Gotowa bibliografia na temat „Adenosine deaminase”

Abstract The role of enzymatic deamination of adenosine monophosphate (AMP) and adenosine in the in vitro growth of the malaria parasite Plasmodium falciparum was investigated by means of human red cells deficient in AMP deaminase to which the adenosine deaminase inhibitor 2′- deoxycoformycin was added. Malaria parasites grew normally in red cells lacking one or both of these enzyme activities. As a further probe of adenosine triphosphate (ATP) catabolism, both infected and uninfected RBCs were incubated with NaF (with and without 2′-deoxycoformycin) and the purine nucleotide/nucleoside content was analyzed by high- performance liquid chromatography (HPLC). Uninfected RBCs lacking either AMP or adenosine deaminase were able to bypass the enzyme block and degrade ATP to hypoxanthine. Uninfected RBCs with both deaminases blocked were unable to produce significant quantities of hypoxanthine. On the other hand, infected RBCs were able to bypass blockade of both deaminases and produce hypoxanthine and adenosine. These findings establish that deamination of adenosine and/or AMP are not essential for plasmodial growth. However, further work will be required to elucidate the pathways that permit the parasites to bypass these catabolic steps.

The role of enzymatic deamination of adenosine monophosphate (AMP) and adenosine in the in vitro growth of the malaria parasite Plasmodium falciparum was investigated by means of human red cells deficient in AMP deaminase to which the adenosine deaminase inhibitor 2′- deoxycoformycin was added. Malaria parasites grew normally in red cells lacking one or both of these enzyme activities. As a further probe of adenosine triphosphate (ATP) catabolism, both infected and uninfected RBCs were incubated with NaF (with and without 2′-deoxycoformycin) and the purine nucleotide/nucleoside content was analyzed by high- performance liquid chromatography (HPLC). Uninfected RBCs lacking either AMP or adenosine deaminase were able to bypass the enzyme block and degrade ATP to hypoxanthine. Uninfected RBCs with both deaminases blocked were unable to produce significant quantities of hypoxanthine. On the other hand, infected RBCs were able to bypass blockade of both deaminases and produce hypoxanthine and adenosine. These findings establish that deamination of adenosine and/or AMP are not essential for plasmodial growth. However, further work will be required to elucidate the pathways that permit the parasites to bypass these catabolic steps.

ABSTRACT Pre-mRNA editing involving the conversion of adenosine to inosine is mediated by adenosine deaminases that act on RNA (ADAR1 and ADAR2). ADARs contain multiple double-stranded RNA(dsRNA)-binding domains in addition to an adenosine deaminase domain. An adenosine deaminase acting on tRNAs, scTad1p (also known as scADAT1), cloned fromSaccharomyces cerevisiae has a deaminase domain related to the ADARs but lacks dsRNA-binding domains. We have identified a gene homologous to scADAT1 in the region of Drosophila melanogaster Adh chromosome II. Recombinant Drosophila ADAT1 (dADAT1) has been expressed in the yeast Pichia pastorisand purified. The enzyme has no activity on dsRNA substrates but is a tRNA deaminase with specificity for adenosine 37 of insect alanine tRNA. dADAT1 shows greater similarity to vertebrate ADARs than to yeast Tad1p, supporting the hypothesis of a common evolutionary origin for ADARs and ADATs. dAdat1 transcripts are maternally supplied in the egg. Zygotic expression is widespread initially and later concentrates in the central nervous system.

Adenosine deaminase was infused into isolated perfused guinea pig hearts to determine its effect on myocardial adenosine levels. The enzyme was administered during constant coronary flow perfusion at 6.11 +/- 0.36 ml.min-1.g-1. Venous adenosine was measured in samples of pulmonary artery effluent; epicardial and endocardial adenosine were measured with the porous nylon disk technique. Infusion of adenosine deaminase at 2.4 and 4.8 U/ml produced adenosine deaminase activity of 0.92 +/- 0.09 and 2.33 +/- 0.15 U/ml, respectively, in epicardial fluid and 1.93 +/- 0.28 and 4.84 +/- 0.47 U/ml, respectively, in endocardial fluid. Aortic pressure was unchanged by infusion of adenosine deaminase at both infusion rates. Adenosine deaminase (data from both infusion rates pooled) reduced epicardial adenosine from 0.327 +/- 0.028 to 0.139 +/- 0.022 microM, endocardial adenosine from 4.61 +/- 0.42 to 1.64 +/- 0.20 microM, and venous adenosine from 0.017 +/- 0.02 to 0.003 +/- 0.001 microM. The data indicate that infused adenosine deaminase reaches the epicardial and endocardial interstitial fluid (ISF) compartments. The absence of any effect on coronary pressure suggests that adenosine may not be involved in resting basal coronary tone. The presence of significant residual adenosine despite adenosine deaminase infusion indicates that adenosine production in the unstressed isolated guinea pig heart exceeds the degradative capacity of infused adenosine deaminase. Previous studies in which it was assumed that almost all of the endogenous adenosine is inactivated by the infusion of adenosine deaminase should be reevaluated in light of these observations.

The adenosine hypothesis of local metabolic control of coronary blood flow was tested in the unstressed heart with adenosine deaminase, which converts adenosine to nonvasoactive inosine. If adenosine is normally an important physiological regulator, then adenosine deaminase should lower coronary blood flow. The left main coronary artery was perfused at constant pressure in anesthetized, closed-chest dogs. Adenosine deaminase was deposited in one region of the left ventricle by selective infusion into a branch of the left coronary artery. Coronary blood flow measured with radioactive microspheres was not lower in the region treated with adenosine deaminase than flow measured simultaneously in an untreated control region of the same heart. This finding is contrary to the prediction of the adenosine hypothesis. Coronary vasodilation elicited by intracoronary adenosine infusion was inhibited in the adenosine deaminase-treated region compared with the control region, indicating that adenosine deaminase lowered adenosine concentration at the vascular adenosine receptor. Inhibition of exogenous adenosine vasodilation was fully reversed by intracoronary infusion of a specific inhibitor of adenosine deaminase. Measurement of adenosine deaminase activity in cardiac lymph provided evidence that adenosine deaminase reached the myocardial interstitial space. These results demonstrate that introducing adenosine deaminase into the interstitial space of the unstressed heart did not lower coronary blood flow. This finding indicates that adenosine is normally below the vasoactive threshold and therefore is not important in mediating local metabolic control of blood flow in the unstressed heart.

