When R > 0.8R 0: fluorescence anisotropy, non-additive intensity, and cluster size.

Assembly and clustering feature in many biological processes and homo-FRET and fluorescence anisotropy can assist in estimating the aggregation state of a system. The distance dependence of resonance energy transfer is well described and tested. Similarly, assessment of cluster size using steady state anisotropy is well described for non-oriented systems when R < 0.8R 0, however, these methods break down when R > 0.8R 0. Fused trimeric DNA clusters labelled with fluorescein were engineered to provide inter-fluorophore distances from 0.7 to 1.6 R/R 0 and intensity and anisotropy were measured. These constructs cover a range where anisotropy effects depend on distance. Analytical expressions were derived for fully labelled and fractionally labelled clusters and the experimental results analysed. The experimental results showed that: (1) the system underwent distance dependent quenching; (2) when incompletely labelled both doubly and triply labelled forms could be assessed to obtain distance dependent intensity factors; (3) the anisotropy behaviour of a multiply labelled cluster of a particular size depends on the behaviour of the fluorophores and their distance in a cluster. This work establishes that when emission intensity data are available the analytically useful range for investigating clusters does not have to be restricted to R < 0.8R 0 and is applicable to cases where the anisotropy of a cluster of N fluorophores is not well approximated by r 1/N.

Imaging lifetime and anisotropy spectra in the frequency domain.

We report the development of a system combining the capabilities of fluorescence imaging spectroscopy (x, lambda, I), fluorescence lifetime (tau) and static and dynamic fluorescence anisotropy (r), enabling the wide-field measurement of the spectroscopic parameters of fluorophores: (x, lambda, I, tau, r). The system employs a frequency domain data collection strategy with a modulated light emitting diode as the light source. A polarization rotator placed in the excitation path after a polarizer allows alternating parallel and perpendicular images to be collected without moving parts. A second polarizer on the emission side serves as the analyzer, leading to estimations of the wavelength-dependent dynamic anisotropies. The spectrograph has a nominal range of 365-920 nm; however, the light-emitting diodes and filter sets used in this study restricted the usable range from about 510 to 700 nm. The system was tested on rhodamine 6G (R6G) solutions containing 0, 15, 37, 45, 59, 74 and 91 glycerol. These experiments gave rotational diffusion results comparing favourably with literature values while also demonstrating a trend towards shorter measured lifetimes at high refractive index. The ability of the system to resolve mixtures was tested on mixtures of anti-human IgG-FITC (gamma-chain-specific) and R6G. These fluorophores have similar lifetimes but could be separated using anisotropy parameters. The imaging capabilities of the system were tested on mixtures of fluorescent beads with glycerol solutions of R6G.

AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers.

Most treatments of frequency domain lifetime measurements indicate that a set of measurements must be made at multiple frequencies in order to determine the lifetimes of the components in a mixture. Although this is the case in general, under special conditions, single-frequency data can resolve multiple lifetimes. Here, data are presented showing several approaches to determining fluorescence lifetimes in two-component mixtures using single-frequency data. Common to all of the procedures presented is exploitation of variations in the relative contributions of a particular fluorophore to the total fluorescence from a mixture of fluorophores. This variation can be produced intentionally by observing a number of samples which contain different relative amounts of the fluorophores. It can be produced fortuitously by observing spatial variations in a mixture of fluorophores in a specimen or set of specimens observed with a lifetime imaging system. It can also be produced by examination of the lifetime spectrum obtained from a fluorophore mixture or by varying the concentration of a quencher in a fluorophore mixture, in which the two fluorophores have different rate constants for quenching. In many instances, the set of approaches presented here will be unsuitable for examination of arbitrary samples of unknown composition for which the multifrequency approach should be used. However, measurements produced using single-frequency methods may be applied to good effect for controlled experiments having defined fluorophores or sets of fluorophores, particularly in the case of biological lifetime imaging studies.

Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data.

