Bacteria and mould components in house dust and children's allergic sensitisation.

It has been suggested that early childhood exposure to microbial agents decreases the risk of allergies in children. The current authors studied the association between microbial agents in house dust and allergic sensitisation in children aged 2-4 yrs. Nested case-control studies were performed within ongoing birth cohort studies in Germany, the Netherlands and Sweden and approximately 180 sensitised and 180 nonsensitised children were selected per country. Levels of bacterial endotoxin, beta(1,3)-glucans and fungal extracellular polysaccharides (EPS) were measured in dust samples from the children's mattresses and the living-room floors. Combined across countries, higher amounts of mattress dust and higher mattress dust loads of endotoxin, beta(1,3)-glucans and EPS were associated with a significantly decreased risk of sensitisation to inhalant allergens. After mutual adjustment, only the protective effect of the amount of mattress dust remained significant (odds ratio (95% confidence interval) 0.57(0.39-0.84)). Higher amounts of mattress dust may decrease the risk of allergic sensitisation to inhalant allergens. The effect might be partly attributable to endotoxin, beta(1,3)-glucans and extracellular polysaccharides, but could also reflect (additional) protective effects of (microbial) agents other than the ones measured. It is not possible to distinguish with certainty which component relates to the effect, since their levels are highly correlated.

Determinants of house dust endotoxin in three European countries - the AIRALLERG study.

UNLABELLED: The comparison of endotoxin levels between study populations and countries is limited as a result of differences in sampling, extraction, and storage procedures. The objective of this study is to assess the levels and determinants of endotoxin in mattress and living room floor dust samples from three European countries, namely, Germany, the Netherlands, and Sweden, using a standardized sampling, storage, and analysis protocol. The mattress and living room floor dust was collected from the homes of 1065 German, Dutch, and Swedish (pre-)school children. All the samples were collected in the cool season and analyzed for endotoxin in a central laboratory. The determinants were assessed by a standardized questionnaire. The endotoxin concentrations in mattress and living room floor dust were found to be the highest in German homes and lowest in the Swedish ones. Differences between the geometric means were small (factor 1.1-1.7). Most of the associations between endotoxin concentrations and potential determinants were not statistically significant and heterogeneous across countries. However, keeping pets and having more than four persons living in the home were consistently associated with up to 1.7-fold higher endotoxin concentrations in mattress and floor dust. Furthermore, having carpets or rugs, and opening the windows frequently was associated with up to 3.4-fold and 1.3-fold higher endotoxin concentrations in living room floor dust, respectively. The proportion of variance explained by the questionnaire variables was generally low. In conclusion, the data on housing characteristics did not accurately predict the endotoxin concentrations in house dust, and could only partly explain the differences between countries. PRACTICAL IMPLICATIONS: The differences between the endotoxin concentrations in German, Dutch, and Swedish homes are small. House dust endotoxin concentrations are associated with a number of housing factors, such as pet-ownership, floor cover, number of persons living in the home, and ventilation. The variability of the endotoxin levels between homes and countries can only be partly explained by these factors.

Comparison of parental reports of smoking and residential air nicotine concentrations in children.

BACKGROUND: Using questionnaires to assess children's residential exposure to environmental tobacco smoke (ETS) may result in misclassification from recall and response bias. Questionnaire data have frequently been validated against urinary cotinine measurements, but rarely against actual measurements of residential air nicotine. OBJECTIVE: To compare questionnaire reported smoking with air nicotine concentrations in a large population of children and with urinary cotinine levels in a subpopulation; and to assess the potential impact of the symptom status of the children on the agreement between different measures of exposure. METHODS: The authors assessed residential exposure to ETS in 347 German, 335 Dutch, and 354 Swedish preschool and schoolchildren by questionnaire and air nicotine measurements, and in a subset of 307 German children by urinary cotinine measurements. They then compared the different measures of ETS exposure. RESULTS: In all countries, air nicotine concentrations increased with increasing questionnaire reported smoking in a dose-response fashion. Specificity and negative predictive values of questionnaire reports for nicotine concentrations were excellent. Sensitivity and positive predictive values were moderate to good. Excluding occasional smokers, the overall percentage of homes misclassified was 6.9%, 6.7%, and 5.1% in Germany, the Netherlands, and Sweden, respectively. Similar results were found for the agreement of urinary cotinine concentrations with questionnaire reports and air nicotine levels. There was no indication of underreporting by parents of symptomatic children. CONCLUSION: Despite some misclassification, questionnaire reports are an inexpensive and valid estimate of residential ETS exposure among preschool and school children.

