Single-chain polymorphism analysis in long QT syndrome using planar waveguide fluorescent biosensors

Rapid detection of single nucleotide polymorphisms (SNPs) has potential applications in both genetic screening and pharmacogenomics. Planar waveguide fluorescent biosensor technology was employed to detect SNPs using a simple hybridization assay with the complementary strand ("capture oligo") immobilized on the waveguide. This technology allows real-time measurements of DNA hybridization kinetics. Under normal conditions, both the wild-type sequence and the SNP-containing sequence will hybridize with the capture oligo, but with different reaction kinetics and equilibrium duplex concentrations. A "design of experiments" approach was used to maximize the differences in the kinetics profiles of the two. Nearly perfect discrimination can be achieved at short times (2 min) with temperatures that destabilize or melt the heteroduplex while maintaining the stability of the homoduplex. The counter ion content of the solvent was shown to have significant effect not only on the melting point of the heteroduplex and the homoduplex but also on the hybridization rate. Changes in both the stability and the difference between the hybridization rates of the hetero- and homoduplex were observed with varying concentrations of three different cations (Na(+), K(+), Mg(2+)). With the difference in hybridization rates maximized, discrimination between the hetero- and the homoduplex can be obtained at lower, less rigorous temperatures at hybridization times of 7.5 min or longer.     

Alteration of the metaphase checkpoint by B chromosomes

The B chromosome polymorphism in Spanish populations of the grasshopper, Eyprepocnemis plorans (Charpentier) is ancient and widespread. Meiocytes containing B chromosomes were analyzed in our laboratory using the 3F3/2 monoclonal antibody, which binds to a kinetochore phosphoepitope whose degree of phosphorylation is sensitive to tension applied to the kinetochore. Further, the tension created by the spindle at metaphase controls a checkpoint (the metaphase checkpoint) that allows the cell to begin anaphase when all chromosomes are aligned at the metaphase plate. Fluorescence patterns of the 3F3/2 phosphoepitope in cells containing B chromosomes were determined using confocal laser scanning microscopy. The phosphorylation pattern of kinetochores in these cells was shown to be different from that of cells without Bs. This suggests that the metaphase checkpoint has been modified in some way. We propose that B chromosomes in these grasshopper populations may have survived during evolution due to an alteration of the metaphase checkpoint, making it more permissive to the presence of misaligned chromosomes.     

Expression and purification of the hormone binding domain of the Drosophila ecdysone and ultraspiracle receptors

Escherichia coli vectors were constructed for the production of a protein complex that mimics the native ecdysone receptor (EcR) isolated from Drosophila. The two steroid receptors, ultraspiracle (USP) and EcR, were expressed as truncations, retaining primarily the hormone binding domains. The recombinant receptor complex was able to mimic the pharmacology of the native receptor with respect to both synthetic and natural agonists. USP and EcR fusion proteins could be expressed in separate cell lines and then recombined following isolation to yield a ligand binding preparation with a dissociation constant (K(D)) for Ponasterone A of 1.5 nM and a total yield of 1.9 pmol ligand binding sites/mg protein. Alternatively, the simultaneous coexpression of both receptors increased yields by several orders of magnitude to 6 nmol ligand binding sites/mg protein with a K(D) of 0.6 nM. Chromatographic analysis under native conditions showed that EcR, when expressed alone, migrated as a variety of complexes, mostly coming out in the void volume as denatured, insoluble, aggregate. In contrast, purified extracts of coexpressed EcR and USP eluted as a single peak with a mobility indicating a heterodimer. The majority of the coexpressed fusion receptors, following purification, formed functional steroid binding sites. A detailed scheme is provided for the expression and isolation of milligram quantities of highly purified receptor dimer.     

First detection of chlorfenapyr (Secure®) resistance in two-spotted spider mite (Acari: Tetranychidae) from nectarines in an Australian orchard

Chlorfenapyr resistance (2.9- and 19.9-fold respectively at LC50 and LC99 level) was detected in Tetranychus urticae Koch causing control failure following a single application of product to nectarines.     

Force probe measurements of antibody-antigen interactions

The surface force apparatus has been used to quantify directly the forces that govern the interactions between proteins and ligands. In this work, we describe the measured interactions between the antigen fluorescein and the Fab' fragment of the monoclonal 4-4-20 anti-fluorescyl IgG antibody. Here we first describe the use of the surface force apparatus to demonstrate directly the impact of the charge composition in the region of the antibody binding site on the antibody interactions. Several approaches are described for immobilizing antigens, antibodies, and proteins in general for direct force measurements. The measured force profiles presented are accompanied by an extensive discussion of protocols used to analyze the force-distance curves and to interpret them in terms of the antibody structure. In addition to long-range electrostatic forces, we also consider short-range forces that can affect the strength of adhesion between the Fab' and immobilized fluorescein. The latter investigations demonstrate the influence of interfacial properties on the recognition of surface-bound antigens.     

