Navegando por Palavras-chave "Rhipicephalus microplus"
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- ItemSomente MetadadadosBmTI-A, a Kunitz type inhibitor from Rhipicephalus microplus able to interfere in vessel formation(Elsevier Science Bv, 2016) Soares, Tatiane S. [UNIFESP]; Oliveira, Felipe [UNIFESP]; Torquato, Ricardo J. S. [UNIFESP]; Sasaki, Sergio D.; Araujo, Mariana S. [UNIFESP]; Paschoalin, Thaysa [UNIFESP]; Tanaka, Aparecida S. [UNIFESP]Rhipicephalus microplus is an ectoparasite responsible for transmissions of babesiosis and anaplasmosis causing large losses to livestock production. To survive R. microplus tick produces several active molecules, such as protease inhibitors. This ectoparasite has been described as a rich source of serine protease inhibitors most of them are Kunitz-BPTI members named BmTIs which have no clear function yet. In the present work, we described the expression and functional characterization of rBmTI-A which showed to be similar to the native BmTI-A, a double-headed Kunitz-BPTI inhibitor, capable to inhibit trypsin, human neutrophil elastase (HNE), human plasma kalikrein (HuPK) and human plasmin. rBmTI-A was able to cause a decrease of HUVEC cell viability. Besides, the rBmTI-A showed to be a potent inhibitor of "in vitro" vessel formation. Our results suggested that BmTI-A may participate in the blood acquisition process interfering in the vessel formation during the tick parasite life stage, around 20 days. In conclusion, BmTI-A is a promising molecule to be used in the drug design and development of new method of R. microplus control. (C) 2016 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosBYC, an atypical aspartic endopeptidase from Rhipicephalus (Boophilus) microplus eggs(Elsevier B.V., 2008-04-01) Nascimento-Silva, Maria Clara L.; Leal, Alexandre T.; Daffre, Sirlei; Juliano, Luiz [UNIFESP]; Silva Vaz, Itabajara da; Paiva-Silva, Gabriela de O.; Oliveira, Pedro L.; Sorgine, Marcos Henrique F.; Universidade Federal do Rio de Janeiro (UFRJ); Univ Fed Rio Grande do Sul; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)An aspartic endopeptidase was purified in our laboratory from Rhipicephalus (Boophilus) microplus eggs [Logullo, C., Vaz, I.S., Sorgine, M.H., Paiva-Silva, G.O., Faria, F.S., Zingali, R.B., de Lima, M.F., Abreu, L., Oliveira, E.F., Alves, E.W, Masuda, H., Gonzales, J.C., Masuda, A., and Oliveira, P.L., 1998. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Rhipicephalus (Boophilus) microplus. Parasitology 116, 525-532]. Boophilus yolk cathepsin (BYC) was tested as component of a protective vaccine against the tick, inducing a significant immune response in cattle [da Silva, VI., Jr., Logullo, C., Sorgine, M., Velloso, F.F., Rosa de Lima, M.F., Gonzales, J.C., Masuda, H., Oliveira, P.L., and Masuda, A., 1998. Immunization of bovines with an aspartic proteinase precursor isolated from Rhipicephalus (Boophilus) microplus eggs. Vet. Immunol. Immunopathol. 66,331-341]. in this work, BYC was cloned and its primary sequence showed high similarity with other aspartic endopeptidases. in spite of this similarity, BYC sequence shows many important differences in relation to other aspartic peptidases, the most important being the lack of the second catalytic Asp residue, considered to be essential for the catalysis of this class of endopeptidases. When we determined BYC cleavage specificity by LC-MS, we found out that it presents a preference for hydrophobic residues in P1 and P1' in accordance to most aspartic endopeptidases. Also, when analyzed by circular dicroism, BYC presented high beta sheet content, also a characteristic of aspartic endopeptidases. On the other hand, although both native and recombinant BYC are catalytically active, they present a very low specific activity, what seems to indicate that this peptidase will digest its natural substrate, vitellin, very slowly. We speculate that such a slow Vn degradative process might constitute an important strategy to preserve egg protein content to the hatching larvae. (c) 2007 Elsevier Inc. All rights reserved.