Separate functional features of proinsulin C-peptide.

Proinsulin C-peptide influences a number of physiological parameters in addition to its well-established role in the parent proinsulin molecule. It is of interest as a candidate for future co-replacement therapy with insulin for patients with diabetes mellitus type 1, but specific receptors have not been identified and additional correlation with functional effects is desirable. Based on comparisons of 22 mammalian proinsulin variants, we have constructed analogues for activity studies, choosing phosphorylation of mitogen-activated protein kinases (MAPKs) in Swiss 3T3 fibroblasts for functional measurements. In this manner, we find that effective phosphorylation of MAPKs is promoted by the presence of conserved glutamic acid residues at positions 3, 11 and 27 of C-peptide and by the presence of helix-promoting residues in the N-terminal segment. Previous findings have ascribed functional roles to the C-terminal pentapeptide segment, and all results combined therefore now show the importance of different segments, suggesting that C-peptide interactions are complex or multiple.

Road traffic noise and annoyance--an increasing environmental health problem.

Traffic noise, which is steadily increasing, is considered to be an important environmental health problem. The aim of this study was to estimate the degree of annoyance and sleep disturbance related to road traffic noise in residential settings in an urban community. The study is based on a questionnaire on environmentally related health effects distributed to a stratified random sample of 1000 individuals, 19-80 years old, in a municipality with heavy traffic in the county of Stockholm. The response rate was 76%. The individual noise exposure was estimated using evaluated noise dispersion models and local noise assessments. Frequent annoyance was reported by 13% of subjects exposed to Leq 24 hr >50 dBA compared to 2% among those exposed to <50 dBA, resulting in a difference of 11% (95% Confidence Interval (CI) 7%, 15%). Sometimes or frequently occurring sleep disturbance was reported by 23% at Leq 24 hr >50 dBA and by 13% at levels <50 dBA, a difference of 11% (95% CI 4%, 18%). A positive exposure- response relation was indicated for annoyance as well as for sleep disturbances when classifying the individuals into four different exposure categories (< 45, 46- 50, 51-55 and >55 dBA Leq 24 hr). There was some habituation to noise for problems related to sleep but not for annoyance. The prevalence of both annoyance and sleep problems was higher when bedroom windows were facing streets. People living in apartments had more sleep problems compared to people living in detached or semi-detached houses. In conclusion traffic noise exposure, even at low levels, was associated with annoyance and sleep disturbance. Access to a quiet side seemed to be a major protective factor for noise related problems.

Differential multiplicity of MDR alcohol dehydrogenases: enzyme genes in the human genome versus those in organisms initially studied.

Screens were made for alcohol dehydrogenase (ADH) of the classical type (the MDR superfamily) in translations of human and other relevant genomes, corresponding to the organism types from which the enzyme was initially purified. Considerable multiplicities were detected in the dimeric enzymes from higher eukaryotes: seven forms in the human (plus three pseudogenes), all genes on chromosome 4, in the order class IV --> class Igamma --> class Ibeta --> class Ialpha --> class V --> class II --> class III, and eight forms in Arabidopsis thaliana (plus one pseudogene). These multiplicity patterns, and the species variability in the animal (human/mouse) and plant (Arabidopsis/pea) lines, suggest parallel but separate duplicatory events, giving rise to three families of dimeric MDR-ADH: class III, the animal non-class III, and the plant non-class III enzymes, with functions in formaldehyde elimination, in alcohol/aldehyde detoxication and in special pathways in higher eukaryotes. Multiplicity, although to a lesser extent, was also noted in tetrameric MDR-ADH, suggesting functional divergence between the dimeric and tetrameric enzymes. Combining these observations, at least five levels of divergence are reflected in the present ADH forms, corresponding to nodes at the SDR/MDR, the dimer/tetramer, the class III/non-class III, the class I/P, and the more recent class splits, each branch associated with separate functional patterns.

