Biomedical subjects

A Blomberg

Publications and source records attributed to A Blomberg.

At least 55 records · Page 3Linked to original sources

PubMed1997

Osmoregulation and protein expression in a pbs2delta mutant of Saccharomyces cerevisiae during adaptation to hypersaline stress.

We deleted the PBS2 gene encoding the MAP kinase activator of the osmosignaling HOG pathway in Saccharomyces cerevisiae and examined the effects on the kinetics of the osmoregulatory glycerol response and protein induction during adaptation to 0.7 M NaCl. Changes in protein expression as analyzed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) demonstrated that for the 29 proteins showing a 6-fold induction in wild-type cells during adaptation to NaCl stress, all displayed a decreased and delayed response in pbs2delta cells. Of the seven proteins that were identified, two were previously not known to be under HOG pathway control: Ald6p, an isoform of aldehyde dehydrogenase and Dak1p, a putative dihydroxyacetone kinase. The presence of a remaining significant induction in pbs2delta cells for about half of the examined proteins indicates existence of alternative osmosignaling pathway(s). Northern analysis of the salt induced transcription of GPD1 and GPP2, encoding the cytosolic glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase involved in the osmostress induced glycerol production, demonstrated an about 20-fold PBS2-dependent transient activation, in agreement with the previously reported transient nature of the signal transduced by the HOG pathway.

Adaptation, Physiological↗

PubMed1997

The osmotic hypersensitivity of the yeast Saccharomyces cerevisiae is strain and growth media dependent: quantitative aspects of the phenomenon.

Osmotic hypersensitivity is manifested as cellular death at magnitudes of osmotic stress that can support growth. Cellular capacity for survival when plated onto high NaCl media was examined for a number of laboratory and industrial strains of Saccharomyces cerevisiae. During respiro-fermentative growth in rich medium with glucose as energy and carbon source, the hypersensitivity phenomenon was fairly strain invariant with a threshold value of about 1 M-NaCl; most strains fell within a 300 mM range in LD10 values (lethal dose yielding 10% survival). Furthermore, all but one of the strains displayed similar differential death responses above the threshold value, i.e. ten-fold decreased viability for every 250 mM increase in salinity. Addition of small amounts of salt to the growth medium drastically improved tolerance and shifted the hypersensitivity threshold to higher NaCl concentrations. This salt-instigated tolerance could partly be reversed by washing in water. The washing procedure depleted cells of the glycerol that they had accumulated under saline growth, and the contribution from glycerol to the improved tolerance was about 50% in the two strains examined. Growth on derepressing carbon sources like galactose, ethanol or glycerol gave strain-dependent responses. The laboratory strain X2180-1A drastically improved tolerance while the bakers' yeast strain Y41 did so only marginally. It was concluded that all strains of S. cerevisiae display the osmotic hypersensitivity phenomenon in qualitative terms while the quantitative values differ. It was also proposed that growth rate does not dictate the level of osmotic hypersensitivity of S. cerevisiae.

Culture Media↗

PubMed1997

Two-dimensional electrophoretic separation of yeast proteins using a non-linear wide range (pH 3-10) immobilized pH gradient in the first dimension; reproducibility and evidence for isoelectric focusing of alkaline (pI > 7) proteins.

The proteome of the yeast Saccharomyces cerevisiae was analysed by two-dimensional (2D) polyacrylamide gel electrophoresis utilizing a non-linear immobilized pH gradient (3-10) in the first-dimensional separation. Cells were labelled by [35S]methionine incorporation in the respiro-fermentative phase during exponential growth on glucose. Gels were run, visualized with phosphoimager technology and all resolved proteins automatically quantified. Proteins were well resolved over the whole pH interval, and evidence for isoelectric focusing on the basic side of the pattern was generated by sequencing of some spots, revealing the 2D positions of Tef1p, Pgk1p, Gpm1p, Tdh1p and Shm2p. Roughly 25% of the spots were resolved at the alkaline side of the pattern (pI > 7). The position reproducibility was high and in the range 1-2 mm in the x- and y-dimension, respectively. No quantitative variation was linked to a certain size or charge class of resolved proteins, and the average quantitative standard deviation was 17 +/- 11%. The obtained immobilized pH gradient based pattern could easily be compared to the old ampholine-based 2D pattern, and the previously reported identifications could thus be transferred. Our yeast pattern currently contains 43 known proteins, all identified by protein sequencing. Utilizing these identified proteins, relevant pI and Mr scales in the pattern were constructed. Normalization of the expression of identified spots by compensating for the number of methionine residues a protein contains allowed stoichiometric comparisons. The most dominant proteins under these growth conditions were Tdh3p, Fba1p, Eno2p and Tef1p/Tef2p, all being expressed at more than 500,000 copies per cell. The differential carbon source response during exponential growth on either glucose, galactose or ethanol was examined for the alkaline proteins identified by micro-sequencing in this study.

