An esterase from the basidiomycete Pleurotus sapidus hydrolyzes feruloylated saccharides

Investigating the secretion of esterases by the basidiomycetous fungus Pleurotus sapidus in a Tween 80-rich nutrient medium, an enzyme was discovered that hydrolyzed the ester bond of feruloylated saccharides. The enzyme was purified by ion exchange and size exclusion chromatography. Polyacrylamide gel electrophoresis analysis showed a monomeric protein of about 55 kDa. The complete coding sequence with an open reading frame of 1,665 bp encoded a protein (Est1) consisting of 554 amino acids. The enzyme showed no significant homology to any published feruloyl esterase sequences, but possessed putative conserved domains of the lipase/esterase superfamily. Substrate specificity studies classified the new enzyme as type-A feruloyl esterase, hydrolyzing methyl ferulate, methyl sinapate, and methyl p-coumarate but no methyl caffeate. The enzyme had a pH optimum of 6 and a temperature optimum at 50 °C. Ferulic acid was efficiently released from ferulated saccharides, and the feruloyl esterase exhibited moderate stability in biphasic systems (50 % toluene or tert-butylmethyl ether).     

A novel missense mutation T101N in the melanocortin-4 receptor gene associated with obesity

Mutations in the melanocortin-4 receptor (MC4R) are associated with severe obesity, independent of their effect on cortisol or thyroid-stimulating hormone levels. We examined a morbidly obese male (BMI = 62 kg/m2) with a binge-eating disorder and eight family members for mutations in the MC4R gene and potential differences in leptin levels. Fifty healthy individuals served as controls. Sequence analysis revealed a novel heterozygous missense mutation (c.302 C>A, p.T101N) located in the second transmembrane domain of the receptor, which was not detected in controls. The Fisher exact test revealed an association between the T101N mutation and history of obesity (P < 0.05) in the family. The Kruskal-Wallis test showed an association between the mutation and the leptin/BMI ratio (P < 0.05), while there was no association between the T101N mutation and diabetes or arterial hypertension in the family. Although the available family was small, we could show a significant association between the heterozygous T101N mutation and obesity.     

Bruton's tyrosine kinase is dispensable for the Toll-like receptor-mediated activation of mast cells

Bruton's tyrosine kinase (Btk) represents an important signaling element downstream of ITAM-containing receptors, e.g. FcepsilonR1 and BCR. Btk is part of the calcium signalosome and thus, critically involved in intracellular calcium mobilization. Loss of Btk or expression of mutant forms results in severe disease phenotypes, X-linked agammaglobulinemia (XLA) and Xid in humans and mice, respectively. Previously, roles for Btk in TLR-mediated signal transduction have been found in monocytes/macrophages. In the present study we show that Btk deficiency moderately enhances or has no influence on the LPS- or lipopeptide-induced secretion of IL-6 and TNF-alpha from murine bone marrow-derived mast cells (BMMCs). Furthermore, activation of p38 kinase, which is required for cytokine production, is comparable in WT and Btk-/- BMMCs. Moreover, stability of the adaptor protein Mal as well as LPS-induced H(2)O(2) production does not vary between WT and Btk-/- cells. Interestingly, PKC-beta deficiency, which results in a Xid-like phenotype as well, has also no negative effect on LPS-induced cytokine secretion, suggesting that proteins of the calcium signalosome are not involved in TLR-mediated BMMC activation. In conclusion, the study reveals that Btk is dispensable for TLR signaling and function in murine BMMCs.     

In vivo characterization of estrogen receptor modulators with reduced genomic versus nongenomic activity in vitro

Estrogen receptor (ER) ligands that are able to prevent postmenopausal bone loss, but have reduced activity in the uterus and the mammary gland might be of great value for hormone therapy. It is well established that the classical ER can activate genomic as well as nongenomic signal transduction pathways. In this study, we analyse the in vivo behaviour of ER ligands that stimulate nongenomic ER effects to the same extent as estradiol, but show clearly reduced activation of genomic ER effects in vitro. Using different readout parameters such as morphological changes, cellular proliferation, and target gene induction, we are able to demonstrate that ER ligands with reduced genomic activity in vitro show a better dissociation of bone versus uterine and mammary gland effects than estradiol that stimulates genomic and nongenomic effects to the same extent. We conclude that pathway-selective ER ligands may represent an interesting option for hormone therapy.     

Biasing Factors for Simple Soil Ingestion Estimates in Mass Balance Studies of Soil Ingestion

Soil ingestion estimates from mass balance soil ingestion studies can be used in Monte Carlo Risk assessment. We develop and describe a simulation model based on four mass balance soil ingestion studies that enables food ingestion, soil ingestion, and transit time to be mimicked. We use the simulation to evaluate potential biases that exist in current estimates of the distribution of daily soil ingestion in children (constructed from subject specific average daily estimates). The simulation identifies the importance of the study duration on the bias in the upper percentile soil ingestion estimates, indicating that the 95% soil ingestion estimate may be positively biased by over 100%. Misspecification of play areas for soil sampling is shown to have no biasing effect, and absorption of trace elements in food of up to 30% is shown to bias the soil ingestion distribution by less than 20 mg/d. The results, based on Al and Si trace element estimates, define the limits of previously published soil ingestion estimates, and provide insight for future study design and estimation methods.     

Soil Ingestion Distributions for Monte Carlo Risk Assessment in Children

Monte Carlo environmental risk assessment requires estimates of the exposure distributions. An exposure of principal concern is often soil ingestion among children. We estimate the long-term (annual) average soil ingestion exposure distribution using daily soil ingestion estimates from children who participated in a mass-balance study at Anaconda, MT. The estimated distribution is accompanied by uncertainty estimates. The estimates take advantage of developing knowledge about bias in soil ingestion estimates and are robust. The estimates account for small particle size soil, use the median trace element estimate for subject days, account for the small sample variance of the median estimates, and use best linear unbiased predictors to estimate the cumulative long term soil ingestion distribution. Bootstrapping is used to estimate the uncertainty of the distribution estimates. The median soil ingestion is estimated as 24?mg/d (sd = 4?mg/d), with the 95 percentile soil ingestion estimated as 91?mg/d (sd = 16.6?mg/d). Strategies are discussed for use of these estimates in Monte Carlo risk assessment.     

Locomotory Responses of the Cave Cyprinid Phreatichthys Andruzzii Chemical Signals from Conspecifics and Related Species: New Findings

Previous research on chemical communication among hypogean fishes have suggested that these species are attracted, as are epigean species, by the odour of non-injured conspecifics. Considerations on the biological meaning of such responses and some doubts about the reliability of the experimental procedure previously utilised, have led us to reconsider the matter and verify the results already obtained in the cave cyprinid Phreatichthys andruzzii. Using a new methodological approach we ascertained not only the absence of positive chemotaxis to odour of known or unknown conspecifics, but also the presence of a strong negative chemotaxis towards the odour of unfamiliar conspecifics and of individuals belonging to the closely related species Barbus conchonius. The biological meaning and the adaptive value of these reactions are briefly discussed.