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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.

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%) 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.

PROQUALI (UFJF) A NTPDase 1 (50 kDa) de promastigotas de Leishmania infantum foi purificada por eletroforese em gel não-desnaturante. A identidade desta proteína ou de seu domínio B conservado (r83-122) foi confirmada por “Western blots” utilizando soros imunes produzidos contra a apirase de batata (SA), r-potDomínioB (SB), um polipeptídeo recombinante derivado do domínio B desta proteína vegetal, e LbB1LJ (SC; r82-103) e LbB2LJ (SD; r102-121), peptídeos sintéticos derivados do domínio B da NTPDase 1 de L. braziliensis. Sua antigenicidade foi evidenciada em “Western blots” pela reatividade com soros de cães com leishmaniose visceral. Os soros imunes SC ou SD inibiram sua atividade (87-99%) em preparação de promastigotas, sugerindo um efeito direto sobre o domínio B. Por ELISA, 45-50% de 38 cães infectados foram soropositivos para LbB1LJ e LbB2LJ confirmando a antigenicidade deste domínio. The NTPDase 1 (50 kDa) of Leishmania infantum promastigotes was purified by nondenaturing gel electrophoresis. The identity of this protein or of its conserved domain B (r83-122) was confirmed by Western blots using immune sera raised against potato apyrase (SA); r-potDomainB (SB), a recombinant polypeptide derived from the B domain of this vegetable protein, and LbB1LJ (SC; r82-103) and LbB2LJ (SD; r102-121), synthetic peptides derived from the B domain of the L. braziliensis NTPDase 1. Its antigenicity was evidenced in Western blots by the reactivity with serum samples from dogs with visceral leishmaniasis. The immune sera SC and SD inhibited its activity (87- 99%) in promastigotes preparation, suggesting a direct effect on the B domain. By ELISA, 45-50% of 38 infected dogs were seropositive for LbB1LJ and LbB2LJ confirming the antigenicity of this domain.

The activities of the enzymes NTPDase (E.C.3.6.1.5, apyrase, ATP diphosphohydrolase, ecto-CD39) and 5′-nucleotidase (E.C.3.1.3.5, CD73) were analyzed in platelets from patients with chronic renal failure (CRF), both undergoing hemodialysis treatment (HD) and not undergoing hemodialysis (ND), as well as from a control group. The results showed an increase in platelet NTPDase activity in CRF patients on HD treatment (52.88%) with ATP as substrate (P

CD39, the mammalian ATP diphosphohydrolase (ATPDase), is thought to contain two transmembrane domains and five "apyrase conserved regions" (ACR) within a large extracellular region. To study the structure of this ectoenzyme, human CD39 was modified by directed mutations within these ACRs or by sequential deletions at both termini. ATPDase activity was well preserved with FLAG tagging, followed by the removal of either of the demonstrated C- or N-transmembrane regions. However, deletions within ACR-1 (aa 54-61) or -4 (aa 212-220), as well as truncation mutants that included ACR-1, -4, or -5 (aa 447-454), resulted in substantive loss of biochemical activity. Intact ACR-1, -4, and -5 within CD39 are therefore required for maintenance of biochemical activity. Native and mutant forms of CD39 lacking TMR were observed to undergo multimerization, associated with the formation of intermolecular disulfide bonds. Limited tryptic cleavage of intact CD39 resulted in two noncovalently membrane-associated fragments (56 and 27 kDa) that substantially augmented ATPDase activity. Glycosylation variation accounted for minor heterogeneity in native and mutant forms of CD39 but did not influence ATPDase function. Enzymatic activity of ATPDase may be influenced by certain posttranslational modifications that are relevant to vascular inflammation.

FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais A esquistossomose é uma doença que acomete cerca de 200 milhões de pessoas no mundo causando grande morbidade. Devido à falta de vacina para essa doença, muitos antígenos são pesquisados. Um domínio antigênico B (r156-195) da ATPDase 2 de Schistosoma mansoni foi previamente descrito, e dois peptídeos sintéticos (SmB1LJ, r155-176; SmB2LJ, r175-194) derivados deste domínio foram obtidos com o objetivo de avaliar a imunogenicidade dessa proteía. O inóculo de cada peptídeo em camundongos BALB/c induziu atividade imunoestimulatória significativa, elevando os níveis de anticorpos IgG1 e IgG2, como detectado por ELISA. Os anticorpos policlonais anti-SmB1LJ e anti-SmB2LJ, quando imobilizados em Proteína A-Sepharose, imunoprecipitaram aproximadamente 42-96% da atividade ADPásica da preparação de verme adulto. Por “Western blots” do complexo imunoprecipitado, três bandas de 76, 63 e 55 kDa foram identificadas pelos dois soros imunes, confirmando a identidade da ATPDase 2, e sugerindo que esta proteína está sujeita a vários processamentos pós-tradução, tais como glicosilação e proteólise, resultando finalmente em uma proteína secretada de 55 kDa. Adicionalmente, anticorpos policlonais anti-SmB2LJ inibiram 62% e 37% das atividades ATPásica e ADPásica, respectivamente, enquanto anticorpos policlonais anti-SmB1LJ inibiram ADPase (92%), mas não atividade ATPásica, sugerindo que o domínio B da ATPDase 2 do S. mansoni está envolvido na regulação catalítica da enzima, sendo um novo alvo para o desenho de inibidores. Imunomarcação com anticorpos policlonais anti-SmB2LJ de cortes de fígado de camundongo infectado revelaram reações positivas na superfície externa do miracídio no ovo de S. mansoni. Fluorescência intensa foi detectada na região entre o miracídio e o lado interno da casca do ovo, localizada no envelope de von Lichtenberg, a qual é uma região de produção de componentes imunogênicos. Fluorescência foi também detectada no lado de fora da casca do ovo, aprisionada pelos microespinhos de superfície, confirmando que esta proteína, como no verme adulto, é também secretada de ovos de S. mansoni. Nenhuma reatividade foi observada nos tecidos granulomatosos circundantes. Amostras de plasmas de camundongos Swiss infectados com S. mansoni foram testadas por ELISA usando SmB1LJ or SmB2LJ como antígeno. Reatividade significativamente maior de anticorpos IgG, IgG1 ou IgG2a foi detectada com SmB1LJ, enquanto somente anticorpo IgG1 foi altamente reativo com SmB2LJ. A reatividade de amostras de plasma (diluídas 1:200) de camundongos Swiss previamente inoculados com apirase de batata ou r-potDomínio B, um polipeptídio com cauda de hexahistidina derivado do domínio B (r78-117) da apirase de batata, e homólogo ao domínio B da ATPDase 2, foi também testada por ELISA usando os peptídeos como antígenos. Anticorpos policlonais IgG e IgG1 anti-apirase de batata reagiram significativamente com SmB1LJ, enquanto somente IgG1 reagiu com SmB2LJ. Anticorpos policlonais anti-r-potDomínio B não reagiram significativamente com SmB2LJ. Comparado aos controles, amostras de plasma (diluídas 1:200) destes animais pré-imunizados com apirase de batata ou r-potDomínio B e desafiados com S. mansoni não reagem significativamente com os peptídeos. Os resultados confirmaram que o domínio B da ATPDase 2 está potencialmente envolvido na resposta imune do hospedeiro, e os peptídeos SmB1LJ e SmB2LJ podem ser usados em estudos da esquistossomose. Schistosomiasis is a disease that affects approximately 200 million people worldwide causing great morbidity. Due to lack of vaccine for this disease, many antigens are searched. An antigenic domain B (r156-195) from the Schistosoma mansoni ATPDase 2 isoform was previously described, and two synthetic peptides (SmB1LJ, r155-176; SmB2LJ, r175-194) belonging to this domain were obtained. Inoculation of each peptide in healthy BALB/c mice induced significant immunostimulatory activity, increasing the IgG1 and IgG2a antibody levels, as detected by ELISA. The polyclonal anti-SmB1LJ and anti-SmB2LJ antibodies, when immobilized on Protein A-Sepharose, immunoprecipitated approximately 42-96% of the ADPase activity from the adult worm preparation. By Western blots of immunoprecipitated complex, three bands of 76, 63 and 55 kDa were identified by the two immune sera, confirming the ATPDase 2 identity, and suggesting that this protein is subject to several posttranslational processing, such as glycosylation and proteolysis, resulting finally in a secreted protein of 55 kDa. Additionally, polyclonal anti-SmB2LJ antibodies inhibited 62% and 37% of the ATPase and ADPase activities, respectively, whereas polyclonal anti-SmB1LJ antibodies inhibited ADPase (92%), but not ATPase activity, suggesting that the domain B from the S. mansoni ATPDase 2 is involved in enzyme catalytic regulation, being a new target for inhibitor design. Immunolabelled cryostat sections of infected mouse liver by the anti-SmB2LJ polyclonal antibodies revealed positive reactions on the external surface from the miracidium in the S. mansoni egg. Intense fluorescence was detected in the region between the miracidium and the inner side of the egg-shell, located in von Lichtenberg’s envelope, which is a region of production of immunogenic components. Fluorescence was also detected in the outer side of the egg-shell and entrapped by the surface microspines, confirming that this protein, like in the adult worm, is also secreted from the S. mansoni eggs. No reactivity was observed in the surrounding granumomatous tissues, which are rich en inflammatory cells. Serum samples from S. mansoni-infected Swiss mice were tested by ELISA using SmB1LJ or SmB2LJ as coating antigen. Significantly higher IgG, IgG1 or IgG2a antibody reactivity was detected against SmB1LJ, whereas only IgG1 antibody was highly reactive against SmB2LJ. The reactivity of plasma samples (diluted 1:200) from Swiss mice previously inoculated with potato apyrase or r-potDomain B, a 6xHis tag polypeptide belonging to the domain B (r78-117) from the potato apyrase, homologue to the domain B from the ATPDase 2, was also tested by ELISA using the peptides as coating antigens. Polyclonal IgG and IgG1 anti-potato apyrase antibodies significantly reacted with SmB1LJ, whereas only IgG1 reacted with SmB2LJ. Polyclonal anti-r-potDomain B antibodies did not significantly react with SmB2LJ. Compared to controls, plasma samples (diluted 1:200) from these animals pre-immunized with potato apyrase or r-potDomain B and challenged with S. mansoni did not react significantly with peptides. The results confirmed that the domain B from the ATPDase 2 is potentially involved in the host immune response, and peptides SmB1LJ and SmB2LJ can be used in schistosomiasis studies.

