The Interconversion between a Flexible β-Sheet and a Fibril β-Arrangement Constitutes the Main Conformational Event during Misfolding of PSD95-PDZ3 Domain

The temperature-induced misfolding pathway of PDZ3, the third PDZ domain of the PSD95 neuronal protein, is populated by a trimeric β-sheet-rich intermediate state that leads to a stepwise and reversible formation of supramacromolecular structures. Using FTIR, we have found that misfolding of this pathway is not due to different ensembles of a variety of precursors, but comes mainly from the interconversion of a flexible β-sheet of the domain to wormlike fibrils. The appearance of the wormlike fibril FTIR component is also accompanied by a slight decrease of the band that corresponds to loops in the native state, whereas the rest of the regular elements of secondary structure are fairly well maintained upon misfolding. Transmission electron microscope micrographs have confirmed the presence of wormlike fibrils upon heating at 60°C, where the trimeric intermediate is maximally populated. Toxicity assays in the human neuroblastoma cell line SH-SY5Y show that cytotoxicity increases as the aggregation pathway proceeds. NMR analysis of chemical shifts as a function of temperature has revealed, as one of the main conformational aspects of such an interconversion at the residue level, that the β-sheet arrangement around strand β3 promotes the change that drives misfolding of the PDZ3 domain.     

Climate change effects on above- and below-ground interactions in a dryland ecosystem

Individual species respond to climate change by altering their abundance, distribution and phenology. Less is known, however, about how climate change affects multitrophic interactions, and its consequences for food-web dynamics. Here, we investigate the effect of future changes in rainfall patterns on detritivore–plant–herbivore interactions in a semiarid region in southern Spain by experimentally manipulating rainfall intensity and frequency during late spring–early summer. Our results show that rain intensity changes the effect of below-ground detritivores on both plant traits and above-ground herbivore abundance. Enhanced rain altered the interaction between detritivores and plants affecting flower and fruit production, and also had a direct effect on fruit and seed set. Despite this finding, there was no net effect on plant reproductive output. This finding supports the idea that plants will be less affected by climatic changes than by other trophic levels. Enhanced rain also affected the interaction between detritivores and free-living herbivores. The effect, however, was apparent only for generalist and not for specialist herbivores, demonstrating a differential response to climate change within the same trophic level. The complex responses found in this study suggest that future climate change will affect trophic levels and their interactions differentially, making extrapolation from individual species'' responses and from one ecosystem to another very difficult.     

Genetic analysis of the individual contribution to virulence of the type III effector inventory of Pseudomonas syringae pv. phaseolicola

Several reports have recently contributed to determine the effector inventory of the sequenced strain Pseudomonas syringae pv. phaseolicola (Pph) 1448a. However, the contribution to virulence of most of these effectors remains to be established. Genetic analysis of the contribution to virulence of individual P. syringae effectors has been traditionally hindered by the lack of phenotypes of the corresponding knockout mutants, largely attributed to a high degree of functional redundancy within their effector inventories. In support of this notion, effectors from Pseudomonas syringae pv. tomato (Pto) DC3000 have been classified into redundant effector groups (REGs), analysing virulence of polymutants in the model plant Nicotiana benthamiana. However, using competitive index (CI) as a virulence assay, we were able to establish the individual contribution of AvrPto1(Pto) (DC3000) to Pto DC3000 virulence in tomato, its natural host, even though typically, contribution to virulence of AvrPto1 is only shown in strains also lacking AvrPtoB (also called HopAB2), a member of its REG. This report raised the possibility that even effectors targeting the same defence signalling pathway may have an individual contribution to virulence, and pointed out to CI assays as the means to establish such a contribution for individual effectors. In this work, we have analysed the individual contribution to virulence of the majority of previously uncharacterised Pph 1448a effectors, by monitoring the development of disease symptoms and determining the CI of single knockout mutants at different stages of growth within bean, its natural host. Despite their potential functional redundancy, we have found individual contributions to virulence for six out of the fifteen effectors analysed. In addition, we have analysed the functional relationships between effectors displaying individual contribution to virulence, highlighting the diversity that these relationships may present, and the interest of analysing their functions within the context of the infection.     

Administration of triclabendazole is safe and effective in controlling fascioliasis in an endemic community of the bolivian altiplano

Background

The Bolivian northern Altiplano is characterized by a high prevalence of Fasciola hepatica infection. In order to assess the feasibility, safety and efficacy of large-scale administration of triclabendazole as an appropriate public health measure to control morbidity associated with fascioliasis, a pilot intervention was implemented in 2008.

Materials and Methods

Schoolchildren from an endemic community were screened for fascioliasis and treated with a single administration of triclabendazole (10 mg/kg). Interviews to assess the occurrence of adverse events were conducted on treatment day, one week later, and one month after treatment. Further parasitological screenings were performed three months after treatment and again two months later (following a further treatment) in order to evaluate the efficacy of the intervention.

Results

Ninety infected children were administered triclabendazole. Adverse events were infrequent and mild. No serious adverse events were reported. Observed cure rates were 77.8% after one treatment and 97.8% after two treatments, while egg reduction rates ranged between 74% and 90.3% after one treatment, and between 84.2% and 99.9% after two treatments. The proportion of high-intensity infections (≥400 epg) decreased from 7.8% to 1.1% after one treatment and to 0% after two treatments.

Conclusion

Administration of triclabendazole is a feasible, safe and efficacious public health intervention in an endemic community in the Bolivian Altiplano, suggesting that preventive chemotherapy can be applied to control of fascioliasis. Further investigations are needed to define the most appropriate frequency of treatment.     

