IgE antibodies from schistosomiasis patients to recognize epitopes in potato apyrase

Abstract INTRODUCTION: High percentages of structural identity and cross-immunoreactivity have been reported between potato apyrase and Schistosoma mansoni ATP diphosphohydrolase (SmATPDases) isoforms, showing the existence of particular epitopes shared between these proteins. METHODS: Potato apyrase was employed using ELISA, western blot, and mouse immunization methods to verify IgE reactivity. RESULTS: Most of the schistosomiasis patient's (75%) serum was seropositive for potato apyrase and this protein was recognized using western blotting, suggesting that parasite and plant proteins share IgE-binding epitopes. C57BL/6 mice immunized with potato apyrase showed increased IgE antibody production. CONCLUSIONS: Potato apyrase and SmATPDases have IgE-binding epitopes.

Hospitalizations for cardiovascular diseases and the coverage by the family health strategy / Internações por doenças cardiovasculares e a cobertura da estratégia saúde da família / Internaciones por enfermedades cardiovasculares y la cobertura de la estrategia salud de la familia

AbstractObjective: to verify the correlation between the rates of hospitalization for primary care-sensitive cardiovascular diseases and the coverage by the Family Health Strategy of residents of the State of Paraná, by regional health divisions, from 2000 to 2011.Method: ecological study developed from data of the Hospital Information System of the Brazilian Unified Health System (SUS) and the Department of Primary Care of the Ministry of Health. The rates of hospitalization for cardiovascular diseases were correlated with the annual coverage by the Family Health Strategy using Pearson's and Spearman's correlation coefficients.Result: there was a strong and negative correlation in the State of Paraná (r=-0.91; p <0.001) and in most regional health divisions, with the highest correlations observed in the Metropolitan and Toledo (r =-0.93; p<0.001) and Paranaguá (r=-0.92, p<0.001) regional health divisions.Conclusion: the results suggest that the increase in the coverage by the Family Health Strategy was an important factor for decrease in the hospitalizations for cardiovascular conditions among residents of the State of Paraná and in most regional health divisions. Other studies should be performed to analyze the factors and causes in regional health divisions where there was no correlation with increase in the Family Health Strategy.

ResumoObjetivo:verificar a correlação entre taxas de internação por doenças cardiovasculares sensíveis à atenção primária e a cobertura da Estratégia Saúde da Família de residentes no estado do Paraná, por regionais de saúde, no período de 2000 a 2011.Método:estudo ecológico, desenvolvido a partir de dados do Sistema de Informações Hospitalares do Sistema Único de Saúde e do Departamento de Atenção Básica do Ministério da Saúde. Correlacionaram-se as taxas de internação por doenças cardiovasculares com as coberturas anuais da Estratégia Saúde da Família, utilizando-se os coeficientes de correlação de Pearson e Spearman.Resultado:houve correlação negativa e forte no estado do Paraná (r=-0,91; p<0,001) e na maioria das regionais de saúde, sendo maior na Metropolitana e Toledo (r=-0,93; p<0,001) e Paranaguá (r=-0,92; p<0,001).Conclusão:os resultados sugerem que o aumento da cobertura da Estratégia Saúde da Família foi fator importante para a diminuição das internações por condições cardiovasculares em residentes no estado do Paraná e na maioria das regionais de saúde. Outros estudos devem ser realizados para analisar fatores e causas nas regiões do estado onde não houve correlação com incremento da Estratégia Saúde da Família.

