Navegando por Palavras-chave "Inibidores De Proteases"
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- ItemSomente MetadadadosDesenvolvimento de novos materiais dentários: efeito do peptídeo autopolimerizante P11-4 e do trimetafosfato de sódio na degradação do colágeno I e na formação de Hidroxiapatita(Universidade Federal de São Paulo (UNIFESP), 2020-09-24) Carvalho, Rafael Guzella De [UNIFESP]; Tersariol, Ivarne Luis Dos Santos [UNIFESP]; Universidade Federal de São PauloObjectives: This study investigated the influence of peptide P11-4 (Ace-QQRFEWEFEQQ-NH2) and sodium trimetaphosphate (STMP) on the rate of calcium phosphate crystal growth, its anti-proteolytic action against the enzymatic degradation of type I collagen, the binding mechanism of P11-4 and STMP to collagen fibrils, and the potential mechanism to induce collagen stabilization. It was also evaluated the cell viability and the gene expression of different major genes involved in mineralization in odontoblast-like cells exposed to different concentrations of P11-4 and STMP. Methods: P11-4 interaction with calcium was analyzed by intrinsic fluorescence analysis. Crystalline particles formation was monitored by light-scattering detectors to estimate pH variation and radial size of the crystalline particles as a function of reaction time (pH 7.4, 25 ºC) in the presence of P11-4 or STMP in supersaturated calcium phosphate solution (Ca/P=1.67). Images were obtained under the atomic force microscopy (AFM) to measure the particle size in the presence of P11-4 or STMP and to measure the width of collagen fibrils in the presence of P11-4. The interaction of P11-4 and collagen I was analyzed by surface plasmon resonance (SPR). The binding mechanism of P11-4 and STMP to collagen fibrils were assessed with intrinsic fluorescence analysis and circular dichroism spectrometry (CD) respectively. Immortalized rat odontoblast cells (MDPC-23) were cultured. Cell viability was assessed by Trypan Blue staining and MTT assay. The changes in gene expression balance induced by P11-4 and STMP were assessed by qRT-PCR assays. Three-point bending test was used to obtain the elastic modulus of fully demineralized dentin beams before and after immersion in STMP solutions. We also performed assays of bond strength to microtensile, nanoinfiltration and in situ zymography to assess the effects of P11-4 on stabilizing the structure of dentine carie-affected. Data was statistically analyzed (α=0.05). Results: Intrinsic fluorescence analysis, dynamical light scattering with pH monitoring and AFM showed P11-4 induces hydroxyapatite (HAP) crystal nucleation process improving the structural match among HAP crystallite aggregates. STMP greatly increased the rate of crystal growth, significantly increasing the average radial crystal size. AFM corroborated the significant increase of STPM treated crystal size. SPR and AFM indicated that P11-4 binds to collagen type I fibers, increasing their width from 214 ± 4 nm to 308 ± 5 nm (P < 0.0001). The CD and intrinsic xvii fluorescence analysis showed the peptide sequence Ace-KGHRGFSGL-NH2 is the binding site of P11-4 and STMP in type I collagen. CD analysis demonstrated changes in the conformational stability after STMP binding to type I collagen. Mineralized collagen I fibrils exhibited less collagenase degradation with lower STMP concentration. P11-4 also increased the resistance of collagen type I fibers against the proteolytic activity of collagenases. We verified from the analysis of the elastic module for the treated substrate STMP and the analysis of nanoinfiltration, bond strength to microtensile and in situ zymography of the caries-affected dentin treated with P11-4 that these biomaterials stabilize the extracellular dentinal matrix. P11-4 and STMP had no significance influence in the cell viability of MDPC-23 cells and only P11-4 had significant influence in gene expression of these cells. Conclusion: The present study observed that P11-4 and STMP induces the nucleation of hydroxyapatite, interacts with collagen type I and increased resistance of collagen against the proteolytic activity of collagenases, improving the mechanical properties of the extracellular dentin matrix.
- 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.