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Background: The amount of allergen necessary to sensitise genetically "at risk" children is unclear. The relation between allergen exposure and asthma is also uncertain., Methods: To ensure a wide range of allergen exposures the data from case-control studies of asthma in children aged 12-14 years attending three schools in Los Alamos, New Mexico and Central Virginia were combined. Skin prick tests to indoor and outdoor allergens and bronchial hyperreactivity to histamine were assessed in children with and without symptoms of asthma. The concentration of mite, cat, and cockroach allergens in dust from the children's homes was used as a marker of exposure., Results: Three hundred and thirty two children (157 with asthmatic symptoms and 175 controls) were investigated. One hundred and eighty three were classified as atopic on the basis of allergen skin prick tests and 68 as asthmatic (symptoms plus bronchial responsiveness). The prevalence and degree of sensitisation to mite and cockroach, but not cat, was strongly associated in atopic children with increasing domestic concentrations of these allergens. Asthma was strongly associated with sensitisation to indoor allergens (p<10(-6)) and weakly to outdoor allergens (p = 0.026). There was an association between current asthma and the concentration of mite allergen amongst atopic children (p = 0.008) but not amongst those who were specifically mite sensitised (p = 0.16)., Conclusions: The domestic reservoir concentration of mite and cockroach, but not cat, allergen was closely related to the prevalence of sensitisation in atopic children. However, the prevalence of current asthma had a limited relationship to these allergen measurements, suggesting that other factors play a major part in determining which allergic individuals develop asthma.

This cross-sectional emergency department study of 70 wheezing children and 59 control subjects (2 mo to 16 yr of age) examined the prevalence of respiratory viruses and their relationship to age, atopic status, and eosinophil markers. Nasal washes were cultured for respiratory viruses, assayed for respiratory syncytial virus (RSV) antigen, and tested for coronavirus and rhinovirus RNA using reverse transcription-PCR (RT-PCR). Also evaluated were eosinophil numbers and eosinophil cationic protein (ECP) in both nasal washes and serum, along with total IgE and specific IgE antibody in serum. Respiratory viruses were detected in 82% (18 of 22) of wheezing infants younger than 2 yr of age and in 83% (40 of 48) of older wheezing children. The predominant pathogens were RSV in infants (detected in 68% of wheezing subjects) and rhinovirus in older wheezing children (71%), and both were strongly associated with wheezing (p < 0.005). RSV was largely limited to wheezing children younger than 24 mo of age, but rhinovirus was detected by RT-PCR in 41% of all infants and in 35% of nonwheezing control subjects older than 2 yr of age. After 2 yr of age the strongest odds for wheezing were observed among those who had a positive RT-PCR test for rhinovirus together with a positive serum radioallergosorbent testing (RAST), nasal eosinophilia, or elevated nasal ECP (odds ratios = 17, 21, and 25, respectively). Results from this study demonstrate that a large majority of emergent wheezing illnesses during childhood (2 to 16 yr of age) can be linked to infection with rhinovirus, and that these wheezing attacks are most likely in those who have rhinovirus together with evidence of atopy or eosinophilic airway inflammation.

The mechanism of uptake of benzoic and 2,4-dichlorobenzoic acid (2,4-DCBA) by Alcaligenes denitrificans BRI 3010 and BRI 6011 and Pseudomonas sp. strain B13, three organisms capable of degrading various isomers of chlorinated benzoic acids, was investigated. In all three organisms, uptake of benzoic acid was inducible. For benzoic acid uptake into BRI 3010, monophasic saturation kinetics with apparent K(infm) and V(infmax) values of 1.4 (mu)M and 3.2 nmol/min/mg of cell dry weight, respectively, were obtained. For BRI 6011, biphasic saturation kinetics were observed, suggesting the presence of two uptake systems for benzoic acid with distinct K(infm) (0.72 and 5.3 (mu)M) and V(infmax) (3.3 and 4.6 nmol/min/mg of cell dry weight) values. BRI 3010 and BRI 6011 accumulated benzoic acid against a concentration gradient by a factor of 8 and 10, respectively. A wide range of structural analogs, at 50-fold excess concentrations, inhibited benzoic acid uptake by BRI 3010 and BRI 6011, whereas with B13, only 3-chlorobenzoic acid was an effective inhibitor. For BRI 3010 and BRI 6011, the inhibition by the structural analogs was not of a competitive nature. Uptake of benzoic acid by BRI 3010 and BRI 6011 was inhibited by KCN, by the protonophore 3,5,3(prm1), 4(prm1)-tetrachlorosalicylanilide (TCS), and, for BRI 6011, by anaerobiosis unless nitrate was present, thus indicating that energy was required for the uptake process. Uptake of 2,4-DCBA by BRI 6011 was constitutive and saturation uptake kinetics were not observed. Uptake of 2,4-DCBA by BRI 6011 was inhibited by KCN, TCS, and anaerobiosis even if nitrate was present, but the compound was not accumulated intracellularly against a concentration gradient. Uptake of 2,4-DCBA by BRI 6011 appears to occur by passive diffusion into the cell down its concentration gradient, which is maintained by the intracellular metabolism of the compound. This process could play an important role in the degradation of xenobiotic compounds by microorganisms.

