Stability of subtilisins and related proteinases (subtilases)

The stability towards thermal and chemical (guanidine hydrochloride, GnHCl) denaturation of six inhibited subtilases (mesentericopeptidase, subtilisins BPN′, Carlsberg and DY, proteinase K and thermitase) has been investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroic and fluorescence spectroscopy. Experiments in the absence and presence of extraneous calcium in the concentration range 2×10^?3-10^?1 M were performed. The presence of calcium in the weak calcium binding site changes the denaturation drastically. The heat- (or GnHCl-) induced unfolding curves obtained using CD spectroscopy show two independent transitions which seem not to have been resolved before. The presence of Ca²⁺ in the second (third in the case of thermitase) binding site increases the T_m, values by 11-21 °C and the δG_D(H₂O) values obtained from denaturation experiments in GnHCl by 6.7-7.2 kcal/mol when an extraneous Ca²⁺ concentration of 2 × 10^?2 M was used. One interpretation is that the initial step of denaturation in the presence of added calcium is the formation of a partially unfolded intermediate form, retaining a highly ordered structure with 60-85% of the a-helix structure of the native enzyme. This intermediate then unfolds at a temperature considerably higher than that of the same proteinases in the absence of added Ca²⁺. The free energy of stabilization of the intermediates is increased by 1.8-2.8 times in comparison with that for the unfolding reactions of the subtilases with empty Ca2/Ca3 binding sites. A second interpretation is that the two steps in the unfolding curves correspond to enzyme without and with calcium in the weak binding site. Fluorescence experiments confirm the mechanism involving the formation of intermediate states. The results are discussed in relation to the X-ray models of the six subtilases.     

Immunoblotting of streptococcal antigens in guttate psoriasis

Guttate psoriasis may be precipitated by acute streptococcal infection, usually of the upper respiratory tract. We have studied the immune response to streptococci in 26 patients presenting with a first or recurrent episode of acute guttate psoriasis (AGP), using immunoblotting. Eighteen of 26 patients studied had a demonstrable response to a wide range of streptococcal antigens using this approach, compared with 14 of 26 patients who demonstrated a response using more conventional anti-streptococcal antibody tests. Patients with AGP had a significantly higher antibody detection score using immunoblotting than did control subjects (P<0.005). We conclude that immunoblotting is a useful technique in studying this condition and may be of benefit in exploring the immunopathogene-sis of AGP.     

An evaluation of the gum elastic bougie

This study was designed to evaluate the routine use of a gum elastic bougie for tracheal intubation. The median time to intubation with the gum elastic bougie while simulating an 'epiglottis only' view was only 10 s longer than the time taken during conventional intubation with an optimum view. Three of the patients required a gum elastic bougie-assisted intubation after attempts at conventional visual intubation had failed. There was no significant difference in the incidence of postoperative sore throat and hoarseness between the two groups. We recommend that anaesthetists should use the gum elastic bougie whenever a good view of the glottis is not immediately obtained.     

The use of propofol and alfentanil by infusion in military anaesthesia

A total intravenous technique using propofol and alfentanil was used successfully in four battlefield casualties treated at a British Military Field Hospital during the recent Gulf conflict. All patients made a rapid recovery of adequate quality for prompt evacuation. We believe that the use of propofol and alfentanil as an induction and maintenance regimen for military anaesthesia merits further evaluation and comparison with established techniques.     

Cardiovascular responses to insertion of the laryngeal mask

We have compared, in 40 healthy patients, the cardiovascular responses induced by laryngoscopy and intubation with those produced by insertion of a laryngeal mask. Anaesthesia was induced with thiopentone and maintained with enflurane and nitrous oxide in oxygen; vecuronium was used for muscle relaxation. Arterial pressure was measured with a Finapres monitor. The mean maximum increase in systolic arterial pressure after laryngoscopy and tracheal intubation was 51.3% compared with 22.9% for laryngeal mask insertion (p less than 0.01). Increases in maximum heart rate were similar, (26.6% v 25.7%) although heart rate remained elevated for longer after tracheal intubation. We conclude that insertion of the laryngeal mask airway is accompanied by smaller cardiovascular responses than those after laryngoscopy and intubation and that its use may be indicated in those patients in whom a marked pressor response would be deleterious.