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.     

Stress in families of children who have ingested poisons.

One hundred families of children under 5 years admitted to Cardiff Hospitals after accidentally ingesting poisons were compared with 100 control families matched for socioeconomic class and age and sex of the child. Questioning about five major stress factors (serious family illness, pregnancy, recent family moves, one parent away from home, anxiety or depression in one or both parents) disclosed significantly more stress in the affected families than in the controls. Thirty of the affected families had more than one major stress factor compared with four of the controls, while 63 of the controls had no major stress factor compared with 24 of the affected families (P less than 0.001). In only four of the affected families was there no stress factor. Fifteen children took poisons in homes other than their own. Unemployment was significantly more prevalent in the affected families than in the general population, though apart from this the socioeconomic backgrounds were similar. There were significantly more accidents and childhood poisonings in the parents and siblings of affected children than in the control families. In 25% of the cases poisoning was with Angiers Junior Aspirin.     

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.     

The growth of nonlymphoid thymic components in vitro: Age-related differences during development

Summary An explant culture procedure has been developed that makes it possible to measure the relative growth capacity of the epithelial and mesenchymal cells of the canine thymus gland. Standardized growth conditions were obtained by size-grading thymic fragments and counting to allow uniform fragment density during culture. After 6 d in culture, outgrowth from the fragments formed colonies that could be classified into epithelial, mixed, or spindle cell type. Uniform fragment size and number in each flask allowed calculation of the total plating efficiency, relative distribution of colony types, and mean colony diameters for thymic fragments collected from fetuses (50 d of gestation), neonates (0 d postpartum), and juveniles (70 d postpartum). Data show age-related changes in the proliferative capacity of the cells in all three colony types. The most significant difference was seen in the epithelium, which showed a 30% reduction in mean colony diameter over the 2 wk between fetal and neonatal ages and a 23% reduction over the postnatal period of 70 d. Significant reductions were seen in the other colony types as well. Because the severity of the effect of many injurious agents is proportional to the rate of growth of the target cells, these data suggest that the thymus gland of the fetus may be more sensitive to physical or chemical injury than is the neonate or adult. Funding was provided by grants NCI CA36456, NCI T15CA09408, NIEHS ES07152 from the National Institutes of Health, Bethesda, MD, and by USDA Animal Health and Disease Program (PL 95-113).     

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.