriptional RNA editing, in tactile allodynia, a hallmark of neuropathic pain. Seven days after L5 spinal nerve transection (SNT) in adult mice, we found an increase in ADAR2 expression and a decrease in ADAR3 expression in the injured, but not in the uninjured, dorsal root ganglions (DRGs). These changes were accompanied by elevated levels of editing at the D site of the serotonin (5-hydroxytryptamine) 2C receptor (5-HT 2C R), at the I/V site of coatomer protein complex subunit α (COPA), and at the R/G site of AMPA receptor subunit GluA2 in the injured DRG. Compared to Adar2 +/+ /Gria2 R/R littermate controls, Adar2 -/- /Gria2 R/R mice completely lacked the increased editing of 5-HT 2C R, COPA, and GluA2 transcripts in the injured DRG and showed attenuated tactile allodynia after SNT. Furthermore, the antidepressant fluoxetine inhibited neuropathic allodynia after injury and reduced the COPA I/V site editing in the injured DRG. These findings suggest that ADAR2 is a mediator of injury-induced tactile allodynia and thus a potential therapeutic target for the treatment of neuropathic pain.-Uchida, H., Matsumura, S., Okada, S., Suzuki, T., Minami, T., Ito, S. RNA editing enzyme ADAR2 is a mediator of neuropathic pain after peripheral nerve injury. © FASEB.

Effects of social isolation and re-socialization on cognition and ADAR1 (p110) expression in mice.

PubMed

Chen, Wei; An, Dong; Xu, Hong; Cheng, Xiaoxin; Wang, Shiwei; Yu, Weizhi; Yu, Deqin; Zhao, Dan; Sun, Yiping; Deng, Wuguo; Tang, Yiyuan; Yin, Shengming

2016-01-01

It has been reported that social isolation stress could be a key factor that leads to cognitive deficit for both humans and rodent models. However, detailed mechanisms are not yet clear. ADAR1 (Adenosine deaminase acting on RNA) is an enzyme involved in RNA editing that has a close relation to cognitive function. We have hypothesized that social isolation stress may impact the expression of ADAR1 in the brain of mice with cognitive deficit. To test our hypothesis, we evaluated the cognition ability of mice isolated for different durations (2, 4, and 8 weeks) using object recognition and object location tests; we also measured ADAR1 expression in hippocampus and cortex using immunohistochemistry and western blot. Our study showed that social isolation stress induced spatial and non-spatial cognition deficits of the tested mice. In addition, social isolation significantly increased both the immunoreactivity and protein expression of ADAR1 (p110) in the hippocampus and frontal cortex. Furthermore, re-socialization could not only recover the cognition deficits, but also bring ADAR1 (p110) immunoreactivity of hippocampus and frontal cortex, as well as ADAR1 (p110) protein expression of hippocampus back to the normal level for the isolated mice in adolescence. In conclusion, social isolation stress significantly increases ADAR1 (p110) expression in the hippocampus and frontal cortex of the mice with cognitive deficit. This finding may open a window to better understand the reasons (e.g., epigenetic change) that are responsible for social isolation-induced cognitive deficit and help the development of novel therapies for the resulted diseases.

Optimising the utility of pleural fluid adenosine deaminase for the diagnosis of adult tuberculous pleural effusion in Hong Kong.

PubMed

Chang, K C; Chan, M C; Leung, W M; Kong, F Y; Mak, C M; Chen, S Pl; Yu, W C

2018-02-01

Pleural fluid adenosine deaminase level can be applied to rapidly detect tuberculous pleural effusion. We aimed to establish a local diagnostic cut-off value for pleural fluid adenosine deaminase to identify patients with tuberculous pleural effusion, and optimise its utility. We retrospectively reviewed the medical records of consecutive adults with pleural fluid adenosine deaminase level measured by the Diazyme commercial kit (Diazyme Laboratories, San Diego [CA], United States) during 1 January to 31 December 2011 in a cluster of public hospitals in Hong Kong. We considered its level alongside early (within 2 weeks) findings in pleural fluid and pleural biopsy, with and without applying Light's criteria in multiple scenarios. For each scenario, we used the receiver operating characteristic curve to identify a diagnostic cut-off value for pleural fluid adenosine deaminase, and estimated its positive and negative predictive values. A total of 860 medical records were reviewed. Pleural effusion was caused by congestive heart failure, chronic renal failure, or hypoalbuminaemia caused by liver or kidney diseases in 246 (28.6%) patients, malignancy in 198 (23.0%), non-tuberculous infection in 168 (19.5%), tuberculous pleural effusion in 157 (18.3%), and miscellaneous causes in 91 (10.6%). All those with tuberculous pleural effusion had a pleural fluid adenosine deaminase level of ≤100 U/L. When analysis was restricted to 689 patients with pleural fluid adenosine deaminase level of ≤100 U/L and early negative findings for malignancy and non-tuberculous infection in pleural fluid, the positive predictive value was significantly increased and the negative predictive value non-significantly reduced. Using this approach, neither additionally restricting analysis to exudates by Light's criteria nor adding closed pleural biopsy would further enhance predictive values. As such, the diagnostic cut-off value for pleural fluid adenosine deaminase is 26.5 U/L, with a

Cell death cascade and molecular therapy in ADAR2-deficient motor neurons of ALS.

PubMed

Yamashita, Takenari; Kwak, Shin

2018-06-23

TAR DNA-binding protein (TDP-43) pathology in the motor neurons is the most reliable pathological hallmark of amyotrophic lateral sclerosis (ALS), and motor neurons bearing TDP-43 pathology invariably exhibit failure in RNA editing at the GluA2 glutamine/arginine (Q/R) site due to down-regulation of adenosine deaminase acting on RNA 2 (ADAR2). Conditional ADAR2 knockout (AR2) mice display ALS-like phenotype, including progressive motor dysfunction due to loss of motor neurons. Motor neurons devoid of ADAR2 express Q/R site-unedited GluA2, and AMPA receptors with unedited GluA2 in their subunit assembly are abnormally permeable to Ca 2+ , which results in progressive neuronal death. Moreover, analysis of AR2 mice has demonstrated that exaggerated Ca 2+ influx through the abnormal AMPA receptors overactivates calpain, a Ca 2+ -dependent protease, that cleaves TDP-43 into aggregation-prone fragments, which serve as seeds for TDP-43 pathology. Activated calpain also disrupts nucleo-cytoplasmic transport and gene expression by cleaving molecules involved in nucleocytoplasmic transport, including nucleoporins. These lines of evidence prompted us to develop molecular targeting therapy for ALS by normalization of disrupted intracellular environment due to ADAR2 down-regulation. In this review, we have summarized the work from our group on the cell death cascade in sporadic ALS and discussed a potential therapeutic strategy for ALS. Copyright © 2018 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Evidence for ADAR-induced hypermutation of the Drosophila sigma virus (Rhabdoviridae).

PubMed

Carpenter, Jennifer A; Keegan, Liam P; Wilfert, Lena; O'Connell, Mary A; Jiggins, Francis M

2009-11-26

ADARs are RNA editing enzymes that target double stranded RNA and convert adenosine to inosine, which is read by translation machinery as if it were guanosine. Aside from their role in generating protein diversity in the central nervous system, ADARs have been implicated in the hypermutation of some RNA viruses, although why this hypermutation occurs is not well understood. Here we describe the hypermutation of adenosines to guanosines in the genome of the sigma virus--a negative sense RNA virus that infects Drosophila melanogaster. The clustering of these mutations and the context in which they occur indicates that they have been caused by ADARs. However, ADAR-editing of viral RNA is either rare or edited viral RNA are rapidly degraded, as we only detected evidence for editing in two of the 104 viral isolates we studied. This is the first evidence for ADARs targeting viruses outside of mammals, and it raises the possibility that ADARs could play a role in the antiviral defences of insects.

A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

PubMed

Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

2011-05-01

A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

Positive correlation between ADAR expression and its targets suggests a complex regulation mediated by RNA editing in the human brain

PubMed Central

Liscovitch, Noa; Bazak, Lily; Levanon, Erez Y; Chechik, Gal

2014-01-01

A-to-I RNA editing by adenosine deaminases acting on RNA is a post-transcriptional modification that is crucial for normal life and development in vertebrates. RNA editing has been shown to be very abundant in the human transcriptome, specifically at the primate-specific Alu elements. The functional role of this wide-spread effect is still not clear; it is believed that editing of transcripts is a mechanism for their down-regulation via processes such as nuclear retention or RNA degradation. Here we combine 2 neural gene expression datasets with genome-level editing information to examine the relation between the expression of ADAR genes with the expression of their target genes. Specifically, we computed the spatial correlation across structures of post-mortem human brains between ADAR and a large set of targets that were found to be edited in their Alu repeats. Surprisingly, we found that a large fraction of the edited genes are positively correlated with ADAR, opposing the assumption that editing would reduce expression. When considering the correlations between ADAR and its targets over development, 2 gene subsets emerge, positively correlated and negatively correlated with ADAR expression. Specifically, in embryonic time points, ADAR is positively correlated with many genes related to RNA processing and regulation of gene expression. These findings imply that the suggested mechanism of regulation of expression by editing is probably not a global one; ADAR expression does not have a genome wide effect reducing the expression of editing targets. It is possible, however, that RNA editing by ADAR in non-coding regions of the gene might be a part of a more complex expression regulation mechanism. PMID:25692240

Positive correlation between ADAR expression and its targets suggests a complex regulation mediated by RNA editing in the human brain.

PubMed

Liscovitch, Noa; Bazak, Lily; Levanon, Erez Y; Chechik, Gal

2014-01-01

A-to-I RNA editing by adenosine deaminases acting on RNA is a post-transcriptional modification that is crucial for normal life and development in vertebrates. RNA editing has been shown to be very abundant in the human transcriptome, specifically at the primate-specific Alu elements. The functional role of this wide-spread effect is still not clear; it is believed that editing of transcripts is a mechanism for their down-regulation via processes such as nuclear retention or RNA degradation. Here we combine 2 neural gene expression datasets with genome-level editing information to examine the relation between the expression of ADAR genes with the expression of their target genes. Specifically, we computed the spatial correlation across structures of post-mortem human brains between ADAR and a large set of targets that were found to be edited in their Alu repeats. Surprisingly, we found that a large fraction of the edited genes are positively correlated with ADAR, opposing the assumption that editing would reduce expression. When considering the correlations between ADAR and its targets over development, 2 gene subsets emerge, positively correlated and negatively correlated with ADAR expression. Specifically, in embryonic time points, ADAR is positively correlated with many genes related to RNA processing and regulation of gene expression. These findings imply that the suggested mechanism of regulation of expression by editing is probably not a global one; ADAR expression does not have a genome wide effect reducing the expression of editing targets. It is possible, however, that RNA editing by ADAR in non-coding regions of the gene might be a part of a more complex expression regulation mechanism.

How We Manage Adenosine Deaminase-Deficient Severe Combined Immune Deficiency (ADA SCID).

PubMed

Kohn, Donald B; Gaspar, H Bobby

2017-05-01

Adenosine deaminase-deficient severe combined immune deficiency (ADA SCID) accounts for 10-15% of cases of human SCID. From what was once a uniformly fatal disease, the prognosis for infants with ADA SCID has improved greatly based on the development of multiple therapeutic options, coupled with more frequent early diagnosis due to implementation of newborn screening for SCID. We review the various treatment approaches for ADA SCID including allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen-matched sibling or family member or from a matched unrelated donor or a haplo-identical donor, autologous HSCT with gene correction of the hematopoietic stem cells (gene therapy-GT), and enzyme replacement therapy (ERT) with polyethylene glycol-conjugated adenosine deaminase. Based on growing evidence of safety and efficacy from GT, we propose a treatment algorithm for patients with ADA SCID that recommends HSCT from a matched family donor, when available, as a first choice, followed by GT as the next option, with allogeneic HSCT from an unrelated or haplo-identical donor or long-term ERT as other options.

Large-scale prediction of ADAR-mediated effective human A-to-I RNA editing.

PubMed

Yao, Li; Wang, Heming; Song, Yuanyuan; Dai, Zhen; Yu, Hao; Yin, Ming; Wang, Dongxu; Yang, Xin; Wang, Jinlin; Wang, Tiedong; Cao, Nan; Zhu, Jimin; Shen, Xizhong; Song, Guangqi; Zhao, Yicheng

2017-08-10

Adenosine-to-inosine (A-to-I) editing by adenosine deaminase acting on the RNA (ADAR) proteins is one of the most frequent modifications during post- and co-transcription. To facilitate the assignment of biological functions to specific editing sites, we designed an automatic online platform to annotate A-to-I RNA editing sites in pre-mRNA splicing signals, microRNAs (miRNAs) and miRNA target untranslated regions (3' UTRs) from human (Homo sapiens) high-throughput sequencing data and predict their effects based on large-scale bioinformatic analysis. After analysing plenty of previously reported RNA editing events and human normal tissues RNA high-seq data, >60 000 potentially effective RNA editing events on functional genes were found. The RNA Editing Plus platform is available for free at https://www.rnaeditplus.org/, and we believe our platform governing multiple optimized methods will improve further studies of A-to-I-induced editing post-transcriptional regulation. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Patterns of developmental expression of the RNA editing enzyme rADAR2.

PubMed

Paupard M-C; O'Connell, M A; Gerber, A P; Zukin, R S

2000-01-01

To date, two structurally related RNA-editing enzymes with adenosine deaminase activity have been identified in mammalian tissue: ADAR1 and ADAR2 [Bass B. I. et al. (1997) RNA 3, 947-949]. In rodents, ADAR2 undergoes alternative RNA splicing, giving rise to two splice variants that differ by the presence or absence of a 10-amino-acid insert in the carboxy-terminal catalytic domain. However, the physiological significance of the splicing and its regional and developmental regulation are as yet unknown. The present study examined spatial and temporal patterns of ADAR2 gene transcripts within specific neuronal populations of rat brain. The two rodent ADAR2 isoforms were expressed at comparable levels at all ages examined. rADAR2 messenger RNA expression was first detectable in the thalamic nuclei formation at embryonic day E19. The rADAR2b insert and rADAR2a splice probes produced images similar to that of the rADAR2 pan probe. At birth, rADAR2a messenger RNA splice variants were abundantly expressed in the thalamic nuclei. No signal for any probe was detectable in other brain regions, including neocortex, hippocampus, striatum and cerebellum at this stage of development. During the first week of postnatal life, rADAR2 messenger RNA expression (detected with the pan probe) increased gradually in several brain regions, with low expression detected at postnatal day P7 in the olfactory bulb, inferior colliculus, and within the pyramidal and granule cell layers of the hippocampus. Hybridization patterns of the rADAR2a variant probe reached peak expression at about the second week of life, while peak expression of the rADAR2b probe was reached at about the third week of life. At the end of the first week of life (P7), expression of both splice variants was strongest in the thalamic nuclei. By P14, rADAR2 messenger RNA expression was more consolidated in the deeper structures, including the thalamic nuclei and the granule cell layer of the cerebellum. By P21, maximal levels

Applying Human ADAR1p110 and ADAR1p150 for Site-Directed RNA Editing-G/C Substitution Stabilizes GuideRNAs against Editing.

PubMed

Heep, Madeleine; Mach, Pia; Reautschnig, Philipp; Wettengel, Jacqueline; Stafforst, Thorsten

2017-01-14

Site-directed RNA editing is an approach to reprogram genetic information at the RNA level. We recently introduced a novel guideRNA that allows for the recruitment of human ADAR2 to manipulate genetic information. Here, we show that the current guideRNA design is already able to recruit another human deaminase, ADAR1, in both isoforms, p110 and p150. However, further optimization seems necessary as the current design is less efficient for ADAR1 isoforms. Furthermore, we describe hotspots at which the guideRNA itself is edited and show a way to circumvent this auto-editing without losing editing efficiency at the target. Both findings are important for the advancement of site-directed RNA editing as a tool in basic biology or as a platform for therapeutic editing.

Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1

NASA Technical Reports Server (NTRS)

Schwartz, T.; Lowenhaupt, K.; Kim, Y. G.; Li, L.; Brown, B. A. 2nd; Herbert, A.; Rich, A.

1999-01-01

Zalpha is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zalpha is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1), a candidate enzyme for nuclear pre-mRNA editing in mammals. Zalpha is conserved in ADAR1 from many species; in each case, there is a second similar motif, Zbeta, separated from Zalpha by a more divergent linker. To investigate the structure-function relationship of Zalpha, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zalpha is part of a domain, Zab, of 229 amino acids (residues 133-361 in human ADAR1). This domain contains both Zalpha and Zbeta as well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133-209), containing only Zalpha, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zbeta, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zalpha and Zbeta act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.

Diagnostic value of serum adenosine deaminase activity in HIV infected patients of Kurdish population.

PubMed

Abdi, Mohammad; Ahmadi, Abbas; Roshany, Daem; Khodadadi, Iraj; Javid, Saman; Shahmohammad-Nezhad, Shiva; Sharifipour, Mozhdeh; Hoseini, Javad

2013-01-01

Adenosine deaminase (ADA) is a hydrolytic enzyme involved in the deamination of adenosine to inosine. ADA is involved in T-lymphocyte differentiation and development. This study was aimed to determine the diagnostic value of the adenosine deaminase (ADA) activity test for the diagnosis of HIV positive patients in the Kurdish population. This descriptive analytical case-control study was performed on 30 healthy and 60 HIV positive subjects. Blood CD4+ cell count was recorded and serum total ADA, and ADA1 and ADA2 isoenzyme activities were determined. Serum total ADA and ADA2 isoenzyme activity was significantly higher in HIV positive patients than in healthy subjects. CD4+ cell counts markedly decreased in all patients and showed a significant inverse correlation with ADA activities. Using a cut-off level of 36.52 U/L and 30.98 U/L for serum total ADA and ADA2, respectively, sensitivity and specificity were 90.9% and 90.27% for total ADA and 93% and 90% for ADA2, respectively. Serum ADA was significantly increased in HIV infected patients. Therefore, because of its low cost and simplicity to perform, ADA activity might be considered a useful diagnostic tool among the other markers in this disease.

Evaluation of the adenosine deaminase (ADA) G22A gene polymorphism with recurrent spontaneous abortion among Egyptian patients.

PubMed

Farhan, Hanan Mohamed; Abu-Gabal, Khadiga; Katta, Maha; Ibrahim, Raghda

2017-01-01

Adenosine and deoxyadenosine metabolism is influenced by adenosine deaminase (ADA) enzyme. ADA increases in different diseases and is considered as one of the markers for cell-mediated immunity. Pregnancy is associated with depressed cell-mediated immunity. The level of ADA expression, which seems to play a key role in maintaining pregnancy, is influenced by adenosine deaminase G22A gene polymorphism. We aimed in our study to evaluate the association of ADA G22A gene polymorphism with recurrent spontaneous abortion (RSA) in Egyptian women. Adenosine deaminase G22A gene polymorphism was genotyped in 40 patients (age range 22-39 years) with a history of RSA, selected from those attending the Gynaecology and Obstetrics Clinic of Beni-Suef University Hospital, and 20 age-matched healthy women as a control group, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. In our study, no statistically significant difference was found between RSA patients and control group as regards ADA G22A genotypes (p = 0.653) and alleles (p = 0.697). A comparison of the frequencies of ADA alleles in RSA patients as regards the below-35-years-old age group revealed that ADA 2(A) allele was associated with a low risk for RSA in patients aged 35 years old or younger (p = 0.008). In conclusion, our study revealed an age-dependent protective value of ADA 2(A) allele in recurrent spontaneous abortions among the Egyptian population.

C. elegans ADARs antagonize silencing of cellular dsRNAs by the antiviral RNAi pathway.

PubMed

Reich, Daniel P; Tyc, Katarzyna M; Bass, Brenda L

2018-02-01

Cellular dsRNAs are edited by adenosine deaminases that act on RNA (ADARs). While editing can alter mRNA-coding potential, most editing occurs in noncoding sequences, the function of which is poorly understood. Using dsRNA immunoprecipitation (dsRIP) and RNA sequencing (RNA-seq), we identified 1523 regions of clustered A-to-I editing, termed editing-enriched regions (EERs), in four stages of Caenorhabditis elegans development, often with highest expression in embryos. Analyses of small RNA-seq data revealed 22- to 23-nucleotide (nt) siRNAs, reminiscent of viral siRNAs, that mapped to EERs and were abundant in adr-1;adr-2 mutant animals. Consistent with roles for these siRNAs in silencing, EER-associated genes (EAGs) were down-regulated in adr-1;adr-2 embryos, and this was dependent on associated EERs and the RNAi factor RDE-4. We observed that ADARs genetically interact with the 26G endogenous siRNA (endo-siRNA) pathway, which likely competes for RNAi components; deletion of factors required for this pathway ( rrf-3 or ergo-1 ) in adr-1;adr-2 mutant strains caused a synthetic phenotype that was rescued by deleting antiviral RNAi factors. Poly(A) + RNA-seq revealed EAG down-regulation and antiviral gene induction in adr-1;adr-2;rrf-3 embryos, and these expression changes were dependent on rde-1 and rde-4 Our data suggest that ADARs restrict antiviral silencing of cellular dsRNAs. © 2018 Reich et al.; Published by Cold Spring Harbor Laboratory Press.

CONTRASTING BEHAVIOR OF CONFORMATIONALLY LOCKED CARBOCYCLIC NUCLEOSIDES OF ADENOSINE AND CYTIDINE AS SUBSTRATES FOR DEAMINASES

PubMed Central

Schroeder, Gottfried K.; Ludek, Olaf R.; Siddiqui, Maqbool A.; Ezzitouni, Abdallah; Wolfenden, Richard

2010-01-01

In addition to the already known differences between adenosine deaminase (ADA) and cytidine deaminase (CDA) in terms of their tertiary structure, the sphere of Zn+2 coordination, and their reverse stereochemical preference, we present evidence that the enzymes also differ significantly in terms of the North/South conformational preferences for their substrates and the extent to which the lack of the O(4’) oxygen affects the kinetics of the enzymatic deamination of carbocyclic substrates. The carbocyclic nucleoside substrates used in this study have either a flexible cyclopentane ring or a rigid bicyclo[3.1.0]hexane scaffold. PMID:20183605

The RNA-Editing Enzyme ADAR1 Controls Innate Immune Responses to RNA

PubMed Central

Mannion, Niamh M.; Greenwood, Sam M.; Young, Robert; Cox, Sarah; Brindle, James; Read, David; Nellåker, Christoffer; Vesely, Cornelia; Ponting, Chris P.; McLaughlin, Paul J.; Jantsch, Michael F.; Dorin, Julia; Adams, Ian R.; Scadden, A.D.J.; Öhman, Marie; Keegan, Liam P.; O’Connell, Mary A.

2014-01-01

Summary The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform. PMID:25456137

The RNA-editing enzyme ADAR1 controls innate immune responses to RNA.

PubMed

Mannion, Niamh M; Greenwood, Sam M; Young, Robert; Cox, Sarah; Brindle, James; Read, David; Nellåker, Christoffer; Vesely, Cornelia; Ponting, Chris P; McLaughlin, Paul J; Jantsch, Michael F; Dorin, Julia; Adams, Ian R; Scadden, A D J; Ohman, Marie; Keegan, Liam P; O'Connell, Mary A

2014-11-20

The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Evaluation of the adenosine deaminase (ADA) G22A gene polymorphism with recurrent spontaneous abortion among Egyptian patients

PubMed Central

Abu-Gabal, Khadiga; Katta, Maha; Ibrahim, Raghda

2017-01-01

Introduction Adenosine and deoxyadenosine metabolism is influenced by adenosine deaminase (ADA) enzyme. ADA increases in different diseases and is considered as one of the markers for cell-mediated immunity. Pregnancy is associated with depressed cell-mediated immunity. The level of ADA expression, which seems to play a key role in maintaining pregnancy, is influenced by adenosine deaminase G22A gene polymorphism. We aimed in our study to evaluate the association of ADA G22A gene polymorphism with recurrent spontaneous abortion (RSA) in Egyptian women. Material and methods Adenosine deaminase G22A gene polymorphism was genotyped in 40 patients (age range 22-39 years) with a history of RSA, selected from those attending the Gynaecology and Obstetrics Clinic of Beni-Suef University Hospital, and 20 age-matched healthy women as a control group, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Results In our study, no statistically significant difference was found between RSA patients and control group as regards ADA G22A genotypes (p = 0.653) and alleles (p = 0.697). A comparison of the frequencies of ADA alleles in RSA patients as regards the below-35-years-old age group revealed that ADA 2(A) allele was associated with a low risk for RSA in patients aged 35 years old or younger (p = 0.008). Conclusions In conclusion, our study revealed an age-dependent protective value of ADA 2(A) allele in recurrent spontaneous abortions among the Egyptian population. PMID:29204093

Conformational change of adenosine deaminase during ligand-exchange in a crystal.

PubMed

Kinoshita, Takayoshi; Tada, Toshiji; Nakanishi, Isao

2008-08-15

Adenosine deaminase (ADA) perpetuates chronic inflammation by degrading extracellular adenosine which is toxic for lymphocytes. ADA has two distinct conformations: open form and closed form. From the crystal structures with various ligands, the non-nucleoside type inhibitors bind to the active site occupying the critical water-binding-position and sustain the open form of apo-ADA. In contrast, substrate mimics do not occupy the critical position, and induce the large conformational change to the closed form. However, it is difficult to predict the binding of (+)-erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), as it possesses characteristic parts of both the substrate and the non-nucleoside inhibitors. The crystal structure shows that EHNA binds to the open form through a novel recognition of the adenine base accompanying conformational change from the closed form of the PR-ADA complex in crystalline state.

The ADAR RNA editing enzyme controls neuronal excitability in Drosophila melanogaster

PubMed Central

Li, Xianghua; Overton, Ian M.; Baines, Richard A.; Keegan, Liam P.; O’Connell, Mary A.

2014-01-01

RNA editing by deamination of specific adenosine bases to inosines during pre-mRNA processing generates edited isoforms of proteins. Recoding RNA editing is more widespread in Drosophila than in vertebrates. Editing levels rise strongly at metamorphosis, and Adar5G1 null mutant flies lack editing events in hundreds of CNS transcripts; mutant flies have reduced viability, severely defective locomotion and age-dependent neurodegeneration. On the other hand, overexpressing an adult dADAR isoform with high enzymatic activity ubiquitously during larval and pupal stages is lethal. Advantage was taken of this to screen for genetic modifiers; Adar overexpression lethality is rescued by reduced dosage of the Rdl (Resistant to dieldrin), gene encoding a subunit of inhibitory GABA receptors. Reduced dosage of the Gad1 gene encoding the GABA synthetase also rescues Adar overexpression lethality. Drosophila Adar5G1 mutant phenotypes are ameliorated by feeding GABA modulators. We demonstrate that neuronal excitability is linked to dADAR expression levels in individual neurons; Adar-overexpressing larval motor neurons show reduced excitability whereas Adar5G1 null mutant or targeted Adar knockdown motor neurons exhibit increased excitability. GABA inhibitory signalling is impaired in human epileptic and autistic conditions, and vertebrate ADARs may have a relevant evolutionarily conserved control over neuronal excitability. PMID:24137011

New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins

PubMed Central

Mannion, Niamh; Arieti, Fabiana; Gallo, Angela; Keegan, Liam P.; O’Connell, Mary A.

2015-01-01

The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. Inosine can have a profound effect on the RNAs that are edited, not only changing the base-pairing properties, but can also result in recoding, as inosine behaves as if it were guanosine. In mammals there are three ADAR proteins and two ADAR-related proteins (ADAD) expressed. All have a very similar modular structure; however, both their expression and biological function differ significantly. Only two of the ADAR proteins have enzymatic activity. However, both ADAR and ADAD proteins possess the ability to bind double-strand RNA. Mutations in ADARs have been associated with many diseases ranging from cancer, innate immunity to neurological disorders. Here, we will discuss in detail the domain structure of mammalian ADARs, the effects of RNA editing, and the role of ADARs in human diseases. PMID:26437436

Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palmer, T.D.; Hock, R.A.; Osborne, W.R.A.

1987-02-01

Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. The authors studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vectormore » containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA/sup -/ fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.« less

Adar3 Is Involved in Learning and Memory in Mice.

PubMed

Mladenova, Dessislava; Barry, Guy; Konen, Lyndsey M; Pineda, Sandy S; Guennewig, Boris; Avesson, Lotta; Zinn, Raphael; Schonrock, Nicole; Bitar, Maina; Jonkhout, Nicky; Crumlish, Lauren; Kaczorowski, Dominik C; Gong, Andrew; Pinese, Mark; Franco, Gloria R; Walkley, Carl R; Vissel, Bryce; Mattick, John S

2018-01-01

The amount of regulatory RNA encoded in the genome and the extent of RNA editing by the post-transcriptional deamination of adenosine to inosine (A-I) have increased with developmental complexity and may be an important factor in the cognitive evolution of animals. The newest member of the A-I editing family of ADAR proteins, the vertebrate-specific ADAR3, is highly expressed in the brain, but its functional significance is unknown. In vitro studies have suggested that ADAR3 acts as a negative regulator of A-I RNA editing but the scope and underlying mechanisms are also unknown. Meta-analysis of published data indicates that mouse Adar3 expression is highest in the hippocampus, thalamus, amygdala, and olfactory region. Consistent with this, we show that mice lacking exon 3 of Adar3 (which encodes two double stranded RNA binding domains) have increased levels of anxiety and deficits in hippocampus-dependent short- and long-term memory formation. RNA sequencing revealed a dysregulation of genes involved in synaptic function in the hippocampi of Adar3 -deficient mice. We also show that ADAR3 transiently translocates from the cytoplasm to the nucleus upon KCl-mediated activation in SH-SY5Y cells. These results indicate that ADAR3 contributes to cognitive processes in mammals.

Investigations into the origin of the molecular recognition of several adenosine deaminase inhibitors.

PubMed

Gillerman, Irina; Fischer, Bilha

2011-01-13

Inhibitors of adenosine deaminase (ADA, EC 3.5.4.4) are potential therapeutic agents for the treatment of various health disorders. Several highly potent inhibitors were previously identified, yet they exhibit unacceptable toxicities. We performed a SAR study involving a series of C2 or C8 substituted purine-riboside analogues with a view to discover less potent inhibitors with a lesser toxicity. We found that any substitution at C8 position of nebularine resulted in total loss of activity toward calf intestinal ADA. However, several 2-substituted-adenosine, 8-aza-adenosine, and nebularine analogues exhibited inhibitory activity. Specifically, 2-Cl-purine riboside, 8-aza-2-thiohexyl adenosine, 2-thiohexyl adenosine, and 2-MeS-purine riboside were found to be competitive inhibitors of ADA with K(i) values of 25, 22, 6, and 3 μM, respectively. We concluded that electronic parameters are not major recognition determinants of ADA but rather steric parameters. A C2 substituent which fits ADA hydrophobic pocket and improves H-bonding with the enzyme makes a good inhibitor. In addition, a gg rotamer about C4'-C5' bond is apparently an important recognition determinant.

Regulation of adenosine deaminase (ADA) on induced mouse experimental autoimmune uveitis (EAU) ‡

PubMed Central

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

2016-01-01

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 have shown that adenosine receptor (AR) agonists can be either anti- or pro-inflammatory. 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 days post-immunization, shortly before EAU expression, but 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 improve the design of ADA- and AR-targeted therapies. PMID:26856700

Increased activity of vascular adenosine deaminase in atherosclerosis and therapeutic potential of its inhibition.

PubMed

Kutryb-Zajac, Barbara; Mateuszuk, Lukasz; Zukowska, Paulina; Jasztal, Agnieszka; Zabielska, Magdalena A; Toczek, Marta; Jablonska, Patrycja; Zakrzewska, Agnieszka; Sitek, Barbara; Rogowski, Jan; Lango, Romuald; Slominska, Ewa M; Chlopicki, Stefan; Smolenski, Ryszard T

2016-11-01

Extracellular nucleotides and adenosine that are formed or degraded by membrane-bound ecto-enzymes could affect atherosclerosis by regulating the inflammation and thrombosis. This study aimed to evaluate a relation between ecto-enzymes that convert extracellular adenosine triphosphate to adenine dinucleotide phosphate, adenosine monophosphate, adenosine, and inosine on the surface of the vessel wall with the severity or progression of experimental and clinical atherosclerosis. Furthermore, we tested whether the inhibition of adenosine deaminase will block the development of experimental atherosclerosis. Vascular activities of ecto-nucleoside triphosphate diphosphohydrolase 1, ecto-5'-nucleotidase, and ecto-adenosine deaminase (eADA) were measured in aortas of apolipoprotein E-/- low density lipoprotein receptor (ApoE-/-LDLR-/-) and wild-type mice as well as in human aortas. Plaques were analysed in the entire aorta, aortic root, and brachiocephalic artery by Oil-Red O and Orcein Martius Scarlet Blue staining and vascular accumulation of macrophages. The cellular location of ecto-enzymes was analysed by immunofluorescence. The effect of eADA inhibition on atherosclerosis progression was studied by a 2-month deoxycoformycin treatment of ApoE-/-LDLR-/- mice. The vascular eADA activity prominently increased in ApoE-/-LDLR-/- mice when compared with wild type already at the age of 1 month and progressed along atherosclerosis development, reaching a 10-fold difference at 10 months. The activity of eADA correlated with atherosclerotic changes in human aortas. High abundance of eADA in atherosclerotic vessels originated from activated endothelial cells and macrophages. There were no changes in ecto-nucleoside triphosphate diphosphohydrolase 1 activity, whereas ecto-5'-nucleotidase was moderately decreased in ApoE-/-LDLR-/- mice. Deoxycoformycin treatment attenuated plaque development in aortic root and brachiocephalic artery of ApoE-/-LDLR-/- mice, suppressed vascular

The effect of adenosine monophosphate deaminase overexpression on the accumulation of umami-related metabolites in tomatoes.

PubMed

Chew, Bee Lynn; Fisk, Ian D; Fray, Rupert; Tucker, Gregory A; Bodi, Zsuzsanna; Ferguson, Alison; Xia, Wei; Seymour, Graham B

2017-01-01

This study highlights the changes in umami-related nucleotide and glutamate levels when the AMP deaminase gene was elevated in transgenic tomato. Taste is perceived as one of a combination of five sensations, sweet, sour, bitter, salty, and umami. The umami taste is best known as a savoury sensation and plays a central role in food flavour, palatability, and eating satisfaction. Umami flavour can be imparted by the presence of glutamate and is greatly enhanced by the addition of ribonucleotides, such as inosine monophosphate (IMP) and guanosine monophosphate (GMP). The production of IMP is regulated by the enzyme adenosine monophosphate (AMP) deaminase which functions to convert AMP into IMP. We have generated transgenic tomato (Solanum lycopersicum) lines over expressing AMP deaminase under the control of a fruit-specific promoter. The transgenic lines showed substantially enhanced levels of AMP deaminase expression in comparison to the wild-type control. Elevated AMP deaminase levels resulted in the reduced accumulation of glutamate and increased levels of the umami nucleotide GMP. AMP concentrations were unchanged. The effects on the levels of glutamate and GMP were unexpected and are discussed in relation to the metabolite flux within this pathway.

Moonlighting adenosine deaminase: a target protein for drug development.

PubMed

Cortés, Antoni; Gracia, Eduard; Moreno, Estefania; Mallol, Josefa; Lluís, Carme; Canela, Enric I; Casadó, Vicent

2015-01-01

Interest in adenosine deaminase (ADA) in the context of medicine has mainly focused on its enzymatic activity. This is justified by the importance of the reaction catalyzed by ADA not only for the intracellular purine metabolism, but also for the extracellular purine metabolism as well, because of its capacity as a regulator of the concentration of extracellular adenosine that is able to activate adenosine receptors (ARs). In recent years, other important roles have been described for ADA. One of these, with special relevance in immunology, is the capacity of ADA to act as a costimulator, promoting T-cell proliferation and differentiation mainly by interacting with the differentiation cluster CD26. Another role is the ability of ADA to act as an allosteric modulator of ARs. These receptors have very general physiological implications, particularly in the neurological system where they play an important role. Thus, ADA, being a single chain protein, performs more than one function, consistent with the definition of a moonlighting protein. Although ADA has never been associated with moonlighting proteins, here we consider ADA as an example of this family of multifunctional proteins. In this review, we discuss the different roles of ADA and their pathological implications. We propose a mechanism by which some of their moonlighting functions can be coordinated. We also suggest that drugs modulating ADA properties may act as modulators of the moonlighting functions of ADA, giving them additional potential medical interest. © 2014 Wiley Periodicals, Inc.

Diagnostic Value of Serum Adenosine Deaminase (ADA) Level for Pulmonary Tuberculosis.

PubMed

Salmanzadeh, Shokrollah; Tavakkol, Heshmatollah; Bavieh, Khalid; Alavi, Seyed Mohammad

2015-03-01

Diagnosis of tuberculosis (TB) is not always easy, thus employing methods with a short duration and acceptable sensitivity and specificity is necessary to diagnose TB. The aim of this study was to investigate the diagnostic value of serum adenosine deaminase (ADA) level for diagnosis of pulmonary tuberculosis. A total of 160 sex and age-matched subjects were included in this study, and were divided to four groups; forty patients with pulmonary tuberculosis (PTB) diagnosed based on the national TB program (NTP), forty patients with non-tuberculosis bacterial pneumonia, forty patients with lung cancer and forty people who were healthy in every respect. Serum adenosine deaminase activity in patients of each group was measured by the Giusti and Galanti calorimetry method using a commercial kit (Diazyme, USA). The ANOVA analysis was used to compare groups for quantitative variables. Mean serum ADA level in the PTB group was clearly higher than the mean serum ADA in the other three groups. Mean serum ADA was 26 IU/L in PTB patients, 19.48 IU/L in patients with pneumonia, 15.8 IU/L in patients with lung cancer, and 10.7 IU/L in the control group (P < 0.05). In regard to the cut off value of 26 IU/L for ADA in patients with PTB sensitivity and specificity was defined as 35% and 91%, respectively. Serum ADA activity with high specificity percentage may be a useful alternative test in restricted resource areas to rule out diagnosis of PTB. However, serum ADA activity is not a useful tool for TB diagnosis.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ku, Min-Je; Lee, Won-Ho; Biotechnology and Genetic Engineering, Korea University, Seoul 136-701

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. Themore » 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%.« less

Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

PubMed Central

Moreno, Estefanía; Canet, Júlia; Gracia, Eduard; Lluís, Carme; Mallol, Josefa; Canela, Enric I.; Cortés, Antoni; Casadó, Vicent

2018-01-01

Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits. PMID:29497379

Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins.

PubMed

Moreno, Estefanía; Canet, Júlia; Gracia, Eduard; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Cortés, Antoni; Casadó, Vicent

2018-01-01

Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A 2A R present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A 2A R and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A 2A R involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A 2A R-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A 2A R). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sadat Hayatshahi, Sayyed Hamed; Abdolmaleki, Parviz; Safarian, Shahrokh

2005-12-16

Logistic regression and artificial neural networks have been developed as two non-linear models to establish quantitative structure-activity relationships between structural descriptors and biochemical activity of adenosine based competitive inhibitors, toward adenosine deaminase. The training set included 24 compounds with known k {sub i} values. The models were trained to solve two-class problems. Unlike the previous work in which multiple linear regression was used, the highest of positive charge on the molecules was recognized to be in close relation with their inhibition activity, while the electric charge on atom N1 of adenosine was found to be a poor descriptor. Consequently, themore » 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%.« less

Adenosine Deaminase Deficiency - More Than Just an Immunodeficiency.

PubMed

Whitmore, Kathryn V; Gaspar, Hubert B

2016-01-01

Adenosine deaminase (ADA) deficiency is best known as a form of severe combined immunodeficiency (SCID) that results from mutations in the gene encoding ADA. Affected patients present with clinical and immunological manifestations typical of a SCID. Therapies are currently available that can target these immunological disturbances and treated patients show varying degrees of clinical improvement. However, there is now a growing body of evidence that deficiency of ADA has significant impact on non-immunological organ systems. This review will outline the impact of ADA deficiency on various organ systems, starting with the well-understood immunological abnormalities. We will discuss possible pathogenic mechanisms and also highlight ways in which current treatments could be improved. In doing so, we aim to present ADA deficiency as more than an immunodeficiency and suggest that it should be recognized as a systemic metabolic disorder that affects multiple organ systems. Only by fully understanding ADA deficiency and its manifestations in all organ systems can we aim to deliver therapies that will correct all the clinical consequences.

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

PubMed

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

2013-08-01

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

Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency

PubMed Central

Kohn, Donald B.; Weinberg, Kenneth I.; Nolta, Jan A.; Heiss, Linda N.; Lenarsky, Carl; Crooks, Gay M.; Hanley, Mary E.; Annett, Geralyn; Brooks, Judith S.; El-Khoureiy, Anthony; Lawrence, Kim; Wells, Susie; Moen, Robert C.; Bastian, John; Williams-Herman, Debora E.; Elder, Melissa; Wara, Diane; Bowen, Thomas; Hershfield, Michael S.; Mullen, Craig A.; Blaese, R. Michael; Parkman, Robertson

2010-01-01

Haematopoietic stem cells in umbilical cord blood are an attractive target for gene therapy of inborn errors of metabolism. Three neonates with severe combined immunodeficiency were treated by retroviral-mediated transduction of the CD34+ cells from their umbilical cord blood with a normal human adenosine deaminase complementary DNA followed by autologous transplantation. The continued presence and expression of the introduced gene in leukocytes from bone marrow and peripheral blood for 18 months demonstrates that umbilical cord blood cells may be genetically modified with retroviral vectors and engrafted in neonates for gene therapy. PMID:7489356

The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis

PubMed Central

Deffit, Sarah N; Yee, Brian A; Manning, Aidan C; Rajendren, Suba; Vadlamani, Pranathi; Wheeler, Emily C; Domissy, Alain; Washburn, Michael C

2017-01-01

ADAR proteins alter gene expression both by catalyzing adenosine (A) to inosine (I) RNA editing and binding to regulatory elements in target RNAs. Loss of ADARs affects neuronal function in all animals studied to date. Caenorhabditis elegans lacking ADARs exhibit reduced chemotaxis, but the targets responsible for this phenotype remain unknown. To identify critical neural ADAR targets in C. elegans, we performed an unbiased assessment of the effects of ADR-2, the only A-to-I editing enzyme in C. elegans, on the neural transcriptome. Development and implementation of publicly available software, SAILOR, identified 7361 A-to-I editing events across the neural transcriptome. Intersecting the neural editome with adr-2 associated gene expression changes, revealed an edited mRNA, clec-41, whose neural expression is dependent on deamination. Restoring clec-41 expression in adr-2 deficient neural cells rescued the chemotaxis defect, providing the first evidence that neuronal phenotypes of ADAR mutants can be caused by altered gene expression. PMID:28925356

Pleural Fluid Adenosine Deaminase (ADA) Predicts Survival in Patients with Malignant Pleural Effusion.

PubMed

Terra, Ricardo Mingarini; Antonangelo, Leila; Mariani, Alessandro Wasum; de Oliveira, Ricardo Lopes Moraes; Teixeira, Lisete Ribeiro; Pego-Fernandes, Paulo Manuel

2016-08-01

Systemic and local inflammations have been described as relevant prognostic factors in patients with cancer. However, parameters that stand for immune activity in the pleural space have not been tested as predictors of survival in patients with malignant pleural effusion. The objective of this study was to evaluate pleural lymphocytes and Adenosine Deaminase (ADA) as predictors of survival in patients with recurrent malignant pleural effusion. Retrospective cohort study includes patients who underwent pleurodesis for malignant pleural effusion in a tertiary center. Pleural fluid protein concentration, lactate dehydrogenase, glucose, oncotic cytology, cell count, and ADA were collected before pleurodesis and analyzed. Survival analysis was performed considering pleurodesis as time origin, and death as the event. Backwards stepwise Cox regression was used to find predictors of survival. 156 patients (out of 196 potentially eligible) were included in this study. Most were female (72 %) and breast cancer was the most common underlying malignancy (53 %). Pleural fluid ADA level was stratified as low (<15 U/L), normal (15 ≤ ADA < 40), and high (≥40). Low and high ADA levels were associated with worse survival when compared to normal ADA (logrank: 0.0024). In multivariable analysis, abnormal ADA (<15 or ADA ≥ 40) and underlying malignancies different from lymphoma, lung, or breast cancer were associated with worse survival. Pleural fluid cell count and lymphocytes number and percentage did not correlate with survival. Pleural fluid Adenosine Deaminase levels (<15 or ≥40 U/L) and neoplasms other than lung, breast, or lymphoma are independent predictors of worse survival in patients with malignant pleural effusion who undergo pleurodesis.

RNA editing in Drosophila melanogaster: new targets and functionalconsequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stapleton, Mark; Carlson, Joseph W.; Celniker, Susan E.

2006-09-05

Adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine in primary mRNA transcripts. These re-coding events affect coding potential, splice-sites, and stability of mature mRNAs. ADAR is an essential gene and studies in mouse, C. elegans, and Drosophila suggest its primary function is to modify adult behavior by altering signaling components in the nervous system. By comparing the sequence of isogenic cDNAs to genomic DNA, we have identified and experimentally verified 27 new targets of Drosophila ADAR. Our analyses lead us to identify new classes of genes whose transcripts are targets of ADAR includingmore » components of the actin cytoskeleton, and genes involved in ion homeostasis and signal transduction. Our results indicate that editing in Drosophila increases the diversity of the proteome, and does so in a manner that has direct functional consequences on protein function.« less

Adenosine Deaminase Deficiency – More Than Just an Immunodeficiency

PubMed Central

Whitmore, Kathryn V.; Gaspar, Hubert B.

2016-01-01

Adenosine deaminase (ADA) deficiency is best known as a form of severe combined immunodeficiency (SCID) that results from mutations in the gene encoding ADA. Affected patients present with clinical and immunological manifestations typical of a SCID. Therapies are currently available that can target these immunological disturbances and treated patients show varying degrees of clinical improvement. However, there is now a growing body of evidence that deficiency of ADA has significant impact on non-immunological organ systems. This review will outline the impact of ADA deficiency on various organ systems, starting with the well-understood immunological abnormalities. We will discuss possible pathogenic mechanisms and also highlight ways in which current treatments could be improved. In doing so, we aim to present ADA deficiency as more than an immunodeficiency and suggest that it should be recognized as a systemic metabolic disorder that affects multiple organ systems. Only by fully understanding ADA deficiency and its manifestations in all organ systems can we aim to deliver therapies that will correct all the clinical consequences. PMID:27579027

Nitric oxide - an activating factor of adenosine deaminase 2 in vitro.

PubMed

Sargisova, Ye G; Andreasyan, N A; Hayrapetyan, H L; Harutyunyan, H A

2012-01-01

In this study we have investigated the effect of reactive oxygen species produced by some chemicals in aqueous solutions on activity of adenosine deaminase 2 (ADA2) purified from human blood plasma. An activating effect on ADA2 was observed in vitro with sodium nitroprusside (SNP), the source of NO (nitrosonium ions NO(-) in aqueous solutions). Not SH-groups of cysteine but other amino acid residues sensitive to NO were responsible for ADA2 activation. The SNP-derived activation was more pronounced when purified ADA2 was preincubated with heparin and different proteins as an experimental model of the protein environment in vivo. The most effective was heparin, which is known for its ability to regulate enzyme and protein functions in extracellular matrix. We conclude that ADA2 is a protein with flexible conformation that is affected by the protein environment, and it changes its activity under oxidative (nitrosative) stress.

A to I editing in disease is not fake news.

PubMed

Bajad, Prajakta; Jantsch, Michael F; Keegan, Liam; O'Connell, Mary

2017-09-02

Adenosine deaminases acting on RNA (ADARs) are zinc-containing enzymes that deaminate adenosine bases to inosines within dsRNA regions in transcripts. In short, structured dsRNA hairpins individual adenosine bases may be targeted specifically and edited with up to one hundred percent efficiency, leading to the production of alternative protein variants. However, the majority of editing events occur within longer stretches of dsRNA formed by pairing of repetitive sequences. Here, many different adenosine bases are potential targets but editing efficiency is usually much lower. Recent work shows that ADAR-mediated RNA editing is also required to prevent aberrant activation of antiviral innate immune sensors that detect viral dsRNA in the cytoplasm. Missense mutations in the ADAR1 RNA editing enzyme cause a fatal auto-inflammatory disease, Aicardi-Goutières syndrome (AGS) in affected children. In addition RNA editing by ADARs has been observed to increase in many cancers and also can contribute to vascular disease. Thus the role of RNA editing in the progression of various diseases can no longer be ignored. The ability of ADARs to alter the sequence of RNAs has also been used to artificially target model RNAs in vitro and in cells for RNA editing. Potentially this approach may be used to repair genetic defects and to alter genetic information at the RNA level. In this review we focus on the role of ADARs in disease development and progression and on their potential use to artificially modify RNAs in a targeted manner.

Gene amplification-associated overexpression of the RNA editing enzyme ADAR1 enhances human lung tumorigenesis.

PubMed

Anadón, C; Guil, S; Simó-Riudalbas, L; Moutinho, C; Setien, F; Martínez-Cardús, A; Moran, S; Villanueva, A; Calaf, M; Vidal, A; Lazo, P A; Zondervan, I; Savola, S; Kohno, T; Yokota, J; Ribas de Pouplana, L; Esteller, M

2016-08-18

The introduction of new therapies against particular genetic mutations in non-small-cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis.

Insulin and adenosine regulate the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes.

PubMed

Kiechle, F L; Sykes, E; Artiss, J D

1995-01-01

Blockade of adenosine receptors by 3-isobutyl-1-methylxanthine or degradation of endogenous adenosine with adenosine deaminase increased the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes, an effect which was suppressed by the phosphatidylethanolamine methyltransferase inhibitor, S-adenosyl-L-homocysteine, and reversed by the adenosine analogue, N6-(L-phenylisopropyl)-adenosine. For example, the addition of N6-(L-phenylisopropyl)-adenosine to adenosine deaminase pretreated plasma membranes rapidly lowered the concentration of phosphatidylcholine by 171 nmol/mg at 30 seconds compared to control. Insulin-induced stimulation of phospholipid methylation in membranes treated with 3-isobutyl-1-methylxanthine or adenosine deaminase was achieved only after the addition of N6-(L-phenylisopropyl)-adenosine. These results suggest that adenosine receptor occupancy inhibits phospholipid methylation, is required for insulin stimulation of phospholipid methylation, and may perhaps activate a phosphatidylcholine-specific phospholipase C or phospholipase D.

Adenosine monophosphate as a mediator of ATP effects at P1 purinoceptors

PubMed Central

Ross, Fiona M; Brodie, Martin J; Stone, Trevor W

1998-01-01

When perfused with a medium containing no added magnesium and 4-aminopyridine (4AP) (50 μM) hippocampal slices generated epileptiform bursts of an interictal nature. We have shown in a previous study that adenosine 5′-triphosphate (ATP) depressed epileptiform activity and that this effect was blocked by the adenosine A1 receptor antagonist cyclopentyltheophylline but was not affected by adenosine deaminase. This implied that ATP might act indirectly at P1 receptors or at a xanthine-sensitive P2 receptor. The aim of the present study was to investigate further the action of ATP on epileptiform activity.ATP can be metabolized by ecto-nucleotidases to adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP) and adenosine, respectively. Each of these metabolites can activate receptors in its own right: P2 receptors for ADP and P1 receptors for AMP and adenosine.We now show that both AMP and ATP (50 μM) significantly decrease epileptiform discharge rate in a rapid and reversible manner. 5′Adenylic acid deaminase (AMP deaminase, AMPase) (0.2 u ml−1), when perfused alone did not significantly alter the discharge rate over the 10 min superfusion period used for drug application. When perfused concurrently with AMP (50 μM), AMP deaminase prevented the depressant effect of AMP on discharge rate.AMP deaminase, at a concentration of 0.2 u ml−1 which annulled the effect of AMP (50 μM), prevented the inhibitory activity of ATP (50 μM). A higher concentration of ATP (200 μM) depressed the frequency of spontaneous bursts to approximately 30% control and this response was also prevented by AMP deaminase.Superfusion of the slices with 5′-nucleotidase also prevented the inhibitory activity of ATP on epileptiform discharges.The results suggest that AMP mediates the inhibitory effects of ATP on epileptiform activity, a conclusion which can explain the earlier finding that cyclopentyltheophylline but not adenosine deaminase inhibited the

Diagnostic yield of adenosine deaminase in bronchoalveolar lavage.

PubMed

Reechaipichitkul, Wipa; Lulitanond, Viraphong; Patjanasoontorn, Boonsong; Boonsawat, Watchara; Phunmanee, Anakapong

2004-09-01

Adenosine deaminase (ADA) activity rises in various body fluids in patients with tuberculosis. A prospective study was conducted to determine the diagnostic value of ADA activity in bronchoalveolar lavage. Between March 2001 and February 2003, 148 patients were enrolled in our study, mean age 55.6 years (SD 14.6), and a male to female ratio of 2.4:1. The mean duration of symptoms was 66.2 days. All patients were either sputum-smear negative for AFB or failed to produce sputum. The final diagnosis resulted in three patient groups: 43 with pulmonary tuberculosis, 70 malignancy, and 35 miscellaneous causes. The mean ADA activity in the bronchoalveolar lavage for the pulmonary tuberculosis, malignancy, and miscellaneous causes groups was 8.98 (95% CI, 3.79-14.17), 7.63 (95% CI, 4.12-11.14), and 11.61 U/l (95% CI, 3.59-19.62), respectively. No difference was detected in the ADA level in the pulmonary tuberculosis vs other groups (p=0.56, one-way ANOVA). A high level of ADA activity was found in non-tuberculous conditions such as bronchogenic carcinoma, pulmonary hemosiderosis, chronic pneumonia with empyema thoracis and chronic myeloid leukemia. We concluded that ADA activity in the bronchoalveolar lavage was not clearly diagnostic of smear-negative pulmonary tuberculosis. Early diagnosis required histopathology of biopsied transbronchial specimens obtained by fiberoptic bronchoscopy.

The Binding Site of Human Adenosine Deaminase for Cd26/Dipeptidyl Peptidase IV

PubMed Central

Richard, Eva; Arredondo-Vega, Francisco X.; Santisteban, Ines; Kelly, Susan J.; Patel, Dhavalkumar D.; Hershfield, Michael S.

2000-01-01

Human, but not murine, adenosine deaminase (ADA) forms a complex with the cell membrane protein CD26/dipeptidyl peptidase IV. CD26-bound ADA has been postulated to regulate extracellular adenosine levels and to modulate the costimulatory function of CD26 on T lymphocytes. Absence of ADA–CD26 binding has been implicated in causing severe combined immunodeficiency due to ADA deficiency. Using human–mouse ADA hybrids and ADA point mutants, we have localized the amino acids critical for CD26 binding to the helical segment 126–143. Arg142 in human ADA and Gln142 in mouse ADA largely determine the capacity to bind CD26. Recombinant human ADA bearing the R142Q mutation had normal catalytic activity per molecule, but markedly impaired binding to a CD26+ ADA-deficient human T cell line. Reduced CD26 binding was also found with ADA from red cells and T cells of a healthy individual whose only expressed ADA has the R142Q mutation. Conversely, ADA with the E217K active site mutation, the only ADA expressed by a severely immunodeficient patient, showed normal CD26 binding. These findings argue that ADA binding to CD26 is not essential for immune function in humans. PMID:11067872

Adenosine Deaminase (ADA)-Deficient Severe Combined Immune Deficiency (SCID): Molecular Pathogenesis and Clinical Manifestations.

PubMed

Bradford, Kathryn L; Moretti, Federico A; Carbonaro-Sarracino, Denise A; Gaspar, Hubert B; Kohn, Donald B

2017-10-01

Deficiency of adenosine deaminase (ADA, EC3.5.4.4), a housekeeping enzyme of purine metabolism encoded by the Ada gene, is a cause of human severe combined immune deficiency (SCID). Numerous deleterious mutations occurring in the ADA gene have been found in patients with profound lymphopenia (T - B - NK - ), thus underscoring the importance of functional purine metabolism for the development of the immune defense. While untreated ADA SCID is a fatal disorder, there are multiple life-saving therapeutic modalities to restore ADA activity and reconstitute protective immunity, including enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) with autologous gene-corrected hematopoietic stem cells (HSC). We review the pathogenic mechanisms and clinical manifestations of ADA SCID.

ADA (adenosine deaminase) gene therapy enters the competition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Culliton, B.J.

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

Regulatory factors governing adenosine-to-inosine (A-to-I) RNA editing.

PubMed

Hong, HuiQi; Lin, Jaymie Siqi; Chen, Leilei

2015-03-31

Adenosine-to-inosine (A-to-I) RNA editing, the most prevalent mode of transcript modification in higher eukaryotes, is catalysed by the adenosine deaminases acting on RNA (ADARs). A-to-I editing imposes an additional layer of gene regulation as it dictates various aspects of RNA metabolism, including RNA folding, processing, localization and degradation. Furthermore, editing events in exonic regions contribute to proteome diversity as translational machinery decodes inosine as guanosine. Although it has been demonstrated that dysregulated A-to-I editing contributes to various diseases, the precise regulatory mechanisms governing this critical cellular process have yet to be fully elucidated. However, integration of previous studies revealed that regulation of A-to-I editing is multifaceted, weaving an intricate network of auto- and transregulations, including the involvement of virus-originated factors like adenovirus-associated RNA. Taken together, it is apparent that tipping of any regulatory components will have profound effects on A-to-I editing, which in turn contributes to both normal and aberrant physiological conditions. A complete understanding of this intricate regulatory network may ultimately be translated into new therapeutic strategies against diseases driven by perturbed RNA editing events. Herein, we review the current state of knowledge on the regulatory mechanisms governing A-to-I editing and propose the role of other co-factors that may be involved in this complex regulatory process.

IgG4-related Pleuritis with Elevated Adenosine Deaminase in Pleural Effusion: A Case Report.

PubMed

Nagayasu, Atsushi; Kubo, Satoshi; Nakano, Kazuhisa; Nakayamada, Shingo; Iwata, Shigeru; Miyagawa, Ippei; Fukuyo, Shunsuke; Saito, Kazuyoshi; Tanaka, Yoshiya

2018-03-09

An 81-year-old man was admitted with bilateral pleural effusion. A clinical examination showed lymphocytic pleura effusion and elevated serum IgG4 levels, so that IgG4-related disease was suggested, whereas tuberculous pleurisy was suspected because of high adenosine deaminase (ADA) levels in the pleural effusion. A surgical pleural biopsy revealed that there were large numbers of IgG4-positive cells and IgG4/IgG positive cell ratio exceeded 40% in several sites. Accordingly, we diagnosed IgG4-related pleuritis and treated with the patient with glucocorticoid therapy. The ADA levels in pleural effusion can increase in IgG4-related pleuritis, and it is therefore important to perform a pleural biopsy.

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

PubMed Central

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

2012-01-01

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 ADA is inhibited by 1,3-DNB as a substrate mimic, thereby preventing adenosine catabolism. ADA was inhibited by 1,3-DNB with an IC50 of 284μM, Hill slope, n = 4.8 ± 0.4. Native gel electrophoresis showed that 1,3-DNB did not denature ADA. Furthermore, adding Triton X-100 (0.01–0.05%, wt/vol), Nonidet P-40 (0.0015–0.0036%, wt/vol), or bovine serum albumin (0.05 mg/ml or changing [ADA] (0.2 and 2nM) did not substantially alter the 1,3-DNB IC50 value. Likewise, dynamic light scattering showed no particle formation over a (1,3-DNB) range of 149–1043μM. Kinetics revealed mixed inhibition with 1,3-DNB binding to ADA (KI = 520 ± 100μM, n = 1 ± 0.6) and the ADA-adenosine complex (KIS = 262 ± 7μM, n = 6 ± 0.6, indicating positive cooperativity). In accord with the kinetics, docking predicted binding of 1,3-DNB to the active site and three peripheral sites. In addition, exposure of DI TNC-1 astrocytes to 10–500μM 1,3-DNB produced concentration-dependent increases in extracellular adenosine at 24 h. Overall, the results demonstrate that 1,3-DNB is a mixed inhibitor of ADA and may thus lead to increases in extracellular adenosine. The finding may provide insights to guide future work on chemically-induced energy deprivation. PMID:22106038

Effect of two imidazolium derivatives of ionic liquids on the structure and activity of adenosine deaminase.

PubMed

Ajloo, Davood; Sangian, Masoomeh; Ghadamgahi, Maryam; Evini, Mina; Saboury, Ali Akbar

2013-04-01

The effect of two ionic liquids, 1-allyl 3-methyl-imidazolium (IL1) and 1-octhyl 3-methyl-imidozolium chlorides (IL2), on the structure and activity of adenosine deaminase (ADA) were described by UV-vis and fluorescence spectrophotometry in phosphate buffer and results were compared with docking and molecular dynamics (MD) simulation studies. All results showed that inhibition of activity and reduction of enzyme tertiary structure are more for octhyl than allyl derivative due to the more hydrophobic property of it. Finally structure parameters obtained from MD simulation showed that ionic liquid reduces intermolecular hydrogen bond and unfold enzyme structure. Calculation results are in good agreement with spectrophotometric studies. Copyright © 2013 Elsevier B.V. All rights reserved.

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

PubMed

Kühl, J S; Schwarz, K; Münch, A; Schmugge, M; Pekrun, A; Meisel, C; Wahn, V; Ebell, W; von Bernuth, H

2011-03-01

Adenosin deaminase (ADA) deficiency is the cause for Severe Combined Immunodeficiency (SCID) in about 15% of patients with SCID, often presenting as T (-)B (-)NK (-)SCID. Treatment options for ADA-SCID are enzyme replacement, bone marrow transplantation or gene therapy. We here describe the first patient with ADA-SCID and fatal hepatic failure despite bone marrow transplantation from a 10/10 HLA identical related donor. As patients with ADA-SCID may be at yet underestimated increased risk for rapid hepatic failure we speculate whether hepatitis in ADA-SCID should lead to the immediate treatment with enzyme replacement by pegylated ADA. © Georg Thieme Verlag KG Stuttgart · New York.

Does adenosine deaminase activity play a role in the early diagnosis of ectopic pregnancy?

PubMed

Turkmen, G G; Karçaaltıncaba, D; Isık, H; Fidancı, V; Kaayalp, D; Tımur, H; Batıoglu, S

2016-01-01

Early diagnosis of ectopic pregnancy (EP) is important due to life-threatening consequences in the first trimester of pregnancy. In this study we aimed to investigate the role of adenosine deaminase (ADA) activity in the prediction of EP. Forty-one patients with unruptured ectopic pregnancy comprised the case group and forty-two first trimester pregnant women with shown foetal heart beating in ultrasound comprised the control group. The mean ADA level in EP (10.9 ± 3.0 IU/L) was higher than that in control group (9.2 ± 3.6 IU/L) (p = 0.018). Receiver operating characteristics or ROC curve identified ADA value of 10.95 IU/L as optimal threshold for the prediction of EP with 56% sensitivity and 67% specificity. High ADA levels are valuable in the early diagnosis of EP. However more comprehensive studies are required.

