Atlantic Cod Trypsins: From Basic Research to Practical Applications

Atlantic cod trypsin I is an appropriate representative of the traditionally classified cold-adapted group I trypsins, and the recombinant form of cod trypsin Y is the only biochemically characterized member of the novel group III trypsins. Trypsin Y is adapted to lower temperatures than all other presently known trypsins. This review describes the basic characteristics of and practical uses for trypsins of Atlantic cod, as well as those of other organisms. Overexpression of the recombinant forms of cod trypsins I and Y in microorganisms is explained as well as the advantages of using site-directed mutagenesis to increase their stability toward autolysis and thermal inactivation. Trypsins appear to play a key role in the nutrition and development of marine fish. We discuss the potential use of cod trypsins as biomarkers to evaluate the nutritional status of cod larvae and describe the industrial applications of cod trypsin I and other trypsins.     

Stickleback fights: why do winners win? Influence of metabolic and morphometric parameters

Pairs of reproductively mature male three-spined stickleback (Gasterosteus aculeatus) were introduced into unfamiliar aquaria and observed until one male became dominant. Skin carotenoid content, morphometric indexes, and metabolic capacities of the axial and pectoral muscles were examined to establish whether morphological or physiological parameters differentiated winners and losers. Stickleback that initiated fights typically won. Quick initiation led to quick victory. Overall, winners and losers differed in few morphological or metabolic characteristics, but these properties and the differences between these attributes for losers and winners of specific fights were linked with initiation time and fight duration. Morphometric indexes of losers were the primary determinants of initiation time and fight duration, whereas for winners muscle metabolic capacities were linked to these fight characteristics. The greater the hepatosomatic index (HSI) of losers, the longer the fight initiation times. Similarly, losers with high HSI and carotenoid levels resisted defeat longer. In winners, initiation time decreased as axial muscle phosphofructokinase levels increased and citrate synthase levels decreased, whereas the metabolic capacities of the pectoral muscle were linked with time to achieve victory. When losers had greater HSI values than the winners of a specific fight, fight initiation was delayed and fights lasted longer. When losers had higher carotenoid levels than winners, fights also lasted longer. On the other hand, when losers had more visceral fat (fat body mass over somatic mass) than winners, both initiation time and combat duration were reduced. These results suggest that male stickleback assess their physiological status and that of their opponents, in particular the HSI, and adjust their combat strategies accordingly.     

Oligonucleotide microarray for identification of Enterococcus species

For detection of most members of the Enterococcaceae, the specificity of a novel oligonucleotide microarray (ECC-PhyloChip) consisting of 41 hierarchically nested 16S or 23S rRNA gene-targeted probes was evaluated with 23 pure cultures (including 19 Enterococcus species). Target nucleic acids were prepared by PCR amplification of a 4.5-kb DNA fragment containing large parts of the 16S and 23S rRNA genes and were subsequently labeled fluorescently by random priming. Each tested member of the Enterococcaceae was correctly identified on the basis of its unique microarray hybridization pattern. The evaluated ECC-PhyloChip was successfully applied for identification of Enterococcus faecium and Enterococcus faecalis in artificially contaminated milk samples demonstrating the utility of the ECC-PhyloChip for parallel identification and differentiation of Enterococcus species in food samples.     

Perivascular cells expressing annexin A5 define a novel mesenchymal stem cell-like population with the capacity to differentiate into multiple mesenchymal lineages

The annexin A5 gene (Anxa5) was recently found to be expressed in the developing and adult vascular system as well as the skeletal system. In this paper, the expression of an Anxa5-lacZ fusion gene was used to define the onset of expression in the vasculature and to characterize these Anxa5-lacZ-expressing vasculature-associated cells. After blastocyst implantation, Anxa5-lacZ-positive cells were first detected in extra-embryonic tissues and in angioblast progenitors forming the primary vascular plexus. Later, expression is highly restricted to perivascular cells in most blood vessels resembling pericytes or vascular smooth muscle cells. Viable Anxa5-lacZ+ perivascular cells were isolated from embryos as well as adult brain meninges by specific staining with fluorescent X-gal substrates and cell-sorting. These purified lacZ+ cells specifically express known markers of pericytes, but also markers characteristic for stem cell populations. In vitro and in vivo differentiation experiments show that this cell pool expresses early markers of chondrogenesis, is capable of forming a calcified matrix and differentiates into adipocytes. Hence, Anxa5 expression in perivascular cells from mouse defines a novel population of cells with a distinct developmental potential.     

