Evaluation of phosphoramidon and three synthetic phosphonates for inhibition of botulinum neurotoxin B catalytic activity

Three putative metalloprotease inhibitors were synthesized and tested for their ability to inhibit the catalytic activity of botulinum neurotoxin B light chain (BoNT/B LC). The compounds were designed to emulate the naturally occurring metalloprotease inhibitor phosphoramidon, which has been reported to be a weak antagonist of BoNT/B action. All three analogs contained the dipeptide Phe-Glu in place of Leu-Trp of phosphoramidon and possessed a phenyl, ethyl or methyl group in place of the rhamnose sugar of the parent compound. The inhibitors were evaluated in a cell-free assay based on the detection of a fluorescent product following cleavage of a 50-mer synaptobrevin peptide ([Pya(88)] S 39-88) by BoNT/B LC. This peptide corresponds to the hydrophilic core of synaptobrevin-2 and contains a fluorescent analog L-pyrenylalanine (Pya) in place of Tyr(88). Cleavage of [Pya(88)] S 39-88 by BoNT/B LC gives rise to fragments of 38 and 12 amino acid residues. Quantification of BoNT/B-mediated substrate cleavage was achieved by separating the 12-mer fragment (FETSAAKLKRK-Pya) that contains the C-terminal fluorophore and measuring fluorescence at 377 nm. The results indicate that the phenyl-substituted synthetic compound ICD 2821 was slightly more active than phosphoramidon, but analogs with methyl or ethyl substitutions were relatively inactive. These findings suggest that phosphonate monoesters may be useful for providing insights into the structural requirement of BoNT/B protease inhibitors.     

Socioeconomics of influenza and influenza vaccination in Europe

Although the effectiveness of influenza vaccination is established vaccination policies and their implementation differ considerably across Europe. Historically the selective policies for influenza vaccination were based on the proven efficacy of influenza vaccine in healthy volunteers, and recognition that influenza complications and death occur mostly in elderly people with chronic medical conditions. Healthcare providers are faced with increasingly aging populations and costly new technologies and are more likely to extend immunisation policies if new initiatives are cost effective compared with accepted measures. Few studies of vaccine effectiveness focus on elderly cohorts with and without high risk conditions. Accordingly, healthcare providers in Denmark, Sweden, The Netherlands and the UK may require further data on vaccine effectiveness in elderly people without high risk conditions before reconsidering their policies. Scandinavian countries may also require data demonstrating benefits in people with diabetes. Review of recent US studies indicates that the available data on vaccine effectiveness in preventing influenza-related hospitalisation and death are applicable in Europe, but vaccine costs and cost effectiveness, and the overall economic burden of inpatient and outpatient care, need to be assessed country by country.     

Improved detection of rhinoviruses in nasal and throat swabs by seminested RT-PCR

A seminested RT-PCR (nRT-PCR) was used to detect picornavirus (PV) RNA in cell cultures inoculated with rhinoviruses (HRVs) and enteroviruses (EVs). PCR tests in which a primary "touchdown" PCR was followed by secondary reactions using PV or HRV specific primers were able to differentiate HRVs of 48 serotypes from EVs. PVnRT-PCR and HRVnRT-PCR were then used to test nasal and throat swabs from adult subjects with naturally acquired respiratory virus infections. The swabs were also analysed for respiratory viruses by cell culture techniques and the rates of PV identification by the two methods were compared. PVnRT-PCR was found to be at least five times more sensitive than cell culture for the detection of PVs in these clinical specimens. Paired acute and convalescent serum samples were tested for complement fixing antibodies to adenovirus, influenza A and B, respiratory syncytial virus, parainfluenza viruses 1, 2, and 3, Myco plasma pneumoniae, and Chlamydia psittaci. An enzyme-linked immunosorbent assay (ELISA) was used to detect rises in antibody level to coronavirus types 229E and OC43. The overall rate of pathogen identification in 159 swabs from adult asthmatics increased from 28% when only cell culture and serology were used to 57% when these methods were supplemented by PVnRT-PCR. © 1993 Wiley-Liss, Inc.     

