Mapping of B-cell epitopes in the N-terminal repeated peptides of Anaplasma marginale major surface protein 1a and characterization of the humoral immune response of cattle immunized with recombinant and whole organism antigens

Major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: Anaplasmataceae) together with MSP1b forms the MSP1 complex. MSP1a has been shown to be involved in adhesion, infection and tick transmission of A. marginale, as well as to contribute to protective immunity in cattle. A differential antibody response to MSP1a and MSP1b was observed in cattle immunized with A. marginale derived from bovine erythrocytes (anti-MSP1a response) or cultured tick cells (anti-MSP1b response). In this study, we further characterized the MSP1a antibody response of cattle using several immunogens, including recombinant MSP1a (rMSP1a) protein, erythrocyte- or tick cell culture-derived A. marginale, or a combination of tick cell culture-derived A. marginale and rMSP1a. The MSP1a antibody response to all these immunogens was directed primarily against the N-terminal region of MSP1a that contains tandemly repeated peptides, whereas low antibody levels were detected against the C-terminal portion. Linear B-cell epitopes of MSP1a were mapped using synthetic peptides representing the entire sequence of the protein that were prepared by SPOT synthesis technology. Only two peptides in the N-terminal repeats were recognized by sera from immunized cattle. These peptides shared the sequence SSAGGQQQESS, which is likely to contain the linear B-cell epitope that was recognized by the pools of bovine sera. The average differential of antibody titers against MSP1a minus those against MSP1b correlated with lower percent reductions in PCV. A preferential antibody response to MSP1a was observed in cattle immunized with erythrocyte-derived, cell culture-derived plus rMSP1a or rMSP1a alone, and the percent reduction PCV was significantly lower in these cattle as compared with the other immunization groups. These results provide insight into the bovine antibody response against A. marginale and the role of MSP1a in protection of cattle against A. marginale infection.     

Growth versus storage: responses of Mediterranean oak seedlings to changes in nutrient and water availabilities

We compare dry mass (DM) and storage of starch (St) and nitrogen (N) in seedlings of three Mediterranean oaks, two evergreens (Quercus coccifera L. and Q. ilex L. subsp. ballota (Desf.) Samp) and one deciduous (Q. faginea Lam.), across different scenarios of nutrient and water availabilities. Three fertilization (5, 50 and 200 mg of N per plant and growing period) and watering (28–39, 55–71 and 70–85 g H₂O 100 g _soil ^?1 gravimetric soil water) treatments were applied to current-year seedlings between May and October 2002 in two independent experiments. The three species showed a similar response to fertilization, storing nitrogen instead of increasing biomass, in agreement with adaptations to nutrient-poor habitats. However, they differed in their responses to water, reflecting the different water requirements in the field: Q. coccifera, from arid zones, showed no response to water regarding DM and St; Q. faginea, from humid zones, required higher water availability to simultaneously increase growth and storage; while Q. ilex, spanning over most of the water availability range, exhibited a balanced increase of both functions when water increased moderately. In the two evergreen species, N concentration increased with water supply, whereas the reverse occurred in Q. faginea. The latter species favoured growth over storage at moderate water supply (according to its more competitive strategy), although it was the species which accumulated more St and N at the end of the experiments (autumn).     

Interactions between physical and biotic factors influence CO2 flux in Antarctic dry valley soils

Soil carbon dioxide (CO₂) flux is an integrative measure of ecosystem functioning representing both biotic and physical controls over carbon (C) balance. In the McMurdo Dry Valleys of Antarctica, soil CO₂ fluxes (approximately −0.1-0.15 μmol m⁻² s⁻¹) are generally low, and negative fluxes (uptake of CO₂) are sometimes observed. A combination of biological respiration and physical mechanisms, driven by temperature and mediated by soil moisture and mineralogy, determine CO₂ flux and, therefore, soil organic C balance. The physical factors important to CO₂ flux are being altered with climate variability in many ecosystems including arid forms such as the Antarctic terrestrial ecosystems, making it critical to understand how climate factors interact with biotic drivers to control soil CO₂ fluxes and C balances. We measured soil CO₂ flux in experimental field manipulations, microcosm incubations and across natural environmental gradients of soil moisture to estimate biotic soil respiration and abiotic sources of CO₂ flux in soils over a range of physical and biotic conditions. We determined that temperature fluctuations were the most important factor influencing diel variation in CO₂ flux. Variation within these diel CO₂ cycles was explained by differences in soil moisture. Increased temperature (as opposed to temperature fluctuations) had little or no effect on CO₂ flux if moisture was not also increased. We conclude that CO₂ flux in dry valley soils is driven primarily by physical factors such as soil temperature and moisture, indicating that future climate change may alter the dry valley soil C cycle. Negative CO₂ fluxes in arid soils have recently been identified as potential net C sinks. We demonstrate the potential for arid polar soils to take up CO₂, driven largely by abiotic factors associated with climate change. The low levels of CO₂ absorption into soils we observed may not constitute a significant sink of atmospheric CO₂, but will influence the interpretation of CO₂ flux for the dry valley soil C cycle and possibly other arid environments where biotic controls over C cycling are secondary to physical drivers.     