Amino Acid Sequence↗

PubMed1997

Osmoresponsive proteins and functional assessment strategies in Saccharomyces cerevisiae.

Cells respond to increased external osmolarities by enhanced accumulation of compatible solutes. In yeast-cells, mainly exemplified by Saccharomyces cerevisiae, the premier compatible solute is the polyhydroxy-alcohol glycerol, the production of which is accompanied by overall metabolic changes. By applying two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled to computerized image quantification, a large body of valuable physiological information relating to this stress-adaptation has been gathered. One of the presumed key-enzymes in the production of glycerol in the cell is glycerol 3-phosphate dehydrogenase encoded by the GPD1 gene. The amount of this protein is enhanced during saline stress, and from 2-D analysis linked to microsequencing it became apparent that the osmo-regulated from contained a putative presequence. Sequence analysis of another salt-induced spot in the 2-D pattern revealed identity to a gene, YER062c, with previously unknown function. Biochemical characterization of this protein, including standard purification via chromatography and subsequent activity/specificity measurements, identified this salt-regulated protein as the missing protein/gene in glycerol production, namely the glycerol 3-phosphatase. The sequence of another salt regulated protein resolved in the 2-D gel revealed identity to a bacterial dihydroxyacetone kinase, thus indicating salt induced glycerol dissimilation. Comparing Northern data to the 2-D generated expression pattern revealed a strong correlation, indicating mainly regulation at the transcriptional level. In addition, altered expression during saline growth of some of the glycolytic enzymes was also apparent. Signalling mutants, either in the cAMP-dependent protein kinase A pathway or in a protein kinase cascade, have been analyzed during osmotic stress via 2-D PAGE, grouping proteins/genes apparently regulated via similar mechanismus. Proteome analysis has proven invaluable in the unravelling of the molecular physiology of yeast cells during adaptation and growth under osmotic stress, identifying vital components not selected by purely genetic approaches.

Adaptation, Physiological↗

PubMed1997

Impaired pulmonary function in patients with hemorrhagic fever with renal syndrome.

Pulmonary and cardiac functions were investigated in 13 patients hospitalized with nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome. As compared with reference values, the patients' diffusion capacity for carbon monoxide was decreased (P = .002) and pulmonary clearance of inhaled technetium-99m-labeled diethylenetriamine pentaacetic acid was increased (P = .002). In four of 11 patients, arterial blood gas analysis disclosed a reduction in partial pressure of O2 (< 10 kPa) and oxygen saturation (< 94%). In three of 13 patients, chest radiography revealed interstitial infiltrates or pleural effusions. Lung volumes and expiratory flow rates of the patients were not significantly changed. By electrocardiography and echocardiography, no significant cardiac dysfunction was demonstrable. The pulmonary dysfunction was best explained by an alveolocapillary lesion. The two hantavirus-caused clinical syndromes, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome, may be pathophysiologically more similar than appears from the clinical presentations.

Adult↗

PubMed1997

Glycolytic flux is conditionally correlated with ATP concentration in Saccharomyces cerevisiae: a chemostat study under carbon- or nitrogen-limiting conditions.