Adenosine deaminase and adenosine deaminase complexing protein have been localized in rabbit brain. Brains fixed in paraformaldehyde or in Clarke's solution were blocked coronally. Blocks from brains fixed in paraformaldehyde were either frozen in liquid nitrogen or embedded in paraffin. Tissue fixed in Clarke's solution was embedded in paraffin. Sections from each block were stained by the peroxidase-antiperoxidase method for adenosine deaminase or complexing protein using affinity-purified goat antibodies. Adenosine deaminase and complexing protein did not co-localize. Adenosine deaminase was detected in oligodendroglia and in endothelial cells lining blood vessels, whereas complexing protein was concentrated in neurons. The subcellular location and appearance of the peroxidase reaction product associated with individual cells was also quite distinctive. The cell bodies of adenosine deaminase-positive oligodendroglia were filled with intense deposits of peroxidase reaction product. In contrast to oligodendroglia, the reaction product associated with most neurons stained for complexing protein was concentrated in granular-appearing cytoplasmic deposits. In some instances, these deposits were clustered about the nuclear membrane. Staining of neurons in the granular layer of cerebellum was an exception. Granule cells were lightly outlined by peroxidase reaction product. Cerebellar islands, also referred to as glomeruli, were stained an intense uniform brown. These results raise the possibility that oligodendroglia and blood vessel endothelia, through the action of adenosine deaminase, might play a role in controlling the concentration of extracellular adenosine in brain. They do not, however, support the suggestion that complexing protein aids in adenosine metabolism by positioning adenosine deaminase on the plasma membrane.

CD73 (ecto-5′-nucleotidase) on human gingival fibroblasts plays a role in the regulation of intracellular cAMP levels through the generation of adenosine, which subsequently activates adenosine receptors. In this study, we examined the involvement of ecto-adenosine deaminase, which can be anchored to CD26 on human gingival fibroblasts, in metabolizing adenosine generated by CD73, and thus attenuating adenosine receptor activation. Ecto-adenosine deaminase expression on fibroblasts could be increased by pre-treatment with a lysate of Jurkat cells, a cell line rich in cytoplasmic adenosine deaminase. Interestingly, the cAMP response to adenosine generated from 5′-AMP via CD73 and the ability of 5′-AMP to induce hyaluronan synthase 1 mRNA were significantly decreased by the pre-treatment of fibroblasts with Jurkat cell lysate. This inhibitory effect was reversed by the specific adenosine deaminase inhibitor. These results suggest that ecto-adenosine deaminase metabolizes CD73-generated adenosine and regulates adenosine receptor activation.

Thesis (MSc (Med)(Microbiology)) -- University of Limpopo, 2012.
Mycobacterium tuberculosis is the most common cause of death world-wide and its incidence has been steadily increasing, which is more evident when comparing the global tuberculosis (T8) incidence of 9.24 million in 2006 to 9.27 million cases in 2007. African countries are the second most affected by the epidemic and South Africa is among the 22 highest burden countries most affected by T8 with a very high number of cases relative to the total population. The early diagnosis of tuberculosis and screening of contacts is the cornerstone for controlling spread of active T8 infection. T8 diagnosis becomes even more challenging in patients with immunosuppression (for example in human immunodeficiency virus (HIV) infected), in the case of latent infection and extra pulmonary T8 such as pleural T8. The definitive diagnosis of pleural T8 depends on the demonstration of M. tuberculosis in sputum, pleural fluid and pleural biopsy. Although acid fast bacilli (AF8) microscopy is a rapid, inexpensive and relatively simple method, it has low sensitivity. The culture method is more sensitive than AF8 microscopy, detecting 25-37% of all pleural tuberculosis cases however it takes 4 to 8 weeks for a visible growth on a solid medium. Therefore it is important to find a rapid and reliable test for the diagnosis of pleural T8 particularly in developing countries such as South Africa where there is a high T8 incidence and HIV infection rate.

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The purinergic system is known to be an important signaling pathway in different tissues. Among the components of this system have adenosine, a modulator of central nervous, circulatory and immune systems. The concentration of adenosine in the host is controlled by the enzyme adenosine deaminase (ADA), present in tissues, cells and fluids. As a result, the objectives of this study were (1) to determine the ADA activity in Trypanosoma evansi, (2) evaluate the activity of ADA in serum, erythrocytes, lymphocytes and brain of infected rats, and (3) determine the concentration of nucleotides and nucleosides in serum and cerebral cortex of rats infected with T. evansi. In the first study two mice were infected with T. evansi. When these animals showed high parasitemia (±108 parasites/uL) was performed with blood collection and separation of trypomastigotes by DEAE-cellulose column for performing the assays. Spectrometry was performed by the biochemical detection of ADA in the form trypomastigotes of T. evansi. In a second study, we used 39 rats divided into three groups: group A and B (infected) and group C (C1 and C2 control group) Samples of blood and brain samples were collected on day 4 PI (A and C1) and 20 PI (B and C2). From the blood (with anticoagulant) were separated lymphocytes and erythrocytes for measurement of ADA activity, since the serum was obtained from blood samples stored in tubes without anticoagulant. The brain was separated into cerebellum, cerebral cortex, hippocampus and striatum to evaluate the ADA activity in each structure. Decrease of ADA activity in serum and erythrocytes in rats infected with T. evansi when compared not-infected (P<0.05). ADA activity in lymphocytes was decreased at day 4 PI and increased in day 20 PI. There was no difference in ADA activity in the cerebellum. In the cerebral cortex caused a reduction of ADA activity on days 4 and 20 PI. Decrease of ADA activity in hippocampus and striatum in the day 4 and day 20 PI, respectively. In a third study, 24 rats were used, 12 used as a negative control and 12 infected with T. evansi. On day 4 (n = 6 per group) and 20 PI (n = 6 per group) were performed to obtain blood samples of serum and cerebral cortex for analysis. The samples were prepared for quantification of ATP, ADP, AMP and adenosine. This study found increased concentrations of ATP, AMP and adenosine in the brain and serum of rats infected with T. evansi in both periods, except that the levels of adenosine decreased on day 4 PI. The ADP concentration did not change in this study. Therefore, the infection by T. evansi purinergic system components can be changed, may be involved in immune response, in anemia and neurological signs.
