Intestinal cellular localization of PCNA protein and CYP1A mRNA in Atlantic salmon Salmo salar L. exposed to a model toxicant

<p>Abstract</p> <p>Background</p> <p>The aim of the study was to examine the intestinal cellular localization of proliferating cell nuclear antigen (PCNA) and cytochrome P450 A1 (CYP1A) expression in Atlantic salmon <it>Salmo salar </it>L. exposed to a model toxicant. The stress response was induced by intraperitoneal injection of four salmon with a single dose (50 mg/kg) of the CYP1A inducer β-naphthoflavone (BNF) and intestinal tissue (mid and distal intestine; MI and DI) was sampled seven days later. Samples for histology and gene transcription analysis were collected from four exposed fish and four control fish. PCNA was assessed by immunohistochemistry, CYP1A mRNA was studied by <it>in situ </it>hybridization (ISH) and finally the transcription of five genes was quantified by real-time quantitative RT-PCR (real-time RT-PCR); two detoxifying genes (CYP1A and glutathione S-transferase; GST), a stress marker gene (heat shock protein 70; HSP70), PCNA and a gene marker of apoptosis (caspase 6A).</p> <p>Results</p> <p>PCNA protein and CYP1A mRNA were successfully localized in the intestinal cells (MI) of both experimental groups. At the cellular level, BNF significantly lowered intestinal cell proliferation and increased the CYP1A mRNA levels compared to the control group. The real-time RT-PCR data, which showed an increased mRNA expression both in the MI and DI of 139- and 62-fold, respectively, confirmed the increased cellular CYP1A mRNA levels detected using ISH. HSP70 expression was also up-regulated in the exposed fish. The other examined genes did not show any differential regulation in the experimental fish group.</p> <p>Conclusion</p> <p>This study showed that CYP1A mRNA had a specific intestinal cellular transcription pattern in Atlantic salmon exposed to BNF. At the cellular level CYP1A mRNA expression was always observed at or around the cell nucleus close to the basolateral cell membrane and at the tissue level CYP1A mRNA expression was most frequently observed in the basal and apex area of the intestinal folds. Taken together, a link between the intestinal detoxification system (CYP1A) and cell renewal system (PCNA) is indicated with these two processes being inversely correlated in BNF exposed fish.</p

Characterization of an Atlantic cod (Gadus morhua) embryonic stem cell cDNA library

<p>Abstract</p> <p>Background</p> <p>The Atlantic cod is an ecologically and economically important North Atlantic fish species and also an emerging aquaculture species. To study gene expression in Atlantic cod embryonic stem (ES) cells, our goal was to generate and analyze expressed sequence tags (ESTs) from an ES cell cDNA library of mRNA consisting of approximately 3,900 ESTs.</p> <p>Results</p> <p>We sequenced 3,935 EST clones using a directional cDNA library made from pooled ES cells harvested at the blastula stage. Quality filtering of these ESTs allowed identification of 2,719 high-quality sequences with an average length of 442 bp containing 368 contigs and 1,276 singletons (1,644 unique sequences). BLASTX searches produced 889 significant (E-value < 10^-3) hits, of which 698 (42.5%) were annotated with Gene Ontology terms (E-value < 10^-6). The number of unknown unique sequences was 946 (57.5%). All the high-quality EST sequences have been deposited in GenBank (GenBank: 2,719 sequences in UniGene library dbEST id: 22,021). Gene discovery and annotations are presented and discussed.</p> <p>Conclusion</p> <p>This set of ESTs represents one of the first attempts to describe mRNA in ES cells from a marine cold-water fish species, and provides a basis for gene expression studies of Atlantic cod ES cells.</p

In vitro toxicity of glyphosate in Atlantic salmon evaluated with a 3D hepatocyte-kidney co-culture model

publishedVersio

Mixture toxicity of chlorpyrifos-methyl, pirimiphos-methyl, and nonylphenol in Atlantic salmon (Salmo salar) hepatocytes