Anaerobic and aerobic chemostat cultures of Saccharomyces cerevisiae were performed at a constant dilution rate of 0.10 h(-1). The glucose concentration was kept constant, whereas the nitrogen concentration was gradually decreasing; i.e., the conditions were changed from glucose and energy limitation to nitrogen limitation and energy excess. This experimental setup enabled the glycolytic rate to be separated from the growth rate. There was an extensive uncoupling between anabolic energy requirements and catabolic energy production when the energy source was present in excess both aerobically and anaerobically. To increase the catabolic activity even further, experiments were carried out in the presence of 5 mM acetic acid or benzoic acid. However, there was almost no effect with acetate addition, whereas both respiratory (aerobically) and fermentative activities were elevated in the presence of benzoic acid. There was a strong negative correlation between glycolytic flux and intracellular ATP content; i.e., the higher the ATP content, the lower the rate of glycolysis. No correlation could be found with the other nucleotides tested (ADP, GTP, and UTP) or with the ATP/ADP ratio. Furthermore, a higher rate of glycolysis was not accompanied by an increasing level of glycolytic enzymes. On the contrary, the glycolytic enzymes decreased with increasing flux. The most pronounced reduction was obtained for HXK2 and ENO1. There was also a correlation between the extent of carbohydrate accumulation and glycolytic flux. A high accumulation was obtained at low glycolytic rates under glucose limitation, whereas nitrogen limitation during conditions of excess carbon and energy resulted in more or less complete depletion of intracellular storage carbohydrates irrespective of anaerobic or aerobic conditions. However, there was one difference in that glycogen dominated anaerobically whereas under aerobic conditions, trehalose was the major carbohydrate accumulated. Possible mechanisms which may explain the strong correlation between glycolytic flux, storage carbohydrate accumulation, and ATP concentrations are discussed.

Adenine Nucleotides↗

PubMed1997

Short-term ozone exposure upregulates P-selectin in normal human airways.

Short-term exposure to ambient levels of ozone induces neutrophilic bronchitis. To investigate the early events contributing to inflammatory cell recruitment in the airways we exposed 12 healthy nonsmoking volunteers to 0.12 ppm ozone or filtered air for 2 h on two separate occasions. Spirometry and fiberoptic bronchoscopy were performed immediately and at 1.5 h after the two exposures, respectively. Total protein, albumin, and total and differential cell counts were performed on the bronchial wash and BAL fluid. Bronchial biopsies were embedded in glycol methacrylate and immunostained for inflammatory cells, including neutrophils, mast cells, total T-cells (CD3), T-cell subset CD8, and leukocyte endothelial adhesion molecules, including VCAM-1, ICAM-1, E-selectin, and P-selectin. No significant changes were observed in FEV1, FVC, or any inflammatory indices in the bronchial wash and BAL fluid. In addition, no significant differences were seen in inflammatory cell numbers or percentages of vessels expressing VCAM-1, E-selectin, or ICAM-1 in the biopsies. The percentage of vessels expressing P-selectin increased significantly after ozone exposure: p = 0.016; median (IQR), 28.76 (26.36-36.94) versus 47.06 (38.14-56.86)%. The upregulation of P-selectin could signify an early inflammatory response to ozone such as margination and rolling of the neutrophils on the vessel wall prior to transendothelial migration.

Adult↗

PubMed1997

The inflammatory effects of 2 ppm NO2 on the airways of healthy subjects.

Nitrogen dioxide (NO2) is a free radical and a common oxidant in polluted air. Here we present data on the time course of inflammation after NO2 exposure, as reflected in bronchial biopsy and airway lavage specimens. Healthy, nonsmoking subjects were exposed to air or 2 ppm NO2 for 4 h in random order on separate occasions. Endobronchial biopsies, bronchial washing (BW), and bronchoalveolar lavage (BAL) were done at 1.5 h (n = 15) or 6 h (n = 15) after exposure. In BW, exposure to NO2 induced a 1.5-fold increase in interleukin-8 (IL-8) (p < 0.05) at 1.5 h and a 2.5-fold increase in neutrophils (p < 0.01) at 6 h. In BAL fluid (BALF), small increases were observed in CD45RO+ lymphocytes, B-cells, and natural killer (NK) cells only. Immunohistologic examination of bronchial biopsy specimens showed no signs of upregulation of adhesion molecules, and failed to reveal any significant changes in inflammatory cells at either time point after NO2 exposure. In summary, NO2 induced a neutrophilic inflammation in the airways that was detectable in BW at 6 h after NO2 exposure. The increase in neutrophils could be related to the enhanced IL-8 secretion observed at 1.5 h after exposure. The absence of adhesion-molecule upregulation or cellular inflammation in mucosal biopsy specimens indicates that the major site of inflammation following exposure to NO2 may be in the smaller airways and not in the alveoli.

