Bud dormancy and vegetative growth in Salix polaris as affected by temperature and photoperiod

Summary Vegetative growth of two ecotypes (lat. 78° 15N and 69°37N) of Salix polaris L. was studied in phytotron experiments. Dormancy of the winter buds was broken by chilling at 0.5°C for 14 to 42 days. Chilling requirement increased with decreasing growth temperature. The optimum temperature for bud break and shoot growth was about 15°C for both ecotypes. Cessation of apical shoot growth and abscission of shoot tip was not prevented by long photoperiods. However, at high temperature, 15°C or more, and in 18 to 24 h photoperiod, two or three growth flushes occurred frequently in both ecotypes. Leaf abscission in the arctic ecotype from lat. 78°N was not affected by photoperiod when grown at 6°C, but was stimulated by short photoperiod when grown at 15°C. In the ecotype from lat. 69°N leaf abscission was enhanced by short photoperiod even at 6°C.     

Inoculation of Infectious Pancreatic Necrosis Virus Serotype Sp did not cause Pancreas Disease in Atlantic Salmon (Salmo salar L.)

Atlantic salmon were selected from a fish farm with no previous record of pancreas disease (PD) or infectious pancreatic necrosis virus (IPNV) infection. Groups of fish were inoculated with either IPNV (strain Sp) from cell culture, organ material from fish with PD or control material as phosphate-buffered saline (PBS). Virological, histological and immunohistochemical examinations were carried out throughout the experiment. None of the fish died or showed clinical symptoms of PD. Histological examination revealed no pathological changes, and immunohistochemical studies were negative. Virus was isolated only sporadically from the group inoculated with organ material, whereas it was isolated consistently from the group inoculated with virus propagated in cell culture, as well as from in-contact control fish after the first week. In a latent carrier test, changes were entirely lacking in the first mentioned group, and were only slight in the last mentioned group. The data suggest that PD is not a transmissible disease, and that IPNV isolated from a PD outbreak does not play any part in the etiology of this disease.     

Characterization of infectious salmon anemia virus, an orthomyxo-like virus isolated from Atlantic salmon (Salmo salar L.).

Infectious salmon anemia (ISA) virus is the cause of infectious salmon anemia in farmed Atlantic salmon. The virus has been shown to contain RNA with structural characteristics similar to those of accepted members of the Orthomyxoviridae. Further biochemical, physiochemical, and morphological characterization of ISA virus was undertaken to clarify its taxonomic position. The virus was found to be sensitive to chloroform, heat, and low pH and agglutinated erythrocytes from fish. Erythrocytes from mammals or birds were not agglutinated. Receptor-destroying enzyme activity was detected, and the nature of this enzyme was suggested to be an acetylesterase. The buoyant density of the virus was 1.18 g/ml in sucrose and CsCl gradients. The maximum rate of virus replication was observed at 15 degrees C, while no virus was produced at 25 degrees C. Actinomycin D inhibited viral replication, and viral antigen was detected in nuclei by immunofluorescence. The addition of trypsin to the culture medium during virus replication had a beneficial effect on virus replication. ISA virus contains four major polypeptides with estimated molecular sizes of 71, 53, 43, and 24 kDa. Electron microscopy revealed structures closely resembling the nucleocapsids of influenza virus. Mushroom-shaped surface projections were a distinctive morphological feature, which differed from the rod-shaped hemagglutinin projections of the influenza viruses. The data reported here support the relationship of ISA virus to the Orthomyxoviridae, although ISA virus differs from influenza viruses in some morphological characteristics and in showing restricted hemagglutination, in different specificity of the receptor-destroying enzyme, in different polypeptide profile, in being unable to replicate at temperatures above 25 degrees C, and in host range.     

Hsp90 inhibition and co‐incubation with pertuzumab induce internalization and degradation of trastuzumab: Implications for use of T‐DM1

The receptor tyrosine kinase HER2 is associated with a number of human malignancies and is an important therapeutic target. The antibody‐drug conjugate trastuzumab emtansine (T‐DM1; Kadcyla^®) is recommended as a first‐line treatment for patients with HER2‐positive metastatic breast cancer. T‐DM1 combines the antibody‐induced effects of the anti‐HER2 antibody trastuzumab (Herceptin^®) with the cytotoxic effect of the tubulin inhibitor mertansine (DM1). For DM1 to have effect, the T‐DM1‐HER2 complex has to be internalized and the trastuzumab part of T‐DM1 has to be degraded. HER2 is, however, considered endocytosis‐resistant. As a result of this, trastuzumab is only internalized to a highly limited extent, and if internalized, it is rapidly recycled. The exact reasons for the endocytosis resistance of HER2 are not clear, but it is stabilized by heat‐shock protein 90 (Hsp90) and Hsp90 inhibitors induce internalization and degradation of HER2. HER2 can also be internalized upon activation of protein kinase C, and contrary to trastuzumab alone, the combination of two or more anti‐HER2 antibodies can induce efficient internalization and degradation of HER2. With intention to find ways to improve the action of T‐DM1, we investigated how different ways of inducing HER2 internalization leads to degradation of trastuzumab. The results show that although both Hsp90 inhibition and activation of protein kinase C induce internalization of trastuzumab, only Hsp90 inhibition induces degradation. Furthermore, we find that antibody internalization and degradation are increased when trastuzumab is combined with the clinically approved anti‐HER2 antibody pertuzumab (Perjeta^®).     

