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Biomedical subjects

A Blomberg

Publications and source records attributed to A Blomberg.

At least 37 records · Page 2Linked to original sources
PubMed2000

Metabolic surprises in Saccharomyces cerevisiae during adaptation to saline conditions: questions, some answers and a model.

This review describes the metabolic alterations and adaptations of yeast cells in response to osmotic stress. The basic theme of the cellular response is known to be exclusion of the extracellular stress agent salt and intracellular accumulation of the compatible solute glycerol. Molecular details of these basic processes are currently rather well known. However, analysis of expression changes during adaptation to salt has revealed a number of metabolic surprises. These include the induced expression of genes involved in glycerol dissimilation as well as trehalose turnover. The physiological rationale for these responses to osmotic stress is discussed. A model is presented in which it is hypothesised that the two pathways function as glycolytic safety valves during adaptation to stress.

Adaptation, Physiological↗
PubMed2000

Airway inflammation following exposure to diesel exhaust: a study of time kinetics using induced sputum.

The adverse health effects of particulate matter pollution are of increasing concern. In a recent bronchoscopic study in healthy volunteers, pronounced airway inflammation was detected following exposure to diesel exhaust (DE). The present study was conducted in order to evaluate the time kinetics of the inflammatory response following exposure to DE using induced sputum from healthy volunteers. Fifteen healthy nonsmoking volunteers were exposed to DE particles with a 50% cut-off aerodynamic diameter of 10 microm 300 microg x m(-3) and air for 1 h on two separate occasions. Sputum induction with hypertonic saline was performed 6 and 24 h after each exposure. Analyses of sputum differential cell counts and soluble protein concentrations were performed. Six hours after exposure to DE, a significant increase was found in the percentage of sputum neutrophils (37.7 versus 26.2% p=0.002) together with increases in the concentrations of interleukin-6 (12.0 versus 6.3 pg x mL(-1), p=0.006) and methylhistamine (0.11 versus 0.12 microg x L(-1), p=0.024). Irrespective of exposure, a significant increase was found in the percentage of sputum neutrophils at 24 as compared to 6 h, indicating that the procedure of sputum induction itself may change the composition of sputum. This study demonstrates that exposure to diesel exhaust induces inflammatory response in healthy human airways, represented by an early increase in interleukin-6 and methylhistamine concentration and the percentage of neutrophils. Induced sputum provides a safe tool for the investigation of the inflammatory effects of diesel exhaust, but care must be taken when interpreting results from repeated sputum inductions.

Adult↗
PubMed2000

Acute exposure to diesel exhaust increases IL-8 and GRO-alpha production in healthy human airways.

We have previously demonstrated that short-term exposure to diesel exhaust (DE) for 1 h induced a marked leukocytic infiltration in the airways of healthy human volunteers involving neutrophils, lymphocytes, and mast cells along with increases in several inflammatory mediators. We hypothesized that the leukocyte infiltration and the various inflammatory responses induced by DE were mediated by enhanced chemokine and cytokine production by resident cells of the airway tissue and lumen. To investigate this, 15 healthy human volunteers were exposed to diluted DE and air on two separate occasions for 1 h each in an exposure chamber. Fiberoptic bronchoscopy was performed 6 h after each exposure to obtain endobronchial biopsies and bronchial wash (BW) cells. Using reverse transcriptase/polymerase chain reaction enzyme-linked immunosorbent assay (RT-PCR ELISA), a novel and sensitive technique to quantify relative amounts of cytokine mRNA gene transcripts, and immunohistochemical staining with computer-assisted image analysis to quantify expression of cytokine protein in the bronchial tissue, we have demonstrated that DE enhanced gene transcription of interleukin-8 (IL-8) in the bronchial tissue and BW cells along with increases in IL-8 and growth-regulated oncogene-alpha (GRO-alpha) protein expression in the bronchial epithelium, and an accompanying trend toward an increase in IL-5 mRNA gene transcripts in the bronchial tissue. There were no significant changes in the gene transcript levels of interleukin-1B (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interferon gamma (IFN-gamma), and granulocyte macrophage colony-stimulating factor (GM-CSF) either in the bronchial tissue or BW cells after DE exposure at this time point. These observations suggest an underlying mechanism for DE-induced airway leukocyte infiltration and offer a possible explanation for the association observed between ambient levels of particulate matter and various respiratory health outcome indices noted in epidemiological studies.

