Atlantic herring (Clupea harengus) population structure in the Northeast Atlantic Ocean.

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 over-exploitation 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).

Connexins during 500 Million Years-From Cyclostomes to Mammals.

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.

Phylogeny of teleost connexins reveals highly inconsistent intra- and interspecies use of nomenclature and misassemblies in recent teleost chromosome assemblies.

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.

Identification of male heterogametic sex-determining regions on the Atlantic herring Clupea harengus genome.

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.

Proteomics made more accessible.

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.

Bacterial species diversity of traditionally ripened sheep legs from the Faroe Islands (skerpikjøt).

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.

Study of the long-finned pilot whale (Globicephala melas) bile content - An indicator of ocean health.

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.

Ischemia induces closure of gap junctional channels and opening of hemichannels in heart-derived cells and tissue.

AIM: 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. METHODS: 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. RESULTS: 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%, p<0.001), while the pannexin-derived peptide, (10)Panx1, did not change infarct/risk ratio. CONCLUSION: 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.

Composition and dynamics of the bacterial communities present in the post-slaughter environment of farmed Atlantic salmon (Salmo salar L.) and correlations to gelatin degrading activity.

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.

Characterization of Glycosaminoglycans in Gaping and Intact Connective Tissues of Farmed Atlantic Salmon (Salmo salar) Fillets by Mass Spectrometry.

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.

Using long and linked reads to improve an Atlantic herring (Clupea harengus) genome assembly.

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.

The metastasis-associated protein S100A4 exists in several charged variants suggesting the presence of posttranslational modifications.

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: peptide mass fingerprinting data analysis.

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.

Protease-dependent fractional mass and peptide properties.

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.

Cutting through the smoke: the diversity of microorganisms in deep-sea hydrothermal plumes.

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. 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.

Microbe biogeography tracks water masses in a dynamic oceanic frontal system.

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.

MassSorter: a tool for administrating and analyzing data from mass spectrometry experiments on proteins with known amino acid sequences.

BACKGROUND: Proteomics is the study of the proteome, and is critical to the understanding of cellular processes. Two central and related tasks of proteomics are protein identification and protein characterization. Many small laboratories are interested in the characterization of a small number of proteins, e.g., how posttranslational modifications change under different conditions. RESULTS: We have developed a software tool called MassSorter for administrating and analyzing data from peptide mass fingerprinting experiments on proteins with known amino acid sequences. It is meant for small scale mass spectrometry laboratories that are interested in posttranslational modifications of known proteins. Several experiments can be compared simultaneously, and the matched and unmatched peak values are clearly indicated. The hits can be sorted according to m/z values (default) or according to the sequence of the protein. Filters defined by the user can mark autolytic protease peaks and other contaminating peaks (keratins, proteins co-migrating with the protein of interest, etc.). Unmatched peaks can be further analyzed for unexpected modifications by searches against a local version of the UniMod database. They can also be analyzed for unexpected cleavages, a highly useful feature for proteins that undergo maturation by proteolytic cleavage, creating new N- or C-terminals. Additional tools exist for visualization of the results, like sequence coverage, accuracy plots, different types of statistics, 3D models, etc. The program and a tutorial are freely available for academic users at http://www.bioinfo.no/software/massSorter. CONCLUSION: MassSorter has a number of useful features that can promote the analysis and administration of MS-data.

Pancreatic trypsin activates human promatrix metalloproteinase-2.

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.

Photochemically enhanced gene transfection increases the cytotoxicity of the herpes simplex virus thymidine kinase gene combined with ganciclovir.

Tumor targeting is an important issue in cancer gene therapy. We have developed a gene transfection method, based on light-inducible photochemical internalization (PCI) of a transgene, to improve gene delivery and expression selectively in illuminated areas, for example, in tumors. In the present work, we demonstrate that PCI improved the nonviral vector polyethylenimine (PEI)-mediated transfection of a therapeutic gene, the 'suicide' gene encoding herpes simplex virus thymidine kinase (HSVtk). In U87MG glioblastoma cells in vitro, the photochemical treatment stimulated expression of the HSVtk transgene, and, consequently, enhanced cell killing by the subsequent treatment with the prodrug ganciclovir (GCV). When relatively low doses of DNA (1 microg/ml) and the PEI vector (N/P 4) were used, HSVtk gene transfection followed by the GCV treatment did not have an effect on cell survival unless the photochemical treatment was performed, which potentiated the cytotoxicity to 90%. These findings indicate that photochemical transfection allows: (i) selective enhancement in gene expression and gene-mediated biological effects (cell killing by the Hsvtk/GCV approach) in response to illumination; (ii) the use of low, suboptimal for the nonviral transfection methods without PCI, doses of both DNA and the vector, which may be relevant and advantageous for therapeutic gene transfer in vivo.

Truncated mouse adenomatous polyposis coli reduces connexin32 content and increases matrilysin secretion from Paneth cells.

Heterozygous mutations in adenomatous polyposis coli (APC) is an early event in inheritable and sporadic colon cancer development. We recently found reduced connexin (Cx43) expression in intestinal cell lines with heterozygous Apc mutation. In this study we investigated Cx expression and the role of one mutated Apc allele in epithelia of multiple intestinal neoplasia (Min) mouse intestines by immunohistochemistry. Cx43 was not expressed in intestinal epithelia of Min and wild-type mice. Cx32 was specifically expressed in enterochromaffin cells in both mice types, and in Paneth cells of wild-type mice. In contrast, Min mice had nearly undetectable level of Cx32 in Paneth cells. Isolated small intestinal crypts from Min mice had markedly increased secretion of both lysozyme and matrilysin compared with wild-type mice. Absence of matrilysin in Min mice reduces adenoma development. Reduced Cx32 and increased matrilysin secretion from Paneth cells could be important to neoplastic development in the intestine.