Purification, characterization, and mode of action of a rhamnogalacturonan hydrolase from Irpex lacteus, tolerant to an acetylated substrate

A novel rhamnogalacturonase (RGase) acting on an acetylated substrate was detected in the commercial preparation Driselase, an enzymatic mixture derived from the basidiomycete Irpex lacteus. The activity was isolated by hydrophobic interaction chromatography, gel filtration, and preparative isoelectric focusing, resulting in the isolation of five different rhamnogalacturonan hydrolases exhibiting various isoelectric points from 6.2 to 7.7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry analyses after trypsin cleavage of the five fractions revealed that the five rhamnogalacturonases have a molar mass of 55 kDa without any divergences in the identified peptides. The RGase with a pI of 7.2 exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C. Its mode of action was analyzed by mass spectrometry of the oligosaccharides produced after hydrolysis of acetylated and nonacetylated rhamnogalacturonan. Oligomers esterified by an acetyl group on the reducing galacturonic acid residue or fully acetylated were detected in the hydrolysate showing that the novel enzyme is able to bind acetylated galacturonic acid in its active site.     

Resolving taxonomic uncertainties using molecular systematics: <Emphasis Type="Italic">Salmo dentex</Emphasis> and the Balkan trout community

The genetic structure of Salmo dentex and its phylogenetic relations to sympatric salmonids in the Neretva and Skadar River basins were evaluated using mitochondrial DNA (mtDNA) control region, eight microsatellites, and somatolactin (SL) gene. In the Neretva River basin of Bosnia–Herzegovina, the results based on mtDNA analysis showed extensive haplotype sharing between S. marmoratus, S. dentex, and S. trutta, and were therefore not conclusive; however, F-statistics and assignment testing based on nuclear DNA markers indicated that S. dentex of the Neretva basin were grouped in a genetically unified cluster with S. marmoratus in the Neretva basin. Using the same analytical approach, S. dentex from the Skadar basin in Montenegro appeared to be genetically distinct from S. marmoratus in the same basin and indistinct from local S. trutta. Molecular data also indicated that S. dentex of the Neretva basin in Bosnia–Herzegovina are not closely related to S. dentex of the Skadar basin in Montenegro. Based on these results, we hypothesize S. dentex to be a particular life history form of S. marmoratus in the Neretva basin and of S. trutta in the Skadar basin. These results clearly demonstrate that S. dentex does not represent a monophyletic lineage and should not be considered a distinct species.     

Contributions of edema factor and protective antigen to the induction of protective immunity by Bacillus anthracis edema toxin as an intranasal adjuvant

We have shown that intranasal coapplication of Bacillus anthracis protective Ag (PA) together with a B. anthracis edema factor (EF) mutant having reduced adenylate cyclase activity (i.e., EF-S414N) enhances anti-PA Ab responses, but also acts as a mucosal adjuvant for coadministered unrelated Ags. To elucidate the role of edema toxin (EdTx) components in its adjuvanticity, we examined how a PA mutant lacking the ability to bind EF (PA-U7) or another mutant that allows the cellular uptake of EF, but fails to efficiently mediate its translocation into the cytosol (PA-dFF), would affect EdTx-induced adaptive immunity. Native EdTx promotes costimulatory molecule expression by macrophages and B lymphocytes, and a broad spectrum of cytokine responses by cervical lymph node cells in vitro. These effects were reduced or abrogated when cells were treated with EF plus PA-dFF, or PA-U7 instead of PA. We also intranasally immunized groups of mice with a recombinant fusion protein of Yersinia pestis F1 and LcrV Ags (F1-V) together with EdTx variants consisting of wild-type or mutants PA and EF. Analysis of serum and mucosal Ab responses against F1-V or EdTx components (i.e., PA and EF) revealed no adjuvant activity in mice that received PA-U7 instead of PA. In contrast, coimmunization with PA-dFF enhanced serum Ab responses. Finally, immunization with native PA and an EF mutant lacking adenylate cyclase activity (EF-K346R) failed to enhance Ab responses. In summary, a fully functional PA and a minimum of adenylate cyclase activity are needed for EdTx to act as a mucosal adjuvant.     

Orally Co-Infected Aedes albopictus from La Reunion Island,Indian Ocean,Can Deliver Both Dengue and Chikungunya Infectious Viral Particles in Their Saliva

Background

First described in humans in 1964, reports of co-infections with dengue (DENV) and chikungunya (CHIKV) viruses are increasing, particularly after the emergence of chikungunya (CHIK) in the Indian Ocean in 2005–2006 due to a new variant highly transmitted by Aedes albopictus. In this geographic area, a dengue (DEN) outbreak transmitted by Ae. albopictus took place shortly before the emergence of CHIK and co-infections were reported in patients. A co-infection in humans can occur following the bite of two mosquitoes infected with one virus or to the bite of a mosquito infected with two viruses. Co-infections in mosquitoes have never been demonstrated in the field or in the laboratory. Thus, we question about the ability of a mosquito to deliver infectious particles of two different viruses through the female saliva.

Methodology/Principal Findings

We orally exposed Ae. albopictus from La Reunion Island with DENV-1 and CHIKV isolated respectively during the 2004–2005 and the 2005–2006 outbreaks on this same island. We were able to show that Ae. albopictus could disseminate both viruses and deliver both infectious viral particles concomitantly in its saliva. We also succeeded in inducing a secondary infection with CHIKV in mosquitoes previously inoculated with DENV-1.

