Ernst Bayer

Bcl-XL protects pancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis

In this study we sought to clarify the role of the pro-apoptotic potential of mitochondria in the death pathway emanating from the TRAIL (APO-2L) and CD95 receptors in pancreatic carcinoma cells. We focused on the role of the Bcl-2 family member Bcl-XL, using three pancreatic carcinoma cell lines as a model system, two of which have high (Panc-1, PancTuI) and one has low (Colo357) Bcl-XL expression. In these cell lines, the expression of Bcl-XL correlated with sensitivity to apoptosis induced by TRAIL or anti-CD95. Flow cytometric analysis revealed cell surface expression of TRAIL-R1 and TRAIL-R2 on PancTuI and Colo357, and TRAIL-R2 on Panc-1 cells. In Colo357 cells retrovirally transduced with Bcl-XL, caspase-8 activation in response to treatment with TRAIL or anti-CD95 antibody was not different from parental cells and EGFP-transfected controls, however, apoptosis was completely suppressed as measured by the mitochondrial transmembrane potential Δψm, caspase-3 activity (PARP cleavage) and DNA-fragmentation. Inhibition of Bcl-XL function by overexpression of Bax or administration of antisense oligonucleotides against Bcl-XL mRNA resulted in sensitization of Panc-1 cells to TRAIL and PancTuI cells to anti-CD95 antibody-induced cell death. The results show that Bcl-XL can protect pancreatic cancer cells from CD95- and TRAIL-mediated apoptosis. Thus, in these epithelial tumour cells the mitochondrially mediated ‘type II’ pathway of apoptosis induction is not only operative regarding the CD95 system but also regarding the TRAIL system.

Recent advances in capillary electrophoresis/electrospray/mass spectrometry

In this review, the progress in hyphenation of capillary electrophoresis (CE) with electrospray ionization‐mass spectrometry (ESI‐MS) since the article of Banks (Banks, J. F., Electrophoresis 1997, 18, 2255–2266) is reported. In all capillary‐based electromigration techniques, such as capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), capillary isoelectric focussing (CIEF), micellar electrokinetic chromatography (MEKC), affinity capillary electrophoresis (ACE), as well as in the hybrid techniques capillary electrochromatography (CEC), and pressurized capillary electrochromatography (pCEC) progress has been made in experimental setups, and for many groups of analytes, such as peptides, proteins, nucleotides, saccharides, drugs and their metabolites, CE/ESI‐MS has been successfully applied. Electromigration is further miniaturized. New preconcentration methods allow the investigation of compounds, which are not sensitively detected with ESI‐MS. Coordination ion spray (CIS) MS is another method for sensitivity enhancement by on‐line formation of charged coordination compounds.

Characterization of bonded phases by solid-state NMR spectroscopy

Structure and dynamics of chemically modified silica gels have been investigated by high-resolution solid-state NMR spectroscopy. 29Si cross-polarization—magic angle spinning (CP-MAS) NMR spectroscopy yields information on variety and quantity of surface species of both pure silica gel and modified silica gel. 13C CP-MAS NMR spectroscopy reveals dynamic properties of the attached alkyl chains. The data from 29Si and 13C solid-state NMR spectroscopy can be correlated with the separation characteristics in high-performance liquid chromatography.

Characterization of chemically modified silica gels by 29Si and 13C cross-polarization and magic angle spinning nuclear magnetic resonance

Abstract Chemically modified silica gels were prepared and examined by use of solid-state 13C and 29Si cross-polarization and magic angle spinning nuclear magnetic resonance spectroscopy. By this method, one can not only distinguish between various structural elements in the surface region, but also differentiate according to their mode of preparation. The results of the original manufacturing procedures for reversed-phase high-performance liquid chromatography materials and their subsequent treatment with trimethylsilylating reagents can be investigated. It is also possible to decide whether the solvents (e.g., methanol) used in the preparation remain adsorbed at the surface or are completely removed by heat treatment. Further developments of this method of investigation may reveal the molecular mechanism of chromatographic separations.