ResumenObjetivo:verificar la correlación entre tasas de internación por enfermedades cardiovasculares sensibles a la atención primaria y la cobertura de la Estrategia Salud de la Familia de residentes en el estado de Paraná, por regionales de salud, en el período de 2000 a 2011.Método:estudio ecológico, desarrollado a partir de datos del Sistema de Informaciones Hospitalarias del Sistema Único de Salud y del Departamento de Atención Básica del Ministerio de la Salud. Se correlacionaron las tasas de internación por enfermedades cardiovasculares con las coberturas anuales de la Estrategia Salud de la Familia, utilizando los coeficientes de correlación de Pearson y Spearman.Resultado:hubo correlación negativa y fuerte en el estado de Paraná (r=-0,91; p<0,001) y en la mayoría de las regionales de salud, siendo mayor en la Metropolitana y Toledo (r=-0,93; p<0,001) y Paranaguá (r=-0,92; p<0,001).Conclusión:los resultados sugieren que el aumento de la cobertura de la Estrategia Salud de la Familia fue un factor importante para la disminución de las internaciones por condiciones cardiovasculares en residentes en el estado de Paraná y en la mayoría de las regionales de salud. Otros estudios deben ser realizados para analizar factores y causas en las regiones del estado en donde no hubo correlación con incremento de la Estrategia Salud de la Familia.

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

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 percent 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 percent 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, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.

Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydro-lase isoform (SmATPDase 2) and immunolocalisation of this protein in the Schistosoma mansoni egg

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.

Detection of IgG1 and IgG4 subtypes reactive against potato apyrase in schistosomiasis patients

In this paper, we showed for the first time that the conserved domains within Schistosoma mansoni ATP diphosphohydrolase isoforms, shared with potato apyrase, possess epitopes for the IgG1 and IgG4 subtypes, as 24 (80 percent) of the 30 schistosomiasis patients were seropositive for this vegetable protein. The analyses for each patient cured (n = 14) after treatment (AT) with praziquantel revealed variable IgG1 and IgG4 reactivity against potato apyrase. Different antigenic epitopes shared between the vegetable and parasite proteins could be involved in susceptibility or resistance to S. mansoni AT with praziquantel and these possibilities should be explored.

Antibody reactivity against potato apyrase, a protein that shares epitopes with Schistosoma mansoni ATP diphosphohydrolase isoforms, in acute and chronically infected mice, after chemotherapy and reinfection

Schistosoma mansoni ATP diphosphohydrolase isoforms and potato apyrase share conserved epitopes. By enzyme-linked immunosorbent assays, elevated levels of IgM, IgG2a and IgG1 antibody reactivity against potato apyrase were observed in S. mansoni-infected BALB/c mice during the acute phase of infection, while only IgM and IgG1 antibody reactivity levels maintained elevated during the chronic phase of infection. Antibody reactivity against potato apyrase was monitored over an 11-month period in chronically-infected mice treated with oxamniquine. Eleven months later, the level of seropositive IgM decreased significantly (~30 percent) compared to the level found in untreated, infected mice. The level of seropositive IgG1 decreased significantly four months after treatment (MAT) (61 percent) and remained at this level even after 11 months. The IgG2a reactivity against potato apyrase, although unchanged during chronic phase to 11 MAT, appeared elevated again in re-infected mice suggesting a response similar to that found during the acute phase. BALB/c mouse polyclonal anti-potato apyrase IgG reacted with soluble egg antigens probably due to the recognition of parasite ATP diphosphohydrolase. This study, for the first time, showed that the IgG2a antibody from S. mansoni-infected BALB mice cross-reacts with potato apyrase and the level of IgG2a in infected mice differentiates disease phases. The results also suggest that different conserved-epitopes contribute to the immune response in schistosomiasis.

