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Molecular characterization of five Portuguese patients with pyrimidine 5’-nucleotidase deficient hemolytic anemia showing three new P5’N-I mutations

Manco, Licínio; Relvas, Luís; Pinto, C. Silva; Pereira, Janet; Almeida, A. Bessa; Ribeiro, M. Letícia
Fonte: Ferrata Storti Foundation Publicador: Ferrata Storti Foundation
Tipo: Artigo de Revista Científica
Português
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Four different gene mutations were identified in five unrelated Portuguese patients with pyrimidine 5’-nucleotidase type I (P5’N-I) deficient chronic hemolytic anemia. Mutations 502G®C (168 Gly®Arg), 773T®C (258Ile®Thr) and the insertion of an Alu element in exon 9, leading to skipping of this exon in the mRNA transcript, are newly described mutations whereas mutation 425T®C (142Leu®Pro) has been previously reported.

Interaction between ecto-5’-nucleotidase and adenosine A2A receptors in nerve terminals of mice prefrontal cortex

Silva, Tiago Soares
Fonte: Universidade de Coimbra Publicador: Universidade de Coimbra
Tipo: Dissertação de Mestrado
Português
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A ativação dos recetores de adenosina A2A (A2AR) é feita através da adenosina que pode ser produzida através do catabolismo do ATP libertado no meio extracelular. A ecto-5’-nucleotidase (e-5’N) desempenha um papel importante na formação de adenosina proveniente do catabolismo do ATP, e subsequentemente na ativação dos A2AR controlando assim a plasticidade sináptica. Após uma lesão cerebral, o ATP é libertado como um sinal aversivo provocando o aumento em simultâneo da atividade da e-5’N e da densidade dos A2AR. Isto levanta a hipótese de que a e-5’N e os A2AR podem encontrar-se co-localizados e poderá haver uma interação funcional entre eles. Este estudo tem como objetivos definir: i) a localização sináptica e sub-sináptica (regiões pré-, pós- e extra-sinápticas) da e-5’N focando principalmente no córtex pré-frontal (PFC) de ratinhos C57Bl/6 adultos, ii) a co-localização da e-5’N com os A2AR em fatias do PFC e em terminais nervosos corticais, iii) se a deleção genética dos A2AR afeta a densidade sináptica da e-5’N em regiões corticais, iv) a função da e-5’N na plasticidade sináptica do PFC, e por fim, v) se o envelhecimento afeta a densidade sináptica da e-5’N em regiões do PFC. A comparação por análise de Western blot da densidade da e-5’N em membranas totais e sinaptossomas do córtex pré-frontal revelou que a e-5’N se encontrava nos terminais nervosos (52.1  2.3%...

AMP hydrolysis in soluble and microsomal rat cardiac cell fractions: kinetic characterization and molecular identification of 5 `-nucleotidase

POCHMANN, Daniela; INNOCENTE, Adrine M.; COTOMACCI, Guilherme; BARRETO-CHAVES, Maria Luiza M.; SARKIS, Joao J. F.
Fonte: PORTLAND PRESS LTD Publicador: PORTLAND PRESS LTD
Tipo: Artigo de Revista Científica
Português
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The present study describes the enzymatic properties and molecular identification of 5`-nucleotidase in soluble and microsomal fractions from rat cardiac ventricles. Using AMP as a substrate, the results showed that the cation and the concentration required for maximal activity in the two fractions was magnesium at a final concentration of 1 mM. The pH optimum for both fractions was 9.5. The apparent K-m (Michaelis constant) values calculated from the Eadie-Hofstee plot were 59.7 +/- 10.4 mu M and 134.8 +/- 32.1 mu M, with V-max values of 6.7 +/- 0.4 and 143.8 +/- 23.8 nmol P-i/min/mg of protein (means +/- S.D., n = 4) from soluble and microsomal fractions respectively. Western blotting analysis of ecto-5`-nucleotidase revealed a 70 kDa protein in both fractions, with the major proportion present in the microsomal fraction. The presence of these enzymes in the heart probably has a physiological function in adenosine signalling. Furthermore, the presence of ecto-5`-nucleotidase in the microsomal fraction could have a role in the modulation of the excitation-contraction-coupling process through involvement of the Ca2+ influx into the sarcoplasmic reticulum. The measurement of maximal enzyme activities in the two fractions highlights the potential capacity of the different pathways of purine metabolism in the heart.; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); CNPq-Brasil; CAPES; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeitos da fenilalanina, fenilpiruvato e alanina sobre as atividades da ATP-difosfoidrolase (EC 3.6.1.5) e 5þ-nucleotidase (EC 3.1.3.5) em sinaptossomas de córtex cerebral de ratos

