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Globicephala melas has been harvested in the Faroe Islands for centuries. Given the distances travelled by this species, tissue/body fluid samples represent unique matrices to be considered as an integration of environmental condition and pollution status of their prey. For the first time, bile samples were analysed for presence of polycyclic aromatic hydrocarbon (PAH) metabolites and protein content. Concentrations of 2- and 3-ring PAH metabolites ranged from 11 to 25 μg mL−1 pyrene fluorescence equivalents. In total, 658 proteins were identified and 61,5 % were common amongst all individuals. Identified proteins were integrated into in silico software and determined that the top predicted disease and functions were neurological diseases, inflammation, and immunological disorders. The metabolism of reactive oxygen species (ROS) was predicted to be dysregulated, which can have consequences to both the protection against ROS produced during dives and contaminant exposures. The obtained data is valuable for understanding metabolism and physiology of G. melas.

The Atlantic herring Clupea harengus L has a vast geographical distribution and a complex population structure with a few very large migratory units and many small local populations. Each population has its own spawning ground and/or time, thereby maintaining their genetic integrity. Several herring populations migrate between common feeding grounds and over-wintering areas resulting in frequent mixing of populations. Thus, many herring fisheries are based on mixed populations of different demographic status. In order to avoid overexploitation of weak populations and to conserve biodiversity, understanding the population structure and population mixing is important for maintaining biologically sustainable herring fisheries. The aim of this study was to investigate the genetic population structure of herring in the Faroese and surrounding waters, and to develop genetic markers for distinguishing between four herring management units (often called stocks), namely the Norwegian spring-spawning herring (NSSH), Icelandic summer-spawning herring (ISSH), North Sea autumn-spawning herring (NSAH), and Faroese autumn-spawning herring (FASH). Herring from the four stocks were sequenced at low coverage, and single nucleotide polymorphisms (SNPs) were called and used for population structure analysis and individual assignment. An ancestry-informative SNP panel with 118 SNPs was developed and tested on 240 individuals. The results showed that all four stocks appeared to be genetically differentiated populations, but at lower levels of differentiation between FASH and ISSH than the other two populations. Overall assignment rate with the SNP panel was 80.7%, and agreement between the genetic and traditional visual assignment was 75.5%. The NSAH and NSSH samples had the highest assignment rate (100% and 98.3%, respectively) and highest agreement between traditional and genetic assignment methods (96.6% and 94.9%, respectively). The FASH and ISSH samples had substantially lower assignment rates (72.9% and 51.7%, respectively) and agreement between traditional and genetic methods (39.5% and 48.4%, respectively)

Background Based on an initial collecting of database sequences from the gap junction protein gene family (also called connexin genes) in a few teleosts, the naming of these sequences appeared variable. The reasons could be (i) that the structure in this family is variable across teleosts, or (ii) unfortunate naming. Rather clear rules for the naming of genes in fish and mammals have been outlined by nomenclature committees, including the naming of orthologous and ohnologous genes. We therefore analyzed the connexin gene family in teleosts in more detail. We covered the range of divergence times in teleosts (eel, Atlantic herring, zebrafish, Atlantic cod, three-spined stickleback, Japanese pufferfish and spotted pufferfish; listed from early divergence to late divergence). Results The gene family pattern of connexin genes is similar across the analyzed teleosts. However, (i) several nomenclature systems are used, (ii) specific orthologous groups contain genes that are named differently in different species, (iii) several distinct genes have the same name in a species, and (iv) some genes have incorrect names. The latter includes a human connexin pseudogene, claimed as GJA4P, but which in reality is Cx39.2P (a delta subfamily gene often called GJD2like). We point out the ohnologous pairs of genes in teleosts, and we suggest a more consistent nomenclature following the outlined rules from the nomenclature committees. We further show that connexin sequences can indicate some errors in two high-quality chromosome assemblies that became available very recently. Conclusions Minimal consistency exists in the present practice of naming teleost connexin genes. A consistent and unified nomenclature would be an advantage for future automatic annotations and would make various types of subsequent genetic analyses easier. Additionally, roughly 5% of the connexin sequences point out misassemblies in the new high-quality chromosome assemblies from herring and cod.

Skerpikjøt is a traditionally ripened sheep leg product from the Faroe Islands, constituting a relatively underexplored microbial ecosystem. The objective of this study is to achieve a deeper understanding of the microbial composition of this artisanal product. Nine ripened hind legs, obtained from three different producers, were assessed regarding their bacterial communities and contents of biogenic amines, including both surface and core samples. Biogenic amine concentrations were generally low, although one sample had a somewhat elevated concentration of cadaverine. Bacterial diversity was investigated by culture-dependent and culture-independent techniques. Gram-positive catalase-positive cocci (GCC) constituted the most abundant group. Within this group, Staphylococcus equorum was the most prevailing species, followed by Kocuria sp., Mammaliicoccus vitulinus, and Staphylococcus saprophyticus. Lactic acid bacteria prevailed in only one sample and were mainly represented by Latilactobacillus curvatus. Enterobacterial communities were characterised by the prevalence of Serratia proteamaculans. Despite the majority of GCC, Clostridium putrefaciens was the most abundant bacterial species in some core samples. Taken together, the culture-dependent and culture-independent identification methods gave complementary results.

In the Atlantic salmon (Salmo salar) aquaculture industry, gaping (the separation of muscle bundles from the connective tissue) is a major quality problem. This study characterized chondroitin sulfate (CS) and heparan sulfate (HS) in the connective tissue of intact and gaping salmon fillets from 30 salmon by mass spectrometry. Statistical difference was detected between gaping and intact tissues only when comparing pairwise samples from the same individual (n = 10). The gaping tissue had a lower content of monosulfated CS disaccharides (p = 0.027), and the relative distribution of CS disaccharides was significantly different (p < 0.05). The HS chains were short (average = 14.09, SD = 4.91), and the intact tissue seemed to have a more uniform HS chain structure compared to the gaping tissue. Time-series samples from the same individuals are recommended for future research to improve the understanding of reasons and implications of these differences.