Human placental ATP diphosphohydrolase (ATP-DPH), has been previously characterized as an azide-sensitive, Ca(2+)- or Mg(2+)-dependent triphospho- and diphosphonucleosidase which migrates as an 82 kDa protein band on SDS-PAGE (Christoforidis, S. et al. (1995) Eur. J. Biochem. 234, 66-74). In this paper we have studied the subcellular localization of placental ATP-DPH by differential centrifugation and flotation experiments. Using specific enzymatic markers it was found that ATP-DPH is localized on plasma membrane. ATP-DPH was found to be a highly N-glycosylated protein which is a common post-translational modification of plasma membrane proteins. Extensive incubation of the native pure enzyme with N-glycosidase F resulted in the elimination of the 82 kDa form and the concurrent formation of a deglycosylated product of 57.5 kDa and four other intermediate products, indicating the presence of at least five N-glycosylation sites within the ATP-DPH molecule. The partially deglycosylated sample retained its activity in solution and in native gel electrophoresis and activity staining. Biochim Biophys Acta

Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) PROQUALI/UFJF Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Nucleoside triphosphate diphosphohydrolase (NTPDase) activity was recently characterized in Leishmania (Viannia) braziliensis promastigotes (Lb), and an antigenic conserved domain (r82-121) from the specific NTPDase 1 isoform was identified. in this work, mouse polyclonal antibodies produced against two synthetic peptides derived from this domain (LbB1LJ, r82-103; LbB2LJ, r102-121) were used. the anti-LbB1LJ or anti-LbB2LJ antibodies were immobilized on protein A-sepharose and immunoprecipitated the NTPDase 1 of 48 kDa and depleted approximately 40% of the phosphohydrolytic activity from detergent-homogenized Lb preparation. Ultrastructural immunocytochemical microscopy identified the NTPDase 1 on the parasite surface and in its subcellular cytoplasmic vesicles, mitochondria, kinetoplast and nucleus. the ATPase and ADPase activities of detergent-homogenized Lb preparation were partially inhibited by anti-LbB1LJ antibody (43-79%), which was more effective than that inhibition (18-47%) by anti-LbB2LJ antibody. in addition, the immune serum anti-LbB1LJ (67%) or anti-LbB2LJ (33%) was cytotoxic, significantly reducing the promastigotes growth in vitro. the results appoint the conserved domain from the L braziliensis NTPDase as an important target for inhibitor design and the potential application of these biomolecules in experimental protocols of disease control. (c) 2012 Elsevier Inc. All rights reserved. Univ Fed Juiz de Fora, Inst Ciencias Biol, Dept Bioquim, BR-36036900 Juiz de Fora, MG, Brazil Univ Fed Alfenas, Inst Ciencias Biomed, Dept Ciencias Biol, Alfenas, MG, Brazil Fiocruz MS, Inst Oswaldo Cruz, Lab Biol Estrut, Rio de Janeiro, RJ, Brazil Universidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, Brazil Universidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, Brazil FAPEMIG: CBB-APQ-01384-09 FAPEMIG: CBB-APQ 00754-09 Web of Science

FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais Um polipeptídio originado do domínio B conservado da apirase de batata (r78-117), homólogo ao domínio B da isoforma ATPDase 2 de Schistosoma mansoni (r156-195), foi obtido como um polipeptídio com cauda de hexahistidina (r-potDomínio B). Anticorpos policlonais anti-r-potDomínio B, quando imobilizados em Proteína A-Sepharose, imunoprecipitaram 67-91% das atividades ATPásica e ADPásica de preparação de vermes adultos. Por “Western blots”, o soro imune reconheceu bandas de 63 e 55 kDa na preparação do parasito e no complexo imunoprecipitado, confirmando a identidade da isoforma ATPDase 2. Adicionalmente, anticorpos policlonais anti-r-potDomínio B inibiram 38-58% das atividades ATPásica e ADPásica, sugerindo que o domínio B da ATPDase 2 de S.mansoni é um