The major conformational IgE-binding epitopes of hevein (Hev b6.02) are identified by a novel chimera-based allergen epitope mapping strategy

A novel approach to localize and reconstruct conformational IgE-binding epitope regions of hevein (Hev b6.02), a major natural rubber latex allergen, is described. An antimicrobial protein (AMP) from the amaranth Amaranthus caudatus was used as an immunologically non-IgE-binding adaptor molecule to which terminal or central parts of hevein were fused. Hevein and AMP share a structurally identical core region but have different N-terminal and C-terminal regions. Only 1 of 16 hevein-allergic patients showed weak IgE binding to purified native or recombinant AMP. Chimeric AMP with the hevein N terminus was recognized by IgE from 14 (88%) patients, and chimeric AMP with the hevein C terminus was recognized by IgE from 6 (38%) patients. In contrast, chimeric AMP containing the hevein core region was recognized by IgE from only two patients. When both the N-terminal and C-terminal regions of hevein were fused with the AMP core, IgE from all 16 patients bound to the chimera. This chimera was also able to significantly inhibit (>70%) IgE binding to the native hevein. On the contrary, linear synthetic peptides corresponding to hevein regions in the AMP chimeras showed no significant IgE binding capacity in either enzyme-linked immunosorbent assay or inhibition enzyme-linked immunosorbent assay. These results suggest that the IgE binding ability of hevein is essentially determined by its N-terminal and C-terminal regions and that major IgE-binding epitopes of hevein are conformational. The chimera-based epitope mapping strategy described here provides a valuable tool for defining structural epitopes and creating specific reagents for allergen immunotherapy.     

Use of forest corridors by boreal Xestia moths

Abstract We studied movements of the noctuid moths Xestia speciosa and X. fennica in a fragmented forest landscape including old-growth spruce forest corridors in eastern Finland. We individually marked and released 1,796 adults of the habitat generalist X. speciosa and 683 individuals of X. fennica, which prefers old-growth spruce forests. Moths were recaptured with 60 sugar-bait traps that were placed within habitat corridors, in the non-habitat matrix and in surrounding old-growth forest patches. Both species favoured the corridors during their movements and tended to avoid entering the matrix. We analysed recapture rates and movements of the recaptured moths. Results suggest that X. speciosa moves longer distances than X. fennica. Though corridors clearly canalized movements by both species, the impact of old-growth forest corridors at the larger landscape scale was less clear in X. fennica. Movement behaviour of the two sexes differed more than that of the two species.     

Forest management is associated with physiological stress in an old-growth forest passerine

We investigated how physiological stress in an area-sensitive old-growth forest passerine, the Eurasian treecreeper (Certhia familiaris), is associated with forest fragmentation and forest structure. We found evidence that the concentrations of plasma corticosterone in chicks were higher under poor food supply in dense, young forests than in sparse, old forests. In addition, nestlings in large forest patches had lower corticosterone levels and a better body condition than in small forest patches. In general, corticosterone levels were negatively related to body condition and survival. We also found a decrease in corticosterone levels within the breeding season, which may have been a result of an increase in food supply from the first to the second broods. Our results suggest that forest fragmentation may decrease the fitness of free-living individual treecreepers.     

Total parenteral nutrition adversely affects gut barrier function in neonatal piglets

Sepsis is the most common morbidity in preterm infants, who often receive total parenteral nutrition (TPN). We hypothesized that gut barrier function is compromised in TPN-fed compared with enterally fed newborn piglets (ENT pigs). Colostrum-deprived newborn pigs were implanted with jugular venous and bladder catheters under general anesthesia. Pigs were either administered TPN (n = 15) or fed formula (ENT pigs, n = 15). After 6 days, pigs were gavaged a solution of mannitol, lactulose, and polyethylene glycol 4000 (PEG 4000) and urine was collected for 24 h. At 7 days, small bowel samples were assayed for myeloperoxidase activity, morphometry, and tight junction protein abundance. Intestinal contents and peripheral organ sites were cultured for bacteria. Urinary recovery (%dose) of mannitol (53 vs. 68) was lower, whereas that of lactulose (2.93 vs. 0.18) and PEG 4000 (12.78 vs. 0.96) were higher in TPN vs. ENT pigs, respectively (P < 0.05). Incidence of translocation was similar in TPN and ENT pigs. Myeloperoxidase activity was increased in TPN vs. ENT pigs in the jejunum (P < 0.001) and was weakly correlated with lactulose (R2 = 0.32) and PEG 4000 (R2 = 0.38) recovery. Goblet cell counts did not change, but intraepithelial lymphocyte numbers decreased with TPN. Only claudin-1 protein abundance was increased in the TPN group. We conclude that TPN is associated with impairment of neonatal gut barrier function as measured by permeability but not translocation.     

