The reduced, denatured somatomedin B domain of vitronectin refolds into a stable, biologically active molecule

The high-affinity binding site in human vitronectin (VN) for plasminogen activator inhibitor-1 (PAI-1) has been localized to the NH(2)-terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44). A number of published structural and biochemical studies show conflicting results for the disulfide bonding pattern and the overall fold of the SMB domain, possibly because this domain may undergo disulfide shuffling and/or conformational changes during handling. Here we show that bacterially expressed recombinant SMB (rSMB) can be refolded to a single form that shows maximal activity in binding to PAI-1 and to a conformation-dependent monoclonal antibody (mAb 153). The oxidative refolding pathway of rSMB can be followed in the presence of glutathione redox buffers. This approach allowed the isolation and analysis of a number of intermediate folding species and of the final stably folded species at equilibrium. Competitive surface plasmon resonance analysis demonstrated that the stably refolded rSMB regained biological activity since it bound efficiently to PAI-1 and to mAb 153. In contrast, none of the folding intermediates bound to PAI-1 or to mAb 153. We also show by NMR analysis that the stably refolded rSMB is identical to the material used for the solution structure determination [Kamikubo et al. (2004) Biochemistry 43, 6519] and that it binds specifically to mAb 153 via an interface that includes the three aromatic side chains previously implicated in binding to PAI-1.     

Depletion of phosphatidylcholine in yeast induces shortening and increased saturation of the lipid acyl chains: evidence for regulation of intrinsic membrane curvature in a eukaryote

To study the consequences of depleting the major membrane phospholipid phosphatidylcholine (PC), exponentially growing cells of a yeast cho2opi3 double deletion mutant were transferred from medium containing choline to choline-free medium. Cell growth did not cease until the PC level had dropped below 2% of total phospholipids after four to five generations. Increasing contents of phosphatidylethanolamine (PE) and phosphatidylinositol made up for the loss of PC. During PC depletion, the remaining PC was subject to acyl chain remodeling with monounsaturated species replacing diunsaturated species, as shown by mass spectrometry. The remodeling of PC did not require turnover by the SPO14-encoded phospholipase D. The changes in the PC species profile were found to reflect an overall shift in the cellular acyl chain composition that exhibited a 40% increase in the ratio of C16 over C18 acyl chains, and a 10% increase in the degree of saturation. The shift was stronger in the phospholipid than in the neutral lipid fraction and strongest in the species profile of PE. The shortening and increased saturation of the PE acyl chains were shown to decrease the nonbilayer propensity of PE. The results point to a regulatory mechanism in yeast that maintains intrinsic membrane curvature in an optimal range.     

Plasticity as a plastic response: how submergence-induced leaf elongation in Rumex palustris depends on light and nutrient availability in its early life stage

Plants may experience different environmental cues throughout their development which interact in determining their phenotype. This paper tests the hypothesis that environmental conditions experienced early during ontogeny affect the phenotypic response to subsequent environmental cues. This hypothesis was tested by exposing different accessions of Rumex palustris to different light and nutrient conditions, followed by subsequent complete submergence. Final leaf length and submergence-induced plasticity were affected by the environmental conditions experienced at early developmental stages. In developmentally older leaves, submergence-induced elongation was lower in plants previously subjected to high-light conditions. Submergence-induced elongation of developmentally younger leaves, however, was larger when pregrown in high light. High-light and low-nutrient conditions led to an increase of nonstructural carbohydrates in the plants. There was a positive correlation between submergence-induced leaf elongation and carbohydrate concentration and content in roots and shoots, but not with root and shoot biomass before submergence. These results show that conditions experienced by young plants modulate the responses to subsequent environmental conditions, in both magnitude and direction. Internal resource status interacts with cues perceived at different developmental stages in determining plastic responses to the environment.     

Marburg Virus VP35 Can Both Fully Coat the Backbone and Cap the Ends of dsRNA for Interferon Antagonism

Filoviruses, including Marburg virus (MARV) and Ebola virus (EBOV), cause fatal hemorrhagic fever in humans and non-human primates. All filoviruses encode a unique multi-functional protein termed VP35. The C-terminal double-stranded (ds)RNA-binding domain (RBD) of VP35 has been implicated in interferon antagonism and immune evasion. Crystal structures of the VP35 RBD from two ebolaviruses have previously demonstrated that the viral protein caps the ends of dsRNA. However, it is not yet understood how the expanses of dsRNA backbone, between the ends, are masked from immune surveillance during filovirus infection. Here, we report the crystal structure of MARV VP35 RBD bound to dsRNA. In the crystal structure, molecules of dsRNA stack end-to-end to form a pseudo-continuous oligonucleotide. This oligonucleotide is continuously and completely coated along its sugar-phosphate backbone by the MARV VP35 RBD. Analysis of dsRNA binding by dot-blot and isothermal titration calorimetry reveals that multiple copies of MARV VP35 RBD can indeed bind the dsRNA sugar-phosphate backbone in a cooperative manner in solution. Further, MARV VP35 RBD can also cap the ends of the dsRNA in solution, although this arrangement was not captured in crystals. Together, these studies suggest that MARV VP35 can both coat the backbone and cap the ends, and that for MARV, coating of the dsRNA backbone may be an essential mechanism by which dsRNA is masked from backbone-sensing immune surveillance molecules.     

