Navegando por Palavras-chave "Aedes aegypti"
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- ItemAcesso aberto (Open Access)Bioprospecção de compostos inibidores da atividade catalítica de homólogos das enzimas conversoras de angiotensina (ECA 1 e ECA 2) de insetos e de mamíferos(Universidade Federal de São Paulo, 2022-07-29) Pederiva, Victoria Perini [UNIFESP]; Lapa, Antonio José [UNIFESP]; Tanae, Mirtes Midori [UNIFESP]; http://lattes.cnpq.br/5151320202625343; http://lattes.cnpq.br/8371393564245194; http://lattes.cnpq.br/2290990422603154Mosquitos transmissores de arboviroses (dengue, zika, chikungunya, febre amarela), ou de malária e filariose, têm apresentado resistência progressiva aos inseticidas sintéticos clássicos de baixa toxicidade humana. A (quase) inexistência de vacinas eficazes e de medicamentos específicos contra essas enfermidades soma-se ao insucesso na pesquisa de novos inseticidas para prevenir surtos de doenças vetoriais. As enzimas conversoras de angiotensina (ECAs) de mamíferos têm importantes ações reguladoras da pressão arterial e foram encontrados homólogos dessas enzimas em insetos, cuja função ainda é pouco conhecida, porém vital. Esta pesquisa visa a obtenção de compostos naturais inibidores das ECAs de insetos que podem agir como inseticidas. A Cecropia glazioui foi a planta selecionada para este estudo. As atividades enzimáticas das ECAs foram determinadas por fluorimetria utilizando o substrato FRET do tipo Abz-FRK(Dnp)P-OH para a ECA-1, e Mca-APK(Dnp) para a ECA-2. A atividade enzimática foi determinada incubando o homogenato de larvas (ou o plasma de ratos) com a fração butanólica (Fbut) (10-1000 ug/mL) ou com os compostos majoritários (0,1-100 uM) da C. glazioui. A Fbut inibiu não seletivamente as ECAs de plasma de rato e de homogenato de larvas, com valores de IC50 muito próximos. Apesar dos compostos serem menos ativos que a Fbut, o ácido clorogênico, as procianidinas B5, B3 e B2, a orientina, a isoorientina e a isovitexina (100 uM) reduziram significativamente a fluorescência emitida pelo substrato Abz para ECA-1 (p<0,0001) Para a prova de conceito in vivo, foram testados a Fbut (0,3-30 mg/mL) e os compostos (0,03-0,3 mg/mL) que apresentaram maior atividade nos testes in vitro: orientina, isoorientina e isovitexina. Nesse ensaio, as larvas de Aedes aegypti foram expostas a diferentes concentrações desses compostos, registrando a mortalidade após 24, 48 e 72h. O desenvolvimento da larva até pupa foi avaliado por duas semanas. A letalidade para os primeiros estágios larvais foi maior e significativamente diferente da observada no estágio final de desenvolvimento da larva. Apesar dos flavonóides não terem mostrado uma ação inseticida in vivo, esses compostos inibiram significativamente o crescimento nos primeiros ínstares. O efeito foi persistente durante o tempo de exposição das larvas aos compostos. É possível que essa atividade esteja relacionada à inibição dos homólogos das ECAs em insetos, porém mecanismos adicionais ainda não foram estudados.
- ItemAcesso aberto (Open Access)Characterization of thrombin inhibitory mechanism of rAaTI, a Kazal-type inhibitor from Aedes aegypti with anticoagulant activity(Elsevier B.V., 2011-03-01) Watanabe, Renata Midori Okuta [UNIFESP]; Tanaka-Azevedo, Anita M.; Araujo, Mariana da Silva [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Tanaka, Aparecida Sadae [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Inst ButantanSaliva of blood-sucking arthropods contains a complex mixture of anti-haemostatic, anti-inflammatory and immune-modulator compounds. Among anti-haemostatic factors, there are anticoagulants, vasodilators and platelet aggregation inhibitors. Previous analyses of the sialotranscriptome of Aedes aegypti showed the potential presence of a Kazal-type serine protease inhibitor in the female salivary glands, carcass and also in the whole male, which inhibitor we named AaTI (A. aegypti thrombin inhibitor). Recently, we expressed and characterized rAaTI as a trypsin inhibitor, and its anticoagulant activity [1]. in this work we characterized the thrombin inhibition mechanism of rAaTI. Recombinant AaTI was able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. in contrast, AaTI Delta (rAaTI truncated form) and C-terminal AaTI acidic tail prolong only thrombin time. in the competition assay, rAaTI, AaTI Delta or C-terminal AaTI acidic tail thrombin interactions seem to be affected by heparin but not by hirudin, suggesting that rAaTI binds to thrombin exosite 2. Finally, the thrombin inhibition assay of rAaTI showed an uncompetitive inhibition mechanism. in conclusion, rAaTI can probably inhibit thrombin by interacting with thrombin exosite 2, and the interaction is not mediated by the AaTI C-terminal region, since the truncated AaTI Delta form also prolongs thrombin time. (C) 2010 Elsevier Masson SAS. All rights reserved.