Conclusions/Significance

In this study, we underline the ability of Ae. albopictus to be orally co-infected with two different arboviruses and furthermore, its capacity to deliver concomitantly infectious particles of CHIKV and DENV in saliva. This finding is of particular concern as Ae. albopictus is still expanding its geographical range in the tropical as well as in the temperate regions. Further studies are needed to try to elucidate the molecular/cellular basis of this phenomenon.     

A phyloproteomic characterization of in vitro autophosphorylation in calcium-dependent protein kinases

Calcium-dependent protein kinases (CDPKs) are a novel class of signaling molecules that have been broadly implicated in relaying specific calcium-mediated responses to biotic and abiotic stress as well as developmental cues in both plants and protists. Calcium-dependent autophosphorylation has been observed in almost all CDPKs examined, but a physiological role for autophosphorylation has not been demonstrated. To date, only a handful of autophosphorylation sites have been mapped to specific residues within CDPK amino acid sequences. In an attempt to gain further insight into this phenomenon, we have mapped autophosphorylation sites and compared these phosphorylation patterns among multiple CDPK isoforms. From eight CDPKs and two CDPK-related kinases from Arabidopsis thaliana and Plasmodium falciparum, 31 new autophosphorylation sites were characterized, which in addition to the previously described sites, allowed the identification of five conserved loci. Of the 35 total sites analyzed approximately one-half were observed in the N-terminal variable domain. Homology models were generated for the protein kinase and calmodulin-like domains, each containing two of the five conserved sites, to allow intelligent speculation regarding subsequent lines of investigation.     

Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1

Cryptococcus neoformans, a fungal pathogen of humans, causes fatal meningitis in immunocompromised patients. Its virulence is mainly determined by the elaboration of a polysaccharide capsule surrounding its cell wall. During its life, C. neoformans is confronted with and responds to dramatic variations in CO2 concentrations; one important morphological change triggered by the shift from its natural habitat (0.033% CO2) to infected hosts (5% CO2) is the induction of capsule biosynthesis. In cells, CO2 is hydrated to bicarbonate in a spontaneous reaction that is accelerated by carbonic anhydrases. Here we show that C. neoformans contains two beta-class carbonic anhydrases, Can1 and Can2. We further demonstrate that CAN2, but not CAN1, is abundantly expressed and essential for the growth of C. neoformans in its natural environment, where CO2 concentrations are limiting. Structural studies reveal that Can2 forms a homodimer in solution. Our data reveal Can2 to be the main carbonic anhydrase and suggest a physiological role for bicarbonate during C. neoformans growth. Bicarbonate directly activates the C. neoformans Cac1 adenylyl cyclase required for capsule synthesis. We show that this specific activation is optimal at physiological pH.     

Degradation and controlled release behavior of epsilon-caprolactone copolymers in biodegradable antifouling coatings

Copolymers of caprolactone with delta-valerolactone and L-lactide were synthesized by ring-opening polymerization in the presence of tetrabutoxytitane in order to decrease the crystallinity of polycaprolactone (PCL) and to enlarge its potential applications. The kinetics of degradation and controlled release of bioactive molecules were investigated in aqueous medium at room temperature for 9 months. The influence of the comonomer structures, their molar ratio, and the presence of fillers on these kinetics were examined. Complementary analytical methods were used (i) to quantify the degradation of the copolymers by titration of products of degradation (lactic acid, hydroxycaproic acid, and hydroxypentanoic acid) and (ii) to reveal the degradation processes by determination of molecular weights and thermal characteristics. After aging, films were observed by scanning electronic microscopy and EDX microanalysis to check their capabilities for the release of bioactive agent. The results showed that the incorporation of a comonomer such as L-lactide or delta-valerolactone led to a faster degradation than that of PCL homopolymer. The release of biocides could be correlated with the degradation of copolymer but depended on the structure of the leached molecule.     

Subunit-specific coupling between gamma-aminobutyric acid type A and P2X2 receptor channels

ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central synapses, where they co-activate excitatory P2X and inhibitory GABAA (GABA type A) receptors. We report here that co-activation of P2X2 and various GABAA receptors, co-expressed in Xenopus oocytes, leads to a functional cross-inhibition dependent on GABAA subunit composition. Sequential applications of GABA and ATP revealed that alphabeta- or alphabetagamma-containing GABAA receptors inhibited P2X2 channels, whereas P2X2 channels failed to inhibit gamma-containing GABAA receptors. This functional cross-talk is independent of membrane potential, changes in current direction, and calcium. Non-additive responses observed between cation-selective GABAA and P2X2 receptors further indicate the chloride independence of this process. Overexpression of minigenes encoding either the C-terminal fragment of P2X2 or the intracellular loop of the beta3 subunit disrupted the functional cross-inhibition. We previously demonstrated functional and physical cross-talk between rho1 and P2X2 receptors, which induced a retargeting of rho1 channels to surface clusters when co-expressed in hippocampal neurons (Boue-Grabot, E., Emerit, M. B., Toulme, E., Seguela, P., and Garret, M. (2004) J. Biol. Chem. 279, 6967-6975). Co-expression of P2X2 and chimeric rho1 receptors with the C-terminal sequences of alpha2, beta3, or gamma2 subunits indicated that only rho1-beta3 and P2X2 channels exhibit both functional cross-inhibition in Xenopus oocytes and co-clustering/retargeting in hippocampal neurons. Therefore, the C-terminal domain of P2X2 and the intracellular loop of beta GABAA subunits are required for the functional interaction between ATP- and GABA-gated channels. This gamma subunit-dependent cross-talk may contribute to the regulation of synaptic activity.