Biomedical subjects

Anders Blomberg

Publications and source records attributed to Anders Blomberg.

At least 19 recordsLinked to original sources

PubMed2006

Ozone enhances the airway inflammation initiated by diesel exhaust.

Exposure to air pollution is associated with adverse health effects, with particulate matter (PM) and ozone (O(3)) both indicated to be of considerable importance. Diesel engine exhaust (DE) and O(3) generate substantial inflammatory effects in the airways. However, as yet it has not been determined whether a subsequent O(3) exposure would add to the diesel-induced airway inflammatory effects. Healthy subjects underwent two separate exposure series: A 1-h DE exposure at a PM-concentration of 300 microg/m(3), followed after 5h by a 2-h exposure to filtered air and 0.2 ppm O(3), respectively. Induced sputum was collected 18 h after the second exposure. A significant increase in the percentage of neutrophils (PMN) and concentration of myeloperoxidase (MPO) was seen in sputum post DE+O(3) vs. DE+air (p<0.05 and <0.05, respectively). Significant associations were observed between the responses in MPO concentration and total PMN cells (p=0.001), and also between MPO and matrix metalloproteinase-9 (MMP-9) (p<0.001). The significant increase of PMN and MPO after the DE+O(3) exposures, compared to DE+air, denotes an O(3)-induced magnification of the DE-induced inflammation. Furthermore, the correlation between responses in MPO and number of PMNs and MMP-9 illustrate that the PMNs are activated, resulting in a more potent inflammatory response. The present study indicates that O(3) exposure adds significantly to the inflammatory response that is established by diesel exhaust. This interaction between exposure to particulate pollution and O(3) in sequence should be taken into consideration when health effects of air pollution are considered.

Adult↗

PubMed2006

PROPHECY--a yeast phenome database, update 2006.

Connecting genotype to phenotype is fundamental in biomedical research and in our understanding of disease. Phenomics--the large-scale quantitative phenotypic analysis of genotypes on a genome-wide scale--connects automated data generation with the development of novel tools for phenotype data integration, mining and visualization. Our yeast phenomics database PROPHECY is available at http://prophecy.lundberg.gu.se. Via phenotyping of 984 heterozygous diploids for all essential genes the genotypes analysed and presented in PROPHECY have been extended and now include all genes in the yeast genome. Further, phenotypic data from gene overexpression of 574 membrane spanning proteins has recently been included. To facilitate the interpretation of quantitative phenotypic data we have developed a new phenotype display option, the Comparative Growth Curve Display, where growth curve differences for a large number of mutants compared with the wild type are easily revealed. In addition, PROPHECY now offers a more informative and intuitive first-sight display of its phenotypic data via its new summary page. We have also extended the arsenal of data analysis tools to include dynamic visualization of phenotypes along individual chromosomes. PROPHECY is an initiative to enhance the growing field of phenome bioinformatics.

Chromosomes, Fungal↗

PubMed2006

Evolutionary constraints on yeast protein size.

BACKGROUND: Despite a strong evolutionary pressure to reduce genome size, proteins vary in length over a surprisingly wide range also in very compact genomes. Here we investigated the evolutionary forces that act on protein size in the yeast Saccharomyces cerevisiae utilizing a system-wide bioinformatics approach. Data on yeast protein size was compared to global experimental data on protein expression, phenotypic pleiotropy, protein-protein interactions, protein evolutionary rate and biochemical classification. RESULTS: Comparing the experimentally determined abundance of individual proteins, highly expressed proteins were found to be consistently smaller than lowly expressed proteins, in accordance with the biosynthetic cost minimization hypothesis. Yeast proteins able to maintain a high expression level despite a large size tended to belong to a very distinct set of protein families, notably nuclear transport and translation initiation/elongation. Large proteins have significantly more protein-protein interactions than small proteins, suggesting that a requirement for multiple interaction domains may constitute a positive selective pressure for large protein size in yeast. The higher frequency of protein-protein interactions in large proteins was not accompanied by a higher phenotypic pleiotropy. Hence, the increase in interactions may not reflect an increase in function differentiation. Proteins of different sizes also evolved at similar rates. Finally, whereas the biological process involved was found to have little influence on protein size the biochemical activity exerted by the protein represented a dominant factor. More than one third of all biochemical activity classes were enriched in one or more size intervals. CONCLUSION: In yeast, there is an inverse relationship between protein size and protein expression such that highly expressed proteins tend to be of smaller size. Also, protein size is moderately affected by protein connectivity and strongly affected by biochemical activity. Phenotypic pleiotropy does not seem to affect protein size.