It was previously shown that the connexin gene family had relatively similar subfamily structures in several vertebrate groups. Still, many details were left unclear. There are essentially no data between tunicates, which have connexins that cannot be divided into the classic subfamilies, and teleosts, where the subfamilies are easily recognized. There are also relatively few data for the groups that diverged between the teleosts and mammals. As many of the previously analyzed genomes have been improved, and many more genomes are available, we reanalyzed the connexin gene family and included species from all major vertebrate groups. The major results can be summarized as follows: (i) The same connexin subfamily structures are found in all Gnathostomata (jawed vertebrates), with some variations due to genome duplications, gene duplications and gene losses. (ii) In contrast to previous findings, birds do not have a lower number of connexins than other tetrapods. (iii) The cyclostomes (lampreys and hagfishes) possess genes in the alpha, beta, gamma and delta subfamilies, but only some of the genes show a phylogenetic affinity to specific genes in jawed vertebrates. Thus, two major evolutionary transformations have occurred in this gene family, from tunicates to cyclostomes and from cyclostomes to jawed vertebrates.

The sex determination system of Atlantic herring Clupea harengus L., a commercially important fish, was investigated. Low coverage whole-genome sequencing of 48 females and 55 males and a genome-wide association study revealed two regions on chromosomes 8 and 21 associated with sex. The genotyping data of the single nucleotide polymorphisms associated with sex showed that 99.4% of the available female genotypes were homozygous, whereas 68.6% of the available male genotypes were heterozygous. This is close to the theoretical expectation of homo/heterozygous distribution at low sequencing coverage when the males are factually heterozygous. This suggested a male heterogametic sex determination system in C. harengus, consistent with other species within the Clupeiformes group. There were 76 protein coding genes on the sex regions but none of these genes were previously reported master sex regulation genes, or obviously related to sex determination. However, many of these genes are expressed in testis or ovary in other species, but the exact genes controlling sex determination in C. harengus could not be identified.

Atlantic herring (Clupea harengus) is one of the most abundant fish species in the world. It is an important economical and nutritional resource, as well as a crucial part of the North Atlantic ecosystem. In 2016, a draft herring genome assembly was published. Being a species of such importance, we sought to independently verify and potentially improve the herring genome assembly. We sequenced the herring genome generating paired-end, mate-pair, linked and long reads. Three assembly versions of the herring genome were generated based on a de novo assembly (A1), which was scaffolded using linked and long reads (A2) and then merged with the previously published assembly (A3). The resulting assemblies were compared using parameters describing the size, fragmentation, correctness, and completeness of the assemblies. Results showed that the A2 assembly was less fragmented, more complete and more correct than A1. A3 showed improvement in fragmentation and correctness compared with A2 and the published assembly but was slightly less complete than the published assembly. Thus, we here confirmed the previously published herring assembly, and made improvements by further scaffolding the assembly and removing low-quality sequences using linked and long reads and merging of assemblies.

Background Microbial analyses performed in connection with the post-slaughter environment of farmed Atlantic salmon (Salmo salar L.) have mostly focused on specific bacteria that may have negative effects on the health of consumers. However, bacteria may also affect other quality variables. The objective of this study was to provide general knowledge about composition and dynamics of the bacterial communities present at slaughter and cold storage of farmed Atlantic salmon, as well as reveal any possible correlations to gelatinase activity, which may affect fillet quality. Thus, these data may provide a basis for optimization opportunities in the aquaculture industry. Methods Samples were taken from the digestive system harvested from 15 salmon immediately after slaughter. Another 17 salmon were taken from the processing line just before the final cleaning stage; of these eight were distributed in three iced storage boxes while the other nine were rinsed an extra time with industrial water before being distributed into another three storage boxes. In the following 6 days, samples were taken of skin mucus, liquids in the abdominal cavity and the storage ice. The compositions of the bacterial communities were analyzed by next-generation sequencing and gelatinase activity was measured in all samples except the storage ice. Results The bacterial communities in the digestive tract samples were dominated by the family Mycoplasmataceae. The genus Aliivibrio was also relatively abundant. Bacterial communities in the abdominal cavity were generally more diverse than the intestinal samples. However, all of the abdominal samples from storage box no. 3 had a high relative abundance of Mycoplasmataceae, and could not be distinguished from the intestinal samples (Q = 1.27, p = 0.633) while being significantly different from the other abdominal samples (Q = 9.02, p = 0.01). In addition, the abdominal samples from storage box no. 3 had a significantly higher gelatin degrading activity (Q = 9.43, p = 0.001) than those from the other storage boxes and similar to the high gelatinase activity in the intestinal samples. This indicated that in storage box no. 3 there was a transfer of intestinal fluids to the abdominal cavities, which was not removed by the cleaning procedure. There was a significant difference of the major phyla detected in the skin mucus of salmon rinsed an additional time, as these salmon had a higher relative amount of Firmicutes (F = 4.76, p = 0.04) and lower amount of Proteobacteria (F = 4.41, p = 0.047). Conclusions The study showed a correlation between intestinal fluids and bacteria left in the abdominal cavity and gelatinase activity. This suggested that intestinal fluids and/or bacteria could enhance the degradation of connective tissue in the abdominal cavity and hence negatively affect the fillet quality. In addition, the study provided general knowledge of the composition and dynamics of bacterial communities present.

Dispersal limitation, not just environmental selection, plays an important role in microbial biogeography. The distance–decay relationship is thought to be weak in habitats where dispersal is high, such as in the pelagic environment, where ocean currents facilitate microbial dispersal. Most studies of microbial community composition to date have observed little geographical heterogeneity on a regional scale (100 km). We present a study of microbial communities across a dynamic frontal zone in the southwest Indian Ocean and investigate the spatial structure of the microbes with respect to the different water masses separated by these fronts. We collected 153 samples of free-living microorganisms from five seamounts located along a gradient from subtropical to subantarctic waters and across three depth layers: (i) the sub-surface chlorophyll maximum (approx. 40 m), (ii) the bottom of the euphotic zone (approx. 200 m), and (iii) the benthic boundary layer (300–2000 m). Diversity and abundance of microbial operational taxonomic units (OTUs) were assessed by amplification and sequencing of the 16S rRNA gene on an Illumina MiSeq platform. Multivariate analyses showed that microbial communities were structured more strongly by depth than by latitude, with similar phyla occurring within each depth stratum across seamounts. The deep layer was homogeneous across the entire survey area, corresponding to the spread of Antarctic intermediate water. However, within both the sub-surface layer and the intermediate depth stratum there was evidence for OTU turnover across fronts. The microbiome of these layers appears to be divided into three distinct biological regimes corresponding to the subantarctic surface water, the convergence zone and subtropical. We show that microbial biogeography across depth and latitudinal gradients is linked to the water masses the microbes persist in, resulting in regional patterns of microbial biogeography that correspond to the regional scale physical oceanography.

