We have developed a new assay that differentiates between indoleacetic acid (IAA)-producing and-nonproducing bacteria on a colony plate lift. Medium supplemented with 5 mM L-tryptophan is inoculated with isolates of interest, overlaid with a nitrocellulose membrane, and then incubated until bacterial colonies reach 1 to 2 mm in diameter. The membrane is removed to a filter paper saturated with Salkowski reagent and incubated until distinct red haloes form around the colonies. The colorimetric reaction to IAA is limited to a region immediately surrounding each colony, is specific to isolates producing IAA, occurs within 1 h after the membrane is placed in the reagent, and is sensitive to as little as 50 pmol of IAA in a 2-mm2 spot. We have used this assay for quantifying epiphytic and endophytic populations of IAA-producing isolates of Pseudomonas syringae subsp. savastanoi and for detecting IAA-producing colonies of other pseudomonads and Erwinia herbicola. The assay provides a rapid and convenient method to screen large numbers of bacteria.

Children raised with extended exposure to environmental tobacco smoke (ETS) experience increased cough and wheeze. This study was designed to determine whether extended ETS exposure enhances citric acid-induced cough and bronchoconstriction in young guinea pigs via a neurokinin-1 (NK-1) receptor mechanism at the first central synapse of lung afferent neurons, the nucleus tractus solitarius. Guinea pigs were exposed to ETS from 1 to 6 weeks of age. At 5 weeks of age, guide cannulae were implanted bilaterally in the medial nucleus tractus solitarius at a site that produced apnea in response to the glutamate agonist D,L-homocysteic acid. At 6 weeks of age, either vehicle or a NK-1 receptor antagonist, SR 140333, was injected into the nucleus tractus solitarius of the conscious guinea pigs who were then exposed to citric acid aerosol. ETS exposure significantly enhanced citric acid-induced cough by 56% and maximal Penh (a measure of airway obstruction) by 43%, effects that were attenuated by the NK-1 receptor antagonist in the nucleus tractus solitarius. We conclude that in young guinea pigs extended exposure to ETS increases citric acid-induced cough and bronchoconstriction in part by an NK-1 receptor mechanism in the nucleus tractus solitarius.

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Acute lung injury induced by reactive oxygen gases such as ozone (O(3)) is focal and site-selective. To define patterns of acute epithelial injury along intrapulmonary airways, we developed a new analytic approach incorporating labeling of permeable cells, airway microdissection, and laser scanning confocal microscopy, and applied it to isolated perfused rat lungs where ventilation and breathing pattern could be controlled. After exposure to O(3) (0, 0.25, 0.5, or 1.0 ppm), lungs were lavaged to assess lactate dehydrogenase (LDH) and protein, or infused with the permeability marker ethidium homodimer-1 (EthD-1) via tracheal cannula, gently lavaged, and fixed by airway infusion. The airway tree of the right middle lobe was exposed by microdissection of the axial pathway down to the terminal bronchioles; the dissection was incubated with a second nuclear dye, YOPRO-1, to label all nuclei; and whole mounts were examined by confocal microscopy. Abundance of EthD-1-positive (injured) cells was estimated as the number per epithelial volume using stereology on Z-series of projected images. For ozone concentrations of 1.0 ppm, lavage fluid LDH and total protein did not increase over controls. Exposure produced a concentration- dependent but nonhomogeneous increase in the abundance of EthD-1-labeled cells in proximal and distal conducting airways both in the main pathway, including terminal bronchioles, and in side branches. Overall, the highest EthD-1 labeling occurred in the side branches of the most proximal part of the airway tree at 1 ppm with the adjacent axial pathway airway having approximately one-third the labeling density. Density of EthD-1-labeled cells was lowest in terminal bronchioles at all O(3) doses. For the model we used, identification of injured epithelial cells by differential permeability and laser confocal microscopy appeared to be highly sensitive and permitted mapping of acute cytotoxicity throughout the airway tree and quantitative comparisons of sites with different branching histories and potential dosimetry rates.

Infants and young children experiencing extended exposure to secondhand smoke (SHS) have an increased occurrence of asthma, as well as increased cough, wheeze, mucus production and airway hyper-reactivity. Plasticity in lung reflex pathways has been implicated in causing these symptoms, as have changes in substance P-related mechanisms. Using whole-cell voltage-clamp recordings and immunohistochemistry in brainstem slices containing anatomically identified second-order lung afferent nucleus tractus solitarius (NTS) neurons, we determined whether extended SHS exposure during the equivalent period of human childhood modified evoked or spontaneous excitatory synaptic transmission, and whether those modifications were altered by endogenous substance P. SHS exposure enhanced evoked synaptic transmission between sensory afferents and the NTS second-order neurons by eliminating synaptic depression of evoked excitatory postsynaptic currents (eEPSCs), an effect reversed by the neurokinin-1-receptor antagonist (SR140333). The recruitment of substance P in enhancing evoked synaptic transmission was further supported by an increased number of substance P-expressing lung afferent central terminals synapsing onto the second-order lung afferent neurons. SHS exposure did not change background spontaneous EPSCs. The data suggest that substance P in the NTS augments evoked synaptic transmission of lung sensory input following extended exposure to a pollutant. The mechanism may help to explain some of the exaggerated respiratory responses of children exposed to SHS.