Analysis of 6-mercaptopurine in human plasma with a high-performance liquid chromatographic method including post-column derivatization and fluorimetric detection

A relatively simple assay with improved reliability and sensitivity for measuring levels of 6-mercaptopurine in human plasma is presented. After extraction of the compound and the added internal standard with phenyl mercury acetate, samples were separated by ion-pair reversed-phase high-performance liquid chromatography. On-line the analytes were oxidized to fluorescent products and detected in a flow-fluorimeter. The within-day coefficient of variation was 3.8% at a concentration of 25 ng/ml. The lower detection limit was 2 ng/ml when 1.0 ml of plasma was used. Mercaptopurine concentration versus time curves of two subjects after a single oral dose of azathioprine are shown.     

Preparation of megabase-sized tomato DNA and separation of large restriction fragments by field inversion gel electrophoresis (FIGE)

The Schwartz and Cantor technique for releasing and fractionating megabase-sized DNA from agarose-embedded cells is beginning to bridge the gap in resoluation between classical genetics and current molecular DNA techniques, particularly in mammalian systems. As yet no conditions have been described for preparing plant DNA that is of sufficient length to allow similar long-range restriction mapping experiments in plant systems. In this report, we describe the application of the Schwartz and Cantor technique for preparing high molecular weight DNA from embedded tomato leaf protoplasts, as well as conditions for generating and fractionating large restriction fragments, by field inversion gel electrophoresis (FIGE). The bulk of DNA released from lysed protoplasts was at least 2 Mb in size and amenable to restriction digestion as shown by hybridizing Southern blots with, among others, a probe for the Adh-2 gene of tomato. Restriction fragments as large as 700 kb were detected. Chloroplast DNA is isolated intact, amenable to restriction analysis and, in its native form, not mobile in FIGE.     

Test-Retest Reliability of Innovated Strength Tests for Hip Muscles

The burden of hip muscles weakness and its relation to other impairments has been well documented. It is therefore a pre-requisite to have a reliable method for clinical assessment of hip muscles function allowing the design and implementation of a proper strengthening program. Motor-driven dynamometry has been widely accepted as the gold-standard for lower limb muscle strength assessment but is mainly related to the knee joint. Studies focusing on the hip joint are less exhaustive and somewhat discrepant with regard to optimal participants position, consequently influencing outcome measures. Thus, we aimed to develop a standardized test setup for the assessment of hip muscles strength, i.e. flexors/extensors and abductors/adductors, with improved participant stability and to define its psychometric characteristics. Eighteen participants performed unilateral isokinetic and isometric contractions of the hip muscles in the sagittal and coronal plane at two separate occasions. Peak torque and normalized peak torque were measured for each contraction. Relative and absolute measures of reliability were calculated using the intraclass correlation coefficient and standard error of measurement, respectively. Results from this study revealed higher levels of between-day reliability of isokinetic/isometric hip abduction/flexion peak torque compared to existing literature. The least reliable measures were found for hip extension and adduction, which could be explained by a less efficient stabilization technique. Our study additionally provided a first set of reference normalized data which can be used in future research.     

Fatty Acid Ethyl Esters Induce Intestinal Epithelial Barrier Dysfunction via a Reactive Oxygen Species-Dependent Mechanism in a Three-Dimensional Cell Culture Model

Background & Aims

Evidence is accumulating that ethanol and its oxidative metabolite, acetaldehyde, can disrupt intestinal epithelial integrity, an important factor contributing to ethanol-induced liver injury. However, ethanol can also be metabolized non-oxidatively generating phosphatidylethanol and fatty acid ethyl esters (FAEEs). This study aims to investigate the effects of FAEEs on barrier function, and to explore the role of oxidative stress as possible mechanism.

Methods

Epithelial permeability was assessed by paracellular flux of fluorescein isothiocyanate-conjugated dextran using live cell imaging. Cell integrity was evaluated by lactate dehydrogenase release. Localization and protein levels of ZO-1 and occludin were analyzed by immunofluorescence and cell-based ELISA, respectively. Intracellular oxidative stress and cellular ATP levels were measured by dichlorofluorescein and luciferase driven bioluminescence, respectively.

Results

In vitro, ethyl oleate and ethyl palmitate dose dependently increased permeability associated with disruption and decreased ZO-1 and occludin protein levels, respectively, and increased intracellular oxidative stress without compromising cell viability. These effects could partially be attenuated by pretreatment with the antioxidant, resveratrol, pointing to the role of oxidative stress in the FAEEs-induced intestinal barrier dysfunction.

Conclusions

These findings show that FAEEs can induce intestinal barrier dysfunction by disrupting the tight junctions, most likely via reactive oxygen species-dependent mechanism.     

Non-rigid deformation to include subject-specific detail in musculoskeletal models of CP children with proximal femoral deformity and its effect on muscle and contact forces during gait

To account for proximal femoral deformities in children with cerebral palsy (CP), subject-specific musculoskeletal models are needed. Non-rigid deformation (NRD) deforms generic onto personalized bone geometry and thereby transforms the muscle points. The goal of this study was to determine to what extent the models and simulation outcomes in CP patients differ when including subject-specific detail using NRD or Magnetic Resonance Imaging (MRI)-based models. The NRD models slightly overestimated hip contact forces compared to MRI models and differences in muscle point positions and moment arm lengths (MALs) remained, although differences were smaller than for the generic model.     

Dimethylsulfoxide reduction by marine sulfate-reducing bacteria

Abstract Dimethylsulfoxide (DMSO) reduction occurred in five out of nine strains of sulfate-reducing bacteria from marine or saline environments, but not in three freshwater isolates. DMSO reduction supported growth in all positive strains. In Desulfovibrio desulfuricans strain PA2805, DMSO reduction occurred simultaneously with sulfate reduction and was not effectively inhibited by molybdate, a specific inhibitor of sulfate reduction. The growth yield per mol lactate was 26% higher with DMSO than with sulfate as electron acceptor. In extracts of cells of strain PA2805 grown on sulfate, a low level of DMSO-reducing activity was present (0.013 μmol (mg protein)⁻ min⁻); higher levels were found in cells grown on DMSO (0.56 μmol (mg protein)⁻ min⁻). In anoxic marine environments DMSO reduction by sulfate-reducing bacteria may lead to enhanced dimethylsulfide emission rates.     

Mouse models for sporadic cancer

Much of the advancement in mouse models for cancer during the past 2 decades can be attributed to our increasing capacity to specifically modify the mouse germ line. The first generations of oncomice and tumor-suppressor gene knockouts are now being succeeded by regulatable or conditional mouse tumor models, which can be utilized more effectively to establish correlations between distinct genetic lesions and specific tumor characteristics and to design and improve therapeutic intervention strategies. In this review we try to give the reader a flavor of how the latest reagents can be utilized.