O sistema purinérgico é conhecido por ser uma via de sinalização importante em diversos tecidos. Entre os componentes desse sistema destacamos a adenosina, um modulador do sistema nervoso central, circulatório e imunológico. A concentração de adenosina no hospedeiro é controlada pela enzima adenosina deaminase (ADA), presentes em tecidos, células e fluidos. Em virtude disso, os objetivos deste estudo foram (1) determinar a atividade da ADA no Trypanosoma evansi; (2) avaliar a atividade da ADA no soro, eritrócitos, linfócitos e encéfalo e (3) determinar a concentração de nucleotídeos e nucleosideos no soro e córtex cerebral de ratos infectados com T. evansi. Para um primeiro estudo foram infectados dois camundongos com T. evansi. Quando estes animais apresentavam elevada parasitemia (±108 parasito/μL) foi realizada a coleta de sangue e separação dos flagelados por coluna de DEAE-celulose, a fim realização dos ensaios enzimáticos no parasito. Atividade da ADA nas formas trypomastigotas de T. evansi foi determinada por espectofotometria. Em um segundo estudo foi utilizado 39 ratos, divididos em três grupos: grupo A e B (infectado) e grupo C (C1 e C2/controle). Amostras de sangue e encéfalo foram colhidas nos dias 4 pós-infecção (PI) (grupos A e C1) e 20 PI (grupos B e C2). A partir do sangue total colhido com anticoagulante foram separados os linfócitos e eritrócitos para mensuração da atividade da ADA, já o soro foi obtido de amostras de sangue armazenadas em tubos sem anticoagulante. O encéfalo foi separado em cerebelo, córtex cerebral, hipocampo e estriado para avaliar a atividade da ADA em cada estrutura. Então, observou-se redução da atividade de ADA no soro e eritrócitos em ratos infectados com T. evansi em comparação com não-infectados (P <0,05). A atividade de ADA em linfócitos estava diminuída no dia 4 PI e aumentou no dia 20 PI. Não houve diferença da ADA no cerebelo. No córtex cerebral, no hipocampo e estriado ocorreu redução da atividade da ADA nos dia 4 e 20 PI, respectivamente. Em todas as estruturas do encéfalo foi detectada a presença do parasito por PCR. Em um terceiro estudo foram utilizados 24 ratos, sendo 12 controles negativos e outros 12 infectados com T. evansi. Nos dias 4 (n=6 por grupo) e 20 (n=6 por grupo) foram realizadas as coletas de sangue para obtenção do soro e amostras do córtex cerebral para mensuração dos níveis de ATP, ADP, AMP e adenosina. Neste estudo, foi constatado aumento das concentrações de ATP, AMP e adenosina no encéfalo e soro de ratos infectados com T. evansi nos dois períodos avaliados, com exceção dos níveis de adenosina que reduziram no dia 4 PI. Não houve alteração na concentração de ADP. Portanto, na infecção por T. evansi os componentes do sistema purinérgico pode ser alterados, podendo estar envolvido na resposta imunológica, na anemia e nos sinais neurológicos.

Na Anemia Falciforme em situações de baixa tensão de oxigênio, a hemoglobina mutante S (HbS) sofre polimerização promovendo a falcização das hemácias, que podem aderir ao endotélio vascular, causando a oclusão de vasos (VO) e isquemia tecidual (crises dolorosas) que caracterizam o quadro clínico da doença. Além disso, os pacientes falciformes apresentam outras manifestações clínicas como o priapismo, alterações ósseas, certas complicações pulmonares entre outros. Além das células eritróides, células endoteliais, leucócitos e plaquetas também desempenham um papel fundamental na fisiopatologia da anemia falciforme. A hidroxiuréia (HU), na anemia falciforme, aumenta a produção de hemoglobina fetal (HbF) em células eritróides, reduzindo a polimerização da HbS, diminuindo os sintomas clínicos dos pacientes. O aumento da HbF, no entanto, não implica necessariamente na melhora clínica, indicando desta forma a potencial ação da HU sobre outros processos. Estudos recentes vêm relacionando priapismo e asma com elevados níveis de adenosina. Devido a esta importância da adenosina relacionada a patologias comuns a AF, tivemos como objetivo identificar polimorfismos em genes de receptores de adenosina e na adenosina deaminase e verificar a possível associação entre as manifestações clínicas, além de investigar o papel da HU na modulação de marcadores envolvido na síntese e degradação da adenosina. Foram analisados diversos sítios polimórficos nos genes que codificam ADORA1, ADORA 2b, ADORA 3 e ADA, seguindo com a genotipagem em pacientes com AF, comparando afetados e não afetados. Em adição foi avaliada a expressão diferencial de mRNA de ADA pela HU em monócitos destes pacientes, comparando tratados e não tratados e também avaliamos por citometria de fluxo a modulação de marcadores de superfície CD39, CD73 e CD26, pela HU. As análises estatísticas foram realizadas utilizando os softwares GenePop 3.4 para análises de associação, cálculo do HWE, GraphPad Prism 5, Arlequin para identificação de desequilíbrio de ligação, haplótipos, heterozigozidade e SAS 9.13 para associação dos haplótipos as características. Os resultados mostraram que os pacientes sob tratamento com HU apresentaram um aumento da expressão de mRNA de ADA, aumento da expressão de CD26 em monócitos e diminuição de CD39 em linfócitos. Sem alterações significativas em relação a CD73. Encontramos também um aumento da freqüência do alelo T do SNP (rs1685103) presente no gene de ADORA 1 associado com pacientes afetados com síndrome torácica aguda. Apesar de não ter sido