The component polypeptide chains of bovine insulin nucleate or inhibit aggregation of the parent protein in a conformation-dependent manner

Amyloid fibrils are typically rigid, unbrariched structures with diameters of similar to 10 nm and lengths up to several micrometres, and are associated with more than 20 diseases including Alzheimer's disease and type II diabetes. Insulin is a small, predominantly alpha-helical protein consisting of 51 residues in two disulfide-linked polypeptide chains that readily assembles into amyloid fibrils under conditions of low PH and elevated temperature. We demonstrate here that both the A-chain and the B-chain of insulin are capable of forming amyloid fibrils in isolation under similar conditions, with fibrillar morphologies that differ from those composed of intact insulin. Both the A-chain and B-chain fibrils were found to be able to cross-seed the fibrillization of the parent protein, although these reactions were substantially less efficient than self-seeding with fibrils composed of full-length insulin. In both cases, the cross-seeded fibrils were morphologically distinct from the seeding, material, but shared common characteristics with typical insulin fibrils, including a very similar helical repeat. The broader distribution of heights of the cross-seeded fibrils compared to typical insulin fibrils, however, indicates that their underling protofilament hierarchy may be subtly different. In addition, and remarkably in view of this seeding behavior, the soluble forms of the A-chain and B-chain peptides were found to be capable of inhibiting insulin fibril formation. Studies using mass spectrometry suggest that this behavior might be attributable to complex formation between insulin and the A-chain and B-chain peptides. The finding that the same chemical form of a polypeptide chain in different physical states can either stimulate or inhibit the conversion of a protein into amyloid fibrils sheds new light on the mechanisms underlying fibril formation, fibril strain propagation and amyloid disease initiation and progression. (c) 2006 Elsevier Ltd. All rights reserved.

Functionalised amyloid fibrils for roles in cell adhesion

We describe experiments designed to explore the possibility of using amyloid fibrils as new nanoscale biomaterials for promoting and exploiting cell adhesion, migration and differentiation in vitro. We created peptides that add the biological cell adhesion sequence (RGD) or a control sequence (RAD) to the C-terminus of an 11-residue peptide corresponding to residues 105-115 of the amyloidogenic protein transthyretin. These peptides readily self-assemble in aqueous solution to form amyloid fibrils, and X-ray fibre diffraction shows that they possess the same strand and sheet spacing in the characteristic cross-beta structure as do fibrils formed by the parent peptide. We report that the fibrils containing the RGD sequence are bioactive and that these fibrils interact specifically with cells via the RGD group displayed on the fibril surface. As the design of such functionalized fibrils can be systematically altered, these findings suggest that it will be possible to generate nanomaterials based on amyloid fibrils that are tailored to promote interactions with a wide variety of cell types. (c) 2007 Elsevier Ltd. All rights reserved.

X-ray scattering study of the effect of hydration on the cross-beta structure of amyloid fibrils

We have investigated the effect of sample hydration on the wide-angle X-ray scattering patterns of amyloid fibrils from two different sources, hen egg white lysozyme (HEWL) and an 11-residue peptide taken from the sequence of transthyretin (TTR105-115). Both samples show an inter-strand reflection at 4.7 Å and an inter-sheet reflection which occurs at 8.8 and 10 Å for TTR105-115 and HEWL fibrils, respectively. The positions, widths, and relative intensities of these reflections are conserved in patterns obtained from dried stalks and hydrated samples over a range of fibril concentrations. In 2D scattering patterns obtained from flow-aligned hydrated samples, the inter-strand and inter-sheet reflections showed, respectively, axial and equatorial alignment relative to the fibril axis, characteristic of the cross-β structure. Our results show that the cross-β structure of the fibrils is not a product of the dehydrating conditions typically employed to produce aligned samples, but is conserved in individual fibrils in hydrated samples under dilute conditions comparable to those associated with other biophysical and spectroscopic techniques. This suggests a structure consisting of a stack of two or more sheets whose interfaces are inaccessible to bulk water.

Large-scale functional expression of WT and truncated human adenosine A(2A) receptor in Pichia pastoris bioreactor cultures

Background: The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 similar to 15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A(2A)R) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures. Results: Bioreactor cultures yielded an approximately five times increase in cell density (OD600 similar to 75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A(2A)R, and therefore more suitable for further functional and structural studies. Conclusion: Large-scale expression of the A(2A)R in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.