- ItemAcesso aberto (Open Access)Os ciclos do dengue em São Paulo e o modelo de intervenção(Universidade Federal de São Paulo (UNIFESP), 2009-03-27) Matos, Marina Ruiz de [UNIFESP]; Goldenberg, Paulete [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Este estudo utiliza o modelo ecológico para avaliar a situação epidemiológica do dengue no Estado de São Paulo de 1985 a 2005. Apresenta como discussão central as dificuldades de aplicação de um modelo de controle que prevê homogeneidade de práticas na diversidade de contextos da vida urbana contemporânea. Para esta análise foram buscadas as origens e utilização do modelo ao mesmo tempo em que se ocorreram mudanças nas relações sócioeconômicas e políticas no espaço urbano do Estado de São Paulo. Foram utilizadas informações da epidemiologia da doença, da instalação e permanência do Aedes aegypti e da ação dos serviços públicos, destacando-se a busca pela participação nas ações de controle e erradicação.
- ItemAcesso aberto (Open Access)Clonagem, expressão e caracterização de um inibidor de tripsina presente no mosquito Aedes aegypti(Universidade Federal de São Paulo (UNIFESP), 2010-02-24) Watanabe, Renata Midori Okuta [UNIFESP]; Tanaka, Aparecida Sadae [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The Aedes aegypti mosquito is the classic vector of yellow fever and dengue fever. In haematophagous insects, blood digestion is carried out by proteolitic enzymes, which can also be envolved in parasite-vector relationship. Blood feeding requires that haematophagous insects are able to inhibit host hemostatic defenses, containing inhibitors of coagulation cascade enzymes, for example. In order to understand the vector bilogy, the aim of this work was expression and characterization of a Kazal-type serine protease inhibitor present in Aedes aegypti mosquito. Recombinant inhibitor (rAaTI) and its truncated form rAaTIΔ (without Cterminal region) were expressed in Pichia pastoris system and purified by affinity chromatography. Purified rAaTI was able to inhibit amidolytic activity of trypsin, plasmin and weakly inhibited thrombin. rAaTI was also able to prolong coagulation time, and there are evidences that inhibitor bind in some region occupied by heparin or antithrombin III. Transcription analyzes showed that AaTI is present in salivary gland and midgut of females 3 and 24 h after blood feeding, and in all larval instars, pupae and males, which suggest that inhibitor may have other functions in mosquito development. rAaTI was also able to strongly inhibit trypsin from 4th instar lavae , and female trypsin 3 and 34 h after blood feeding, suggesting that rAaTI may be involved in trypsin modulation in larval phase and in the end of blood difgestion.