Unravelling below-ground plant distributions: a real-time polymerase chain reaction method for quantifying species proportions in mixed root samples

Knowledge on below-ground plant distributions is almost lacking to date, despite the fact that such information would be very valuable in understanding below-ground competition and species-specific interactions, processes that are expected to shape community structure. Methods available so far for below-ground species determination have drawbacks that we tried to challenge. Some methods make use of differences in the chemical composition between species, but this is highly variable upon environmental factors. DNA-based techniques - far less dependent on chemical composition - such as polymerase chain reaction on internal transcribed spacer (ITS) primers can so far only determine presence-absence of a species in a mixed root sample. Here, we present a quantitative DNA-based technique that allows investigation of relative species abundances in experimental mixed root samples. We used quantitative real-time polymerase chain reaction (PCR) on species-specific markers obtained from intersimple sequence repeat (ISSR) analyses in root samples. This molecular technique is novel in the field of root ecology and its development overcame three challenges: (i) determination of species-specific DNA fragments, (ii) development and optimization of the real time PCR protocol, (iii) designing a data treatment method based on a modified delta-delta-cycle threshold (CT) analysis. The method gained robustness from using relative DNA abundances in species mixtures rather than absolute concentration readings. This requires accurate multispecies reference series as a calibration. Test samples with different known biomass ratios of all species showed proof of concept of this method. The pro's and contra's of this method are discussed in the light of its contribution to advancing ecological research on below-ground plant-plant interactions.     

Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS

Previously, a 2D gel electrophoresis approach was developed for the Escherichia coli inner membrane, which detects membrane protein complexes that are stable in sodium dodecyl sulfate (SDS) at room temperature, and dissociate under the influence of trifluoroethanol [R. E. Spelbrink et al., J. Biol. Chem. 280 (2005), 28742-8]. Here, the method was applied to the evolutionarily related mitochondrial inner membrane that was isolated from the yeast Saccharomyces cerevisiae. Surprisingly, only very few proteins were found to be dissociated by trifluoroethanol of which Lpd1p, a component of multiple protein complexes localized in the mitochondrial matrix, is the most prominent. Usage of either milder or more stringent conditions did not yield any additional proteins that were released by fluorinated alcohols. This strongly suggests that membrane protein complexes in yeast are less stable in SDS solution than their E. coli counterparts, which might be due to the overall reduced hydrophobicity of mitochondrial transmembrane proteins.     

Improving the scale and precision of hypotheses to explain root foraging ability

Background

Numerous hypotheses have been proposed to explain the wide variation in the ability of plants to forage for resources by proliferating roots in soil nutrient patches. Comparative analyses have found little evidence to support many of these hypotheses, raising the question of what role resource-foraging ability plays in determining plant fitness and community structure.

Scope

In the present viewpoint, we respond to Grime''s (2007; Annals of Botany 99: 1017–1021) suggestion that we misinterpreted the scope of the scale–precision trade-off hypothesis, which states that there is a trade-off between the spatial scale over which plant species forage and the precision with which they are able to proliferate roots in resource patches. We use a meta-analysis of published foraging scale–precision correlations to demonstrate that there is no empirical support for the scale–precision trade-off hypothesis. Based on correlations between foraging precision and various plant morphological and ecophysiological traits, we found that foraging precision forms part of the ‘fast’ suite of plant traits related to rapid growth rates and resource uptake rates.

Conclusions

We suggest there is a need not only to examine correlations between foraging precision and other plant traits, but to expand our notion of what traits might be important in determining the resource-foraging ability of plants. By placing foraging ability in the broader context of plant traits and resource economy strategies, it will be possible to develop a new and empirically supported framework to understand how plasticity in resource uptake and allocation affect plant fitness and community structure.Key words: Root foraging, phenotypic plasticity, scale, precision, resource uptake strategies, traits     

The influence of savanna trees on nutrient,water and light availability and the understorey vegetation

In an East African savanna herbaceous layer productivity and species composition were studied around Acacia tortilis trees of three different age classes, as well as around dead trees and in open grassland patches. The effects of trees on nutrient, light and water availability were measured to obtain an insight into which resources determine changes in productivity and composition of the herbaceous layer. Soil nutrient availability increased with tree age and size and was lowest in open grassland and highest under dead trees. The lower N:P ratios of grasses from open grassland compared to grasses from under trees suggested that productivity in open grassland was limited by nitrogen, while under trees the limiting nutrient was probably P. N:P ratios of grasses growing under bushes and small trees were intermediate between large trees and open grassland indicating that the understorey of Acacia trees seemed to change gradually from a N-limited to a P-limited vegetation. Soil moisture contents were lower under than those outside of canopies of large Acacia trees suggesting that water competition between trees and grasses was important. Species composition of the herbaceous layer under Acacia trees was completely different from the vegetation in open grassland. Also the vegetation under bushes of Acacia tortilis was different from both open grassland and the understorey of large trees. The main factor causing differences in species composition was probably nutrient availability because species compositions were similar for stands of similar soil nutrient concentrations even when light and water availability was different. Changes in species composition did not result in differences in above-ground biomass, which was remarkably similar under different sized trees and in open grassland. The only exception was around dead trees where herbaceous plant production was 60% higher than under living trees. The results suggest that herbaceous layer productivity did not increase under trees by a higher soil nutrient availability, probably because grass production was limited by competition for water. This was consistent with the high plant production around dead trees because when trees die, water competition disappears but the high soil nutrient availability remains. Hence, in addition to tree soil nutrient enrichment, below-ground competition for water appears to be an important process regulating tree-grass interactions in semi-arid savanna.