The dual roles of AlgG in C-5-epimerization and secretion of alginate polymers in Pseudomonas aeruginosa

Pseudomonas aeruginosa strains causing chronic pulmonary infections in cystic fibrosis patients produce high levels of alginate, an exopolysaccharide that confers a mucoid phenotype. Alginate is a linear polymer of d-mannuronate (M) and variable amounts of its C-5-epimer, l-guluronate (G). AlgG is a periplasmic C-5-epimerase that converts poly d-mannuronate to the mixed M+G sequence of alginate. To understand better the role and mechanism of AlgG activity, a mutant was constructed in the mucoid strain FRD1 with a defined non-polar deletion of algG. Instead of producing poly mannuronate, the algG deletion mutant secreted dialysable uronic acids, as does a mutant lacking the periplasmic protein AlgK. High levels of unsaturated ends and the nuclear magnetic resonance spectroscopy pattern revealed that the small, secreted uronic acids were the products of extensive polymer digestion by AlgL, a periplasmic alginate lyase co-expressed with AlgG and AlgK. Thus, AlgG is bifunctional with (i) epimerase activity and (ii) a role in protecting alginate from degradation by AlgL during transport through the periplasm. AlgK appears to share the second role. AlgG and AlgK may be part of a periplasmic protein complex, or scaffold, that guides alginate polymers to the outer membrane secretin (AlgE). To characterize the epimerase activity of AlgG further, the algG4 allele of poly mannuronate-producing FRD462 was shown to encode a protein lacking only the epimerase function. The sequence of algG4 has a Ser-272 to Asn substitution in a serine-threonine-rich and conserved region of AlgG, which revealed a critical residue for C-5-epimerase activity.     

Mutational spectrum in the cardioauditory syndrome of Jervell and Lange-Nielsen

Jervell and Lange-Nielsen syndrome (JLNS) is an autosomal recessive syndrome characterised by profound congenital sensorineural deafness and prolongation of the QT interval on the electrocardiogram, representing abnormal ventricular repolarisation. In a study of ten British and Norwegian families with JLNS, we have identified all of the mutations in the KCNQ1 gene, including two that are novel. Of the nine mutations identified in this group of 10 families, five are nonsense or frameshift mutations. Truncation of the protein proximal to the recently identified C-terminal assembly domain is expected to preclude assembly of KCNQ1 monomers into tetramers and explains the recessive inheritance of JLNS. However, study of a frameshift mutation, with a dominant effect phenotypically, suggests the presence of another assembly domain nearer to the N-terminus.     

Molecular Modeling of the NPY Binding Site on the Y1 Receptor

A three-dimensional model of the neuropeptide Y (NPY) - rat Y₁ (rY₁) receptor complex and of the NPY _13-36 - rY₁ receptor complex was constructed by molecular modeling based on the electron density projection map of rhodopsin and on site-directed mutagenesis studies of neuropeptide receptors. In order to further guide the modeling, the nucleotide sequences encoding Trp287, Cys295 and His297 in the third extracellular loop of the rY₁ receptor, were altered by site-directed mutagenesis experiments. Single-point mutated receptors were expressed in COS-7 cells, and tested for their ability to bind radio labelled NPY (³H-NPY). Mutations of Trp287 and His297 completely abolished binding of ³H-NPY. The Cys295Ser mutation only slightly decreased the binding of ³H-NPY, suggesting that the involvement of Cys295 in a disulphide bond is not essential for maintaining the correct three-dimensional structure of the binding site for NPY. Molecular dynamics simulations of NPY-rY₁ receptor interactions suggested that Asp199, Asp103 and Asp286 in the receptor interact, respectively, with Lys4, Arg33 and Arg35 of NPY. The simulations also suggested that His297 acts as a hydrogen acceptor from Arg35 in NPY, and that Tyr1 of NPY interacts with a binding pocket on the receptor formed by Asn115, Asp286, Trp287 and His297. Tyr36 in NPY interacted both with Thr41 and Tyr99 via hydrogen bonds, and also with Asn296, His297 and Phe301. The present study suggests that amino acid residues at the extracellular end of the transmembrane helices and in the extracellular loops are strongly involved in binding to NPY and NPY_13-36.Electronic Supplementary Material available.     

