N-glycosylation analysis of the human Tweety family of putative chloride ion channels supports a penta-spanning membrane arrangement: impact of N-glycosylation on cellular processing of Tweety homologue 2 (TTYH2)

The Tweety proteins are a family of recently identified putative Cl(-) channels predicted to be modified by N-glycosylation and, controversially, to contain five or six membrane-spanning domains, leading to the contentious proposal that members of this family do not share the same topology at the plasma membrane. In humans, three family members have been identified, designated TTYH1 (Tweety homologue 1), TTYH2 and TTYH3. To gain greater insight into the arrangement of membrane-spanning domains and cellular processing of Tweety proteins, in the present study we have examined the sequence homology, hydrophobicity and N-glycan content of members of this family and performed N-glycosylation site-mutagenesis studies on TTYH2 and TTYH3. Based on these observations we propose a structure for Tweety family proteins which incorporates five membrane-spanning domains with a topology at the cell surface in which the N-terminus is located extracellularly and the C-terminus cytoplasmically. Our results also suggest that N-glycosylation is important, but not essential, in the processing of members of the Tweety family with results indicating that, although incomplete N-glycosylation mediates reduced expression and increased ubiquitination of TTYH2, N-glycosylation is not the determining factor for TTYH2 trafficking to the plasma membrane. This information will be important for the characterization of Tweety family proteins in normal physiology and disease.     

Toe-out gait in patients with knee osteoarthritis partially transforms external knee adduction moment into flexion moment during early stance phase of gait: a tri-planar kinetic mechanism

Altered gait kinematics and kinetics are observed in patients with medial compartment knee osteoarthritis. Although various kinematic adaptations are proposed to be compensatory mechanisms that unload the knee, the nature of these mechanisms is presently unclear. We hypothesized that an increased toe-out angle during early stance phase of gait shifts load away from the knee medial compartment, quantified as the external adduction moment about the knee. Specifically, we hypothesized that by externally rotating the lower limb anatomy, primarily about the hip joint, toe-out gait alters the lengths of ground reaction force lever arms acting about the knee joint in the frontal and sagittal planes and transforms a portion of knee adduction moment into flexion moment. To test this hypothesis, gait data from 180 subjects diagnosed with medial compartment knee osteoarthritis were examined using two frames of reference. The first frame was attached to the tibia (reporting actual toe-out) and the second frame was attached to the laboratory (simulating no-toe-out). Four measures were compared within subjects in both frames of reference: the lengths of ground reaction force lever arms acting about the knee joint in the frontal and sagittal planes, and the adduction and flexion components of the external knee moment. The mean toe-out angle was 11.4 degrees (S.D. 7.8 degrees , range -2.2 degrees to 28.4 degrees ). Toe-out resulted in significant reductions in the frontal plane lever arm (-6.7%) and the adduction moment (-11.7%) in early stance phase when compared to the simulated no-toe-out values. These reductions were coincident with significant increases in the sagittal plane lever arm (+33.7%) and flexion moment (+25.0%). Peak adduction lever arm and moment were also reduced significantly in late stance phase (by -22.9% and -34.4%, respectively) without a corresponding increase in sagittal plane lever arm or flexion moment. These results indicate that toe-out gait in patients with medial compartment knee osteoarthritis transforms a portion of the adduction moment into flexion moment in early stance phase, suggesting that load is partially shifted away from the medial compartment to other structures.     

Parallel synthesis of 3-aryloxy-2-propanolamines and evaluation as dual affinity 5-HT(1A) and 5-HT re-uptake ligands

A solution phase synthesis for the preparation of 3-aryloxy-2-propanolamine libraries has been developed. This resulted in the identification of 5 as a ligand with dual affinity for 5-HT1A and serotonin reuptake receptors which shows excellent pharmacokinetic parameters.     

Lilac alcohol epoxide: a linalool derivative in Actinidia arguta flowers

Lilac alcohol epoxide (2-(5-methyl-5-(oxiran-2-yl)-tetrahydrofuran-2-yl)propan-1-ol), a previously unreported monoterpene, was identified in the solvent extract of the flowers of seven Actinidia arguta genotypes. The diastereomeric lilac alcohol epoxides co-occurred with the lilac aldehydes and alcohols. Another compound, the lilac diol (2-(5-(1-hydroxyethyl)-5-methyl-tetrahydrofuran-2-yl)propan-1-ol) was synthesised as part of our efforts to identify the lilac alcohol epoxide.     

