BACKGROUNDWe recently reported that allergic lung inflammation in guinea pigs became steroid resistant in the presence of latent adenoviral infection.OBJECTIVEWe sought to investigate the molecular mechanisms that underlie steroid resistance in adenoviral infection.METHODSGuinea pigs with a latent adenoviral infection were sensitized and challenged with ovalbumin (OVA) and given daily injections of budesonide (20 mg/kg administered intraperitoneally). Sham-infected animals received either saline challenge without budesonide injection or OVA challenge with or without budesonide. The inflammatory response in the lung was measured by means of quantitative histology. Eotaxin, monocyte chemoattractant protein 1 (MCP-1), and RANTES expression in the lung were analyzed by means of Northern blotting, and the binding activity of activator protein 1 (AP-1) and nuclear factor kappaB in nuclear extracts from the lung was analyzed with electrophoretic mobility shift assays.RESULTSOVA challenge increased eosinophil infiltration and eotaxin and MCP-1 mRNA expression in the lungs, and glucocorticoids reduced these increases in the sham-infected, but not the adenovirus-infected, animals. Changes in binding activity of AP-1, but not nuclear factor kappaB, paralleled changes in eotaxin and MCP-1 mRNA.CONCLUSIONWe conclude that latent adenoviral infection inhibits the anti-inflammatory effects of glucocorticoids on allergen-induced eotaxin and MCP-1 expression through AP-1, leading to steroid-resistant allergic lung inflammation.

Sphingomyelins obtained from bovine brain, chicken egg yolk and bovine milk fat were analysed. Separation was performed by reversed-phase high-performance liquid chromatography utilizing a binary solvent gradient consisting of n-butanol-water-1-propanol-isooctane. Detection was accomplished by light scattering and on-line discharge-assisted thermospray (plasmaspray) ionization mass spectrometry. The positive ion mass spectra exhibit prominent ions related to the amine base structure and fragments which can be utilized for identification of molecular species. The abundance of the fragments containing the fatty acid reflects relatively well the fatty acid composition of natural sphingomyelin mixtures. Bovine brain sphingomyelin has, for example, two distinctive major molecular species composed of C24:1 and C18:0 acids as indicated by the m/z 630.6 and m/z 548.5 ions, respectively. While egg yolk exhibits ions of m/z 520.6 which indicate the presence of C16:0, bovine milk sphingomyelin has several prominent ions of m/z 632.5, 618.6 and 604.7, reflecting the proportions of C24:0, C23:0 and C22:0, respectively.

Class separation of common glycerolipids on a diol-phase liquid chromatography column with a gradient of the mixed solvents hexane/isopropanol/acetic acid/triethylamine is compatible with on-line plasmaspray ionization mass spectrometry. The positive ion mass spectra exhibit prominent diacylglycerol and monoacylglycerol derived fragments, which can be utilized for quantification. In the selected-ion monitoring mode, the detection limit for phospholipids is in the low nanogram range. The abundance of the diacylglycerol and monoacylglycerol fragments reflects the relative fatty acid composition of the phospholipid classes. Collision-induced decomposition of diacylglycerol fragments with argon as collision gas unambiguously reveals the chain length and degree of unsaturation of the esterified fatty acids in the native lipid.

Chromatographic methods were evaluated concerning their effectiveness in the detection of bacterial contaminations of cultures of Leuconostoc mesenteroides, a Gram-positive bacterium used for industrial production of dextran. Three classes of cellular components were studied: fatty acids, amino acids and carbohydrates. Proportions of Staphylococcus and Bacillus as small as 0.25% (w/w), and 0.5% Streptococcus, could be detected in L. mesenteroides cell suspensions by determination of fatty acid chemical markers. In addition, the effect of bacterial contamination on chromatographic profiles was studied by comparing the profiles of non-contaminated L. mesenteroides with those of L. mesenteroides contaminated with a strain of Enterobacter cloacae isolated at a dextran-producing industry. The noted differences in the profiles were evaluated using a multivariate classification program (SIMCA). The results indicate that sensitive chromatographic methods for the determination of cellular components may be versatile tools for the early detection of bacterial contamination in biotechnical processes.

Effects of rhizosphere bacteria (RB), and rhizosphere bacteria with bacteria-feeding nematodes (RBN), on the composition of root exudate were examined after 2 weeks in gnotobiotic culture systems with rape seedlings (Brassica napus (L.)). The amounts of low molecular weight carbohydrates and of some free amino acids, per unit root dry weight, in the exudates were lower in the RB and RBN treatments than in the axenic control (R treatment). The growth of nematodes implied a production of bacterial cells in the RBN treatment 2.6 times that in the RB treatment. The bacterial growth in the RB treatment and the bacterial growth in combination with grazing by nematodes in the RBN treatment implied 24 and 63 times as much exudation of organic carbon, respectively, as in the R treatment.

Gas chromatography/mass spectrometry was used to detect the presence of Enterobacter cloacae in cultures of Leuconostoc mesenteroides, an organism used in an industrial process for production of dextrane. The penta-fluorobenzoyl-methyl ester derivative of 3-hydroxy-myristic acid, a characteristic compound of gram-negative bacteria, was used as the analyte. By using gas chromatography/mass spectrometry with selected ion monitoring, E. cloacae was determined over the range of 1 ppm to 1% in cultures of L. mesenteroides. The proposed analytical approach represents a useful alternative to conventional methods for determining contaminating organisms in industrial fermentation processes.

