Effects of Fish-farm Activity on the Limnetic Community Structure of Brown Trout, Salmo trutta, and Arctic Charr, Salvelinus alpinus

Establishment of four fish-farms during the period 1971 to 1994 in the oligotrophic lake Skogseidvatnet affected Arctic charr, Salvelinus alpinus, but not brown trout, Salmo trutta. From 1971 to 1987, an increase in mean individual size of Arctic charr was recorded, while the mean individual size of brown trout remained stable. Arctic charr were found to use deeper benthic areas than brown trout. Approximately 8% of the Arctic charr population (>26cm), were found to switch to waste food from fish-farms, resulting in a novel feeding habitat for the species. They were, however, found in gillnets distant from the fish farm cages, indicating high mobility. The habitat segregation between the two species can most likely be explained by selective differences and asymmetric competition with brown trout as the dominant species. Based on the present results, changes in the Arctic charr population may be due to increased food availability and due to a new habitat use as a waste food feeder. The reason for the brown trout population to have remained stable with respect to mean size, growth pattern and habitat use, may be due to a different diet choice than Arctic charr in this lake. Brown trout were found to feed mainly on terrestrial insects, while Arctic charr fed mainly on zooplankton and on waste food.     

Replication studies using genotype 1a subgenomic hepatitis C virus replicons

Recently, cell-based replicon systems for hepatitis C virus (HCV), in which the nonstructural proteins stably replicate subgenomic viral RNA in Huh7 cells, were developed. To date, one limitation of using these replicon systems to advance drug discovery is the inability of other genotypic derivatives, beyond those of two distinct strains of genotype 1b (HCV-N and Con1), to stably replicate in Huh7 cells. In this report, we evaluated a series of replicon genotype 1a-1b chimeras, as well as a complete genotype 1a replicon clone. A subgenomic replicon construct containing only type 1a sequences failed to generate stable colonies in Huh7 cells even after repeated attempts. Furthermore, addition of an NS5A adaptive mutation (S2204I) which enhances type 1b replicon efficiency was insufficient to confer replication to the wild-type 1a replicon. This subgenomic replicon was subsequently found to be inefficiently translated in Huh7 cells compared to a type 1b replicon, and the attenuation of translation mapped to the N-terminal region of NS3. Therefore, to ensure efficient translation and thereby support replication of the 1a genome, the coding sequence for first 75 residues from type 1a were replaced with the type 1b (strain Con 1) NS3 coding sequence. Although nonstructural proteins were expressed at lower levels with this replicon than with type 1b and although the amount of viral RNA was also severalfold lower (150 copies of positive-strand RNA per cell), the replicon stably replicated in Huh7 cells. Notwithstanding this difference, the ratio of positive- to negative-strand RNA of 26 was similar to that found with the type 1b replicon. Similar results were found for a 1b replicon expressing the type 1a RNA-dependent RNA polymerase. These 1a hybrid replicons maintained sensitivity to alpha interferon (IFN-alpha), albeit with an eightfold-higher 50% inhibitory concentration than type 1b replicons. Evidence is provided herein to confirm that this differential response to IFN-alpha may be attributed directly to the type 1a polymerase.     

COX-1 dependent biosynthesis of 15-hydroxyeicosatetraenoic acid in human mast cells

15-hydroxyeicosatetraenoic acid (15-HETE) is an arachidonic acid derived lipid mediator which can originate both from 15-lipoxygenase (15-LOX) activity and cyclooxygenase (COX) activity. The enzymatic source determines the enantiomeric profile of the 15-HETE formed. 15-HETE is the most abundant arachidonic acid metabolite in the human lung and has been suggested to influence the pathophysiology of asthma. Mast cells are central effectors in asthma, but there are contradictory reports on whether 15-HETE originates from 15-LOX or COX in human mast cells. This prompted the current study where the pathway of 15-HETE biosynthesis was examined in three human mast cell models; the cell line LAD2, cord blood derived mast cells (CBMC) and tissue isolated human lung mast cells (HLMC). Levels and enantiomeric profiles of 15-HETE and levels of the downstream metabolite 15-KETE, were analyzed by UPLC-MS/MS after stimulation with anti-IgE or calcium ionophore A23187 in the presence and absence of inhibitors of COX isoenzymes. We found that 15-HETE was produced by COX-1 in human mast cells under these experimental conditions. Unexpectedly, chiral analysis showed that the 15(R) isomer was predominant and gradually accumulated, whereas the 15(S) isomer was metabolized by the 15-hydroxyprostaglandin dehydrogenase. We conclude that during physiological conditions, i.e., without addition of exogenous arachidonic acid, both enantiomers of 15-HETE are produced by COX-1 in human mast cells but that the 15(S) isomer is selectively depleted by undergoing further metabolism. The study highlights that 15-HETE cannot be used as an indicator of 15-LOX activity for cellular studies, unless chirality and sensitivity to pharmacologic inhibition is determined.     

Microbially influenced corrosion and rust tubercle formation on sheet piles in freshwater systems

The extent of how complex natural microbial communities contribute to metal corrosion is still not fully resolved, especially not for freshwater environments. In order to elucidate the key processes, we investigated rust tubercles forming massively on sheet piles along the river Havel (Germany) applying a complementary set of techniques. In-situ microsensor profiling revealed steep gradients of O₂, redox potential and pH within the tubercle. Micro-computed tomography and scanning electron microscopy showed a multi-layered inner structure with chambers and channels and various organisms embedded in the mineral matrix. Using Mössbauer spectroscopy we identified typical corrosion products including electrically conductive iron (Fe) minerals. Determination of bacterial gene copy numbers and sequencing of 16S rRNA and 18S rRNA amplicons supported a densely populated tubercle matrix with a phylogenetically and metabolically diverse microbial community. Based on our results and previous models of physic(electro)chemical reactions, we propose here a comprehensive concept of tubercle formation highlighting the crucial reactions and microorganisms involved (such as phototrophs, fermenting bacteria, dissimilatory sulphate and Fe(III) reducers) in metal corrosion in freshwaters.     

Leukotriene-dependent airway anaphylaxis in guinea pigs

The antiallergic efficacy of the selective leukotriene synthesis inhibitor, piriprost, was evaluated in two models of airway anaphylaxis in sensitized guinea pigs. Contractions of lung strips evoked by cumulative challenge with allergen were resistant to mepyramine and enhanced by indomethacin. On the other hand, piriprost shifted the dose-response curve markedly to the right, causing more than 50 % inhibition at the highest dose of allergen. The bronchoconstrictor response evoked by cumulative challenge with aerosols of allergen in anesthetized animals, also enhanced by indomethacin, had a distinct mepyramine-sensitive component. Aerosols of piriprost blocked almost completely the allergic bronchoconstriction remaining after indomethacin and mepyramine. These findings indicate that leukotrienes, but not cyclooxygenase products, are major mediators of the acute airway response to allergen in guinea pigs.     

A procedure for isolation of spontaneous mutants with temperature sensitive synthesis of RNA and/or protein

Summary A procedure for the isolation of spontaneous temperature sensitive mutants of Escherichia coli has been developed. They are selected as survivors at high temperature against the combined killing effects exerted by a temperature inducible lambda prophage and either streptomycin plus ampillicin or ampicillin plus cycloserine. The mutants so obtained are blocked in vivo in the synthesis of RNA or protein or both at restrictive temperature.