Berti, Simone Luisa
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Dissertação Formato: application/pdf
Português
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Estudos anteriores realizados no Laboratório de Erros Inatos do Metabolismo demonstraram que fenilalanina (Phe) 2,0 mM ou fenilpiruvato (PP) 5,0 mM, um metabólito desaminado da Phe, reduzem a atividade da ATP-difosfoidrolase em sinaptossomas de córtex cerebral de ratos. Já estudos do efeito da Phe e do fenilactato sobre a atividade da Na+K+-ATPase, enzima envolvida na manutenção do potencial de membrana e na integridade celular no cérebro, demonstraram seus efeitos inibitórios, bem como a reversão destes efeitos pela alanina (Ala), competitivamente. Por outro lado, quando testada isoladamente, a alanina não altera a atividade da enzima. Resultados semelhantes foram observados em relação à fosforilação de proteínas de citoesqueleto neuronal na presença de Phe e/ou Ala. O presente estudo teve como principal objetivo caracterizar a inibição causada por Phe ou PP sobre a atividade da ATP-difosfoidrolase. Considerando que a ATP- difosfoidrolase participa com a 5’-nucleotidase na síntese de adenosina a partir do ATP e do ADP, decidimos investigar também os efeitos “in vitro” da Phe ou do PP sobre a atividade da 5’-nucleotidase na mesma fração. Além disso, considerando que a alanina reverte o efeito inibitório da Phe sobre algumas enzimas...

Erythropoetin-Sekretion bei Ekto-5-Nukleotidase (CD 73)- Knockout Mäusen; Erythropoetin-secretion in Ecto-5-Nucleotidase (CD 73)- knockout mice

Wietersheim, Simone von
Fonte: Universität Tübingen Publicador: Universität Tübingen
Tipo: Dissertation; info:eu-repo/semantics/doctoralThesis
Português
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Seit langem wird kontrovers diskutiert, ob Adenosin bei der Regulation der EPO-Produktion in der Niere eine bedeutsame Rolle spielt. Zur näheren Beleuchtung dieser Fragestellung wurde in der vorliegenden Arbeit durch den Einsatz von Ekto5´-Nukleotidase-Knockout (Ekto-5-NT-KO)-Mäusen der wichtigste Produktionsweg für Adenosin in der Niere ohne die Verwendung pharmakologischer Substanzen ausgeschaltet. Die KO- Mäuse und ihre entsprechenden Wildtypen wurden zur Anregung der EPO-Produktion einer funktionellen Hypoxie bei 400 ppm und 750ppm CO (4h), einer hypoxischen Hypoxie (8% O2) (4h), sowie einer 24 Stunden dauernden Anämie ausgesetzt. Anschließend wurden die renale EPO-mRNA, die EPO-Serumkonzentration sowie die mRNA der vier Ado-Rezeptor-Subtypen in der Niere bestimmt. Die Ergebnisse dieser Versuche lassen sich folgendermaßen zusammenfassen: 1. Ekto5´-NT-KO- und -WT-Mäuse hatten unter normoxischen Bedingungen identische basale EPO-mRNA-Werte sowie gleich hohe EPO- Serumkonzentrationen. 2. Nach vierstündiger CO-Exposition bei 400 ppm war die EPO-mRNA um das 20fache und bei 750 ppm um das 35 – 54fache im Vergleich zu Normoxie angestiegen, wobei sich die EPO-mRNA-Konzentrationen bei KO- und WT- Mäusen nicht signifikant voneinander unterschieden. 3. Die Serum-EPO-Konzentration erhöhte sich parallel dazu bei 400 ppm CO/4h um das 25-33fache und bei 750 ppm CO/4h um das 75-90fache. Auch hier war kein signifikanter Unterschied zwischen Mutanten und Wildtypen erkennbar. 4. Nach 8%iger Sauerstoffexposition über vier Stunden (hypoxische Hypoxie) waren die EPO-Serumkonzentrationen 22 – 34fach und die EPO- mRNA 21-23fach im Vergleich zur Normoxie erhöht. Die Werte der KO- und WT-Mäuse waren wieder identisch. 5. 24 Stunden Anämie durch Blutentnahmen führten bei KO- und WT- Mäusen ohne signifikanten Unterschied zu einem 76-97fachen Anstieg der EPO- Serumkonzentrationen und einem 22-25fachen Anstieg der EPO-mRNA im Vergleich zu Normoxie. 6. Die mRNA der vier Adenosin-Rezeptor-Subtypen wurde unter normoxischen Bedingungen sowie nach den verschiedenen Stimuli in KO- und WT-Mäusen gleich stark exprimiert. Unsere Ergebnisse sprechen somit gegen eine regulatorische Funktion von Adenosin und seinen Rezeptoren bei der EPO-Produktion in der Niere unter normoxischen...