BackgroundMicrobial analyses performed in connection with the post-slaughter environment of farmed Atlantic salmon (Salmo salarL.) have mostly focused on specific bacteria that may have negative effects on the health of consumers. However, bacteria may also affect other quality variables. The objective of this study was to provide general knowledge about composition and dynamics of the bacterial communities present at slaughter and cold storage of farmed Atlantic salmon, as well as reveal any possible correlations to gelatinase activity, which may affect fillet quality. Thus, these data may provide a basis for optimization opportunities in the aquaculture industry.MethodsSamples were taken from the digestive system harvested from 15 salmon immediately after slaughter. Another 17 salmon were taken from the processing line just before the final cleaning stage; of these eight were distributed in three iced storage boxes while the other nine were rinsed an extra time with industrial water before being distributed into another three storage boxes. In the following 6 days, samples were taken of skin mucus, liquids in the abdominal cavity and the storage ice. The compositions of the bacterial communities were analyzed by next-generation sequencing and gelatinase activity was measured in all samples except the storage ice.ResultsThe bacterial communities in the digestive tract samples were dominated by the familyMycoplasmataceae.The genusAliivibriowas also relatively abundant. Bacterial communities in the abdominal cavity were generally more diverse than the intestinal samples. However, all of the abdominal samples from storage box no. 3 had a high relative abundance ofMycoplasmataceae, and could not be distinguished from the intestinal samples (Q = 1.27,p = 0.633) while being significantly different from the other abdominal samples (Q = 9.02,p = 0.01). In addition, the abdominal samples from storage box no. 3 had a significantly higher gelatin degrading activity (Q = 9.43,p = 0.001) than those from the other storage boxes and similar to the high gelatinase activity in the intestinal samples. This indicated that in storage box no. 3 there was a transfer of intestinal fluids to the abdominal cavities, which was not removed by the cleaning procedure. There was a significant difference of the major phyla detected in the skin mucus of salmon rinsed an additional time, as these salmon had a higher relative amount ofFirmicutes(F = 4.76,p = 0.04) and lower amount ofProteobacteria(F = 4.41,p = 0.047).ConclusionsThe study showed a correlation between intestinal fluids and bacteria left in the abdominal cavity and gelatinase activity. This suggested that intestinal fluids and/or bacteria could enhance the degradation of connective tissue in the abdominal cavity and hence negatively affect the fillet quality. In addition, the study provided general knowledge of the composition and dynamics of bacterial communities present.

There are still notable gaps regarding the detailed distribution of microorganisms between and within insular habitats such as deep-sea hydrothermal vents. This study investigates the community composition of black smoker vent microorganisms in the Southern Hemisphere, and changes thereof along a spatial and chemical gradient ranging from the vent plume to surrounding waters. We sampled two hydrothermal vent fields, one at the South West Indian Ridge (SWIR), the other at the East Scotia Ridge (ESR). Samples were collected across vent fields at varying vertical distances from the origin of the plumes. The microbial data were sequenced on an Illumina MiSeq platform for the 16SrRNA gene. A substantial amount of vent-specific putative chemosynthetic microorganisms were found, particularly in samples from focused hydrothermal venting. Common vent-specific organisms from both vent fields were the generaArcobacter,CaminibacterandSulfurimonasfrom the Epsilonproteobacteria and the SUP05 group from the Gammaproteobacteria. There were no major differences in microbial composition between SWIR and ESR for focused plume samples. However, within the ESR the diffuse flow and focused samples differed significantly in microbial community composition and relative abundance. For Epsilonproteobacteria, we found evidence of niche-specificity to hydrothermal vent environments. This taxon decreased in abundance by three orders of magnitude from the vent orifice to background water. Epsilonproteobacteria distribution followed a distance–decay relationship as vent-effluents mixed with the surrounding seawater. This study demonstrates strong habitat affinity of vent microorganisms on a metre scale with distinct environmental selection.

Dispersal limitation, not just environmental selection, plays an important role in microbial biogeography. The distance–decay relationship is thought to be weak in habitats where dispersal is high, such as in the pelagic environment, where ocean currents facilitate microbial dispersal. Most studies of microbial community composition to date have observed little geographical heterogeneity on a regional scale (100 km).We present a study of microbial communities across a dynamic frontal zone in the South West Indian Ocean and investigate the spatial structure of the microbes with respect to the different water masses separated by these fronts.We collected 153 samples of free-living microorganisms from five seamounts located along a gradient from subtropical to subantarctic waters and across three depth layers, (i) the sub-surface chlorophyll maximum (approx. 40 m), (ii) the bottom of the euphotic zone (approx. 200 m) and (iii) the benthic boundary layer (300–2000 m). Diversity and abundance of microbial operational taxonomic units (OTUs) was assessed by amplification and sequencing of the 16S rRNA gene on an Illumina MiSeq platform.Multivariate analyses showed that microbial communities were structured more strongly by depth than by latitude, with similar phyla occurring within each depth stratum across seamounts. The deep layer was homogeneous across the entire survey area, corresponding to the spread of Antarctic intermediate water. However, within both the sub-surface layer and the intermediate depth stratum there was evidence for OTU turnover across fronts. The microbiome of these layers appears to be divided into three distinct biological regimes corresponding to the subantarctic surface water, the convergence zone and subtropical. We show that microbial biogeography across depth and latitudinal gradients is linked to the water-masses the microbes persist in, resulting in regional patterns of microbial biogeography, that correspond to the regional scale physical oceanography.