Bronchoalveolar Lavage Fluid↗
PubMed1999

Identification and specificities of N-terminal acetyltransferases from Saccharomyces cerevisiae.

N-terminal acetylation can occur cotranslationally on the initiator methionine residue or on the penultimate residue if the methionine is cleaved. We investigated the three N-terminal acetyltransferases (NATs), Ard1p/Nat1p, Nat3p and Mak3p. Ard1p and Mak3p are significantly related to each other by amino acid sequence, as is Nat3p, which was uncovered in this study using programming alignment procedures. Mutants deleted in any one of these NAT genes were viable, but some exhibited diminished mating efficiency and reduced growth at 37 degrees C, and on glycerol and NaCl-containing media. The three NATs had the following substrate specificities as determined in vivo by examining acetylation of 14 altered forms of iso-1-cytochrome c and 55 abundant normal proteins in each of the deleted strains: Ard1p/Nat1p, subclasses with Ser-, Ala-, Gly- and Thr-termini; Nat3p, Met-Glu- and Met-Asp- and a subclass of Met-Asn-termini; and Mak3p subclasses with Met-Ile- and Met-Leu-termini. In addition, a special subclass of substrates with Ser-Glu- Phe-, Ala-Glu-Phe- and Gly-Glu-Phe-termini required all three NATs for acetylation.

Acetylation↗
PubMed1999

Altered lung antioxidant status in patients with mild asthma.

Lung lining fluid ascorbic acid (vitamin C) and alpha-tocopherol (vitamin E) concentrations are low in patients with mild asthma even though blood levels are normal or increased. These findings, along with the presence of increased amounts of oxidised glutathione in their airways, indicate that patients with asthma are subject to increased oxidative stress.

Antioxidants↗
PubMed1999

Quantitative aspects of the use of bacterial chloramphenicol acetyltransferase as a reporter system in the yeast Saccharomyces cerevisiae.

The quantitative aspects of the use of the reporter system chloramphenicol acetyltransferase (CAT) has been evaluated in the yeast Saccharomyces cerevisiae. It was found that the CAT activity measured with a radiosotopic fluor diffusion assay was strongly dependent on the amount of yeast extract applied, both when CAT was expressed endogenously and when a purified Escherichia coli enzyme was investigated. Desalting the yeast extract by gel filtration partly eliminated the problem, indicating that some low-molecular-weight compound was involved in the phenomenon. However, the extract still exhibited stability problems on ice. An immunological CAT assay was tested and found to yield satisfactory quantitative result.

Chloramphenicol O-Acetyltransferase↗
PubMed1999

Thiamine repression and pyruvate decarboxylase autoregulation independently control the expression of the Saccharomyces cerevisiae PDC5 gene.

The Saccharomyces cerevisiae gene PDC5 encodes the minor isoform of pyruvate decarboxylase (Pdc). In this work we show that expression of PDC5 but not that of PDC1, which encodes the major isoform, is repressed by thiamine. Hence, under thiamine limitation both PDC1 and PDC5 are expressed. PDC5 also becomes strongly expressed in a pdc1delta mutant. Two-dimensional gel electrophoresis of whole protein extracts shows that thiamine limitation stimulates the production of THI gene products and of Pdc5p. Deletion of PDC1 only stimulates production of Pdc5p. We conclude that the stimulation of PDC5 expression in a pdc1delta mutant is not due to a response to thiamine limitation.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1999

Characterization of Saccharomyces cerevisiae CBS 7764 isolated from rainbow trout intestine.

