Enzyme activity and dynamics in near-anhydrous conditions

Water is widely assumed to be essential for life 1, although the exact molecular basis of this requirement is unclear 2-4. Water facilitates protein motions 5-9 and although enzyme activity has been demonstrated at low hydrations in organic solvents 10-13, such non-aqueous solvents may allow the necessary motions for catalysis. To examine enzyme function in the absence of solvation and bypass diffusional constraints we have tested the ability of an esterase to catalyse alcoholysis as an anhydrous powder, using a closed reaction system in which the substrates and products of the enzyme reaction are gaseous 14-15, and where the water content can be well defined 16. At hydrations equivalent to 3 (±2) molecules of water per molecule of enzyme, activity is observed that is several orders of magnitude greater than non-enzymatic catalysis. Neutron spectroscopy indicates that the fast (≤nanosecond) global anharmonic dynamics of the anhydrous functional enzyme are heavily suppressed. The results indicate that neither hydration water nor the solvent-activated fast anharmonic dynamics are required for enzyme function. An implication of these results is that one of the essential requirements of water for life may lie with its role as a diffusion medium rather than any of its more specific properties

From powder to solution: hydration dependence of human hemoglobin dynamics correlated to body temperature

A transition in hemoglobin (Hb), involving partial unfolding and aggregation, has been shown previously by various biophysical methods. The correlation between the transition temperature and body temperature for Hb from different species, suggested that it might be significant for biological function. In order to focus on such biologically relevant human Hb dynamics, we studied the protein internal picosecond motions as a response to hydration, by elastic and quasielastic neutron scattering. Rates of fast diffusive motions were found to be significantly enhanced with increasing hydration from fully hydrated powder to concentrated Hb solution. In concentrated protein solution, the data revealed that amino acid side-chains can explore larger volumes above body temperature than expected from normal temperature dependence. The body temperature transition in protein dynamics was absent in fully hydrated powder, indicating that picosecond protein dynamics responsible for the transition is activated only at a sufficient level of hydration. A collateral result from the study is that fully hydrated protein powder samples do not accurately describe all aspects of protein picosecond dynamics that might be necessary for biological function

口唇トレーニングの多方位口唇閉鎖力に対する影響─健常若年成人および健常高齢者を対象として─

The topical application of sunscreen and cosmetic products that afford protection from terrestrial ultraviolet radiation to reduce the risk of developing skin cancer is an integral part of modern sun protection. The current sunscreen formulations use inorganic zinc oxide (ZnO) and titanium dioxide (TiO ), that are highly photocatalytic, with the ability to penetrate the skin, and exhibit potential toxicity. In this study, we demonstrate a novel methodology to increase the sun protection factor (SPF) of encapsulated metal oxide (Fe O ) nanoparticles in poly(lactic acid) (PLA) microspheres as ultraviolet filters for sunscreen applications. The combination of these two materials results in a nanocomposite with increased UV attenuation and without significant cytotoxic effects during in vitro evaluation. A commercial sunscreen formulation was used to show the benefits of the developed nanocomposites by replacing the harmful ZnO particles by the Fe O -PLA ones, and a throughout comparison between commercial and the new sunscreen formulation was performed. By adding a small amount of the novel Fe O -PLA particles, a remarkable increase in the SPF performance, from 40 (commercial ZnO formulation) to 50 was achieved just by replacing the ZnO particles with 5 wt% of Fe O -PLA ones, resulting in a high level of UV protection, coupled with lower dangerous photocatalytic activity when compared with commercial ZnO nanoparticles. The encapsulation of metal oxide nanoparticles by PLA could provide a route to improve the sunscreen efficacy and enhance its SPF by using more environmentally friendly materials. 2 2 3 2 3 2 3 2

Establishment of novel long-term cultures from EpCAM positive and negative circulating tumour cells from patients with metastatic gastroesophageal cancer

Circulating tumour cell (CTC) enumeration and profiling has been established as a valuable clinical tool in many solid malignancies. A key challenge in CTC research is the limited number of cells available for study. Ex vivo CTC culture permits expansion of these rare cell populations for detailed characterisation, functional assays including drug sensitivity testing, and investigation of the pathobiology of metastases. We report for the first time the establishment and characterisation of two continuous CTC lines from patients with gastroesophageal cancer. The two cell lines (designated UWG01CTC and UWG02CTC) demonstrated rapid tumorigenic growth in immunodeficient mice and exhibit distinct genotypic and phenotypic profiles which are consistent with the tumours of origin. UWG02CTC exhibits an EpCAM+, cytokeratin+, CD44+ phenotype, while UWG01CTC, which was derived from a patient with metastatic neuroendocrine cancer, displays an EpCAM−, weak cytokeratin phenotype, with strong expression of neuroendocrine markers. Further, the two cell lines show distinct differences in drug and radiation sensitivity which match differential cancer-associated gene expression pathways. This is strong evidence implicating EpCAM negative CTCs in metastasis. These novel, well characterised, long-term CTC cell lines from gastroesophageal cancer will facilitate ongoing research into metastasis and the discovery of therapeutic targets

Protein interactions and behaviour in multi-protein complexes

Understanding how proteins interact and behave in large dynamic multi-protein complexes is an important area of significance in many human diseases that are associated with faults in these interactions. Increasing understanding of the well-characterized bacterial DNA replication machinery (the replisome) will continue to enrich our understanding of other dynamic complexes, including those that carry out human DNA replication. This research focussed on pairwise interactions that involve one of the key organizational centres of the bacterial replisome, the DnaI helicase loader and the DnaB replicative helicase. AINSE postgraduate scholar Mr Flynn Hill measured the dynamics of the hydrogenated DnaI, DnaB and the DnaI-DnaB complex at the Institut Laue Langevin (France, Grenoble) with the IN6 scientist Marek Koza

Water in extremophiles [L\u27eau dans les extremophiles]

La cellule est considérée comme l’unité élémentaire de la vie. Les analyses génétiques ont permis de regrouper les cellules en trois royaumes distincts : les eucaryotes, les eubactéries et les archébactéries. Les extrêmophiles, organismes des environnements extrêmes, sont représentés dans ces trois royaumes. L’environnement de l’intérieur d’une cellule est très encombré par différentes macromolécules dont les protéines composent le type le plus d’important, tant par leur quantité que par leur variété de fonctions. Dans cette matrice hétérogène à la structure vulnérable, l’eau est très présente (elle compose environ 70 % de l’intérieur de la cellule). Elle constitue un élément crucial – les interactions entre macromolécules et eau sont fondamentales pour la fonction biologique et la viabilité de la cellule – qu’il est important de comprendre

Adaptation de la dynamique moléculaire aux conditions extrêmes

GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Specific cellular water dynamics observed in vivo by neutron scattering and NMR.

Neutron scattering, by using deuterium labelling, revealed how intracellular water dynamics, measured in vivo in E. coli, human red blood cells and the extreme halophile, Haloarcula marismortui, depends on the cell type and nature of the cytoplasm. The method uniquely permits the determination of motions on the molecular length (Ba˚ ngstrøm) and time (pico- to nanosecond) scales. In the bacterial and human cells, intracellular water beyond the hydration shells of cytoplasmic macromolecules and membrane faces flows as freely as liquid water. It is not ‘‘tamed’’ by confinement. In contrast, in the extreme halophile archaeon, in addition to free and hydration water an intracellular water component was observed with significantly slowed down translational diffusion. The results are discussed and compared to observations in E. coli and Haloarcula marismortui by deuteron spin relaxation in NMR—a method that is sensitive to water rotational dynamics on a wide range of time scales