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URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/11847
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Item Extracellular nucleotide metabolism in Leishmania: influence of adenosine in the establishment of infection(Microbes and Infection, 2008-07) Fietto, Juliana Lopes Rangel; Marques-da-Silva, Eduardo de Almeida; Oliveira, Jamile Camargos de; Figueiredo, Amanda Braga; Lima Júnior, Djalma de Souza; Carneiro, Cláudia Martins; Afonso, Luís Carlos CroccoLeishmaniasis is a parasitic disease with a variety of clinical forms, which are related to the Leishmania species involved. In the murine model, Leishmania amazonensis causes chronic non-healing lesions in Leishmania braziliensis- or Leishmania major-resistant mouse strains. In this study, we investigated the involvement of the pathway of extracellular nucleotide hydrolysis, with special focus on the role of extracellular adenosine, in the establishment of Leishmania infection. Our results show that the more virulent parasite—L. amazonensis—hydrolyzes higher amounts of ATP, ADP and AMP than the two other species, probably due to the higher expression of membrane NTPDase. Corroborating the idea that increased production of adenosine is important to lesion development and establishment of tissue parasitism, we observed that increased 5′-nucleotidase activity in L. braziliensis or addition of adenosine at the moment of infection with this parasite resulted in an increase in lesion size and parasitism as well as a delay in lesion healing. Furthermore, inhibition of adenosine receptor A2B led to decreased lesion size and parasitism. Thus, our results suggest that the conversion of ATP, a molecule with pro-inflammatory activity, into adenosine, which possesses immunomodulatory properties, may contribute to the establishment of infection by Leishmania.Item E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) of Leishmania amazonensis inhibits macrophage activation(Microbes and Infection, 2014-12-30) Souza Vasconcellos, Raphael de; Gomes, Rodrigo Saar; Carvalho, Luana Cristina Faria de; Fietto, Juliana Lopes Rangel; Afonso, Luís Carlos CroccoLeishmania amazonensis, the causal agent of diffuse cutaneous leishmaniasis, is known for its ability to modulate the host immune response. Because a relationship between ectonucleotidase activity and the ability of Leishmania to generate injury in C57BL/6 mice has been demonstrated, in this study we evaluated the involvement of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) activity of L. amazonensis in the process of infection of J774-macrophages. Our results show that high-activity parasites show increased survival rate in LPS/IFN-γ-activated cells, by inhibiting the host-cell NO production. Conversely, inhibition of E-NTPDase activity reduces the parasite survival rates, an effect associated with increased macrophage NO production. E-NTPDase activity generates substrate for the production of extracellular adenosine, which binds to A2B receptors and reduces IL-12 and TNF-α produced by activated macrophages, thus inhibiting NO production. These results indicate that E-NTPDase activity is important for survival of L. amazonensis within macrophages, showing the role of the enzyme in modulating macrophage response and lower NO production, which ultimately favors infection. Our results point to a new mechanism of L. amazonensis infection that may pave the way for the development of new treatments for this neglected disease.Item The Genome of Anopheles darlingi, the main neotropical Malaria vector(Nucleic Acids Research, 2013-06-12) Maciel, Talles Eduardo Ferreira; Fietto, Juliana Lopes Rangel; Carvalho, Carlos Roberto de; Pereira, Maristela; Ribeiro, Carlos Alexandre Gomes; Neves, Rogério de Oliveira; Astolfi-Filho, Spartaco; Marinotti, Osvaldo; Cerqueira, Gustavo C.; Almeida, Luiz Gonzaga Paula de; Ferro, Maria Inês Tiraboschi; Loreto, Elgion Lucio da Silva; Zaha, Arnaldo; Teixeira, Santuza M. R.; Wespiser, Adam R.; Almeida e Silva, Alexandre; Schlindwein, Aline Daiane; Pacheco, Ana Carolina Landim; Silva, Artur Luiz da Costa da; et al.Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector–human and vector–parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible atItem Potential antileukemia effect and structural analyses of SRPK inhibition by N-(2- (Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl) isonicotinamide (SRPIN340)(Plos One, 2014-04-08) Siqueira, Raoni Pais; Barbosa, Éverton de Almeida Alves; Polêto, Marcelo Depólo; Righetto, Germanna Lima; Seraphim, Thiago Vargas; Salgado, Rafael Locatelli; Ferreira, Joana Gasperazzo; Oliveira, Leandro Licursi de; Laranjeira, Angelo Brunelli Albertoni; Almeida, Márcia Rogéria; Fietto, Juliana Lopes Rangel; Kobarg, Jörg; Oliveira, Eduardo Basílio de; Teixeira, Robson Ricardo; Borges, Júlio César; Silva Júnior, Abelardo; Bressan, Gustavo Costa; et al.Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.Item Retrospective study on Porcine circovirus-2 by nested pcr and real time pcr in archived tissues from 1978 in brazil(Brazilian Journal of Microbiology, 2011-03-14) Silva, Fernanda Miquelitto Figueira da; Silva Júnior, Abelardo; Peternelli, Ethel Fernandes de Oliveira; Viana, Vinícius Winter; Chiarelli Neto, Orlando; Fietto, Juliana Lopes Rangel; Vargas, Marlene Izabel; Nero, Luís Augusto; Almeida, Márcia Rogéria dePorcine circovirus-2 (PCV-2) infection is currently considered an important disease of swine. The pathogenic agent was first described in Brazil in 2000. This study detected the PCV-2 DNA in four Brazilian pig tissues collected between 1978 and 1979. This observation is the oldest description of this virus in Brazil.Item Trypanosoma cruzi nucleoside triphosphate diphosphohydrolase 1 (TcNTPDase-1) biochemical characterization, immunolocalization and possible role in host cell adhesion(Acta Tropica, 2013-11-19) Mariotini-Moura, Christiane; Bastos, Matheus Silva e; Castro, Felipe Freitas de; Trindade, Mellina Lanna; Vasconcellos, Raphael de Souza; Neves-do-Valle, Myrian Augusta Araújo; Moreira, Bernardo Pereira; Santos, Ramon de Freitas; Oliveira, Claudia Miranda de; Cunha, Luana Celina Seraphim; Souto, Xênia Macedo; Bressan, Gustavo Costa; Silva-Júnior, Abelardo; Baqui, Munira Muhammad Abdel; Bahia, Maria Terezinha; Almeida, Márcia Rogéria de; Meyer-Fernandes, José Roberto; Fietto, Juliana Lopes RangelPrevious work has suggested that Trypanosoma cruzi diphosphohydrolase 1 (TcNTPDase-1) may be involved in the infection of mammalian cells and serve as a potential target for rational drug design. In this work, we produced recombinant TcNTPDase-1 and evaluated its nucleotidase activity, cellular localization and role in parasite adhesion to mammalian host cells. TcNTPDase-1 was able to utilize a broad range of triphosphate and diphosphate nucleosides. The enzyme's Km for ATP (0.096 mM) suggested a capability to influence the host's ATP-dependent purinergic signaling. The use of specific polyclonal antibodies allowed us to confirm the presence of TcNTPDase-1 at the surface of parasites by confocal and electron microscopy. In addition, electron microscopy revealed that TcNTPDase-1 was also found in the flagellum, flagellum insertion region, kinetoplast, nucleus and intracellular vesicles. The presence of this enzyme in the flagellum insertion region and vesicles suggests that it may have a role in nutrient acquisition, and the widespread distribution of TcNTPDase-1 within the parasite suggests that it may be involved in other biological process. Adhesion assays using anti-TcNTPDase-1 polyclonal antibodies as a blocker or purified recombinant TcNTPDase-1 as a competitor revealed that the enzyme has a role in parasite–host cell adhesion. These data open new frontiers to future studies on this specific parasite–host interaction and other unknown functions of TcNTPDase-1 related to its ubiquitous localization.Item Recombinant Leishmania (Leishmania) infantum Ecto-Nucleoside Triphosphate Diphosphohydrolase NTPDase-2 as a new antigen in canine visceral leishmaniasis diagnosis(Acta Tropica, 2012-09-26) Souza, Ronny Francisco de; Santos, Yaro Luciolo dos; Vasconcellos, Raphael de Souza; Borges-Pereira, Lucas; Almeida, Márcia Rogéria de; Fietto, Juliana Lopes Rangel; Caldas, Ivo Santana; Bahia, Maria TerezinhaCanine visceral leishmaniasis is an important public health concern. In the epidemiological context of human visceral leishmaniasis, dogs are considered the main reservoir of Leishmania parasites; therefore, dogs must be epidemiologically monitored constantly in endemic areas. Furthermore, dog to human transmission has been correlated with emerging urbanization and increasing rates of leishmaniasis infection worldwide. Leishmania (Leishmania) infantum (L. chagasi) is the etiologic agent of visceral leish-maniasis in the New World. In this work, a new L. (L.) infantum (L. chagasi) recombinant antigen, named ATP diphosphohydrolase (rLic-NTPDase-2), intended for use in the immunodiagnosis of CVL was produced and validated. The extracellular domain of ATP diphosphohydrolase was cloned and expressed in the pET21b-Escherichia coli expression system. Indirect ELISA assays were used to detect the purified rLic-NTPDase-2 antigen using a standard canine sera library. This library contained CVL-positive samples, leishmaniasis-negative samples and samples from Trypanosoma cruzi-infected dogs. The results show a high sensitivity of 100% (95% CI = 92.60–100.0%) and a high specificity of 100% (95% CI = 86.77–100.0%),with a high degree of confidence (k = 1). These findings demonstrate the potential use of this recombinant protein in immune diagnosis of canine leishmaniasis and open the possibility of its application to other diagnostic approaches, such as immunochromatography fast lateral flow assays and human leishmaniasis diagnosis.