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.     

A Note on a Simple and Rapid Method of Bacteriological Sampling by Means of Agar Sausages

A rapid and simple method is described for the bacteriological examination of contaminated surfaces. It uses an imprint process with 'agar sausages' sterilized in polyamide casings.     

The role of glucose in ajmalicine production by catharanthus roseus cell cultures

The role of glucose in ajmalicine production by Catharanthus roseus was investigated in the second stage of a two-stage batch process. Activities of tryptophan decar-boxylate (TDC) and anthranilate synthase (AS), two enzymes In the pathway leading to ajmalicine, were higher after induction with 40 g/L glucose than after induction with 60 or 80 g/L glucose. Experiments with different media containing mixtures of glucose and the nonpermeating osmotic agent xylose, and using an already induced culture as inoculum, revealed that a minimum amount of glucose is required to support ajmalicine production after enzyme induction. This requirement was not an osmotic effect. The relation between the glucose concentration and the specific ajmalicine production rate, q(p), was investigated in seven (fed-)batch cultures with constant glucose concentrations: 23, 29, 35, 53, 57, 75, and 98 g/L. In the cultures with a low glucose concentration (23, 29, and 35 g/L) the q(p) was 2.7-times higher than the cultures with 53 and 57 g/L, and almost six times higher than the cultures with a high glucose concentration (75 and 98 g/L). A glucose perturbation experiment (from 53 to 32 g/L) demonstrated that the ajmalicine production rate was adjusted without much delay. A kinetic equation is proposed for the relationship between the glucose concentration and q(p). Differences in enzyme induction and ajmalicine production at different glucose levels could not be explained by the intracellular concentrations of glucose, fructose, sucrose, or starch. (c) 1995 John Wiley & Sons Inc.     

Measurement of bone displacement in a macerated human skull induced by orthodontic forces; A holographic study

A macerated human skull was subjected to orthodontic forces from 6.25 to 7.0 N per side. Double-exposure holographic interferograms (5 mW HeNe laser) were made frontally and oblique laterally. These were complemented with observations from a real time holographic interferogram. The displacements of the maxilla and the zygomatic bone were quantified. Special attention was paid to the reaction of the zygomaticomaxillary suture.     

A multiplicity of erythrocyte glycolipids of the neolacto series revealed by immuno-thin-layer chromatography with monoclonal anti-I and anti-i antibodies

The thin-layer-chromatography immunostaining procedure was applied to human erythrocyte glycolipids using monoclonal anti-i and anti-I antibodies which are directed against epitopes on linear and branched carbohydrate chains of the neolacto (poly-N-acetyl-lactosamine) series. An examination of native and mild-acid-treated glycolipids from normal adult (I_{adul t} antigen type), neonatal (i_cord) , and I-antigen-deficient adult (i_adult) erythrocytes enabled certain structural inferences to be made as follows: (a) cells of both I and i phenotypes contain a multiplicity of glycolipids of the neolacto series whose backbones consist of 8 or more sugar residues; (b) the octasaccharide backbones are predominantly linear in cells of i phenotype and branched in those of I type; and (c) more complex glycolipids having decasaccharide and larger backbones with both linear and branched sequences occur in erythrocytes of both phenotypes, 0     

Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines.

Using fluorescence in situ hybridization and Southern blot analysis, we show that three clonally derived cell lines transformed with bovine papillomavirus (BPV), including ID13, the cell line commonly employed for BPV replication studies, are heterogeneous populations having extensive cell-to-cell variation in both the distribution and amount of BPV DNA. Different subclones of ID13 were found to differ in the form and amount of BPV DNA they contain. Most subclones showed no detectable BPV sequences; some contained either extrachromosomal BPV molecules distributed throughout the nucleus or BPV sequences integrated at discrete chromosomal sites, while others contained both integrated and plasmid forms. The results of density gradient analysis of BPV DNA from individual homogeneous subclones showed replication of the extrachromosomal BPV plasmids in a random-choice mode. In all cell lines studied, the presence after one round of chromosomal DNA replication of unreplicated BPV DNA and of BPV DNA having two postreplicative strands was independent of the presence of high-BPV-copy-number ("jackpot") cells. Our results substantiate the earlier conclusion that extrachromosomal BPV molecules replicate randomly and not according to a once-per-cell-cycle mechanism.     

Chromosomal localization of transfected genes by a combination of hot banding and fluorescence in situ hybridization.

We describe the combination of hot banding with fluorescence in situ hybridization as a rapid and efficient method to identify integration sites of transfected DNA sequences in chromosomes. As a test system we used SW480 EJ2, a clonal cell line obtained after transfection of SW480 with pSV2neoEJ, a plasmid containing a point-mutated, c-Ha-RAS oncogene. Nick-translated probes were compared with random primed-labeled probes to evaluate their relative efficiency in fluorescence in situ hybridization. The fluorescence signals were quantified in interphase nuclei by confocal scanning laser microscopy. Nick-translated probes were found to yield better results. Hot banding followed by fluorescence in situ hybridization localized the integration site of pSV2neoEJ in SW480 EJ2 at the site of a translocation on a marker chromosome Xp+. The combination of fluorescence in situ hybridization and hot banding can be used to (a) rapidly and efficiently analyze integration sites in large numbers of transfectants, (b) assess the clonality of transfected cell lines, and (c) localize the site of integration of transfected genes in the recipient genome.     

Differential responses of freshwater wetland soils to sulphate pollution

Sulphate (SO₄ ^2-)reduction rates are generally low in freshwaterwetlands and are regulated by the scarceavailability of the ion. Increasedconcentrations of this electron acceptor due tosulphur (S) pollution of groundwater andsurface water may, however, lead to highSO₄ ^2- reduction rates now regulatedby the availability of appropriate electrondonors. Due to variations in this availability,the response to S pollution (e.g. from surfacewater or groundwater) is expected to differbetween soils. This hypothesis was tested inlaboratory mesocosm experiments by comparingtwo wetland soil types with distinctlydifferent humus profiles: a Hydromoder and aRhizomull type. In the first type, expected tohave a higher availability of degradable soilorganic matter (SOM), SO₄ ^2-availability appeared to be rate limiting forSO₄ ^2- reduction. In the Rhizomullsoils, in contrast, the electron acceptor didnot limit SO₄ ^2- reduction rates athigher concentrations. These differences inresponse could not, however, be attributed todifferences in the various SOM fractions or inSOM densities. Eutrophication and free sulphideaccumulation, two major biogeochemical problemscaused by SO₄ ^2- pollution, occurredin both types. The absolute extent ofphosphorus mobilisation was determined by theconcentration of this element in the soil (C/Pratio), while the level of sulphideaccumulation was governed by the concentrationof dissolved iron in the pore water. It wastherefore concluded that neither the humusprofile nor the concentrations of different SOMfractions in the soils are reliable indicatorsfor the sensitivity of wetland types to Spollution.