There are still notable gaps regarding the detailed distribution of microorganisms between and within insular habitats such as deep-sea hydrothermal vents. This study investigates the community composition of black smoker vent microorganisms in the Southern Hemisphere, and changes thereof along a spatial and chemical gradient ranging from the vent plume to surrounding waters. We sampled two hydrothermal vent fields, one at the South West Indian Ridge (SWIR), the other at the East Scotia Ridge (ESR). Samples were collected across vent fields at varying vertical distances from the origin of the plumes. The microbial data were sequenced on an Illumina MiSeq platform for the 16SrRNA gene. A substantial amount of vent-specific putative chemosynthetic microorganisms were found, particularly in samples from focused hydrothermal venting. Common vent-specific organisms from both vent fields were the genera Arcobacter, Caminibacter and Sulfurimonas from the Epsilonproteobacteria and the SUP05 group from the Gammaproteobacteria. There were no major differences in microbial composition between SWIR and ESR for focused plume samples. However, within the ESR the diffuse flow and focused samples differed significantly in microbial community composition and relative abundance. or Epsilonproteobacteria, we found evidence of niche-specificity to hydrothermal vent environments. This taxon decreased in abundance by three orders of magnitude from the vent orifice to background water. Epsilonproteobacteria distribution followed a distance–decay relationship as vent-effluents mixed with the surrounding seawater. This study demonstrates strong habitat affinity of vent microorganisms on a metre scale with distinct environmental selection.

Gap junction intercellular communication (GJIC) and hemichannel permeability may have important roles during an ischemic insult. Our aim was to evaluate the effect of ischemia on gap junction channels and hemichannels.We used neonatal rat heart myofibroblasts and simulated ischemia with a HEPES buffer with high potassium, low pH, absence of glucose, and oxygen tension was reduced by dithionite. Microinjection, western blot, immunofluorescence, cell viability and dye uptake were used to evaluate the effects induced by dithionite. Isolated perfused rat hearts were used to analyse infarct size.Short period with simulated ischemia reduced the ability to transfer a dye between neighbouring cells, which indicated reduced GJIC. Prolonged exposure to simulated ischemia caused opening of hemichannels, and cell death was apparent while gap junction channels remained closed. Connexin 43 became partially dephosphorylated and the total amount decreased during simulated ischemia. We were not able to detect the alternative hemichannel-forming protein, Pannexin 1, in these cells. The potential importance of Connexin 43 or Pannexin 1 hemichannels in ischemia-induced infarct in the intact heart was studied by perfusion of the heart in the presence of peptides that block one or the other type of hemichannels. The connexin-derived peptide, Gap26, significantly reduced the infract/risk zone ratio (control 48.7±4.2% and Gap26 19.4±4.1%, p0.001), while the pannexin-derived peptide, (10)Panx1, did not change infarct/risk ratio.Connexin 43 is most likely responsible for both closure of gap junction channels and opening of hemichannels during simulated ischemia in neonatal rat heart myofibroblasts. Opening of connexin 43 hemichannels during ischemia-reperfusion seems to be an important mechanism for ischemia-reperfusion injury in the heart. By preventing the opening of these channels during early ischemia-reperfusion the infarct size becomes significantly reduced.

Mass spectrometric analyses of peptides mainly rely on cleavage of proteins with proteases that have a defined specificity. The specificities of the proteases imply that there is not a random distribution of amino acids in the peptides. The physico–chemical effects of this distribution have been partly analyzed for tryptic peptides, but to a lesser degree for other proteases. Using all human proteins in Swiss-Prot, the relationships between peptide fractional mass, pI and hydrophobicity were investigated. The distribution of the fractional masses and the average regression lines for the fractional masses were similar, but not identical, for the peptides generated by the proteases trypsin, chymotrypsin and gluC, with the steepest regression line for gluC. The fractional mass regression lines for individual proteins showed up to ±100 ppm in mass difference from the average regression line and the peptides generated showed protease-dependent properties. We here show that the fractional mass and some other properties of the peptides are dependent on the protease used for generating the peptides. With the increasing accuracy of mass spectrometry instruments, it is possible to exploit the information embedded in the fractional mass of unknown peaks in peptide mass fingerprint spectra.

We suggest an extension ofconnexinorthology relationships across the major vertebrate lineages. We first show that the conserved domains of mammalianconnexins(encoding the N-terminus, four transmembrane domains and two extracellular loops) are subjected to a considerably more strict selection pressure than the full-length sequences or the variable domains (the intracellular loop and C-terminal tail). Therefore, the conserved domains are more useful for the study of family relationships over larger evolutionary distances. The conserved domains ofconnexinswere collected from chicken,Xenopus tropicalis, zebrafish, pufferfish, green spotted pufferfish,Ciona intestinalisandHalocynthia pyriformis(two tunicates). A total of 305connexinsequences were included in this analysis. Phylogenetic trees were constructed, from which the orthologies and the presumed evolutionary relationships between the sequences were deduced. The tunicateconnexinsstudied had the closest, but still distant, relationships to vertebrateconnexin36,39.2,43.4,45and47. The main structure in theconnexinfamily known from mammals pre-dates the divergence of bony fishes, but some additional losses and gains ofconnexinsequences have occurred in the evolutionary lineages of subsequent vertebrates. Thus, theconnexingene family probably originated in the early evolution of chordates, and underwent major restructuring with regard to gene and subfamily structures (including the number of genes in each subfamily) during early vertebrate evolution.

In contrast to the prevalent view in the literature hitherto, the present study shows that pancreatic trypsin can activate human promatrix metalloproteinase-2 (proMMP-2). It is shown that trypsin's ability to activate proMMP-2 is dependent on various environmental factors such as the level of exogenously added Ca(2+) and Brij-35, temperature, as well as trypsin concentration. The activation occurred as a sequential processing of the proenzyme, initially generating an active 62kDa species. This was followed by successive truncation of the C-terminal domain, giving rise to active species of 56kDa, 52kDa and 50kDa. Tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) prevented the trypsin-mediated C-terminal truncation, without affecting the generation of the 62kDa species, while the presence of EDTA increased the rate of the trypsin-mediated activation of proMMP-2. MALDI-TOF MS analysis of the 50kDa form indicated that trypsin generated active forms with either Lys87 or Trp90 as the N-terminal residue and Arg538 as a C-terminal residue. The trypsin-activated MMP-2 was active in solution against both synthetic and physiologic substrates, and the steady-state kinetic coefficients k(cat), K(m) and k(cat)/K(m) were determined for the enzyme activated either by APMA, membrane-type 1 matrix metalloproteinase (MT1-MMP) or trypsin. The trypsin-activated MMP-2 exhibited slightly lower k(cat) and k(cat)/K(m) values as well as a slightly higher K(i) value against TIMP-1 compared to the enzyme activated by APMA or MT1-MMP. Docking studies of TIMP-1 revealed that the slightly weaker binding of the inhibitor to the trypsin-activated MMP-2 could be attributed to its shorter N terminus (Lys87/Trp90 versus Tyr81), as Phe83 and Arg86 interacted directly with the inhibitor. Our results suggest that the trypsin-activated MMP-2 possesses the catalytic and regulatory potential to be of significance in vivo.

