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La responsabilidad contractual de los organizadores y los detallistas de viajes combinados

Con la entrada en vigor de la Ley 21/1995, de 6 de julio, reguladora de los viajes combinados (LVC) se instaura una nuevo régimen de responsabilidad contractual del organizador y el detallista. Ahora el consumidor de esos viajes no sólo mantiene una relación contractual con el organizador si no también, en su caso, el detallista, cuya actuación deja de tener carácter representativo. Ambos sujetos responderán frente al consumidor "en función de las obligaciones que les correspondan por su ámbito respectivo de gestión del viaje combinado..." (art. 11.1), circunstancia que plantea algunas dificultades en orden a delimitar el alcance de su responsabilidad. Esa misma norma establece la responsabilidad del organizador por los actos de los prestadores de los servicios del viaje utilizados como auxiliares en el cumplimiento contractual. Analizados los sujetos responsables, la presente tesis doctoral estudia los supuestos de incumplimiento del contrato de viaje, las partidas de daños resarcibles y su extensión. El trabajo propone distintos criterios en orden a calcular el valor de los daños por lesión al llamado interés de prestación, los daños corporales, los daños ocasionados por el extravío, destrucción o deterioro de objetos del consumidor, los daños por gastos inútiles y costes de negocios de reemplazo, los daños no patrimoniales y los daños por ganancias dejadas de obtener. Cada una de esas partidas e daños merece un estudio pormenorizado. Así, por ejemplo, se constatan enormes problemas en cuanto a la identificación e indemnización separada de los daños por frustración o pérdida de vacaciones, así como la nesesidad de establecer unas circunstancias a las que los jueces deban acogerse para llevar a cabo su valoración discrecional. La tesis propone que dichas circunstanciasd sean el alcance del incumplimiento del contrato de viaje, las condiciones personales del consumidor, el tipo de vacaciones objeto del viaje contratado y el valor residual de las vacaciones.

Studying reactive processes with classical dynamics: rebinding dynamics in MbNO

A new surface-crossing algorithm suitable for describing bond-breaking and bond-forming processes in molecular dynamics simulations is presented. The method is formulated for two intersecting potential energy manifolds which dissociate to different adiabatic states. During simulations, crossings are detected by monitoring an energy criterion. If fulfilled, the two manifolds are mixed over a finite number of time steps, after which the system is propagated on the second adiabat and the crossing is carried out with probability one. The algorithm is extensively tested (almost 0.5 mu s of total simulation time) for the rebinding of NO to myoglobin. The unbound surface ((FeNO)-N-...) is represented using a standard force field, whereas the bound surface (Fe-NO) is described by an ab initio potential energy surface. The rebinding is found to be nonexponential in time, in agreement with experimental studies, and can be described using two time constants. Depending on the asymptotic energy separation between the manifolds, the short rebinding timescale is between 1 and 9 ps, whereas the longer timescale is about an order of magnitude larger. NO molecules which do not rebind within 1 ns are typically found in the Xenon-4 pocket, indicating the high affinity of NO to this region in the protein.

Ligand dynamics in myoglobin: calculation of infrared spectra for photodissociated NO

We present molecular dynamics simulations of the photodissociated state of MbNO performed at 300 K using a fluctuating charge model for the nitric oxide (NO) ligand. After dissociation, NO is observed to remain mainly in the centre of the distal haem pocket, although some movement towards the primary docking site and the xenon-4 pocket can be seen. We calculate the NO infrared spectrum for the photodissociated ligand within the haem pocket and find a narrow peak in the range 1915-1922 cm(-1). The resulting blue shift of 1 to 8 cm(-1) compared to gas-phase NO is much smaller than the red shifts calculated and observed for carbon monoxide (CO) in Mb. A small splitting, due to NO in the xenon-4 pocket, is also observed. At lower temperatures, the spectra and conformational space explored by the ligand remain largely unchanged, but the electrostatic interactions with residue His64 become increasingly significant in determining the details of the ligand orientation within the distal haem pocket. The investigation of the effect of the L29F mutation reveals significant differences between the behaviour of NO and that of CO, and suggests a coupling between the ligand and the protein dynamics due to the different ligand dipole moments.