Señales purinérgicas / Purinergic signals

En la última década se ha aportado clara evidencia de que tanto nucleósidos como nucleótidos de adenina y uridina pueden funcionar como factores de señalización extracelular. Su acción es mediada por dos tipos principales de receptores de superficie denominados purinérgicos. Los receptores P1 se activan por adenosina, y son todos metabotrópicos, mientras que los receptores de nucleótidos (ATP, ADP, UTP y UDP) y nucleótidos-azúcares (UDP-glucosa y UDP-galactosa) pueden ser metabotrópicos (P2Y) o ionotrópicos (P2X). La importancia y complejidad de este sistema de señalización se evidencia por la diversidad de mecanismos de liberación de nucleótidos al medio extracelular y por la distribución ubicua de varios grupos de ectonucleotidasas capaces de catalizar la degradación y conversión de nucleótidos. Hasta el momento se han descrito y clonado una veintena de estos receptores que modulan una variedad de respuestas, como el impulso nervioso, la respuesta inflamatoria, la secreción de insulina, la regulación del tono vascular y la percepción del dolor. En la presente revisión se describen las características estructurales y farmacológicas de los receptores purinérgicos y se analiza la interacción dinámica entre estos receptores, los nucleósidos y nucleótidos, y las ectonucleotidasas, con especial atención a la dinámica de la agregación plaquetaria, la respuesta inmune y la hidratación de las mucosas respiratorias.

In the last decade evidence accumulated that nucleosides and nucleotides of both uridine and adenine can act as extracellular signaling factors. Their action is mediated by two main types of surface receptors commonly known as purinergic. P1 receptors are metabotropic and activated by adenosine, whereas receptors for nucleotides (ATP, ADP, UTP and UDP) and nucleotide-sugars (UDP-glucose and UDP-galactose) can be either metabotropic (P2Y) or ionotropic (P2X). The importance and complexity of this signaling system is evidenced by various mechanisms of nucleotide release, as well as by the ibiquitous distribution of various types of ectonucleotidases which catalyze and convert extracellular nucleotides. Up to now about twenty receptors have been cloned and found to modulate the nerve impulse, inflammatory response, insuline secretion, the regulation of the vascular tone and nociception, among other processes. In the present review we describe the main structural and pharmacological features of purinergic receptors, and analyze how the dynamic interaction between these receptors, nucleotides and nucleosides, and ectonucleotidases modulate several biological responses. Particular focus is given to platelet aggregation and thrombus formation, the immune response and the hydration of the mucosal linings of the respiratory tract.

Mapeamento dos domínios antigênicos compartilhados entre a apirase de batata e as isoformas de ATP difosfohidrolase de Schistosoma mansoni

Usando a apirase de batata como antígeno, as propriedades imunogênicas das isoformas de ATP difosfohidrolase de S. mansoni foram inicialmente exploradas em esquistossomose experimental. Elevada reatividade de anticorpos IgG2a e IgG1 contra esta proteína vegetal foi observada em soro de camundongos BALB/c na fase aguda da infecção (8-9ª semana pós-infecção). Elevada reatividade de anticorpos IgG1 com a apirase de batata, mas não de IgG2a, foi encontrada na fase crônica da infecção (17ª semana pós-infecção), diferenciando sorologicamente as fases da infecção. Adicionalmente, foi observado que o inóculo de apirase de batata em camundongos BALB/c saudáveis tem marcante atividade estimulatória, aumentando significativamente os níveis de anticorpos IgG1 e IgG2a específicos. O subtipo IgG1 de camundongo, mas não IgG2a, reage com a ATP difosfohidrolase presente na preparação de SEA. A reatividade de anticorpos contra a apirase de batata foi monitorada por um período de 11 meses em camundongos BALB/c tratados com oxaminiquina na fase crônica da doença.

A reatividade de IgM, IgG total ou IgG1 contra a apirase de batata reduziu significativamente (~60%) após 11 meses, enquanto IgG2a, que esteve elevada na fase aguda, perde a significância na fase crônica e permanece inalterada na etapa do pós tratamento. Após a quimioterapia, os camundongos foram re-infectados com 100 cercárias, e nenhuma diferença significativa foi observada na soropositividade de IgG1, mas uma elevação significativa de IgG2a foi detectada nos camundongos re-infectados sugerindo uma nova fase aguda e sua participação nos mecanismos de proteção contra o Schistosoma. Estudos in silico demonstraram uma íntima relação estrutural e evolucionária entre a apirase de batata e as isoformas de ATP difosfohidrolases de S. mansoni. A predição de modelos tridimensionais sugeriu que os domínios conservados podem estar expostos e disponíveis para a ligação com anticorpos. O perfil de reatividade de anticorpos contra a apirase de batata foi, então, estudado em amostras de soros obtidas de pacientes com esquistossomose, moradores de três áreas endêmicas diferentes. Amostras de soros de grupos de adultos e crianças mostraram alta reatividade contra a apirase de batata, com elevação dos níveis de anticorpos IgA, IgE, IgG1 ou IgG4, com diferenças significativas entre eles. Após a quimioterapia, redução significativa ou ausência de reatividade de anticorpos contra a apirase de batata foi observada nestes pacientes. Esses achados podem estar associados à resistência ou susceptibilidade

