First isolation of Vibrio tapetis and an atypical strain of Aeromonas salmonicida from skin ulcerations in common dab (Limanda limanda) in the North Sea.

Skin ulcerations rank amongst the most prevalent lesions affecting wild common dab (Limanda limanda) with an increase in prevalence of up to 3.5% in the Belgian part of the North Sea. A complex aetiology of these ulcerations is suspected, and many questions remain on the exact factors contributing to these lesions. To construct the aetiological spectrum of skin ulcerations in flatfish, a one-day monitoring campaign was undertaken in the North Sea. Fifteen fish presented with one or more ulcerations on the pigmented and/or non-pigmented side. Pathological features revealed various stages of ulcerations with loss of epidermal and dermal tissue, inflammatory infiltrates and degeneration of the myofibers bordering the ulceration, albeit in varying degrees. Upon bacteriological examination, pure cultures of Vibrio tapetis were retrieved in high numbers from five fish and of Aeromonas salmonicida in one fish. The V. tapetis isolates showed cross-reactivity with the sera against the representative strain of serotype O2 originating form a carpet-shell clam (Ruditapes descussatus). Moreover, the A. salmonicida isolates displayed a previously undescribed vapA gene sequence (A-layer type) with possible specificity towards common dab. Further research is necessary to pinpoint the exact role of these agents in the development of skin ulcerations in common dab.

Vibrio tapetis isolated from vesicular skin lesions in Dover sole Solea solea.

Vibrio tapetis is primarily known as the causative agent for brown ring disease in bivalves, although it has been isolated from cultivated fish during mortalities on farms. Here we describe the first isolation of V. tapetis from wild-caught and subsequently captive-held Dover sole Solea solea. Pathological features consisted of multifocal circular greyish-white skin discolourations evolving into vesicular lesions and subsequent ulcerations on the pigmented side. On the non-pigmented side, multiple circular lesions-white at the center and red at the edges-were evident. Histological examination of the vesicular lesions revealed dermal fluid-filled spaces, collagen tissue necrosis and a mixed inflammatory infiltrate, with large numbers of small rod-shaped bacteria. In the deep skin lesions, loss of scales and dermal connective tissue, with degeneration and fragmentation of the myofibres bordering the ulceration, were noted. Serotyping, DNA-DNA hybridization and REP- and ERIC-PCR techniques showed that the retrieved isolates displayed a profile similar to the representative strain of genotype/serotype O2 which originally was isolated from carpet-shell clam Venerupis decussata and to which isolates obtained from wedge sole Dicologoglossa cuneata were also closely related.

Serum albumin is essential for in vitro growth of activated human lymphocytes.

The effect of human plasma, the plasma protein fractions of Cohn, and crystallized serum albumin on the in vitro growth of human lymphocytes activated by concanavalin A (Con A) or bacterial lipopolysaccharide was compared. It was found that fraction V or serum albumin (SA) is essential for growth of activated T and B lymphocytes. The other plasma proteins have no effect. The growth response of Con A-activated T lymphocytes to increasing concentrations of SA is similar to the response to increasing equivalent concentrations of plasma suggesting but not proving that SA is the only growth-stimulating factor in plasma when added to a protein-free culture medium. The growth-promoting effect of SA is not due to the fatty acids or hormones bound to SA but is attributed to the albumin molecule itself or to a factor tightly bound to it. SA can also effectively replace plasma to stimulate proliferation of lymphocytes activated by allogeneic lymphocytes or purified protein derivative of tuberculin.

Mitigating seafloor disturbance of bottom trawl fisheries for North Sea sole Solea solea by replacing mechanical with electrical stimulation.

