Gradual adaptive changes of a protein facing high salt concentrations

Several experimental techniques were applied to unravel fine molecular details of protein adaptation to high salinity. We compared four homologous enzymes, which suggested a new halo-adaptive state in the process of molecular adaptation to high-salt conditions. Together with comparative functional studies, the structure of malate dehydrogenase from the eubacterium Salinibacter ruber shows that the enzyme shares characteristics of a halo-adapted archaea-bacterial enzyme and of non-halo-adapted enzymes from other eubacterial species. The S. ruber enzyme is active at the high physiological concentrations of KCl but, unlike typical halo-adapted enzymes, remains folded and active at low salt concentrations. Structural aspects of the protein, including acidic residues at the surface, solvent-exposed hydrophobic surface, and buried hydrophobic surface, place it between the typical halo-adapted and non-halo-adapted proteins. The enzyme lacks inter-subunit ion-binding sites often seen in halo-adapted enzymes. These observations permit us to suggest an evolutionary pathway that is highlighted by subtle trade-offs to achieve an optimal compromise among solubility, stability, and catalytic activity.     

PepLine: a software pipeline for high-throughput direct mapping of tandem mass spectrometry data on genomic sequences

PepLine is a fully automated software which maps MS/MS fragmentation spectra of trypsic peptides to genomic DNA sequences. The approach is based on Peptide Sequence Tags (PSTs) obtained from partial interpretation of QTOF MS/MS spectra (first module). PSTs are then mapped on the six-frame translations of genomic sequences (second module) giving hits. Hits are then clustered to detect potential coding regions (third module). Our work aimed at optimizing the algorithms of each component to allow the whole pipeline to proceed in a fully automated manner using raw nucleic acid sequences (i.e., genomes that have not been "reduced" to a database of ORFs or putative exons sequences). The whole pipeline was tested on controlled MS/MS spectra sets from standard proteins and from Arabidopsis thaliana envelope chloroplast samples. Our results demonstrate that PepLine competed with protein database searching softwares and was fast enough to potentially tackle large data sets and/or high size genomes. We also illustrate the potential of this approach for the detection of the intron/exon structure of genes.     

Genomic islands targeting dusA in Vibrio species are distantly related to Salmonella Genomic Island 1 and mobilizable by IncC conjugative plasmids

Salmonella Genomic Island 1 (SGI1) and its variants are significant contributors to the spread of antibiotic resistance among Gammaproteobacteria. All known SGI1 variants integrate at the 3’ end of trmE, a gene coding for a tRNA modification enzyme. SGI1 variants are mobilized specifically by conjugative plasmids of the incompatibility groups A and C (IncA and IncC). Using a comparative genomics approach based on genes conserved among members of the SGI1 group, we identified diverse integrative elements distantly related to SGI1 in several species of Vibrio, Aeromonas, Salmonella, Pokkaliibacter, and Escherichia. Unlike SGI1, these elements target two alternative chromosomal loci, the 5’ end of dusA and the 3’ end of yicC. Although they share many features with SGI1, they lack antibiotic resistance genes and carry alternative integration/excision modules. Functional characterization of IMEVchUSA3, a dusA-specific integrative element, revealed promoters that respond to AcaCD, the master activator of IncC plasmid transfer genes. Quantitative PCR and mating assays confirmed that IMEVchUSA3 excises from the chromosome and is mobilized by an IncC helper plasmid from Vibrio cholerae to Escherichia coli. IMEVchUSA3 encodes the AcaC homolog SgaC that associates with AcaD to form a hybrid activator complex AcaD/SgaC essential for its excision and mobilization. We identified the dusA-specific recombination directionality factor RdfN required for the integrase-mediated excision of dusA-specific elements from the chromosome. Like xis in SGI1, rdfN is under the control of an AcaCD-responsive promoter. Although the integration of IMEVchUSA3 disrupts dusA, it provides a new promoter sequence and restores the reading frame of dusA for proper expression of the tRNA-dihydrouridine synthase A. Phylogenetic analysis of the conserved proteins encoded by SGI1-like elements targeting dusA, yicC, and trmE gives a fresh perspective on the possible origin of SGI1 and its variants.     

