In vivo non-specific binding of lambda CI and Cro repressors is significant

We propose a thermodynamic model that includes the non-specific binding of the lambda phage regulatory proteins CI and Cro. By fitting the model to experimental in vivo data on activities of the two promoters P(RM) and P(R) versus concentration, we estimate the free energy upon non-specific binding to be -4.1+/-0.9 kcal/mol for CI and -4.2+/-0.8 kcal/mol for Cro. For concentrations >100 nM of CI or Cro, we find that >50% of these proteins are non-specifically bound. In particular, in a lysogen (approximately 250 CI monomeric equivalents per cell) nearly 90% of CI is non-specifically bound.     

Degradation of chitosans with chitinase B from Serratia marcescens. Production of chito-oligosaccharides and insight into enzyme processivity

Family 18 chitinases such as chitinase B (ChiB) from Serratia marcescens catalyze glycoside hydrolysis via a mechanism involving the N-acetyl group of the sugar bound to the -1 subsite. We have studied the degradation of the soluble heteropolymer chitosan, to obtain further insight into catalysis in ChiB and to experimentally assess the proposed processive action of this enzyme. Degradation of chitosans with varying degrees of acetylation was monitored by following the size-distribution of oligomers, and oligomers were isolated and partly sequenced using (1)H-NMR spectroscopy. Degradation of a chitosan with 65% acetylated units showed that ChiB is an exo-enzyme which degrades the polymer chains from their nonreducing ends. The degradation showed biphasic kinetics: the faster phase is dominated by cleavage on the reducing side of two acetylated units (occupying subsites -2 and -1), while the slower kinetic phase reflects cleavage on the reducing side of a deacetylated and an acetylated unit (bound to subsites -2 and -1, respectively). The enzyme did not show preferences with respect to acetylation of the sugar bound in the +1 subsite. Thus, the preference for an acetylated unit is absolute in the -1 subsite, whereas substrate specificity is less stringent in the -2 and +1 subsites. Consequently, even chitosans with low degrees of acetylation could be degraded by ChiB, permitting the production of mixtures of oligosaccharides with different size distributions and chemical composition. Initially, the degradation of the 65% acetylated chitosan almost exclusively yielded oligomers with even-numbered chain lengths. This provides experimental evidence for a processive mode of action, moving the sugar chain two residues at a time. The results show that nonproductive binding events are not necessarily followed by substrate release but rather by consecutive relocations of the sugar chain.     

Development and application of a model for chitosan hydrolysis by a family 18 chitinase

Hydrolysis of partially deacetylated chitosans by ChitinaseB (ChiBeta) from Serratia marcescens results in mixtures of oligosaccharides typically between 2 and 20 sugar residues. The amounts of different oligomer fractions depend on the degree of acetylation of the starting chitosans. We have used experimentally determined distributions of hydrolysis products to develop a model for chitosan hydrolysis by ChiB. Important elements of the model include interaction parameters for acetylated/deacetylated units in each of the six subsites in the active cleft and degree of processivity (multiple attack). The hydrolysis reaction is described as a chemical reaction with an activation barrier that depends on the substrate sequence presented to the enzyme subsites. Using a Monte Carlo approach, the interaction parameters were refined by minimizing the difference between observed and predicted amounts of hydrolysis products obtained upon degradation of chitosan with a degree of acetylation of 65%. The final model can accurately predict complex patterns of oligosaccharides produced in the hydrolysis of chitosans with various degrees of acetylation, as well as patterns observed during reactions with chito-oligosaccharides. The behavior of a ChiB mutant with a mutation in subsite +2 (Gly188Asp), which reduces the affinity for an acetylated sugar, could be predicted correctly by introducing one single change in the model parameters (the interaction energy for an acetylated unit in the +2 subsite). The proposed model may be used to explore degradation products for different enzyme-substrates combinations and to optimize conditions for preparation of specific oligosaccharides. In addition, the model provides insight into subsite interaction parameters and the degree of processivity, which complements previous experimental studies on the mode of action of ChiB.     

A second-generation genetic linkage map of tilapia (Oreochromis spp.)

We constructed a second-generation linkage map of tilapia from the F(2) progeny of an interspecific cross between Oreochromis niloticus and Oreochromis aureus. The map reported here contains 525 microsatellite and 21 gene-based markers. It spans 1311 cM in 24 linkage groups, for an average marker spacing of 2.4 cM. We detected associations of sex and red color with markers on linkage group 3. This map will enable mapping and selective breeding of quantitative traits important to the economic culture of tilapia as a food fish and will contribute to the study of closely related cichlids that have undergone explosive adaptive radiation in the lakes of East Africa.     

