Long-term stability and effective population size in North Sea and Baltic Sea cod (Gadus morhua)

DNA from archived otoliths was used to explore the temporal stability of the genetic composition of two cod populations, the Moray Firth (North Sea) sampled in 1965 and 2002, and the Bornholm Basin (Baltic Sea) sampled in 1928 and 1997. We found no significant changes in the allele frequencies for the Moray Firth population, while subtle but significant genetic changes over time were detected for the Bornholm Basin population. Estimates of the effective population size ( N _e ) generally exceeded 500 for both populations when employing a number of varieties of the temporal genetic method. However, confidence intervals were very wide and N _e 's most likely range in the thousands. There was no apparent loss of genetic variability and no evidence of a genetic bottleneck for either of the populations. Calculations of the expected levels of genetic variability under different scenarios of N _e showed that the number of alleles commonly reported at microsatellite loci in Atlantic cod is best explained by N _e 's exceeding thousand. Recent fishery-induced bottlenecks can, however, not be ruled out as an explanation for the apparent discrepancy between high levels of variability and recently reported estimates of N _e  << 1000. From life history traits and estimates of survival rates in the wild, we evaluate the compatibility of the species' biology and extremely low N _e / N ratios. Our data suggest that very small N _e 's are not likely to be of general concern for cod populations and, accordingly, most populations do not face any severe threat of losing evolutionary potential due to genetic drift.     

CD4+CD25+ regulatory T cells in HIV infection

The immune system faces the difficult task of discerning between foreign, potentially pathogen-derived antigens and self-antigens. Several mechanisms, including deletion of self-reactive T cells in the thymus, have been shown to contribute to the acceptance of self-antigens and the reciprocal reactivity to foreign antigens. Over the last decade it has become increasingly clear that CD4(+)CD25(+) T(Reg) cells are crucial for maintenance of T cell tolerance to self-antigens in the periphery, and to avoid development of autoimmune disorders. Recently, evidence has also emerged that demonstrates that CD4(+)CD25(+) T(Reg) cells can also suppress T cell responses to foreign pathogens, including viruses such as HIV. In this article we review the current knowledge and potential role of CD4(+)CD25(+) T(Reg) cells in HIV infection.     

Probiotic effect of lactic acid bacteria in the feed on growth and survival of fry of Atlantic cod (Gadus morhua)

A growing concern for the high consumption of antibiotics in aquaculture has initiated a search for alternative methods of disease control. Improved resistance against infectious diseases can be achieved by the use of probiotics. Probiotics are live microorganisms supplemented in food or feed which give beneficial effects on the intestinal microbial balance. In the present study a dry feed containing lactic acid bacteria (Carnobacterium divergens) isolated from Atlantic cod (Gadus morhua)intestines was given to cod fry. After three weeks of feeding the fry was exposed to a virulent strain of Vibrio anguillarum. The death rate was recorded during further three weeks of feeding with lactic acid bacteria supplemented feed. A certain improvement of disease resistance was obtained, and at the end of the experiment lactic acid bacteria dominated the intestinal flora in surviving fish given feed supplemented with lactic acid bacteria. No obvious growth inhibition of V. anguillarum was observed in an in vitro mixed culture of this bacterium and the C. divergens isolated from cod intestines. This revised version was published online in August 2006 with corrections to the Cover Date.     

Functional expression of horseradish peroxidase in E. coli by directed evolution.

In an effort to develop a bacterial expression system for horseradish peroxidase (HRP), we inserted the gene encoding HRP into the pET-22b(+) vector (Novagen) as a fusion to the signal peptide PelB. A similar construct for cytochrome c peroxidase (CcP) leads to high CcP activity in the supernatant. Expression of the wild-type HRP gene in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) yielded no detectable activity against ABTS (azinobis(ethylbenzthiazoline sulfonate)). However, weak peroxidase activity was detected in the supernatant in the absence of IPTG. The HRP gene was subjected to directed evolution: random mutagenesis and gene recombination followed by screening in a 96-well microplate format. From 12 000 clones screened in the first generation, one was found that showed 14-fold higher HRP activity than wild-type, amounting to approximately 110 microg of HRP/L, which is similar to that reported from laborious in vitro refolding. No further improvement was obtained in subsequent generations of directed evolution. This level of expression has nonetheless enabled us to carry out further directed evolution to render the enzyme more thermostable and more resistant toward inactivation by H2O2. These results show that directed evolution can identify mutations that assist proteins to fold more efficiently in Escherichia coli. This approach will greatly facilitate efforts to "fine-tune" those many enzymes that are promising industrial biocatalysts, but for which suitable bacterial or yeast expression systems are currently lacking.     

Genetic population structure of turbot (Scophthalmus maximus L.) supports the presence of multiple hybrid zones for marine fishes in the transition zone between the Baltic Sea and the North Sea

Genetic population structure of turbot (Scophthalmus maximus L.) in the Northeast Atlantic was investigated using eight highly variable microsatellite loci. In total 706 individuals from eight locations with temporal replicates were assayed, covering an area from the French Bay of Biscay to the Aaland archipelago in the Baltic Sea. In contrast to previous genetic studies of turbot, we found significant genetic differentiation among samples with a maximum pairwise FST of 0.032. Limited or no genetic differentiation was found among samples within the Atlantic/North Sea area and within the Baltic Sea, suggesting high gene flow among populations in these areas. In contrast, there was a sharp cline in genetic differentiation going from the low saline Baltic Sea to the high saline North Sea. The data were explained best by two divergent populations connected by a hybrid zone; however, a mechanical mixing model could not be ruled out. A significant part of the genetic variance could be ascribed to variation among years within locality. Nevertheless, the population structure was relatively stable over time, suggesting that the observed pattern of genetic differentiation is biologically significant. This study suggests that hybrid zones are a common phenomenon for marine fishes in the transition area between the North Sea and the Baltic Sea and highlights the importance of using interspecific comparisons for inferring population structure in high gene flow species such as most marine fishes.     