Metabolic fingerprinting reveals differences between northern and southern strains of the cryptic diatom Chaetoceros socialis

Morphology and molecular phylogeny constitute the structural elements of diatom taxonomy. These approaches do not, however, give information on the functioning of taxa. Additional methods to serve a more integrated and wide-ranging taxonomy have therefore been called for. Metabolic fingerprinting is one approach used within the field of metabolomics, often applied in classification of samples. Here we apply metabolic fingerprinting in a taxonomic study of a cryptic diatom species. Strains of the cosmopolitan diatom Chaetoceros socialis from two geographical areas; the north-east Atlantic and Arctic and the Gulf of Naples, were cultivated at three different temperatures; 2.5, 8 and 13°C. The strains from the two different geographical areas exhibited different growth rates as well as different photosynthetic efficiencies. Algal extracts, collected at the end of the growth experiments, were analysed by Ultra-Performance Liquid Chromatography High Resolution Mass Spectrometry. The two groups of strains were separated by principal component analysis of their metabolic fingerprints. Analysis of the data revealed both qualitative and quantitative differences in metabolite markers. These phenotypic differences reinforce differences also found for morphology, phylogenetic markers and growth rates, and point at different adaptive characteristics in organisms living under different temperature regimes.     

The Cbl-interacting protein TULA inhibits dynamin-dependent endocytosis

The Cbl- and ubiquitin-interacting protein T-cell ubiquitin ligand (TULA) has been demonstrated to inhibit endocytosis and downregulation of ligand-activated EGF receptor (EGFR) by impairing Cbl-induced ubiquitination. We presently report that TULA additionally inhibited clathrin-dependent endocytosis in general, as both uptake of transferrin (Tf) and low-density lipoprotein (LDL) was inhibited. Additionally, endocytosis of the raft proteins CD59 and major histocompatibility complex class I (MHC-I), which we demonstrate were mainly endocytosed clathrin-independently, but dynamin-dependently, was blocked in cells overexpressing TULA. By contrast, the uptake of ricin, which is mainly endocytosed clathrin- and dynamin-independently, was not affected by overexpressed TULA. Consistently, TULA and dynamin co-immunoprecipitated and colocalized intracellularly, and upon overexpression of dynamin the TULA-mediated inhibitory effect on endocytosis of Tf, LDL, CD59 and MHC-I was counteracted. Overexpressed dynamin did not restore ubiquitination of the EGFR, and consistently dynamin did not rescue endocytosis of the EGFR in cells overexpressing TULA. We conclude that TULA inhibits both clathrin-dependent and clathrin-independent endocytic pathways by functionally sequestering dynamin via the SH3 domain of TULA binding proline-rich sequences in dynamin.     

Studying properties of neurotransmitter receptors by non-stationary noise analysis of spontaneous postsynaptic currents and agonist-evoked responses in outside-out patches

Chemical synaptic transmission depends on neurotransmitter-gated ion channels concentrated in the postsynaptic membrane of specialized synaptic contacts. The functional characteristics of these neurotransmitter receptor channels are important for determining the properties of synaptic transmission. Whole-cell recording of postsynaptic currents (PSCs) and outside-out patch recording of transmitter-evoked currents are important tools for estimating the single-channel conductance and the number of receptors contributing to the PSC activated by a single transmitter quantum. When single-channel activity cannot be directly resolved, non-stationary noise analysis is a valuable tool for determining these parameters. Peak-scaled non-stationary noise analysis can be used to compensate for quantal variability in synaptic currents. Here, we present detailed protocols for conventional and peak-scaled non-stationary noise analysis of spontaneous PSCs and responses in outside-out patches. In addition, we include examples of computer code for individual functions used in the different stages of non-stationary noise analysis. These analysis procedures require 3-8 h.     

Protection of atlantic salmon Salmo salar against infectious pancreatic necrosis after DNA vaccination

Although vaccines against infectious pancreatic necrosis (IPN) based on inactivated virus or recombinant structural viral proteins are commercially available, the protection is not complete and the disease is still a problem for the Atlantic salmon Salmo salar farming industry. In the present study, 5 different plasmids that expressed whole or parts of the large open reading frames (ORF) of Segment A of the IPN virus (IPNV) were constructed. The plasmids were shown to express proteins in cell cultures and in zebrafish Danio rerio in vivo. The specificities of the expressed proteins were confirmed by staining with IPNV-specific monoclonal antibodies (MAb) The plasmids were then used alone or in different combinations to vaccinate groups of Atlantic salmon, which subsequently were challenged in an experimental assay for IPN. A high level of protection was induced only by the plasmid combination that contained a plasmid expressing all the large ORF polyprotein.     

Activation of MK5/PRAK by the atypical MAP kinase ERK3 defines a novel signal transduction pathway

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK), which is regulated by protein stability. However, its function is unknown and no physiological substrates for ERK3 have yet been identified. Here we demonstrate a specific interaction between ERK3 and MAPK-activated protein kinase-5 (MK5). Binding results in nuclear exclusion of both ERK3 and MK5 and is accompanied by ERK3-dependent phosphorylation and activation of MK5 in vitro and in vivo. Endogenous MK5 activity is significantly reduced by siRNA-mediated knockdown of ERK3 and also in fibroblasts derived from ERK3-/- mice. Furthermore, increased levels of ERK3 protein detected during nerve growth factor-induced differentiation of PC12 cells are accompanied by an increase in MK5 activity. Conversely, MK5 depletion causes a dramatic reduction in endogenous ERK3 levels. Our data identify the first physiological protein substrate for ERK3 and suggest a functional link between these kinases in which MK5 is a downstream target of ERK3, while MK5 acts as a chaperone for ERK3. Our findings provide valuable tools to further dissect the regulation and biological roles of both ERK3 and MK5.