- ItemAcesso aberto (Open Access)Produção e Caracterização in silico da Proteína Inibidora de elastase de neutrófilos (NEI) recombinante do carrapato Rhipicephalus microplus: uma candidata para tratamento de desordens inflamatórias(Universidade Federal de São Paulo, 2021-06-02) Leite, Gabriela Karen [UNIFESP]; Campos, Claudia Barbosa Ladeira de [UNIFESP]; Anatriello, Elen; http://lattes.cnpq.br/6295907986341326; http://lattes.cnpq.br/8867737169235839; http://lattes.cnpq.br/4772381910969440Os carrapatos, tais quais o Rhipicephalus microplus, são artrópodes ectoparasitas hematófagos, que precisam permanecer fixos aos seus hospedeiros durante todo o tempo de vida para se alimentarem. Para tal, secretam diversas moléculas em sua saliva a fim de evadir a resposta imune do hospedeiro. Ao estudar essas diferentes moléculas, observa-se uma grande gama de potenciais imunobiológicos que podem atuar como efetores farmacológicos contra desordens inflamatórias causadas ou não por patógenos. Um dos desafios está em estabelecer formas de produção destas moléculas com baixo custo e grande rendimento. O objetivo do trabalho foi a produção da proteína inibidora de elastase de neutrófilo (NEI) recombinante em Escherichia coli e em Pichia pastoris, cuja sequência primária foi derivada do sialotranscriptoma do carrapato R. microplus, e a sua caracterização in silico. Conduzimos esse projeto, comparando os dois sistemas de produção de proteínas recombinantes, em pequena e larga escala em laboratório. Também foram realizados métodos de purificação como a cromatografia por afinidade e por gel filtração, para posteriormente ser utilizada na investigação do seu potencial farmacológico em modelos experimentais murinos. Para validar esse potencial, comparamos a nossa proteína de interesse com proteínas já caracterizadas como Inibidoras de elastase de carrapatos como R. microplus, A. monolakensis, I. scapularis, H. asiaticum, I. persulcatus, O. parkeri, H. dromedarii H. marginatum rufipe, A. delacruzi, A. triste, e A. cajennense e A. parvvum assim como do mosquito C. tarsalis. Esse comparativo foi feito via comparações filogenéticas e estruturais em vários níveis (verificando estruturas primarias, secundarias e tridimensionais). Nesse projeto, revistando os principais processos biotecnológicos de produção de proteínas recombinantes usando técnicas como crescimento em meio LB, usando a via do OperonLac e para concentração de proteínas por diálise usado no processo de produção via E. coli competentes, assim como crescendo em meios específicos com protocolos de produção proteica otimizados, ativando a produção da proteína de interesse usando metanol e usando instrumentos como NanoDrop para garantir a concentração a proteína produzida conseguiu-se definir o protocolo de P. pastoris como o de melhor rendimento e fácil purificação para utilização em modelos in vivo. Os resultados deste trabalho podem contribuir com um potencial candidata para tratamento de doenças inflamatórias com exacerbação da ação neutrofílica.
- ItemAcesso aberto (Open Access)Rmcystatin3, a cysteine protease inhibitor from Rhipicephalus microplus hemocytes involved in immune response(Elsevier B.V., 2014-11-01) Lu, Stephen [UNIFESP]; Soares, Tatiane Sanches [UNIFESP]; Vaz Junior, Itabajara S.; Lovato, Diogo Ventura [UNIFESP]; Tanaka, Aparecida Sadae [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Fed Rio Grande do SulThe Rhipicephalus microplus tick is responsible for losses in the livestock production estimated in 2 billions USD. Despite its economical importance the knowledge in tick's physiology is sparse. in order to contribute to this scenario we describe the characterization of a cysteine proteinase inhibitor named Rmcystatin-3. Purified recombinant Rmcystatin-3 was able to inhibit cathepsin L (Ki = 2.5 nM), BmCl1 (Ki = 1.8 nM) and cathepsin B (Ki = 136 nM). Western blot and quantitative PCR analysis revealed the presence of Rmcystatin-3 in fat body, salivary gland but mainly in hemocytes. the mRNA levels of Rmcystatin-3 during bacterial challenge are drastically down-regulated. in order to define the Rmcystatin-3 possible role in tick immunity, the cystatin gene was knockdown by RNA interference with and without Escherichia coli infection. Our results showed that the Rmcystatin-3 silenced group was more immune competent to control bacterial infection than the group injected with non-related dsRNA. Taking together, our data strongly suggested an important role of Rmcystatin-3 in tick immunity. (C) 2014 Elsevier B.V. and Societe francaise de biochimie et biologie Moleculaire (SFBBM). All rights reserved.
- ItemSomente MetadadadosRmKK, a tissue kallikrein inhibitor from Rhipicephalus microplus eggs(Elsevier B.V., 2014-06-20) Abreu, Patricia A. [UNIFESP]; Soares, Tatiane S. [UNIFESP]; Buarque, Diego S. [UNIFESP]; Torquato, Ricardo S. [UNIFESP]; Tanaka, Aparecida S. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Rhipicephalus microplus is an important ectoparasite that is responsible for transmission of anaplasmosis and babesiosis to cattle. Tissue kallikrein inhibitors might play an important role in R. microplus eggs. in the present work, we purified and characterized, a tissue kallikrein inhibitor presents in R. microplus eggs (RmKK), a protein which contains two Kunitz domain in tandem. Purified inhibitor was confirmed by amino terminal determination and its dissociation constant (K-i) for bovine trypsin and porcine pancreatic kallikrein were 0.6 nM and 91.5 nM, respectively. Using a cDNA library from R. microplus midgut, we cloned the cDNA fragment encoding mature RmKK and expressed the protein in Pichia pastoris system. Recombinant RmKK was purified by ion exchange chromatography and presented molecular mass of 16.3 kDa by MALDI-TOF analysis. Moreover, RmKK showed a tight binding inhibition for serine proteases as bovine trypsin (K-i = 0.2 nM) and porcine pancreatic kallikrein (PPK) (K-i = 300 nM). We performed, for the first time, the characterization of a tissue kallikrein inhibitor presents in R. microplus eggs, which the transcript is produced in the adult female gut. BmKK seems to be the strongest PPK inhibitor among all BmTIs present in the eggs and larvae (Andreotti et al., 2001; Sasaki et al., 2004). This data suggests that BmKK may participate in the development of tick egg and larvae phase. (C) 2014 Elsevier Inc. All rights reserved.