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The Baltic Sea is among the most polluted seas worldwide. Anthropogenic contaminants are mainly introduced via riverine discharge and atmospheric deposition. Regional and international measures have successfully been employed to reduce concentrations of several legacy contaminants. However, current Baltic Sea monitoring programs do not address compounds of emerging concern. Hence, potentially harmful pharmaceuticals, UV filters, polar pesticides, estrogenic compounds, per- and polyfluoroalkyl substances, or naturally produced algal toxins are not taken into account during the assessment of the state of the Baltic Sea. Herein, we conducted literature searches based on systematic approaches and compiled reported data on these substances in Baltic Sea surface water and on methodological advances for sample processing and chemical as well as effect-based analysis of these analytically challenging marine pollutants. Finally, we provide recommendations for improvement of future contaminant and risk assessment in the Baltic Sea, which revolve around a combination of both chemical and effect-based analyses.

For half a decade, the Atlantic salmon in the Baltic Sea has been facing severe health issues. Clinical signs like haemorrhage, erosions and ulcerative/necrotic skin conditions in returning adults have been reported from different Swedish rivers. These primary disease signs precede a secondary, terminal fungal infection. As initial investigations of the disease did not provide conclusive answers regarding the pathogenesis, this study was initiated to gain insight into a possible link between this so‐called Red Skin Disease and anthropogenic influences. Therefore, returning salmon were caught in rivers along the Swedish coast and different tissues were sampled. The focus was put on the measurements of a battery of biomarkers as well as biochemical and haematological parameters, which were analysed using multivariate statistics. The main findings were a severe osmotic haemodilution, an immune response and an alteration of the carbohydrate metabolism in diseased fish. Furthermore, oxidative stress does not seem to be a likely factor in the pathogenesis. Concluding, certain changes in physiological parameters were shown to be indicative for the disease patterns, while others were ruled out as significant factors. Thus, this study contributes to the understanding of the Red Skin Disease and may act as a hypothesis generator for future studies.

Bisphenol A is a widely used compound found in large amount of consumer products. As concerns have been raised about its toxicological and public health effect, the use of alternatives to bisphenol A are now increasing. Bisphenol S is one of the analogues being used as a replacement for bisphenol A despite the fact that little is known about the effects of bisphenol S on living organisms. In this study, we investigated the potential endocrine and genotoxic effects of bisphenol A and bisphenol S in juvenile brown trout (Salmo trutta). The fish were exposed to the compounds for either 2 weeks or 8 weeks via sustained-release cholesterol implants containing doses of 2 mg/kg fish or 20 mg/kg fish of the substances. The effects on the thyroid hormone levels and the estrogenic disrupting marker vitellogenin were evaluated, along with the genotoxic markers micronucleated cells and erythrocyte nuclear abnormalities. An increase in plasma vitellogenin was observed in fish exposed to the high dose of bisphenol A for 2 weeks. At this experimental time the level of the thyroid hormone triiodothyronine (T3) in plasma was elevated after bisphenol S exposure at the high concentration, and paralleled by an increase of micronucleated cells. Moreover, bisphenol A induced an increase of micronuclei frequency in fish erythrocytes after the exposure at the lowest dose tested. Taken together the results indicate that both bisphenol A and its alternative bisphenol S cause endocrine disrupting and genotoxic effects in brown trout, although suggesting two different mechanisms of damage underlying bisphenol A and bisphenol S activity.

The textile industry, while of major importance in the world economy, is a toxic industry utilizing and emitting thousands of chemical substances into the aquatic environment. The aim of this project was to study the potentially harmful effects associated with the leaching of chemical residues from three different types of textiles: sportswear, children’s bath towels, and denim using different fish models (cell lines, fish larvae and juvenile fish). A combination of in vitro and in vivo test systems was used. Numerous biomarkers, ranging from gene expression, cytotoxicity and biochemical analysis to behavior, were measured to detect effects of leached chemicals. Principle findings indicate that leachates from all three types of textiles induced cytotoxicity on fish cell lines (RTgill-W1). Leachates from sportswear and towels induced mortality in zebrafish embryos, and chemical residues from sportswear reduced locomotion responses in developing larval fish. Sportswear leachate increased Cyp1a mRNA expression and EROD activity in liver of exposed brown trout. Leachates from towels induced EROD activity and VTG in rainbow trout, and these effects were mitigated by the temperature of the extraction process. All indicators of toxicity tested showed that exposure to textile leachate can cause adverse reactions in fish. These findings suggested that chemical leaching from textiles from domestic households could pose an ecotoxicological threat to the health of the aquatic environment.

