Intracellular oxygen measurements of mouse liver cells using quantitative fluorescence video microscopy.

Our currently developed fluorescence video microscope can measure fluorescence intensities with an error of +/- 1.5% of full scale in 65536 different positions of a microscope field. With a video frame freeze acquisition time of 33 ms, time-dependent changes of this order of time or slower can be followed. Using cells which have absorbed pyrene-1-butyrate to an intracellular concentration of 0.05 to 1 mM, the changes in fluorescence intensity with oxygen concentration are easily measured. The spatial resolution for data collection is 0.5 micron when a 54X objective is used. The individual Stern-Volmer quenching constants of each individual pixel were measured for agar slices and mouse liver cells treated with pyrenebutyric acid. The distribution of quenching constants for agar follows a normal curve about a mean value of 16 . 10(-4) torr-1. The data for mouse liver cells gave a non-normal distribution of quenching constants with a mean value of 18 . 10(-4) torr-1. The greater spread of the data from cells is interpreted as evidence for a real biological variation in the solubility coefficient of oxygen in different locations within the cell. In all the cells examined, this distribution has been observed to be non-random and appears to be associated with specific cell structures.

Inhaled magnesium sulfate in the treatment of acute asthma.

BACKGROUND: Asthma exacerbations can be frequent and range in severity from relatively mild to status asthmaticus. The use of magnesium sulfate (MgSO4) is one of numerous treatment options available during acute exacerbations. While the efficacy of intravenous MgSO4 has been demonstrated, little is known about inhaled MgSO4. OBJECTIVES: To examine the efficacy of inhaled MgSO4 in the treatment asthma exacerbations. SEARCH STRATEGY: Randomised controlled trials were identified from the Cochrane Airways Group "Asthma and Wheez*" register. These trials were supplemented with trials found in the reference list of published studies, studies found using extensive electronic search techniques, as well as a review of the gray literature and conference proceedings. SELECTION CRITERIA: Randomised (or pseudo-randomised) controlled trials were eligible for inclusion. Studies were included if patients were treated with nebulised MgSO4 alone or in combination with beta2-agonist and where compared to beta2-agonist alone or inactive control. DATA COLLECTION AND ANALYSIS: Trial selection, data extraction and methodological quality were assessed by two independent reviewers. Efforts were made to collect missing data from authors. Results from fixed effects models are presented as standardized mean differences (SMD) for pulmonary functions and relative risks (RR) for hospital admission; both are displayed with their 95% confidence intervals (95% CI). MAIN RESULTS: Six trials involving 296 patients were included. Four studies compared nebulised MgSO4 with beta2-agonist to beta2-agonist and two studies compared MgSO4 to beta2-agonist alone. Three studies enrolled only adults and 2 enrolled exclusively pediatric patients; three of the studies enrolled severe asthmatics. Overall, there was a significant difference in pulmonary function between patients whose treatments included nebulised MgSO4 in addition to beta2-agonist (SMD: 0.37; 95% CI: 0.1 to 0.63; 4 studies); however, hospitalizations were similar between the groups (RR: 0.64; 95% CI: 0.40 to 1.04; 3 studies). Subgroup analyses did not demonstrate significant differences in lung function improvement between adults and children, or between severe and mild to moderate asthmatics. Conclusions regarding treatment with nebulised MgSO4 alone are difficult to draw due to lack of studies in this area. AUTHORS' CONCLUSIONS: Nebulised inhaled magnesium sulfate in addition to beta2-agonist in the treatment of an acute asthma exacerbation, appears to have benefits with respect to improved pulmonary function and there is a trend towards benefit in hospital admission. Heterogeneity between trials included in this review precludes a more definitive conclusion.

Inhaled magnesium sulfate in the treatment of acute asthma.

