TLR9-independent activation of B lymphocytes by bacterial DNA

The intracellular Toll-like receptor 9 (TLR9) is unique in its ability to recognize single-stranded DNA unmethylated at CpG motifs. Work from this laboratory showed that plasmid DNA is spontaneously internalized in B lymphocytes. This event is followed by the upregulation of costimulatory molecules and the acquisition of antigen presenting function by these cells. However, it is not known whether this phenomenon depends on TLR9. Because of the relevant role played by DNA-based drugs in immunotherapy and vaccination, and the central role of TLR9 signaling by CpG motifs, we decided to investigate whether signaling through TLR9 is a prerequisite for spontaneous transgenesis of lymphocytes. Here we found that transgene expression and upregulation of CD40 and CD86 costimulatory molecules was not inhibited by chloroquine treatment. Spontaneous transgenesis also occurred in B lymphocytes from TLR9-/- mice, and the injection of TLR9-/- transgenic B lymphocytes in C57Bl/6 mice induced both CD4 and CD8 T cell responses comparable to those induced by wild-type B lymphocytes. Collectively, these results suggest that plasmid DNA activates mammalian B lymphocytes through a TLR9 independent pathway.     

A drug-sensitive genetic network masks fungi from the immune system

Fungal pathogens can be recognized by the immune system via their beta-glucan, a potent proinflammatory molecule that is present at high levels but is predominantly buried beneath a mannoprotein coat and invisible to the host. To investigate the nature and significance of "masking" this molecule, we characterized the mechanism of masking and consequences of unmasking for immune recognition. We found that the underlying beta-glucan in the cell wall of Candida albicans is unmasked by subinhibitory doses of the antifungal drug caspofungin, causing the exposed fungi to elicit a stronger immune response. Using a library of bakers' yeast (Saccharomyces cerevisiae) mutants, we uncovered a conserved genetic network that is required for concealing beta-glucan from the immune system and limiting the host response. Perturbation of parts of this network in the pathogen C. albicans caused unmasking of its beta-glucan, leading to increased beta-glucan receptor-dependent elicitation of key proinflammatory cytokines from primary mouse macrophages. By creating an anti-inflammatory barrier to mask beta-glucan, opportunistic fungi may promote commensal colonization and have an increased propensity for causing disease. Targeting the widely conserved gene network required for creating and maintaining this barrier may lead to novel broad-spectrum antimycotics.     

Site Mapping and Characterization of O-Glycan Structures on α-Dystroglycan Isolated from Rabbit Skeletal Muscle

The main extracellular matrix binding component of the dystrophin-glycoprotein complex, α-dystroglycan (α-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown α-DG to be modified by both O-GalNAc- and O-mannose-initiated glycan structures. O-Mannosylation, which accounts for up to 30% of the reported O-linked structures in certain tissues, has been rarely observed on mammalian proteins. Mutations in multiple genes encoding defined or putative glycosyltransferases involved in O-mannosylation are causal for various forms of congenital muscular dystrophy. Here, we explore the glycosylation of purified rabbit skeletal muscle α-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on α-DG isolated from rabbit skeletal muscle. Additionally, we demonstrate the use of tandem mass spectrometry-based workflows to directly analyze glycopeptides generated from the purified protein. By combining glycomics and tandem mass spectrometry analysis of 91 glycopeptides from α-DG, we were able to assign 21 different residues as being modified by O-glycosylation with differing degrees of microheterogeneity; 9 sites of O-mannosylation and 14 sites of O-GalNAcylation were observed with only two sites definitively exhibiting occupancy by either type of glycan. The distribution of identified sites of O-mannosylation suggests a limited role for local primary sequence in dictating sites of attachment.     

Adaptive protein evolution and regulatory divergence in Drosophila

Two recent studies demonstrated a positive correlation between divergence in gene expression and protein sequence in Drosophila. This correlation could be driven by positive selection or variation in functional constraint. To distinguish between these alternatives, we compared patterns of molecular evolution for 1,862 genes with two previously reported estimates of expression divergence in Drosophila. We found a slight negative trend (nonsignificant) between positive selection on protein sequence and divergence in expression levels between Drosophila melanogaster and Drosophila simulans. Conversely, shifts in expression patterns during Drosophila development showed a positive association with adaptive protein evolution, though as before the relationship was weak and not significant. Overall, we found no strong evidence for an increase in the incidence of positive selection on protein-coding regions in genes with divergent expression in Drosophila, suggesting that the previously reported positive association between protein and regulatory divergence primarily reflects variation in functional constraint.     

Venom evolution widespread in fishes: a phylogenetic road map for the bioprospecting of piscine venoms

Knowledge of evolutionary relationships or phylogeny allows for effective predictions about the unstudied characteristics of species. These include the presence and biological activity of an organism's venoms. To date, most venom bioprospecting has focused on snakes, resulting in six stroke and cancer treatment drugs that are nearing U.S. Food and Drug Administration review. Fishes, however, with thousands of venoms, represent an untapped resource of natural products. The first step involved in the efficient bioprospecting of these compounds is a phylogeny of venomous fishes. Here, we show the results of such an analysis and provide the first explicit suborder-level phylogeny for spiny-rayed fishes. The results, based on approximately 1.1 million aligned base pairs, suggest that, in contrast to previous estimates of 200 venomous fishes, >1,200 fishes in 12 clades should be presumed venomous. This assertion was corroborated by a detailed anatomical study examining potentially venomous structures in >100 species. The results of these studies not only alter our view of the diversity of venomous fishes, now representing >50% of venomous vertebrates, but also provide the predictive phylogeny or "road map" for the efficient search for potential pharmacological agents or physiological tools from the unexplored fish venoms.     