Ilimaquinone (IQ) and brefeldin A (BFA) disrupt the Golgi complex structure and block protein transport to the plasma membrane, and inhibit gap junctional communication. HeLa cells expressing rat connexin26, 32, or 43, or mouse connexin31, 36, 45, or 57, were used to study the response patterns of gap junctional communication (dye transfer) to ilimaquinone, brefeldin, and the potent protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA). 12-O-Tetradecanoylphorbol-13-acetate (followed for 2 h) caused dose- and time-dependent decreases in communication for five of seven connexins, the unresponsive being connexin45 and 57. Brefeldin (followed for 6 h) caused dose- and time-dependent decreases in communication for six of seven connexins, the exception being connexin26. These results are consistent with Golgi-mediated transport to the cell membrane for all connexins except connexin26. In contrast, ilimaquinone (followed for 6 h) caused a rapid (15–30 min) and nearly complete inhibition of dye transfer through connexin43 channels. For the other connexins, there was a slow and weak response for connexin26, 31, and 32, reaching 65–70% of control communication level, while connexin36, 45, and 57 were unresponsive. Thus, among the tested connexins, ilimaquinone has a strong specificity for connexin43, and the mechanism appears independent of the Golgi complex and of protein kinase C.

The open reading frames of 17 connexins from Syrian hamster (using tissues) and 16 connexins from the Chinese hamster cell line V79, were fully (Cx30, Cx31, Cx37, Cx43 and Cx45) or partially sequenced. We have also detected, and partially sequenced, seven rat connexins that previously were unavailable. The expression of connexin genes was examined in some hamster organs and cultured hamster cells, and compared with wild-type mouse and the cancer-prone Min mouse. Although the expression patterns were similar for most organs and connexins in hamster and mouse, there were also some prominent differences (Cx29 and 30.3 in testis; Cx31.1 and 32 in eye; Cx46 in brain, kidney and testis; Cx47 in kidney). This suggests that some connexins have species-specific expression profiles. In contrast, there were minimal differences in expression profiles between wild type and Min mice. Species-specific expression profiles should be considered in attempts to make animal models of human connexin-associated diseases.

Brefeldin A and ilimaquinone are compounds known to affect Golgi structure and function. In particular, the transport of proteins is blocked either at the level of exit from endoplasmic reticulum (brefeldin) or at cis-Golgi (ilimaquinone). Brefeldin caused a slow decrease in gap-junctional communication and a slow loss of all phosphorylated forms of connexin43 in hamster and rat fibroblasts, while ilimaquinone caused an abrupt decrease in gap-junctional communication and rapid loss of only the slowest migrating phosphorylated connexin43 band (P2). Ilimaquinone caused these effects prior to any significant Golgi fragmentation, especially in hamster fibroblasts. Concurrently, ilimaquinone minimally affected protein secretion, while brefeldin caused an instantaneous decrease. These results show that ilimaquinone inhibits gap-junctional communication in connexin43-expressing cells by a mechanism not dependent on Golgi fragmentation or block in protein transport.

Reversible down-regulation of gap junctional intercellular communication (GJIC) is proposed to be an important cellular mechanism in tumor promotion. Gap junction function is modified by a variety of tumor promoters, including the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment of cells with TPA results in the activation and subsequent depletion of the TPA-responsive protein kinase C (PKC) isoforms. TPA-induced degradation of the PKC isoforms alpha, delta and epsilon was recently shown to occur via the ubiquitin-proteasome pathway. In the present study we investigated the role of the proteasome in the TPA-induced modification of GJIC in IAR20 rat liver epithelial cells. TPA exposure of IAR20 cells induced hyperphosphorylation of gap junction protein connexin43 and inhibition of GJIC. Prolonged TPA treatment induced down-regulation of PKCalpha, delta and epsilon and a reduction in the total PKC activity, which was associated with recovery of GJIC. Co-treatment of IAR20 cells with TPA and the proteasomal inhibitor MG132 suppressed down-regulation of PKCalpha, delta and epsilon and caused prolonged PKC activity. Under these conditions, the recovery of GJIC was blocked. The general PKC inhibitor GF109203X reversed the effect of MG132, indicating that the prolonged TPA-induced inhibition of GJIC caused by MG132 was due to the prolonged PKC activity. These results indicate that proteasomal degradation of PKC is one mechanism by which the recovery of GJIC after TPA treatment is regulated.

A large number of experimental studies have linked the S100A4 gene product to the metastatic phenotype of cancer cells and clinical evidence indicates a correlation between S100A4 expression and poor prognosis in several cancer types. The aim of the present study was to analyse the expression of the S100A4 protein in colorectal cancer. Paraffin-embedded samples from 277 colorectal cancer patients were immunostained with anti-S100A4 antibody. Cytoplasmic staining was observed in 178 of 277 samples (64%), whereas, unexpectedly, nuclear expression of S100A4 was found in 88 of 277 of the samples (32%). This novel finding was confirmed by western blot analysis of nuclear fractions isolated from frozen tumour tissue. Statistical analysis revealed a significant correlation between nuclear expression of S100A4 and tumour stage at diagnosis, while there was no such correlation between cytoplasmic staining and tumour stage. The nuclear localization of S100A4 in colorectal cancer and its relationship to tumour stage suggest that this protein may be involved in gene regulatory pathways of relevance to the metastatic phenotype of cancer cells.

Several phorbol esters are potent activators of protein kinase C. They down-regulate gap junctional intercellular communication and induce phosphorylation of connexin43, but the sensitivity and extent of responses vary much between systems. We asked whether the total protein kinase C enzyme activity or the protein kinase C isoenzyme constitution was of importance for such variations. Some fibroblastic culture systems were compared. It was concluded that the total protein kinase C enzyme activity did not determine the sensitivity to phorbol esters. Furthermore, the use of isotype-specific inhibitors of protein kinase C indicated that protein kinase C alpha, delta, and epsilon may be involved to different extents in different fibroblastic systems in the response to phorbol esters.

