The parasite species complex Anisakis simplex sensu lato (Anisakis simplex sensu stricto; (A. simplex s.s.), A. pegreffii, A. simplex C) is the main cause of severe anisakiasis (allergy) worldwide and is now an important health matter. In this study, the relationship of this Anisakis species complex and their allergenic capacities is assessed by studying the differences between the two most frequent species (A. simplex s.s., A. pegreffii) and their hybrid haplotype by studying active L3 larvae parasiting Merluccius merluccius. They were compared by 2D gel electrophoresis and parallel Western blot (2DE gels were hybridized with pools of sera from Anisakis allergenic patients). Unambiguous spot differences were detected and protein assignation was made by MALDI-TOF/TOF analysis or de novo sequencing. Seventy-five gel spots were detected and the corresponding proteins were identified. Differentially expressed proteins for A. simplex s.s., A. pegreffii, and their hybrid are described and results are statistically supported. Twenty-eight different allergenic proteins are classified according to different families belonging to different biological functions. These proteins are described for the first time as antigenic and potentially new allergens in Anisakis. Comparative proteomic analyses of allergenic capacities are useful for diagnosis, epidemiological surveys, and clinical research. All MS data have been deposited in the ProteomeXchange with identifier PXD000662 (http://proteomecentral.proteomexchange.org/dataset/PXD000662).
Titania (TiO?)-based nanocomposites subjected to light excitation are remarkably effective in eliciting microbial death. However, the mechanism by which these materials induce microbial death and the effects that they have on microbes are poorly understood. Here, we assess the low dose radical-mediated TiO? photocatalytic action of such nanocomposites and evaluate the genome/proteome-wide expression profiles of Pseudomonas aeruginosa PAO1 cells after two minutes of intervention. The results indicate that the impact on the gene-wide flux distribution and metabolism is moderate in the analysed time span. Rather, the photocatalytic action triggers the decreased expression of a large array of genes/proteins specific for regulatory, signalling and growth functions in parallel with subsequent selective effects on ion homeostasis, coenzyme-independent respiration and cell wall structure. The present work provides the first solid foundation for the biocidal action of titania and may have an impact on the design of highly active photobiocidal nanomaterials.
Euryarchaea from the genus Halorhabdus have been found in hypersaline habitats worldwide, yet are represented by only two isolates: Halorhabdus utahensis?AX-2(T) from the shallow Great Salt Lake of Utah, and Halorhabdus tiamatea?SARL4B(T) from the Shaban deep-sea hypersaline anoxic lake (DHAL) in the Red Sea. We sequenced the H.?tiamatea genome to elucidate its niche adaptations. Among sequenced archaea, H.?tiamatea features the highest number of glycoside hydrolases, the majority of which were expressed in proteome experiments. Annotations and glycosidase activity measurements suggested an adaptation towards recalcitrant algal and plant-derived hemicelluloses. Glycosidase activities were higher at 2% than at 0% or 5% oxygen, supporting a preference for low-oxygen conditions. Likewise, proteomics indicated quinone-mediated electron transport at 2% oxygen, but a notable stress response at 5% oxygen. Halorhabdus tiamatea furthermore encodes proteins characteristic for thermophiles and light-dependent enzymes (e.g. bacteriorhodopsin), suggesting that H.?tiamatea evolution was mostly not governed by a cold, dark, anoxic deep-sea habitat. Using enrichment and metagenomics, we could demonstrate presence of similar glycoside hydrolase-rich Halorhabdus members in the Mediterranean DHAL Medee, which supports that Halorhabdus species can occupy a distinct niche as polysaccharide degraders in hypersaline environments.