The use of deslorelin implants for the synchronization of estrous in diestrous bitches

A novel approach to estrous induction in diestrous bitches is described. Twelve spontaneously cycling anestrous bitches served as controls. Thirteen anestrous and 15 diestrous bitches were induced to come into synchronous estrous using prostaglandin (diestrous bitches only) and deslorelin implants (Ovuplant). Implants contained either 2.1 or 1.05 mg deslorelin and were administered beneath the vestibular submucosa. All treated bitches came into estrous, regardless of implant size. Whereas all anestrous bitches ovulated, one of six diestrous bitches treated with the larger implant and three of nine treated with the smaller implant failed to ovulate. Induced bitches generally produced fewer corpora lutea than controls. Sixty-seven percent of control bitches became pregnant, with 0.63 fetuses per corpus luteum, whereas the pregnancy rate and fetuses per corpus luteum were 67 and 70% and 0.42 and 0.55 in the anestrous bitches induced with 1.05 and 2.1 mg deslorelin implants, respectively (not different from controls). Only 2 of 15 induced diestrous bitches conceived a detectable pregnancy, one of which was resorbed. In conclusion, although ovulatory estrous can be induced in bitches that had their most recent ovulation 40-100 days ago, these bitches are very unlikely to become pregnant during the induced estrous. The reason for the poor fertility in these diestrous bitches requires further study.     

Application of sexed semen technology to in vitro embryo production in cattle

Use of sexed semen in conjunction with in vitro embryo production is a potentially efficient means of obtaining offspring of predetermined sex. For thousands of years, livestock owners have desired a methodology to predetermine the sex of offspring for their herds. The ability to sort individual sperm cells into viable X- and Y-chromosome-bearing fractions made producers' sex selection dreams reality in the 1990s and now semen can be sexed with greater than 90% accuracy with use of a flow cytometric cell sorter. Several concerns regarding the implementation of sexed semen technology include the apparent lower fertility of sorted sperm, the lower survival of sorted sperm after cryopreservation and the reduced number of sperm that could be separated in a specified time period. These issues are discussed in this review. There are also a number of issues that appear to influence the success rates of using sexed semen to produce bovine embryos in vitro. These issues include reductions in fertilization rates, lower cleavage rates, blastocyst rates and pregnancy rates, partial capacitation of the sperm, dilute sperm samples and sire variation. These subjects are also addressed in this paper. Finally, we will describe a recent field trial in which female Holstein embryos produced using the combined technologies of sex-selected semen and microfluidics were transferred either as single or bilateral twin embryos into beef cattle recipients, demonstrating these technologies' contributions to viable embryo production. The results indicate that large-scale transfer of in vitro produced, Holstein heifer embryos to beef recipients is a feasible production scheme.     

Acyl-homoserine lactones modulate the settlement rate of zoospores of the marine alga Ulva intestinalis via a novel chemokinetic mechanism

Bacteria utilize quorum sensing to regulate the expression of cell density-dependant phenotypes such as biofilm formation and virulence. Zoospores of the marine alga Ulva intestinalis exploit the acyl-homoserine lactone (AHL) quorum sensing system to identify bacterial biofilms for preferential settlement. Here, we demonstrate that AHLs act as strong chemoattractants for Ulva zoospores. Chemoattraction does not involve a chemotactic orientation towards the AHL source. Instead, it occurs through a chemokinesis in which zoospore swimming speed is rapidly decreased in the presence of AHLs. The chemoresponse to AHLs was dependant on the nature of the acyl side chain, with N-(3-oxododecanoyl)-homoserine lactone (30-C12-HSL) being the most effective signal molecule. Mean zoospore swimming speed decreased more rapidly over wild-type biofilms of the marine bacteria Vibrio anguillarum relative to biofilms of the vanM mutant, in which AHL synthesis is disrupted. These data implicate a role for AHL-mediated chemokinesis in the location and preferential settlement of Ulva zoospores on marine bacterial assemblages. Exposure to AHLs did not inhibit the negative phototaxis of Ulva zoospores, indicating that chemoattraction to bacterial biofilms does not preclude the response to a light stimulus in substrate location.     

Alkali pH directly activates ATP-sensitive K+ channels and inhibits insulin secretion in beta-cells

Glucose stimulation of pancreatic beta-cells is reported to lead to sustained alkalization, while extracellular application of weak bases is reported to inhibit electrical activity and decrease insulin secretion. We hypothesize that beta-cell K(ATP) channel activity is modulated by alkaline pH. Using the excised patch-clamp technique, we demonstrate a direct stimulatory action of alkali pH on recombinant SUR1/Kir6.2 channels due to increased open probability. Bath application of alkali pH similarly activates native islet beta-cell K(ATP) channels, leading to an inhibition of action potentials, and hyperpolarization of membrane potential. In situ pancreatic perfusion confirms that these cellular effects of alkali pH are observable at a functional level, resulting in decreases in both phase 1 and phase 2 glucose-stimulated insulin secretion. Our data are the first to report a stimulatory effect of a range of alkali pH on K(ATP) channel activity and link this to downstream effects on islet beta-cell function.