Mapeamento dos domínios antigênicos compartilhados entre a apirase de batata e as isoformas de ATP difosfohidrolase de Schistosoma mansoni / Mapping of the conserved antigenic domains shared between potato apyrase and ATP diphosphohydrolase isoforms from Schistosoma mansoni

Usando a apirase de batata como antígeno, as propriedades imunogênicas das isoformas de ATP difosfohidrolase de S. mansoni foram inicialmente exploradas em esquistossomose experimental. Elevada reatividade de anticorpos IgG2a e IgG1 contra esta proteína vegetal foi observada em soro de camundongos BALB/c na fase aguda da infecção (8-9ª semana pós-infecção). Elevada reatividade de anticorpos IgG1 com a apirase de batata, mas não de IgG2a, foi encontrada na fase crônica da infecção (17ª semana pós-infecção), diferenciando sorologicamente as fases da infecção. Adicionalmente, foi observado que o inóculo de apirase de batata em camundongos BALB/c saudáveis tem marcante atividade estimulatória, aumentando significativamente os níveis de anticorpos IgG1 e IgG2a específicos. O subtipo IgG1 de camundongo, mas não IgG2a, reage com a ATP difosfohidrolase presente na preparação de SEA. A reatividade de anticorpos contra a apirase de batata foi monitorada por um período de 11 meses em camundongos BALB/c tratados com oxaminiquina na fase crônica da doença.

A reatividade de IgM, IgG total ou IgG1 contra a apirase de batata reduziu significativamente (~60%) após 11 meses, enquanto IgG2a, que esteve elevada na fase aguda, perde a significância na fase crônica e permanece inalterada na etapa do pós tratamento. Após a quimioterapia, os camundongos foram re-infectados com 100 cercárias, e nenhuma diferença significativa foi observada na soropositividade de IgG1, mas uma elevação significativa de IgG2a foi detectada nos camundongos re-infectados sugerindo uma nova fase aguda e sua participação nos mecanismos de proteção contra o Schistosoma. Estudos in silico demonstraram uma íntima relação estrutural e evolucionária entre a apirase de batata e as isoformas de ATP difosfohidrolases de S. mansoni. A predição de modelos tridimensionais sugeriu que os domínios conservados podem estar expostos e disponíveis para a ligação com anticorpos. O perfil de reatividade de anticorpos contra a apirase de batata foi, então, estudado em amostras de soros obtidas de pacientes com esquistossomose, moradores de três áreas endêmicas diferentes. Amostras de soros de grupos de adultos e crianças mostraram alta reatividade contra a apirase de batata, com elevação dos níveis de anticorpos IgA, IgE, IgG1 ou IgG4, com diferenças significativas entre eles. Após a quimioterapia, redução significativa ou ausência de reatividade de anticorpos contra a apirase de batata foi observada nestes pacientes. Esses achados podem estar associados à resistência ou susceptibilidade

Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae)

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3 - 10 days was approximately 1.08 ± 0.04 æg/female and 0.1 ± 0.05 æg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.

