A novel tool for measuring ankle dorsiflexion: A study of its reliability in patients following ankle fractures.

BACKGROUND: Assessment of ankle joint movement in a weight bearing position has important clinical implications. The lunge ankle dorsiflexion measurement device (LAD) has been developed with the aim of facilitating ease of and standardisation of the measurement of ankle joint movement. The literature lacks studies evaluating the reliability of weight bearing measurements of the ankle joint in study groups with ankle disabilities. The objective of this study was to examine the intra- and inter-tester reliability of ankle dorsiflexion measured with the novel LAD in patients following a fracture of the ankle. METHOD: This study was a randomized intra- and inter-tester reliability study with blinding of testers and participants. All participants were tested twice by each tester, with the order of testers randomized. The intra- and inter-tester reliability was assessed by the calculation of interclass correlation coefficients (ICC). RESULTS: The study sample consisted of 24 patients: 15 females and nine males post-immobilisation following surgery for ankle fractures. The mean age was 51.0 years, ranging from 22 to 92 years. All patients had sustained an AO classification 44- fracture of the ankle. The mean follow-up time was 9.3 months (16.2 SD) after the time of fracture. The inter-tester reliability was high, with an ICC of 0.984 (95%CI: 0.963-0.993) and SEmeas of 0.14cm. The ICC for Tester A was 0.989 (95%CI: 0.974-0.995) and SEmeas 0.10cm. The ICC for Tester B was 0.990 (95%CI: 0.977-0.996) and SEmeas 0.09cm. CONCLUSION: This study shows a high inter- and intra-tester reliability for measuring ankle dorsiflexion with the LAD following a fracture of the ankle.

Characterization of the prion protein 3F4 epitope and its use as a molecular tag.

The monoclonal antibody (MAb) 3F4 has for nearly two decades been one of the most commonly used tools in prion research. This MAb has contributed significantly to our understanding of the normal cell biology of the prion protein (PrP(C)), as well as the disease related abnormalities occurring in prion diseases. The 3F4 antibody binds strongly to human and hamster PrP, with a specific requirement of two Met residues at positions 109 and 112 in the human PrP. Other species in which PrP lack one of the Met residues, like cattle and sheep, or both, like rat and mouse, do not react with the 3F4 antibody. These and other observations have led to the commonly accepted notion that the 3F4 epitope consists of the tetra-peptide Met-Lys-His-Met. In this study, we have identified the minimal epitope for 3F4 by studying its binding to synthetic peptides and by analysis of mutated ovine PrP::GFP constructs expressed in cell culture. We have found that the 3F4 epitope consists of a hepta-peptide (Lys-Thr-Asn-Met-Lys-His-Met), which in sheep encompass residues 109-115. We found that Lys 109 is critically important for 3F4 binding, as omission, or substitution of this residue to Ala resulted in no binding. We also demonstrate that the hepta-peptide constituting the minimal 3F4 epitope, can be used as a discrete, moveable high-affinity molecular tag. Thus, the 3F4 antibody can find its use beyond prion research.

Alternative translation initiation generates cytoplasmic sheep prion protein.

Cytoplasmic localization of the prion protein (PrP) has been observed in different species and cell types. We have investigated this poorly understood phenomenon by expressing fusion proteins of sheep prion protein and green fluorescent protein ((GFP)PrP) in N2a cells, with variable sequence context surrounding the start codon Met(1). (GFP)PrP expressed with the wild-type sequence was transported normally through the secretory pathway to the cell surface with acquisition of N-glycan groups, but two N-terminal fragments of (GFP)PrP were detected intracellularly, starting in frame from Met(17). When (GFP)PrP was expressed with a compromised Kozak sequence ((GFP)PrP*), dispersed intracellular fluorescence was observed. A similar switch from pericellular to intracellular PrP localization was seen when analogous constructs of sheep PrP, without inserted GFP, were expressed, showing that this phenomenon is not caused by the GFP tag. Western blotting revealed a reduction in glycosylated forms of (GFP)PrP*, whereas the N-terminal fragments starting from Met(17) were still present. Formation of these N-terminal fragments was completely abolished when Met(17) was replaced by Thr, indicating that leaky ribosomal scanning occurs for normal sheep PrP and that translation from Met(17) is the cause of the aberrant cytoplasmic localization observed for a fraction of the protein. In contrast, the same phenomenon was not detected upon expression of similar constructs for mouse PrP. Analysis of samples from sheep brain allowed immunological detection of N-terminal PrP fragments, indicating that sheep PrP is subject to similar processing mechanisms in vivo.

Proteolytic processing of the ovine prion protein in cell cultures.

The cellular compartment and purpose of the proteolytic processing of the prion protein (PrP) are still under debate. We have studied ovine PrP constructs expressed in four cell lines; murine neuroblastoma cells (N2a), human neuroblastoma cells (SH-SY5Y), dog kidney epithelial cells (MDCK), and human furin-deficient colon cancer cells (LoVo). Cleavage of PrP in LoVo cells indicates that the processing is furin independent. Neither is it reduced by some inhibitors of lysosomal proteinases, proteasomes or zinc-metalloproteinases, but incubation with bafilomycin A1, an inhibitor of vacuolar H+/ATPases, increases the amount of uncleaved PrP in the apical medium of MDCK cells. Mutations affecting the putative cleavage site near amino acid 113 reveal that the cleavage is independent of primary structure at this site. Absence of glycosylphosphatidylinositol anchor and glycan modifications does not influence the proteolytic processing of PrP. Our data indicate that PrP is cleaved during transit to the cell membrane.