Identification of olive (Olea europaea) seed and pulp proteins by nLC-MS/MS via combinatorial peptide ligand libraries

Different types of extraction protocols are described for identifying proteins in seed and pulp of olive (Olea europea), by employing both conventional extraction methods and capture with ProteoMiner as well as with in house-made combinatorial peptide ligand libraries (HM-CPLLs) at pH 7.4 and at pH 2.2. Thanks to the use of CPLLs, able to dramatically amplify the signal of low-abundance species, a quite large number of compounds has been indeed identified: 61 in the seed (vs. only four reported in current literature) and 231 in the pulp (vs. 56 described so far), the deepest investigation up to the present of the olive proteome. In the seed, it highlights the presence of seed storage proteins, oleosins and histones. In the pulp, the allergenic thaumatin-like protein (Ole e 13) was confirmed, among the other 231, as the most abundant protein in the olive pulp. The present research has also been undertaken with the aim of identifying proteins in olive oil and ascertaining the relative contribution of seed and pulp proteins in their presence, if any, in oils.     

Probing a polar cluster in the retinal binding pocket of bacteriorhodopsin by a chemical design approach

Bacteriorhodopsin has a polar cluster of amino acids surrounding the retinal molecule, which is responsible for light harvesting to fuel proton pumping. From our previous studies, we have shown that threonine 90 is the pivotal amino acid in this polar cluster, both functionally and structurally. In an attempt to perform a phenotype rescue, we have chemically designed a retinal analogue molecule to compensate the drastic effects of the T90A mutation in bacteriorhodopsin. This analogue substitutes the methyl group at position C(13) of the retinal hydrocarbon chain by and ethyl group (20-methyl retinal). We have analyzed the effect of reconstituting the wild-type and the T90A mutant apoproteins with all-trans-retinal and its 20-methyl derivative (hereafter, 13-ethyl retinal). Biophysical characterization indicates that recovering the steric interaction between the residue 90 and retinal, eases the accommodation of the chromophore, however it is not enough for a complete phenotype rescue. The characterization of these chemically engineered chromoproteins provides further insight into the role of the hydrogen bond network and the steric interactions involving the retinal binding pocket in bacteriorhodopsin and other microbial sensory rhodopsins.     

Three-dimensional reconstruction of tarantula myosin filaments suggests how phosphorylation may regulate myosin activity

Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLCs). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free head and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens revealed that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal in this study was to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction revealed intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC and fitted an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 (subfragment 2) crystal structure to the reconstruction. The fitting suggests one intramolecular interaction, between the cardiomyopathy loop of the free head and its own S2, and two intermolecular interactions, between the cardiac loop of the free head and the essential light chain of the blocked head and between the Leu305-Gln327 interaction loop of the free head and the N-terminal fragment of the RLC of the blocked head. These interactions, added to those previously described, would help switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament.     

Influence of proline on the thermostability of the active site and membrane arrangement of transmembrane proteins

Proline residues play a fundamental and subtle role in the dynamics, structure, and function in many membrane proteins. Temperature derivative spectroscopy and differential scanning calorimetry have been used to determine the effect of proline substitution in the structural stability of the active site and transmembrane arrangement of bacteriorhodopsin. We have analyzed the Pro-to-Ala mutation for the helix-embedded prolines Pro⁵⁰, Pro⁹¹, and Pro¹⁸⁶ in the native membrane environment. This information has been complemented with the analysis of the respective crystallographic structures by the FoldX force field. Differential scanning calorimetry allowed us to determine distorted membrane arrangement for P50A and P186A. The protein stability was severely affected for P186A and P91A. In the case of Pro⁹¹, a single point mutation is capable of strongly slowing down the conformational diffusion along the denaturation coordinate, becoming a barrier-free downhill process above 371 K. Temperature derivative spectroscopy, applied for first time to study thermal stability of proteins, has been used to monitor the stability of the active site of bacteriorhodopsin. The mutation of Pro⁹¹ and Pro¹⁸⁶ showed the most striking effects on the retinal binding pocket. These residues are the Pro in closer contact to the active site (activation energies for retinal release of 60.1 and 76.8 kcal/mol, respectively, compared to 115.8 kcal/mol for WT). FoldX analysis of the protein crystal structures indicates that the Pro-to-Ala mutations have both local and long-range effects on the structural stability of residues involved in the architecture of the protein and the active site and in the proton pumping function. Thus, this study provides a complete overview of the substitution effect of helix-embedded prolines in the thermodynamic and dynamic stability of a membrane protein, also related to its structure and function.     

Gamma-lactones alpha,beta- and beta,gamma-fused to carbocycles as novel antiproliferative drugs

A set of gamma-lactones alpha,beta-fused and beta,gamma-fused to carbocycles have been synthesized and evaluated for their in vitro antiproliferative activities using the human cancer cell lines SW1573 (lung), T-47D (breast) and WiDr (colon). The compounds are obtained by intramolecular ring closing metathesis of the corresponding dienes. Active compounds exhibited GI(50) values in the range 8-18 microM. A structure-activity relationship is also discussed.     