A wild-type Saccharomyces cerevisiae, strain CBS 7764, isolated from the intestine of rainbow trout, was analyzed with respect to general growth parameters and global protein expression. Characterization of this strain was of interest since previous data show non-typical S. cerevisiae cell surface properties and because data suggest a probiotic potential of CBS 7764. The heat production rate (dQ/dt), monitored by microcalorimetry, showed that the typical growth phases resulting from diauxic growth on glucose were present in the fish isolate. However, CBS 7764 differentiated from a reference strain by becoming limited in the respiratory phase as demonstrated by a plateau in the dQ/dt signal. The global protein expression, as studied by two-dimensional gel electrophoresis (2D-PAGE), revealed a large degree of resemblance of the fish isolate to the reference strain, however, also clear qualitative and quantitative expression differences were detected; e.g. 14% of the proteins differed in expression level by a factor of at least 2. In addition, the fish isolate expressed 12 unique proteins. The heat shock proteins, which for other organisms have been identified as important in mucosal colonization, were generally expressed to a higher level in CBS 7764.

Animals↗
PubMed1999

Bronchoalveolar inflammation after exposure to diesel exhaust: comparison between unfiltered and particle trap filtered exhaust.

OBJECTIVES: Air pollution particulates have been identified as having adverse effects on respiratory health. The present study was undertaken to further clarify the effects of diesel exhaust on bronchoalveolar cells and soluble components in normal healthy subjects. The study was also designed to evaluate whether a ceramic particle trap at the end of the tail pipe, from an idling engine, would reduce indices of airway inflammation. METHODS: The study comprised three exposures in all 10 healthy never smoking subjects; air, diluted diesel exhaust, and diluted diesel exhaust filtered with a ceramic particle trap. The exposures were given for 1 hour in randomised order about 3 weeks apart. The diesel exhaust exposure apperatus has previously been carefully developed and evaluated. Bronchoalveolar lavage was performed 24 hours after exposures and the lavage fluids from the bronchial and bronchoalveolar region were analysed for cells and soluble components. RESULTS: The particle trap reduced the mean steady state number of particles by 50%, but the concentrations of the other measured compounds were almost unchanged. It was found that diesel exhaust caused an increase in neutrophils in airway lavage, together with an adverse influence on the phagocytosis by alveolar macrophages in vitro. Furthermore, the diesel exhaust was found to be able to induce a migration of alveolar macrophages into the airspaces, together with reduction in CD3+CD25+ cells. (CD = cluster of differentiation) The use of the specific ceramic particle trap at the end of the tail pipe was not sufficient to completely abolish these effects when interacting with the exhaust from an idling vehicle. CONCLUSIONS: The current study showed that exposure to diesel exhaust may induce neutrophil and alveolar macrophage recruitment into the airways and suppress alveolar macrophage function. The particle trap did not cause significant reduction of effects induced by diesel exhaust compared with unfiltered diesel exhaust. Further studies are warranted to evaluate more efficient treatment devices to reduce adverse reactions to diesel exhaust in the airways.

Adult↗
PubMed1999

Persistent airway inflammation but accommodated antioxidant and lung function responses after repeated daily exposure to nitrogen dioxide.

Nitrogen dioxide (NO2) is a common indoor and outdoor air pollutant that may induce deterioration of respiratory health. In this study the effects of repeated daily exposure to NO2 on airway antioxidant status, inflammatory cell and mediator responses, and lung function were examined. Healthy nonsmoking subjects were exposed under controlled conditions to air (once) and to 2 ppm of NO2 for 4 h on four consecutive days. Lung function measurements were made before and immediately after the end of each exposure. Bronchoscopy with endobronchial biopsies, bronchial wash (BW), and bronchoalveolar lavage (BAL) was carried out 1.5 h after the air exposure and after the last exposure to NO2. Repeated NO2 exposure resulted in a decrease in neutrophil numbers in the bronchial epithelium. The BW revealed a twofold increase in content of neutrophils (p < 0.05) and a 1.5-fold increase in myeloperoxidase (MPO) (p < 0.01) indicative of both migration and activation of neutrophils in the airways. After the fourth NO2 exposure, antioxidant status of the airway fluid was unchanged. Significant decrements in FEV1 and FVC were found after the first exposure to NO2, but these attenuated with repeated exposures. Together, these data indicate that four sequential exposures to NO2 result in a persistent neutrophilic inflammation in the airways, whereas changes in pulmonary function and airway antioxidants are resolved. We conclude that NO2 is a proinflammatory air pollutant under conditions of repeated exposure.