- ItemAcesso aberto (Open Access)The component of Carica papaya seed toxic to A-aegypti and the identification of tegupain, the enzyme that generates it(Elsevier B.V., 2013-07-01) Nunes, Natalia Neto dos Santos [UNIFESP]; Santana, Lucimeire Aparecida de [UNIFESP]; Sampaio, Misako Uemura [UNIFESP]; Lemos, Francisco J. A.; Oliva, Maria Luiza Vilela [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Estadual Norte FluminenseAs Aedes aegypti transmits the etiologic agents of both yellow and dengue fever; vector control is considered essential to minimise their incidence. the aim of this work was to identify the component of Carica papaya seed toxic to A. aegypti, and the identification of tegupain, the enzyme that generates it. Aqueous extracts (1%, w/v) of the seed tegument and cotyledon of C. papaya are not larvicidal isolately. However, a mixture of 17 mu g mL(-1) tegument extract and 27 mu g mL(-1) cotyledon extract caused 100% larval mortality in a bioassay. the mixture was no longer larvicidal after the tegument extract was pre-treated at 100 degrees C for 10 min. the enzyme tegupain efficiently hydrolysed the substrate Z-Phe-Arg-pNan (K-m 58.8 mu M, K-cat 28020 s(-1), K-cat/K-m 5 x 10(8) M-1 s(-1)), and its activity increased with 2 mM dithiothreitol (DTT), at 37 degrees C, pH 5.0. the chelating agent EDTA did not modify the enzyme activity. Inhibition of tegupain by cystatin (K-iapp 2.43 nM), E64 (3.64 nM, 83% inhibition), and the propeptide N-terminal sequence indicate that the toxic activity is due to a novel cysteine proteinase-like enzyme, rendered active upon the hydrolysis of a cotyledon component of C. papaya seeds. (c) 2013 Elsevier B.V. All rights reserved.
- ItemAcesso aberto (Open Access)Estudos moleculares de enzimas do tipo tripsina presentes no intestino médio de larvas de Aedes aegypti(Universidade Federal de São Paulo (UNIFESP), 2009-07-29) Soares, Tatiane Sanches [UNIFESP]; Tanaka, Aparecida Sadae [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Aedes aegypti is the most important vector of human arboviral diseases and it is responsible for dengue and urban yellow fever transmissions. Trypsin-like enzymes plays an important role in the Ae. aegypti adult and larval life stages digestion. In the present work, we identified the two major trypsin-like enzymes of Ae. aegypti larval midgut through the trypsin cDNA fragments library construction. They are AAEL005607 and AAEL006371, with expression frequencies of 29.3% and 20%, respectively. Semi quantitative PCR analysis showed that the AAEL005607 was transcripted in all larval instars, but AAEL006371 appeared only in 3rd and 4th larval instars. In order to confirm the transcription data, trypsin-like enzymes from 4th instar larvae of Ae. aegypti midgut were purified by affinity, ionic exchange and reversedphase chromatographies. Purified trypsin presented molecular mass of 28 kDa by SDS-PAGE. Its partial amino acid sequence allowed us to suggest that the trypsin activity is encoding by AAEL005607 sequence. The purified trypsin (AAEL005607) showed Km value of 36.4 μM for Tosyl-Gly-Pro-Arg-pNa substrate and was strongly inhibited by AaTI and HiTI, both trypsin inhibitors, with Ki of 0.94 pM and 160 pM, respectively. In conclusion, for the first time, the major digestive enzyme of 4th larval instar of Ae. aegypti was identified and characterized.
- ItemSomente MetadadadosFunctional characterization of a serine protease inhibitor modulated in the infection of the Aedes aegypti with dengue virus(Elsevier France-Editions Scientifiques Medicales Elsevier, 2018) Soares, Tatiane Sanches [UNIFESP]; Rodriguez Gonzalez, Boris Luis [UNIFESP]; Soares Torquato, Ricardo Jose [UNIFESP]; Alves Lemos, Francisco Jose; Costa-da-Silva, Andre L.; Capurro Guimaraes, Margareth de Lara; Tanaka, Aparecida Sadae [UNIFESP]During feeding with blood meal, female Aedes aegypti can transmit infectious agents, such as dengue, yellow fever, chikungunya and Zika viruses. Dengue virus causes human mortality in tropical regions of the world, and there is no specific treatment or vaccine with maximum efficiency being used for these infections. In the vector-virus interaction, the production of several molecules is modulated by both mosquitoes and invading agents. However, little information is available about these molecules in the Ae. aegypti mosquito during dengue infection. Inhibitors of the pacifastin family have been described to participate in the immune response of insects and Pac2 is the only gene of this family present in Ae. aegypti being then chosen for investigation. Pac2 was expressed in E. coli, purified and analyzed by mass spectrometry and SDS-PAGE. The Pac2 transcript was detected by qPCR, and its protein levels were assessed by Western blotting. The inhibitory activity of Pac2 was measured using its K-i, IC50 and zymography. Mosquito infections with DENV were introduced with the Brazilian ACS-46 DENV-2 strain propagated in C6/36 cells. In the present work, we showed that it is possibly involved in the interaction of the mosquitoes with the dengue virus. The Pac2 transcript was detected in larvae and in both the salivary gland and midgut of Ae. aegypti females, while the native protein was identified in females 3 h post-blood meal. Pac2 is a strong inhibitor of trypsin-like and thrombin-like proteases, which are present in 4th instar larvae midgut and females 24 h after blood meal. During DENV infection, up regulation of Pac2 expression occurs in the salivary gland and midgut. Pac2 is the first Pacifastin inhibitor member described in mosquitoes. Our results suggest that Pac2 acts on mosquito serine proteases, mainly the trypsin-like type, and is under transcriptional control by virus infection signals to allow its survival in the vector or by the mosquito as a defense mechanism against virus infection. (C) 2017 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.
