Mode of Action of the Natural Insecticide, Decaleside Involves Sodium Pump Inhibition.

Decalesides are a new class of natural insecticides which are toxic to insects by contact via the tarsal gustatory chemosensilla. The symptoms of their toxicity to insects and the rapid knockdown effect suggest neurotoxic action, but the precise mode of action and the molecular targets for decaleside action are not known. We have presented experimental evidence for the involvement of sodium pump inhibition in the insecticidal action of decaleside in the cockroach and housefly. The knockdown effect of decaleside is concomitant with the in vivo inhibition of Na+, K+ -ATPase in the head and thorax. The lack of insecticidal action by experimental ablation of tarsi or blocking the tarsal sites with paraffin correlated with lack of inhibition of Na+- K+ ATPase in vivo. Maltotriose, a trisaccharide, partially rescued the toxic action of decaleside as well as inhibition of the enzyme, suggesting the possible involvement of gustatory sugar receptors. In vitro studies with crude insect enzyme preparation and purified porcine Na+, K+ -ATPase showed that decaleside competitively inhibited the enzyme involving the ATP binding site. Our study shows that the insecticidal action of decaleside via the tarsal gustatory sites is causally linked to the inhibition of sodium pump which represents a unique mode of action. The precise target(s) for decaleside in the tarsal chemosensilla and the pathway linked to inhibition of sodium pump and the insecticidal action remain to be understood.

Porcine sperm bind to specific 6-sialylated biantennary glycans to form the oviduct reservoir.

After mating, many female mammals store a subpopulation of sperm in the lower portion of the oviduct, forming a reservoir. The reservoir lengthens sperm lifespan, regulates sperm capacitation, controls polyspermy, and selects normal sperm. It is believed that sperm bind to glycans on the oviduct epithelium to form the reservoir, but the specific adhesion molecules that retain sperm are unclear. Herein, using a glycan array to test 377 glycans for their ability to bind porcine sperm, we found two glycan motifs in common among all glycans with sperm-binding ability: the Lewis X trisaccharide and biantennary structures containing a mannose core with 6-sialylated lactosamine at one or more termini. Binding to both motifs was specific; isomers of each motif did not bind sperm. Further work focused on sialylated lactosamine. Sialylated lactosamine was found abundantly on the apical side of epithelial cells collected from the oviduct isthmus, among N-linked and O-linked glycans. Sialylated lactosamine bound to the head of sperm, the region that interacts with the oviduct epithelium. After capacitation, sperm lost affinity for sialylated lactosamine. Receptor modification may contribute to release from the reservoir so that sperm can move to the site of fertilization. Sialylated lactosamine was required for sperm to bind oviduct cells. Simbucus nigra agglutinin or an antibody specific to sialylated lactosamine with a preference for Neu5Acalpha2-6Gal rather than Neu5Acalpha2-3Gal reduced sperm binding to oviduct isthmic cells, as did occupying putative receptors on sperm with sialylated biantennary glycans. These results demonstrate that sperm binding to oviduct 6-sialylated biantennary glycans is necessary for normal adhesion to the oviduct.

Oxidative coupling of a feruloyl-arabinoxylan trisaccharide (FAXX) in the walls of living maize cells requires endogenous hydrogen peroxide and is controlled by a low-Mr apoplastic inhibitor.

Feruloyl-polysaccharides can be oxidatively coupled in isolated cell walls by peroxidase plus exogenous H(2)O(2) in vitro, but the extent to which similar reactions may occur in the apoplast in vivo was unclear. Numerous cellular factors potentially control feruloyl coupling in vivo, and their net controlling influence is not readily studied in vitro. Therefore, we have monitored apoplastic feruloyl coupling in cultured maize cells in vivo using a radiolabelled model substrate, 5-O-feruloyl-alpha-L: -arabinofuranosyl-(1-->3)-beta-D: -xylopyranosyl-(1-->4)-D: -xylose (FAXX). FAXX was expected to permeate the wall and to undergo reactions analogous to those normally exhibited by apoplastic feruloyl-polysaccharides in vivo. Little difference was found between the fates of [feruloyl-(14)C]FAXX and [pentosyl-(3)H]FAXX, indicating negligible apoplastic hydrolase or transferase activities. Very little radioactivity entered the protoplasm. Maize cells that had recently been washed in fresh medium were able to bind most of the FAXX (90%) in their cell walls, regardless of the age of the culture. During wall-binding, the [(14)C]feruloyl groups were converted to [(14)C]dehydrodiferulates and larger coupling products, as revealed by TLC after alkaline hydrolysis. As expected for an oxidative reaction, wall-binding was delayed by added anti-oxidants (ascorbate, ferulate, sinapate, chlorogenate or rutin). It was also completely inhibited by iodide, an H(2)O(2)-scavenger, indicating a role for peroxidase rather than oxidase. The observations indicate that oxidative coupling of feruloyl groups occurred within the cell wall, dependent on endogenous apoplastic H(2)O(2) and wall-localised peroxidase, in vivo. Cells that had not recently been washed in fresh medium were much less able to bind FAXX, indicating the presence in the apoplast of an endogenous inhibitor of oxidative coupling. This inhibitor was of low M(r), was destroyed by heating, and remained in the aqueous phase (pH approximately 3.5) when shaken with ethyl acetate. Its effectiveness was not altered by ascorbate oxidase. It is thus a small, heat-labile, hydrophilic inhibitor (not ascorbate) which we suggest plays a natural role in the control of wall cross-linking, and thus potentially in the control of cell growth.

Olefin self-metathesis as a new entry into xenotransplantation antagonists bearing the Galili antigen.

A hexameric disaccharide cluster bearing the terminal Gal alpha related xenotransplantation antigen was constructed using a sequence of ruthenium carbenoid catalyzed olefin self-metathesis of monoallylated tribenzyl pentaerythritol followed, after interconversion of benzyl ethers into para-iodobenzyl ethers, by a single step Sonogashira cross-coupling of six prop-2-ynyl glycosides onto a hexameric aryl iodide scaffold.

Site-directed selection of oligonucleotide antagonists by competitive elution.

Oligonucleotide ligands that bind a protein or a small molecule of interest are readily isolated by in vitro selection and amplification of rare sequences from combinatorial libraries of sequence-randomized oligonucleotides (Gold et al., 1995). Classic systematic evolution of ligands by exponential enrichment (SELEX) protocols are affinity based (Tuerk and Gold, 1990), but because many problems and applications require antagonists, protocols for selecting inhibitors are both desirable and valuable. A widely applicable approach for isolating inhibitors is competitive elution with a molecule that binds the targeted molecule's active or binding site. We have used this approach to isolate antagonists of wheat germ agglutinin (WGA) from a library of 2'NH2-pyrimidine, 2'OH-purine oligonucleotides by elution with N N' N"-triacetylchitotriose, (GlcNAc)3. The highest affinity aptamers have equilibrium dissociation constants of 1 nM-20 nM for WGA, a 10(3)-10(4)-fold improvement relative to (GlcNAc)3, and unlike the carbohydrate, are highly specific. In addition to competing for binding with (GlcNAc)3, aptamers inhibit WGA-mediated agglutination of sheep erythrocytes, demonstrating that they are able to compete with natural ligands presented on the surfaces of cells. These results illustrate the feasibility of isolating high-affinity, high-specificity antagonists by competitive elution with low molecular weight, relatively low-affinity, and low-specificity small molecules.