12-O:-tetradecanoylphorbol-13-acetate (TPA) inhibits gap junctional communication in many cell culture systems, but TPA-induced phosphorylation of the gap junction protein connexin43 (Cx43) varies much between systems. We have here studied whether these responses and their sensitivities can be correlated with total protein kinase C (PKC) enzyme activity and if specific PKC isoenzymes are involved. Rat R6 fibroblasts transfected with the cDNA sequence encoding PKC beta I (R6-PKC3) had a total PKC activity 7- to 16-fold higher than the corresponding control cells (R6-C1), depending on the selection pressure (G418 concentration). Still, R6-PKC3 cells were no more sensitive than R6-C1 cells to TPA-induced down-regulation of communication, except at the highest selection pressure (500 micrograms/ml G418). Thus, total PKC activity does not indicate absolute sensitivity of a cell system to TPA-induced suppression of communication, but within a certain cell system increasing PKC activity may enhance the sensitivity to TPA in this respect. The results also suggest that PKC beta I is of minor importance for TPA-induced regulation of communication. Experiments with the Lilly compound 379196, a PKC beta-specific inhibitor, further supported this conclusion. Except for PKC beta I in R6-PKC3 cells, both cell lines contained the TPA-responsive PKC isoenzymes alpha, delta, epsilon and mu. Long-term treatment with TPA caused strong down-regulation of PKC alpha, delta and epsilon, but little down-regulation of PKC mu. Concurrently, the cells became refractory to repeated exposure to TPA, indicating that PKC mu is of minor importance. Experiments with the general PKC inhibitor GF109203X and the PKC alpha (and beta/gamma) inhibitor Gö6976 suggested that both classical (alpha) and novel PKCs (delta and epsilon) might be involved in TPA-induced suppression of intercellular communication, while phosphorylation of Cx43 may mainly be mediated by PKC alpha in the present systems.

A large fraction of chemicals observed to cause cancer in experimental animals is devoid of mutagenic activity. It is therefore of importance to develop methods that can be used to detect and study environmental carcinogenic agents that do not interact directly with DNA. Previous studies have indicated that induction of in vitro cell transformation and inhibition of gap junction intercellular communication are endpoints that could be useful for the detection of non-genotoxic carcinogens. In the present work, 13 compounds [chlordane, Arochlor 1260, di(2-ethylhexyl)phthalate, 1,1,1-trichloro-2, 2-bis(4-chlorophenyl)ethane, limonene, sodium fluoride, ethionine, o-anisidine, benzoyl peroxide, o-vanadate, phenobarbital, 12-O-tetradecanoylphorbol 13-acetate and clofibrate] have been tested for their ability to induce morphological transformation and affect intercellular communication in Syrian hamster embryo cells. The substances were selected on the basis of being proven or suspected non-genotoxic carcinogens, and thus difficult to detect in short-term tests. The data show that nine of the 13 compounds induced morphological transformation, and seven of the 13 inhibited intercellular communication in hamster embryo cells. Taken together, 12 of the 13 substances either induced transformation or caused inhibition of communication. The data suggest that the combined use of morphological transformation and gap junction intercellular communication in Syrian hamster embryo cells may be beneficial when screening for non-genotoxic carcinogens.

12-O-Tetradecanoylphorbol-13-acetate (TPA) caused strong suppression of gap junctional intercellular communication, altered phosphorylation status of the gap junction protein, connexin43, and disappearance of immunorecognizible connexin43-containing gap junction plaques in V79 fibroblasts. When TPA was removed, all parameters normalized during a 3- to 4-h period. The normalizations were independent of protein synthesis, suggesting the possible involvement of phosphatases. None of the phosphatase inhibitors okadaic acid, calyculin A, cyclosporin A, or FK506 affected intercellular communication or connexin43 phosphorylation status on their own. In sequential exposures to TPA and phosphatase inhibitors, only the protein-phosphatase 2B (PP2B) inhibitors cyclosporin A and FK506 delayed the recovery of the studied parameters. Rapamycin binds to the same set of proteins as does FK506, but without inhibiting PP2B. Rapamycin did not affect the recovery of intercellular communication, but it delayed the normalization of connexin43 band pattern and immunorecognition of gap junction plaques. Dephosphorylation of immunoprecipitated connexin43 was studied using PP1, 2A, 2B, and 2C. PP2A was the most efficient (by 100-fold on a molar basis). Connexin43 immunoprecipitated from TPA-exposed cells was a poor substrate for PP1, 2B, and 2C. Thus, PP2B appeared to play a role in normalization of intercellular communication, but not necessarily in direct dephosphorylation of connexin43. Peptidyl-prolyl isomerase activity of cyclosporin/FK506/rapamycin-binding proteins may promote the dephosphorylation of connexin43 in cells.

A monoclonal antibody, Zymed 13-8300, was previously reported to only detect nonphosphorylated connexin43 (Nagy et al., Exp. Cell Res. 236, 127–136, 1997). We show that 13-8300 can detect several phosphorylated species of connexin43 in Western blots after stimulation of two fibroblast cell systems with fresh growth medium, 12- O -tetradecanoyl phorbol-13-acetate, pervanadate, or permolybdate. In one of the cell systems, at least three forms of phosphorylated connexin43 could migrate at the same position during electrophoresis. The comigration of differentially phosphorylated species may complicate the molecular and functional analysis of phosphorylation sites in Cx43. Immunofluorescence experiments indicated that the newly generated phosphorylated Cx43 forms mainly had a perinuclear location. Also, in cells treated with brefeldin A for 8 h, in which the majority of connexin43 was intracellular, phosphorylation was induced by the agents. Phosphorylation of intracellular connexin43 can therefore be induced by several stimuli.