Efficacy of orally administered flumequine in the treatment of vibriosis caused by Listonella anguillarum in atlantic cod Gadus morhua

The efficacy of orally administered flumequine in the treatment of experimentally induced vibriosis in Atlantic cod Gadus morhua was investigated. Cod (mean +/- SD, 120 +/- 30 g) were randomly distributed to twelve tanks and bath challenged for 1 h with Listonella anguillarum serotype O2alpha, strain HI-610, using a dose of 9.2 x 10(6) CFU ml(-1). At 3 d post-challenge, medication was introduced in 10 of the groups at doses of 2.5, 5, 10, 15 and 25 mg flumequine kg(-1) body weight d(-1) in duplicate. The medication was administered on Days 1, 2, 4, 6, 8, and 10 after the initiation of treatment. In challenged unmedicated fish, mortality started on Day 4 post-challenge, reaching a final cumulative mortality of 82% at Day 18. In the medicated groups, mortality started on Days 3 to 5 post-challenge, reaching final cumulative mortalities of 42, 49, 37, 37 and 23% respectively for the fish treated with 2.5, 5, 10, 15 and 25 mg flumequine kg(-1) body weight d(-1). Survival of medicated fish in all groups was significantly greater than in challenged unmedicated fish (p < 0.001). Twenty-four h following the final medication, HPLC analysis found a linear relationship between doses and mean concentrations of the drug in plasma, muscle and liver.     

Exogenous lysophospholipids with large head groups perturb clathrin‐mediated endocytosis

In this study, we have investigated how clathrin‐dependent endocytosis is affected by exogenously added lysophospholipids (LPLs). Addition of LPLs with large head groups strongly inhibits transferrin (Tf) endocytosis in various cell lines, while LPLs with small head groups do not. Electron and total internal reflection fluorescence microscopy (EM and TIRF) reveal that treatment with lysophosphatidylinositol (LPI) with the fatty acyl group C18:0 leads to reduced numbers of invaginated clathrin‐coated pits (CCPs) at the plasma membrane, fewer endocytic events per membrane area and increased lifetime of CCPs. Also, endocytosis of Tf becomes dependent on actin upon LPI treatment. Thus, our results demonstrate that one can regulate the kinetics and properties of clathrin‐dependent endocytosis by addition of LPLs in a head group size‐ and fatty acyl‐dependent manner. Furthermore, studies performed with optical tweezers show that less force is required to pull membrane tubules outwards from the plasma membrane when LPI is added to the cells. The results are in agreement with the notion that insertion of LPLs with large head groups creates a positive membrane curvature which might have a negative impact on events that require plasma membrane invagination, while it may facilitate membrane bending toward the cell exterior.      

Genetic versus Rearing-Environment Effects on Phenotype: Hatchery and Natural Rearing Effects on Hatchery- and Wild-Born Coho Salmon

With the current trends in climate and fisheries, well-designed mitigative strategies for conserving fish stocks may become increasingly necessary. The poor post-release survival of hatchery-reared Pacific salmon indicates that salmon enhancement programs require assessment. The objective of this study was to determine the relative roles that genotype and rearing environment play in the phenotypic expression of young salmon, including their survival, growth, physiology, swimming endurance, predator avoidance and migratory behaviour. Wild- and hatchery-born coho salmon adults (Oncorhynchus kisutch) returning to the Chehalis River in British Columbia, Canada, were crossed to create pure hatchery, pure wild, and hybrid offspring. A proportion of the progeny from each cross was reared in a traditional hatchery environment, whereas the remaining fry were reared naturally in a contained side channel. The resulting phenotypic differences between replicates, between rearing environments, and between cross types were compared. While there were few phenotypic differences noted between genetic groups reared in the same habitat, rearing environment played a significant role in smolt size, survival, swimming endurance, predator avoidance and migratory behaviour. The lack of any observed genetic differences between wild- and hatchery-born salmon may be due to the long-term mixing of these genotypes from hatchery introgression into wild populations, or conversely, due to strong selection in nature—capable of maintaining highly fit genotypes whether or not fish have experienced part of their life history under cultured conditions.     

Effects of hydrographic variability on the spatial, seasonal and diel diving patterns of southern elephant seals in the eastern Weddell Sea

Weddell Sea hydrography and circulation is driven by influx of Circumpolar Deep Water (CDW) from the Antarctic Circumpolar Current (ACC) at its eastern margin. Entrainment and upwelling of this high-nutrient, oxygen-depleted water mass within the Weddell Gyre also supports the mesopelagic ecosystem within the gyre and the rich benthic community along the Antarctic shelf. We used Conductivity-Temperature-Depth Satellite Relay Data Loggers (CTD-SRDLs) to examine the importance of hydrographic variability, ice cover and season on the movements and diving behavior of southern elephant seals in the eastern Weddell Sea region during their overwinter feeding trips from Bouvetøya. We developed a model describing diving depth as a function of local time of day to account for diel variation in diving behavior. Seals feeding in pelagic ice-free waters during the summer months displayed clear diel variation, with daytime dives reaching 500-1500 m and night-time targeting of the subsurface temperature and salinity maxima characteristic of CDW around 150–300 meters. This pattern was especially clear in the Weddell Cold and Warm Regimes within the gyre, occurred in the ACC, but was absent at the Dronning Maud Land shelf region where seals fed benthically. Diel variation was almost absent in pelagic feeding areas covered by winter sea ice, where seals targeted deep layers around 500–700 meters. Thus, elephant seals appear to switch between feeding strategies when moving between oceanic regimes or in response to seasonal environmental conditions. While they are on the shelf, they exploit the locally-rich benthic ecosystem, while diel patterns in pelagic waters in summer are probably a response to strong vertical migration patterns within the copepod-based pelagic food web. Behavioral flexibility that permits such switching between different feeding strategies may have important consequences regarding the potential for southern elephant seals to adapt to variability or systematic changes in their environment resulting from climate change.