Ecdysteroid-Responsive Genes in a Drosophila Cell Line

SYNOPSIS. In the Drosophila Kc-H cell line ecdysteroids causerapid increases in the synthesis of two groups of polypeptideswhich we call ecdysteroid-inducible polypeptides. We have shownthat these changes occur at least in part at the level of mRNAtiters and we have isolated cDNA clones corresponding to themRNAs for these polypeptides. The increases in these mRNAs maywell be among the primary hormone effects.     

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.     

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.     

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.     

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.      

The abundance of an invasive freshwater snail Tarebia granifera (Lamarck, 1822) in the Nseleni River,South Africa

The invasive freshwater snail Tarebia granifera (Lamarck, 1822) was first reported in South Africa in 1999 and it has become widespread across the country, with some evidence to suggest that it reduces benthic macroinvertebrate biodiversity. The current study aimed to identify the primary abiotic drivers behind abundance patterns of T. granifera, by comparing the current abundance of the snail in three different regions, and at three depths, of the highly modified Nseleni River in KwaZulu-Natal, South Africa. Tarebia granifera was well established throughout the Nseleni River system, with an overall preference for shallow waters and seasonal temporal patterns of abundance. Although it is uncertain what the ecological impacts of the snail in this system are, its high abundances suggest that it should be controlled where possible and prevented from invading other systems in the region.     

Multiple nuclear-gene phylogenies: application to pinnipeds and comparison with a mitochondrial DNA gene phylogeny

Phylogenetic analyses of closely related species should use information from multiple, independent genes with relatively high rates of sequence evolution. To investigate species for which there are few prior sequence data for single-copy nuclear (scnDNA) genes, primers for gene amplification can be designed to highly conserved regions of exons in order to amplify both coding (exons) and noncoding (introns) sequences. We have explored this approach in a phylogenetic analysis of six species of pinnipeds that, together with terrestrial carnivore outgroups, encompass divergence times < or = 40-50 Mya. We sequenced one intron from each of the aldolase A (ALD-A), aldolase C (ALD-C), and histone H2AF genes; one exon from the major-histocompatibility-complex DQA gene; a H2AF processed pseudogene (psi H2AF); and, for comparison with the nuclear genes, the 5' portion of the mitochondrial DNA (mtDNA) control region. The pinniped psi H2AF genes were found to be of limited use because they were paralogous with the gene in the outgroup. The rate of silent substitution in scnDNA (primarily introns) was 5-10-fold lower than that for mtDNA control region I, and scnDNA sequence divergence increased linearly with time < or = 40-50 Mya. Alleles at three polymorphic scnDNA loci (ALD-A, H2AF, and DQA) in the southern elephant seal were paraphyletic with respect to the allele from the closely related northern elephant seal, while the more numerous mtDNA alleles were monophyletic. This we attribute to the consequences of a higher mutation rate rather than to a lower effective population size of mtDNA compared with scnDNA. Within the short (i.e., < 500-bp) sequences of individual scnDNA sequences, phylogenetically informative variation was insufficient to obtain robust phylogenies. However, the combined scnDNA sequences produced a well-supported phylogeny congruent with that derived from mtDNA. This analysis illustrates the high resolution of mtDNA sequences compared with a similar length of scnDNA sequence, but it also demonstrates the utility of combining information from multiple short scnDNA sequences obtained using broadly applicable primers.      

Local spread of classical swine fever upon virus introduction into The Netherlands: Mapping of areas at high risk

Background

In the recent past, the introduction of Classical Swine Fever Virus (CSFV) followed by between-herd spread has given rise to a number of large epidemics in The Netherlands and Belgium. Both these countries are pork-exporting countries. Particularly important in these epidemics has been the occurrence of substantial "neighborhood transmission" from herd to herd in the presence of base-line control measures prescribed by EU legislation. Here we propose a calculation procedure to map out "high-risk areas" for local between-herd spread of CSFV as a tool to support decision making on prevention and control of CSFV outbreaks. In this procedure the identification of such areas is based on an estimated inter-herd distance dependent probability of neighborhood transmission or "local transmission". Using this distance-dependent probability, we derive a threshold value for the local density of herds. In areas with local herd density above threshold, local transmission alone can already lead to epidemic spread, whereas in below-threshold areas this is not the case. The first type of area is termed 'high-risk' for spread of CSFV, while the latter type is termed 'low-risk'.

Results

As we show for the case of The Netherlands, once the distance-dependent probability of local transmission has been estimated from CSFV outbreak data, it is possible to produce a map of the country in which areas of high-risk herds and of low-risk herds are identified. We made these maps even more informative by estimating border zones between the two types of areas. In these border zones the risk of local transmission of infection to a nearby high-risk area exceeds a certain level.

Conclusion

The risk maps provide an easily understandable visualization of the spatial heterogeneities in transmission risk. They serve as a tool for area-specific designs of control strategies, and possibly also for spatial planning of areas where livestock farming is allowed. Similar risk maps can in principle be constructed for other highly-transmissible livestock infections that spread via neighborhood transmission.

     

Effects of fixation on routine,special and immunohistochemical stains: introduction to a symposium for the Biological Stain Commission

Orcein was first proposed as an elastic fiber stain by Taenzer in 1890, and has proved very useful for the purpose. It is an important constituent of the author's “Quad” stain. Unfortunately not all orcein samples have proved equally satisfactory, whether they are derived from the lichens from which the dye was originally prepared or have been manufactured by a synthetic process. At the present time several brands are available, and two of the brands of the synthetic product investigated have not proved to be the same thing; one of the latter proves only fair as an elastin stain, the other is one of the best samples the author has ever tried.