novo alvo para o desenho de inibidores. Amostras de soros de pacientes com esquistossomose foram testados em ELISA usando r-potDomínio B como antígeno. A soropositividade da subclasse IgG1 (27%, 8/30), IgG2 (27%, 8/30), IgG3 (50%, 15/30) ou IgG4 (17%, 5/30) sugeriu que o domínio B da ATPDase 2 está potencialmente envolvido na resposta imune do hospedeiro. A apirase de batata ou r-potDomínio B, emulsificado em adjuvantes Completo e Incompleto de Freund, foi inoculado em camundongos Suíços, aumentando a produção de anticorpos IgG, os quais foram também reativos com antígenos solúveis de ovos (SEA). Imunidade protetora induzida por cada biomolécula foi avaliada 60 dias após o desafio com cercárias. Nenhuma redução de vermes ou do número de ovos foi observada. Camundongos imunizados com apirase de batata desenvolveram altos níveis de anticorpos IgG1 reativos com as biomoléculas e SEA, enquanto anticorpos IgG2a foram produzidos em menor proporção, sugerindo uma tendência de resposta imune do tipo Th2. O r-potDomínio B foi capaz de induzir maior produção de anticorpos IgG2a reativos com as biomoléculas e SEA, uma resposta imune possivelmente associada ao tipo Th1. Análises histológicas de cortes de fígado corados por hematoxilina-eosina, obtidos de camundongos imunizados com apirase de batata, mas não com r-potDomínio B, mostraram uma redução significativa na área do granuloma, como evidenciado pela redução do número de células e substituição por fibras de colágeno. Estudos futuros destas biomoléculas poderão contribuir para um melhor entendimento da interação parasito e hospedeiro, e poderão ser exploradas como novas ferramentas de estudo da esquistossomose. A polypeptide belonging to the conserved domain B (r78-117) from the potato apyrase, homologue to the domain B (r156-195) from the S. mansoni ATPDase 2 isoform, was obtained as a 6xHis tag polypeptide (r-potDomain B). Polyclonal anti-r-potDomain B antibodies, when immobilized on Protein A-Sepharose, immunoprecipitated 67-91% of the ATPase and ADPase activities from the adult worm preparation. By Western blots, the immune serum recognized band of 63 and 55 kDa in both parasite preparation and immunoprecipitated complex, confirming the ATPDase 2 identity. Additionally, polyclonal anti-r-potDomain B antibodies inhibited 38-58% of the ATPase and ADPase activities, suggesting that the domain B from the S.mansoni ATPDase 2 is a new target for inhibitor design. Serum samples from patients with schistosomiasis were tested in ELISA using r-potDomain B as coating antigen. The seropositivity of the subclass IgG1 (27%, 8/30), IgG2 (27%, 8/30), IgG3 (50%, 15/30) or IgG4 (17%, 5/30) suggested that the domain B from the ATPDase 2 is potentially involved in the host immune response. The potato apyrase or r-potDomain B, emulsified in Freund Complete and Incomplete adjuvants, was inoculated in Swiss mice, increasing the IgG antibodies production, which were also reactive with soluble egg antigens (SEA). Protective immunity induced by each biomolecule was evaluated 60 days after challenge infection. No worm burden or egg number reduction was observed. Mice immunized with potato apyrase developed high IgG1 antibody levels, reactive with either biomolecules or SEA, while IgG2a antibody levels were produced in lower proportion, suggesting a tendency of a Th2 type immune response. The r-potDomain B was able to induce higher IgG2a antibody production, an immune response possibly associated to the Th1 type. Histological analyses of haematoxylin-eosin-sections obtained from mice immunized with potato apyrase, but not with r-potDomain B, showed a significant reduction in liver granuloma area as evidenced by the cells number reduction and substitution by collagen fibbers. Further studies of these biomolecules could contribute to a better understanding of host-parasite interactions, and may to be explored as new tools in schistosomiasis research.

