Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1

Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding V_H CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long V_H CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long V_H CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.     

Modelling nicotine intake in smokers and snuff users using biological fluid nicotine metabolites

Data from two clinical studies involving smokers and snuff users were analysed to address the estimation of nicotine intake using urinary and salivary nicotine metabolites. Comprehensive regression modelling is performed to determine which combinations of urinary nicotine metabolites provide better estimation of nicotine intake in these subjects than the predominant practice of basing nicotine intake on urinary cotinine analysis alone. Within-subject and between-subject variability is examined with regard to reliability of measurement and replicate sampling. Salivary cotinine models are compared to urinary metabolite models. Results suggest that estimation of nicotine intake is greatly improved by measuring urinary cotinine and additional metabolites (trans-3´-hydroxycotinine, and glucuronide conjugates) rather than measuring only cotinine. Analyses also indicate that replicate sampling on subjects greatly improves the reliability of the measurement. Based on these data, a model to predict nicotine equivalents based solely on saliva cotinine was severely inferior to any of the urinary models, including that of urinary cotinine alone.     

Metabolic effects of paraquat onSaccharomyces cerevisiae

Paraquat-tolerant cells ofSaccharomyces cerevisiae were selected by growing the cells in medium supplemented with paraquat either in a continuous selection culture with UV irradiation of the cells or in shaker flasks. When tranferred to fresh medium containing 1.0 mM paraquat, the selected cells showed growth rates and product formation patterns very similar to those of normal cells grown in a medium without paraquat. In the presence of paraquat, cells that had developed a tolerance were able to metabolize the fermentation products formed from glucose. Normal cells were unable to do so for considerable time.     

Transformation of photoinactive to photoactive protochlorophyllide in isolated prolamellar bodies of wheat (Triticum aestivum) exposed to low pH and ATP

Isolated prolamellar bodies from the etioplasts of dark-grown wheat ( Triticum aestivum L. cv. Walde, Weibull) contain the enzyme NADPH-protochlorophyllide oxidoreductase. The organisation of this enzyme in a pigment-protein complex results in fluorescence emission maxima at 633 and 657 nm. Isolated prolamellar bodies stored in darkness for 24 or 48 h at 4°C (pH 7.2) in the presence of NADPH showed a fluorescence emission ratio 657/633 nm around 4 at −196°C. With acidic conditions this fluorescence ratio increased, with an optimum at pH 5.5. Such an increase was even more pronounced in the presence of ATP and NADPH with ratios up to 8, but was completely blocked when the sulfhydryl inhibitor, dithiobis-nitrobenzoic acid, was added. As shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis the amount of NADPH-protochlorophyllide oxidoreductase in the prolamellar bodies did not change during storage for 24 or 48 h.
The total amount of protochlorophyllide measured in acetone extracts did not change significantly during storage for 48 h. The values were similar for storage at pH 7.2 and 5.5, but at lower pH (around 5) the pigment content decreased to a third.
The most plausible explanation for the increase in fluorescence ratio is that low pH and ATP give rise to a change in conformation, which results in transformation of the short wavelength (633 nm) fluorescing protochlorophyllide to the long wavelength (657 nm) fluorescing form.     

The mechanism of the Ni-Fe hydrogenases: a quantum chemical perspective

The catalytic cycle for the heterolytic splitting of H2 by Ni-Fe hydrogenase has been investigated in four recent quantum chemical studies. The mechanisms proposed are described and compared. Although there are clear differences in these mechanisms and in the assignments of the different states observed experimentally, there are also important points of concensus.     

The reliability of histochemical fibre typing of human necropsy muscles

Summary The reliability of muscle fibre typing of post mortem specimens was investigated with special reference to the influence of time and temperature. In specimens stored at +4° C, muscle fibre typing could be reliably performed up to at least ten and fifteen days post mortem for the masseter and biceps brachii muscles respectively. The corresponding figures for storage at room temperature were three and six days. The difference in the preservation of enzyme activity between masticatory and limb muscles might be related to the demonstrated difference in the fibre type composition and thus the enzyme content and energy sources.     

Author index to volume 154

Horseradish peroxidase-catalyzed N-demethylation of aminopyrine and dimethylaniline results in generation of free radical intermediates which can interact with glutathione (GSH) to form a glutathione radical. This can either dimerize to yield glutathione disulfide or react with O₂ to form oxygenated products of glutathione. Ethylmorphine is not a substrate in the peroxidase-mediated reaction, and free radical intermediates which react with GSH, are not formed from aminopyrine and dimethylaniline when the horseradish peroxidase/H₂O₂ system is replaced by liver microsomes and NADPH. Therefore, it appears unlikely that formation of free radical intermediates can be responsible for the depletion of GSH observed during N-demethylation of several drugs in isolated liver cells.     

A 5, 6-dimethoxylated flavone in the leaf resin of Adenostoma sparsifolium

A new flavone 5,6-dimethoxy-3,7,4′-trihydroxyflavone was isolated from the external leaf resin of Adenostoma sparsifolium and identified by spect