Diseases of farmed Atlantic salmon Salmo salar associated with infections by the microsporidian Paranucleospora theridion

The microsporidian Paranucleospora theridion was discovered in Atlantic salmon Salmo salar suffering from proliferative gill disease in a marine farm in western Norway in 2008. The parasite develops in cells of the reticuloendothelial system, cells important for normal immune function. The aim of this study was to see if P. theridion could play a part in some of the diseases with unclear causes in salmon production in Norway, i.e. proliferative gill disease (PGI), pancreas disease (PD), heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS). P. theridion was present in all areas with salmon farming in Norway, but high prevalence and densities of the parasite in salmon and salmon lice were only seen in southern Norway. This region is also the main area for PGI and PD in Norway. Quantification of pathogens associated with PGI, PD, HSMI and CMS diagnoses showed that P. theridion levels are high in southern Norway, and may therefore play a role in susceptibility and disease development. However, among the different diagnoses, fish with PGI are particularly heavily infected with P. theridion. Therefore, P. theridion appears as a possible primary agent in cases with high mortality in connection with PGI in western Norway.     

Vegetation dynamics following trampling in grassland and heathland in Sølendet Nature Reserve, a boreal upland area in Central Norway

Trampling is recognised as a cause of disturbance in grassland and heathland vegetation along two public nature trails in Sølendet nature reserve in central Norway. The effects of trampling by visitors and of controlled trampling were monitored over a period of 5–7 years in permanent plots. A retrogressive development was recorded as vegetation cover and height were reduced and a substantial loss of species richness and biomass took place. Some species, mainly graminoids such as Agrostis capillaris and Carex vaginata , initially appeared to be more tolerant, but were eventually reduced as well. Low, geophytic and prostrate habits of growth and hardy leaf tissues seemed to be beneficial for tolerance to trampling. Moist grassland and lichen-dominated heathland were more vulnerable to trampling than dry grassland. Even though 350–1500 visitors walked through the permanent plots along the trails each summer, effects on the soil surface remained moderate and only a minor furrow was visible on the ground.     

Proteome-derived peptide libraries allow detailed analysis of the substrate specificities of N(alpha)-acetyltransferases and point to hNaa10p as the post-translational actin N(alpha)-acetyltransferase

The impact of N(α)-terminal acetylation on protein stability and protein function in general recently acquired renewed and increasing attention. Although the substrate specificity profile of the conserved enzymes responsible for N(α)-terminal acetylation in yeast has been well documented, the lack of higher eukaryotic models has hampered the specificity profile determination of N(α)-acetyltransferases (NATs) of higher eukaryotes. The fact that several types of protein N termini are acetylated by so far unknown NATs stresses the importance of developing tools for analyzing NAT specificities. Here, we report on a method that implies the use of natural, proteome-derived modified peptide libraries, which, when used in combination with two strong cation exchange separation steps, allows for the delineation of the in vitro specificity profiles of NATs. The human NatA complex, composed of the auxiliary hNaa15p (NATH/hNat1) subunit and the catalytic hNaa10p (hArd1) and hNaa50p (hNat5) subunits, cotranslationally acetylates protein N termini initiating with Ser, Ala, Thr, Val, and Gly following the removal of the initial Met. In our studies, purified hNaa50p preferred Met-Xaa starting N termini (Xaa mainly being a hydrophobic amino acid) in agreement with previous data. Surprisingly, purified hNaa10p preferred acidic N termini, representing a group of in vivo acetylated proteins for which there are currently no NAT(s) identified. The most prominent representatives of the group of acidic N termini are γ- and β-actin. Indeed, by using an independent quantitative assay, hNaa10p strongly acetylated peptides representing the N termini of both γ- and β-actin, and only to a lesser extent, its previously characterized substrate motifs. The immunoprecipitated NatA complex also acetylated the actin N termini efficiently, though displaying a strong shift in specificity toward its known Ser-starting type of substrates. Thus, complex formation of NatA might alter the substrate specificity profile as compared with its isolated catalytic subunits, and, furthermore, NatA or hNaa10p may function as a post-translational actin N(α)-acetyltransferase.     