Tissue-selective restriction of RNA editing of CaV1.3 by splicing factor SRSF9.

PubMed

Huang, Hua; Kapeli, Katannya; Jin, Wenhao; Wong, Yuk Peng; Arumugam, Thiruma Valavan; Koh, Joanne Huifen; Srimasorn, Sumitra; Mallilankaraman, Karthik; Chua, John Jia En; Yeo, Gene W; Soong, Tuck Wah

2018-05-04

Adenosine DeAminases acting on RNA (ADAR) catalyzes adenosine-to-inosine (A-to-I) conversion within RNA duplex structures. While A-to-I editing is often dynamically regulated in a spatial-temporal manner, the mechanisms underlying its tissue-selective restriction remain elusive. We have previously reported that transcripts of voltage-gated calcium channel CaV1.3 are subject to brain-selective A-to-I RNA editing by ADAR2. Here, we show that editing of CaV1.3 mRNA is dependent on a 40 bp RNA duplex formed between exon 41 and an evolutionarily conserved editing site complementary sequence (ECS) located within the preceding intron. Heterologous expression of a mouse minigene that contained the ECS, intermediate intronic sequence and exon 41 with ADAR2 yielded robust editing. Interestingly, editing of CaV1.3 was potently inhibited by serine/arginine-rich splicing factor 9 (SRSF9). Mechanistically, the inhibitory effect of SRSF9 required direct RNA interaction. Selective down-regulation of SRSF9 in neurons provides a basis for the neuron-specific editing of CaV1.3 transcripts.

Elevated Adenosine Induces Placental DNA Hypomethylation Independent of A2B Receptor Signaling in Preeclampsia.

PubMed

Huang, Aji; Wu, Hongyu; Iriyama, Takayuki; Zhang, Yujin; Sun, Kaiqi; Song, Anren; Liu, Hong; Peng, Zhangzhe; Tang, Lili; Lee, Minjung; Huang, Yun; Ni, Xin; Kellems, Rodney E; Xia, Yang

2017-07-01

Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear. Using 2 independent experimental models of preeclampsia, adenosine deaminase-deficient mice and a pathogenic autoantibody-induced mouse model of preeclampsia, we demonstrate that elevated placental adenosine not only induces hallmark features of preeclampsia but also causes placental DNA hypomethylation. The use of genetic approaches to express an adenosine deaminase minigene specifically in placentas, or adenosine deaminase enzyme replacement therapy, restored placental adenosine to normal levels, attenuated preeclampsia features, and abolished placental DNA hypomethylation in adenosine deaminase-deficient mice. Genetic deletion of CD73 (an ectonucleotidase that converts AMP to adenosine) prevented the elevation of placental adenosine in the autoantibody-induced preeclampsia mouse model and ameliorated preeclampsia features and placental DNA hypomethylation. Immunohistochemical studies revealed that elevated placental adenosine-mediated DNA hypomethylation predominantly occurs in spongiotrophoblasts and labyrinthine trophoblasts and that this effect is independent of A2B adenosine receptor activation in both preeclampsia models. Extending our mouse findings to humans, we used cultured human trophoblasts to demonstrate that adenosine functions intracellularly and induces DNA hypomethylation without A2B adenosine receptor activation. Altogether, both mouse and human studies reveal novel mechanisms underlying placental DNA hypomethylation and potential therapeutic approaches for preeclampsia. © 2017 American Heart Association, Inc.

ADAR1-mediated 3' UTR editing and expression control of antiapoptosis genes fine-tunes cellular apoptosis response.

PubMed

Yang, Chang-Ching; Chen, Yi-Tung; Chang, Yi-Feng; Liu, Hsuan; Kuo, Yu-Ping; Shih, Chieh-Tien; Liao, Wei-Chao; Chen, Hui-Wen; Tsai, Wen-Sy; Tan, Bertrand Chin-Ming

2017-05-25

Adenosine-to-inosine RNA editing constitutes a crucial component of the cellular transcriptome and critically underpins organism survival and development. While recent high-throughput approaches have provided comprehensive documentation of the RNA editome, its functional output remains mostly unresolved, particularly for events in the non-coding regions. Gene ontology analysis of the known RNA editing targets unveiled a preponderance of genes related to apoptosis regulation, among which proto-oncogenes XIAP and MDM2 encode two the most abundantly edited transcripts. To further decode this potential functional connection, here we showed that the main RNA editor ADAR1 directly targets this 3' UTR editing of XIAP and MDM2, and further exerts a negative regulation on the expression of their protein products. This post-transcriptional silencing role was mediated via the inverted Alu elements in the 3' UTR but independent of alteration in transcript stability or miRNA targeting. Rather, we discovered that ADAR1 competes transcript occupancy with the RNA shuttling factor STAU1 to facilitate nuclear retention of the XIAP and MDM2 mRNAs. As a consequence, ADAR1 may acquire functionality in part by conferring spatial distribution and translation efficiency of the target transcripts. Finally, abrogation of ADAR1 expression or catalytic activity elicited a XIAP-dependent suppression of apoptotic response, whereas ectopic expression reversed this protective effect on cell death. Together, our results extended the known functions of ADAR1 and RNA editing to the critical fine-tuning of the intracellular apoptotic signaling and also provided mechanistic explanation for ADAR1's roles in development and tumorigenesis.

Homozygosity for a newly identified missense mutation in a patient with very severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID).

PubMed Central

Hirschhorn, R; Chakravarti, V; Puck, J; Douglas, S D

1991-01-01

We have identified a previously unrecognized missense mutation in a patient with severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID). The mutation is a G646-to-A transition at a CG dinucleotide and predicts a glycine-to-arginine substitution at codon 216. Computer analysis of secondary structure predicts a major alteration with loss of a beta-pleated sheet in a highly conserved region of the protein. The basepair substitution also generates a new site for the restriction enzyme BstXI in exon 7 of the genomic DNA. Digestion of genomic DNA from the patient and from his parents revealed that he was homozygous for the mutation and that his mother and father were carriers. This mutation in homozygous form appears to be associated with very severe disease, since the patient had perinatal onset of clinical manifestations of SCID, the highest concentration of the toxic metabolite deoxyATP in nine patients studied, and a relatively poor immunologic response during the initial 2 years of therapy with polyethylene glycol-adenosine deaminase. Analysis of DNA from 21 additional patients with ADA-SCID and from 19 unrelated normals revealed that, while none of the normal individuals showed the abnormal restriction fragment, two of the 21 patients studied were heterozygous for the G646-to-A mutation. Images Figure 2 PMID:1680289

Inherited variants affecting RNA editing may contribute to ovarian cancer susceptibility: results from a large-scale collaboration.

PubMed

Permuth, Jennifer B; Reid, Brett; Earp, Madalene; Chen, Y Ann; Monteiro, Alvaro N A; Chen, Zhihua; Chenevix-Trench, Georgia; Fasching, Peter A; Beckmann, Matthias W; Lambrechts, Diether; Vanderstichele, Adriaan; Van Niewenhuyse, Els; Vergote, Ignace; Rossing, Mary Anne; Doherty, Jennifer Anne; Chang-Claude, Jenny; Moysich, Kirsten; Odunsi, Kunle; Goodman, Marc T; Shvetsov, Yurii B; Wilkens, Lynne R; Thompson, Pamela J; Dörk, Thilo; Bogdanova, Natalia; Butzow, Ralf; Nevanlinna, Heli; Pelttari, Liisa; Leminen, Arto; Modugno, Francesmary; Edwards, Robert P; Ness, Roberta B; Kelley, Joseph; Heitz, Florian; Karlan, Beth; Lester, Jenny; Kjaer, Susanne K; Jensen, Allan; Giles, Graham; Hildebrandt, Michelle; Liang, Dong; Lu, Karen H; Wu, Xifeng; Levine, Douglas A; Bisogna, Maria; Berchuck, Andrew; Cramer, Daniel W; Terry, Kathryn L; Tworoger, Shelley S; Poole, Elizabeth M; Bandera, Elisa V; Fridley, Brooke; Cunningham, Julie; Winham, Stacey J; Olson, Sara H; Orlow, Irene; Bjorge, Line; Kiemeney, Lambertus A; Massuger, Leon; Pejovic, Tanja; Moffitt, Melissa; Le, Nhu; Cook, Linda S; Brooks-Wilson, Angela; Kelemen, Linda E; Gronwald, Jacek; Lubinski, Jan; Wentzensen, Nicolas; Brinton, Louise A; Lissowska, Jolanta; Yang, Hanna; Hogdall, Estrid; Hogdall, Claus; Lundvall, Lene; Pharoah, Paul D P; Song, Honglin; Campbell, Ian; Eccles, Diana; McNeish, Iain; Whittemore, Alice; McGuire, Valerie; Sieh, Weiva; Rothstein, Joseph; Phelan, Catherine M; Risch, Harvey; Narod, Steven; McLaughlin, John; Anton-Culver, Hoda; Ziogas, Argyrios; Menon, Usha; Gayther, Simon; Ramus, Susan J; Gentry-Maharaj, Aleksandra; Pearce, Celeste Leigh; Wu, Anna H; Kupryjanczyk, Jolanta; Dansonka-Mieszkowska, Agnieszka; Schildkraut, Joellen M; Cheng, Jin Q; Goode, Ellen L; Sellers, Thomas A

2016-11-08

RNA editing in mammals is a form of post-transcriptional modification in which adenosine is converted to inosine by the adenosine deaminases acting on RNA (ADAR) family of enzymes. Based on evidence of altered ADAR expression in epithelial ovarian cancers (EOC), we hypothesized that single nucleotide polymorphisms (SNPs) in ADAR genes modify EOC susceptibility, potentially by altering ovarian tissue gene expression. Using directly genotyped and imputed data from 10,891 invasive EOC cases and 21,693 controls, we evaluated the associations of 5,303 SNPs in ADAD1, ADAR, ADAR2, ADAR3, and SND1. Unconditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI), with adjustment for European ancestry. We conducted gene-level analyses using the Admixture Maximum Likelihood (AML) test and the Sequence-Kernel Association test for common and rare variants (SKAT-CR). Association analysis revealed top risk-associated SNP rs77027562 (OR (95% CI)= 1.39 (1.17-1.64), P=1.0x10-4) in ADAR3 and rs185455523 in SND1 (OR (95% CI)= 0.68 (0.56-0.83), P=2.0x10-4). When restricting to serous histology (n=6,500), the magnitude of association strengthened for rs185455523 (OR=0.60, P=1.0x10-4). Gene-level analyses revealed that variation in ADAR was associated (P<0.05) with EOC susceptibility, with PAML=0.022 and PSKAT-CR=0.020. Expression quantitative trait locus analysis in EOC tissue revealed significant associations (P<0.05) with ADAR expression for several SNPs in ADAR, including rs1127313 (G/A), a SNP in the 3' untranslated region. In summary, germline variation involving RNA editing genes may influence EOC susceptibility, warranting further investigation of inherited and acquired alterations affecting RNA editing.

Inherited variants affecting RNA editing may contribute to ovarian cancer susceptibility: results from a large-scale collaboration

PubMed Central

Permuth, Jennifer B.; Reid, Brett; Earp, Madalene; Chen, Y. Ann; Monteiro, Alvaro N.A.; Chen, Zhihua; Group, AOCS Study; Chenevix-Trench, Georgia; Fasching, Peter A.; Beckmann, Matthias W.; Lambrechts, Diether; Vanderstichele, Adriaan; Niewenhuyse, Els Van; Vergote, Ignace; Rossing, Mary Anne; Doherty, Jennifer Anne; Chang-Claude, Jenny; Moysich, Kirsten; Odunsi, Kunle; Goodman, Marc T.; Shvetsov, Yurii B.; Wilkens, Lynne R.; Thompson, Pamela J.; Dörk, Thilo; Bogdanova, Natalia; Butzow, Ralf; Nevanlinna, Heli; Pelttari, Liisa; Leminen, Arto; Modugno, Francesmary; Edwards, Robert P.; Ness, Roberta B.; Kelley, Joseph; Heitz, Florian; Karlan, Beth; Lester, Jenny; Kjaer, Susanne K.; Jensen, Allan; Giles, Graham; Hildebrandt, Michelle; Liang, Dong; Lu, Karen H.; Wu, Xifeng; Levine, Douglas A.; Bisogna, Maria; Berchuck, Andrew; Cramer, Daniel W.; Terry, Kathryn L.; Tworoger, Shelley S.; Poole, Elizabeth M.; Bandera, Elisa V.; Fridley, Brooke; Cunningham, Julie; Winham, Stacey J.; Olson, Sara H.; Orlow, Irene; Bjorge, Line; Kiemeney, Lambertus A.; Massuger, Leon; Pejovic, Tanja; Moffitt, Melissa; Le, Nhu; Cook, Linda S.; Brooks-Wilson, Angela; Kelemen, Linda E.; Gronwald, Jacek; Lubinski, Jan; Wentzensen, Nicolas; Brinton, Louise A.; Lissowska, Jolanta; Yang, Hanna; Hogdall, Estrid; Hogdall, Claus; Lundvall, Lene; Pharoah, Paul D.P.; Song, Honglin; Campbell, Ian; Eccles, Diana; McNeish, Iain; Whittemore, Alice; McGuire, Valerie; Sieh, Weiva; Rothstein, Joseph; Phelan, Catherine M.; Risch, Harvey; Narod, Steven; McLaughlin, John; Anton-Culver, Hoda; Ziogas, Argyrios; Menon, Usha; Gayther, Simon; Ramus, Susan J.; Gentry-Maharaj, Aleksandra; Pearce, Celeste Leigh; Wu, Anna H.; Kupryjanczyk, Jolanta; Dansonka-Mieszkowska, Agnieszka; Schildkraut, Joellen M.; Cheng, Jin Q.; Goode, Ellen L.; Sellers, Thomas A.

2016-01-01

RNA editing in mammals is a form of post-transcriptional modification in which adenosine is converted to inosine by the adenosine deaminases acting on RNA (ADAR) family of enzymes. Based on evidence of altered ADAR expression in epithelial ovarian cancers (EOC), we hypothesized that single nucleotide polymorphisms (SNPs) in ADAR genes modify EOC susceptibility, potentially by altering ovarian tissue gene expression. Using directly genotyped and imputed data from 10,891 invasive EOC cases and 21,693 controls, we evaluated the associations of 5,303 SNPs in ADAD1, ADAR, ADAR2, ADAR3, and SND1. Unconditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI), with adjustment for European ancestry. We conducted gene-level analyses using the Admixture Maximum Likelihood (AML) test and the Sequence-Kernel Association test for common and rare variants (SKAT-CR). Association analysis revealed top risk-associated SNP rs77027562 (OR (95% CI)= 1.39 (1.17-1.64), P=1.0×10−4) in ADAR3 and rs185455523 in SND1 (OR (95% CI)= 0.68 (0.56-0.83), P=2.0×10−4). When restricting to serous histology (n=6,500), the magnitude of association strengthened for rs185455523 (OR=0.60, P=1.0×10−4). Gene-level analyses revealed that variation in ADAR was associated (P<0.05) with EOC susceptibility, with PAML=0.022 and PSKAT-CR=0.020. Expression quantitative trait locus analysis in EOC tissue revealed significant associations (P<0.05) with ADAR expression for several SNPs in ADAR, including rs1127313 (G/A), a SNP in the 3′ untranslated region. In summary, germline variation involving RNA editing genes may influence EOC susceptibility, warranting further investigation of inherited and acquired alterations affecting RNA editing. PMID:27911851

Determination of serum adenosine deaminase and xanthine oxidase activity in Kangal dogs with maternal cannibalism.

PubMed

Ercan, N; Koçkaya, M; Kapancik, S; Bakir, D

2017-11-01

Kangal dogs, known as guard dogs in many countries of the world, have been found to eat their own puppies during their first 24 h following birth, which is called as maternal cannibalism. Adenosine deaminase (ADA) and xanthine oxidase (XO) are important enzymes for purine metabolism. In this study, the aim is to evaluate ADA and XO activities in Kangal dogs with maternal cannibalism. The material of the study consists of the blood sera of Kangal dog breed with and without maternal cannibalism in the breeders around Sivas city and its districts. ADA and XO activities in blood serum of these animals were investigated by spectrophotometric method. ADA activities in Kangal dogs with maternal cannibalism were increased to the control group without maternal cannibalism (p<0.01). Postnatal measurement of ADA activity in dogs may be useful in assessing maternal cannibalism.

RNA editing is induced by type I interferon in esophageal squamous cell carcinoma.

PubMed

Zhang, Jinyao; Chen, Zhaoli; Tang, Zefang; Huang, Jianbing; Hu, Xueda; He, Jie

2017-07-01

In recent years, abnormal RNA editing has been shown to play an important role in the development of esophageal squamous cell carcinoma, as such abnormal editing is catalyzed by ADAR (adenosine deaminases acting on RNA). However, the regulatory mechanism of ADAR1 in esophageal squamous cell carcinomas remains largely unknown. In this study, we investigated ADAR1 expression and its association with RNA editing in esophageal squamous cell carcinomas. RNA sequencing applied to esophageal squamous cell carcinoma clinical samples showed that ADAR1 expression was correlated with the expression of STAT1, STAT2, and IRF9. In vitro experiments showed that the abundance of ADAR1 protein was associated with the induced activation of the JAK/STAT pathway by type I interferon. RNA sequencing results showed that treatment with type I interferon caused an increase in the number and degree of RNA editing in esophageal squamous cell carcinoma cell lines. In conclusion, the activation of the JAK/STAT pathway is a regulatory mechanism of ADAR1 expression and causes abnormal RNA editing profile in esophageal squamous cell carcinoma. This mechanism may serve as a new target for esophageal squamous cell carcinoma therapy.

Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum*

PubMed Central

Cassera, María B.; Hazleton, Keith Z.; Riegelhaupt, Paul M.; Merino, Emilio F.; Luo, Minkui; Akabas, Myles H.; Schramm, Vern L.

2008-01-01

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum. PMID:18799466

Erythrocytic adenosine monophosphate as an alternative purine source in Plasmodium falciparum.

PubMed

Cassera, María B; Hazleton, Keith Z; Riegelhaupt, Paul M; Merino, Emilio F; Luo, Minkui; Akabas, Myles H; Schramm, Vern L

2008-11-21

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.

Adenosine deaminase polymorphism affects sleep EEG spectral power in a large epidemiological sample.

PubMed

Mazzotti, Diego Robles; Guindalini, Camila; de Souza, Altay Alves Lino; Sato, João Ricardo; Santos-Silva, Rogério; Bittencourt, Lia Rita Azeredo; Tufik, Sergio

2012-01-01

Slow wave oscillations in the electroencephalogram (EEG) during sleep may reflect both sleep need and intensity, which are implied in homeostatic regulation. Adenosine is strongly implicated in sleep homeostasis, and a single nucleotide polymorphism in the adenosine deaminase gene (ADA G22A) has been associated with deeper and more efficient sleep. The present study verified the association between the ADA G22A polymorphism and changes in sleep EEG spectral power (from C3-A2, C4-A1, O1-A2, and O2-A1 derivations) in the Epidemiologic Sleep Study (EPISONO) sample from São Paulo, Brazil. Eight-hundred individuals were subjected to full-night polysomnography and ADA G22A genotyping. Spectral analysis of the EEG was carried out in all individuals using fast Fourier transformation of the signals from each EEG electrode. The genotype groups were compared in the whole sample and in a subsample of 120 individuals matched according to ADA genotype for age, gender, body mass index, caffeine intake status, presence of sleep disturbance, and sleep-disturbing medication. When compared with homozygous GG genotype carriers, A allele carriers showed higher delta spectral power in Stage 1 and Stages 3+4 of sleep, and increased theta spectral power in Stages 1, 2 and REM sleep. These changes were seen both in the whole sample and in the matched subset. The higher EEG spectral power indicates that the sleep of individuals carrying the A allele may be more intense. Therefore, this polymorphism may be an important source of variation in sleep homeostasis in humans, through modulation of specific components of the sleep EEG.

Autoimmune Dysregulation and Purine Metabolism in Adenosine Deaminase Deficiency

PubMed Central

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

2012-01-01

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

Adenosine monophosphate deaminase 3 activation shortens erythrocyte half-life and provides malaria resistance in mice.

PubMed

Hortle, Elinor; Nijagal, Brunda; Bauer, Denis C; Jensen, Lora M; Ahn, Seong Beom; Cockburn, Ian A; Lampkin, Shelley; Tull, Dedreia; McConville, Malcolm J; McMorran, Brendan J; Foote, Simon J; Burgio, Gaetan

2016-09-01

The factors that determine red blood cell (RBC) lifespan and the rate of RBC aging have not been fully elucidated. In several genetic conditions, including sickle cell disease, thalassemia, and G6PD deficiency, erythrocyte lifespan is significantly shortened. Many of these diseases are also associated with protection from severe malaria, suggesting a role for accelerated RBC senescence and clearance in malaria resistance. Here, we report a novel, N-ethyl-N-nitrosourea-induced mutation that causes a gain of function in adenosine 5'-monophosphate deaminase (AMPD3). Mice carrying the mutation exhibit rapid RBC turnover, with increased erythropoiesis, dramatically shortened RBC lifespan, and signs of increased RBC senescence/eryptosis, suggesting a key role for AMPD3 in determining RBC half-life. Mice were also found to be resistant to infection with the rodent malaria Plasmodium chabaudi. We propose that resistance to P. chabaudi is mediated by increased RBC turnover and higher rates of erythropoiesis during infection. © 2016 by The American Society of Hematology.

A case of red-cell adenosine deaminase overproduction associated with hereditary hemolytic anemia found in Japan.

PubMed

Miwa, S; Fujii, H; Matsumoto, N; Nakatsuji, T; Oda, S; Asano, H; Asano, S

1978-01-01

A case of red cell adenosine deaminase (ADA) overproduction associated with hereditary hemolytic anemia is reported here. This appears to be the second report. Proband is a 38-year-old Japanese male who had hemoglobin, 15.8 g/100 ml; reticulocyte count, 4.5%; serum indirect bilirubin, 4.9 mg/100 ml; 51Cr-labeled red cell half-life, 12 days; red cells showed moderate stomatocytosis. His red cell ADA activity showed 40-fold increase while that of the mother showed 4-fold increase. The mother was hematologically normal. The father had a normal enzyme activity. The proband and the mother showed slightly high serum uric acid levels. The proband's red cell showed: ATP, 628 nmoles/ml (normal, 1,010--1,550); adenine nucleotide pool, 46% of the normal mean; 2,3-diphosphoglycerate content, 3,782 nmoles/ml (normal 4,170--5,300); increased oxygen affinity of hemoglobin, P50 of intact erythrocytes being 21.8 mmHg (normal, 24.1--26.1). Red cell glycolytic intermediates in the proband were low in general, and the rate of lactate production was low. Kinetic studies using crude hemolysate revealed a normal Km for adenosine, normal electrophoretic mobility but slightly abnormal pH curve and slightly low utilization of 2-deoxyadenosine. The ADA activity of lymphocytes was nearly normal.

Dyschromatosis symmetrica hereditaria with chilblains due to a novel two-amino-acid deletion in the double-stranded RNA binding domain of ADAR1.

PubMed

Kono, M; Suganuma, M; Shimada, T; Ishikura, Y; Watanabe, S; Takeichi, T; Muro, Y; Akiyama, M

2018-05-18

Dyschromatosis symmetrica hereditaria (DSH) is an autosomal dominant skin disease caused by a heterozygous mutation of ADAR1. 1 DSH is characterized by a mixture of hyper- and hypo-pigmented small macules in the extremities. Among the mutations, the pathogenicity of in-frame deletion in regions other than the deaminase domain has not been clarified in DSH. 2 This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

The rate of the AMP/adenosine substrate cycle in concanavalin-A-stimulated rat lymphocytes.

PubMed Central

Szondy, Z; Newsholme, E A

1989-01-01

The effect of adenosine on the metabolism of prelabelled adenine nucleotides was investigated in concanavalin-A-stimulated rat lymphocytes. Adenosine in the presence of the adenosine deaminase inhibitor, deoxycoformycin, caused a 2-fold increase in the ATP concentration. This effect was, in part, countereacted by an increased rate of adenine nucleotide catabolism, which could be explained by a stimulation of AMP deaminase (EC 3.5.4.6). At the same time a continuous rate of labelled adenosine production was found, which was not affected by the increased ATP concentration and which could only be detected by the trapping effect of a high concentration of added unlabelled adenosine. It is concluded that the rate of the substrate cycle between AMP and adenosine is low (1.9 +/- 0.2 nmol/h per 10(7) cells) in comparison to the rate of AMP deamination. PMID:2552990

[Diagnosis of tuberculosis meningitis by detection of adenosine deaminase activity and amplification of nucleotide sequences with PCR].

PubMed

Correa, M F; Armas, E; Díaz, D; de Elguezabal, K; De la Rosa, M L; Calles, G; Adjounian, H; Pedroza, R

2001-01-01

Tuberculous meningitis (TBM) is the most severe and lethal form of tuberculosis. The rapid bacteriological diagnosis with the conventional techniques is nearly impossible in TBM. There for many patients are treated with anti-TBC drugs without a definitive diagnosis. A more fast and accurate diagnostic method is necessary, in order to initiate the treatment on time to prevent the irreversible neurologic sequel or death. We evaluated the use of two rapid methods: Adenosine deaminase activity (ADA) and polymerase chain reaction (PCR) for IS6110 and mtp40 sequences on cerebrospinal fluid (CSF) from chronic meningitis patients. For ADA activity > 8.0 U/L the sensibility and specificity was 80% and 91%. PCR sensibility was 80% and specificity 97%. ADA activity and PCR on CSF could be specially useful as complementary tools in the early diagnosis of TBM.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larson, E.T.; Deng, W.; Krumm, B.E.

Plasmodium and other apicomplexan parasites are deficient in purine biosynthesis, relying instead on the salvage of purines from their host environment. Therefore, interference with the purine salvage pathway is an attractive therapeutic target. The plasmodial enzyme adenosine deaminase (ADA) plays a central role in purine salvage and, unlike mammalian ADA homologs, has a further secondary role in methylthiopurine recycling. For this reason, plasmodial ADA accepts a wider range of substrates, as it is responsible for deamination of both adenosine and 5{prime}-methylthioadenosine. The latter substrate is not accepted by mammalian ADA homologs. The structural basis for this natural difference in specificitymore » between plasmodial and mammalian ADA has not been well understood. We now report crystal structures of Plasmodium vivax ADA in complex with adenosine, guanosine, and the picomolar inhibitor 2{prime}-deoxycoformycin. These structures highlight a drastic conformational change in plasmodial ADA upon substrate binding that has not been observed for mammalian ADA enzymes. Further, these complexes illuminate the structural basis for the differential substrate specificity and potential drug selectivity between mammalian and parasite enzymes.« less

Nuclear factor 90 uses an ADAR2-like binding mode to recognize specific bases in dsRNA.

PubMed

Jayachandran, Uma; Grey, Heather; Cook, Atlanta G

2016-02-29

Nuclear factors 90 and 45 (NF90 and NF45) form a protein complex involved in the post-transcriptional control of many genes in vertebrates. NF90 is a member of the dsRNA binding domain (dsRBD) family of proteins. RNA binding partners identified so far include elements in 3' untranslated regions of specific mRNAs and several non-coding RNAs. In NF90, a tandem pair of dsRBDs separated by a natively unstructured segment confers dsRNA binding activity. We determined a crystal structure of the tandem dsRBDs of NF90 in complex with a synthetic dsRNA. This complex shows surprising similarity to the tandem dsRBDs from an adenosine-to-inosine editing enzyme, ADAR2 in complex with a substrate RNA. Residues involved in unusual base-specific recognition in the minor groove of dsRNA are conserved between NF90 and ADAR2. These data suggest that, like ADAR2, underlying sequences in dsRNA may influence how NF90 recognizes its target RNAs. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Coronary Artery Disease: A Study on the Joint Role of Birth Weight, Adenosine Deaminase, and Gender

PubMed Central

Gloria-Bottini, F.; Banci, M.; Saccucci, P.; Lucarini, N.; Ianniello, F.; Paradisi, G.; Magrini, A.; Bottini, E.

2009-01-01

An inverse relationship between birth weight and coronary artery diseases is well documented but it remains unclear which exposure in early life might underlie such association. Recently it has been reported an association between adenosine deaminase genetic polymorphism and coronary artery diseases. Gender differences in the degree of this association have been also observed. These observations prompted us to study the possible joint effects of BW, ADA, and gender on the susceptibility to coronary artery diseases. 222 subjects admitted to hospital for nonfatal coronary artery diseases, and 762 healthy consecutive newborns were studied. ADA genotypes were determined by DNA analysis. A highly significant complex relationship has emerged among ADA, birth weight, and gender concerning their role on susceptibility to coronary artery diseases in adult life. Odds ratio analysis suggests that low birth weight is more important in females than in males. ADA∗2 allele appears protective in males, while in females such effect is obscured by birth weight. PMID:20428226

A-to-I RNA Editing Up-regulates Human Dihydrofolate Reductase in Breast Cancer.

PubMed

Nakano, Masataka; Fukami, Tatsuki; Gotoh, Saki; Nakajima, Miki

2017-03-24

Dihydrofolate reductase (DHFR) plays a key role in folate metabolism and is a target molecule of methotrexate. An increase in the cellular expression level of DHFR is one of the mechanisms of tumor resistance to methotrexate. The present study investigated the possibility that adenosine-to-inosine RNA editing, which causes nucleotide conversion by adenosine deaminase acting on RNA (ADAR) enzymes, might modulate DHFR expression. In human breast adenocarcinoma-derived MCF-7 cells, 26 RNA editing sites were identified in the 3'-UTR of DHFR. Knockdown of ADAR1 decreased the RNA editing levels of DHFR and resulted in a decrease in the DHFR mRNA and protein levels, indicating that ADAR1 up-regulates DHFR expression. Using a computational analysis, miR-25-3p and miR-125a-3p were predicted to bind to the non-edited 3'-UTR of DHFR but not to the edited sequence. The decrease in DHFR expression by the knockdown of ADAR1 was restored by transfection of antisense oligonucleotides for these miRNAs, suggesting that RNA editing mediated up-regulation of DHFR requires the function of these miRNAs. Interestingly, we observed that the knockdown of ADAR1 decreased cell viability and increased the sensitivity of MCF-7 cells to methotrexate. ADAR1 expression levels and the RNA editing levels in the 3'-UTR of DHFR in breast cancer tissues were higher than those in adjacent normal tissues. Collectively, the present study demonstrated that ADAR1 positively regulates the expression of DHFR by editing the miR-25-3p and miR-125a-3p binding sites in the 3'-UTR of DHFR, enhancing cellular proliferation and resistance to methotrexate. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The adenosine deaminases of Plasmodium vivax and Plasmodium falciparum exhibit surprising differences in ligand specificity

PubMed Central

Ivanov, Andrei A.; Matsumura, Ichiro

2012-01-01

Plasmodium vivax and P. falciparum cause malaria, so proteins essential for their survival in vivo are potential anti-malarial drug targets. Adenosine deaminases (ADA) catalyze the irreversible conversion of adenosine into inosine, and play a critical role in the purine salvage pathways of Plasmodia and their mammalian hosts. Currently, the number of selective inhibitors of Plasmodium ADAs is limited. One potent and widely used inhibitor of the human ADA (hADA), erythro-9-(2-hydroxy-3-nonly)adenine (EHNA), is a very weak inhibitor (Ki = 120uM) of P. falciparum ADA (pfADA). EHNA-like compounds are thus excluded from consideration as potential inhibitors of Plasmodium ADA in general. However, EHNA activity in P. vivax ADA (pvADA) has not been reported. Here we applied computational molecular modeling to identify the mechanisms of the ligand recognition unique for P. vivax and P. falciparum ADA. Based on the computational studies, we performed molecular biology experiments to show that EHNA is at least 60-fold more potent against pvADA (Ki = 1.9uM) than against pfADA. The D172A pvADA mutant is bound even more tightly (Ki = 0.9uM). These results improve our understanding of the mechanisms of ADA ligand recognition and species-selectivity, and facilitate the rational design of novel EHNA-based ADA inhibitors as anti-malarial drugs. To demonstrate a practical application of our findings we have computationally predicted a novel potential inhibitor of pvADA selective versus the human ADA. PMID:22481078

MicroRNA profiling of human primary macrophages exposed to dengue virus identifies miRNA-3614-5p as antiviral and regulator of ADAR1 expression

PubMed Central

Echavarría-Consuegra, Liliana; Flipse, Jacky; Fernández, Geysson Javier; Kluiver, Joost; van den Berg, Anke; Urcuqui-Inchima, Silvio; Smit, Jolanda M.

2017-01-01

Background Due to the high burden of dengue disease worldwide, a better understanding of the interactions between dengue virus (DENV) and its human host cells is of the utmost importance. Although microRNAs modulate the outcome of several viral infections, their contribution to DENV replication is poorly understood. Methods and principal findings We investigated the microRNA expression profile of primary human macrophages challenged with DENV and deciphered the contribution of microRNAs to infection. To this end, human primary macrophages were challenged with GFP-expressing DENV and sorted to differentiate between truly infected cells (DENV-positive) and DENV-exposed but non-infected cells (DENV-negative cells). The miRNAome was determined by small RNA-Seq analysis and the effect of differentially expressed microRNAs on DENV yield was examined. Five microRNAs were differentially expressed in human macrophages challenged with DENV. Of these, miR-3614-5p was found upregulated in DENV-negative cells and its overexpression reduced DENV infectivity. The cellular targets of miR-3614-5p were identified by liquid chromatography/mass spectrometry and western blot. Adenosine deaminase acting on RNA 1 (ADAR1) was identified as one of the targets of miR-3614-5p and was shown to promote DENV infectivity at early time points post-infection. Conclusion/Significance Overall, miRNAs appear to play a limited role in DENV replication in primary human macrophages. The miRNAs that were found upregulated in DENV-infected cells did not control the production of infectious virus particles. On the other hand, miR-3614-5p, which was upregulated in DENV-negative macrophages, reduced DENV infectivity and regulated ADAR1 expression, a protein that facilitates viral replication. PMID:29045406

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

PubMed

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 < 0·05) increased in NP, HP, GDM and HIP groups when compared with the CG, while the addition of 10 µM of the same agonist caused significant (p < 0·05) elevations in HP, GDM and HIP groups when compared with CG. Furthermore, ADA activity was significantly (p < 0·05) enhanced in NP, HP, GDM and HIP groups when compared with CG. In this study, the increased platelet aggregation and ADA activity in 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. Copyright © 2016 John Wiley & Sons, Ltd.

Defective B cell tolerance in adenosine deaminase deficiency is corrected by gene therapy

PubMed Central

Sauer, Aisha V.; Morbach, Henner; Brigida, Immacolata; Ng, Yen-Shing; Aiuti, Alessandro; Meffre, Eric

2012-01-01

Adenosine deaminase (ADA) gene defects are among the most common causes of SCID. Restoration of purine metabolism and immune functions can be achieved by enzyme replacement therapy, or more effectively by bone marrow transplant or HSC gene therapy (HSC-GT). However, autoimmune complications and autoantibody production, including anti-nuclear antibodies (ANAs), frequently occur in ADA-SCID patients after treatment. To assess whether ADA deficiency affects the establishment of B cell tolerance, we tested the reactivity of recombinant antibodies isolated from single B cells of ADA-SCID patients before and after HSC-GT. We found that before HSC-GT, new emigrant/transitional and mature naive B cells from ADA-SCID patients contained more autoreactive and ANA-expressing clones, indicative of defective central and peripheral B cell tolerance checkpoints. We further observed impaired B cell receptor (BCR) and TLR functions in B cells after ADA inhibition, which may underlie the defects in B cell tolerance. Strikingly, after HSC-GT, ADA-SCID patients displayed quasi-normal early B cell tolerance checkpoints, as evidenced by restored removal of developing autoreactive and ANA-expressing B cells. Hence, ADA plays an essential role in controlling autoreactive B cell counterselection by regulating BCR and TLR functions. PMID:22622038

AZIN1 RNA editing confers cancer stemness and enhances oncogenic potential in colorectal cancer.

PubMed

Shigeyasu, Kunitoshi; Okugawa, Yoshinaga; Toden, Shusuke; Miyoshi, Jinsei; Toiyama, Yuji; Nagasaka, Takeshi; Takahashi, Naoki; Kusunoki, Masato; Takayama, Tetsuji; Yamada, Yasuhide; Fujiwara, Toshiyoshi; Chen, Leilei; Goel, Ajay

2018-06-21

Adenosine-to-inosine (A-to-I) RNA editing, a process mediated by adenosine deaminases that act on the RNA (ADAR) gene family, is a recently discovered epigenetic modification dysregulated in human cancers. However, the clinical significance and the functional role of RNA editing in colorectal cancer (CRC) remain unclear. We have systematically and comprehensively investigated the significance of the expression status of ADAR1 and of the RNA editing levels of antizyme inhibitor 1 (AZIN1), one of the most frequently edited genes in cancers, in 392 colorectal tissues from multiple independent CRC patient cohorts. Both ADAR1 expression and AZIN1 RNA editing levels were significantly elevated in CRC tissues when compared with corresponding normal mucosa. High levels of AZIN1 RNA editing emerged as a prognostic factor for overall survival and disease-free survival and were an independent risk factor for lymph node and distant metastasis. Furthermore, elevated AZIN1 editing identified high-risk stage II CRC patients. Mechanistically, edited AZIN1 enhances stemness and appears to drive the metastatic processes. We have demonstrated that edited AZIN1 functions as an oncogene and a potential therapeutic target in CRC. Moreover, AZIN1 RNA editing status could be used as a clinically relevant prognostic indicator in CRC patients.

Spectroscopy and computational studies on the interaction of octyl, dodecyl, and hexadecyl derivatives of anionic and cationic surfactants with adenosine deaminase.

PubMed

Ajloo, Davood; Mahmoodabadi, Najmeh; Ghadamgahi, Maryam; Saboury, Ali Akbar

2016-07-01

Effects of sodium (octyl, dodecyl, hexadecyl) sulfate and their cationic analogous on the structure of adenosine deaminase (ADA) were investigated by fluorescence and circular dichroism spectroscopy as well as molecular dynamics simulation and docking calculation. Root-mean-square derivations, radius of gyration, solvent accessible surface area, and radial distribution function were obtained. The results showed that anionic and cationic surfactants reduce protein stability. Cationic surfactants have more effect on the ADA structure in comparison with anionic surfactants. More concentration and longer surfactants are parallel to higher denaturation. Furthermore, aggregation in the presence of anionic surfactants is more than cationic surfactants. Docking data showed that longer surfactants have more interaction energy and smaller ones bound to the active site.

Diagnostic value of sputum adenosine deaminase (ADA) level in pulmonary tuberculosis.

PubMed

Binesh, Fariba; Jalali, Hadi; Zare, Mohammad Reza; Behravan, Farhad; Tafti, Arefeh Dehghani; Behnaz, Fatemah; Tabatabaee, Mohammad; Shahcheraghi, Seyed Hossein

2016-06-01

Tuberculosis is still a considerable health problem in many countries. Rapid diagnosis of this disease is important, and adenosine deaminase (ADA) has been used as a diagnostic test. The aim of this study was to assess the diagnostic value of ADA in the sputum of patients with pulmonary tuberculosis. The current study included 40 patients with pulmonary tuberculosis (culture positive, smear ±) and 42 patients with non tuberculosis pulmonary diseases (culture negative). ADA was measured on all of the samples. The median value of ADA in non-tuberculosis patients was 2.94 (4.2) U/L and 4.01 (6.54) U/L in tuberculosis patients, but this difference was not statistically significant (p=0.100). The cut-off point of 3.1 U/L had a sensitivity of 61% and a specificity of 53%, the cut-off point of 2.81 U/L had a sensitivity of 64% and a specificity of 50% and the cut-off point of 2.78 U/L had a sensitivity of 65% and a specificity of 48%. The positive predictive values for cut-off points of 3.1, 2.81 and 2.78 U/L were 55.7%, 57.44% and 69.23%, respectively. The negative predictive values for the abovementioned cut-off points were 56.75%, 57.14% and 55.88%, respectively. Our results showed that sputum ADA test is neither specific nor sensitive. Because of its low sensitivity and specificity, determination of sputum ADA for the diagnosis of pulmonary tuberculosis is not recommended.

Absence of the Fragile X Mental Retardation Protein results in defects of RNA editing of neuronal mRNAs in mouse

PubMed Central

Filippini, Alice; Bonini, Daniela; Lacoux, Caroline; Zingariello, Maria; Sancillo, Laura; Bosisio, Daniela; Salvi, Valentina; Mingardi, Jessica; La Via, Luca; Zalfa, Francesca; Bagni, Claudia

2017-01-01

ABSTRACT The fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the absence of FMRP, a protein regulating RNA metabolism. Recently, an unexpected function of FMRP in modulating the activity of Adenosine Deaminase Acting on RNA (ADAR) enzymes has been reported both in Drosophila and Zebrafish. ADARs are RNA-binding proteins that increase transcriptional complexity through a post-transcriptional mechanism called RNA editing. To evaluate the ADAR2-FMRP interaction in mammals we analyzed several RNA editing re-coding sites in the fmr1 knockout (KO) mice. Ex vivo and in vitro analysis revealed that absence of FMRP leads to an increase in the editing levels of brain specific mRNAs, indicating that FMRP might act as an inhibitor of editing activity. Proximity Ligation Assay (PLA) in mouse primary cortical neurons and in non-neuronal cells revealed that ADAR2 and FMRP co-localize in the nucleus. The ADAR2-FMRP co-localization was further observed by double-immunogold Electron Microscopy (EM) in the hippocampus. Moreover, ADAR2-FMRP interaction appeared to be RNA independent. Because changes in the editing pattern are associated with neuropsychiatric and neurodevelopmental disorders, we propose that the increased editing observed in the fmr1-KO mice might contribute to the FXS molecular phenotypes. PMID:28640668

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

PubMed

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

A-to-I RNA editing promotes developmental stage–specific gene and lncRNA expression

PubMed Central

Goldstein, Boaz; Agranat-Tamir, Lily; Light, Dean; Ben-Naim Zgayer, Orna; Fishman, Alla; Lamm, Ayelet T.

2017-01-01

A-to-I RNA editing is a conserved widespread phenomenon in which adenosine (A) is converted to inosine (I) by adenosine deaminases (ADARs) in double-stranded RNA regions, mainly noncoding. Mutations in ADAR enzymes in Caenorhabditis elegans cause defects in normal development but are not lethal as in human and mouse. Previous studies in C. elegans indicated competition between RNA interference (RNAi) and RNA editing mechanisms, based on the observation that worms that lack both mechanisms do not exhibit defects, in contrast to the developmental defects observed when only RNA editing is absent. To study the effects of RNA editing on gene expression and function, we established a novel screen that enabled us to identify thousands of RNA editing sites in nonrepetitive regions in the genome. These include dozens of genes that are edited at their 3′ UTR region. We found that these genes are mainly germline and neuronal genes, and that they are down-regulated in the absence of ADAR enzymes. Moreover, we discovered that almost half of these genes are edited in a developmental-specific manner, indicating that RNA editing is a highly regulated process. We found that many pseudogenes and other lncRNAs are also extensively down-regulated in the absence of ADARs in the embryo but not in the fourth larval (L4) stage. This down-regulation is not observed upon additional knockout of RNAi. Furthermore, levels of siRNAs aligned to pseudogenes in ADAR mutants are enhanced. Taken together, our results suggest a role for RNA editing in normal growth and development by regulating silencing via RNAi. PMID:28031250

A-to-I RNA editing promotes developmental stage-specific gene and lncRNA expression.

PubMed

Goldstein, Boaz; Agranat-Tamir, Lily; Light, Dean; Ben-Naim Zgayer, Orna; Fishman, Alla; Lamm, Ayelet T

2017-03-01

A-to-I RNA editing is a conserved widespread phenomenon in which adenosine (A) is converted to inosine (I) by adenosine deaminases (ADARs) in double-stranded RNA regions, mainly noncoding. Mutations in ADAR enzymes in Caenorhabditis elegans cause defects in normal development but are not lethal as in human and mouse. Previous studies in C. elegans indicated competition between RNA interference (RNAi) and RNA editing mechanisms, based on the observation that worms that lack both mechanisms do not exhibit defects, in contrast to the developmental defects observed when only RNA editing is absent. To study the effects of RNA editing on gene expression and function, we established a novel screen that enabled us to identify thousands of RNA editing sites in nonrepetitive regions in the genome. These include dozens of genes that are edited at their 3' UTR region. We found that these genes are mainly germline and neuronal genes, and that they are down-regulated in the absence of ADAR enzymes. Moreover, we discovered that almost half of these genes are edited in a developmental-specific manner, indicating that RNA editing is a highly regulated process. We found that many pseudogenes and other lncRNAs are also extensively down-regulated in the absence of ADARs in the embryo but not in the fourth larval (L4) stage. This down-regulation is not observed upon additional knockout of RNAi. Furthermore, levels of siRNAs aligned to pseudogenes in ADAR mutants are enhanced. Taken together, our results suggest a role for RNA editing in normal growth and development by regulating silencing via RNAi. © 2017 Goldstein et al.; Published by Cold Spring Harbor Laboratory Press.

Discovery of a cAMP Deaminase That Quenches Cyclic AMP-Dependent Regulation

PubMed Central

Goble, Alissa M.; Feng, Youjun; Raushel, Frank M.; Cronan, John E.

2013-01-01

An enzyme of unknown function within the amidohydrolase superfamily was discovered to catalyze the hydrolysis of the universal second messenger, cyclic-3’, 5’-adenosine monophosphate (cAMP). The enzyme, which we have named CadD, is encoded by the human pathogenic bacterium Leptospira interrogans. Although CadD is annotated as an adenosine deaminase, the protein specifically deaminates cAMP to cyclic-3’, 5’-inosine monophosphate (cIMP) with a kcat/Km of 2.7 ± 0.4 × 105 M−1 s−1 and has no activity on adenosine, adenine, or 5’-adenosine monophosphate (AMP). This is the first identification of a deaminase specific for cAMP. Expression of CadD in Escherichia coli mimics the loss of adenylate cyclase in that it blocks growth on carbon sources that require the cAMP-CRP transcriptional activator complex for expression of the cognate genes. The cIMP reaction product cannot replace cAMP as the ligand for CRP binding to DNA in vitro and cIMP is a very poor competitor of cAMP activation of CRP for DNA binding. Transcriptional analyses indicate that CadD expression represses expression of several cAMP-CRP dependent genes. CadD adds a new activity to the cAMP metabolic network and may be a useful tool in intracellular study of cAMP-dependent processes. PMID:24074367

Tandem mass spectrometry, but not T-cell receptor excision circle analysis, identifies newborns with late-onset adenosine deaminase deficiency.

PubMed

la Marca, Giancarlo; Canessa, Clementina; Giocaliere, Elisa; Romano, Francesca; Duse, Marzia; Malvagia, Sabrina; Lippi, Francesca; Funghini, Silvia; Bianchi, Leila; Della Bona, Maria Luisa; Valleriani, Claudia; Ombrone, Daniela; Moriondo, Maria; Villanelli, Fabio; Speckmann, Carsten; Adams, Stuart; Gaspar, Bobby H; Hershfield, Michael; Santisteban, Ines; Fairbanks, Lynette; Ragusa, Giovanni; Resti, Massimo; de Martino, Maurizio; Guerrini, Renzo; Azzari, Chiara

2013-06-01

Adenosine deaminase (ADA)-severe combined immunodeficiency (SCID) is caused by genetic variants that disrupt the function of ADA. In its early-onset form, it is rapidly fatal to infants. Delayed or late-onset ADA-SCID is characterized by insidious progressive immunodeficiency that leads to permanent organ damage or death. Quantification of T-cell receptor excision circles (TRECs) or tandem mass spectrometry (tandem-MS) analysis of dried blood spots (DBSs) collected at birth can identify newborns with early-onset ADA-SCID and are used in screening programs. However, it is not clear whether these analyses can identify newborns who will have delayed or late-onset ADA-SCID before symptoms appear. We performed a retrospective study to evaluate whether tandem-MS and quantitative TREC analyses of DBSs could identify newborns who had delayed-onset ADA-SCID later in life. We tested stored DBSs collected at birth from 3 patients with delayed-onset ADA-SCID using tandem-MS (PCT EP2010/070517) to evaluate levels of adenosine and 2'-deoxyadenosine and real-time PCR to quantify TREC levels. We also analyzed DBSs from 3 newborns with early-onset ADA-SCID and 2 healthy newborn carriers of ADA deficiency. The DBSs taken at birth from the 3 patients with delayed-onset ADA-SCID had adenosine levels of 10, 25, and 19 μmol/L (normal value, <1.5 μmol/L) and 2'-deoxyadenosine levels of 0.7, 2.7, and 2.4 μmol/L (normal value, <0.07 μmol/L); the mean levels of adenosine and 2'-deoxyadenosine were respectively 12.0- and 27.6-fold higher than normal values. DBSs taken at birth from all 3 patients with delayed-onset ADA deficiency had normal TREC levels, but TRECs were undetectable in blood samples taken from the same patients at the time of diagnosis. Tandem-MS but not TREC quantification identifies newborns with delayed- or late-onset ADA deficiency. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Adenosine Deaminase activity and HLA-DRB as diagnostic markers for Rheumatoid Arthritis.

PubMed

Valadbeigi, Shirin; Ebrahimi-Rad, Mina; Khatami, Shohreh; Akhbari, Hadi; Saghiri, Reza

2018-04-05

Rheumatoid Arthritis (RA) is a chronic multi systemic disorder with the unclarified ethiopathology. Although several markers have been presented for recognition of RA, but none of them has been specific. New markers such as HLA typing and activity of Adenosine deaminase (ADA) isoenzymes could be useful and specific. The aim of this study is to evaluate the pattern of ADA isoenzymes activity and HLA typing in both RA patients and healthy cases. Blood samples were collected from 55 RA patients and 60 healthy subjects, over a period of 6 months. Levels of C-reactive protein (CRP), rheumatoid factor (RF) and ADA (ADA1, ADA2, total ADA) were measured using AVITEX kit and HITACHI Auto Analyzer. In addition, HLA-DRB1*1,*04 and *010 was detected using PCR-SSP. ADA activity, particularly ADA2 level, was significantly higher among RA group (P<0.05). The concentrations of tADA in patients with RF and CRP positive were significantly higher (P <0.05). The allele prevalence of DRB1*10 and *01 was significantly higher in RA patients (8.3% and 13.1%, respectively) compared with control group (2.51% and 5.5%, respectively) (P <0.05). Calculated sensitivity and specificity for diagnostic tests in this study are listed as: CRP (75%), RF (80%), ADA (84%) and RF (90%), ADA (83%), CRP (72%), respectively. Increase tADA level and the frequency of DRB1*010 and *01 caused to susceptibility to RA. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

RNA Editing Modulates Human Hepatic Aryl Hydrocarbon Receptor Expression by Creating MicroRNA Recognition Sequence.

PubMed

Nakano, Masataka; Fukami, Tatsuki; Gotoh, Saki; Takamiya, Masataka; Aoki, Yasuhiro; Nakajima, Miki

2016-01-08

Adenosine to inosine (A-to-I) RNA editing is the most frequent type of post-transcriptional nucleotide conversion in humans, and it is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes. In this study we investigated the effect of RNA editing on human aryl hydrocarbon receptor (AhR) expression because the AhR transcript potentially forms double-stranded structures, which are targets of ADAR enzymes. In human hepatocellular carcinoma-derived Huh-7 cells, the ADAR1 knockdown reduced the RNA editing levels in the 3'-untranslated region (3'-UTR) of the AhR transcript and increased the AhR protein levels. The ADAR1 knockdown enhanced the ligand-mediated induction of CYP1A1, a gene downstream of AhR. We investigated the possibility that A-to-I RNA editing creates miRNA targeting sites in the AhR mRNA and found that the miR-378-dependent down-regulation of AhR was abolished by ADAR1 knockdown. These results indicated that the ADAR1-mediated down-regulation of AhR could be attributed to the creation of a miR-378 recognition site in the AhR 3'-UTR. The interindividual differences in the RNA editing levels within the AhR 3'-UTR in a panel of 32 human liver samples were relatively small, whereas the differences in ADAR1 expression were large (220-fold). In the human liver samples a significant inverse association was observed between the miR-378 and AhR protein levels, suggesting that the RNA-editing-dependent down-regulation of AhR by miR-378 contributes to the variability in the constitutive hepatic expression of AhR. In conclusion, this study uncovered for the first time that A-to-I RNA editing modulates the potency of xenobiotic metabolism in the human liver. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Left-handed Z-DNA: structure and function

NASA Technical Reports Server (NTRS)

Herbert, A.; Rich, A.

1999-01-01

Z-DNA is a high energy conformer of B-DNA that forms in vivo during transcription as a result of torsional strain generated by a moving polymerase. An understanding of the biological role of Z-DNA has advanced with the discovery that the RNA editing enzyme double-stranded RNA adenosine deaminase type I (ADAR1) has motifs specific for the Z-DNA conformation. Editing by ADAR1 requires a double-stranded RNA substrate. In the cases known, the substrate is formed by folding an intron back onto the exon that is targeted for modification. The use of introns to direct processing of exons requires that editing occurs before splicing. Recognition of Z-DNA by ADAR1 may allow editing of nascent transcripts to be initiated immediately after transcription, ensuring that editing and splicing are performed in the correct sequence. Structural characterization of the Z-DNA binding domain indicates that it belongs to the winged helix-turn-helix class of proteins and is similar to the globular domain of histone-H5.

RNA editing with CRISPR-Cas13.

PubMed

Cox, David B T; Gootenberg, Jonathan S; Abudayyeh, Omar O; Franklin, Brian; Kellner, Max J; Joung, Julia; Zhang, Feng

2017-11-24

Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13. We profiled type VI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology. Copyright © 2017, American Association for the Advancement of Science.

ADAR RNA editing in human disease; more to it than meets the I.

PubMed

Gallo, Angela; Vukic, Dragana; Michalík, David; O'Connell, Mary A; Keegan, Liam P

2017-09-01

We review the structures and functions of ADARs and their involvements in human diseases. ADAR1 is widely expressed, particularly in the myeloid component of the blood system, and plays a prominent role in promiscuous editing of long dsRNA. Missense mutations that change ADAR1 residues and reduce RNA editing activity cause Aicardi-Goutières Syndrome, a childhood encephalitis and interferonopathy that mimics viral infection and resembles an extreme form of Systemic Lupus Erythmatosus (SLE). In Adar1 mouse mutant models aberrant interferon expression is prevented by eliminating interferon activation signaling from cytoplasmic dsRNA sensors, indicating that unedited cytoplasmic dsRNA drives the immune induction. On the other hand, upregulation of ADAR1 with widespread promiscuous RNA editing is a prominent feature of many cancers and particular site-specific RNA editing events are also affected. ADAR2 is most highly expressed in brain and is primarily required for site-specific editing of CNS transcripts; recent findings indicate that ADAR2 editing is regulated by neuronal excitation for synaptic scaling of glutamate receptors. ADAR2 is also linked to the circadian clock and to sleep. Mutations in ADAR2 could contribute to excitability syndromes such as epilepsy, to seizures, to diseases involving neuronal plasticity defects, such as autism and Fragile-X Syndrome, to neurodegenerations such as ALS, or to astrocytomas or glioblastomas in which reduced ADAR2 activity is required for oncogenic cell behavior. The range of human disease associated with ADAR1 mutations may extend further to include other inflammatory conditions while ADAR2 mutations may affect psychiatric conditions.

Postspreading rifting in the Adare Basin, Antarctica: Regional tectonic consequences

NASA Astrophysics Data System (ADS)

Granot, R.; Cande, S. C.; Stock, J. M.; Davey, F. J.; Clayton, R. W.

2010-08-01

Extension during the middle Cenozoic (43-26 Ma) in the north end of the West Antarctic rift system (WARS) is well constrained by seafloor magnetic anomalies formed at the extinct Adare spreading axis. Kinematic solutions for this time interval suggest a southward decrease in relative motion between East and West Antarctica. Here we present multichannel seismic reflection and seafloor mapping data acquired within and near the Adare Basin on a recent geophysical cruise. We have traced the ANTOSTRAT seismic stratigraphic framework from the northwest Ross Sea into the Adare Basin, verified and tied to DSDP drill sites 273 and 274. Our results reveal three distinct periods of tectonic activity. An early localized deformational event took place close to the cessation of seafloor spreading in the Adare Basin (˜24 Ma). It reactivated a few normal faults and initiated the formation of the Adare Trough. A prominent pulse of rifting in the early Miocene (˜17 Ma) resulted in normal faulting that initiated tilted blocks. The overall trend of structures was NE-SW, linking the event with the activity outside the basin. It resulted in major uplift of the Adare Trough and marks the last extensional phase of the Adare Basin. Recent volcanic vents (Pliocene to present day) tend to align with the early Miocene structures and the on-land Hallett volcanic province. This latest phase of tectonic activity also involves near-vertical normal faulting (still active in places) with negligible horizontal consequences. The early Miocene extensional event found within the Adare Basin does not require a change in the relative motion between East and West Antarctica. However, the lack of subsequent rifting within the Adare Basin coupled with the formation of the Terror Rift and an on-land and subice extension within the WARS require a pronounced change in the kinematics of the rift. These observations indicate that extension increased southward, therefore suggesting that a major change in

Phosphatidic acid and phosphatidylinositol labelling in adipose tissue. The role of endogenously formed adenosine.

PubMed

Schimmel, R J; Honeyman, T W; McMahon, K K

1983-05-15

Incorporation of [32P]Pi into phosphatidic acid and phosphatidylinositol of hamster epididymal adipocytes was partially inhibited by 3-isobutyl-1-methylxanthine. This effect of 3-isobutyl-1-methylxanthine was antagonized by isopropyl-N6-phenyladenosine but not by 2',5'-dideoxyadenosine, prostaglandin E1 or clonidine. N6-Phenylisopropyladenosine did not affect incorporation of [32P]Pi into phosphatidic acid or phosphatidylinositol when 3-isobutyl-1-methylxanthine was not present. In contrast with 3-isobutyl-1-methylxanthine inhibition of [32P]Pi incorporation into phospholipids, which was blocked only by N6-phenylisopropyladenosine, accelerated lipolysis was blocked by prostaglandin E1, clonidine and 2',5'-dideoxyadenosine as well as by N6-phenylisopropyladenosine. Phospholipid labelling was also decreased in the presence of adenosine deaminase, but not in the presence of isoprenaline (isoproterenol). The stimulatory effect of N6-phenylisopropyladenosine on [32P]Pi incorporation into phospholipids in cells exposed to 3-isobutyl-1-methylxanthine was evident as soon as 3 min after addition of the adenosine analogue and maximum 10 min after its addition. As observed by others, [32P]Pi incorporation into phospholipids was increased by the alpha 1-selective agonist methoxamine. The stimulatory effect of methoxamine occurred with a time course similar to that of N6-phenylisopropyladenosine and was present at nearly equal magnitude in the absence or presence of 3-isobutyl-1-methylxanthine. The inhibitory effects of 3-isobutyl-1-methylxanthine and adenosine deaminase on phospholipid labelling are attributed to blockade of the action, or to the enzymic removal, of adenosine formed in and released from the fat-cells during their incubation. Supporting this view is the selective reversal of the actions of 3-isobutyl-1-methylxanthine and of adenosine deaminase by N6-phenylisopropyladenosine. These findings suggest an important role for endogenous adenosine in regulation of

Activity of cholinesterases and adenosine deaminase in blood and serum of rats experimentally infected with Trypanosoma cruzi

PubMed Central

DA SILVA, A S; PIMENTEL, V C; FIORENZA, A M; FRANÇA, R T; TONIN, A A; JAQUES, J A; LEAL, C A M; DA SILVA, C B; MORSCH, V; SCHETINGER, M R C; LOPES, S T A; MONTEIRO, S G

2011-01-01

This study aimed to evaluate the activity of cholinesterases and adenosine deaminase (ADA) in blood and serum of rats infected with Trypanosoma cruzi. Twelve adult rats were used in the experiment divided into two uniform groups. Rodents from group A (control group) were non-infected and animals from group B served as infected, receiving intraperitoneally 3.3×107 trypomastigotes/each. Blood collection was performed at days 60 and 120 post-infection (PI) in order to evaluate the hemogram, blood activity of acetylcholinesterase, and serum butyrylcholinesterase and ADA activities. Hematological parameters did not differ between groups. A significant increase (P<0.05) of acetylcholinesterase activity was observed in blood while butyrylcholinesterase had a significant reduction (P<0.01) in serum of infected rats at days 60 and 120 PI. ADA activity in serum showed an inhibition in infected animals when compared to non-infected at day 120 PI. Based on these results, it is possible to conclude that the activity of cholinesterases and ADA were changed in animals infected with T. cruzi. The possible causes of these alterations will be discussed in this paper. PMID:21929880

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

PubMed

Garca, Mehmet Fatih; Demir, Halit; Turan, Mahfuz; Bozan, Nazım; Kozan, Ahmet; Belli, Şeyda Bayel; Arslan, Ayşe; Cankaya, Hakan

2014-06-01

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

Structural and conformational analysis of pentostatin (2'-deoxycoformycin), a potent inhibitor of adenosine deaminase.

PubMed

Cox, M B; Arjunan, P; Arora, S K

1990-08-01

X-ray, NMR and molecular mechanics studies on pentostatin (C11H16N4O4), a potent inhibitor of the enzyme adenosine deaminase, have been carried out to study the structure and conformation. The crystals belong to the monoclinic space group P21 with the cell dimensions of a = 4.960(1), b = 10.746(3), c = 11.279(4)A, beta = 101.18(2) degrees and Z = 2. The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.047 for 997 reflections. The trihydrodiazepine ring is nonplanar and adopts a distorted sofa conformation with C(7) deviated from the mean plane by 0.66A. The deoxyribose ring adopts a C3'-endo conformation, different from coformycin where the sugar has a C2'-endo conformation. The observed glycosidic torsion angle (chi = -119.5 degrees) is in the anti range. The conformation about the C(4')-C(5') bond is gauche+. The conformation of the molecule is compared with that of coformycin and 2-azacoformycin. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. The conformation of deoxyribose in solution is approximately 70% S and 30% N. Molecular mechanics studies were performed to obtain the energy minimized conformation, which is compared with X-ray and NMR results.

DNA sequence polymorphism within the bovine adenosine monophosphate deaminase 1 (AMPD1) is associated with production traits in Chinese cattle.

PubMed

Wei, C-B; Wang, J-Q; Chen, F-Y; Niu, H; Li, K

2015-02-06

The objectives of the present study were to detect an 18-bp deletion mutation in the bovine adenosine monophosphate deaminase 1 (AMPD1) gene and analyze its effect on growth traits in 2 Chinese cattle breeds using DNA sequencing and agarose electrophoresis. The five 19-bp polymerase chain reaction products of the AMPD1 gene exhibited 3 genotypes and 2 alleles: WW: homozygote genotype (wild-type); DD: homozygote genotype (mutant-type); WD: heterozygote genotype. Frequencies of the W allele varied from 66.15-70.35%. The associations between the 18-bp deletion mutation in the AMPD1 gene with production traits in 226 Jia-Xian red cattle was analyzed. The animals with genotype WW showed significantly higher heart girth and body weight than those with genotypes WD and DD at 24 months (P < 0.01). Our results indicate that the deletion mutation in the AMPD1 gene is associated with production traits, and may be used for marker-assisted selection in beef cattle breeding programs.