Evaluation and detection of Duchenne's and Becker's muscular dystrophy carriers by manual muscle testing

Manual muscle testing of mothers of patients with X-linked muscular dystrophy has demonstrated patterns of proximal muscle weakness. The degree of weakness usually has little effect on activities of daily living but can be detected by standardized "break" testing with manual stabilization of body parts, elimination of synergistic muscles, and mechanical advantage given to the patient. Manual muscle testing is a valuable adjunct to the clinical and biochemical tools available for detecting carriers.     

A successful transport scenario for the health sector in developing countries

The role and operation of transport in the health sector in developing countries is important, costly but often taken for granted. This article suggests the need for a fresh look at the policy, planning and management of transport through the analysis of the essential components of a successful transport scenario for health services in developing countries i.e. transport and health planning; transport and organisational responsibility; the role of health sector donors; decision-making and procurement of transport and spares; transport and human resources; monitoring and control of transport and information; maintenance and repair; the budget. The article concludes with a checklist of key questions that may be used in assessing the contribution of transport to the health services.     

Cephaloridine-induced nephrotoxicity in the Fischer 344 rat: proton NMR spectroscopic studies of urine and plasma in relation to conventional clinical chemical and histopathological assessments of nephronal damage

The acute toxicological effects of the nephrotoxic antibiotic cephaloridine (CPH, 0–1500 mg/kg) in male Fischer 344 (F344) rats, have been investigated over 48 h using clinical chemistry, histopathology and proton nuclear magnetic resonance (¹H NMR) spectroscopy of urine and plasma. High field (400 and 600 MHz)¹H NMR urinalysis revealed increased excretion of lactic acid, acetoacetate, alanine, valine, lysine, glutamine and glutamate and a severe, time-dependent glycosuria. A major change observed in urine of CPH-treated animals was the dose-dependent increase in HB which may relate to altered energy metabolism. CPH also caused dose-dependent decreases in the urinary excretion of hippurate, allantoin and protein (conventional assay). This abnormal metabolic profile is consistent with a functional defect in the S₁/S₂ regions of the proximal tubule, and was confirmed by histologypost mortem. Functional changes observed included elevations in blood urea nitrogen (BUN) and urine flow rate (UFR) and dose-related decreases in urine osmolality. Spin-echo¹H NMR spectroscopic analysis of lyophilised plasma, reconstituted with²H₂O revealed an abnormal phase modulation of the methyl signal from free alanine and it is postulated that this is due to the release of transaminases from damaged tissue which via a reversible conversion to pyruvate, cause variable deuteration of alanine at the -CH position. This observation suggests that¹H NMR spectral patterns are also dependent on the level of plasma transaminases and this may provide a novel indicator of tissue damage.     

Effect of a metabolite of diazepam, 3-hydroxydiazepam (temazepam), on sleep in man

1 The effect of 3-hydroxydiazepam (temazepam, 10 mg and 20 mg) on sleep was studied in six healthy adult males using electroencephalography for sleep measures, and analogue scales for subjective assessments of well-being and sleep quality. The effects were compared with diazepam (5 mg and 10 mg).

2 Effect on total sleep time was restricted to the night of ingestion. There was no change in total sleep time after temazepam (10 mg), but with 20 mg total sleep time was increased (P = 0.01). Sleep onset latencies and awakenings were markedly reduced.

3 Temazepam reduced the duration (min) of stage 0 (P = 0.05) and stage 1 (P = 0.01) sleep, and the effect on stage 1 was seen during each two hourly interval of sleep (P = 0.05). No effects were observed with stage 3, 3+4 and REM sleep, except that the appearance of the first REM period was delayed with temazepam (20 mg) (P = 0.001).

4 The subjects, as a group, reported improved sleep, but subjective assessments of well-being were not altered. Correlations were calculated for sleep measures and subjective assessments.