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.     

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.     

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.

     

Scorpion alpha and alpha-like toxins differentially interact with sodium channels in mammalian CNS and periphery

Scorpion alpha-toxins from Leiurus quinquestriatus hebraeus, LqhII and LqhIII, are similarly toxic to mice when administered by a subcutaneous route, but in mouse brain LqhII is 25-fold more toxic. Examination of the two toxins effects in central nervous system (CNS), peripheral preparations and expressed sodium channels revealed the basis for their differential toxicity. In rat brain synaptosomes, LqhII binds with high affinity, whereas LqhIII competes only at high concentration for LqhII-binding sites in a voltage-dependent manner. LqhII strongly inhibits sodium current inactivation of brain rBII subtype expressed in HEK293 cells, whereas LqhIII is weakly active at 2 microM, suggesting that LqhIII affects sodium channel subtypes other than rBII in the brain. In the periphery, both toxins inhibit tetrodotoxin-sensitive sodium current inactivation in dorsal root ganglion neurons, and are strongly active directly on the muscle and on expressed muI channels. Only LqhII, however, induced repetitive end-plate potentials in mouse phrenic nerve-hemidiaphragm muscle preparation by direct effect on the motor nerve. Thus, rBII and sodium channel subtypes expressed in peripheral nervous system (PNS) serve as the main targets for LqhII but are mostly not sensitive to LqhIII. Toxicity of both toxins in periphery may be attributed to the direct effect on muscle. Our data elucidate, for the first time, how different toxins affect mammalian central and peripheral excitable cells, and reveal unexpected subtype specificity of toxins that interact with receptor site 3.