Oxygen cost of exercise hyperpnea: measurement.

To quantitate the O2 cost of maximal exercise hyperpnea, we required eight healthy adult subjects to mimic, at rest, the important mechanical components of submaximal and maximal exercise hyperpnea. Expired minute ventilation (VE), transpulmonary and transdiaphragmatic (Pdi) pressures, and end-expiratory lung volume (EELV) were measured during exercise at 70 and 100% of maximal O2 uptake. At rest, subjects were given visual feedback of their exercise transpulmonary pressure-tidal volume loop (WV), breathing frequency, and EELV, which they mimicked repeatedly for 5 min per trial over several trials, while hypocapnia was prevented. The change in total body O2 uptake (VO2) was measured and presumed to represent the O2 cost of the hyperpnea. In 61 mimicking trials with VE of 115-167 l/min and WV of 124-544 J/min, VE, WV, duty cycle of the breath, and expiratory gastric pressure (Pga) integrated with respect to time (integral of Pga.dt/min) were not different from those observed during maximum exercise. integral of Pdi.dt/min was 14% less and EELV was 6% greater during maximum exercise than during mimicking. The O2 cost measurements within a subject were reproducible over 3-12 trials (coefficient of variation +/- 10% range 5-16%). The O2 costs of hyperpnea correlated highly and positively with VE and WV and less, but significantly, with integral of Pdi.dt and integral of Pga.dt. The O2 cost of VE rose out of proportion to the increasing hyperpnea, so that between 70 and 100% of maximal VO2 delta VO2/delta VE increased 40-60% (1.8 +/- 0.2 to 2.9 +/- 0.1 ml O2/l VE) as VE doubled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of the gene coding for Caenorhabditis elegans Rac2 homologue, a Ras-related small GTP-binding protein

When screening a Caenorhabditis elegans genomic library using the human Racl cDNA as probe, a hybridizing fragment of 2.7 kb was isolated which contained four exons with high sequence similarity to CeRacl, coding for the nematode homologue of the Ras-related small GTP-binding protein Racl. The putative translational product of 195 amino acids (aa) from the exons displayed 88% identity to the sequence of CeRacl. Interestingly, three alterations were found in the N-terminal ‘effector domain’ (residues 22–45) which hitherto was identical among all known Rac p21s, suggesting that CeRac2 might have different targets/functions for nematode development. Additionally, an insertion of 4 as was found in the hypervariable region at the C terminus of CeRac2.     

Series-to-parallel transition in the filament lattice of airway smooth muscle.

Force-velocity curves measured at different times during tetani of sheep trachealis muscle were analyzed to assess whether velocity slowing could be explained by thick-filament lengthening. Such lengthening increases force by placing more cross bridges in parallel on longer filaments and decreases velocity by reducing the number of filaments spanning muscle length. From 2 s after the onset of stimulation, when force had achieved 42% of it final value, to 28 s, when force had been at its tetanic plateau for approximately 15 s, velocity decreases were exactly matched by force increases when force was adjusted for changes in activation, as assessed from the maximum power value in the force-velocity curves. A twofold change in velocity could be quantitatively explained by a series-to-parallel change in the filament lattice without any need to postulate a change in cross-bridge cycling rate.     

Effect of osmotic compression on the force-velocity properties of glycerinated rabbit skeletal muscle cells

The force-velocity relations of single glycerinated rabbit psoas muscle fibers at 5 degrees C were studied at maximum and half-maximum activation in the presence of 0 (control) and 39-145 g/liter dextran T-70. Resting fiber diameter decreased progressively to approximately 70% of the nondextran control as the dextran concentration was increased. Isometric force at full activation increased to a maximum of 136% of control at 111 g/liter dextran and then fell to 80% of control in 145 g/liter dextran. Maximum velocity, which fell to 49% of the control value in the highest concentration of dextran, was nearly constant at approximately 65% control over the range of 58-111 g/liter dextran. Relative maximum power, which gives an estimate of changes in intermediate velocity, was not significantly reduced by dextran concentrations up to 76 g/liter, but then fell progressively to 62% of control in the highest concentration of dextran. At half-maximum activation, maximum velocity and relative maximum power were not significantly different from the values at full activation. The results obtained at partial activation indicate that the decline of velocity seen in the presence of dextran is not due to a passive internal load and that the dextran does not cause a viscous resistance to shortening. The increased velocity in the absence of dextran can be explained by the reduced ability of cross-bridges to resist shortening, as proposed by Goldman (1987. Biophys. J. 51:57).     

Time dependence of series elasticity in tracheal smooth muscle

The stress-strain curve for the series elastic component (SEC) of tracheal smooth muscle was obtained by quick releasing the muscle from isometric tension to various afterloads and measuring the elastic recoils (SEC lengths) at a specific time after stimulation. A family of such curves was obtained by releasing the muscle at different points in time during contraction. Stiffnesses of the SEC (slopes of the stress-strain curves) at a specific stress level calculated from these curves (constant-stress stiffness) showed significant difference from one another. The same difference can also be characterized by the slope of the linear stiffness-stress curve, the constant A. The constant A during a 10-s isometric contraction was maximal at 2 s. It then decreased with time. This stiffness behavior is only seen when the effect of stress is held constant or eliminated. If stress is allowed to increase with time as it does during a tetanus then stiffness appears to increase monotonically. The SEC stiffness during active contraction was found to vary within the boundaries of the stiffness of muscle in rigor (upper limit) and that at resting state (lower limit).     

Relationship between myosin phosphorylation and contractile capability of canine airway smooth muscle.