estatisticamente significante, concorda com dados da literatura. No gene ADORA 2B, verificamos associação do SNP 1007 C>T no desenvolvimento de STA indicando o alelo T como fator de risco e o alelo C para alterações ósseas. Para o SNP 968 G>T houve associação com alterações ósseas. Na análise haplotípica entre os SNPs 968 G>T e 1007 C>T encontramos associação dos haplótipos ht2 e ht3 com STA, como fator de risco, ht2 para hipertensão pulmonar. ht1 para priapismo, alterações ósseas e estenose/AVC. Os haplótipos formados pelos três SNPs 968 G>T, 1007 C>T e rs16851030, encontramos associação entre ht1, ht3 e ht4 entre os afetados com priapismo, caracterizando-o como haplótipo de risco e também ht1 e ht6 associados à estenose/AVC. Concluímos, que a hidroxiuréia participa na modulação da expressão da adenosina deaminase, de CD26 em monócitos e CD39 em linfócitos. Além disso, mostrou-se a importância de sítios polimórfico presente no gene ADORA 2B e ADORA1 envolvido na fisiopatologia das manifestações clínicas da doença falciforme. Associações dos SNPs em ADORA 1 e ADA, devem ser melhor estudados em um número maior de pacientes. A determinação destes polimorfismos associados com diferentes características clínicas pode levar a um melhor entendimento dos processos fisiopatológicos da anemia falciforme, levando à identificação de pacientes de risco, possibilitando um manejamento racional dos mesmos, em termos de cuidados específicos, ou mesmo à determinação de alvos para o desenvolvimento de terapias alternativas.
In sickle cell disease in low oxygen tension, mutant hemoglobin S (HbS) undergoes polymerization promoting sickling of red blood cells that can adhere to vascular endothelium, causing vessel occlusion (VO) and tissue ischemia (painful crises) that characterize the clinical disease. In addition, sickle cell patients have other clinical manifestations such as priapism, bone disorders, certain pulmonary complications among others. In addition to the erythroid cells, endothelial cells, white cells and platelets also play a key role in the pathophysiology of sickle cell anemia. Hydroxyurea (HU) in sickle cell anemia, increases the production of fetal hemoglobin (HbF) in erythroid cells, reducing the HbS polymerization, reducing the clinical symptoms of patients. The increase in HbF, however, does not necessarily imply clinical improvement, thus indicating the potential effects of HU on other processes. Recent studies relating asthma and priapism with high levels of adenosine. Due to this importance of adenosine-related pathologies common to AF, we aimed to identify gene polymorphisms in adenosine receptors and adenosine deaminase and verify the possible association between clinical manifestations, and to investigate the role of HU in the modulation of markers involved synthesis and degradation of adenosine. We analyzed several polymorphic sites in genes that encode ADORA1, ADORA 2b, 3 and ADORA ADA, according to the genotype in patients with AF, comparing affected and unaffected. In addition we assessed the differential expression of ADA mRNA by HU in monocytes of these patients, comparing treated and untreated, and also evaluated by flow cytometry modulation of surface markers CD39, CD73 and CD26 by HU. Statistical analysis was performed using the software GenePop 3.4 for association analysis, calculation of HWE, GraphPad Prism 5, Arlequin for identification of linkage disequilibrium, haplotypes, heterozygosity and SAS 9.13 for association of haplotypes features. The results showed that patients treated with HU showed an increase in mRNA expression of ADA, increased expression of CD26 on monocytes and decreased CD39 on lymphocytes. No significant changes in relation to CD73. We also found an increased frequency of allele T (SNP rs1685103) present in a gene associated with ADORA affected patients with acute chest syndrome. Although not statistically significant, agrees with literature data. ADORA 2B gene, we found association of the SNP 1007 C> T in the development of STA indicating the T allele as a risk factor for the C allele and bone changes. For the SNP 968 G> T was associated with bone disorders. In haplotype analysis between SNPs 968 G> T and 1007 C> T found association of haplotypes ht2 and HT3 with STA as a risk factor for pulmonary hypertension ht2. ht1 for priapism, stenosis and bone disorders / stroke. The three haplotypes formed by SNPs 968 G> T, 1007 C> T and rs16851030, we found association between ht1, HT3 and HT4 among those affected with priapism, characterizing it as a risk haplotype and also ht1 ht6 associated with renal and / AVC. We conclude that hydroxyurea participates in modulating the expression of adenosine deaminase of CD26 on monocytes and CD39 on lymphocytes. Moreover, he showed the importance of polymorphic sites in this gene and ADORA 2B ADORA1 involved in the pathophysiology of clinical manifestations of sickle cell disease. Associations of SNPs in ADORA 1 and ADA should be better studied in a larger number of patients. The determination of these polymorphisms associated with different clinical characteristics can lead to a better understanding of the pathophysiological processes of sickle cell anemia, leading to the identification of patients at risk, enabling a rational handling of the same in terms of specific care, or even the determination of targets for the development of alternative therapies.