Adenosine and the Auditory System

PubMed Central

Vlajkovic, Srdjan M; Housley, Gary D; Thorne, Peter R

2009-01-01

Adenosine is a signalling molecule that modulates cellular activity in the central nervous system and peripheral organs via four G protein-coupled receptors designated A1, A2A, A2B, and A3. This review surveys the literature on the role of adenosine in auditory function, particularly cochlear function and its protection from oxidative stress. The specific tissue distribution of adenosine receptors in the mammalian cochlea implicates adenosine signalling in sensory transduction and auditory neurotransmission although functional studies have demonstrated that adenosine stimulates cochlear blood flow, but does not alter the resting and sound-evoked auditory potentials. An interest in a potential otoprotective role for adenosine has recently evolved, fuelled by the capacity of A1 adenosine receptors to prevent cochlear injury caused by acoustic trauma and ototoxic drugs. The balance between A1 and A2A receptors is conceived as critical for cochlear response to oxidative stress, which is an underlying mechanism of the most common inner ear pathologies (e.g. noise-induced and age-related hearing loss, drug ototoxicity). Enzymes involved in adenosine metabolism, adenosine kinase and adenosine deaminase, are also emerging as attractive targets for controlling oxidative stress in the cochlea. Other possible targets include ectonucleotidases that generate adenosine from extracellular ATP, and nucleoside transporters, which regulate adenosine concentrations on both sides of the plasma membrane. Developments of selective adenosine receptor agonists and antagonists that can cross the blood-cochlea barrier are bolstering efforts to develop therapeutic interventions aimed at ameliorating cochlear injury. Manipulations of the adenosine signalling system thus hold significant promise in the therapeutic management of oxidative stress in the cochlea. PMID:20190966

Myeloperoxidase and adenosine-deaminase levels in the pleural fluid leakage induced by carrageenan in the mouse model of pleurisy.

PubMed Central

Fröde, T S; Medeiros, Y S

2001-01-01

BACKGROUND: Although myeloperoxidase (MPO) and adenosine-deaminase (ADA) levels are markers of activated leukocytes, both enzymes have not been currently addressed in inflammation models. AIMS: This study evaluates whether the concentrations of these enzymes are significantly correlated with the content of leukocytes in a pleurisy model. METHODS: The pleurisy was induced by carrageenan (1%) in mice, and the parameters analyzed 4 and 48 h after. RESULTS: After the induction of inflammation (4h), MPO and ADA levels peaked in parallel to neutrophils (p<0.01). Regarding the second phase of pleurisy (48 h), the highest concentrations of ADA were detected in parallel to the highest levels of mononuclears (p<0.01). At this time, MPO levels and neutrophils remained elevated, although at lower levels than those found at 4 h. A significant positive correlation was found among neutrophiLs and MPO, and mononuclears and ADA (p<0.01). CONCLUSIONS: These findings support the evidence that both enzymes are markers of the inflammatory process, and provide new tools for a better understanding of the immunoregulatory pathways that occur in inflammation. PMID:11577999

Exploiting the pyrazolo[3,4-d]pyrimidin-4-one ring system as a useful template to obtain potent adenosine deaminase inhibitors.

PubMed

La Motta, Concettina; Sartini, Stefania; Mugnaini, Laura; Salerno, Silvia; Simorini, Francesca; Taliani, Sabrina; Marini, Anna Maria; Da Settimo, Federico; Lavecchia, Antonio; Novellino, Ettore; Antonioli, Luca; Fornai, Matteo; Blandizzi, Corrado; Del Tacca, Mario

2009-03-26

A number of pyrazolo[3,4-d]pyrimidin-4-ones bearing either alkyl or arylalkyl substituents in position 2 of the nucleus were synthesized and tested for their ability to inhibit adenosine deaminase (ADA) from bovine spleen. The 2-arylalkyl derivatives exhibited excellent inhibitory activity, showing Ki values in the nanomolar/subnanomolar range. The most active compound, 1-(4-((4-oxo-4,5-dihydropyrazolo[3,4-d]pyrimidin-2-yl)methyl)phenyl)-3-(4-(trifluoromethyl)phenyl)urea, 14d, was tested in rats with colitis induced by 2,4-dinitrobenzenesulfonic acid to assess its efficacy to attenuate bowel inflammation. The treatment with 14d induced a significant amelioration of both systemic and intestinal inflammatory alterations in animals with experimental colitis. Docking simulations of the synthesized compounds into the ADA catalytic site were also performed to rationalize the structure-activity relationships observed and to highlight the key pharmacophoric elements of these products, thus prospectively guiding the design of novel ADA inhibitors.

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

PubMed

Gui, Xuwei; Xiao, Heping

2014-01-01

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

CD26 expression and adenosine deaminase activity in regulatory T cells (Treg) and CD4+ T effector cells in patients with head and neck squamous cell carcinoma

PubMed Central

Mandapathil, Magis; Szczepanski, Miroslaw; Harasymczuk, Malgorzata; Ren, Jin; Cheng, Dongmei; Jackson, Edwin K.; Gorelik, Elieser; Johnson, Jonas; Lang, Stephan; Whiteside, Theresa L

2012-01-01

Adenosine deaminase (ADA) is responsible for the deamination of immunosuppressive adenosine to inosine. In human T lymphocytes, ADA is associated with dipeptidyl peptidase IV (CD26). ADA expression and activity were evaluated in regulatory T cells (Treg) and CD4+ T effector cells (Teff) of patients with head and neck squamous cell cancer (HNSCC). CD4+CD39+ and CD4+CD39neg T cells were isolated by single-cell sorting from the peripheral blood of 15 HNSCC patients and 15 healthy donors (NC). CD26/ADA expression in these cells was studied by multicolor flow cytometry, confocal microscopy, RT-PCR and immunohistochemistry in tumor tissues. ADA activity was evaluated by mass spectrometry, suppression of Teff proliferation in CFSE assays and cytokine production by Luminex. CD4+CD39+ Treg had low and CD4+CD39neg Teff high CD26/ADA expression and ADA activity in NC or HNSCC. The frequency and suppressor activity of CD39+CD26neg Treg were elevated in patients relative to NC (p < 0.01). However, ADA activity in patients’ CD4+CD39neg Teff was decreased (p < 0.05), resulting in extracellular adenosine accumulation. Also, patients’ Teff were more sensitive to inhibitory signals delivered via adenosine receptors. IL-2, IL12 and INFγ upregulated ADA expression and activity in CD4+CD39neg Teff, whereas IL-10, PGE2 and CADO downregulated it. The differentially expressed CD26/ADA can serve as surface markers for functionally-active CD39+CD26neg Treg. PMID:22934258

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

ERIC Educational Resources Information Center

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

2012-01-01

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

Excess adenosine in murine penile erectile tissues contributes to priapism via A2B adenosine receptor signaling

PubMed Central

Mi, Tiejuan; Abbasi, Shahrzad; Zhang, Hong; Uray, Karen; Chunn, Janci L.; Xia, Ling Wei; Molina, Jose G.; Weisbrodt, Norman W.; Kellems, Rodney E.; Blackburn, Michael R.; Xia, Yang

2008-01-01

Priapism, abnormally prolonged penile erection in the absence of sexual excitation, is associated with ischemia-mediated erectile tissue damage and subsequent erectile dysfunction. It is common among males with sickle cell disease (SCD), and SCD transgenic mice are an accepted model of the disorder. Current strategies to manage priapism suffer from a poor fundamental understanding of the molecular mechanisms underlying the disorder. Here we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected priapic activity. ADA enzyme therapy successfully corrected the priapic activity both in vivo and in vitro, suggesting that it was dependent on elevated adenosine levels. Further genetic and pharmacologic evidence demonstrated that A2B adenosine receptor–mediated (A2BR-mediated) cAMP and cGMP induction was required for elevated adenosine–induced prolonged penile erection. Finally, priapic activity in SCD transgenic mice was also caused by elevated adenosine levels and A2BR activation. Thus, we have shown that excessive adenosine accumulation in the penis contributes to priapism through increased A2BR signaling in both Ada–/– and SCD transgenic mice. These findings provide insight regarding the molecular basis of priapism and suggest that strategies to either reduce adenosine or block A2BR activation may prove beneficial in the treatment of this disorder. PMID:18340377

Ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) and adenosine deaminase (ADA) activities in prostate cancer patients: influence of Gleason score, treatment and bone metastasis.

PubMed

Battisti, Vanessa; Maders, Liési D K; Bagatini, Margarete D; Battisti, Iara E; Bellé, Luziane P; Santos, Karen F; Maldonado, Paula A; Thomé, Gustavo R; Schetinger, Maria R C; Morsch, Vera M

2013-04-01

The relation between adenine nucleotides and cancer has already been described in literature. Considering that the enzymes ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) and adenosine deaminase (ADA) act together to control nucleotide levels, we aimed to investigate the role of these enzymes in prostate cancer (PCa). E-NPP and ADA activities were determined in serum and platelets of PCa patients and controls. We also verified the influence of the Gleason score, bone metastasis and treatment in the enzyme activities. Platelets and serum E-NPP activity increased, whereas ADA activity in serum decreased in PCa patients. In addition, Gleason score, metastasis and treatment influenced E-NPP and ADA activities. We may propose that E-NPP and ADA are involved in the development of PCa. Moreover, E-NPP and ADA activities are modified in PCa patients with distinct Gleason score, with bone metastasis, as well as in patients under treatment. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Clinical efficacy of gene-modified stem cells in adenosine deaminase-deficient immunodeficiency.

PubMed

Shaw, Kit L; Garabedian, Elizabeth; Mishra, Suparna; Barman, Provaboti; Davila, Alejandra; Carbonaro, Denise; Shupien, Sally; Silvin, Christopher; Geiger, Sabine; Nowicki, Barbara; Smogorzewska, E Monika; Brown, Berkley; Wang, Xiaoyan; de Oliveira, Satiro; Choi, Yeong; Ikeda, Alan; Terrazas, Dayna; Fu, Pei-Yu; Yu, Allen; Fernandez, Beatriz Campo; Cooper, Aaron R; Engel, Barbara; Podsakoff, Greg; Balamurugan, Arumugam; Anderson, Stacie; Muul, Linda; Jagadeesh, G Jayashree; Kapoor, Neena; Tse, John; Moore, Theodore B; Purdy, Ken; Rishi, Radha; Mohan, Kathey; Skoda-Smith, Suzanne; Buchbinder, David; Abraham, Roshini S; Scharenberg, Andrew; Yang, Otto O; Cornetta, Kenneth; Gjertson, David; Hershfield, Michael; Sokolic, Rob; Candotti, Fabio; Kohn, Donald B

2017-05-01

Autologous hematopoietic stem cell transplantation (HSCT) of gene-modified cells is an alternative to enzyme replacement therapy (ERT) and allogeneic HSCT that has shown clinical benefit for adenosine deaminase-deficient (ADA-deficient) SCID when combined with reduced intensity conditioning (RIC) and ERT cessation. Clinical safety and therapeutic efficacy were evaluated in a phase II study. Ten subjects with confirmed ADA-deficient SCID and no available matched sibling or family donor were enrolled between 2009 and 2012 and received transplantation with autologous hematopoietic CD34+ cells that were modified with the human ADA cDNA (MND-ADA) γ-retroviral vector after conditioning with busulfan (90 mg/m2) and ERT cessation. Subjects were followed from 33 to 84 months at the time of data analysis. Safety of the procedure was assessed by recording the number of adverse events. Efficacy was assessed by measuring engraftment of gene-modified hematopoietic stem/progenitor cells, ADA gene expression, and immune reconstitution. With the exception of the oldest subject (15 years old at enrollment), all subjects remained off ERT with normalized peripheral blood mononuclear cell (PBMC) ADA activity, improved lymphocyte numbers, and normal proliferative responses to mitogens. Three of nine subjects were able to discontinue intravenous immunoglobulin replacement therapy. The MND-ADA vector was persistently detected in PBMCs (vector copy number [VCN] = 0.1-2.6) and granulocytes (VCN = 0.01-0.3) through the most recent visits at the time of this writing. No patient has developed a leukoproliferative disorder or other vector-related clinical complication since transplant. These results demonstrate clinical therapeutic efficacy from gene therapy for ADA-deficient SCID, with an excellent clinical safety profile. ClinicalTrials.gov NCT00794508. Food and Drug Administration Office of Orphan Product Development award, RO1 FD003005; NHLBI awards, PO1 HL73104 and Z01 HG000122; UCLA

Good Laboratory Practice Preclinical Safety Studies for GSK2696273 (MLV Vector-Based Ex Vivo Gene Therapy for Adenosine Deaminase Deficiency Severe Combined Immunodeficiency) in NSG Mice.

PubMed

Carriglio, Nicola; Klapwijk, Jan; Hernandez, Raisa Jofra; Vezzoli, Michela; Chanut, Franck; Lowe, Rhiannon; Draghici, Elena; Nord, Melanie; Albertini, Paola; Cristofori, Patrizia; Richards, Jane; Staton, Hazel; Appleby, Jonathan; Aiuti, Alessandro; Sauer, Aisha V

2017-03-01

GSK2696273 (autologous CD34+ cells transduced with retroviral vector that encodes for the human adenosine deaminase [ADA] enzyme) is a gamma-retroviral ex vivo gene therapy of bone marrow-derived CD34+ cells for the treatment of adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCID). ADA-SCID is a severe monogenic disease characterized by immunologic and nonimmunologic symptoms. Bone-marrow transplant from a matched related donor is the treatment of choice, but it is available for only a small proportion of patients. Ex vivo gene therapy of patient bone-marrow CD34+ cells is an alternative treatment. In order to prepare for a marketing authorization application in the European Union, preclinical safety studies in mice were requested by the European Medicines Agency (EMA). A pilot study and a main biodistribution study were performed according to Good Laboratory Practice (GLP) at the San Raffaele Telethon Institute for Gene Therapy test facility. In the main study, human umbilical cord blood (UCB)-derived CD34+ cells were transduced with gamma-retroviral vector used in the production of GSK2696273. Groups of 10 male and 10 female NOD-SCID gamma (NSG) mice were injected intravenously with a single dose of transduced- or mock-transduced UCB CD34+ cells, and they were observed for 4 months. Engraftment and multilineage differentiation of blood cells was observed in the majority of animals in both groups. There was no significant difference in the level of chimerism between the two groups. In the gene therapy group, vector was detectable in lymphohemopoietic and nonlymphohemopoietic tissues, consistent with the presence of gene-modified human hematopoietic donor cells. Given the absence of relevant safety concerns in the data, the nonclinical studies and the clinical experience with GSK2696273 supported a successful application for market authorization in the European Union for the treatment of ADA-SCID patients, for whom no suitable human leukocyte

Gene therapy for adenosine deaminase-deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans.

PubMed

Candotti, Fabio; Shaw, Kit L; Muul, Linda; Carbonaro, Denise; Sokolic, Robert; Choi, Christopher; Schurman, Shepherd H; Garabedian, Elizabeth; Kesserwan, Chimene; Jagadeesh, G Jayashree; Fu, Pei-Yu; Gschweng, Eric; Cooper, Aaron; Tisdale, John F; Weinberg, Kenneth I; Crooks, Gay M; Kapoor, Neena; Shah, Ami; Abdel-Azim, Hisham; Yu, Xiao-Jin; Smogorzewska, Monika; Wayne, Alan S; Rosenblatt, Howard M; Davis, Carla M; Hanson, Celine; Rishi, Radha G; Wang, Xiaoyan; Gjertson, David; Yang, Otto O; Balamurugan, Arumugam; Bauer, Gerhard; Ireland, Joanna A; Engel, Barbara C; Podsakoff, Gregory M; Hershfield, Michael S; Blaese, R Michael; Parkman, Robertson; Kohn, Donald B

2012-11-01

We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34(+) cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m(2)). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.

Effects of different concentrations of metal ions on degradation of adenosine triphosphate in common carp (Cyprinus carpio) fillets stored at 4°C: An in vivo study.

PubMed

Li, Dapeng; Qin, Na; Zhang, Longteng; Lv, Jian; Li, Qingzheng; Luo, Yongkang

2016-11-15

The impact of different concentrations of Na(+), K(+), Ca(2+), Mg(2+), Fe(2+), and Zn(2+) on the degradation of adenosine triphosphate (ATP) and the influence of these ions on the activity of adenosine monophosphate deaminase (AMP-deaminase) and acid phosphatase (ACP) in common carp fillets (in vivo) during 4°C storage was examined. The content of ATP, inosine monophosphate (IMP), and hypoxanthine (Hx), and the activity of AMP-deaminase and ACP were determined. Results indicated that the effects of different concentrations of six kinds of metal ions on AMP-deaminase and ACP were not the same. Na(+), K(+), Fe(2+), and Zn(2+) enhanced AMP-deaminase activity, which led to the rapid degradation of ATP and to the generation of a large quantity of IMP within a short time. Ca(2+) and Mg(2+) delayed the change in AMP-deaminase and ACP activity in carp and caused a further delay in the degradation of ATP. Fe(2+) and Zn(2+) inhibited ACP activity, which reduced the decomposition of IMP and the formation of Hx. Copyright © 2016 Elsevier Ltd. All rights reserved.

A rapid enzymatic assay for high-throughput screening of adenosine-producing strains

PubMed Central

Dong, Huina; Zu, Xin; Zheng, Ping; Zhang, Dawei

2015-01-01

Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3, the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples. PMID:25580842

[Features of influence adenosine, AMP and hyperadrenalinemiya on the immune status, metabolic enzymes of purine nucleotides and the antioxidant defense system].

PubMed

Tapbergenov, S O; Sovetov, B S; Tapbergenov, A T

2016-11-01

Administration of a large dose of adrenaline (4 mg/kg 60 min before analysis) increased blood levels of total leukocytes, lymphocytes, decreased T-cell suppressors, leukocyte migration inhibition reaction (LMIR) and NBT test, but increased the level of conjugated dienes (CD). Administration of AMPand adenosine increased levels of total leukocytes, lymphocytes, T- lymphocytes, T-helpers, decreased the level of malondialdehyde (MDA), LMIR, and T-cell suppressors. Sympathetic hyperactivation induced by administration of a large dose of adrenaline (4 mg/kg 60 min before analysis) was accompanied by an increase in heart and liver activities of glutathione peroxidase (GPx), catalase, AMP deaminase (AMPD), and adenosine deaminase (AD). Administration of AMP or adenosine caused a decrease in activities of glutathione reductase (GR), GPx, catalase, a decrease in the MDA level and an increase in activities of AMPD and AD in the heart. In the liver AMP and adenosine also caused a decrease in activities of glutathione reductase (GR), GPx, a decrease in the MDA level and an increase in activities of AMPD and AD. The data obtained suggest that administration of adrenaline, AMP, and adenosine influences activity of enzymes involved in purine nucleotide metabolism. However, in contrast to adrenaline, administration of AMP or adenosine does not provoke stress reaction.

Alternative Splicing of STAT3 Is Affected by RNA Editing.

PubMed

Goldberg, Lior; Abutbul-Amitai, Mor; Paret, Gideon; Nevo-Caspi, Yael

2017-05-01

A-to-I RNA editing, carried out by adenosine deaminase acting on RNA (ADAR) enzymes, is an epigenetic phenomenon of posttranscriptional modifications on pre-mRNA. RNA editing in intronic sequences may influence alternative splicing of flanking exons. We have previously shown that conditions that induce editing result in elevated expression of signal transducer and activator of transcription 3 (STAT3), preferentially the alternatively-spliced STAT3β isoform. Mechanisms regulating alternative splicing of STAT3 have not been elucidated. STAT3 undergoes A-to-I RNA editing in an intron residing in proximity to the alternatively spliced exon. We hypothesized that RNA editing plays a role in regulating alternative splicing toward STAT3β. In this study we extend our observation connecting RNA editing to the preferential induction of STAT3β expression. We study the involvement of ADAR1 in STAT3 editing and reveal the connection between editing and alternative splicing of STAT3. Deferoaxamine treatment caused the induction in STAT3 RNA editing and STAT3β expression. Silencing ADAR1 caused a decrease in STAT3 editing and expression with a preferential decrease in STAT3β. Cells transfected with a mutated minigene showed preferential splicing toward the STAT3β transcript. Editing in the STAT3 intron is performed by ADAR1 and affects STAT3 alternative splicing. These results suggest that RNA editing is one of the molecular mechanisms regulating the expression of STAT3β.

A novel mouse model for sudden unexpected death in epilepsy (SUDEP): role of impaired adenosine clearance.

PubMed

Shen, Hai-Ying; Li, Tianfu; Boison, Detlev

2010-03-01

Sudden unexpected death in epilepsy (SUDEP) is a significant cause of mortality in people with epilepsy. Two postulated causes for SUDEP, cardiac and respiratory depression, can both be explained by overstimulation of adenosine receptors. We hypothesized that SUDEP is a consequence of a surge in adenosine as a result of prolonged seizures combined with deficient adenosine clearance; consequently, blockade of adenosine receptors should prevent SUDEP. Here we induced impaired adenosine clearance in adult mice by pharmacologic inhibition of the adenosine-removing enzymes, adenosine kinase and deaminase. Combination of impaired adenosine clearance with kainic acid-induced seizures triggered sudden death in all animals. Most importantly, the adenosine receptor antagonist caffeine, when given after seizure onset, increased survival from 23.75 +/- 1.35 min to 54.86 +/- 6.59 min (p < 0.01). Our data indicate that SUDEP is due to overactivation of adenosine receptors and that caffeine treatment after seizure onset might be beneficial.

Magnetic anomalies northeast of Cape Adare, northern Victoria Land (Antarctica), and their relation to onshore structures

USGS Publications Warehouse

Damaske, D.; Läufer, A.L.; Goldmann, F.; Möller, H.-D.; Lisker, F.

2007-01-01

An aeromagnetic survey was flown over the offshore region northeast of Cape Adare and the magnetic anomalies compared to onshore structures between Pennell Coast and Tucker Glacier. The magnetic anomalies show two nearly orthogonal major trends. NNW-SSE trending anomalies northeast of Cape Adare represent seafloor spreading within the Adare Trough. A connection of these anomalies to the Northern Basin of the Ross Sea is not clear. Onshore faults are closely aligned to offshore anomalies. Main trends are NW-SE to NNW-SSE and NE-SW to NNESSW. NNW-SSE oriented dextral-transtensional to extensional faults parallel the Adare Peninsula and Adare Trough anomalies. NE-SW trending normal faults appear to segment the main Hallett volcanic bodies.

Can pleural adenosine deaminase (ADA) levels in pleural tuberculosis predict the presence of pulmonary tuberculosis? A CT analysis.

PubMed

Koh, Myung Je; Lee, In Jae; Kim, Joo-Hee

2016-06-01

To assess the relationship between imaging features of pulmonary tuberculosis at computed tomography (CT) and adenosine deaminase (ADA) values via pleural fluid analysis in patients with pleural tuberculosis. This retrospective study enrolled 60 patients who underwent fluid analysis for ADA and chest CT and were diagnosed with tuberculosis by culture or polymerase chain reaction of pleural fluid and sputum. The presence of centrilobular nodules, consolidation, cavitation, and mediastinal lymphadenopathy at CT were evaluated. The relationship between ADA values and the pattern of pulmonary involvement of tuberculosis was analysed. Pulmonary involvement was seen in 42 of the 60 patients. A centrilobular nodular pattern was seen in 37 and consolidation in 22. In 17 patients, both findings were identified. A centrilobular nodular pattern was more common than consolidation or cavitary lesions. When ADA values were high, pulmonary involvement was more frequent (p=0.002). Comparing low and high ADA groups using an obtained cut-off value of 80 IU/l, the high group had more frequent pulmonary involvement (p<0.001). Patients with tuberculous pleurisy who had high ADA values had a higher probability of manifesting pulmonary tuberculosis. High ADA values may help predict contagious pleuroparenchymal tuberculosis. The most common pulmonary involvement of tuberculous pleurisy showed a centrilobular nodular pattern. Copyright © 2016 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

Seed specific expression and analysis of recombinant human adenosine deaminase (hADA) in three host plant species.

PubMed

Doshi, Ketan M; Loukanina, Natalia N; Polowick, Patricia L; Holbrook, Larry A

2016-10-01

The plant seed is a leading platform amongst plant-based storage systems for the production of recombinant proteins. In this study, we compared the activity of human adenosine deaminase (hADA) expressed in transgenic seeds of three different plant species: pea (Pisum sativum L.), Nicotiana benthamiana L. and tarwi (Lupinus mutabilis Sweet). All three species were transformed with the same expression vector containing the hADA gene driven by the seed-specific promoter LegA2 with an apoplast targeting pinII signal peptide. During the study, several independent transgenic lines were generated and screened from each plant species and only lines with a single copy of the gene of interest were used for hADA expression analysis. A stable transgenic canola line expressing the ADA protein, under the control of 35S constitutive promoter was used as both as a positive control and for comparative study with the seed specific promoter. Significant differences were detected in the expression of hADA. The highest activity of the hADA enzyme (Units/g seed) was reported in tarwi (4.26 U/g) followed by pea (3.23 U/g) and Nicotiana benthamiana (1.69 U/g). The expression of mouse ADA in canola was very low in both seed and leaf tissue compared to other host plants, confirming higher activity of seed specific promoter. Altogether, these results suggest that tarwi could be an excellent candidate for the production of valuable recombinant proteins.

Sitagliptin attenuates sympathetic innervation via modulating reactive oxygen species and interstitial adenosine in infarcted rat hearts.

PubMed

Lee, Tsung-Ming; Chen, Wei-Ting; Yang, Chen-Chia; Lin, Shinn-Zong; Chang, Nen-Chung

2015-02-01

We investigated whether sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, attenuates arrhythmias through inhibiting nerve growth factor (NGF) expression in post-infarcted normoglycemic rats, focusing on adenosine and reactive oxygen species production. DPP-4 bound adenosine deaminase has been shown to catalyse extracellular adenosine to inosine. DPP-4 inhibitors increased adenosine levels by inhibiting the complex formation. Normoglycemic male Wistar rats were subjected to coronary ligation and then randomized to either saline or sitagliptin in in vivo and ex vivo studies. Post-infarction was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham. Compared with vehicle, infarcted rats treated with sitagliptin significantly increased interstitial adenosine levels and attenuated oxidative stress. Sympathetic hyperinnervation was blunted after administering sitagliptin, as assessed by immunofluorescent analysis and western blotting and real-time quantitative RT-PCR of NGF. Arrhythmic scores in the sitagliptin-treated infarcted rats were significantly lower than those in vehicle. Ex vivo studies showed a similar effect of erythro-9-(2-hydroxy-3-nonyl) adenine (an adenosine deaminase inhibitor) to sitagliptin on attenuated levels of superoxide and NGF. Furthermore, the beneficial effects of sitagliptin on superoxide anion production and NGF levels can be reversed by 8-cyclopentyl-1,3-dipropulxanthine (adenosine A1 receptor antagonist) and exogenous hypoxanthine. Sitagliptin protects ventricular arrhythmias by attenuating sympathetic innervation via adenosine A1 receptor and xanthine oxidase-dependent pathways, which converge through the attenuated formation of superoxide in the non-diabetic infarcted rats. © 2014 The Authors. Journal of Cellular and

Ticagrelor Compared with Clopidogrel Increased Adenosine and Cyclic Adenosine Monophosphate Plasma Concentration in Acute Coronary Syndrome Patients.

PubMed

Li, Xiaoye; Wang, Qibing; Xue, Ying; Chen, Jiahui; Lv, Qianzhou

2017-06-01

Ticagrelor produces a more potent antiplatelet effect than clopidogrel and inhibits cellular uptake of adenosine, which is associated with several cardiovascular consequences. We aimed to explore the correlation between adenosine and cyclic adenosine monophosphate (cAMP) plasma concentration and antiplatelet effect by clopidogrel or ticagrelor in patients with acute coronary syndrome (ACS) receiving dual antiplatelet therapy (DAPT). We conducted a prospective, observational and single-centre cohort study enrolling 68 patients with non-ST-segment elevation ACS from January 2016 to May 2016. We monitored the inhibition of platelet aggregation (IPA) and assessed adenosine, adenosine deaminase (ADA) and cAMP plasma concentrations by immunoassay on admission and 48 hr after coronary angiography. The demographic and clinical data were collected by reviewing their medical records. The two groups exhibited similar baseline characteristics including adenosine, ADA and cAMP. The mean IPA in patients receiving ticagrelor was significantly higher than that in patients receiving clopidogrel (93.5% versus 67.2%; p = 0.000). Also, we observed that patients treated with ticagrelor had a significantly higher increase in levels of adenosine and cAMP compared with those treated with clopidogrel (1.04 (0.86; 1.41) versus 0.04 (-0.25; 0.26); p = 0.029 and 0.78 (-1.67; 1.81) versus 0.60 (-1.91; 4.60); p = 0.037, respectively). And there was a weak correlation between IPA and adenosine as well as cAMP plasma concentration (r = 0.390, p = 0.001 and r = 0.335, p = 0.005, respectively). Ticagrelor increased adenosine and cAMP plasma concentration compared with clopidogrel in patients with ACS. © 2017 The Authors. Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Circadian variations of adenosine and of its metabolism. Could adenosine be a molecular oscillator for circadian rhythms?

PubMed

Chagoya de Sánchez, V

1995-03-01

The present review describes the biological implications of the periodic changes of adenosine concentrations in different tissues of the rat. Adenosine is a purine molecule that could have been formed in the prebiotic chemical evolution and has been preserved. The rhythmicity of this molecule, as well as its metabolism and even the presence of specific receptors, suggests a regulatory role in eukaryotic cells and in multicellular organisms. Adenosine may be considered a chemical messenger and its action could take place at the level of the same cell (autocrine), the same tissue (paracrine), or on separate organs (endocrine). Exploration of the circadian variations of adenosine was planned considering the liver as an important tissue for purine formation, the blood as a vehicle among tissues, and the brain as the possible acceptor for hepatic adenosine or its metabolites. The rats used in these studies were adapted to a dark-light cycle of 12 h with an unrestrained feeding and drinking schedule. The metabolic control of adenosine concentration in the different tissues studied through the 24-h cycle is related to the activity of adenosine-metabolizing enzyme: 5'-nucleotidase adenosine deaminase, adenosine kinase, and S-adenosylhomocysteine hydrolase. Some possibilities of the factors modulating the activity of these enzymes are commented upon. The multiphysiological action of adenosine could be mediated by several actions: (i) by interaction with extracellular and intracellular receptors and (ii) through its metabolism modulating the methylation pathway, possibly inducing physiological lipoperoxidation, or participating in the energetic homeostasis of the cell. The physiological meaning of the circadian variations of adenosine and its metabolism was focused on: maintenance of the energetic homeostasis of the tissues, modulation of membrane structure and function, regulation of fasting and feeding metabolic pattern, and its participation in the sleep-wake cycle. From

Analysis of serum adenosine deaminase (ADA) and ADA1 and ADA2 isoenzyme activities in HIV positive and HIV-HBV co-infected patients.

PubMed

Khodadadi, Iraj; Abdi, Mohammad; Ahmadi, Abbas; Wahedi, Mohammad Saleh; Menbari, Shahoo; Lahoorpour, Fariba; Rahbari, Rezgar

2011-08-01

To determine adenosine deaminase (ADA) activity as a possible diagnostic marker in HIV and HIV-HBV co-infected patients. Blood samples were collected from 72 healthy, 33 HIV positive and 30 HIV-HBV co-infected subjects. Blood CD4+ cell count was recorded and serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total ADA, and ADA1 and ADA2 isoenzyme activities were determined. Serum ALT, AST, total ADA and ADA2 isoenzyme activities were significantly higher in HIV positive and HIV-HBV co-infected groups compare to the control (p<0.05), whereas serum ALP showed no differences between groups. CD4+ cell counts markedly decreased in all patients and showed a significant inverse correlation with ADA activities (R(2)=0.589, p<0.001). Serum ADA was significantly increased in HIV and HIV-HBV co-infections. Therefore, because of its low cost and simplicity to perform, ADA activity might be considered as a useful diagnostic tool among the other markers in these diseases. Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

A2B Adenosine Receptor–Mediated Induction of IL-6 Promotes CKD

PubMed Central

Dai, Yingbo; Zhang, Weiru; Wen, Jiaming; Zhang, Yujin; Kellems, Rodney E.

2011-01-01

Chronic elevation of adenosine, which occurs in the setting of repeated or prolonged tissue injury, can exacerbate cellular dysfunction, suggesting that it may contribute to the pathogenesis of CKD. Here, mice with chronically elevated levels of adenosine, resulting from a deficiency in adenosine deaminase (ADA), developed renal dysfunction and fibrosis. Both the administration of polyethylene glycol–modified ADA to reduce adenosine levels and the inhibition of the A2B adenosine receptor (A2BR) attenuated renal fibrosis and dysfunction. Furthermore, activation of A2BR promoted renal fibrosis in both mice infused with angiotensin II (Ang II) and mice subjected to unilateral ureteral obstruction (UUO). These three mouse models shared a similar profile of profibrotic gene expression in kidney tissue, suggesting that they share similar signaling pathways that lead to renal fibrosis. Finally, both genetic and pharmacologic approaches showed that the inflammatory cytokine IL-6 mediates adenosine-induced renal fibrosis downstream of A2BR. Taken together, these data suggest that A2BR-mediated induction of IL-6 contributes to renal fibrogenesis and shows potential therapeutic targets for CKD. PMID:21511827

Membrane lipid-protein interactions modify the regulatory role of adenosine-deaminase complexing protein: a phase fluorometry study of a malignancy marker

NASA Astrophysics Data System (ADS)

Parola, Abraham H.; Porat, Nurith; Caiolfa, Valeria R.; Gill, David; Kiesow, Lutz A.; Weisman, Mathew; Nemschitz, S.; Yaron, Dahlia; Singer, Karen; Solomon, Ethel

1990-05-01

The role of membrane lipid-protein interactions in malignant cell transformation was examined with adenosine deaminase (ADA) as a representative membrane protein. ADA's activity changes dramatically in transformed cells and accordingly it is a malignancy marker. Yet, the mechanisms controlling its variable activity are unknown. We undertook the spectroscopic deciphering of its interactions with its lipidic environment in normal and malignant cells. ADA exists in two interconvertible forms, small (45 KD) and large (21OKD). The large form consists of two small catalytic subunits (55-ADA) and a dimeric complexing protein ADCP. The physiological role of ADCP was not known either. Our studies were carried out at three levels.: 1. Solution enzyme kinetics, 2. The interaction of 55-ADA with ADCP reconstituted in liposomes: Effect of cholesterol and 3. Multifrequency phase modulation spectrofluorometry of pyrene-labeled 55-ADA bound to ADCP on the membranes of normal and RSV or RSV Ts68 transformed chick embryo fibroblasts. We found: 1. ADCP has an allosteric regulatory role on 55-ADA, which may be of physiological relevance: It inhibits 55-ADA activity at low physiological adenosine concentrations but accelerates deamination at high substrate concentration. 2. When reconstituted in DMPC liposomes, it retains 55-ADA activity (in its absence the activity is lost) and upon rigidification with cholesterol, a three fold increase in 55-ADA activity is attained, contrary to ADCP's regulatory activity when free of lipids. 3. The reduced ADA activity in transformed chick embryo fibroblasts is associated with increased membrane lipid fluidity (reduced order parameter), reduced accessibility of ADCP and increase rotational dynamics of the complex. We thus obtained spectroscopic deciphering of the vertical motion of ADCP, controlled by lipid-protein interaction, resulting in variable activity of this malignancy marker.

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

PubMed

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. Copyright © 2016 John Wiley & Sons, Ltd.

Aflatoxins produced by Aspergillus parasiticus present in the diet of quails increase the activities of cholinesterase and adenosine deaminase.

PubMed

da Silva, Aleksandro Schafer; Santurio, Janio M; Roza, Lenilson F; Bottari, Nathieli B; Galli, Gabriela M; Morsch, Vera M; Schetinger, Maria Rosa C; Baldissera, Matheus D; Stefani, Lenita M; Radavelli, Willian M; Tomasi, Thainã; Boiago, Marcel M

2017-06-01

The aim of this study was to evaluate the effects of aflatoxins on cholinesterases (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and adenosine deaminase (ADA) activities in quails. For this, twenty male quails were randomly distributed into two groups (n = 10 each): the group A was composed by quails that received feed without aflatoxin (the control group); while the group B was composed by quails that received feed contaminated with 200 ppm/kg of feed of aflatoxin. On day 20, the animals were euthanized to measure the activities of AChE (total blood and brain), BChE (serum) and ADA (serum, liver, and brain), as well as for histopathological analyses (liver and intestine). AChE, BChE, and ADA levels increased in animals intoxicated by aflatoxin compared to the control group. The presence of aflatoxin lead to severe hydropic degeneration of hepatocytes and small focus of hepatocyte necrosis. In conclusion, aflatoxins poisoning increased AChE, BChE, and ADA activities, suggesting the involvement of these enzymes during this type of intoxication, in addition to the fact that they are well known molecules that participate in physiological and pathological events as inflammatory mediators. In summary, increased AChE, BChE and ADA activities contribute directly to the inflammatory process and tissue damage, and they might be involved in disease development. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of the swine adipocyte A1 adenosine receptor using an optimized assay system.

PubMed

Dong, Q; Schuchman, J; Carey, G B

1994-07-01

The radioligand binding assay of A1 adenosine receptors in adipocyte crude plasma membrane from Yucatan miniature swine was optimized by evaluating 17 factors involved in the assay. Significant effects of CHAPS, adenosine deaminase, EDTA, pre-rinsing glass fiber filters and pH were found for the binding measurements. Using the optimized procedure, [3H]8-cyclopentyl-1,3-dipropylxanthine, ([3H]-DPCPX) binding to A1 adenosine receptors in swine subcutaneous adipocyte crude plasma membrane was measured; Bmax and Kd values were 479 +/- 77 fmol/mg protein and 0.87 +/- 0.10 nM, respectively. Values for mesenteric adipose tissue from sedentary swine and subcutaneous adipose tissue from exercise-trained swine were also measured.

Adenosine kinase regulation of cardiomyocyte hypertrophy

PubMed Central

Fassett, John T.; Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Chen, Yingjie

2011-01-01

There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 μM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 μM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PE-induced phosphosignaling pathways revealed that CADO treatment did not reduce AKTSer473 phosphorylation but did attenuate sustained phosphorylation of RafSer338 (24–48 h), mTORSer2448 (24–48 h), p70S6kThr389 (2.5–48 h), and ERKThr202/Tyr204 (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PE-induced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6kThr389 phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERKThr202/Tyr204 and AKTSer473. Reduction of Raf-induced p70S6kThr389 phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte hypertrophy, which acts, at least in part, through inhibition of

Adenosine kinase regulation of cardiomyocyte hypertrophy.

PubMed

Fassett, John T; Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Chen, Yingjie; Bache, Robert J

2011-05-01

There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 μM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 μM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PE-induced phosphosignaling pathways revealed that CADO treatment did not reduce AKT(Ser⁴⁷³) phosphorylation but did attenuate sustained phosphorylation of Raf(Ser³³⁸) (24-48 h), mTOR(Ser²⁴⁴⁸) (24-48 h), p70S6k(Thr³⁸⁹) (2.5-48 h), and ERK(Thr²⁰²/Tyr²⁰⁴) (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PE-induced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6k(Thr³⁸⁹) phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERK(Thr202/Tyr204) and AKT(Ser⁴⁷³). Reduction of Raf-induced p70S6k(Thr³⁸⁹) phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte

Utility of adenosine deaminase (ADA), PCR & thoracoscopy in differentiating tuberculous & non-tuberculous pleural effusion complicating chronic kidney disease.

PubMed

Kumar, Sravan; Agarwal, Ritesh; Bal, Amanjit; Sharma, Kusum; Singh, Navneet; Aggarwal, Ashutosh N; Verma, Indu; Rana, Satyawati V; Jha, Vivekanand

2015-03-01

Pleural effusion is a common occurrence in patients with late-stage chronic kidney disease (CKD). In developing countries, many effusions remain undiagnosed after pleural fluid analysis (PFA) and patients are empirically treated with antitubercular therapy. The aim of this study was to evaluate the role of adenosine deaminase (ADA), nucleic acid amplification tests (NAAT) and medical thoracoscopy in distinguishing tubercular and non-tubercular aetiologies in exudative pleural effusions complicating CKD. Consecutive stage 4 and 5 CKD patients with pleural effusions underwent PFA including ADA and PCR [65 kDa gene; multiplex (IS6110, protein antigen b, MPB64)]. Patients with exudative pleural effusion undiagnosed after PFA underwent medical thoracoscopy. All 107 patients underwent thoracocentesis with 45 and 62 patients diagnosed as transudative and exudative pleural effusions, respectively. Twenty six of the 62 patients underwent medical thoracoscopy. Tuberculous pleurisy was diagnosed in six while uraemic pleuritis was diagnosed in 20 subjects. The sensitivity and specificity of pleural fluid ADA, 65 kDa gene PCR, and multiplex PCR were 66.7 and 90 per cent, 100 and 50 per cent, and 100 and 100 per cent, respectively. Thoracoscopy was associated with five complications in three patients. Uraemia remains the most common cause of pleural effusion in CKD even in high TB prevalence country. Multiplex PCR and thoracoscopy are useful investigations in the diagnostic work-up of pleural effusions complicating CKD while the sensitivity and/or specificity of ADA and 65 kDa gene PCR is poor.

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

PubMed Central

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

2002-01-01

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

Deoxycytidine Deaminase-Deficient Escherichia coli Strains Display Acute Sensitivity to Cytidine, Adenosine, and Guanosine and Increased Sensitivity to a Range of Antibiotics, Including Vancomycin

PubMed Central

Kang, Tina Manzhu; Yuan, Jessica; Zhou, Alice; Beppler, Casey

2014-01-01

We show here that deoxycytidine deaminase (DCD)-deficient mutants of Escherichia coli are hypersensitive to killing by exogenous cytidine, adenosine, or guanosine, whereas wild-type cells are not. This hypersensitivity is reversed by exogenous thymidine. The mechanism likely involves the allosteric regulation of ribonucleotide reductase and severe limitations of the dTTP pools, resulting in thymineless death, the phenomenon of cell death due to thymidine starvation. We also report here that DCD-deficient mutants of E. coli are more sensitive to a series of different antibiotics, including vancomycin, and we show synergistic killing with the combination of vancomycin and cytidine. One possibility is that a very low, subinhibitory concentration of vancomycin enters Gram-negative cells and that this concentration is potentiated by chromosomal lesions resulting from the thymineless state. A second possibility is that the metabolic imbalance resulting from DCD deficiency affects the assembly of the outer membrane, which normally presents a barrier to drugs such as vancomycin. We consider these findings with regard to ideas of rendering Gram-negative bacteria sensitive to drugs such as vancomycin. PMID:24633874

Direct measurement of adenosine release during hypoxia in the CA1 region of the rat hippocampal slice

PubMed Central

Dale, Nicholas; Pearson, Tim; Frenguelli, Bruno G

2000-01-01

We have used an enzyme-based, twin-barrelled sensor to measure adenosine release during hypoxia in the CA1 region of rat hippocampal slices in conjunction with simultaneous extracellular field recordings of excitatory synaptic transmission. When loaded with a combination of adenosine deaminase, nucleoside phosphorylase and xanthine oxidase, the sensor responded linearly to exogenous adenosine over the concentration range 10 nM to 20 μM. Without enzymes, the sensor when placed on the surface of hippocampal slices recorded a very small net signal during hypoxia of 40 ± 43 pA (mean ±s.e.m.; n = 7). Only when one barrel was loaded with the complete sequence of enzymes and the other with the last two in the cascade did the sensor record a large net difference signal during hypoxia (1226 ± 423 pA; n = 7). This signal increased progressively during the hypoxic episode, scaled with the hypoxic depression of the simultaneously recorded field excitatory postsynaptic potential and was greatly reduced (67 ± 6.5 %; n = 9) by coformycin (0.5-2 μM), a selective inhibitor of adenosine deaminase, the first enzyme in the enzymic cascade within the sensor. For 5 min hypoxic episodes, the sensor recorded a peak concentration of adenosine of 5.6 ± 1.2 μM (n = 16) with an IC50 for the depression of transmission of approximately 3 μM. In slices pre-incubated for 3-6 h in nominally Ca2+-free artificial cerebrospinal fluid, 5 min of hypoxia resulted in an approximately 9-fold greater release of adenosine (48.9 ± 17.7 μM; n = 6). High extracellular Ca2+ (4 mM) both reduced the adenosine signal recorded by the sensor during hypoxia (3.5 ± 0.6 μM; n = 4) and delayed the hypoxic depression of excitatory synaptic transmission. PMID:10878107

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

PubMed

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. Copyright © 2008 Elsevier Ltd. All rights reserved.

In vitro effect of adenosine agonist GR79236 on the insulin sensitivity of glucose utilisation in rat soleus and human rectus abdominus muscle.

PubMed

Webster, J M; Heseltine, L; Taylor, R

1996-06-07

The dose-response effects of a new adenosine agonist, GR79236, were examined in isolated rat soleus muscle strips and human rectus abdominus muscle strips. Effects on the insulin sensitivity of carbohydrate metabolism were examined, in particular upon insulin stimulated glycogen synthesis and glycolytic flux. In the presence of adenosine deaminase (ADA), GR79236 increased insulin sensitivity of pyruvate release from rat soleus muscle strips by 24% from 82.5 +/- 10.0 to 102.5 +/- 10.0 (P < 0.01), by 27% to 105.0 +/- 12.5 (P < 0.01) and by 24% to 102.5 +/- 10.0 (P < 0.01) nmol/25 mg per h at 0.1 and 10 microM GR79236, respectively. Rates of lactate release followed a similar but non-significant trend. Addition of GR79236 in the presence of ADA had no effect on rates of glycogen synthesis. Insulin stimulated rates of pyruvate or lactate release or of glycogen synthesis were unaffected by the addition of adenosine deaminase or GR79236 in human rectus abdominus muscle strips. Adenosine agonists may act indirectly to modulate insulin sensitivity of carbohydrate metabolism.

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

PubMed Central

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

2015-01-01

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

Serum Adenosine Deaminase (ADA) Activity: A Novel Screening Test to Differentiate HIV Monoinfection From HIV-HBV and HIV-HCV Coinfections.

PubMed

Abdi, Mohammad; Rahbari, Rizgar; Khatooni, Zahed; Naseri, Nima; Najafi, Adel; Khodadadi, Iraj

2016-05-01

CD4(+) cell count, the common HIV infection screening test, is costly and unable to differentiate HIV monoinfection from its concurrent infection with hepatitis B or C virus. We aimed to ascertain diagnostic value of serum adenosine deaminase (ADA) activity as a useful tool to differentiate HIV mono- and co-infection. Blood samples were collected from 30 HIV-HBV and 30 HIV-HCV coinfected patients, 33 HIV positive subjects, and 72 controls. CD4(+) cell count, serum total ADA (tADA), and ADA1, and ADA2 isoenzyme activities were determined and their sensitivity and specificity were computed. tADA and ADA2 activities were significantly higher and CD4(+) counts were markedly lower in all patients compared with controls. Strong inverse agreements between CD4(+) cell counts and both tADA and ADA2 activities were observed. Serum tADA and ADA1 activities showed the highest specificity and the highest sensitivity, respectively, for differentiating HIV monoinfection from HIV-HBV and HIV-HCV coinfections. We showed strong agreement and correlation between CD4(+) cell count and ADA enzyme activity. Based on high ADA sensitivity and specificity, it is concluded that determination of ADA activity might be a novel diagnostic tool to distinguish of HIV monoinfection from its coinfection with HBV or HCV. © 2015 Wiley Periodicals, Inc.

Preclinical Demonstration of Lentiviral Vector-mediated Correction of Immunological and Metabolic Abnormalities in Models of Adenosine Deaminase Deficiency

PubMed Central

Carbonaro, Denise A; Zhang, Lin; Jin, Xiangyang; Montiel-Equihua, Claudia; Geiger, Sabine; Carmo, Marlene; Cooper, Aaron; Fairbanks, Lynette; Kaufman, Michael L; Sebire, Neil J; Hollis, Roger P; Blundell, Michael P; Senadheera, Shantha; Fu, Pei-Yu; Sahaghian, Arineh; Chan, Rebecca Y; Wang, Xiaoyan; Cornetta, Kenneth; Thrasher, Adrian J; Kohn, Donald B; Gaspar, H Bobby

2014-01-01

Gene transfer into autologous hematopoietic stem cells by γ-retroviral vectors (gRV) is an effective treatment for adenosine deaminase (ADA)–deficient severe combined immunodeficiency (SCID). However, current gRV have significant potential for insertional mutagenesis as reported in clinical trials for other primary immunodeficiencies. To improve the efficacy and safety of ADA-SCID gene therapy (GT), we generated a self-inactivating lentiviral vector (LV) with a codon-optimized human cADA gene under the control of the short form elongation factor-1α promoter (LV EFS ADA). In ADA−/− mice, LV EFS ADA displayed high-efficiency gene transfer and sufficient ADA expression to rescue ADA−/− mice from their lethal phenotype with good thymic and peripheral T- and B-cell reconstitution. Human ADA-deficient CD34+ cells transduced with 1–5 × 107 TU/ml had 1–3 vector copies/cell and expressed 1–2x of normal endogenous levels of ADA, as assayed in vitro and by transplantation into immune-deficient mice. Importantly, in vitro immortalization assays demonstrated that LV EFS ADA had significantly less transformation potential compared to gRV vectors, and vector integration-site analysis by nrLAM-PCR of transduced human cells grown in immune-deficient mice showed no evidence of clonal skewing. These data demonstrated that the LV EFS ADA vector can effectively transfer the human ADA cDNA and promote immune and metabolic recovery, while reducing the potential for vector-mediated insertional mutagenesis. PMID:24256635

Characterization of adenosine receptors in guinea-pig isolated left atria.

PubMed Central

Jahnel, U.; Nawrath, H.

1989-01-01

1. The effects of purinergic stimulation on action potential, force of contraction, 86Rb efflux and 45Ca uptake were investigated in guinea-pig left atria. 2. Adenosine exerted a negative inotropic effect which was antagonized by adenosine deaminase but enhanced by dipyridamole. 3. The negative inotropic effect of adenosine was mimicked by 5'-(N-ethyl)-carboxamido-adenosine (NECA) and the isomers of N6-(phenyl-isopropyl)-adenosine, R-PIA and S-PIA. NECA and R-PIA were about 100 times more potent than adenosine, whereas R-PIA was about 100 times more potent than S-PIA. 4. The inotropic effects of adenosine (in the presence of dipyridamole), NECA, R-PIA and S-PIA were competitively antagonized either by theophylline (pA2 about 4.5) or 8-phenyltheophylline (pA2 about 6.3). 5. NECA and R-PIA shortened the action potential duration and increased the rate constant of the efflux of 86Rb in a concentration-dependent manner with no differences in potency; the effects were competitively antagonized by 8-phenyltheophylline. 6. Barium ions reduced the efflux of 86Rb under control conditions and antagonized the increase induced by NECA and R-PIA. 7. NECA and R-PIA significantly reduced 45Ca uptake in beating preparations. 8. It is concluded that adenosine, NECA and R-PIA activate a common receptor population (P1 or A3) on the outside of the cell membrane of atrial heart muscle to increase the potassium conductance and to reduce the action potential and, thereby, calcium influx and force of contraction. PMID:2790380

IL-17 receptor A and adenosine deaminase 2 deficiency in siblings with recurrent infections and chronic inflammation.

PubMed

Fellmann, Florence; Angelini, Federica; Wassenberg, Jacqueline; Perreau, Matthieu; Arenas Ramirez, Natalia; Simon, Gregoire; Boyman, Onur; Demaria, Olivier; Christen-Zaech, Stephanie; Hohl, Daniel; Belfiore, Marco; von Scheven-Gete, Annette; Gilliet, Michel; Bochud, Pierre-Yves; Perrin, Yannick; Beck Popovic, Maya; Bart, Pierre-Alexandre; Beckmann, Jacques S; Martinet, Danielle; Hofer, Michaël

2016-04-01

Data on patients affected by chronic mucocutaneous candidiasis underscore the preponderant role of IL-17 receptor A (IL-17RA) in preserving mucocutaneous immunity. Little is known about the role of adenosine deaminase (ADA) 2 in regulation of immune responses, although recent reports linked ADA2 deficiency with inflammation and vasculitis. We sought to investigate the mechanisms of chronic inflammation and vasculitis in a child lacking IL-17RA and ADA2 to identify therapeutic targets. We report a family with 2 siblings who have had recurrent mucocutaneous infections with Candida albicans and Staphylococcus aureus and chronic inflammatory disease and vasculitis since early childhood, which were refractory to classical treatments. Array-based comparative genomic hybridization analysis showed that both siblings are homozygous for a 770-kb deletion on chr22q11.1 encompassing both IL17RA and cat eye critical region 1 (CECR1). Immunologic studies were carried out by means of flow cytometry, ELISA, and RIA. As expected, in the affected child we found a lack of IL-17RA expression, which implies a severe malfunction in the IL-17 signaling pathway, conferring susceptibility to recurrent mucocutaneous infections. Surprisingly, we detected an in vitro and in vivo upregulation of proinflammatory cytokines, notably IL-1β and TNF-α, which is consistent with the persistent systemic inflammation. This work emphasizes the utility of whole-genome analyses combined with immunologic investigation in patients with unresolved immunodeficiency. This approach is likely to provide an insight into immunologic pathways and mechanisms of disease. It also provides molecular evidence for more targeted therapies. In addition, our report further corroborates a potential role of ADA2 in modulating immunity and inflammation. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Evaluation of adenosine deaminase in saliva and serum, and salivary α-amylase, in canine pyometra at diagnosis and after ovariohysterectomy.

PubMed

Tecles, F; Escribano, D; Contreras-Aguilar, M D; Rubio, C P; Szczubiał, M; Cerón, J J; Dąbrowski, R; Tvarijonaviciute, A

2018-06-01

An assay for adenosine deaminase (ADA) was validated in serum and saliva in dogs. Changes in ADA and salivary α-amylase activities were analysed in 26 bitches diagnosed with pyometra and compared with activities in 19 healthy bitches. All animals were classified according to the American Society of Anaesthesiologists (ASA) scoring for physical status. In the validation study, the ADA assay had an imprecision<12% and determination coefficients>0.90 in linearity under dilution experiments, with recoveries of 99.2-114.4%. On the day of presentation, salivary ADA activity was significantly higher in dogs with pyometra than in healthy dogs (median values 7.1IU/L vs. 0.8IU/L, respectively; P<0.01). ADA had a moderate positive correlation with leucocyte and band neutrophil counts, haptoglobin, salivary α-amylase and ASA score, and a low positive correlation with C-reactive protein. There were no significant differences in salivary α-amylase activity between dogs with pyometra and healthy dogs (57.3IU/L vs. 27.4IU/L, respectively). Salivary α-amylase had a low correlation with ASA grade, and leucocyte and band neutrophil counts. In 7/26 bitches with pyometra that were sampled 3 and 10days after ovariohysterectomy, there were no significant changes in α-amylase or ADA activities. These results indicate that ADA activity is increased in the saliva of bitches with pyometra, probably related to systemic inflammation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Inhibition of Transient Receptor Potential Channel Mucolipin-1 (TRPML1) by Lysosomal Adenosine Involved in Severe Combined Immunodeficiency Diseases*

PubMed Central

Zhong, Xi Zoë; Zou, Yuanjie; Sun, Xue; Dong, Gaofeng; Cao, Qi; Pandey, Aditya; Rainey, Jan K.; Zhu, Xiaojuan; Dong, Xian-Ping

2017-01-01

Impaired adenosine homeostasis has been associated with numerous human diseases. Lysosomes are referred to as the cellular recycling centers that generate adenosine by breaking down nucleic acids or ATP. Recent studies have suggested that lysosomal adenosine overload causes lysosome defects that phenocopy patients with mutations in transient receptor potential channel mucolipin-1 (TRPML1), a lysosomal Ca2+ channel, suggesting that lysosomal adenosine overload may impair TRPML1 and then lead to subsequent lysosomal dysfunction. In this study, we demonstrate that lysosomal adenosine is elevated by deleting adenosine deaminase (ADA), an enzyme responsible for adenosine degradation. We also show that lysosomal adenosine accumulation inhibits TRPML1, which is rescued by overexpressing ENT3, the adenosine transporter situated in the lysosome membrane. Moreover, ADA deficiency results in lysosome enlargement, alkalinization, and dysfunction. These are rescued by activating TRPML1. Importantly, ADA-deficient B-lymphocytes are more vulnerable to oxidative stress, and this was rescued by TRPML1 activation. Our data suggest that lysosomal adenosine accumulation impairs lysosome function by inhibiting TRPML1 and subsequently leads to cell death in B-lymphocytes. Activating TRPML1 could be a new therapeutic strategy for those diseases. PMID:28087698

Interleukin-6 Contributes to Inflammation and Remodeling in a Model of Adenosine Mediated Lung Injury

PubMed Central

Pedroza, Mesias; Schneider, Daniel J.; Karmouty-Quintana, Harry; Coote, Julie; Shaw, Stevan; Corrigan, Rebecca; Molina, Jose G.; Alcorn, Joseph L.; Galas, David; Gelinas, Richard; Blackburn, Michael R.

2011-01-01

Background Chronic lung diseases are the third leading cause of death in the United States due in part to an incomplete understanding of pathways that govern the progressive tissue remodeling that occurs in these disorders. Adenosine is elevated in the lungs of animal models and humans with chronic lung disease where it promotes air-space destruction and fibrosis. Adenosine signaling increases the production of the pro-fibrotic cytokine interleukin-6 (IL-6). Based on these observations, we hypothesized that IL-6 signaling contributes to tissue destruction and remodeling in a model of chronic lung disease where adenosine levels are elevated. Methodology/Principal Findings We tested this hypothesis by neutralizing or genetically removing IL-6 in adenosine deaminase (ADA)-deficient mice that develop adenosine dependent pulmonary inflammation and remodeling. Results demonstrated that both pharmacologic blockade and genetic removal of IL-6 attenuated pulmonary inflammation, remodeling and fibrosis in this model. The pursuit of mechanisms involved revealed adenosine and IL-6 dependent activation of STAT-3 in airway epithelial cells. Conclusions/Significance These findings demonstrate that adenosine enhances IL-6 signaling pathways to promote aspects of chronic lung disease. This suggests that blocking IL-6 signaling during chronic stages of disease may provide benefit in halting remodeling processes such as fibrosis and air-space destruction. PMID:21799929

Evaluation of a diagnostic flow chart applying medical thoracoscopy, adenosine deaminase and T-SPOT.TB in diagnosis of tuberculous pleural effusion.

PubMed

He, Y; Zhang, W; Huang, T; Wang, X; Wang, M

2015-10-01

To evaluate a diagnostic flow chart applying medical thoracoscoy (MT), adenosine deaminase (ADA) and T-SPOT.TB in diagnosis of tuberculous pleural effusion (TPE) at a high TB burden country. 136 patients with pleural effusion (PE) were enrolled and divided into TPE and Non-TPE group. MT (histology), PE ADA and T-SPOT.TB were conducted on all patients. ROC analysis was performed for the best cut-off value of PE ADA in detection of TPE. The diagnostic flow chart applying MT, ADA and T-SPOT.TB was evaluated for improving the limitations of each diagnostic method. ROC analysis showed that the best cut-off value of PE ADA was 30U/L. The sensitivity and specificity of these tests were calculated respectively to be: 71.4% (58.5%-81.6%) and 100% (95.4-100.0%) for MT, 92.9% (83.0-97.2%) and 68.8% (57.9-77.9%) for T-SPOT.TB, and 80.0% (69.6-88.1%) and 92.9% (82.7-98.0%) for PE ADA. The sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, positive predictive value and negative predictive value of the diagnostic flow chart were 96.4% (87.9-99.0%), 96.3% (89.6-98.7%), 25.714, 0.037, 97.4 and 94.9, respectively. The diagnostic flow chart applying MT, ADA and T-SPOT.TB is an accurate and rapid diagnostic method in detection of TPE.

Curcumin improves episodic memory in cadmium induced memory impairment through inhibition of acetylcholinesterase and adenosine deaminase activities in a rat model.

PubMed

Akinyemi, Ayodele Jacob; Okonkwo, Princess Kamsy; Faboya, Opeyemi Ayodeji; Onikanni, Sunday Amos; Fadaka, Adewale; Olayide, Israel; Akinyemi, Elizabeth Olufisayo; Oboh, Ganiyu

2017-02-01

Curcumin, the main polyphenolic component of turmeric (Curcuma longa) rhizomes has been reported to exert cognitive enhancing potential with limited scientific basis. Hence, this study sought to evaluate the effect of curcumin on cerebral cortex acetylcholinesterase (AChE) and adenosine deaminase (ADA) activities in cadmium (Cd)-induced memory impairment in rats. Animals were divided into six groups (n = 6): saline/vehicle, saline/curcumin 12.5 mg/kg, saline/curcumin 25 mg/kg, Cd/vehicle, Cd/curcumin 12.5 mg/kg, and Cd/curcumin 25 mg/kg. Rats received Cd (2.5 mg/kg) and curcumin (12.5 and 25 mg/kg, respectively) by gavage for 7 days. The results of this study revealed that cerebral cortex AChE and ADA activities were increased in Cd-poisoned rats, and curcumin co-treatment reversed these activities to the control levels. Furthermore, Cd intoxication increased the level of lipid peroxidation in cerebral cortex with a concomitant decreased in functional sulfuhydryl (-SH) group and nitric oxide (NO), a potent neurotransmitter and neuromodulatory agent. However, the co-treatment with curcumin at 12.5 and 25 mg/kg, respectively increased the non-enzymatic antioxidant status and NO in cerebral cortex with a decreased in malondialdehyde (MDA) level. Therefore, inhibition of AChE and ADA activities as well as increased antioxidant status by curcumin in Cd-induced memory dysfunction could suggest some possible mechanism of action for their cognitive enhancing properties.

Valerian extract Ze 911 inhibits postsynaptic potentials by activation of adenosine A1 receptors in rat cortical neurons.

PubMed

Vissiennon, Z; Sichardt, K; Koetter, U; Brattström, A; Nieber, K

2006-06-01

In this study we evaluated the adenosine A1 receptor-mediated effect of valerian extract (Ze 911) on postsynaptic potentials (PSPs) in pyramidal cells of the rat cingulate cortex in a slice preparation. We first observed that N6-cyclopentyladenosine (CPA, 0.01 - 10 microM), an adenosine A1 receptor agonist, inhibited PSPs in a concentration-dependent manner. The CPA (10 microM)-induced inhibition was antagonized by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1 microM), an adenosine A1 receptor antagonist. Ze 911 concentration dependently (0.1 - 15 mg/mL) inhibited PSPs in the presence of the adenosine A2A receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC, 0.2 microM) and adenosine deaminase (1 U/mL). The maximal inhibition induced by 10 mg/mL was completely antagonised by DPCPX (0.1 microM), an A1 receptor blocker. The data suggest that activation of adenosine A1 receptors is involved in the pharmacological effects of the valerian extract Ze 911.

