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.

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.

In vitro induction of the expression of multiple IgA isotype genes in rabbit B cells by TGF-beta and IL-2.

The rabbit genome has 13 different Calpha genes that are expressed at different levels in mucosal tissues. To analyze the factors involved in the differential expression of these Calpha genes, we cloned and sequenced the promoters of the Ialpha regions that control the expression of sterile mRNA. We found that all Calpha genes, including Calpha3 and Calpha8, which are not expressed, and Calpha4, which is expressed at high levels, have similar nucleotide sequences in the Ialpha region, and all contain the recognition elements for TGF-beta in the promoter. B lymphocytes from popliteal lymph nodes or Peyer's patch activated in vitro could be induced by TGF-beta to express sterile IgA transcripts of all IgA isotypes, except Calpha2, Calpha3, and Calpha8. Many single B lymphocytes transcribed sterile mRNA of more than one IgA isotype, which demonstrates that transcription of sterile mRNA alone does not regulate the IgA isotype switch. The addition of IL-2 led to the expression of transcripts of mature IgA of all isotypes, except Calpha2, Calpha3, and Calpha8. The predominantly expressed isotype in these experiments was Calpha4. With the use of an IgA4-specific mAb we found that IgA4+ plasma cells are unevenly distributed throughout the small intestine such that many of the IgA+ plasma cells in the duodenum-jejunum produced IgA4, whereas in the lower part of the ileum IgA4-producing cells were almost absent. Because the microbial flora varies throughout the intestine, we suggest that the microbial flora creates different local environments and thus affects either isotype switching or homing of IgA-expressing cells.

Carotid-Cavernous Fistula Associated with Ehlers-Danlos Syndrome Type IV. A Case Report and Review of Literature.

SUMMARY: A case of traumatic, direct, carotid cavernous fistula (CCF) associated with Ehlers - Danlos syndrome (EDS) Type IV is reported along with a review of the literature. Excluding the present case, three similar cases associated with EDSTypeJV have already been reported by Gerard M. Debrun et Al(l). Despite the risks associated with endovascular manipulation, the fistula was successfully closed by intravascular embolisation but the patient expired a few days later because of underlying disease-associated vascular and visceral complications.

Trans-chromosomal recombination within the Ig heavy chain switch region in B lymphocytes.

Somatic DNA rearrangements in B lymphocytes, including V(D)J gene rearrangements and isotype switching, generally occur in cis, i. e., intrachromosomally. We showed previously, however, that 3 to 7% of IgA heavy chains have the VH and Calpha regions encoded in trans. To determine whether the trans-association of VH and Calpha occurred by trans-chromosomal recombination, by trans-splicing, or by trans-chromosomal gene conversion, we generated and analyzed eight IgA-secreting rabbit hybridomas with trans-associated VH and Calpha heavy chains. By ELISA and by nucleotide sequence analysis we found that the VH and Calpha regions were encoded by genes that were in trans in the germline. We cloned the rearranged VDJ-Calpha gene from a fosmid library of one hybridoma and found that the expressed VH and Calpha genes were juxtaposed. Moreover, the juxtaposed VH and Calpha genes originated from different IgH alleles. From the same hybridoma, we also identified a fosmid clone with the other expected product of a trans-chromosomal recombination. The recombination breakpoint occurred within the Smicro/Salpha region, indicating that the trans-association of VH and Calpha genes occurred by trans-chromosomal recombination during isotype switching. We conclude that trans-chromosomal recombination occurs at an unexpectedly high frequency (7%) within the IgH locus of B lymphocytes in normal animals, which may explain the high incidence of B-cell tumors that arise from oncogene translocation into the IgH locus.

Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas.

During the last 15 years several laboratories have attempted to generate rabbit monoclonal antibodies, mainly because rabbits recognize antigens and epitopes that are not immunogenic in mice or rats, two species from which monoclonal antibodies are usually generated. Monoclonal antibodies from rabbits could not be generated, however, because a plasmacytoma fusion partner was not available. To obtain a rabbit plasmacytoma cell line that could be used as a fusion partner we generated transgenic rabbits carrying two transgenes, c-myc and v-abl. These rabbits developed plasmacytomas, and we obtained several plasmacytoma cell lines from which we isolated hypoxanthine/aminopterin/thymidine-sensitive clones. One of these clones, when fused with spleen cells of immunized rabbits, produced stable hybridomas that secreted antibodies specific for the immunogen. The hybridomas can be cloned and propagated in nude mice, and they can be frozen without change in their ability to secrete specific monoclonal antibodies. These rabbit-rabbit hybridomas will be useful not only for production of monoclonal antibodies but also for studies of immunoglobulin gene rearrangements and isotype switching.

