Effects of predator exposure on baseline and stress-induced glucocorticoid hormone concentrations in pumpkinseed Lepomis gibbosus

We compared baseline and maximal cortisol concentrations between predator exposure and prey blood samples in pumpkinseed Lepomis gibbosus, captured using a standardised fishing event underneath osprey Pandion haliaetus nests and away from osprey nests. We did not detect differences in cortisol or glucose between sites. These findings suggest that predictable sources of predation risk may not confer stress-related costs in teleosts.     

Fatty acids differentiate consumers despite variation within prey fatty acid profiles

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Binding of SeqA protein to DNA requires interaction between two or more complexes bound to separate hemimethylated GATC sequences

The SeqA protein binds to the post-replicative forms of the origins of replication of the Escherichia coli chromosome (oriC) and the P1 plasmid (P1oriR) at hemimethylated GATC adenine methylation sites. It appears to regulate replication by preventing premature reinitiation. However, SeqA binding is not exclusive to replication origins: different fragments with hemimethylated GATC sites can bind SeqA in vitro when certain rules apply. Most notably, more than one such site must be present on a bound fragment. The protein appears to recognize individual hemimethylated sites, but must undergo an obligate cooperative interaction with a nearby bound protein for stable binding. SeqA contacts both DNA strands in a discrete patch at each hemimethylated GATC sequence. All four GATC bases are contacted and are essential for binding. Although the recognized sequence is symmetrical, the footprint on the methylated strand is always broader, suggesting that the bound protein is positioned asymmetrically with its orientation dictated by the position of the unique methyl group. Studies of alternative spacings and relative orientations of adjacent sites suggest that each site may be recognized by a symmetrical dimer with an induced asymmetry in one of the subunits similar to that seen with certain type II restriction endonucleases.     

Ascorbate distribution during hibernation is independent of ascorbate redox state

Distribution of ascorbate into tissues is an essential process in ascorbate antioxidant defense. Hibernating animals are studied as a model of tolerance to ischemia-reperfusion because of their tolerance to fluctuations in blood flow associated with prolonged torpor and periodic arousal episodes. Throughout hibernation, plasma ascorbate concentration ([Asc](p)) repetitively increases during torpor, then falls during periodic arousal bouts. We previously proposed that high [Asc](p) provides a ready source of antioxidant protection for distribution to the central nervous system and peripheral tissues during arousal. Here we tested whether deliberate oxidation of plasma ascorbate by intravenous administration of ascorbate oxidase (AO), prior to arousal, compromised tissue levels of ascorbate or the other water-soluble antioxidants, glutathione (GSH) and urate. Although AO decreased [Asc](p) to below the level of detection during torpor and after arousal, ascorbate oxidation did not decrease post-arousal tissue levels of reduced ascorbate, glutathione, or urate in any tissue examined, except liver. The data imply that ascorbate is taken up equally well into brain and other tissues as either ascorbate or its oxidized product dehydroascorbate, with subsequent intracellular reduction of dehydroascorbate. Lack of effect of ascorbate oxidation on tissue levels of GSH or urate indicates that dehydroascorbate uptake and reduction do not compromise tissue concentrations of these other water-soluble antioxidants. Thus, we show equal availability of reduced and oxidized plasma ascorbate during metabolically demanding thermogenesis and reperfusion associated with arousal from hibernation.     

Role of hyperhomocysteinemia in endothelial dysfunction and atherothrombotic disease

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease, including ischemic heart disease, stroke, and peripheral vascular disease. Mutations in the enzymes responsible for homocysteine metabolism, particularly cystathionine beta-synthase (CBS) or 5,10-methylenetetrahydrofolate reductase (MTHFR), result in severe forms of HHcy. Additionally, nutritional deficiencies in B vitamin cofactors required for homocysteine metabolism, including folic acid, vitamin B6 (pyridoxal phosphate), and/or B12 (methylcobalamin), can induce HHcy. Studies using animal models of genetic- and diet-induced HHcy have recently demonstrated a causal relationship between HHcy, endothelial dysfunction, and accelerated atherosclerosis. Dietary enrichment in B vitamins attenuates these adverse effects of HHcy. Although oxidative stress and activation of proinflammatory factors have been proposed to explain the atherogenic effects of HHcy, recent in vitro and in vivo studies demonstrate that HHcy induces endoplasmic reticulum (ER) stress, leading to activation of the unfolded protein response (UPR). This review summarizes the current role of HHcy in endothelial dysfunction and explores the cellular mechanisms, including ER stress, that contribute to atherothrombosis.     

Pre-clinical development of cord blood-derived progenitor cell graft expanded ex vivo with cytokines and the polyamine copper chelator tetraethylenepentamine

BACKGROUND: We have previously demonstrated that the copper chelator tetraethylenepentamine (TEPA) enables preferential expansion of early hematopoietic progenitor cells (CD34+CD38-, CD34+CD38-Lin-) in human umbilical cord blood (CB)-derived CD34+ cell cultures. This study extends our previous findings that copper chelation can modulate the balance between self-renewal and differentiation of hematopoietic progenitor cells. METHODS: In the present study we established a clinically applicative protocol for large-scale ex vivo expansion of CB-derived progenitors. Briefly, CD133+ cells, purified from CB using Miltenyi Biotec's (Bergisch Gladbach, Germany) CliniMACS separation device and the anti-CD133 reagent, were cultured for 3 weeks in a clinical-grade closed culture bag system, using the chelator-based technology in combination with early-acting cytokines (SCF, thrombopoietin, IL-6 and FLT-3 ligand). This protocol was evaluated using frozen units derived from accredited cord blood banks. RESULTS: Following 3 weeks of expansion under large-scale culture conditions that were suitable for clinical manufacturing, the median output value of CD34+ cells increase by 89-fold, CD34+CD38- increase by 30-fold and CFU cells (CFUc) by 172-fold over the input value. Transplantation into sublethally irradiated non-obese diabetic (NOD/SCID) mice indicated that the engraftment potential of the ex vivo expanded CD133+ cells was significantly superior to that of unexpanded cells: 60+/-5.5% vs. 21+/-3.5% CD45+ cells, P=0.001, and 11+/-1.8% vs. 4+/-0.68% CD45+CD34+ cells, P=0.012, n=32, respectively. DISCUSSION: Based on these large-scale experiments, the chelator-based ex vivo expansion technology is currently being tested in a phase 1 clinical trial in patients undergoing CB transplantation for hematological malignancies.     

Auto-validation of fluorescent primer extension genotyping assay using signal clustering and neural networks

Background

SNP genotyping typically incorporates a review step to ensure that the genotype calls for a particular SNP are correct. For high-throughput genotyping, such as that provided by the GenomeLab SNPstream^® instrument from Beckman Coulter, Inc., the manual review used for low-volume genotyping becomes a major bottleneck. The work reported here describes the application of a neural network to automate the review of results.

Results

We describe an approach to reviewing the quality of primer extension 2-color fluorescent reactions by clustering optical signals obtained from multiple samples and a single reaction set-up. The method evaluates the quality of the signal clusters from the genotyping results. We developed 64 scores to measure the geometry and position of the signal clusters. The expected signal distribution was represented by a distribution of a 64-component parametric vector obtained by training the two-layer neural network onto a set of 10,968 manually reviewed 2D plots containing the signal clusters.

Conclusion

The neural network approach described in this paper may be used with results from the GenomeLab SNPstream instrument for high-throughput SNP genotyping. The overall correlation with manual revision was 0.844. The approach can be applied to a quality review of results from other high-throughput fluorescent-based biochemical assays in a high-throughput mode.