Bath exposure of Atlantic halibut (Hippoglossus hippoglossus L.) yolk sac larvae to bacterial lipopolysaccharide (LPS): absorption and distribution of the LPS and effect on fish survival

Radiolabelled bacterial lipopolysaccharide (3H-LPS) obtained from Aeromonas salmonicida subsp. salmonicida was added to the petri dishes containing yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.). The larvae were exposed either to 6.25, 12.5, 25, 50 or 100 micrograms 3H-LPS ml-1. The uptake was both dependent on the LPS concentration and the time of exposure. After 5 days of exposure, each larva contained 1.8-7.4 ng 3H-LPS dependent on the initial concentration. After 10 days of exposure each larva contained 7.0-12.4 ng LPS and after 15 days they contained 18.3-34.9 ng 3H-LPS. Fluorescence microscopic analysis of sections obtained from larvae exposed to FITC-LPS (25, 50 and 100 micrograms ml-1) for 5, 10 and 15 days, revealed fluorescence in intestinal epithelial cells, cells in the connective tissue adjacent to the intestine, in cells located between the integumental layer and yolk sac, and in some epithelial cells in the integument. By use of immunohistochemical techniques, LPS was confined to intestinal epithelial cells, lumen of excretory duct and in numerous cells in the epidermal layer. Control specimens did not contain fluorescence or were immunohistochemically negative for LPS. In groups of larvae exposed to 12.5, 25, 50 and 100 micrograms LPS ml-1, the survival was significantly increased after exposure to 50 and 100 micrograms LPS ml-1 from day 20 (96 d degree) and throughout the yolk sac period compared to untreated larvae.     

Expanded in vivo substrate profile of the yeast N-terminal acetyltransferase NatC

N-terminal acetylation is a conserved protein modification among eukaryotes. The yeast Saccharomyces cerevisiae is a valuable model system for studying this modification. The bulk of protein N-terminal acetylation in S. cerevisiae is catalyzed by the N-terminal acetyltransferases NatA, NatB, and NatC. Thus far, proteome-wide identification of the in vivo protein substrates of yeast NatA and NatB has been performed by N-terminomics. Here, we used S. cerevisiae deleted for the NatC catalytic subunit Naa30 and identified 57 yeast NatC substrates by N-terminal combined fractional diagonal chromatography analysis. Interestingly, in addition to the canonical N-termini starting with ML, MI, MF, and MW, yeast NatC substrates also included MY, MK, MM, MA, MV, and MS. However, for some of these substrate types, such as MY, MK, MV, and MS, we also uncovered (residual) non-NatC NAT activity, most likely due to the previously established redundancy between yeast NatC and NatE/Naa50. Thus, we have revealed a complex interplay between different NATs in targeting methionine-starting N-termini in yeast. Furthermore, our results showed that ectopic expression of human NAA30 rescued known NatC phenotypes in naa30Δ yeast, as well as partially restored the yeast NatC Nt-acetylome. Thus, we demonstrate an evolutionary conservation of NatC from yeast to human thereby underpinning future disease models to study pathogenic NAA30 variants. Overall, this work offers increased biochemical and functional insights into NatC-mediated N-terminal acetylation and provides a basis for future work to pinpoint the specific molecular mechanisms that link the lack of NatC-mediated N-terminal acetylation to phenotypes of NatC deletion yeast.     

N-Terminal Acetylation by NatC Is Not a General Determinant for Substrate Subcellular Localization in Saccharomyces cerevisiae

N-terminal acetylation has been suggested to play a role in the subcellular targeting of proteins, in particular those acetylated by the N-terminal acetyltransferase complex NatC. Based on previous positional proteomics data revealing N-terminal acetylation status and the predicted NAT substrate classes, we selected 13 suitable NatC substrates for subcellular localization studies in Saccharomyces cerevisiae. Fluorescence microscopy analysis of GFP-tagged candidates in the presence or absence of the NatC catalytic subunit Naa30 (Mak3) revealed unaltered localization patterns for all 13 candidates, thus arguing against a general role for the N-terminal acetyl group as a localization determinant. Furthermore, all organelle-localized substrates indicated undisrupted structures, thus suggesting that absence of NatC acetylation does not have a vast effect on organelle morphology in yeast.