Modulating human proteinase activated receptor 2 with a novel antagonist (GB88) and agonist (GB110).

BACKGROUND AND PURPOSE: Many cells express proteinase activated receptor 2 (PAR2) on their plasma membrane. PAR2 is activated by proteolytic enzymes, such as trypsin and tryptase that cleave the receptor N-terminus, inititating signalling to intracellular G proteins. Studies on PAR2 have relied heavily upon activating effects of proteases and peptide agonists that lack stability and bioavailability in vivo. EXPERIMENTAL APPROACH: A novel small molecule agonist GB110 and an antagonist GB88 were characterized in vitro against trypsin, peptide agonists, PAR2 antibody, PAR1 agonists and flow cytometry,in seven cell lines using intracellular Ca(2+) mobilization and examined in vivo against PAR2- and PAR1-induced rat paw oedema. KEY RESULTS: GB110 is a potent non-peptidic agonist activating PAR2-mediated Ca(2+) release in HT29 cells (EC(50) ∼200 nM) and six other human cell lines, inducing PAR2 internalization. GB88 is a unique PAR2 antagonist, inhibiting PAR2 activated Ca(2+) release (IC(50) ∼2 µM) induced by native (trypsin) or synthetic peptide and non-peptide agonists. GB88 was a competitive and surmountable antagonist of agonist 2f-LIGRLO-NH(2), a competitive but insurmountable antagonist of agonist GB110, and a non-competitive insurmountable antagonist of trypsin. GB88 was orally active and anti-inflammatory in vivo, inhibiting acute rat paw oedema elicited by agonist GB110 and proteolytic or peptide agonists of PAR2 but not by corresponding agonists of PAR1 or PAR4. CONCLUSIONS AND IMPLICATIONS: The novel PAR2 agonist and antagonist modulate intracellular Ca(2+) and rat paw oedema, providing novel molecular tools for examining PAR2-mediated diseases.

Silver nanoparticles and magnetic beads with electrochemical measurement as a platform for immunosensing devices.

The simplicity and analytical utility of silver nanoparticles used as immunolabels with screen-printed measurement electrodes is illustrated by demonstrating an appropriate analytical signal for myoglobin (a protein marker for muscle damage) across a range of concentrations of physiological interest for distinguishing potential myocardial infarctions from normal background levels in serum. Silver nanoparticles were used as labels on one of a pair of anti-myoglobin clones while the other clone was covalently attached to magnetic beads. The two clones were selected so as to bind to different sites on the target protein and allow the formation of complexes containing both magnetic beads and silver nanoparticles. The magnetic beads enabled protein captured from test samples to be separated from other components, while the silver nanoparticles enabled the protein to be quantified. An oxidising potential, applied to screen-printed carbon electrodes, was used to dissolve silver without the need for an external oxidising agent. Silver ions released in the process were subsequently accumulated at the measurement electrodes by applying a suitable reducing potential and, finally, analytical signals were obtained by integrating the charges passed when accumulated silver was stripped from the electrodes by applying a potential ramp. The magnitudes of the measured charges were indicative of the concentrations of myoglobin in each of the test solutions.

Enhancement by Copper(II) of the voltammetric signal of vitamin B2 applied to its determination in breakfast cereals.

Addition of copper(II) to breakfast cereal samples was shown to significantly enhance the analytical signal obtained by electrochemical reduction of vitamin B(2) using linear sweep voltammetry on disposable carbon electrodes. The enhancement was observed only when dissolved oxygen was present. In model solutions the analytical signal was linear in the concentration range 6-150 ng/mL with a calculated limit of detection of 5 ng/mL (S/N = 3). This compared favorably with earlier work using a similar measurement approach--but in the absence of copper--in which the limit of detection was calculated to be 900 ng/mL. The effects of potential interferents commonly found in cereals were examined. In addition to signal attenuation by both sugar and starch (already reported), folic acid was found to increase (+6%) and iron to decrease (-11%) the analytical signal when present in the maximum concentration ratios, with respect to vitamin B(2), that are normally found in breakfast cereals. Nevertheless, the simplicity of the approach was potentially attractive for near-line quality control applications in manufacturing. The utility of the measurement approach was demonstrated by the addition of excess copper(II) sulfate to determine vitamin B(2) in aqueous extracts of breakfast cereals. The results agreed well with those provided by the cereal manufacturer who used an established HPLC method.

Hexacyanoferrate(III) as a mediator in the determination of total iron in potable waters as iron(II)-1,10-phenanthroline at a single-use screen-printed carbon sensor device.

Hexacyanoferrate(III) was used as a mediator in the determination of total iron, as iron(II)-1,10-phenanthroline, at a screen-printed carbon sensor device. Pre-reduction of iron(III) at -0.2V versus Ag/AgCl (1M KCl) in the presence of hexacyanoferrate(II) and 1,10-phenanthroline (pH 3.5-4.5), to iron(II)-1,10-phenanthroline, was complete at the unmodified carbon electrode surface. Total iron was then determined voltammetrically by oxidation of the iron(II)-1,10-phenanthroline at +0.82V, with a detection limit of 10microg l(-1). In potable waters, iron is present in hydrolysed form, and it was found necessary to change the pH to 2.5-2.7 in order to reduce the iron(III) within 30s. A voltammetric response was not found at lower pH values owing to the non-formation of the iron(II)-1,10-phenanthroline complex below pH 2.5. Attempts to incorporate all the relevant reagents (1,10-phenanthroline, potassium hexacyanoferrate(III), potassium hydrogen sulphate, sodium acetate, and potassium chloride) into a modifying coated PVA film were partially successful. The coated electrode behaved very satisfactorily with freshly-prepared iron(II) and iron(III) solutions but with hydrolysed iron, the iron(III) signal was only 85% that of iron(II).

Disposable sensor for measurement of vitamin B(2) in nutritional premix, cereal, and milk powder.

Methodologies for the determination of vitamin B(2) in food matrixes and a premix using simple sample conditioning steps coupled with a convenient and cheap electrochemical sensing device are presented. Electrochemical analysis based on differential pulse voltammetry (DPV) coupled to carbon electrodes gave a well-defined reduction peak at -0.42 V versus a Ag/AgCl quasi-reference electrode. Using a straightforward sample preparation step, vitamin B(2) can be measured successfully in a nutritional premix and food products. Standard additions of riboflavin were used to confirm the analyte concentrations and to provide precision data.

Protective effect of glucose on the anoxic myocardium of old and young mice.

To compare the function of old and young hearts contracting under identical conditions, isolated hearts of young and old C57BL/6J mice were perfused using a Langendorff preparation. During a 3-min period of anoxia some hearts showed a decrease in systolic pressure, while other hearts developed contracture. The incidence and magnitude of contracture was greater in the old hearts and they also showed a significantly larger decline in contractility. Increasing the glucose concentration improved the performance of both age groups but the performance of the old hearts was still inferior to that of the young. The glycogen content and utilization were virtually the same in the two age groups. When iodoacetate was added, all hearts developed contracture and the magnitude of the contracture was greater than in the absence of iodoacetate; during the period of reoxygenation, the young hearts recovered but, the old hearts developed a second contracture. A brief period of anoxia is more debilitating to old C57BL/6J hearts than to young ones.