Molecular and immunological characterization of a wheat serine proteinase inhibitor as a novel allergen in baker's asthma

IgE-mediated sensitization to wheat flour belongs to the most frequent causes of occupational asthma. A cDNA library from wheat seeds was constructed and screened with serum IgE from baker's asthma patients. One IgE-reactive phage clone contained a full-length cDNA coding for an allergen with a molecular mass of 9.9 kDa and an isoelectric point of 6. According to sequence analysis it represents a member of the potato inhibitor I family, a group of serine proteinase inhibitors, and thus is the first allergen belonging to the group 6 pathogenesis-related proteins. The recombinant wheat seed proteinase inhibitor was expressed in Escherichia coli and purified to homogeneity. According to circular dichroism analysis, it represented a soluble and folded protein with high thermal stability containing mainly beta-sheets, random coils, and an alpha-helical element. The recombinant allergen showed allergenic activity in basophil histamine release assays and reacted specifically with IgE from 3 of 22 baker's asthma patients, but not with IgE from grass pollen allergic patients or patients suffering from food allergy to wheat. Allergen-specific Abs were raised to localize the allergen by immunogold electron microscopy in the starchy endosperm and the aleuron layer. The allergen is mainly expressed in mature wheat seeds and, despite an approximately 50% sequence identity, showed no relevant cross-reactivity with allergens from other plant-derived food sources such as maize, rice, beans, or potatoes. Recombinant wheat serine proteinase inhibitor, when used in combination with other specific allergens, may be useful for the diagnosis and therapy of IgE-mediated baker's asthma.     

Environmental impacts of bioenergy wood production from poplar short‐rotation coppice grown at a marginal agricultural site in Germany

For avoiding competition with food production, marginal land is economically and environmentally highly attractive for biomass production with short‐rotation coppices (SRCs) of fast‐growing tree species such as poplars. Herein, we evaluated the environmental impacts of technological, agronomic, and environmental aspects of bioenergy production from hybrid poplar SRC cultivation on marginal land in southern Germany. For this purpose, different management regimes were considered within a 21‐year lifetime (combining measurements and modeling approaches) by means of a holistic Life Cycle Assessment (LCA). We analyzed two coppicing rotation lengths (7 × 3 and 3 × 7 years) and seven nitrogen fertilization rates and included all processes starting from site preparation, planting and coppicing, wood chipping, and heat production up to final stump removal. The 7‐year rotation cycles clearly resulted in higher biomass yields and reduced environmental impacts such as nitrate (NO₃) leaching and soil nitrous oxide (N₂O) emissions. Fertilization rates were positively related to enhanced biomass accumulation, but these benefits did not counterbalance the negative impacts on the environment due to increased nitrate leaching and N₂O emissions. Greenhouse gas (GHG) emissions associated with the heat production from poplar SRC on marginal land ranged between 8 and 46 kg CO₂‐eq. GJ^?1 (or 11–57 Mg CO₂‐eq. ha^?1). However, if the produced wood chips substitute oil heating, up to 123 Mg CO₂‐eq. ha^?1 can be saved, if produced in a 7‐year rotation without fertilization. Dissecting the entire bioenergy production chain, our study shows that environmental impacts occurred mainly during combustion and storage of wood chips, while technological aspects of establishment, harvesting, and transportation played a negligible role.     

Diazotrophic community structure and function in two successional stages of biological soil crusts from the Colorado Plateau and Chihuahuan Desert