A web 2.0 user interface for wide-area resource monitoring

Resource monitoring in distributed systems is required to understand the 'health' of the overall system and to help identify particular problems, such as dysfunctional hardware or faulty system or application software. Monitoring systems such as GridRM provide the ability to connect to any number of different types of monitoring agents and provide different views of the system, based on a client's particular preferences. Web 2.0 technologies, and in particular 'mashups', are emerging as a promising technique for rapidly constructing rich user interfaces, that combine and present data in intuitive ways. This paper describes a Web 2.0 user interface that was created to expose resource data harvested by the GridRM resource monitoring system.

Local orientational disorder in peptide fibrils probed by a combination of residue-specific 13C-18O labelling, polarised infrared spectroscopy and molecular combing

A novel combination of site-specific isotope labelling, polarised infrared spectroscopy and molecular combing reveal local orientational ordering in the fibril-forming peptide YTIAALLSPYSGGRADS. Use of 13C-18O labelled alanine residues demonstrates that the Nterminal end of the peptide is incorporated into the cross-beta structure, while the C-terminal end shows orientational disorder

Rapid assessment of historic, current and future habitat quality for biodiversity around UK Natura 2000 sites

Changes in landscape composition and structure may impact the conservation and management of protected areas. Species that depend on specific habitats are at risk of extinction when these habitats are degraded or lost. Designing robust methods to evaluate landscape composition will assist decision- and policy-making in emerging landscapes. This paper describes a rapid assessment methodology aimed at evaluating landcover quality for birds, plants, butterflies and bees around seven UK Natura 2000 sites. An expert panel assigned quality values to standard Coordination of Information on the Environment (CORINE) landcover classes for each taxonomic group. Quality was assessed based on historical (1950, 1990), current (2000) and future (2030) land-cover data, the last projected using three alternative scenarios: a growth applied strategy (GRAS), a business-as-might-beusual (BAMBU) scenario, and sustainable European development goal (SEDG) scenario. A quantitative quality index weighted the area of each land-cover parcel with a taxa-specific quality measure. Land parcels with high quality for all taxonomic groups were evaluated for temporal changes in area, size and adjacency. For all sites and taxonomic groups, the rate of deterioration of land-cover quality was greater between 1950 and 1990 than current rates or as modelled using the alternative future scenarios (2000– 2030). Model predictions indicated land-cover quality stabilized over time under the GRAS scenario, and was close to stable for the BAMBU scenario. The SEDG scenario suggested an ongoing loss of quality, though this was lower than the historical rate of c. 1% loss per decade. None of the future scenarios showed accelerated fragmentation, but rather increases in the area, adjacency and diversity of high quality land parcels in the landscape.

Intercomparison and evaluation of global aerosol microphysical properties among AeroCom models of a range of complexity

Many of the next generation of global climate models will include aerosol schemes which explicitly simulate the microphysical processes that determine the particle size distribution. These models enable aerosol optical properties and cloud condensation nuclei (CCN) concentrations to be determined by fundamental aerosol processes, which should lead to a more physically based simulation of aerosol direct and indirect radiative forcings. This study examines the global variation in particle size distribution simulated by 12 global aerosol microphysics models to quantify model diversity and to identify any common biases against observations. Evaluation against size distribution measurements from a new European network of aerosol supersites shows that the mean model agrees quite well with the observations at many sites on the annual mean, but there are some seasonal biases common to many sites. In particular, at many of these European sites, the accumulation mode number concentration is biased low during winter and Aitken mode concentrations tend to be overestimated in winter and underestimated in summer. At high northern latitudes, the models strongly underpredict Aitken and accumulation particle concentrations compared to the measurements, consistent with previous studies that have highlighted the poor performance of global aerosol models in the Arctic. In the marine boundary layer, the models capture the observed meridional variation in the size distribution, which is dominated by the Aitken mode at high latitudes, with an increasing concentration of accumulation particles with decreasing latitude. Considering vertical profiles, the models reproduce the observed peak in total particle concentrations in the upper troposphere due to new particle formation, although modelled peak concentrations tend to be biased high over Europe. Overall, the multi-model-mean data set simulates the global variation of the particle size distribution with a good degree of skill, suggesting that most of the individual global aerosol microphysics models are performing well, although the large model diversity indicates that some models are in poor agreement with the observations. Further work is required to better constrain size-resolved primary and secondary particle number sources, and an improved understanding of nucleation and growth (e.g. the role of nitrate and secondary organics) will improve the fidelity of simulated particle size distributions.