Ecosystem effects of bottom trawl fisheries are of major concern. Although it is prohibited to catch fish using electricity in European Union waters, a number of beam trawlers obtained a derogation and switched to pulse trawling to explore the potential to reduce impacts. Here we analyse whether using electrical rather than mechanical stimulation results in an overall reduction in physical disturbance of the seafloor in the beam-trawl fishery for sole Solea solea. We extend and apply a recently developed assessment framework to the Dutch beam-trawl fleet and show that the switch to pulse trawling substantially reduced benthic impacts when exploiting the total allowable catch of sole in the North Sea. Using Vessel Monitoring by Satellite and logbook data from 2009 to 2017, we estimate that the trawling footprint decreased by 23%, the precautionary impact indicator of the benthic community decreased by 39%, the impact on median longevity of the benthic community decreased by 20%, the impact on benthic biomass decreased by 61%, and the amount of sediment mobilised decreased by 39%. The decrease in impact is due to the replacement of tickler chains by electrode arrays, a lower towing speed and higher catch efficiency for sole. The effort and benthic physical disturbance of the beam-trawl fishery targeting plaice Pleuronectes platessa in the central North Sea increased with the recovery of the plaice stock. Our study illustrates the utility of a standardized methodological framework to assess the differences in time trends and physical disturbance between gears.

Effects of epidermal growth factor on protein degradation the translocation of non-histone proteins to the nucleus and DNA synthesis.

Stimulation of resting Chang liver or monkey kidney cells, prelabeled with [3H]leucine, by epidermal growth factor (EGF), caused inhibition of cellular protein degradation and a parallel increase nuclear translocation of 3H-labeled non-histone proteins and DNA synthesis. Nuclear translocation of these proteins was independent of protein synthesis. Fractionation of the nuclear 3H-labeled non-histone proteins in a pH gradient of 2.5-6.5 showed that the protein fractions with a high degree of proteolysis in resting cells corresponded to the protein fractions with a high extent of translocation in stimulated cells, suggesting that degradation and translocation of these proteins may be related. EGF inhibited cellular uptake of [3H]chloroquine, suggesting that EGF inhibits non-histone protein degradation via the lysosomal pathway. These observations support the hypothesis that EGF induces non-histone protein translocation to the nucleus by inhibiting lysosomal degradation of these proteins.

The effects of amino acids on protein degradation and translocation of non-histone proteins to the nucleus in lymphocytes.

Tryptophan, phenylalanine and leucine have two parallel effects in cultured lymphocytes, they inhibit cellular proteolysis and increase the translocation of non-histone proteins to the nucleus. The latter is associated with an increased cellular binding of [3H]actinomycin D, indicating an altered structure of chromatin. The amino acids also inhibit the cellular uptake of [3H]chloroquine, suggesting that inhibited protein degradation is lysosomal. Several amine catabolites of tryptophan and phenylalanine, some of which are known to play a role as biogenic amines, have similar actions, and can explain, at least in part, the effects of their parent amino acids. Fractionation of the nuclear 3H-labeled non-histone proteins according to pH 2.5-6.5 shows that such proteins with a high rate of degradation in untreated cells correspond to the 3H-labeled non-histone proteins with a high rate of translocation in tryptophan treated cells. These data suggest that the degradation and the translocation of the non-histone proteins are linked and that the increased translocation of the non-histone proteins to the nucleus may be the consequence of inhibited lysosomal degradation of these proteins by the amino acids.

Lymphoid and non-lymphoid tumors in E kappa-myc transgenic rabbits.

We developed transgenic rabbits with a DNA construct containing the proto-oncogene c-myc conjugated to the Ig kappa-chain enhancer gene, E kappa. One of four transgenic rabbits was mated to a normal rabbit and we used the offspring to develop a colony of rabbits carrying the E kappa-myc transgene in their germline. Of a total of 19 E kappa-myc transgenic rabbits, eight developed tumors. The tumors were characterized histologically and four were diagnosed as lymphoma, and one each was diagnosed as embryonic carcinoma, hepatoma, ovarian carcinoma and basal cell carcinoma. By Southern analysis, we showed the four lymphomas were of B-lymphoid lineage and by nucleotide sequence analysis we found three of them most likely used VH1 in their VDJ gene rearrangements. Cells from the embryonic carcinoma, the hepatoma and two of the B-lymphomas were adapted to tissue culture. We discuss the possibility that tumors of non-lymphoid origin develop in the E kappa-myc transgenic rabbits because of the potential for NF-kappa B to activate the kappa-enhancer in cells other than B-lymphoid lineage cells.

