A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Chick embryo fibroblasts produce two forms of hyaluronidase

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.     

Physiological consequences of captive conditions in water voles (Arvicola terrestris)

Re-introductions of captive-bred animals are increasingly common in wildlife conservation and it is important that they fulfil their potential. To foster this goal we examined variations in stress levels in a captive-bred population of water voles Arvicola terrestris in response to housing conditions and radio-collaring, using weight loss and leukocyte coping capacity (LCC) as measures of relative stress, to investigate the impacts of housing conditions, handling and radio-collaring on this species. Thirty-eight water voles (22 males and 16 females) were used in the investigation, 25 housed in outdoor enclosures and 13 in laboratory cages. During the 6-week study, LCC, body weight and urine refractive index (URI, an indicator of hydration levels) were recorded once a week for each individual in weeks 1, 2, 4 and 6. After the first sample, radio-collars were attached to 20 individuals (10 males and 10 females) taken from both housing types. Throughout the experiment laboratory-cage housed voles weighed less, had lower LCC scores – indicating a reduced ability to combat infection – and had higher URIs than outdoor-enclosure voles. This suggests that the laboratory-cage voles were more stressed and dehydrated than the outdoor-enclosure voles. Weights and LCC scores of both housing groups decreased as the study progressed, suggesting that elements of the study, such as repeated handling, may have caused stress to both groups. Evidence suggested a short-term effect of radio-collaring on immuno-competence. We conclude that captive housing conditions, repeated manipulation and radio-collaring had demonstrable physiological effects on the water voles studied. We recommend that the effects of husbandry and tagging practices upon captive-bred mammals be closely studied as part of the quest to improve the success of the re-introductions to which they contribute.     

Comparison of the anti-allergic activity of Syk inhibitors with optimized Syk siRNAs in FcεRI-activated RBL-2H3 basophilic cells

Spleen tyrosine kinase (Syk) binds ITAM-bearing receptors in a wide variety of cell types. One such example is the activation of mast cells, basophils and eosinophils via the stimulation of the FcεRI receptor by IgE/allergen complexes. The possible role of Syk in inflammatory signaling cascades has led to the development of pharmacological agents designed to block the Syk catalytic domain as potential novel therapeutics. Whilst the enzymatic activity of Syk lends towards the design of small-molecule inhibitors, other attention has focused on the possibility of targeting Syk expression using anti-sense oligonucleotides as an alternate means of anti-inflammatory therapy. In this study, we compared the ability of multiple optimized Syk siRNA sequences and small-molecule Syk inhibitors to block FcεRI-mediated signal transduction, degranulation and TNFα secretion in the basophilic cell line RBL-2H3. We also characterized the specificity of each siRNA sequence with regards to off-target induction of the interferon-inducible gene IFIT1. We identified a single siRNA sequence, which displayed a favorable profile of efficient Syk knockdown, blockage of FcεRI-mediated signal transduction, degranulation and TNFα secretion and a lack of IFIT1 induction. The effect of this siRNA was comparable to that of the Syk kinase domain inhibitors BAY61-3606 and R406. The identification of an active and specific Syk siRNA could be a basis for the development of therapeutic Syk siRNAs against inflammatory diseases.     

Measuring animal welfare within a reintroduction: an assessment of different indices of stress in water voles Arvicola amphibius

Reintroductions are an increasingly common conservation restoration tool; however, little attention has hitherto been given to different methods for monitoring the stress encountered by reintroduced individuals. We compared ten potential measures of stress within four different categories (neuroendocrine, cell function, body condition and immune system function) as proxies for animal welfare in water voles being reintroduced to the Upper Thames region, Oxfordshire, UK. Captive-bred voles were assessed pre-release, and each month post-release for up to five months. Wild-born voles were captured in the field and assessed from two months post-release. Plasma corticosteroid, hydration and body condition of captive-bred voles differed between their pre-release measures and both their first ("short-term") recapture, and their final recapture ("long-term" release), however only body condition and immunocompetence measured using the Nitroblue Tetrazolium (NBT) test were significantly different post-release between the first and last recaptures. Captive-bred animals had lower fat reserves, higher weight/length ratios and better immunocompetence (NBT) than did wild-born voles. Captive-bred males had higher ectoparasite burdens compared to wild-born males and, as reintroduction site quality decreased, became less hydrated. These observations indicate that some methods can identify changes in the stress response in individuals, highlighting areas of risk in a reintroduction programme. In addition, a single measure may not provide a full picture of the stress experienced; instead, a combination of measures of different physiological systems may give a more complete indication of stress during the reintroduction process. We highlight the need to monitor stress in reintroductions using measures from different physiological systems to inform on possible animal welfare improvements and thus the overall success rate of reintroductions.     

Biological staining: mechanisms and theory

New staining techniques continue to be introduced, and older ones continue to be used and improved. Several factors control specificity, selectivity and visibility of the end product in any procedure using dyes, fluorochromes, inorganic reagents or histochemical reactions applied to sections or similar preparations. Local concentration of the tissue target often determines the intensity of the observed color, as does the fine structure within the object being stained, which may facilitate or impede diffusion of dyes and other reagents. Several contributions to affinity control the specificity of staining. These include electrical forces, which result in accumulation of dye ions in regions of oppositely charged tissue polyions. Weaker short-range attractions (hydrogen bonding, van der Waals forces or hydrophobic bonding, depending on the solvent) hold dyes ions and histochemical end products in contact with their macromolecular substrates. Nonionic forces can also increase visibility of stained sites by causing aggregation of dye molecules. Covalent bonds between dye and tissue result in the strongest binding, such as in methods using Schiff's reagent and possibly also some mordant dyes. The rate at which a reagent gains access to or is removed from targets in a section or other specimen affect what is stained, especially when more then one dye is used, together or sequentially. Rate-controlled staining is greatly influenced by the presence and type of embedding medium, such as a resin, that infiltrates the tissue. The rates of chemical reactions are major determinants of outcome in many histochemical techniques. Selective staining of different organelles within living cells is accomplished mainly with fluorochromes and is controlled by mechanisms different from those that apply to fixed tissues. Quantitative structure-activity relations (QSAR) of such reagents can be derived from such molecular properties as hydrophilic-hydrophobic balance, extent of conjugated bond systems, acid-base properties and ionic charge. The QSAR correlates with staining of endoplasmic reticulum, lysosomes, mitochondria, DNA, or the plasma membranes of living cells.     

MAGI: Methylation analysis using genome information

By incorporating annotation information into the analysis of next-generation sequencing DNA methylation data, we provide an improvement in performance over current testing procedures. Methylation analysis using genome information (MAGI) is applicable for both unreplicated and replicated data, and provides an effective analysis for studies with low sequencing depth. When compared with current tests, the annotation-informed tests provide an increase in statistical power and offer a significance-based interpretation of differential methylation.