Biomembrane force probe investigation of RNA dissociation

Investigations into the energy pathways of biomolecular interactions by use of dynamic force spectroscopy are limited by the range of loading rates accessible with a single technique. In the work discussed in this paper, this range has been extended for a previously studied system by using the biomembrane force probe (BFP). This work builds on our previous single-molecule atomic force microscopy (AFM) study of the dissociation of a bulge-motif-containing RNA complex. The disparity observed, at high loading rates, between the dissociation of a 12-base pair complex with and without a central three-base pair bulge was not observed at low rates. This suggests that the two species share a similar outer barrier to dissociation and that inclusion of the bulge motif creates an additional barrier at a distance closer to the bound state. Experiments performed in different buffer environments yielded similar results. The results, when combined with those of previous studies, suggest that the shared outer barrier to dissociation is that due to a rearrangement and fraying of the ends of the helix.     

Picomolar nitric oxide signals from central neurons recorded using ultrasensitive detector cells

Nitric oxide (NO) is a widespread signaling molecule with potentially multifarious actions of relevance to health and disease. A fundamental determinant of how it acts is its concentration, but there remains a lack of coherent information on the patterns of NO release from its sources, such as neurons or endothelial cells, in either normal or pathological conditions. We have used detector cells having the highest recorded NO sensitivity to monitor NO release from brain tissue quantitatively and in real time. Stimulation of NMDA receptors, which are coupled to activation of neuronal NO synthase, routinely generated NO signals from neurons in cerebellar slices. The average computed peak NO concentrations varied across the anatomical layers of the cerebellum, from 12 to 130 pm. The mean value found in the hippocampus was 200 pm. Much variation in the amplitudes recorded by individual detector cells was observed, this being attributable to their location at variable distances from the NO sources. From fits to the data, the NO concentrations at the source surfaces were 120 pm to 1.4 nm, and the underlying rates of NO generation were 36-350 nm/s, depending on area. Our measurements are 4-5 orders of magnitude lower than reported by some electrode recordings in cerebellum or hippocampus. In return, they establish coherence between the NO concentrations able to elicit physiological responses in target cells through guanylyl cyclase-linked NO receptors, the concentrations that neuronal NO synthase is predicted to generate locally, and the concentrations that neurons actually produce.     

Historical tests of the toxicity of pesticides to Typhlodromus pyri (Acari: Phytoseiidae) and their relevance to current pest management in New Zealand apple orchards 2. Short-term field tests

A two-part review is presented relating historical tests of the toxicity of pesticides to Typhlodromus pyri and their relevance to modern pest management in New Zealand pome-fruit orchards. Over the past 30 years, the initial need for T. pyri to be resistant to broad-spectrum pesticides has substantially declined as a growing array of new selective chemicals have come into use. In Part 2, a short-term field test is described for determining the toxicity of single applications of pesticides at recommended rates to European red mite (ERM), Panonychus ulmi, and its predator, an organophosphate (OP)-resistant strain of T. pyri on apples in New Zealand. For each pesticide, changes in mite density were measured from pre-treatment to 2, 7 and up to 25 days post-treatment compared with a water-sprayed control. Density was recorded and analysed for live adult and immature ERM, and live and dead eggs, larvae, nymphs and adults of T. pyri. Fifteen acaricides, 17 fungicides and 17 insecticides were evaluated. Chemicals more toxic to T. pyri than ERM were aminocarb, amitraz, binapacryl, chlordimeform, etrimphos, fenvalerate + azinphos-methyl, mancozeb + dinocap, methidathion, methiocarb, omethoate, oxamyl, pirimiphos-methyl and pyrazophos. Chemicals equally or less toxic to T. pyri than to ERM were acequinocyl, azocyclotin, benzoximate, bromopropylate, chlorpyrifos, clofentezine, cycloprate, cyhexatin, dinocap, mineral oil, propargite, triazophos and vamidothion. The remaining 23 chemicals (primarily fungicides and OP insecticides) had slight or no toxicity to ERM and T.pyri. The short-term field tests provided a useful guide to the long-term effects on ERM and T. pyri populations of almost all the pesticides. However, the potential disruptive effect of pyrazophos was not found in long-term field trials, and conversely, the apparently harmless dithiocarbamate fungicides were later shown to be highly disruptive when repeatedly sprayed, as in commercial practice. Most of the chemicals tested are no longer used in commercial pome-fruit orchards in New Zealand, all of which now practise integrated fruit production or organic fruit production based on selective pest management methods. The tested pesticides of continuing importance are identified and discussed with special emphasis on the current need to retest for dithiocarbamate resistance in T. pyri, some populations of which have been exposed to these compounds for up to 40 years. This and the changes in pesticide use in New Zealand are paralleled by similar developments in most pome-fruit growing areas of the world.     