- ItemSomente MetadadadosHigh-resolution structure of a Kazal-type serine protease inhibitor from the dengue vector Aedes aegypti(Int Union Crystallography, 2017) Torquato, Ricardo J. S. [UNIFESP]; Lu, Stephen [UNIFESP]; Martins, Nadia Helena; Tanaka, Aparecida S. [UNIFESP]; Barbosa Pereira, Pedro JoseBlood-feeding exoparasites are rich sources of protease inhibitors, and the mosquito Aedes aegypti, which is a vector of Dengue virus, Yellow fever virus, Chikungunya virus and Zika virus, is no exception. AaTI is a single-domain, noncanonical Kazal-type serine proteinase inhibitor from A. aegypti that recognizes both digestive trypsin-like serine proteinases and the central protease in blood clotting, thrombin, albeit with an affinity that is three orders of magnitude lower. Here, the 1.4 angstrom resolution crystal structure of AaTI is reported from extremely tightly packed crystals (similar to 22% solvent content), revealing the structural determinants for the observed inhibitory profile of this molecule.
- ItemSomente MetadadadosA novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombin coagulant inhibitory activity(Elsevier B.V., 2010-08-01) Watanabe, Renata M. O. [UNIFESP]; Soares, Tatiane S. [UNIFESP]; Morais-Zani, Karen; Tanaka-Azevedo, Anita M.; Maciel, Ceres; Capurro, Margareth L.; Torquato, Ricardo J. S. [UNIFESP]; Tanaka, Aparecida S. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Inst Butantan; Universidade de São Paulo (USP)Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. in this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI (A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity with insect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptase inhibitor from leech Hirudo medicinalis (LDTI). in this work we expressed, purified and characterized the recombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant (K(i)) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. the rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquito's development. (C) 2010 Elsevier Masson SAS. All rights reserved.
- ItemAcesso aberto (Open Access)Triagem sorológica e influência do conhecimento sobre a dengue em pacientes do ambulatório de especialidades do SUS(Sociedade Brasileira de Patologia ClínicaSociedade Brasileira de PatologiaSociedade Brasileira de Citopatologia, 2011-04-01) Vilas Boas, Viviane Aparecida; Rocha, Katya Cristina; Oliveira, Claudia Giorgia Bronzatti De; Sant'anna, Aleksandra Vanessa Lambiasi; Azzalis, Ligia Ajaime [UNIFESP]; Beltrame, Registila Libânia; Junqueira, Virginia Berlanga Campos [UNIFESP]; Fonseca, Fernando Luiz Affonso [UNIFESP]; Faculdade de Medicina do ABC programa de pós-graduação em Ciências da Saúde; FMABC; FMABC Laboratório de Análises Clínicas; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); FMABC curso de Ciências FarmacêuticasINTRODUCTION: Dengue is a public health problem in Brazil and worldwide. OBJECTIVE: To determine dengue seropositivity of patients from SUS specialty laboratory by correlating the results with socio-demographic data. METHODS: One hundred and eighty-four questionnaires on socio-demographic assessment and dengue transmission awareness were filled out. Enzyme linked immunosorbent assay (ELISA) method was performed for the detection of IgM and IgG antibodies against dengue virus. RESULTS: Fifteen percent of patients had IgG against dengue virus without the presence of IgM. CONCLUSION: All patients showed disease and prevention awareness regardless of their socio-economic background. The asymptomatic infection must be evaluated mainly in cases of disease with fever.