Graphical representation of fluorescence lifetime imaging microscopy data demonstrates that a mixture of two components with single exponential decays can be resolved by single frequency measurements. We derive a method based on linear fitting that allows the calculation of the fluorescence lifetimes of the two components. We show that introduction of proper error-weighting results in a non-linear method that is mathematically identical to a global analysis algorithm that was recently derived. The graphical approach was applied to cellular data obtained from a lifetime-based phosphorylation assay for the epidermal growth factor receptor and yielded results similar to those obtained by a global analysis algorithm.

A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images.

A programmable array microscope (PAM) incorporates a spatial light modulator (SLM) placed in the primary image plane of a widefield microscope, where it is used to define patterns of illumination and/or detection. We describe the characteristics of a special type of PAM collecting two images simultaneously. The conjugate image (Ic) is formed by light originating from the object plane and returning along the optical path of the illumination light. The non-conjugate image (Inc) receives light from only those regions of the SLM that are not used for illuminating the sample. The dual-signal PAM provides much more time-efficient excitation than the confocal laser scanning microscope (CLSM) and greater utilization of the available emission light. It has superior noise characteristics in comparison to single-sided instruments. The axial responses of the system under a variety of conditions were measured and the behaviour of the novel Inc image characterized. As in systems in which only Ic images are collected (Nipkow-disc microscopes, and previously characterized PAMs), the axial response to thin fluorescent films showed a sharpening of the axial response as the unit cell of the repetitive patterns decreased in size. The dual-signal PAM can be adapted to a wide range of data analysis and collection strategies. We investigated systematically the effects of patterns and unit cell dimensions on the axial response. Sufficiently sparse patterns lead to an Ic image formed by the superposition of the many parallel beams, each of which is equivalent to the single scanning spot of a CLSM. The sectioning capabilities of the system, as given by its axial responses, were similar for a given scan pattern and for processed pseudorandom sequence (PRS) scans with the same size of the unit cell. For the PRS scans, optical sectioning was achieved by a subtraction of an Inc image or, alternatively, a scaled widefield image from the Ic image. Based on the comparative noise levels of the two methods, the non-conjugate subtraction was significantly superior. A point spread function for Ic and Inc was simulated and properties of the optical transfer functions (OTFs) were compared. Simulations of the OTF in non-conjugate imaging did not suffer from the missing cone problem, enabling a high quality deconvolution of the non-conjugate side alone. We also investigated the properties of images obtained by subjecting the Ic and Inc data to a combined maximum likelihood deconvolution.

Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression.

BACKGROUND: Frequency-domain fluorescence lifetime imaging microscopy (FLIM) is finding increasing use in the analysis of biological systems. However, the calibration, determination of resolvable lifetime differences, and evaluation of artifacts have not been extensively treated. We describe a multi-point method for calibrating a frequency-domain FLIM system, characterize the minimum detectable heterogeneity and intra- and inter-image lifetime differences, discuss the statistical treatment of FLIM data, and suggest methods for minimizing artifacts. METHODS: A set of solutions exhibiting single-component lifetimes suffice for accurately calibrating a reference material with a single-component lifetime, even in the absence of accurate data on the lifetimes of the individual solutions or the reference material. We used a set of rhodamine 6G solutions quenched with varying concentrations of iodide, leading to lifetimes of 0.5--4.0 ns, to calibrate a 1 microM reference solution of rhodamine 6G in water. RESULTS: We measured a value of 4.11 ns with an estimated absolute error of +/-0.05 ns for the rhodamine 6G reference solution. With 57.7 MHz modulation, the minimum detectable inter-image lifetime difference was 0.1--0.15 ns and the minimum detectable intra-image lifetime difference was 4--5 ps, allowing solutions differing in lifetime by 40 and 70 ps to be easily distinguished. The minimum detectable lifetime heterogeneity was 50--80 ps. Evaluation of replicate measurements of single solutions demonstrated that inter-image instrument errors exceeded those predicted from intra-image statistics by more than an order of magnitude. We also measured lifetimes and heterogeneity in 4 GFP variants (WTGFP, EGFP, S65T, and EYFP) with the technique. CONCLUSION: The multi-point calibration method is applicable to any system consisting of single-component lifetimes. Applying the method in our FLIM microscope allowed us to demonstrate a previously unreported degree of lifetime resolution in a FLIM microscope. Cytometry 43:248-260;2001.