Adenosine deaminase (ADA) severe combined immunodeficiency (SCED) is a life-threatening condition resulting from lack of the ADA enzyme. Consequences include immunodeficiency and non-immunological symptoms such as neurological abnormalities. Bone marrow transplantation (BMT) from a haploidentical donor usually results in complete restoration of immune function. However, the majority of patients do not have a matched donor and are therefore treated with enzyme replacement therapy (PEG-ADA). This treatment is not always fully effective, it is expensive and needs to be administered throughout life. Gene therapy is an alternative treatment, and previous trials for ADA deficiency have shown that it can significantly improve immunological function. Immune recovery was assessed in three ADA-SCID patients treated with PEG-ADA by analysis of lymphocyte counts and emergence of naive T cells. One patient was not responding well to PEG-ADA and was enrolled in a Phase I clinical gene therapy trial. A gammaretroviral vector encoding ADA was constructed and tested extensively on cell lines and patient cells and a CD34+ cell transduction protocol was optimised. The gene therapy procedure was based on previous successful trials, and involved withdrawal of PEG-ADA prior to treatment to provide selective growth advantage for transduced cells, and mild conditioning to encourage engraftment. Assessments of immune function were then performed in a similar manner to patients treated with PEG-AD A. Recent evidence from studies of ADA deficiency indicates that it is a multi-organ disease. However, gene therapy using CD34+ cells may only correct the immunodeficiency without ameliorating non-immunological symptoms. Hence, studies were performed to develop systemic gene therapy for ADA-SCID, involving the use of CD34+ cells and mesenchymal stem cells (MSCs). MSCs were isolated from bone marrow, and their multipotential nature was assessed prior to and following gene transfer using a cloned ADA lentiviral vector. Transduced MSCs maintained their ability to undergo differentiation and transgene expression was not affected by this. These clinical and preclinical in vitro studies demonstrate that gene therapy holds therapeutic potential for treatment of ADA-SCID.

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Nucleotide- and nucleoside-degrading enzymes are present in the surface of platelets, blood cells involved in clotting disturbances of Trypanosoma evansi-infected animals. Thus, this study was aimed at evaluating the activity of the enzymes NTPDase, 5 - nucleotidase and adenosine deaminase in platelets of rats experimentally infected by T. evansi. Animals were divided into four groups, according to the degree of parasitemia. Samples were collected at days 3 (group A: at the beginning of parasitemia), 5 (group B: high parasitemia) and 15 (group C: chronic infection). Group D (control group) was composed of non-infected animals. Blood samples with citrate as the anticoagulant were collected and used for platelet separation and enzymatic assays. NTPDase, 5 - nucleotidase and adenosine deaminase (ADA) activities were decreased (p<0.05) in platelets from rats of groups A and B, when compared to the control group. In group C, only NTPDase and 5 -nucleoside activities were decreased (p<0.001), observed by ADP and AMP hydrolysis. The correlation between platelet count and nucleotide and nucleoside hydrolysis was positive and statistically significant (p<0.05) in groups A and B. Platelet aggregation of all infected groups was decreased in comparison to the control group (p<0.05). Based upon the results, it is concluded that the alterations observed in the activity of the enzymes NTPDase, 5 -nucleotidase and adenosine deaminase in platelets of T. evansi-infected animals might be related to thrombocytopenia.
Nucleotide- and nucleoside-degrading enzymes are present in the surface of platelets, blood cells involved in clotting disturbances of Trypanosoma evansi-infected animals. Thus, this study was aimed at evaluating the activity of the enzymes NTPDase, 5 - nucleotidase and adenosine deaminase in platelets of rats experimentally infected by T. evansi. Animals were divided into four groups, according to the degree of parasitemia. Samples were collected at days 3 (group A: at the beginning of parasitemia), 5 (group B: high parasitemia) and 15 (group C: chronic infection). Group D (control group) was composed of non-infected animals. Blood samples with citrate as the anticoagulant were collected and used for platelet separation and enzymatic assays. NTPDase, 5 - nucleotidase and adenosine deaminase (ADA) activities were decreased (p<0.05) in platelets from rats of groups A and B, when compared to the control group. In group C, only NTPDase and 5 -nucleoside activities were decreased (p<0.001), observed by ADP and AMP hydrolysis. The correlation between platelet count and nucleotide and nucleoside hydrolysis was positive and statistically significant (p<0.05) in groups A and B. Platelet aggregation of all infected groups was decreased in comparison to the control group (p<0.05). Based upon the results, it is concluded that the alterations observed in the activity of the enzymes NTPDase, 5 -nucleotidase and adenosine deaminase in platelets of T. evansi-infected animals might be related to thrombocytopenia.