Amino Acid Sequence↗

PubMed2006

Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae.

Large-scale protein overexpression phenotype screens provide an important complement to the more common gene knockout screens. Here, we have targeted the so far poorly understood Saccharomyces cerevisiae membrane proteome and report growth phenotypes for a strain collection overexpressing approximately 600 C-terminally tagged integral membrane proteins grown both under normal and three different stress conditions. Although overexpression of most membrane proteins reduce the growth rate in synthetic defined medium, we identify a large number of proteins that, when overexpressed, confer specific resistance to various stress conditions. Our data suggest that regulation of glycosylphosphatidylinositol anchor biosynthesis and the Na(+)/K(+) homeostasis system constitute major downstream targets of the yeast PKA/RAS pathway and point to a possible connection between the early secretory pathway and the cells' response to oxidative stress. We also have quantified the expression levels for >550 membrane proteins, facilitating the choice of well expressing proteins for future functional and structural studies.

Caffeine↗

PubMed2006

Vitamin supplementation does not protect against symptoms in ozone-responsive subjects.

Vitamin supplements have been reported to reduce the magnitude of symptoms in subjects exposed to oxidant air pollution. To confirm whether supplementation with vitamins C and E could reduce lung function decrements, airway inflammation, and epithelial injury in subjects sensitive to ozone, a double-blinded, crossover control study was performed. Fourteen ozone-responsive subjects were randomly exposed to both air and ozone (0.2 ppm for 2 h) after 7 days of either placebo treatment or supplementation with vitamin C (500 mg/day) and E (100 mg/day). Lung function was assessed pre- and immediately postexposure and blood samples were taken at set intervals. Inflammatory, tissue injury, and antioxidant responses were examined in lavage fluid obtained by bronchoscopy 6 h postexposure. Exposure to ozone resulted in significant (P < 0.01) decrements in FEV1 with no protection observed following vitamin supplementation (-8.5%) versus placebo (-7.3%) treatment. Similarly, ozone-induced neutrophilia were of a similar magnitude after both treatments (P < 0.05). This lack of protection was observed despite elevated plasma vitamin C (+60.1%) and vitamin E (+51.4%) concentrations following supplementation, and increased vitamin C concentrations in the airways after supplementation following ozone exposure. These data do not therefore support the contention that acute ozone-induced symptoms can be attenuated through the use of dietary antioxidants in well-nourished individuals.

Air Pollutants↗

PubMed2006

Involvement of yeast YOL151W/GRE2 in ergosterol metabolism.

The Saccharomyces cerevisiae gene YOL151W/GRE2 is widely used as a model gene in studies on yeast regulatory responses to osmotic and oxidative stress. Nevertheless, information concerning the physiological role of this enzyme, a distant homologue of mammalian 3-beta-hydroxysteroid dehydrogenases, is scarce. Combining quantitative phenotypic profiling and protein expression analysis studies, we here report the involvement of yeast Gre2p in ergosterol metabolism. Growth was significantly and exclusively reduced in gre2Delta strains subjected to environmental stress straining the cell membrane. Furthermore, whereas no compensatory mechanisms were activated due to loss of Gre2p during growth in favourable conditions (synthetic defined media, no stress), a striking and highly specific induction of the ergosterol biosynthesis pathway, represented by the enzymes Erg10p, Erg19p and Erg6p, was observed in gre2Delta during growth in a stress condition in which lack of Gre2p significantly affects growth. Involvement of Gre2p in ergosterol metabolism was confirmed by application of an array of selective inhibitors of lipid biosynthesis, as gre2Delta displayed vastly impaired tolerance exclusively to agents targeting the ergosterol biosynthesis. The approach outlined here, combining broad-spectrum phenotypic profiling, expression analysis during conditions reducing the growth of the mutant and functional confirmation by application of highly selective inhibitors, may prove a valuable tool in gene functional analysis.