Proteínas das glândulas salivares do Anopheles dirus B (Diptera: Culicidae), vetor da malária humana foram determinadas e analisadas. A quantidade de proteínas das glândulas salivares em mosquitos com três a 10 dias de idade foi de aproximadamente 1,08 ± 0,04 æg/ fêmea e de 0,1 ± 0,05 æg/macho. As glândulas salivares de ambos os sexos mostraram organização morfológica semelhante à de outros mosquitos anofelinos. Em fêmeas, apirase acumula-se nas regiões distais, enquanto alfa-glucosidase foi encontrada na região proximal dos lóbulos laterais. Esta distribuição diferencial das enzimas analisadas reflete a especialização de diferentes regiões para alimentação de açucares e sangue. Análise SDS-PAGE revelou que pelo menos sete proteínas foram encontradas nas glândulas salivares de fêmeas, das quais cada região morfológica continha diferentes proteínas principais. Perfis eletroforéticos de proteínas semelhantes foram detectados comparando-se mosquitos não alimentados e alimentados por sangue, sugerindo que não existe proteína específica induzida pelo mesmo. Análise por gel poliacrilamida bi-dimensional mostrou a mais abundante proteína de glândulas salivares com aproximadamente 35 kilodaltons de massa molecular e ponto isoelétrico de aproximadamente 4,0. Estes resultados dão informações básicas que levariam a estudos adicionais sobre o papel das proteínas salivares do An. dirus B na transmissão da doença e hematofagia.

Cross-immunoreactivity between anti-potato apyrase antibodies and mammalian ATP diphosphohydrolases: potential use of the vegetal protein in experimental schistosomiasis

We have previously showed that Schistosoma mansoni ATP-diphosphohydrolase and Solanum tuberosum potato apyrase share epitopes and the vegetable protein has immunostimulatory properties. Here, it was verified the in situ cross-immunoreactivity between mice NTPDases and anti-potato apyrase antibodies produced in rabbits, using confocal microscopy. Liver samples were taken from Swiss Webster mouse 8 weeks after infection with S. mansoni cercariae, and anti-potato apyrase and TRITC-conjugated anti-rabbit IgG antibody were tested on cryostat sections. The results showed that S. mansoni egg ATP diphosphohydrolase isoforms, developed by anti-potato apyrase, are expressed in miracidial and egg structures, and not in granulomatous cells and hepatic structures (hepatocytes, bile ducts, and blood vessels). Therefore, purified potato apyrase when inoculated in rabbit generates polyclonal sera containing anti-apyrase antibodies that are capable of recognizing specifically S. mansoni ATP diphosphohydrolase epitopes, but not proteins from mammalian tissues, suggesting that autoantibodies are not induced during potato apyrase immunization. A phylogenetic tree obtained for the NTPDase family showed that potato apyrase had lower homology with mammalian NTPDases 1-4, 7, and 8. Further analysis of potato apyrase epitopes could implement their potential use in schistosomiasis experimental models.

Ingestion of saliva during carbohydrate feeding by Lutzomyia longipalpis (Diptera; Psychodidae)

The aim of this study was to obtain experimental evidence that phlebotomine saliva is actually ingested during the carbohydrate ingestion phase (before and after blood digestion). The ingestion of carbohydrate was simulated as it occurs in the field by offering the insects balls of cotton soaked in sucrose, sucrose crystals or orange juice cells. The results obtained here showed that ingestion occurred under each condition investigated, as indicated by the presence of apyrase, an enzyme used as a marker to detect saliva in the insect gut and/or carbohydrate sources. Saliva ingestion by phlebotomine during the carbohydrate ingestion phase is important to explain how it could promote starch digestion and to trigger Leishmania promastigotes to follow a differentiation pathway as proposed previously by some authors.

Construção e caracterização de mini-bibliotecas de EST geradas com RT-PCR de baixa estringencia e clonagem de uma apirase de Schistosoma mansoni / Construction and characterization of EST minilibraries generated with low stringency RT-PCR and cloning of apyrase of Schistosoma mansoni

Este trabalho demonstrou a construção de minibibliotecas de "Expressed Sequence Tags" (EST) com o uso de RT-PCR de baixa estringencia e "primer" consenso-degenerado. Através do estudo de parâmetros críticos como concentração de sais, temperatura e velocidade de ciclagem, composição dos "primers" e qualidade do RNA mensageiro, foi possível padronizar um protocolo. Tal protocolo permitiu um aumento do número de seqüências por minibibliotecas em relação a protocolos similares existentes na literatura (Dias Neto et al., 1997 e 2000) sem perda de características desejáveis como amplificação preferencial do centro dos genes e normalização das mensagens...