Spatial dynamics of Norwegian tetraonid populations

Different species in a given site or population of a given species in different sites may fluctuate in synchrony if they are affected similarly by factors such as spatially autocorrelated climate, predation, or by dispersal between populations of one species. We used county wise time series of hunting bag records of four Norwegian tetraonid species covering 24 years to examine patterns of interspecific and intraspecific synchrony. We estimated synchrony at three spatial scales; national, regional (consisting of counties with similar climate), and county level. Ecologically related species with overlapping distributions exhibited strong synchrony across Norway, but there was much variation between the different regions and counties. Regions with a long coastline to both the North Sea and the Norwegian Ocean exhibited an overall stronger synchrony than those consisting of more continental areas. Intraspecific synchrony was generally low across all counties, but stronger synchrony between counties within regions defined by climatic conditions. Synchrony was negatively related to distance between populations in three of four species. Only the synchrony in willow ptarmigan showed a clear negative relationship with distance, while the other species had both strong positive and negative correlations at short distances. Strong interspecific synchrony between some species pairs within regions and weak intraspecific synchrony across counties within regions suggest a stronger synchronizing effect from environmental factors such as weather or predation and less effect from dispersal. Our results suggest that the complete tetraonid community is structured by environmental factors affecting the different species similarly and causes widespread interspecific synchrony. Local factors affecting the population dynamics nevertheless frequently forces neighbouring populations out of phase.     

Novel Rumen Bacterial Diversity in Two Geographically Separated Sub-Species of Reindeer

Svalbard reindeer (Rangifer tarandus platyrhynchus) live under austere nutritional conditions on the high-arctic archipelago of Svalbard, while semi-domesticated Norwegian reindeer (R. tarandus tarandus) migrate between lush coastal summer pastures and inland winter pastures with lichens on mainland Norway. Svalbard reindeer are known to have high rumen concentrations of cellulolytic bacteria, ranging from 15% of the viable population in summer to 35% in winter, compared to only 2.5% in Norwegian reindeer. Their rumen bacterial diversity was investigated through comparative analyses of 16S rRNA gene sequences (∼1.5 kb in length) generated from clone libraries (n = 121) and bacterial isolates (n = 51). LIBSHUFF comparisons of the composition of the two 16S rRNA libraries from Norwegian reindeer showed a significant effect of artificial feeding compared to natural pasture, but failed to yield significant differences between libraries from Norwegian reindeer and Svalbard reindeer. The combined sequences from reindeer were not significantly different from those reported in wild Thompson’s gazelle in Kenya but did differ from those reported in domestic cattle in Japan. A total of 90 distinct operational taxonomic units (OTUs) were identified by employing a criterion of 97% similarity, while the Chao1 index estimated the reindeer bacterial rumen population richness at 698 OTUs. The majority of the clone library sequences (92.5%) represented novel strains with <97% identity to any known sequence in the public database, most of them affiliated with the bacterial phylum Firmicutes (low G+C Gram-positives) related to the order Clostridiales (76.7%), while Gram-negative bacteria in the Bacteriodales (Prevotella–Bacteroides group) contributed to 22.5%. Also, six of the isolates were putatively novel strains, possibly representing new species in the Clostridium subphylum (cluster XIVa), Actinomyces and Butyrivibrio.     