BACKGROUND: Asthma exacerbations can be frequent and range in severity from relatively mild to status asthmaticus. The use of magnesium sulfate (MgSO4) is one of numerous treatment options available during acute exacerbations. While the efficacy of intravenous MgSO4 has been demonstrated, little is known about inhaled MgSO4. OBJECTIVES: To examine the efficacy of inhaled MgSO4 in the treatment asthma exacerbations. SEARCH STRATEGY: Randomised controlled trials were identified from the Cochrane Airways Group "Asthma and Wheez*" register. These trials were supplemented with trials found in the reference list of published studies, studies found using extensive electronic search techniques, as well as a review of the gray literature and conference proceedings. SELECTION CRITERIA: Randomised (or pseudo-randomised) controlled trials were eligible for inclusion. Studies were included if patients were treated with nebulised MgSO4 alone or in combination with beta(2)-agonist and where compared to beta2-agonist alone or inactive control. DATA COLLECTION AND ANALYSIS: Trial selection, data extraction and methodological quality were assessed by two independent reviewers. Efforts were made to collect missing data from authors. Results from fixed effects models are presented as standardized mean differences (SMD) for pulmonary functions and relative risks (RR) for hospital admission; both are displayed with their 95% confidence intervals (95% CI). MAIN RESULTS: Six trials involving 296 patients were included. Four studies compared nebulised MgSO4 with beta2-agonist to beta2-agonist and two studies compared MgSO4 to beta2-agonist alone. Three studies enrolled only adults and 2 enrolled exclusively pediatric patients; three of the studies enrolled severe asthmatics. Overall, there was a significant difference in pulmonary function between patients whose treatments included nebulised MgSO4 in addition to beta2-agonist (SMD: 0.30; 95% CI: 0.03 to 0.56; 4 studies); however, hospitalizations were similar between the groups (RR: 0.69; 95% CI: 0.42 to 1.12; 3 studies). Subgroup analyses did not demonstrate significant differences in lung function improvement between adults and children, but were significantly different between severe and mild to moderate asthmatics (SMD: 0.69; 95% CI 0.13 to 1.25). Conclusions regarding treatment with nebulised MgSO4 alone are difficult to draw due to lack of studies in this area. AUTHORS' CONCLUSIONS: Nebulised inhaled magnesium sulfate in addition to beta2-agonist in the treatment of an acute asthma exacerbation, appears to have benefits with respect to improved pulmonary function and there is a trend towards benefit in hospital admission. The benefit is significantly greater in more severe asthma exacerbations. Heterogeneity between trials included in this review precludes a more definitive conclusion.

Inhaled magnesium sulfate in the treatment of acute asthma.

BACKGROUND: Asthma exacerbations can be frequent and range in severity from relatively mild to status asthmaticus. The use of magnesium sulfate (MgSO4) is one of numerous treatment options available during acute exacerbations. While the efficacy of intravenous MgSO4 has been demonstrated, little is known about inhaled MgSO4. OBJECTIVES: To examine the efficacy of inhaled MgSO4 in the treatment asthma exacerbations. SEARCH STRATEGY: Randomised controlled trials were identified from the Cochrane Airways Group "Asthma and Wheez*" register. These trials were supplemented with trials found in the reference list of published studies, studies found using extensive electronic search techniques, as well as a review of the gray literature and conference proceedings. SELECTION CRITERIA: Randomised (or pseudo-randomised) controlled trials were eligible for inclusion. Studies were included if patients were treated with nebulised MgSO4 alone or in combination with beta2-agonist and where compared to beta2-agonist alone or inactive control. DATA COLLECTION AND ANALYSIS: Trial selection, data extraction and methodological quality were assessed by two independent reviewers. Efforts were made to collect missing data from authors. Results from fixed effects models are presented as standardized mean differences (SMD) for pulmonary functions and relative risks (RR) for hospital admission; both are displayed with their 95% confidence intervals (95% CI). MAIN RESULTS: Six trials involving 296 patients were included. Four studies compared nebulised MgSO4 with beta2-agonist to beta2-agonist and two studies compared MgSO4 to beta2-agonist alone. Three studies enrolled only adults and 2 enrolled exclusively pediatric patients; three of the studies enrolled severe asthmatics. Overall, there was a non significant improvement in pulmonary function between patients whose treatments included nebulised MgSO4 in addition to beta2-agonist (SMD: 0.23; 95% CI: -0.03 to 0.50; 4 studies). Hospitalizations were similar between the groups (RR: 0.69; 95% CI: 0.42 to 1.12; 3 studies). Subgroup analyses did not demonstrate significant differences in lung function improvement between adults and children, but in severe asthmatics the lung function difference was significant (SMD: 0.55; 95% CI: 0.12 to 0.98). Conclusions regarding treatment with nebulised MgSO4 alone are difficult to draw due to lack of studies in this area. AUTHORS' CONCLUSIONS: Nebulised inhaled magnesium sulfate in addition to beta2-agonist in the treatment of an acute asthma exacerbation, appears to have benefits with respect to improved pulmonary function in patients with severe asthma and there is a trend towards benefit in hospital admission. Heterogeneity between trials included in this review precludes a more definitive conclusion.

Influence of PaO2 on cerebral macro- and microcirculation as observed by light reflection: time course of changes.

A silicon intensified target camera was used to study cerebral cortical vessels of the cat through a skull window implant, and red cell content changes were measured by light reflectance. Red cell content changes were observed in cerebral arterioles, capillaries, and venules when the PaO2 was decreased by lowering the PiO2. The time course of change in the diameter of the arterioles and venules was measured by selecting a cross section of the vessel plus some surrounding tissue. From the averaged cross-sectional reflectance signal, the change in vessel diameter was followed as a function of time following the PiO2 change. All vessels of greater than 10 microns were observed in focus. Substantial areas where no vessels could be discriminated would contain only capillaries, and changes in light reflectance from such areas would indicate changes in capillary red cell content. The time course of these changes following a step decrease in PiO2 was recorded. Results show that the sequence of red cell content increase in cerebral microcirculation during hypoxia is capillary before venule and arteriole. The times of initial red cell content increase are 37.9 +/- 7 s, 59.7 +/- 7.9 s, and 60.8 +/- 9.1 s, respectively. These results suggest an increase in the capillary bed red cell content as the initial response to hypoxia, but venules and arterioles change only on longer exposure to hypoxia. The sequence of the increase in red cell content suggests the capillaries rather than the arterioles are the vessels which respond to the oxygen autoregulation signal.