Metabolismo energético y esteroidogénico de la plancenta humana / Energetic and steroidogenic metabolism at the human placenta

Las células del sinciciotrofoblasto obtienen el ATP a través de la glucólisis anaerobia. Sin embargo, aunque las mitocondrias de la placenta sintetizan ATP, éste no participa en los procesos citoplasmáticos. Nuestros datos muestran la presencia de una ATP-difosfohidrolsa (apirasa) asociada a las mitocondrias de la placenta, que se inhibe por vanadato y FSBA. En este trabajo proponemos la hipótesis de que la apirasa y el ATP que sintetizan las mitocondrias de las células del sinciciotrofoblasto están asociados al transporte de colesterol necesario para la síntesis de progesterona, y que el uso del ATP y la actividad de la apirasa están asociados a los puntos de unión mitocondriales.

Construçäo de bibliotecas de cDNA com PCR de baixa estringência e primers híbridos e clonagem de uma apirase de Schistosoma mansoni / Construction of cDNA libraries with low stringency RT-PCR and hybrid primers and cloning of an apyrase of Schistosoma mansoni

Este trabalho mostrou a purificação de uma apirase em extratos de tegumentos de vermes adultos de S. mansoni com razão de hidrólise de ADP/ATP aproximadamente 2. Análise da amostra por MALDI-TOF evidenciou a presença de helicase e carboxilesterase, sendo que a atividade de hidrólise de ADP ainda não foi descrita para estas proteínas. Através de ®Western blot¼ mostrou-se que a proteína purificada não tem epítopos reconhecíveis por anticorpo anti-apirase de batata, e que a imunoreatividade cruzada com apirase da família CD39 está presente na fração insolúvel que não foi utilizada no processo de purificação. Esta tese caracteriza também a utilização de ®primers¼ consenso degenerados na construção de bibliotecas de cDNA com PCR de baixa estringência...

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

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, and returned to control levels after 10 days. Ischemic preconditioning cancelled the effects of 10-min ischemia on the enzymatic activities. These results indicate that brain ischemia and ischemic preconditioning induce peripheral effects on ecto-enzymes from rat platelets involved in nucleotide metabolism. Thus, ATP, ADP and AMP degradation and probably the generation of adenosine in the circulation may be altered, leading to regulation of microthrombus formation since ADP aggregates platelets and adenosine is an inhibitor of platelet aggregation

Eficacia del clopidogrel como inhibidor de la agregación plaquetaria dependiente de ADP. un estudio en individuos con enfermedad arteral coronaria / Eficacy of clopidogrel as inhibitor of ADP-Induced platelet aggregation. A study in patients with coronary artery disease

El ácido acetil-salicílico, al inhibir la producción de tromboxano A2, reduce entre 20 y 25 por ciento el riesgo de eventos oclusivos arteriales. La ticlopidina inhibe la agregación plaquetaria dependiente de adenosín-difosfato, y reduce el mismo riesgo en 30 a 35 por ciento, pero tiene efectos indeseables. Su derivado, el clopidogrel, tiene el mismo mecanismo de acción y disminuye indirectamente la expresión del receptor de fibrinógeno en las plaquetas. Conserva la eficacia clínica de la ticlopidina, con menor frecuencia de efectos adversos. En este estudio, evaluamos la acción de 75 mg diarios de clopidogrel sobre la función plaquetaria de 33 individuos con enfermedad arterial coronaria; se les practicó agregometría plaquetaria inducida con adenosín-difosfato 5 mM y colágena 20 mg/mL, tiempo de hemorragia y fibrinógeno, antes del tratamiento y a las semanas 6 y 12. La agregación plaquetaria inducida con adenosín-difosfato fue de 90.7 por ciento ñ 13.2, 54.6 por ciento ñ 23.2 y 49.2 por ciento ñ 23.7 en las muestras basal y a las semanas 6 y 12, lo que representó una reducción significativa de 38.6 por ciento y 44.4 por ciento. La agregación plaquetaria inducida con colágena no disminuyó significativamente. El tiempo de hemorragia se prolongó de 4.1 ñ 1.6 a 15.43 ñ 13.1 y 14.6 ñ 14.4 minutos (3.7-3.5 veces). No se observaron modificaciones en la concentración de fibrinógeno. No se presentaron complicaciones hemorrágicas. Ocurrieron molestias digestivas con una frecuencia menor al 3 por ciento. Se concluye que el clopidogrel reduce eficazmente la agregación plaquetaria dependiente de adenosin-difosfato, y prolonga el tiempo de hemorragia. El perfil de seguridad clínico y de laboratorio son adecuados.

Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.

Comparison of the biochemical properties, regulation and function of ATP-diphosphohydrolase from human placenta and rat kidney

ATP-diphosphohydrolase (apyrase, EC 3.6.1.5) has both ATPase and ADPase activity that are stimulated by bivalent metais, with Ca2+ being the most effective. The possible physiological function of this enzyme, associated with placental and renal microvilli, is related to the extracellular metabolism of nucleotides. A comparison of the biochemical properties of human placenta and rat kidney apyrase is presented, showing similaiities in Mr, bivalent metal stimulation, nucleotide nonspecificity, insensitivity towards specifjc ATPase inhibitors, and lack of essential sulfhydryl and aliphatic hydroxyl groups. We describe the treatment of membrane preparations from both tissues with different detergents and the isoelectric focusing of the solubilized proteins to partially purify apyrase. An ectoenzyme localization is assigned both in microvillus membranes and in the vasculature on the basis of organ perfusion experiments with nucleotides in the presence of antibodies. Placental and kidney microvillus membranes inhibited ADP-induced platelet aggregation, in agreement with an extracellular role. Initial studies on enzyme regulation suggested the existence of at least two types of modulatory proteins: an activating protein in the cytosol of both tissues, and an inhibitory protein associated with placental microsomes. Possible hormonal regulation was investigated in kidneys using in vivo estradiol treatment, but only slight changes in total apyrase activity were observed.

ATP diphosphohydrolase activity in synaptosomes from cerebral cortex of rats subjected to chemically induced phenylketonuria

ATP diphosphohydrolase (apyrase)(EC3.6.1.5) activity was measured in synaptosomes from cerebral cortex of Wistar rats of both sexes subjected to experimental phenylketonuria, i.e., chemical hyperphenylaninemia induced by subcutaneous administration of 5.2 µmol phenylalanine/g body weight (twice a day) plus 0.9 µmol p-chlorophenylalanine/g body weight (once a day). ATP diphosphohydrolase specific activity (nmol Pi min-1 mg protein-1) of synaptosomes was significantly decreased compared to controls for both ATp (from 147.6 to 129.9) and ADP (from 70.2 to 63.1) hydrolysis one hour after single administration of the drugs to 35-day old rats. Chronic treatment was performed from the 6th to the 28th postpartum day. The enzyme specific activity of synaptosomes was measured one week after the last administration of the drugs and was significantly reduced compared to controls for both ATP (from 164.1 to 150.2) and ADP (from 76.3 to 62.1) hydrolysis. The in vitro effects of the drugs on the synaptosome enzyme specific activity were also investigated. Phenylalnine alone or associated with p-chlorophenylalanine significantly reduced enzyme specific activity for both ATP (from 150.2 to 136.0) and ADP (from 70.5 to 59.3) nucleotides as substrates. Since ATP diphosphohrolase seems to play an important role in neurotransmission, these findings may be related to the neurological dysfunction characteristic of human phenylketonuria