Thyroid hormones are involved in 5 '-nucleotidase modulation in soluble fraction of cardiac tissue

Cotomacci, Guilherme; Sarkis, Joao J. F.; Fuerstenau, Cristina R.; Chaves, Maria Luiza Morais Barreto de
Fonte: PERGAMON-ELSEVIER SCIENCE LTD; OXFORD Publicador: PERGAMON-ELSEVIER SCIENCE LTD; OXFORD
Tipo: Artigo de Revista Científica
Português
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Aims: To investigate the role of TH (thyroid hormones) in 5'-nucleotidase activity and expression in cardiac soluble fraction (SF). Main methods: Male Wistar rats received daily injections of 14 (10, 25 or 50 mu g T4/100 g body weight) for 14 days to develop a hyperthyroidism condition. Thyroidectomy was performed in other animals to mimic hypothyroidism, and 14 days after surgery they were submitted to TH replacement therapy. Key findings: T4 reduced the 5'-nucleotidase activity (T4-25. P<0.05 and 14-50, P<0.01) in the SF. Conversely, hypothyroidism significantly increased the 5'-nucleotidase activity in this fraction (P<0.001) and TH replacement therapy reversed the latter result (P<0.001 compared to hypothyroid group). The analysis of protein expression in the SF showed that 5'-nucleotidase was more expressed in hypothyroid than in the control group and that the phosphorylated state of PKC observed in this condition may contribute to a possible mechanism of 5'-nucleotidase modulation by thyroid status. Significance: Taken together, these data reveal that TH can influence adenosine production by modulating 5'-nucleotidase activity and expression, which may contribute to the cardioprotective effect and the maintenance of cardiac function under TH privation. (C) 2012 Elsevier Inc. All rights reserved.; Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP); Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP); Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq-Brazil); Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)...

Immunogold labeling and cerium cytochemistry of the enzyme ecto-5'-nucleotidase in promastigote forms of Leishmania species

Corte-Real,Suzana; Porrozzi,Renato; Meirelles,Maria de Nazareth Leal de
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/1993 Português
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We have applied both enzyme cytochemistry and immunological labeling techniques to characterize the enzyme 5'-nucleotidase (5'-Nase), at the ultrastructural level, in promastigote forms of four Leishmania species: Leishmania amazonensis, Leishmania mexicana, Leishmania donovani and Leishmania chagasi. The cerium phosphate staining was localized at the surface of the cell body, the flagellum and the flagellar pocket membranes of all the parasites studied. The immunogold labelling technique confirmed these results. In this report we localized 5'-Nase in L. chagasi and L. amazonensis which have been implicated respectively in visceral and cutaneous forms of leishmaniasis. In addition, we confirmed the localization of this phosphomonoesterase in the other two species studied. The superior quality of the images, obtained with both methodologies, confirms that these parasites possess mechanisms capable of hydrolyzing nucleotide monophosphates, and that the expression of 5'-Nase is associated with the outer surface of the plasma membrane.

Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis

Frasson,Amanda Piccoli; Charão,Mariele Feiffer; Rosemberg,Denis Broock; Souza,Ana Paula de; Garcia,Solange Cristina; Bonorino,Cristina; Bogo,Maurício Reis; De Carli,Geraldo Attilio; Tasca,Tiana
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/03/2012 Português
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Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover...