Effects of fibroblastic growth factor on protein degradation, the migration of non-histone proteins to the nucleus and DNA synthesis in diploid fibroblasts.

Stimulation of resting WI38 cells, prelabeled with [3H]leucine, with fibroblastic growth factor (FGF) or serum, caused increased nuclear translocation of [3H]non-histone proteins [( 3H]NHP) and DNA synthesis, and a parallel decrease of proteolysis. [3H]NHP migration was independent of protein synthesis. Fractionation of the nuclear proteins in a pH gradient of 2.5-6.5 showed that [3H]NHP fractions with high degradation rates in resting cells corresponded to the [3H]NHP fractions with high migration rates in stimulated cells, suggesting that degradation and migration of [3H]NHP are linked. FGF inhibited cellular uptake of [3H]chloroquine, suggesting that FGF inhibits NHP degradation via lysosomes. The lysosomotropic amine eserine had similar effects as FGF. It is proposed that FGF induces NHP migration to the nucleus by inhibiting their lysosomal degradation. FGF also caused migration of [3H]histones, however, the mechanism is not clear.

The effects of concanavalin A and other agents on protein degradation and migration of non-histone proteins (NHP) to the nucleus in lymphocytes.

Concanavalin A (conA) inhibits the degradation of [3H]leucine-labeled cellular proteins of human lymphocytes. The lectin also stimulates the migration of non-histone proteins (NHP) from the cytoplasm to the nucleus. The increased nuclear level of NHP is associated with increased cellular binding of [3H]actinomycin D [(3H]AD). Decreased protein breakdown and increased migration of NHP are parallel events, i.e. both changes occur as a function of the lectin concentration and display a similar time course, suggesting that these events could be related. Similar effects are observed with fluoride, chloroquine and iodoacetate: these agents simultaneously decrease proteolysis and increase the nuclear level of NHP, associated with increased cellular [3H]AD binding. Fractionation of the acidic NHP according to pH 2.5-6.5 shows that proteins with a high degree of degradation in unstimulated cells correspond to proteins with a high degree of migration in conA-stimulated cells. A similar correlation was observed in fluoride-treated lymphocytes. conA, fluoride and iodoacetate decrease cellular [3H]chloroquine [(3H]CQ) accumulation, indicating a lysosomotropic effect. These and previously reported data suggest, but do not prove that conA inhibits degradation of cellular proteins via the lysosomal pathway. Ammonium chloride, methylamine and sodium azide also inhibit proteolysis and increase cellular [3H]AD binding; however, their effects are weak. On the basis of these observations it appears that lysosomal degradation and migration of NHP to the nucleus are linked; however, the mechanism of the linkage is unknown.

The effects of lysosomotropic amines on protein degradation, migration of nonhistone proteins to the nucleus, and cathepsin D in lymphocytes.

Various lysosomotropic amines have two parallel effects in human lymphocytes: they inhibit the degradation of cellular proteins and increase the migration of nonhistone proteins (NHP) from the cytoplasm to the nucleus. The increased nuclear level of NHP is associated with increased cellular binding of [3H] actinomycin D, indicating an altered structure of chromatin. The agents inhibit the degradation of short- and long-lived proteins equally. Fractionation of the [3H] NHP of the nucleus according to pH 2.5-6.5 shows that [3H] NHP with a high rate of degradation in untreated cells correspond to [3H] NHP with a high rate of migration in cells treated with the agents. Eserine, amantadine, nicotine, atropine, benzylamine, and propranolol inhibit cathepsin D in concentrations causing proteolytic inhibition in cell cultures or in concentrations believed to be attained in lysosomes. The agents strongly inhibit the cellular accumulation of [3H] chloroquine. The data support the proposal that the migration of NHP from the cytoplasm to the nucleus is the direct consequence of inhibited degradation of these proteins in lysosomes by the amines.

Effects of platelet-derived growth factor on degradation, nuclear translocation of nonhistone proteins, and DNA synthesis.

