Separation of friend erythroleukaemic cell histones and high-mobility-group proteins by reversed-phase high-performance liquid chromatography

A procedure for the rapid separation of histones and high-mobility-group (HMG) proteins from Friend erythroleukaemic cells (line F4N) by reversed-phase high-performance liquid chromatography is reported. By using a Nucleosil 300-5 C₄, column and a multistep water—acetonitrile gradient containing 0.1% trifluoroacetic acid, the HMG-1 and HMG-2 proteins, several his subtractions including H1⁰, H4, H2B, two H2A variants and two H3 subtractions were separated. Under changed conditions, by applying a varied acetonitrile gradient system, even two H2B variants were fractionated. The methods described seem to be a real alternative to the time-consuming polyacrylamide gel electrophoresis.     

Quantification of free and total salicylic acid in plants by solid-phase extraction and isocratic high-performance anion-exchange chromatography

Salicylic acid (SA) is an important signaling compound in plants and is involved in various defense responses. Here we report a new method for quantification of free and total soluble SA in Arabidopsis thaliana with 5-fluorosalicylic acid (5-FSA) as internal standard. The SA was isolated from leaf extracts by solid-phase extraction with phenyl-phase cartridges and selectively eluted as the cationic iron(III)-complex. Recoveries of SA and 5-FSA were equal and exceeded 90%. Free SA was subsequently released from the iron(III)-complex by addition of 2,2-bipyridyl and high-performance anion-exchange chromatography was performed on an NH₂ column. The SA appeared as last peak with a retention time of 15 min, baseline-separated from other substances. On-line detection was performed fluorimetrically for both SA and 5-FSA at an excitation wavelength of 300 nm and an emission wavelength of 410 nm, because both substances give similar fluorescence spectra. The detection limit for SA was 5 ng g^–1 FW for a sample size of 100 mg. Thus the main advantages of the method are highly selective sample preparation, increased sensitivity, reduced analysis time compared with reversed-phase HPLC, and use of a novel internal standard detectable under the same conditions as SA. The techniques described are applicable to other plant materials.     

Structural and Chemical Analysis of Materials with High Spatial Resolution

 An understanding of the correlation between microstructures and properties of materials require the characterization of the material on many different length scales. Often the properties depend primarily on the atomistics of defects, such as dislocations and interfaces. The different techniques of transmission electron microscopy allow the characterization of the structure and of the chemical composition of materials with high spatial resolution to the atomic level: high resolution transmission electron microscopy allows the determination of the position of the columns of atoms (ions) with high accuracy. The accuracy which can be achieved in these measurements depends not only on the instrumentation but also on the quality of the transmitted specimen and on the scattering power of the atoms (ions) present in the analyzed column. The chemical composition can be revealed from investigations by analytical microscopy which includes energy dispersive X-ray spectroscopy, mainly quantitatively applied for heavy elements, and electron energy-loss spectroscopy. Furthermore, the energy-loss near-edge structure of EELS data results in information on the local band structure of unoccupied states of the excited atoms and, therefore, on bonding. A quantitative evaluation of convergent beam electron diffraction results in information on the electron charge density distribution of the bulk (defect-free) material. The different techniques are described and applied to different problems in materials science. It will be shown that nearly atomic resolution can be achieved in high resolution electron microscopy and in analytical electron microscopy. Recent developments in electron microscopy instrumentation will result in atomic resolution in the foreseeable future.     

Electron microprobe analysis of micro amount powder samples: A case study on high purity coal

For the major, minor and trace element analysis of the inorganic compounds of a Ruhr-Saar coal different preparation techniques are investigated with X-ray fluorescence analysis, electron microprobe and classical wet chemical methods minimizing sample weight at standard preparation times and analytical accuracies. Considering accuracy as well as preparation efforts, determinations by electron microprobe on small sample amounts (<50 mg) proved to be superior to the other methods.Abbreviations AAS atomic absorbtion spectrometry - EMA electron microprobe analysis - IC ion chromatography - ISE ion sensitive electrode - PHOT photometry - WCA wet chemical analysis - XRF X-ray fluorescence analysis - LTA low temperature plasma ashing - HTA high temperature ashing - l.o.i. loss on ignition     

Average local electrostatic potential and the core-valency separation in atoms

The average local electrostatic potential function, V(r)/ ρ(r), is calculated for 87 atoms, Li-Ac, in the ground state using the nonrelativistic average-over-configuration numerical Hartree-Fock density. It is found empirically that in a given atom the shell boundaries are expressed as the successively increasing maxima in V(r)/ ρ(r) and the outermost maximum presents good approximate estimates of the core-valence separation in atoms. The likeness in behavior of V(r)/ ρ(r) at each shell boundary with the maximum hardness principle is discussed. The single-exponent-fit parameters for the electron density in the valency region are provided for all atoms. © 1995 John Wiley & Sons, Inc.     

Cytotoxicity and Antiproliferative Effect of Hypericin and Derivatives after Photosensitization

The toxicity on three human tumor cell lines (A431, HeLa and MCF7) of five phenanthroperylenequinones (hypericin and derivatives) and two perylenequinones (cercosporin and calphostin C) was investigated after photosensitization (4 J/cm²). Furthermore, the antiproliferative effect on HeLa cells was studied for the phenanthroperylenequinones. Hypericin, 2,5-dibromohypericin, 2,5,9,12-tetrabromohypericin and perylenequinones displayed a potent cytotoxic and antiproliferative effect in the nanomolar range. Hypericin dicarboxylic acid exhibited no photoactivity. In general, the antiproliferative activity correlated well with the photocytotoxicity. However, the nonphotocytotoxic compound hexamethylhypericin showed potent antiproliferative activity in the nanomolar range, probably exerting its action by protein kinase C inhibition. Without light irradiation, no cytotoxic and antiproliferative effect was observed for any photocytotoxic phenanthroperylenequinone compound. Furthermore, confocal laser microscopy revealed that the subcellular localization in A431 cells was similar for the photoactive compounds; the photosensitizers were mainly concentrated in the perinuclear region, probably corresponding with the Golgi apparatus and the endoplasmic reticulum. In addition, the accumulation of the photosensitizers in HeLa cells was investigated. All compounds except hypericin dicarboxylic acid were found to concentrate to a large extent in the cells. The compound 2,5,9,12-tetrabromohypericin seemed intrinsically more effective than hypericin since the intracellular concentration of the bromoderivative was a magnitude of order lower than that of hypericin although both compounds showed similar photobiological activity.