Green fluorescent protein as a reporter of prion protein folding

Background: The amino terminal half of the cellular prion protein PrPc is implicated in both the binding of copper ions and the conformational changes that lead to disease but has no defined structure. However, as some structure is likely to exist we have investigated the use of an established protein refolding technology, fusion to green fluorescence protein (GFP), as a method to examine the refolding of the amino terminal domain of mouse prion protein. Results: Fusion proteins of PrPc and GFP were expressed at high level in E. coli and could be purified to near homogeneity as insoluble inclusion bodies. Following denaturation, proteins were diluted into a refolding buffer whereupon GFP fluorescence recovered with time. Using several truncations of PrPc the rate of refolding was shown to depend on the prion sequence expressed. In a variation of the format, direct observation in E. coli, mutations introduced randomly in the PrPc protein sequence that affected folding could be selected directly by recovery of GFP fluorescence. Conclusion: Use of GFP as a measure of refolding of PrPc fusion proteins in vitro and in vivo proved informative. Refolding in vitro suggested a local structure within the amino terminal domain while direct selection via fluorescence showed that as little as one amino acid change could significantly alter folding. These assay formats, not previously used to study PrP folding, may be generally useful for investigating PrPc structure and PrPc-ligand interaction.

Comparison of the bacterial Enhancin-like proteins from Yersinia and Bacillus spp. with a baculovirus Enhancin

Enhancins are a class of metalloproteases found in some baculoviruses that enhance viral infection by degrading the peritrophic, membrane (PM) of the insect midgut. However, sequencing has revealed enhancin-like genes with 24-25% homology to viral enhancins, in the genomes of Yersinia pestis and Bacillus anthracis. AcMNPV does not encode enhancin therefore recombinant AcMNPV budded viruses (BVs) and polyhedra inclusion bodies (PIBs) were generated expressing the bacterial Enhancins. Bacterial Enhancins were found to be cytotoxic when compared to viral enhancin, however, larval bioassays suggested that the bacterial Enhancins did not enhance infection in the same way as viral Enhancin. This suggests that the bacterial Enhancins may have evolved a distinct biochemical function. (c) 2005 Elsevier Inc. All rights reserved.

'Roman' or 'native' bodies in Britain: the evidence of late Roman nail-cleaner strap-ends

This paper reviews late Roman `nail-cleaner strap-ends', a group of objects first discussed by Hawkes and Dunning (1961). The precise function of these objects is unclear as their shape suggests use as toilet instruments but the split socket suggests that they were part of belt-fittings. We suggest a detailed typology and discuss the dating evidence and the spatial distribution of the type. Regardless of their precise function, it is argued in this paper that nail-cleaner strap-ends of this type are unique to late Roman Britain and thus represent a distinct regional type. The use of nail-cleaner strap-ends can be viewed in the context of gender associations, military status and religious beliefs.

Promoting resilience and protective factors in the Children's Fund: supporting children and young people's pathways towards social inclusion?

As part of the broader prevention and social inclusion agenda, concepts of risk, resilience, and protective factors inform a range of U.K. Government initiatives targeted towards children and young people in England, including Sure Start, the Children's Fund, On Track, and Connexions. This paper is based on findings from a large qualitative dataset of interviews conducted with children and their parents or caregiver who accessed Children's Fund services as part of National Evaluation of the Children's Fund research.1 Drawing on the notion of young people's trajectories, the paper discusses how Children's Fund services support children's and young people's pathways towards greater social inclusion. While many services help to build resilience and protective factors for individual children, the paper considers the extent to which services also promote resilience within the domains of the family, school, and wider community and, hence, attempt to tackle the complex, multi-dimensional aspects of social exclusion affecting children, young people, and their families.

Inclusion, social networks and resilience: strategies, practices and outcomes for disabled children and their families

This paper explores the strategies of service providers and the benefits reported by disabled children and their parents/carers in three Children's Fund programmes in England. Based on National Evaluation of the Children's Fund research, we discuss how different understandings of ‘inclusion’ informed the diverse strategies and approaches service providers adopted. While disabled children and families perceived the benefits of services predominantly in terms of building individual children's resilience and social networks, the paper highlights the need for holistic approaches which have a broad view of inclusion, support children's networks and tackle disabling barriers within all the spheres of children's lives.

Promoting inclusion: facilitating access to the Silchester 'town life' project

It is becoming increasingly difficult to resource field archaeology outside the developer-funded context. Consequently it is difficult to engage the wider public in understanding the nature of archaeology and in the writing of its history from the study of material and environmental evidence. This paper describes a project funded by the UK's Heritage Lottery Fund designed to increase access by several means to a well-established and long-running archaeological excavation at the Iron Age and Roman Town at Silchester, Hampshire (UK).

Effects of dietary vitamin A concentration of roasted soybean inclusion on marbling, adipose cellularity, and fatty acid composition of beef

A feedlot trial was conducted to determine the effect of dietary vitamin A concentration and roasted soybean (SB) inclusion on carcass characteristics, adipose tissue cellularity, and muscle fatty acid composition. Angus-crossbred steers (n = 168; 295 +/- 1.8 kg) were allotted to 24 pens (7 steers each). Four treatments, in a 2 x 2 factorial arrangement, were investigated: no supplemental vitamin A, no roasted soybeans (NANS); no vitamin A, roasted SB (20% of the diet on a DM basis; NASB); with supplemental (2,700 IU/kg) vitamin A, no roasted SB (WANS); and with supplemental vitamin A, roasted SB (WASB). Diets included high moisture corn, 5% corn silage, 10 to 20% supplement, and 20% roasted SB in the SB treatments on a DM basis. The calculated vitamin A concentration in the basal diet was < 1,300 IU/kg of DM. Blood samples (2 steers/pen) were collected for serum vitamin A determination. Steers were slaughtered after 168 d on feed. Carcass characteristics and LM composition were determined. Fatty acid composition of LM was analyzed, and adipose cellularity in the i.m. and s.c. depots was determined. No vitamin A x SB interactions were detected (P > 0.10) for cattle performance, carcass composition, or muscle fatty acid composition. Low vitamin A diets (NA) did not affect (P > 0.05) ADG, DMI, or G:F. Quality grade tended (P = 0.07) to be greater in NA steers. Marbling scores and the percentage of carcasses grading > or = Choice(-) were 10% greater for NA steers, although these trends were not significant (P = 0.11 and 0.13, respectively). Backfat thickness and yield grade were not affected (P > 0.26) by vitamin A supplementation. Composition of the LM was not affected (P > 0.15) by vitamin A or SB supplementation. Serum retinol at slaughter was 44% lower (P < 0.01) for steers fed NA than for steers supplemented with vitamin A (23.0 vs. 41.1 microg/dL). A vitamin A x SB interaction occurred (P < 0.05) for adipose cellularity in the i.m. depot; when no SB was fed, vitamin A supplementation decreased cell density and increased cell size. However, when SB was fed, vitamin A supplementation did not affect adipose cellularity. Adipose cellularity at the s.c. depot was not affected (P > 0.18) by vitamin A or SB treatments. Fatty acid profile of the LM was not affected by vitamin A (P > 0.05), but SB increased (P < 0.05) PUFA (7.88 vs. 4.30 g/100 g). It was concluded that feeding NA tended to increase marbling without affecting back-fat and yield grade. It appeared that NA induced hyperplasia in the i.m. but not in the s.c. fat depot.