Homogeneous time resolved fluorescence assay to measure histamine release

Histamine is a biogenic amine synthesized by the enzymatic decarboxylation of histidine. Implication of histamine in allergy is well described but histamine is also found in some specific neurones, functions as a neurotransmitter and regulates sleep/wake cycles, hormonal secretion, cardiovascular control and thermo-regulation. We have developed a TR-FRET histamine assay, based on the competition between sample histamine and allophycocyanine (XL665) labelled histamine for binding to a Europium cryptate (EuK) labelled antibody. As histamine is a small monoamine molecule, high affinity antibodies have been raised against carrier protein conjugated histamine. Therefore, sample histamine needs to be derivatized in the same way as the conjugated histamine, so that the antibody will have a similar affinity for both molecules. This acylation step is performed directly in wells and does not need to be done in separate vials, making handling easier for large numbers of samples. The incubation takes place at room temperature for 3 hours. The assay covers a measurement range of 1.56 to 400 nM and shows an analytical sensitivity of 1.3nM. We have shown that miniaturization of sample and reagents volumes down to 20 micro l does not alter these performances. This histamine release assay provides a particularly well adapted procedure for HTS and secondary screening compared to current heterogeneous methods.     

The effect of β-carotene supplementation on the pharmacokinetics of nelfinavir and its active metabolite M8 in HIV-1-infected patients

β-Carotene supplements are often taken by individuals living with HIV-1. Contradictory results from in vitro studies suggest that β-carotene may inhibit or induce cytochrome P450 enzymes and transporters. The study objective was to investigate the effect of β-carotene on the steady-state pharmacokinetics of nelfinavir and its active metabolite M8 in HIV-1 infected individuals. Twelve hour nelfinavir pharmacokinetic analysis was conducted at baseline and after 28 days of β-carotene supplementation (25,000 IU twice daily). Nelfinavir and M8 concentrations were measured with validated assays. Non-compartmental methods were used to calculate the pharmacokinetic parameters. Geometric mean ratios comparing day 28 to day 1 area under the plasma concentration-time curve (AUC(0-12 h)), maximum (C(max)) and minimum (C(min)) concentrations of nelfinavir and M8 are presented with 90% confidence intervals. Eleven subjects completed the study and were included in the analysis. There were no significant differences in nelfinavir AUC(0-12 h) and C(min) (-10%, +4%) after β-carotene supplementation. The M8 C(min) was increased by 31% while the M8 AUC(0-12 h) and C(max) were unchanged. During the 28 day period, mean CD4+ % and CD4+:CD8+ ratio increased significantly (p < 0.01). β-carotene supplementation increased serum carotene levels but did not cause any clinically significant difference in the nelfinavir and M8 exposure.     

A new acylated triterpene with antimicrobial activity from the leaves of <Emphasis Type="Italic">Rauvolfia vomitoria</Emphasis>

A new acylated tritrepene, 3β-hexadecanoyloxy-lup-20(29)-en-21-ol (1), along with seven known compounds, lupeol (2), betulinic acid (3), ursonic acid (4), -sitosterol (5), β-stigmasterol (6), 3-O-β-D-glucopyranosyl-β-stigmasterol (7), and palmitic acid (8), were isolated from the leaves of Rauvolfia vomitoria (Apocynaceae). Their structures were established on the basis of spectroscopic analysis and chemical evidence. The new acylated triterpene exhibited interesting antimicrobial activity against Candida albicans (a yeast) with the MIC value 64 μg/mL.     