Cannabidiol Reverses Deficits in Hippocampal LTP in a Model of Alzheimer’s Disease

Here we demonstrate for the first time that cannabidiol (CBD) acts to protect synaptic plasticity in an in vitro model of Alzheimer’s disease (AD). The non-psycho active component of Cannabis sativa, CBD has previously been shown to protect against the neurotoxic effects of beta amyloid peptide (Aβ) in cell culture and cognitive behavioural models of neurodegeneration. Hippocampal long-term potentiation (LTP) is an activity dependent increase in synaptic efficacy often used to study cellular mechanisms related to memory. Here we show that acute application of soluble oligomeric beta amyloid peptide (Aβ_1–42) associated with AD, attenuates LTP in the CA₁ region of hippocampal slices from C57Bl/6 mice. Application of CBD alone did not alter LTP, however pre-treatment of slices with CBD rescued the Aβ_1–42 mediated deficit in LTP. We found that the neuroprotective effects of CBD were not reversed by WAY100635, ZM241385 or AM251, demonstrating a lack of involvement of 5HT_1A, adenosine (A_2A) or Cannabinoid type 1 (CB₁) receptors respectively. However in the presence of the PPARγ antagonist GW9662 the neuroprotective effect of CBD was prevented. Our data suggests that this major component of Cannabis sativa, which lacks psychoactivity may have therapeutic potential for the treatment of AD.

     

Chronic intrauterine pulmonary hypertension increases capacitative calcium entry in fetal pulmonary artery smooth muscle cells

Oxygen causes perinatal pulmonary dilatation. Although fetal pulmonary artery smooth muscle cells (PA SMC) normally respond to an acute increase in oxygen (O2) tension with a decrease in cytosolic calcium ([Ca2+]i), an acute increase in O2 tension has no net effect on [Ca(2+)](i) in PA SMC derived from lambs with chronic intrauterine pulmonary hypertension (PHTN). The present experimental series tests the hypothesis that an acute increase in O2 tension decreases capacitative calcium entry (CCE) in normal, but not hypertensive, fetal PA SMC. PA SMC were isolated from late-gestation fetal lambs after either ligation of the ductus arteriosus (PHTN) or sham (control) operation at 127 days gestation. PA SMC were isolated from the distal PA (>or=4th generation) and maintained under hypoxic conditions ( approximately 25 Torr) in primary culture. After fura 2 loading, apparent [Ca2+]i in PA SMC was determined as the ratio of 340- to 380-nm fluorescence intensity. Under both hypoxic and normoxic conditions, cyclopiazonic acid (CPA) increased [Ca2+]i more in PHTN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca2+ and intracellular store depletion with CPA, followed by superfusion with Ca2+-containing solution, in the presence of the voltage-operated calcium channel blockade. CCE was increased in PHTN compared with control PA SMC under conditions of both acute and sustained normoxia. Transient receptor potential channel gene expression was greater in control compared with PHTN PA SMC. PHTN may compromise perinatal pulmonary vasodilation, in part, by modulating PA SMC CCE.     

Anthranilamide inhibitors of factor Xa

SAR about the B-ring of a series of N(2)-aroyl anthranilamide factor Xa (fXa) inhibitors is described. B-ring o-aminoalkylether and B-ring p-amine probes of the S1' and S4 sites, respectively, afforded picomolar fXa inhibitors that performed well in in vitro anticoagulation assays.     

Invasive plants and their ecological strategies: prediction and explanation of woody plant invasion in New England

Effective management of introduced species requires the early identification of species that pose a significant threat of becoming invasive. To better understand the invasive ecology of species in New England, USA, we compiled a character data set with which to compare non-native species that are known invaders to non-native species that are not currently known to be invasive. In contrast to previous biological trait-based models, we employed a Bayesian hierarchical analysis to identify sets of plant traits associated with invasiveness for each of three growth forms (vines, shrubs, and trees). The resulting models identify a suite of 'invasive traits' highlighting the ecology associated with invasiveness for each of three growth forms. The most effective predictors of invasiveness that emerged from our model were 'invasive elsewhere', 'fast growth rate', 'native latitudinal range', and 'growth form'. The contrast among growth forms was pronounced. For example, 'wind dispersal' was positively correlated with invasiveness in trees, but negatively correlated in shrubs and vines. The predictive model was able to correctly classify invasive plants 67% of the time (22/33), and non-invasive plants 95% of the time (204/215). A number of potential future invasive species in New England that deserve management consideration were identified.