Uma atividade NTPDásica foi previamente caracterizada em formas promastigotas de L. (V.) braziliensis (Lb). Neste trabalho, sua localização ultraestrutural foi obtida por técnicas de citoquímica, usando um microscópio eletrônico JEM-1011. A atividade enzimática foi encontrada distribuída como um depósito de fosfato de cério eletrodenso na superfície da membrana plasmática, membrana mitocondrial, cinetoplasto, bolsa flagelar e flagelo de Lb, quando foram incubados em meio de reação padrão com ATP, ADP ou GDP, e na presença de inibidores clássicos de ATPases, fosfatase e nucleotidase. Um polipeptídio originado do domínio B conservado da apirase de batata, homólogo ao domínio B da isoforma NTPDase 1 de Lb, foi então obtido como um polipeptídio com cauda de hexahistidina (r-potDomínio B), o qual foi inoculado em camundongos BALB/c. Anticorpos policlonais anti-r-potDomínio B, quando imobilizados em Proteína A-Sepharose, imunoprecipitaram 43-46% das atividades ATPásica e ADPásica de preparação de Lb. Por ―Western blots‖, o soro imune reconheceu banda de 48 kDa na preparação de Lb e no complexo imunoprecipitado, confirmando a identidade da isoforma NTPDase 1. Amostras de soros de pacientes com leishmaniose cutânea (ACL) foram testados em ELISA, usando r-potDomínio B como antígeno. A reatividade de anticorpos IgG1 (0,077 0,059) ou IgG3 (0,020 0,024) de pacientes com ACL foi significativamente (P < 0,05) maior quando comparado ao controle (IgG1, 0,047 0,029, ponto de corte, 0,105; IgG3, 0,010 0.009, ponto de corte, 0,029), e 9-10 (30-33%) dos 30 pacientes foram soropositivos para r-potDomínio B. A reatividade de anticorpos IgG2 (0,054 0,064; 17% de soropositividade, 5/30) ou IgG4 (0,077 0,031; 23% de soropositividade, 7/30) de pacientes com ACL foi similar ao controle (IgG2, 0,050 0,021, ponto de corte, 0,092; IgG4, 0,059 0,023, ponto de corte, 0,105). Estes resultados sugeriram que o domínio B (r82-121) da NTPDase 1 de L. (V.) braziliensis está potencialmente envolvido na resposta imune do hospedeiro. Em estudo ultraestrutural e imunocitoquímico, os anticorpos policlonais anti-r-potDomínio B identificaram a isoforma NTPDase 1 na superfície do parasito e, também, na membrana mitocondrial, cinetoplasto, bolsa flagelar e flagelo. Estes resultados sugeriram que a atividade fosfohidrolítica da NTPDase 1 do parasito está possivelmente associada à recuperação de nutrientes e/ou a mecanismo protetor contra o organismo hospedeiro sob condições que envolvam nucleotídeos. Adicionalmente, anticorpos policlonais anti-r-potDomínio B (A) inibiram 22-72% das atividades ATPásica e ADPásica da preparação total de Lb; e (B) após inativação do sistema complemento, e usados em ensaios de proliferação celular pelo método MTT, inibiram significativamente 42% do aumento de promastigotas após 24 h de incubação, quando comparado à cultura na ausência de soro, ou na presença de soro de camundongo ou coelho saudável. Estes resultados sugerem que o domínio B (r82-121) da NTPDase 1 de L. (V.) braziliensis é um novo alvo terapêutico. O polipeptídio recombinante r-potDomínio B e anticorpos produzidos contra ele poderão ser úteis em estudos desta proteína do parasito e da leishmaniose cutânea. An NTPDase activity was previously characterized in L. (V.) braziliensis promastigote forms (Lb). In this work, its localization ultrastructural was obtained by cytochemical techniques, using a JEM-1011 electron microscope. Enzyme activity was found distributed as an electron dense cerium phosphate deposit at the surface of the plasma membrane, at mitochondrial membrane, kinetoplast, flagellar pocket and flagellum of Lb when they were incubated in standard reaction medium with ATP, ADP or GDP, and in the presence of classical ATPase, phosphatase and nucleotidase inhibitors. A polypeptide belonging to the conserved domain B from the potato apyrase, homologue to the domain B from the Lb NTPDase 1 isoform, was then obtained as a 6xHis tag polypeptide (r-potDomain B), which was inoculated in BALB/c mice. Polyclonal anti-r-potDomínio B antibodies, when immobilized on Protein A-Sepharose, immunoprecipitated 43-46% of the ATPase and ADPase activities from the parasite preparation. By Western blots, the immune serum recognized band of 48 kDa in both parasite preparations and immunoprecipitated complex, confirming the NTPDase 1 identity. Serum samples of American cutaneous leishmaniasis (ACL) patients were tested in ELISA using r-potDomain B as coating antigen. The IgG1 (0.077 0.059) or IgG3 (0.020 0.024) antibody reactivity of ACL patients was significantly (P < 0.05) higher when compared to the control (IgG1, 0.047 0.029, cut-off, 0.105; IgG3, 0.010 0.009, cut-off, 0.029), and 9-10 (30-33%) of the 30 patients were seropositive for r-potDomain B. The IgG2 (0.054 0.064; 17% seropositivity, 5/30) or IgG4 (0.077 0.031; 23% seropositivity, 7/30) antibody reactivity of ACL patients was similar to the control (IgG2, 0.050 0.021, cut-off, 0.092; IgG4, 0.059 0.023, cut-off, 0.105). These results suggested that the domain B (r82-121) from the L. (V.) braziliensis NTPDase 1 is potentially involved in the host immune response. In ultrastructural and immunocytochemical study, polyclonal anti-r-potDomain B antibodies identified the NTPDase 1 isoform on the parasite surface and, also, at mitochondrial membrane, kinetoplast, flagellar pocket and flagellum. These results suggested that phosphohydrolytic activity from the parasite NTPDase 1 is possibly associated with nutrient recuperation and/or as a protective mechanism against the host organism under conditions that involve nucleotides. Additionally, polyclonal anti-r-potDomain B antibodies (A) inhibited 22-72% of the ATPase and ADPase activities from the total Lb preparation, and (B) after complement system inactivation, and used in MTT cell proliferation assays, significantly inhibited 42% of the promastigote growth after 24 h of incubation, when compared to the cell growth in medium alone, or in the presence of serum from healthy mouse or rabbit. The domain B (r82-121) from the L. (V.) braziliensis NTPDase 1 is a new therapeutic target. The recombinant r-potDomain B polypeptide and antibodies produced against it could be useful in studies of this parasite protein and American cutaneous leishmaniasis.