Neisseria meningitidis SD1C exhibited a low tolerance to penicillin G (0.03 microgram/ml). Loss of viability in the absence of polyvinylpyrrolidone-40 and horse serum was independent of the concentration of antibiotic above the minimum inhibitory concentration, whereas the rate of bacteriolysis was concentration dependent. Penicillin-induced lysis was a secondary event in this organism. At low levels of penicillin G, growth characteristics, i.e., absorbancy changes, respiratory rate, and uptake of Mg2+, appeared normal during the first 90 min in penicillin; however, viability dropped dramatically. Additionally, total cell numbers remained constant while cell mass continued to increase at a rate normal for the population. The increase in cellular mass in the absence of cell division could be observed microscopically. Only one ultrastructural change induced by penicillin correlated with the loss in viability: the loss in continuity of the outer membrane with the peptidoglycan but only at the site of septum formation. This lesion did not occur when cells were grown in media supplemented with the protective agents polyvinylpyrrolidone-40 and horse serum. Under these conditions of growth and with relatively high levels of penicillin, constant viability was maintained, but cell division no longer occurred. Cell populations treated with penicillin in the presence of the protective agents became increasingly more dependent on the presence of these agents for total viability even in the absence of penicillin in the culture.

Pseudomonas aeruginosa grows at an apparent reduced rate at 46 C as compared with the rate at 37 C, when growth is measured as an increase in absorbance. Cells at 46 C are long, plasmolyzed, nonmotile filaments. The filaments contain phase-dark material that may be chromosomal in nature. When the 46 C culture is shifted to 37 C, the filaments fragment at polar ends after flagella form, and the final number of cells is equal to the number of chromosomal "packets" observed within the filament. The outer envelope of the filament appears to be structurally complete as determined by biochemical, thin section, and freeze-etch examination. When filaments are treated with lysozyme, they form large spheroplasts, suggesting that the outer wall and the cytoplasmic membrane are continuous within the filament. Filaments produce little or no periplasm-located alkaline phosphatase (APase), but activity appears immediately after a shift to 37 C. Cells grown at 37 C and shifted to 46 C remain as single, nonmotile, rods or doublets, and the APase formed at 37 C remains stable at 46 C. The addition of APase or inorganic phosphate is partially or completely effective as an inducer of filament fragmentation at 46 C. The results suggest that periplasm-located APase is an important enzyme in the final stages of cell division when P. aeruginosa is cultured on inorganic phosphate-limiting media.

Both reaction-product localization and ferritin-coupled antibody studies have shown that alkaline phosphatase is evenly distributed throughout the peri-plasmic space of Escherichia coli and a marine pseudomonad. This space is not locally enlarged except in cases where plasmolysis has occurred.

Ingram, Jordan M. (Michigan State University, East Lansing), and W. A. Wood. Enzymatic basis for d-arabitol production by Saccharomyces rouxii. J. Bacteriol. 89:1186-1194. 1965.-The enzymatic steps in d-arabitol synthesis by Saccharomyces rouxii were studied. The fermentation of d-glucose-6-C(14) gave rise to d-arabitol labeled at C-5; d-ribose of ribonucleic acid had the same isotope pattern. Crude extracts were able to reduce d-ribulose with reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)) and d-xylulose with reduced nicotinamide adenine dinucleotide (NADH(2)). These extracts also oxidized d-arabitol with nicotinamide adenine dinucleotide phosphate and xylitol with nicotinamide adenine dinucleotide. No reduction of d-ribulose-5-phosphate or d-xylulose-5-phosphate was observed. An enzyme which reduced d-xylulose with NADH(2) was purified 33-fold and characterized as a xylitol (--> d-xylulose) dehydrogenase. Similarly, an enzyme reducing d-ribulose with NADPH(2) was purified 12-fold and characterized as a d-arabitol (--> d-ribulose) dehydrogenase. Alkaline and acid phosphatases were purified 50- and 40-fold, respectively, and their specificities were determined. Only the acid phosphatase had detectable activity on d-ribulose-5-phosphate. The data support the postulate that d-arabitol arises by dephosphorylation of d-ribulose-5-phosphate and reduction of d-ribulose by a NADPH(2)-linked d-arabitol (--> d-ribulose) dehydrogenase.