CO migration in native and mutant myoglobin: atomistic simulations for the understanding of protein function

Molecular dynamics simulations of the events after the photodissociation of CO in the myoglobin mutant L29F in which leucine is replaced by phenylalanine are reported. Using both classical and mixed quantum-classical molecular dynamics calculations, we observed the rapid motion of CO away from the distal heme pocket to other regions of the protein, in agreement with recent experimental results. The experimentally observed and calculated infrared spectra of CO after dissociation are also in good agreement. We compared the results with data from simulations of WT myoglobin. As the time resolution of experimental techniques is increased, theoretical methods and models can be validated at the atomic scale by direct comparison with experiment.

Differential recognition of Staphylococcus aureus quorum-sensing signals depends on both extracellular loops 1 and 2 of the transmembrane sensor AgrC.

Virulence in Staphylococcus aureus is regulated via agr-dependent quorum sensing in which an autoinducing peptide (AIP) activates AgrC, a histidine protein kinase. AIPs are usually thiolactones containing seven to nine amino acid residues in which the thiol of the central cysteine is linked to the alpha-carboxyl of the C-terminal amino acid residue. The staphylococcal agr locus has diverged such that the AIPs of the four different S. aureus agr groups self-activate but cross-inhibit. Consequently, although the agr system is conserved among the staphylococci, it has undergone significant evolutionary divergence whereby to retain functionality, any changes in the AIP-encoding gene (agrD) that modifies AIP structure must be accompanied by corresponding changes in the AgrC receptor. Since AIP-1 and AIP-4 only differ by a single amino acid, we compared the transmembrane topology of AgrC1 and AgrC4 to identify amino acid residues involved in AIP recognition. As only two of the three predicted extracellular loops exhibited amino acid differences, site-specific mutagenesis was used to exchange the key AgrC1 and AgrC4 amino acid residues in each loop either singly or in combination. A novel lux-based agrP3 reporter gene fusion was constructed to evaluate the response of the mutated AgrC receptors. The data obtained revealed that while differential recognition of AIP-1 and AIP-4 depends primarily on three amino acid residues in loop 2, loop 1 is essential for receptor activation by the cognate AIP. Furthermore, a single mutation in the AgrC1 loop 2 resulted in conversion of (Ala5)AIP-1 from a potent antagonist to an activator, essentially resulting in the forced evolution of a new AIP group. Taken together, our data indicate that loop 2 constitutes the predicted hydrophobic pocket that binds the AIP thiolactone ring while the exocyclic amino acid tail interacts with loop 1 to facilitate receptor activation.

Evolutionary analysis of novel serine proteases in the venom gland transcriptome of Bitis gabonica rhinoceros

Background: Serine proteases are major components of viper venom and target various stages of the blood coagulation system in victims and prey. A better understanding of the diversity of serine proteases and other enzymes present in snake venom will help to understand how the complexity of snake venom has evolved and will aid the development of novel therapeutics for treating snake bites. Methodology and Principal Findings: Four serine protease-encoding genes from the venom gland transcriptome of Bitis gabonica rhinoceros were amplified and sequenced. Mass spectrometry suggests the four enzymes corresponding to these genes are present in the venom of B. g. rhinoceros. Two of the enzymes, rhinocerases 2 and 3 have substitutions to two of the serine protease catalytic triad residues and are thus unlikely to be catalytically active, though they may have evolved other toxic functions. The other two enzymes, rhinocerases 4 and 5, have classical serine protease catalytic triad residues and thus are likely to be catalytically active, however they have glycine rather than the more typical aspartic acid at the base of the primary specificity pocket (position 189). Based on a detailed analysis of these sequences we suggest that alternative splicing together with individual amino acid mutations may have been involved in their evolution. Changes within amino acid segments which were previously proposed to undergo accelerated change in venom serine proteases have also been observed. Conclusions and Significance: Our study provides further insight into the diversity of serine protease isoforms present within snake venom and discusses their possible functions and how they may have evolved. These multiple serine protease isoforms with different substrate specificities may enhance the envenomation effects and help the snake to adapt to new habitats and diets. Our findings have potential for helping the future development of improved therapeutics for snake bites.