Adult↗

PubMed1997

Differential inhibition of inflammatory cytokine release from cultured alveolar macrophages from smokers and non-smokers by NO2.

Human alveolar macrophages (AMs) obtained from smokers and non-smokers by bronchoalveolar lavage (BAL) were subjected to various concentrations of NO2 in an inverted monolayer exposure model. Culture supernatants were collected 4 h after the exposure and assayed for secreted TNF-alpha, IL-1 beta, IL-8 and MIP-1 alpha. The steady state levels of the mRNAs for these cytokines were also analysed in the cells. The adherence of BAL cells to plastic prior to exposure to the gas elevated the steady state mRNA levels of all four cytokines tested in smoker's cells and that of TNF-alpha and IL-1 beta, but not IL-8 (MIP-1 alpha not tested), in non-smoker's cells. Interestingly, adherent cells from non-smokers released circa 15-, 3-, 1.5- and 3-fold the amounts of IL-1 beta, IL-8, TNF-alpha and MIP-1 alpha, respectively, than smoker's cells during control incubation or exposure to air. A 20 min exposure to NO2 (5 or 20 p.p.m.) did not increase the secretion of any of the cytokines from either cell type. In contrast, NO2 caused a concentration-dependent inhibition of the secretion of all cytokines except IL-1 beta from smoker's cells. Additionally, NO2 greatly diminished the release of all cytokines in response to further treatment with lipopolysaccharide (LPS). In contrast, only the secretion of TNF-alpha from non-smoker's cells was inhibited by the gas in a concentration-dependent manner, whilst LPS-induced secretion of the cytokines was not affected by the gas. The steady state levels of the respective mRNAs for each of the cytokines were not significantly affected in smoker's cells by exposure to NO2, except for a negative, dose-dependent trend in the case of TNF-alpha. Nitrogen dioxide also failed to elevate the levels of the mRNAs in non-smoker's cells but, again, tended to diminish the levels, particularly of IL-1 beta mRNA. However, exposure to the gas inhibited LPS-induced accumulation of cytokine mRNAs in smoker's cells only. The data suggest that macrophage-derived cytokine mediators of the sepsis response may not play a role in the generation of NO2-induced inflammation in the human lung. Conversely, the gas seems to non-specifically inhibit the release and/or production of cytokines, particularly from smoker's cells, at the post-transcriptional level, and impairs the ability of the cells to increase the transcription and release of the cytokines in response to bacterial LPS. The fact that NO2 seriously impaired the already diminished capacity of smoker's cells to release several important pro-inflammatory cytokines, both under control conditions and in response to LPS, strongly suggest that the inhalation of NO2 in cigarette smoke may contribute to impairing host defence against infection in the lung.

Adult↗

PubMed1996

Purification and characterization of two isoenzymes of DL-glycerol-3-phosphatase from Saccharomyces cerevisiae. Identification of the corresponding GPP1 and GPP2 genes and evidence for osmotic regulation of Gpp2p expression by the osmosensing mitogen-activated protein kinase signal transduction pathway.