Brefeldin A↗

PubMed2006

Genetic pleiotropy in Saccharomyces cerevisiae quantified by high-resolution phenotypic profiling.

Genetic pleiotropy, the ability of a mutation in a single gene to give rise to multiple phenotypic outcomes, constitutes an important but incompletely understood biological phenomenon. We used a high-resolution and high-precision phenotypic profiling approach to quantify the fitness contribution of genes on the five smallest yeast chromosomes during different forms of environmental stress, selected to probe a wide diversity of physiological features. We found that the extent of pleiotropy is much higher than previously claimed; 17% of the yeast genes were pleiotropic whereof one-fifth were hyper-pleiotropic. Pleiotropic genes preferentially participate in functions related to determination of protein fate, cell growth and morphogenesis, signal transduction and transcription. Contrary to what has earlier been proposed we did not find experimental evidence for slower evolutionary rate of pleiotropic genes/proteins. We also refute the existence of phenotypic islands along chromosomes but report on a remarkable loss both of pleiotropy and of phenotypic penetrance towards chromosomal ends. Thus, the here reported features of pleiotropy both have implications on our understanding of evolutionary processes as well as the mechanisms underlying disease.

Genes, Fungal↗

PubMed2006

Dihydroxyacetone detoxification in Saccharomyces cerevisiae involves formaldehyde dissimilation.

To investigate Saccharomyces cerevisiae physiology during growth on the conditionally toxic triose dihydroxyacetone (DHA), protein expression was studied in strains overexpressing either of the two dihydroxyacetone kinase isogenes, DAK1 or DAK2, that grow well utilizing DHA as a carbon and energy source. DHA metabolism was found mostly similar to ethanol utilization, involving a strong component of glucose derepression, but also involved DHA-specific regulatory changes. A specific and strong (10- to 30-fold induction of formaldehyde dehydrogenase, Fdhlp, indicated activation of the formaldehyde dissimilation pathway in DHA medium. The importance of this pathway was further supported by impaired adaptation to DHA growth and DHA survival in a glutathione-dependent formaldehyde dehydrogenase (SFA1) deletion mutant. Glutathione synthase (GSH1) deletion led to decreased DHA survival in agreement with the glutathione cofactor requirement for the SFA1-encoded activity. DHA toxicity did, however, not solely appear related to formaldehyde accumulation, because SFA1 overexpression only enhanced formaldehyde but not DHA tolerance. In further agreement with a low DHA-to-formaldehyde flux, GSH supplements in the low microM range also fully suppressed the DHA sensitivity of a gsh1Delta strain. Under growth reduction on high (100 mM) DHA medium we report increased levels of advanced glycation end-product (AGE) formation on total protein. Under these high-DHA conditions expression of several stress-related proteins, e.g. a heat-shock protein (Hsp104p) and the oxidative stress indicator, alkyl hydroperoxide reductase (Ahp1p) was also found induced. However, hallmark determinants of oxidative stress tolerance (e.g. YAP1, SKN7, HYR1/GPX3 and SOD2) were redundant for DHA tolerance, thus indicating mechanisms of DHA toxicity largely independent of central oxidative stress defence mechanisms. We conclude that mechanisms for DHA growth and detoxification appear complex and that the evolutionary strive to minimize detrimental effects of this intracellular metabolite links to both formaldehyde and glutathione metabolism.

Aldehyde Oxidoreductases↗

PubMed2006

Physiological importance and identification of novel targets for the N-terminal acetyltransferase NatB.

