The effectiveness of a pyriprole (125 mg/ml) and a metaflumizone (150 mg/ml) combined with amitraz (150 mg/ml) spot-on treatment in preventing Phlebotomus perniciosus from feeding on dogs

A controlled clinical trial was performed to assess the effectiveness of a pyriprole (125 mg/ml) and a metaflumizone (150 mg/ml) combined with amitraz (150 mg/ml) spot-on treatment (recommended dosage) in preventing adult female sandflies (Phlebotomus perniciosus) from feeding on dogs. Sandfly mortality was also assessed. Twelve beagle dogs were used in the study. Prior to treatment they were checked for their attractiveness to sandflies, ranked accordingly to generate partner triplets of equivalent sensitivity to sandflies: four control dogs, four treated with the pyriprole and four with the metaflumizone spot-on. The dogs were challenged with 50 unfed adult female sandflies (8-10 days old), in cages for one hour on Day 1 and Day 7. The sandflies were checked for blood feeding and mortality at one hour, 24 hours and 48 hours after exposure to the dogs. A very poor anti-feeding effect (near 7%) was seen on sandflies with the metaflumizone combined with amitraz and no antifeeding effect was seen with pyriprole. The sandfly mortality effect as a result of exposure to treated dogs was under 20% for the two spot-on. The two formulations could not be proposed in a leishmaniosis prevention program.     

Intranasal midazolam in piglets: pharmacodynamics (0.2 vs 0.4 mg/kg) and pharmacokinetics (0.4 mg/kg) with bioavailability determination

Intranasal midazolam was studied in two series of piglets: series 1, n = 20 (18 +/- 3 kg), a randomized double blind pharmacodynamic study to compare doses of 0.2 mg/kg and 0.4 mg/kg; series 2, n = 9 (42 +/- 8 kg), a pharmacokinetic study with a 0.4 mg/kg dose administered either intravenously (i.v.) or intranasally (i.n.) in a cross-over protocol with a one-week wash-out period between each. In series 1, midazolam caused significant anxiolysis and sedation within 3 to 4 min, without a significant difference between 0.2 and 0.4 mg/kg doses for any of the studied parameters. In series 2, after intranasal midazolam administration of 0.4 mg/kg, plasma concentrations attained a maximum (Cmax) of 0.13 +/- 0.04 mg/l at 5 min (median Tmax) and remained higher than 0.04 mg/l until 60 min. The bioavailability factor (F) in this study was F = 0.64 +/- 0.17 by the intranasal route. The terminal half-life (T1/2 lambda z) = 145 +/- 138 min was comparable with the i.v. administration half-life (158 +/- 127 min). In conclusion, optimal intranasal midazolam dose in piglets was 0.2 mg/kg, which procures rapid and reliable sedation, adapted to laboratory piglets.     

Relative biological effectiveness of high-energy photons (up to 50 MV) and electrons (50 MeV)

In vitro studies of the relative biological effectiveness (RBE) of 50-MV X rays have shown an RBE of 1.1 relative to 4-MV X rays. This will be important in clinical radiotherapy. The aim of this study was to verify these results and to investigate whether photonuclear processes might cause the difference in RBE. To do so, 50- and 20-MV X rays and 50-MeV electrons were investigated with respect to RBE. Chinese hamster V79 cells were irradiated in a specially designed system which allows for a high reproducibility of geometry and dosimetry. Fractionation experiments were also carried out to establish the RBE at the clinically relevant dose level, 2 Gy. Fricke dosimetry was used, and the results were confirmed with ionization chamber measurements. The RBE for 50-MV X rays was estimated to be 1.14 at a surviving fraction of 0.1 and 1.12 at a surviving fraction of 0.01. The RBEs for the other qualities were equal to one. The RBE calculated for the 2 Gy/fraction experiments was 1.17.     

Studies on poly(L-lysine50, L-tyrosine50)-DNA interaction

Interaction between poly(Lys⁵⁰, Tyr⁵⁰) and DNA has been studied by absorption, circular dichroism (CD), and fluorescence spectroscopy and thermal denaturation in 0.001M Tris, pH 6.8. The binding of this copolypeptide to DNA results in an absorbance enhancement and fluorescence quenching on tyrosine. There is also an increase in the tyrosine CD at 230 nm. The CD of DNA above 250 nm is slightly shifted to the longer wavelength which is qualitatively similar to, but quantitatively much smaller than, that induced by polylysine binding. At physiological pH the poly(Lys⁵⁰, Tyr⁵⁰)–DNA complex is soluble until there is one lysine and one tyrosine per nucleotide in the complex. The same ratio of amino acid residues to nucleotide has also been observed in copolypeptide-bound regions of the complex. The addition of more poly(Lys⁵⁰, Tyr⁵⁰) to DNA yields a constant melting temperature, T_m′, for bound base pairs at 90°C which is close to that of polylysine-bound DNA under the same condition. The melting temperature, T_m, of free base pairs at about 60°C on the other hand, is increased by 10°C as more copolypeptide is bound to DNA. As the temperature is raised, both absorption and CD spectra of the complexes with high coverage are changed, suggesting structural alteration, perhaps deprotonation, on bound tyrosine. The results in this report also suggest that intercalation of tyrosine in DNA is unlikely to be the mode of binding.     

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH₅₀ is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH₅₀ tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH₅₀ level will be zero; if one or more components of the classical pathway are decreased, the CH₅₀ will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.Download video file.^{(81M, mp4)}     

Evidence for ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) self-association through PDZ-PDZ interactions

Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a versatile membrane-cytoskeleton linking protein that binds to the COOH-tail of specific integral membrane proteins through its two PDZ domains. These EBP50 binding interactions have been implicated in sequestering interactive sets of proteins into common microdomains, regulating the activity of interacting proteins, and modulating membrane protein trafficking. With only two PDZ domains, it is unclear how EBP50 forms multiprotein complexes. Other PDZ proteins increase their breadth and diversity of protein interactions through oligomerization. Hypothesizing that EBP50 self-associates to amplify its functional capacity, far-Western blotting of cholangiocyte epithelial cell proteins with EBP50 fusion protein revealed that EBP50 binds to a 50-kDa protein. Far-Western blotting of EBP50 isolated by two-dimensional gel electrophoresis or immunoprecipitation demonstrates that the 50-kDa binding partner is itself EBP50. Further, co-transfection/co-precipitation studies show the self-association can occur in an intracellular environment. In vitro analysis of the EBP50-EBP50 binding interaction indicates it is both saturable and of relatively high affinity. Analysis of truncated EBP50 proteins indicates EBP50 self-association is mediated through its PDZ domains. The ability to self-associate provides a mechanism for EBP50 to expand its capacity to form multiprotein complexes and regulate membrane transport events.