Identification and characterisation of two allelic forms of human alcohol dehydrogenase 2.

The human alcohol dehydrogenase system is comprised of multiple forms that catalyse the oxidation/reduction of a large variety of alcohols and aldehydes. A transition that results in an Ile308Val substitution was identified in the human ADH2 gene by single-strand conformation polymorphism analysis. Screening a Swedish population revealed that Val308 was the most frequent allele (73%), and site-directed mutagenesis was used to obtain both allelozymes, which were expressed in Escherichia coli for characterisation. Thermostability was assayed by activity measurements and circular dichroism spectroscopy. The results showed that the 308Val substitution decreases protein stability, as compared to the Ile308 variant, an effect also demonstrated during prolonged storage. Ethanol, octanol, 12-hydroxydodecanoic acid and all-trans retinol were used as model substrates and, generally, slightly higher Km values were observed with Val at position 308. Finally, homology modelling, from mouse ADH2, further supported the decreased stability of the Val308 variant and located position 308 in the subunit interface of the molecule and in the vicinity of the active-site pocket entrance. In conclusion, the Ile308Val substitution represents a novel functional polymorphism within the human alcohol dehydrogenase gene cluster that may affect the metabolism of ethanol and other substrates.

Structural role of conserved Asn179 in the short-chain dehydrogenase/reductase scaffold.

Short-chain dehydrogenases/reductases (SDR) constitute a large family of enzymes found in all forms of life. Despite a low level of sequence identity, the three-dimensional structures determined display a nearly superimposable alpha/beta folding pattern. We identified a conserved asparagine residue located within strand betaF and analyzed its role in the short-chain dehydrogenase/reductase architecture. Mutagenetic replacement of Asn179 by Ala in bacterial 3beta/17beta-hydroxysteroid dehydrogenase yields a folded, but enzymatically inactive enzyme, which is significantly more resistant to denaturation by guanidinium hydrochloride. Crystallographic analysis of the wild-type enzyme at 1.2-A resolution reveals a hydrogen bonding network, including a buried and well-ordered water molecule connecting strands betaE to betaF, a common feature found in 16 of 21 known three-dimensional structures of the family. Based on these results, we hypothesize that in mammalian 11beta-hydroxysteroid dehydrogenase the essential Asn-linked glycosylation site, which corresponds to the conserved segment, displays similar structural features and has a central role to maintain the SDR scaffold.

Human type 10 17 beta-hydroxysteroid dehydrogenase: molecular modelling and substrate docking.

17 beta-hydroxysteroid dehydrogenases catalyze the oxidoreduction of hydroxy/oxo groups at position C17 of steroid hormones, thereby constituting a prereceptor control mechanism of hormone action. At present, 11 different mammalian 17 beta-hydroxysteroid dehydrogenases have been identified, catalyzing the cell- and steroid-specific activation and inactivation of estrogens and androgens. The human type 10 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD-10) is a multifunctional mitochondrial enzyme that efficiently catalyzes the oxidative inactivation at C17 of androgens and estrogens. However, it also mediates oxidation of 3 alpha-hydroxy groups of androgens, thereby reactivating androgen metabolites. Finally, it is involved in beta-oxidation of fatty acids by catalyzing the L-hydroxyacyl CoA dehydrogenase reaction of the beta-oxidation cycle. These features and expression profiles suggest a critical role of 17 beta-HSD-10 in neurodegenerative and steroid-dependent cancer forms. Since no three-dimensional structure of 17 beta-HSD-10 is available, homology modelling was carried out to understand the molecular basis of these substrate specificities. The structure obtained displays the properties of a one-domain, alpha/beta fold enzyme of the SDR family. The active site is located within a large, hydrophobic cleft, which forms optimal contacts with the different steroid surfaces. The data provide explanations for the substrate specificities toward the various classes of sex steroid hormones. The model is suitable to explore substrate and inhibitor characteristics that may be used in the development of novel strategies in the treatment of degenerative or malignant diseases.