Conflict RNA modification, host-parasite co-evolution, and the origins of DNA and DNA-binding proteins1.

PubMed

McLaughlin, Paul J; Keegan, Liam P

2014-08-01

Nearly 150 different enzymatically modified forms of the four canonical residues in RNA have been identified. For instance, enzymes of the ADAR (adenosine deaminase acting on RNA) family convert adenosine residues into inosine in cellular dsRNAs. Recent findings show that DNA endonuclease V enzymes have undergone an evolutionary transition from cleaving 3' to deoxyinosine in DNA and ssDNA to cleaving 3' to inosine in dsRNA and ssRNA in humans. Recent work on dsRNA-binding domains of ADARs and other proteins also shows that a degree of sequence specificity is achieved by direct readout in the minor groove. However, the level of sequence specificity observed is much less than that of DNA major groove-binding helix-turn-helix proteins. We suggest that the evolution of DNA-binding proteins following the RNA to DNA genome transition represents the major advantage that DNA genomes have over RNA genomes. We propose that a hypothetical RNA modification, a RRAR (ribose reductase acting on genomic dsRNA) produced the first stretches of DNA in RNA genomes. We discuss why this is the most satisfactory explanation for the origin of DNA. The evolution of this RNA modification and later steps to DNA genomes are likely to have been driven by cellular genome co-evolution with viruses and intragenomic parasites. RNA modifications continue to be involved in host-virus conflicts; in vertebrates, edited cellular dsRNAs with inosine-uracil base pairs appear to be recognized as self RNA and to suppress activation of innate immune sensors that detect viral dsRNA.

ADAR1 regulates ARHGAP26 gene expression through RNA editing by disrupting miR-30b-3p and miR-573 binding.

PubMed

Wang, Qiong; Hui, Haipeng; Guo, Zhendong; Zhang, Weina; Hu, Yaou; He, Tao; Tai, Yanhong; Peng, Peng; Wang, Li

2013-11-01

Rho GTPase activating protein 26 (ARHGAP26) is a negative regulator of the Rho family that converts the small G proteins RhoA and Cdc42 to their inactive GDP-bound forms. It is essential for the CLIC/GEEC endocytic pathway, cell spreading, and muscle development. The present study shows that ARHGAP26 mRNA undergoes extensive A-to-I RNA editing in the 3' UTR that is specifically catalyzed by ADAR1. Furthermore, the mRNA and protein levels of ARHGAP26 were decreased in cells in which ADAR1 was knocked down. Conversely, ADAR1 overexpression increased the abundance of ARHGAP26 mRNA and protein. In addition, we found that both miR-30b-3p and miR-573 target the ARHGAP26 gene and that RNA editing of ARHGAP26 mediated by ADAR1 abolished the repression of its expression by miR-30b-3p or miR-573. When ADAR1 was overexpressed, the reduced abundance of ARHGAP26 protein mediated by miR-30b-3p or miR-573 was rescued. Importantly, we also found that knocking down ADAR1 elevated RhoA activity, which was consistent with the reduced level of ARHGAP26. Conversely, when ADAR1 was overexpressed, the amount of RhoA-GTP decreased. The similar expression patterns of ARHGAP26 and ADAR1 in human tissue samples further confirmed our findings. Taken together, our results suggest that ADAR1 regulates the expression of ARHGAP26 through A-to-I RNA editing by disrupting the binding of miR-30b-3p and miR-573 within the 3' UTR of ARHGAP26. This study provides a novel insight into the mechanism by which ADAR1 and its RNA editing function regulate microRNA-mediated modulation of target genes.

Inhibition of AMP deaminase as therapeutic target in cardiovascular pathology.

PubMed

Zabielska, Magdalena A; Borkowski, Tomasz; Slominska, Ewa M; Smolenski, Ryszard T

2015-08-01

AMP deaminase (AMPD; EC 3.5.4.6) catalyzes hydrolysis of the amino group from the adenine ring of AMP resulting in production of inosine 5'-monophosphate (IMP) and ammonia. This reaction helps to maintain healthy cellular energetics by removing excess AMP that accumulates in energy depleted cells. Furthermore, AMPD permits the synthesis of guanine nucleotides from the larger adenylate pool. This enzyme competes with cytosolic 5'-nucleotidases (c5NT) for AMP. Adenosine, a product of c5NT is a vasodilator, antagonizes inotropic effects of catecholamines and exerts anti-platelet, anti-inflammatory and immunosuppressive activities. The ratio of AMPD/c5NT defines the amount of adenosine produced in adenine nucleotide catabolic pathway. Inhibition of AMPD could alter this ratio resulting in increased adenosine production. Besides the potential effect on adenosine production, elevation of AMP due to inhibition of AMPD could also lead to activation of AMP regulated protein kinase (AMPK) with myriad of downstream events including enhanced energetic metabolism, mitochondrial biogenesis and cytoprotection. While the benefits of these processes are well appreciated in cells such as skeletal or cardiac myocytes its role in protection of endothelium could be even more important. Therapeutic use of AMPD inhibition has been limited due to difficulties with obtaining compounds with adequate characteristics. However, endothelium seems to be the easiest target as effective inhibition of AMPD could be achieved at much lower concentration than in the other types of cells. New generation of AMPD inhibitors has recently been established and its testing in context of endothelial and organ protection could provide important basic knowledge and potential therapeutic tools. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Hepatitis D virus RNA editing is inhibited by a GFP fusion protein containing a C-terminally deleted delta antigen.

PubMed

Shih, Ko-Nien; Chuang, Ya-Ting; Liu, Hsuan; Lo, Szecheng J

2004-04-01

During its life cycle, hepatitis D virus (HDV) produces two forms of delta antigen (HDAg), small delta antigen (SDAg) and large delta antigen (LDAg), which differ in their C-terminal 19 amino acids. Host enzymes termed ADARs (adenosine deaminases that act on double-stranded RNA) are required for LDAg production. These enzymes change the stop codon (UAG) of SDAg to a tryptophan codon (UGG). However, the temporal and spatial regulation of HDV RNA editing is largely unknown. In this study, we constructed three GFP fusion proteins containing different lengths of SDAg and characterized their cellular localization and effects on HDV replication. One of these fusion proteins, designated D(1-88)-GFP, inhibited LDAg but not SDAg production, suggesting that D(1-88)-GFP inhibits HDV RNA editing. Two experiments further supported this supposition: (i). RT-PCR analysis combined with NcoI restriction enzyme digestion revealed that HDV RNA editing was reduced by 42% in HeLa-D(1-88)-GFP when compared with HeLa cells; and (ii). the ratio of SDAg/LDAg production from the reporter RNAs was reduced in cells co-transfected with ADAR-expressing and reporter plasmids in the presence of D(1-88)-GFP. Double fluorescence microscopy found that D(1-88)-GFP was either associated with SC-35 or was adjacent to PML (premyelocytic leukaemia antigen) at nuclear speckles, but D(1-88)-GFP was not co-localized with ADAR, which was mainly located in the nucleolus. In situ hybridization showing co-localization of HDV RNA with D(1-88)-GFP at nuclear speckles suggested that HDV RNA editing might occur in the nuclear speckles and require other nuclear factor(s), in addition to ADAR.

Extracellular adenosine production by ecto-5′-nucleotidase (CD73) enhances radiation-induced lung fibrosis

PubMed Central

Wirsdörfer, Florian; de Leve, Simone; Cappuccini, Federica; Eldh, Therese; Meyer, Alina V.; Gau, Eva; Thompson, Linda F.; Chen, Ning-Yuan; Karmouty-Quintana, Harry; Fischer, Ute; Kasper, Michael; Klein, Diana; Ritchey, Jerry W.; Blackburn, Michael R.; Westendorf, Astrid M.; Stuschke, Martin; Jendrossek, Verena

2016-01-01

Radiation-induced pulmonary fibrosis is a severe side effect of thoracic irradiation, but its pathogenesis remains poorly understood and no effective treatment is available. In this study, we investigated the role of the extracellular adenosine as generated by the ecto-5'-nucleotidase CD73 in fibrosis development after thoracic irradiation. Exposure of wild-type C57BL/6 mice to a single dose (15 Gray) of whole thorax irradiation triggered a progressive increase in CD73 activity in the lung between 3 and 30 weeks post-irradiation. In parallel, adenosine levels in bronchoalveolar lavage fluid (BALF) were increased by approximately three-fold. Histological evidence of lung fibrosis was observed by 25 weeks after irradiation. Conversely, CD73-deficient mice failed to accumulate adenosine in BALF and exhibited significantly less radiation-induced lung fibrosis (P<0.010). Furthermore, treatment of wild-type mice with pegylated adenosine deaminase (PEG-ADA) or CD73 antibodies also significantly reduced radiation-induced lung fibrosis. Taken together, our findings demonstrate that CD73 potentiates radiation-induced lung fibrosis, suggesting that existing pharmacological strategies for modulating adenosine may be effective in limiting lung toxicities associated with the treatment of thoracic malignancies. PMID:26921334

Chicken embryo fibroblasts exposed to weak, time-varying magnetic fields share cell proliferation, adenosine deaminase activity, and membrane characteristics of transformed cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parola, A.H.; Porat, N.; Kiesow, L.A.

1993-01-01

Chicken embryo fibroblasts (CEF) exposed to a sinusoidally varying magnetic field (SVMF) (100 Hz, 700 microT, for 24 h) showed a remarkable rise of segmental rotational relaxation rate of adenosine deaminase (ADA, EC 3.5.4.4) as determined by multifrequency phase fluorometry. Pyrene-labeled, small subunit ADA was applied to cultured (normal) CEF, which have available and abundant ADA complexing protein (ADCP) on their plasma membranes. Sine-wave-modulated fluorometry of the pyrene yielded a profile of phase angle vs. modulation frequency. In SVMF-treated cells and in Rous-sarcoma-virus (RSV) transformed cells the differential phase values at low modulation frequencies of the excitation are remarkably reduced.more » This effect is magnetic rather than thermal, because the temperature was carefully controlled and monitored; nevertheless to further check this matter we studied CEF, infected by the RSV-Ts68 temperature-sensitive mutant (36 degrees C transformed, 41 degrees C revertant). When grown at 36 degrees C in the SVMF, cells did not show the slightest trend towards reversion, as would be expected had there been local heating. Concomitant with the increased segmental rotational relaxation rate of ADA, there was a decrease in fluorescence lifetime and a slight, yet significant, increase in membrane lipid microfluidity. These biophysical observations prompted us to examine the effect of SVMF on cell proliferation and ADA activity (a malignancy marker): higher rates of cell proliferation and reduced specific activity of ADA were observed.« less

The Role of G22 A Adenosine Deaminase 1 Gene Polymorphism and the Activities of ADA Isoenzymes in Fertile and Infertile Men.

PubMed

Fattahi, Amir; Khodadadi, Iraj; Amiri, Iraj; Latifi, Zeinab; Ghorbani, Marzieh; Tavilani, Heidar

2015-10-01

To evaluate frequency distribution of adenosine deaminase 1 (ADA1) G22 A alleles and genotypes in fertile and infertile men. In this study we evaluate frequency distribution of ADA1 G22 A alleles and genotypes in 200 fertile and 200 infertile men. The polymerase chain reaction-restriction fragment length polymorphism technique was used for determining ADA1 G22 A variants. In addition, ADA isoenzymes activities (ADA1 and ADA2) were measured using colorimetric method. The frequency of GG genotype was significantly higher and GA genotype was lower in infertile males compared with fertile men (P = .048 and P = .045, respectively). However, there was not any noticeable difference in allele distribution between groups (P >.05). Based on logistic regression analysis, the GA genotype has a protective role and can decrease the risk of male infertility 1.7 times (P = .046). There were significantly higher activities of ADAT and its isoenzymes in infertile males compared with fertile men (P <.05). Also, the ADA1 activity with GG genotype was higher than GA carriers in all population (P = .001). Our results revealed that the activity of ADA isoenzymes and distribution of ADA1 G22 A genotypes were different among fertile and infertile men and more likely the GA genotype, which had lower ADA1 activity and was higher in fertile men is a protective factor against infertility. Copyright © 2015 Elsevier Inc. All rights reserved.

A decision tree for the genetic diagnosis of deficiency of adenosine deaminase 2 (DADA2): a French reference centres experience.

PubMed

Rama, Mélanie; Duflos, Claire; Melki, Isabelle; Bessis, Didier; Bonhomme, Axelle; Martin, Hélène; Doummar, Diane; Valence, Stéphanie; Rodriguez, Diana; Carme, Emilie; Genevieve, David; Heimdal, Ketil; Insalaco, Antonella; Franck, Nathalie; Queyrel-Moranne, Viviane; Tieulie, Nathalie; London, Jonathan; Uettwiller, Florence; Georgin-Lavialle, Sophie; Belot, Alexandre; Koné-Paut, Isabelle; Hentgen, Véronique; Boursier, Guilaine; Touitou, Isabelle; Sarrabay, Guillaume

2018-04-23

Deficiency of adenosine deaminase 2 (DADA2) is a recently described autoinflammatory disorder. Genetic analysis is required to confirm the diagnosis. We aimed to describe the identifying symptoms and genotypes of patients referred to our reference centres and to improve the indications for genetic testing. DNA from 66 patients with clinically suspected DADA2 were sequenced by Sanger or next-generation sequencing. Detailed epidemiological, clinical and biological features were collected by use of a questionnaire and were compared between patients with and without genetic confirmation of DADA2. We identified 13 patients (19.6%) carrying recessively inherited mutations in ADA2 that were predicted to be deleterious. Eight patients were compound heterozygous for mutations. Seven mutations were novel (4 missense variants, 2 predicted to affect mRNA splicing and 1 frameshift). The mean age of the 13 patients with genetic confirmation was 12.7 years at disease onset and 20.8 years at diagnosis. Phenotypic manifestations included fever (85%), vasculitis (85%) and neurological disorders (54%). Features best associated with a confirmatory genotype included fever with neurologic or cutaneous attacks (odds ratio [OR] 10.71, p = 0.003 and OR 10.9, p < 0.001), fever alone (OR 8.1, p = 0.01), and elevated C-reactive protein (CRP) level with neurologic involvement (OR 6.63, p = 0.017). Our proposed decision tree may help improve obtaining genetic confirmation of DADA2 in the context of autoinflammatory symptoms. Prerequisites for quick and low-cost Sanger analysis include one typical cutaneous or neurological sign, one marker of inflammation (fever or elevated CRP level), and recurrent or chronic attacks in adults.

Urtica dioica inhibits cell growth and induces apoptosis by targeting Ornithine decarboxylase and Adenosine deaminase as key regulatory enzymes in adenosine and polyamines homeostasis in human breast cancer cell lines.

PubMed

Fattahi, Sadegh; Ghadami, Elham; Asouri, Mohsen; Motevalizadeh Ardekanid, Ali; Akhavan-Niaki, Haleh

2018-02-28

Breast cancer is a heterogeneous and multifactorial disease with variable disease progression risk, and treatment response. Urtica dioica is a traditional herb used as an adjuvant therapeutic agent in cancer. In the present study, we have evaluated the effects of the aqueous extract of Urtica dioica on Adenosine deaminase (ADA) and Ornithine decarboxylase (ODC1) gene expression in MCF-7, MDA-MB-231, two breast cancer cell lines being estrogen receptor positive and estrogen receptor negative, respectively.  Cell lines were cultured in suitable media. After 24 h, different concentrations of the extract were added and after 72 h, ADA and ODC1 gene expression as well as BCL2 and BAX apoptotic genes were assessed by Taqman real time PCR assay. Cells viability was assessed by MTT assay, and apoptosis was also evaluated at cellular level. The intra and extracellular levels of ODC1 and ADA enzymes were evaluated by ELISA. Results showed differential expression of ADA and ODC1 genes in cancer cell lines. In MCF-7 cell line, the expression level of ADA was upregulated in a dose-dependent manner but its expression did not change in MDA-MB cell line. ODC1 expression was increased in both examined cell lines. Also, increased level of the apoptotic BAX/BCL-2 ratio was detected in MCF-7 cells. These results demonstrated that Urtica dioica induces apoptosis in breast cancer cells by influencing ODC1 and ADA genes expression, and estrogen receptors. The different responses observed with these cell lines could be due to the interaction of Urtica dioica as a phytoestrogen with the estrogen receptor.

A biochemical landscape of A-to-I RNA editing in the human brain transcriptome

PubMed Central

Sakurai, Masayuki; Ueda, Hiroki; Yano, Takanori; Okada, Shunpei; Terajima, Hideki; Mitsuyama, Toutai; Toyoda, Atsushi; Fujiyama, Asao; Kawabata, Hitomi; Suzuki, Tsutomu

2014-01-01

Inosine is an abundant RNA modification in the human transcriptome and is essential for many biological processes in modulating gene expression at the post-transcriptional level. Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosines to inosines (A-to-I editing) in double-stranded regions. We previously established a biochemical method called “inosine chemical erasing” (ICE) to directly identify inosines on RNA strands with high reliability. Here, we have applied the ICE method combined with deep sequencing (ICE-seq) to conduct an unbiased genome-wide screening of A-to-I editing sites in the transcriptome of human adult brain. Taken together with the sites identified by the conventional ICE method, we mapped 19,791 novel sites and newly found 1258 edited mRNAs, including 66 novel sites in coding regions, 41 of which cause altered amino acid assignment. ICE-seq detected novel editing sites in various repeat elements as well as in short hairpins. Gene ontology analysis revealed that these edited mRNAs are associated with transcription, energy metabolism, and neurological disorders, providing new insights into various aspects of human brain functions. PMID:24407955

DNA and RNA editing of retrotransposons accelerate mammalian genome evolution.

PubMed

Knisbacher, Binyamin A; Levanon, Erez Y

2015-04-01

Genome evolution is commonly viewed as a gradual process that is driven by random mutations that accumulate over time. However, DNA- and RNA-editing enzymes have been identified that can accelerate evolution by actively modifying the genomically encoded information. The apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBECs) are potent restriction factors that can inhibit retroelements by cytosine-to-uridine editing of retroelement DNA after reverse transcription. In some cases, a retroelement may successfully integrate into the genome despite being hypermutated. Such events introduce unique sequences into the genome and are thus a source of genomic innovation. adenosine deaminases that act on RNA (ADARs) catalyze adenosine-to-inosine editing in double-stranded RNA, commonly formed by oppositely oriented retroelements. The RNA editing confers plasticity to the transcriptome by generating many transcript variants from a single genomic locus. If the editing produces a beneficial variant, the genome may maintain the locus that produces the RNA-edited transcript for its novel function. Here, we discuss how these two powerful editing mechanisms, which both target inserted retroelements, facilitate expedited genome evolution. © 2015 New York Academy of Sciences.

Rare ADAR and RNASEH2B variants and a type I interferon signature in glioma and prostate carcinoma risk and tumorigenesis.

PubMed

Beyer, Ulrike; Brand, Frank; Martens, Helge; Weder, Julia; Christians, Arne; Elyan, Natalie; Hentschel, Bettina; Westphal, Manfred; Schackert, Gabriele; Pietsch, Torsten; Hong, Bujung; Krauss, Joachim K; Samii, Amir; Raab, Peter; Das, Anibh; Dumitru, Claudia A; Sandalcioglu, I Erol; Hakenberg, Oliver W; Erbersdobler, Andreas; Lehmann, Ulrich; Reifenberger, Guido; Weller, Michael; Reijns, Martin A M; Preller, Matthias; Wiese, Bettina; Hartmann, Christian; Weber, Ruthild G

2017-12-01

In search of novel germline alterations predisposing to tumors, in particular to gliomas, we studied a family with two brothers affected by anaplastic gliomas, and their father and paternal great-uncle diagnosed with prostate carcinoma. In this family, whole-exome sequencing yielded rare, simultaneously heterozygous variants in the Aicardi-Goutières syndrome (AGS) genes ADAR and RNASEH2B co-segregating with the tumor phenotype. AGS is a genetically induced inflammatory disease particularly of the brain, which has not been associated with a consistently increased cancer risk to date. By targeted sequencing, we identified novel ADAR and RNASEH2B variants, and a 3- to 17-fold frequency increase of the AGS mutations ADAR,c.577C>G;p.(P193A) and RNASEH2B,c.529G>A;p.(A177T) in the germline of familial glioma patients as well as in test and validation cohorts of glioblastomas and prostate carcinomas versus ethnicity-matched controls, whereby rare RNASEH2B variants were significantly more frequent in familial glioma patients. Tumors with ADAR or RNASEH2B variants recapitulated features of AGS, such as calcification and increased type I interferon expression. Patients carrying ADAR or RNASEH2B variants showed upregulation of interferon-stimulated gene (ISG) transcripts in peripheral blood as seen in AGS. An increased ISG expression was also induced by ADAR and RNASEH2B variants in tumor cells and was blocked by the JAK inhibitor Ruxolitinib. Our data implicate rare variants in the AGS genes ADAR and RNASEH2B and a type I interferon signature in glioma and prostate carcinoma risk and tumorigenesis, consistent with a genetic basis underlying inflammation-driven malignant transformation in glioma and prostate carcinoma development.

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

PubMed

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

2014-02-01

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

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

PubMed Central

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

Alterations in the brain adenosine metabolism cause behavioral and neurological impairment in ADA-deficient mice and patients

PubMed Central

Sauer, Aisha V.; Hernandez, Raisa Jofra; Fumagalli, Francesca; Bianchi, Veronica; Poliani, Pietro L.; Dallatomasina, Chiara; Riboni, Elisa; Politi, Letterio S.; Tabucchi, Antonella; Carlucci, Filippo; Casiraghi, Miriam; Carriglio, Nicola; Cominelli, Manuela; Forcellini, Carlo Alberto; Barzaghi, Federica; Ferrua, Francesca; Minicucci, Fabio; Medaglini, Stefania; Leocani, Letizia; la Marca, Giancarlo; Notarangelo, Lucia D.; Azzari, Chiara; Comi, Giancarlo; Baldoli, Cristina; Canale, Sabrina; Sessa, Maria; D’Adamo, Patrizia; Aiuti, Alessandro

2017-01-01

Adenosine Deaminase (ADA) deficiency is an autosomal recessive variant of severe combined immunodeficiency (SCID) caused by systemic accumulation of ADA substrates. Neurological and behavioral abnormalities observed in ADA-SCID patients surviving after stem cell transplantation or gene therapy represent an unresolved enigma in the field. We found significant neurological and cognitive alterations in untreated ADA-SCID patients as well as in two groups of patients after short- and long-term enzyme replacement therapy with PEG-ADA. These included motor dysfunction, EEG alterations, sensorineural hypoacusia, white matter and ventricular alterations in MRI as well as a low mental development index or IQ. Ada-deficient mice were significantly less active and showed anxiety-like behavior. Molecular and metabolic analyses showed that this phenotype coincides with metabolic alterations and aberrant adenosine receptor signaling. PEG-ADA treatment corrected metabolic adenosine-based alterations, but not cellular and signaling defects, indicating an intrinsic nature of the neurological and behavioral phenotype in ADA deficiency. PMID:28074903

Alterations in the brain adenosine metabolism cause behavioral and neurological impairment in ADA-deficient mice and patients.

PubMed

Sauer, Aisha V; Hernandez, Raisa Jofra; Fumagalli, Francesca; Bianchi, Veronica; Poliani, Pietro L; Dallatomasina, Chiara; Riboni, Elisa; Politi, Letterio S; Tabucchi, Antonella; Carlucci, Filippo; Casiraghi, Miriam; Carriglio, Nicola; Cominelli, Manuela; Forcellini, Carlo Alberto; Barzaghi, Federica; Ferrua, Francesca; Minicucci, Fabio; Medaglini, Stefania; Leocani, Letizia; la Marca, Giancarlo; Notarangelo, Lucia D; Azzari, Chiara; Comi, Giancarlo; Baldoli, Cristina; Canale, Sabrina; Sessa, Maria; D'Adamo, Patrizia; Aiuti, Alessandro

2017-01-11

Adenosine Deaminase (ADA) deficiency is an autosomal recessive variant of severe combined immunodeficiency (SCID) caused by systemic accumulation of ADA substrates. Neurological and behavioral abnormalities observed in ADA-SCID patients surviving after stem cell transplantation or gene therapy represent an unresolved enigma in the field. We found significant neurological and cognitive alterations in untreated ADA-SCID patients as well as in two groups of patients after short- and long-term enzyme replacement therapy with PEG-ADA. These included motor dysfunction, EEG alterations, sensorineural hypoacusia, white matter and ventricular alterations in MRI as well as a low mental development index or IQ. Ada-deficient mice were significantly less active and showed anxiety-like behavior. Molecular and metabolic analyses showed that this phenotype coincides with metabolic alterations and aberrant adenosine receptor signaling. PEG-ADA treatment corrected metabolic adenosine-based alterations, but not cellular and signaling defects, indicating an intrinsic nature of the neurological and behavioral phenotype in ADA deficiency.

Diagnosis of tuberculous pleurisy with combination of adenosine deaminase and interferon-γ immunospot assay in a tuberculosis-endemic population

PubMed Central

Xu, Han-Yan; Li, Cheng-Ye; Su, Shan-Shan; Yang, Li; Ye, Min; Ye, Jun-Ru; Ke, Pei-Pei; Chen, Cheng-Shui; Xie, Yu-Peng; Li, Yu-Ping

2017-01-01

Abstract The aim of this study was to identify the optimal cut-off value of T cell enzyme-linked immunospot assay for tuberculosis (T-SPOT.TB) and evaluate its diagnostic performance alone (in the peripheral blood) or in combination with the adenosine deaminase (ADA) activity test (in peripheral blood and the pleural fluid) in patients with tuberculous pleurisy. Adult patients presenting with pleural effusion were included in this prospective cohort study. Tuberculous pleurisy was diagnosed by T-SPOT.TB in peripheral blood and a combination of T-SPOT.TB and ADA activity test in pleural fluid and peripheral blood. Receiver operating characteristic (ROC) curve in combination with multivariate logistic regression was used to evaluate the diagnostic performance of the assays. Among a total of 189 patients with suspected tuberculous pleurisy who were prospectively enrolled in this study, 177 patients were validated for inclusion in the final analysis. ROC analysis revealed that the area under the ROC curve (AUC) for T-SPOT.TB in pleural fluid and peripheral blood was 0.918 and 0.881, respectively, and for the ADA activity test in pleural fluid was 0.944. In addition, 95.5 spot-forming cells (SFCs)/2.5 × 105 cells were determined as the optimal cut-off value for T-SPOT.TB in pleural fluid. Parallel combination of T-SPOT.TB and ADA activity test in pleural fluid showed increased sensitivity (96.9%) and specificity (87.5%), whereas serial combination showed increased specificity (97.5%). The combination of 3 assays had the highest sensitivity at 97.9%, with an AUC value of 0.964. T-SPOT.TB in pleural fluid performed better than that in peripheral blood and the ADA activity test in pleural fluid for tuberculous pleurisy diagnosis. The optimal cut-off value of T-SPOT.TB in pleural fluid was 95.5 SFCs/2.5 × 105 cells. Combination of 3 assays might be a promising approach for tuberculous pleurisy diagnosis. PMID:29381918

Adenosine-to-Inosine Editing of MicroRNA-487b Alters Target Gene Selection After Ischemia and Promotes Neovascularization.

PubMed

van der Kwast, Reginald V C T; van Ingen, Eva; Parma, Laura; Peters, Hendrika A B; Quax, Paul H A; Nossent, A Yaël

2018-02-02

Adenosine-to-inosine editing of microRNAs has the potential to cause a shift in target site selection. 2'-O-ribose-methylation of adenosine residues, however, has been shown to inhibit adenosine-to-inosine editing. To investigate whether angiomiR miR487b is subject to adenosine-to-inosine editing or 2'-O-ribose-methylation during neovascularization. Complementary DNA was prepared from C57BL/6-mice subjected to hindlimb ischemia. Using Sanger sequencing and endonuclease digestion, we identified and validated adenosine-to-inosine editing of the miR487b seed sequence. In the gastrocnemius muscle, pri-miR487b editing increased from 6.7±0.4% before to 11.7±1.6% ( P =0.02) 1 day after ischemia. Edited pri-miR487b is processed into a novel microRNA, edited miR487b, which is also upregulated after ischemia. We confirmed editing of miR487b in multiple human primary vascular cell types. Short interfering RNA-mediated knockdown demonstrated that editing is adenosine deaminase acting on RNA 1 and 2 dependent. Using reverse-transcription at low dNTP concentrations followed by quantitative-PCR, we found that the same adenosine residue is methylated in mice and human primary cells. In the murine gastrocnemius, the estimated methylation fraction increased from 32.8±14% before to 53.6±12% 1 day after ischemia. Short interfering RNA knockdown confirmed that methylation is fibrillarin dependent. Although we could not confirm that methylation directly inhibits editing, we do show that adenosine deaminase acting on RNA 1 and 2 and fibrillarin negatively influence each other's expression. Using multiple luciferase reporter gene assays, we could demonstrate that editing results in a complete switch of target site selection. In human primary cells, we confirmed the shift in miR487b targeting after editing, resulting in a edited miR487b targetome that is enriched for multiple proangiogenic pathways. Furthermore, overexpression of edited miR487b, but not wild-type miR487b, stimulates

RNA Editing with CRISPR-Cas13

PubMed Central

Cox, David B.T.; Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Franklin, Brian; Kellner, Max J.; Joung, Julia; Zhang, Feng

2017-01-01

Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided RNases Cas13. Here, we profile Type VI systems to engineer a Cas13 ortholog capable of robust knockdown and demonstrate RNA editing by using catalytically-inactive Cas13 (dCas13) to direct adenosine to inosine deaminase activity by ADAR2 to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineer this system to create a high specificity variant, REPAIRv2, that is 919 times more specific than REPAIRv1 as well as minimize the system to ease viral delivery. REPAIR presents a promising RNA editing platform with broad applicability for research, therapeutics, and biotechnology. PMID:29070703

Deficits in Endogenous Adenosine Formation by Ecto-5′-Nucleotidase/CD73 Impair Neuromuscular Transmission and Immune Competence in Experimental Autoimmune Myasthenia Gravis

PubMed Central

Cristina Costa, Ana; Guerra-Gomes, Sónia; Ferreirinha, Fátima; Magalhães-Cardoso, Maria Teresa; Correia-de-Sá, Paulo

2015-01-01

AMP dephosphorylation via ecto-5′-nucleotidase/CD73 is the rate limiting step to generate extracellular adenosine (ADO) from released adenine nucleotides. ADO, via A2A receptors (A2ARs), is a potent modulator of neuromuscular and immunological responses. The pivotal role of ecto-5′-nucleotidase/CD73, in controlling extracellular ADO formation, prompted us to investigate its role in a rat model of experimental autoimmune myasthenia gravis (EAMG). Results show that CD4+CD25+FoxP3+ regulatory T cells express lower amounts of ecto-5′-nucleotidase/CD73 as compared to controls. Reduction of endogenous ADO formation might explain why proliferation of CD4+ T cells failed upon blocking A2A receptors activation with ZM241385 or adenosine deaminase in EAMG animals. Deficits in ADO also contribute to neuromuscular transmission failure in EAMG rats. Rehabilitation of A2AR-mediated immune suppression and facilitation of transmitter release were observed by incubating the cells with the nucleoside precursor, AMP. These findings, together with the characteristic increase in serum adenosine deaminase activity of MG patients, strengthen our hypothesis that the adenosinergic pathway may be dysfunctional in EAMG. Given that endogenous ADO formation is balanced by ecto-5′-nucleotidase/CD73 activity and that A2ARs exert a dual role to restore use-dependent neurocompetence and immune suppression in myasthenics, we hypothesize that stimulation of the two mechanisms may have therapeutic potential in MG. PMID:25691808

Genome-wide identification and analysis of A-to-I RNA editing events in bovine by transcriptome sequencing

PubMed Central

Salehi, Abdolreza; Rivera, Rocío Melissa

2018-01-01

RNA editing increases the diversity of the transcriptome and proteome. Adenosine-to-inosine (A-to-I) editing is the predominant type of RNA editing in mammals and it is catalyzed by the adenosine deaminases acting on RNA (ADARs) family. Here, we used a largescale computational analysis of transcriptomic data from brain, heart, colon, lung, spleen, kidney, testes, skeletal muscle and liver, from three adult animals in order to identify RNA editing sites in bovine. We developed a computational pipeline and used a rigorous strategy to identify novel editing sites from RNA-Seq data in the absence of corresponding DNA sequence information. Our methods take into account sequencing errors, mapping bias, as well as biological replication to reduce the probability of obtaining a false-positive result. We conducted a detailed characterization of sequence and structural features related to novel candidate sites and found 1,600 novel canonical A-to-I editing sites in the nine bovine tissues analyzed. Results show that these sites 1) occur frequently in clusters and short interspersed nuclear elements (SINE) repeats, 2) have a preference for guanines depletion/enrichment in the flanking 5′/3′ nucleotide, 3) occur less often in coding sequences than other regions of the genome, and 4) have low evolutionary conservation. Further, we found that a positive correlation exists between expression of ADAR family members and tissue-specific RNA editing. Most of the genes with predicted A-to-I editing in each tissue were significantly enriched in biological terms relevant to the function of the corresponding tissue. Lastly, the results highlight the importance of the RNA editome in nervous system regulation. The present study extends the list of RNA editing sites in bovine and provides pipelines that may be used to investigate the editome in other organisms. PMID:29470549

Short-term hyperthyroidism modulates adenosine receptors and catalytic activity of adenylate cyclase in adipocytes.

PubMed Central

Rapiejko, P J; Malbon, C C

1987-01-01

The effects of short-term hyperthyroidism in vivo on the status of the components of the fat-cell hormone-sensitive adenylate cyclase were investigated. The number of beta-adrenergic receptors was elevated by about 25% in membranes of fat-cells isolated from hyperthyroid rats as compared with euthyroid rats, but their affinity for radioligand was unchanged. Membranes of hyperthyroid-rat fat-cells displayed less than 65% of the normal complement of receptors for [3H]cyclohexyladenosine. The affinity of the receptors for this ligand was normal. In contrast with the marked increase in the amounts of the alpha-subunits of the guanine nucleotide-binding proteins Gi (Mr 41,000) and Go (Mr 39,000) observed in the hypothyroid state [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564], the amounts of alpha-Gi, alpha-Go as well as alpha-Gs subunits [Mr 42,000 (major) and 46,000/48,000 (minor)] were not changed by hyperthyroidism. Adenylate cyclase activity in response to forskolin, guanosine 5'-[gamma-thio]triphosphate or isoprenaline, in contrast, was decreased by 30-50% in fat-cell membranes from hyperthyroid rats. Fat-cells isolated from hyperthyroid rats accumulated cyclic AMP to less than 50% of the extent in their euthyroid counterparts in the presence of adenosine deaminase and either adrenaline or forskolin, suggesting a decrease in the amount or activity of the catalytic subunit of adenylate cyclase. In the absence of exogenous adenosine deaminase, cyclic AMP accumulation in response to adrenaline was elevated rather than decreased in fat-cells from hyperthyroid rats. The inhibitory influence of adenosine is apparently limited in the hyperthyroid state by the decreased complement of inhibitory R-site purinergic receptors in these fat-cells. Short-term hyperthyroidism modulates the fat-cell adenylate cyclase system at the receptor level (beta-receptor number increased, R-site purinergic-receptor number decreased) and the catalytic subunit of adenylate

A continuous spectrophotometric assay for monitoring adenosine 5'-monophosphate production.

PubMed

First, Eric A

2015-08-15

A number of biologically important enzymes release adenosine 5'-monophosphate (AMP) as a product, including aminoacyl-tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5'-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5'-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD(+)) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses. Copyright © 2015 Elsevier Inc. All rights reserved.

The regulation of ATP release from the urothelium by adenosine and transepithelial potential.

PubMed

Dunning-Davies, Bryony M; Fry, Christopher H; Mansour, Dina; Ferguson, Douglas R

2013-03-01

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Stretch of the urothelium, as occurs during bladder filling, is associated with a release of ATP that is postulated to act as a sensory neurotransmitter. The regulation of ATP release is poorly understood and in particular if there is a feedback mechanism provided by ATP itself. Adenosine, a breakdown product of ATP, is a potent inhibitor of stretch-induced ATP release, acting through and A1 receptor; endogenous levels are about 0.6μM. Data are consistent with ATP release relying on the rise of intracellular Ca2+. Transepithelial potential also controls ATP release, also acting via an A1 receptor-dependent pathway. To test the hypothesis that distension-induced ATP release from the bladder urothelium is regulated by adenosine as well as changes to transurothelial potential (TEP). To examine the role of changes to intracellular [Ca(2+) ] in ATP release. Rabbit urothelium/suburothelium membranes were used in an Ussing chamber system. Distension was induced by fluid removal from the chamber bathing the serosal (basolateral) membrane face. The TEP and short-circuit current were measured. ATP was measured in samples aspirated from the serosal chamber by a luciferin-luciferase assay. Intracellular [Ca(2+) ] was measured in isolated urothelial cells using the fluorochrome Fura-2. All experiments were performed at 37°C. Distension-induced ATP release was decreased by adenosine (1-10 μm) and enhanced by adenosine deaminase and A1- (but not A2-) receptor antagonists. Distension-induced ATP release was reduced by 2-APB, nifedipine and capsazepine; capsaicin induced ATP release in the absence of distension. ATP and capsaicin, but not adenosine, generated intracellular Ca(2+) transients; adenosine did not affect the ATP-generated Ca(2+) transient. ATP release was dependent on a finite transepithelial potential. Changes to TEP, in the absence of distension, generated ATP release that was in turn reduced by adenosine

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

PubMed

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

2013-01-01

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

AMP deaminase histochemical activity and immunofluorescent isozyme localization in rat skeletal muscle

NASA Technical Reports Server (NTRS)

Thompson, J. L.; Sabina, R. L.; Ogasawara, N.; Riley, D. A.

1992-01-01

The cellular distribution of AMP deaminase (AMPda) isozymes was documented for rat soleus and plantaris muscles, utilizing immunofluorescence microscopy and immunoprecipitation methods. AMPda is a ubiquitous enzyme existing as three distinct isozymes, A, B and C, which were initially purified from skeletal muscle, liver (and kidney), and heart, respectively. AMPda-A is primarily concentrated subsarcolemmally and intermyofibrillarly within muscle cells, while isozymes B and C are concentrated within non-myofiber elements of muscle tissue. AMPda-B is principally associated with connective tissues surrounding neural elements and the muscle spindle capsule, and AMPda-C is predominantly associated with circulatory elements, such as arterial and venous walls, capillary endothelium, and red blood cells. These specific localizations, combined with documented differences in kinetic properties, suggest multiple functional roles for the AMPda isozymes or temporal segregation of similar AMPda functions. Linkage of the AMPda substrate with adenosine production pathways at the AMP level and the localization of isozyme-C in vascular tissue suggest a regulatory role in the microcirculation.

Myoadenylate deaminase deficiency, hypertrophic cardiomyopathy and gigantism syndrome.

PubMed

Skyllouriotis, M L; Marx, M; Bittner, R E; Skyllouriotis, P; Gross, M; Wimmer, M

1997-07-01

We report a 20-year-old man with gigantism syndrome, hypertrophic cardiomyopathy, muscle weakness, exercise intolerance, and severe psychomotor retardation since childhood. Histochemical and biochemical analysis of skeletal muscle biopsy revealed myoadenylate deaminase deficiency; molecular genetic analysis confirmed the diagnosis of primary (inherited) myoadenylate deaminase deficiency. Plasma, urine, and muscle carnitine concentrations were reduced. L-Carnitine treatment led to gradual improvement in exercise tolerance and cognitive performance; plasma and tissue carnitine levels returned to normal, and echocardiographic evidence of left ventricular hypertrophy disappeared. The combination of inherited myoadenylate deaminase deficiency, gigantism syndrome and carnitine deficiency has not previously been described.

Clinical value of jointly detection serum lactate dehydrogenase/pleural fluid adenosine deaminase and pleural fluid carcinoembryonic antigen in the identification of malignant pleural effusion.

PubMed

Zhang, Fan; Hu, Lijuan; Wang, Junjun; Chen, Jian; Chen, Jie; Wang, Yumin

2017-09-01

Limited data are available for the diagnostic value, and for the diagnostic sensitivity and specificity of joint detection of serum lactate dehydrogenase (sLDH)/pleural fluid adenosine deaminase (pADA) and pleural fluid carcinoembryonic antigen (pCEA) in malignant pleural effusion (MPE). We collected 987 pleural effusion specimens (of which 318 were malignant pleural effusion, 374 were tubercular pleural effusion, and 295 were parapneumonic effusion specimens) from the First Affiliated Hospital of Wenzhou Medical University from July 2012 to March 2016. The pADA, sLDH, pleural fluid LDH (pLDH), serum C-reactive protein (sCRP), pleural fluid protein, pCEA, white blood cell (WBC), and red blood cell (RBC) were analyzed, and the clinical data of each group were collected for statistical analysis. The level of sLDH/pADA, pCEA, and RBC from the MPE group was markedly higher than the tuberculosis pleural effusion (TB) group (Mann-Whitney U=28422.000, 9278.000, 30518, P=.000, .000, .000) and the parapneumonic pleural fluid group (Mann-Whitney U=5972.500, 7113.000, 36750.500, P=.000, .000, .000). The receiver operating characteristic curve ROC showed that the area under the ROC curve (AUC) (=0.924, 0.841) of pCEA and sLDH/pADA (cutoff=4.9, 10.6) were significantly higher than other markers for the diagnosis of MPE. Thus, joint detection of pCEA and sLDH/pADA suggested that the sensitivity, specificity, and AUC was 0.94, 81.70, and 94.32 at the cutoff 0.16 and diagnostic performance was higher than pCEA or sLDH/pADA. Joint detection of sLDH/pADA and pCEA can be used as a good indicator for the identification of benign and MPE with higher sensitivity and specificity than pCEA or sLDH/pADA. © 2016 Wiley Periodicals, Inc.

The rise and fall of an ancient Adélie penguin `supercolony' at Cape Adare, Antarctica

NASA Astrophysics Data System (ADS)

Emslie, Steven D.; McKenzie, Ashley; Patterson, William P.

2018-04-01

We report new discoveries and radiocarbon dates on active and abandoned Adélie penguin (Pygoscelis adeliae) colonies at Cape Adare, Antarctica. This colony, first established at approximately 2000 BP (calendar years before present, i.e. 1950), is currently the largest for this species with approximately 338 000 breeding pairs, most located on low-lying Ridley Beach. We hypothesize that this colony first formed after fast ice began blocking open-water access by breeding penguins to the Scott Coast in the southern Ross Sea during a cooling period also at approximately 2000 BP. Our results suggest that the new colony at Cape Adare continued to grow, expanding to a large upper terrace above Ridley Beach, until it exceeded approximately 500 000 breeding pairs (a `supercolony') by approximately 1200 BP. The high marine productivity associated with the Ross Sea polynya and continental shelf break supported this growth, but the colony collapsed to its present size for unknown reasons after approximately 1200 BP. Ridley Beach will probably be abandoned in the near future due to rising sea level in this region. We predict that penguins will retreat to higher elevations at Cape Adare and that the Scott Coast will be reoccupied by breeding penguins as fast ice continues to dissipate earlier each summer, restoring open-water access to beaches there.

The rise and fall of an ancient Adélie penguin 'supercolony' at Cape Adare, Antarctica.

PubMed

Emslie, Steven D; McKenzie, Ashley; Patterson, William P

2018-04-01

We report new discoveries and radiocarbon dates on active and abandoned Adélie penguin ( Pygoscelis adeliae ) colonies at Cape Adare, Antarctica. This colony, first established at approximately 2000 BP (calendar years before present, i.e. 1950), is currently the largest for this species with approximately 338 000 breeding pairs, most located on low-lying Ridley Beach. We hypothesize that this colony first formed after fast ice began blocking open-water access by breeding penguins to the Scott Coast in the southern Ross Sea during a cooling period also at approximately 2000 BP. Our results suggest that the new colony at Cape Adare continued to grow, expanding to a large upper terrace above Ridley Beach, until it exceeded approximately 500 000 breeding pairs (a 'supercolony') by approximately 1200 BP. The high marine productivity associated with the Ross Sea polynya and continental shelf break supported this growth, but the colony collapsed to its present size for unknown reasons after approximately 1200 BP. Ridley Beach will probably be abandoned in the near future due to rising sea level in this region. We predict that penguins will retreat to higher elevations at Cape Adare and that the Scott Coast will be reoccupied by breeding penguins as fast ice continues to dissipate earlier each summer, restoring open-water access to beaches there.

Changes in Adenosine Deaminase Activity in Patients with Type 2 Diabetes Mellitus and Effect of DPP-4 Inhibitor Treatment on ADA Activity.

PubMed

Lee, Jae-Geun; Kang, Dong Gu; Yu, Jung Re; Kim, Youngree; Kim, Jinsoek; Koh, Gwanpyo; Lee, Daeho

2011-04-01

Dipeptidyl peptidase 4 (DPP-4, also known as CD26) binds with adenosine deaminase (ADA) to activate T lymphocytes. Here, we investigated whether ADA activity is specifically affected by treatment with DPP-4 inhibitor (DPP4I) compared with other anti-diabetic agents. Fasting ADA activity, in addition to various metabolic and biochemical parameters, were measured in 262 type 2 diabetes mellitus (T2DM) patients taking various anti-diabetic agents and in 46 non-diabetic control subjects. ADA activity was increased in T2DM patients compared with that in non-diabetic control subjects (mean±standard error, 23.1±0.6 U/L vs. 18.6±0.8 U/L; P<0.05). ADA activity was correlated with fasting plasma glucose (r=0.258, P<0.05), HbA1c (r=0.208, P<0.05), aspartate aminotransferase (r=0.325, P<0.05), and alanine aminotransferase (r=0.248, P<0.05). Compared with the well-controlled T2DM patients (HbA1c<7%), the poorly controlled group (HbA1c>9%) showed significantly increased ADA activity (21.1±0.8 U/L vs. 25.4±1.6 U/L; P<0.05). The effect of DPP4I on ADA activity in T2DM patients did not differ from those of other oral anti-diabetic agents or insulin. T2DM patients on metformin monotherapy showed a lower ADA activity (20.9±1.0 U/L vs. 28.1±2.8 U/L; P<0.05) compared with that of those on sulfonylurea monotherapy. Our results show that ADA activity is increased in T2DM patients compared to that in non-diabetic patients, is positively correlated with blood glucose level, and that DPP4I has no additional specific effect on ADA activity, except for a glycemic control- or HbA1c-dependent effect.

Changes in Adenosine Deaminase Activity in Patients with Type 2 Diabetes Mellitus and Effect of DPP-4 Inhibitor Treatment on ADA Activity

PubMed Central

Lee, Jae-Geun; Kang, Dong Gu; Yu, Jung Re; Kim, Youngree; Kim, Jinsoek; Koh, Gwanpyo

2011-01-01

Background Dipeptidyl peptidase 4 (DPP-4, also known as CD26) binds with adenosine deaminase (ADA) to activate T lymphocytes. Here, we investigated whether ADA activity is specifically affected by treatment with DPP-4 inhibitor (DPP4I) compared with other anti-diabetic agents. Methods Fasting ADA activity, in addition to various metabolic and biochemical parameters, were measured in 262 type 2 diabetes mellitus (T2DM) patients taking various anti-diabetic agents and in 46 non-diabetic control subjects. Results ADA activity was increased in T2DM patients compared with that in non-diabetic control subjects (mean±standard error, 23.1±0.6 U/L vs. 18.6±0.8 U/L; P<0.05). ADA activity was correlated with fasting plasma glucose (r=0.258, P<0.05), HbA1c (r=0.208, P<0.05), aspartate aminotransferase (r=0.325, P<0.05), and alanine aminotransferase (r=0.248, P<0.05). Compared with the well-controlled T2DM patients (HbA1c<7%), the poorly controlled group (HbA1c>9%) showed significantly increased ADA activity (21.1±0.8 U/L vs. 25.4±1.6 U/L; P<0.05). The effect of DPP4I on ADA activity in T2DM patients did not differ from those of other oral anti-diabetic agents or insulin. T2DM patients on metformin monotherapy showed a lower ADA activity (20.9±1.0 U/L vs. 28.1±2.8 U/L; P<0.05) compared with that of those on sulfonylurea monotherapy. Conclusion Our results show that ADA activity is increased in T2DM patients compared to that in non-diabetic patients, is positively correlated with blood glucose level, and that DPP4I has no additional specific effect on ADA activity, except for a glycemic control- or HbA1c-dependent effect. PMID:21738897

Adenosine and inosine exert cytoprotective effects in an in vitro model of liver ischemia-reperfusion injury

PubMed Central

MÓDIS, KATALIN; GERŐ, DOMOKOS; STANGL, RITA; ROSERO, OLIVÉR; SZIJÁRTÓ, ATTILA; LOTZ, GÁBOR; MOHÁCSIK, PETRA; SZOLECZKY, PETRA; COLETTA, CIRO; SZABÓ, CSABA

2013-01-01

Liver ischemia represents a common clinical problem. In the present study, using an in vitro model of hepatic ischemia-reperfusion injury, we evaluated the potential cytoprotective effect of the purine metabolites, such as adenosine and inosine, and studied the mode of their pharmacological actions. The human hepatocellular carcinoma-derived cell line HepG2 was subjected to combined oxygen-glucose deprivation (COGD; 0-14-24 h), followed by re-oxygenation (0-4-24 h). Adenosine or inosine (300–1,000 μM) were applied in pretreatment. Cell viability and cytotoxicity were measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and lactate dehydrogenase methods, respectively. The results showed that both adenosine and inosine exerted cytoprotective effects, and these effects were not related to receptor-mediated actions, since they were not prevented by selective adenosine receptor antagonists. On the other hand, the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA, 10 μM) markedly and almost fully reversed the protective effect of adenosine during COGD, while it did not influence the cytoprotective effect of inosine in the same assay conditions. These results suggest that the cytoprotective effects are related to intracellular actions, and, in the case of adenosine also involve intracellular conversion to inosine. The likely interpretation of these findings is that inosine serves as an alternative source of energy to produce ATP during hypoxic conditions. The protective effects are also partially dependent on adenosine kinase, as the inhibitor 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d]pyrimidine, 2HCl (ABT 702, 30 μM) significantly reversed the protective effect of both adenosine and inosine during hypoxia and re-oxygenation. Collectively, the current results support the view that during hypoxia, adenosine and inosine exert cytoprotective effects via receptor

Adenosine and inosine exert cytoprotective effects in an in vitro model of liver ischemia-reperfusion injury.

PubMed

Módis, Katalin; Gerő, Domokos; Stangl, Rita; Rosero, Olivér; Szijártó, Attila; Lotz, Gábor; Mohácsik, Petra; Szoleczky, Petra; Coletta, Ciro; Szabó, Csaba

2013-02-01

Liver ischemia represents a common clinical problem. In the present study, using an in vitro model of hepatic ischemia-reperfusion injury, we evaluated the potential cytoprotective effect of the purine metabolites, such as adenosine and inosine, and studied the mode of their pharmacological actions. The human hepatocellular carcinoma-derived cell line HepG2 was subjected to combined oxygen-glucose deprivation (COGD; 0-14-24 h), followed by re-oxygenation (0-4-24 h). Adenosine or inosine (300-1,000 µM) were applied in pretreatment. Cell viability and cytotoxicity were measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and lactate dehydrogenase methods, respectively. The results showed that both adenosine and inosine exerted cytoprotective effects, and these effects were not related to receptor-mediated actions, since they were not prevented by selective adenosine receptor antagonists. On the other hand, the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA, 10 µM) markedly and almost fully reversed the protective effect of adenosine during COGD, while it did not influence the cytoprotective effect of inosine in the same assay conditions. These results suggest that the cytoprotective effects are related to intracellular actions, and, in the case of adenosine also involve intracellular conversion to inosine. The likely interpretation of these findings is that inosine serves as an alternative source of energy to produce ATP during hypoxic conditions. The protective effects are also partially dependent on adenosine kinase, as the inhibitor 4-amino-5-(3-bromophenyl)-7-(6‑morpholino-pyridin-3-yl)pyrido[2,3-d]pyrimidine, 2HCl (ABT 702, 30 µM) significantly reversed the protective effect of both adenosine and inosine during hypoxia and re-oxygenation. Collectively, the current results support the view that during hypoxia, adenosine and inosine exert cytoprotective effects via receptor

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA

PubMed Central

Cui, Yalei; Huang, Tianzhi; Zhang, Xiaobo

2015-01-01

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex. PMID:26674414

Cell type-specific in vivo expression of genes encoding signalling molecules in the brain in response to chronic mild stress and chronic treatment with fluoxetine.

PubMed

Dong, Lu; Li, Baoman; Verkhratsky, Alexei; Peng, Liang

2015-08-01

Previously, we reported that chronic treatment with fluoxetine increased gene expression of 5-hydroxytryptamine receptor 2B (5-HT2BR), cytosolic phospholipase 2α (cPLA2α), glutamate receptor, ionotropic kainate 2 (GluK2) and adenosine deaminase acting on RNA 2 (ADAR2), in cultured astrocytes and astrocytes freshly isolated from transgenic mice tagged with an astrocyte-specific marker. In contrast, neurones isolated from transgenic mice tagged with a neurone-specific marker and exposed to fluoxetine showed an increase in gene expression of glutamate receptor, ionotropic kainate 4 (GluK4) and 5-hydroxytryptamine receptor 2C (5-HT2CR). In a mouse model of anhedonia, the downregulation of 5-HT2BR, cPLA2α, ADAR2 and GluK4 but not GluK2 and 5-HT2CR was detected. To investigate the effects of chronic mild stress (CMS) and/or fluoxetine treatment on gene expression of 5-HT2BR, 5-HT2CR, cPLA2α, ADAR2, GluK2 and GluK4 specifically in astrocytes and neurones. Transgenic mice tagged with either astrocyte- or neurone-specific markers were exposed to the CMS. Real-time PCR was applied to determine expression of messenger RNA (mRNA). We found that (i) mRNAs of the 5-HT2BR and cPLA2α in astrocytes and GluK4 in neurones were significantly reduced in mice that became anhedonic; the mRNA levels were restored by fluoxetine treatment; (ii) ADAR2 in astrocytes was decreased by the CMS but showed no response to fluoxetine in anhedonic animals; (iii) neither GluK2 expression in astrocytes nor 5-HT2CR expression in neurones were affected in anhedonic animals, although expression of 5-HT2CR mRNA was upregulated by fluoxetine. Our results indicate that the effects of chronic treatment with fluoxetine are not only dependent on the cell type studied but also on the development of anhedonia. This suggests that fluoxetine may affect major depression (MD) patients and healthy people in a different manner.

The rise and fall of an ancient Adélie penguin ‘supercolony’ at Cape Adare, Antarctica

PubMed Central

McKenzie, Ashley; Patterson, William P.

2018-01-01

We report new discoveries and radiocarbon dates on active and abandoned Adélie penguin (Pygoscelis adeliae) colonies at Cape Adare, Antarctica. This colony, first established at approximately 2000 BP (calendar years before present, i.e. 1950), is currently the largest for this species with approximately 338 000 breeding pairs, most located on low-lying Ridley Beach. We hypothesize that this colony first formed after fast ice began blocking open-water access by breeding penguins to the Scott Coast in the southern Ross Sea during a cooling period also at approximately 2000 BP. Our results suggest that the new colony at Cape Adare continued to grow, expanding to a large upper terrace above Ridley Beach, until it exceeded approximately 500 000 breeding pairs (a ‘supercolony’) by approximately 1200 BP. The high marine productivity associated with the Ross Sea polynya and continental shelf break supported this growth, but the colony collapsed to its present size for unknown reasons after approximately 1200 BP. Ridley Beach will probably be abandoned in the near future due to rising sea level in this region. We predict that penguins will retreat to higher elevations at Cape Adare and that the Scott Coast will be reoccupied by breeding penguins as fast ice continues to dissipate earlier each summer, restoring open-water access to beaches there. PMID:29765656

Adenine formation from adenosine by mycoplasmas: adenosine phosphorylase activity.

PubMed Central

Hatanaka, M; Del Giudice, R; Long, C

1975-01-01

Mammalian cells have enzymes to convert adenosine to inosine by deamination and inosine to hypoxanthine by phosphorolysis, but they do not possess the enzymes necessary to form the free base, adenine, from adenosine. Mycoplasmas grown in broth or in cell cultures can produce adenine from adenosine. This activity was detected in a variety of mycoplasmatales, and the enzyme was shown to be adenosine phosphorylase. Adenosine formation from adenine and ribose 1-phosphate, the reverse reaction of adenine formation from adenosine, was also observed with the mycoplasma enzyme. Adenosine phosphorylase is apparently common to the mycoplasmatales but it is not universal, and the organisms can be divided into three groups on the basis of their use of adenosine as substrate. Thirteen of 16 Mycoplasma, Acholeplasma, and Siroplasma species tested exhibit adenosine phosphorylase activity. M. lipophilium differed from the other mycoplasmas and shared with mammalian cells the ability to convert adenosine to inosine by deamination. M. pneumoniae and the unclassified M. sp. 70-159 showed no reaction with adenosine. Adenosine phosphorylase activity offers an additional method for the detection of mycoplasma contamination of cells. The patterns of nucleoside metabolism will provide additional characteristics for identification of mycoplasmas and also may provide new insight into the classification of mycoplasmas. PMID:236559

Diagnosis of tuberculous pleurisy with combination of adenosine deaminase and interferon-γ immunospot assay in a tuberculosis-endemic population: A prospective cohort study.

PubMed

Xu, Han-Yan; Li, Cheng-Ye; Su, Shan-Shan; Yang, Li; Ye, Min; Ye, Jun-Ru; Ke, Pei-Pei; Chen, Cheng-Shui; Xie, Yu-Peng; Li, Yu-Ping

2017-11-01

The aim of this study was to identify the optimal cut-off value of T cell enzyme-linked immunospot assay for tuberculosis (T-SPOT.TB) and evaluate its diagnostic performance alone (in the peripheral blood) or in combination with the adenosine deaminase (ADA) activity test (in peripheral blood and the pleural fluid) in patients with tuberculous pleurisy.Adult patients presenting with pleural effusion were included in this prospective cohort study. Tuberculous pleurisy was diagnosed by T-SPOT.TB in peripheral blood and a combination of T-SPOT.TB and ADA activity test in pleural fluid and peripheral blood. Receiver operating characteristic (ROC) curve in combination with multivariate logistic regression was used to evaluate the diagnostic performance of the assays.Among a total of 189 patients with suspected tuberculous pleurisy who were prospectively enrolled in this study, 177 patients were validated for inclusion in the final analysis. ROC analysis revealed that the area under the ROC curve (AUC) for T-SPOT.TB in pleural fluid and peripheral blood was 0.918 and 0.881, respectively, and for the ADA activity test in pleural fluid was 0.944. In addition, 95.5 spot-forming cells (SFCs)/2.5 × 10 cells were determined as the optimal cut-off value for T-SPOT.TB in pleural fluid. Parallel combination of T-SPOT.TB and ADA activity test in pleural fluid showed increased sensitivity (96.9%) and specificity (87.5%), whereas serial combination showed increased specificity (97.5%). The combination of 3 assays had the highest sensitivity at 97.9%, with an AUC value of 0.964.T-SPOT.TB in pleural fluid performed better than that in peripheral blood and the ADA activity test in pleural fluid for tuberculous pleurisy diagnosis. The optimal cut-off value of T-SPOT.TB in pleural fluid was 95.5 SFCs/2.5 × 10 cells. Combination of 3 assays might be a promising approach for tuberculous pleurisy diagnosis. Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc

Basal Adare volcanics, Robertson Bay, North Victoria Land, Antarctica: Late Miocene intraplate basalts of subaqueous origin

USGS Publications Warehouse

Mortimer, N.; Dunlap, W.J.; Isaac, M.J.; Sutherland, R.P.; Faure, K.

2007-01-01

Late Cenozoic lavas and associated hyaloclastite breccias of the Adare volcanics (Hallett volcanic province) in Robertson Bay, North Victoria Land rest unconformably on Paleozoic greywackes. Abundant hyaloclastite breccias are confined to a paleovalley; their primary geological features, and the stable isotope ratios of secondary minerals, are consistent with eruption in a subaqueous environment with calcite formation probably involving seawater. In contrast, the lavas which stratigraphically overlie the hyaloclastites on Mayr Spur probably were erupted subaerially. K-Ar dating of eight samples from this basal sequence confirms the known older age limit (Late Miocene) of the Hallett volcanic province. Geochemical data reveal an ocean island basalt-like affinity, similar to other Cenozoic igneous rocks of the Hallett volcanic province. If a submarine eruptive paleoenvironment is accepted then there has been net tectonic or isostatic post-Late Miocene uplift of a few hundred metres in the Robertson Bay-Adare Peninsula area

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

PubMed Central

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

2013-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2013-01-01

AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) ( P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) ( P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.

Coupling to protein kinases A and C of adenosine A2B receptors involved in the facilitation of noradrenaline release in the prostatic portion of rat vas deferens.

PubMed

Queiroz, Glória; Quintas, Clara; Talaia, Carlos; Gonçalves, Jorge

2004-08-01

In the prostatic portion of rat vas deferens, the non-selective adenosine receptor agonist NECA (0.1-30 microM), but not the A(2A) agonist CGS 21680 (0.001-10 microM), caused a facilitation of electrically evoked noradrenaline release (up to 43 +/- 4%), when inhibitory adenosine A(1) receptors were blocked. NECA-elicited facilitation of noradrenaline release was prevented by the A(2B) receptor-antagonist MRS 1754, enhanced by preventing cyclic-AMP degradation with rolipram, abolished by the protein kinase A inhibitors H-89, KT 5720 and cyclic-AMPS-Rp and attenuated by the protein kinase C inhibitors Ro 32-0432 and calphostin C. The adenosine uptake inhibitor NBTI also elicited a facilitation of noradrenaline release; an effect that was abolished by adenosine deaminase and attenuated by MRS 1754, by inhibitors of the extracellular nucleotide metabolism and by blockade of alpha(1)-adrenoceptors and P2X receptors with prazosin and NF023, respectively. It was concluded that adenosine A(2B) receptors are involved in a facilitation of noradrenaline release in the prostatic portion of rat vas deferens that can be activated by adenosine formed by extracellular catabolism of nucleotides. The receptors seem to be coupled to the adenylyl cyclase-protein kinase A pathway but activation of the protein kinase C by protein kinase A, may also contribute to the adenosine A(2B) receptor-mediated facilitation of noradrenaline release.

On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells.

PubMed

Costa, M Adelina; Barbosa, A; Neto, E; Sá-e-Sousa, A; Freitas, R; Neves, J M; Magalhães-Cardoso, T; Ferreirinha, F; Correia-de-Sá, P

2011-05-01

Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation. Copyright © 2010 Wiley-Liss, Inc.

Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID.

PubMed

Sauer, Aisha V; Brigida, Immacolata; Carriglio, Nicola; Hernandez, Raisa Jofra; Scaramuzza, Samantha; Clavenna, Daniela; Sanvito, Francesca; Poliani, Pietro L; Gagliani, Nicola; Carlucci, Filippo; Tabucchi, Antonella; Roncarolo, Maria Grazia; Traggiai, Elisabetta; Villa, Anna; Aiuti, Alessandro

2012-02-09

Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA-treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA-treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.

Maintaining Genome Stability: The Role of Helicases and Deaminases

DTIC Science & Technology

2008-07-01

deaminases. The cells response to different forms of damage is fundamental to its ability to repair itself when challenged by environmental or...Page 4 of 12 INTRODUCTION Genomic DNA stores all the information for living organisms. The faithful duplication the maintanance of DNA are...maturation of the immune system by modifying enzymes called deaminases. The cells response to different forms of damage is fundamental to its ability to

Genetic, Phenotypic, and Interferon Biomarker Status in ADAR1-Related Neurological Disease.

PubMed

Rice, Gillian I; Kitabayashi, Naoki; Barth, Magalie; Briggs, Tracy A; Burton, Annabel C E; Carpanelli, Maria Luisa; Cerisola, Alfredo M; Colson, Cindy; Dale, Russell C; Danti, Federica Rachele; Darin, Niklas; De Azua, Begoña; De Giorgis, Valentina; De Goede, Christian G L; Desguerre, Isabelle; De Laet, Corinne; Eslahi, Atieh; Fahey, Michael C; Fallon, Penny; Fay, Alex; Fazzi, Elisa; Gorman, Mark P; Gowrinathan, Nirmala Rani; Hully, Marie; Kurian, Manju A; Leboucq, Nicolas; Lin, Jean-Pierre S-M; Lines, Matthew A; Mar, Soe S; Maroofian, Reza; Martí-Sanchez, Laura; McCullagh, Gary; Mojarrad, Majid; Narayanan, Vinodh; Orcesi, Simona; Ortigoza-Escobar, Juan Dario; Pérez-Dueñas, Belén; Petit, Florence; Ramsey, Keri M; Rasmussen, Magnhild; Rivier, François; Rodríguez-Pombo, Pilar; Roubertie, Agathe; Stödberg, Tommy I; Toosi, Mehran Beiraghi; Toutain, Annick; Uettwiller, Florence; Ulrick, Nicole; Vanderver, Adeline; Waldman, Amy; Livingston, John H; Crow, Yanick J

2017-06-01

We investigated the genetic, phenotypic, and interferon status of 46 patients from 37 families with neurological disease due to mutations in ADAR1 . The clinicoradiological phenotype encompassed a spectrum of Aicardi-Goutières syndrome, isolated bilateral striatal necrosis, spastic paraparesis with normal neuroimaging, a progressive spastic dystonic motor disorder, and adult-onset psychological difficulties with intracranial calcification. Homozygous missense mutations were recorded in five families. We observed a p.Pro193Ala variant in the heterozygous state in 22 of 23 families with compound heterozygous mutations. We also ascertained 11 cases from nine families with a p.Gly1007Arg dominant-negative mutation, which occurred de novo in four patients, and was inherited in three families in association with marked phenotypic variability. In 50 of 52 samples from 34 patients, we identified a marked upregulation of type I interferon-stimulated gene transcripts in peripheral blood, with a median interferon score of 16.99 (interquartile range [IQR]: 10.64-25.71) compared with controls (median: 0.93, IQR: 0.57-1.30). Thus, mutations in ADAR1 are associated with a variety of clinically distinct neurological phenotypes presenting from early infancy to adulthood, inherited either as an autosomal recessive or dominant trait. Testing for an interferon signature in blood represents a useful biomarker in this context. Georg Thieme Verlag KG Stuttgart · New York.

Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamat, S.S.; Swaminathan, S.; Bagaria, A.

2011-03-22

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with kcat and kcat/Km values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. Themore » apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism

Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Kamat; A Bagaria; D Kumaran

2011-12-31

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k{sub cat} and k{sub cat}/K{sub m} values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{supmore » -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical

Characterization of pterin deaminase from Mucor indicus MTCC 3513

NASA Astrophysics Data System (ADS)

Thandeeswaran, M.; Karthika, P.; Mahendran, R.; Palaniswamy, M.; Angayarkanni, J.

2018-03-01

Pterin deaminase is an amidohydrolase enzyme which hydrolyses pteridines to produce lumazine derivatives and ammonia. Even though the enzyme was shown as early as 1959 for its anticancer efficacy there was a long gap in the communique after that which was in 2013. In our study we have chosen Mucor indicus MTCC 3513 which was a promising strain for production of different industrial products.The pterin deaminase enzyme was harvested and extracellular from M. indicus. The extracellular sample was partially purified by using ethanol precipitation and ion exchange column (Hi-Trap QFF) in Fast Protein Liquid Chromatography. The molecular weight of the purified pterin deaminase enzyme was apparently determined by SDS-PAGE. The purified enzyme was further biochemically characterized. Molecular docking studies with the predicted sequence showed higher binding affinity towards folic acid interaction. The structure of this protein may open the windows for new drug targets for cancer therapy.

Bilateral striatal necrosis caused by ADAR mutations in two siblings with dystonia and freckles-like skin changes that should be differentiated from Leigh syndrome.

PubMed

Piekutowska-Abramczuk, Dorota; Mierzewska, Hanna; Bekiesińska-Figatowska, Monika; Ciara, Elżbieta; Trubicka, Joanna; Pronicki, Maciej; Rokicki, Dariusz; Rydzanicz, Małgorzata; Płoski, Rafał; Pronicka, Ewa

2016-01-01

Pathogenic molecular variants in the ADAR gene are a known cause of rare diseases, autosomal recessive Aicardi- Goutières syndrome type 6, severe infantile encephalopathy with intracranial calcifications and dominant dyschromatosis symmetrica hereditaria, demonstrated mainly in Asian adults. Recently, they have been also found in patients with nonsyndromic bilateral striatal necrosis accompanied by skin changes of the freckles-like type. Here, we present Polish siblings with acute onset and slowly progressive extrapyramidal syndrome with preserved intellectual abilities and basal ganglia changes found in MRI. A Leigh syndrome was considered for a long time as the most frequent cause of such lesions in children. Finally, two molecular variants in non-mitochondria-related ADAR gene c.3202+1G>A (p.?) and c.577C>G (p.Pro193Ala) were revealed by whole exome sequencing. We suggest that bilateral striatal necrosis should be always differentiated from LS to prevent the diagnosis delay. The striatal involvement accompanied by the presence of freckles-like skin changes should direct differential diagnosis to the ADAR gene mutations screening.

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA.

PubMed

Cui, Yalei; Huang, Tianzhi; Zhang, Xiaobo

2015-12-01

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex. © 2015 The Authors.

Genotype-specific enrichment of ACC deaminase-positive bacteria in winter wheat rhizospheres

USDA-ARS?s Scientific Manuscript database

Bacteria that produce ACC deaminase promote plant growth and development by lowering levels of the stress hormone ethylene through deamination of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene. Therefore, it is hypothesized that ACC deaminase positive (ACC+) bacteri...

Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID

PubMed Central

Sauer, Aisha V.; Brigida, Immacolata; Carriglio, Nicola; Jofra Hernandez, Raisa; Scaramuzza, Samantha; Clavenna, Daniela; Sanvito, Francesca; Poliani, Pietro L.; Gagliani, Nicola; Carlucci, Filippo; Tabucchi, Antonella; Roncarolo, Maria Grazia; Traggiai, Elisabetta; Villa, Anna

2012-01-01

Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)–mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA–treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA−/− Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA–treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA–treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID. Trials were registered at www.clinicaltrials.gov as NCT00598481/NCT00599781. PMID:22184407

Adenosine and adenosine receptors in the pathogenesis and treatment of rheumatic diseases.

PubMed

Cronstein, Bruce N; Sitkovsky, Michail

2017-01-01

Adenosine, a nucleoside derived primarily from the extracellular hydrolysis of adenine nucleotides, is a potent regulator of inflammation. Adenosine mediates its effects on inflammatory cells by engaging one or more cell-surface receptors. The expression and function of adenosine receptors on different cell types change during the course of rheumatic diseases, such as rheumatoid arthritis (RA). Targeting adenosine receptors directly for the treatment of rheumatic diseases is currently under study; however, indirect targeting of adenosine receptors by enhancing adenosine levels at inflamed sites accounts for most of the anti-inflammatory effects of methotrexate, the anchor drug for the treatment of RA. In this Review, we discuss the regulation of extracellular adenosine levels and the role of adenosine in regulating the inflammatory and immune responses in rheumatic diseases such as RA, psoriasis and other types of inflammatory arthritis. In addition, adenosine and its receptors are involved in promoting fibrous matrix production in the skin and other organs, and the role of adenosine in fibrosis and fibrosing diseases is also discussed.

Adenosine and preeclampsia.

PubMed

Salsoso, Rocío; Farías, Marcelo; Gutiérrez, Jaime; Pardo, Fabián; Chiarello, Delia I; Toledo, Fernando; Leiva, Andrea; Mate, Alfonso; Vázquez, Carmen M; Sobrevia, Luis

2017-06-01

Adenosine is an endogenous nucleoside with pleiotropic effects in different physiological processes including circulation, renal blood flow, immune function, or glucose homeostasis. Changes in adenosine membrane transporters, adenosine receptors, and corresponding intracellular signalling network associate with development of pathologies of pregnancy, including preeclampsia. Preeclampsia is a cause of maternal and perinatal morbidity and mortality affecting 3-5% of pregnancies. Since the proposed mechanisms of preeclampsia development include adenosine-dependent biological effects, adenosine membrane transporters and receptors, and the associated signalling mechanisms might play a role in the pathophysiology of preeclampsia. Preeclampsia associates with increased adenosine concentration in the maternal blood and placental tissue, likely due to local hypoxia and ischemia (although not directly demonstrated), microthrombosis, increased catecholamine release, and platelet activation. In addition, abnormal expression and function of equilibrative nucleoside transporters is described in foetoplacental tissues from preeclampsia; however, the role of adenosine receptors in the aetiology of this disease is not well understood. Adenosine receptors activation may be related to abnormal trophoblast invasion, angiogenesis, and ischemia/reperfusion mechanisms in the placenta from preeclampsia. These mechanisms may explain only a low fraction of the associated abnormal transformation of spiral arteries in preeclampsia, triggering cellular stress and inflammatory mediators release from the placenta to the maternal circulation. Although increased adenosine concentration in preeclampsia may be a compensatory or adaptive mechanism favouring placental angiogenesis, a poor angiogenic state is found in preeclampsia. Thus, preeclampsia-associated complications might affect the cell response to adenosine due to altered expression and activity of adenosine receptors, membrane transporters

Removal of interfering nucleotides from brain extracts containing substance p. Effect of drugs on brain concentrations of substance p

PubMed Central

Laszlo, I.

1963-01-01

Several methods for removing interfering nucleotides, adenosine-5'-monophosphate and adenosine 5'-triphosphate from brain extracts have been studied. An enzymic method, using adenylic acid deaminase, has been found suitable. This deaminates adenosine monophosphate to 5'-inosinic acid, an inactive compound which does not influence the estimations of substance P. Owing to the adenosine triphosphatase content of the enzyme extract, adenosine triphosphate was also inactivated. For the estimation of adenosine monophosphate-deaminase activity, a simple colorimetric method is described which measures the ammonia liberated from adenosine monophosphate. Substance P in mouse brain extracts was estimated after treatment of the animals with various drugs, and after the enzymic removal of interfering nucleotides from the brain extracts. The drugs had no effect on the substance P content of mouse brain. The effect of drugs on the contractions of the guinea-pig ileum induced by substance P was also investigated, and the effect of drugs on the estimations of substance P in brain extracts is discussed. PMID:14066136

Delay of flower senescence by bacterial endophytes expressing 1-aminocyclopropane-1-carboxylate deaminase.

PubMed

Ali, S; Charles, T C; Glick, B R

2012-11-01

The ability of 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing plant growth-promoting bacterial (PGPB) endophytes Pseudomonas fluorescens YsS6 and Pseudomonas migulae 8R6, their ACC deaminase minus mutants and the rhizospheric plant growth-promoting bacterium Pseudomonas putida UW4 to delay the senescence of mini carnation cut flowers was assessed. Fresh cut flowers were incubated with either a bacterial cell suspension, the ethylene precursor ACC, the ethylene inhibitor l-α-(aminoethoxyvinyl)-glycine or 0·85% NaCl at room temperature for 11 days. Levels of flower senescence were recorded every other day. To verify the presence of endophytes inside the plant tissues, scanning electron microscopy was performed. Among all treatments, flowers treated with wild-type ACC deaminase-containing endophytic strains exhibited the most significant delay in flower senescence, while flowers treated with the ACC deaminase minus mutants senesced at a rate similar to the control. Flowers treated with Ps. putida UW4 senesced more rapidly than untreated control flowers. The only difference between wild-type and mutant bacterial endophytes was ACC deaminase activity so that it may be concluded that this enzyme is directly responsible for the significant delay in flower senescence. Despite containing ACC deaminase activity, Ps. putida UW4 is not taken up by the cut flowers and therefore has no effect on prolonging their shelf life. The world-wide cut flower industry currently uses expensive and potentially environmentally dangerous chemical inhibitors of ethylene to prolong the shelf life of cut flowers. The use of PGPB endophytes with ACC deaminase activity has the potential to replace the chemicals that are currently used by the cut flower industry. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Role of Host-Driven Mutagenesis in Determining Genome Evolution of Sigma Virus (DMelSV; Rhabdoviridae) in Drosophila melanogaster.

PubMed

Piontkivska, Helen; Matos, Luis F; Paul, Sinu; Scharfenberg, Brian; Farmerie, William G; Miyamoto, Michael M; Wayne, Marta L

2016-10-05

Sigma virus (DMelSV) is ubiquitous in natural populations of Drosophila melanogaster. Host-mediated, selective RNA editing of adenosines to inosines (ADAR) may contribute to control of viral infection by preventing transcripts from being transported into the cytoplasm or being translated accurately; or by increasing the viral genomic mutation rate. Previous PCR-based studies showed that ADAR mutations occur in DMelSV at low frequency. Here we use SOLiD TM deep sequencing of flies from a single host population from Athens, GA, USA to comprehensively evaluate patterns of sequence variation in DMelSV with respect to ADAR. GA dinucleotides, which are weak targets of ADAR, are strongly overrepresented in the positive strand of the virus, consistent with selection to generate ADAR resistance on this complement of the transient, double-stranded RNA intermediate in replication and transcription. Potential ADAR sites in a worldwide sample of viruses are more likely to be "resistant" if the sites do not vary among samples. Either variable sites are less constrained and hence are subject to weaker selection than conserved sites, or the variation is driven by ADAR. We also find evidence of mutations segregating within hosts, hereafter referred to as hypervariable sites. Some of these sites were variable only in one or two flies (i.e., rare); others were shared by four or even all five of the flies (i.e., common). Rare and common hypervariable sites were indistinguishable with respect to susceptibility to ADAR; however, polymorphism in rare sites were more likely to be consistent with the action of ADAR than in common ones, again suggesting that ADAR is deleterious to the virus. Thus, in DMelSV, host mutagenesis is constraining viral evolution both within and between hosts. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

An efficient system for selectively altering genetic information within mRNAs

PubMed Central

Montiel-González, Maria Fernanda; Vallecillo-Viejo, Isabel C.; Rosenthal, Joshua J. C.

2016-01-01

Site-directed RNA editing (SDRE) is a strategy to precisely alter genetic information within mRNAs. By linking the catalytic domain of the RNA editing enzyme ADAR to an antisense guide RNA, specific adenosines can be converted to inosines, biological mimics for guanosine. Previously, we showed that a genetically encoded iteration of SDRE could target adenosines expressed in human cells, but not efficiently. Here we developed a reporter assay to quantify editing, and used it to improve our strategy. By enhancing the linkage between ADAR's catalytic domain and the guide RNA, and by introducing a mutation in the catalytic domain, the efficiency of converting a UAG premature termination codon (PTC) to tryptophan (UGG) was improved from ∼11 % to ∼70 %. Other PTCs were edited, but less efficiently. Numerous off-target edits were identified in the targeted mRNA, but not in randomly selected endogenous messages. Off-target edits could be eliminated by reducing the amount of guide RNA with a reduction in on-target editing. The catalytic rate of SDRE was compared with those for human ADARs on various substrates and found to be within an order of magnitude of most. These data underscore the promise of site-directed RNA editing as a therapeutic or experimental tool. PMID:27557710

Heat shock protein 70 (Hsp70)-stimulated deoxycytidine deaminases from a human lymphoma cell but not the activation-induced cytidine deaminase (AID) from Ramos 6.4 human Burkitt's lymphoma cells

PubMed Central

2010-01-01

Deoxycytidine deaminase enzyme activity was reduced in lysates of human leukemic THP1 cells 24 h after transfection with siRNA designed to inhibit cell synthesis of heat shock protein 70 (Hsp70)1a and Hsp701b. The cytidine deaminase enzyme activity from the cell lysates was purified from an affinity column which contained bound single-stranded oligodeoxycytidylic acid. Deficient enzyme activity in certain elution fractions from the siRNA-transfected cells was restored by including recombinant HSP 70 in the assays. Enzyme activity in some other fractions was increased after siRNA transfection. Activation-induced cytidine deaminase (AID) is a central factor in the immune response. A more specific assay for AID was used to study the influence of Hsp70 on AID activity. Unlike Hsp70's ability to stimulate certain enzymes of DNA base excision repair and other cytidine deaminases, it had little effect on AID activity in vitro, or was weakly inhibitory. PMID:20680536

Purification and Properties of Cytidine Deaminase from Normal and Leukemic Granulocytes

PubMed Central

Chabner, Bruce A.; Johns, David G.; Coleman, C. Norman; Drake, James C.; Evans, Warren H.

1974-01-01

Cytidine deaminase, an enzyme that catalyses the deamination of both cytidine and its nucleoside analogues including the antineoplastic agents cytosine arabinoside (ara-C) and 5-azacytidine (5-azaC), has been partially purified from normal and leukemic human granulocytes. The purification procedure included heat precipitation at 70°C, ammonium sulfate precipitation, calcium phosphate gel ion exchange, and Sephadex G-150 gel filtration. The enzyme has mol wt 51,000, isoelectric pH of 4.8, and maximum activity over a broad pH range of 5-9.5. The enzyme is stabilized by the presence of the sulfhydryl reagent, dithiothreitol. Cytidine deaminase from normal human granulocytes has a greater affinity for its physiologic substrate cytidine (Km = 1.1 × 10−5 M) than for ara-C (8.8 × 10−5 M) or 5-azaC (4.3 × 10−4 M). Halogenated analogues such as 5-fluorocytidine and 5-bromo-2′-deoxycytidine also exhibited substrate activity, with maximum velocities greater than that of the physiologic substrates cytidine and deoxycytidine. No activity was observed with nucleotides or deoxynucleotides. The relative maximum velocity of the enzyme for cytidine and its nucleoside analogues remained constant during purification, indicating that a single enzyme was responsible for deamination of these substrates. Tetrahydrouridine (THU) was found to be a strong competitive inhibitor of partially purified deaminase with a Ki of 5.4 × 10−8 M. The biochemical properties of partially purified preparations of cytidine deaminase from normal and leukemic cells were compared with respect to isoelectric pH, molecular weight, and substrate and inhibitor kinetic parameters, and no differences were observed. However, normal circulating granulocytes contained a significantly greater concentration of cytidine deaminase (3.52±1.86 × 103/mg protein) than chronic myelocytic leukemia (CML) cells (1.40±0.70 × 103 U/mg protein) or acute myelocytic leukemia (AML) cells (0.19±0.17 × 103 U

Functional coupling between adenosine A1 receptors and G-proteins in rat and postmortem human brain membranes determined with conventional guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding or [35S]GTPγS/immunoprecipitation assay.

PubMed

Odagaki, Yuji; Kinoshita, Masakazu; Ota, Toshio; Meana, J Javier; Callado, Luis F; Matsuoka, Isao; García-Sevilla, Jesús A

2018-06-01

Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A 1 receptor and G-protein was assessed by means of two [ 35 S]GTPγS binding assays, i.e., conventional filtration method and [ 35 S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A 1 receptor-mediated Gα i-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [ 35 S]GTPγS binding determined with conventional assay derives from functional activation of Gα i/o proteins (not restricted only to Gα i-3 ) coupled to adenosine A 1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [ 35 S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %E max values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A 1 receptor/G-protein interaction in postmortem human brain tissue.

A novel adenosine precursor 2',3'-cyclic adenosine monophosphate inhibits formation of post-surgical adhesions.

PubMed

Forman, Mervyn B; Gillespie, Delbert G; Cheng, Dongmei; Jackson, Edwin K

2014-09-01

Intraperitoneal adenosine reduces abdominal adhesions. However, because of the ultra-short half-life and low solubility of adenosine, optimal efficacy requires multiple dosing. Here, we compared the ability of potential adenosine prodrugs to inhibit post-surgical abdominal adhesions after a single intraperitoneal dose. Abdominal adhesions were induced in mice using an electric toothbrush to damage the cecum. Also, 20 μL of 95 % ethanol was applied to the cecum to cause chemically induced injury. After injury, mice received intraperitoneally either saline (n = 18) or near-solubility limit of adenosine (23 mmol/L; n = 12); 5'-adenosine monophosphate (75 mmol/L; n = 11); 3'-adenosine monophosphate (75 mmol/L; n = 12); 2'-adenosine monophosphate (75 mmol/L; n = 12); 3',5'-cyclic adenosine monophosphate (75 mmol/L; n = 19); or 2',3'-cyclic adenosine monophosphate (75 mmol/L; n = 20). After 2 weeks, adhesion formation was scored by an observer blinded to the treatments. In a second study, intraperitoneal adenosine levels were measured using tandem mass spectrometry for 3 h after instillation of 2',3'-cyclic adenosine monophosphate (75 mmol/L) into the abdomen. The order of efficacy for attenuating adhesion formation was: 2',3'-cyclic adenosine monophosphate > 3',5'-cyclic adenosine monophosphate ≈ adenosine > 5'-adenosine monophosphate ≈ 3'-adenosine monophosphate ≈ 2'-adenosine monophosphate. The groups were compared using a one-factor analysis of variance, and the overall p value for differences between groups was p < 0.000001. Intraperitoneal administration of 2',3'-cAMP yielded pharmacologically relevant levels of adenosine in the abdominal cavity for >3 h. Administration of 2',3'-cyclic adenosine monophosphate into the surgical field is a unique, convenient and effective method of preventing post-surgical adhesions by acting as an adenosine prodrug.

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

EPA Science Inventory

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

Adenosine and Ischemic Preconditioning

PubMed Central

Liang, Bruce T.; Swierkosz, Tomasz A.; Herrmann, Howard C.; Kimmel, Stephen; Jacobson, Kenneth A.

2012-01-01

Adenosine is released in large amounts during myocardial ischemia and is capable of exerting potent cardioprotective effects in the heart. Although these observations on adenosine have been known for a long time, how adenosine acts to achieve its anti-ischemic effect remains incompletely understood. However, recent advances on the chemistry and pharmacology of adenosine receptor ligands have provided important and novel information on the function of adenosine receptor subtypes in the cardiovascular system. The development of model systems for the cardiac actions of adenosine has yielded important insights into its mechanism of action and have begun to elucidate the sequence of signalling events from receptor activation to the actual exertion of its cardioprotective effect. The present review will focus on the adenosine receptors that mediate the potent anti-ischemic effect of adenosine, new ligands at the receptors, potential molecular signalling mechanisms downstream of the receptor, mediators for cardioprotection, and possible clinical applications in cardiovascular disorders. PMID:10607860

The role of microorganisms in the degradation of adenosine triphosphate (ATP) in chill-stored common carp (Cyprinus carpio) fillets.

PubMed

Li, Dapeng; Zhang, Longteng; Song, Sijia; Wang, Zhiying; Kong, Chunli; Luo, Yongkang

2017-06-01

Biochemical and microbial changes after harvest strongly affect the final quality and shelf life of fish and fish products. In this study, the role of microbes in the degradation of adenosine triphosphate (ATP), and the origin of adenosine monophosphate deaminase (AMPD) and acid phosphatase (ACP) in common carp fillets during different stages of chilled storage (at 4°C) were investigated. The content of ATP, ADP, AMP, IMP, HxR, and Hx, the activity of AMPD and ACP, and the total count of viable, Aeromonas, Pseudomonas, H 2 S-producing bacteria, and lactic acid bacteria were examined. Results indicated that the population of microbial communities in control samples increased with storage time, and Pseudomonas peaked on the 10th day of storage. Changes in AMPD activity were less related to the abundance of microbes during the entire storage period. However, ACP was derived from both fish muscle and microbial secretion during the middle and late stages of storage. Degradation of ATP to IMP was not affected by spoilage bacteria, but the hydrolysis of IMP, and the transformation of HxR to Hx was affected considerably by the spoilage bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

AID/APOBEC cytosine deaminase induces genome-wide kataegis

PubMed Central

2012-01-01

Clusters of localized hypermutation in human breast cancer genomes, named “kataegis” (from the Greek for thunderstorm), are hypothesized to result from multiple cytosine deaminations catalyzed by AID/APOBEC proteins. However, a direct link between APOBECs and kataegis is still lacking. We have sequenced the genomes of yeast mutants induced in diploids by expression of the gene for PmCDA1, a hypermutagenic deaminase from sea lamprey. Analysis of the distribution of 5,138 induced mutations revealed localized clusters very similar to those found in tumors. Our data provide evidence that unleashed cytosine deaminase activity is an evolutionary conserved, prominent source of genome-wide kataegis events. Reviewers This article was reviewed by: Professor Sandor Pongor, Professor Shamil R. Sunyaev, and Dr Vladimir Kuznetsov. PMID:23249472

Activation of adenosine low-affinity A3 receptors inhibits the enteric short interplexus neural circuit triggered by histamine.

PubMed

Bozarov, Andrey; Wang, Yu-Zhong; Yu, Jun Ge; Wunderlich, Jacqueline; Hassanain, Hamdy H; Alhaj, Mazin; Cooke, Helen J; Grants, Iveta; Ren, Tianhua; Christofi, Fievos L

2009-12-01

We tested the novel hypothesis that endogenous adenosine (eADO) activates low-affinity A3 receptors in a model of neurogenic diarrhea in the guinea pig colon. Dimaprit activation of H2 receptors was used to trigger a cyclic coordinated response of contraction and Cl(-) secretion. Contraction-relaxation was monitored by sonomicrometry (via intracrystal distance) simultaneously with short-circuit current (I(sc), Cl(-) secretion). The short interplexus reflex coordinated response was attenuated or abolished by antagonists at H2 (cimetidine), 5-hydroxytryptamine 4 receptor (RS39604), neurokinin-1 receptor (GR82334), or nicotinic (mecamylamine) receptors. The A1 agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) abolished coordinated responses, and A1 antagonists could restore normal responses. A1-selective antagonists alone [8-cyclopentyltheophylline (CPT), 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX), or 8-cyclopentyl-N(3)-[3-(4-(fluorosulfonyl)benzoyloxy)propyl]-xanthine (FSCPX)] caused a concentration-dependent augmentation of crypt cell secretion or contraction and acted at nanomolar concentrations. The A3 agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) abolished coordinated responses and the A3 antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191) could restore and further augment responses. The IB-MECA effect was resistant to knockdown of adenosine A1 receptor with the irreversible antagonist FSCPX; the IC(50) for IB-MECA was 0.8 microM. MRS1191 alone could augment or unmask coordinated responses to dimaprit, and IB-MECA suppressed them. MRS1191 augmented distension-evoked reflex I(sc) responses. Adenosine deaminase mimicked actions of adenosine receptor antagonists. A3 receptor immunoreactivity was differentially expressed in enteric neurons of different parts of colon. After tetrodotoxin, IB-MECA caused circular muscle relaxation. The data support the novel concept that

Activation of adenosine low-affinity A3 receptors inhibits the enteric short interplexus neural circuit triggered by histamine

PubMed Central

Bozarov, Andrey; Wang, Yu-Zhong; Yu, Jun Ge; Wunderlich, Jacqueline; Hassanain, Hamdy H.; Alhaj, Mazin; Cooke, Helen J.; Grants, Iveta; Ren, Tianhua

2009-01-01

We tested the novel hypothesis that endogenous adenosine (eADO) activates low-affinity A3 receptors in a model of neurogenic diarrhea in the guinea pig colon. Dimaprit activation of H2 receptors was used to trigger a cyclic coordinated response of contraction and Cl− secretion. Contraction-relaxation was monitored by sonomicrometry (via intracrystal distance) simultaneously with short-circuit current (Isc, Cl− secretion). The short interplexus reflex coordinated response was attenuated or abolished by antagonists at H2 (cimetidine), 5-hydroxytryptamine 4 receptor (RS39604), neurokinin-1 receptor (GR82334), or nicotinic (mecamylamine) receptors. The A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA) abolished coordinated responses, and A1 antagonists could restore normal responses. A1-selective antagonists alone [8-cyclopentyltheophylline (CPT), 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX), or 8-cyclopentyl-N3-[3-(4-(fluorosulfonyl)benzoyloxy)propyl]-xanthine (FSCPX)] caused a concentration-dependent augmentation of crypt cell secretion or contraction and acted at nanomolar concentrations. The A3 agonist N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) abolished coordinated responses and the A3 antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1191) could restore and further augment responses. The IB-MECA effect was resistant to knockdown of adenosine A1 receptor with the irreversible antagonist FSCPX; the IC50 for IB-MECA was 0.8 μM. MRS1191 alone could augment or unmask coordinated responses to dimaprit, and IB-MECA suppressed them. MRS1191 augmented distension-evoked reflex Isc responses. Adenosine deaminase mimicked actions of adenosine receptor antagonists. A3 receptor immunoreactivity was differentially expressed in enteric neurons of different parts of colon. After tetrodotoxin, IB-MECA caused circular muscle relaxation. The data support the novel concept that eADO acts at

RNA editing of non-coding RNA and its role in gene regulation.

PubMed

Daniel, Chammiran; Lagergren, Jens; Öhman, Marie

2015-10-01

It has for a long time been known that repetitive elements, particularly Alu sequences in human, are edited by the adenosine deaminases acting on RNA, ADAR, family. The functional interpretation of these events has been even more difficult than that of editing events in coding sequences, but today there is an emerging understanding of their downstream effects. A surprisingly large fraction of the human transcriptome contains inverted Alu repeats, often forming long double stranded structures in RNA transcripts, typically occurring in introns and UTRs of protein coding genes. Alu repeats are also common in other primates, and similar inverted repeats can frequently be found in non-primates, although the latter are less prone to duplex formation. In human, as many as 700,000 Alu elements have been identified as substrates for RNA editing, of which many are edited at several sites. In fact, recent advancements in transcriptome sequencing techniques and bioinformatics have revealed that the human editome comprises at least a hundred million adenosine to inosine (A-to-I) editing sites in Alu sequences. Although substantial additional efforts are required in order to map the editome, already present knowledge provides an excellent starting point for studying cis-regulation of editing. In this review, we will focus on editing of long stem loop structures in the human transcriptome and how it can effect gene expression. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

[Isolation, identification and characterization of ACC deaminase-containing endophytic bacteria from halophyte Suaeda salsa].

PubMed

Teng, Songshan; Liu, Yanping; Zhao, Lei

2010-11-01

We Isolated and characterized 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing endophytic bacteria from halophyte Suaeda salsa to understand the interactions between endophytes and halophyte. ACC deaminase-containing endophytic bacteria were isolated from root, stalk and leaf of Suaeda salsa and were identified based on morphological, physiological-biochemical properties, API and 16S rRNA sequence analysis. Isolates were evaluated for their ACC deaminase, antifungal, protease activity, siderophores and phytohormones, such as indole-3-acetic acid, gibberellic acid and abscisic acid production, as well as atmospheric nitrogen fixation and phosphate solubilization. Four ACC deaminase-containing endophytic bacteria strains named as LP11, SS12, TW1 and TW2 were isolated and identified as Pseudomonas oryzihabitans, Pseudomonas sp., Pantoea agglomerans and Pseudomonas putida respectively. All the strains possessed the phosphate-solubilizing ability and could produce siderophores and phytohormones more or less. None of them could fix atmospheric nitrogen or produce protease. Only strain SS12 showed antagonism against two phytopathogenic fungi viz Fusarium oxysporum f. sp. conglutinans and F. oxysporum f. sp. cucumerinum. ACC deaminase-containing endophytic bacteria of Pseudomonas sp. and Pantoea sp. isolated from halophyte Suaeda salsa have abundant biological characteristics related to plant growth promotion, stress homeostasis regulation and biocontrol activity.

The structures of non-CG-repeat Z-DNAs co-crystallized with the Z-DNA-binding domain, hZ alpha(ADAR1).

PubMed

Ha, Sung Chul; Choi, Jongkeun; Hwang, Hye-Yeon; Rich, Alexander; Kim, Yang-Gyun; Kim, Kyeong Kyu

2009-02-01

The Z-DNA conformation preferentially occurs at alternating purine-pyrimidine repeats, and is specifically recognized by Z alpha domains identified in several Z-DNA-binding proteins. The binding of Z alpha to foreign or chromosomal DNA in various sequence contexts is known to influence various biological functions, including the DNA-mediated innate immune response and transcriptional modulation of gene expression. For these reasons, understanding its binding mode and the conformational diversity of Z alpha bound Z-DNAs is of considerable importance. However, structural studies of Z alpha bound Z-DNA have been mostly limited to standard CG-repeat DNAs. Here, we have solved the crystal structures of three representative non-CG repeat DNAs, d(CACGTG)(2), d(CGTACG)(2) and d(CGGCCG)(2) complexed to hZ alpha(ADAR1) and compared those structures with that of hZ alpha(ADAR1)/d(CGCGCG)(2) and the Z alpha-free Z-DNAs. hZ alpha(ADAR1) bound to each of the three Z-DNAs showed a well conserved binding mode with very limited structural deviation irrespective of the DNA sequence, although varying numbers of residues were in contact with Z-DNA. Z-DNAs display less structural alterations in the Z alpha-bound state than in their free form, thereby suggesting that conformational diversities of Z-DNAs are restrained by the binding pocket of Z alpha. These data suggest that Z-DNAs are recognized by Z alpha through common conformational features regardless of the sequence and structural alterations.

Extra Double-stranded RNA Binding Domain (dsRBD) in a Squid RNA Editing Enzyme Confers Resistance to High Salt Environment*

PubMed Central

Palavicini, Juan Pablo; Correa-Rojas, Rodrigo A.; Rosenthal, Joshua J. C.

2012-01-01

A-to-I RNA editing is particularly common in coding regions of squid mRNAs. Previously, we isolated a squid editing enzyme (sqADAR2) that shows a unique structural feature when compared with other ADAR2 family members: an additional double-stranded RNA (dsRNA) binding domain (dsRBD). Alternative splicing includes or excludes this motif, generating a novel or a conventional variant termed sqADAR2a and sqADAR2b, respectively. The extra dsRBD of sqADAR2a increases its editing activity in vitro. We hypothesized that the high activity is due to an increase in the affinity of the enzyme for dsRNA. This may be important because protein-RNA interactions can be influenced by physical factors. We became particularly interested in analyzing the effects of salt on interactions between sqADAR2 and RNA because squid cells have a ∼3-fold higher ionic strength and proportionally more Cl− than vertebrate cells. To date, in vitro biochemical analyses of adenosine deamination have been conducted using vertebrate-like ionic strength buffers containing chloride as the major anion, although the vast majority of cellular anions are known to be organic. We found that squid-like salt conditions severely impair the binding affinity of conventional ADAR2s for dsRNA, leading to a decrease in nonspecific and site-specific editing activity. Inhibition of editing was mostly due to high Cl− levels and not to the high concentrations of K+, Na+, and organic anions like glutamate. Interestingly, the extra dsRBD in sqADAR2a conferred resistance to the high Cl− levels found in squid neurons. It does so by increasing the affinity of sqADAR2 for dsRNA by 30- or 100-fold in vertebrate-like or squid-like conditions, respectively. Site-directed mutagenesis of squid ADAR2a showed that its increased affinity and editing activity are directly attributable to the RNA binding activity of the extra dsRBD. PMID:22457361

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

PubMed

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

2013-12-20

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

Dynamic landscape and regulation of RNA editing in mammals

PubMed Central

Tan, Meng How; Li, Qin; Shanmugam, Raghuvaran; Piskol, Robert; Kohler, Jennefer; Young, Amy N.; Liu, Kaiwen Ivy; Zhang, Rui; Ramaswami, Gokul; Ariyoshi, Kentaro; Gupte, Ankita; Keegan, Liam P.; George, Cyril X.; Ramu, Avinash; Huang, Ni; Pollina, Elizabeth A.; Leeman, Dena S.; Rustighi, Alessandra; Sharon Goh, Y. P.; Chawla, Ajay; Del Sal, Giannino; Peltz, Gary; Brunet, Anne; Conrad, Donald F.; Samuel, Charles E.; O’Connell, Mary A.; Walkley, Carl R.; Nishikura, Kazuko; Li, Jin Billy

2017-01-01

Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules1. Although many editing sites have recently been discovered2–7, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood8–10. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing. PMID:29022589

Dynamic landscape and regulation of RNA editing in mammals.

PubMed

Tan, Meng How; Li, Qin; Shanmugam, Raghuvaran; Piskol, Robert; Kohler, Jennefer; Young, Amy N; Liu, Kaiwen Ivy; Zhang, Rui; Ramaswami, Gokul; Ariyoshi, Kentaro; Gupte, Ankita; Keegan, Liam P; George, Cyril X; Ramu, Avinash; Huang, Ni; Pollina, Elizabeth A; Leeman, Dena S; Rustighi, Alessandra; Goh, Y P Sharon; Chawla, Ajay; Del Sal, Giannino; Peltz, Gary; Brunet, Anne; Conrad, Donald F; Samuel, Charles E; O'Connell, Mary A; Walkley, Carl R; Nishikura, Kazuko; Li, Jin Billy

2017-10-11

Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.

A genome-wide identification and analysis of the DYW-deaminase genes in the pentatricopeptide repeat gene family in cotton (Gossypium spp.)

PubMed Central

Liu, Guoyuan; Li, Xue; Guo, Liping; Zhang, Xuexian; Qi, Tingxiang; Wang, Hailin; Tang, Huini; Qiao, Xiuqin; Zhang, Jinfa; Xing, Chaozhu; Wu, Jianyong

2017-01-01

The RNA editing occurring in plant organellar genomes mainly involves the change of cytidine to uridine. This process involves a deamination reaction, with cytidine deaminase as the catalyst. Pentatricopeptide repeat (PPR) proteins with a C-terminal DYW domain are reportedly associated with cytidine deamination, similar to members of the deaminase superfamily. PPR genes are involved in many cellular functions and biological processes including fertility restoration to cytoplasmic male sterility (CMS) in plants. In this study, we identified 227 and 211 DYW deaminase-coding PPR genes for the cultivated tetraploid cotton species G. hirsutum and G. barbadense (2n = 4x = 52), respectively, as well as 126 and 97 DYW deaminase-coding PPR genes in the ancestral diploid species G. raimondii and G. arboreum (2n = 26), respectively. The 227 G. hirsutum PPR genes were predicted to encode 52–2016 amino acids, 203 of which were mapped onto 26 chromosomes. Most DYW deaminase genes lacked introns, and their proteins were predicted to target the mitochondria or chloroplasts. Additionally, the DYW domain differed from the complete DYW deaminase domain, which contained part of the E domain and the entire E+ domain. The types and number of DYW tripeptides may have been influenced by evolutionary processes, with some tripeptides being lost. Furthermore, a gene ontology analysis revealed that DYW deaminase functions were mainly related to binding as well as hydrolase and transferase activities. The G. hirsutum DYW deaminase expression profiles varied among different cotton tissues and developmental stages, and no differentially expressed DYW deaminase-coding PPRs were directly associated with the male sterility and restoration in the CMS-D2 system. Our current study provides an important piece of information regarding the structural and evolutionary characteristics of Gossypium DYW-containing PPR genes coding for deaminases and will be useful for characterizing the DYW deaminase gene

Metabolite signatures in hydrophilic extracts of mouse lungs exposed to cigarette smoke revealed by 1H NMR metabolomics investigation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Jian Z.; Wang, Xuan; Feng, Ju

Herein, 1H-NMR metabolomics are carried out to evaluate the changes of metabolites in lungs of mice exposed to cigarette smoke. It is found that the concentrations of adenosine derivatives (i.e. ATP, ADP and AMP), inosine and uridine are significantly fluctuated in the lungs of mice exposed to cigarette smoke compared with those of controls regardless the mouse is obese or regular weight. The decreased ATP, ADP, AMP and elevated inosine predict that the deaminases in charge of adenosine derivatives to inosine derivatives conversion are altered in lungs of mice exposed to cigarette smoke. Transcriptional analysis reveals that the concentrations ofmore » adenosine monophosphate deaminase and adenosine deaminase are different in the lungs of mice exposed to cigarette smoke, confirming the prediction from metabolomics studies. We also found, for the first time, that the ratio of glycerophosphocholine (GPC) to phosphocholine (PC) is significantly increased in the lungs of obese mice compared with regular weight mice. The ratio of GPC/PC is further elevated in the lungs of obese group by cigarette smoke exposure. Since GPC/PC ratio is a known biomarker for cancer, these results may suggest that obese group is more susceptible to lung cancer when exposed to cigarette smoke.« less

Metabolite signatures in hydrophilic extracts of mouse lungs exposed to cigarette smoke revealed by 1H NMR metabolomics investigation

DOE PAGES

Hu, Jian Z.; Wang, Xuan; Feng, Ju; ...

2015-05-12

Herein, 1H-NMR metabolomics are carried out to evaluate the changes of metabolites in lungs of mice exposed to cigarette smoke. It is found that the concentrations of adenosine derivatives (i.e. ATP, ADP and AMP), inosine and uridine are significantly fluctuated in the lungs of mice exposed to cigarette smoke compared with those of controls regardless the mouse is obese or regular weight. The decreased ATP, ADP, AMP and elevated inosine predict that the deaminases in charge of adenosine derivatives to inosine derivatives conversion are altered in lungs of mice exposed to cigarette smoke. Transcriptional analysis reveals that the concentrations ofmore » adenosine monophosphate deaminase and adenosine deaminase are different in the lungs of mice exposed to cigarette smoke, confirming the prediction from metabolomics studies. We also found, for the first time, that the ratio of glycerophosphocholine (GPC) to phosphocholine (PC) is significantly increased in the lungs of obese mice compared with regular weight mice. The ratio of GPC/PC is further elevated in the lungs of obese group by cigarette smoke exposure. Since GPC/PC ratio is a known biomarker for cancer, these results may suggest that obese group is more susceptible to lung cancer when exposed to cigarette smoke.« less

Adenosine receptor desensitization and trafficking.

PubMed

Mundell, Stuart; Kelly, Eamonn

2011-05-01

As with the majority of G-protein-coupled receptors, all four of the adenosine receptor subtypes are known to undergo agonist-induced regulation in the form of desensitization and trafficking. These processes can limit the ability of adenosine receptors to couple to intracellular signalling pathways and thus reduce the ability of adenosine receptor agonists as well as endogenous adenosine to produce cellular responses. In addition, since adenosine receptors couple to multiple signalling pathways, these pathways may desensitize differentially, while the desensitization of one pathway could even trigger signalling via another. Thus, the overall picture of adenosine receptor regulation can be complex. For all adenosine receptor subtypes, there is evidence to implicate arrestins in agonist-induced desensitization and trafficking, but there is also evidence for other possible forms of regulation, including second messenger-dependent kinase regulation, heterologous effects involving G proteins, and the involvement of non-clathrin trafficking pathways such as caveolae. In this review, the evidence implicating these mechanisms is summarized for each adenosine receptor subtype, and we also discuss those issues of adenosine receptor regulation that remain to be resolved as well as likely directions for future research in this field. Copyright © 2010 Elsevier B.V. All rights reserved.

AMP and adenosine are both ligands for adenosine 2B receptor signaling.

PubMed

Holien, Jessica K; Seibt, Benjamin; Roberts, Veena; Salvaris, Evelyn; Parker, Michael W; Cowan, Peter J; Dwyer, Karen M

2018-01-15

Adenosine is considered the canonical ligand for the adenosine 2B receptor (A 2B R). A 2B R is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A 2B R signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A 2B R. The computational modeling suggested that AMP interacts with more favorable energy to A 2B R compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A 2B R, indicating preferential signaling via the G q pathway. Therefore, a putative AMP-A 2B R interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential G q activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI. Copyright © 2017 Elsevier Ltd. All rights reserved.

Extracellular formation and uptake of adenosine during skeletal muscle contraction in the rat: role of adenosine transporters

PubMed Central

Lynge, J; Juel, C; Hellsten, Y

2001-01-01

The existence of adenosine transporters in plasma membrane giant vesicles from rat skeletal muscles and in primary skeletal muscle cell cultures was investigated. In addition, the contribution of intracellularly or extracellularly formed adenosine to the overall extracellular adenosine concentration during muscle contraction was determined in primary skeletal muscle cell cultures. In plasma membrane giant vesicles, the carrier-mediated adenosine transport demonstrated saturation kinetics with Km= 177 ± 36 μm and Vmax= 1.9 ± 0.2 nmol ml−1 s−1 (0.7 nmol (mg protein)−1 s−1). The existence of an adenosine transporter was further evidenced by the inhibition of the carrier-mediated adenosine transport in the presence of NBMPR (nitrobenzylthioinosine; 72 % inhibition) or dipyridamol (64 % inhibition; P < 0.05). In primary skeletal muscle cells, the rate of extracellular adenosine accumulation was 5-fold greater (P < 0.05) with electrical stimulation than without electrical stimulation. Addition of the adenosine transporter inhibitor NBMPR led to a 57 % larger (P < 0.05) rate of extracellular adenosine accumulation in the electro-stimulated muscle cells compared with control cells, demonstrating that adenosine is taken up by the skeletal muscle cells during contractions. Inhibition of ecto-5′-nucleotidase with AOPCP in electro-stimulated cells resulted in a 70 % lower (P < 0.05) rate of extracellular adenosine accumulation compared with control cells, indicating that adenosine to a large extent is formed in the extracellular space during contraction. The present study provides evidence for the existence of an NBMPR-sensitive adenosine transporter in rat skeletal muscle. Our data furthermore demonstrate that the increase in extracellular adenosine observed during electro-stimulation of skeletal muscle is due to production of adenosine in the extracellular space of skeletal muscle and that adenosine is taken up rather than released by the skeletal muscle cells

ADA genetic polymorphism and the effect of smoking on neonatal bilirubinemia and developmental parameters.

PubMed

Gloria-Bottini, F; Magrini, A; Cozzoli, E; Bergamaschi, A; Bottini, E

2008-11-01

Genetic variability of metabolic enzymes may influence the effect of cigarette smoking on intrauterine development and on early neonatal events. To investigate the role of adenosine deaminase genetic polymorphism on the effect of smoking on neonatal bilirubinemia and developmental parameters. Analysis of association between adenosine deaminase phenotypes and neonatal developmental parameters. Prospective study of serum bilirubin level in relation to adenosine deaminase phenotype. We have studied 360 consecutive newborn infants from the Caucasian population of Rome. Serum bilirubin concentration was determined at birth and every 24 h for the first five days. Overall maternal smoking is associated with a slight decrease in the incidence of phototherapy (13.4% in non smoking vs 11.7% in smoking mothers) and with a reduction of birth weight (3374 g in non smoking mothers vs 3133 g in smoking mothers). There is a significant interaction between smoke and adenosine deaminase. While in non smoking mothers the incidence of phototherapy in carriers of ADA 2 allele is higher than in ADA 1 phenotype, in infants from smoking mothers the pattern is reversed and the incidence of phototherapy in carriers of ADA 2 allele is lower than in infants with ADA 1 phenotype. Other neonatal bilirubin parameters follow a similar pattern of interaction between smoking and ADA. The negative effect of smoke on birth weight is much more evident in infant with ADA 1 phenotype than in those carrying the ADA 2 allele. The data suggest that ADA phenotype modifies the effect of smoking on developmental and bilirubin parameters.

Performance comparison between multienzymes loaded single and dual electrodes for the simultaneous electrochemical detection of adenosine and metabolites in cancerous cells.

PubMed

Hussain, Khalil K; Akhtar, Mahmood H; Kim, Moo-Hyun; Jung, Dong-Keun; Shim, Yoon-Bo

2018-06-30

The analytical performance of the multi enzymes loaded single electrode sensor (SES) and dual electrode sensor (DES) was compared for the detection of adenosine and metabolites. The SES was fabricated by covalent binding of tri-enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and xanthine oxidase (XO) along with hydrazine (Hyd) onto a functionalized conducting polymer [2,2:5,2-terthiophene-3-(p-benzoic acid)] (pTTBA). The enzyme reaction electrode in DES was fabricated by covalent binding of ADA and PNP onto pTTBA coated on Au nanoparticles. The detection electrode in DES was constructed by covalent binding of XO and Hyd onto pTTBA coated on porous Au. Due to the higher amount (3.5 folds) of the immobilized enzymes and Hyd onto the DES than SES, and the lower Michaelis constant (Km) value for DES (28.7 µM) compared to SES (36.1 µM), the sensitivity was significantly enhanced for the DES (8.2 folds). The dynamic range obtained using DES was from 0.5 nM to 120.0 µM with a detection limit of 1.43 nM ± 0.02, 0.76 nM ± 0.02, and 0.48 nM ± 0.01, for adenosine (AD), inosine (IN), and hypoxanthine (Hypo) respectively. Further, the DES was coupled with an electrochemical potential modulated microchannel for the separation and simultaneous detection of AD, IN, and Hypo in an extracellular matrix of cancerous (A549) and non-cancerous (Vero) cells. The sensor probe confirms a higher basal level of extracellular AD and its metabolites in cancer cells compared to normal cells. In addition, the effect of dipyridamole on released adenosine in A549 cells was investigated. Copyright © 2018 Elsevier B.V. All rights reserved.

Detection of canonical A-to-G editing events at 3′ UTRs and microRNA target sites in human lungs using next-generation sequencing

PubMed Central

Soundararajan, Ramani; Stearns, Timothy M.; Griswold, Anthony J.; Mehta, Arpit; Czachor, Alexander; Fukumoto, Jutaro; Lockey, Richard F.; King, Benjamin L.; Kolliputi, Narasaiah

2015-01-01

RNA editing is a post-transcriptional modification of RNA. The majority of these changes result from adenosine deaminase acting on RNA (ADARs) catalyzing the conversion of adenosine residues to inosine in double-stranded RNAs (dsRNAs). Massively parallel sequencing has enabled the identification of RNA editing sites in human transcriptomes. In this study, we sequenced DNA and RNA from human lungs and identified RNA editing sites with high confidence via a computational pipeline utilizing stringent analysis thresholds. We identified a total of 3,447 editing sites that overlapped in three human lung samples, and with 50% of these sites having canonical A-to-G base changes. Approximately 27% of the edited sites overlapped with Alu repeats, and showed A-to-G clustering (>3 clusters in 100 bp). The majority of edited sites mapped to either 3′ untranslated regions (UTRs) or introns close to splice sites; whereas, only few sites were in exons resulting in non-synonymous amino acid changes. Interestingly, we identified 652 A-to-G editing events in the 3′ UTR of 205 target genes that mapped to 932 potential miRNA target binding sites. Several of these miRNA edited sites were validated in silico. Additionally, we validated several A-to-G edited sites by Sanger sequencing. Altogether, our study suggests a role for RNA editing in miRNA-mediated gene regulation and splicing in human lungs. In this study, we have generated a RNA editome of human lung tissue that can be compared with other RNA editomes across different lung tissues to delineate a role for RNA editing in normal and diseased states. PMID:26486088

Detection of canonical A-to-G editing events at 3' UTRs and microRNA target sites in human lungs using next-generation sequencing.

PubMed

Soundararajan, Ramani; Stearns, Timothy M; Griswold, Anthony L; Mehta, Arpit; Czachor, Alexander; Fukumoto, Jutaro; Lockey, Richard F; King, Benjamin L; Kolliputi, Narasaiah

2015-11-03

RNA editing is a post-transcriptional modification of RNA. The majority of these changes result from adenosine deaminase acting on RNA (ADARs) catalyzing the conversion of adenosine residues to inosine in double-stranded RNAs (dsRNAs). Massively parallel sequencing has enabled the identification of RNA editing sites in human transcriptomes. In this study, we sequenced DNA and RNA from human lungs and identified RNA editing sites with high confidence via a computational pipeline utilizing stringent analysis thresholds. We identified a total of 3,447 editing sites that overlapped in three human lung samples, and with 50% of these sites having canonical A-to-G base changes. Approximately 27% of the edited sites overlapped with Alu repeats, and showed A-to-G clustering (>3 clusters in 100 bp). The majority of edited sites mapped to either 3' untranslated regions (UTRs) or introns close to splice sites; whereas, only few sites were in exons resulting in non-synonymous amino acid changes. Interestingly, we identified 652 A-to-G editing events in the 3' UTR of 205 target genes that mapped to 932 potential miRNA target binding sites. Several of these miRNA edited sites were validated in silico. Additionally, we validated several A-to-G edited sites by Sanger sequencing. Altogether, our study suggests a role for RNA editing in miRNA-mediated gene regulation and splicing in human lungs. In this study, we have generated a RNA editome of human lung tissue that can be compared with other RNA editomes across different lung tissues to delineate a role for RNA editing in normal and diseased states.

Carbamazepine and oxcarbazepine, but not eslicarbazepine, enhance excitatory synaptic transmission onto hippocampal CA1 pyramidal cells through an antagonist action at adenosine A1 receptors.

PubMed

Booker, Sam A; Pires, Nuno; Cobb, Stuart; Soares-da-Silva, Patrício; Vida, Imre

2015-06-01

This study assessed the anticonvulsant and seizure generation effects of carbamazepine (CBZ), oxcarbazepine (OXC) and eslicarbazepine (S-Lic) in wild-type mice. Electrophysiological recordings were made to discriminate potential cellular and synaptic mechanisms underlying anti- and pro-epileptic actions. The anticonvulsant and pro-convulsant effects were evaluated in the MES, the 6-Hz and the Irwin tests. Whole-cell patch-clamp recordings were used to investigate the effects on fast excitatory and inhibitory synaptic transmission in hippocampal area CA1. The safety window for CBZ, OXC and eslicarbazepine (ED50 value against the MES test and the dose that produces grade 5 convulsions in all mice), was 6.3, 6.0 and 12.5, respectively. At high concentrations the three drugs reduced synaptic transmission. CBZ and OXC enhanced excitatory postsynaptic currents (EPSCs) at low, therapeutically-relevant concentrations. These effects were associated with no change in inhibitory postsynaptic currents (IPSCs) resulting in altered balance between excitation and inhibition. S-Lic had no effect on EPSC or IPSC amplitudes over the same concentration range. The CBZ mediated enhancement of EPSCs was blocked by DPCPX, a selective antagonist, and occluded by CCPA, a selective agonist of the adenosine A1 receptor. Furthermore, reduction of endogenous adenosine by application of the enzyme adenosine deaminase also abolished the CBZ- and OXC-induced increase of EPSCs, indicating that the two drugs act as antagonists at native adenosine receptors. In conclusion, CBZ and OXC possess pro-epileptic actions at clinically-relevant concentrations through the enhancement of excitatory synaptic transmission. S-Lic by comparison has no such effect on synaptic transmission, explaining its lack of seizure exacerbation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Wireless Instantaneous Neurotransmitter Concentration System–based amperometric detection of dopamine, adenosine, and glutamate for intraoperative neurochemical monitoring

PubMed Central

Agnesi, Filippo; Tye, Susannah J.; Bledsoe, Jonathan M.; Griessenauer, Christoph J.; Kimble, Christopher J.; Sieck, Gary C.; Bennet, Kevin E.; Garris, Paul A.; Blaha, Charles D.; Lee, Kendall H.

2009-01-01

Object In a companion study, the authors describe the development of a new instrument named the Wireless Instantaneous Neurotransmitter Concentration System (WINCS), which couples digital telemetry with fast-scan cyclic voltammetry (FSCV) to measure extracellular concentrations of dopamine. In the present study, the authors describe the extended capability of the WINCS to use fixed potential amperometry (FPA) to measure extracellular concentrations of dopamine, as well as glutamate and adenosine. Compared with other electrochemical techniques such as FSCV or high-speed chronoamperometry, FPA offers superior temporal resolution and, in combination with enzyme-linked biosensors, the potential to monitor nonelectroactive analytes in real time. Methods The WINCS design incorporated a transimpedance amplifier with associated analog circuitry for FPA; a microprocessor; a Bluetooth transceiver; and a single, battery-powered, multilayer, printed circuit board. The WINCS was tested with 3 distinct recording electrodes: 1) a carbon-fiber microelectrode (CFM) to measure dopamine; 2) a glutamate oxidase enzyme-linked electrode to measure glutamate; and 3) a multiple enzyme-linked electrode (adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase) to measure adenosine. Proof-of-principle analyses included noise assessments and in vitro and in vivo measurements that were compared with similar analyses by using a commercial hardwired electrochemical system (EA161 Picostat, eDAQ; Pty Ltd). In urethane-anesthetized rats, dopamine release was monitored in the striatum following deep brain stimulation (DBS) of ascending dopaminergic fibers in the medial forebrain bundle (MFB). In separate rat experiments, DBS-evoked adenosine release was monitored in the ventrolateral thalamus. To test the WINCS in an operating room setting resembling human neurosurgery, cortical glutamate release in response to motor cortex stimulation (MCS) was monitored using a large-mammal animal

Wireless Instantaneous Neurotransmitter Concentration System-based amperometric detection of dopamine, adenosine, and glutamate for intraoperative neurochemical monitoring.

PubMed

Agnesi, Filippo; Tye, Susannah J; Bledsoe, Jonathan M; Griessenauer, Christoph J; Kimble, Christopher J; Sieck, Gary C; Bennet, Kevin E; Garris, Paul A; Blaha, Charles D; Lee, Kendall H

2009-10-01

In a companion study, the authors describe the development of a new instrument named the Wireless Instantaneous Neurotransmitter Concentration System (WINCS), which couples digital telemetry with fast-scan cyclic voltammetry (FSCV) to measure extracellular concentrations of dopamine. In the present study, the authors describe the extended capability of the WINCS to use fixed potential amperometry (FPA) to measure extracellular concentrations of dopamine, as well as glutamate and adenosine. Compared with other electrochemical techniques such as FSCV or high-speed chronoamperometry, FPA offers superior temporal resolution and, in combination with enzyme-linked biosensors, the potential to monitor nonelectroactive analytes in real time. The WINCS design incorporated a transimpedance amplifier with associated analog circuitry for FPA; a microprocessor; a Bluetooth transceiver; and a single, battery-powered, multilayer, printed circuit board. The WINCS was tested with 3 distinct recording electrodes: 1) a carbon-fiber microelectrode (CFM) to measure dopamine; 2) a glutamate oxidase enzyme-linked electrode to measure glutamate; and 3) a multiple enzyme-linked electrode (adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase) to measure adenosine. Proof-of-principle analyses included noise assessments and in vitro and in vivo measurements that were compared with similar analyses by using a commercial hardwired electrochemical system (EA161 Picostat, eDAQ; Pty Ltd). In urethane-anesthetized rats, dopamine release was monitored in the striatum following deep brain stimulation (DBS) of ascending dopaminergic fibers in the medial forebrain bundle (MFB). In separate rat experiments, DBS-evoked adenosine release was monitored in the ventrolateral thalamus. To test the WINCS in an operating room setting resembling human neurosurgery, cortical glutamate release in response to motor cortex stimulation (MCS) was monitored using a large-mammal animal model, the pig. The

Potentiation of adenosine triphosphate-induced contractile responses of the guinea-pig isolated vas deferens by adenosine monophosphate and adenosine 5'-monophosphorothioate.

PubMed Central

Fedan, J. S.

1987-01-01

The effects of incubating the guinea-pig isolated vas deferens in the presence of adenine nucleotides (adenosine triphosphate, ATP; adenosine diphosphate, ADP; and adenosine monophosphate, AMP), or in the presence of their phosphorothioate analogues (adenosine 5'-O-(3-thiotriphosphate), ATP gamma S; adenosine 5'-O-(2-thiodiphosphate), ADP beta S; and adenosine 5'-monophosphorothioate, AMP alpha S), on contractile responses to ATP were compared. After challenge with a low (1 microM) or high (300 microM) concentration of ATP to obtain control responses, one vas deferens of a pair was incubated for 5 min with one of the adenine nucleotides, while the contralateral preparation was incubated with the corresponding phosphorothioate analogue. At the conclusion of the incubation the preparations were challenged again with ATP. Incubation with AMP or AMP alpha S resulted in a transient potentiation of responses to 1 microM and 300 microM ATP. The potentiation following incubation with AMP alpha S was larger than that produced by AMP. After incubation with ADP, ADP beta S, ATP and ATP gamma S, responses to 1 microM ATP were decreased, while those to 300 microM ATP were unaffected. Thus, incubation with AMP and AMP alpha S results in potentiation, rather than inhibition, of ATP-induced responses. On the other hand, 5'-diphosphate, 5'-triphosphate, 5'-O-(2-thiodiphosphate) and 5'-O-(3-thiotriphosphate) moieties on adenosine have no effect or cause autoinhibition. These results indicate that AMP exerts a potentiating effect on reactivity to exogenous ATP. AMP arising from the enzymatic degradation of ATP might modulate the level of response to ATP released endogenously as a cotransmitter. PMID:3038248

Replication protein A (RPA) hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA.

PubMed

Lada, Artem G; Waisertreiger, Irina S-R; Grabow, Corinn E; Prakash, Aishwarya; Borgstahl, Gloria E O; Rogozin, Igor B; Pavlov, Youri I

2011-01-01

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

The Role of cGMP on Adenosine A1 Receptor-mediated Inhibition of Synaptic Transmission at the Hippocampus

PubMed Central

Pinto, Isa; Serpa, André; Sebastião, Ana M.; Cascalheira, José F.