Efeito de diferentes componentes da matriz extracelular sobre a ecto-5'-nucleotidase, proliferação, adesão e migração celular na linhagem de células de glioma humano U138-MG

Cappellari, Angélica Regina
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Dissertação Formato: application/pdf
Português
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Glioblastoma multiforme é a forma mais comum e agressiva de tumor cerebral que apresenta um severo crescimento e um comportamento altamente invasivo. Linhagens de células de glioma em cultura apresentam alta atividade da enzima ecto-5’-nucleotidase que metaboliza AMP em adenosina. Em adição, ela também interage com componentes da matriz extracelular como molécula adesiva. Neste trabalho, nós avaliamos o efeito de diferentes componentes da matriz extracelular sobre a atividade da ecto-5’-nucleotidase, proliferação, adesão e migração celular na linhagem de células de glioma humano U138-MG. Os resultados obtidos mostraram uma inibição da atividade enzimática da ecto-5’- nucleotidase quando tratada com laminina sozinha e com fibronectina ou laminina em co-tratamento com dextran sulfato. O dextran sulfato mostrou reduzir a proliferação em 37%. O mesmo efeito foi observado para os co-tratamentos entre dextran/laminina (29%) e dextran/colágeno (28%). A presença de adenosina diminuiu a adesão celular em torno de 40% e o APCP aumentou a adesão em 75%. Laminina inibiu a adesão celular, já a condroitina sulfato aumentou em 70%. As células U138 apresentaram uma redução da adesão e migração celular quando tratadas apenas com dextran e também no co-tratamento deste com adenosina e APCP. Diante dos resultados podemos sugerir a modulação da atividade da ecto5’-nucleotidase e assim uma modulação da produção de adenosina por moléculas da matriz extracelular...

Brain ischemia alters platelet ATP disphosphohydrolase and 5'-nucleotidase activities in naive and preconditioned rats

Frasseto, Silvana Soriano; Schetinger, Maria Rosa Chitolina; Schierholt, Rejane Cristina; Webber, Analupe; Bonan, Carla Denise; Wyse, Angela Terezinha de Souza; Dias, Renato Dutra; Netto, Carlos Alexandre; Sarkis, João José Freitas
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Artigo de Revista Científica Formato: application/pdf
Português
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The effects of transient forebrain ischemia, reperfusion and ischemic preconditioning on rat blood platelet ATP diphosphohydrolase and 5'- nucleotidase activities were evaluated. Adult Wistar rats were submitted to 2 or 10 min of single ischemic episodes, or to 10 min of ischemia 1 day after a 2-min ischemic episode (ischemic preconditioning) by the four-vessel occlusion method. Rats submitted to single ischemic insults were reperfused for 60 min and for 1, 2, 5, 10 and 30 days after ischemia; preconditioned rats were reperfused for 60 min 1 and 2 days after the long ischemic episode. Brain ischemia (2 or 10 min) inhibited ATP and ADP hydrolysis by platelet ATP diphosphohydrolase. On the other hand, AMP hydrolysis by 5'-nucleotidase was increased after 2, but not 10, min of ischemia. Ischemic preconditioning followed by 10 min of ischemia caused activation of both enzymes. Variable periods of reperfusion distinctly affected each experimental group. Enzyme activities returned to control levels in the 2-min group. However, the decrease in ATP diphosphohydrolase activity was maintained up to 30 days of reperfusion after 10-min ischemia. 5'-Nucleotidase activity was decreased 60 min and 1 day following 10-min ischemia; interestingly, enzymatic activity was increased after 2 and 5 days of reperfusion...

Estudo do papel da ecto-5'-nucleotidase/CD73 na proliferação de gliomas

Bavaresco, Luci
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Dissertação Formato: application/pdf
Português
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Os gliomas são os tumores primários mais comuns e devastadores que atingem o sistema nervoso central. O prognóstico para pacientes com estes tumores é ruim, e apesar de intensos esforços para o desenvolvimento de novas terapias, agentes efetivos ainda não estão disponíveis. A ecto-5‟-nucleotidase/CD73 (ecto-5‟-NT/CD73) regula os níveis extracelulares de AMP e adenosina, a qual tem sido amplamente descrita como fator indutor de proliferação celular. A ecto-5‟-NT/CD73 per se tem sido relatada como proteína envolvida no controle dos processos de crescimento, maturação, diferenciação, invasão e migração celular, além do processo de formação de metástases. No presente estudo nós avaliamos a atividade enzimática e as funções da ecto-5‟-NT/CD73 durante o processo proliferativo em linhagens de glioma C6 e U138-MG. Os resultados obtidos demonstram que ocorre um aumento da atividade da ecto-5‟-NT/CD73 com o aumento da confluência celular, quer seja esta obtida por semeadura de crescentes densidades celulares ou por crescentes dias de cultivo, em ambas as linhagens estudadas. As análises por RT-PCR e citometria de fluxo revelaram um aumento dos níveis de mRNA e proteína da ecto-5‟-NT/CD73, respectivamente quando comparadas culturas confluentes com culturas subconfluentes em linhagem de glioma humano U138-MG. Nesta mesma linhagem...