An alginate-layer technique for culture of Brassica oleracea L. protoplasts

Ten accessions belonging to the Brassica oleracea subspecies alba and rubra, and to B. oleracea var. sabauda were used in this study. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants. The influence of selected factors on the yield, viability, and mitotic activity of protoplasts immobilized in calcium alginate layers was investigated. The efficiency of protoplast isolation from hypocotyls was lower (0.7 ± 0.1 × 10⁶ ml⁻¹) than for protoplasts isolated from leaf mesophyll tissue (2 ± 0.1 × 10⁶ ml⁻¹). High (70–90%) viabilities of immobilized protoplasts were recorded, independent of the explant sources. The highest proportion of protoplasts undergoing divisions was noted for cv. Reball F1, both from mesophyll (29.8 ± 2.2%) and hypocotyl (17.5 ± 0.3%) tissues. Developed colonies of callus tissue were subjected to regeneration and as a result plants from six accessions were obtained.     

An error analysis for two-state protein-folding kinetic parameters and phi-values: progress toward precision by exploring pH dependencies on Leffler plots

The interpretation of φ-values has led to an understanding of the folding transition state ensemble of a variety of proteins. Although the main guidelines and equations for calculating φ are well established, there remains some controversy about the quality of the numerical values obtained. By analyzing a complete set of results from kinetic experiments with the SH3 domain of α-spectrin (Spc-SH3) and applying classical error methods and error-propagation formulas, we evaluated the uncertainties involved in two-state-folding kinetic experimental parameters and the corresponding calculated φ-values. We show that kinetic constants in water and m values can be properly estimated from a judicious weighting of fitting errors and describe some procedures to calculate the errors in Gibbs energies and φ-values from a traditional two-point Leffler analysis. Furthermore, on the basis of general assumptions made with the protein engineering method, we show how to generate multipoint Leffler plots via the analysis of pH dependencies of kinetic parameters. We calculated the definitive φ-values for a collection of single mutations previously designed to characterize the folding transition state of the α-spectrin SH3 domain. The effectiveness of the pH-scanning procedure is also discussed in the context of error analysis. Judging from the magnitudes of the error bars obtained from two-point and multipoint Leffler plots, we conclude that the precision obtained for φ-values should be ∼25%, a reasonable limit that takes into account the propagation of experimental errors.     

Unraveling the mechanisms of tryptophan fluorescence quenching in the triosephosphate isomerase from Giardia lamblia

In the native state several proteins exhibit a quenching of fluorescence of their tryptophans. We studied triosephosphate isomerase from Giardia lamblia (GlTIM) to dissect the mechanisms that account for the quenching of fluorescence of its Trp. GlTIM contains four Trp per monomer (Trp75, Trp162, Trp173, and Trp196) distributed throughout the 3D structure. The fluorescence of the denatured enzyme is 3-fold higher than that of native GlTIM. To ascertain the origin of this phenomenon, single and triple mutants of Trp per Phe were made. The intrinsic fluorescence was determined, and the data were interpreted on the basis of the crystal structure of the enzyme. Our data show that the fluorescence of all Trp residues is quenched through two different mechanisms. In one, fluorescence is quenched by aromatic-aromatic interactions due to the proximity and orientation of the indole groups of Trp196 and Trp162. The magnitude of the quenching of fluorescence in Trp162 is higher than in the other three Trp. Fluorescence quenching is also due to energy transfer to the charged residues that surround Trp 75, 173 and 196. Further analysis of the fluorescence of GlTIM showed that, among TIMs from other parasites, Trp at position 12 exhibits rather unique properties.     

Evidence for cooperative effects in the binding of polyvalent metal ions to pure phosphatidylcholine bilayer vesicle surfaces

An internal NMR monitor for the study of lanthanide ion (Ln³⁺) binding to phospholipid bilayer membranes has been developed. The dimethylphosphate anion, DMP^?, forms labile complexes with Ln³⁺ in aqueous solution and in solutions also containing bilayer dispersions. The hyperfine shift in the DMP^? resonance induced by Pr³⁺ ions has been used to determine the overall thermodynamic formation constants for the Pr(DMP)²⁺ and Pr(DMP)₂⁺ complexes: 81 (M^?1) and 349 (M^?2) at 52°C; the limiting hyperfine shift (³¹P) at 52°C is 91.5 ppm downfield. These parameters, applied to the observed DMP^? hyperfine shift in the presence of the membrane, establish both the free Pr³⁺ concentration and the amount of Pr³⁺ bound to the phospholipid surface. Extensive data for the binding of Pr³⁺ to the outer surfaces of sonicated vesicles yield a limiting hyperfine shift per Pr³⁺ of 181.6 ppm downfield for the dipalmitoylphosphatidylcholine ³¹P resonance at 52°C, clearly demonstrating that the binding stoichiometry is two DPPCs per Pr³⁺. A Hill analysis indicates that the binding data are more anti-cooperative than a realistic Langmuir isotherm, yet more cooperative than a Stern isotherm incorporating electrostatic considerations at the Debye-Hückel level. Fittings to specific models lead to a cooperative model in which tense (T) sites, with low affinity for Pr³⁺, present in the absence of metal ions, quickly give way to relaxed (R) sites (two DPPCs per site), with much higher affinity for Pr³⁺, as the amount of Pr³⁺ bound to the surface increases. The intrinsic equilibrium constants for the binding of Pr³⁺ to DPPC vesicles are 2 M^?1 and 3 000 M^?1 for the T and R sites, respectively, at 52°C. The distribution coefficient between these sites ([R]/[T]) in the absence of Ln³⁺ is 0.14 at 52°C. We picture the binding site conversion as a head-group conformational change involving mostly the choline moiety. Sketchy results for binding on the inside vesicle surface indicate that the overall affinity for Pr³⁺ is significantly greater and suggest that the site stoichiometry may be different.