The existence of specific dl-glycerol-3-phosphatase (EC 3.1.3.21) activity in extracts of Saccharomyces cerevisiae was confirmed by examining strains lacking nonspecific acid and alkaline phosphatase activities. During purification of the glycerol-3-phosphatase, two isozymes having very similar molecular weights were isolated by gel filtration and anion exchange chromatography. By microsequencing of trypsin-generated peptides the corresponding genes were identified as previously sequenced open reading frames of unknown function. The two genes, GPP1 (YIL053W) and GPP2 (YER062C) encode proteins that show 95% amino acid identity and have molecular masses of 30.4 and 27.8 kDa, respectively. The intracellular concentration of Gpp2p increases in cells subjected to osmotic stress, while the production of Gpp1p is unaffected by changes of external osmolarity. Both isoforms have a high specificity for dl-glycerol-3-phosphate, pH optima at 6.5, and KmG3P in the range of 3-4 mM. The osmotic induction of Gpp2p is blocked in cells that are defective in the HOG-mitogen-activated protein kinase pathway, indicating that GPP2 is a target gene for this osmosensing signal transduction pathway. Together with DOG1 and DOG2, encoding two highly homologous enzymes that dephosphorylate 2-deoxyglucose-6-phosphate, GPP1 and GPP2 constitute a new family of genes for low molecular weight phosphatases.

Amino Acid Sequence↗

PubMed1996

Protein expression during exponential growth in 0.7 M NaCl medium of Saccharomyces cerevisiae.

Saccharomyces cerevisiae exponentially growing in basic or 0.7 M NaCl medium were isotopically labelled with 35S-methionine, followed by protein separation and quantification by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with computerised image analysis. The electrophoretic separation resolved about 650 proteins of which 13 displayed significant and at least 2-fold changes in rate of synthesis during saline growth. By sequencing of 2D-PAGE resolved proteins, one of the 8 induced spot, p42.9/5.5, was shown to correspond to the full length (containing the N-terminal extension) product of the GPD1 gene encoding the cytoplasmic glycerol 3-phosphate dehydrogenase. The expression of the TDH3 gene, glyceraldehyde 3-phosphate dehydrogenase, and the ENO2 gene, enolase, decreased during growth in NaCl medium, declines hypothesised to have an impact on the flux to glycerol.

Amino Acid Sequence↗

PubMed1996

Antioxidant kinetics in lung lavage fluid following exposure of humans to nitrogen dioxide.

To determine if nitrogen dioxide (NO2), a gaseous free radical, modifies the protective antioxidant pool present in respiratory tract lining fluids, a random, double-blind study utilizing flexible fiberoptic bronchoscopy with bronchial and bronchoalveolar lavage was performed. Healthy, nonsmoking, asymptomatic subjects were exposed to filtered air and 2 ppm NO2 for 4 h on separate occasions. To examine the kinetics of the NO2-induced antioxidant reactions, 44 subjects were randomly assigned to one of three groups. Bronchoscopy was performed 1.5 h (group 1), 6 h (group 2) or 24 h (group 3) after each exposure. Reduced glutathione (GSH), uric acid, and ascorbic acid concentrations were determined in both bronchial and bronchoalveolar lavage fluid fractions. In addition, bronchoalveolar lavage fluid was screened for malondialdehyde as a marker of lipid peroxidation. Exposure to NO2 resulted in a rapid (1.5 h) loss of uric acid from the bronchial region, however by 6 h after exposure it had increased significantly above control uric acid concentration in this region. At 24 h after exposure, uric acid concentration had returned to the control level. A similar response of uric acid to NO2 was seen in the bronchoalveolar region. Ascorbic acid was also decreased in bronchial and bronchoalveolar lavage fluids 1.5 h after exposure to NO2, but returned to control values by 6 h. In marked contrast, significant increases in GSH concentration were seen at 1.5 and 6 h in bronchial lavage fluid after exposure to NO2, which subsequently returned to control levels by 24 h. No change in bronchoalveolar lavage fluid GSH concentration or malondialdehyde content was seen after NO2 exposure. These data support the view that antioxidants present in lung fluids react with, and hence modulate the impact of, NO2 on the lung.

Adult↗

PubMed1995

Gene linkage of two-dimensional polyacrylamide gel electrophoresis resolved proteins from isogene families in Saccharomyces cerevisiae by microsequencing of in-gel trypsin generated peptides.