Subcellular distribution and movement of 5'-nucleotidase in rat cells.

Stanley, K K; Edwards, M R; Luzio, J P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/01/1980 Português
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1. Cell-surface 5'-nucleotidase was assayed by incubating whole-cell suspensions with 5'[3H]-AMP in iso-osmotic buffer and measuring [3H]adenosine production. The activity of cell-surface 5'-nucleotidase in hepatocytes, adipocytes and lymphocytes isolated from the rat was 15.0, 0.5 and 0.8pmol/min per cell at 37 degrees C respectively. 2. Disruption of the cells by vigorous mechanical homogenization or detergent treatment exposed additional 5'-nucleotidase activity, which represented 52%, 25% and 21% of the total activity in the three cell types respectively. This increase in 5'-nucleotidase activity which occurred when the cells were homogenized was due to a second pool of 5'-nucleotidase within the cell, rather than activation of the cell-surface enzyme. 3. In hepatocytes the intracellular 5'-nucleotidase activity was membrane-bound, indistinguishable from cell-surface 5'-nucleotidase in its inhibition by rabbit anti-(rat liver 5'-nucleotidase) serum and its kinetics with AMP, and was located on the extracytoplasmic face of vesicles within the cell. 4. The cell-surface 5'-nucleotidase of rat hepatocytes was rapidly inhibited when rabbit anti-(rat liver 5'-nucleotidase) serum or concanavalin A was added to the medium at 37 degrees C. Incubation with antiserum for 5 min at 37 degrees C inhibited 83 +/- 3% of the cell-surface enzyme. 5. Incubation of hepatocytes with exogenous antiserum or concanavalin A for 30 min at 37 degrees C resulted in over 50% inhibition of the intracellular enzyme. This inhibition was not prevented by disruption of the cytoskeleton or by ATP depletion. 6. Incubation of hepatocytes with exogenous antiserum or concanavalin A for up to 2h at 0 degrees C caused little or no inhibition of the intracellular enzyme...

The soluble 'low-Km' 5'-nucleotidase of rat kidney represents solubilized ecto-5'-nucleotidase.

Piec, G; Le Hir, M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/01/1991 Português
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A soluble 'low-Km' 5'-nucleotidase has been described previously in several organs. It has been presumed to be of cytosolic origin and thus to play a role in the intracellular production of adenosine. Its catalytic properties are similar to those of the ecto-5'-nucleotidase of cell membranes. In the present study we compared molecular properties of the two enzymes in the kidney of the rat. The Mr of the main peak of soluble 'low-Km' 5'-nucleotidase in gel-filtration chromatography was similar to that of the ecto-5'-nucleotidase solubilized by a phosphatidylinositol-specific phospholipase C from renal brush-border membranes. In phase-partition experiments using Triton X-114, the soluble enzyme appeared to be hydrophobic. Its hydrophobicity was decreased on treatment with a phosphatidylinositol-specific phospholipase C, suggesting that the soluble 'low-Km' 5'-nucleotidase contains the phosphatidylinositol anchor which is characteristic for the ecto-enzyme. An anti-ecto-5'-nucleotidase antiserum provoked an almost complete inhibition of the soluble enzyme. Immunoblotting using anti-ecto-5'-nucleotidase antiserum revealed in the high-speed supernatants a polypeptide with a similar Mr to the subunit of the ecto-5'-nucleotidase. The soluble 'low-Km' 5'-nucleotidase...

Uptake of free adenosine and adenosine from adenosine monophosphate by human peripheral blood lymphocytes: possible kinetic role for ecto-5'-nucleotidase in the regulation of intracellular adenosine.

