Proteolysis of SNAP-25 isoforms by botulinum neurotoxin types A, C, and E: domains and amino acid residues controlling the formation of enzyme-substrate complexes and cleavage.

Tetanus toxin and the seven serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at nerve endings through the action of their light chains (L chains), which proteolytically cleave VAMP (vesicle-associated membrane protein)/synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa), or syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP-25. Here, we demonstrate that cleavage of SNAP-25 occurs between Arg198 and Ala199, depends on the presence of regions Asn93 to Glu145 and Ile156 to Met202, and requires about 1,000-fold higher L chain concentrations in comparison with BoNT/A and BoNT/E. Analyses of the BoNT/A and BoNT/E cleavage sites revealed that changes in the carboxyl-terminal residues, in contrast with changes in the amino-terminal residues, drastically impair proteolysis. A proteolytically inactive BoNT/A L chain mutant failed to bind to VAMP/synaptobrevin and syntaxin, but formed a stable complex (KD = 1.9 x 10(-7) M) with SNAP-25. The minimal essential domain of SNAP-25 required for cleavage by BoNT/A involves the segment Met146-Gln197, and binding was optimal only with full-length SNAP-25. Proteolysis by BoNT/E required the presence of the domain Ile156-Asp186. Murine SNAP-23 was cleaved by BoNT/E and, to a reduced extent, by BoNT/A, whereas human SNAP-23 was resistant to all clostridial L chains. Lys185Asp or Pro182Arg mutations of human SNAP-23 induced susceptibility toward BoNT/E or toward both BoNT/A and BoNT/E, respectively.

General practitioner's knowledge of when to refer deaths to a coroner.

BACKGROUND: In 1992 about 179,000 deaths were reported to coroners in England and Wales and these represented 32% of the total number of registered deaths. Many of these cases were referred to coroners by general practitioners who certify the vast majority of deaths which occur outside hospitals. The safeguards to society which are provided by the coroner system in England and Wales are undermined if doctors fail to recognize those deaths which should be reported for further investigation. AIM: A study was undertaken to assess the ability of general practitioners to recognize deaths which require referral to a coroner. METHOD-A postal questionnaire consisting of 12 fictitious case histories was sent to all 323 general practitioners in Sheffield and the senior staff of the local coroner's office (two coroner's officers and two deputy coroners). Ten of the case histories contained a clear indication for referral to the coroner. RESULTS: A total fo 196 general practitioners (61%) and all the coroner's office staff returned the questionnaire. General practitioners correctly identified whether or not referral was indicated, with reasons, in a mean of 8.5 cases (range 4-12). Only six general practitioners (3%) were correct in all 12 cases. All of the coroner's staff were correct in all cases. CONCLUSION: General practitioners may be failing to bring certain categories of cases to the attention of coroners because of misconceptions of ignorance of their medico-legal responsibilities. General practitioner education in this area, and a closer working relationship between general practitioners and coroners may improve the situation.

Clinicians and the coronial system: ability of clinicians to recognise reportable deaths.

OBJECTIVE: To assess the ability of clinicians to recognise deaths which require referral to the coroner. DESIGN: Postal questionnaire consisting of 16 fictitious case histories, 14 of which contained a clear indication for referral to the coroner. SETTING: Large teaching hospital. Coroner's office. SUBJECTS: 200 clinicians from general medical and surgical firms and senior staff of the local coroner's office (two coroner's officers and the two deputy coroners). MAIN OUTCOME MEASURES: Number of correct assessments on questionnaire. RESULTS: The mean recognition score for the clinicians was 9.11 (range 3-14) with no difference between the clinical grades. All of the coroner's senior staff recorded maximum recognition scores of 16. CONCLUSIONS: The study highlights several features of the coronial system which are poorly understood by clinicians and provides the basis for an initiative to improve the medicolegal education of all clinicians.