The N-terminal acetyltransferase NatB in Saccharomyces cerevisiae consists of the catalytic subunit Nat3p and the associated subunit Mdm20p. We here extend our present knowledge about the physiological role of NatB by a combined proteomics and phenomics approach. We found that strains deleted for either NAT3 or MDM20 displayed different growth rates and morphologies in specific stress conditions, demonstrating that the two NatB subunits have partly individual functions. Earlier reported phenotypes of the nat3Delta strain have been associated with altered functionality of actin cables. However, we found that point mutants of tropomyosin that suppress the actin cable defect observed in nat3Delta only partially restores wild-type growth and morphology, indicating the existence of functionally important acetylations unrelated to actin cable function. Predicted NatB substrates were dramatically overrepresented in a distinct set of biological processes, mainly related to DNA processing and cell cycle progression. Three of these proteins, Cac2p, Pac10p, and Swc7p, were identified as true NatB substrates. To identify N-terminal acetylations potentially important for protein function, we performed a large-scale comparative phenotypic analysis including nat3Delta and strains deleted for the putative NatB substrates involved in cell cycle regulation and DNA processing. By this procedure we predicted functional importance of the N-terminal acetylation for 31 proteins.

Acetylation↗

PubMed2006

Exploring the time dependence of serum clara cell protein as a biomarker of pulmonary injury in humans.

We have previously demonstrated Clara cell protein (CC16) [secretoglobin 1A1] in serum to be a highly sensitive biomarker of altered lung epithelial permeability after ozone challenge. As a previous experimental study has indicated a diurnal variation in serum CC16 in humans, the aims of the present investigation were to confirm this observation and to attempt to model the diurnal variation in CC16 concentrations. In 18 healthy nonsmoking subjects, peripheral blood samples were drawn at six sampling points over a 15-h period and repeated twice within 3 to 4 weeks. A clear within-day variation was revealed in serum CC16 concentrations, falling significantly from baseline levels between the 11:30 am and 10:00 pm time points (p = 0.000). Furthermore, it was shown that this within-day variation was reproducible regardless of subject or day, enabling the diurnal variation in serum CC16 to be modeled and fitted a second-degree polynomial for the observed time span. In conclusion, the present data demonstrate a pronounced time-dependent diurnal variation in serum levels of CC16, which can be mathematically compensated for, when addressing the issue of an air pollution-induced effect on CC16 in field studies.

Adult↗

PubMed2005

Diesel exhaust inhalation causes vascular dysfunction and impaired endogenous fibrinolysis.

BACKGROUND: Although the mechanisms are unknown, it has been suggested that transient exposure to traffic-derived air pollution may be a trigger for acute myocardial infarction. The study aim was to investigate the effects of diesel exhaust inhalation on vascular and endothelial function in humans. METHODS AND RESULTS: In a double-blind, randomized, cross-over study, 30 healthy men were exposed to diluted diesel exhaust (300 microg/m3 particulate concentration) or air for 1 hour during intermittent exercise. Bilateral forearm blood flow and inflammatory factors were measured before and during unilateral intrabrachial bradykinin (100 to 1000 pmol/min), acetylcholine (5 to 20 microg/min), sodium nitroprusside (2 to 8 microg/min), and verapamil (10 to 100 microg/min) infusions 2 and 6 hours after exposure. There were no differences in resting forearm blood flow or inflammatory markers after exposure to diesel exhaust or air. Although there was a dose-dependent increase in blood flow with each vasodilator (P<0.0001 for all), this response was attenuated with bradykinin (P<0.05), acetylcholine (P<0.05), and sodium nitroprusside (P<0.001) infusions 2 hours after exposure to diesel exhaust, which persisted at 6 hours. Bradykinin caused a dose-dependent increase in plasma tissue plasminogen activator (P<0.0001) that was suppressed 6 hours after exposure to diesel (P<0.001; area under the curve decreased by 34%). CONCLUSIONS: At levels encountered in an urban environment, inhalation of dilute diesel exhaust impairs 2 important and complementary aspects of vascular function in humans: the regulation of vascular tone and endogenous fibrinolysis. These important findings provide a potential mechanism that links air pollution to the pathogenesis of atherothrombosis and acute myocardial infarction.

Adult↗

PubMed2005

Diesel exhaust activates redox-sensitive transcription factors and kinases in human airways.

