Identification of immunogenic proteins of Waddlia chondrophila

Evidence is growing for a role of Waddlia chondrophila as an agent of adverse pregnancy outcomes in both humans and ruminants. This emerging pathogen, member of the order Chlamydiales, is also implicated in bronchiolitis and lower respiratory tract infections. Until now, the serological diagnosis of W. chondrophila infection has mainly relied on manually intensive tests including micro-immunofluorescence and Western blotting. Thus, there is an urgent need to establish reliable high throughput serological assays. Using a combined genomic and proteomic approach, we detected 57 immunogenic proteins of W. chondrophila, of which 17 were analysed by mass spectrometry. Two novel hypothetical proteins, Wim3 and Wim4, were expressed as recombinant proteins in Escherichia coli, purified and used as antigens in an ELISA test. Both proteins were recognized by sera of rabbits immunized with W. chondrophila as well as by human W. chondrophila positive sera but not by rabbit pre-immune sera nor human W. chondrophila negative sera. These results demonstrated that the approach chosen is suitable to identify immunogenic proteins that can be used to develop a serological test. This latter will be a valuable tool to further clarify the pathogenic potential of W. chondrophila.     

Chromatin as an expansive canvas for chemical biology

Chromatin is extensively chemically modified and thereby acts as a dynamic signaling platform controlling gene function. Chromatin regulation is integral to cell differentiation, lineage commitment and organism development, whereas chromatin dysregulation can lead to age-related and neurodegenerative disorders as well as cancer. Investigating chromatin biology presents a unique challenge, as the issue spans many disciplines, including cell and systems biology, biochemistry and molecular biophysics. In recent years, the application of chemical biology methods for investigating chromatin processes has gained considerable traction. Indeed, chemical biologists now have at their disposal powerful chemical tools that allow chromatin biology to be scrutinized at the level of the cell all the way down to the single chromatin fiber. Here we present recent examples of how this rapidly expanding palette of chemical tools is being used to paint a detailed picture of chromatin function in organism development and disease.     

High variation in foliage and leaf litter chemistry among 45 tree species of a neotropical rainforest community

Distinct ecosystem level carbon : nitrogen : phosphorus (C : N : P) stoichiometries in forest foliage have been suggested to reflect ecosystem-scale selection for physiological strategies in plant nutrient use. Here, this hypothesis was explored in a nutrient-poor lowland rainforest in French Guiana. Variation in C, N and P concentrations was evaluated in leaf litter and foliage from neighbour trees of 45 different species, and the litter concentrations of major C fractions were also measured. Litter C ranged from 45.3 to 52.4%, litter N varied threefold (0.68-2.01%), and litter P varied seven-fold (0.009-0.062%) among species. Compared with foliage, mean litter N and P concentrations decreased by 30% and 65%, respectively. Accordingly, the range in mass-based N : P shifted from 14 to 55 in foliage to 26 to 105 in litter. Resorption proficiencies indicated maximum P withdrawal in most species, but with a substantial increase in variation in litter P compared with foliage. These data suggest that constrained ecosystem-level C : N : P ratios do not preclude the evolution of highly diversified strategies of nutrient use and conservation among tropical rainforest tree species. The resulting large variation in litter quality will influence stoichiometric constraints within the decomposer food web, with potentially far-ranging consequences on nutrient dynamics and plant-soil feedbacks.     

Serial protein labeling with infrared maleimide dyes to identify cysteine modifications

Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification and quantification of growing importance. However, saturation labeling of thiols with fluorescent dyes results in poor protein recuperation and therefore requires the use of large quantities of starting material. This is especially important in sequential dye-labeling steps when applied for an identification of cysteine modifications. First, we studied the effects of different detergents during labeling procedure, i.e. Tween 20, Triton X-100 and CHAPS, on protein yield and composition. Tween 20 and Triton X-100 resulted in yields of around 50% labeled proteins compared to only 10% with PBS alone and a most diversified 2-DE protein pattern. Secondly, Tween 20 was used for serial protein labeling with maleimid fluorophores, first to conjugate to accessible thiols and after a reduction to label with another fluorophore previously masked di-sulphide and/or oxidized proteins in frontal cortex autopsy tissue of a subject with mild Alzheimer's disease. Two-DE DIGE revealed a complex protein pattern of readily labeled thiols and di-sulphide and/or oxidized proteins. Seventeen proteins were identified by MALDI-TOF and by peptide fingerprints. Several proteins were oxidized and involved in Alzheimer's disease. However methionine oxidation was prevalent. Infrared DIGE may provide an additional tool for an identification of oxidation susceptible proteins.     