The objective of this study was to characterize the community structure and activity of N2-fixing microorganisms in mature and poorly developed biological soil crusts from both the Colorado Plateau and Chihuahuan Desert. Nitrogenase activity was approximately 10 and 2.5 times higher in mature crusts than in poorly developed crusts at the Colorado Plateau site and Chihuahuan Desert site, respectively. Analysis of nifH sequences by clone sequencing and the terminal restriction fragment length polymorphism technique indicated that the crust diazotrophic community was 80 to 90% heterocystous cyanobacteria most closely related to Nostoc spp. and that the composition of N2-fixing species did not vary significantly between the poorly developed and mature crusts at either site. In contrast, the abundance of nifH sequences was approximately 7.5 times greater (per microgram of total DNA) in mature crusts than in poorly developed crusts at a given site as measured by quantitative PCR. 16S rRNA gene clone sequencing and microscopic analysis of the cyanobacterial community within both crust types demonstrated a transition from a Microcoleus vaginatus-dominated, poorly developed crust to mature crusts harboring a greater percentage of Nostoc and Scytonema spp. We hypothesize that ecological factors, such as soil instability and water stress, may constrain the growth of N2-fixing microorganisms at our study sites and that the transition to a mature, nitrogen-producing crust initially requires bioengineering of the surface microenvironment by Microcoleus vaginatus.     

Crystal structure of aphrodisin, a sex pheromone from female hamster

We have solved the crystal structure of aphrodisin, a pheromonal protein inducing a copulatory behaviour in male hamster, using MAD methods with selenium, at 1.63 A resolution. The monomeric protein belongs to the lipocalin family, and possesses a disulfide bridge in a loop between strands 2 and 3. This disulfide bridge is characteristic of a family of lipocalins mainly identified in rodents, and is analogous to the fifth disulfide bridge of the long neurotoxins, such as alpha cobratoxin. An elongated electron density was found inside the buried cavity, which might represent a serendipitous ligand of unknown origin. The analysis of the water accessible surfaces of the side-chains bordering the cavity indicates that Phe76 may be the door for the natural ligand to access the cavity. This residue defines the entry of the cavity as belonging to the consensus for lipocalins. The face bearing Phe76 might also serve for the interaction with the receptor.     

Geranylgeranylated SNAREs are dominant inhibitors of membrane fusion

Exocytosis in yeast requires the assembly of the secretory vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) Sncp and the plasma membrane t-SNAREs Ssop and Sec9p into a SNARE complex. High-level expression of mutant Snc1 or Sso2 proteins that have a COOH-terminal geranylgeranylation signal instead of a transmembrane domain inhibits exocytosis at a stage after vesicle docking. The mutant SNARE proteins are membrane associated, correctly targeted, assemble into SNARE complexes, and do not interfere with the incorporation of wild-type SNARE proteins into complexes. Mutant SNARE complexes recruit GFP-Sec1p to sites of exocytosis and can be disassembled by the Sec18p ATPase. Heterotrimeric SNARE complexes assembled from both wild-type and mutant SNAREs are present in heterogeneous higher-order complexes containing Sec1p that sediment at greater than 20S. Based on a structural analogy between geranylgeranylated SNAREs and the GPI-HA mutant influenza virus fusion protein, we propose that the mutant SNAREs are fusion proteins unable to catalyze fusion of the distal leaflets of the secretory vesicle and plasma membrane. In support of this model, the inverted cone-shaped lipid lysophosphatidylcholine rescues secretion from SNARE mutant cells.     

Cutting edge: preferentially the R-stereoisomer of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates immune cells through a toll-like receptor 2- and MyD88-dependent signaling pathway

Mycoplasmas and their membranes are potent activators of macrophages, the active principle being lipoproteins and lipopeptides. Two stereoisomers of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 (MALP-2) differing in the configuration of the lipid moiety were synthesized and compared in their macrophage-activating potential, the R-MALP being >100 times more active than the S-MALP in stimulating the release of cytokines, chemokines, and NO. To assess the role of the Toll-like receptor (TLR) family in mycoplasmal lipopeptide signaling, the MALP-2-mediated responses were analyzed using macrophages from wild-type, TLR2-, TLR4-, and MyD88-deficient mice. TLR2- and MyD88-deficient cells showed severely impaired cytokine productions in response to R- and S-MALP. The MALP-induced activation of intracellular signaling molecules was fully dependent on both TLR2 and MyD88. There was a strong preference for the R-MALP in the recognition by its functional receptor, TLR2.     