Shah, T; Simpson, R J; Webster, A D; Peters, T J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1986 Português
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The role of 5'-nucleotidase in the uptake of adenosine from AMP was investigated in lymphocytes from normal subjects and patients with common variable hypogammaglobulinaemia (CVH) and chronic lymphatic leukaemia (CLL). At physiological pH, the Km values for the uptake of adenosine and of adenosine from AMP by intact cells were one order of magnitude higher than the Km values for 5'-nucleotidase. The Vmax values for the hydrolysis of AMP by 5'-nucleotidase were two orders of magnitude greater than for the uptake of adenosine itself or the uptake of adenosine from AMP by normal lymphocytes. 5'-Nucleotidase activity is clearly not rate-limiting in normal lymphocytes for uptake of adenosine from AMP in steady state conditions. Patients with common variable hypogammaglobulinaemia showed a low Vmax for 5'-nucleotidase assayed at pH 7.4 in intact cells as compared to values from control subjects. Michaelis constants (Km) for the uptake of free adenosine and adenosine from AMP as well as 5'-nucleotidase were similar compared to those obtained for controls. The uptake of adenosine moiety from AMP in CLL lymphocytes with a low Vmax for 5'-nucleotidase was also reduced, although not to the same extent as the reduction in 5'-nucleotidase activity. One CLL patient with supranormal levels of 5'-nucleotidase activity showed elevated uptake of adenosine moiety from AMP and of free adenosine. These results suggest that 5'-nucleotidase can influence the salvage of purine by lymphocytes from extracellular nucleotides but only when the enzyme activity is greatly reduced.

Evidence for deactivation of both ectosolic and cytosolic 5'-nucleotidase by adenosine A1 receptor activation in the rat cardiomyocytes.

Kitakaze, M; Hori, M; Minamino, T; Takashima, S; Komamura, K; Node, K; Kurihara, T; Morioka, T; Sato, H; Inoue, M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1994 Português
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Adenosine, an important regulator of many cardiac functions, is produced by ectosolic and cytosolic 5'-nucleotidase. The activity of these enzymes is influenced by several ischemia-sensitive metabolic factors, e.g., ATP, ADP, H+, and inorganic phosphate. However, there is no clear evidence that adenosine itself affects 5'-nucleotidase activity. This study tested whether adenosine decreases the activity of ectosolic and cytosolic 5'-nucleotidase. Cardiomyocytes were isolated from adult male Wistar rats and suspended in the modified Hepes-Tyrode buffer solution. After stabilization, isolated cardiomyocytes were incubated with and without adenosine (10(-9) - 10(-4) M). Ectosolic and cytosolic 5'-nucleotidase activity was decreased by exogenous adenosine (ectosolic 5'-nucleotidase activity, 20.6 +/- 2.3 vs. 8.6 +/- 1.6 mumol/min per 10(6) cells [P < 0.05]; cytosolic 5'-nucleotidase activity, 2.47 +/- 0.58 vs. 1.61 +/- 0.54 mumol/min per 10(6) cells [P < 0.05] at 10(-6) M adenosine) after 30 min. The decrease in ectosolic and cytosolic 5'-nucleotidase activity was inhibited by 8-phenyltheophylline and pertussis toxin, and was mimicked by N6-cyclohexyladenosine, an adenosine A1 receptor agonist. Neither CGS21680C, and A2 receptor agonist...

Distribution of 5'-nucleotidase in human lymphoid tissues.

Edwards, N L; Gelfand, E W; Burk, L; Dosch, H M; Fox, I H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1979 Português
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Low activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) in T lymphoblasts may explain the marked sensitivity of this cell to deoxynucleotide accumulation when compared to B lymphoblasts. The relevance of such observations with cultured cells to the normal immune system requires the demonstration of similar differences in the 5'-nucleotidase activity of normal human lymphocyte subpopulations. Sheep erythrocyte (E) rosette-forming cells from normal thymus, tonsil, and peripheral mononuclear cells have 5'-nucleotidase activities of 1.7, 11.3, and 21.2 nmol/hr per 10(6) cells. Non-E-rosette forming cells from the peripheral blood or tonsil have 5'-nucleotidase activity comparable to the higher levels found in the peripheral E-RFC. Increased levels of 5'-nucleotidase activity may be a marker for post-thymic T lymphocytes. T lymphoblasts have 5'-nucleotidase activity similar to values demonstrated for E-RFC in thymus, whereas cultured B lymphoblasts have 5'-nucleotidase activity 15 times greater than that of T lymphoblasts. On the basis of these observations, the 5'-nucleotidase deficiency in congenital agammaglobulinemia has been reevaluated. In these patients the data indicate that peripheral E-rosette forming cells have the enzyme deficiency...

