Kinetic characterization of a novel cysteine peptidase from the protozoan Babesia bovis, a potential target for drug design

C1A cysteine peptidases have been shown to play an important role during apicomplexan invasion and egress of host red blood cells (RBCs) and therefore have been exploited as targets for drug development, in which peptidase specificity is deterministic. Babesia bovis genome is currently available and from the 17 putative cysteine peptidases annotated four belong to the C1A subfamily. In this study, we describe the biochemical characterization of a C1A cysteine peptidase, named here BbCp (B. bovis cysteine peptidase) and evaluate its possible participation in the parasite asexual cycle in host RBCs. The recombinant protein was obtained in bacterial inclusion bodies and after a refolding process, presented typical kinetic features of the cysteine peptidase family, enhanced activity in the presence of a reducing agent, optimum pH between 6.5 and 7.0 and was inhibited by cystatins from R. microplus. Moreover, rBbCp substrate specificity evaluation using a peptide phage display library showed a preference for Val > Leu > Phe. Finally, antibodies anti-rBbCp were able to interfere with B. bovis growth in vitro, which highlights the BbCp as a potential target for drug design.Instituto de PatobiologíaFil: Lu, Stephen. Universidade Federal de São Paulo. Escola Paulista de Medicina. Department of Biochemistry; BrasilFil: Ascencio, Mariano E. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Torquato, Ricardo J.S. Universidade Federal de São Paulo. Escola Paulista de Medicina. Department of Biochemistry; BrasilFil: Florin-Christensen, Monica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiologia; ArgentinaFil: Florin-Christensen, Monica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Tanaka, Aparecida S. Universidade Federal de São Paulo. Escola Paulista de Medicina. Department of Biochemistry; BrasilFil: Tanaka, Aparecida S. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular; Brasi

Rmcystatin3, a cysteine protease inhibitor from Rhipicephalus microplus hemocytes involved in immune response

The 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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)INCT-Entomologia Molecular, BrazilUniversidade Federal de São Paulo, Dept Biochem, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniv Fed Rio Grande do Sul, Ctr Biotecnol Estado Rio Grande do Sul, Porto Alegre, RS, BrazilUniversidade Federal de São Paulo, Dept Biochem, Escola Paulista Med, BR-04044020 São Paulo, BrazilFAPESP: 05/03514-9FAPESP: 09/17589-1FAPESP: 12/03657-8Web of Scienc

Novel pseudo-aspartic peptidase from the midgut of the tick Rhipicephalus microplus

The characterization of Rhipicephalus microplus tick physiology can support efforts to develop and improve the efficiency of control methods. A sequence containing a domain with similarity to one derived from the aspartic peptidase family was isolated from the midgut of engorged female R. microplus. The lack of the second catalytic aspartic acid residue suggest that it may be a pseudoaspartic peptidase, and it was named RmPAP. In this work we confirm the lack of proteolytic activity of RmPAP and investigate it’s non-proteolytic interaction with bovine hemoglobin by Surface Plasmon Resonance and phage display. Moreover we carried out RNAi interference and artificial feeding of ticks with anti-RmPAP antibodies to assess it’s possible biological role, although no changes were observed in the biological parameters evaluated. Overall, we hypothesize that RmPAP may act as a carrier of hemoglobin/heme between the tick midgut and the ovaries

A novel type 1 cystatin involved in the regulation of Rhipicephalus microplus midgut cysteine proteases