Ingestion has been proposed as a prominent exposure route for plastic debris in aquatic organisms, including fish. While the consequences of ingestion of large plastic litter are mostly understood, the impacts resulting from ingestion of microplastics (MPs) are largely unknown. We designed a study that aimed to assess impacts of MPs on fish intestinal physiology and examined integrity of extrinsic, physical and immunological barriers. Rainbow trout were exposed to polystyrene (PS) MPs (100-400 μm) via feed for a period of 4 weeks. Fish were fed four types of diets: control, diets containing virgin PS particles, or particles exposed to two different environmental matrices (sewage or harbor effluent). Extrinsic barrier disturbance in intestinal tissue was evaluated via histology. The paracellular permeability toward ions and molecules was examined using Ussing chambers and mRNA expression analysis of tight junction proteins. Active transport was monitored as transepithelial potential difference, short-circuits current and uptake rate of amino acid

The environment contains a multitude of man-made chemicals, some of which can act as endocrine disruptors (EDCs), while others can be immunotoxic. We evaluated thyroid disruption and immunotoxic effects in wild female perch (Perca fluviatilis) collected from two contaminated areas in Sweden; one site contaminated with perand polyfluoroalkyl substances (PFASs) and two sites contaminated with polychlorinated biphenyls (PCBs), with one reference site included for each area. The hepatic mRNA expression of thyroid receptors alpha and beta, and the thyroid hormone metabolising iodothyronine deiodinases (dio1, dio2 and dio3) were measured using real-time PCR, while the levels of thyroid hormone T3 in plasma was analysed using a radioimmunoassay. In addition, lymphocytes, granulocytes, and thrombocytes were counted microscopically. Our results showed lower levels of T3 as well as lower amounts of lymphocytes and granulocytes in perch collected from the PFAS-contaminated site compared to reference sites. In addition, expressions of mRNA coding for thyroid hormone metabolising enzymes (dio2 and dio3) and thyroid receptor alpha (thra) were significantly different in these fish compared to their reference site. For perch collected at the two PCB-contaminated sites, there were no significant differences in T3 levels or in expression levels of the thyroid-related genes, compared to the reference fish. Fish from one of the PCB-contaminated sites had higher levels of thrombocytes compared with both the second PCB lake and their reference lake; hence PCBs are unlikely to be the cause of this effect. The current study suggests that lifelong exposure to PFASs could affect both the thyroid hormone status and immune defence of perch in the wild.

Perch (Perca fluviatilis) has been used in biological effect monitoring in a program for integrated coastal fish monitoring at the reference site Kvädöfjärden along the Swedish east coast, which is a site characterized by no or minor local anthropogenic influences. Using a set of physiological and biochemical endpoints (i.e., biomarkers), clear time trends for “early warning” signs of impaired health were noted in the perch from this site, possibly as a result of increased baseline pollution. The data sets also showed relatively large variations among years. To identify additional temporal variation in biological parameters, global mRNA expression studies using RNA sequencing was performed. Perch collected in 2010 and 2014 were selected, as they showed variations in several biomarkers, such as the activity of the detoxification enzyme CYP1A (EROD), the plasma levels of vitellogenin, markers for oxidative stress, white blood cells count and gonad sizes. The RNA sequencing study identified approximately 4800 genes with a significantly difference in mRNA expression levels. A gene ontology enrichment analysis showed that these differentially expressed genes were involved in biological processes such as complement activation, iron ion homeostasis and cholesterol biosynthetic process. In addition, differences in immune system parameters and responses to the exposure of toxic substances have now been verified in two different biological levels (mRNA and protein) in perch collected in 2010 and 2014. Markedly higher mRNA expression of the membrane transporter (MATE) and the detoxification enzyme COMT, together with higher concentrations of bioactive naturally produced brominated compounds, such as brominated indoles and carbazoles, seem to indicate that the perch collected in 2014 had been exposed to macro- and microalga blooming to a higher degree than did perch from 2010. These results and the differential mRNA expression between the 2 years in genes related to immune and oxidative stress parameters suggest that attention must be given to algae blooming when elucidating the well-being of the perch at Kvädöfjärden and other Baltic coastal sites.