Diesel exhaust (DE) is a major component of airborne particulate matter. In previous studies we have described the acute inflammatory response of the human airway to inhaled DE. This was characterized by neutrophil, mast cell, and lymphocyte infiltration into the bronchial mucosa with enhanced epithelial expression of IL-8, Gro-alpha, and IL-13. In the present study, we investigated whether redox-sensitive transcription factors were activated as a consequence of DE exposure, consistent with oxidative stress triggering airway inflammation. In archived biopsies from 15 healthy subjects exposed to DE [particulates with a mass median diameter of <10 mum, 300 microg/m3] and air, immunohistochemical staining was used to quantify the expression of the transcription factors NF-kappaB (p65) and AP-1 (c-jun and c-fos), as well their upstream MAPKs, p38 and JNK, in the bronchial epithelium. In addition, phosphorylation of tyrosine residues was examined. DE induced a significant increase in the nuclear translocation of NF-kappaB (P = 0.02), AP-1 (P = 0.02), phosphorylated JNK (P = 0.04), and phosphorylated p38 (P = 0.01), as well as an increase in total (cytoplasmic + nuclear) immunostaining of phosphorylated p38 (P = 0.03). A significant increase in nuclear phosphorylated tyrosine was also observed (P < 0.05). These observations demonstrate that DE activates redox-sensitive transcription factors in vivo consistent with oxidative stress triggering the increased synthesis of proinflammatory cytokines.

Adult↗

PubMed2005

PROPHECY--a database for high-resolution phenomics.

The rapid recent evolution of the field phenomics--the genome-wide study of gene dispensability by quantitative analysis of phenotypes--has resulted in an increasing demand for new data analysis and visualization tools. Following the introduction of a novel approach for precise, genome-wide quantification of gene dispensability in Saccharomyces cerevisiae we here announce a public resource for mining, filtering and visualizing phenotypic data--the PROPHECY database. PROPHECY is designed to allow easy and flexible access to physiologically relevant quantitative data for the growth behaviour of mutant strains in the yeast deletion collection during conditions of environmental challenges. PROPHECY is publicly accessible at http://prophecy.lundberg.gu.se.

Computer Graphics↗

PubMed2004

The stress-induced Tfs1p requires NatB-mediated acetylation to inhibit carboxypeptidase Y and to regulate the protein kinase A pathway.

The Saccharomyces cerevisiae N-terminal acetyltransferase NatB consists of the subunits Nat3p and Mdm20p. We found by two-dimensional PAGE analysis that nat3Delta exhibited protein expression during growth in basal medium resembling protein expression in salt-adapted wild-type cells. The stress-induced carboxypeptidase Y (CPY) inhibitor and phosphatidylethanolamine-binding protein family member Tfs1p was identified as a novel NatB substrate. The N-terminal acetylation status of Tfs1p, Act1p, and Rnr4p in both wild type and nat3Delta was confirmed by tandem mass spectrometry. Furthermore it was found that unacetylated Tfs1p expressed in nat3Delta showed an approximately 100-fold decrease in CPY inhibition compared with the acetylated form, indicating that the N-terminal acetyl group is essential for CPY inhibition by Tfs1p. Phosphatidylethanolamine-binding proteins in other organisms have been reported to be involved in the regulation of cell signaling. Here we report that a number of proteins, whose expression has been shown previously to be dependent on the activity in the protein kinase A (PKA) signaling pathway, was found to be regulated in line with low PKA activity in the nat3Delta strain. The involvement of Nat3p and Tfs1p in PKA signaling was supported by caffeine growth inhibition studies. First, growth inhibition by caffeine addition (resulting in enhanced cAMP levels) was suppressed in tfs1Delta. Second, this suppression by tfs1Delta was abolished in the nat3Delta background, indicating that Tfs1p was not functional in the nat3Delta strain possibly because of a lack of N-terminal acetylation. We conclude that the NatB-dependent acetylation of Tfs1p appears to be essential for its inhibitory activity on CPY as well its role in regulating the PKA pathway.

Acetylation↗

PubMed2004

An in vitro and in vivo investigation of the effects of diesel exhaust on human airway lining fluid antioxidants.