Acclimation of cuttings from different willow genotypes to flooding depth level

Climate change will increase the frequency of extreme rain events, causing more flooding episodes. Willows are usually planted in marginal lands like flood prone areas. For willow plantations developed from rootless cuttings, the establishment phase is crucial, because the cuttings are still developing a shoot and root system and have a higher vulnerability to stress. A flooding episode during this early period may have a negative effect upon plants. We analyzed the responses to flooding of eight willow genotypes, representing important species from the economic and ecological point of view (Salix alba, S. matsudana, S. amygdaloides and S. matsudana × S. nigra hybrids). The treatments started when the plants were 2 months old and lasted for 3 weeks. They were identified as: Control (watered to field capacity); F10 (plants submerged 10 cm above soil surface) and F50 (plants submerged 50 cm above soil surface). The F50 treatment showed a greater growth reduction than the F10 treatment in most clones, either measured in height, diameter or total biomass. Both flooding treatments reduced significantly the root-to-shoot ratio compared to control plants. The F50 treatment increased the leaf nitrogen content and specific leaf area in all genotypes. Both treatments changed plant and leaf traits in different ways according to the depth of the floodwater. These changes may have lasting effects on growth recovery in the post-flooding period. Willow clones with a fast initial growth will be the best option to minimize growth reduction in areas prone to experience flooding episodes.     

Detecting interaction(s) and assessing the impact of component subsets in a chemical mixture using fixed-ratio mixture ray designs

An important environmental and regulatory issue is the protection of human health from potential adverse effects of cumulative exposure to multiple chemicals. Earlier literature suggested restricting inference to specific fixed-ratio rays of interest. Based on appropriate definitions of additivity, single chemical data are used to predict the relationship among the chemicals under the zero-interaction case. Parametric comparisons between the additivity model and the model fit along the fixed-ratio ray(s) are used to detect departure from additivity. Collection of data along reduced fixed-ratio rays, where subsets of chemicals of interest are removed from the mixture and the remaining compounds are at the same relative ratios as considered in the full ray, allow researchers to make inference about the effect of the removed chemicals. Methods for fitting simultaneous confidence bands about the difference between the best fitting model and the model predicted under additivity are developed to identify regions along the rays where significant interactions occur. This general approach is termed the “single chemicals required” (SCR) method of analysis. A second approach, termed “single chemicals not required” (SCNR) method of analysis, is based on underlying assumptions about the parameterization of the response surface. Under general assumptions, polynomial terms for models fit along fixed-ratio rays are associated with interaction terms. Consideration is given to the case where only data along the mixture rays are available. Tests of hypotheses, which consider interactions due to subsets of chemicals, are also developed.     

Rapid wastage of Alaska glaciers and their contribution to rising sea level

We have used airborne laser altimetry to estimate volume changes of 67 glaciers in Alaska from the mid-1950s to the mid-1990s. The average rate of thickness change of these glaciers was -0.52 m/year. Extrapolation to all glaciers in Alaska yields an estimated total annual volume change of -52 +/- 15 km3/year (water equivalent), equivalent to a rise in sea level (SLE) of 0.14 +/- 0.04 mm/year. Repeat measurements of 28 glaciers from the mid-1990s to 2000-2001 suggest an increased average rate of thinning, -1.8 m/year. This leads to an extrapolated annual volume loss from Alaska glaciers equal to -96 +/- 35 km3/year, or 0.27 +/- 0.10 mm/year SLE, during the past decade. These recent losses are nearly double the estimated annual loss from the entire Greenland Ice Sheet during the same time period and are much higher than previously published loss estimates for Alaska glaciers. They form the largest glaciological contribution to rising sea level yet measured.     

Ordered and dynamic assembly of single spliceosomes

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.