Pervanadate and permolybdate are irreversible protein-tyrosine phosphatase inhibitors, with IC50values of 0.3 and 20 μm, respectively, in intact cells. Maximal inhibition was obtained within 1 min at higher concentrations of the compounds. They induced prominent changes in the phosphorylation status of the gap junction protein, connexin43. These effects were utilized as model systems to assess the stability and inactivation of the compounds. Although the concentrated stock solutions were relatively stable, the diluted compounds were unstable. The biological activity had decreased to 20–30% after 6 h of incubation in a phosphate buffer, 1 h in phosphate buffer with 10% fetal calf serum, and 1–3 minutes in culture medium. Thiols reacted rapidly with the compounds and inactivated them (initial reaction rates with cysteine: permolybdate > pervanadate > H2O2). Catalase inactivated the compounds, and permolybdate was the more sensitive. The cells inactivated permolybdate faster than pervanadate. Cellular inactivation of permolybdate, and to a lesser degree pervanadate, appeared to be partly dependent on catalase and thiols. However, a general decrease in cellular thiols was not the mediator of the biological effects of pervanadate or permolybdate. Mathematical modeling of the thiol reactivity suggested that monoperoxovanadate at maximum could possess 20% of the biological activity of diperoxovanadate.

Effects of 12-O-tetradecanoyl phorbol 13-acetate (TPA) and the hepatic peroxisome proliferators (HPPs) clofibrate, methyl clofenapate (MCP), di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) were studied in 2 gap junctional intercellular communication (GJIC) systems, metabolic cooperation in V79 cells and microinjection/dye transfer in Syrian hamster embryo (SHE) cells and V79 cells. TPA inhibited GJIC in both systems but was considerably more potent in V79 cells. SHE cells showed a rapid and transient inhibition of GJIC after exposure to HPPs, with maximal inhibition occurring at 5-15 min. The transient inhibition could be caused by metabolization of the compounds. Clofibrate and MEHP produced strong inhibition of metabolic cooperation in V79 cells at high concentrations, while the effect of MCP and DEHP was lower. However, DEHP, MEHP and clofibrate strongly inhibited dye transfer in V79 cells after a 30 min exposure. Clofibrate also showed a dose- and time-dependent effect on dye transfer in V79 cells. The phosphorylation status of the gap junction protein connexin43 (Cx43) changed minimally in SHE cells after exposure to TPA or HPPs. Cx43 from V79 cells was strongly affected by TPA, but not by HPPs. Immunofluorescence of Cx43 disappeared in both cell types when they were exposed to TPA and MEHP, but not to the other HPPs. Thus, there is no direct correlation between the inhibition of GJIC and changes in the phosphorylation status of Cx43 or the appearance of Cx43 in immunofluorescence experiments. The discrepancies may partly be explained by binding of accessory proteins to Cx43. We point out sequences that may be involved in such binding.

Biological and analytical characterizations of permolybdate (a mixture of H2O2 and molybdate) were done. Molybdate (10 mM) and molybdenum(V) chloride (3 mM) did not affect gap junctional intercellular communication (GJIC), phosphorylation status of connexin43 (Cx43) or cellular tyrosine phosphorylation in early passage hamster embryonic cells (mainly fibroblast-like). High concentrations of H2O2 (3–10 mM) affected some of the parameters. Acidified permolybdate was clearly more stable than the unadjusted permolybdate. The maximum biological potency of acidified permolybdate was found at a molar ratio of 2:1 (H2O2:molybdate). The mixtures of molybdenum(V) chloride and H2O2 gave a maximum effect at 4:1 molar ratio (H2O2:molybdenum(V)). This can be explained by decomposition of H2O2 and by the generation of less biologically active compounds. Spectrophotometric analyses of the mixtures corroborated the biological results. The Mo(V) electron spin resonance spectrum disappeared upon addition of H2O2 to Mo(V) solutions, and no spectrum appeared when H2O2 was mixed with Mo(VI). Thus, permolybdate is probably diperoxomolybdate, a Mo(VI) compound. Regardless of the parent metal salt, the H2O2/metal salt mixtures showed concentration-dependent biphasic responses with an initial decrease in GJIC followed by an increase. A dissociation between alteration in Cx43 phosphorylation status and GJIC was obtained under certain conditions. The biological activities of permolybdate were only partially mimicked by phenylarsine oxide, an alternative protein tyrosine phosphatase inhibitor.

The protein-tyrosine phosphatase inhibitors pervanadate, permolybdate, H2O2, and to a much lesser extent vanadate, increased the amount of cellular phosphotyrosine and induced tyrosine phosphorylation of connexin43 (Cx43) in early passage hamster embryo fibroblasts. The presence of phosphotyrosine in Cx43 immunoprecipitates from pervanadate-treated cells was shown by a phosphotyrosine-specific antibody and a phosphotyrosine-specific phosphatase. Pervanadate-induced Cx43 tyrosine phosphorylation was further verified by phosphoamino acid analysis, while no phosphotyrosine was present in control cells. This is the first observation of tyrosine phosphorylation of connexins in normal cells.

— Seven phosphatase inhibitors were used to investigate the possible importance of phosphatases in the regulation of gap junctional intercellular communication (GJIC). Only the tyrosine phosphatase inhibitor, pervanadate, was found to inhibit GJIC and to alter the band pattern of connexin43 (Cx43) in Western blots. Only the unphosphorylated band of Cx43 was seen after alkaline phosphatase treatment of pervanadate-exposed cells. Application of a tyrosine-specific phosphatase and a phosphotyrosine antibody clearly indicated that pervanadate induced tyrosine phosphorylation in Cx43.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), 12-deoxyphorbol-13-phenylacetate (DOPP), 12-deoxyphorbol-13-phenylacetate-20-acetate (DOPP A), sapintoxin D (SAP D) and sapintoxin A (SAP A) on the decrease in [125I]epidermal growth factor (EGF) binding (indicating protein kinase C activation), suppression of gap junctional intercellular communication (GJIC) and induction of morphological cell transformation (MCT) in Syrian hamster embryo (SHE) cells were investigated. All five phorbol esters were found to reduce [125I]EGF binding in early passage SHE cells at comparable concentrations. DOPP A was approximately 10-fold less potent in decreasing GJIC compared to the other phorbol esters in early passage SHE cells, while the compounds showed less difference in suppressing GJIC in the phorbol ester sensitive SHE cell line BPNi. The decreases in [125I]EGF binding and GJIC were found to be transient in the continuous presence of phorbol esters. All phorbol esters induced MCT in early passage SHE cells, but DOPP and DOPP A were approximately 10-fold less potent than TPA, SAP D and SAP A. Thus, there seems to be some degree of correlation, but not to a full extent, between the ability of the phorbol esters to activate PKC, decrease GJIC and to induce MCT. The results do not suggest a simple relationship between PKC activation, inhibition of GJIC and the reported tumor-promoting activities of the compounds.

