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991.
Giampiero Pietrocola Joan A. Geoghegan Simonetta Rindi Antonella Di Poto Antonino Missineo Valerio Consalvi Timothy J. Foster Pietro Speziale 《PloS one》2013,8(6)
Staphylococcus pseudintermedius, a commensal and pathogen of dogs and occasionally of humans, expresses surface proteins potentially involved in host colonization and pathogenesis. Here, we describe the cloning and characterization of SpsD, a surface protein of S. pseudintermedius reported as interacting with extracellular matrix proteins and corneocytes. A ligand screen and Western immunoblotting revealed that the N-terminal A domain of SpsD bound fibrinogen, fibronectin, elastin and cytokeratin 10. SpsD also interfered with thrombin-induced fibrinogen coagulation and blocked ADP-induced platelet aggregation. The binding site for SpsD was mapped to residues 395–411 in the fibrinogen γ-chain, while binding sites in fibronectin were localized to the N- and C-terminal regions. SpsD also bound to glycine- and serine-rich omega loops within the C-terminal tail region of cytokeratin 10. Ligand binding studies using SpsD variants lacking the C-terminal segment or containing an amino-acid substitution in the putative ligand binding site provided insights into interaction mechanism of SpsD with the different ligands. Together these data demonstrate the multi-ligand binding properties of SpsD and illustrate some interesting differences in the variety of ligands bound by SpsD and related proteins from S. aureus. 相似文献
992.
Antonella Capozzi Olimpia Vincentini Pietro Gizzi Alessandra Porzia Agostina Longo Cristina Felli Vincenzo Mattei Fabrizio Mainiero Marco Silano Maurizio Sorice Roberta Misasi 《PloS one》2013,8(6)
Celiac Disease (CD) is a chronic inflammatory enteropathy, triggered in genetically susceptible individuals by dietary gluten. Gluten is able to elicit proliferation of specific T cells and secretion of inflammatory cytokines in the small intestine. In this study we investigated the possibility that p10-mer, a decapeptide from durum wheat (QQPQDAVQPF), which was previously shown to prevent the activation of celiac peripheral lymphocytes, may exert an inhibitory effect on peptic-tryptic digested gliadin (PT-Gly)-stimulated intestinal carcinoma CACO-2 cells. In these cells, incubated with PT-Gly or p31-43 α-gliadin derived peptide in the presence or in the absence of p10-mer, IRAK1 activation and NF-kB, ERK1/2 and p38 MAPK phosphorylation were measured by immunoblotting, Cyclooxigenase 2 (COX-2) activity by PGE-2 release assay, and production of cytokines in the cell supernatants by ELISA. Our results showed that pre-treatment of CACO-2 cells with p10-mer significantly inhibited IRAK1 activation and NF-kB, ERK1/2 and p38 MAPK phosphorylation, as well as COX-2 activity (i.e. PGE-2 release) and production of the IL-6 and IL-8 pro-inflammatory cytokines, induced by gliadin peptides. These findings demonstrate the inhibitory effect of the p10-mer peptide on inflammatory response in CACO-2 cells. The results of the present study show that this p10-mer peptide can modulate "in vitro" the inflammatory response induced by gliadin peptides, allowing to move towards new therapeutic strategies. Turning off the inflammatory response, may in fact represent a key target in the immunotherapy of celiac disease. 相似文献
993.
Mammalian genomes are replicated under a flexible program, with random use of origins and variable fork rates, and many details of the process must be still unraveled. Molecular combing provides a set of direct data regarding the replication profile of eukaryotic cells: fork rates; organization of the replication clusters; proportion of unidirectional forks; and fork dynamics. In this study the replication profiles of different primary and immortalized non-cancer human cells (lymphocytes, lymphoblastoid cells, fibroblasts) were evaluated at the whole-genome level or within reference genomic regions harboring coding genes. It emerged that these different cell types are characterized by specific replication profiles. In primary fibroblasts, a remarkable fraction of the mammalian genome was found to be replicated by unidirectional forks, and interestingly, the proportion of unidirectional forks further increased in the replicating genome along the population divisions. A second difference concerned in the proportion of paused replication forks, again more frequent in primary fibroblasts than in PBL/lymphoblastoid cells. We concluded that these patterns, whose relevance could escape when genomic methods are applied, represent normal replication features. In single-locus analyses, unidirectional and paused replication forks were highly represented in all genomic regions considered with respect to the average estimates referring to the whole-genome. In addition, fork rates were significantly lower than whole-genome estimates. Instead, when considering the specificities of each genomic region investigated (early to late replication, normal or fragile site) no further differentiating features of replication profiles were detected. These data, representing the integration of genome-wide and single-locus analyses, highlight a large heterogeneity of replication profiles among cell types and within the genome, which should be considered for the correct use of replication datasets. 相似文献
994.
Filipe Madeira Antonella Di Lascio Pasquale Carlino Maria Letizia Costantini Xavier Pons 《Entomologia Experimentalis et Applicata》2013,148(3):287-296
Orius majusculus Reuter (Hemiptera: Anthocoridae) is an important component of the pest predatory complex in arable crops in Mediterranean areas. It moves between crops searching for prey, and improving knowledge on its dispersal abilities will help to develop conservation biological control strategies. Stable isotope ratios may be used as a tool for tracking insect movements, as the isotopic composition of insect tissues changes to reflect that of their diet when they undergo dietary shifts on moving between isotopically distinct crops. We carried out laboratory diet switch experiments with a stable isotope approach to infer information on dispersal of O. majusculus individuals among C3 and C4 crops to better understand isotopic field data collections. Switching the aphid food source caused a quick change in δ13C signatures, regardless of the original and final food source. Changes in the δ13C ratio of O. majusculus after diet switching fitted with an exponential model that showed similar turnover rates, and thus half‐lives, between shifting diets up to 20 days. Subsequently, whereas individuals feeding on C4 aphids did not survive, turnover rate decreased in individuals that switched from C4 to C3 aphids. However, δ13C traces from the original source remained in the predator until 25 days after switching, and this is enough time to help determine the movement of O. majusculus between crops in the field and to plan the timing of predator sampling and crop practices that may enhance predator ecological services. Orius majusculus that switched to a maize aphid diet showed different turnover rates between sexes, although this did not influence the pattern of switchover. 相似文献
995.
996.
Valentina Bosello-Travain Marcus Conrad Giorgio Cozza Alessandro Negro Silvia Quartesan Monica Rossetto Antonella Roveri Stefano Toppo Fulvio Ursini Mattia Zaccarin Matilde Maiorino 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.Methods
Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.Results
Oxidation of the CP is fast (k+ 1 > 103 M− 1 s− 1), however the rate of reduction by GSH is slow (k′+ 2 = 12.6 M− 1 s− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized CP can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k+ 1 > 103 M− 1 s− 1), but not by Trx. By surface plasmon resonance analysis, a KD = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.Conclusions
GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.General significance
In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH. 相似文献997.
Antonella Sgarbossa Susanna Monti Francesco Lenci Emilia Bramanti Ranieri Bizzarri Vincenzo Barone 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Current research has indicated that small natural compounds could interfere with β-amyloid fibril growth and have the ability to disassemble preformed folded structures. Ferulic acid (FA), which possesses both hydrophilic and hydrophobic moieties and binds to peptides/proteins, is a potential candidate against amyloidogenesis. The molecular mechanisms connected to this action have not been elucidated in detail yet.Methods
Here the effects of FA on preformed fibrils are investigated by means of a concerted experimental–computational approach. Spectroscopic techniques, such as FTIR, fluorescence, size exclusion chromatography and confocal microscopy in combination with molecular dynamics simulations are used to identify those features which play a key role in the destabilization of the aggregates.Results
Experimental findings highlight that FA has disruptive effects on the fibrils. The computational analysis suggests that dissociation of peptides from the amyloid superstructures could take place along the fibril axis and be primarily determined by the cooperative rupture of the backbone hydrogen bonds and of the Asp-Lys salt bridges.Conclusion
FA clusters could induce a sort of stabilization and tightening of the fibril structure in the short term and its disruption in the long term, inhibiting further fibril re-assembly through FA screening effects.General significance
The combination of experimental and computational techniques could be successfully used to identify the disrupting action of FA on preformed Aβ fibrils in water solution. 相似文献998.
999.
Francesco Pini Benjamin Frage Lorenzo Ferri Nicole J. De Nisco Saswat S. Mohapatra Lucilla Taddei Antonella Fioravanti Frederique Dewitte Marco Galardini Matteo Brilli Vincent Villeret Marco Bazzicalupo Alessio Mengoni Graham C. Walker Anke Becker Emanuele G. Biondi 《Molecular microbiology》2013,90(1):54-71
Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen‐fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis. 相似文献
1000.
Filippo Pullara Jennifer Guerrero-Santoro Monica Calero Qiangmin Zhang Ye Peng Henrik Spåhr Guy L. Kornberg Antonella Cusimano Hilary P. Stevenson Hugo Santamaria-Suarez Shelley L. Reynolds Ian S. Brown Satdarshan P.S. Monga Bennett Van Houten Vesna Rapić-Otrin Guillermo Calero Arthur S. Levine 《Protein expression and purification》2013,87(2):111-119
Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, more soluble, non-human proteins and limits the number of potential “druggable” targets. In this study we present a highly reproducible protocol that introduces the systematic use of an extensive number of detergents to solubilize aggregated proteins expressed in bacterial and eukaryotic systems. We validate the usefulness of this protocol by solubilizing traditionally difficult human protein targets to milligram quantities and confirm their biological activity. We use this method to solubilize monomeric or multimeric components of multi-protein complexes and demonstrate its efficacy to reconstitute large cellular machines. This protocol works equally well on cytosolic, nuclear and membrane proteins and can be easily adapted to a high throughput format. 相似文献