Novel isosorbide di-ester compounds as inhibitors of acetylcholinesterase

We report herein that a variety of isosorbide di-esters, previously reported to be novel substrates for butyrylcholinesterase (BuChE, EC 3.1.1.8), are in fact inhibitors of the homologous enzyme acetylcholinesterase (AChE), with IC(50) values in the micromolar range. In vitro studies show that they are mixed inhibitors of the enzyme, and thus the ternary enzyme-inhibitor-substrate complex can form in acetylcholinesterase. This is rationalised by molecular modelling which shows that the compounds bind in the mid-gorge area. In this position, simultaneous substrate binding might be possible, but the hydrolysis of this substrate is prevented. The di-esters dock within the butyrylcholinesterase gorge in a very different manner, with the ester sidechain at the 5-position occupying the acyl pocket at residues Leu286 and Val288, and the 2-ester binding to Trp82. The carbonyl group of the 2-ester is susceptible to nucleophilic attack by Ser198 of the catalytic triad. The larger residues of the acyl pocket in acetylcholinesterase prevent binding in this manner. The results complement each other and explain the differing behaviours of the esters in the cholinesterase enzymes. These findings may prove very significant for future work.     

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 degrees C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.     

Dissociation between cardiomyocyte function and remodeling with beta-adrenergic receptor blockade in isolated canine mitral regurgitation

The low-pressure volume overload of isolated mitral regurgitation (MR) is associated with increased adrenergic drive, left ventricular (LV) dilatation, and loss of interstitial collagen. We tested the hypothesis that beta1-adrenergic receptor blockade (beta1-RB) would attenuate LV remodeling after 4 mo of MR in the dog. beta1-RB did not attenuate collagen loss or the increase in LV mass in MR dogs. Using MRI and three-dimensional (3-D) analysis, there was a 70% increase in the LV end-diastolic (LVED) volume-to-LV mass ratio, a 23% decrease in LVED midwall circumferential curvature, and a >50% increase in LVED 3-D radius/wall thickness in MR dogs that was not attenuated by beta1-RB. However, beta1-RB caused a significant increase in LVED length from the base to apex compared with untreated MR dogs. This was associated with an increase in isolated cardiomyocyte length (171+/-5 microm, P<0.05) compared with normal (156+/-3 microm) and MR (165+/-4 microm) dogs. Isolated cardiomyocyte fractional shortening was significantly depressed in MR dogs compared with normal dogs (3.73+/-0.31 vs. 5.02+/-0.26%, P<0.05) and normalized with beta1-RB (4.73+/-0.48%). In addition, stimulation with the beta-adrenergic receptor agonist isoproterenol (25 nM) increased cardiomyocyte fractional shortening by 215% (P<0.05) in beta1-RB dogs compared with normal (56%) and MR (50%) dogs. In summary, beta1-RB improved LV cardiomyocyte function and beta-adrenergic receptor responsiveness despite further cell elongation. The failure to attenuate LV remodeling associated with MR could be due to a failure to improve ultrastructural changes in extracellular matrix organization.     

Lead dynamics in the forest floor and mineral soil in south-central Ontario

Recent studies have suggested that the residence time of Pb in the forest floor may not be as long as previously thought, and there is concern that the large pulse of atmospheric Pb deposited in the 1960s and early 1970s may move rapidly through mineral soils and eventually contaminate groundwater. In order to assess Pb mobility at a woodland (JMOEC) in south-central Ontario, a stable Pb isotope tracer ²⁰⁷Pb (8?mg?m^?2) was added to the forest floor in white pine (Pinus strobus) and sugar maple (Acer saccharum) stands, respectively, and monitored over a 2-year period. Excess ²⁰⁷Pb was rapidly lost from the forest floor. Applying first-order rate coefficients (k) of 0.57 (maple) and 0.32 (pine) obtained from the tracer study, and estimates of Pb deposition in the region, current predicted Pb concentrations in the forest floor are 1.5–3.1 and 2.1–5.8?mg?kg^?1 in the maple and pine plots, respectively. These values compare favorably with measured concentrations (corrected for mineral soil contamination) of 3.1–4.3?mg?kg^?1 in the maple stand and 2.6–3.6?mg?kg^?1 in the pine stand. The response time (1/k) of Pb in the forest floor at the sugar maple and white pine plots was estimated to be 1.8 and 3.1 years, respectively. The rapid loss of Pb from the forest floor at the JMOEC is much greater than previously reported, and is probably due to the rapid rate of litter turnover that is characteristic of forests with mull-type forest floors. In a survey of 23 forested sites that border the Precambrian Shield in south-central Ontario, Pb concentrations in the forest floor increased exponentially with decreasing soil pH. Lead concentrations in the forest floor at the most acidic survey sites, which exhibited mor-type forest floors, were approximately 10 times higher (～80?mg?kg^?1) than at the JMOEC, and pollution Pb burdens were up to 25 times greater. Despite the rapid loss of Pb from the forest floor at the JMOEC, the highest pollution Pb concentrations were found in the upper (0–1?cm) mineral soil horizon. Lead concentrations in the upper 30?cm of mineral soil were strongly correlated with organic matter content, indicating that pollution Pb does not move as a pulse down the soil profile, but instead is linked with organic matter distribution, indicating groundwater contamination is unlikely.     