2016-01-01

Both adenosine A1 receptor and cGMP inhibit synaptic transmission at the hippocampus and recently it was found that A1 receptor increased cGMP levels in hippocampus, but the role of cGMP on A1 receptor-mediated inhibition of synaptic transmission remains to be established. In the present work we investigated if blocking the NOS/sGC/cGMP/PKG pathway using nitric oxide synthase (NOS), protein kinase G (PKG), and soluble guanylyl cyclase (sGC) inhibitors modify the A1 receptor effect on synaptic transmission. Neurotransmission was evaluated by measuring the slope of field excitatory postsynaptic potentials (fEPSPs) evoked by electrical stimulation at hippocampal slices. N6-cyclopentyladenosine (CPA, 15 nM), a selective A1 receptor agonist, reversibly decreased the fEPSPs by 54 ± 5%. Incubation of the slices with an inhibitor of NOS (L-NAME, 200 μM) decreased the CPA effect on fEPSPs by 57 ± 9% in female rats. In males, ODQ (10 μM), an sGC inhibitor, decreased the CPA inhibitory effect on fEPSPs by 23 ± 6%, but only when adenosine deaminase (ADA,1 U/ml) was present; similar results were found in females, where ODQ decreased CPA-induced inhibition of fEPSP slope by 23 ± 7%. In male rats, the presence of the PKG inhibitor (KT5823, 1 nM) decreased the CPA effect by 45.0 ± 9%; similar results were obtained in females, where KT5823 caused a 32 ± 9% decrease on the CPA effect. In conclusion, the results suggest that the inhibitory action of adenosine A1 receptors on synaptic transmission at hippocampus is, in part, mediated by the NOS/sGC/cGMP/PKG pathway. PMID:27148059

Adenosine-Associated Delivery Systems

PubMed Central

Kazemzadeh-Narbat, Mehdi; Annabi, Nasim; Tamayol, Ali; Oklu, Rahmi; Ghanem, Amyl; Khademhosseini, Ali

2016-01-01

Adenosine is a naturally occurring purine nucleoside in every cell. Many critical treatments such as modulating irregular heartbeat (arrhythmias), regulation of central nervous system (CNS) activity, and inhibiting seizural episodes can be carried out using adenosine. Despite the significant potential therapeutic impact of adenosine and its derivatives, the severe side effects caused by their systemic administration have significantly limited their clinical use. In addition, due to adenosine’s extremely short half-life in human blood (less than 10 s), there is an unmet need for sustained delivery systems to enhance efficacy and reduce side effects. In this paper, various adenosine delivery techniques, including encapsulation into biodegradable polymers, cell-based delivery, implantable biomaterials, and mechanical-based delivery systems, are critically reviewed and the existing challenges are highlighted. PMID:26453156

Improving the accuracy of synovial fluid analysis in the diagnosis of prosthetic joint infection with simple and inexpensive biomarkers: C-reactive protein and adenosine deaminase.

PubMed

Sousa, R; Serrano, P; Gomes Dias, J; Oliveira, J C; Oliveira, A

2017-03-01

The aims of this study were to increase the diagnostic accuracy of the analysis of synovial fluid in the differentiation of prosthetic joint infection (PJI) by the addition of inexpensive biomarkers such as the levels of C-reactive protein (CRP), adenosine deaminase (ADA), alpha-2-macrogloblulin (α2M) and procalcitonin. Between January 2013 and December 2015, synovial fluid and removed implants were requested from 143 revision total joint arthroplasties. A total of 55 patients met inclusion criteria of the receipt of sufficient synovial fluid, tissue samples and removed implants for analysis. The diagnosis of PJI followed the definition from a recent International Consensus Meeting to create two groups of patients; septic and aseptic. Using receiver operating characteristic curves we determined the cutoff values and diagnostic accuracy for each marker. There were 23 PJIs and 32 patients with aseptic loosening. The levels of total leucocyte count, proportion of polymorphonuclear leucocytes (PMNs), CRP, ADA and α2M in the synovial fluid were all significantly higher in those with a PJI than in those with aseptic loosening. The levels of procalcitonin were comparable in the two groups. Cutoff values for the optimal performance in the diagnosis of infection were: total leucocyte count > 1463 cells/μL (sensitivity (Sens) 100%, specificity (Spec) 71.9%, positive predictive value (PPV) 71.9%, negative predictive value (NPV) 100%); proportion of PMNs > 81% (Sens 78.3%, Spec 75.0%, PPV 69.2%, NPV 82.8%); CRP > 6.7mg/L (Sens 78.3%, Spec 93.8%, PPV 90.0%, NPV 85.7%); ADA > 61U/L (Sens 78.3%, Spec 96.9%, PPV 94.7%, NPV 86.1%) and α2M > 958 mg/L (Sens 47.8%, Spec 96.9%, PPV 91.7%, NPV 72.1%). The addition of a raised level of CRP or ADA to the total leukocyte count increased the specificity: total leukocyte count > 1463 cells/μL and CRP > 6.7mg/L (Sens 78.3%, Spec 100%, PPV 100%, NPV 86.5%) or with ADA > 61U/L (Sens 78.3%, Spec 96.9%, PPV 94.7%, NPV 86.1%). The total

Time-resolved photoelectron spectroscopy of adenosine and adenosine monophosphate photodeactivation dynamics in water microjets

NASA Astrophysics Data System (ADS)

Williams, Holly L.; Erickson, Blake A.; Neumark, Daniel M.

2018-05-01

The excited state relaxation dynamics of adenosine and adenosine monophosphate were studied at multiple excitation energies using femtosecond time-resolved photoelectron spectroscopy in a liquid water microjet. At pump energies of 4.69-4.97 eV, the lowest ππ* excited state, S1, was accessed and its decay dynamics were probed via ionization at 6.20 eV. By reversing the role of the pump and probe lasers, a higher-lying ππ* state was excited at 6.20 eV and its time-evolving photoelectron spectrum was monitored at probe energies of 4.69-4.97 eV. The S1 ππ* excited state was found to decay with a lifetime ranging from ˜210 to 250 fs in adenosine and ˜220 to 250 fs in adenosine monophosphate. This lifetime drops with increasing pump photon energy. Signal from the higher-lying ππ* excited state decayed on a time scale of ˜320 fs and was measureable only in adenosine monophosphate.

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

PubMed

Dieni, Christopher A; Storey, Kenneth B

2008-04-22

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. 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 approximately 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 degrees C) and by the presence/absence of the natural cryoprotectant (250 mM glucose) that accumulates during freezing

Molecular Recognition of 6′-N-5-Hexynoate Kanamyin A and RNA 1×1 Internal Loops Containing CA Mismatches

PubMed Central

Tran, Tuan; Disney, Matthew D.

2011-01-01

In our previous study to identify the RNA internal loops that bind an aminoglycoside derivative, we determined that 6′-N-5-hexynoate kanamycin A prefers to bind 1×1 nucleotide internal loops containing C•A mismatches. In this present study, the molecular recognition between a variety of RNAs that are mutated around the C•A loop and the ligand was investigated. Studies show that both loop nucleotides and loop closing pairs affect binding affinity. Most interestingly, it was shown that there is a correlation between the thermodynamic stability of the C•A internal loops and ligand affinity. Specifically, C•A loops that had relatively high or low stability bound the ligand most weakly whereas loops with intermediate stability bound the ligand most tightly. In contrast, there is no correlation between the likelihood that a loop forms a C-A+ pair at lower pH and ligand affinity. It was also found that a 1×1 nucleotide C•A loop that bound to the ligand with the highest affinity is identical to the consensus site in RNAs that are edited by adenosine deaminases acting on RNA type 2 (ADAR2). These studies provide a detailed investigation of factors affecting small molecule recognition of internal loops containing C•A mismatches, which are present in a variety of RNAs that cause disease. PMID:21207945

Downregulation of adenosine and adenosine 1 receptor contributes to neuropathic pain in resiniferatoxin neuropathy.

PubMed

Kan, Hung-Wei; Chang, Chin-Hong; Lin, Chih-Lung; Lee, Yi-Chen; Hsieh, Sung-Tsang; Hsieh, Yu-Lin

2018-04-16

The neurochemical effects of adenosine signaling in small-fiber neuropathy leading to neuropathic pain are yet to be explored in a direct manner. This study examined this system at the level of ligand (via the ectonucleotidase activity of prostatic acid phosphatase, PAP) and adenosine A1 receptors (A1Rs) in resiniferatoxin (RTX) neuropathy, a peripheral neurodegenerative disorder which specifically affects nociceptive nerves expressing transient receptor potential vanilloid type 1 (TRPV1). We conducted immunohistochemistry on dorsal root ganglion neurons (DRG), high-performance liquid chromatography (HPLC) for functional assays, and pharmacological interventions to alter PAP and A1Rs in mice with RTX neuropathy. In DRG of RTX neuropathy, PAP(+) neurons were reduced compared with vehicle-treated mice (P = 0.002) . Functionally, PAP ectonucleotidase activity was consequently reduced (i.e., the content of adenosine in DRG, P = 0.012). PAP(+) neuronal density was correlated with the degree of mechanical allodynia, which was reversed by intrathecal lumbar puncture (i.t.) injection of recombinant PAP with a dose-dependent effect. Furthermore, A1Rs were downregulated (P = 0.002), and this downregulation was colocalized with the TRPV1 receptor (31.0% ± 2.8%). Mechanical allodynia was attenuated in a dose-dependent response by i.t. injection of the A1R ligand, adenosine; however, no analgesia was evident when an exogenous adenosine was blocked by A1R antagonist. This study demonstrated dual mechanisms of neuropathic pain in TRPV1-induced neuropathy, involving a reduced adenosine system at both the ligand (adenosine) and receptor (A1Rs) levels.

Adenosine and sleep

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yanik, G.M. Jr.

Behavioral and biochemical approaches have been used to determine the relative contribution of endogenous adenosine and adenosine receptors to the sleep-wake cycle in the rat. Adenosine concentrations in specific areas of the rat brain were not affected by 24 hours of total sleep deprivation, or by 24 or 48 hours of REM sleep deprivation. In order to assess the effect of REM sleep deprivation on adenosine A/sub 1/ receptors, /sup 3/H-L-PIA binding was measured. The Bmax values for /sup 3/H-L-PIA binding to membrane preparations of the cortices and corpus striata from 48 hour REM sleep-deprived animals were increased 14.8% andmore » 23%, respectively. These increases were not maintained following the cessation of sleep deprivation and recovered within 2 hours. The results of a 96 hour REM deprivation experiment were similar to those of the 48 hour REM sleep deprivation experiment. However, these increases were not evident in similar structures taken from stress control animals, and conclusively demonstrated that the changes in /sup 3/H-L-PIA binding resulted from REM sleep deprivation and not from stress.« less

Adenosine 5'-monophosphate ameliorates D-galactosamine/lipopolysaccharide-induced liver injury through an adenosine receptor-independent mechanism in mice.

PubMed

Zhan, Y; Wang, Z; Yang, P; Wang, T; Xia, L; Zhou, M; Wang, Y; Wang, S; Hua, Z; Zhang, J

2014-01-09

D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced lethality and acute liver failure is dependent on endogenously produced inflammatory cytokines. Adenosine has been proven to be a central role in the regulation of inflammatory response. It is not entirely clear that which adenosine action is actually crucial to limiting inflammatory tissue destruction. Here we showed that GalN/LPS challenge elevated hepatic adenosine and induced lethality in adenosine receptor-deficient mice with equal efficiency as wild-type mice. In GalN/LPS-treated mice, pretreatment with adenosine 5'-monophosphate (5'-AMP) significantly elevated hepatic adenosine level and reduced mortality through decreasing cytokine and chemokine production. In RAW264.7 cells, 5'-AMP treatment inhibited the production of inflammatory cytokines, which is not mediated through adenosine receptors. 5'-AMP failed to attenuate LPS-induced nuclear factor-κB (NF-κB) p65 nuclear translocation, but reduced LPS-induced recruitment of NF-κB p65 to inflammatory gene promoters and decreased LPS-induced enrichment of H3K4 dimethylation at the tumor necrosis factor-α (TNF-α) promoter, which was involved in 5'-AMP-induced elevation of cellular adenosine and a decline of methylation potential. In vitro biochemical analysis revealed that adenosine directly attenuated recruitment of NF-κB to the TNF-α and interleukin-6 promoters. Our findings demonstrate that 5'-AMP-inhibiting inflammatory response is not mediated by adenosine receptors and it may represent a potential protective agent for amelioration of LPS-induced liver injury.

Adenosine Kinase: Exploitation for Therapeutic Gain

PubMed Central

2013-01-01

Adenosine kinase (ADK; EC 2.7.1.20) is an evolutionarily conserved phosphotransferase that converts the purine ribonucleoside adenosine into 5′-adenosine-monophosphate. This enzymatic reaction plays a fundamental role in determining the tone of adenosine, which fulfills essential functions as a homeostatic and metabolic regulator in all living systems. Adenosine not only activates specific signaling pathways by activation of four types of adenosine receptors but it is also a primordial metabolite and regulator of biochemical enzyme reactions that couple to bioenergetic and epigenetic functions. By regulating adenosine, ADK can thus be identified as an upstream regulator of complex homeostatic and metabolic networks. Not surprisingly, ADK dysfunction is involved in several pathologies, including diabetes, epilepsy, and cancer. Consequently, ADK emerges as a rational therapeutic target, and adenosine-regulating drugs have been tested extensively. In recent attempts to improve specificity of treatment, localized therapies have been developed to augment adenosine signaling at sites of injury or pathology; those approaches include transplantation of stem cells with deletions of ADK or the use of gene therapy vectors to downregulate ADK expression. More recently, the first human mutations in ADK have been described, and novel findings suggest an unexpected role of ADK in a wider range of pathologies. ADK-regulating strategies thus represent innovative therapeutic opportunities to reconstruct network homeostasis in a multitude of conditions. This review will provide a comprehensive overview of the genetics, biochemistry, and pharmacology of ADK and will then focus on pathologies and therapeutic interventions. Challenges to translate ADK-based therapies into clinical use will be discussed critically. PMID:23592612

Pain-relieving prospects for adenosine receptors and ectonucleotidases

PubMed Central

Zylka, Mark J.

2010-01-01

Adenosine receptor agonists have potent antinociceptive effects in diverse preclinical models of chronic pain. In contrast, the efficacy of adenosine or adenosine receptor agonists at treating pain in humans is unclear. Two ectonucleotidases that generate adenosine in nociceptive neurons were recently identified. When injected spinally, these enzymes have long-lasting adenosine A1 receptor (A1R)-dependent antinociceptive effects in inflammatory and neuropathic pain models. Furthermore, recent findings indicate that spinal adenosine A2A receptor activation can enduringly inhibit neuropathic pain symptoms. Collectively, these studies suggest the possibility of treating chronic pain in humans by targeting specific adenosine receptor subtypes in anatomically defined regions with agonists or with ectonucleotidases that generate adenosine. PMID:21236731

Adenosine Deaminase Enhances the Immunogenicity of Human Dendritic Cells from Healthy and HIV-Infected Individuals

PubMed Central

Massanella, Marta; Rodríguez-García, Marta; Blanco, Julià; Gatell, José M.; García, Felipe; Gallart, Teresa; Lluis, Carme; Mallol, Josefa

2012-01-01

ADA is an enzyme implicated in purine metabolism, and is critical to ensure normal immune function. Its congenital deficit leads to severe combined immunodeficiency (SCID). ADA binding to adenosine receptors on dendritic cell surface enables T-cell costimulation through CD26 crosslinking, which enhances T-cell activation and proliferation. Despite a large body of work on the actions of the ecto-enzyme ADA on T-cell activation, questions arise on whether ADA can also modulate dendritic cell maturation. To this end we investigated the effects of ADA on human monocyte derived dendritic cell biology. Our results show that both the enzymatic and non-enzymatic activities of ADA are implicated in the enhancement of CD80, CD83, CD86, CD40 and CCR7 expression on immature dendritic cells from healthy and HIV-infected individuals. These ADA-mediated increases in CD83 and costimulatory molecule expression is concomitant to an enhanced IL-12, IL-6, TNF-α, CXCL8(IL-8), CCL3(MIP1-α), CCL4(MIP-1β) and CCL5(RANTES) cytokine/chemokine secretion both in healthy and HIV-infected individuals and to an altered apoptotic death in cells from HIV-infected individuals. Consistently, ADA-mediated actions on iDCs are able to enhance allogeneic CD4 and CD8-T-cell proliferation, globally yielding increased iDC immunogenicity. Taken together, these findings suggest that ADA would promote enhanced and correctly polarized T-cell responses in strategies targeting asymptomatic HIV-infected individuals. PMID:23240012

A-to-I RNA editing is developmentally regulated and generally adaptive for sexual reproduction in Neurospora crassa

PubMed Central

Li, Yang; Chen, Daipeng; Qi, Zhaomei; Wang, Qinhu; Wang, Jianhua; Jiang, Cong; Xu, Jin-Rong

2017-01-01

Although fungi lack adenosine deaminase acting on RNA (ADAR) enzymes, adenosine to inosine (A-to-I) RNA editing was reported recently in Fusarium graminearum during sexual reproduction. In this study, we profiled the A-to-I editing landscape and characterized its functional and adaptive properties in the model filamentous fungus Neurospora crassa. A total of 40,677 A-to-I editing sites were identified, and approximately half of them displayed stage-specific editing or editing levels at different sexual stages. RNA-sequencing analysis with the Δstc-1 and Δsad-1 mutants confirmed A-to-I editing occurred before ascus development but became more prevalent during ascosporogenesis. Besides fungal-specific sequence and secondary structure preference, 63.5% of A-to-I editing sites were in the coding regions and 81.3% of them resulted in nonsynonymous recoding, resulting in a significant increase in the proteome complexity. Many genes involved in RNA silencing, DNA methylation, and histone modifications had extensive recoding, including sad-1, sms-3, qde-1, and dim-2. Fifty pseudogenes harbor premature stop codons that require A-to-I editing to encode full-length proteins. Unlike in humans, nonsynonymous editing events in N. crassa are generally beneficial and favored by positive selection. Almost half of the nonsynonymous editing sites in N. crassa are conserved and edited in Neurospora tetrasperma. Furthermore, hundreds of them are conserved in F. graminearum and had higher editing levels. Two unknown genes with editing sites conserved between Neurospora and Fusarium were experimentally shown to be important for ascosporogenesis. This study comprehensively analyzed A-to-I editing in N. crassa and showed that RNA editing is stage-specific and generally adaptive, and may be functionally related to repeat induced point mutation and meiotic silencing by unpaired DNA. PMID:28847945

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

PubMed

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

2010-08-01

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

Prevention of adenosine A2A receptor activation diminishes beat-to-beat alternation in human atrial myocytes.

PubMed

Molina, Cristina E; Llach, Anna; Herraiz-Martínez, Adela; Tarifa, Carmen; Barriga, Montserrat; Wiegerinck, Rob F; Fernandes, Jacqueline; Cabello, Nuria; Vallmitjana, Alex; Benitéz, Raúl; Montiel, José; Cinca, Juan; Hove-Madsen, Leif

2016-01-01

Atrial fibrillation (AF) has been associated with increased spontaneous calcium release from the sarcoplasmic reticulum and linked to increased adenosine A2A receptor (A2AR) expression and activation. Here we tested whether this may favor atrial arrhythmogenesis by promoting beat-to-beat alternation and irregularity. Patch-clamp and confocal calcium imaging was used to measure the beat-to-beat response of the calcium current and transient in human atrial myocytes. Responses were classified as uniform, alternating or irregular and stimulation of Gs-protein coupled receptors decreased the frequency where a uniform response could be maintained from 1.0 ± 0.1 to 0.6 ± 0.1 Hz; p < 0.01 for beta-adrenergic receptors and from 1.4 ± 0.1 to 0.5 ± 0.1 Hz; p < 0.05 for A2ARs. The latter was linked to increased spontaneous calcium release and after-depolarizations. Moreover, A2AR activation increased the fraction of non-uniformly responding cells in HL-1 myocyte cultures from 19 ± 3 to 51 ± 9 %; p < 0.02, and electrical mapping in perfused porcine atria revealed that adenosine induced electrical alternans at longer cycle lengths, doubled the fraction of electrodes showing alternation, and increased the amplitude of alternations. Importantly, protein kinase A inhibition increased the highest frequency where uniform responses could be maintained from 0.84 ± 0.12 to 1.86 ± 0.11 Hz; p < 0.001 and prevention of A2AR-activation with exogenous adenosine deaminase selectively increased the threshold from 0.8 ± 0.1 to 1.2 ± 0.1 Hz; p = 0.001 in myocytes from patients with AF. In conclusion, A2AR-activation promotes beat-to-beat irregularities in the calcium transient in human atrial myocytes, and prevention of A2AR activation may be a novel means to maintain uniform beat-to-beat responses at higher beating frequencies in patients with atrial fibrillation.

Adenosine 5′-monophosphate ameliorates D-galactosamine/lipopolysaccharide-induced liver injury through an adenosine receptor-independent mechanism in mice

PubMed Central

Zhan, Y; Wang, Z; Yang, P; Wang, T; Xia, L; Zhou, M; Wang, Y; Wang, S; Hua, Z; Zhang, J

2014-01-01

D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced lethality and acute liver failure is dependent on endogenously produced inflammatory cytokines. Adenosine has been proven to be a central role in the regulation of inflammatory response. It is not entirely clear that which adenosine action is actually crucial to limiting inflammatory tissue destruction. Here we showed that GalN/LPS challenge elevated hepatic adenosine and induced lethality in adenosine receptor-deficient mice with equal efficiency as wild-type mice. In GalN/LPS-treated mice, pretreatment with adenosine 5′-monophosphate (5′-AMP) significantly elevated hepatic adenosine level and reduced mortality through decreasing cytokine and chemokine production. In RAW264.7 cells, 5′-AMP treatment inhibited the production of inflammatory cytokines, which is not mediated through adenosine receptors. 5′-AMP failed to attenuate LPS-induced nuclear factor-κB (NF-κB) p65 nuclear translocation, but reduced LPS-induced recruitment of NF-κB p65 to inflammatory gene promoters and decreased LPS-induced enrichment of H3K4 dimethylation at the tumor necrosis factor-α (TNF-α) promoter, which was involved in 5′-AMP-induced elevation of cellular adenosine and a decline of methylation potential. In vitro biochemical analysis revealed that adenosine directly attenuated recruitment of NF-κB to the TNF-α and interleukin-6 promoters. Our findings demonstrate that 5′-AMP-inhibiting inflammatory response is not mediated by adenosine receptors and it may represent a potential protective agent for amelioration of LPS-induced liver injury. PMID:24407238

AMP is an adenosine A1 receptor agonist.

PubMed

Rittiner, Joseph E; Korboukh, Ilia; Hull-Ryde, Emily A; Jin, Jian; Janzen, William P; Frye, Stephen V; Zylka, Mark J

2012-02-17

Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5'-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5'-monophosphonate, ACP) directly activated the adenosine A(1) receptor (A(1)R). In contrast, AMP only activated the adenosine A(2B) receptor (A(2B)R) after hydrolysis to adenosine by ecto-5'-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A(1)R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A(1)R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A(1)R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A(1)R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine.

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

PubMed

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

2008-05-01

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

Caffeine and adenosine.

PubMed

Ribeiro, Joaquim A; Sebastião, Ana M

2010-01-01

Caffeine causes most of its biological effects via antagonizing all types of adenosine receptors (ARs): A1, A2A, A3, and A2B and, as does adenosine, exerts effects on neurons and glial cells of all brain areas. In consequence, caffeine, when acting as an AR antagonist, is doing the opposite of activation of adenosine receptors due to removal of endogenous adenosinergic tonus. Besides AR antagonism, xanthines, including caffeine, have other biological actions: they inhibit phosphodiesterases (PDEs) (e.g., PDE1, PDE4, PDE5), promote calcium release from intracellular stores, and interfere with GABA-A receptors. Caffeine, through antagonism of ARs, affects brain functions such as sleep, cognition, learning, and memory, and modifies brain dysfunctions and diseases: Alzheimer's disease, Parkinson's disease, Huntington's disease, Epilepsy, Pain/Migraine, Depression, Schizophrenia. In conclusion, targeting approaches that involve ARs will enhance the possibilities to correct brain dysfunctions, via the universally consumed substance that is caffeine.

[Diagnostic performance of T-SPOT.TB on peripheral blood in combination with adenosine deaminase on pleural fluid for the diagnosis of tuberculous pleurisy within different age group].

PubMed

Xu, H Y; Zhang, D Q; Ye, J R; Su, S S; Xie, Y P; Chen, C S; Li, Y P

2017-06-27

Objective: To evaluate the performance of T cell enzyme-linked immuno-spot assay (T-SPOT) on peripheral blood in combination with adenosine deaminase (ADA) on pleural fluid for diagnosis of tuberculous (TB) pleurisy within different age groups. Methods: The data of patients with pleural effusion from the Department of Pulmonary and Critical Care Medicine of the First Affiliated Hospital of Wenzhou Medical University from April 2012 to November 2016 were retrospectively analyzed, and the diagnoses of these patients were histopathologically confirmed through medical thoracoscopy. The cases who had confirmed diagnosis, in the same time, received peripheral blood T-SPOT.TB were enrolled. The performance of peripheral blood T-SPOT.TB in combination with pleural fluid ADA on diagnosing TB pleurisy in the younger patients (16-59 years old) and elderly patients (≥60 years old) were analyzed respectively. The sensitivity, specificity and the receiver operating characteristic (ROC) curve were adopted for statistical analysis. Results: A total of 448 cases were finally enrolled, 341(76.1%) confirmed with TB pleurisy, 224 males, 117 females, (47±19) years old; and 107 (23.9%) classified as non-TB pleurisy, 65 males, 42 females, (61±14) years old. There were 285 cases who were classified as younger group, and the other 163 cases were classified as elderly group. The sensitivity and specificity of peripheral blood T-SPOT.TB were 85.4% (204/239) and 71.7% (33/46) in the younger patients, 76.5% (78/102) and 59.0% (36/61) respectively in the elderly patients. The sensitivity of peripheral blood T-SPOT.TB in the younger patients was significantly higher than that in the elderly patients ( P =0.047). The sensitivity and specificity were 99.2% and 95.7% in combination with peripheral blood T-SPOT.TB and pleural fluid ADA respectively in the younger patients. The area under ROC curve (AUC) of T-SPOT.TB in the younger patients was 0.833, AUC of T-SPOT.TB combined with ADA was 0

AMP Is an Adenosine A1 Receptor Agonist*

PubMed Central

Rittiner, Joseph E.; Korboukh, Ilia; Hull-Ryde, Emily A.; Jin, Jian; Janzen, William P.; Frye, Stephen V.; Zylka, Mark J.

2012-01-01

Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5′-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5′-monophosphonate, ACP) directly activated the adenosine A1 receptor (A1R). In contrast, AMP only activated the adenosine A2B receptor (A2BR) after hydrolysis to adenosine by ecto-5′-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A1R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A1R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A1R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A1R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine. PMID:22215671

The Transcription Elongation Complex Directs Activation-Induced Cytidine Deaminase-Mediated DNA Deamination†

PubMed Central

Besmer, Eva; Market, Eleonora; Papavasiliou, F. Nina

2006-01-01

Activation-induced cytidine deaminase (AID) is a single-stranded DNA deaminase required for somatic hypermutation of immunoglobulin (Ig) genes, a key process in the development of adaptive immunity. Transcription provides a single-stranded DNA substrate for AID, both in vivo and in vitro. We present here an assay which can faithfully replicate all of the molecular features of the initiation of hypermutation of Ig genes in vivo. In this assay, which detects AID-mediated deamination in the context of transcription by Escherichia coli RNA polymerase, deamination targets either strand and declines in efficiency as the distance from the promoter increases. We show that AID binds DNA exposed by the transcribing polymerase, implicating the polymerase itself as the vehicle which distributes AID on DNA as it moves away from the promoter. PMID:16705187

Evolution of vitamin B2 biosynthesis: structural and functional similarity between pyrimidine deaminases of eubacterial and plant origin.

PubMed

Fischer, Markus; Römisch, Werner; Saller, Sabine; Illarionov, Boris; Richter, Gerald; Rohdich, Felix; Eisenreich, Wolfgang; Bacher, Adelbert

2004-08-27

The Arabidopsis thaliana open reading frame At4g20960 predicts a protein whose N-terminal part is similar to the eubacterial 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate deaminase domain. A synthetic open reading frame specifying a pseudomature form of the plant enzyme directed the synthesis of a recombinant protein which was purified to apparent homogeneity and was shown by NMR spectroscopy to convert 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate at a rate of 0.9 micromol mg(-1) min(-1). The substrate and product of the enzyme are both subject to spontaneous anomerization of the ribosyl side chain as shown by (13)C NMR spectroscopy. The protein contains 1 eq of Zn(2+)/subunit. The deaminase activity could be assigned to the N-terminal section of the plant protein. The deaminase domains of plants and eubacteria share a high degree of similarity, in contrast to deaminases from fungi. These data show that the riboflavin biosynthesis in plants proceeds by the same reaction steps as in eubacteria, whereas fungi use a different pathway.

Total-Body Irradiation Followed By Cyclosporine and Mycophenolate Mofetil in Treating Patients With Severe Combined Immunodeficiency Undergoing Donor Bone Marrow Transplant

ClinicalTrials.gov

2017-07-12

Adenosine Deaminase Deficiency; Autosomal Recessive Disorder; Immune System Disorder; Purine-Nucleoside Phosphorylase Deficiency; Severe Combined Immunodeficiency; Severe Combined Immunodeficiency With Absence of T and B Cells; X-Linked Severe Combined Immunodeficiency

An RNA electrophoretic mobility shift and mutational analysis of rnp-4f 5′-UTR intron splicing regulatory proteins in Drosophila reveals a novel new role for a dADAR protein isoform

PubMed Central

Lakshmi, G. Girija; Ghosh, Sushmita; Jones, Gabriel P.; Parikh, Roshni; Rawlins, Bridgette A.; Vaughn, Jack C.

2014-01-01

Alternative splicing greatly enhances the diversity of proteins encoded by eukaryotic genomes, and is also important in gene expression control. In contrast to the great depth of knowledge as to molecular mechanisms in the splicing pathway itself, relatively little is known about the regulatory events behind this process. The 5′-UTR and 3′-UTR in pre-mRNAs play a variety of roles in controlling eukaryotic gene expression, including translational modulation, and nearly 4,000 of the roughly 14,000 protein coding genes in Drosophila contain introns of unknown functional significance in their 5′-UTR. Here we report the results of an RNA electrophoretic mobility shift analysis of Drosophila rnp-4f 5′-UTR intron 0 splicing regulatory proteins. The pre-mRNA potential regulatory element consists of an evolutionarily-conserved 177-nt stem-loop arising from pairing of intron 0 with part of adjacent exon 2. Incubation of in vitro transcribed probe with embryo protein extract is shown to result in two shifted RNA-protein bands, and protein extract from a dADAR null mutant fly line results in only one shifted band. A mutated stem-loop in which the conserved exon 2 primary sequence is changed but secondary structure maintained by introducing compensatory base changes results in diminished band shifts. To test the hypothesis that dADAR plays a role in intron splicing regulation in vivo, levels of unspliced rnp-4f mRNA in dADAR mutant were compared to wild-type via real-time qRT-PCR. The results show that during embryogenesis unspliced rnp-4f mRNA levels fall by up to 85% in the mutant, in support of the hypothesis. Taken together, these results demonstrate a novel role for dADAR protein in rnp-4f 5′-UTR alternative intron splicing regulation which is consistent with a previously proposed model. PMID:23026215

A study of three polymorphic sites of ADA gene in colon cancer.

PubMed

Spina, C; Saccucci, P; Cozzoli, E; Bottini, E; Gloria-Bottini, F

2010-12-01

Adenosine inhibits the immune response in tumors. Adenosine deaminase (ADA) controls adenosine level and as ecto-enzyme acts as costimulatory molecule of adenosine receptors and/or CD26. We examined ADA₁, ADA₂, ADA₆ polymorphic sites of ADA gene in 109 subjects with colon cancer from Rome's population and in 246 blood donors as controls from the same population. In colon cancer ADA₁*2/ADA₂*1 haplotype is more represented, while ADA₁*2/ADA₂*2 is less represented than in controls. ADA₂*2/ADA₆*2 is less represented in patients than in controls. Polymorphic sites of ADA might influence cell-mediated anti-tumor immune responses controlling adenosine level and extraenzymatic protein functions.

Adenosine and inflammation: what's new on the horizon?

PubMed

Antonioli, Luca; Csóka, Balázs; Fornai, Matteo; Colucci, Rocchina; Kókai, Endre; Blandizzi, Corrado; Haskó, György

2014-08-01

Adenosine contributes to the maintenance of tissue integrity by modulating the immune system. Encouraging results have emerged with adenosine receptor ligands for the management of several inflammatory conditions in preclinical and clinical settings. However, therapeutic applications of these drugs are sometimes complicated by the occurrence of serious adverse effects. The scientific community is making intensive efforts to design novel adenosine receptor ligands endowed with greater selectivity or to develop innovative compounds acting as allosteric receptor modulators. In parallel, research is focusing on novel pharmacological entities (designated as adenosine-regulating agents) that can increase, in a site- and event-specific manner, adenosine concentrations at the inflammatory site, thereby minimizing the adverse systemic effects of adenosine. Copyright © 2014 Elsevier Ltd. All rights reserved.

Metabolism of Exogenous Purine Bases and Nucleosides by Salmonella typhimurium

PubMed Central

Hoffmeyer, J.; Neuhard, J.

1971-01-01

Purine-requiring mutants of Salmonella typhimurium LT2 containing additional mutations in either adenosine deaminase or purine nucleoside phosphorylase have been constructed. From studies of the ability of these mutants to utilize different purine compounds as the sole source of purines, the following conclusions may be drawn. (i) S. typhimurium does not contain physiologically significant amounts of adenine deaminase and adenosine kinase activities. (ii) The presence of inosine and guanosine kinase activities in vivo was established, although the former activity appears to be of minor significance for inosine metabolism. (iii) The utilization of exogenous purine deoxyribonucleosides is entirely dependent on a functional purine nucleoside phosphorylase. (iv) The pathway by which exogenous adenine is converted to guanine nucleotides in the presence of histidine requires a functional purine nucleoside phosphorylase. Evidence is presented that this pathway involves the conversion of adenine to adenosine, followed by deamination to inosine and subsequent phosphorolysis to hypoxanthine. Hypoxanthine is then converted to inosine monophosphate by inosine monophosphate pyrophosphorylase. The rate-limiting step in this pathway is the synthesis of adenosine from adenine due to lack of endogenous ribose-l-phosphate. PMID:4928005

Cultured astrocytes do not release adenosine during hypoxic conditions

PubMed Central

Fujita, Takumi; Williams, Erika K; Jensen, Tina K; Smith, Nathan A; Takano, Takahiro; Tieu, Kim; Nedergaard, Maiken

2012-01-01

Recent reports based on a chemiluminescent enzymatic assay for detection of adenosine conclude that cultured astrocytes release adenosine during mildly hypoxic conditions. If so, astrocytes may suppress neural activity in early stages of hypoxia. The aim of this study was to reevaluate the observation using high-performance liquid chromatography (HPLC). The HPLC analysis showed that exposure to 20 or 120 minutes of mild hypoxia failed to increase release of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine from cultured astrocytes. Similar results were obtained using a chemiluminescent enzymatic assay. Moreover, since the chemiluminescent enzymatic assay relies on hydrogen peroxide generation, release of free-radical scavengers from hypoxic cells can interfere with the assay. Accordingly, adenosine added to samples collected from hypoxic cultures could not be detected using the chemiluminescent enzymatic assay. Furthermore, addition of free-radical scavengers sharply reduced the sensitivity of adenosine detection. Conversely, use of a single-step assay inflated measured values due to the inability of the assay to distinguish adenosine and its metabolite inosine. These results show that cultured astrocytes do not release adenosine during mild hypoxia, an observation consistent with their high resistance to hypoxia. PMID:21989480

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

PubMed Central

Worrell, V E; Nagle, D P

1990-01-01

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaminase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines. PMID:2345148

N6-(2-Hydroxyethyl)-Adenosine Exhibits Insecticidal Activity against Plutella xylostella via Adenosine Receptors.

PubMed

Fang, Ming; Chai, Yiqiu; Chen, Guanjv; Wang, Huidong; Huang, Bo

The diamondback moth, Plutella xylostella, is one of the most important pests of cruciferous crops. We have earlier shown that N6-(2-hydroxyethyl)-adenosine (HEA) exhibits insecticidal activity against P. xylostella. In the present study we investigated the possible mechanism of insecticidal action of HEA on P. xylostella. HEA is a derivative of adenosine, therefore, we speculated whether it acts via P. xylostella adenosine receptor (PxAdoR). We used RNAi approach to silence PxAdoR gene and used antagonist of denosine receptor (AdoR) to study the insecticidal effect of HEA. We cloned the whole sequence of PxAdoR gene. A BLAST search using NCBI protein database showed a 61% identity with the Drosophila adenosine receptor (DmAdoR) and a 32-35% identity with human AdoR. Though the amino acids sequence of PxAdoR was different compared to other adenosine receptors, most of the amino acids that are known to be important for adenosine receptor ligand binding and signaling were present. However, only 30% binding sites key residues was similar between PxAdoR and A1R. HEA, at a dose of 1 mg/mL, was found to be lethal to the second-instar larvae of P. xylostella, and a significant reduction of mortality and growth inhibition ratio were obtained when HEA was administered to the larvae along with PxAdoR-dsRNA or antagonist of AdoR (SCH58261) for 36, 48, or 60 h. Especially at 48 h, the rate of growth inhibition of the PxAdoR knockdown group was 3.5-fold less than that of the HEA group, and the corrected mortality of SCH58261 group was reduced almost 2-fold compared with the HEA group. Our findings show that HEA may exert its insecticidal activity against P. xylostella larvae via acting on PxAdoR.

A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase.

PubMed

Li, Z; Chang, S; Lin, L; Li, Y; An, Q

2011-08-01

1-Aminocyclopropane-1-carboxylate (ACC) deaminase activity is an efficient marker for bacteria to promote plant growth by lowering ethylene levels in plants. We aim to develop a method for rapidly screening bacteria containing ACC deaminase, based on a colorimetric ninhydrin assay of ACC. A reliable colorimetric ninhydrin assay was developed to quantify ACC using heat-resistant polypropylene chimney-top 96-well PCR plates, having the wells evenly heated in boiling water, preventing accidental contamination from boiling water and limiting evaporation. With this method to measure bacterial consumption of ACC, 44 ACC-utilizing bacterial isolates were rapidly screened out from 311 bacterial isolates that were able to grow on minimal media containing ACC as the sole nitrogen source. The 44 ACC-utilizing bacterial isolates showed ACC deaminase activities and belonged to the genus Burkholderia, Pseudomonas or Herbaspirillum. Determination of bacterial ACC consumption by the PCR-plate ninhydrin-ACC assay is a rapid and efficient method for screening bacteria containing ACC deaminase from a large number of bacterial isolates. The PCR-plate ninhydrin-ACC assay extends the utility of the ninhydrin reaction and enables a rapid screening of bacteria containing ACC deaminase from large numbers of bacterial isolates. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

Tween 20-stabilized gold nanoparticles combined with adenosine triphosphate-BODIPY conjugates for the fluorescence detection of adenosine with more than 1000-fold selectivity.

PubMed

Hung, Szu-Ying; Shih, Ya-Chen; Tseng, Wei-Lung

2015-02-01

This study describes the development of a simple, enzyme-free, label-free, sensitive, and selective system for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles (Tween 20-AuNPs) as an efficient fluorescence quencher for boron dipyrromethene-conjugated adenosine 5'-triphosphate (BODIPY-ATP) and as a recognition element for adenosine. BODIPY-ATP can interact with Tween 20-AuNPs through the coordination between the adenine group of BODIPY-ATP and Au atoms on the NP surface, thereby causing the fluorescence quenching of BODIPY-ATP through the nanometal surface energy transfer (NSET) effect. When adenosine attaches to the NP surface, the attached adenosine exhibits additional electrostatic attraction to BODIPY-ATP. As a result, the presence of adenosine enhances the efficiency of AuNPs in fluorescence quenching of BODIPY-ATP. The AuNP-induced fluorescence quenching of BODIPY-ATP progressively increased with an increase in the concentration of adenosine; the detection limit at a signal-to-noise ratio of 3 for adenosine was determined to be 60nM. The selectivity of the proposed system was more than 1000-fold for adenosine over any adenosine analogs and other nucleotides. The proposed system combined with a phenylboronic acid-containing column was successfully applied to the determination of adenosine in urine. Copyright © 2014 Elsevier B.V. All rights reserved.

Adenosine through the A2A adenosine receptor increases IL-1β in the brain contributing to anxiety

PubMed Central

Chiu, Gabriel S.; Darmody, Patrick T.; Walsh, John P.; Moon, Morgan L.; Kwakwa, Kristin A.; Bray, Julie K.; McCusker, Robert H.; Freund, Gregory G.

2014-01-01

Anxiety is one of the most commonly reported psychiatric conditions, but its pathogenesis is poorly understood. Ailments associated with activation of the innate immune system, however, are increasingly linked to anxiety disorders. In adult male mice, we found that adenosine doubled caspase-1 activity in brain by a pathway reliant on ATP-sensitive potassium (KATP) channels, protein kinase A (PKA) and the A2A adenosine receptor (AR). In addition, adenosine-dependent activation of caspase-1 increased interleukin (IL)-1β in the brain by two-fold. Peripheral administration of adenosine in wild-type (WT) mice led to a 2.3-fold increase in caspase-1 activity in the amygdala and to a 33% and 42% reduction in spontaneous locomotor activity and food intake, respectively, that were not observed in caspase-1 knockout (KO), IL-1 receptor type 1 (IL-1R1) KO and A2A AR KO mice or in mice administered a caspase-1 inhibitor centrally. Finally, adenosine administration increased anxiety-like behaviors in WT mice by 28% in the open field test and by 55% in the elevated zero-maze. Caspase-1 KO mice, IL-1R1 KO mice, A2A AR KO mice and WT mice treated with the KATP channel blocker, glyburide, were resistant to adenosine-induced anxiety-like behaviors. Thus, our results indicate that adenosine can act as an anxiogenic by activating caspase-1 and increasing IL-1β in the brain. PMID:24907587

Adenosine signaling promotes hematopoietic stem and progenitor cell emergence

PubMed Central

Jing, Lili; Tamplin, Owen J.; Chen, Michael J.; Deng, Qing; Patterson, Shenia; Kim, Peter G.; Durand, Ellen M.; McNeil, Ashley; Green, Julie M.; Matsuura, Shinobu; Ablain, Julien; Brandt, Margot K.; Schlaeger, Thorsten M.; Huttenlocher, Anna; Daley, George Q.; Ravid, Katya

2015-01-01

Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1+/cmyb+ HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl+ hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP–protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates. PMID:25870200

Structural features of antiviral DNA cytidine deaminases.

PubMed

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

2013-11-01

The APOBEC3 (A3) family of cytidine deaminases plays a vital role for innate defense against retroviruses. Lentiviruses such as HIV-1 evolved the Vif protein that triggers A3 protein degradation. There are seven A3 proteins, A3A-A3H, found in humans. All A3 proteins can deaminate cytidines to uridines in single-stranded DNA (ssDNA), generated during viral reverse transcription. A3 proteins have either one or two cytidine deaminase domains (CD). The CDs coordinate a zinc ion, and their amino acid specificity classifies the A3s into A3Z1, A3Z2, and A3Z3. A3 proteins occur as monomers, dimers, and large oligomeric complexes. Studies on the nature of A3 oligomerization, as well as the mode of interaction of A3s with RNA and ssDNA are partially controversial. High-resolution structures of the catalytic CD2 of A3G and A3F as well as of the single CD proteins A3A and A3C have been published recently. The NMR and X-ray crystal structures show globular proteins with six α-helices and five β sheets arranged in a characteristic motif (α1-β1-β2/2'-α2-β3-α3-β4-α4-β5-α5-α6). However, the detailed arrangement and extension of individual structure elements and their relevance for A3 complex formation and activity remains a matter of debate and will be highlighted in this review.

Is adenosine associated with sudden death in schizophrenia? A new framework linking the adenosine pathway to risk of sudden death.

PubMed

Gadelha, Ary; Zugman, André; Calzavara, Mariana Bendlin; de Mendonça Furtado, Remo Holanda; Scorza, Fulvio Alexandre; Bressan, Rodrigo Afonsecca

2018-01-01

Schizophrenia is associated with an increased mortality from cardiovascular disease. Relatively few studies have assessed the putative association of schizophrenia pathophysiology with sudden death. Low adenosine levels have been associated with schizophrenia. In cardiology, increased mortality among patients with congestive heart failure has been associated with genetic polymorphisms that potentially lead to lower adenosine levels. Thus, we hypothesize that adenosine could link schizophrenia and cardiovascular mortality, with decreased adenosine levels leading to increased vulnerability to hyperexcitability following hypoxic insults, increasing the odds of fatal arrhythmias. Low adenosine levels might also lead to a small increase in overall mortality rates and a major increase in the sudden death rate. This hypothesis paves the way for further investigation of the increased cardiac mortality associated with schizophrenia. Potentially, a better characterization of adenosine-related mechanisms of sudden death in schizophrenia could lead to new evidence of factors leading to sudden death in the general population. Copyright © 2017. Published by Elsevier Ltd.

[Screening and identification of an endophytic bacterium with 1-aminocyclopropane-1-carboxylate deaminase activity from Panax ginseng and its effect on host growth].

PubMed

Tian, Lei; Jiang, Yun; Chen, Changqing; Zhang, Guanjun; Li, Tong; Tong, Bin; Xu, Peng

2014-07-04

This study aimed to screen endophytic bacteria with 1-aminocyclopropane-1-carboxylate deaminase activity from Panax ginseng and test the capability of growth promotion to its host. In total 120 endophytic bacterial strains isolated from Panax ginseng were screened for 1-aminocyclopropane-1-carboxylate deaminase activity using the qualitative and quantitative methods. The obtained strain was also tested for its ability of nitrogen fixation using the Ashby agar plates and the gene of nifH, for its ability of phosphate solubilization using the Pikovaskaia's plates and quantitative analysis of Mo-Sb-Ascrobiology acid colorimetry, for its ability of producing siderophores using the method of Chrome azurol S detecting, and its effect on promoting growth of Panax ginseng by laboratory and field experiments. The bacterial strain with ACC deaminase was identified based on morphology, physiological and biochemical traits, and 16S rRNA sequence analysis. The bacterial stain JJ8-3 with the ability of producing ACC deaminase activity was obtained through screening, which its ACC deaminase activity was alpha-ketobutyric acid 6.7 micromol/(mg x h). Strain JJ8-3 had other traits of phosphate solubilizing, nitrogen fixation, producing siderophores, and the ability of promoting growth of Panax ginseng. Strain JJ8-3 was identified as Pseudomonas fluorescens. Strain JJ8-3 of endophytic bacterium with ACC deaminase activity from Panax ginseng was obtained and would lay the foundation for its further study and application on plant growth promotion.

Staphylococcus aureus synthesizes adenosine to escape host immune responses

PubMed Central

Thammavongsa, Vilasack; Kern, Justin W.; Missiakas, Dominique M.

2009-01-01

Staphylococcus aureus infects hospitalized or healthy individuals and represents the most frequent cause of bacteremia, treatment of which is complicated by the emergence of methicillin-resistant S. aureus. We examined the ability of S. aureus to escape phagocytic clearance in blood and identified adenosine synthase A (AdsA), a cell wall–anchored enzyme that converts adenosine monophosphate to adenosine, as a critical virulence factor. Staphylococcal synthesis of adenosine in blood, escape from phagocytic clearance, and subsequent formation of organ abscesses were all dependent on adsA and could be rescued by an exogenous supply of adenosine. An AdsA homologue was identified in the anthrax pathogen, and adenosine synthesis also enabled escape of Bacillus anthracis from phagocytic clearance. Collectively, these results suggest that staphylococci and other bacterial pathogens exploit the immunomodulatory attributes of adenosine to escape host immune responses. PMID:19808256

A High Affinity Adenosine Kinase from Anopheles gambiae

PubMed Central

Cassera, María B.; Ho, Meng-Chiao; Merino, Emilio F.; Burgos, Emmanuel S.; Rinaldo-Matthis, Agnes; Almo, Steven C.; Schramm, Vern L.

2011-01-01

Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (Km 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap4A (2.0 Å resolution) reveals interactions for adenosine, ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg2+ ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layered α/β/α sandwich, and a small cap domain in contact with adenosine. The specificity and tight-binding for adenosine arises from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168 and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64 and Asn68 and the ribosyl 2′- and 3′-hydroxyl groups. The structure is more similar to human adenosine kinase (48% identity) than to AK from Toxoplasma gondii (31% identity). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role of this enzyme to maintain the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects. PMID:21247194

A High-Affinity Adenosine Kinase from Anopheles Gambiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

M Cassera; M Ho; E Merino

2011-12-31

Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K{sub m} = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap{sub 4}A (2.0 {angstrom} resolution) reveals interactionsmore » for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg{sup 2+} ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer {alpha}/{beta}/{alpha} sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and the ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.« less

Ischaemic tolerance in aged mouse myocardium: the role of adenosine and effects of A1 adenosine receptor overexpression

PubMed Central

Headrick, John P; Willems, Laura; Ashton, Kevin J; Holmgren, Kirsten; Peart, Jason; Matherne, G Paul

2003-01-01

The genesis of the ischaemia intolerant phenotype in aged myocardium is poorly understood. We tested the hypothesis that impaired adenosine-mediated protection contributes to ischaemic intolerance, and examined whether this is countered by A1 adenosine receptor (A1AR) overexpression. Responses to 20 min ischaemia and 45 min reperfusion were assessed in perfused hearts from young (2–4 months) and moderately aged (16–18 months) mice. Post-ischaemic contractility was impaired by ageing with elevated ventricular diastolic (32 ± 2 vs. 18 ± 2 mmHg in young) and reduced developed (37 ± 3 vs. 83 ± 6 mmHg in young) pressures. Lactate dehydrogenase (LDH) loss was exaggerated (27 ± 2 vs. 16 ± 2 IU g−1in young) whereas the incidence of tachyarrhythmias was similar in young (15 ± 1 %) and aged hearts (16 ± 1 %). Functional analysis confirmed equipotent effects of 50 μm adenosine at A1 and A2 receptors in young and aged hearts. Nonetheless, while 50 μm adenosine improved diastolic (5 ± 1 mmHg) and developed pressures (134 ± 7 mmHg) and LDH loss (6 ± 2 IU g−1) in young hearts, it did not alter these variables in the aged group. Adenosine did attenuate arrhythmogenesis for both ages (to ∼10 %). In contrast to adenosine, 50 μm diazoxide reduced ischaemic damage and arrhythmogenesis for both ages. Contractile and anti-necrotic effects of adenosine were limited by 100 μm 5-hydroxydecanoate (5-HD) and 3 μm chelerythrine. Anti-arrhythmic effects were limited by 5-HD but not chelerythrine. Non-selective (100 μm 8-sulfophenyltheophylline) and A1-selective (150 nm 8-cyclopentyl-1,3-dipropylxanthine) adenosine receptor antagonism impaired ischaemic tolerance in young but not aged hearts. Quantitative real-time PCR and radioligand analysis indicated that impaired protection is unrelated to changes in A1AR mRNA transcription, or receptor density (∼8 fmol mg−1 protein in both age groups). However, A1AR overexpression improved tolerance for both ages, restoring

Adenosine regulation of microtubule dynamics in cardiac hypertrophy.

PubMed

Fassett, John T; Xu, Xin; Hu, Xinli; Zhu, Guangshuo; French, Joel; Chen, Yingjie; Bache, Robert J

2009-08-01

There is evidence that endogenous extracellular adenosine reduces cardiac hypertrophy and heart failure in mice subjected to chronic pressure overload, but the mechanism by which adenosine exerts these protective effects is unknown. Here, we identified a novel role for adenosine in regulation of the cardiac microtubule cytoskeleton that may contribute to its beneficial effects in the overloaded heart. In neonatal cardiomyocytes, phenylephrine promoted hypertrophy and reorganization of the cytoskeleton, which included accumulation of sarcomeric proteins, microtubules, and desmin. Treatment with adenosine or the stable adenosine analog 2-chloroadenosine, which decreased hypertrophy, specifically reduced accumulation of microtubules. In hypertrophied cardiomyocytes, 2-chloroadenosine or adenosine treatment preferentially targeted stabilized microtubules (containing detyrosinated alpha-tubulin). Consistent with a role for endogenous adenosine in reducing microtubule stability, levels of detyrosinated microtubules were elevated in hearts of CD73 knockout mice (deficient in extracellular adenosine production) compared with wild-type mice (195%, P < 0.05). In response to aortic banding, microtubules increased in hearts of wild-type mice; this increase was exaggerated in CD73 knockout mice, with significantly greater amounts of tubulin partitioning into the cold-stable Triton-insoluble fractions. The levels of this stable cytoskeletal fraction of tubulin correlated strongly with the degree of heart failure. In agreement with a role for microtubule stabilization in promoting cardiac dysfunction, colchicine treatment of aortic-banded mice reduced hypertrophy and improved cardiac function compared with saline-treated controls. These results indicate that microtubules contribute to cardiac dysfunction and identify, for the first time, a role for adenosine in regulating cardiomyocyte microtubule dynamics.

Differential distribution of adenosine receptors in rat cochlea.

PubMed

Vlajkovic, Srdjan M; Abi, Shukri; Wang, Carol J H; Housley, Gary D; Thorne, Peter R

2007-06-01

Adenosine is a constitutive cell metabolite that can be released from cells via specific bi-directional transporters and is an end-point for nucleotide hydrolysis. In the extracellular space, adenosine becomes a signalling molecule for P1 (adenosine) receptors that modulate physiological responses in a wide range of mammalian tissues. Whereas adenosine signalling has been implicated in the regulation of cochlear blood flow and in cochlear protection from oxidative damage, the potential roles for adenosine signalling in the modulation of sound transduction and auditory neurotransmission have not been established. We have characterised the expression and distribution of adenosine receptors in the rat cochlea. mRNA transcripts for all four subtypes of adenosine receptors (A(1), A(2A), A(2B) and A(3)) were detected in dissected cochlear tissue by using reverse transcription/polymerase chain reaction analysis. The protein distribution for the A(1), A(2A) and A(3) receptor subtypes was identified by immunoperoxidase histochemistry and confocal immunofluorescence labelling. These receptors were differentially expressed in the organ of Corti, spiral ganglion neurones, lateral wall tissues and cochlear blood vessels. The distribution of adenosine receptors in sensory and neural tissues and in the vasculature coincided with other elements of purinergic signalling (P2X and P2Y receptors, ectonucleotidases), consistent with the integrative regulation of many physiological processes in the cochlea by extracellular nucleotides and nucleosides. Our study provides a framework for further investigation of adenosine signalling in the inner ear, including putative roles in oxidative stress responses.

Mechanism-specific effects of adenosine on ventricular tachycardia.

PubMed

Lerman, Bruce B; Ip, James E; Shah, Bindi K; Thomas, George; Liu, Christopher F; Ciaccio, Edward J; Wit, Andrew L; Cheung, Jim W; Markowitz, Steven M

2014-12-01

There is no universally accepted method by which to diagnose clinical ventricular tachycardia (VT) due to cAMP-mediated triggered activity. Based on cellular and clinical data, adenosine termination of VT is thought to be consistent with a diagnosis of triggered activity. However, a major gap in evidence mitigates the validity of this proposal, namely, defining the specificity of adenosine response in well-delineated reentrant VT circuits. To this end, we systematically studied the effects of adenosine in a model of canine reentrant VT and in human reentrant VT, confirmed by 3-dimensional, pace- and substrate mapping. Adenosine (12 mg [IQR 12-24]) failed to terminate VT in 31 of 31 patients with reentrant VT due to structural heart disease, and had no effect on VT cycle length (age, 67 years [IQR 53-74]); ejection fraction, 35% [IQR 20-55]). In contrast, adenosine terminated VT in 45 of 50 (90%) patients with sustained focal right or left outflow tract tachycardia. The sensitivity of adenosine for identifying VT due to triggered activity was 90% (95% CI, 0.78-0.97) and its specificity was 100% (95% CI, 0.89-1.0). Additionally, reentrant circuits were mapped in the epicardial border zone of 4-day-old infarcts in mongrel dogs. Adenosine (300-400 μg/kg) did not terminate sustained VT or have any effect on VT cycle length. These data support the concept that adenosine's effects on ventricular myocardium are mechanism specific, such that termination of VT in response to adenosine is diagnostic of cAMP-mediated triggered activity. © 2014 Wiley Periodicals, Inc.

Reticulocyte Count Test

MedlinePlus

... Acid-Fast Bacillus (AFB) Testing Activated Clotting Time Acute Viral Hepatitis Panel Adenosine Deaminase Adrenocorticotropic Hormone (ACTH) ... Guillain-Barré Syndrome Hashimoto Thyroiditis Heart Attack and Acute Coronary Syndrome Heart Disease Hemochromatosis Hemoglobin Abnormalities Hepatitis ...

WBC (White Blood Cell) Differential Count

MedlinePlus

... Acid-Fast Bacillus (AFB) Testing Activated Clotting Time Acute Viral Hepatitis Panel Adenosine Deaminase Adrenocorticotropic Hormone (ACTH) ... Guillain-Barré Syndrome Hashimoto Thyroiditis Heart Attack and Acute Coronary Syndrome Heart Disease Hemochromatosis Hemoglobin Abnormalities Hepatitis ...

Index of Glossary Terms

MedlinePlus

... Acid-Fast Bacillus (AFB) Testing Activated Clotting Time Acute Viral Hepatitis Panel Adenosine Deaminase Adrenocorticotropic Hormone (ACTH) ... Guillain-Barré Syndrome Hashimoto Thyroiditis Heart Attack and Acute Coronary Syndrome Heart Disease Hemochromatosis Hemoglobin Abnormalities Hepatitis ...

BCR-ABL1: Test

MedlinePlus

... Acid-Fast Bacillus (AFB) Testing Activated Clotting Time Acute Viral Hepatitis Panel Adenosine Deaminase Adrenocorticotropic Hormone (ACTH) ... Guillain-Barré Syndrome Hashimoto Thyroiditis Heart Attack and Acute Coronary Syndrome Heart Disease Hemochromatosis Hemoglobin Abnormalities Hepatitis ...

White Blood Cell Count

MedlinePlus

... Acid-Fast Bacillus (AFB) Testing Activated Clotting Time Acute Viral Hepatitis Panel Adenosine Deaminase Adrenocorticotropic Hormone (ACTH) ... Guillain-Barré Syndrome Hashimoto Thyroiditis Heart Attack and Acute Coronary Syndrome Heart Disease Hemochromatosis Hemoglobin Abnormalities Hepatitis ...

N6-(2-Hydroxyethyl)-Adenosine Exhibits Insecticidal Activity against Plutella xylostella via Adenosine Receptors

PubMed Central

Fang, Ming; Chai, Yiqiu; Chen, Guanjv; Wang, Huidong; Huang, Bo

2016-01-01

The diamondback moth, Plutella xylostella, is one of the most important pests of cruciferous crops. We have earlier shown that N6-(2-hydroxyethyl)-adenosine (HEA) exhibits insecticidal activity against P. xylostella. In the present study we investigated the possible mechanism of insecticidal action of HEA on P. xylostella. HEA is a derivative of adenosine, therefore, we speculated whether it acts via P. xylostella adenosine receptor (PxAdoR). We used RNAi approach to silence PxAdoR gene and used antagonist of denosine receptor (AdoR) to study the insecticidal effect of HEA. We cloned the whole sequence of PxAdoR gene. A BLAST search using NCBI protein database showed a 61% identity with the Drosophila adenosine receptor (DmAdoR) and a 32–35% identity with human AdoR. Though the amino acids sequence of PxAdoR was different compared to other adenosine receptors, most of the amino acids that are known to be important for adenosine receptor ligand binding and signaling were present. However, only 30% binding sites key residues was similar between PxAdoR and A1R. HEA, at a dose of 1 mg/mL, was found to be lethal to the second-instar larvae of P. xylostella, and a significant reduction of mortality and growth inhibition ratio were obtained when HEA was administered to the larvae along with PxAdoR-dsRNA or antagonist of AdoR (SCH58261) for 36, 48, or 60 h. Especially at 48 h, the rate of growth inhibition of the PxAdoR knockdown group was 3.5-fold less than that of the HEA group, and the corrected mortality of SCH58261 group was reduced almost 2-fold compared with the HEA group. Our findings show that HEA may exert its insecticidal activity against P. xylostella larvae via acting on PxAdoR. PMID:27668428

Genetics Home Reference: adenosine deaminase 2 deficiency

MedlinePlus

... not fully understand how a loss of this enzyme's function leads to the features of ADA2 deficiency. They speculate that a lack of this enzyme may disrupt the balance between pro-inflammatory and ...

Enzymatic regeneration of adenosine triphosphate cofactor

NASA Technical Reports Server (NTRS)

Marshall, D. L.

1974-01-01

Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

Role of adenosine receptors in caffeine tolerance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holtzman, S.G.; Mante, S.; Minneman, K.P.

1991-01-01

Caffeine is a competitive antagonist at adenosine receptors. Receptor up-regulation during chronic drug treatment has been proposed to be the mechanism of tolerance to the behavioral stimulant effects of caffeine. This study reassessed the role of adenosine receptors in caffeine tolerance. Separate groups of rats were given scheduled access to drinking bottles containing plain tap water or a 0.1% solution of caffeine. Daily drug intake averaged 60-75 mg/kg and resulted in complete tolerance to caffeine-induced stimulation of locomotor activity, which could not be surmounted by increasing the dose of caffeine. 5'-N-ethylcarboxamidoadenosine (0.001-1.0 mg/kg) dose dependently decreased the locomotor activity ofmore » caffeine-tolerant rats and their water-treated controls but was 8-fold more potent in the latter group. Caffeine (1.0-10 mg/kg) injected concurrently with 5-N-ethylcarboxamidoadenosine antagonized the decreases in locomotor activity comparably in both groups. Apparent pA2 values for tolerant and control rats also were comparable: 5.05 and 5.11. Thus, the adenosine-antagonist activity of caffeine was undiminished in tolerant rats. The effects of chronic caffeine administration on parameters of adenosine receptor binding and function were measured in cerebral cortex. There were no differences between brain tissue from control and caffeine-treated rats in number and affinity of adenosine binding sites or in receptor-mediated increases (A2 adenosine receptor) and decreases (A1 adenosine receptor) in cAMP accumulation. These results are consistent with theoretical arguments that changes in receptor density should not affect the potency of a competitive antagonist. Experimental evidence and theoretical considerations indicate that up-regulation of adenosine receptors is not the mechanism of tolerance to caffeine-induced stimulation of locomotor activity.« less

APOBEC3 Cytidine Deaminases in Double-Strand DNA Break Repair and Cancer Promotion

PubMed Central

Nowarski, Roni; Kotler, Moshe

2013-01-01

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

Administration of exogenous adenosine triphosphate to ischemic skeletal muscle induces an energy-sparing effect: role of adenosine receptors.