Pesticide formulations typically contain adjuvants added to enhance the performance of the active ingredient. Adjuvants may modify the bioavailability and toxicity of pesticides. In this study, the aim was to examine to which degree nonylphenol (NP) may interfere with the toxicity of two organophosphorus pesticides found in aquafeeds, chlorpyrifos-methyl (CPM) and pirimiphos-methyl (PPM). Atlantic salmon liver cells were exposed to these compounds singly or in combinations for 48 h using 3D cell cultures. Cytotoxicity, gene expression (RT-qPCR), and lipidomics endpoints were used to assess toxicity. The dose-response assessment showed that NP was the most toxic compound at equimolar concentrations (100 μM). Shotgun lipidomics pointed to a general pattern of elevated levels of saturated 18:0 fatty acids and declined levels of 18:1 monounsaturated fatty acids by the combined treatment. All three compounds had a distinct effect on membrane phospholipids, in particular on phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Lipid species patterns predicted inhibited stearoyl CoA desaturase (SCD) activity and increased Δ6 desaturase (D6D) activity in co-treated cells. While all three compounds alone mitigated increased triacylglycerol (TAG) accumulation, combined treatment resulted in lower total TAG in the cells. Multivariate analysis with PLS regression showed significant combined effects for nine genes (d5d, d6d, scd, srebf2, vtg, esr1, cyp1, ugt1a, and cat) and four lipid species (FFA 22:5, LPC 18:0, TAG52:1-FA16:0, and TAG52:1-FA18:0). In summary, this study demonstrates that the adjuvant can be the main contributor to the toxicity of a mixture of two organophosphorus pesticides with relatively low toxicity in fish cells.publishedVersio

Selection of reference genes for qRT-PCR examination of wild populations of Atlantic cod Gadus morhua

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Bioaccumulation of mercury and transcriptional responses in tusk (Brosme brosme), a deep-water fish from a Norwegian fjord

High concentrations of mercury (Hg) have been documented in deep-water fish species from some Norwegian fjords. In this study, tusk (Brosme brosme) was sampled from four locations in the innermost parts of Sognefjorden in Western Norway. Total Hg and methylmercury (MeHg) levels were measured in liver tissue. To search for potential sublethal effects of Hg, we characterized the hepatic transcriptome in tusk with high and low levels of Hg bioaccumulation using global transcriptomics analysis (RNA-seq). The results showed that there was a significant correlation between fish weight and accumulated concentrations of MeHg but not total Hg. MeHg accounted for 30–40% of total Hg in liver of most of the fish, although at concentrations above 2–3 mg Hg/kg wet weight the percentage of MeHg dropped considerably. Transcriptome analysis resulted in hundreds of differentially expressed genes in the liver of tusk with high Hg levels. Functional enrichment analysis suggested that the top affected pathways are associated with protein folding, adipogenesis, notch signaling, and lipid metabolism (beta-oxidation and phospholipids). Based on transcriptional responses pointing to well-known effects of Hg compounds in fish, the study suggests that tusk in Sognefjorden could be negatively impacted by Hg bioaccumulation.publishedVersio

The new England code : controlling female agency in contemporary American tv drama

Masteroppgave i engelsk - Universitetet i Agder 2008In an attempt to contextualize the DC observations, the overarching super-genre of melo-drama has been given particular attention. The relevance of melodrama is evident as the main ‘corpus’ of the survey, Cold Case, embodies a range of melodramatic traits. Furthermore, as will be seen in Chapters 1 and 3 particularly, melodramatic and Puritan value sets tend to overlap, especially in the attitude towards family. All chapters include analyses of individual Cold Case episodes, and each analysis comes with a description of scenes within the episode written in a slightly more subjective language. This has been done in order to provide the reader with an opportunity to assess the author’s subjective experience of each episode, which has bearing on the analysis. Chapter 1 introduces Cold Case, evaluating the show in terms of melodramatic content, comparing traits; drawing further comparisons between the show’s main character Lily Rush and the heroines of classical melodrama. Puritan attitudes towards sex and New England legislation are given a brief examination. In Chapter 2 the results of the survey are analyzed to determine whether the DC can be corroborated statistically, as well as presenting a few of the premises for the data collection. Chapter 3 examines the Victorian and Modern ‘fallen woman’ narratives, the former bearing close resemblance to the observations leading up to this thesis, entertaining a ‘death correspondence’ of its own. Chapter 3 also incorporates a discussion of values as they seem to be expressed through the DC, and a comparison between the early American ‘execution narrative’ and the way some DC occurrences in Cold Case are structured. Finally, Chapter 4 looks at the values expressed in the first American written code for upholding ‘decency’ in the movies, compounding it with examinations of how the DC relate to sex professionals and gay persons