It has been demonstrated by differential centrifugation methods and separation of membranes on a sucrose gradient that wheat membrane apyrase is bound

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A antigenicidade das isoformas de ATP difosfohidrolase, a solúvel e a associada à membrana, foi recentemente descrita. Imunoreatividade cruzada entre a apirase de batata e anticorpos IgG de camundongos infectados com S. mansoni ou pacientes com esquistossomose sugeriu fortemente que os epitopos compartilhados entre as proteínas do vegetal e parasito são antigênicos. Por análises de bioinformática, um estudo comparativo e estratégico dos domínios compartilhados entre a isoforma solúvel de ATP difosfohidrolase e a apirase de batata permitiu a predição de um domínio homólogo e antigênico disponível para a ligação de anticorpos e moléculas HLA-DR. Peptídeos sintéticos derivados do domínio das proteínas do parasito (Sm) e da batata (pot) foram então obtidos. Usando ELISA, amostras de soro de pacientes com esquistossomose (n= 146) mostraram alta soropositividade de anticorpos das subclasses IgG1 (58%) e IgG4 (58%) para apirase de batata. Pacientes soropositivos (n= 35) foram então selecionados para a análise da reatividade de anticorpos contra os peptídeos sintéticos. Soropositividade significativa de IgG (potB1LJ, 9%; potB1MP, 54%; potB2LJ, 54%; SmB1LJ, 23%; SmB1MP, 86%; SmB2LJ, 34%), e de IgG1 (potB1LJ, 57%; potB1MP, 67%; potB2LJ, 46%; SmB1LJ, 57%; SmB1MP, 10%; SmB2LJ, 31%) ou IgG4 (potB1LJ, 71%; potB1MP, 40%; potB2LJ, 71%; SmB1LJ, 54%; SmB1MP, 63%; SmB2LJ, 49%) foi observada para os peptídeos homólogos confirmando a existência de epitopos antigênicos compartilhados entre as proteínas do parasito e vegetal, e demonstrando que na esquistossomose o domínio r155-194 da isoforma solúvel de ATP difosfohidrolase é rica em epitopos para células B. A reatividade de anticorpos IgG1 e IgG4 de amostras de soros de pacientes (n= 26) curados após a quimioterapia com o praziquantel, foi analisada com potB1LJ e potB2LJ, antes e 6 meses após o tratamento. Análise individual revelou reatividade variável de IgG1 e IgG4 com cada peptídeo, sugerindo que diferentes epitopos antigênicos poderiam estar envolvidos na susceptibilidade ou resistência à reinfecção por S. mansoni. Usando ELISA, anticorpos policlonais contra apirase de batata de camundongos BALB/c ou CBA/J reagem com potB1LJ, potB1MP e/ou potB2LJ. Reatividade similar foi também observada entre estes soros imune e SmB1LJ, SmB1MP e/ou SmB2LJ, confirmando novamente a imunoreatividade cruzada entre as proteínas do parasito e vegetal. Adicionalmente, o inóculo de cada peptídeo em camundongos Swiss saudáveis teve significativa atividade imunoestimulatória, elevando a quantidade de anticorpos IgG e/ou IgG1 e IgG2, como observado por ELISA e/ou “Dot blots”, sugerindo que cada peptídeo tem dois epitopos distintos capazes de induzir resposta imune do tipo Th1 ou Th2. Por “Western blots”, o soro imune anti-SmB2LJ reconheceu 2 polipeptídeos de aproximadamente 63 e 55 kDa em SEA e SWAP, indicando a presença da isoforma solúvel de ATP difosfohidrolase nestas preparações antigênicas, bem como a sensibilidade e especificidade deste soro imune. A apirase de batata e o potB1LJ foram avaliados em reação de hipersensibilidade tardia (DTH) usando a ovalbumina (OVA) como antígeno padrão, por medidas de edema de pata, titulação de subclasses de anticorpos por ELISA, e análise histológica. Aparentemente, a reação inflamatória foi similar entre a apirase de batata e a OVA, enquanto potB1LJ inibiu a reação de DTH. Estudos futuros destas novas biomoléculas poderão contribuir para um melhor entendimento da interação parasito e hospedeiro, e poderão ser exploradas como novas ferramentas de estudo da esquistossomose. The antigenicity of both soluble S. mansoni ATP diphosphohydrolase and membraneassociated isoforms has been recently described. Cross-immunoreactivity between potato apyrase and IgG antibody from S. mansoni-infected mice or schistosomiasis patients strongly suggested that the epitopes shared between the vegetable and parasite proteins are antigenic. By bioinformatics analysis, a strategic comparative study of the conserved domains shared between the soluble ATP diphosphohydrolase isoform and potato apyrase predicted a homologue antigenic-domain available for antibody and HLA-DR binding. Synthetic peptides belonging to the domain of the parasite (Sm) and vegetable (pot) proteins were then obtained. By ELISA, the serum samples from the schistosomiasis patients (n= 146) showed high seropositivity of IgG1 (58%) and IgG4 (58%) antibody sub-classes for potato apyrase. Seropositive patients (n= 35) were then selected for analyses of antibody reactivity against the synthetic peptides. Significant seropositivity of IgG (potB1LJ, 9%; potB1MP, 54%; potB2LJ, 54%; SmB1LJ, 23%; SmB1MP, 86%; SmB2LJ, 34%), and IgG1 (potB1LJ, 57%; potB1MP, 67%; potB2LJ, 46%; SmB1LJ, 57%; SmB1MP, 10%; SmB2LJ, 31%) or IgG4 (potB1LJ, 71%; potB1MP, 40%; potB2LJ, 71%; SmB1LJ, 54%; SmB1MP, 63%; SmB2LJ, 49%) was observed for the homologue peptides in accordance with the existence of shared antigenic epitopes between the parasite and vegetable proteins, and demonstrating that in schistosomiasis the domain r155-194 from soluble ATP diphosphohydrolase isoform is rich in B-cell epitopes. The reactivity of the IgG1 and IgG4 antibody from the serum samples of patients (n= 26) cured after chemotherapy with praziquantel was analyzed against potB1LJ e potB2LJ, before and six months after treatment. Individual analysis revealed variable IgG1 and IgG4 reactivity against each peptide, suggesting that different antigenic epitopes could be involved in susceptibility or resistance to S. mansoni reinfection. By ELISA, polyclonal antibody to potato apyrase of BALB/c or CBA/J mice reacts with potB1LJ, potB1MP and/or potB2LJ. Similar reactivity was also observed between these immune sera and SmB1LJ, SmB1MP and/or SmB2LJ, confirming again the cross-immunoreactivity between parasite and vegetable proteins. In addition, inoculation of each peptide in healthy Swiss mice had significant immunostimulatory activity, increasing the amount of total IgG and/or IgG1 and IgG2a antibody, as observed by ELISA and/or Dot blots, suggesting that each peptide has two distinct epitopes capable of inducing a Th1 or Th2-type immune response. By Western blots, serum immune anti-SmB2LJ recognized two polypeptides of approximately 63 and 55 kDa in SEA or SWAP, indicating the presence of the soluble isoform of ATP diphosphohydrolase in these antigenic preparations, as well as the sensitivity and specificity of this immune serum. The potato apyrase and potB1LJ were evaluated in delayed-type hypersensitivity reaction (DTH), using ovalbumin (OVA) as standard antigen, by footpad thickness measurement, titration of antibody sub-class by ELISA, and histological analysis. Seemingly, the inflammatory reaction was similar between potato apyrase and OVA, while potB1J inhibited the DTH reaction. Further studies of these new biomolecules could contribute to a better understanding of host-parasite interactions, and may to be explored as new tools in schistosomiasis research.

