Aims: To produce a method of distinguishing between type 1 and 2 skeletal muscle fibres that would be more economical and reproducible than the standard ATPase method and be applicable to both fixed and frozen tissue. Because the ATPase method has been accepted as the basis for fibre identification for the past 50 years, the new method should not give significantly different results. Methods: Isoforms of myosin correlate with isoforms of myofibrillar ATPase and an immunohistochemical (IHC) double labelling protocol was devised using monoclonal antibodies to fast and slow myosin. This required one tissue section rather than four. The results of the two methods were compared by means of morphometric analysis of skeletal muscle biopsies from 20 normal healthy volunteers. Results: There were no significant differences (p = 0.57) in the percentages of type 1 (46% using the IHC method v 48% using ATPase) or type 2 fibres (54% v 52%, respectively). The 2a and 2b subtypes were distinguished easily. Analysis of variance revealed that cross sectional area (µm 2 ), diameter (µm), form factor, and density of fibre staining (a measure of substrate-enzyme or protein) were all similar. The method worked equally well on fixed material. Conclusion: An IHC method based on the fast and slow isoforms of myosin shows no significant differences in fibre type analysis from the standard ATPase method although it provides important advantages because it is applicable to fixed (including archival) material, is economical and reproducible, and yields a permanent preparation.

A system for continuous culture of the hyperthermophilic archaeum Pyrococcus furiosus in the absence of elemental sulphur has been developed. An all-glass "gas-lift" bioreactor was used to provide high mass transfer at low shear forces, whilst eliminating the potential for corrosion. Steady-state cell densities of P.furiosus were found to increase with higher inert gas flow rates, reaching a maximum in this system with 0.5 vol. v o l -1 m i n -1 of nitrogen (N2). N2 permitted higher cell densities than the other inert gases tested (argon, helium and sulphur hexafluoride) under equivalent conditions. At 0.5 vol. vo1-1 min -1 of N2 a cell density in excess of 3 x 10 9 ml -1 could be maintained indefinitely at a dilution rate of 0.2 h -1. Higher dilution rates gave progressively lower steady-state cell densities. The biomass production was maximal, however, at a dilution rate of 0.4 h -1. At this dilution rate the bioreactor was able to generate more than 1.5 g wet weight of cells h -1 1-1 culture volume.They also appear to possess unusual metabolic pathways giving rise to the possibility of novel biotransformations. The major limitation to their study has been the small amounts of biomass that can be generated.

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Proline-specific dipeptidyl peptidases (DPPs) are emerging targets for drug development. DPP4 inhibitors are approved in many countries, and other dipeptidyl peptidases are often referred to as DPP4 activity- and/or structure-homologues (DASH). Members of the DASH family have overlapping substrate specificities, and, even though they share low sequence identity, therapeutic or clinical cross-reactivity is a concern. Here, we report the structure of human DPP7 and its complex with a selective inhibitor Dab-Pip (L-2,4-diaminobutyryl-piperidinamide) and compare it with that of DPP4. Both enzymes share a common catalytic domain (α/β-hydrolase). The catalytic pocket is located in the interior of DPP7, deep inside the cleft between the two domains. Substrates might access the active site via a narrow tunnel. The DPP7 catalytic triad is completely conserved and comprises Ser162, Asp418 and His443 (corresponding to Ser630, Asp708 and His740 in DPP4), while other residues lining the catalytic pockets differ considerably. The “specificity domains” are structurally also completely different exhibiting a β-propeller fold in DPP4 compared to a rare, completely helical fold in DPP7. Comparing the structures of DPP7 and DPP4 allows the design of specific inhibitors and thus the development of less cross-reactive drugs. Furthermore, the reported DPP7 structures shed some light onto the evolutionary relationship of prolyl-specific peptidases through the analysis of the architectural organization of their domains.