Inhibition of antigen-specific T cell proliferation and cytokine production by protein kinase A type I

cAMP inhibits biochemical events leading to T cell activation by triggering of an inhibitory protein kinase A (PKA)-C-terminal Src kinase pathway assembled in lipid rafts. In this study, we demonstrate that activation of PKA type I by Sp-8-bromo-cAMPS (a cAMP agonist) has profound inhibitory effects on Ag-specific immune responses in peripheral effector T cells. Activation of PKA type I inhibits both cytokine production and proliferative responses in both CD4(+) and CD8(+) T cells in a concentration-dependent manner. The observed effects of cAMP appeared to occur endogenously in T cells and were not dependent on APC. The inhibition of responses was not due to apoptosis of specific T cells and was reversible by a PKA type I-selective cAMP antagonist. This supports the notion of PKA type I as a key enzyme in the negative regulation of immune responses and a potential target for inhibiting autoreactive T cells.     

The Microenvironment of Cervical Carcinoma Xenografts: Associations with Lymph Node Metastasis and Its Assessment by DCE-MRI

Poor disease-free and overall survival rates in locally advanced cervical cancer are associated with a tumor micro-environment characterized by extensive hypoxia, interstitial hypertension, and high lactate concentrations. The potential of gadolinium diethylenetriamine pentaacetic acid-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing the microenvironment and microenvironment-associated aggressiveness of cervical carcinomas was investigated in this preclinical study. CK-160 and TS-415 cervical carcinoma xenografts were used as tumor models. DCE-MRI was carried out at 1.5 T, and parametric images of K^trans and v_e were produced by pharmacokinetic analysis of the DCE-MRI series. Pimonidazole was used as a marker of hypoxia. A Millar catheter was used to measure tumor interstitial fluid pressure (IFP). The concentrations of glucose, adenosine triphosphate (ATP), and lactate were measured by induced metabolic bioluminescence imaging. High incidence of lymph node metastases was associated with high hypoxic fraction and high lactate concentration in CK-160 tumors and with high IFP and high lactate concentration in TS-415 tumors. Low K^trans was associated with high hypoxic fraction, low glucose concentration, and high lactate concentration in tumors of both lines and with high incidence of metastases in CK-160 tumors. Associations between v_e and microenvironmental parameters or metastatic propensity were not detected in any of the tumor lines. Taken together, this preclinical study suggests that K^trans is a potentially useful biomarker for poor outcome of treatment in advanced cervical carcinoma. The possibility that K^trans may be used to identify patients with cervical cancer who are likely to benefit from particularly aggressive treatment merits thorough clinical investigations.     

Fertility of gynogenetic Atlantic cod (Gadus morhua L.)

In order to investigate whether meiotic gynogenetic Atlantic cod is fertile and able to produce viable offspring, meiotic gynogenetic females were produced in spring 2010 by activating cod eggs using irradiated sperm. The extrusion of the second polar body was prevented by the application of hydrostatic pressure (56.6 MPa) 36 min after fertilization. In February 2012, their mean round weight was 972 g, and 2580 g in March 2013. In 2012, when the fish were 2 years old, about 52% were mature, 33% were immature, and 13% had undifferentiated gonads. One year later, 77% were mature, 11% were immature, and 11% had undifferentiated gonads. Several of the mature females had malformed gonads, with only one developed ovary lobe or with the two lobes fused. The mean gonadosomic index (GSI) of the 2‐year‐old mature females was 5.2%, with an estimated relative fecundity of 581 000 eggs kg ovary‐free wet weight^?1. Females were stripped for eggs when 2 and 3 years old (2012 and 2013), and fertilized with sperm from normal males. Offspring were obtained from 12 of 17 and 12 of 15 egg batches incubated in 2012 and 2013, respectively, proving that the gynogenetic females are fertile. Furthermore, larvae in all but one of the hatched groups from 2013 had commenced feeding 2 h after being startfed using rotifers 4 days after hatch, indicating viable offspring.     

Reduced immunocompetence and cost of reproduction in common eiders

Immunocompetence may be especially important in long-lived species where infectious organisms may have detrimental effects upon future reproductive value of hosts. The resource demand for immunocompetence may reduce resource availability for reproduction and a trade-off between these traits has therefore been proposed. Capital breeders, such as the common eider (Somateria mollissima), rely upon accumulated body reserves during reproduction. Eiders lose more than 40% of pre-breeding body mass during egglaying and incubation and many females abandon their ducklings to other females after hatching. Results from our observational study show that levels of leukocytes (i.e., lymphocytes, heterophils and heterophil/lymphocyte ratio) are not related to body mass early in the incubation period. However, eider females with low initial body mass showed signs of immunosuppression (i.e., decreased late levels of lymphocytes) and increased response towards stressors (i.e., increased heterophil/lymphocyte ratio) later in the incubation period. Moreover, females with low lymphocyte levels more frequently abandoned their brood, and females abandoning young had an increased return rate to the next breeding season. However, among brood abandoning females return rate was lower for the females with low lymphocyte levels. These results suggest that immunosuppression, as indicated by low lymphocyte levels, is a reproductive cost that may be partly compensated for by abandoning young.