PubMed

Maldonado, Claudio; Pushpakumar, Sathnur B; Perez-Abadia, Gustavo; Arumugam, Sengodagounder; Lane, Andrew N

2013-05-01

Ischemia-reperfusion injury is a devastating complication that occurs in allotransplantation and replantation of limbs. Over the years, several preservation strategies have been used to conserve the critical levels of intracellular adenosine triphosphate (ATP) during ischemia to sustain the ion gradients across the membranes and thus the tissue viability. The administration of exogenous ATP to ischemic tissues is known to provide beneficial effects during reperfusion, but it is unclear whether it provides protection during ischemia. The purpose of the present study was to determine the effect of ATP administration on high-energy phosphate levels in ischemic skeletal muscle and to examine the role of purinergic and adenosine receptors in mediating the response to exogenous ATP. The extensor digitorum longus muscles of Fischer rats were subjected to ischemia and treated with different concentrations of ATP with or without purinergic and adenosine receptor blockers. Phosphorus-31 nuclear magnetic resonance spectroscopy was used to measure the rate of decay of ATP, phosphocreatine (PCr), and the formation of adenosine monophosphate and acidification. Phosphorylated compounds were analyzed using a simple model of energy metabolism, and the PCr half-life was used as an index of internal depletion of ATP to distinguish between intracellular and extracellular ATP. PCr decay was rapid in all muscle groups and was followed by gradual ATP decay. The half-life of PCr was significantly longer in the ATP-treated muscles than in the vehicle controls and was maximally prolonged by treating with slow hydrolyzing adenosine 5'-O-(3-thio)triphosphate. Purinoceptor (P2X) blockade with ATP treatment significantly increased the half-life of PCr, and adenosine receptor blockers blunted the response. Administration of adenosine to ischemic muscles significantly increased the half-life of PCr compared with that in the vehicle controls. Exogenous ATP administration to ischemic skeletal

Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells.

PubMed

Yamaguchi, Hiroshi; Maruyama, Toshihiko; Urade, Yoshihiro; Nagata, Shigekazu

2014-03-25

Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5'-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a 'calm down' signal. DOI: http://dx.doi.org/10.7554/eLife.02172.001.

An Adenosine-Mediated Glial-Neuronal Circuit for Homeostatic Sleep.

PubMed

Bjorness, Theresa E; Dale, Nicholas; Mettlach, Gabriel; Sonneborn, Alex; Sahin, Bogachan; Fienberg, Allen A; Yanagisawa, Masashi; Bibb, James A; Greene, Robert W

2016-03-30

Sleep homeostasis reflects a centrally mediated drive for sleep, which increases during waking and resolves during subsequent sleep. Here we demonstrate that mice deficient for glial adenosine kinase (AdK), the primary metabolizing enzyme for adenosine (Ado), exhibit enhanced expression of this homeostatic drive by three independent measures: (1) increased rebound of slow-wave activity; (2) increased consolidation of slow-wave sleep; and (3) increased time constant of slow-wave activity decay during an average slow-wave sleep episode, proposed and validated here as a new index for homeostatic sleep drive. Conversely, mice deficient for the neuronal adenosine A1 receptor exhibit significantly decreased sleep drive as judged by these same indices. Neuronal knock-out of AdK did not influence homeostatic sleep need. Together, these findings implicate a glial-neuronal circuit mediated by intercellular Ado, controlling expression of homeostatic sleep drive. Because AdK is tightly regulated by glial metabolic state, our findings suggest a functional link between cellular metabolism and sleep homeostasis. The work presented here provides evidence for an adenosine-mediated regulation of sleep in response to waking (i.e., homeostatic sleep need), requiring activation of neuronal adenosine A1 receptors and controlled by glial adenosine kinase. Adenosine kinase acts as a highly sensitive and important metabolic sensor of the glial ATP/ADP and AMP ratio directly controlling intracellular adenosine concentration. Glial equilibrative adenosine transporters reflect the intracellular concentration to the extracellular milieu to activate neuronal adenosine receptors. Thus, adenosine mediates a glial-neuronal circuit linking glial metabolic state to neural-expressed sleep homeostasis. This indicates a metabolically related function(s) for this glial-neuronal circuit in the buildup and resolution of our need to sleep and suggests potential therapeutic targets more directly related to

Purinoceptor modulation of noradrenaline release in rat tail artery: tonic modulation mediated by inhibitory P2Y- and facilitatory A2A-purinoceptors.

PubMed Central

Gonçalves, J.; Queiroz, G.

1996-01-01

1. The effects of analogues of adenosine and ATP on noradrenaline release elicited by electrical stimulation (5 Hz, 2700 pulses) were studied in superfused preparations of rat tail artery. The effects of purinoceptor antagonists, of adenosine deaminase and of adenosine uptake blockade were also examined. Noradrenaline was measured by h.p.l.c. electrochemical detection. 2. The A1-adenosine receptor agonist, N6-cyclopentyladenosine (CPA; 0.1-100 nM) reduced, whereas the A2A-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 3-30 nM) increased evoked noradrenaline overflow. These effects were antagonized by the A1-adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 20 nM) and the A2-adenosine receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX; 100 nM), respectively. The P2Y-purinoceptor agonist, 2-methylthio-ATP (1-100 microM) reduced noradrenaline overflow, an effect prevented by the P2-purinoceptor antagonist, cibacron blue 3GA (100 microM) and suramin (100 microM). 3. Adenosine deaminase (2 u ml-1), DMPX (100 nM) and inhibition of adenosine uptake with S-(p-nitrobenzyl)-6-thioinosine (NBTI; 50 nM) decreased evoked noradrenaline overflow. DPCPX alone did not change noradrenaline overflow but prevented the inhibition caused by NBTI. The P2Y-purinoceptor antagonist, cibacron blue 3GA (100 microM) increased evoked noradrenaline overflow as did suramin, a non-selective P2-antagonist. 4. It is concluded that, in rat tail artery, inhibitory (A1 and P2Y) and facilitatory (A2A) purinoceptors are present and modulate noradrenaline release evoked by electrical stimulation. Endogenous purines tonically modulate noradrenaline release through activation of inhibitory P2Y and facilitatory A2A purinoceptors, whereas a tonic activation of inhibitory A1 purinoceptors seems to be prevented by adenosine uptake. PMID:8825357

Increased cortical extracellular adenosine correlates with seizure termination.

PubMed

Van Gompel, Jamie J; Bower, Mark R; Worrell, Gregory A; Stead, Matt; Chang, Su-Youne; Goerss, Stephan J; Kim, Inyong; Bennet, Kevin E; Meyer, Fredric B; Marsh, W Richard; Blaha, Charles D; Lee, Kendall H

2014-02-01

Seizures are currently defined by their electrographic features. However, neuronal networks are intrinsically dependent on neurotransmitters of which little is known regarding their periictal dynamics. Evidence supports adenosine as having a prominent role in seizure termination, as its administration can terminate and reduce seizures in animal models. Furthermore, microdialysis studies in humans suggest that adenosine is elevated periictally, but the relationship to the seizure is obscured by its temporal measurement limitations. Because electrochemical techniques can provide vastly superior temporal resolution, we test the hypothesis that extracellular adenosine concentrations rise during seizure termination in an animal model and humans using electrochemistry. White farm swine (n = 45) were used in an acute cortical model of epilepsy, and 10 human epilepsy patients were studied during intraoperative electrocorticography (ECoG). Wireless Instantaneous Neurotransmitter Concentration Sensor (WINCS)-based fast scan cyclic voltammetry (FSCV) and fixed potential amperometry were obtained utilizing an adenosine-specific triangular waveform or biosensors, respectively. Simultaneous ECoG and electrochemistry demonstrated an average adenosine increase of 260% compared to baseline, at 7.5 ± 16.9 s with amperometry (n = 75 events) and 2.6 ± 11.2 s with FSCV (n = 15 events) prior to electrographic seizure termination. In agreement with these animal data, adenosine elevation prior to seizure termination in a human patient utilizing FSCV was also seen. Simultaneous ECoG and electrochemical recording supports the hypothesis that adenosine rises prior to seizure termination, suggesting that adenosine itself may be responsible for seizure termination. Future work using intraoperative WINCS-based FSCV recording may help to elucidate the precise relationship between adenosine and seizure termination. Wiley Periodicals, Inc. © 2014 International League Against

A halotolerant Enterobacter sp. displaying ACC deaminase activity promotes rice seedling growth under salt stress.

PubMed

Sarkar, Anumita; Ghosh, Pallab Kumar; Pramanik, Krishnendu; Mitra, Soumik; Soren, Tithi; Pandey, Sanjeev; Mondal, Monohar Hossain; Maiti, Tushar Kanti

2018-01-01

Agricultural productivity is proven to be hampered by the synthesis of reactive oxygen species (ROS) and production of stress-induced ethylene under salinity stress. One-aminocyclopropane-1-carboxylic acid (ACC) is the direct precursor of ethylene synthesized by plants. Bacteria possessing ACC deaminase activity can use ACC as a nitrogen source preventing ethylene production. Several salt-tolerant bacterial strains displaying ACC deaminase activity were isolated from rice fields, and their plant growth-promoting (PGP) properties were determined. Among them, strain P23, identified as an Enterobacter sp. based on phenotypic characteristics, matrix-assisted laser desorption ionization-time of flight mass spectrometry data and the 16S rDNA sequence, was selected as the best-performing isolate for several PGP traits, including phosphate solubilization, IAA production, siderophore production, HCN production, etc. Enterobacter sp. P23 was shown to promote rice seedling growth under salt stress, and this effect was correlated with a decrease in antioxidant enzymes and stress-induced ethylene. Isolation of an acdS mutant strain enabled concluding that the reduction in stress-induced ethylene content after inoculation of strain P23 was linked to ACC deaminase activity. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Autophagy occurs within an hour of adenosine triphosphate treatment after nerve cell damage: the neuroprotective effects of adenosine triphosphate against apoptosis

PubMed Central

Lu, Na; Wang, Baoying; Deng, Xiaohui; Zhao, Honggang; Wang, Yong; Li, Dongliang

2014-01-01

After hypoxia, ischemia, or inflammatory injuries to the central nervous system, the damaged cells release a large amount of adenosine triphosphate, which may cause secondary neuronal death. Autophagy is a form of cell death that also has neuroprotective effects. Cell Counting Kit assay, monodansylcadaverine staining, flow cytometry, western blotting, and real-time PCR were used to determine the effects of exogenous adenosine triphosphate treatment at different concentrations (2, 4, 6, 8, 10 mmol/L) over time (1, 2, 3, and 6 hours) on the apoptosis and autophagy of SH-SY5Y cells. High concentrations of extracellular adenosine triphosphate induced autophagy and apoptosis of SH-SY5Y cells. The enhanced autophagy first appeared, and peaked at 1 hour after treatment with adenosine triphosphate. Cell apoptosis peaked at 3 hours, and persisted through 6 hours. With prolonged exposure to the adenosine triphosphate treatment, the fraction of apoptotic cells increased. These data suggest that the SH-SY5Y neural cells initiated autophagy against apoptosis within an hour of adenosine triphosphate treatment to protect themselves against injury. PMID:25368646

Fast-scan Cyclic Voltammetry for the Characterization of Rapid Adenosine Release

PubMed Central

Nguyen, Michael D.; Venton, B. Jill

2014-01-01

Adenosine is a signaling molecule and downstream product of ATP that acts as a neuromodulator. Adenosine regulates physiological processes, such as neurotransmission and blood flow, on a time scale of minutes to hours. Recent developments in electrochemical techniques, including fast-scan cyclic voltammetry (FSCV), have allowed direct detection of adenosine with sub-second temporal resolution. FSCV studies have revealed a novel mode of rapid signaling that lasts only a few seconds. This rapid release of adenosine can be evoked by electrical or mechanical stimulations or it can be observed spontaneously without stimulation. Adenosine signaling on this time scale is activity dependent; however, the mode of release is not fully understood. Rapid adenosine release modulates oxygen levels and evoked dopamine release, indicating that adenosine may have a rapid modulatory role. In this review, we outline how FSCV can be used to detect adenosine release, compare FSCV with other techniques used to measure adenosine, and present an overview of adenosine signaling that has been characterized using FSCV. These studies point to a rapid mode of adenosine modulation, whose mechanism and function will continue to be characterized in the future. PMID:26900429

Fast-scan Cyclic Voltammetry for the Characterization of Rapid Adenosine Release.

PubMed

Nguyen, Michael D; Venton, B Jill

2015-01-01

Adenosine is a signaling molecule and downstream product of ATP that acts as a neuromodulator. Adenosine regulates physiological processes, such as neurotransmission and blood flow, on a time scale of minutes to hours. Recent developments in electrochemical techniques, including fast-scan cyclic voltammetry (FSCV), have allowed direct detection of adenosine with sub-second temporal resolution. FSCV studies have revealed a novel mode of rapid signaling that lasts only a few seconds. This rapid release of adenosine can be evoked by electrical or mechanical stimulations or it can be observed spontaneously without stimulation. Adenosine signaling on this time scale is activity dependent; however, the mode of release is not fully understood. Rapid adenosine release modulates oxygen levels and evoked dopamine release, indicating that adenosine may have a rapid modulatory role. In this review, we outline how FSCV can be used to detect adenosine release, compare FSCV with other techniques used to measure adenosine, and present an overview of adenosine signaling that has been characterized using FSCV. These studies point to a rapid mode of adenosine modulation, whose mechanism and function will continue to be characterized in the future.

Adenosine receptors and caffeine in retinopathy of prematurity.

PubMed

Chen, Jiang-Fan; Zhang, Shuya; Zhou, Rong; Lin, Zhenlang; Cai, Xiaohong; Lin, Jing; Huo, Yuqing; Liu, Xiaoling

2017-06-01

Retinopathy of prematurity (ROP) is a major cause of childhood blindness in the world and is caused by oxygen-induced damage to the developing retinal vasculature, resulting in hyperoxia-induced vaso-obliteration and subsequent delayed retinal vascularization and hypoxia-induced pathological neovascularization driven by vascular endothelial growth factor (VEGF) signaling pathway in retina. Current anti-VEGF therapy has shown some effective in a clinical trial, but is associated with the unintended effects on delayed eye growth and retinal vasculature development of preterm infants. Notably, cellular responses to hypoxia are characterized by robust increases in extracellular adenosine production and the markedly induced adenosine receptors, which provide a novel target for preferential control of pathological angiogenesis without affecting normal vascular development. Here, we review the experimental evidence in support of adenosine receptor-based therapeutic strategy for ROP, including the aberrant adenosine signaling in oxygen-induced retinopathy and the role of three adenosine receptor subtypes (A 1 R, A 2A R, A 2B R) in development and treatment of ROP using oxygen-induced retinopathy models. The clinical and initial animal evidence that implicate the therapeutic effect of caffeine (a non-selective adenosine receptor antagonist) in treatment of ROP are highlighted. Lastly, we discussed the translational potential as well therapeutic advantage of adenosine receptor- and caffeine-based therapy for ROR and possibly other proliferative retinopathy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Adenosine receptors and caffeine in retinopathy of prematurity

PubMed Central

Chen, Jiang-Fan; Zhang, Shuya; Zhou, Rong; Lin, Zhenlang; Cai, Xiaohong; Lin, Jing; Huo, Yuqing; Liu, Xiaoling

2017-01-01

Retinopathy of prematurity (ROP) is a major cause of childhood blindness in the world and is caused by oxygen-induced damage to the developing retinal vasculature, resulting in hyperoxia-induced vaso-obliteration and subsequent delayed retinal vascularization and hypoxia-induced pathological neovascularization driven by vascular endothelial growth factor (VEGF) signaling pathway in retina. Current anti-VEGF therapy has shown some effective in a clinical trial, but is associated with the unintended effects on delayed eye growth and retinal vasculature development of preterm infants. Notably, cellular responses to hypoxia are characterized by robust increases in extracellular adenosine production and the markedly induced adenosine receptors, which provide a novel target for preferential control of pathological angiogenesis without affecting normal vascular development. Here, we review the experimental evidence in support of adenosine receptor-based therapeutic strategy for ROP, including the aberrant adenosine signaling in oxygen-induced retinopathy and the role of three adenosine receptor subtypes (A1R, A2AR, A2BR) in development and treatment of ROP using oxygen-induced retinopathy models. The clinical and initial animal evidence that implicate the therapeutic effect of caffeine (a non-selective adenosine receptor antagonist) in treatment of ROP are highlighted. Lastly, we discussed the translational potential as well therapeutic advantage of adenosine receptor- and caffeine-based therapy for ROR and possibly other proliferative retinopathy. PMID:28088487

Application of the newly developed Japanese adenosine normal database for adenosine stress myocardial scintigraphy.

PubMed

Harata, Shingo; Isobe, Satoshi; Morishima, Itsuro; Suzuki, Susumu; Tsuboi, Hideyuki; Sone, Takahito; Ishii, Hideki; Murohara, Toyoaki

2015-10-01

The currently available Japanese normal database (NDB) in stress myocardial perfusion scintigraphy recommended by the Japanese Society of Nuclear Medicine (JSNM-NDB) is created based on the data from exercise tests. The newly developed adenosine normal database (ADS-NDB) remains to be validated for patients undergoing adenosine stress test. We tested whether the diagnostic accuracy of adenosine stress test is improved by the use of ADS-NDB (Kanazawa University). Of 233 consecutive patients undergoing (99m)Tc-MIBI adenosine stress test, 112 patients were tested. The stress/rest myocardial (99m)Tc-MIBI single-photon emission computed tomography (SPECT) images were analyzed by AutoQUANT 7.2 with both ADS-NDB and JSNM-NDB. The summed stress score (SSS) and summed difference score (SDS) were calculated. The agreements of the post-stress defect severity between ADS-NDB and JSNM-NDB were assessed using a weighted kappa statistic. In all patients, mean SSSs of all, right coronary artery (RCA), left anterior descending (LAD), and left circumflex (LCx) territories were significantly lower with ADS-NDB than those with JSNM-NDB. Mean SDSs in all, RCA, and LAD territories were significantly lower with ADS-NDB than those with JSNM-NDB. In 28 patients with significant coronary stenosis, the mean SSS in the RCA territory was significantly lower with ADS-NDB than that with JSNM-NDB. In 84 patients without ischemia, both mean SSSs and SDSs in all, RCA, LAD, and LCx territories were significantly lower with ADS-NDB than those with JSNM-NDB. Weighted kappa values of all patients, patients with significant stenosis, and patients without ischemia were 0.89, 0.83, and 0.92, respectively. Differences were observed between results from ADS-NDB and JSNM-NDB. The diagnostic accuracy of adenosine stress myocardial perfusion scintigraphy may be improved by reducing false-positive results.

Adenosine signaling contributes to ethanol-induced fatty liver in mice

PubMed Central

Peng, Zhongsheng; Borea, Pier Andrea; Wilder, Tuere; Yee, Herman; Chiriboga, Luis; Blackburn, Michael R.; Azzena, Gianfranco; Resta, Giuseppe; Cronstein, Bruce N.

2009-01-01

Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the biochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5′-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5′-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver. PMID:19221436

Adenosine signaling in normal and sickle erythrocytes and beyond.

PubMed

Zhang, Yujin; Xia, Yang

2012-08-01

Sickle cell disease (SCD) is a debilitating hemolytic genetic disorder with high morbidity and mortality affecting millions of individuals worldwide. Although SCD was discovered more than a century ago, no effective mechanism-based prevention and treatment are available due to poorly understood molecular basis of sickling, the fundamental pathogenic process of the disease. SCD patients constantly face hypoxia. One of the best-known signaling molecules to be induced under hypoxic conditions is adenosine. Recent studies demonstrate that hypoxia-mediated elevated adenosine signaling plays an important role in normal erythrocyte physiology. In contrast, elevated adenosine signaling contributes to sickling and multiple life threatening complications including tissue damage, pulmonary dysfunction and priapism. Here, we summarize recent research on the role of adenosine signaling in normal and sickle erythrocytes, progression of the disease and therapeutic implications. In normal erythrocytes, both genetic and pharmacological studies demonstrate that adenosine can enhance 2,3-bisphosphoglycerate (2,3-BPG) production via A(2B) receptor (ADORA2B) activation, suggesting that elevated adenosine has an unrecognized role in normal erythrocytes to promote O(2) release and prevent acute ischemic tissue injury. However, in sickle erythrocytes, the beneficial role of excessive adenosine-mediated 2,3-BPG induction becomes detrimental by promoting deoxygenation, polymerization of sickle hemoglobin and subsequent sickling. Additionally, adenosine signaling via the A(2A) receptor (ADORA2A) on invariant natural killer T (iNKT) cells inhibits iNKT cell activation and attenuates pulmonary dysfunction in SCD mice. Finally, elevated adenosine coupled with ADORA2BR activation is responsible for priapism, a dangerous complication seen in SCD. Overall, the research reviewed here reveals a differential role of elevated adenosine in normal erythrocytes, sickle erythrocytes, iNK cells and

Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells

PubMed Central

Yamaguchi, Hiroshi; Maruyama, Toshihiko; Urade, Yoshihiro; Nagata, Shigekazu

2014-01-01

Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5’-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a ‘calm down’ signal. DOI: http://dx.doi.org/10.7554/eLife.02172.001 PMID:24668173

Reaction kinetics and inhibition of adenosine kinase from Leishmania donovani.

PubMed

Bhaumik, D; Datta, A K

1988-04-01

The reaction kinetics and the inhibitor specificity of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) from Leishmania donovani, have been analysed using homogeneous preparation of the enzyme. The reaction proceeds with equimolar stoichiometry of each reactant. Double reciprocal plots of initial velocity studies in the absence of products yielded intersecting lines for both adenosine and Mg2+-ATP. AMP is a competitive inhibitor of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. In contrast, ADP was a noncompetitive inhibitor with respect to both adenosine and ATP, with inhibition by ADP becoming uncompetitive at very high concentration of ATP. Parallel equilibrium dialysis experiments against [3H]adenosine and [gamma-32P]ATP resulted in binding of adenosine to fre enzyme. Tubercidin (7-deazaadenosine) and 6-methyl-mercaptopurine riboside acted as substrates for the enzyme and were found to inhibit adenosine phosphorylation competitively in vitro. 'Substrate efficiency (Vmax/Km)' and 'turnover numbers (Kcat)' of the enzyme with respect to specific analogs were determined. Taken together the results suggest that (a) the kinetic mechanism of adenosine kinase is sequential Bi-Bi, (b) AMP and ADP may regulate enzyme activity in vivo and (c) tubercidin and 6-methylmercaptopurine riboside are monophosphorylated by the parasite enzyme.

Role of central and peripheral adenosine receptors in the cardiovascular responses to intraperitoneal injections of adenosine A1 and A2A subtype receptor agonists.

PubMed

Schindler, Charles W; Karcz-Kubicha, Marzena; Thorndike, Eric B; Müller, Christa E; Tella, Srihari R; Ferré, Sergi; Goldberg, Steven R

2005-03-01

1. The cardiovascular effects of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) were investigated in rats implanted with telemetry transmitters for the measurement of blood pressure and heart rate. 2. Intraperitoneal (i.p.) injections of the adenosine A1 receptor agonist CPA led to dose-dependent decreases in both blood pressure and heart rate. These effects of 0.3 mg kg(-1) CPA were antagonized by i.p. injections of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine (CPT), but not by i.p. injections of the adenosine A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3). Injections (i.p.) of the peripherally acting nonselective adenosine antagonist 8-sulfophenyltheophylline (8-SPT) and the purported nonselective adenosine antagonist caffeine also antagonized the cardiovascular effects of CPA. 3. The adenosine A2A agonist CGS 21680 given i.p. produced a dose-dependent decrease in blood pressure and an increase in heart rate. These effects of 0.5 mg kg(-1) CGS 21680 were antagonized by i.p. injections of the adenosine A2A receptor antagonist MSX-3, but not by i.p. injections of the antagonists CPT, 8-SPT or caffeine. 4. Central administration (intracerebral ventricular) of CGS 21680 produced an increase in heart rate, but no change in blood pressure. MSX-3 given i.p. antagonized the effects of the central injection of CGS 21680. 5. These results suggest that adenosine A1 receptor agonists produce decreases in blood pressure and heart rate that are mediated by A1 receptors in the periphery, with little or no contribution of central adenosine A1 receptors to those effects. 6. The heart rate increasing effect of adenosine A2A agonists appears to be mediated by adenosine A2A receptors in the central nervous system. The blood pressure decreasing

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

PubMed

Nowarski, Roni; Kotler, Moshe

2013-06-15

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

Correlation of transient adenosine release and oxygen changes in the caudate-putamen

PubMed Central

Wang, Ying; Venton, B. Jill

2016-01-01

Adenosine is an endogenous nucleoside that modulates important physiological processes, such as vasodilation, in the central nervous system. A rapid, 2–4 seconds, mode of adenosine signaling has been recently discovered, but the relationship between this type of adenosine and blood flow change has not been characterized. In this study, adenosine and oxygen changes were simultaneously measured using fast-scan cyclic voltammetry. Oxygen changes occur when there is an increase in local cerebral blood flow and thus are a measure of vasodilation. About 34% of adenosine transients in the rat caudate-putamen are correlated with a subsequent transient change in oxygen. The amount of oxygen was correlated with the concentration of adenosine release and larger adenosine transients (over 0.4 μM) always had subsequent oxygen changes. The average duration of adenosine and oxygen transients were 3.2 seconds and 3.5 seconds, respectively. On average, the adenosine release starts and peaks 0.2 seconds prior to the oxygen. The A2a antagonist, SCH442416, decreased the number of both adenosine and oxygen transient events by about 32%. However, the A1 antagonist, DPCPX, did not significantly affect simultaneous adenosine and oxygen release. The nitric oxide (NO) synthase inhibitor L-NAME also did not affect the concentration or number of adenosine and oxygen release events. These results demonstrate that both adenosine and oxygen release are modulated via A2a receptors. The correlation of transient concentrations, time delay between adenosine and oxygen peaks, and effect of A2a receptors suggests adenosine modulates blood flow on a rapid, sub-second time scale. PMID:27314215

Ticagrelor and Rosuvastatin Have Additive Cardioprotective Effects via Adenosine.

PubMed

Birnbaum, Yochai; Birnbaum, Gilad D; Birnbaum, Itamar; Nylander, Sven; Ye, Yumei

2016-12-01

Ticagrelor inhibits the equilibrative-nucleoside-transporter-1 and thereby, adenosine cell re-uptake. Ticagrelor limits infarct size (IS) in non-diabetic rats and the effect is adenosine-dependent. Statins, via ecto-5'-nucleotidase activation, also increase adenosine levels and limit IS. Ticagrelor and rosuvastatin have additive effects on myocardial adenosine levels, and therefore, on IS and post-reperfusion activation of the NLRP3-inflammasome. Diabetic ZDF rats received via oral gavage; water (control), ticagrelor (150 mg/kg/d), prasugrel (7.5 mg/kg/d), rosuvastatin (5 mg/kg/d), ticagrelor + rosuvastatin and prasugrel + rosuvastatin for 3d. On day 4, rats underwent 30 min coronary artery occlusion and 24 h of reperfusion. Two additional groups received, ticagrelor + rosuvastatin or water in combination with CGS15943 (CGS, an adenosine receptor antagonist, 10 mg/kg i.p. 1 h before ischemia). Both ticagrelor and rosuvastatin increased myocardial adenosine levels with an additive effect of the combination whereas prasugrel had no effect. Similarly, both ticagrelor and rosuvastatin significantly reduced IS with an additive effect of the combination whereas prasugrel had no effect. The effect on IS was adenosine dependent as CGS15943 reversed the effect of ticagrelor + rosuvastatin. The ischemia-reperfusion injury increased myocardial mRNA levels of NLRP3, ASC, IL-1β and IL-6. Ticagrelor and rosuvastatin, but not prasugrel, significantly decreased these pro-inflammatory mediators with a trend to an additive effect of the combination. The combination also increased the levels of anti-inflammatory 15-epilipoxin A 4 . Ticagrelor and rosuvastatin when given in combination have an additive effect on local myocardial adenosine levels in the setting of ischemia reperfusion. This translates into an additive cardioprotective effect mediated by adenosine-induced effects including downregulation of pro- but upregulation of anti-inflammatory mediators.

Detrimental effects of adenosine signaling in sickle cell disease

PubMed Central

Zhang, Yujin; Dai, Yingbo; Wen, Jiaming; Zhang, Weiru; Grenz, Almut; Sun, Hong; Tao, Lijian; Lu, Guangxiu; Alexander, Danny C; Milburn, Michael V; Carter-Dawson, Louvenia; Lewis, Dorothy E; Zhang, Wenzheng; Eltzschig, Holger K; Kellems, Rodney E; Blackburn, Michael R; Juneja, Harinder S; Xia, Yang

2016-01-01

Hypoxia can act as an initial trigger to induce erythrocyte sickling and eventual end organ damage in sickle cell disease (SCD). Many factors and metabolites are altered in response to hypoxia and may contribute to the pathogenesis of the disease. Using metabolomic profiling, we found that the steady-state concentration of adenosine in the blood was elevated in a transgenic mouse model of SCD. Adenosine concentrations were similarly elevated in the blood of humans with SCD. Increased adenosine levels promoted sickling, hemolysis and damage to multiple tissues in SCD transgenic mice and promoted sickling of human erythrocytes. Using biochemical, genetic and pharmacological approaches, we showed that adenosine A2B receptor (A2BR)-mediated induction of 2,3-diphosphoglycerate, an erythrocyte-specific metabolite that decreases the oxygen binding affinity of hemoglobin, underlies the induction of erythrocyte sickling by excess adenosine both in cultured human red blood cells and in SCD transgenic mice. Thus, excessive adenosine signaling through the A2BR has a pathological role in SCD. These findings may provide new therapeutic possibilities for this disease. PMID:21170046

Detrimental effects of adenosine signaling in sickle cell disease.

PubMed

Zhang, Yujin; Dai, Yingbo; Wen, Jiaming; Zhang, Weiru; Grenz, Almut; Sun, Hong; Tao, Lijian; Lu, Guangxiu; Alexander, Danny C; Milburn, Michael V; Carter-Dawson, Louvenia; Lewis, Dorothy E; Zhang, Wenzheng; Eltzschig, Holger K; Kellems, Rodney E; Blackburn, Michael R; Juneja, Harinder S; Xia, Yang

2011-01-01

Hypoxia can act as an initial trigger to induce erythrocyte sickling and eventual end organ damage in sickle cell disease (SCD). Many factors and metabolites are altered in response to hypoxia and may contribute to the pathogenesis of the disease. Using metabolomic profiling, we found that the steady-state concentration of adenosine in the blood was elevated in a transgenic mouse model of SCD. Adenosine concentrations were similarly elevated in the blood of humans with SCD. Increased adenosine levels promoted sickling, hemolysis and damage to multiple tissues in SCD transgenic mice and promoted sickling of human erythrocytes. Using biochemical, genetic and pharmacological approaches, we showed that adenosine A(2B) receptor (A(2B)R)-mediated induction of 2,3-diphosphoglycerate, an erythrocyte-specific metabolite that decreases the oxygen binding affinity of hemoglobin, underlies the induction of erythrocyte sickling by excess adenosine both in cultured human red blood cells and in SCD transgenic mice. Thus, excessive adenosine signaling through the A(2B)R has a pathological role in SCD. These findings may provide new therapeutic possibilities for this disease.

Elevated placental adenosine signaling contributes to the pathogenesis of preeclampsia.

PubMed

Iriyama, Takayuki; Sun, Kaiqi; Parchim, Nicholas F; Li, Jessica; Zhao, Cheng; Song, Anren; Hart, Laura A; Blackwell, Sean C; Sibai, Baha M; Chan, Lee-Nien L; Chan, Teh-Sheng; Hicks, M John; Blackburn, Michael R; Kellems, Rodney E; Xia, Yang

2015-02-24

Preeclampsia is a prevalent hypertensive disorder of pregnancy and a leading cause of maternal and neonatal morbidity and mortality worldwide. This pathogenic condition is speculated to be caused by placental abnormalities that contribute to the maternal syndrome. However, the specific factors and signaling pathways that lead to impaired placentas and maternal disease development remain elusive. Using 2 independent animal models of preeclampsia (genetically engineered pregnant mice with elevated adenosine exclusively in placentas and a pathogenic autoantibody-induced preeclampsia mouse model), we demonstrated that chronically elevated placental adenosine was sufficient to induce hallmark features of preeclampsia, including hypertension, proteinuria, small fetuses, and impaired placental vasculature. Genetic and pharmacological approaches revealed that elevated placental adenosine coupled with excessive A₂B adenosine receptor (ADORA2B) signaling contributed to the development of these features of preeclampsia. Mechanistically, we provided both human and mouse evidence that elevated placental CD73 is a key enzyme causing increased placental adenosine, thereby contributing to preeclampsia. We determined that elevated placental adenosine signaling is a previously unrecognized pathogenic factor for preeclampsia. Moreover, our findings revealed the molecular basis underlying the elevation of placental adenosine and the detrimental role of excess placental adenosine in the pathophysiology of preeclampsia, and thereby, we highlight novel therapeutic targets. © 2014 American Heart Association, Inc.

Activities of purine converting enzymes in heart, liver and kidney mice LDLR-/- and Apo E-/.

PubMed

Rybakowska, I M; Kutryb-Zając, B; Milczarek, R; Łukasz, B; Slominska, E M; Smolenski, R T

2018-05-21

Nucleotide metabolism plays a major role in a number of vital cellular processes such as energetics. This, in turn, is important in pathologies such as atherosclerosis. Three month old atherosclerotic mice with knock outs for LDLR and apolipoprotein E (ApoE) were used for the experiments. Activities of AMP-deaminase (AMPD), ecto5'-nucleotidase (e5NT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) were measured in heart, liver and kidney cortex and medulla by analysing conversion of substrates into products using HPLC. The activity of ecto5'-nucleotidase differ in hearts of LDLR -/- and ApoE -/- mice with no differences in ADA and AMPD activity. We noticed highest activity of e5NT in kidney medulla of the models. This model of atherosclerosis characterize with an inhibition of enzyme responsible for production of protective adenosine in heart but not in other organs and different metabolism of nucleotides in kidney medulla.

Genetic mapping uncovers cis-regulatory landscape of RNA editing.

PubMed

Ramaswami, Gokul; Deng, Patricia; Zhang, Rui; Anna Carbone, Mary; Mackay, Trudy F C; Li, Jin Billy

2015-09-16

Adenosine-to-inosine (A-to-I) RNA editing, catalysed by ADAR enzymes conserved in metazoans, plays an important role in neurological functions. Although the fine-tuning mechanism provided by A-to-I RNA editing is important, the underlying rules governing ADAR substrate recognition are not well understood. We apply a quantitative trait loci (QTL) mapping approach to identify genetic variants associated with variability in RNA editing. With very accurate measurement of RNA editing levels at 789 sites in 131 Drosophila melanogaster strains, here we identify 545 editing QTLs (edQTLs) associated with differences in RNA editing. We demonstrate that many edQTLs can act through changes in the local secondary structure for edited dsRNAs. Furthermore, we find that edQTLs located outside of the edited dsRNA duplex are enriched in secondary structure, suggesting that distal dsRNA structure beyond the editing site duplex affects RNA editing efficiency. Our work will facilitate the understanding of the cis-regulatory code of RNA editing.

Adenosine uptake by the isolated epithelium of guine pig jejunum.

PubMed

Kolassa, N; Stengg, R; Turnheim, K

1977-10-01

The uptake of [8-14C]adenosine by the isolated epithelium of guinea pig jejunum was faster than that of inosine, hypoxanthine, or adenine. The initial velocity of adenosine uptake from both the luminal and the antiluminal side of the epithelium exhibited saturation kinetics. The apparent Km, V, and passive permeability of luminal adenosine uptake were all lower than the corresponding values of antiluminal uptake. p-Nitrobenzyl-thioguanosine inhibited adenosine uptake from both the luminal and the antiluminal side, whilst hexobendine decreased the uptake only from the antiluminal side of the epithelium. The results suggest that adenosine enters the intestinal epithelium by a carrier-mediated process in addition to passive diffusion. The antiluminal transport system for adenosine seems similar to that of other tissues with respect to hexobendine inhibition; the luminal transport mechanism, however, exhibits different properties, being insensitive to hexobendine.

Ab initio ONIOM-molecular dynamics (MD) study on the deamination reaction by cytidine deaminase.

PubMed

Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

2007-08-23

We applied the ONIOM-molecular dynamics (MD) method to the hydrolytic deamination of cytidine by cytidine deaminase, which is an essential step of the activation process of the anticancer drug inside the human body. The direct MD simulations were performed for the realistic model of cytidine deaminase by calculating the energy and its gradient by the ab initio ONIOM method on the fly. The ONIOM-MD calculations including the thermal motion show that the neighboring amino acid residue is an important factor of the environmental effects and significantly affects not only the geometry and energy of the substrate trapped in the pocket of the active site but also the elementary step of the catalytic reaction. We successfully simulate the second half of the catalytic cycle, which has been considered to involve the rate-determining step, and reveal that the rate-determining step is the release of the NH3 molecule.

EFFECTS OF X IRRADIATION ON ENZYME SYNTHESIS DURING LIVER REGENERATION

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myers, D.K.

1962-05-01

Twenty-four different enzymes or enzyme systems were assayed in regenerating rat liver from control and irradiated animals at various times after partial hepatectomy. X irradiation, either of the whole liver region or of an exteriorized liver lobule, interfered with the accumulation of only three of these enzymes: deoxycytidylate deaminase, thymidine phosphorylase, and NAD pyrophosphorylase. Irradiation did not affect the synthesis of related enzymes such as adenosine and guanine deaminases, and inosine and uridine phosphorylases. The effects of irradiation on enzyme synthesis in regenerating liver would appear to be highly selective. (auth)

Reentry Tachycardia in Children: Adenosine Can Make It Worse.

PubMed

Hien, Maximilian D; Benito Castro, Fernando; Fournier, Philippe; Filleron, Anne; Tran, Tu-Anh

2016-10-08

We report on a rare but severe complication of adenosine use in a child with reentry tachycardia. Treatment with adenosine, which is the standard medical therapy of atrioventricular reentry tachycardia, led to the development of an irregular wide complex tachycardia, caused by rapid ventricular response to atrial fibrillation. The girl was finally stabilized with electrical cardioversion. We analyze the pathomechanism and discuss possible treatment options. Atrial fibrillation, as well as its conduction to the ventricles, can be caused by adenosine. Rapid ventricular response in children with Wolff-Parkinson-White syndrome is more frequent than previously believed. A patient history of atrial fibrillation is a contraindication for cardioversion with adenosine and needs to be assessed in children with reentry tachycardia. High-risk patients may potentially profit from prophylactic comedication with antiarrhythmic agents, such as flecainide, ibutilide, or vernakalant, before adenosine administration.

LDL-cholesterol reduction in patients with hypercholesterolemia by modulation of adenosine triphosphate-citrate lyase and adenosine monophosphate-activated protein kinase.

PubMed

Filippov, Sergey; Pinkosky, Stephen L; Newton, Roger S

2014-08-01

To review the profile of ETC-1002, as shown in preclinical and clinical studies, including LDL-cholesterol (LDL-C)-lowering activity and beneficial effects on other cardiometabolic risk markers as they relate to the inhibition of adenosine triphosphate-citrate lyase and the activation of adenosine monophosphate-activated protein kinase. ETC-1002 is an adenosine triphosphate-citrate lyase inhibitor/adenosine monophosphate-activated protein kinase activator currently in Phase 2b clinical development. In seven Phase 1 and Phase 2a clinical studies, ETC-1002 dosed once daily for 2-12 weeks has lowered LDL-C and reduced high-sensitivity C-reactive protein by up to 40%, with neutral to positive effects on glucose levels, blood pressure, and body weight. Importantly, use of ETC-1002 in statin-intolerant patients has shown statin-like lowering of LDL-C without the muscle pain and weakness responsible for discontinuation of statin use by many patients. ETC-1002 has also been shown to produce an incremental benefit, lowering LDL-C as an add-on therapy to a low-dose statin. In over 300 individuals in studies of up to 12 weeks, ETC-1002 has been well tolerated with no serious adverse effects. Because adenosine triphosphate-citrate lyase and adenosine monophosphate-activated protein kinase play central roles in regulating lipid and glucose metabolism, pharmacological modulation of these two enzymes could provide an important therapeutic alternative for statin-intolerant patients with hypercholesterolemia.

TOR-induced resistance to toxic adenosine analogs in Leishmania brought about by the internalization and degradation of the adenosine permease.

PubMed

Detke, Siegfried

2007-05-15

TOR is an atypical multidrug resistance protein present in the human protozoan parasite, Leishmania. Resistance to the toxic adenosine analog tubercidin was brought about by redirecting the adenosine permease from the plasma membrane to the multivesicular tubule lysosome. The cells became resistant to tubercidin because they were unable to take up and accumulate this toxic purine. The domain, which was recognized by TOR in this internalization pathway, was identified by expressing portions of this transporter in Leishmania and assessing whether they were capable of hindering the multidrug resistance capability of TOR. This approach identified the adenosine permease region spanning Met289 to Trp305. This region was also the epitope recognized by the internalization mechanism. An internal deletion mutant lacking Met289-Trp305 was functionally active but could no longer be internalized in cells with high TOR levels. The internalization and altered trafficking of the adenosine permease by TOR was observed in yeast and human embryonic kidney cells co-expressing these two Leishmania proteins indicating that the internalization process was conserved in evolutionary diverse organisms. The inability of Saccharomyces with a temperature-sensitive ubiquitin ligase to internalize adenosine permease suggested that ubiquitination was involved in this altered trafficking.

Beneficial and detrimental role of adenosine signaling in diseases and therapy

PubMed Central

Liu, Hong

2015-01-01

Adenosine is a major signaling nucleoside that orchestrates cellular and tissue adaptation under energy depletion and ischemic/hypoxic conditions by activation of four G protein-coupled receptors (GPCR). The regulation and generation of extracellular adenosine in response to stress are critical in tissue protection. Both mouse and human studies reported that extracellular adenosine signaling plays a beneficial role during acute states. However, prolonged excess extracellular adenosine is detrimental and contributes to the development and progression of various chronic diseases. In recent years, substantial progress has been made to understand the role of adenosine signaling in different conditions and to clarify its significance during the course of disease progression in various organs. These efforts have and will identify potential therapeutic possibilities for protection of tissue injury at acute stage by upregulation of adenosine signaling or attenuation of chronic disease progression by downregulation of adenosine signaling. This review is to summarize current progress and the importance of adenosine signaling in different disease stages and its potential therapeutic effects. PMID:26316513

Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes

PubMed Central

Taylor, Benjamin JM; Wu, Yee Ling; Rada, Cristina

2014-01-01

Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors. DOI: http://dx.doi.org/10.7554/eLife.03553.001 PMID:25237741

Low-dose adenosine stress echocardiography: detection of myocardial viability.

PubMed

Djordjevic-Dikic, Ana; Ostojic, Miodrag; Beleslin, Branko; Nedeljkovic, Ivana; Stepanovic, Jelena; Stojkovic, Sinisa; Petrasinovic, Zorica; Nedeljkovic, Milan; Saponjski, Jovica; Giga, Vojislav

2003-06-03

The aim of this study was to evaluate the diagnostic potential of low-dose adenosine stress echocardiography in detection of myocardial viability. Vasodilation through low dose dipyridamole infusion may recruit contractile reserve by increasing coronary flow or by increasing levels of endogenous adenosine. Forty-three patients with resting dyssynergy, due to previous myocardial infarction, underwent low-dose adenosine (80, 100, 110 mcg/kg/min in 3 minutes intervals) echocardiography test. Gold standard for myocardial viability was improvement in systolic thickening of dyssinergic segments of >or= 1 grade at follow-up. Coronary angiography was done in 41 pts. Twenty-seven patients were revascularized and 16 were medically treated. Echocardiographic follow up data (12 +/- 2 months) were available in 24 revascularized patients. Wall motion score index improved from rest 1.55 +/- 0.30 to 1.33 +/- 0.26 at low-dose adenosine (p < 0.001). Of the 257 segments with baseline dyssynergy, adenosine echocardiography identified 122 segments as positive for viability, and 135 as necrotic since no improvement of systolic thickening was observed. Follow-up wall motion score index was 1.31 +/- 0.30 (p < 0.001 vs. rest). The sensitivity of adenosine echo test for identification of viable segments was 87%, while specificity was 95%, and diagnostic accuracy 90%. Positive and negative predictive values were 97% and 80%, respectively. Low-dose adenosine stress echocardiography test has high diagnostic potential for detection of myocardial viability in the group of patients with left ventricle dysfunction due to previous myocardial infarction. Low dose adenosine stress echocardiography may be adequate alternative to low-dose dobutamine test for evaluation of myocardial viability.

Low-dose adenosine stress echocardiography: Detection of myocardial viability

PubMed Central

Djordjevic-Dikic, Ana; Ostojic, Miodrag; Beleslin, Branko; Nedeljkovic, Ivana; Stepanovic, Jelena; Stojkovic, Sinisa; Petrasinovic, Zorica; Nedeljkovic, Milan; Saponjski, Jovica; Giga, Vojislav

2003-01-01

Objective The aim of this study was to evaluate the diagnostic potential of low-dose adenosine stress echocardiography in detection of myocardial viability. Background Vasodilation through low dose dipyridamole infusion may recruit contractile reserve by increasing coronary flow or by increasing levels of endogenous adenosine. Methods Forty-three patients with resting dyssynergy, due to previous myocardial infarction, underwent low-dose adenosine (80, 100, 110 mcg/kg/min in 3 minutes intervals) echocardiography test. Gold standard for myocardial viability was improvement in systolic thickening of dyssinergic segments of ≥ 1 grade at follow-up. Coronary angiography was done in 41 pts. Twenty-seven patients were revascularized and 16 were medically treated. Echocardiographic follow up data (12 ± 2 months) were available in 24 revascularized patients. Results Wall motion score index improved from rest 1.55 ± 0.30 to 1.33 ± 0.26 at low-dose adenosine (p < 0.001). Of the 257 segments with baseline dyssynergy, adenosine echocardiography identified 122 segments as positive for viability, and 135 as necrotic since no improvement of systolic thickening was observed. Follow-up wall motion score index was 1.31 ± 0.30 (p < 0.001 vs. rest). The sensitivity of adenosine echo test for identification of viable segments was 87%, while specificity was 95%, and diagnostic accuracy 90%. Positive and negative predictive values were 97% and 80%, respectively. Conclusion Low-dose adenosine stress echocardiography test has high diagnostic potential for detection of myocardial viability in the group of patients with left ventricle dysfunction due to previous myocardial infarction. Low dose adenosine stress echocardiography may be adequate alternative to low-dose dobutamine test for evaluation of myocardial viability. PMID:12812523

Enzymatic properties of Staphylococcus aureus adenosine synthase (AdsA)

PubMed Central

2011-01-01

Background Staphylococcus aureus is a human pathogen that produces extracellular adenosine to evade clearance by the host immune system, an activity attributed to the 5'-nucleotidase activity of adenosine synthase (AdsA). In mammals, conversion of adenosine triphosphate to adenosine is catalyzed in a two-step process: ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTDPases) hydrolyze ATP and ADP to AMP, whereas 5'-nucleotidases hydrolyze AMP to adenosine. NTPDases harbor apyrase conserved regions (ACRs) that are critical for activity. Results NTPDase ACR motifs are absent in AdsA, yet we report here that recombinant AdsA hydrolyzes ADP and ATP in addition to AMP. Competition assays suggest that hydrolysis occurs following binding of all three substrates at a unique site. Alanine substitution of two amino acids, aspartic acid 127 and histidine 196 within the 5'-nucleotidase signature sequence, leads to reduced AMP or ADP hydrolysis but does not affect the binding of these substrates. Conclusion Collectively, these results provide insight into the unique ability of AdsA to produce adenosine through the consecutive hydrolysis of ATP, ADP and AMP, thereby endowing S. aureus with the ability to modulate host immune responses. PMID:22035583

Metabolic changes of cultured DRG neurons induced by adenosine using confocal microscopy imaging

NASA Astrophysics Data System (ADS)

Zheng, Liqin; Huang, Yimei; Chen, Jiangxu; Wang, Yuhua; Yang, Hongqin; Zhang, Yanding; Xie, Shusen

2012-12-01

Adenosine exerts multiple effects on pain transmission in the peripheral nervous system. This study was performed to use confocal microscopy to evaluate whether adenosine could affect dorsal root ganglia (DRG) neurons in vitro and test which adenosine receptor mediates the effect of adenosine on DRG neurons. After adding adenosine with different concentration, we compared the metabolic changes by the real time imaging of calcium and mitochondria membrane potential using confocal microscopy. The results showed that the effect of 500 μM adenosine on the metabolic changes of DRG neurons was more significant than others. Furthermore, four different adenosine receptor antagonists were used to study which receptor mediated the influences of adenosine on the cultured DRG neurons. All adenosine receptor antagonists especially A1 receptor antagonist (DPCPX) had effect on the Ca2+ and mitochondria membrane potential dynamics of DRG neurons. The above studies demonstrated that the effect of adenosine which may be involved in the signal transmission on the sensory neurons was dose-dependent, and all the four adenosine receptors especially the A1R may mediate the transmission.

Adenosine signaling promotes regeneration of pancreatic β-cells in vivo

PubMed Central

Andersson, Olov; Adams, Bruce A.; Yoo, Daniel; Ellis, Gregory C.; Gut, Philipp; Anderson, Ryan M.; German, Michael S.; Stainier, Didier Y. R.

2012-01-01

Diabetes can be controlled with insulin injections, but a curative approach that restores the number of insulin-producing β-cells is still needed. Using a zebrafish model of diabetes, we screened ~7000 small molecules to identify enhancers of β-cell regeneration. The compounds we identified converge on the adenosine signaling pathway and include exogenous agonists and compounds that inhibit degradation of endogenously produced adenosine. The most potent enhancer of β-cell regeneration was the adenosine agonist 5′-N-Ethylcarboxamidoadenosine (NECA), which acting through the adenosine receptor A2aa increased β-cell proliferation and accelerated restoration of normoglycemia in zebrafish. Despite markedly stimulating β-cell proliferation during regeneration, NECA had only a modest effect during development. The proliferative and glucose-lowering effect of NECA was confirmed in diabetic mice, suggesting an evolutionarily conserved role for adenosine in β-cell regeneration. With this whole-organism screen, we identified components of the adenosine pathway that could be therapeutically targeted for the treatment of diabetes. PMID:22608007

Mediation of tubuloglomerular feedback by adenosine: evidence from mice lacking adenosine 1 receptors.

PubMed

Sun, D; Samuelson, L C; Yang, T; Huang, Y; Paliege, A; Saunders, T; Briggs, J; Schnermann, J

2001-08-14

Adenosine is a determinant of metabolic control of organ function increasing oxygen supply through the A2 class of adenosine receptors and reducing oxygen demand through A1 adenosine receptors (A1AR). In the kidney, activation of A1AR in afferent glomerular arterioles has been suggested to contribute to tubuloglomerular feedback (TGF), the vasoconstriction elicited by elevations in [NaCl] in the macula densa region of the nephron. To further elucidate the role of A1AR in TGF, we have generated mice in which the entire A1AR coding sequence was deleted by homologous recombination. Homozygous A1AR mutants that do not express A1AR mRNA transcripts and do not respond to A1AR agonists are viable and without gross anatomical abnormalities. Plasma and urinary electrolytes were not different between genotypes. Likewise, arterial blood pressure, heart rates, and glomerular filtration rates were indistinguishable between A1AR(+/+), A1AR(+/-), and A1AR(-/-) mice. TGF responses to an increase in loop of Henle flow rate from 0 to 30 nl/min, whether determined as change of stop flow pressure or early proximal flow rate, were completely abolished in A1AR(-/-) mice (stop flow pressure response, -6.8 +/- 0.55 mmHg and -0.4 +/- 0.2 in A1AR(+/+) and A1AR(-/-) mice; early proximal flow rate response, -3.4 +/- 0.4 nl/min and +0.02 +/- 0.3 nl/min in A1AR(+/+) and A1AR(-/-) mice). Absence of TGF responses in A1AR-deficient mice suggests that adenosine is a required constituent of the juxtaglomerular signaling pathway. A1AR null mutant mice are a promising tool to study the functional role of A1AR in different target tissues.

Ethanol Tolerance Affects Endogenous Adenosine Signaling in Mouse Hippocampus

PubMed Central

Zhang, Dali; Xiong, Wei; Jackson, Michael F.

2016-01-01

Ethanol has many pharmacological effects, including increases in endogenous adenosine levels and adenosine receptor activity in brain. Ethanol consumption is associated with both positive and negative health outcomes, but tolerance to the behavioral effects of ethanol can lead to increased consumption, which increases the risk of negative health outcomes. The present study was performed to test whether a 7-day treatment with ethanol is linked to reduced adenosine signaling and whether this is a consequence of reduced ecto-5′-nucleotidase activity. Wild-type (CD73+/+) and ecto-5′-nucleotidase-deficient (CD73−/−) mice were treated with ethanol (2 g/kg) or saline for 7 days. In CD73+/+ mice, repeated ethanol treatment reduced the hypothermic and ataxic effects of acute ethanol, indicating the development of tolerance to the acute effects of ethanol. In CD73+/+ mice, this 7-day ethanol treatment led to increased hippocampal synaptic activity and reduced adenosine A1 receptor activity under both basal and low Mg2+ conditions. These effects of ethanol tolerance were associated with an 18% decrease in activity of ecto-5′-nucleotidase activity in hippocampal cell membranes. In contrast, ethanol treatment was not associated with changes in synaptic activity or adenosine signaling in hippocampus from CD73−/− mice. These data indicate that ethanol treatment is associated with a reduction in adenosine signaling through adenosine A1 receptors in hippocampus, mediated, at least in part, via reduced ecto-5′-nucleotidase activity. PMID:27189965

Ethanol Tolerance Affects Endogenous Adenosine Signaling in Mouse Hippocampus.

PubMed

Zhang, Dali; Xiong, Wei; Jackson, Michael F; Parkinson, Fiona E

2016-07-01

Ethanol has many pharmacological effects, including increases in endogenous adenosine levels and adenosine receptor activity in brain. Ethanol consumption is associated with both positive and negative health outcomes, but tolerance to the behavioral effects of ethanol can lead to increased consumption, which increases the risk of negative health outcomes. The present study was performed to test whether a 7-day treatment with ethanol is linked to reduced adenosine signaling and whether this is a consequence of reduced ecto-5'-nucleotidase activity. Wild-type (CD73(+/+)) and ecto-5'-nucleotidase-deficient (CD73(-/-)) mice were treated with ethanol (2 g/kg) or saline for 7 days. In CD73(+/+) mice, repeated ethanol treatment reduced the hypothermic and ataxic effects of acute ethanol, indicating the development of tolerance to the acute effects of ethanol. In CD73(+/+) mice, this 7-day ethanol treatment led to increased hippocampal synaptic activity and reduced adenosine A1 receptor activity under both basal and low Mg(2+) conditions. These effects of ethanol tolerance were associated with an 18% decrease in activity of ecto-5'-nucleotidase activity in hippocampal cell membranes. In contrast, ethanol treatment was not associated with changes in synaptic activity or adenosine signaling in hippocampus from CD73(-/-) mice. These data indicate that ethanol treatment is associated with a reduction in adenosine signaling through adenosine A1 receptors in hippocampus, mediated, at least in part, via reduced ecto-5'-nucleotidase activity. Copyright © 2016 The Author(s).

Estimation of skeletal muscle interstitial adenosine during forearm dynamic exercise in humans

NASA Technical Reports Server (NTRS)

Costa, F.; Heusinkveld, J.; Ballog, R.; Davis, S.; Biaggioni, I.

2000-01-01

It has been proposed that adenosine is a metabolic signal that triggers activation of muscle afferents involved in the exercise pressor reflex. Furthermore, exogenous adenosine induces sympathetic activation that mimics the exercise pressor reflex, and blockade of adenosine receptors inhibits sympathetic activation induced by exercise. Thus, we hypothesize that adenosine is released locally by the muscle during exercise. We used microdialysis probes, placed in the flexor digitorium superficialis muscle, to estimate muscle interstitial adenosine levels in humans. We estimated resting in vivo muscle interstitial adenosine concentrations (0.292+/-0.058 micromol/L, n=4) by perfusing increasing concentrations of adenosine to determine the gradient produced in the dialysate. Muscle interstitial adenosine concentrations increased from 0.23+/-0.04 to 0.82+/-0.14 micromol/L (n=14, P<0.001) during intermittent dynamic exercise at 50% of maximal voluntary contraction. Lactate increased from 0.8+/-0.1 to 2.3+/-0.3 mmol/L (P<0.001). Lower intensity (15% maximal voluntary contraction) intermittent dynamic exercise increased adenosine concentrations from 0.104+/-0.02 to 0.42+/-0.16 micromol/L (n=7). The addition of ischemia to this low level of exercise produced a greater increase in adenosine (from 0.095+/-0.02 to 0.48+/-0.2 micromol/L) compared with nonischemic exercise (0. 095+/-0.02 to 0.25+/-0.12 micromol/L). These results indicate that microdialysis is useful in estimating adenosine concentrations and in reflecting changes in muscle interstitial adenosine during dynamic exercise in humans.

Extracellular cyclic AMP-adenosine pathway in isolated adipocytes and adipose tissue.

PubMed

Strouch, Marci B; Jackson, Edwin K; Mi, Zaichuan; Metes, Nicole A; Carey, Gale B

2005-06-01

Our goal was to evaluate the presence and lipolytic impact of the extracellular cyclic adenosine monophosphate (AMP)-adenosine pathway in adipose tissue. Sixteen miniature Yucatan swine (Sus scrofa) were used for these in vitro and in situ experiments. Four microdialysis probes were implanted into subcutaneous adipose tissue and perfused at 2 microL/min with Ringer's solution containing no addition, varying levels of cyclic AMP, 10 microM isoproterenol, or 10 microM isoproterenol plus 1 mM alpha,beta-methylene adenosine 5'-diphosphate (AMPCP), a 5'-nucleotidase inhibitor. Dialysate was assayed for AMP, adenosine, inosine, hypoxanthine, and glycerol. Freshly isolated adipocytes were incubated with buffer, 1 microM isoproterenol, or 1 microM isoproterenol plus 0.1 mM AMPCP, and extracellular levels of AMP, adenosine, inosine, hypoxanthine, and glycerol were measured. Perfusion of adipose tissue with exogenous cyclic AMP caused a significant increase in AMP and adenosine appearance. Perfusion with AMPCP, in the presence or absence of isoproterenol, significantly increased the levels of AMP and glycerol, whereas it significantly reduced the level of adenosine and its metabolites. However, the AMPCP-provoked increase in lipolysis observed in situ and in vitro was not temporally associated with a decrease in adenosine. These data suggest the existence of a cyclic AMP-adenosine pathway in adipocytes and adipose tissue. The role of this pathway in the regulation of lipolysis remains to be clarified.

TOR induced resistance to toxic adenosine analogs in Leishmania brought about by the internalization and degradation of the adenosine permease

PubMed Central

Detke, Siegfried

2007-01-01

TOR is an atypical multidrug resistance protein present in the human protozoan parasite, Leishmania. Resistance to the toxic adenosine analog tubercidin was brought about by redirecting the adenosine permease from the plasma membrane to the multivesicular tubule lysosome. The cells became resistant to tubercidin because they were unable to take up and accumulate this toxic purine. The domain which was recognized by TOR in this internalization pathway was identified by expressing portions of this transporter in Leishmania and assessing whether they were capable of hindering the multidrug resistance capability of TOR. This approach identified the adenosine permease region spanning Met289 to Trp305. This region was also the epitope recognized by the internalization mechanism. An internal deletion mutant lacking Met289-Trp305 was functionally active but could no longer be internalized in cells with high TOR levels. The internalization and altered trafficking of the adenosine permease by TOR was observed in yeast and human embryonic kidney cells co-expressing these two Leishmania proteins indicating that the internalization process was conserved in evolutionary diverse organisms. The inability of Saccharomyces with a temperature sensitive ubiquitin ligase to internalize adenosine permease suggested that ubiquitination was involved in this altered trafficking. PMID:17428463

The catalase activity of diiron adenine deaminase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamat S. S.; Swaminathan S.; Holmes-Hampton, G. P.

2011-12-01

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

Endogenous adenosine produced during hypoxia attenuates neutrophil accumulation: coordination by extracellular nucleotide metabolism.

PubMed

Eltzschig, Holger K; Thompson, Linda F; Karhausen, Jorn; Cotta, Richard J; Ibla, Juan C; Robson, Simon C; Colgan, Sean P

2004-12-15

Hypoxia is a well-documented inflammatory stimulus and results in tissue polymorphonuclear leukocyte (PMN) accumulation. Likewise, increased tissue adenosine levels are commonly associated with hypoxia, and given the anti-inflammatory properties of adenosine, we hypothesized that adenosine production via adenine nucleotide metabolism at the vascular surface triggers an endogenous anti-inflammatory response during hypoxia. Initial in vitro studies indicated that endogenously generated adenosine, through activation of PMN adenosine A(2A) and A(2B) receptors, functions as an antiadhesive signal for PMN binding to microvascular endothelia. Intravascular nucleotides released by inflammatory cells undergo phosphohydrolysis via hypoxia-induced CD39 ectoapyrase (CD39 converts adenosine triphosphate/adenosine diphosphate [ATP/ADP] to adenosine monophosphate [AMP]) and CD73 ecto-5'-nucleotidase (CD73 converts AMP to adenosine). Extensions of our in vitro findings using cd39- and cd73-null animals revealed that extracellular adenosine produced through adenine nucleotide metabolism during hypoxia is a potent anti-inflammatory signal for PMNs in vivo. These findings identify CD39 and CD73 as critical control points for endogenous adenosine generation and implicate this pathway as an innate mechanism to attenuate excessive tissue PMN accumulation.

Adenosine for postoperative analgesia: A systematic review and meta-analysis

PubMed Central

2017-01-01

Purpose Perioperative infusion of adenosine has been suggested to reduce the requirement for inhalation anesthetics, without causing serious adverse effects in humans. We conducted a meta-analysis of randomized controlled trials evaluating the effect of adenosine on postoperative analgesia. Methods We retrieved articles in computerized searches of Scopus, Web of Science, PubMed, EMBASE, and Cochrane Library databases, up to July 2016. We used adenosine, postoperative analgesia, and postoperative pain(s) as key words, with humans, RCT, and CCT as filters. Data of eligible studies were extracted, which included pain scores, cumulative opioid consumption, adverse reactions, and vital signs. Overall incidence rates, relative risk (RR), and 95% confidence intervals (CI) were calculated employing fixed-effects or random-effects models, depending on the heterogeneity of the included trials. Results In total, 757 patients from 9 studies were included. The overall effect of adenosine on postoperative VAS/VRS scores and postoperative opioid consumption was not significantly different from that of controls (P >0.1). The occurrence of PONV and pruritus was not statistically significantly different between an adenosine and nonremifentanil subgroup (P >0.1), but the rate of PONV occurrence was greater in the remifentanil subgroup (P <0.01). Time to first postoperative analgesic requirement in the adenosine group was not significantly difference from that of the saline group (SMD = 0.07, 95%CI: −0.28 to 0.41, P = 0.71); but this occurred significantly later than with remifentanil (SMD = 1.10, 95%CI: 2.48 to 4.06, P < 0.01). Time to hospital discharge was not significantly different between the control and adenosine groups (P = 0.78). The perioperative systolic blood pressure was significantly lower in the adenosine than in the control group in the mannitol subgroup (P < 0.01). The incidence of bradycardia, transient first- degree atrioventricular block, and tachycardia was not

Adenosine-Induced Atrial Fibrillation: Localized Reentrant Drivers in Lateral Right Atria due to Heterogeneous Expression of Adenosine A1 Receptors and GIRK4 Subunits in the Human Heart.