Walking speed and area utilization of red king crab (<Emphasis Type="Italic">Paralithodes camtschaticus</Emphasis>) introduced to the Barents Sea coastal ecosystem

The red king crab (Paralithodes camtschaticus) was introduced in the Barents Sea in the 1960s and soon established a viable population. Proper management and exploitation of the Barents Sea king crab stock require better understanding of the spatial dynamics at different scales. This study examines the small-scale movement patterns of seven adult male crabs tracked for a period of up to one month from mid July to mid August at 150 m depth in a semi-enclosed fjord on the Russo-Norwegian border. The crabs were tagged with acoustic transmitters and their movements monitored with an acoustic positioning system. Low walking speeds (<0.01 m s⁻¹) were most frequent but the crabs could move at a maximum speed of 0.15 m s⁻¹ and walk an actual distance of up to 270 m over a period of one hour. However, the crabs usually moved within a relatively restricted area with mean hourly longest rectilinear distance varying from 26 to 64 m. The crabs alternated between periods of low and high activity, which could reflect feeding in and movements between food patches. The lack of a diel activity rhythm may be due to high light levels during the polar summer night, or a chemically mediated food search strategy.     

Syndecan-4 Is a Major Syndecan in Primary Human Endothelial Cells In Vitro,Modulated by Inflammatory Stimuli and Involved in Wound Healing

Syndecans are important cell surface proteoglycans with many functions; yet, they have not been studied to a very large extent in primary human endothelial cells. The purpose of this study was to investigate syndecan-4 expression in cultured human umbilical vein endothelial cells (HUVECs) and assess its role in inflammatory reactions and experimental wound healing. qRT-PCR analysis revealed that syndecan-3 and syndecan-4 were highly expressed in HUVECs, whereas the expression of syndecan-1 and -2 was low. HUVECs were cultured with the inflammatory mediators lipopolysaccharide (LPS) and interleukin 1β (IL-1β). As a result, syndecan-4 expression showed a rapid and strong increase. Syndecan-1 and -2 expressions decreased, whereas syndecan-3 was unaffected. Knockdown of syndecan-4 using siRNA resulted in changes in cellular morphology and focal adhesion sites, delayed wound healing and tube formation, and increased secretion of the pro-inflammatory and angiogenic chemokine, CXCL8. These data suggest functions for syndecan-4 in inflammatory reactions, wound healing and angiogenesis in primary human endothelial cells.     

Real-time ichthyoplankton drift in Northeast Arctic cod and Norwegian spring-spawning herring

Background

Individual-based biophysical larval models, initialized and parameterized by observations, enable numerical investigations of various factors regulating survival of young fish until they recruit into the adult population. Exponentially decreasing numbers in Northeast Arctic cod and Norwegian Spring Spawning herring early changes emphasizes the importance of early life history, when ichthyoplankton exhibit pelagic free drift. However, while most studies are concerned with past recruitment variability it is also important to establish real-time predictions of ichthyoplankton distributions due to the increasing human activity in fish habitats and the need for distribution predictions that could potentially improve field coverage of ichthyoplankton.

Methodology/Principal Findings

A system has been developed for operational simulation of ichthyoplankton distributions. We have coupled a two-day ocean forecasts from the Norwegian Meteorological Institute with an individual-based ichthyoplankton model for Northeast Arctic cod and Norwegian Spring Spawning herring producing daily updated maps of ichthyoplankton distributions. Recent years observed spawning distribution and intensity have been used as input to the model system. The system has been running in an operational mode since 2008. Surveys are expensive and distributions of early stages are therefore only covered once or twice a year. Comparison between model and observations are therefore limited in time. However, the observed and simulated distributions of juvenile fish tend to agree well during early fall. Area-overlap between modeled and observed juveniles September 1^st range from 61 to 73%, and 61 to 71% when weighted by concentrations.

Conclusions/Significance

The model system may be used to evaluate the design of ongoing surveys, to quantify the overlap with harmful substances in the ocean after accidental spills, as well as management planning of particular risky operations at sea. The modeled distributions are already utilized during research surveys to estimate coverage success of sampled biota and immediately after spills from ships at sea.     

The putative endoglucanase PcGH61D from Phanerochaete chrysosporium is a metal-dependent oxidative enzyme that cleaves cellulose

Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61), some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33), this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency.