FGF2 effects in periosteal fibroblasts bearing the FGFR2 receptor Pro253 Arg mutation

AIM: A growing number of mutations mapped in the receptor gene for fibroblast growth factor have been implicated in several cranial development disorders including the Apert and Crouzon syndromes. The present paper investigated cellular mechanisms underlying Apert phenotype, by analyzing the effects of FGF2 in primary cultures of Apert periosteal fibroblasts carrying the FGFR2 Pro253Arg mutation. RESULTS: FGF2 administration significantly decreased extracellular matrix production in mutant cells by stimulating degradative enzymatic activities. Gene expression analysis revealed that decorin and biglycan, two proteoglycans involved in collagen fibrillogenesis, were more expressed in mutant cells and down-regulated by FGF2. FGF2 receptor binding showed little differences in high affinity receptor counts between mutant and wild-type cells, while we showed for the first time that low affinity receptors are significantly fewer in mutant cells. Differences were found in Crouzon syndrome, where both high and low affinity receptor counts were up-regulated. CONCLUSIONS: The different mutation and low affinity receptor regulation in mutant receptors support the hypothesis that the impact on the activity of the ligand-receptor complex could allow distinct modes of FGF2 activation in Apert and Crouzon syndromes, which interfere with the FGFR2 signalling cascade.     

Taste receptors in the gastrointestinal tract. IV. Functional implications of bitter taste receptors in gastrointestinal chemosensing

Changes in the luminal contents of the gastrointestinal tract modulate gastrointestinal functions, including absorption of nutrients, food intake, and protection against harmful substances. The current notion is that mucosal enteroendocrine cells act as primary chemoreceptors by releasing signaling molecules in response to changes in the luminal environment, which in turn activate nerve terminals. The recent discovery that taste receptors and G protein subunits alpha-gustducin and alpha-transducin, involved in gustatory signal transduction, are expressed in the gastrointestinal mucosa supports the concept of a chemosensory machinery in the gastrointestinal tract. An understanding of luminal sensing processes responsible for the generation of the appropriate functional response to specific nutrients and nonnutrients is of clinical importance since aberrant or unsteady responses to changes in luminal contents might result in disease states ranging from intoxication to feeding disorders and inflammation. The purpose of this theme article is to discuss the functional implications of bitter taste signaling molecules in the gastrointestinal tract deduced by their localization in selected populations of epithelial cells and their relationship with neural pathways responsible for the generation of specific responses to luminal contents.     

The Shigella flexneri OmpA amino acid residues 188EVQ190 are essential for the interaction with the virulence factor PhoN2

Shigella flexneri is an intracellular pathogen that deploys an arsenal of virulence factors promoting host cell invasion, intracellular multiplication and intra- and inter-cellular dissemination. We have previously reported that the interaction between apyrase (PhoN2), a periplasmic ATP-diphosphohydrolase, and the C-terminal domain of the outer membrane (OM) protein OmpA is likely required for proper IcsA exposition at the old bacterial pole and thus for full virulence expression of Shigella flexneri (Scribano et al., 2014). OmpA, that is the major OM protein of Gram-negative bacteria, is a multifaceted protein that plays many different roles both in the OM structural integrity and in the virulence of several pathogens. Here, by using yeast two-hybrid technology and by constructing an in silico 3D model of OmpA from S. flexneri 5a strain M90T, we observed that the OmpA residues ₁₈₈EVQ₁₉₀ are likely essential for PhoN2-OmpA interaction. The ₁₈₈EVQ₁₉₀ amino acids are located within a flexible region of the OmpA protein that could represent a scaffold for protein-protein interaction.     

Relative quantification of membrane proteins in wild-type and prion protein (PrP)-knockout cerebellar granule neurons

Approximately 25% of eukaryotic proteins possessing homology to at least two transmembrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass spectrometry-based comparison of membrane protein expression in cerebellar granule neurons grown in primary culture that were isolated from wild-type mice and mice lacking the cellular prion protein. This protein is a cell-surface glycoprotein that is mainly expressed in the central nervous system and is involved in several neurodegenerative disorders, though its physiological role is unclear. We used a low specificity enzyme α-chymotrypsin to digest membrane proteins preparations that had been separated by SDS-PAGE. The resulting peptides were labeled with tandem mass tags and analyzed by MS. The differentially expressed proteins identified using this approach were further analyzed by multiple reaction monitoring to confirm the expression level changes.     

Pollution Impacts on Bacterioplankton Diversity in a Tropical Urban Coastal Lagoon System

Despite a great number of published studies addressing estuarine, freshwater and marine bacterial diversity, few have examined urban coastal lagoons in tropical habitats. There is an increasing interest in monitoring opportunistic pathogens as well as indigenous microbial community members in these water bodies by current molecular and microbiological approaches. In this work, bacterial isolates were obtained through selective plate dilution methods to evaluate antibiotic resistances. In addition, 16S rRNA gene libraries were prepared from environmental waters and mixed cultures grown in BHI medium inoculated with Jacarepaguá lagoon waters. Denaturing gradient gel electrophoresis (DGGE) analyses showed distinct community profiles between environmental communities from each studied site and their cultured counterparts. A total of 497 bacterial sequences were analyzed by MOTHUR, yielding 245 operational taxonomic units (OTUs) grouped at 97% similarity. CCA diagrams showcased how several environmental variables affect the distribution of 18 bacterial orders throughout the three distinct habitats. UniFrac metrics and Venn diagrams revealed that bacterial communities retrieved through each experimental approach were significantly different and that only one OTU, closely related to Vibrio cholerae, was shared between them. Potentially pathogenic bacteria were isolated from most sampled environments, fifty percent of which showed antibiotic resistance.     