To better understand excitation-contraction coupling in smooth muscle, myosin phosphorylation and force-velocity properties of canine tracheal muscle were compared during the rise and early plateau of force in electrically stimulated tetani. Velocity reached a peak of approximately 1.5 times plateau value when force had risen to approximately 45% of its maximum value and then declined progressively. Except early in the tetanus, when phosphorylation rose rapidly, maximum power and phosphorylation had nearly parallel time courses, reaching peaks of 1.2-1.3 times reference at 6-8 s before declining to the plateau level at approximately 12 s. Force, velocity, maximum power, and phosphorylation fell somewhat during the plateau, with the closest correlation between phosphorylation and power. These results suggest that 1) early velocity slowing is not associated with light chain dephosphorylation and 2) maximum power, which we use to signal changes in activation, is closely correlated with the degree of light chain phosphorylation, at least when phosphorylation level is not changing rapidly. Dissociation of these two properties would be expected early in the tetanus if phosphorylation precedes mechanical activity.     

Does a fibroblast membrane proteinase affect the binding, uptake and metabolism of epidermal growth factor?

Pre-treatment of human fibroblasts to inhibit a cell-surface growth-related proteinase inhibits the mitogenic action of epidermal growth factor. It also reduces the binding of epidermal growth factor to these cells, and lowers the rate of internalisation and degradation of the growth factor, but quantitative considerations render it unlikely that these parameters contribute directly to the inhibition of mitogenesis.     

Volatile sulphur compounds and pathways of L-methionine catabolism in Williopsis yeasts

Volatile sulphur compounds (VSCs) are important to the food industry due to their high potency and presence in many foods. This study assessed for the first time VSC production and pathways of L: -methionine catabolism in yeasts from the genus Williopsis with a view to understanding VSC formation and their potential flavour impact. Five strains of Williopsis saturnus (var. saturnus, var. subsufficiens, var. suavolens, var. sargentensis and var. mrakii) were screened for VSC production in a synthetic medium supplemented with L: -methionine. A diverse range of VSCs were produced including dimethyl disulphide, dimethyl trisulphide, 3-(methylthio)-1-propanal (methional), 3-(methylthio)-1-propanol (methionol), 3-(methylthio)-1-propene, 3-(methylthio)-1-propyl acetate, 3-(methylthio)-1-propanoic acid (methionic acid) and ethyl 3-(methylthio)-1-propanoate, though the production of these VSCs varied between yeast strains. W. saturnus var. saturnus NCYC22 was selected for further studies due to its relatively high VSC production. VSC production was characterised step-wise with yeast strain NCYC22 in coconut cream at different L: -methionine concentrations (0.00-0.20%) and under various inorganic sulphate (0.00-0.20%) and nitrogen (ammonia) supplementation (0.00-0.20%), respectively. Optimal VSC production was obtained with 0.1% of L: -methionine, while supplementation of sulphate had no significant effect. Nitrogen supplementation showed a dramatic inhibitory effect on VSC production. Based on the production of VSCs, the study suggests that the Ehrlich pathway of L: -methionine catabolism is operative in W. saturnus yeasts and can be manipulated by adjusting certain nutrient parameters to control VSC production.     

Oligopeptide-mediated gene transfer into mouse corneal endothelial cells: expression,design optimization,uptake mechanism and nuclear localization

Gene transfer to the corneal endothelium has potential in preventing corneal transplant rejection. In this study, we transfected mouse corneal endothelial cells (MCEC) with a class of novel arginine-rich oligopeptides. The peptides featured a tri-block design and mediated reporter gene expression in MCEC more efficiently than the commercial polyethylenimine standard. The functionality of each block was demonstrated to critically influence the performance of the peptide. Results from confocal imaging and flow cytometry then showed that energy-dependent endocytosis was the dominant form of uptake and multiple pathways were involved. Additionally, uptake was strongly dependent on interactions with cell-surface heparan sulphate. Fluorescence resonance energy transfer studies revealed that the peptide/DNA entered cells as an associated complex and some will have dissociated by 8.5 h. Large-scale accumulation of uncondensed DNA within the nucleus can also be observed by 26 h. Finally, as a proof of biological relevance, we transfected MCEC with plasmids encoding for the functional indoleamine 2,3-dioxygenase (IDO) enzyme. We then demonstrated that the expressed IDO could catalyse the degradation of l-tryptophan, which in turn suppressed the growth of CD4⁺ T-cells in a proliferation assay.     

Nitrate respiration protects hypoxic Mycobacterium tuberculosis against acid- and reactive nitrogen species stresses

There are strong evidences that Mycobacterium tuberculosis survives in a non-replicating state in the absence of oxygen in closed lesions and granuloma in vivo. In addition, M. tuberculosis is acid-resistant, allowing mycobacteria to survive in acidic, inflamed lesions. The ability of M. tuberculosis to resist to acid was recently shown to contribute to the bacillus virulence although the mechanisms involved have yet to be deciphered. In this study, we report that M. tuberculosis resistance to acid is oxygen-dependent; whereas aerobic mycobacteria were resistant to a mild acid challenge (pH 5.5) as previously reported, we found microaerophilic and hypoxic mycobacteria to be more sensitive to acid. In hypoxic conditions, mild-acidity promoted the dissipation of the protonmotive force, rapid ATP depletion and cell death. Exogenous nitrate, the most effective alternate terminal electron acceptor after molecular oxygen, protected hypoxic mycobacteria from acid stress. Nitrate-mediated resistance to acidity was not observed for a respiratory nitrate reductase NarGH knock-out mutant strain. Furthermore, we found that nitrate respiration was equally important in protecting hypoxic non-replicating mycobacteria from radical nitrogen species toxicity. Overall, these data shed light on a new role for nitrate respiration in protecting M. tuberculosis from acidity and reactive nitrogen species, two environmental stresses likely encountered by the pathogen during the course of infection.