Ocean acidification and rising seawater temperature are environmental stressors resulting from the continuous increase of the atmospheric CO2 concentration due to anthropogenic activities. As a consequence, marine fish are expected to undergo conditions outside of their tolerance range, leading to physiological challenges with possible detrimental implications. Our research group has previously shown that exposure to elevated CO2 modulated the immune system of the Atlantic halibut. To further investigate this finding, we analysed non-specific immune components in blood plasma of Atlantic halibut (Hippoglossus hippoglossus) juveniles acclimated to six different temperatures (5, 10, 12, 14, 16 and 18 °C), and to water pH of 8.0 (control) or 7.6 (predicted for year 2100) for three months. Plasma ions (K+, Na+, Ca++, Cl-) and lactate concentrations were also measured. The analysis of plasma ions did not show any trends related to temperature or CO2 exposure, and the majority of the experimental fish were able to maintain ionic balance. The results show that both innate immune components (lysozyme and alternative complement system) had increased activities in response to elevated CO2, representing a CO2-related impact on the halibut's immune system. The increased activity of lysozyme and complement system is possibly part of the acclimatization process, and might be protective.

The presence of diclofenac in the aquatic environment and the risks for aquatic wildlife, especially fish, have been raised in several studies. One way to manage risks without enforcing improved wastewater treatment would be to substitute diclofenac (when suitable from a clinical perspective) with another non-steroidal anti-inflammatory drug (NSAID) associated with less environmental risk. While there are many ecotoxicity-studies of different NSAIDs, they vary extensively in set-up, species studied, endpoints and reporting format, making direct comparisons difficult. We previously published a comprehensive study on the effects of diclofenac in the three-spined stickleback (Gasterosteus aculeatus). Our present aim was to generate relevant effect data for another NSAID (naproxen) using a very similar setup, which also allowed direct comparisons with diclofenac regarding hazards and risks. Sticklebacks were therefore exposed to naproxen in flow-through systems for 27 days. Triplicate aquaria with 20 fish per aquarium were used for each concentration (0, 18, 70, 299 or 1232 μg/L). We investigated bioconcentration, hepatic gene expression, jaw lesions, kidney and liver histology. On day 21, mortalities in the highest exposure concentration group unexpectedly reached ≥ 25 % in all three replicate aquaria, leading us to terminate and sample that group the same day. On the last day (day 27), the mortality was also significantly increased in the second highest exposure concentration group. Increased renal hematopoietic hyperplasia was observed in fish exposed to 299 and 1232 μg/L. This represents considerably higher concentrations than those expected in surface waters as a result of naproxen use. Such effects were observed already at 4.6 μg/L in the experiment with diclofenac (lowest tested concentration). Similar to the responses to diclofenac, a concentration-dependent increase in both relative hepatic gene expression of c7 (complement component 7) and jaw lesions were observed, again at concentrations considerably higher than expected in surface waters. Naproxen bioconcentrated less than diclofenac, in line with the observed effect data. An analysis of recent sales data and reported concentrations in treated sewage effluent in Sweden suggest that despite higher dosages used for naproxen, a complete substitution would only be expected to double naproxen emissions. In summary, naproxen and diclofenac produce highly similar effects in fish but the environmental hazards and risks are clearly lower for naproxen. Hence, if there are concerns for environmental risks to fish with diclofenac, a substitution would be advisable when naproxen presents an adequate alternative from a clinical point-of-view.

Global climate change is predicted to result in increases in water temperature in the polar regions, but the full consequences of this for marine fish species are not understood, especially with regard to cellular mechanisms underlying oxidative stress. Warmer temperatures could potentially result in increased oxidative stress, and it is not known whether stenothermal fish can cope with this on a cellular and physiological level. In order to address this, we exposed bald notothen ( Pagothenia borchgrevinki ), a fish species endemic to Antarctica, to an increase in temperature from − 1.6 °C to 4 °C and measured the effects on oxidative stress including antioxidant defenses, oxidative damage in proteins and lipids, and transcriptional regulation of involved genes. We show that the fish responds to an acute (12 h) temperature increase with increased antioxidant defenses. However, these antioxidant defenses were similar to basal levels following long-term (3 weeks) exposure to the higher temperature and moreover, these individuals also had higher levels of oxidative damage. These results indicate that this species has the ability to alter levels of endogenous antioxidants, but that this response is transient and insufficient to protect against oxidative damage. These effects may have serious consequences for these fish in a warmer future since long-term consequences of this accumulation of damaged lipids and proteins are associated with aging and known to include decreased cellular function, disease and eventually cell death.