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.

Preferential VH gene usage in rabbit Ig-secreting heterohybridomas.

B cells from leukemic rabbits preferentially use a single VH gene, VH1, in their VDJ gene rearrangements. To determine whether Ig-secreting B cells from normal rabbits also preferentially use VH1, we generated rabbit X mouse heterohybridomas that stably secreted rabbit Ig that expressed VHa allotypic specificities and analyzed the VH genes used in their VDJ gene rearrangements. We cloned the VDJ genes from nine heterohybridomas, and by comparing the restriction map of the DNA immediately 5' of the translational start site of these VDJ genes to that of the same region 5' of VH1, we showed that eight of the nine heterohybridoma clones use VH1. Comparison of the nucleotide sequences of the eight VH1-using VDJ genes with the nucleotide sequence of germ-line VH1 showed that each of them had somatically diversified. The diversified regions included clustered nucleotide changes and codon insertions and deletions, such as would be expected if the diversification process involved somatic gene conversion. We searched our database of germ-line VH genes for genes that could serve as donors for the gene conversion events, and we identified potential VH donor genes for five regions of diversification. One of these regions of diversification spanned at least 132 bp and included a codon insertion as well as 15 nucleotide changes. The data confirm that Ab diversity may be generated by a somatic gene conversion-like mechanism. The results directly demonstrate that Ig-secreting B cells from normal rabbits preferentially use VH1 in their VDJ gene rearrangements.

Differential expression of 13 IgA-heavy chain genes in rabbit lymphoid tissues.

Molecular cloning techniques have recently demonstrated that rabbit has 13 different IgA C alpha H chain genes. This is in contrast to human and mouse that have only two and one C alpha heavy chain genes, respectively. In previous studies, nucleotide sequence analysis indicated that the 13 rabbit C alpha genes were potentially functional, and in vitro expression experiments showed that at least 12 of these genes were expressible. To understand the role of these multiple IgA isotypes we analyzed RNA of various lymphoid tissues for the presence of mRNA representing each of the multiple C alpha genes. We used the RNase protection assay with probes that are specific for the 13 different C alpha genes and we consistently found that at least 10 of the C alpha genes are expressed, albeit at different levels, in gut (small intestines), appendix, mesenteric lymph node, and mammary tissue. However, in salivary gland (submandibular), only seven of these genes are expressed at significant levels and in lung and tonsil only one C alpha gene, C alpha 4, is expressed at a level comparable to its expression in other tissues. Analysis of RNA of Peyer's patch showed differences in the level of C alpha gene expression between different animals and between different Peyer's patches of the same rabbit; in some cases, most of the C alpha genes were expressed, but in some cases only C alpha 4 was expressed at a significant level. Inasmuch as C alpha 4 is the 5' most C alpha gene we propose that IgA-producing cells are derived from B cells that have initially undergone isotype switching to C alpha 4 and we discuss various mechanisms that could explain switching to the more 3' C alpha genes.

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.

Cytochalasin D inhibits nuclear translocation of nonhistone proteins induced by lectin and other agents in lymphocytes.

Cytochalasin D (CD), known to interfere with microfilament activity, inhibits RNA and protein synthesis and the cellular uptake of [3H]actinomycin D in concanavalin A-stimulated lymphocytes. It also inhibits the nuclear translocation of nonhistone proteins (NHP) induced by the lectin. Since NHP mediate RNA and protein synthesis and [3H]actinomycin D binding, inhibited nuclear translocation of NHP can explain the inhibition of the former events. CD also inhibits nuclear translocation of NHP caused by NaF or chloroquine. The mechanism of how CD inhibits nuclear translocation of the NHP is obscure. Since NaF and chloroquine enter cells by diffusion, it would appear that CD acts on an intracellular target, rather than abrogating a cell surface-mediated signal generated by binding of the lectin to a cell surface receptor.

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.

Rabbit major histocompatibility complex. IV. Expression of major histocompatibility complex class II genes.

The rabbit MHC class II DP, DQ, and DR alpha and beta chain genes were transfected into murine B lymphoma cells. The transfected cells expressed R-DQ and R-DR molecules on the cell surface but they did not express the R-DP genes either on the cell surface or at the level of mRNA. Northern blot analyses showed that the R-DP genes were expressed, albeit at low levels, in rabbit spleen. Similar analyses showed that the R-DQ and R-DR genes were expressed at high levels in rabbit spleen. A new monoclonal anti-rabbit class II antibody, RDR34, has been developed and shown to react with the R-DR transfected cells and not with the R-DQ transfected cells. The previously described monoclonal anti-rabbit class II antibody, 2C4, reacted with the R-DQ transfected cells and not with the R-DR transfected cells. Thus, 2C4 and RDR34 MAb's are specific for the R-DQ and R-DR molecules, respectively. Each of the antibodies reacted with approximately 50% of rabbit spleen cells as shown by immunofluorescent antibody studies.