Balancing Structure and Learning in an Open Prison

Leira Prison is a branch of Trondheim Prison, functioning as a relatively small,open prison with a maximum capacity of only 29 inmates. Leira Prison appliesthe method ‘consequence pedagogy.’ This article aims to pinpoint howconsequence pedagogy is executed at Leira. 50% of the Leira inmates arereleased back into society, while new ones enter the prison. It is thereforeinteresting to see how they balance structure and at the same time adjust tochanges, enabling Leira Prison to continue as a learning organization. This articleidentifies three items, consequence pedagogy and the view of humans,maintenance of the philosophy and coherence in the community, and selfregulationof justice through interaction. The use of consequence pedagogyis deeply aligned to their positive view of humans and has generated a constructiveorganization based on empowerment and involvement of both staffand inmates. Consequently, management, staff and inmates maintain thephilosophy of consequence pedagogy through interaction and self-regulation.However, questions regarding the fundamentals of the consequence pedagogyare not raised

Do anesthetics and sampling strategies affect transcription analysis of fish tissues?

<p>Abstract</p> <p>Background</p> <p>The aim of the current examination was to evaluate if sedation and anesthetic treatment techniques affect the quality of RNA extracted from liver, gill, head kidney and brain tissues in Atlantic salmon <it>Salmo salar </it>L. Blood parameters were measured and tissue specimens sampled in six groups of fish; one control group (0 minutes), two groups kept in pure seawater in 90 liter tanks for 30 and 120 minutes, two groups treated with the anesthetic isoeugenol for 30 and 120 minutes, and one group kept in pure seawater for 105 minutes and then anaesthetized with metacaine for 15 minutes. RNA quality was assessed with the NanoDrop ND-1000 spectrophotometer (260/280 and 260/230 nm ratios) and with the Agilent Bioanalyzer (28S/18S ratio and RIN data) in samples either preserved in liquefied nitrogen (N₂) or in RNA<it>later</it>. In addition, the transcriptional levels of two fast-responding genes were quantified in gill and brain tissues.</p> <p>Results</p> <p>The results show that physiological stress during sampling does not affect the quality of RNA extracted from fish specimens. However, prolonged sedation (2 hours) resulted in a metabolic alkalosis that again affected the transcriptional levels of genes involved in ionoregulation and respiration. In gills, <it>Na</it>⁺-<it>K</it>⁺-<it>ATPase α1b </it>was significantly downregulated and <it>hypoxia inducible factor 1 </it>(<it>HIF1</it>) significantly upregulated after two hours of treatment with isoeugenol, suggesting that this commonly used sedative affects osmo-regulation and respiration in the fish. The results also suggest that for tissue preservation in general it is better to flash-freeze fish specimens in liquefied N₂than to use RNA<it>later</it>.</p> <p>Conclusion</p> <p>Prolonged sedation may affect the transcription of fast-responding genes in tissues of fish. Two hours of sedation with isoeugenol resulted in downregulation of the <it>Na</it>⁺-<it>K</it>⁺-<it>ATPase α1b </it>gene and upregulation of the <it>HIF1 </it>gene in gills of Atlantic salmon. The quality of RNA extracted from tissue specimens, however, was not affected by sedation treatment. Flash-freezing of tissue specimens seems to be the preferred preservation technique, when sampling fish tissue specimens for RNA extraction.</p