Medetomidine is under evaluation for use as an antifouling agent, and its effects on non-target aquatic organisms are therefore of interest. In this study, rainbow trout was exposed to low (0.5 and 5.0nM) concentrations of medetomidine for up to 54 days. Recently we have reported on effects on paleness and melanophore aggregation of medetomidine in these fish. Here, specific growth rates were investigated together with a broad set of physiological parameters including plasma levels of growth hormone (GH), insulin-like growth factor-I (IGF-I) and leptin, glucose and haemoglobin (Hb), hematocrit (Ht), condition factor, liver and heart somatic indexes (LSI, HSI). Hepatic enzyme activities of CYP1A (EROD activity), glutathione S-transferases (GST) and glutathione reductase (GR) were also measured. Additionally, hepatic mRNA expression was analysed through microarray and quantitative PCR in fish sampled after 31 days of exposure. Medetomidine at both concentrations significantly lowered blood glucose levels and the higher concentration significantly reduced the LSI. The mRNA expression analysis revealed few differentially expressed genes in the liver and the false discovery rate was high. Taken together, the results suggest that medetomidine at investigated concentrations could interfere with carbohydrate metabolism of exposed fish but without any clear consequences for growth.

To increase our understanding of possible chemical impacts on coastal fish populations in the Baltic Sea, Kattegat and Skagerrak, the viviparous eelpout (Zoarces viviparus) was used as sentinel species in two major sampling campaigns (spring and autumn) in 16 different coastal sites. Condition factor (CF), liver somatic index (LSI), gonad somatic index (GSI) were measured and the activity of the hepatic enzymes ethoxyresorufin-O-deethylase (EROD), glutathione reductase GR), glutathione S-transferase (GST), catalase (CAT) and muscular activity of acetylcholinesterase (AChE) were assessed. PAH metabolites in bile were also analyzed. The most notable finding in the data set was the low EROD activity in eelpouts collected at the relatively polluted region in Germany compared to the other regions, which could be due to an inhibition of the CYP1A-system or to adaptation to chronic exposure of pollutants in this area. Additionally, low AChE activity was noted in the German region in the autumn campaign and low AChE activity detected in the Danish region in the spring campaign. These differences suggest possible season-specific differences in the use and release of AChE-inhibiting chemicals in the Danish and German regions. Clustering of biomarkers on site level indicated a relationship between CF and GSI and suggested that sites with a high CF contained eelpout that put a larger effort into their larvae development. Clustering of the oxidative stress markers GR, GST and CAT on the individual level reflected a possible coordinated regulation of these enzymes. Overall, the results support the importance of taking into account general regional differences and seasonal variation in biomarker activity when monitoring and assessing the effects of pollution. Despite the expected seasonal variation for most of the measured endpoint, several markers (GSI, EROD and CF) vary similarly between all selected sites in both spring and autumn. This suggests that the differences between sites for these endpoints are independent of season.

The entire cDNA corresponding to the C-terminal cysteine-rich domain of the human MUC2 apomucin, after the serine- and threonine-rich tandem repeat, was expressed in Chinese-hamster ovary-K1 cells and in the human colon carcinoma cell line, LS 174T. The C-terminus was expressed as a fusion protein with the green fluorescent protein and mycTag sequences and the murine immunoglobulin κ-chain signal sequence to direct the protein to the secretory pathway. Pulse–chase studies showed a rapid conversion of the C-terminal monomer into a dimer in both Chinese-hamster ovary-K1 and LS 174T cells. Disulphide-bond-stabilized dimers secreted into the media of both cell lines had a higher apparent molecular mass compared with the intracellular forms. The MUC2 C-terminus was purified from the spent culture medium and visualized by molecular electron microscopy. The dimer nature of the molecule was visible clearly and revealed that each monomer was attached to the other by a large globular domain. Gold-labelled antibodies against the mycTag or green fluorescent protein revealed that these were localized to the ends opposite to the parts responsible for the dimerization. The C-terminus expressed in LS 174T cells formed heterodimers with the full-length wild-type MUC2, but not with the MUC5AC mucin, normally expressed in LS 174T cells. The homodimers of the MUC2 C-termini were secreted continuously from the LS 174T cells, but no wild-type MUC2 secretion has been observed from these cells. This suggests that the information for sorting the MUC2 mucin into the regulated secretory pathway in cells having this ability is present in parts other than the C-terminus of MUC2.