MS-based proteomics is a bioinformatic-intensive field. Additionally, the instruments and instrument-related and analytic software are expensive. Some free Internet-based proteomics tools have gained wide usage, but there have not been any single bioinformatic framework that in an easy and intuitive way guided the user through the whole process from analyses to submission. Together, these factors may have limited the expansion of proteomics analyses, and also the secondary use (reanalyses) of proteomic data. Vaudel et al. (Proteomics 2014, 14, 1001-1005) are now describing their Compomics framework that guides the user through all the main steps, from the database generation, via the analyses and validation, and through the submission process to PRIDE, a proteomic data bank. Vaudel et al. partly base the framework on tools that they have developed themselves, and partly they are integrating other freeware tools into the workflow. One of the most interesting aspects with the Compomics framework is the possibility of extending MS-based proteomics outside the MS laboratory itself. With the Compomics framework, any laboratory can handle large amounts of proteomic data, thereby facilitating collaboration and in-depth data analyses. The described software also opens the potential for any laboratory to reanalyze data deposited in PRIDE.

The effects of NiSO4, K2CrO4, PbAc2, CrCl3 and CdAc2 on gap junctional intercellular communication (GJIC) were studied. Two exposure procedures were used — A: an established monolayer of cells was exposed to the compound, and B: the compound was mixed with the cells before seeding. The apparent inhibition of GJIC by CrCl3 and CdAc2 could be accounted for by cytotoxicity, i.e. disruption of physical contacts between cells. PbAc2 had no effect on GJIC. The effects of NiSO4 and K2CrO4 depended on the exposure procedure and cell type studied. Specifically, K2CrO4 induced a significant increase in GJIC in BPNi cells by procedure A, and a decrease by procedure B. The cytotoxic effects of NiSO4 were examined in more detail. The toxic concentrations varied widely between the methods used. The results indicate that the effect of compounds on GJIC may vary with different exposure procedures, and that inhibition of GJIC is not due to general cytotoxic effects.

MassSorter is a software tool that sorts, systemizes, and analyzes data from peptide mass fingerprinting (PMF) experiments on proteins with known amino acid sequences. Several experiments can be simultaneously analyzed for sequence coverage and posttranslational modifications occurring during sample handling, induced chemical modifications, and unexpected cleavages. Experimental m/z values are compared with m/z values from an in silico digestion, taking modifications into account. Filters can be defined by users for marking autolytic protease peaks and other contaminating peaks. MassSorter functions as a database of all the detected peptides. It includes tools for visualization of the results, such as sequence coverage, accuracy plots, statistics, and 3D models.

Background S100A4 is a metastasis-associated protein which has been linked to multiple cellular events, and has been identified extracellularly, in the cytoplasm and in the nucleus of tumor cells; however, the biological implications of subcellular location are unknown. Associations between a variety of posttranslational protein modifications and altered biological functions of proteins are becoming increasingly evident. Identification and characterization of posttranslationally modified S100A4 variants could thus contribute to elucidating the mechanisms for the many cellular functions that have been reported for this protein, and might eventually lead to the identification of novel drugable targets. Methods S100A4 was immuoprecipitated from a panel of in vitro and in vivo sources using a monoclonal antibody and the samples were separated by 2D-PAGE. Gels were analyzed by western blot and silver staining, and subsequently, several of the observed spots were identified as S100A4 by the use of MALDI-TOF and MALDI-TOF/TOF. Results A characteristic pattern of spots was observed when S100A4 was separated by 2D-PAGE suggesting the presence of at least three charge variants. These charge variants were verified as S100A4 both by western immunoblotting and mass spectrometry, and almost identical patterns were observed in samples from different tissues and subcellular compartments. Interestingly, recombinant S100A4 displayed a similar pattern on 2D-PAGE, but with different quantitative distribution between the observed spots. Conclusion Endogenously expressed S100A4 were shown to exist in several charge variants, which indicates the presence of posttranslational modifications altering the net charge of the protein. The different variants were present in all subcellular compartments and tissues/cell lines examined, suggesting that the described charge variants is a universal phenomenon, and cannot explain the localization of S100A4 in different subcellular compartments. However, the identity of the specific posttranslational modification and its potential contribution to the many reported biological events induced by S100A4, are subject to further studies.

MassSorter is a software tool that sorts, systemizes, and analyzes data from peptide mass fingerprinting (PMF) experiments on proteins with known amino acid sequences. Several experiments can be simultaneously analyzed for sequence coverage and posttranslational modifications occurring during sample handling, induced chemical modifications, and unexpected cleavages. Experimental m/z values are compared with m/z values from an in silico digestion, taking modifications into account. Filters can be defined by users for marking autolytic protease peaks and other contaminating peaks. MassSorter functions as a database of all the detected peptides. It includes tools for visualization of the results, such as sequence coverage, accuracy plots, statistics, and 3D models.

Several hepatic peroxisome proliferators (HHPs) such as di(2-ethylhexyl)phthalate (DEHP), mono(2-ethylhexyl)-phthalate, clofibrate and tiadenol, induce morphological transformation of Syrian hamster embryo (SHE) cells in vitro. According to one hypothesis, the hepatocarcinogenic effect of HPPs is caused by an oxidative stress due to increased H2O2-production from the strongly induced peroxisomal beta-oxidation of fatty acids. Thus, increased transformation frequencies by HPPs should be obtained when catalase was inhibited by 3-amino-1,2,4-triazole (amitrole). However, co-exposure to HPPs and amitrole did not enhance the transformation frequencies for any of the HPPs. The sensitivity of SHE cells for oxidative agents was studied by using menadione and H2O2. Menadione only induced transformation at a toxic concentration, while H2O2 induced transformation at non-toxic concentrations. To study the generation of oxidative radicals in SHE cells, electron spin resonance was employed. No oxidative radical formation was detected in tiadenol- or DEHP-exposed SHE cells. When menadione or H2O2 were added during the measurements, oxidative radicals were found. A transmission electron microscopic study showed a small number of peroxisomes, and did not reveal any increase in the number of peroxisomes in clofibrate-treated SHE cells.