Structure of the asparagine-linked sugar chains of porcine kidney and human urine cerebroside sulfate activator protein

The specific sugar residues and their linkages in the oligosaccharides from pig kidney and human urine cerebroside sulfate activator proteins (saposin B), although previously hypothesized, have been unambiguously characterized. Exhaustive sequential exoglycosidase digestion of the trimethyl-p-aminophenyl derivatives, followed by either matrix-assisted laser desorption/ionization and/or mass spectrometry, was used to define the residues and their linkages. The oligosaccharides were enzymatically released from the proteins by treatment with peptidyl-N-glycosidase F and separated from the proteins by reversed-phase high-performance liquid chromatography (HPLC). Reducing termini were converted to the trimethyl-p-aminophenyl derivative and the samples were further purified by normal-phase HPLC. The derivatized carbohydrates were then treated sequentially with a series of exoglycosidases of defined specificity, and the products of each digestion were examined by mass spectrometry. The pentasaccharides from pig kidney and human urine protein were shown to be of the asparagine-linked complex type composed of mannose-alpha 1-6-mannose-beta 1-4-N-acetylglucosamine-N-acetylglucosamine(alpha 1-6-fucose). This highly degraded structure probably represents the final product of intra-lysosomal exoglycosidase digestion. Oligosaccharide sequencing by specific exoglycosidase degradation coupled with mass spectrometry is more rapid than conventional oligosaccharide sequencing. The procedures developed will be useful for sequencing other oligosaccharides including those from other members of the lipid-binding protein class to which cerebroside sulfate activator belongs. (c) 2000 John Wiley & Sons, Ltd.     

Chemiluminescence immunoassay for the detection and quantification of methyltestosterone residues in muscle tissue

Chemiluminescence as a detection method for immunoassay has successfully been applied to the measurement of methyltestosterone (MT) residues in muscle tissue. The sample is digested enzymatically, extracted with diethyl ether and purified on a Lipidex-5000 column. An optional clean-up utilized disposable C18 columns. As the luminescent label the N-(4-aminobutyl)-N-ethylisoluminol conjugate of MT was used. The antiserum was raised in a rabbit against MT-3-carboxymethyloxime-bovine serum albumin. The detection limit of the assay was 14 +/- 7 pg (n = 13), with a limit of quantification in muscle tissue of 0.125 ppb.     

Electrochemical oxidation of ethyl 3-oxo-3-phenyl-2-phenylhydrazonopropionate

The electrochemical oxidation of ethyl 3-oxo-3-phenyl-2-phenylhydrazonopropionate has been studied in the pH range 3.0–11.0 at a pyrolytic graphite electrode by linear and cyclic sweep voltammetry, coulometry and spectral studies. The results indicate that the 2 e⁻, 2 H⁺ oxidation of this compound gives phenol and ethyl 3-phenyl-2,3-dioxopropionate as the major products of electrooxidation.     

Gas chromatographic quantitation of underivatized amines in the determination of their octanol-0.1 M sodium hydroxide partition coefficients by the shake-flask method

The elution behaviour of a selection of sequence variant and modified insulins has been compared on a C18 reversed-phase column. Observed elution times were compared with those expected from the nature of the differences from bovine insulin. In some cases prediction rules established for peptides are adequate to explain the observed elution and in others detailed knowledge of the structure of the protein is of considerable importance in understanding elution behaviour.     

Synthese, structure cristalline et spectres infrarouges de l''acide β-alanyl amino methyl phosphonique

β-Alanyl aminomethylphosphonic acid (β-ala AMPh) has been studied in the solid state by X-ray diffraction and vibration spectrometry. It crystallizes (space group P1&;#x0304; with Z = 4) as a zwitterion with, in the asymétrie unit, two independent molecules the conformations of which are quite different. The hydrogen bonding network is very complicated. Because the infrared spectra are very complicated, only a partial correlation of the vibrations of ammonium, amide and phosphonic groups is proposed.     

Determination of Taxotere in human plasma by a semi-automated high-performance liquid chromatographic method.

A rapid, selective and reproducible high-performance liquid chromatographic (HPLC) method with ultraviolet detection was developed for the determination of the anti-cancer agent Taxotere in biological fluids. The method involves a solid-phase extraction step (C2 ethyl microcolumns) using a Varian Advanced Automated Sample Processor (AASP) followed by reversed-phase HPLC. The validated quantitation range of the method is 10-2500 ng/ml in plasma with coefficients of variation < or = 11%. The method is also suitable for the determination of Taxotere in urine samples under the same conditions. The method was applied in a phase I tolerance study of Taxotere in cancer patients, allowing the pharmacokinetic profile of Taxotere to be established.     

Polymérisation anionique amorcée par les composés lamellaires du graphite et du lithium

In the present work, we use the binary insertion compound LiC₁₂ to polymerize styrene, methyl methacrylate, butadiene, isoprene, and to copolymerize isoprene and styrene in various hydrocarbon solvents (aromatics and aliphatic) and etheral solvents. We show that the styrene polymerization in aromatic solvents gives better yields than in the etheral solvents, the polymer being atactic. Methyl methacrylate does not polymerize in toluene but does so completely in DME. More generally, the yields of polymerization are better with KC₃₇ than with LiC₁₂ because of the different capacities of the monomer to get into the carbon layers. The polymerization of dienes with LiC₁₂ shows that the microstructures of the polymer obtained in π-or n-donor solvents are similar to the ones obtained by homogenous polymerization with Li cation in such solvents. However, for isoprene in cyclohexane, the results are different. The isoprene styrene copolymers are statistical ones and the mean length of styrene blocks is less than 5. The monomer interaction with the insertion compound and the growing chain geometry between the carbon layers are the facts which control either the stereospecificity of the polymerization or the selectivity of the copolymerization.     

Supercritical fluid extraction for liquid chromatographic determination of carotenoids in Spirulina Pacifica algae: a chemometric approach

An experimental design procedure was used to investigate the effects of some operating parameters on the supercritical fluid extraction of carotenoids beta-carotene, beta-cryptoxanthin and zeaxanthin from Spirulina Pacifica algae, a carotenoid-rich dietary product. Variables tested were temperature and pressure of the supercritical fluid, dynamic extraction time and percentage of ethanol added as the modifier. Each variable was tested at three levels; 31 experiments were performed in random order. Analyses of the extracts were performed by high-performance liquid chromatography with UV-Vis photodiode array detection. Analytical responses (chromatographic peak areas) were processed by using a stepwise multiple regression analysis, in order to find polynomial functions describing the relationships between variables and responses. For all the analytes the experimental conditions providing the highest extraction yield inside the experimental domain considered were found. Supercritical fluid extraction results obtained in these conditions were compared with those obtained by performing solvent extraction in order to evaluate the effectiveness of the supercritical fluid extraction procedure.