Pseudomonas aeruginosa ATCC 9027 contains an inducible alkaline phosphatase. The enzyme is readily removed from 14-hr cells by washes in 0.2 m MgCl(2), pH 8.4. Similar washes in tris(hydroxymethyl)aminomethane buffer, 20% sucrose, monovalent ions, or water partially release enzyme from the cells. The release of alkaline phosphatase is correlated with an increased release of protein and retention of internal enzymes. The effect of 0.2 m MgCl(2) washing upon the cells is minimal since both viability and growth rates remain unchanged as compared to water washing. Although cells are plasmolyzed in both 0.2 m MgCl(2) and 20% sucrose, it is evident that plasmolysis alone is unable to account for total enzyme release and that a divalent metal, i.e. Mg(2+), augments the release pattern. Growing cells in the presence of increasing concentrations of MgCl(2) or at increased pH values results in an almost total secretion of the enzyme to the culture filtrate. The findings suggest that P. aeruginosa alkaline phosphatase is linked to the exocytoplasmic region through divalent metal ion, presumably Mg(2+), bridges.

Cells of Pseudomonas aeruginosa suspended in 0.2 M Mg(2+), 20% sucrose, 0.01 M tris(hydroxymethyl)aminomethane, or water partially release lipopolysaccharide. The release of alkaline phosphatase from the periplasmic space and the ability to form spheroplasts on lysozyme treatment is directly related to the lipopolysaccharide released during treatment with 0.2 M Mg(2+), 20% sucrose, or other agents. The synthesis of ribonucleic acid (RNA) by intact cells, magnesium-lysozyme spheroplasts, or 20% sucrose-lysozyme spheroplasts is not sensitive to actinomycin D, whereas RNA synthesis by intact cells or spheroplasts in the presence of ethylene-diaminetetraacetic acid (EDTA) is sensitive to actinomycin D. EDTA alone has an inhibitory effect on RNA synthesis by whole cell, by magnesium-lysozyme spheroplasts, and by 20% sucrose-lysozyme spheroplasts. The experimental data indicate that, although the cell wall is damaged by 0.2 M Mg(2+) or 20% sucrose treatment in the presence of lysozyme, the treated cells or spheroplasts are still resistant to actinomycin D. These results suggest that the cytoplasmic membrane should be considered as the final and determinative barrier to this antibiotic in this organism.

Spheroplasts prepared by lysozyme treatment of cells of Pseudomonas aeruginosa, suspended in 20% sucrose or 0.2 m MgCl(2), were examined in detail. Preparation of spheroplasts in the presence of 0.2 m Mg(2+) released periplasmic alkaline phosphatase, whereas preparation in the presence of 20% sucrose did not, even though untreated cells released phosphatase when suspended in sucrose in the absence of lysozyme. Biochemical characterizations of the sucrose-lysozyme preparations indicated that lysozyme mediated a reassociation of the released phosphatase with the spheroplasts. In addition, the enzyme released from whole cells suspended in 20% sucrose (which represents 20 to 40% of the cell-bound phosphatase) reassociates with the cells in the presence of lysozyme. Electron microscopic examinations of various preparations revealed that phosphatase released in sucrose reassociated with the external cell wall layers in the presence of lysozyme, that sucrose-lysozyme prepared spheroplasts did not dissociate phosphatase which remained in the periplasm of sucrose-washed cells, and that phosphatase was never observed to be associated with the cytoplasmic membrane. A model to account for the binding of P. aeruginosa alkaline phosphatase to the internal portion of the tripartite layer of the cell wall rather than to the cytoplasmic membrane or peptidoglycan layer is presented.