Made available in DSpace on 2016-10-10T03:52:51Z (GMT). No. of bitstreams: 5 Mapping of the conserved antigenic domains shared between.PDF: 249741 bytes, checksum: 6ac6ab0d5b30165c912bd06b1e723817 (MD5) license_url: 52 bytes, checksum: 2f32edb9c19a57e928372a33fd08dba5 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 24623 bytes, checksum: 378d22d8fe50e084ee2f354be78cbe62 (MD5) license.txt: 1887 bytes, checksum: 445d1980f282ec865917de35a4c622f6 (MD5) Previous issue date: 2008 Evolutionary and closer structural relationships are demonstrated by phylogenetic analysis, peptide prediction and molecular modelling between Solanum tuberosum apyrase, Schistosoma mansoni SmATPase 2 and Leishmania braziliensis NDPase. Specific protein domains are suggested to be potentially involved in the immune response, and also seem to be conserved during host and parasite co-evolution. Significant IgG antibody reactivity was observed in sera from patients with American cutaneous leishmaniasis (ACL) and schistosomiasis using potato apyrase as antigen in ELISA. S. mansoni adult worm or egg, L. braziliensis promastigote (Lb) and Trypanosoma cruzi epimastigote (EPI) have ATP diphosphohydrolases, and antigenic preparations of them were evaluated. In ACL patients, IgG seropositivity was about 43% and 90% for Lb and potato apyrase, respectively, while IgM was lower (40%) or IgG (100%) seropositivity for both soluble egg (SEA) and adult worm (SWAP) antigens was higher than that found for potato apyrase (IgM=10%; IgG=39%). In Chagas disease, IgG seropositivity for EPI and potato apyrase was 97% and 17%, respectively, while the IgM was low (3%) for both antigens. The study of the conserved domains from both parasite proteins and potato apyrase could lead to the development of new drug targets or molecular markers. Sim Publicado

Uma ATP difosfohidrolase foi identificada em promastigotas de Leishmania braziliensis, e resultado similar foi encontrado em outra cepa de Leishmania, pertencente ao Complexo Leishmania braziliensis. Hidrólise de nucleosídeos di- e trifosfatados sob estímulo de íons bivalentes, associada à inibição por azida sódica, sugeriram que atividade ATP difosfohidrolásica está presente em promastigotas. Western blots desenvolvidos com anticorpos anti-apirase de batata produzidos em coelho revelaram bandas de 48 e 43 kDa, possivelmente sub-unidades da mesma proteína. Estes anticorpos imobilizados em Proteína A-Sepharose depletaram aproximadamente 85% da atividade ATPásica ou ADPásica da ATP difosfohidrolase solubilizada em C12E9. O complexo imunoprecipitado (resina-anticorpo-antígeno) foi separado por SDS-PAGE. Através de western blots desenvolvidos com anticorpos anti-apirase de batata produzidos em camundongos, a mesma banda de 48 kDa foi identificada nas duas cepas, confirmando que as proteínas do parasito e da batata compartilham epítopos. Além disso, o peso molecular coincidente sugeriu que esta ATP difosfohidrolase corresponde a um nucleosídeo difosfatase identificada no genoma de L. braziliensis como pertencente à família das NTPDases. Estreita relação estrutural e evolucionária foram então demonstradas entre a apirase de batata e a NDPase de L. braziliensis por cladograma, alinhamento de seqüências de aminoácidos, predição de epítopos e modelagem molecular. Domínios específicos parecem estar potencialmente envolvidos na resposta imune do hospedeiro, conservados entre estes diferentes organismos. Devido a esta imunoreatividade cruzada observada entre a apirase de batata e a ATP difosfohidrolase de L. braziliensis, também sugerido por análises in silico, western blots foram desenvolvidos com soros de pacientes com LTA, e os resultados sugeriram que estes epítopos compartilhados são antigênicos para o humano. Os níveis de anticorpos contra a proteína vegetal ou contra o homogeneizado de L. braziliensis foram então analisados em 21 amostras de soros de pacientes com LTA. Aproximadamente 84% deles têm soropositividade de IgG para Lb ou apirase de batata. Correlação positiva foi observada entre os níveis de IgG e os níveis elevados dos subtipos IgG1 e IgG3 contra Lb, ambos com 76% de soropositividade em pacientes com LTA. Interessantemente, aumentos significativos dos níveis de IgG1 (71% de soropositividade) e IgG3 (43% soropositividade) contra a apirase de batata foram também observados. Elevada soropositividade de IgG4 para Lb (62%) e apirase de batata (48%), mas não de IgG2 (