Rhipicephalus microplus is a cattle ectoparasite found in tropical and subtropical regions around the world with great impact on livestock production. R. microplus can also harbor pathogens, such as Babesia sp. and Anaplasma sp. which further compromise cattle production. Blood meal acquisition and digestion are key steps for tick development. In ticks, digestion takes place inside midgut cells and is mediated by aspartic and cysteine peptidases and, therefore, regulated by their inhibitors. Cystatins are a family of cysteine peptidases inhibitors found in several organisms and have been associated in ticks with blood acquisition, blood digestion, modulation of host immune response and tick immunity. In this work, we characterized a novel R. microplus type 1 cystatin, named Rmcystatin-1b. The inhibitor transcripts were found to be highly expressed in the midgut of partially and fully engorged females and they appear to be modulated at different days post-detachment. Purified recombinant Rmcystatin-1b displayed inhibitory activity towards typical cysteine peptidases with high affinity. Moreover, rRmcystatin-1b was able to inhibit native R. microplus cysteine peptidases and RNAi-mediated knockdown of the cystatin transcripts resulted in increased proteolytic activity. Moreover, rRmcystatin-1b was able to interfere with B. bovis growth in vitro. Taken together our data strongly suggest that Rmcystatin-1b is a regulator of blood digestion in R. microplus midgut.Fil: Lu, Stephen. Universidade Federal de Sao Paulo; BrasilFil: da Rocha, Leticia A.. Universidade Federal de Sao Paulo; BrasilFil: Torquato, Ricardo J.S.. Universidade Federal de Sao Paulo; BrasilFil: da Silva Vaz Junior, Itabajara. Universidade Federal do Rio Grande do Sul; BrasilFil: Jacobsen, Monica Ofelia. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Patobiologia Veterinaria. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Patobiologia Veterinaria.; ArgentinaFil: Tanaka, Aparecida S.. Universidade Federal de Sao Paulo; Brasi

A Treatment with a Protease Inhibitor Recombinant from the Cattle Tick (Rhipicephalus Boophilus microplus) Ameliorates Emphysema in Mice

Aims: To determine whether a serine protease inhibitor treatment can prevent or minimize emphysema in mice.Methods: C57BL/6 mice were subjected to porcine pancreatic elastase (PPE) nasal instillation to induce emphysema and were treated with a serine protease inhibitor (rBmTI-A) before (Protocol 1) and after (Protocol 2) emphysema development. in both protocols, we evaluated lung function to evaluate the airway resistance (Raw), tissue damping (Gtis) and tissue elastance (Htis). the inflammatory profile was analyzed in the bronchoalveolar lavage (BALF) and through the use of morphometry; we measured the mean linear intercept (Lm) (to verify alveolar enlargement), the volume proportion of collagen and elastic fibers, and the numbers of macrophages and metalloprotease 12 (MMP-12) positive cells in the parenchyma. We showed that at both time points, even after the emphysema was established, the rBmTI-A treatment was sufficient to reverse the loss of elastic recoil measured by Htis, the alveolar enlargement and the increase in the total number of cells in the BALF, with a primary decrease in the number of macrophages. Although, the treatment did not control the increase in macrophages in the lung parenchyma, it was sufficient to decrease the number of positive cells for MMP-12 and reduce the volume of collagen fibers, which was increased in PPE groups. These findings attest to the importance of MMP-12 in PPE-induced emphysema and suggest that this metalloprotease could be an effective therapeutic target.Laboratorios de Investigacao Medica do Hospital das Clinicas da Faculdade de Medicina da USP (LIM/HC)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ São Paulo, Dept Med, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, São Paulo, BrazilUNIFESP EPM, Dept Bioquim, São Paulo, BrazilUFABC, Ctr Ciencias Nat & Humanas, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, São Paulo, BrazilUNIFESP EPM, Dept Bioquim, São Paulo, BrazilWeb of Scienc

Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

Peer reviewedPublisher PD

Avaliação dos registros de enfermagem em hemoterapia de um hospital geral

Objetivo: avaliar os registros de enfermagem em hemoterapia nas unidades de internação de um hospital geral.Métodos: Estudo descritivo e exploratório, realizado no período de julho a outubro de 2009, em um hospital de grande porte no interior de Minas Gerais, Brasil. Foram auditados 606 prontuários de pacientes que se submeteram ao tratamento hemoterápico. Doze quesitos foram analisados nos registros de enfermagem.Resultados: Foram analisadas 7.272 anotações. Dos itens avaliados, 65,5% estavam em conformidade. Dentre as não conformidades auditadas, as que apresentaram maior frequência foram: os “Sinais Vitais Pós-Transfusional” (83,8%), o “Registro de Observação nos 10 minutos iniciais” (73,6%), o “Número da Bolsa” (61,2%), “Horário de Término” e “Sinais Vitais Início” (57,3%). Os procedimentos que obtiveram melhor resultado foram: “Assinatura” (0,7%), “Carimbo” (1,5%) e “Cabeçalho” (2,8%).Conclusão: Apesar de necessitar melhorias, a qualidade dos registros foi considerada boa, pois a maioria dos registros sobre hemoterapia estava em conformidade