Enantiomer labelling, a method for the quantitative analysis of amino acids

Enantiomer labelling a method for the quntitative analysis of optically active natural compounds by gas chromatography, involves the use of the unnatural enantiomer as an internal standard. With Chirasil-Val, a chiral stationary phase that is thermally stable up to up to 240 degrees, the enantiomers of amino acids and a variety of other compounds can be separated and quantitated. Incomplete recovery from the sample, incomplete derivatization, hydrolysis and thermal decomposition of the derivative and shifting response factors can be compensated for by adding the unnatural enantiomer. The accuracy of amino acid analysis by enantiomer labelling is equal or superior to that of hitherto known methods. The procedure affords a complete analysis of peptides with respect to both amino acid composition and the optical purity of each amino acid.

Pressurized gradient electro-high-performance liquid chromatography

A microbore liquid chromatographic system was developed for gradient elution using capillary columns with inner diameters of 50 and 100 μm. In addition, voltage gradients up to 30 000 V can be applied across the length of the column. The dramatic improvement of reversed-phase separations of charged analytes is demonstrated by a separation of detritylated oligonucleotides on 5 μm C18 reversed-phase silica gel. Several examples are given, illustrating the influence of applied voltage gradients up to 400 V/cm upon the separation in both isocratic and gradient elution modes.

Evaluation of the parameters determining the performance of electrochromatography in packed capillary columns

Abstract Electrochromatography is a varient of reversed-phase liquid chromatography performed in capillary columns whereby transport of the eluent is accomplished by applying an electric field across the length of the column. Column-length restrictions arising from resistance to flow encountered with conventional pumping are absent in electrochromatography, allowing separation in capillaries packed with 1.5-μm stationary phases up to 50 cm, thus rendering efficiencies of more than 100 000 plates per column. Practical problems, however, have restricted the number of successful applications reported. A number of parameters determining the electrochromatographic performance are investigated: eluent, frits, packed columns and optional supplementary pressure. These factors are investigated separately and then combined stepwise. With thoroughly degassed eluent, frits made of sintered silica gel, and the use of supplementary pressure, stable and reproducible conditions may be readily obtained.

On-line coupling of high-performance liquid chromatography and nuclear magnetic resonance

Abstract High-performance liquid chromatography—nuclear magnetic resonance (HPLC—NMR) on-line coupling is a possible solution to the problemof universal detectors in liquid chromatogrphy. By use of a newly developed 1 H-FT-NMR* flow cell, 1 H-NMR spectra of flowing systems cn be obtained. Although sensitivity and resolution are somewhat lower than in the conventional system, the 1 H-MNMR spectra are of sufficient quality to enable classification of unknown compounds. The performed HPLC separation with on-line NMR-measurement shows the capabilities of the applied arrangement.

Chiral recognition in the resolution of enantiomers by GLC

SummaryChiral recognition of many enantiomeric solutes by a chiral amide stationary phase is based mainly on hydrogen bonding. A chiral-recognition-factor CHI is proposed, given by the difference of the enthalpy change in the enantiomer discrimination, standardized with respect to the specific interaction of the solutes with the diamide core of the stationary phase. The röle of the entropy part is also discussed. By extrapolation of the retention behaviour to elevated temperature, peak inversion of enantiomers is predicted.

Gas chromatographic—mass spectrometric analysis of optically active metabolites and drugs on a novel chiral stationary phase

Chirasil-Val, a novel chiral polysiloxane-type stationary phase is capable of separating the enantiomers of optically active drugs and metabolites of several compound classes; alpha-amino acides, alpha-amino alcohols, glycols, aromatic and aliphatic alpha-hydroxy carboxylic acids and amines. Due to their high thermal stability, columns coated with Chirasil-Val may be coupled to a mass spectrometer. Potential applications of the new stationary phase include analysis of the optical purity of enantiomeric drugs, determination of the configuration of metabolites, and quantitation of optically active drugs and metabolites using the unnatural enantiometer as internal standard. Direct separation of enantimoers on Chirasil-Val is especially useful if only minute amounts of the optically active compounds are availalbe for analysis.