Adult↗
PubMed1999

Acute inflammatory responses in the airways and peripheral blood after short-term exposure to diesel exhaust in healthy human volunteers.

Several epidemiologic studies have demonstrated a consistent association between levels of particulate matter (PM) in the ambient air with increases in cardiovascular and respiratory mortality and morbidity. Diesel exhaust (DE), in addition to generating other pollutants, is a major contributor to PM pollution in most places in the world. Although the epidemiologic evidence is strong, there are as yet no established biological mechanisms to explain the toxicity of PM in humans. To determine the impact of DE on human airways, we exposed 15 healthy human volunteers to air and diluted DE under controlled conditions for 1 h with intermittent exercise. Lung functions were measured before and after each exposure. Blood sampling and bronchoscopy were performed 6 h after each exposure to obtain airway lavages and endobronchial biopsies. While standard lung function measures did not change following DE exposure, there was a significant increase in neutrophils and B lymphocytes in airway lavage, along with increases in histamine and fibronectin. The bronchial biopsies obtained 6 h after DE exposure showed a significant increase in neutrophils, mast cells, CD4+ and CD8+ T lymphocytes along with upregulation of the endothelial adhesion molecules ICAM-1 and VCAM-1, with increases in the numbers of LFA-1+ cells in the bronchial tissue. Significant increases in neutrophils and platelets were observed in peripheral blood following DE exposure. This study demonstrates that at high ambient concentrations, acute short-term DE exposure produces a well-defined and marked systemic and pulmonary inflammatory response in healthy human volunteers, which is underestimated by standard lung function measurements.

Adult↗
PubMed1999

Interleukin-5 production by human airway epithelial cells.

Interleukin (IL)-5 is a pleiotropic cytokine that exhibits biologic activity on cells of diverse hemopoieitic lineages. IL-5 enhances mediator release from human basophils and plays a pivotal role in the chemoattraction, proliferation, differentiation, survival, and activation of eosinophils. Th2- and Tc2-like T cells, mast cells, basophils, and eosinophils are the known cellular sources of this cytokine. Using a sensitive and novel reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay system, we found that IL-5 messenger RNA (mRNA) was constitutively expressed in bronchial biopsies obtained from healthy individuals, and that the levels of IL-5 mRNA expression decreased 1. 5 h after exposure to 0.12 ppm ozone for 2 h. Because the oxidative effects of ozone are confined to the epithelial cell surface and it is known that ozone induces epithelial damage and shedding, we hypothesized that epithelial cells might be a source of IL-5 mRNA. We demonstrate here that both transformed human bronchial epithelial cell lines (A549 and 16HBE14o-) and primary human bronchial and nasal epithelial cells grown in culture constitutively express IL-5 mRNA, which is upregulated on stimulation with tumor necrosis factor (TNF)-alpha. Culture supernatants derived from A549 cells exposed to TNF-alpha and interferon-gamma demonstrated detectable levels of IL-5 protein, and immunostaining of bronchial biopsies obtained from healthy human airways revealed the presence of IL-5 protein localized to the bronchial epithelium. To our knowledge, this is the first report demonstrating IL-5 production by human airway epithelial cells. This observation provides further evidence for the role of airway epithelium in regulating airway immune responses, in particular enhancing chemotaxis, activation, and survival of eosinophils, which could play an important role in the pathogenesis of bronchial asthma.

Adult↗
PubMed1999

Ozone-induced lung function decrements do not correlate with early airway inflammatory or antioxidant responses.

