Performance evaluation of a whole blood propofol analyser.

The authors evaluated an analyser for the determination of propofol concentrations in whole blood. The Pelorus 1000 (Sphere Medical) measures propofol concentrations in around 5 min without the requirement for sample preparation. The performance of the analyser was characterised with respect to linearity, precision in control solutions and whole blood and method comparison to an HPLC based reference method. In addition, the effects of substances considered to potentially affect the assay method were investigated. The analyser was found to be linear up to 12 µg/ml (R2 = 0.9993), with a lower limit of quantification of 0.75 µg/ml. Total within device imprecision in control solutions was 0.11 µg/ml at 5.32 µg/ml and 0.17 µg/ml at 10.3 µg/ml. Within run precision in whole blood was 0.04 µg/ml at 2.84 µg/ml and 0.08 µg/ml at 6.68 µg/ml and for the reference method was 0.06 µg/ml and 0.12 µg/ml respectively. In comparison to the reference method, the overall bias of the Pelorus 1000 system over the range is estimated to be 0.15 µg/ml (95% confidence interval -0.11-0.41 µg/ml). The only cross interference of note is to a highly elevated level of conjugated bilirubin, while low haematocrit levels lead to a 0.13 µg/ml under reading with respect to the HPLC reference. The system fulfils the requirements for measurement of propofol concentrations in whole blood samples with precision and accuracy suitable for elucidating propofol pharmacokinetics at clinically relevant concentrations. With no requirement for sample preparation and a fast time to results, the analyser opens up the possibility of studies to measure and respond to blood propofol concentrations in patients in close to real time.

A structural study of the interaction between the Dr haemagglutinin DraE and derivatives of chloramphenicol.

Dr adhesins are expressed on the surface of uropathogenic and diffusely adherent strains of Escherichia coli. The major adhesin subunit (DraE/AfaE) of these organelles mediates attachment of the bacterium to the surface of the host cell and possibly intracellular invasion through its recognition of the complement regulator decay-accelerating factor (DAF) and/or members of the carcinoembryonic antigen (CEA) family. The adhesin subunit of the Dr haemagglutinin, a Dr-family member, additionally binds type IV collagen and is inhibited in all its receptor interactions by the antibiotic chloramphenicol (CLM). In this study, previous structural work is built upon by reporting the X-ray structures of DraE bound to two chloramphenicol derivatives: chloramphenicol succinate (CLS) and bromamphenicol (BRM). The CLS structure demonstrates that acylation of the 3-hydroxyl group of CLM with succinyl does not significantly perturb the mode of binding, while the BRM structure implies that the binding pocket is able to accommodate bulkier substituents on the N-acyl group. It is concluded that modifications of the 3-hydroxyl group would generate a potent Dr haemagglutinin inhibitor that would not cause the toxic side effects that are associated with the normal bacteriostatic activity of CLM.

Decay-accelerating factor must bind both components of the complement alternative pathway C3 convertase to mediate efficient decay.

Decay-accelerating factor (DAF; CD55) inhibits the complement (C) cascade by dissociating the multimolecular C3 convertase enzymes central to amplification. We have previously demonstrated using surface plasmon resonance (Biacore International) that DAF mediates decay of the alternative pathway C3 convertase, C3bBb, but not of the inactive proenzyme, C3bB, and have shown that the major site of interaction is with the larger cleavage subunit factor B (Bb) subunit. In this study, we dissect these interactions and demonstrate that the second short consensus repeat (SCR) domain of DAF (SCR2) interacts only with Bb, whereas SCR4 interacts with C3b. Despite earlier studies that found SCR3 to be critical to DAF activity, we find that SCR3 does not directly interact with either subunit. Furthermore, we demonstrate that properdin, a positive regulator of the alternative pathway, does not directly interact with DAF. Extending from studies of binding to decay-accelerating activity, we show that truncated forms of DAF consisting of SCRs 2 and 3 bind the convertase stably via SCR2-Bb interactions but have little functional activity. In contrast, an SCR34 construct mediates decay acceleration, presumably due to SCR4-C3b interactions demonstrated above, because SCR3 alone has no binding or functional effect. We propose that DAF interacts with C3bBb through major sites in SCR2 and SCR4. Binding to Bb via SCR2 increases avidity of binding, concentrating DAF on the active convertase, whereas more transient interactions through SCR4 with C3b directly mediate decay acceleration. These data provide new insights into the mechanisms involved in C3 convertase decay by DAF.

Structural and functional insights into the interaction of echoviruses and decay-accelerating factor.

Many enteroviruses bind to the complement control protein decay-accelerating factor (DAF) to facilitate cell entry. We present here a structure for echovirus (EV) type 12 bound to DAF using cryo-negative stain transmission electron microscopy and three-dimensional image reconstruction to 16-A resolution, which we interpreted using the atomic structures of EV11 and DAF. DAF binds to a hypervariable region of the capsid close to the 2-fold symmetry axes in an interaction that involves mostly the short consensus repeat 3 domain of DAF and the capsid protein VP2. A bulge in the density for the short consensus repeat 3 domain suggests that a loop at residues 174-180 rearranges to prevent steric collision between closely packed molecules at the 2-fold symmetry axes. Detailed analysis of receptor interactions between a variety of echoviruses and DAF using surface plasmon resonance and comparison of this structure (and our previous work; Bhella, D., Goodfellow, I. G., Roversi, P., Pettigrew, D., Chaudhry, Y., Evans, D. J., and Lea, S. M. (2004) J. Biol. Chem. 279, 8325-8332) with reconstructions published for EV7 bound to DAF support major differences in receptor recognition among these viruses. However, comparison of the electron density for the two virus.receptor complexes (rather than comparisons of the pseudo-atomic models derived from fitting the coordinates into these densities) suggests that the dramatic differences in interaction affinities/specificities may arise from relatively subtle structural differences rather than from large-scale repositioning of the receptor with respect to the virus surface.