Three-dimensional spectral imaging by hadamard transform spectroscopy in a programmable array microscope.

We report the acquisition and deconvolution of three-dimensional spectrally resolved images in a programmable array microscope implementing a Hadamard transform fluorescence spectroscopy system with adjustable spectral resolution. A stack of 16 two-dimensional spectral images was collected at 400 nm intervals along the optical axis. The specimen consisted of a polytene chromosome spread from Drosophila melanogaster doubly labelled for the Polyhomeotic protein by indirect immunofluorescence labelling with Alexa594 and for DNA with YOYO-1. The resulting four-dimensional data set consisted of the xyz spatial dimensions (898 x 255 x 16) with a 26-point spectrum at each spatial location. The total exposure time to the sample was 34 min. The system requires the acquisition of multiple images, and thus works best with fluorophores that are resistant to photobleaching. Image deconvolution reduced the amount of out-of-focus blur by up to a factor of 8, resulting in a dramatic improvement in the visualization of the chromosome backbone and localization of the specific Polyhomeotic domains.

An optical sectioning programmable array microscope implemented with a digital micromirror device.

The defining feature of a programmable array microscope (PAM) is the presence of a spatial light modulator in the image plane. A spatial light modulator used singly or as a matched pair for both illumination and detection can be used to generate an optical section. Under most conditions, the basic optical properties of an optically sectioning PAM are similar to those of rotating Nipkow discs. The method of pattern generation, however, is fundamentally different and allows arbitrary illumination patterns to be generated under programmable control, and sectioning strategies to be changed rapidly in response to specific experimental conditions. We report the features of a PAM incorporating a digital micromirror device, including the axial sectioning response to fluorescent thin films and the imaging of biological specimens. Three axial sectioning strategies were compared: line scans, dot lattice scans and pseudo-random sequence scans. The three strategies varied widely in light throughput, sectioning strength and robustness when used on real biological samples. The axial response to thin fluorescent films demonstrated a consistent decrease in the full width at half maximum (FWHM), accompanied by an increase in offset, as the unit cells defining the patterns grew smaller. Experimental axial response curves represent the sum of the response from a given point of illumination and cross-talk from neighbouring points. Cross-talk is minimized in the plane of best focus and when measured together with the single point response produces a decrease in FWHM. In patterns having constant throughput, there appears to be tradeoff between the FWHM and the size of the offset. The PAM was compared to a confocal laser scanning microscope using biological samples. The PAM demonstrated higher signal levels and dynamic range despite a shorter acquisition time. It also revealed more structures in x-z sections and less intensity drop-off with scanning depth.

Application of energy-resolved measurements to Laue diffraction: determination of unit-cell parameters, deconvolution of harmonics and assignment of systematic absences.

The use of energy-resolved area detection of Laue diffraction patterns for the determination of unit-cell parameters and systematic absences is demonstrated. Seven different crystals having previously known unit cells were re-examined using Laue diffraction methods. These crystals included four different crystal systems including cubic, orthorhombic, tetragonal and monoclinic cells. The crystals had cell sizes ranging from 179.4 to 4588.3 A(3). Comparison of known and re-determined cells showed good agreement (ratio of known to measured cells = 0.987 +/- 0.18). A single procedure was suitable for all unit-cell determinations. The accuracy of the method is presently limited by the quality of the available energy measurements. Some of the crystals represent space groups containing systematic absences normally obscured by harmonic overlap when using the Laue method. These include absences due to 2(1) screw axes (h, k or 1 = 2n + 1) and cell centering (h + k = 2n + 1). All systematic absences were identified using a combination of multiple linear regression with either stepwise elimination or stepwise inclusion and an F test for assignment of systematic absence. The methods are discussed in detail and simulations are used to evaluate critical tolerances for future systems.

A foil-mask spectrometer for laue pattern imaging: simultaneous position, intensity and energy.

An X-ray spectrometer for simultaneous position, intensity and energy determinations suitable for Laue diffraction applications is described. The foil-mask spectrometer consists of a series of metal foils of varying composition and thickness which are used to modulate the energy distribution of an incident X-ray source. Three modes of operation are described: a high-resolution spectrometer for measurement of nearly monochromatic X-rays, an intensity discriminator for partitioning the intensity from a small number of spatially overlapped monochromatic X-ray sources, and a low-resolution spectrometer for polychromatic X-rays with broad spectral features. The first mode of operation is designed to allow the energy of monochromatic Laue reflections to be measured with a resolution suitable for determination of unit-cell parameters. The second mode of operation is designed to allow the intensity of each component in a spatial region containing overlapping orders or spatially overlapped reflections to be discriminated for use in refinements or space-group assignment. The third mode of operation is described for completeness. The theory behind each mode of operation is described. The energy resolution of the spectrometer improves with the square root of the intensity of the incident beam. It also increases linearly with the change in energy with respect to transmission efficiency of a particular foil. In theory, the resolution of the spectrometer can readily exceed 50 eV over a wide range of energies depending on the foils used and the incident X-ray photon flux. Determinations of the energies of Mo Kalpha and Cu Kalpha radiation using a first-generation ten-foil spectrometer gave values of 17.5 +/- 0.1 and 8.08 +/- 0.05 keV, respectively. Treatment of random error shows good correspondence with a Poisson model. The use of this spectrometer is demonstrated using a sample of tetraphenylphosphonium tetrachlorooxomolybdenum(V). Comparison of predicted and observed energies shows good agreement over a wide range of energies. The ratio of predicted to measured energy for the first 50 measurements was 0.9918+/-0.0344. Up to three components of a position having harmonic overlap were separated. This work demonstrates the feasibility of using Laue diffraction to completely determine the crystal structure of a molecule without recourse to monochromatic methods.

Evaluation of charge-injection devices for use in laue diffraction imaging.

Charge-injection devices (CIDs) are versatile detectors having a number of features which recommend them for use in the imaging of X-ray diffraction patterns. They have a flexible nondestructive readout allowing for analysis of image quality during data collection and rapid readout of selected portions of the device. CIDs have full-well capacities in the range of 10(6) charge carriers giving them a high dynamic range for both direct and indirect imaging of X-rays. CIDs have peak quantum efficiencies in the optical region over 50% allowing for their incorporation into indirect detection systems. Rapid random single-pixel address allows for their use as single X-ray photon counters with energy discrimination. Three types of position-sensitive detectors for X-rays have been developed using CIDs. Two CID formats, the CID 17PPRA (388 x 256) and the CID 38SG (512 x 512), were incorporated into systems performing indirect imaging, direct imaging and single X-ray photon counting with energy discrimination. Indirect images of the Laue diffraction patterns from tetraphenylphosphonium tetrachlorooxomolybdenum(V) and natural MoS(2) were collected using a phosphor sheet to convert X-rays into optical photons which were detected with the CID 38SG. Directly detected images of spots from the Laue diffraction pattern of MoS(2) were recorded with the CID 17PPRA. Single photon counting with energy discrimination is demonstrated with the CID 17PPRA using a reflection from the Laue diffraction pattern of MoS(2). Useful information could be obtained from a single pixel at read rates over 7 kHz. Complete energy-dispersive analysis suitable for determination of space groups from Laue diffraction is currently limited due to incomplete charge collection and/or split events.

Oxidant and acid aerosol exposure in healthy subjects and subjects with asthma. Part I: Effects of oxidants, combined with sulfuric or nitric acid, on the pulmonary function of adolescents with asthma.

Both peak flow decrements in children at summer camps and increased hospital admissions for asthma have been associated with summer "acid haze," which is composed of ozone and various acidic species. The objective of this study was to investigate the pulmonary effects of acid summer haze in a controlled laboratory setting. Twenty-eight adolescent subjects with allergic asthma, exercise-induced bronchospasm, and a positive response to a standardized methacholine challenge enrolled in the study; 22 completed the study. Each subject inhaled one of four test atmospheres by mouthpiece on two consecutive days. The order of exposure to the four test atmospheres was assigned via a random protocol: air, oxidants (0.12 parts per million [ppm]* ozone plus 0.30 ppm nitrogen dioxide), oxidants plus sulfuric acid at 70 micrograms/m3 of air, or oxidants plus 0.05 ppm nitric acid. Exposure to each of the different atmospheres was separated by at least one week. The exposures were carried out during alternating 15-minute periods of rest and moderate exercise for a total exposure period of 90 minutes per day. Pulmonary function was measured before and after exposure on both test days and again on the third day as a follow-up measurement. A postexposure methacholine challenge was performed on Day 3. Low methacholine concentrations were chosen for the postexposure challenge to avoid provoking a response. The protocol was designed to detect subtle changes in airway reactivity. The statistical significance of the pulmonary function values was tested using paired t tests. First, we compared the difference between baseline and postexposure measurements after air exposure on Day 1 with the differences between baseline and postexposure measurements after Day 1 exposure to each of the other three atmospheres. Second, we compared the difference between baseline and postexposure measurements after the Day 2 air exposure with the differences between baseline and postexposure measurements after the Day 2 exposure to each of the pollutant atmospheres. Third, we compared the difference between baseline measurements on Day 1 of each exposure atmosphere with measurements after exposure to the same atmosphere on Day 2 to detect delayed effects. No changes in any of the pulmonary function parameters were statistically significant when compared with changes after clean air exposure. Six subjects left the study because of uncomfortable symptoms associated with the exposures. These all occurred after exposure to pollutant atmospheres and not after exposure to clean air.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of sulfur dioxide exposure on African-American and Caucasian asthmatics.

There is concern that air pollution may be causing increases in asthma morbidity and mortality, especially among African-Americans. It is possible that there may be ethnic differences in susceptibility. To evaluate this speculation, a comparative pilot study of respiratory function in 10 African American and 12 Caucasian methacholine positive asthmatic males was conducted. Subjects were exposed to pure air or 1 ppm SO2 while breathing inside a polycarbonate head dome, for 10 min of rest and 10 min of exercise. Baseline and postexposure pulmonary function measurements were recorded, and nasal lavage fluid samples were collected and processed for epithelial and white blood counts. Although significant increases were seen in total respiratory resistance following SO2 exposure in both groups (P = 0.04), no ethic-based difference in response was seen. No significant differences were found in pulmonary or nasal measurements after exposure to SO2 between African-American and Caucasian subjects. No significant changes in epithelial or white blood cell count were found either when data were analyzed from the entire group or separately from the two subject groups. Even though there were no significant group changes, some individuals were particularly responsive to SO2. Three Caucasian and 5 African-American subjects showed greater than 20% increases in total respiratory resistance.

The effects of ozone exposure on lactate dehydrogenase release from human and primate respiratory epithelial cells.

Ozone is the most persistent, wide-spread air pollutant in the United States. Over one half of the population of the US lives in cities or suburban areas which do not meet the National Ambient Air Quality Standard for ozone which is 0.12 ppm averaged over 1 h. Controlled laboratory exposures of human subjects have shown that ozone exposure produces decreased pulmonary function, hyperresponsiveness to inhaled methacholine, inspiratory pain, and airway inflammation as assessed by bronchoalveolar lavage. However, the cellular mechanisms responsible for such effects are incompletely known. The present study examined the effects of ozone exposure at 0.50 ppm for 3 h on three types of cultured respiratory epithelial cells; primary cultures of human nasal cells and primate bronchial cells, and the A549 type II pneumocyte-derived cell line. Cells were grown to confluent monolayers in plastic 6-well plates and then exposed to ozone or filtered air on a tilting platform over a heated water bath. Lactose dehydrogenase release was significantly increased following ozone exposure of all cell types; a 75% increase from human nasal cells (P = 0.0002), a 79% increase from primate bronchial cells (P = 0.003), and a 69% increase from A549 cells (P = 0.02). These data suggest that even brief ozone exposure causes membrane injury to cultured human respiratory epithelial cells.