The total cellular extract of proteins from the yeast Saccharomyces cerevisiae was resolved by preparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) where 500 micrograms was loaded per gel, and a number of proteins in isogene families were selected for microsequencing analysis. Peptides were generated from resolved proteins by in-gel trypsin digestion, and fractionated by reversed phase-high performance liquid chromatography (RP-HPLC). Subsequent sequencing of peptides yielded internal amino acid sequences which unambiguously identified the selected proteins spots as gene products from PCD1, ENO1, ENO2, ADH1, HXK2, TDH2, TDH3, SSB1 and SSB2. The chromatograms obtained from RP-HPLC of related proteins were utilized to distinguish discriminating peptide fractions. With this approach two out of four amino acid differences between Ssb1p and Ssb2p were allocated. We estimate that by pooling five preparative gels, at least one hundred protein spots in the 2-D pattern of S. cerevisiae will be obtained in sequencable amounts.

Amino Acid Sequence↗

PubMed1995

Interlaboratory reproducibility of yeast protein patterns analyzed by immobilized pH gradient two-dimensional gel electrophoresis.

An interlaboratory comparison was conducted on the positional and quantitative reproducibility of yeast proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) using isoelectric focusing with immobilized pH gradient (pH 4-7) in the first dimension. The basic experimental set-up was as follows: one laboratory prepared and distributed a [35S]methionine-labeled total yeast protein extract (Göteborg, Sweden), another laboratory prepared the IPG strips to be used by all labs in this study (Munich, Germany), the third laboratory (Aarhus, Denmark) circulated the protocols and coordinated the modest attempts to unify them. Samples were run horizontally in the first dimension and vertically in the second. The gels were sent to Göteborg for processing by phosphoimager technology and computerized image analysis (PDQuest), and the 2-D PAGE resolved proteins were located and quantified automatically. A subset of 470 spots was manually matched in all gels out of an average of 1328 resolved proteins. The positional interlaboratory comparison revealed great pattern reproducibility, the correlation coefficient in no case being less than 0.9994. In absolute terms an average deviation of 2.8 mm (x-position) and 1.8 mm (y-position) were obtained for all nine gels (three gels per lab). The interlaboratory comparison of protein quantitation displayed higher variability, and the best correlation coefficient generated was 0.975. An average standard deviation of 34.5% was calculated for protein quantitation including all three labs, a value slightly higher than the intralaboratory variation (range 20-28%). Thus, despite differences in protocols, chemicals and equipment, the immobilized pH gradient technology gave extremely high positional and quantitative reproducibility. This will greatly facilitate the exchange of data and the establishment of multi-user image-based 2-D gel databases.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1995

Cloning and characterization of GPD2, a second gene encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae, and its comparison with GPD1.

We have cloned and characterized a homologue of the previously isolated GPD1 gene, encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae. This second gene, called GPD2, encodes a protein of 384 amino acids that shares 69% sequence identity with GPD1. Like GPD1 it has an amino-terminal extension of unknown function. GPD2 is located on chromosome VII and cross-hybridizes with GPD1 at chromosome IV as well as with an unknown homologue at chromosome XV. Disruption of the GPD2 gene did not reveal any observable phenotypic effects, whereas overexpression resulted in a slight, but significant, increase of GPD enzyme activity in wild-type cells. Analysis of gene transcription by a CAT-reporter gene fused to the GPD promoters revealed decreased transcriptional activity of the GPD2 promoter in cells grown on nonfermentable as opposed to fermentable carbon sources, and no induction in cells exposed to high osmolarity or heat shock. Similar analysis of GPD1 demonstrated an 8-17-fold higher basal level of transcription compared to GPD2. Furthermore, such analysis revealed that the GPD1 promoter was induced by increased osmolarity essentially independent of the type of stress solute used, the level of GPD1 transcription being increased about sevenfold in cells growing at 1.4 M NaCl.

Amino Acid Sequence↗

PubMed1995

Global changes in protein synthesis during adaptation of the yeast Saccharomyces cerevisiae to 0.7 M NaCl.