This study sought to clarify the early events occurring within the airways of healthy human subjects performing moderate intermittent exercise following ozone challenge. Thirteen healthy nonsmoking subjects were exposed in a single blinded, crossover control fashion to 0.2 parts per million (ppm) O3 and filtered air for 2 h, using a standard intermittent exercise and rest protocol. Lung function was assessed pre- and immediately post-exposure. Bronchoscopy was performed with endobronchial mucosal biopsies, bronchial wash (BW) and bronchoalveolar lavage (BAL) 1.5 h after the end of the exposure period. Respiratory tract lining fluid (RTLF) redox status was assessed by measuring a range of antioxidants and oxidative damage markers in BW and BAL fluid samples. There was a significant upregulation after O3 exposure in the expression of vascular endothelial P-selectin (p<0.005) and intercellular adhesion molecule-1 (p<0.005). This was associated with a 2-fold increase in submucosal mast cells (p<0.005) in biopsy samples, without evidence of neutrophilic inflammation, and a decrease in BAL fluid macrophage numbers (1.6-fold, p<0.005), with an activation of the remaining macrophage subset (2.5-fold increase in % human leukocyte antigen (HLA)-DR+ cells, p<0.005). In addition, exposure led to a 4.5-fold and 3.1-fold increase of reduced glutathione (GSH) concentrations, in BW and BAL fluid respectively (p<0.05), with alterations in urate and alpha-tocopherol plasma/RTLF partitioning ratios (p<0.05). Spirometry showed reductions in forced vital capacity (p<0.05) and forced expiratory volume in one second (p<0.01), with evidence of small airway narrowing using forced expiratory flow values (p<0.005). Evidence was found of O3-induced early adhesion molecule upregulation, increased submucosal mast cell numbers and alterations to the respiratory tract lining fluid redox status. No clear relationship was demonstrable between changes in these early markers and the lung function decrements observed. The results therefore indicate that the initial lung function decrements are not predictive of, or causally related to the O3-induced inflammatory events in normal human subjects.

Adult↗
PubMed1999

Antioxidant consumption and repletion kinetics in nasal lavage fluid following exposure of healthy human volunteers to ozone.

To obtain information on the real-time events occurring within human respiratory tract lining fluids (RTLFs) during ozone exposure, sequential nasal lavage was performed on 13 human volunteers exposed on separate occasions to 0.2 parts per million O3 and filtered air (2-h exposures, with intermittent exercise). Nasal lavage was performed and blood samples obtained at four time points throughout each exposure: pre-exposure (Pre-E), 1 h into exposure (1h-E), immediately post-exposure (0h-PE) and 1 h post-exposure (1h-PE). Endobronchial mucosal biopsies were obtained at 1.5 h-post exposure (1.5h-PE). Nasal RTLF neutrophilia was not apparent during, or 1.5 h after, 03 exposure. Furthermore, activation of the pre-existing neutrophil population did not occur. Airway permeability was not altered by this 03 exposure regimen. Sequential lavage resulted in significant washout of RTLF ascorbic acid, reduced glutathione, extracellular superoxide dismutase and myeloperoxidase at 1h-E, 0h-PE and 1.5h-PE relative to baseline Pre-E values. In contrast, RTLF uric acid (UA), total protein and albumin concentrations did not display washout kinetics. Of the antioxidants examined, only UA was clearly depleted by 03, concentrations, falling by 6.22 micromol x L(-1) at 1h-E, compared with 1.61 micromol x L(-1) (p<0.01) during control air exposure. The establishment of a new pseudo-steady-state concentration of RTLF UA (70% of Pre-E values) during the second hour of O3 exposure was coincident with a small but significant increase in plasma UA concentration (19.27 (O3) versus 1.95 micromol x L(-1) (air), p<0.05). These data demonstrate that inhalation of 0.2 parts per million 03 results in the depletion of nasal respiratory tract lining fluid uric acid and that this regional loss of uric acid leads to a small increase in plasma uric acid concentration. Whilst the reaction of uric acid with inspired 03 may confer protection locally, the role of upper airway uric acid as a sink for inhaled O3 is not supported by these findings.

Adult↗
PubMed1998

Amino acid uptake is strongly affected during exponential growth of Saccharomyces cerevisiae in 0.7 M NaCl medium.