Pulmonary function changes in children associated with fine particulate matter.

During winter months many neighborhoods in the Seattle metropolitan area are heavily affected by particulate matter from residential wood burning. A study was conducted to investigate the relationship between fine particulate matter and pulmonary function in young children. The subjects were 326 elementary school children, including 24 asthmatics, who lived in an area with high particulate concentrations predominantly from residential wood burning. FEV1 and FVC were measured before, during and after the 1988-1989 and 1989-1990 winter heating seasons. Fine particulate matter was assessed using a light-scattering instrument. Analysis of the relationship between light scattering and lung function indicated that an increase in particulate air pollution was associated with a decline in asthmatic children's pulmonary function. FEV1 and FVC in the asthmatic children dropped an average of 34 and 37 ml respectively for each 10(-4) m-1 increase in sigma sp. This sigma sp increase corresponds to an increase in PM2.5 of 20 micrograms/m3. It is concluded that fine particulate matter from wood burning is significantly associated with acute respiratory irritation in young asthmatic children.

Respiratory effects from the inhalation of hydrogen chloride in young adult asthmatics.

Almost no human data exist from controlled studies using low levels of hydrogen chloride (HCl), and, with no existing HCl ambient standards in the United States, the need for human health effects research is evident. In this study, five female and five male 18 to 25-year-old asthmatic subjects were exposed to filtered air, 0.8 ppm and 1.8 ppm HCl while wearing half-face masks, during three separate 45-minute experimental sessions involving 15 minutes exercise (treadmill walking), 15 minutes rest, followed again by exercise. Baseline and postexposure pulmonary function measurements were taken including forced expiratory volume in 1 second (FEV1), forced expiratory volume (FVC), maximal flow at 50% of expired vital capacity (Vmax50), maximal flow at 75% of expired vital capacity (Vmax75), and total respiratory resistance as well as peak flow. Nasal work of breathing and oral ammonia levels also were measured preexposure and postexposure. No significant pulmonary effects were found at these HCl concentrations and exposure duration. Nasal power showed no significant differences between test atmospheres; however, in isolation a significant decrease (P less than .01) was found in measurements of inspiration with exposure to 0.80 ppm. Ammonia levels showed a significant rise postexposure after both concentrations of HCl (paired t test, (P less than .01)), not seen after air exposure. In summary, the asthmatic subjects in this study showed no adverse respiratory health effects of inhalation of low concentrations of HCl.

Response of young asthmatic patients to inhaled sulfuric acid.

The intent of this study was to explore the effects of inhalation of [H+] defined here as acid airborne particles at near ambient concentrations on the pulmonary function of adolescent asthmatic subjects. During rest and exercise, 22 adolescent asthmatic subjects inhaled atmospheres containing either clean air or sulfuric acid particles (H2SO4) through a mouthpiece. The concentration of hydrogen ion at the mouthpiece ([H+]) ranged from 1.18 to 3.59 mumol/m3 (51 to 176 micrograms/m3 of H2SO4). The lower range of [H+] is near the peak values measured during the summer months in the eastern United States and Canada. Pulmonary function and oral ammonia levels were measured before and after exposure in all subjects. Significant group responses to [H+] were seen in FEV1 (p = 0.016) and FVC (p = 0.039) measured 2 to 3 min post-exposure. Also, the slopes of the change in pulmonary function versus [H+] were computed for each subject. The slopes of changes in FEV1 and Vmax50 and Vmax75 versus [H+] were related to the subject's response to a standard exercise treadmill test, specifically to the subject's percentage decrease in FEV1 after exercise challenge. Pulmonary function changes 20 min postexposure did not show a significant group response to [H+] exposure; however, the relationship between percentage FEV1 decrease after exercise and the individual slopes of Vmax50 and Vmax75 persisted for at least 20 min after exposure.