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.

Transgenic rabbits with lymphocytic leukemia induced by the c-myc oncogene fused with the immunoglobulin heavy chain enhancer.

Transgenic rabbits with the rabbit c-myc oncogene fused with the rabbit immunoglobulin heavy chain enhancer region (E mu) DNA were developed by microinjecting pronuclei of single cell zygotes with the gene construct and implanting the microinjected eggs into pseudopregnant females. At age 17-20 days, 3 of 21 offspring born to seven females were found to have peripheral blood leukocyte counts of greater than 100,000 per mm3. Histology analyses showed extensive lymphocytic infiltration in the liver and kidney, indicating that these rabbits had a malignant lymphocytic leukemia. Genomic DNA analyses of thymus and peripheral blood lymphocytes revealed that the leukemic rabbits were transgenic and that both immunoglobulin heavy and kappa light chain genes were rearranged in the leukemic cells; thus, the leukemic cells are of B-cell lineage. One to four heavy and light chain gene rearrangements were observed, suggesting that the B-cell tumors were oligoclonal. Stable tissue culture cell lines from the bone marrow and peripheral blood of one of the transgenic rabbits have been developed. The development of B-cell leukemias in rabbits with the E mu-myc transgene contrasts with the development of B-cell lymphomas in mice carrying a similar transgene. The lymphomas in mice develop in adults and are monoclonal in origin. The leukemias in rabbits develop in juveniles, less than 3 weeks after birth, and appear oligoclonal in origin. The leukemias seem to develop in rabbit at a specific stage of development, and we suggest that a normal developmental signal may be involved in the oncogenesis.

Demonstration that some of the nonhistone proteins, inducible to translocate into the nucleus, are glycosylated.

Con A, NaF, and eserine (lysosomotropic agents) induced marked translocation of acidic [3H] nonhistone proteins (NHP) from the cytoplasm to the nucleus in lymphocytes prelabeled with [3H]-2-mannose. The nuclear [3H] NHP contents were 38-120% higher in cells treated with these agents than in control cells. Tunicamycin, a strong inhibitor of N-glycosylation via the dolichol pathway, caused a concentration-dependent inhibition of [3H]-2-mannose incorporation into the nuclear [3H] NHP. Considerable amounts of nuclear [3H] NHP from lymphocytes labeled with either [3H]-2-mannose or [3H] leucine, bound specifically to Con A-Sepharose and could be eluted by alpha-methyl mannoside. Con A and NaF caused also nuclear translocation of acidic [3H] NHP in cells labeled with [3H] glucosamine, [3H] galactose, or [3H] fucose. Fractionation of the nuclear proteins by isoelectric focusing in a pH gradient of 2.5-6.5 showed that multiple species of acidic NHP were labeled with each of the four 3H-sugars. These results indicate that a fraction of the acidic nuclear NHP are N-glycosylated proteins and that gene activation and mitogenesis are associated with the translocation of these glycoproteins to the nucleus. Considering the known intracellular traffic of nascent glycoproteins our results suggest that at least some of the acidic NHP are synthesized and glycosylated in the endoplasmic reticulum and the Golgi (secretory pathway). It is likely that these proteins, after completion of synthesis and glycosylation, emerge from the trans-stack of the Golgi packaged in vesicles and accumulate in the cytoplasm. Induction of nuclear translocation of such NHP by various agents may be mediated by a vesicular transport mechanism.

Effects of serum and conditioned medium on protein degradation, migration of nonhistone proteins to the nucleus, and DNA synthesis in transformed cells.

Stimulation of resting transformed cells (Chang liver cells), prelabeled with [3H] leucine, with fetal calf serum, caused increased nuclear translocation of [3H] nonhistone proteins ([3H] NHP) and DNA synthesis and a parallel inhibition of proteolysis of cellular proteins. [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. Conditioned medium (COM) produced by Chang cells had similar effects as serum, suggesting that factors produced by these transformed cells, control cell growth by a mechanism that is similar to serum. The lysosomotropic amine eserine had similar effects as serum and COM. Based on the similarity of the effects, it would appear that serum and COM inhibit lysosomal proteolysis. It is proposed that serum and COM induce NHP migration to the nucleus by inhibiting lysosomal degradation of these proteins. Serum and COM caused also migration of [3H] histones to the nucleus, however the mechanism is not clear.