The importance of B-cells and ecto-5′nucleotidase in Mycoplasma fermentans infection and the relevance to rheumatoid arthritis

Johnson, Sheena M
Fonte: Blackwell Science Inc Publicador: Blackwell Science Inc
Tipo: Artigo de Revista Científica
Publicado em /02/2008 Português
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The aim of this work was to discover if Mycoplasma fermentans, which is known to infect B cells, could be the cause of the raised ecto-5′-nucleotidase observed in the synovial fluid of rheumatoid arthritis patients. The ecto-5′-nucleotidase activity in the patients' serum has been shown to correlate with the erythrocyte sedimentation rate and DNA from the mycoplasma has been found in the synovial fluid. B lymphoblastoid cell lines were exposed to 16 strains of Mycoplasma fermentans and their ecto-5′-nucleotidase, CD73, was measured both biochemically and by mouse antibodies to human ecto 5′-nucleotidase using the fluorescence activated cell sorter. The type strain, PG 18, did not grow with the B cells. Some of the mycoplasma strains (9/15) increased the cellular ecto-5′-nucleotidase activity from twice to 17 fold, and usually showed 5′-nucleotidase activity themselves. At least one strain, M106, induced human 5′-nucleotidase on the normally 5′-nucleotidase negative Daudi and Raji Burkitt's lymphoma cell lines, and increased sevenfold the 5′-nucleotidase on the monocyte/macrophage cell line THP-1. Growing the cells in aged medium increased the level of mycoplasma infection. This mycoplasma-induced enzyme showed a conformational change and an increase in activity with a glycosylation change involving mannose groups. The other group of strains...

Brain ischemia alters platelet ATP diphosphohydrolase and 5'-nucleotidase activities in naive and preconditioned rats

Frassetto,S.S.; Schetinger,M.R.C.; Schierholt,R.; Webber,A.; Bonan,C.D.; Wyse,A.T.; Dias,R.D.; Netto,C.A.; Sarkis,J.J.F.
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/11/2000 Português
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37.306257%
The effects of transient forebrain ischemia, reperfusion and ischemic preconditioning on rat blood platelet ATP diphosphohydrolase and 5'-nucleotidase activities were evaluated. Adult Wistar rats were submitted to 2 or 10 min of single ischemic episodes, or to 10 min of ischemia 1 day after a 2-min ischemic episode (ischemic preconditioning) by the four-vessel occlusion method. Rats submitted to single ischemic insults were reperfused for 60 min and for 1, 2, 5, 10 and 30 days after ischemia; preconditioned rats were reperfused for 60 min 1 and 2 days after the long ischemic episode. Brain ischemia (2 or 10 min) inhibited ATP and ADP hydrolysis by platelet ATP diphosphohydrolase. On the other hand, AMP hydrolysis by 5'-nucleotidase was increased after 2, but not 10, min of ischemia. Ischemic preconditioning followed by 10 min of ischemia caused activation of both enzymes. Variable periods of reperfusion distinctly affected each experimental group. Enzyme activities returned to control levels in the 2-min group. However, the decrease in ATP diphosphohydrolase activity was maintained up to 30 days of reperfusion after 10-min ischemia. 5'-Nucleotidase activity was decreased 60 min and 1 day following 10-min ischemia; interestingly, enzymatic activity was increased after 2 and 5 days of reperfusion...

Analysis of the ntpdase and ecto-5´-nucleotidase profiles in serum-limited trichomonas vaginalis

Frasson, Amanda Piccoli; Charão, Mariele Feiffer; Rosemberg, Denis Broock; Souza, Ana Paula de; Garcia, Solange Cristina; Bonorino, Cristina Beatriz Cazabuena; Bogo, Mauricio Reis; De Carli, Geraldo Attilio; Tasca, Tiana
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Artigo de Revista Científica Formato: application/pdf
Português
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37.383132%
Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family mem- bers, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been char- acterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis tropho- zoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by f low cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5’-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover...

Stimulation of ecto-5'-nucleotidase in human umbilical vein endothelial cells by lipopolysaccharide

Li, Rachel; Man, R.Y.K.; Vanhoutte, Paul M.; Leung, George P. H.
Fonte: American Physiological Society Publicador: American Physiological Society
Tipo: Artigo de Revista Científica
Português
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37.042686%
The involvement of ecto-5′-nucleotidase (E-5′Nu) in the elevation of extracellular adenosine during inflammation is unclear. In the present study, the effect of lipopolysaccharide (LPS), an inflammation inducer, was investigated on E-5′Nu in human u