A high-throughput mass spectrometric assay for discovery of human lipoxygenase inhibitors and allosteric effectors

Lipoxygenases (LOXs) regulate inflammation through the production of a variety of molecules whose specific downstream effects are not entirely understood due to the complexity of the inflammation pathway. The generation of these biomolecules can potentially be inhibited and/or allosterically regulated by small synthetic molecules. The current work describes the first mass spectrometric high-throughput method for identifying small molecule LOX inhibitors and LOX allosteric effectors that change the substrate preference of human lipoxygenase enzymes. Using a volatile buffer and an acid-labile detergent, enzymatic products can be directly detected using high-performance liquid chromatography–mass spectrometry (HPLC–MS) without the need for organic extraction. The method also reduces the required enzyme concentration compared with traditional ultraviolet (UV) absorbance methods by approximately 30-fold, allowing accurate binding affinity measurements for inhibitors with nanomolar affinity. The procedure was validated using known LOX inhibitors and the allosteric effector 13(S)-hydroxy-9Z,11E-octadecadienoic acid (13-HODE).     

Fluorescence quenching studies of apolipoprotein A-I in solution and in lipid-protein complexes: protein dynamics

Fluorescence lifetime and intensity quenching studies of human plasma apolipoprotein A-I (apo A-I) in aqueous solution and in recombinant lipoprotein complexes with dimyristoylphosphatidylcholine (DMPC) indicate differences in conformational dynamics. In aqueous solution, the bimolecular quenching constants (k*) for lipid-free apo A-I fluorescence quenching by oxygen and acrylamide are 2.4 X 10(9) and 0.38 X 10(9) M-1 s-1, respectively. These values are independent of the oligomeric form of the protein. There is no correlation between the relatively small k* for apo A-I, which reflects rapid, low-amplitude protein fluctuations, and the labile conformational changes of apo A-I folding reactions, like denaturation, which occur on a slower time scale. In recombinant DMPC/apo A-I complexes (100:1 molar ratio) the protein increases in amphiphilic alpha-helical structure as it blankets the lipid matrix. The apparent k* for oxygen quenching of apo A-I fluorescence in the complex is large and increases in a temperature-dependent manner. We have introduced a two-compartment model, which discriminates the source of quencher molecules as aqueous or lipid, to describe oxygen quenching of DMPC/apo A-I fluorescence. The magnitude and temperature dependence of the apparent k* predominantly reflect the partitioning of oxygen between the two phases rather than being a probe of the lipid physical state. Calculations of the helical hydrophobic moment in apo A-I indicate that tryptophan residues 8 and 72 occur at the lipid-protein interface of amphiphilic alpha-helices, whereas the other two tryptophan residues (50, 108) lie on the nonpolar faces of amphiphilic helices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aza-BODIPY: Improved synthesis and interaction with soluble Aβ1–42 oligomers

Dye-binding assays that are used to evaluate anti-aggregation ability of small molecule inhibitors towards amyloids are known to be prone to false-positive effects due to spectral overlaps between the dye and the inhibitor. Aza-BODIPY dye, which has both excitation and emission maxima above 600 nm, exhibits a significant increase in its fluorescence intensity in the presence of soluble oligomers of Aβ1–42. These results indicate that aza-BODIPY could serve as a near-IR probe for detecting conformational changes of Aβ1–42 soluble oligomers in vitro, and it should eliminate false-positive effects that are associated with currently utilized thioflavin T-based dyes. In addition, a facile synthesis of aza-BODIPY has been developed, which might further expand the applications of this dye.     

Acute toxicity effects of ibuprofen on behaviour and haematological parameters of African catfish Clarias gariepinus (Burchell, 1822)

Indiscriminate discharge of pharmaceutical waste into the aquatic ecosystem may pose serious health challenges to aquatic biota. The effect of acute exposure to ibuprofen was evaluated using changes in behaviour and haematological parameters under static bio-assay method in Clarias gariepinus. Test specimens were exposed to acute concentrations of ibuprofen (0.28, 0.33, 0.38, 0.43 and 0.48 mg l^?1) for 24, 48, 72 and 96 h durations respectively. Behavioural and phenotypic changes were observed in surviving fish. There were significant (p < 0.05) concentration and duration-dependent increases in erythrocyte (RBC), haemoglobin (Hb), pack cell volume (PCV) and leukocytes (WBC) in treated fish compared to the control. Insignificant decreases (p > 0.05) in mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were observed in treated fish compared to the control. Ibuprofen elicited dose and duration- dependent decrease in neutrophil counts with the decreases being significant (p < 0.05) in the higher doses of 0.43 and 0.48 mg l^?1. Ibuprofen did not elicit any significant changes in monocytes, basophils and eosinophils. Changes observed in this study showed that ibuprofen negatively affected the health of the fish and we recommend that discharge of ibuprofen into the aquatic environment should be monitored and controlled.     

Context-dependent memory: colour versus odour

An olfactory stimulus and a visual stimulus were employed in a context- dependent memory study using a prose passage as the to-be-remembered item. Ninety-five university students (aged 17-35 years) learned the passage of prose in the presence of one of the stimuli and were then asked to recall the passage with the original context either reinstated or not reinstated. The results revealed a significant context-dependent memory effect for the olfactory cue but not for the visual cue. They demonstrate support for the effectiveness of odours as context cues and it is suggested that context-dependent memory processes may underlie the formation and retrieval of odour-evoked autobiographical memories.