A broad biomarker approach was applied to study the effects of marine pollution along the Swedish west coast using the teleost eelpout (Zoarces viviparus) as the sentinel species. Measurements were performed on different biological levels, from the molecular to the organismal, including measurements of messenger RNA (mRNA), proteins, cellular and tissue changes, and reproductive success. Results revealed that eelpout captured in Stenungsund had significantly higher hepatic ethoxyresorufin O‐deethylase activity, high levels of both cytochrome P4501A and diablo homolog mRNA, and high prevalence of dead larvae and nuclear damage in erythrocytes. Eelpout collected in Göteborg harbor displayed extensive macrovesicular steatosis, whereby the majority of hepatocytes were affected throughout the liver, which could indicate an effect on lipid metabolism. Results also indicate that eelpouts collected at polluted sites might have an affected immune system, with lower mRNA expression of genes involved in the innate immune system and a higher number of lymphocytes. Biomarker assessment also was performed on livers dissected from unborn eelpout larvae collected from the ovary of the females. No significant differences were noted, which might indicate that the larvae to some extent are protected from effects of environmental pollutants. In conclusion, usage of the selected set of biological markers, covering responses from gene to organism, has demonstrated site‐specific biomarker patterns that provided a broad and comprehensive picture of the impact of environmental stressors. Environ Toxicol Chem 2015;34:1511–1523. © 2015 The Authors. Published by SETAC.

Loss of pericytes from the capillary wall is a hallmark of diabetic retinopathy, however, the pathogenic significance of this phenomenon is unclear. In previous mouse gene knockout models leading to pericyte deficiency, prenatal lethality has so far precluded analysis of postnatal consequences in the retina. We now report that endothelium-restricted ablation of platelet-derived growth factor-B generates viable mice with extensive inter- and intra-individual variation in the density of pericytes throughout the CNS. We found a strong inverse correlation between pericyte density and the formation of a range of retinal microvascular abnormalities strongly reminiscent of those seen in diabetic humans. Proliferative retinopathy invariably developed when pericyte density was

The colon adenocarcinoma cell line COLO 205 secretes L-CanAg, a mucin-like glycoprotein carrying the carcinoma-associated sialyl-Lewis a carbohydrate epitope. In an attempt to identify its apoprotein, an NH2-terminal peptide sequence was obtained from purified L-CanAg. In all interpretable positions, this sequence showed 100% identity to the NH2-terminal of human CD43 (leukosialin, sialophorin), a plasma membrane-bound sialoglycoprotein hitherto only identified in leukocytes and other hematopoietic cells. An antiserum against deglycosylated L-CanAg and an anti-CD43 antiserum both immunoprecipitated a 61-kDa band, interpreted as the CD43 precursor, from COLO 205 cells as well as from the known CD43-expressing cell line HL-60. Results from immunoprecipitations following pulse-chase experiments and tunicamycin treatments were in agreement with earlier studies on the CD43 precursor. RNA blot analysis confirmed the expression of CD43 by the COLO 205 cell line, whereas three other colon carcinoma cell lines were negative. The glycosylation-dependent monoclonal antibody Leu-22, which recognizes leukocyte CD43, failed to bind L-CanAg, probably due to its much more extensive glycosylation. We conclude that L-CanAg is the secreted extracellular domain of a novel glycoform of CD43 and that CD43, if expressed in other carcinoma cells, may have escaped notice in studies relying on glycosylation-dependent monoclonal antibodies against leukocyte CD43.