Exponentially growing Saccharomyces cerevisiae was challenged to increased salinity by transfer to 0.7 M NaCl medium, and changes in protein synthesis were examined during the 1st h of adaptation by use of two-dimensional gel electrophoresis coupled to computerized quantification. An impressive number of proteins displayed changes in the relative rate of synthesis, with most differences from nonstressed cells being found at between 20 and 40 min. During this period, 18 proteins exhibited more than eightfold increases in their rates of synthesis and were classified as highly NaCl responsive. Only two proteins were repressed to the same level. Most of these highly NaCl-responsive proteins seemed to constitute gene products not earlier reported to respond to dehydration. Applying a selection criterion to subsequent samples of a twofold change in the relative rate of synthesis, 14 different regulatory patterns were discerned. Most identified glycolytic enzymes exhibited a delayed response, and their rates of synthesis did not change until the middle phase of adaptation, with only a minor decrease in the rate of production. A slight salt-stimulated response was observed for some members of the HSP70 gene family. Overall, the data presented indicate complex intracellular signalling as well as involvement of diverse regulatory mechanisms during the period of adaptation to NaCl.

Adaptation, Physiological↗

PubMed1995

Growth of Escherichia coli K88 in piglet ileal mucus: protein expression as an indicator of type of metabolism.

The physiological and molecular responses of enterotoxigenic Escherichia coli K88 strain Bd 1107/7508 during growth in piglet ileal mucus and lipids extracted from mucus were studied in terms of growth rate, protein expression, and rate of heat production. E. coli K88 multiplied at maximum speed in mucus and in lipids extracted from mucus. By two-dimensional gel electrophoresis of [35S]methionine-labelled cells, it was demonstrated that the synthesis of a subclass of 13 proteins was changed at least fourfold during exponential growth in mucus compared with growth in M9 minimal medium. Ten of these proteins were repressed, while three were induced, and one of the induced proteins was identified as heat shock protein GroEL. Furthermore, two-dimensional analysis of E. coli K88 cells grown on lipids extracted from mucus revealed a set of lipid utilization-associated proteins. None of these was induced fourfold during exponential growth in mucus. Microcalorimetric measurements (monitoring the rate of heat production) of E. coli K88 grown in mucus indicated metabolic shifts in the stationary phase, in which five of the lipid utilization-associated proteins were expressed at a higher level. An isogenic E. coli K88 fadAB mutant deficient in fatty acid degradation genes grew as well as the wild type on mucus and mucus lipids. The heat production rate curve of the mutant grown in mucus differed from that of the wild type only during the stationary phase. From these results it was concluded that protein expression is influenced when E. coli K88 is grown in piglet ileal mucus rather than in M9 minimal medium. Lipids extracted from ileal mucus can serve as a substrate for E. coli K88 but appear not to be utilized during exponential growth in mucus. Stationary-phase cells metabolize fatty acids; however, the functional purpose of this is unclear.

Anaerobiosis↗

PubMed1995

Nitrogen dioxide exposure impairs the frequency of the mucociliary activity in healthy subjects.

In previous studies, we have reported bronchoalveolar lavage (BAL) findings following single and repeated exposures to NO2. Here, we present the first data on measurements of the frequency of the mucociliary activity in healthy subjects following exposure to NO2. Twenty four healthy nonsmoking volunteers underwent fibreoptic bronchoscopy, during which mucociliary activity was measured using an in vivo method based on a two-way fibreoptic system, to obtain baseline data. Two weeks later, the subjects were reinvestigated following NO2 exposure. Eight volunteers were investigated 45 min after a 20 min exposure to 1.5 ppm NO2 (Group A); eight subjects 45 min after a 20 min exposure to 3.5 ppm NO2 (Group B); and eight subjects 24 h after a 4 h exposure to 3.5 ppm NO2 (Group C). No activity was detected in Group A or B 45 min after exposure, in contrast to the easily identifiable mucociliary activity waves at the reference bronchoscopy. In group C, a significant elevation of the frequency of the mucociliary activity waves was seen at 24 h. It is concluded that short-term exposure to NO2 in man produces a significant reduction in the mucociliary activity 45 min after exposure. The effect ceases within 24 h. Further studies are needed to evaluate the effects of repeated exposures with NO2 on mucociliary activity in man.

Adult↗