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.     

Parental and hybrid Daphnia from the D. longispina complex: long‐term dynamics in genetic structure and significance of overwintering modes

In recent decades, hybridization has become a focus of attention because of its role in evolutionary processes. However, little is known about changes in genetic structure within and between parental species and hybrids over time. Here, we studied processes of genetic change in parental species and hybrids from the Daphnia longispina complex (Crustacea, Cladocera) over a period of six years across ten habitats. These cyclical parthenogens respond to fluctuating environments by switching from asexual to sexual reproduction. Importantly, sexually produced diapausing eggs, which resist extreme conditions such as low temperatures and serve as dispersal stages, are produced to a lower extent by hybrids. Long‐term microsatellite data revealed clear differences between hybrids and parental species. In hybrids, clonal diversity values were lower, whereas heterozygosity and linkage disequilibrium values were higher compared to parental species. Clonal diversity of hybrids responded to the strength of the winter, with cold winters resulting in few genotypes in the following spring. In time windows when only asexual hybrid females survive, priority effects will favour the establishment of the hybrid offspring before hatchlings from parental diapause eggs can enter the community. The constant high levels of heterozygosity maintained by clonal reproduction in hybrids might lead to their successful establishment over time, when they are able to escape competition from both parental species. Although we found evidence that hybrids diversity depends on fluctuating environments, a direct link between hybrid abundance and the strength of winter was missing. Because of reduced adaptability in clonally reproducing hybrids, multiple factors must contribute to promoting their long‐term success in fluctuating environments.     

Nucleic acids exert a sequence-independent cooperative effect on sequence-dependent activation of Toll-like receptor 9

Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. Although cell stimulation experiments demonstrate the preferential activation of TLR9 by CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions with respect to the ability of this receptor to bind nucleic acids in a sequence-specific manner. To address this apparent discrepancy, we report the purification of the soluble ectodomain of human TLR9 with characterization of its ligand binding properties. We observe that TLR9 has a high degree of specificity in its ability to bind nucleic acids that contain CpG dinucleotides as well as higher order motifs that mediate species-specific activation. However, TLR9 is also functionally influenced by nucleic acids in a sequence-independent fashion as both stimulatory and nonstimulatory nucleic acids sensitize TLR9 for in vitro ligand binding as well as in vivo activation. We propose a model in which receptor activation is achieved in a sequence-dependent manner, and sensitivity is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. This model bears resemblance to that recently proposed for Toll in that activation is a two-step process in which formation of a ligand-bound monomer precedes formation of the activated dimer. In each model receptor sensitivity is determined within the second step with the crucial distinction that Toll undergoes negative cooperativity, whereas TLR9 is sensitized through a positive cooperative effect.     

Targeting with bovine CD154 enhances humoral immune responses induced by a DNA vaccine in sheep

CD40-CD154 interactions play an important role in regulating humoral and cell-mediated immune responses. Recently, these interactions have been exploited for the development of therapeutic and preventive treatments. The objective of this study was to test the ability of bovine CD154 to target a plasmid-encoded Ag to CD40-expressing APCs. To achieve this, a plasmid coding for bovine CD154 fused to a truncated secreted form of bovine herpesvirus 1 glycoprotein D (tgD), pSLIAtgD-CD154, was constructed. The chimeric tgD-CD154 was expressed in vitro in COS-7 cells and reacted with both glycoprotein D- and CD154-specific Abs. Both tgD and tgD-CD154 were capable of binding to epithelial cells, whereas only tgD-CD154 bound to B cells. Furthermore, dual-labeling of ovine PBMCs revealed that tgD-CD154 was bound by primarily B cells. The functional integrity of the tgD-CD154 chimera was confirmed by the induction of both IL-4-dependent B cell proliferation and tgD-specific lymphoproliferative responses in vitro. Finally, sheep immunized with pSLIAtgD-CD154 developed a more rapid primary tgD-specific Ab response and a significantly stronger tgD-specific secondary response when compared with animals immunized with pSLIAtgD and control animals. Similarly, virus-neutralizing Ab titers were significantly higher after secondary immunization with pSLIAtgD-CD154. These results demonstrate that using CD154 to target plasmid-expressed Ag can significantly enhance immune responses induced by a DNA vaccine.     

Differential display analysis of gene expression during the induction of mucosal immunity

One approach to understanding the physiologically relevant events during the induction of an immune response is to identify genes that are expressed when the immune system first encounters antigen. Such an investigation requires a naive but fully functional immune system, and the fetal lamb provides these conditions during the last trimester of gestation. 'Intestinal segments,' containing a jejunal Peyer's patch, were surgically prepared in fetal lambs (>120 days gestation) and individual 'intestinal segments' were injected with either culture medium or infectious bovine rotavirus. Peyer's patch tissue was collected 18 h postinfection. Histology and virus culture confirmed that bovine rotavirus had infected the mucosal epithelium. RNA was extracted from jejunal Peyer's patch tissue and mRNA differential display was used to identify genes expressed following rotavirus infection. Ten cDNAs were identified by differential display and these cDNAs were isolated, cloned, and sequenced. One of the cDNAs sequenced, displayed homology to the gene encoding the sperm surface protein Sp17. Differential expression of this gene in antigen-exposed jejunal Peyer's patches was confirmed by Northern blot and RT-PCR. The complete sequence for sheep Sp17 mRNA was obtained from a lambda cDNA library, prepared from the jejunal Peyer's patch of a young lamb. Sp17 expression was detected by RT-PCR in a variety of mucosa-associated lymphoid tissues but not in primary or other secondary lymphoid tissues. Thus, the fetal lamb model may be appropriate for identifying genes relevant to mucosal immunity.     

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.     

On impulse response functions computed from dynamic contrast-enhanced image data by algebraic deconvolution and compartmental modeling

Concentration-time courses measured by dynamic contrast-enhanced (DCE) imaging can be described by a convolution of the arterial input with an impulse response function, Q_T(t), characterizing tissue microcirculation. Data analysis is based on two different approaches: computation of Q_T(t) by algebraic deconvolution (AD) and subsequent evaluation according to the indicator dilution theory (IDT) or parameterization of Q_T(t) by analytical expressions derived by compartmental modeling. Pitfalls of both strategies will be addressed in this study.Tissue data acquired by DCE-CT in patients with head-and-neck cancer and simulated by a reference model (MMID4) were analyzed by a two-compartment model (TCM), a permeability-limited two-compartment model (PL-TCM) and AD. Additionally, MMID4 was used to compute the ‘true’ response function that corresponds to the simulated tumor data.TCM and AD yielded accurate fits, whereas PL-TCM performed worse. Nevertheless, the corresponding response functions diverge markedly. The response curves obtained by TCM decrease exponentially in the early perfusion phase and overestimate the tissue perfusion, Q_T(0). AD also resulted in response curves starting with a negative slope and not – as the ‘true’ response function in accordance with the IDT – with a horizontal plateau. They are thus not valid responses in the sense of the IDT that can be used unconditionally for parameter estimation.Response functions differing considerably in shape can result in virtually identical tissue curves. This non-uniqueness makes a strong argument not to use algebraic but rather analytical deconvolution to reduce the class of solutions to representatives that are in accordance with a-priori knowledge. To avoid misinterpretations and systematic errors, users must be aware of the pitfalls inherent to the different concepts.     

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.