Evaluation of 23S rRNA PCR primers for use in phylogenetic studies of bacterial diversity

The availability of a diverse set of 23S rRNA gene sequences enabled evaluation of the specificity of 39 previously published and 4 newly designed primers specific for bacteria. An extensive clone library constructed using an optimized primer pair resulted in similar gene richness but slightly differing coverage of some phylogenetic groups, compared to a 16S rRNA gene library from the same environmental sample.     

Identification of pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of Met kinase

An amide library derived from the pyrrolo[2,1-f][1,2,4]triazine scaffold led to the identification of modest inhibitors of Met kinase activity. Introduction of polar side chains at C-6 of the pyrrolotriazine core provided significant improvements in in vitro potency. The amide moiety could be replaced with acylurea and malonamide substituents to give compounds with improved potency in the Met-driven GTL-16 human gastric carcinoma cell line. Acylurea pyrrolotriazines with substitution at C-5 demonstrated single digit nanomolar kinase activity. X-ray crystallography revealed that the C-5 substituted pyrrolotriazines bind to the Met kinase domain in an ATP-competitive manner.     

Baroreflex responsiveness during ventilatory acclimatization in humans

We tested the hypothesis that the decline in muscle sympathetic activity during and after 8 h of poikilocapnic hypoxia (Hx) was associated with a greater sympathetic baroreflex-mediated responsiveness. In 10 healthy men and women (n=2), we measured beat-to-beat blood pressure (Portapres), carotid artery distension (ultrasonography), heart period, and muscle sympathetic nerve activity (SNA; microneurography) during two baroreflex perturbations using the modified Oxford technique before, during, and after 8 h of hypoxia (84% arterial oxygen saturation). The integrated baroreflex response [change of SNA (DeltaSNA)/change of diastolic blood pressure (DeltaDBP)], mechanical (Deltadiastolic diameter/DeltaDBP), and neural (DeltaSNA/Deltadiastolic diameter) components were estimated at each time point. Sympathetic baroreflex responsiveness declined throughout the hypoxic exposure and further declined upon return to normoxia [pre-Hx, -8.3+/-1.2; 1-h Hx, -7.2+/-1.0; 7-h Hx, -4.9+/-1.0; and post-Hx: -4.1+/-0.9 arbitrary integrated units (AIU) x min(-1) x mmHg(-1); P<0.05 vs. previous time point for 1-h, 7-h, and post-Hx values]. This blunting of baroreflex-mediated efferent outflow was not due to a change in the mechanical transduction of arterial pressure into barosensory stretch. Rather, the neural component declined in a similar pattern to that of the integrated reflex response (pre-Hx, -2.70+/-0.53; 1-h Hx, -2.59+/-0.53; 7-h Hx, -1.60+/-0.34; and post-Hx, -1.34+/-0.27 AIU x min(-1) x microm(-1); P < 0.05 vs. pre-Hx for 7-h and post-Hx values). Thus it does not appear as if enhanced baroreflex function is primarily responsible for the reduced muscle SNA observed during intermediate duration hypoxia. However, the central transduction of baroreceptor afferent neural activity into efferent neural activity appears to be reduced during the initial stages of peripheral chemoreceptor acclimatization.     

A novel mechanism by which silica defends grasses against herbivory

Background and Aims

Previous studies have shown that silica in grass leaves defends them against small herbivores, which avoid high-silica grasses and digest them less efficiently. This study tested the idea that silica can reduce digestibility by preventing the mechanical breakdown of chlorenchyma cells.

Methods

Both the percentage of total chlorophyll liberated from high- and low-silica grass leaves by mechanical grinding and the chlorophyll content of locust faeces were measured.

Key Results

High-silica grasses released less chlorophyll after grinding and retained more after passing through the gut of locusts, showing that silica levels correlated with increased mechanical protection.

Conclusions

These results suggest that silica may defend grasses at least in part by reducing mechanical breakdown of the leaf, and that mechanical protection of resources in chlorenchyma cells is a novel and potentially important mechanism by which silica protects grasses.Key words: Grass, silica, locust, digestibility, defence, Lolium perenne, Festuca ovina