Increased genotoxic susceptibility of breast epithelial cells to ethylene oxide

This study was carried out with the aim of elucidating the organ-specific effects of ethylene oxide in comparison with the sensitivity of cells from different tissues. An increased incidence of leukemia and lymphoma has been observed in workers exposed to ethylene oxide. However, contradictory findings exist regarding its ability to induce other tumor types, such as breast cancer. We characterized the genotoxicity of ethylene oxide by means of the alkaline version of comet assay in in vitro systems, in order to investigate the hypothesized role of this substance in the development of breast cancer. For this study, we used primary and secondary cultures of lymphoblasts (well-known target cells of the genotoxicity of ethylene oxide), breast epithelial cells (hypothesized target), peripheral blood lymphocytes (cells commonly used in biomonitoring), and of keratinocytes and cervical epithelial cells. DNA damage was measured and expressed as tail DNA, tail length, and tail moment. In the concentration range 0-100 microM, ethylene oxide induced a dose-dependent increase of DNA damage in the investigated cell types without notable cytotoxicity. A statistically significant increase of DNA damage could be observed after treatment with 20 microM ethylene oxide in lymphoblasts (51% increase of tail moment over the background), breast epithelial cells (26% increase) and peripheral lymphocytes (71% increase). In keratinocytes (5% increase) and cervical epithelial cells (5% increase) significant DNA damage could not be detected at this dose, but at higher concentrations (50-100 microM), such an increase was observed. These results are indicative of an increased sensitivity of breast epithelial cells towards genotoxic insults of ethylene oxide. Our observations provide additional data to evaluate the hypothesis that exposure to ethylene oxide may play a role in breast cancer, and the findings may contribute to the development of screening tests for monitoring an early response to genotoxic insults in occupational settings.     

A phase I vaccination study with tyrosinase in patients with stage II melanoma using recombinant modified vaccinia virus Ankara (MVA-hTyr)

A significant percentage of patients with stage II melanomas suffer a relapse after surgery and therefore need the development of adjuvant therapies. In the study reported here, safety and immunological response were analyzed after vaccination in an adjuvant setting with recombinant modified vaccinia virus Ankara carrying the cDNA for human tyrosinase (MVA-hTyr). A total of 20 patients were included and vaccinated three times at 4-week intervals with 5×10⁸ IU of MVA-hTyr each time. The responses to the viral vector, to known HLA class I–restricted tyrosinase peptides, and to dendritic cells transfected with tyrosinase mRNA, were investigated by ELISpot assay on both ex vivo T cells and on T cells stimulated in vitro prior to testing. The delivery of MVA-hTyr was safe and did not cause any side effects above grade 2. A strong response to the viral vector was achieved, indicated by an increase in the frequency of MVA-specific CD4⁺ and CD8⁺ T cells and an increase in virus-specific antibody titers. However, no tyrosinase-specific T-cell or antibody response was observed with MVA-hTyr in any of the vaccinated patients. Although MVA-hTyr provides a safe and effective antigen-delivery system, it does not elicit a measurable immune response to its transgene product in patients with stage II melanoma after repeated combined intradermal and subcutaneous vaccination. We presume that modification of the antigen and/or prime-boost vaccination applying different approaches to antigen delivery may be required to induce an effective tyrosinase-specific immune response.     

Cutting edge: CTLA-4 (CD152) differentially regulates mitogen-activated protein kinases (extracellular signal-regulated kinase and c-Jun N-terminal kinase) in CD4+ T cells from receptor/ligand-deficient mice

Although CTLA-4 (CD152) has potent inhibitory effects on T cell function, the signaling events affected by this coreceptor remain to be fully defined. Mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) act as crucial regulators of multiple aspects of cell function. Ab ligation studies have reported an inhibitory effect of CTLA-4 on TCR-induced ERK and JNK activation. In this study, we have re-examined the specificity of CTLA-4 inhibition of MAPKs by using natural ligand with ex vivo-purified CD4(+) T cells deficient in CD80 and CD86 (double knockout), or CTLA-4, CD80, and CD86 (triple knockout). Under these conditions, CTLA-4 ligation was found to up-regulate and sustain JNK activation, while inhibiting ERK activity. At the same time, JNK activation could not account for CTLA-4 induction of TGF-beta production. Our findings demonstrate that CTLA-4 cosignaling is more complex than previously appreciated, with an ability to differentially regulate members of the MAPK family in T cells.