PubMed

Li, Ning; Csepe, Thomas A; Hansen, Brian J; Sul, Lidiya V; Kalyanasundaram, Anuradha; Zakharkin, Stanislav O; Zhao, Jichao; Guha, Avirup; Van Wagoner, David R; Kilic, Ahmet; Mohler, Peter J; Janssen, Paul M L; Biesiadecki, Brandon J; Hummel, John D; Weiss, Raul; Fedorov, Vadim V

2016-08-09

Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor and its downstream GIRK (G protein-coupled inwardly rectifying potassium channels) channels (IK,Ado). We applied biatrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and nonfailing human hearts (n=37). Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10-100 µmol/L) produced more significant shortening of action potential durations in RA (from 290±45 to 239±41 ms, 17.3±10.4%; P<0.01) than LA (from 307±24 to 286±23 ms, 6.7±6.6%; P<0.01). In 10 hearts, adenosine induced AF (317±116 s) that, when sustained (≥2 minutes), was primarily maintained by 1 to 2 localized reentrant drivers in lateral RA. Tertiapin (10-100 nmol/L), a selective GIRK channel blocker, counteracted adenosine-induced action potential duration shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher adenosine A1 receptor (2.7±1.7-fold; P<0.01) and GIRK4 (1.7±0.8-fold; P<0.05) protein expression than lateral/posterior LA. This study revealed a 3-fold RA-to-LA adenosine A1 receptor protein expression gradient in the human heart, leading to significantly greater RA versus LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest adenosine A1 receptor/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine. © 2016 American Heart Association, Inc.

ST 1535: a preferential A2A adenosine receptor antagonist.

PubMed

Stasi, Maria Antonietta; Borsini, Franco; Varani, Katia; Vincenzi, Fabrizio; Di Cesare, Maria Assunta; Minetti, Patrizia; Ghirardi, Orlando; Carminati, Paolo

2006-10-01

Antagonism of the A2A adenosine function has proved beneficial in the treatment of Parkinson's disease, in that it increases L-dopa therapeutical effects without concomitant worsening of its side-effects. In this paper we describe a preferential A2A adenosine antagonist, ST 1535, with long-lasting pharmacodynamic effects. It competitively antagonizes the effects of the A2A adenosine agonist NECA on cAMP in cells cloned with the human A2A adenosine receptor (IC50=353+/-30 nM), and the effects of the A1 adenosine agonist CHA on cAMP in cells cloned with the human A1 adenosine receptor (IC50=510+/-38 nM). ST 1535, at oral doses of 5 and 10 mg/kg, antagonizes catalepsy induced by intracerebroventricular administration of the A2A adenosine agonist CGS 21680 (10 microg/5 microl) in mice. At oral doses ranging between 5 and 20 mg/kg, ST 1535 induces hypermotility and antagonizes haloperidol-induced catalepsy in mice up to 7 h. Oral ST 1535, at 1.25 and 2.5 mg/kg, potentiates L-dopa effects in reducing haloperidol-induced catalepsy. ST 1535 represents a potential new compound, with long-lasting activity, for the treatment of Parkinson's disease.

Gene therapy for adenosine deaminase–deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans

PubMed Central

Candotti, Fabio; Shaw, Kit L.; Muul, Linda; Carbonaro, Denise; Sokolic, Robert; Choi, Christopher; Schurman, Shepherd H.; Garabedian, Elizabeth; Kesserwan, Chimene; Jagadeesh, G. Jayashree; Fu, Pei-Yu; Gschweng, Eric; Cooper, Aaron; Tisdale, John F.; Weinberg, Kenneth I.; Crooks, Gay M.; Kapoor, Neena; Shah, Ami; Abdel-Azim, Hisham; Yu, Xiao-Jin; Smogorzewska, Monika; Wayne, Alan S.; Rosenblatt, Howard M.; Davis, Carla M.; Hanson, Celine; Rishi, Radha G.; Wang, Xiaoyan; Gjertson, David; Yang, Otto O.; Balamurugan, Arumugam; Bauer, Gerhard; Ireland, Joanna A.; Engel, Barbara C.; Podsakoff, Gregory M.; Hershfield, Michael S.; Blaese, R. Michael; Parkman, Robertson

2012-01-01

We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)–deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34+ cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m2). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency. PMID:22968453

Adenosine formation and release from neonatal-rat heart cells in culture.

PubMed Central

Meghji, P; Holmquist, C A; Newby, A C

1985-01-01

The incorporation of [3H]adenosine (10 microM) into neonatal-rat heart cell nucleotides was inhibited in a concentration-dependent manner, such that 50% inhibition was obtained with 0.75 microM-dipyridamole, 0.26 microM-hexobendine or 0.22 microM-dilazep. Adenosine formation was accelerated 2.5-fold to 2.1 +/- 0.3 nmol/10(7) cells in 10 min when cells were incubated with a combination of 30 mM-2-deoxyglucose and 2 micrograms of oligomycin/ml. Of the newly formed adenosine, 6 +/- 2% was in the cells. Dipyridamole, hexobendine or dilazep (10 microM) increased the amount of adenosine in the cells and decreased that in the medium such that 45-50% of the newly formed adenosine was in the cells. Antibodies which inhibited ecto-5'-nucleotidase by 98.7 +/- 0.3% did not alter the rate of adenosine formation or its distribution between cells and medium. We conclude that adenosine was formed in the cytoplasm during catabolism of cellular ATP and was released via the dipyridamole-sensitive symmetric nucleoside transporter. PMID:2996488

Changing genetic information through RNA editing

NASA Technical Reports Server (NTRS)

Maas, S.; Rich, A.

2000-01-01

RNA editing, the post-transcriptional alteration of a gene-encoded sequence, is a widespread phenomenon in eukaryotes. As a consequence of RNA editing, functionally distinct proteins can be produced from a single gene. The molecular mechanisms involved include single or multiple base insertions or deletions as well as base substitutions. In mammals, one type of substitutional RNA editing, characterized by site-specific base-modification, was shown to modulate important physiological processes. The underlying reaction mechanism of substitutional RNA editing involves hydrolytic deamination of cytosine or adenosine bases to uracil or inosine, respectively. Protein factors have been characterized that are able to induce RNA editing in vitro. A supergene family of RNA-dependent deaminases has emerged with the recent addition of adenosine deaminases specific for tRNA. Here we review the developments that have substantially increased our understanding of base-modification RNA editing over the past few years, with an emphasis on mechanistic differences, evolutionary aspects and the first insights into the regulation of editing activity.

Involvement of steroids in anti-inflammatory effects of PK11195 in a murine model of pleurisy.

PubMed Central

da Silva, Marcelo Barreto Spillere; Farges, Roseli Coimbra; Fröde, Tânia Silvia

2004-01-01

BACKGROUND: Studies on peripheral benzodiazepine receptor function have yielded a diverse list of activities of which the anti-inflammatory effects need to be further examined. AIMS: To evaluate the role of steroids, nitric oxide and adenosine-deaminase in the anti-inflammatory effect of PK11195. METHODS: Pleurisy was induced by intrapleural injection of carrageenan in mice pre-treated or not with PK11195. Leukocytes, exudation, adenosine-deaminase (ADA) activity and nitric oxide (NO) level were measured. Steroid involvement was evaluated by pre-treatment with D,L-aminogluthetimide before PK11195. RESULTS: Leukocytes, exudation and NO levels were reduced by PK11195 in the early (4 h) phase. In the late (48 h) phase, PK11195 decreased leukocytes and ADA activity. D,L-aminogluthetimide reversed the effect of PK11195 on exudate (4 h), as well as total and differential leukocytes and NO levels (48 h). CONCLUSIONS: Steroids, NO and ADA are implicated in the anti-inflammatory action of PK11195. PMID:15203550

Acetate supplementation modulates brain adenosine metabolizing enzymes and adenosine A₂A receptor levels in rats subjected to neuroinflammation.

PubMed

Smith, Mark D; Bhatt, Dhaval P; Geiger, Jonathan D; Rosenberger, Thad A

2014-06-04

Acetate supplementation reduces neuroglia activation and pro-inflammatory cytokine expression in rat models of neuroinflammation and Lyme neuroborreliosis. Because single-dose glyceryl triacetate (GTA) treatment increases brain phosphocreatine and reduces brain AMP levels, we postulate that GTA modulates adenosine metabolizing enzymes and receptors, which may be a possible mechanism to reduce neuroinflammation. To test this hypothesis, we quantified the ability of GTA to alter brain levels of ecto-5'-nucleotidase (CD73), adenosine kinase (AK), and adenosine A2A receptor using western blot analysis and CD73 activity by measuring the rate of AMP hydrolysis. Neuroinflammation was induced by continuous bacterial lipopolysaccharide (LPS) infusion in the fourth ventricle of the brain for 14 and 28 days. Three treatment strategies were employed, one and two where rats received prophylactic GTA through oral gavage with LPS infusion for 14 or 28 days. In the third treatment regimen, an interventional strategy was used where rats were subjected to 28 days of neuroinflammation, and GTA treatment was started on day 14 following the start of the LPS infusion. We found that rats subjected to neuroinflammation for 28 days had a 28% reduction in CD73 levels and a 43% increase in AK levels that was reversed with prophylactic acetate supplementation. CD73 activity in these rats was increased by 46% with the 28-day GTA treatment compared to the water-treated rats. Rats subjected to neuroinflammation for 14 days showed a 50% increase in levels of the adenosine A2A receptor, which was prevented with prophylactic acetate supplementation. Interventional GTA therapy, beginning on day 14 following the induction of neuroinflammation, resulted in a 67% increase in CD73 levels and a 155% increase in adenosine A2A receptor levels. These results support the hypothesis that acetate supplementation can modulate brain CD73, AK and adenosine A2A receptor levels, and possibly influence purinergic

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

PubMed

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

2012-09-01

Ocimum sanctum grown as rain-fed crop, is known to be poorly adapted to waterlogged conditions. Many a times the crop suffers extreme damages because of anoxia and excessive ethylene generation due to waterlogging conditions present under heavy rain. The usefulness of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth promoting rhizobacteria was investigated under waterlogging stress. The comparison of herb yield and stress induced biochemical changes of waterlogged and non-waterlogged plants with and without ACC deaminase-containing microbiological treatments were monitored in this study. Ten plant growth promoting rhizobacteria strains containing ACC-deaminase were isolated and characterized. Four selected isolates Fd2 (Achromobacter xylosoxidans), Bac5 (Serratia ureilytica), Oci9 (Herbaspirillum seropedicae) and Oci13 (Ochrobactrum rhizosphaerae) had the potential to protect Ocimum plants from flood induced damage under waterlogged glass house conditions. Pot experiments were conducted to evaluate the potential of these ACC deaminase-containing selected strains for reducing the yield losses caused by waterlogging conditions. Bacterial treatments protected plants from waterlogging induced detrimental changes like stress ethylene production, reduced chlorophyll concentration, higher lipid peroxidation, proline concentration and reduced foliar nutrient uptake. Fd2 (A. xylosoxidans) induced maximum waterlogging tolerance as treated waterlogged plants recorded maximum growth and herb yield (46.5% higher than uninoculated waterlogged plants) with minimum stress ethylene levels (53% lower ACC concentration as compared to waterlogged plants without bacterial inoculation) whereas under normal non-waterlogged conditions O. rhizosphaerae was most effective in plant growth promotion. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

PubMed Central

Chiavegatti, T; Costa, V L; Araújo, M S; Godinho, R O

2007-01-01

Background and purpose: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. Experimental approach: cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. Key results: Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5′-AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5′-AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. Conclusions and implications: Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine. PMID:18157164

Adenosine receptor subtypes in the airways responses to 5'-adenosine monophosphate inhalation of sensitized guinea-pigs.

PubMed

Smith, N; Broadley, K J

2008-09-01

Endogenous adenosine levels are raised in the lungs during asthma attacks. 5'-adenosine monophosphate (5'-AMP) inhalation in asthmatics causes bronchoconstriction and in sensitized guinea-pigs induces early (EAR) and late asthmatic responses (LAR), airway hyper-reactivity (AHR) and inflammatory cell recruitment to the lungs. The aim of this study was to investigate the roles of A(1), A(2A), A(2B) and A(3) adenosine receptors in these responses to inhaled 5'-AMP in sensitized guinea-pigs. Comparisons were made with the effect of dexamethasone treatment on 5'-AMP-induced responses. Functional airways responses to inhaled 5'-AMP (3 and 300 mM) of actively sensitized, conscious guinea-pigs were determined by whole-body plethysmography following administration of selective adenosine receptor antagonists or their vehicles. AHR to inhaled histamine (1 mM) and inflammatory cell influx in bronchoalveolar lavage fluid were determined. 5'-AMP at 3 mM caused an immediate bronchoconstriction (EAR), whereas 300 mM caused bronchodilatation. Both responses were followed at 6 h by a LAR, together with inflammatory cell influx and AHR to histamine. The A(2A) receptor antagonist, ZM241385, further enhanced cell influx after 5'-AMP inhalation (3 and 300 mM), and blocked the immediate bronchodilator response to 300 mM 5'-AMP, exposing an EAR. The A(2B) receptor antagonist, MRS1706 (in the presence of ZM241385), inhibited the LAR, AHR and cell influx, following inhalation of 5'-AMP (300 mM). The A(3) receptor antagonist, MRS1220, inhibited 5'-AMP-induced inflammatory cell influx. The A(1) receptor antagonist, DPCPX (in the presence of ZM241385), inhibited the EAR following 5'-AMP inhalation (300 mM). Dexamethasone inhibited the LAR, AHR and cell influx following inhalation of 5'-AMP (300 mM). All four adenosine receptor subtypes play various roles in the airways responses to inhaled 5'-AMP in sensitized guinea-pigs.

Intracoronary Adenosine: Dose-Response Relationship With Hyperemia.

PubMed

Adjedj, Julien; Toth, Gabor G; Johnson, Nils P; Pellicano, Mariano; Ferrara, Angela; Floré, Vincent; Di Gioia, Giuseppe; Barbato, Emanuele; Muller, Olivier; De Bruyne, Bernard

2015-09-01

The present study sought to establish the dosage of intracoronary (IC) adenosine associated with minimal side effects and above which no further increase in flow can be expected. Despite the widespread adoption of IC adenosine in clinical practice, no wide-ranging, dose-response study has been conducted. A recurring debate still exists regarding its optimal dose. In 30 patients, Doppler-derived flow velocity measurements were obtained in 10 right coronary arteries (RCAs) and 20 left coronary arteries (LCAs) free of stenoses >20% in diameter. Flow velocity was measured at baseline and after 8 ml bolus administrations of arterial blood, saline, contrast medium, and 9 escalating doses of adenosine (4 to 500 μg). The hyperemic value was expressed in percent of the maximum flow velocity reached in a given artery (Q/Qmax, %). Q/Qmax did not increase significantly beyond dosages of 60 μg for the RCA and 160 μg for LCA. Heart rate did not change, whereas mean arterial blood pressure decreased by a maximum of 7% (p < 0.05) after bolus injections of IC adenosine. The incidence of transient A-V blocks was 40% after injection of 100 μg in the RCA and was 15% after injection of 200 μg in the LCA. The duration of the plateau reached 12 ± 13 s after injection of 100 μg in the RCA and 21 ± 6 s after the injection of 200 μg in the LCA. A progressive prolongation of the time needed to return to baseline was observed. Hyperemic response after injection of 8 ml of contrast medium reached 65 ± 36% of that achieved after injection of 200 μg of adenosine. This wide-ranging, dose-response study indicates that an IC adenosine bolus injection of 100 μg in the RCA and 200 μg in the LCA induces maximum hyperemia while being associated with minimal side effects. Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Rapid Detection of Cell-Free Mycobacterium tuberculosis DNA in Tuberculous Pleural Effusion.

PubMed

Che, Nanying; Yang, Xinting; Liu, Zichen; Li, Kun; Chen, Xiaoyou

2017-05-01

Tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, but its diagnosis remains difficult. In this study, we report for the first time on the detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and an evaluation of a newly developed molecular assay for the detection of cell-free Mycobacterium tuberculosis DNA. A total of 78 patients with pleural effusion, 60 patients with tuberculous pleurisy, and 18 patients with alternative diseases were included in this study. Mycobacterial culture, the Xpert MTB/RIF assay, the adenosine deaminase assay, the T-SPOT.TB assay, and the cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. The cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities of 75.0% and 68.3%, respectively, than mycobacterial culture and the Xpert MTB/RIF assay, which had sensitivities of 26.7% and 20.0%, respectively ( P < 0.01). All four of these tests showed good specificities: 88.9% for the adenosine deaminase assay and 100% for the remaining three assays. The T-SPOT.TB assay with pleural effusion showed the highest sensitivity of 95.0% but the lowest specificity of 38.9%. The cell-free Mycobacterium tuberculosis DNA assay detected as few as 1.25 copies of IS 6110 per ml of pleural effusion and showed good accordance of the results between repeated tests ( r = 0.978, P = 2.84 × 10 -10 ). These data suggest that the cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology. Copyright © 2017 American Society for Microbiology.

Rapid Detection of Cell-Free Mycobacterium tuberculosis DNA in Tuberculous Pleural Effusion

PubMed Central

Yang, Xinting; Liu, Zichen; Li, Kun

2017-01-01

ABSTRACT Tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, but its diagnosis remains difficult. In this study, we report for the first time on the detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and an evaluation of a newly developed molecular assay for the detection of cell-free Mycobacterium tuberculosis DNA. A total of 78 patients with pleural effusion, 60 patients with tuberculous pleurisy, and 18 patients with alternative diseases were included in this study. Mycobacterial culture, the Xpert MTB/RIF assay, the adenosine deaminase assay, the T-SPOT.TB assay, and the cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. The cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities of 75.0% and 68.3%, respectively, than mycobacterial culture and the Xpert MTB/RIF assay, which had sensitivities of 26.7% and 20.0%, respectively (P < 0.01). All four of these tests showed good specificities: 88.9% for the adenosine deaminase assay and 100% for the remaining three assays. The T-SPOT.TB assay with pleural effusion showed the highest sensitivity of 95.0% but the lowest specificity of 38.9%. The cell-free Mycobacterium tuberculosis DNA assay detected as few as 1.25 copies of IS6110 per ml of pleural effusion and showed good accordance of the results between repeated tests (r = 0.978, P = 2.84 × 10−10). These data suggest that the cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology. PMID:28275073

Delayed production of adenosine underlies temporal modulation of swimming in frog embryo

PubMed Central

Dale, Nicholas

1998-01-01

To investigate the dynamics of adenosine production in the spinal cord during motor activity, and its possible contribution to the temporal modulation of motor patterns, a sensor sensitive to adenosine at concentrations as low as 10 nm was devised.When pressed against the outside of the spinal cord, the sensor detected slow changes in the levels of adenosine during fictive swimming that ranged from 10 to 650 nm. In four embryos where particularly large signals were recorded due to favourable probe placement, the adenosine levels continued to rise for up to a minute following cessation of activity before slowly returning to baseline. In the remaining thirteen embryos, levels of adenosine started to return slowly to baseline almost immediately after activity had stopped.Inhibitors of adenosine uptake increased the magnitude of the signal recorded and slowed the recovery following cessation of activity.A realistic computational model of the spinal circuitry was combined with models of extracellular breakdown of ATP to adenosine. ATP and adenosine inhibited, as in the real embryo, the voltage-gated K+ and Ca2+ currents, respectively. The model reproduced the temporal run-down of motor activity seen in the real embryo suggesting that synaptic release of ATP together with its extracellular breakdown to adenosine is sufficient to exert time-dependent control over motor pattern generation.The computational analysis also suggested that the delay in the rise of adenosine levels is likely to result from feed-forward inhibition of the 5′-ectonucleotidase in the spinal cord. This inhibition is a key determinant of the rate of run-down. PMID:9679180

Neutrophil-derived 5′-Adenosine Monophosphate Promotes Endothelial Barrier Function via CD73-mediated Conversion to Adenosine and Endothelial A2B Receptor Activation

PubMed Central

Lennon, Paul F.; Taylor, Cormac T.; Stahl, Gregory L.; Colgan, Sean P.

1998-01-01

During episodes of inflammation, polymorphonuclear leukocyte (PMN) transendothelial migration has the potential to disturb vascular barrier function and give rise to intravascular fluid extravasation and edema. However, little is known regarding innate mechanisms that dampen fluid loss during PMN-endothelial interactions. Using an in vitro endothelial paracellular permeability model, we observed a PMN-mediated decrease in endothelial paracellular permeability. A similar decrease was elicited by cell-free supernatants from activated PMN (FMLP 10−6 M), suggesting the presence of a PMN-derived soluble mediator(s). Biophysical and biochemical analysis of PMN supernatants revealed a role for PMN-derived 5′-adenosine monophosphate (AMP) and its metabolite, adenosine, in modulation of endothelial paracellular permeability. Supernatants from activated PMN contained micromolar concentrations of bioactive 5′-AMP and adenosine. Furthermore, exposure of endothelial monolayers to authentic 5′-AMP and adenosine increased endothelial barrier function more than twofold in both human umbilical vein endothelial cells and human microvascular endothelial cells. 5′-AMP bioactivity required endothelial CD73-mediated conversion of 5′-AMP to adenosine via its 5′-ectonucleotidase activity. Decreased endothelial paracellular permeability occurred through adenosine A2B receptor activation and was accompanied by a parallel increase in intracellular cAMP. We conclude that activated PMN release soluble mediators, such as 5′-AMP and adenosine, that promote endothelial barrier function. During inflammation, this pathway may limit potentially deleterious increases in endothelial paracellular permeability and could serve as a basic mechanism of endothelial resealing during PMN transendothelial migration. PMID:9782120

Neuronal transporter and astrocytic ATP exocytosis underlie activity-dependent adenosine release in the hippocampus

PubMed Central

Wall, Mark J; Dale, Nicholas

2013-01-01

The neuromodulator adenosine plays an important role in many physiological and pathological processes within the mammalian CNS. However, the precise mechanisms of how the concentration of extracellular adenosine increases following neural activity remain contentious. Here we have used microelectrode biosensors to directly measure adenosine release induced by focal stimulation in stratum radiatum of area CA1 in mouse hippocampal slices. Adenosine release was both action potential and Ca2+ dependent and could be evoked with low stimulation frequencies and small numbers of stimuli. Adenosine release required the activation of ionotropic glutamate receptors and could be evoked by local application of glutamate receptor agonists. Approximately 40% of stimulated-adenosine release occurred by translocation of adenosine via equilibrative nucleoside transporters (ENTs). This component of release persisted in the presence of the gliotoxin fluoroacetate and thus results from the direct release of adenosine from neurons. A reduction of adenosine release in the presence of NTPDase blockers, in slices from CD73−/− and dn-SNARE mice, provides evidence that a component of adenosine release arises from the extracellular metabolism of ATP released from astrocytes. This component of release appeared to have slower kinetics than the direct ENT-mediated release of adenosine. These data suggest that activity-dependent adenosine release is surprisingly complex and, in the hippocampus, arises from at least two distinct mechanisms with different cellular sources. PMID:23713028

The inclusion of ADA-SCID in expanded newborn screening by tandem mass spectrometry.

PubMed

la Marca, Giancarlo; Giocaliere, Elisa; Malvagia, Sabrina; Funghini, Silvia; Ombrone, Daniela; Della Bona, Maria Luisa; Canessa, Clementina; Lippi, Francesca; Romano, Francesca; Guerrini, Renzo; Resti, Massimo; Azzari, Chiara

2014-01-01

Severe combined immunodeficiency due to adenosine-deaminase defect (ADA-SCID) is usually deadly in childhood because of severe recurrent infections. When clinical diagnosis is done, permanent damages due to infections or metabolite accumulation are often present. Gene therapy, bone marrow transplantation or enzyme replacement therapy may be effective if started early. The aim of this study was to set-up a robust method suitable for screening with a minimized preparation process and with inexpensive running costs, for diagnosing ADA-SCID by tandem mass spectrometry. ADA-SCID satisfies all the criteria for inclusion in a newborn screening program. We describe a protocol revised to incorporate adenosine and 2-deoxyadenosine testing into an expanded newborn screening program. We assessed the effectiveness of this approach testing dried blood spots from 4 genetically confirmed early-onset and 5 delayed-onset ADA-SCID patients. Reference values were established on 50,000 healthy newborns (deoxyadenosine <0.09μmol/L, adenosine <1.61μmol/L). We also developed a second tier test to distinguish true positives from false positives and improve the positive predictive value of an initial abnormal result. In the first 18 months, the pilot project has identified a newborn with a genetically confirmed defect in adenosine deaminase (ADA) gene. The results show that the method having great simplicity, low cost and low process preparations can be fully applicable to a mass screening program. Copyright © 2013 Elsevier B.V. All rights reserved.

Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass

PubMed Central

Davidson, Jesse A.; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan

2016-01-01

Rationale Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. Objectives 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Methods and Research Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Conclusions Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post

Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass.

PubMed

Davidson, Jesse A; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan; Wischmeyer, Paul E; Klawitter, Jelena

2016-01-01

Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post-operative alkaline phosphatase activity leads to

Adenosine transiently modulates stimulated dopamine release in the caudate putamen via A1 receptors

PubMed Central

Ross, Ashley E.; Venton, B. Jill

2014-01-01

Adenosine modulates dopamine in the brain via A1 and A2A receptors, but that modulation has only been characterized on a slow time scale. Recent studies have characterized a rapid signaling mode of adenosine that suggests a possible rapid modulatory role. Here, fast-scan cyclic voltammetry was used to characterize the extent to which transient adenosine changes modulate stimulated dopamine release (5 pulses at 60 Hz) in rat caudate putamen brain slices. Exogenous adenosine was applied and dopamine concentration monitored. Adenosine only modulated dopamine when it was applied 2 or 5 s before stimulation. Longer time intervals and bath application of 5 µM adenosine did not decrease dopamine release. Mechanical stimulation of endogenous adenosine 2s before dopamine stimulation also decreased stimulated dopamine release by 41 ± 7 %, similar to the 54 ± 6 % decrease in dopamine after exogenous adenosine application. Dopamine inhibition by transient adenosine was recovered within 10 minutes. The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) blocked the dopamine modulation, whereas dopamine modulation was unaffected by the A2A receptor antagonist SCH 442416. Thus, transient adenosine changes can transiently modulate phasic dopamine release via A1 receptors. These data demonstrate that adenosine has a rapid, but transient, modulatory role in the brain. PMID:25219576

The Role of Extracellular Adenosine Triphosphate in Ischemic Organ Injury.

PubMed

Zhao, Hailin; Kilgas, Susan; Alam, Azeem; Eguchi, Shiori; Ma, Daqing

2016-05-01

Ischemic tissue injury contributes to significant morbidity and mortality and is implicated in a range of pathologic conditions, including but not limited to myocardial infarction, ischemic stroke, and acute kidney injury. The associated reperfusion phase is responsible for the activation of the innate and adaptive immune system, further accentuating inflammation. Adenosine triphosphate molecule has been implicated in various ischemic conditions, including stroke and myocardial infarction. Adenosine triphosphate is a well-defined intracellular energy transfer and is commonly referred to as the body's "energy currency." However, Laboratory studies have demonstrated that extracellular adenosine triphosphate has the ability to initiate inflammation and is therefore referred to as a damage-associated molecular pattern. Purinergic receptors-dependent signaling, proinflammatory cytokine release, increased Ca influx into cells, and subsequent apoptosis have been shown to form a common underlying extracellular adenosine triphosphate molecular mechanism in ischemic organ injury. In this review, we aim to discuss the molecular mechanisms behind adenosine triphosphate-mediated ischemic tissue injury and evaluate the role of extracellular adenosine triphosphate in ischemic injury in specific organs, in order to provide a greater understanding of the pathophysiology of this complex process. We also appraise potential future therapeutic strategies to limit damage in various organs, including the heart, brain, kidneys, and lungs.

Evidence for an A2/Ra adenosine receptor in the guinea-pig trachea

PubMed Central

Brown, C.M.; Collis, M.G.

1982-01-01

1 An attempt was made to determine whether the extracellular adenosine receptor that mediates relaxation in the guinea-pig trachea is of the A1/Ri or A2/Ra subtype. 2 Dose-response curves to adenosine and a number of 5′- and N6-substituted analogues were constructed for the isolated guinea-pig trachea, contracted with carbachol. 3 The 5′-substituted analogues of adenosine were the most potent compounds tested, the order of potency being 5′-N-cyclopropylcarboxamide adenosine (NCPCA) > 5′-N-ethylcarboxamide adenosine (NECA) > 2-chloroadenosine > L-N6-phenylisopropyladenosine (L-PIA) > adenosine > D-N6-phenylisopropyladenosine (D-PIA). 4 The difference in potency between the stereoisomers D- and L-PIA on the isolated trachea was at the most five fold. 5 Responses to low doses of adenosine and its analogues were attenuated after treatment with either theophylline or 8-phenyltheophylline. The responses to 2-chloroadenosine were affected to a lesser extent than were those to the other purines. 6 Adenosine transport inhibitors, dipyridamole and dilazep, potentiated responses to adenosine, did not affect those to NCPCA, NECA, L-PIA and D-PIA but significantly reduced the responses to high doses of 2-chloroadenosine. 7 Relaxations evoked by 9-β-D-xylofuranosyladenosine which can activate intracellular but not extracellular adenosine receptors, were attenuated by dipyridamole but unaffected by 8-phenyltheophylline. 8 The results support the existence of an extracellular A2/Ra subtype of adenosine receptor and an intracellular purine-sensitive site, both of which mediate relaxation. PMID:6286021

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

PubMed

Magnucka, Elżbieta G; Pietr, Stanisław J

2015-12-01

The study evaluates the effect of rhizobacteria having 1-aminocyclopropane-1-carboxylate deaminase (ACCd) on the development of wheat seedlings. This enzyme has been proposed to play a key role in microbe-plant association. Three fluorescent pseudomonads containing this deaminase were selected from 70 strains of pseudomonads isolated from rhizosphere of wheat (Triticum aestivum L.) and rape (Brassica napus L.). These bacteria, varied significantly in the ability to both biosynthesize auxins and hydrolyze ACC. Among them, Pseudomonas brassicacearum subsp. brassicacearum strain RZ310 presented the highest activities of ACC deaminase during 96h of growth in liquid Dworkin and Foster (DF) salt medium. Additionally, this rape rhizosphere strain did not produce indoles. Two other isolates, Pseudomonas sp. PO283 and Pseudomonas sp. PO366, secreted auxins only in the presence of their precursor. Phylogenetic analysis of the 16S rRNA gene and four other protein-encoding genes indicated that these wheat rhizosphere isolates belonged to the fluorescent Pseudomonas group. Moreover, the effects of these strains on wheat seedling growth under in vitro conditions were markedly dependent on both their cell suspensions used to grain inoculation and nutrient conditions. Strains tested had beneficial influence on wheat seedlings mainly at low cell densities. In addition, access to nutrients markedly changed bacteria action on cereal growth. Their presence generally favored the positive effects of pseudomonads on length and the estimated biomasses of wheat coleoptiles. Despite these general rules, impacts of each isolate on the growth parameters of cereal seedlings were unique. Copyright © 2015 Elsevier GmbH. All rights reserved.

Diversification of AID/APOBEC-like deaminases in metazoa: multiplicity of clades and widespread roles in immunity.

PubMed

Krishnan, Arunkumar; Iyer, Lakshminarayan M; Holland, Stephen J; Boehm, Thomas; Aravind, L

2018-04-03

AID/APOBEC deaminases (AADs) convert cytidine to uridine in single-stranded nucleic acids. They are involved in numerous mutagenic processes, including those underpinning vertebrate innate and adaptive immunity. Using a multipronged sequence analysis strategy, we uncover several AADs across metazoa, dictyosteliida, and algae, including multiple previously unreported vertebrate clades, and versions from urochordates, nematodes, echinoderms, arthropods, lophotrochozoans, cnidarians, and porifera. Evolutionary analysis suggests a fundamental division of AADs early in metazoan evolution into secreted deaminases (SNADs) and classical AADs, followed by diversification into several clades driven by rapid-sequence evolution, gene loss, lineage-specific expansions, and lateral transfer to various algae. Most vertebrate AADs, including AID and APOBECs1-3, diversified in the vertebrates, whereas the APOBEC4-like clade has a deeper origin in metazoa. Positional entropy analysis suggests that several AAD clades are diversifying rapidly, especially in the positions predicted to interact with the nucleic acid target motif, and with potential viral inhibitors. Further, several AADs have evolved neomorphic metal-binding inserts, especially within loops predicted to interact with the target nucleic acid. We also observe polymorphisms, driven by alternative splicing, gene loss, and possibly intergenic recombination between paralogs. We propose that biological conflicts of AADs with viruses and genomic retroelements are drivers of rapid AAD evolution, suggesting a widespread presence of mutagenesis-based immune-defense systems. Deaminases like AID represent versions "institutionalized" from the broader array of AADs pitted in such arms races for mutagenesis of self-DNA, and similar recruitment might have independently occurred elsewhere in metazoa. Copyright © 2018 the Author(s). Published by PNAS.

The 2′,3′-cAMP-adenosine pathway

PubMed Central

2011-01-01

Our recent studies employing HPLC-tandem mass spectrometry to analyze venous perfusate from isolated, perfused kidneys demonstrate that intact kidneys produce and release into the extracellular compartment 2′,3′-cAMP, a positional isomer of the second messenger 3′,5′-cAMP. To our knowledge, this represents the first detection of 2′,3′-cAMP in any cell/tissue/organ/organism. Nuclear magnetic resonance experiments with isolated RNases and experiments in isolated, perfused kidneys suggest that 2′,3′-cAMP likely arises from RNase-mediated transphosphorylation of mRNA. Both in vitro and in vivo kidney experiments demonstrate that extracellular 2′,3′-cAMP is efficiently metabolized to 2′-AMP and 3′-AMP, both of which can be further metabolized to adenosine. This sequence of reactions is called the 2′,3′-cAMP-adenosine pathway (2′,3′-cAMP → 2′-AMP/3′-AMP → adenosine). Experiments in rat and mouse kidneys show that metabolic poisons increase extracellular levels of 2′,3′-cAMP, 2′-AMP, 3′-AMP, and adenosine; however, little is known regarding the pharmacology of 2′,3′-cAMP, 2′-AMP, and 3′-AMP. What is known is that 2′,3′-cAMP facilitates activation of mitochondrial permeability transition pores, a process that can lead to apoptosis and necrosis, and inhibits proliferation of vascular smooth muscle cells and glomerular mesangial cells. In summary, there is mounting evidence that at least some types of cellular injury, by triggering mRNA degradation, engage the 2′,3′-cAMP-adenosine pathway, and therefore this pathway should be added to the list of biochemical pathways that produce adenosine. Although speculative, it is possible that the 2′,3′-cAMP-adenosine pathway may protect against some forms of acute organ injury, for example acute kidney injury, by both removing an intracellular toxin (2′,3′-cAMP) and increasing an extracellular renoprotectant (adenosine). PMID:21937608

Opiate-induced Changes in Brain Adenosine Levels and Narcotic Drug Responses

PubMed Central

Wu, Manhong; Sahbaie, Peyman; Zheng, Ming; Lobato, Robert; Boison, Detlev; Clark, J. David; Peltz, Gary

2012-01-01

We have very little information about the metabolomic changes that mediate neurobehavioral responses, including addiction. It was possible that opioid-induced metabolomic changes in brain could mediate some of the pharmacodynamic effects of opioids. To investigate this, opiate-induced brain metabolomic responses were profiled using a semi-targeted method in C57BL/6 and 129Sv1 mice, which exhibit extreme differences in their tendency to become opiate dependent. Escalating morphine doses (10–40 mg/kg) administered over a 4-day period selectively induced a two-fold decrease (p<0.00005) in adenosine abundance in the brainstem of C57BL/6 mice, which exhibited symptoms of narcotic drug dependence; but did not decrease adenosine abundance in 129Sv1 mice, which do not exhibit symptoms of dependence. Based on this finding, the effect of adenosine on dependence was investigated in genetically engineered mice with alterations in adenosine tone in the brain and in pharmacologic experiments. Morphine withdrawal behaviors were significantly diminished (P<0.0004) in genetically engineered mice with reduced adenosine tone in the brainstem, and by treatment with an adenosine receptor1 (A1) agonist (2-chloro-N6-cyclopentyladenosine, 0.5 mg/kg) or an A2a receptor (A2a) antagonist (SCH 58261 1 mg/kg). These results indicate that adenosine homeostasis plays a crucial role in narcotic drug responses. Opiate-induced changes in brain adenosine levels may explain many important neurobehavioral features associated with opiate addiction and withdrawal. PMID:23098802

Systemic Adenosine Triphosphate Impairs Neutrophil Chemotaxis and Host Defense in Sepsis.

PubMed

Li, Xiaoou; Kondo, Yutaka; Bao, Yi; Staudenmaier, Laura; Lee, Albert; Zhang, Jingping; Ledderose, Carola; Junger, Wolfgang G

2017-01-01

Sepsis remains an unresolved clinical problem. Therapeutic strategies focusing on inhibition of neutrophils (polymorphonuclear neutrophils) have failed, which indicates that a more detailed understanding of the underlying pathophysiology of sepsis is required. Polymorphonuclear neutrophil activation and chemotaxis require cellular adenosine triphosphate release via pannexin-1 channels that fuel autocrine feedback via purinergic receptors. In the current study, we examined the roles of endogenous and systemic adenosine triphosphate on polymorphonuclear neutrophil activation and host defense in sepsis. Prospective randomized animal investigation and in vitro studies. Preclinical academic research laboratory. Wild-type C57BL/6 mice, pannexin-1 knockout mice, and healthy human subjects used to obtain polymorphonuclear neutrophils for in vitro studies. Wild-type and pannexin-1 knockout mice were treated with suramin or apyrase to block the endogenous or systemic effects of adenosine triphosphate. Mice were subjected to cecal ligation and puncture and polymorphonuclear neutrophil activation (CD11b integrin expression), organ (liver) injury (plasma aspartate aminotransferase), bacterial spread, and survival were monitored. Human polymorphonuclear neutrophils were used to study the effect of systemic adenosine triphosphate and apyrase on chemotaxis. Inhibiting endogenous adenosine triphosphate reduced polymorphonuclear neutrophil activation and organ injury, but increased the spread of bacteria and mortality in sepsis. By contrast, removal of systemic adenosine triphosphate improved bacterial clearance and survival in sepsis by improving polymorphonuclear neutrophil chemotaxis. Systemic adenosine triphosphate impairs polymorphonuclear neutrophil functions by disrupting the endogenous purinergic signaling mechanisms that regulate cell activation and chemotaxis. Removal of systemic adenosine triphosphate improves polymorphonuclear neutrophil function and host defenses

Use of bacterial acc deaminase to increase oil (especially poly aromatic hydrocarbons) phytoremediation efficiency for maize (zea mays) seedlings.

PubMed

Rezvani Borujeni, Samira; Khavazi, Kazem; Asgharzadeh, Ahmad; Rezvani Borujeni, Iraj

2018-04-16

Oil presence in soil, as a stressor, reduces phytoremediation efficiency through an increase in the plant stress ethylene. Bacterial 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, as a plant stress ethylene reducer, was employed to increase oil phytoremediation efficiency. For this purpose, the ability of ACC deaminase-producing Pseudomonas strains to grow in oil-polluted culture media and withstand various concentrations of oil and also their ability to reduce plant stress ethylene and enhance some growth characteristics of maize and finally their effects on increasing phytoremediation efficiency of poly aromatic hydrocarbons (PAHs) in soil were investigated. Based on the results, of tested strains just P9 and P12 were able to perform oil degradation. Increasing oil concentration from 0 to 10% augmented these two strains population, 15.7% and 12.9%, respectively. The maximum increase in maize growth was observed in presence of P12 strain. Results of high-performance liquid chromatography (HPLC) revealed that PAHs phytoremediation efficiency was higher for inoculated seeds than uninoculated. The highest plant growth and PAHs removal percentage (74.9%) from oil-polluted soil was observed in maize inoculated with P12. These results indicate the significance of ACC deaminase producing bacteria in alleviation of plant stress ethylene in oil-polluted soils and increasing phytoremediation efficiency of such soils.

Analgesic and anti-inflammatory effects of A-286501, a novel orally active adenosine kinase inhibitor.

PubMed

Jarvis, Michael F; Yu, Haixia; McGaraughty, Steve; Wismer, Carol T; Mikusa, Joe; Zhu, Chang; Chu, Katharine; Kohlhaas, Kathy; Cowart, Marlon; Lee, Chih Hung; Stewart, Andrew O; Cox, Bryan F; Polakowski, James; Kowaluk, Elizabeth A

2002-03-01

Adenosine (ADO) is an inhibitory neuromodulator that can increase nociceptive thresholds in response to noxious stimulation. Inhibition of the ADO-metabolizing enzyme, adenosine kinase (AK) increases extracellular ADO concentrations at sites of tissue trauma and AK inhibitors may have therapeutic potential as analgesic and anti-inflammatory agents. N7-((1'R,2'S,3'R,4'S)-2',3'-dihydroxy-4'-amino-cyclopentyl)-4-amino-5-bromo-pyrrolo[2,3-a]pyrimidine (A-286501) is a novel and potent (IC50=0.47 nM) carbocyclic nucleoside AK inhibitor that has no significant activity (IC50 >100 microM) at other sites of ADO interaction (A1, A2A, A3 receptors, ADO transporter, and ADO deaminase) or other (IC50 value >10 microM) neurotransmitter and peptide receptors, ion channel proteins, neurotransmitter reuptake sites and enzymes, including cyclooxygenases-1 and -2. A-286501 showed equivalent potency to inhibit AK from several mammalian species and kinetic studies revealed that A-286501 was a reversible and competitive inhibitor with respect to ADO and non-competitive with respect to MgATP2-. A-286501 was orally effective to reduce nociception in animal models of acute (thermal), inflammatory (formalin and carrageenan), and neuropathic (L5/L6 nerve ligation and streptozotocin-induced diabetic) pain. A-286501 was particularly potent (ED50=1 micromol/kg, p.o.) to reduce carrageenan-induced inflammatory thermal hyperalgesia as compared to its analgesic actions in other pain models (acute and neuropathic) and its ability to alter hemodynamic function and motor performance. A-286501 was also effective to reduce carrageenan-induced paw edema and myeloperoxidase activity, a measure of neutrophil influx (ED50=10 micromol/kg, p.o.), in the injured paw. The anti-nociceptive effects of A-286501 in the L5/L6 nerve injury model of neuropathic pain (ED50=20 micromol/kg, p.o.) were not blocked by the opioid antagonist naloxone, but were blocked by the ADO receptor antagonist, theophylline. Following

Purification and properties of adenosine kinase from rat brain.

PubMed

Yamada, Y; Goto, H; Ogasawara, N

1980-12-04

Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified to apparent homogeneity from rat brain by (NH4)2SO4 fractionation, affinity chromatography on AMP-Sepharose 4B, gel filtration with Sephadex G-100, and DE-52 cellulose column chromatography. The yield was 56% of the initial activity with a final specific activity of 7.8 mumol/min per mg protein. The molecular weight was estimated as 38 000 by gel filtration with Sephadex G-100 and 41 000 by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme catalyzed the phosphorylation of adenosine, deoxyadenosine, arabinoadenosine, inosine and ribavirin. The activity of deoxyadenosine phosphorylation was 20% that of adenosine phosphorylation. The pH optimum profile was biphasic; a sharp pH optimum at pH 5.5 and a broad pH optimum at pH 7.5-8.5. The Km value for adenosine was 0.2 microM and the maximum activity was observed at 0.5 microM. At higher concentrations of adenosine, the activity was strongly inhibited. The Km value for ATP was 0.02 mM and that for Mg2+ was 0.1 mM. GTP, dGTP, dATP and UTP were also proved to be effective phosphate donors. Co2+ was as effective as Mg2+, and Ca2+, Mn2+ or Ni2+ showed about 50% of the activity for Mg2+. The kinase is quite unstable, but stable in the presence of a high concentration of salt; e.g., 0.15 M KCl.

[High performance liquid chromatogram (HPLC) determination of adenosine phosphates in rat myocardium].

PubMed

Miao, Yu; Wang, Cheng-long; Yin, Hui-jun; Shi, Da-zhuo; Chen, Ke-ji

2005-04-18

To establish method for the quantitative determination of adenosine phosphates in rat myocardium by optimized high performance liquid chromatogram (HPLC). ODS HYPERSIL C(18) column and a mobile phase of 50 mmol/L tribasic potassium phosphate buffer solution (pH 6.5), with UV detector at 254 nm were used. The average recovery rates of myocardial adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were 99%-107%, 96%-104% and 95%-119%, respectively; relative standard deviations (RSDs) of within-day and between-days were less than 1.5% and 5.1%, respectively. The method is simple, rapid and accurate, and can be used to analyse the adenosine phosphates in myocardium.

Adenosine-Induced Atrial Fibrillation: Localized Reentrant Drivers in Lateral Right Atria due to Heterogeneous Expression of Adenosine A1 Receptors and GIRK4 Subunits in the Human Heart

PubMed Central

Li, Ning; Csepe, Thomas A.; Hansen, Brian J.; Sul, Lidiya V.; Kalyanasundaram, Anuradha; Zakharkin, Stanislav O.; Zhao, Jichao; Guha, Avirup; Van Wagoner, David R.; Kilic, Ahmet; Mohler, Peter J; Janssen, Paul ML; Biesiadecki, Brandon; Hummel, John D; Weiss, Raul; Fedorov, Vadim V.

2016-01-01

Background Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor (A1R) and its downstream GIRK channels (IK,Ado). Methods We applied bi-atrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and non-failing human hearts (n=37). Results Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10–100μM) produced more significant shortening of action potential durations (APD80) in RA (from 290±45ms to 239±41ms, 17.3±10.4%; p<0.01) than LA (from 307±24ms to 286±23ms, 6.7±6.6%; p<0.01). In ten hearts, adenosine induced AF (317±116 sec) that, when sustained (≥2 min), was primarily maintained by one/two localized reentrant drivers in lateral RA. Tertiapin (10–100nM), a selective GIRK channel blocker, counteracted adenosine-induced APD shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher A1R (2.7±1.7 fold; p<0.01) and GIRK4 (1.7±0.8 fold; p<0.05) protein expression than lateral/posterior LA. Conclusions This study revealed a three-fold RA-to-LA A1R protein expression gradient in the human heart, leading to significantly greater RA vs. LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest A1R/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine. PMID:27462069

Treatment of out-of-hospital supraventricular tachycardia: adenosine vs verapamil.

PubMed

Brady, W J; DeBehnke, D J; Wickman, L L; Lindbeck, G

1996-06-01

To compare the use of adenosine and the use of verapamil as out-of-hospital therapy for supraventricular tachycardia (SVT). A period of prospective adenosine use (March 1993 to February 1994) was compared with a historical control period of verapamil use (March 1990 to February 1991) for SVT. Data were obtained for SVT patients treated in a metropolitan, fire-department-based paramedic system serving a population of approximately 1 million persons. Standard drug protocols were used and patient outcomes (i.e., conversion rates, complications, and recurrences) were monitored. During the adenosine treatment period, 105 patients had SVT; 87 (83%) received adenosine, of whom 60 (69%) converted to a sinus rhythm (SR). Vagal maneuvers (VM) resulted in restoration of SR in 8 patients (7.6%). Some patients received adenosine for non-SVT rhythms: 7 sinus tachycardia, 18 atrial fibrilation, 7 wide-complex tachycardia (WCT), and 2 ventricular tachycardia; no non-SVT rhythm converted to SR and none of these patients experienced an adverse effect. Twenty-five patients were hemodynamically unstable (systolic blood pressure < 90 mm Hg), with 20 receiving drug and 13 converting to SR; 8 patients required electrical cardioversion. Four patients experienced adverse effects related to adenosine (chest pain dyspnea, prolonged bradycardia, and ventricular tachycardia). In the verapamil period, 106 patients had SVT: 52 (49%) received verapamil (p < 0.001, compared with the adenosine period), of whom 43 (88%) converted to SR (p = 0.11). Two patients received verapamil for WCT; neither converted to SR and both experienced cardiovascular collapse. VM resulted in restoration of SR in 12 patients (11.0%) (p = 0.52). Sixteen patients were hemodynamically unstable, with 5 receiving drug (p = 0.005) and 5 converting to SR; 9 patients required electrical cardioversion (p = 0.48). Four patients experienced adverse effects related to verapamil (hypotension ventricular tachycardia, ventricular

PAP and NT5E inhibit nociceptive neurotransmission by rapidly hydrolyzing nucleotides to adenosine

PubMed Central

2011-01-01

Background Prostatic acid phosphatase (PAP) and ecto-5'-nucleotidase (NT5E, CD73) produce extracellular adenosine from the nucleotide AMP in spinal nociceptive (pain-sensing) circuits; however, it is currently unknown if these are the main ectonucleotidases that generate adenosine or how rapidly they generate adenosine. Results We found that AMP hydrolysis, when measured histochemically, was nearly abolished in dorsal root ganglia (DRG) neurons and lamina II of spinal cord from Pap/Nt5e double knockout (dKO) mice. Likewise, the antinociceptive effects of AMP, when combined with nucleoside transport inhibitors (dipyridamole or 5-iodotubericidin), were reduced by 80-100% in dKO mice. In addition, we used fast scan cyclic voltammetry (FSCV) to measure adenosine production at subsecond resolution within lamina II. Adenosine was maximally produced within seconds from AMP in wild-type (WT) mice but production was reduced >50% in dKO mice, indicating PAP and NT5E rapidly generate adenosine in lamina II. Unexpectedly, we also detected spontaneous low frequency adenosine transients in lamina II with FSCV. Adenosine transients were of short duration (<2 s) and were reduced (>60%) in frequency in Pap-/-, Nt5e-/- and dKO mice, suggesting these ectonucleotidases rapidly hydrolyze endogenously released nucleotides to adenosine. Field potential recordings in lamina II and behavioral studies indicate that adenosine made by these enzymes acts through the adenosine A1 receptor to inhibit excitatory neurotransmission and nociception. Conclusions Collectively, our experiments indicate that PAP and NT5E are the main ectonucleotidases that generate adenosine in nociceptive circuits and indicate these enzymes transform pulsatile or sustained nucleotide release into an inhibitory adenosinergic signal. PMID:22011440

Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay

PubMed Central

Jin, Zhao; Di Rienzi, Sara C.; Janzon, Anders; Werner, Jeff J.; Angenent, Largus T.; Dangl, Jeffrey L.; Fowler, Douglas M.

2015-01-01

Metagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen to Escherichia coli in vitro. The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate. PMID:26637602

Inoculation of Soil with Plant Growth Promoting Bacteria Producing 1-Aminocyclopropane-1-Carboxylate Deaminase or Expression of the Corresponding acdS Gene in Transgenic Plants Increases Salinity Tolerance in Camelina sativa

PubMed Central

Heydarian, Zohreh; Yu, Min; Gruber, Margaret; Glick, Bernard R.; Zhou, Rong; Hegedus, Dwayne D.

2016-01-01

Camelina sativa (camelina) is an oilseed crop touted for use on marginal lands; however, it is no more tolerant of soil salinity than traditional crops, such as canola. Plant growth-promoting bacteria (PGPB) that produce 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase) facilitate plant growth in the presence of abiotic stresses by reducing stress ethylene. Rhizospheric and endophytic PGPB and the corresponding acdS- mutants of the latter were examined for their ability to enhance tolerance to salt in camelina. Stimulation of growth and tolerance to salt was correlated with ACC deaminase production. Inoculation of soil with wild-type PGPB led to increased shoot length in the absence of salt, and increased seed production by approximately 30–50% under moderately saline conditions. The effect of ACC deaminase was further examined in transgenic camelina expressing a bacterial gene encoding ACC deaminase (acdS) under the regulation of the CaMV 35S promoter or the root-specific rolD promoter. Lines expressing acdS, in particular those using the rolD promoter, showed less decline in root length and weight, increased seed production, better seed quality and higher levels of seed oil production under salt stress. This study clearly demonstrates the potential benefit of using either PGPB that produce ACC deaminase or transgenic plants expressing the acdS gene under the control of a root-specific promoter to facilitate plant growth, seed production and seed quality on land that is not normally suitable for the majority of crops due to high salt content. PMID:28018305

Threonine deaminase from extremely halophilic bacteria - Cooperative substrate kinetics and salt dependence.

NASA Technical Reports Server (NTRS)

Lieberman, M. M.; Lanyi, J. K.

1972-01-01

The effect of salt on the activity, stability, and allosteric properties of catabolic threonine deaminase from Halobacterium cutirubrum was studied. The enzyme exhibits sigmoidal kinetics with the substrate, threonine. The Hill slope is 1.55 at pH 10. The enzyme is activated by ADP at low substrate concentrations. In the presence of this effector, sigmoidal kinetics are no longer observed. At pH 10, in the absence of ADP, enzyme activity increases with increasing NaCl concentration from 0 to 4 M.

Thermal Inactivation as a Means of Inhibiting the Serum-Associated Deamination of 9-β-d-Arabinofuranosyladenine in Tissue Culture Media

PubMed Central

Schwartz, Pauline M.; Shipman, Charles; Carlson, Roger H.; Drach, John C.

1974-01-01

9-β-d-Arabinofuranosyladenine (ara-A) was deaminated to 9-β-d-arabinofuranosylhypoxanthine by adenosine deaminase present in fetal bovine serum, newborn calf serum, and calf serum used to supplement tissue culture media. Heating newborn calf serum or calf serum for 12 h at 56 C completely eliminated the enzymatic deamination of ara-A. The deaminase activity associated with fetal bovine serum was more refractory to heating, requiring 24 h for complete inactivation. The nutritive value of heat-inactivated calf serum did not differ significantly from that of unheated serum based on considerations of population doubling times, deoxyribonucleic acid synthesis, and relative cloning efficiencies of KB cells. PMID:4840442

Astrocyte-derived adenosine is central to the hypnogenic effect of glucose

PubMed Central

Scharbarg, Emeric; Daenens, Marion; Lemaître, Frédéric; Geoffroy, Hélène; Guille-Collignon, Manon; Gallopin, Thierry; Rancillac, Armelle

2016-01-01

Sleep has been hypothesised to maintain a close relationship with metabolism. Here we focus on the brain structure that triggers slow-wave sleep, the ventrolateral preoptic nucleus (VLPO), to explore the cellular and molecular signalling pathways recruited by an increase in glucose concentration. We used infrared videomicroscopy on ex vivo brain slices to establish that glucose induces vasodilations specifically in the VLPO via the astrocytic release of adenosine. Real-time detection by in situ purine biosensors further revealed that the adenosine level doubles in response to glucose, and triples during the wakefulness period. Finally, patch-clamp recordings uncovered the depolarizing effect of adenosine and its A2A receptor agonist, CGS-21680, on sleep-promoting VLPO neurons. Altogether, our results provide new insights into the metabolically driven release of adenosine. We hypothesise that adenosine adjusts the local energy supply to local neuronal activity in response to glucose. This pathway could contribute to sleep-wake transition and sleep intensity. PMID:26755200

The Brain In Vivo Expresses the 2′,3′-cAMP-Adenosine Pathway

PubMed Central

Verrier, Jonathan D.; Jackson, Travis C.; Bansal, Rashmi; Kochanek, Patrick M.; Puccio, Ava M.; Okonkwo, David O.; Jackson, Edwin K.

2012-01-01

Although multiple biochemical pathways produce adenosine, studies suggest that the 2′,3′-cAMP-adenosine pathway (2′,3′-cAMP → 2′-AMP/3′-AMP → adenosine) contributes to adenosine production in some cells/tissues/organs. To determine whether the 2′,3′-cAMP-adenosine pathway exists in vivo in the brain, we delivered to the brain (gray matter and white matter separately) via the inflow perfusate of a microdialysis probe either 2′,3′-cAMP, 3′,5′-cAMP, 2′-AMP, 3′-AMP, or 5′-AMP and measured the recovered metabolites in the microdialysis outflow perfusate with mass spectrometry. In both gray and white matter, 2′,3′-cAMP increased 2′-AMP, 3′-AMP and adenosine, and 3′,5′-cAMP increased 5′-AMP and adenosine. In both brain regions, 2′-AMP, 3-AMP and 5′-AMP were converted to adenosine. Microdialysis experiments in 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) wild-type mice demonstrated that traumatic brain injury (TBI; controlled cortical impact model) activated the brain 2,3′-cAMP-adenosine pathway; similar experiments in CNPase knockout mice indicated that CNPase was involved in the metabolism of endogenous 2′,3′-cAMP to 2′-AMP and to adenosine. In CSF from TBI patients, 2′,3′-cAMP was significantly increased in the initial 12 hours after injury and strongly correlated with CSF levels of 2′-AMP, 3′-AMP, adenosine and inosine. We conclude that in vivo, 2′,3′-cAMP is converted to 2′-AMP/3′-AMP, and these AMPs are metabolized to adenosine. This pathway exists endogenously in both mice and humans. PMID:22360621

Oral tremor induced by the muscarinic agonist pilocarpine is suppressed by the adenosine A2A antagonists MSX-3 and SCH58261, but not the adenosine A1 antagonist DPCPX.

PubMed

Collins, Lyndsey E; Galtieri, Daniel J; Brennum, Lise T; Sager, Thomas N; Hockemeyer, Jörg; Müller, Christa E; Hinman, James R; Chrobak, James J; Salamone, John D

2010-02-01

Tremulous jaw movements in rats, which can be induced by dopamine (DA) antagonists, DA depletion, and cholinomimetics, have served as a useful model for studies of tremor. Although adenosine A(2A) antagonists can reduce the tremulous jaw movements induced by DA antagonists and DA depletion, there are conflicting reports about the interaction between adenosine antagonists and cholinomimetic drugs. The present studies investigated the ability of adenosine antagonists to reverse the tremorogenic effect of the muscarinic agonist pilocarpine. While the adenosine A(2A) antagonist MSX-3 was incapable of reversing the tremulous jaw movements induced by the 4.0mg/kg dose of pilocarpine, both MSX-3 and the adenosine A(2A) antagonist SCH58261 reversed the tremulous jaw movements elicited by 0.5mg/kg pilocarpine. Systemic administration of the adenosine A(1) antagonist DPCPX failed to reverse the tremulous jaw movements induced by either an acute 0.5mg/kg dose of the cholinomimetic pilocarpine or the DA D2 antagonist pimozide, indicating that the tremorolytic effects of adenosine antagonists may be receptor subtype specific. Behaviorally active doses of MSX-3 and SCH 58261 showed substantial in vivo occupancy of A(2A) receptors, but DPCPX did not. The results of these studies support the use of adenosine A(2A) antagonists for the treatment of tremor. Copyright 2009 Elsevier Inc. All rights reserved.

[Effects of dopamine and adenosine on regulation of water-electrolyte exchange in Amoeba proteus].

PubMed

Bagrov, Ia Iu; Manusova, N B

2014-01-01

Dopamine and adenosine both regulate transport of sodium chloride in the renal tubules in mammals. We have studied the effect of dopamine and adenosine on spontaneous activity of contractile vacuole of Amoeba proteous. Both substances stimulated contractile vacuole. The effect of dopamine was suppressed by D2 receptor antagonist, haloperidol, but not by D1 antagonist, SCH 39166. Adenylate cyclase inhibitor, 2.5-dideoxyadenosine, suppressed the effect of dopamine, but not of adenosine. Inhibitor of protein kinase C, staurosporine, in contrast, blocked the effect of adenosine, but not dopamine. Notably, dopamine opposed effect of adenosine and vice versa. These results suggest that similar effects of dopamine and adenosine could be mediated by different intracellulare mechanisms.

Prevention of methotrexate-induced nephrotoxicity by concomitant administration of garlic aqueous extract in rat.

PubMed

Ahmed, Walaa; Zaki, Amr; Nabil, Taghred

2015-01-01

Methotrexate (MTX) has been widely used for treatment of cancer and rheumatoid arthritis, but its use has been limited by its nephrotoxicity. This study was carried out to determine whether garlic exerts a protective effect against MTX-induced nephrotoxicity. Nephrotoxicity was induced in rats after a single i.p. injection of MTX (20 mg/kg). Garlic extract (1 mL/100 g b.w.) was given orally for 7 days before and after MTX administration. Serum samples were collected to evaluate urea, creatinine, sodium, phosphorous, potassium, and calcium. Reduced glutathione, catalase, adenosine deaminase, nitric oxide, and malondialdehyde were measured in renal tissue. Tubular injury was evaluated by histopathological examination. MTX increased urea and creatinine levels and led to imbalances in some electrolytes. It also depleted renal antioxidant enzyme levels and increased malondialdehyde, adenosine deaminase, and nitric oxide levels. Histopathological examination showed glomerular and tubular alterations. Pretreatment with garlic significantly improved renal function and increased renal antioxidant enzyme activities. Furthermore, garlic reduced renal oxidative stress and prevented alterations in renal morphology. Garlic treatment has a reversible biochemical and histological effect upon MTX-induced nephrotoxicity.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Guan-Jing; Li, Lan-Fen; Li, Dan

2007-09-01

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

Why do premature newborn infants display elevated blood adenosine levels?

PubMed

Panfoli, Isabella; Cassanello, Michela; Bruschettini, Matteo; Colella, Marina; Cerone, Roberto; Ravera, Silvia; Calzia, Daniela; Candiano, Giovanni; Ramenghi, Luca

2016-05-01

Our preliminary data show high levels of adenosine in the blood of very low birth weight (VLBW) infants, positively correlating to their prematurity (i.e. body weight class). This prompted us to look for a mechanism promoting such impressive adenosine increase. We hypothesized a correlation with oxygen challenge. In fact, it is recognized that either oxygen lack or its excess contribute to the pathogenesis of the injuries of prematurity, such as retinopathy (ROP) and periventricular white matter lesions (PWMI). The optimal concentration of oxygen for resuscitation of VLBW infants is currently under revision. We propose that the elevated adenosine blood concentrations of VLBW infants recognizes two sources. The first could be its activity-dependent release from unmyelinated brain axons. Adenosine in this respect would be an end-product of the hypometabolic VLBW newborn unmyelinated axon intensely firing in response to the environmental stimuli consequent to premature birth. Adenosine would be eventually found in the blood due to blood-brain barrier immaturity. In fact, adenosine is the primary activity-dependent signal promoting differentiation of premyelinating oligodendrocyte progenitor cells (OPC) into myelinating cells in the Central Nervous System, while inhibiting their proliferation and inhibiting synaptic function. The second, would be the ecto-cellular ATP synthesized by the endothelial cell plasmalemma exposed to ambient oxygen concentrations due to premature breathing, especially in lung. ATP would be rapidly transformed into adenosine by the ectonucleotidase activities such as NTPDase I (CD39), and NT5E (CD73). An ectopic extra-mitochondrial aerobic ATP synthetic ability was reported in many cell plasma-membranes, among which endothelial cells. The potential implications of the cited hypotheses for the neonatology area would be great. The amount of oxygen administration for reviving of newborns would find a molecular basis for its assessment. VLBW