Breathing high concentrations of diesel exhaust (DE) induces pulmonary inflammation, bronchoconstriction, increased airway reactivity, and oxidative stress in healthy subjects. To examine if these responses occur at environmentally relevant concentrations of DE, we exposed 25 healthy subjects to DE (PM(10) 100 microg/m(3), 0.6 ppm NO(2) for 2-h) and filtered air on separate occasions. Immediately following DE exposure, subjects displayed an increase in subjective symptoms and a mild bronchoconstriction. Six hours following the cessation of DE exposure neither airway inflammation, nor antioxidant depletion (ascorbate, urate, and reduced glutathione), was seen at any level of the respiratory tract. Instead, an increased flux of reduced glutathione into the bronchial (p < 0.01) and nasal airways (p < 0.05) was observed. In separate, in vitro experiments, DE was found to have comparable oxidative activity to the transition metal rich residual oil fly ash (ROFA) particle, significantly depleting lung lining fluid ascorbic acid and reduced glutathione in a transition metal and superoxide-dependent mechanism. Together, these data indicate that even though DE has marked oxidative activity, this effect is not observed to any great extent in the airways of healthy subjects. We interpret these findings as being indicative that the antioxidant network at the air-lung interface in healthy subjects is capable of dealing with the oxidative challenge posed by DE at ambient concentrations.

Adult↗

PubMed2004

Statistical exploration of variation in quantitative two-dimensional gel electrophoresis data.

Two-dimensional gel electrophoresis is a major technique in global analysis at the protein level. This paper presents an examination of spot volume data from three gel sets with radioactively labeled yeast Saccharomyces cerevisiae proteins. A strong variance versus mean dependence in data was found to be stabilized by applying a shifted logarithmic transformation. However, transformed data showed a remaining substantial variance heterogeneity for different proteins. Furthermore, examination of studentized residuals revealed that transformed data were approximately normally distributed and that there were spatial correlations among the measurement errors in the gel.

Analysis of Variance↗

PubMed2004

Schizosaccharomyces pombe ER oxidoreductin-like proteins SpEro1a p and SpEro1b p.

Endoplasmic reticulum oxidoreductins (Ero proteins) are essential for oxidation of protein disulphide isomerase (Pdi), which introduces disulphide bonds in target proteins. Contrary to the situation in Saccharomyces cerevisiae, with a single Ero protein (Ero1p), the genomes of Schizosaccharomyces pombe and of humans encode two Ero-like proteins. Here we show that both Sz. pombe proteins (SpEro1a p and SpEro1b p) are N-glycosylated and firmly associated with membranes of the secretory pathway. Surprisingly, only expression of SpEro1b p completely restores growth of the temperature-sensitive S. cerevisiae ero1-1 mutant, whereas SpEro1a p only partially complements this mutation. Upon expression in S. cerevisiae wild-type cells, SpEro1b p leads to a significantly increased resistance to reductive stress by dithiothreitol, whereas SpEro1a p has only a marginal effect. These data suggest that SpEro1b p is a functional homologue of the S. cerevisiae Ero1p.

Amino Acid Sequence↗

PubMed2004

Diesel exhaust exposure enhances the expression of IL-13 in the bronchial epithelium of healthy subjects.

Epidemiological studies have demonstrated adverse health effects of environmental pollution. Diesel exhaust (DE) is an important contributor to ambient particulate matter pollution. DE exposure has been shown to induce a pronounced inflammatory response in the airways, with an enhanced epithelial expression of IL-8, and Gro-alpha in healthy subjects. The present investigation was aimed to further characterise the epithelial response to DE in vivo, with particular reference to possible TH2 response, in non-atopic healthy subjects. To determine this response, 15 healthy, non-atopic non-smoking subjects with normal lung function were exposed to DE (PM10 300 microg/m3) and filtered air during 1 h on two separate randomised occasions. Bronchoscopy sampling of bronchial mucosal biopsies was performed 6 h after exposure. Immunohistochemical staining were performed using mAb for IL-10, IL-13 and IL-18 expression. DE exposure induced a significant increase in the expression of IL-13 in the bronchial epithelium cells, 2.1 (1.35-4.88) Md (Q1-Q3) vs. air 0.94 (0.53-1.23); P = 0.009. No significant changes were seen in IL-10 and IL-18 expression. This finding suggests an TH2-inflammatory response in the airways of non-atopic healthy individuals.

Adult↗