Descriptive biogeography of Tomicus (Coleoptera: Scolytidae) species in Spain

Aim, location Tomicus (Coleoptera, Scolytidae) species are some of the principal pests of Eurasian forest and are represented by three coexisting species in Spain, Tomicus piniperda (Linnaeus, 1758), Tomicus destruens (Wollaston, 1865) and Tomicus minor (Harting, 1834). The distribution of two taxa are unknown as they have until recently been considered separate species. Therefore, we model the potential distribution centres and establish the potential distribution limits of Tomicus species in Iberia. We also assess the effectiveness of different models by comparing predicted results with observed data. These results will have application in forest pest management. Methods Molecular and morphological techniques were used to identify species from 254 specimens of 81 plots. For each plot, a Geographical Information System was used to extract a set of 14 environmental (one topographic, six climatic) and biotic variables (seven host tree distributions). General Additive Models and Ecological Niche Factor Analysis models are applied for modelling and predicting the potential distribution of the three especies of Tomicus. Results The results of both modelling methodologies are in agreement. Tomicus destruens is the predominant species in Spain, living in low and hot areas. Tomicus piniperda occurs in lower frequency and prefers wet and cold areas of north‐central Spain. We detected sympatric populations of T. destruens and T. piniperda in Northern coast of Spain, infesting mainly P. pinaster. Tomicus minor is the rarest species, and it occupies a fragmented distribution located in high and wet areas. The remarkable biotic variable is the distribution of P. sylvestris, incorporated into the models of T. destruens and T. piniperda. Main conclusions These results indicate that in wet areas of north‐central Spain where T. piniperda occurs (and possibly the high altitudes of the southern mountains), T. destruens has a climatic distribution limit. In the northern border of this area, both species overlap their distributions and some co‐occurrences were detected. Tomicus minor potentially occurs in high and wet fragmented areas.     

Study of amide-proton exchange of Escherichia coli melibiose permease by attenuated total reflection-Fourier transform infrared spectroscopy: evidence of structure modulation by substrate binding.

The accessibility of Escherichia coli melibiose permease to aqueous solvent was studied following hydrogen-deuterium exchange kinetics monitored by attenuated total reflection-Fourier transform infrared spectroscopy under four distinct conditions where MelB forms different complexes with its substrates (H(+), Na(+), melibiose). Analysis of the amide II band upon (2)H(2)O exposure discloses a significant sugar protection of the protein against aqueous solvent, resulting in an 8% less exchange of the corresponding H(+)*melibiose*MelB complex compared with the protein in the absence of sugar. Investigation of the amide I exchange reveals clear substrate effects on beta-sheet accessibility, with the complex H(+)*melibiose*MelB being the most protected state against exchange, followed by Na(+)*melibiose*MelB. Although of smaller magnitude, similar changes in alpha-helices plus non-ordered structures are detected. Finally, no differences are observed when analyzing reverse turn structures. The results suggest that sugar binding induces a remarkable compactness of the carrier's structure, affecting mainly beta-sheet domains of the transporter, which, according to secondary structure predictions, may include cytoplasmic loops 4-5 and 10-11. A possible catalytic role of these two loops in the functioning of MelB is hypothesized.     

Sulfide fluxes in a microbial mat from the Ebro Delta, Spain

The sulfur cycle of Ebro Delta microbial mats was studied in order to determine sulfide production and sulfide consumption. Vertical distribution of two major functional groups involved in the sulfur cycle, anoxygenic phototrophic bacteria and dissimilatory sulfate-reducing bacteria (SRB), was also studied. The former reached up to 2.2×10⁸ cfu cm^–3 sediment in the purple layer, and the latter reached about 1.8×10⁵ SRB cm^–3 sediment in the black layer. From the changes in sulfide concentrations under light-dark cycles it can be inferred that the rate of H₂S production was 6.2 μmol H₂S cm^–3 day^–1 at 2.6 mm, and 7.6 μmol H₂S cm^–3 day^–1 at 6 mm. Furthermore, sulfide consumption was also assessed, determining rates of 0.04, 0.13 and 0.005 mmol l^–1 of sulfide oxidized at depths of 2.6, 3 and 6 mm, respectively. Electronic Publication     

Homecare monitoring system: A technical proposal for the safety of the elderly experimented in an Alzheimer's care unit

Today, the monitoring of dependent people is a real challenge. This is mainly due to the increase in the number of elderly and reduction of medical staff. However, works done in recent years in France are faced with obstacle of clinical and industrial validation. In this paper, we propose a monitoring system for dependent persons living alone at home or in an institution 24 h/24. This system is based on a presence multisensor network deployed in the living environment of the monitored person coupled with a wireless identification system. A learning algorithm is implemented in order to define a personalized behavioural model (motions deviation, nocturnal activity, falls, mobility). This model allows the nursing staff monitoring the behaviour through a web application accessed remotely, and also intervention in case of dangerous situations thanks to an alert system. In further, we describe the experiments conducted at the Caussade Local Hospital in France with Alzheimer patients and we present the results of data obtained. Finally, we propose the way for an economic model that would allow to industrialize the system with prospects of clinical validation on a larger cohort of patients.