Micellar electrokinetic chromatography and laser induced fluorescence detection of botulinum neurotoxin type A activity using a dual-labelled substrate

The activity of Botulinum neurotoxin type A (BoNT A) can be measured by monitoring the toxin's endopeptidase reaction with its peptide substrate. In this report, a sensitive and simple capillary electrophoresis (CE) method for analysing BoNT A activity was developed using a peptide substrate labelled with Fluorescein isothiocynate (FITC) at the N-terminal and biotin at the C-terminal. This dual labelling enables not only highly sensitive laser induced fluorescence (LIF) detection of the reaction product, but also good analytical separation of the product from the peptide substrate by Micellar Electrokinetic Chromatography (MEKC). The separation between the product peak and the substrate peak was approximately 5 min using the dual-labelled substrate, while just about 1 min using the FITC-labelled substrate without biotinylation. Using the current assay method, BoNT A with concentration as low as 0.1 ng ml^?1 (3.6 U mL^?1 in mouse LD₅₀) in water was detected with a S:N ratio of 3 (RSD <19%) and a linear range of four orders of magnitude. With CE's advantages of very small sample volume needed, this method may find particular applications as in assays of BoNT A activity in water samples and kinetic analyses of toxin activity.     

Concomitant detection of CYP1A1 enzymatic activity and CYP1A1 protein in individual cells of a human urothelial cell line using a bilayer microfluidic device

To understand molecular networking at the cellular level, analyses of processes and effects at the single-cell level are most appropriate. Usual biochemical or molecular biological analyses are based on integrated signals of numerous cells which differ, however, in their expression and activity profiles. Here we show that it is possible to determine different types of properties of individual cells by means of a specifically designed microfluidic device. As part of investigations to characterize the human urothelial cell line 5637 as a potential model system for studies of toxic and carcinogenic effects on urothelial cells, we use this cell line to assign cytochrome P450 activity, and expression of the enzymes involved, to individual cells. It is shown that the cell population is very heterogeneous with respect to the extent and kinetics of CYP1A1-dependent ethoxyresorufin O-deethylase (EROD). This is also true for the cells’ CYP1A1 protein content. With some exceptions, the EROD activity largely coincides with the presence of CYP1A1 protein in the cells. The results obtained with the microfluidic device are promising and open up new perspectives with regard to multi-property determinations in individual cells and to studies focusing on the biochemical and molecular heterogeneity of cells. Figure Formation of fluorescent resorufin from ethoxyresorufin by cytochrome P450 activity in urothelial cells attached within the chamber of a microfluidic device     

Convenient preparation of pinometostat and related 5′-deoxy-5′-amino adenosine derivatives as well as their activity against DOT1L

From adenosine and 2′-C-Me adenosine, a 3-step route towards nucleoside DOT1L inhibitors, including pinometostat, EPZ5677, and FED1, was established. With useful structural-activity relationship information, the newly prepared 2′-C-Me adenosine derivatives contribute to the limited repertoire of ribose-modified nucleoside DOT1L inhibitors. In general, this new synthetic method will facilitate not only the study of nucleoside DOT1L inhibitors, but also the synthetic and medicinal chemistry research of 5′-deoxy-5′-amino adenosine derivatives.     

In vivo evaluation of CYP1A2, CYP2A6, NAT-2 and xanthine oxidase activities in a Greek population sample by the RP-HPLC monitoring of caffeine metabolic ratios

A RP-HPLC method was developed for the assessment of caffeine and its metabolites in urine and was used for the evaluation of the CYP1A2, CYP2A6, xanthine oxidase (XO) and N-acetyl-transferase-2 (NAT-2) in vivo activities in 44 Greek volunteers (21 men, 23 women). Spot urine samples were analyzed 6 h after 200 mg caffeine consumption, following a 30 h methylxantine-free diet. The major urinary caffeine metabolites are 1-methyluric acid (1U), 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 1-methylxanthine (1X), 1,7-dimethyluric acid (17U) and 1,7-dimethylxanthine (17X). CYP1A2, CYP2A6, XO and NAT-2 activities were estimated from the metabolic ratios (AFMU + 1U + 1X)/17U, 17U/17X, 1U/(1X + 1U) and AFMU/(AFMU + 1U + 1X), respectively. Metabolites and internal standard were extracted with chloroform/isopropanol (85:15, v/v) and separated on a C18 column by an isocratic HPLC system using a two-step elution with manual switch from solvent A (0.1% acetic acid-methanol-acetonitrile, 92:4:5 v/v) to solvent B (0.1% acetic acid-methanol, 60:40, v/v), and detected at 280 nm. The method exhibited adequate metabolite separation (resolution factors >1.48), accuracy (94.1-106.3%) and intraday and interday precision <8.02 and <8.78%, respectively (n = 6). Smoking affected only CYP1A2, whereas gender had no effect in any enzyme activity. NAT-2 exhibited bimodal distribution, 63.6% of volunteers being slow acetylators. The developed RP-HPLC method was fully validated and successfully applied for the evaluation of CYP1A2, CYP2A6, XO and NAT-2 activities.