Identification of grapevine aquaporins and expression analysis in developing berries

Aquaporins are membrane water channels that play critical roles in controlling the water content of cells and tissues. In this work, nine full-length cDNAs encoding putative aquaporins were isolated from grape berry cDNA libraries. A phylogenetic analysis conducted with 28 aquaporin genes identified in the grapevine genome and previously characterized aquaporins from Arabidopsis indicates that three cDNAs encode putative tonoplast aquaporins (TIPs) whereas six cDNAs belong to the plasma membrane aquaporin subfamily (PIPs). Specific probes designed on the 3' untranslated regions of each cDNA were used for the preparation of cDNA macroarray filters and in situ hybridization experiments. Macroarray data indicate that expression levels of most TIP and PIP genes depend on grape berry developmental stages and point out to a global decrease of aquaporin gene expression during berry ripening. In young berries, high expression of aquaporin genes was preferentially observed in dividing and elongating cells and in cells involved in water and solutes transport. Taken together, the data provided in this paper indicate that aquaporins are implicated in various physiological aspects of grape berry development.     

The Cauchy problem for one-dimensional spiking neuron models

I consider spiking neuron models defined by a one-dimensional differential equation and a reset—i.e., neuron models of the integrate-and-fire type. I address the question of the existence and uniqueness of a solution on for a given initial condition. It turns out that the reset introduces a countable and ordered set of backward solutions for a given initial condition. I discuss the implications of these mathematical results in terms of neural coding and spike timing precision.

Roles for replichores and macrodomains in segregation of the Escherichia coli chromosome

Recent work has highlighted two main levels of global organization of the Escherichia coli chromosome. Macrodomains are large domains inferred from structural data consisting of loci showing the same intracellular positioning. Replichores, defined by base composition skews, coincide with the replication arms in normal cells. We used chromosome inversions to show that the dif site, which resolves chromosome dimers, only functions when located at the junction of the replichores, whatever their size. This is the first evidence that replichore polarization has a role in chromosome segregation. We also show that disruption of the Ter macrodomain provokes a cell-cycle defect independent from dimer resolution. This confirms the existence of the Ter macrodomain and suggests a role in chromosome dynamics.     

From hydrocolloids to high specific surface area porous supports for catalysis

Polysaccharide hydrogels are effective supports for heterogeneous catalysts. Their use in solvents different from water has been hampered by their instability upon drying. While the freeze-drying process or air-drying of hydrocolloid gels led to compact solids with a low surface area, drying the gel in CO2 beyond the critical point provided mesoporous materials with a high specific surface area. Their effectiveness as a support for catalysis was exemplified in the reaction of substitution of an allyl carbonate with morpholine catalyzed by the hydrosoluble Pd(TPPTS)3 complex. The influence of water on the catalytic activity and the properties of the support was evidenced.     

Characterization of 5-HT7-receptor-mediated gastric relaxation in conscious dogs

We aimed to evaluate the gastric relaxant capacity of the 5-HT(1/7)-receptor agonist 5-carboxamidotryptamine (5-CT) in conscious dogs and to clarify the mechanism of action by use of selective antagonists, vagotomy, and in vitro experiments. A barostat enabled us to monitor the intragastric volume in response to different treatments (intravenously administered) before and after supradiaphragmatic vagotomy [results presented as the maximum volume change after treatment (mean; n = 5-11)]. In vitro experiments were performed with isolated muscle strips cut from four different stomach regions of the vagotomized dogs [results were fitted to the operational model of agonism to determine the efficacy parameter tau (n = 5)]. 5-CT (0.5-10 microg/kg) caused a dose-dependent gastric relaxation (29-267 ml) that was completely blocked by the selective 5-HT(7)-receptor antagonist SB-269970 (50 microg/kg). After vagotomy, the relaxation to 10 microg/kg 5-CT was significantly less pronounced (73 vs. 267 ml; P < 0.05) but still blocked by SB-269970, whereas the response to the nitric oxide donor nitroprusside was similar to that before vagotomy (178 vs. 218 ml). In vitro, 5-CT concentration dependently inhibited the PGF(2alpha)-contracted muscle strips before and after vagotomy. Although before and after vagotomy the response in every region was mediated by 5-HT(7) receptors (apparent affinity dissociation constant: SB-269970, 8.2-8.6 vs. 8.3-8.6, respectively), the response after vagotomy was less efficacious (log tau: 1.9 to 0.5 vs. 1.4 to -0.1). The results indicate that the 5-CT-induced proximal stomach relaxation in conscious dogs before and after vagotomy is mediated via 5-HT(7) receptors. The decreased efficacy of 5-CT in vitro after vagotomy is probably related to vagotomy-induced changes in receptor density or coupling efficiency and provides a possible explanation for the decreased in vivo response to 5-CT after vagotomy.