Labelling of Saccharomyces cerevisiae grown in 0.7 M NaCl with 35S-methionine revealed a 5-6 fold lowering of the methionine incorporation into protein, which could not be attributed solely to the approximately 50% longer generation time of cells grown in 0.7 M NaCl. Subsequent studies of the high affinity methionine uptake system showed a strongly reduced uptake of methionine during growth in 0.7 M NaCl medium. This reduced uptake was shown to be strain-independent and caused mainly by an approximately 20-fold lowered maximum velocity (Vmax) of the transport system, while the substrate affinity (Km) displayed only a minor change. A salt-instigated reduction of uptake was furthermore demonstrated for the leucine and histidine high affinity uptake systems and also for a mixture of 15 different amino acids. We therefore suggest that the reduced amino acid uptake is a general phenomenon observed in salt-grown cells.

Amino Acids↗
PubMed1998

Nasal cavity lining fluid ascorbic acid concentration increases in healthy human volunteers following short term exposure to diesel exhaust.

To determine if diesel exhaust (DE) exposure modifies the antioxidant defense network within the respiratory tract lining fluids, a randomized, single blinded, crossover control study using nasal lavage and flexible video bronchoscopy with bronchial and bronchoalveolar lavage was performed. Fifteen healthy, non-smoking, asymptomatic subjects were exposed to filtered air or diluted diesel exhaust (300mg m(-3) particulates, 1.6ppm nitrogen dioxide) for one hour on 2 separate occasions, at least three weeks apart. To examine the kinetics of any DE-induced antioxidant reactions, nasal lavage fluid and blood samples were collected prior to, immediately after, and 5 1/2 hours post exposure. Bronchoscopy was performed 6 hours after the end of DE exposure. Ascorbic acid, uric acid and reduced glutathione (GSH) concentrations were determined in nasal, bronchial, bronchoalveolar lavage and plasma samples. Malondialdehyde (MDA) and protein carbonyl concentrations were determined in plasma and bronchoalveolar lavage samples. Nasal lavage ascorbic acid concentration increased 10-fold during DE exposure [1.02 (0.26-2.09) Vs 7.13 (4.66-10.79) micromol/L(-1)], but returned to basal levels 5.5 hours post-exposure [0.75 (0.26-1.51) micromol/L(-1)]. There was no significant effect of DE exposure on nasal lavage uric acid or GSH concentration. DE exposure did not influence plasma, bronchial wash, or bronchoalveolar lavage antioxidant concentrations and no change in MDA or protein carbonyl concentrations were found. The physiological response to acute DE exposure is an increase in the level of ascorbic acid in the nasal cavity. This response appears to be sufficient to prevent further oxidant stress in the respiratory tract of normal individuals.

Adult↗
PubMed1997

Metabolic and regulatory changes associated with growth of Saccharomyces cerevisiae in 1.4 M NaCl. Evidence for osmotic induction of glycerol dissimilation via the dihydroxyacetone pathway.

The salt-instigated protein expression of Saccharomyces cerevisiae during growth in either 0.7 or 1.4 M NaCl was studied by two-dimensional polyacrylamide gel electrophoresis. The 73 protein spots that were identified as more than 3-fold responsive in 1.4 M NaCl were further grouped by response class (halometric, low-salt, and high-salt regulation). Roughly 40% of these responsive proteins were found to decrease in expression, while at higher magnitudes of change (>8-fold) only induction was recorded. Enolase 1 (Eno1p) was the most increasing protein by absolute numbers per cell, but not by -fold change, and the enzymes involved in glycerol synthesis, Gpd1p and Gpp2p, were also induced to a similar degree as Eno1p. We furthermore present evidence for salt induction of glycerol dissimilation via dihydroxyacetone and also identify genes putatively encoding the two enzymes involved; dihydroxyacetone kinase (DAK1 and DAK2) and glycerol dehydrogenase (YPR1 and GCY1). The GPD1, GPP2, GCY1, DAK1, and ENO1 genes all displayed a halometric increase in the amount of transcript. This increase was closely linked to the salt-induced rate of protein synthesis of the corresponding proteins, indicating mainly transcriptional regulation of expression for these genes. A consensus element with homology to the URS sequence of the ENO1 promoter was found in the promoters of the GPD1, GPP2, GCY1, and DAK1 genes.

Amino Acid Sequence↗