hCLE/C14orf166 is a nuclear and cytoplasmic protein that interacts with the RNAP II, modulates nuclear RNA metabolism and is present in cytoplasmic RNA granules involved in localized translation. Here we have studied whether hCLE shares common interactors in the nucleus and the cytosol, which could shed light on its participation in the sequential phases of RNA metabolism. Nuclear and cytoplasmic purified hCLE-associated factors were identified and proteins involved in mRNA metabolism, motor-related proteins, cytoskeletal and translation-related factors were found. Purified hCLE complexes also contain RNAs and as expected some hCLE-interacting proteins (DDX1, HSPC117, FAM98B) were found both in the nucleus and the cytoplasm. Moreover, endogenous hCLE fractionates in protein complexes together with DDX1, HSPC117 and FAM98B and silencing of hCLE down-regulates their nuclear and cytosolic accumulation levels. Using a photoactivatable hCLE-GFP protein, nuclear import and export of hCLE was observed indicating that hCLE is a shuttling protein. Interestingly, hCLE nuclear import required active transcription, as did the import of DDX1, HSPC117 and FAM98B proteins. The data indicate that hCLE probably as a complex with DDX1, HSPC117 and FAM98B shuttles between the nucleus and the cytoplasm transporting RNAs suggesting that this complex has a prominent role on nuclear and cytoplasmic RNA fate.
Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis--the paradigm of mesophilic hydrocarbonoclastic bacteria--O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.
O-GlcNAcylation is a dynamic protein modification which has been studied mainly in metazoans. We reported previously that an Arabidopsis thaliana O-GlcNAc transferase modifies at least two threonine residues of the Plum pox virus (PPV) capsid protein (CP). Now, six additional residues were shown to be involved in O-GlcNAc modification of PPV CP. CP O-GlcNAcylation was abolished in the PPV CP7-T/A mutant, in which seven threonines were mutated. PPV CP7-T/A infected Nicotiana clevelandii, Nicotiana benthamiana, and Prunus persica without noticeable defects. However, defects in infection of A. thaliana were readily apparent. In mixed infections of wild-type arabidopsis, the CP7-T/A mutant was outcompeted by wild-type virus. These results indicate that CP O-GlcNAcylation has a major role in the infection process. O-GlcNAc modification may have a role in virion assembly and/or stability as the CP of PPV CP7-T/A was more sensitive to protease digestion than that of the wild-type virus.
The functional characterization of Open Reading Frames (ORFs) from sequenced genomes remains a bottleneck in our effort to understand microbial biology. In particular, the functional characterization of proteins with only remote sequence homology to known proteins can be challenging, as there may be few clues to guide initial experiments. Affinity enrichment of proteins from cell lysates, and a global perspective of protein function as provided by COMBREX, affords an approach to this problem. We present here the biochemical analysis of six proteins from Helicobacter pylori ATCC 26695, a focus organism in COMBREX. Initial hypotheses were based upon affinity capture of proteins from total cellular lysate using derivatized nano-particles, and subsequent identification by mass spectrometry. Candidate genes encoding these proteins were cloned and expressed in Escherichia coli, and the recombinant proteins were purified and characterized biochemically and their biochemical parameters compared with the native ones. These proteins include a guanosine triphosphate (GTP) cyclohydrolase (HP0959), an ATPase (HP1079), an adenosine deaminase (HP0267), a phosphodiesterase (HP1042), an aminopeptidase (HP1037), and new substrates were characterized for a peptidoglycan deacetylase (HP0310). Generally, characterized enzymes were active at acidic to neutral pH (4.0-7.5) with temperature optima ranging from 35 to 55°C, although some exhibited outstanding characteristics.