Stimulation of resting normal rat kidney fibroblasts, prelabeled with [3H]leucine, by platelet-derived growth factor (PDGF) caused inhibition of cellular protein degradation and a parallel increased nuclear translocation of 3H-labeled nonhistone proteins (3H-NHP) and DNA synthesis. Nuclear translocation of these proteins was independent of protein synthesis. Fractionation of the nuclear 3H-NHP in a pH gradient of 2.5-6.5 showed that the protein fractions with a high degree of proteolysis in resting cells corresponded to the protein fractions with a high extent of translocation in stimulated cells, suggesting that degradation and translocation of these proteins may be related. PDGF inhibited cellular uptake of [3H]chloroquine, suggesting that PDGF inhibits NHP degradation via the lysosomal pathway. These observations support the hypothesis that PDGF induces NHP translocation to the nucleus by inhibiting lysosomal degradation of these proteins.

Epidermal growth factor stimulates DNA synthesis while inhibiting cell multiplication of A-431 carcinoma cells.

Epidermal growth factor (EGF) has been shown to inhibit the multiplication of the human epidermoid carcinoma cell line A-431. In the present report it is shown that, despite growth inhibition, EGF caused a marked synthesis of DNA and nonhistone proteins, without progression into mitosis. This event was associated with a retraction of the monolayer into colonies of cells. This suggests that the cell cycle of A-431 cells is controlled by two surface membrane signals: one generated by EGF stimulating the synthetic events of the G1 and S phases; a second signal, leading to progression into mitosis appears either not to be generated or to be inhibited by EGF.

Endocytosed non-histone proteins translocate in part to the nucleus in lymphocytes.

[3H]Non-histone proteins ([3H]NHP), dissolved in the culture medium, are endocytosed by lymphocytes and equilibrate rapidly between the cytoplasm and the nucleus. During incubation, the proteins are gradually degraded in the lysosomes. The lysosomotropic agents conA, NaF, eserine and atropine have two parallel effects on resting lymphocytes, after they have endocytosed [3H]NHP: inhibition of degradation and increased translocation of [3H]NHP from the cytoplasm to the nucleus. This indicates that lysosomal degradation and translocation of [3H]NHP to the nucleus are linked and suggests that this translocation may be the result of inhibited lysosomal degradation of the [3H]NHP. The behaviour of endocytosed [3H]NHP appears similar to that of endogenous [3H]NHP in cells prelabeled with [3H]leucine, when subjected to the same lysosomotropic agents, reported previously (Polet, H, Exp cell res 148 (1983) 345). This observation may provide a model to study the mechanism(s) controlling nucleo-cytoplasmic traffic of NHP.

Role of the cell membrane in the uptake of 3H-actinomycin D by mammalian cells in vitro.

The characteristics of the uptake of 3H-Actinomycin D (3H-AMD) by mammalian cells was studied in vitro. Chang liver (CH) cells accululated 3H-AMD over 100 times above extracellular levels. CH cells accumulated and released 3H-AMD at a slow rate. Treatment of cells with ethanol-acetone or Tween 80 significantly increased the rates of drug uptake and release by cells, indicating that the cell membrane effectively slows down passage of 3H-AMD in and out of cells. Cellular 3H-AMD uptake is temperature dependent and not energy dependent. The rates of 3H-AMD uptake after short and prolonged incubation suggest that AMD entry into cells consists of two phases, an initial phase of rate-limiting diffusion process followed by a second phase of binding to deoxyribonucleic acid. Although lymphocytes take up less 3H-AMD than CH cells, the differential inhibitory effect of AMD on nucleic acid synthesis in the two cell types is small. The time required for cells to be fully saturated with 3H-AMD varies with the cell type and is based on permeability differences of the cell membrane for 3H-AMD. The time required for lymphocytes and CH cells to be fully saturated with 3H-AMD is reflected in the differential effect of the drug on nucleic acid synthesis. The physiological basis of AMD resistance can be explained on the basis of impaired diffusion of the drug into cells.