Adenosine deaminase acting on transfer RNA (ADAT) is a human heterodimeric enzyme that catalyzes the deamination of adenosine (A) to inosine (I) at the first position of the anticodon of transfer RNAs (tRNAs) (position 34, or wobble position); one of the few essential post-transcriptional modifications on tRNAs (1-5). Inosine 34 allows the recognition of three different nucleotides: cytidine, uridine and adenosine, at the third position of the codon, thus increasing the decoding capacity of tRNAs to more than one messenger RNA (mRNA) codon (adenosine 34 can in principle only pair with codons with uridine at the third position) (6, 7). This alters the tRNA pool available for each codon and it has been proved to align the correlation between codon usage and tRNA gene copy number (8). It has also been suggested to improve fidelity and efficiency of translation (8, 9), especially for mRNAs enriched in codons translated by modified tRNAs (10, 11). Monitoring ADAT-mediated deamination is crucial for the characterization of the enzyme in terms of activity, substrates, regulation, as well as for drug discovery purposes. However, this analysis is often challenging, laborious and lacks quantitativeness. We developed an in vitro deamination assay based on restriction fragment length polymorphism (RFLP) analyses to monitor ADAT activity in an efficient, cost-effective, and semiquantitative manner (12). To overcome a limitation of the method being the need of reverse transcription and amplification of the tRNA, we designed a direct method to quantify I34 formation in vitro using the first fluorescent analogs of nucleic acids that have been reported to undergo enzymatic deaminations (13-15). ADAT has been conserved over the evolution with the acquisition of multi-substrate specificity. Whereas its bacterial homolog TadA deaminates exclusively tRNAArg (2), the human enzyme deaminates eight different tRNAs (3, 16). However, the mechanisms that drove this evolution remain unknown. While the substrate recognition in TadA has been well studied, in the eukaryotic ADAT is poorly understood. Through in vitro enzymatic activity assays with different variants of tRNAArg and tRNAAla, we elucidated the most important features for efficient A34-to-I34 conversion and characterized the substrate recognition of the human enzyme. We also proposed a new potential mechanism of control of ADAT deamination activity by human tRNA-derived fragments, which provides new insights into the regulation of ADAT function and may open a door for the development of new strategies to modulate ADAT activity. A missense mutation (V128M) in one of the two subunits of the human ADAT enzyme causes intellectual disability and strabismus, but the molecular bases of the pathology are unknown (17, 18). We characterized human ADAT in terms of kinetics and structure, and investigated the effect of the V128M mutation. We found that this substitution decreases ADAT deamination activity, and severely affects the stability of the quaternary structure of the enzyme. In this regard, we discovered small molecules with the ability to activate the enzyme, which could potentially recover the defective tRNA editing caused by the mutation. References 1. Gerber AP, Keller W. An adenosine deaminase that generates inosine at the wobble position of tRNAs. Science. 1999;286(5442):1146-9. Epub 1999/11/05. 2. Wolf J, Gerber AP, Keller W. tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli. The EMBO journal. 2002;21(14):3841-51. Epub 2002/07/12. 3. Torres AG, Pineyro D, Rodriguez-Escriba M, Camacho N, Reina O, Saint-Leger A, et al. Inosine modifications in human tRNAs are incorporated at the precursor tRNA level. Nucleic acids research. 2015;43(10):5145-57. Epub 2015/04/29. 4. Zhou W, Karcher D, Bock R. Identification of enzymes for adenosine-to-inosine editing and discovery of cytidine-to-uridine editing in nucleus-encoded transfer RNAs of Arabidopsis. Plant physiology. 2014;166(4):1985-97. Epub 2014/10/16. 5. Tsutsumi S, Sugiura R, Ma Y, Tokuoka H, Ohta K, Ohte R, et al. Wobble inosine tRNA modification is essential to cell cycle progression in G(1)/S and G(2)/M transitions in fission yeast. J Biol Chem. 2007;282(46):33459-65. Epub 2007/09/19. 6. Crick FH. Codon--anticodon pairing: the wobble hypothesis. Journal of molecular biology. 1966;19(2):548-55. Epub 1966/08/01. 7. Torres AG, Pineyro D, Filonava L, Stracker TH, Batlle E, Ribas de Pouplana L. A-to-I editing on tRNAs: biochemical, biological and evolutionary implications. FEBS Lett. 2014;588(23):4279-86. Epub 2014/09/30. 8. Novoa EM, Pavon-Eternod M, Pan T, Ribas de Pouplana L. A role for tRNA modifications in genome structure and codon usage. Cell. 2012;149(1):202-13. Epub 2012/04/03. 9. Schaub M, Keller W. RNA editing by adenosine deaminases generates RNA and protein diversity. Biochimie. 2002;84(8):791-803. Epub 2002/11/30. 10. Rafels-Ybern A, Attolini CS, Ribas de Pouplana L. Distribution of ADAT-Dependent Codons in the Human Transcriptome. International journal of molecular sciences. 2015;16(8):17303- 14. Epub 2015/08/01. 11. Rafels-Ybern A, Torres AG, Grau-Bove X, Ruiz-Trillo I, de Pouplana LR. Codon adaptation to tRNAs with Inosine modification at position 34 is widespread among Eukaryotes and present in two Bacterial phyla. RNA biology. 2017:0. Epub 2017/09/08. 12. Wulff TF, Arguello RJ, Molina Jordan M, Roura Frigole H, Hauquier G, Filonava L, et al. Detection of a Subset of Posttranscriptional Transfer RNA Modifications in Vivo with a Restriction Fragment Length Polymorphism-Based Method. Biochemistry. 2017;56(31):4029-38. Epub 2017/07/14. 13. Sinkeldam RW, McCoy LS, Shin D, Tor Y. Enzymatic interconversion of isomorphic fluorescent nucleosides: adenosine deaminase transforms an adenosine analogue into an inosine analogue. Angew Chem Int Ed Engl. 2013;52(52):14026-30. Epub 2013/11/30. 14. McCoy LS, Shin D, Tor Y. Isomorphic emissive GTP surrogate facilitates initiation and elongation of in vitro transcription reactions. Journal of the American Chemical Society. 2014;136(43):15176-84. Epub 2014/09/26. 15. Rovira AR, Fin A, Tor Y. Chemical Mutagenesis of an Emissive RNA Alphabet. J Am Chem Soc. 2015;137(46):14602-5. Epub 2015/11/03. 16. Juhling F, Morl M, Hartmann RK, Sprinzl M, Stadler PF, Putz J. tRNAdb 2009: compilation of tRNA sequences and tRNA genes. Nucleic acids research. 2009;37(Database issue):D159-62. Epub 2008/10/30. 17. Alazami AM, Hijazi H, Al-Dosari MS, Shaheen R, Hashem A, Aldahmesh MA, et al. Mutation in ADAT3, encoding adenosine deaminase acting on transfer RNA, causes intellectual disability and strabismus. Journal of medical genetics. 2013;50(7):425-30. Epub 2013/04/27. 18. El-Hattab AW, Saleh MA, Hashem A, Al-Owain M, Asmari AA, Rabei H, et al. ADAT3- related intellectual disability: Further delineation of the phenotype. American journal of medical genetics Part A. 2016;170A(5):1142-7. Epub 2016/02/05.