Mesophilic Crenarchaeota have recently been thought to be significant contributors to nitrogen (N) and carbon (C) cycling. In this study, we examined the vertical distribution of ammonia-oxidizing Crenarchaeota at offshore site in Southern Tyrrhenian Sea. The median value of the crenachaeal cell to amoA gene ratio was close to one suggesting that virtually all deep-sea Crenarchaeota possess the capacity to oxidize ammonia. Crenarchaea-specific genes, nirK and ureC, for nitrite reductase and urease were identified and their affiliation demonstrated the presence of deep-sea clades distinct from shallow representatives. Measured deep-sea dark CO(2) fixation estimates were comparable to the median value of photosynthetic biomass production calculated for this area of Tyrrhenian Sea, pointing to the significance of this process in the C cycle of aphotic marine ecosystems. To elucidate the pivotal organisms in this process, we targeted known marine crenarchaeal autotrophy-related genes, coding for acetyl-CoA carboxylase (accA) and 4-hydroxybutyryl-CoA dehydratase (4-hbd). As in case of nirK and ureC, these genes are grouped with deep-sea sequences being distantly related to those retrieved from the epipelagic zone. To pair the molecular data with specific functional attributes we performed [(14)C]HCO(3) incorporation experiments followed by analyses of radiolabeled proteins using shotgun proteomics approach. More than 100 oligopeptides were attributed to 40 marine crenarchaeal-specific proteins that are involved in 10 different metabolic processes, including autotrophy. Obtained results provided a clear proof of chemolithoautotrophic physiology of bathypelagic crenarchaeota and indicated that this numerically predominant group of microorganisms facilitate a hitherto unrecognized sink for inorganic C of a global importance.
Mitogen-activated protein kinases (MAPKs) are a superfamily of cytoplasmic serine/threonine kinases that transduce many types of extracellular stimuli into cellular responses. p38MAPK is a member of this family with its active form in a diphosphorylated state (p38MAPKdiP). Two strong anti-p38MAPKdiP immunoreactive bands (apparent molecular weight 38 and 34 kDa) were detected by Western blotting in cultured astrocytes. Using a specific antibody and employing immunoprecipitation procedures and SELDI-TOF analysis, the 34 kDa band was found to correspond to Mxi2, a splice variant of p38MAPK; cultured astrocytes therefore express Mxi2. Separate protein extractions of different subcellular fractions, and fluorescent immunovisualisation employing confocal microscopy, showed Mxi2 to have a non-nuclear, cytosolic distribution in the studied cells. ERK1/2, protein whose intracellular distribution is influenced by Mxi2, showed the same cytoplasmic pattern than Mxi2.
The aim of this work was to establish protein profiles in serum and nasal epithelial cells of cystic fibrosis individuals in comparison with controls, asthma and chronic obstructive pulmonary disease patients for specific biomarker signatures identification.
Aquaporin-4 (AQP4) is a water channel protein mainly located in the astroglial plasma membrane, the precise function of which in the brain edema that accompanies hepatic encephalopathy (HE) is unclear. Since ammonia is the main pathogenic agent in HE, its effect on AQP4 expression and distribution in confluent primary astroglial cultures was examined via their exposure to ammonium chloride (1, 3 and 5 mM) for 5 and 10 days. Ammonia induced the general inhibition of AQP4 mRNA synthesis except in the 1 mM/5 day treatment. However, the AQP4 protein content measured was dependent on the method of analysis; an apparent increase was recorded in treated cells in in-cell Western assays, while an apparent reduction was seen with the classic Western blot method, perhaps due to differences in AQP4 aggregation. Ammonia might therefore induce the formation of insoluble AQP4 aggregates in the astroglial plasma membrane. The finding of AQP4 in the pellet of classic Western blot samples, plus data obtained via confocal microscopy, atomic force microscopy (using immunolabeled cells with gold nanoparticles) and scanning electron microscopy, all corroborate this hypothesis. The effect of ammonia on AQP4 seems not to be due to any osmotic effect; identical osmotic stress induced by glutamine and salt had no significant effect on the AQP4 content. AQP4 functional analysis (subjecting astrocytes to a hypo-osmotic medium and using flow cytometry to measure cell size) demonstrated a smaller water influx in ammonia-treated astrocytes suggesting that AQP4 aggregates are representative of an inactive status; however, more confirmatory studies are required to fully understand the functional status of AQP4 aggregates. The present results suggest that ammonia affects AQP4 expression and distribution, and that astrocytes change their expression of AQP4 mRNA as well as the aggregation status of the ensuing protein depending on the ammonia concentration and duration of exposure.
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