Millisecond measurement of transport during and after an electroporation pulse.

Electroporation involves the application of an electric field pulse that creates transient aqueous pathways in lipid bilayer membranes. Transport through these pathways can occur by different mechanisms during and after a pulse. To determine the time scale of transport and the mechanism(s) by which it occurs, efflux of a fluorescent molecule, calcein, across erythrocyte ghost membranes was measured with a fluorescence microscope photometer with millisecond time resolution during and after electroporation pulses several milliseconds in duration. One of four outcomes was typically observed. Ghosts were: (1) partially emptied of calcein, involving efflux primarily after the pulse; (2) completely emptied of calcein, involving efflux primarily after the pulse; (3) completely emptied of calcein, involving efflux both during and after the pulse; or (4) completely emptied of calcein, involving efflux primarily during the pulse. Partial emptying, involving significant efflux during the pulse, was generally not observed. We conclude that under some conditions transport caused by electroporation occurs predominantly by electrophoresis and/or electroosmosis during a pulse, although under other conditions transport occurs in part or almost completely by diffusion within milliseconds to seconds after a pulse.     

A provisional model to predict blood pressure response to biofeedback-assisted relaxation

Blood pressure (BP) response to biofeedback-assisted relaxation is not uniform among hypertensive individuals. The purpose of this exploratory study was to determine if selected psychophysiological variables could be used to identify individuals able to lower blood pressure using biofeedback-assisted relaxation. Responders were defined using a preset criterion of 5 mm Hg or greater decrease in mean arterial pressure. A logistic regression model derived from five variables (heart rate, finger temperature, forehead muscle tension, plasma renin response to furosemide, and mean arterial pressure response to furosemide) provided significant predictive power for BP response, exhibiting a sensitivity of 84.6% and a specificity of 80.0%. With future validation, the proposed model may provide useful information to identify patients likely to benefit from biofeedback-assisted relaxation.     

Future looks bleak for many Ontario hospitals

Ontario will soon begin to experience some of the hospital closures that are already well known in many other provinces. A recent report called for the closure of 12 hospitals in Metropolitan Toronto and a 13% cut in the number of hospital beds. Strong campaigns against some of the proposed closures are already being mounted.     

Coding sequence composition flanking either signal element alters V(D)J recombination efficiency.

Lymphoid V(D)J rearrangement is targeted by recombination signal sequences (RSS) bordering V, D or J exons. We demonstrate that the DNA composition of flanking coding positions, particularly poly(A) or poly(T) stretches at one or both RSS, diminishes V(D)J recombination up to 100-fold. Positionally correct cleavages occur in the inhibited reactions, since the junctions formed show the same frequency of precision as uninhibited reactions. Open/shut cleavage/rejoining is not increased at a normal RSS in substrates containing inhibitory A/T homopolymers versus random sequence at a second RSS. Thus recombinase action at both cleavage sites is severely disrupted by modified coding sequences.     

Characterization and temporal aspects of haemolymph juvenile hormone esterase in adult cockroach,Periplaneta americana

Daily variations in the in vitro haemolymph juvenile hormone esterase activity (hJHE) of adult male and female Periplaneta americana were monitored over a 2 week period from the time of adult emergence and throughout the first reproductive cycle of the adult female. Kinetic analysis of hJHE from females indicated an apparent K(m) JH III of 5.59+/-1.75&mgr;M (V(max)=140pmol JH III hydrolysedh(-1) per &mgr;l haemolymph). In females the mean rate of JH III metabolism in diluted haemolymph shortly after emergence was 27.5+/-1.5pmolh(-1)&mgr;l(-1) (n=16) and remained relatively low (16-32pmolh(-1)&mgr;l(-1)) over much of early adult development. Activity remained at this level during the first two days of the 4 day reproductive cycle, but showed a much increased broad peak of activity (74-93pmolh(-1)&mgr;l(-1)) at 60-72h post-extrusion. This peak lags behind the whole body JH titre peak, which could suggest that elevated levels of JH III may bring about the induction of JH esterase(s). A different pattern of JHE activity was observed in adult males. hJHE rates in males at emergence were almost twice as high (81.5+/-15.8pmolh(-1)&mgr;l(-1), n=16) as those found in females at this time, but thereafter showed a downturn to moderate levels (44-68pmolh(-1)&mgr;l(-1)) that were maintained for the remainder of the study. Rapid (FPLC) DEAE-sepharose ion exchange chromatography, ultrafiltration and fast-flow superose gel filtration chromatography were employed to achieve an efficient partial purification (166-fold) of the hJHE from cell-free plasma of reproductively active female P. americana 48-72h post-ootheca extrusion. Gel filtration and SDS-polyacrylamide gel electrophoresis (PAGE) revealed an enzyme having apparent molecular mass of between 60 and 70kDa, whilst non-denaturing PAGE and iso-electrofocusing resolved a single acidic enzyme peak with a pI of 4.9.     

Activity and Differential Induction of Chitinase Isozymes in Soybean Cultivars Resistant or Susceptible to Root-knot Nematodes

Host physiological events in relation to infestation by parasitic nematodes are not well documented. Soybean plant responses to Meloidogyne incognita infestation were compared to resistant (Bryan) and susceptible (Brim) cultivars at 0, 1, 3, 10, 20, and 34 days after infestation (DAI). The resistant cultivar had higher chitinase activity than the susceptible cultivar at every sample time beginning at 3 DAI. Results from isoelectric focusing gel electrophoresis analyses indicated that three acidic chitinase isozymes with isoelectric points (pIs) of 4.8, 4.4, and 4.2 accumulated to a greater extent in the resistant compared to the susceptible cultivar following challenge. SDS-PAGE analysis of root proteins revealed that two proteins with molecular weights of approximately 31 and 46 kD accumulated more rapidly and to a higher level in the resistant than in the susceptible cultivar. Additionally, three major protein bands (33, 22, and 20 kD) with chitinase activity were detected with a modified SDS-PAGE analysis in which glycolchitin was added into the gel matrix. These results indicate that higher chitinase activity and early induction of specific chitinase isozymes may be associated with resistance to root-knot nematode in soybean.     

Lysophosphatidic Acid Increases Tight Junction Permeability in Cultured Brain Endothelial Cells

Abstract: Brain capillary endothelial cells are coupled by a continuous belt of complex high-electrical-resistance tight junctions that are largely responsible for the blood-brain barrier. We have investigated mechanisms regulating tight junction permeability in brain endothelial cells cultured to maintain high-resistance junctions. The phospholipid lysophosphatidic acid (LPA) was found to cause a rapid, reversible, and dose-dependent decrease in transcellular electrical resistance in brain endothelial cells. LPA also increased the paracellular flux of sucrose, which, together with the resistance decrease, indicated increased tight junction permeability. Activation of protein kinase C attenuated the effect of LPA, suggesting that it was mediated by activation of a signalling pathway. LPA did not cause any obvious relocalization of adherens junction- or tight junction-associated proteins. However, it did stimulate the formation of stress fibres, the recruitment of focal adhesion components, and the appearance of tyrosine phosphorylated protein at focal contacts. Our study shows that LPA is a modulator of tight junction permeability in brain endothelial cells in culture and raises the possibility that it triggers blood-brain barrier permeability changes under (patho)physiological conditions.     

Nitrification and utilization of ammonium and nitrate during oil bioremediation at different soil water potentials

Bioremediation of petroleum spills requires aerobic soil conditions and readily available N, which may be susceptible to leaching. Our objectives were to determine the influence of soil water potential on nitrification in the presence of crude oil, the toxicity of oil to NHj‐oxidizing bacteria, and the preferences of microorganisms for NH⁺ ₄ or NO^? ₃. A Weswood clay loam was amended with crude oil to contain 0, 5, and 10% by soil dry weight, and N was added to achieve C:N ratios of 90:1 and 120:1. Soil water potentials were maintained at ‐0.02, ‐0.1, and ‐1.0 kJ/kg or allowed to fluctuate between ‐0.02 and ‐3 kJ/kg. Concentrations of NH⁺ ₄ and NO₃ ^?were measured during an incubation period of 40 d. Nitrification in soil not amended with oil was rapid at water potentials of ‐0.02 and ‐0.1 kJ/kg but inactive at a water potential of ‐1.0 kJ/kg. Oil reduced nitrification rates and populations of NH⁺ ₄‐oxidizing bacteria. Little NO^? ₃ accumulated when the C:N ratio was 120:1, but when the C:N ratio was 90:1, up to 150 μg of NO₃‐N/g of soil accumulated at a soil water potential of ‐0.02 kJ/kg. Soil water potential in the range used did not greatly influence the extent of oil bioremediation but significantly influenced nitrification. Ammonium was preferentially used over NO^? ₃ by microorganisms during oil bioremediation. Nitrate accumulation from urea applied to stimulate oil bioremediation was low when N application matched requirements for oil bioremediation, and nitrification was restricted by controlling soil water content.