Red Blood Cell Invasion by Plasmodium vivax: Structural Basis for DBP Engagement of DARC

Plasmodium parasites use specialized ligands which bind to red blood cell (RBC) receptors during invasion. Defining the mechanism of receptor recognition is essential for the design of interventions against malaria. Here, we present the structural basis for Duffy antigen (DARC) engagement by P. vivax Duffy binding protein (DBP). We used NMR to map the core region of the DARC ectodomain contacted by the receptor binding domain of DBP (DBP-RII) and solved two distinct crystal structures of DBP-RII bound to this core region of DARC. Isothermal titration calorimetry studies show these structures are part of a multi-step binding pathway, and individual point mutations of residues contacting DARC result in a complete loss of RBC binding by DBP-RII. Two DBP-RII molecules sandwich either one or two DARC ectodomains, creating distinct heterotrimeric and heterotetrameric architectures. The DARC N-terminus forms an amphipathic helix upon DBP-RII binding. The studies reveal a receptor binding pocket in DBP and critical contacts in DARC, reveal novel targets for intervention, and suggest that targeting the critical DARC binding sites will lead to potent disruption of RBC engagement as complex assembly is dependent on DARC binding. These results allow for models to examine inter-species infection barriers, Plasmodium immune evasion mechanisms, P. knowlesi receptor-ligand specificity, and mechanisms of naturally acquired P. vivax immunity. The step-wise binding model identifies a possible mechanism by which signaling pathways could be activated during invasion. It is anticipated that the structural basis of DBP host-cell engagement will enable development of rational therapeutics targeting this interaction.     

Reducing morbidity in the radial forearm flap donor site

The radial forearm flap, although widely used, has been criticized for the poor quality of its donor site. To investigate the causes of morbidity, 100 radial artery free-flap donor sites have been reviewed. Sixty-seven patients required skin grafting (group 1), and the remaining 33 patients were closed directly (group 2). Seventeen patients in the series had compound osteocutaneous flaps (group 3). Wound healing proved to be a significant problem in groups 1 and 3, and fracture of the radius occurred in 4 of the 17 patients in group 3 and was the most significant cause of morbidity. The radial artery was reconstructed in 12 patients, but only 6 of the arteries (50 percent) were patent at the time of review. Subjective assessment on a scale of 0 to 10 demonstrated a relatively pain-free donor site with low pain scores (2.5 of 10). The cosmetic result was acceptable in men (1.5 of 10) but was less so in women (4 of 10). Angulated fracture of the radius produced an unacceptable cosmetic result (7 of 10). In light of this experience, we no longer reconstruct the radial artery as a matter of routine. The donor defect is closed directly wherever possible using an ulnar artery-based transposition flap when required. A "boat shaped" osteotomy is used in preference to right-angled bone cuts when harvesting a segment of radius to avoid the complications and sequelae of fracture. These changes in surgical technique have improved the acceptability and minimized the problems associated with this donor site.     

Amelioration of PXR-mediated CYP3A4 induction by mGluR2 modulators

This work describes the rational amelioration of Cytochrome P450 4/5 (CYP3A4/5) induction through the Pregnane-X Receptor (PXR) pathway in a series of compounds that modulate the metabotropic glutamate Receptor 2 (mGluR2) via an allosteric mechanism. The compounds were initially shown to induce CYP3A4/5 via the gold-standard induction assay measured in primary human hepatocytes. This was followed up by testing the compounds in a PXR assay which correlated well with the assay in primary cells. Further, one of the compounds was crystallized with PXR (pdb code 6DUP). Analysis of this co-crystal structure, together with previously published PXR co-crystal structures, lead to modification ideas. The compounds synthesized based on these ideas were shown not to be CYP3A4/5 inducers. The mGluR2 activity of the resulting compounds was maintained.