L’adenosina deaminasa específica per RNA de transferència (ADAT) és un enzim humà heterodimèric que catalitza la reacció de deaminació de l’adenosina (A) a inosina (I) a la primera posició de l’anticodó de RNAs de transferència (tRNAs) (també anomenada posició 34 o posició de balanceig); una de les poques modificacions post-transcripcionals essencials en tRNAs (1-5). La inosina 34 permet el reconeixement de tres nucleòtids diferents: citidina, uridina i adenosina, a la tercera posició del codó, augmentant per tant la capacitat de descodificació dels tRNAs a més d’un codó en l’RNA missatger (mRNA) (l’adenosina 34 en principi únicament pot aparellar-se amb uridina en la tercera posició) (6, 7). Això altera els nivells de tRNAs disponibles per cada codó i s’ha demostrat que alinea la correlació entre l’ús de codons i número de còpies gèniques de cada tRNA (8). També s’ha suggerit que millora la fidelitat i eficiència de la traducció (8, 9), especialment per mRNAs enriquits en codons traduïts per tRNA modificats (10, 11). Monitoritzar la deaminació produïda per ADAT és clau per la caracterització de l’enzim en termes d’activitat, substrats, regulació, així com també pel disseny de fàrmacs. No obstant, aquest anàlisi és sovint complex, laboriós i poc quantitatiu. Per això, hem desenvolupat un assaig de deaminació in vitro basat en el polimorfisme de longitud dels fragments de restricció (RFLP) amb el propòsit de monitoritzar l’activitat d’ADAT de manera eficient, cost-efectiva i semiquantitativa (12). Per superar una limitació del mètode essent la necessitat de transcripció reversa i amplificació del tRNA, hem dissenyat un mètode directe per quantificar la formació d’I34 in vitro utilitzant els primers anàlegs fluorescents d’àcids nucleics que són substrats de deaminacions enzimàtiques descrits fins al moment (13-15). ADAT ha estat conservat en l’evolució amb l’adquisició de multi-especificitat de substrat. Mentre que el seu homòleg bacterià TadA deamina exclusivament tRNAArg (2), l’enzim humà deamina vuit tRNAs diferents (3, 16). Tot i així, els mecanismes que van conduir a aquesta evolució romanen desconeguts. Mentre que el reconeixement de substrat en TadA es coneix bé, en ADAT eucariòtic ha estat poc estudiat. A través d’assajos enzimàtics in vitro amb diferents variants de tRNAArg i tRNAAla, hem elucidat els trets més importants per a una conversió eficient d’A34 a I34 i hem caracteritzat el reconeixement de substrat per part de l’enzim humà. També proposem un nou potencial mecanisme de control de l’activitat d’ADAT per part de fragments derivats de tRNAs humans, el qual ofereix noves perspectives en la regulació de la funció d’ADAT i que pot obrir la porta al desenvolupament de noves estratègies per modular-ne l’activitat. Una mutació sense sentit (V128M) en una de les dues subunitats en l’enzim ADAT humà s’ha associat a retard mental i estrabisme, encara que les bases moleculars de la patologia es desconeixen (17, 18). Hem caracteritzat ADAT humà en termes de cinètica enzimàtica i estructura, i n’hem investigat l’efecte de la mutació V128M. Hem descobert que la substitució de valina 128 per metionina redueix l’activitat deaminatòria d’ADAT i que altera severament l’estabilitat de l’estructura quaternària de l’enzim. En aquest aspecte, hem descobert molècules amb l’habilitat d’activar l’enzim, la qual cosa podria revertir potencialment la reducció en l’activitat enzimàtica causada per la mutació. References 1. Gerber AP, Keller W. An adenosine deaminase that generates inosine at the wobble position of tRNAs. Science. 1999;286(5442):1146-9. Epub 1999/11/05. 2. Wolf J, Gerber AP, Keller W. tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli. The EMBO journal. 2002;21(14):3841-51. Epub 2002/07/12. 3. Torres AG, Pineyro D, Rodriguez-Escriba M, Camacho N, Reina O, Saint-Leger A, et al. Inosine modifications in human tRNAs are incorporated at the precursor tRNA level. Nucleic acids research. 2015;43(10):5145-57. Epub 2015/04/29. 4. Zhou W, Karcher D, Bock R. Identification of enzymes for adenosine-to-inosine editing and discovery of cytidine-to-uridine editing in nucleus-encoded transfer RNAs of Arabidopsis. Plant physiology. 2014;166(4):1985-97. Epub 2014/10/16. 5. Tsutsumi S, Sugiura R, Ma Y, Tokuoka H, Ohta K, Ohte R, et al. Wobble inosine tRNA modification is essential to cell cycle progression in G(1)/S and G(2)/M transitions in fission yeast. J Biol Chem. 2007;282(46):33459-65. Epub 2007/09/19. 6. Crick FH. Codon--anticodon pairing: the wobble hypothesis. Journal of molecular biology. 1966;19(2):548-55. Epub 1966/08/01. 7. Torres AG, Pineyro D, Filonava L, Stracker TH, Batlle E, Ribas de Pouplana L. A-to-I editing on tRNAs: biochemical, biological and evolutionary implications. FEBS Lett. 2014;588(23):4279-86. Epub 2014/09/30. 8. Novoa EM, Pavon-Eternod M, Pan T, Ribas de Pouplana L. A role for tRNA modifications in genome structure and codon usage. Cell. 2012;149(1):202-13. Epub 2012/04/03. 9. Schaub M, Keller W. RNA editing by adenosine deaminases generates RNA and protein diversity. Biochimie. 2002;84(8):791-803. Epub 2002/11/30. 10. Rafels-Ybern A, Attolini CS, Ribas de Pouplana L. Distribution of ADAT-Dependent Codons in the Human Transcriptome. International journal of molecular sciences. 2015;16(8):17303- 14. Epub 2015/08/01. 11. Rafels-Ybern A, Torres AG, Grau-Bove X, Ruiz-Trillo I, de Pouplana LR. Codon adaptation to tRNAs with Inosine modification at position 34 is widespread among Eukaryotes and present in two Bacterial phyla. RNA biology. 2017:0. Epub 2017/09/08. 