Estimating age from adult occlusal wear: A modification of the miles method

The Miles method of age estimation relies on molar wear to estimate age and is widely used in bioarcheological contexts. However, because the method requires physical seriation and a sample of subadults to estimate wear rates it cannot be applied to many samples. Here, we modify the Miles method by scoring occlusal wear and estimating molar wear rates from adult wear gradients in 311 hunter‐gatherers and provide formulae to estimate the error associated with each age estimate. A check of the modified method in a subsample (n = 22) shows that interval estimates overlap in all but one case with age categories estimated from traditional methods; this suggests that the modifications have not hampered the ability of the Miles method to estimate age even in heterogeneous samples. As expected, the error increases with age and in populations with smaller sample sizes. These modifications allow the Miles method to be applied to skeletal samples of adult crania that were previously only amenable to cranial suture age estimation, and importantly, provide a measure of uncertainty for each age estimate. Am J Phys Anthropol 149:181–192, 2012. © Wiley Periodicals, Inc.     

Modeling the isoprene emission rate from leaves

The leaves of many plants emit isoprene (2-methyl-1,3-butadiene) to the atmosphere, a process which has important ramifications for global and regional atmospheric chemistry. Quantitation of leaf isoprene emission and its response to environmental variation are described by empirically derived equations that replicate observed patterns, but have been linked only in some cases to known biochemical and physiological processes. Furthermore, models have been proposed from several independent laboratories, providing multiple approaches for prediction of emissions, but with little detail provided as to how contrasting models are related. In this review we provide an analysis as to how the most commonly used models have been validated, or not, on the basis of known biochemical and physiological processes. We also discuss the multiple approaches that have been used for modeling isoprene emission rate with an emphasis on identifying commonalities and contrasts among models, we correct some mathematical errors that have been propagated through the models, and we note previously unrecognized covariances within processes of the models. We come to the conclusion that the state of isoprene emission modeling remains highly empirical. Where possible, we identify gaps in our knowledge that have prevented us from achieving a greater mechanistic foundation for the models, and we discuss the insight and data that must be gained to fill those gaps.     

Radiographic evaluation of children with growth disorders

Short stature as well as tall stature can have a wide variety of causes. Tall stature is usually experienced as a less important problem than short stature, but for both clinical presentations it is important to make a correct diagnosis as to etiology. The identification of the diagnosis frequently relies on radiological criteria. However, no international uniformity exists with respect to the radiographic evaluation of children with growth problems. We recommend that in patients with a possible diagnosis of a skeletal dysplasia a skeletal survey must be performed. In patients with a proportionate stature, radiographic analysis of the hand and wrist will be sufficient in most cases. However, whenever there are clinical abnormalities with a possible underlying bone anomaly, a modified skeletal survey is appropriate. The combination of clinical and biochemical features and an appropriate skeletal survey can often lead to the correct diagnosis and/or guide the subsequent molecular analysis.     

Carbohydrate-active enzymes identified by metagenomic analysis of deep-sea sediment bacteria

Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psychrophilic aerobic bacteria on cellulose, xylan, chitin, and starch. A variety of species belonging to Alpha- and Gammaproteobacteria and to Flavobacteria were isolated from sediment depths between 12 and 42 mbsf. Metagenomic DNA purified from the pooled enrichments was sequenced and analyzed for phylogenetic composition and presence of genes encoding carbohydrate-active enzymes. More than 200 open reading frames coding for glycoside hydrolases were identified, and more than 60 of them relevant for enzymatic degradation of lignocellulose. Four genes encoding β-glucosidases with less than 52 % identities to characterized enzymes were chosen for recombinant expression in Escherichia coli. In addition one endomannanase, two endoxylanases, and three β-xylosidases were produced recombinantly. All genes could be actively expressed. Functional analysis revealed discrepancies and additional variability for the recombinant enzymes as compared to the sequence-based predictions.