Large is fast, small is tight: determinants of speed and affinity in subunit capture by a periplasmic chaperone

The chaperone/usher pathway assembles surface virulence organelles of Gram-negative bacteria, consisting of fibers of linearly polymerized protein subunits. Fiber subunits are connected through 'donor strand complementation': each subunit completes the immunoglobulin (Ig)-like fold of the neighboring subunit by donating the seventh β-strand in trans. Whereas the folding of Ig domains is a fast first-order process, folding of Ig modules into the fiber conformation is a slow second-order process. Periplasmic chaperones separate this process in two parts by forming transient complexes with subunits. Interactions between chaperones and subunits are also based on the principle of donor strand complementation. In this study, we have performed mutagenesis of the binding motifs of the Caf1M chaperone and Caf1 capsular subunit from Yersinia pestis and analyzed the effect of the mutations on the structure, stability, and kinetics of Caf1M-Caf1 and Caf1-Caf1 interactions. The results suggest that a large hydrophobic effect combined with extensive main-chain hydrogen bonding enables Caf1M to rapidly bind an early folding intermediate of Caf1 and direct its partial folding. The switch from the Caf1M-Caf1 contact to the less hydrophobic, but considerably tighter and less dynamic Caf1-Caf1 contact occurs via the zip-out-zip-in donor strand exchange pathway with pocket 5 acting as the initiation site. Based on these findings, Caf1M was engineered to bind Caf1 faster, tighter, or both faster and tighter. To our knowledge, this is the first successful attempt to rationally design an assembly chaperone with improved chaperone function.

Sequence and phylogenetic analysis of viper venom serine proteases

Snakebites are a major neglected tropical disease responsible for as many as 95000 deaths every year worldwide. Viper venom serine proteases disrupt haemostasis of prey and victims by affecting various stages of the blood coagulation system. A better understanding of their sequence, structure, function and phylogenetic relationships will improve the knowledge on the pathological conditions and aid in the development of novel therapeutics for treating snakebites. A large dataset for all available viper venom serine proteases was developed and analysed to study various features of these enzymes. Despite the large number of venom serine protease sequences available, only a small proportion of these have been functionally characterised. Although, they share some of the common features such as a C-terminal extension, GWG motif and disulphide linkages, they vary widely between each other in features such as isoelectric points, potential N-glycosylation sites and functional characteristics. Some of the serine proteases contain substitutions for one or more of the critical residues in catalytic triad or primary specificity pockets. Phylogenetic analysis clustered all the sequences in three major groups. The sequences with substitutions in catalytic triad or specificity pocket clustered together in separate groups. Our study provides the most complete information on viper venom serine proteases to date and improves the current knowledge on the sequence, structure, function and phylogenetic relationships of these enzymes. This collective analysis of venom serine proteases will help in understanding the complexity of envenomation and potential therapeutic avenues.

In vivo study of the biocompatibility of a novel compressed collagen hydrogel scaffold for artificial corneas

The experiments were designed to evaluate the biocompatibility of a plastically compressed collagen scaffold (PCCS). The ultrastructure of the PCCS was observed via scanning electron microscopy. Twenty New Zealand white rabbits were randomly divided into experimental and control groups that received corneal pocket transplantation with PCCS and an amniotic membrane, respectively. And the contralateral eye of the implanted rabbit served as the normal group. On the 1st, 7th, 14th, 21st, 30th, 60th, 90th, and 120th postoperative day, the eyes were observed via a slit lamp. On the 120th postoperative day, the rabbit eyes were enucleated to examine the tissue compatibility of the implanted stroma. The PCCS was white and translucent. The scanning electron microscopy results showed that fibers within the PCCS were densely packed and evenly arranged. No edema, inflammation, or neovascularization was observed on ocular surface under a slit lamp and few lymphocytes were observed in the stroma of rabbit cornea after histological study. In conclusion, the PCCS has extremely high biocompatibility and is a promising corneal scaffold for an artificial cornea. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.