Navegando por Palavras-chave "Cystatin"
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- ItemAcesso aberto (Open Access)Avaliação do ritmo de filtração glomerular(Sociedade Brasileira de Patologia ClínicaSociedade Brasileira de PatologiaSociedade Brasileira de Citopatologia, 2007-08-01) Mastroianni Kirsztajn, Gianna [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Glomerular filtration rate (GFR) determination is the most frequently used laboratorial test to evaluate renal function. Indirect markers as blood determination of creatinine and cystatin C are used with this purpose, as well as the direct determination of GFR, with indicators like inulin; iodated contrasts, radioactive or not; and others. Serum creatinine is the test that is most commonly performed in order to evaluate GFR in the clinical pathology laboratory. However, in some conditions, aiming at the adequate interpretation of the test, the result of serum creatinine must be corrected (by using formulas that include individual characteristics of the subjects). In fact, inulin is still seen as the ideal marker of glomerular filtration, but its use is not directed to clinical practice; then the search for appropriate tests for routine use continues.
- ItemSomente MetadadadosCharacterization of a novel cystatin type 2 from Rhipicephalus microplus midgut(Elsevier France-Editions Scientifiques Medicales Elsevier, 2017) Cardoso, Thyago H. S. [UNIFESP]; Lu, Stephen [UNIFESP]; Gonzalez, Boris R. G. [UNIFESP]; Torquato, Ricardo J. S. [UNIFESP]; Tanaka, Aparecida S. [UNIFESP]The Rhipicephalus (Boophilus) microplus is an exclusive bovine ectoparasite responsible for the transmission of pathogens that decrease meat, leather and milk productions. Cattle vaccination is an alternative to control tick infestations, but the discovery of potential antigens is still a challenge for researchers. Recently, our group performed a midgut transcriptome of engorged R. microplus tick, and out of 800-ESTs sequences one cystatin-coding sequence was identified and named Rmcystatin-4. In order to understand the physiological role of Rmcystatin-4, the aim of this work was the expression, purification and functional characterization of a novel type 2 cystatin from the tick R. microplus. Rmcystatin-4 gene expression was identified mostly in tick midgut suggesting its possible role in blood digestion control. Our data showed that rRmcystatin-4 was successfully expressed in active form using Pichia pastoris system and the purified inhibitor presented high selectivity to BmCl-1 (Ki = 0.046 nM). Moreover, rRmcystatin-4 was able to impaired BmCl-1 activity towards bovine hemoglobin. (C) 2017 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.
- ItemSomente MetadadadosExpressão heteróloga e avaliação in vitro da atividade de proteínas recombinantes de Haementeria vizottoi sobre os mecanismos de coagulação sanguínea, com foco em aplicação biotecnológica(Universidade Federal de São Paulo (UNIFESP), 2020-03-05) Linhares, Debora Do Carmo [UNIFESP]; Tavassi, Ana Marisa Chudzinski [UNIFESP]; Universidade Federal de São PauloSalivary secretions from hematophagous animals have been an important source of proteins with biotechnological potential, since there are a number of protein inhibitors that can be isolated and used to modulate the action of enzymes of interest. Many of these inhibitors have evolved to specifically disable key proteases from their hosts, leading to delayed blood coagulation (anticoagulants) and minor immune response, providing the hematophagous with the conditions for successful feeding. Based on transcriptomics of salivary glands of the leech Haementeria vizottoi, were identified sequences of three genes whose predicted proteins have signatures of potential biological activity as protease inhibitors. The aim of this prospective study was to clone, express and characterize these three H. vizottoi derived proteins, namely: Hviz 1276 (hemerythrin-like), Hviz 78 (antistasin-like) and Hviz 340 (cystatinlike). Due to their structural characteristics, antistin and cystatin-like proteins were expressed in P. pastoris (Komagataella pastoris) and hemerythrin-like protein was expressed in E. coli. The proteins of interest were recovered from the supernatant medium or cell extract, and purified by molecular exclusion chromatography and / or affinity chromatography with different resins. To determine their effects on the coagulation cascade, activated thromboplastin time (aPTT) and prothrombin time (PT) were determined using diagnostic kits. Depending on the result, they were also evaluated for the potential to inhibit specific proteases such as FXa, thrombin, elastase, kallikrein, papain and cathepsin L. Hviz 1276 did not demonstrate the expected biological activity and its study was discontinued. The application of partially purified Hviz 78, in the order of 1 µmol/L, was enough to extend the intrinsic-initiated coagulation time by more than 700%. It is still necessary to work on the refinement of purification of this protein and to perform new function tests, since no specific inhibitory activity against any of the proteases tested was identified. The most promising results were obtained with Hviz 340, purified and identified as inhibitor of cysteine proteases papain and cathepsin L, for the latter with ki=7.9 nmol/L. This activity represents new opportunities to evaluate the potential of Hviz 340 as a modulator of cellular activity related to the immune response, possibly anti-inflammatory, since cathepsins are critical for antigen processing and presentation.
- 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.