Synthesis of nonluminescent lanthanide(III) chelates tethered to an aminooxy group and their applicability to biomolecule derivatization

Synthesis of nonluminescent lanthanide(III) chelates tethered to an aminooxy group (i.e., 1-[4-(6-aminooxyhexamido)benzyl]diethylenetriaminetetraacetic acid lanthanides(III), 6a-d, where Ln(3+) is Eu, Dy, Sm, and Tb) is described. Their applicability to biomolecule derivatization is demonstrated by allowing them to react with a synthetic oligopeptide, a protein, two synthetic drugs, and a steroid. The oligopeptide and protein were linked to 6 after preoxidation of their N-terminal serine residues, while the drugs and the steroid reacted via their ketone functionality. Also some application data is included.     

Mixed-phase hybridization of short oligodeoxyribonucleotides on microscopic polymer particles: effect of one-base mismatches on duplex stability

Hybridization of short oligonucleotides (10- and 11-mers) to complementary probes immobilized to microscopic polymer particles was quantified by a sandwich type mixed-phase hybridization assay based on a time-resolved fluorometric measurement of a photoluminescent europium(III) chelate from the surface of a single particle. Among the 54 sequences that were studied, 21 were fully complementary to the particle-bound probes, while 33 contained an internal one-base mismatch. The observed affinities were compared to those predicted by the nearest-neighbor model. In addition, various factors, such as the pore size of the particle, the linker structure, the charge type of the probe, and the efficiency of agitation, that might be expected to affect the kinetics of mixed-phase hybridization have been examined.     

Construction of Gene-Targeting Vectors: a Rapid Mu in vitro DNA Transposition-Based Strategy Generating Null,Potentially Hypomorphic,and Conditional Alleles

Gene targeting into mammalian genomes by means of homologous recombination is a powerful technique for analyzing gene function through generation of transgenic animals. Hundreds of mouse strains carrying targeted alleles have already been created and recent modifications of the technology, in particular generation of conditional alleles, have extended the usefulness of the methodology for a variety of special purposes. Even though the standard protocols, including the construction of gene-targeting vector plasmids, are relatively straightforward, they typically involve time-consuming and laborious gene mapping and/or sequencing steps. To produce various types of gene-targeting constructions rapidly and with minimum effort, we developed a strategy, that utilizes a highly efficient in vitro transposition reaction of phage Mu, and tested it in a targeting of the mouse Kcc2 gene locus. A vast number and different types of targeting constructions can be generated simultaneously with little or no prior sequence knowledge of the gene locus of interest. This quick and efficient general strategy will facilitate easy generation of null, potentially hypomorphic, and conditional alleles. Especially useful it will be in the cases when effects of several exons within a given gene are to be studied, a task that necessarily will involve generation of multiple constructions. The strategy extends the use of diverse recombination reactions for advanced genome engineering and complements existing recombination-based approaches for generation of gene-targeting constructions.     

Flexible work hours, health and well-being in the European Union: preliminary data from a SALTSA project

Demand for flexible work hours (FWH) is increasing in Europe aimed at increasing the number of production hours on one hand, and, on the other, reducing individual working hours and/or increasing autonomy and control on them. In view of the lack of knowledge of the effects of FWH on health and safety, we started a pilot project, funded by the Joint Programme for Working Life Research in Europe (SALTSA), aimed at: a) comparing the most relevant national legislation and how the EU Directive 93/104 "concerning certain aspects of working time" has been implemented in the member States; b) reporting prevalence and trend of FWH in Europe according to the three EU Surveys on Working Conditions carried out in the last decade; c) collecting practical examples of innovative FWH; d) evaluating their impact on health and safety in relation to work sectors, job demands, social life, aging and gender. Consequent actions are going to include information and consultancy for pertinent authorities and social parties involved, as well as training programmes for Union officials and similar groups concerning the organisation of FWH according to ergonomic principles.