Recovery of gel-separated proteins for in-solution digestion and mass spectrometry.

A protocol for mass spectrometry of gel-separated proteins resulting in significantly increased sequence coverage and in improved possibilities for detection and identification of posttranslational modifications was developed. In relation to the standard in-gel digestion procedure, the sequence coverage using a combination of matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry was on the average increased by 30%. The method involves electroblotting of the gel-separated proteins to a poly(vinylidene difluoride) membrane. The proteins are extracted from the membrane using a solution of 1% trifluoroacetic acid in 70% acetonitrile and lyophilized. After reconstitution of the protein extract in digestion buffer, proteolytic cleavage is carried out in-solution as opposed to the standard in-gel digestion procedure. This allows recovery of large and hydrophobic peptides for mass spectrometry and reduces the risk for entrapment of proteolytic peptides in the gel matrix. The method was applied to proteins in the 30-40-kDa range with highly different structural properties. The improved ability to localize and determine protein modifications is shown for N-terminal acetylation and methylation of a histidine residue. Furthermore, the method enables fast screening of homologous protein sequences.

Human liver class I alcohol dehydrogenase gammagamma isozyme: the sole cytosolic 3beta-hydroxysteroid dehydrogenase of iso bile acids.

3beta-Hydroxy (iso) bile acids are formed during enterohepatic circulation from 3alpha-hydroxy bile acids and constitute normal compounds in plasma but are virtually absent in bile. Isoursodeoxycholic acid (isoUDCA) is a major metabolite of UDCA. In a recent study it was found that after administration of isoUDCA, UDCA became the major acid in bile. Thus, epimerization of the 3beta-hydroxy to a 3alpha-hydroxy group, catalyzed by 3beta-hydroxysteroid dehydrogenases (HSD) and 3-oxo-reductases must occur. The present study aims to characterize the human liver bile acid 3beta-HSD. Human liver cytosol and recombinant alcohol dehydrogenase (ADH) betabeta and gammagamma isozymes were subjected to native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. Activity staining with oxidized nicotinamide adenine dinucleotide (NAD(+)) or oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)) as cofactors and various iso bile acids as substrates was used to screen for 3beta-HSD activity. Reaction products were identified and quantified by gas chromotography/mass spectrometry (GC/MS). Computer-assisted substrate docking of isoUDCA to the active site of a 3-dimensional model of human class I gammagamma ADH was performed. ADH gammagamma isozyme was identified as the iso bile acid 3beta-HSD present in human liver cytosol, with NAD(+) as a cofactor. Values for k(cat)/K(m) were in the rank order isodeoxycholic acid (isoDCA), isochenodeoxycholic acid (isoCDCA), isoUDCA, and isolithocholic acid (isoLCA) (0.10, 0.09, 0.08, and 0. 05 min(-1) x micromol/L(-1), respectively). IsoUDCA fits as substrate to the 3-dimensional model of the active-site of ADH gammagamma. ADH gammagamma isozyme was defined as the only bile acid 3beta-HSD in human liver cytosol. Hydroxysteroid dehydrogenases are candidates for the binding and transport of 3alpha-hydroxy bile acids. We assume that ADH gammagamma isozyme is involved in cytosolic bile acid binding and transport processes as well.