Multiple Origins and Replication Proteins Influence Biological Properties of β-Lactamase-Producing Plasmids from Neisseria gonorrhoeae

The beta-lactamase-producing Asia-type plasmid pJD4 of Neisseria gonorrhoeae is a 7.4-kb, broad-host-range plasmid. It is part of a family of plasmids which are structurally related yet vary in size, found in both N. gonorrhoeae and Haemophilus ducreyi. Branch-point analysis by electron microscopy indicates that pJD4 carries three clustered but distinguishable origins of replication, which we named ori1, ori2, and ori3. Although pJD4 belongs to incompatibility (Inc) group W, it also carries a silent IncFII determinant which is expressed when ori2 and ori3 are absent. The Africa-type plasmid pJD5, a naturally occurring deletion derivative of pJD4, carries only ori1, belongs to the IncFII group, and, in contrast to pJD4, requires DNA polymerase I (Pol I) for replication. Plasmids constructed from pJD4 which lack ori1 but carry ori2 and ori3 do not require Pol I and are incompatible with IncW plasmids, suggesting that the ori2 or ori3 region contains the IncW determinant. We have cloned a replication initiation protein (RepB) that is necessary for ori2 and ori3 to function. This Rep protein is distinct from RepA, which is necessary for ori1. Thus, pJD4 is unique because it is the smallest plasmid characterized containing three origins of replication and two unique Rep proteins.     

Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo.

The mouse develops five pairs of mammary glands that arise during mid-gestation from five pairs of placodes of ectodermal origin. We have investigated the molecular mechanisms of mammary placode development using Lef1 as a marker for the epithelial component of the placode, and mice deficient for Fgf10 or Fgfr2b, both of which fail to develop normal mammary glands. Mammary placode induction involves two different signaling pathways, a FGF10/FGFR2b-dependent pathway for placodes 1, 2, 3 and 5 and a FGF10/FGFR2b-independent pathway for placode 4. Our results also suggest that FGF signaling is involved in the maintenance of mammary bud 4, and that Fgf10 deficient epithelium can undergo branching morphogenesis into the mammary fat pad precursor.     

Development of an Accelerometer-Linked Online Intervention System to Promote Physical Activity in Adolescents

Most adolescents do not achieve the recommended levels of moderate-to-vigorous physical activity (MVPA), placing them at increased risk for a diverse array of chronic diseases in adulthood. There is a great need for scalable and effective interventions that can increase MVPA in adolescents. Here we report the results of a measurement validation study and a preliminary proof-of-concept experiment testing the impact of Zamzee, an accelerometer-linked online intervention system that combines proximal performance feedback and incentive motivation features to promote MVPA. In a calibration study that parametrically varied levels of physical activity in 31 12-14 year-old children, the Zamzee activity meter was shown to provide a valid measure of MVPA (sensitivity in detecting MVPA = 85.9%, specificity = 97.5%, and r = .94 correspondence with the benchmark RT3 accelerometer system; all p < .0001). In a subsequent randomized controlled multi-site experiment involving 182 middle school-aged children assessed for MVPA over 6 wks, intent-to-treat analyses found that those who received access to the Zamzee intervention had average MVPA levels 54% greater than those of a passive control group (p < 0.0001) and 68% greater than those of an active control group that received access to a commercially available active videogame (p < .0001). Zamzee’s effects on MVPA did not diminish significantly over the course of the 6-wk study period, and were statistically significant in both females and males, and in normal- vs. high-BMI subgroups. These results provide promising initial indications that combining the Zamzee activity meter with online proximal performance feedback and incentive motivation features can positively impact MVPA levels in adolescents.     

Early steps in the assembly of vesicular stomatitis virus nucleocapsids in infected cells.

The assembly of nucleocapsids is an essential step in the replicative cycle of vesicular stomatitis virus (VSV). In this study, we have examined the early events of vesicular stomatitis virus nucleocapsid assembly in BHK-21 cells. Nuclease-resistant intracellular nucleocapsids were isolated at various stages of assembly and analyzed for RNA and protein contents. The smallest ribonucleoprotein complex formed during nucleocapsid assembly contains the 5'-terminal 65 nucleotides of nascent viral RNA complexed with the viral proteins N and NS. Elongation of the assembling nucleocapsids proceeds unidirectionally towards the 3' terminus by the sequential addition of viral proteins which incrementally protect short stretches of the growing RNA chain. Pulse-chase studies show that the assembling nucleocapsids can be chased into full-length nucleocapsids which are incorporated into mature virions. Our results also suggest an involvement of the cytoskeletal framework during nucleocapsid assembly.