Nest preference of laying hens (Gallus gallus domesticus) and their motivation to exert themselves to gain nest access

In connection with the development of a test method for the attractiveness and appropriateness of nests for laying hens, we carried out an investigation by using a preference test and motivation measuring with the help of push doors. Hens were offered two different nest sites either consisting of a tray filled with wood shavings (litter tray) or a wooden nest box plus wood shavings (nest box). Hens were individually housed in pens (2.0 m × 2.0 m) and had free access to the nest sites until they laid their 15th egg. From that day the hens had to overcome a push door to reach the nest sites. Resistance for entrance was also increased stepwise at the door leading to the hens’ nest. The experiment ended when a hen stopped to lay in her usual nest site for four consecutive days (postexperimental period). The behaviour during the last hour before oviposition was video taped at a level of resistance of 3.5, 6.0, 7.5 and 10.0 N.The hens were categorized into nest and litter layers depending on nest choice. All but one hen pushed maximum resistances between 11.5 and 18.0 N with no differences between nest and litter layers. Behaviour did not significantly change with increasing levels of resistance, but there were significant differences between nest and litter layers. Nest layers spent more time nesting than litter layers while the latter showed a strong tendency to more exploring behaviour. For litter layers, more entries through the push door leading to their nest site and more unsuccessful pushes were detected than for nest layers. According to our results, two types of layers could be distinguished whereas the two were equally motivated to access their nest site.     

No correlation of anthropometry and race performance in ultra-endurance swimmers at a 12-hours-swim

BACKGROUND: In swimmers, the effects of the anthropometric factors, upper extremity length, hand length and body height, on performance over 100 m have been shown, but no data exist about the influence of anthropometric factors on performance in ultra-endurance swimmers. SUBJECTS AND METHODS: Twelve male Caucasian ultra-swimmers participated in our study at the 9th edition in 2007 of the 12-hours-swim in Zurich, Switzerland. We determined body mass, length of arms and legs, body height, circumferences of extremities, skeletal muscle mass and fat mass in order to correlate with the covered distance and to find an effect on race performance. RESULTS: The 12 swimmers achieved an average distance of 29.4 +/- 5.1 km, varying from 22.8 km to 39.1 km during these 12 hours. There was no correlation between body mass, length of arms and legs, body height, circumferences of extremities, skeletal muscle mass and fat mass to race performance. CONCLUSION: In these 12 male ultra-endurance swimmers no effect of the anthropometric parameters body mass, body height, BMI, circumferences of extremities, length of arms and legs, skeletal muscle mass and fat mass, on performance in a 12-hours-swim has been found.     

Regulation and Adaptive Evolution of Lactose Operon Expression in Lactobacillus delbrueckii

Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis are both used in the dairy industry as homofermentative lactic acid bacteria in the production of fermented milk products. After selective pressure for the fast fermentation of milk in the manufacture of yogurts, L. delbrueckii subsp. bulgaricus loses its ability to regulate lac operon expression. A series of mutations led to the constitutive expression of the lac genes. A complex of insertion sequence (IS) elements (ISL4 inside ISL5), inserted at the border of the lac promoter, induced the loss of the palindromic structure of one of the operators likely involved in the binding of regulatory factors. A lac repressor gene was discovered downstream of the beta-galactosidase gene of L. delbrueckii subsp. lactis and was shown to be inactivated by several mutations in L. delbrueckii subsp. bulgaricus. Regulatory mechanisms of the lac gene expression of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis were compared by heterologous expression in Lactococcus lactis of the two lac promoters in front of a reporter gene (beta-glucuronidase) in the presence or absence of the lac repressor gene. Insertion of the complex of IS elements in the lac promoter of L. delbrueckii subsp. bulgaricus increased the promoter's activity but did not prevent repressor binding; rather, it increased the affinity of the repressor for the promoter. Inactivation of the lac repressor by mutations was then necessary to induce the constitutive expression of the lac genes in L. delbrueckii subsp. bulgaricus.     

Leaf-atmosphere NH(3) exchange of white clover (Trifolium repens L.) in relation to mineral N nutrition and symbiotic N(2) fixation.

Plant-atmosphere NH(3) exchange was studied in white clover (Trifolium repens L. cv. Seminole) growing in nutrient solution containing 0 (N(2) based), 0.5 (low N) or 4.5 (high N) mM NO(3)(-). The aim was to show whether the NH(3) exchange potential is influenced by the proportion of N(2) fixation relative to NO(3)(-) supply. During the treatment, inhibition of N(2) fixation by NO(3)(-) was followed by in situ determination of total nitrogenase activity (TNA), and stomatal NH(3) compensation points (chi(NH(3))) were calculated on the basis of apoplastic NH4(+) concentration ([NH4(+)]) and pH. Whole-plant NH(3) exchange, transpiration and net CO(2) exchange were continuously recorded with a controlled cuvette system. Although shoot total N concentration increased with the level of mineral N application, tissue and apoplastic [NH4(+)] as well as chi(NH(3)) were equal in the three treatments. In NH(3)-free air, net NH(3) emission rates of <1 nmol m(-2) s(-1) were observed in both high-N and N(2)-based plants. When plants were supplied with air containing 40 nmol mol(-1) NH(3), the resulting net NH(3) uptake was higher in plants which acquired N exclusively from symbiotic N(2) fixation, compared to NO(3)(-) grown plants. The results indicate that symbiotic N(2) fixation and mineral N acquisition in white clover are balanced with respect to the NH4(+) pool leading to equal chi(NH(3)) in plants growing with or without NO(3)(-). At atmospheric NH(3) concentrations exceeding chi(NH(3)), the NH(3) uptake rate is controlled by the N demand of the plants.