CD39/ATP diphosphohydrolase is expressed on B lymphocytes, cytotoxic T lymphocytes, monocytes, platelets, and endothelial cells, and it has a critical role in the inhibition of platelet responsiveness. To determine whether strenuous exercise could acutely change expression of CD39 in platelets and lymphocytes, eight healthy sedentary men, 34 yr old (SD 7), and eight physically active men, 34 yr old (SD 6), performed graded upright cycle ergometry to volitional exhaustion. Blood samples collected both at baseline and after exercise test were employed to measure CD39 expression in platelets and lymphocytes. The percentage of circulating platelet-platelet aggregates, the “in vitro” ADP and collagen-induced platelet aggregation, and the expression of both platelet glycoprotein IIb-IIIa (PAC-1) and P-selectin (CD62) were also considered markers of platelet activation. After strenuous exercise, all subjects demonstrated significant platelet activation as judged by the increased percentage of platelet-platelet aggregates. The in vitro ADP-induced platelet aggregation and the expression of CD62P on ADP-stimulated platelets significantly increased in sedentary but not in active subjects. After exercise, all of the subjects showed a significant reduction of CD39 expression in platelet [sedentary: from 2.2 (SD 0.8) to 1.1% (SD 0.8), P = 0.008; active: from 0.6 (SD 0.2) to 0.35% (SD 0.1), P = 0.009] and an increase of CD39 expression in B lymphocytes [sedentary: from 47 (SD 13) to 60% (SD 11), P = 0.0039; active: from 46 (SD 11) to 59% (SD 11), P = 0.0038]. Taken together, these findings confirm the critical role of this ADPase in inhibition of platelet responsiveness, also suggesting a possible role of B lymphocytes in thromboregulation mechanism.

There is growing pharmacological evidence from several animal models of seizure disorders that adenosine possesses endogenous anticonvulsant activity. Apart from being released from cells, adenosine can be produced by the degradation of adenine nucleotides by ectoenzymes or soluble nucleotidases. These enzymes constitute an important mechanism in synaptic modulation, as they hydrolyze ATP, an excitatory neurotransmitter, to adenosine, a neuroprotective compound. We recently demonstrated an increase in ectoenzyme activity in rat brain synaptosomes after pentylenetetrazol-kindling in rats resistant to kindling, suggesting a role for ectonucleotidases in the seizure control. The present work investigates the effect of seizures induced by pentylenetetrazol kindling on the enzymes that could be playing a role in ATP, ADP and AMP hydrolysis to adenosine in rat blood serum. Animals received injections of PTZ (30 mg/kg, i.p., dissolved in 0.9% saline) once every 48 h, totaling 10 stimulations and the controls animals were injected with saline. The hydrolysis of ATP, ADP and AMP were significantly increased (42, 40, and 45%, respectively), while phosphodiesterase activity was unchanged. These results suggest once more that an increase in the ATP diphosphohydrolase and 5'-nucleotidase activities and, possibly, in adenosine levels, could represent an important compensatory mechanism in the development of chronic epilepsy. Moreover, the fact that this increase can also be measured in serum could mean that these enzymes might be useful as plasma markers of seizures in epilepsy.

Compelling evidence has indicated the involvement of extracellular ATP and adenosine in the mechanisms of synaptic plasticity and memory formation. In the present study, adult rats were trained in a step-down inhibitory avoidance task (IA) or submitted to isolated foot-shock (IF) (0.4 mA) before measuring ectonucleotidase activities in the synaptosomes of the anterior and posterior cingulate cortex (AC and PC, respectively) and the medial precentral area (Fr2). IA increased ATP and ADP hydrolysis immediately after training in the synaptosomes of PC and AC, respectively, (P

1. Studies have shown that adenosine transport and adenosine A1 receptors in rat brain are subjected to regulation by thyroid hormone levels. Since the ectonucleotidase pathway is an important source of adenosine extracellular, in the present study the in vitro action of T3 and T4 hormones on ectonucleotidase activities in hippocampal synaptosomes was evaluated. 2. T3 (Triiodo-l-thyronine) significantly inhibited, in an uncompetitive manner, the ATP and ADP hydrolysis promoted by ATP diphosphohydrolase activity in hippocampal synaptosomes of adult rats. 3. In contrast, T4 (Thyroxine) only inhibited ATP hydrolysis in an uncompetitive mechanism, at the concentrations tested (100-500 microM), but at the same time did not affect ADP hydrolysis. 4. In the present study, we also investigate the in vitro effect of T3 and T4 on 5'-nucleotidase activity. However, there are no changes in the activity of this enzyme in the presence of T3 and T4 in the hippocampal synaptosomes of rats. 5. These results suggest that thyroid hormones could be involved in the regulation of ectonucleotidase activities, such as ecto-ATP diphosphohydrolase and ecto-ATPase, possibly exerting a modulatory role in extracellular adenosine levels.

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In Shigella flexneri and enteroinvasive Escherichia coli (EIEC) the expression of the virulence-plasmid(pINV)-carried potential pathogenesis-associated apy gene, which encodes apyrase (ATP diphosphohydrolase), is regulated by the same regulators that govern the expression of virulence genes. To understand the transcriptional organization of the apy gene, the authors sequenced an 8023 bp PstI fragment of the pINV of EIEC strain HN280, which encompasses apy as well as its adjacent genes. The PstI fragment displays 99% identity with the corresponding fragment of pWR100, the pINV of S. flexneri strain M90T, and contains four genes. One of these genes, ospB, encodes a secreted protein of unknown activity and is located immediately upstream of apy. Analyses of sequence, Northern hybridization, RT-PCR and primer extension data and transcriptional fusions indicated that ospB and apy are co-transcribed as a 2 kb bicistronic, temperature-regulated mRNA from an upstream promoter that precedes ospB. The 2 kb mRNA is post-transcriptionally processed in the intercistronic ospB-apy region, leading to the considerable accumulation of a more stable 1 kb apy-specific mRNA (half-life of 2.2+/-0.3 min, versus 27+/-4 s for the 2 kb transcript). Upon temperature induction, peak expression of the ospB-apy operon occurs when bacteria enter into the late phases of bacterial growth, where the apy-specific transcript was found to be much more prevalent if compared to the ospB-apy transcript.