Acute pulmonary effects of nitrogen dioxide exposure during exercise in competitive athletes.

The acute pulmonary responses of athletes after short-term exposure to ambient concentrations of NO2 during heavy exercise have been examined. Intercollegiate male athletes were screened for history of cardiac disease, respiratory disease, allergic conditions and extensive exposure to pollutants. After completion of serum IgE level determination, exercise tolerance test and methacholine challenge test with normal results, nine healthy subjects 18 to 23 years of age were exposed to filtered air and to 0.18 and 0.30 ppm NO2 for 30 min on different days while exercising on a treadmill. Pulmonary function parameters were measured before and after each exposure. In this study, no statistically significant changes were observed in FEV1, RT PEFR, and Vmax50% after exposure to 0.18 and 0.30 ppm NO2. For these selected healthy athletes, short-term exposure to ambient NO2 levels during heavy exercise does not affect adversely the pulmonary function.

Prior exposure to ozone potentiates subsequent response to sulfur dioxide in adolescent asthmatic subjects.

The objective of this study was to test whether prior exposure to a low concentration of ozone (120 ppb) would condition airways in asthmatic subjects to respond to a subthreshold concentration of sulfur dioxide (100 ppb). Eight male and five female subjects 12 to 18 yr of age participated. They all had allergic asthma and exercise-induced bronchospasm. Subjects were exposed to three test atmosphere sequences during intermittent moderate exercise (a 45-min exposure to one pollutant followed by a 15-min exposure to the second pollutant). The sequences were: air followed by 100 ppb SO2, 120 ppb O3 followed by 120 ppb O3, and 120 ppb O3 followed by 100 ppb SO2. The pulmonary function measurements assessed were FEV1, total respiratory resistance (RT), and maximal flow (Vmax50). Air-SO2 and O3-O3 exposures did not cause significant changes in pulmonary function. On the other hand, exposure to 100 ppb SO2 after a 45-min exposure to 120 ppb O3 caused a significant (8%) decrease in FEV1 (p = 0.046), a significant (19%) increase in RT (p = 0.048), and a significant (15%) decrease in Vmax50 (p = 0.008). It is concluded that prior O3 exposure increased bronchial hyperresponsiveness in these subjects such that they responded to an ordinarily subthreshold concentration of SO2. These data suggest that assessment of pulmonary changes to single pollutant challenges overlooks the interactive effects of common coexisting or sequentially occurring air pollutants.

Nitric oxide and nitrous oxide production and cycling during dissimilatory nitrite reduction by Pseudomonas perfectomarina.

The denitrifier Pseudomonas perfectomarina reduced nitrite under conditions of kinetic competition between cells and gas sparging for extracellular dissolved nitric and nitrous oxides, NOaq and N2Oaq, in a chemically defined marine medium. Time courses of nitrite reduction and NOg and N2Og alpha removal were integrated to give NOg and N2Og yields. At high sparging rates, the NOg yield was greater than 50% of nitrite-N reduced, and the yield of NOg + N2Og was approximately 75%. Hence interrupted denitrification yields NOaq and N2Oaq as major products. The yields varied with sparging rates in agreement with a quantitative model of denitrification (Betlach, M. P., and Tiedje, J.M. (1981) Appl. Environ. Microbiol. 42, 1074-1084) that applies simplified Michaelis-Menten kinetics to NO2-----NOaq----N2Oaq----N2. The fit gave an estimate of the maximum scavengeable NOaq yield of 73 +/- 8% of nitrite-N. Thus a minor path independent of NOaq is also required. The fit of the model to data at lower sparging rates, where normal denitrification products predominate, implies that the extracellular NOaq pool yield is independent of gas sparging rate. Thus in P. perfectomarina NOaq and N2Oaq are intermediates, or facilely equilibrate with true intermediates, during complete denitrification. The recovery of most nitrite-N as NO and/or N2O under perturbed conditions is not an artifact of irreversible product removal, but an attribute of denitrification in this species, and most probably it is characteristic of denitrification in other species as well.