Plasma membranes isolated from HeLa cells cultivated in suspension cultures supplemented with 3.5% fetal bovine serum or 2% of the commercially available serum substitute Ultroser G contained the same amounts of protein, cholesterol, and phosphate on a cellular basis. Minor differences in the plasma membrane fatty acid composition were seen, with the most pronounced alteration observed for palmitic acid, which amounted to 27 and 20% in fetal bovine serum- and Ultroser G-supplemented cells, respectively. Plasma membranes from cells growth with Ultroser G contained almost twice as much phosphatidylethanolamine and displayed two thirds of the phosphatidylcholine content, compared to plasma membranes obtained from fetal bovine serum supplemented cells. The former membranes also showed a 3 times higher specific [3H]acetate labeling of cholesterol, indicating a higherde novo synthesis of cholesterol. Both quantitative and qualitative alterations were revealed among the plasma membrane polypeptides when these were subjected to immuno- and lectin blottings. Fluorescence anisotropy measurements at different temperatures produced similar results irrespective of the growth medium supplement when the plasma membrane specific probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene was used on intact cells. However, the average cellular rigidity was higher for Ultroser G supplemented cells, determined with 1,6-diphenyl-1,3,5-hexatriene as a probe.

The phospholipid fatty acid composition and poly-β-hydroxybutyrate (PHB) content of initially adhered and free-living cells of a Pseudomonas sp. isolated from the rape plant Brassica napus were examined with gas chromatography (GC). Five different adhesion experiments were made including variations in surface charge (hydrophilic and lipophilic), temperature, media composition and time of adhesion. Lipids and poly-β-hydroxybutyrate (PHB) were extracted with a chloroform-methanol-water mixture, hydrolyzed and esterified with pentafluorobenzyl bromide. Analysis was performed with capillary gas chromatography and flame ionization detection. A pronounced difference in both the ratio saturated/unsaturated fatty acids and in PHB content between free-living and adhered bacteria were found. The free-living bacteria has a significantly smaller ratio of saturated/unsaturated C16 and C18 fatty acids and also a smaller ratio of total C18/total C16 fatty acids. Bacteria adhered to the lipophilic surface had a higher ratio of saturated to unsaturated C16 fatty acids than at the hydrophilic surface. There were no major differences between the treatments regarding the amount of bacteria adhered to the surface or their lipid composition.

Liquid chromatographic class separations of common cellular phospholipids combined with plasma spray ionization of the effluents were investigated. Comparison with true thermospray ionization involving ammonium acetate buffering revealed a gain in total ionization in the plasma spray of a factor of approximately 10 using a cation-exchange column and a solvent mixture consisting of acetonitrile-methanol-water (400:100:15, v/v). Plasma spray ionization studies of bovine brain polyphosphoinositides interrelated by the phosphate content in the inositol moeity showed almost identical monoglyceride and diglyceride ion clusters, indicating possibilities of studying the biochemical turnover of such phospholipids. Plasma spray ionization liquid chromatography-mass spectrometry of bacterial membrane phospholipids (Pseudomonas fluorescens) revealed possibilities of obtaining indications of individual fatty acid compositions from the spectra of the phosphatidylinositol and phosphatidylethanolamine fractions present. Conventional gas chromatographic fatty acid analysis agreed with the direct mass spectrometric structure elucidations. Interestingly, the two phospholipid classes had different relative fatty acid compositions with a significantly higher degree of cyclic fatty acids in the phosphatidyl ethanolamines. Plasma spray ionization yielded linear dose-response curves for both the monoglyceride and diglyceride fragment signals in the selected-ion monitoring mode. The detection limit for the monoglyceride and diglyceride species of phosphatidylcholine under the chromatographic and mass spectrometric conditions used was found to be in the picogram range.

A new system to study bacterial responses is described. The method utilizes a device which consists of a beaker into which funnels with attached membranes are inserted. One funnel exudes a test solution and the other funnel a control solution. Samples were retrieved aseptically at a position one cm below the control and the attractant funnels. A positive chemotactic response by a starved marine Gram-negative bacterium towards mixture of low molecular weight substances was observed. This response was detected during the initial phase of starvation. The model allows the experiments to be carried out under conditions which reflect the situation in natural oligotrophic waters. The possibility to measure subcellular quantities in response to a nutrient concentration was demonstrated by the determinationa of the bacterial fatty acid profile.

An autoclavable all-glass system for studying microbial dynamics at permeable surfaces is described. Standard hydrophobic or hydrophilic membranes (46-mm diameter) of various pore sizes were supported on a glass frit through which nutrient solutions were pumped by a peristaltic pump. The pump provided a precisely controlled flow at speeds of 0.5 to 500 ml of defined or natural cell exudates per h, which passed through the membrane into a receiving vessel. The construction allowed a choice of membranes, which could be modified. The system was tested with a bacterium, isolated from rape plant roots (Brassica napus L.), that was inoculated on a hydrophilic membrane filter and allowed to develop into a biofilm. A defined medium with a composition resembling that of natural rape root exudate was pumped through the membrane at 0.5 ml/h. Scanning electron microscopic examinations indicated that the inoculum formed microcolonies embedded in exopolymers evenly distributed over the membrane surface. The lipid composition and content of poly-beta-hydroxybutyrate in free-living and adhered cells were determined by gas chromatography. The bacterial consumption of amino acids in the exudate was also studied.