A newborn piglet study of moderate hypoxic-ischemic brain injury by 1H-MRS and MRI

Cerebral hypoxia-ischemia (HI) is an important cause of perinatal brain damage in the term newborn. The areas most affected are the parasagittal regions of the cerebral cortex and, in severe situations, the basal ganglia. The aim of this study was to show that the newborn piglet model can be used to produce neuropathology resulting from moderate HI insult and to monitor damage for 7 days. Two acute cerebral HI were induced in newborn Large White piglets by reducing the inspired oxygen fraction to 4% and occluding the carotid arteries. Newborn piglets were resuscitated, extubated and monitored for 7 days. (31)P magnetic resonance spectroscopy (MRS) offers the ability to monitor the severity of the HI insults. Lactate (Lac) was detected in the HI group at 2 h, 3 days and 5 days after insult by (1)H MRS. Lac/n-acetylaspartate and Lac/choline and Lac/creatine ratios increased significantly (p < 0.01) in the HI group 2 h after HI insults and remained high over 7 days. For the HI group, mean T(2) values increased significantly in the parietal white matter (subcortical) for 5 days after HI insult [117.5 (+/-7.4) to 158.5 (+/-19.2) at T+3 days, 167.7 (+/-15.4) at T+5 days and 160.9 (+/-10.1) at T+7 days (p < 0.01)]. This newborn piglet model of moderate HI brain injury with reproducible cerebral damage could be use as reference for the study of neuroprotective strategy for a period of 7 days.     

A Convenient Synthesis of 5-Bromo-2-Pentanone

During a series of studies aimed at the total synthesis of pentacyclic triterpenes¹ we have examined the reaction of enollactones of type 1 with a variety of Grignard reagents. ^1c,2 In one of our problems we required the organometallic reagent 2a derived from the title compound 2b. A previous literature report for the preparation of this compound involved the reaction of α-acetylbutyrolactone 3 with aqueous HBr followed by steam distillation.³ In our hands the yields of 2b via this route were non-reproducible and at best, less than 15%.     

Evaluation of well‐balanced bore‐capturing schemes for 2D wetting and drying processes

We consider numerical solutions of the two‐dimensional non‐linear shallow water equations with a bed slope source term. These equations are well‐suited for the study of many geophysical phenomena, including coastal engineering where wetting and drying processes are commonly observed. To accurately describe the evolution of moving shorelines over strongly varying topography, we first investigate two well‐balanced methods of Godunov‐type, relying on the resolution of non‐homogeneous Riemann problems. But even if these schemes were previously proved to be efficient in many simulations involving occurrences of dry zones, they fail to compute accurately moving shorelines. From this, we investigate a new model, called SURF_WB, especially designed for the simulation of wave transformations over strongly varying topography. This model relies on a recent reconstruction method for the treatment of the bed‐slope source term and is able to handle strong variations of topography and to preserve the steady states at rest. In addition, the use of the recent VFRoe‐ncv Riemann solver leads to a robust treatment of wetting and drying phenomena. An adapted ‘second order’ reconstruction generates accurate bore‐capturing abilities.This scheme is validated against several analytical solutions, involving varying topography, time dependent moving shorelines and convergences toward steady states. This model should have an impact in the prediction of 2D moving shorelines over strongly irregular topography. Copyright © 2006 John Wiley & Sons, Ltd.     

Optical properties of InGaN quantum dots

We present time-resolved and spatially-resolved photoluminescence (PL) measurements of InGaN inclusions in a GaN matrix. The structures were grown by metal-organic chemical vapor deposition on sapphire and Si(111) substrates. Nonresonant pulsed excitation yields a broad PL peak, while resonant excitation into the nonresonant PL intensity maximum results in an evolution of a sharp resonant PL peak, having a spectral shape defined by the excitation laser pulse and a radiative decay time close to that revealed for PL under nonresonant excitation. Observation of a resonantly excited narrow PL line gives clear proof of the quantum dot (QD) nature of luminescence in InGaN–GaN samples. Cathodoluminescence (CL) and micro-PL measurements demonstrate sharp emission lines from single QD states. The recombination dynamics of single QD’s and the whole QD ensemble were investigated. Monoexponential decay was observed for the PL of single QD’s. For similar transition energies different time constants were obtained. Therefore the nonexponential decay observed for the whole ensemble is attributed to the coexistence of QD’s having similar ground-state transition energies, but significantly different electron–hole overlap.