The physiology and reproductive performance of eelpout (Zoarces viviparus) have been monitored along the Swedish coast for more than three decades. In this study, transcriptomic profiling was applied for the first time as an exploratory tool to search for new potential candidate biomarkers and to investigate possible stress responses in fish collected from a chronically polluted area. An oligonucleotide microarray with more than 15,000 sequences was used to assess differentially expressed hepatic mRNA levels in female eelpout collected from the contaminated area at Göteborg harbor compared to fish from a national reference site, Fjällbacka. Genes involved in apoptosis and DNA damage (e.g., SMAC/diablo homolog and DDIT4/DNA-damage-inducible protein transcript 4) had higher mRNA expression levels in eelpout from the harbor compared to the reference site, whereas mRNA expression of genes involved in the innate immune system (e.g., complement components and hepcidin) and protein transport/folding (e.g., signal recognition particle and protein disulfide-isomerase) were expressed at lower levels. Gene Ontology enrichment analysis revealed that genes involved biological processes associated with protein folding, immune responses and complement activation were differentially expressed in the harbor eelpout compared to the reference site. The differential mRNA expression of selected genes involved in apoptosis/DNA damage and in the innate immune system was verified by quantitative PCR, using the same fish in addition to eelpout captured four years later. Thus, our approach has identified new potential biomarkers of pollutant exposure and has generated hypotheses on disturbed physiological processes in eelpout. Despite a higher mRNA expression of genes related to apoptosis (e.g., diablo homolog) in eelpout captured in the harbor there were no significant differences in the number of TUNEL-positive apoptotic cells between sites. The mRNA level of genes involved in apoptosis/DNA damage and the status of the innate immune system in fish species captured in polluted environments should be studied in more detail to lay the groundwork for future biomonitoring studies.

Diclofenac is a nonsteroidal anti-inflammatory drug frequently found in the aquatic environment. Previous studies have reported histological changes in the liver, kidney, and gills of fish at concentrations similar to those measured in treated sewage effluents (approximately 1 µg/L). Analyses or predictions of blood plasma levels in fish allow a direct comparison with human therapeutic plasma levels and may therefore be used to indicate a risk for pharmacological effects in fish. To relate internal exposure to a pharmacological interaction, we investigated global hepatic gene expression together with bioconcentration in blood plasma and liver of rainbow trout (Oncorhynchus mykiss) exposed to waterborne diclofenac. At the highest exposure concentration (81.5 µg/L), the fish plasma concentration reached approximately 88% of the human therapeutic levels (C(max) ) after two weeks. Using an oligonucleotide microarray followed by quantitative PCR, we found extensive effects on hepatic gene expression at this concentration, and some genes were found to be regulated down to the lowest exposure concentration tested (1.6 µg/L), corresponding to a plasma concentration approximately 1.5% of the human C(max) . Thus, at concentrations detected in European surface waters, diclofenac can affect the expression of multiple genes in exposed fish. Functional analysis of differentially expressed genes revealed effects on biological processes such as inflammation and the immune response, in agreement with the mode of action of diclofenac in mammals. In contrast to some previously reported results, the bioconcentration factor was found to be stable (4.02 ± 0.75 for blood plasma and 2.54 ± 0.36 for liver) regardless of the water concentration. Environ. Toxicol. Chem. 2011;30:2126-2134. © 2011 SETAC.

SummaryWe combined reverse and chemical genetics to identify targets and compounds modulating blood vessel development. Through transcript profiling in mice, we identified 150 potentially druggable microvessel-enriched gene products. Orthologs of 50 of these were knocked down in a reverse genetic screen in zebrafish, demonstrating that 16 were necessary for developmental angiogenesis. In parallel, 1280 pharmacologically active compounds were screened in a human cell-based assay, identifying 28 compounds selectively inhibiting endothelial sprouting. Several links were revealed between the results of the reverse and chemical genetic screens, including the serine/threonine (S/T) phosphatases ppp1ca, ppp1cc, and ppp4c and an inhibitor of this gene family; Endothall. Our results suggest that the combination of reverse and chemical genetic screens, in vertebrates, is an efficient strategy for the identification of drug targets and compounds that modulate complex biological systems, such as angiogenesis.

Here we report a new isolation method for mouse glomeruli. The method is fast and simple and allows for the isolation of virtually all glomeruli present in the adult mouse kidney with minimal contamination of nonglomerular cells. Mice were perfused through the heart with magnetic 4.5- micro m diameter Dynabeads. Kidneys were minced into small pieces, digested by collagenase, filtered, and collected using a magnet. The number of glomeruli retrieved from one adult mouse was 20,131 +/- 4699 (mean +/- SD, n = 14) with a purity of 97.5 +/- 1.7%. The isolated glomeruli retained intact morphology, as confirmed by light and electron microscopy, as well as intact mRNA integrity, as confirmed by Northern blot analysis. The method was applicable also to newborn mice, which allows for the isolation of immature developmental stage glomeruli. This method makes feasible transcript profiling and proteomic analysis of the developing, healthy and diseased mouse glomerulus.

