The Manual Work Instability Scale: development and validation.

BACKGROUND: Increasing awareness of the burden of absenteeism and reduced performance at work highlights the importance of early identification of individuals experiencing work instability (WI), a mismatch between functional and cognitive abilities and job demands. AIMS: To develop and validate a screening questionnaire to measure WI in manual workers. METHODS: Questionnaire items were generated via qualitative interviews with manual workers and a draft survey instrument was completed by workers in a variety of fields. Rasch analysis was used interactively to assess the psychometric aspects of the emerging scale, including unidimensionality and absence of item bias (differential item functioning). RESULTS: A total of 17 qualitative interviews generated 110 potential items for the questionnaire. The item set resolved to a 25-item scale, which satisfied model expectations (item residual mean = -0.13, SD = 1.04; person residual mean = -0.29, SD = 0.75), had good reliability (alpha = 0.86) and strict unidimensionality (t-test 7.5% confidence interval 3.8-11.2). CONCLUSIONS: The Manual Work Instability Scale is a short psychometrically robust questionnaire based on the concept of WI, which incorporates both musculoskeletal symptoms and relevant psychosocial factors. It may prove effective in screening and identifying WI in workers in predominantly physical occupations.

The dodecameric vanadium-dependent haloperoxidase from the marine algae Corallina officinalis: cloning, expression, and refolding of the recombinant enzyme.

The dodecameric vanadium-dependent bromoperoxidase from Corallina officinalis has been cloned and over-expressed in Escherichia coli. However, the enzyme was found to be predominantly in the form of inclusion bodies. This protein presents a challenging target for refolding, both due to the size (768kDa) and quaternary structure (12x64kDa). Successful refolding conditions have been established which result in an increase in the final yield of active bromoperoxidase from 0.5mg to 40mg per litre of culture. The refolded protein has been characterised and compared to the native enzyme and was shown to be stable at temperatures of 80 degrees C, over a pH range 5.5-10 and in organic solvents such as ethanol, acetonitrile, methanol, and acetone. The novel refolding approach reported in this paper opens up the full potential of this versatile enzyme for use in large scale biotransformation studies.

Inhibitors of bacterial enoyl acyl carrier protein reductase (FabI): 2,9-disubstituted 1,2,3,4-tetrahydropyrido[3,4-b]indoles as potential antibacterial agents.

An SAR study of a screening lead has led to the identification of 2,9-disubstituted 1,2,3,4-tetrahydropyrido[3,4-b]indoles as inhibitors of Staphylococcus aureus enoyl acyl carrier protein reductase (FabI).

Molecular basis for triclosan activity involves a flipping loop in the active site.

The crystal structure of the Escherichia coli enoyl reductase-NAD+-triclosan complex has been determined at 2.5 A resolution. The Ile192-Ser198 loop is either disordered or in an open conformation in the previously reported structures of the enzyme. This loop adopts a closed conformation in our structure, forming van der Waals interactions with the inhibitor and hydrogen bonds with the bound NAD+ cofactor. The opening and closing of this flipping loop is likely an important factor in substrate or ligand recognition. The closed conformation of the loop appears to be a critical feature for the enhanced binding potency of triclosan, and a key component in future structure-based inhibitor design.

Reduced pH causes structural changes in the potent mitogenic toxin of Pasteurella multocida.

Pasteurella multocida toxin is a potent mitogen that is believed to act intracellularly. On transverse urea gradient gels at pH 8.0 the toxin displayed one major unfolding transition at 4 M urea. However, at pH 6.1 the unfolding transition took place at 3.5 M urea. Circular dichroism spectra also indicated that a structural change took place at acidic pH. In addition it was found that the toxin that had been denatured in 8 M urea refolded in solution with a high recovery of biological activity. These findings are discussed in terms of the likely domain structure of the P. multocida toxin.

3D reconstruction of the ATP-bound form of CCT reveals the asymmetric folding conformation of a type II chaperonin.

The type II chaperonin CCT (chaperonin containing Tcp-1) of eukaryotic cytosol is a heteromeric 16-mer particle composed of eight different subunits. Three-dimensional reconstructions of apo-CCT and ATP-CCT have been obtained at 28 A resolution by cryo-electron microscopy. Binding of ATP generates an asymmetric particle; one ring has a slightly different conformation from the apo-CCT ring, while the other has undergone substantial movements in the apical domains. Upon ATP binding the apical domains rotate and point towards the cylinder axis, so that the helical protrusions present at their tips could act as a lid closing the ring cavity.

ATP binding induces large conformational changes in the apical and equatorial domains of the eukaryotic chaperonin containing TCP-1 complex.

The chaperonin-containing TCP-1 complex (CCT) is a heteromeric particle composed of eight different subunits arranged in two back-to-back 8-fold pseudo-symmetric rings. The structural and functional implications of nucleotide binding to the CCT complex was addressed by electron microscopy and image processing. Whereas ADP binding to CCT does not reveal major conformational differences when compared with nucleotide-free CCT, ATP binding induces large conformational changes in the apical and equatorial domains, shifting the latter domains up to 40 degrees (with respect to the inter-ring plane) compared with 10 degrees for nucleotide-free CCT or ADP-CCT. This equatorial ATP-induced shift has no counterpart in GroEL, its prokaryotic homologue, which suggests differences in the folding mechanism for CCT.

The potent mitogen Pasteurella multocida toxin is highly resistant to proteolysis but becomes susceptible at lysosomal pH.

The susceptibility of the potent mitogen Pasteurella multocida toxin (PMT) to various proteases was investigated. PMT at a toxin to protease molar ratio of 1:1 was resistant to 8 of the 11 proteases tested after one hour. With longer incubation, PMT remained resistant to 7 proteases, and this correlated with a retention of biological activity, indicating that PMT might not require proteolytic cleavage at least until it bound to a cell receptor. Previous evidence had suggested that PMT is processed in the cell via an endosome or lysosome. We have shown that PMT became susceptible to proteolysis when the pH was lowered to 5 or below. This supports the previous suggestion that PMT is processed via a low pH compartment in the cell.