A potential role of Escherichia coli pathobionts in the pathogenesis of pediatric inflammatory bowel disease

Through genomic analysis of mucosa-associated Escherichia coli strains, we found a close genetic association among isolates from pediatric inflammatory bowel disease (IBD) patients. A specific E. coli pathovar, adherent-invasive E. coli (AIEC), was found in Crohn's disease (CD) adult patients - this pathovar has enhanced adhesive and invasive properties, mainly due to the mannose-bonding FimH protein. We aimed to characterize 52 mucosa-associated E. coli strains isolated from pediatric IBD and non-IBD patients. Eleven E. coli strains, showing a strong similarity in fimH gene sequence to that of E. coli AIEC LF82, were characterized for fimH gene sequence, genomic profiling, adhesive and invasive ability, and phylogrouping. The results were compared with E. coli strains AIEC LF82 and MG1655. The 11 E. coli isolates showed 82.4% ± 1.4% fimH sequence similarity and 80.6% ± 1.3% genomic similarity to strain AIEC LF82. All these strains harbored V27A and S78N FimH mutations, as found in LF82. Nine of them belonged to the more virulent B2 and D phylogroups. Neuraminidase treatment, mimicking inflamed mucosa, enhanced adhesion of all 11 strains by 3.5-fold, but none showed invasion ability. It could be argued that the 11 selected strains could be a branch of an E. coli subpopulation (pathobionts), that could take advantage in an inflamed context because of a suitable genomic and (or) genetic backdrop.     

Structural basis for the interaction of isoDGR with the RGD-binding site of alphavbeta3 integrin

Asparagine deamidation at the NGR sequence in the 5th type I repeat of fibronectin (FN-I5) generates isoDGR, an alphavbeta3 integrin-binding motif regulating endothelial cell adhesion and proliferation. By NMR and molecular dynamics studies, we analyzed the structure of CisoDGRC (isoDGR-2C), a cyclic beta-peptide mimicking the FN-I5 site, and compared it with NGR, RGD, or DGR-containing cyclopeptides. Docking experiments show that isoDGR, exploiting an inverted orientation as compared with RGD, favorably interacts with the RGD-binding site of alphavbeta3, both recapitulating canonical RGD-alphavbeta3 contacts and establishing additional polar interactions. Conversely, NGR and DGR motifs lack the fundamental pharmacophoric requirements for high receptor affinity. Therefore, unlike NGR and DGR, isoDGR is a new natural recognition motif of the RGD-binding pocket of alphavbeta3. These findings contribute to explain the different functional properties of FN-I5 before and after deamidation, and provide support for the hypothesis that NGR --> isoDGR transition can work as a molecular timer for activating latent integrin-binding sites in proteins, thus regulating protein function.     

Species-level identification of <Emphasis Type="Italic">Bacillus</Emphasis> strains isolates from marine sediments by conventional biochemical, 16S rRNA gene sequencing and inter-tRNA gene sequence lengths analysis

The aim of this study was to compare the ability of commonly used conventional biochemical tests, sequencing analysis of 16S rRNA genes and tDNA-intergenic spacer length polymorphism (tDNA-PCR) to identify species of the genus Bacillus recovered from marine sediments. While biochemical tests were not sufficiently sensitive to distinguish between the 23 marine strains analyzed, partial 16S rRNA gene sequences allowed a correct identification, clustering them into four species belonging to Bacillus licheniformis (n = 6), Bacillus cereus (n = 9), Bacillus subtilis (n = 7) and Bacillus pumilus (n = 1). The identification results obtained with 16S rRNA sequencing were validated by tDNA-PCR analysis of 23 marine isolates that were identified by the similarities of their fingerprints to those of reference strains. tDNA-PCR fingerprinting was as discriminatory as 16S rRNA sequencing analysis. Although it was not able to distinguish among the species of the B. cereus and B. subtilis groups, it should be considered a rapid and easy approach for the reliable identification of unknown Bacillus isolates or at least for the primary differentiation of Bacillus groups.     

Association of MT1A haplotype with cardiovascular disease and antioxidant enzyme defense in elderly Greek population: comparison with an Italian cohort

Metallothioneins (MT), the antioxidant zinc-binding proteins, seem to mediate cardioprotection. It has been postulated that zinc homeostasis and MT function may be altered, as a consequence of oxidative stress, in cardiovascular disease (CVD), with a potential implication of MT genetic polymorphisms. The present study explores the role of +647A/C and +1245A/G MT1A polymorphisms on the susceptibility to CVD, zinc status and enzyme antioxidant activity, in the Greek and Italian populations. The country selection was based on the lower zinc status and the reduced zinc dietary intake in Greece than in Italy despite the similar Mediterranean dietary pattern. A total of 464 old, healthy control subjects and 369 old CVD patients more than 70 years of age were studied. Logistic regression model indicated that +1245 MT1A G+ genotype significantly increased the risk of CVD in Greece (34.4% vs. 23.2%; odds ratio=1.88, 95% confidence interval=1.14–3.08; P=.013) but not in Italy. Haplotype analysis showed an increment of CG haplotype frequency in CVD Greek patients (17.4% vs. 10.6%, P<.05). Differential country-related frequency distribution was also recorded. Applying a multivariate regression model, +647/+1245 MT1A haplotype was associated with a modulation of enzyme antioxidant activities in both countries. Decreased plasma zinc and reduced intracellular Zn release, as well as increased enzyme antioxidant activity, were more apparent in Greek healthy donors than in Italy. In conclusion, +1245 MT1A polymorphism and +647/+1245 MT1A haplotype are implicated in CVD in Greece but not in Italy, suggesting a role of gene–diet interaction in the disease predisposition.