Biosynthetic studies on the human MUC5AC mucin were performed by immunoprecipitations with antisera recognizing only the non-O-glycosylated apomucin in the colon adenocarcinoma cell line LS 174T. Pulse–chase studies and subcellular fractionations showed that MUC5AC formed dimers in the rough endoplasmic reticulum within 15 min of the initiation of biosynthesis. No non-O-glycosylated species larger than dimers were identified. The dimerization was N-glycosylation-dependent, because tunicamycin treatment significantly lowered the rate of dimerization. When the biosynthesis of MUC5AC apomucin was compared with that of MUC2 apomucin, also produced in the LS 174T cell line, both apomucins were assembled in similar ways with respect to their rates of dimerization with and without inhibition of N-glycosylation. No heterodimerization was observed between the human MUC5AC and the MUC2 apomucins despite the extensive sequence similarities in the positions of the cysteine residues in the C-termini proposed to be involved in mucin dimerization.

Pulse-chase experiments in the colon cell line LS 174T combined with subcellular fractionation by sucrose density gradient centrifugation showed that the initial dimerization of the MUC2 apomucin started directly after translocation of the apomucin into the rough endoplasmic reticulum as detected by calnexin reactivity. As the mono- and dimers were chased, O-glycosylated MUC2 mono- and dimers were precipitated using an O-glycosylation-insensitive antiserum against the N-terminal domain of the MUC2 mucin. These O-glycosylated species were precipitated from the fractions that comigrated with the galactosyltransferase activity during the subcellular fractionation, indicating that not only MUC2 dimers but also a significant amount of monomers are transferred into the Golgi apparatus. Inhibition of N-glycosylation with tunicamycin treatment slowed down the rate of dimerization and introduced further oligomerization of the MUC2 apomucin in the endoplasmic reticulum. Results of two-dimensional gel electrophoresis demonstrated that these oligomers (putative tri- and tetramers) were stabilized by disulfide bonds. The non-N-glycosylated species of the MUC2 mucin were retained in the endoplasmic reticulum because no O-glycosylated species were precipitated after inhibition by tunicamycin. This suggests that N-glycans of MUC2 are necessary for the correct folding and dimerization of the MUC2 mucin.

The MUC2 mucin is the major gel-forming mucin in the small and large intestine. Due to its sequence similarities with the von Willebrand factor, it has been suggested to dimerize in the endoplasmic reticulum and polymerize in the trans-Golgi network. Using an O-glycosylation-sensitive MUC2 antiserum, a dimerization has been shown to occur in the endoplasmic reticulum of LS 174T cells (Asker, N., Axelsson, M. A. B., Olofsson, S.-O., and Hansson, G. C. (1998) J. Biol. Chem. 273, 18857-18863). Using an antiserum immunoprecipitating O-glycosylated MUC2 mucin, monomers and dimers were shown to occur in soluble form in the lysate of LS 174T cells. The amount of O-glycosylated dimer was small, and no larger species were found even after long chase periods. However, most of the labeled MUC2 mucin was found in pelleted debris of the cell lysate. This insoluble MUC2 mucin was recovered by immunoprecipitation after reduction of disulfide bonds. Analysis by agarose gel electrophoresis revealed two bands, of which the smaller migrated as the O-glycosylated monomer and the larger migrated as the O-glycosylated dimer of the cell lysis supernatant. Mucins insoluble in 6 M guanidinium chloride could also be obtained from LS 174T cells. Such mucins have earlier been found in the small intestine (Carlstedt, I., Herrmann, A., Karlsson, H., Sheehan, J., Fransson, L. -A., and Hansson, G. C. (1993) J. Biol. Chem. 268, 18771-18781). Reduction of the mucins followed by purification by isopycnic density gradient ultracentrifugation and analysis by agarose gel electrophoresis revealed two bands reacting with an anti-MUC2 tandem repeat antibody after deglycosylation. These bands migrated identically to the bands shown by metabolic labeling, and they could also be separated by rate zonal ultracentrifugation. These results suggest that the MUC2 mucin is forming nonreducible intermolecular bonds early in biosynthesis, but after initial O-glycosylation.

