Navegando por Palavras-chave "Hematophagous"

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    Biochemical characterization of a Kunitz type inhibitor similar to dendrotoxins produced by Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) hemocytes
    (Elsevier B.V., 2010-02-10) Lima, Cassia A. [UNIFESP]; Torquato, Ricardo J. S. [UNIFESP]; Sasaki, Sergio D. [UNIFESP]; Justo, Giselle Z. [UNIFESP]; Tanaka, Aparecida S. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade Estadual de Campinas (UNICAMP)
    A novel chymotrypsin inhibitor identified in fat body and hemocyte cDNA libraries of Boophilus microplus was named BmCI (B. microplus Chymotrypsin Inhibitor) (Genbank EU636772). the putative BmCI amino acid sequence presented a 22-residue-signal peptide and 58-residue-mature protein. BmCI amino acid sequence analysis allowed its classification as a Kunitz-BPTI inhibitor with six cysteine residues, a theoretical pI of 7.8, and the presence of Tyr at P1 position in the putative reactive site, suggesting inhibitory activity toward chymotrypsin. in this work, we reported the biochemical characterization of BmCI. the recombinant BmCI expressed in yeast Pichia pastoris was purified by size exclusion and reverse phase chromatographies. rBmCI expression yield was of I mg L-1 of culture. Purified rBmCI confirmed its chymotrypsin inhibitory activity with a low K-i (6.2 pM). the BmCI gene expression analysis by semi-quantitative RT-PCR indicated its transcription in the hemocytes, salivary gland and ovary. the cytotoxic activity of purified rBmCI was demonstrated in BALB/c 3T3 mouse fibroblasts. As assessed by the MTT reduction assay, rBMCI induced a dose-dependent decrease in 3T3 fibroblasts viability (IC50 = 8 mu M). Moreover, flow cytometry analysis revealed that rBmCI is able to induce apoptosis, whereas no effect was observed on cell cycle progression. in conclusion, we demonstrated that rBmCI is cytotoxic against mammalian cells and obtained evidence that this growth inhibition is caused by an apoptosis-inducing activity. (C) 2009 Published by Elsevier B.V.
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    The first pacifastin elastase inhibitor characterized from a blood sucking animal
    (Elsevier B.V., 2010-07-01) Marco, Renato de [UNIFESP]; Lovato, Diogo V. [UNIFESP]; Torquato, Ricardo J. S. [UNIFESP]; Clara, Renan O. [UNIFESP]; Buarque, Diego S. [UNIFESP]; Tanaka, Aparecida S. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Pacifastin-like protease inhibitors belong to a recent classified protease inhibitor family and they are the smallest protease inhibitors described in animals. in this work, we purified and characterized, for the first time, two neutrophil elastase inhibitors belonging to the pacifastin family from the blood sucking insect Triatoma infestans eggs. the inhibitors showed the same N-terminal sequences, molecular masses of 4257 and 4024 Da by MALDI-TOF mass spectrometry and dissociation constants (Ki) for neutrophil elastase of 0.52 and 0.29 nM, respectively. Using a fat body cDNA library, we cloned a pacifastin precursor containing two protease inhibitor domains similar to locust pacifastins. the first pacifastin domain translated to T. infestans purified protein, named TIPI1. Recombinant TIPI1 expressed in Pichia pastoris system showed similar inhibitory activities compared to the native inhibitor. Its precursor, called TIPP1, is mainly expressed in fat body, and it is up-regulated after blood feeding. the immune challenges of 1(a) instar T. infestans nymph with bacteria or dsRNA strongly stimulated TiPP1 expression in fat body, suggesting a possible role of TiPP1 in T. infestans immunity. This work is the first to characterize a blood feeding insect pacifastin inhibitor. (C) 2010 Elsevier Inc. All rights reserved.