Giardia duodenalis: protein substrates degradation by trophozoite proteases

The present investigation was undertaken to identify and characterize trophozoite proteases of five axenic strains of Giardia duodenalis isolated in Brazil and the reference strain Portland 1 isolated in the United States. Trophozoite cell lysates of each strain were analysed for the pattern of proteins and for proteolytic activity. Samples were tested in SDS-polyacrylamide gel electrophoresis for the protein profiles, and the detection of proteases in cell lysates was performed using substrate gel electrophoresis [gelatin, collagen, bovine serum albumin (BSA) and haemoglobin] and azocasein assays. Indeed, synthetic inhibitors were included in the assays to characterize the protease classes. Differences on the hydrolysis patterns of protein substrates were observed in relation to the substrate composition as much as the Giardia trophozoite strain. The substrate-containing gels revealed hydrolysis bands with molecular masses ranging from > 97 to 20-15 kDa, and most zones were common to the five strains. However, some pronounced differences could be detected in the BTU-11 pattern. Azocasein was also degraded; however, depending on the lysate assayed, the degree of substrate degradation was variable. It was observed that inhibitory effects are substrate-dependent since the activity was predominantly due to cysteine proteases against gelatin, collagen, BSA and azocasein substrates and due to serine against haemoglobin. The presence of aspartic protease and aminopeptidase activity in the lysates was also indicated.

Performance and carcass characteristics of Nellore young bulls fed different sources of oils, protected or not from rumen degradation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of different lipid levels on protozoa population, microbial protein synthesis and rumen degradability in cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Role of ROC1 protein in the control of cyclin D1 protein expression in skin melanomas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Optimization of incubation time of protein Tc85 in the construction of biosensor: Is the EIS a good tool?

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of phosphoric acid on the degradation of human dentin matrix

This study determined if dentin proteases are denatured by phosphoric acid (PA) used in etch-and-rinse dentin adhesives. Dentin beams were completely demineralized with EDTA for 30 days. We acid-etched experimental groups by exposing the demineralized dentin beams to 1, 10, or 37 mass% PA for 15 sec or 15 min. Control beams were not exposed to PA but were incubated in simulated body fluid for 3 days to assay their total endogenous telopeptidase activity, by their ability to solubilize C-terminal crosslinked telopeptides ICTP and CTX from insoluble dentin collagen. Control beams released 6.1 ± 0.8 ng ICTP and 0.6 ± 0.1 ng CTX/mg dry-wt/3 days. Positive control beams pre-incubated in p-aminophenylmercuric acetate, a compound known to activate proMMPs, released about the same amount of ICTP peptides, but released significantly less CTX. Beams immersed in 1, 10, or 37 mass% PA for 15 sec or 15 min released amounts of ICTP and CTX similar to that released by the controls (p > 0.05). Beams incubated in galardin, an MMP inhibitor, or E-64, a cathepsin inhibitor, blocked most of the release of ICTP and CTX, respectively. It is concluded that PA does not denature endogenous MMP and cathepsin activities of dentin matrices. © 2013 International & American Associations for Dental Research.

Paracoccidoides brasiliensis 30 kDa Adhesin: Identification as a 14-3-3 Protein, Cloning and Subcellular Localization in Infection Models

Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis. © 2013 Silva et al.

Green tea extract and its major constituent, epigallocatechin-3-gallate, induce epithelial beta-defensin secretion and prevent beta-defensin degradation by Porphyromonas gingivalis

Background and Objective: Antimicrobial peptides, such as beta-defensins, secreted by gingival epithelial cells, are thought to play a major role in preventing periodontal diseases. In the present study, we investigated the ability of green tea polyphenols to induce human beta-defensin (hBD) secretion in gingival epithelial cells and to protect hBDs from proteolytic degradation by Porphyromonas gingivalis.Material and Methods: Gingival epithelial cells were treated with various amounts (25-200 mu g/mL) of green tea extract or epigallocatechin-3-gallate (EGCG). The secretion of hBD1 and hBD2 was measured using ELISAs, and gene expression was quantified by real-time PCR. The treatments were also carried out in the presence of specific kinase inhibitors to identify the signaling pathways involved in hBD secretion. The ability of green tea extract and EGCG to prevent hBD degradation by proteases of P. gingivalis present in a bacterial culture supernatant was evaluated by ELISA.Results: The secretion of hBD1 and hBD2 was up-regulated, in a dose-dependent manner, following the stimulation of gingival epithelial cells with a green tea extract or EGCG. Expression of the hBD gene in gingival epithelial cells treated with green tea polyphenols was also increased. EGCG-induced secretion of hBD1 and hBD2 appeared to involve extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. Lastly, green tea extract and EGCG prevented the degradation of recombinant hBD1 and hBD2 by a culture supernatant of P. gingivalis.Conclusion: Green tea extract and EGCG, through their ability to induce hBD secretion by epithelial cells and to protect hBDs from proteolytic degradation by P. gingivalis, have the potential to strengthen the epithelial antimicrobial barrier. Future clinical studies will indicate whether these polyphenols represent a valuable therapeutic agent for treating/preventing periodontal diseases.

A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation

Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development.