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.     

Automated Water Analyser Computer Supported System (AWACSS) Part II: Intelligent, remote-controlled, cost-effective, on-line, water-monitoring measurement system

A novel analytical system AWACSS (Automated Water Analyser Computer Supported System) based on immunochemical technology has been evaluated that can measure several organic pollutants at low nanogram per litre level in a single few-minutes analysis without any prior sample pre-concentration or pre-treatment steps. Having in mind actual needs of water-sector managers related to the implementation of the Drinking Water Directive (DWD) [98/83/EC, 1998. Council Directive (98/83/EC) of 3 November 1998 relating to the quality of water intended for human consumption. Off. J. Eur. Commun. L330, 32-54] and Water Framework Directive (WFD) [2000/60/EC, 2000. Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy. Off. J. Eur. Commun. L327, 1-72], drinking, ground, surface, and waste waters were major media used for the evaluation of the system performance. The first part article gave the reader an overview of the aims and scope of the AWACSS project as well as details about basic technology, immunoassays, software, and networking developed and utilised within the research project. The second part reports on the system performance, first real sample measurements, and an international collaborative trial (inter-laboratory tests) to compare the biosensor with conventional anayltical methods. The systems' capability for analysing a wide range of environmental organic micro-pollutants, such as modern pesticides, endocrine disrupting compounds and pharmaceuticals in surface, ground, drinking and waste water is shown. In addition, a protocol using reconstitution of extracts of solid samples, developed and applied for analysis of river sediments and food samples, is presented. Finally, the overall performance of the AWACSS system in comparison to the conventional analytical techniques, which included liquid and gas chromatographic systems with diode-array UV and mass spectrometric detectors, was successfully tested in an inter-laboratory collaborative trial among six project partners.     

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.     

Plasma urea, creatinine, and urea: creatinine ratio in reindeer (Rangifer tarandus tarandus) and in Svalbard reindeer (Rangifer tarandus platyrhynchus) during defined feeding conditions and in the field.

Variation in plasma urea and creatinine concentration and plasma urea:creatinine ratio (U:C) were studied in semidomestic free-ranging reindeer (Rangifer tarandus tarandus) on the Norwegian mainland, in wild Svalbard reindeer (Rangifer tarandus platyrhynchus), and in captive reindeer maintained either on a lichen-based diet or a protein-rich concentrate to investigate whether these parameters could be used as indicators of the nutritional status of reindeer. In the mainland animals, plasma creatinine concentration was high in winter and early spring and decreased by two-thirds toward the summer. The overall range in mean plasma creatinine concentration (+/-SE) was from 90+/-1.26 to 280+/-2.88 micromol/L. Mean plasma urea concentration (+/-SE) varied from 2.46+/-0.10 in winter up to 17.44+/-0.29 mmol/L in summer and autumn. Month of sampling explained 65% and 90% of the variation in plasma urea and creatinine concentrations, respectively, indicating that seasonality in the diet had the greatest influence on these parameters. Reindeer given lichens as the only feed showed an increase in plasma creatinine and a decrease in plasma urea concentration. Food restriction caused a temporary elevation in urea level but had no significant effect on plasma creatinine concentration. The slight effect of energy intake on urea and creatinine levels was supported by the fact that severe undernutrition in the Svalbard reindeer population had only a small effect on plasma urea and creatinine levels. Protein-rich pellet feed increased plasma urea from around 3 mmol/L to above 10 mmol/L and reduced creatinine concentrations to less than 100 micromol/L, suggesting that the protein content of forage is an important determinant of these blood parameters. Mean U:C ratio (+/-SE) in plasma varied from 8.9+/-0.28 to 120.8+/-1.88. Ratios above 20 appeared when protein intake was low and energy intake was restricted or when protein intake was high. Low ratios occurred when protein intake was low but energy intake adequate. Plasma urea and creatinine concentrations and the U:C ratio showed complex dynamics that were affected by both season and the protein and feed intake. We conclude that they appear to be difficult to interpret as single measures of nutritional status of reindeer.     

Population genetic substructure in blue whiting based on allozyme data

The genetic population structure of the pelagic gadoid blue whiting Micromesistius poutassou throughout its east Atlantic distribution range was explored using polymorphisms at the tissue enzyme loci IDHP-2 * and PGM-1 *. The study included 5025 individuals from 65 locations in the east Atlantic and the Mediterranean. Significant geographic heterogeneity in allele frequencies was demonstrated at both loci ( IDHP-2 *: P=0·030, PGM-1 *: P=0·005). The degree of genetic differentiation ( F _ST= 1·1%) in blue whiting appears to be at the same level as for the demersal gadoids cod and haddock. Several separate reproductive units were indicated at the fringes of the distribution range, i.e. in the Barents Sea, and in one Norwegian fjord (Romsdalsfjord).     

Kinetic study of the chemical reactivity of α-acetolactate as a function of pH in water, and in fresh and fermented culture media used for Lactococcus lactis spp. lactis bv. diacetylactis cultivation

Summary Spontaneous oxidative decarboxylation of -acetolactic acid (ALA) to diacetyl has been assessed under anaerobiosis as a function of pH in water, and in fresh and filtered Lactococcus lactis spp. lactis bv. diacetylactis SD 933 fermented culture media. Whatever the reaction medium, ALA was shown to be potentially reactive, depending on the pH of medium. Diacetyl production mechanism by this strain is discussed on the basis of these kinetic data.     

Consistent melanophore spot patterns allow long‐term individual recognition of Atlantic salmon Salmo salar

The present study shows that permanent melanophore spot patterns in Atlantic salmon Salmo salar make it possible to use images of the operculum to keep track of individual fish over extended periods of their life history. Post‐smolt S. salar (n = 246) were initially photographed at an average mass of 98 g and again 10 months later after rearing in a sea cage, at an average mass of 3088 g. Spots that were present initially remained and were the most overt (largest) 10 months later, while new and less overt spots had developed. Visual recognition of spot size and position showed that fish with at least four initial spots were relatively easy to identify, while identifying fish with less than four spots could be challenging. An automatic image analysis method was developed and shows potential for fast match processing of large numbers of fish. The current findings promote visual recognition of opercular spots as a welfare‐friendly alternative to tagging in experiments involving salmonid fishes.     

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.