The binding of 8-anilino-1-naphthalene sulfonate (ANS) to fish myosin and the effect of salts on the thermal transitions of fish myosin-ANS complex.

Myosin prepared from tilapia (Serotherodon aureus) was complexed with 8-anilino-1-naphthalene sulfonate (ANS) and continuously heated at 1 degree C/min. A large increase in fluorescence was observed with a transition temperature of 34 degrees C. The effect of several salts on the transition temperature was tested. A plot based on the equation of E. E. Schrier and E. B. Schrier [(1967) J. Phys. Chem. 71, 1851-1860] gave a value of less than or equal to 500 cal/mol-deg for the change in enthalpy per residue due to exposure to solvent. The ratio of hydrophobic group to amide group exposure to solvent was intermediate compared with the ratio of RNase and gelatin. Fluorescence titrations yielded one high affinity site with a Kb of 2 X 10(6) M-1 and at least 200 low affinity sites with an average value of 1 X 10(5) M-1. The parameters did not change significantly with temperature. We propose that the increase in ANS fluorescence reflects changes in conformation of myosin as monitored by these low affinity sites, resulting in an increase in surface hydrophobicity and representing a small enthalpic change in the conformation of the myosin molecule. As a consequence, the change in conformation accelerates polymerization of myosin oligomers.

Measurement of tissue oxygen with a fluorescent probe.

Evidence is presented that pyrenebutyric acid is nontoxic at the concentrations required for the measurement of fluorescence quenching by physiological concentrations of oxygen. It appears that this molecule can be used to measure tissue oxygen since it does not interfere with oxygen consumption. Equally, tissue components do not interfere with the quenching of fluorescence to such an extent as to invalidate the method. The spatial resolution of the technique is limited by some tissue geometrical factors which do not, however, appear to be great enough to prevent its use to measure intercapillary oxygen gradients.

The Hypersensitive Reaction of Tobacco to Pseudomonas syringae pv. pisi: Activation of a Plasmalemma K/H Exchange Mechanism.

Net electrolyte efflux from suspension-cultured tobacco cells undergoing the hypersensitive reaction to Pseudomonas syringae pv. pisi resulted from a specific efflux of K(+) which was accompanied by an equimolar net influx of H(+). These fluxes began 60 to 90 minutes after inoculation of tobacco cells with bacteria, reached maximum rates of 6 to 9 micromoles per gram fresh weight tobacco cells per hour within 2.5 to 3 hours, and dropped below 4 micromoles per gram per hour within 5 hours. Tobacco cells lost approximately 35% of total K(+) during this period, and average cellular pH declined by approximately 0.75 pH unit. These events were accompanied by a 30% decrease in cellular ATP. K(+) and H(+) fluxes were inhibited by the protonophore (p-trifluoromethoxy)carbonyl cyanide phenylhydrazone and by increasing the K(+) concentration of the external solution. Tobacco leaf discs inoculated with the bacterium also exhibited a specific net K(+) efflux and H(+) influx. These results suggest that induction of the hypersensitive reaction in tobacco proceeds through the activation of a passive plasmalemma K(+)/H(+) exchange mechanism. It is hypothesized that activation of this exchange is a major contributing factor in hypersensitive plant cell death.

Use of an encapsulated fluorescent probe to measure intracellular PO2.

The objectives of this investigation were to produce a reliable, sensitive probe to measure intracellular PO2 with a high degree of resolution and to apply this technique to biological systems. A fluorescent molecule, pyrene dissolved in paraffin oil, was encapsulated in polyacrylamide to form a probe of nanometer dimensions. The quantitative and microscopic oxygen values were determined by analyzing the quenching of the fluorescence of the probe by oxygen, as displayed on a television monitor by a silicon-intensified-target camera. The nanocapsules had a sensitivity of approximately 1 mm PO2, a spatial resolution of 0.5 micrometer, and a temporal resolution of milliseconds. Calibrated nanocapsules within nonrespiring Amoeba proteus responded to ambient partial pressures of oxygen. At two different ambient partial pressures, nanocapsules engulfed by respiring amoebas indicated an intracellular PO2 28 mm Hg less than extracellular PO2. The capsules retained their sensitivity to oxygen for at least 8 months.