12. Wulff TF, Arguello RJ, Molina Jordan M, Roura Frigole H, Hauquier G, Filonava L, et al. Detection of a Subset of Posttranscriptional Transfer RNA Modifications in Vivo with a Restriction Fragment Length Polymorphism-Based Method. Biochemistry. 2017;56(31):4029-38. Epub 2017/07/14. 13. Sinkeldam RW, McCoy LS, Shin D, Tor Y. Enzymatic interconversion of isomorphic fluorescent nucleosides: adenosine deaminase transforms an adenosine analogue into an inosine analogue. Angew Chem Int Ed Engl. 2013;52(52):14026-30. Epub 2013/11/30. 14. McCoy LS, Shin D, Tor Y. Isomorphic emissive GTP surrogate facilitates initiation and elongation of in vitro transcription reactions. Journal of the American Chemical Society. 2014;136(43):15176-84. Epub 2014/09/26. 15. Rovira AR, Fin A, Tor Y. Chemical Mutagenesis of an Emissive RNA Alphabet. J Am Chem Soc. 2015;137(46):14602-5. Epub 2015/11/03. 16. Juhling F, Morl M, Hartmann RK, Sprinzl M, Stadler PF, Putz J. tRNAdb 2009: compilation of tRNA sequences and tRNA genes. Nucleic acids research. 2009;37(Database issue):D159-62. Epub 2008/10/30. 17. Alazami AM, Hijazi H, Al-Dosari MS, Shaheen R, Hashem A, Aldahmesh MA, et al. Mutation in ADAT3, encoding adenosine deaminase acting on transfer RNA, causes intellectual disability and strabismus. Journal of medical genetics. 2013;50(7):425-30. Epub 2013/04/27. 18. El-Hattab AW, Saleh MA, Hashem A, Al-Owain M, Asmari AA, Rabei H, et al. ADAT3- related intellectual disability: Further delineation of the phenotype. American journal of medical genetics Part A. 2016;170A(5):1142-7. Epub 2016/02/05.

Diabetes mellitus (DM) is a metabolic disorder of multiple etiology characterized by chronic hyperglycemia resulting from deficiency of insulin production and/or action. This state of hyperglycemia may cause a variety of cardiovascular, renal, neurological and eye complications. Adenosine deaminase (ADA) is an important enzyme responsible for regulation the levels of adenosine (ado) an important component of the system purinergic nucleoside. Changes in ADA activity has been demonstrated in several diseases, including DM. The Rutin (RT) is an abundant polyphenolic flavonoid found in food that exhibits multiple pharmacological activities including antibacterial, antitumoural, vasodilator and hepatoprotective activities. The objective of this study was to investigate the effect of RT on the activity of ADA in serum, tissues and biochemical parameters in models of diabetes induced by streptozotocin (STZ). Diabetes was induced in rats by an intraperitoneal injection of streptozotocin (STZ). RT (100 mg/kg/day) and glibenclamide (10mg/kg/day) were administered for 30 days, except for control groups (non diabetic and diabetic). Six groups of rats were used in the study and grouped based on fasting blood glucose levels after diabetes induction. The results showed an increase in ADA activity in serum and liver of diabetic rats, like transaminases (AST, ALT), -glutamyltransferase (-GT) and glucose. The RT at a concentration of 100 mg/kg was able to reduce the ADA activity in serum and liver tissue when compared with the diabetic control. The protective effect of RT was also observed increases the activity of enzymes ALT and -GT. Significant reductions were also observed in total cholesterol and LDL-cholesterol as well as in blood glucose levels in the diabetic group treated with RT. The results suggest that RT can improve hyperglycemia and hyperlipidemia, and restoring damaged liver function, as well as prevents the increase in ADA activity in serum and liver tissue on diabetic rats treated with this flavonoid.
O Diabetes mellitus (DM) é uma disfunção metabólica de múltipla etiologia caracterizado por hiperglicemia crônica resultante da deficiência da produção e/ou ação da insulina. Esse estado de hiperglicemia pode provocar uma série de complicações cardiovasculares, renais, neurológicas e oculares. A Adenosina deaminase (ADA) é uma importante enzima responsável por regular os níveis de adenosina (ado), um importante nucleosídeo componente do sistema purinérgico. Alterações na atividade da ADA têm sido demonstradas em várias doenças, incluindo o DM. A rutina (RT) é um flavonoide polifenólico abundante nos alimentos que exibe múltiplas atividades farmacológicas como atividade antibacteriana, antitumoral, vasodilatadora e hepatoprotetora. O objetivo deste estudo foi verificar o efeito da RT sobre a atividade da ADA sérica e tecidual e parâmetros bioquímicos em modelos de diabetes induzidos por estreptozotocina (STZ). O diabetes foi induzido através de injeção única intraperitoneal (i.p.) de 55 mg/kg de STZ. A RT (100 mg / kg / dia) e a glibenclamida (10mg/kg/dia) foram administradas durante 30 dias, com exceção dos grupos controles (não diabéticos e diabéticos). Seis grupos de ratos foram utilizados no estudo e agrupados com base nos níveis de glicose em jejum após a indução de diabetes. Os resultados demonstraram um aumento na atividade da ADA no soro e no fígado de ratos diabéticos, assim como das transaminases (AST, ALT), -glutamiltransferase (-GT) e glicose. A RT na concentração de 100 mg/kg foi capaz de reduzir a atividade sérica e em tecido hepático da ADA quando comparado com o controle. O efeito protetor da RT também foi observado sobe a atividade das enzimas ALT e -GT. Reduções significativas foram observadas no colesterol total e LDL-colesterol, bem como, na concentração sérica de glicose no grupo diabético tratado com RT. Os resultados sugerem que a RT pode melhorar a hiperglicemia e dislipidemia, restabelecer danos à função hepática, bem como é capaz de prevenir o aumento da atividade da ADA no soro e no fígado de ratos diabéticos tratados com este flavonoide.