The largest high-glycosylated domain, glycopeptide A, of the "insoluble' mucin complex of the rat small intestine has earlier been purified and characterized (Carlstedt et al., 1993, J Biol Chem 268: 18771-81). A rabbit antiserum raised against deglycosylated glycopeptide A was used to clone part of a mucin showing homology to the human MUC2 mucin (Hansson et al., 1994, Biochem Biophys Res Commun 198. 181-90). This serum specifically stained goblet cells (paranuclear) in the mouse small intestine. The size of the coding sequence of glycopeptide A was estimated by using reversed transcriptase PCR of mRNA from an inbred rat strain (GOT-W) using primers in the unique central and C-terminal parts of the proposed rat Muc2 sequences. The PCR and Southern blot of the PCR products showed a fragment of about 5.5 kb corresponding to about 1700 amino acids when the known Cys-rich sequences used for the primers were subtracted. This is slightly larger than the size estimated earlier by biochemical studies. The mRNA encoding the rat Muc2 was slightly smaller than the mRNA encoding the human MUC2 in a colorectal cell line. Although the size of glycopeptide A estimated from biochemical results and by PCR is not identical, the results obtained here further support that the "insoluble' mucin of the rat small intestine is encoded by the Muc2 gene. Most of the oligosaccharides in glycopeptide A were either neutral (40%) or sialylated (40%). The remaining ones were sulfated with the sulfate group attached to C-6 of N-acetylglucosamine linked to C-6 of the N-acetylgalactosaminitol as revealed by tandem mass spectrometry of the perdeuteroacetylated oligosaccharides. Eighteen oligosaccharides were found of which fourteen were characterized and found to be mostly novel. Our findings thus expand the current knowledge of the core peptide of the rat intestinal goblet cell mucin and provide a relatively complete picture of the glycosylation of a defined mucin domain.

Rabbit antiserum against a synthetic peptide corresponding to a tandemly repeated amino acid sequence in the human intestinal mucin apoprotein MUC2 was used in immunoprecipitation to study the biosynthesis of MUC2 in the colon-carcinoma cell line LS 174T. Under non-reducing conditions, two bands were precipitated, the smaller with an apparent size of about 700 kDa on SDS/PAGE. When analysed by two-dimensional electrophoresis after reduction, the larger band migrated to the same position as the smaller band and was interpreted as a putative disulphide-bond-stabilized dimer. Pulse-chase experiments showed only the monomer after 5 min and the appearance of the putative dimer after 30 min. The MUC2 apoprotein was also precipitated by antisera against the HF-deglycosylated peptides of the two highly glycosylated domains of the ‘insoluble’ mucin complex of rat small intestine [Carlstedt, Herrmann, Karlsson, Sheehan, Fransson and Hansson (1993) J. Biol. Chem. 268, [18771-18781]. Endoprotease Lys-C cleavage of the immunopurified apoprotein gave a large fragment of about 250 kDa that was detected by both the antiserum against the MUC2 tandem repeat and one of the glycopeptide antisera. This supports the view that the ‘insoluble’ mucin of rat small intestine is encoded by the Muc2 gene, as recently indicated by a partial cDNA sequence [Hansson, Baeckström, Carlstedt and Klinga-Levan (1994) Biochem. Biophys. Res. Commun. 198, 181-190] and that parts of the apoprotein are conserved between the species. A lectin from the snail Helix pomatia that detects terminal alpha-GalNAc residues did not bind to the monomer or putative dimer, suggesting that O-glycosylation starts after dimerization. The results indicate that the biosynthetic pathway of the MUC2 mucin may be similar to that of the von Willebrand factor with which MUC2 shares sequence similarities at its C- and N-termini.

Background The teleost Zoarces viviparus (eelpout) lives along the coasts of Northern Europe and has long been an established model organism for marine ecology and environmental monitoring. The scarce information about this species genome has however restrained the use of efficient molecular-level assays, such as gene expression microarrays. Results In the present study we present the first comprehensive characterization of the Zoarces viviparus liver transcriptome. From 400,000 reads generated by massively parallel pyrosequencing, more than 50,000 pieces of putative transcripts were assembled, annotated and functionally classified. The data was estimated to cover roughly 40% of the total transcriptome and homologues for about half of the genes of Gasterosteus aculeatus (stickleback) were identified. The sequence data was consequently used to design an oligonucleotide microarray for large-scale gene expression analysis. Conclusion Our results show that one run using a Genome Sequencer FLX from 454 Life Science/Roche generates enough genomic information for adequate de novo assembly of a large number of genes in a higher vertebrate. The generated sequence data, including the validated microarray probes, are publicly available to promote genome-wide research in Zoarces viviparus.