Effect of Escherichia coli lipopolysaccharide on the glucagon and insulin binding to isolated rat hepatocytes

Summary Number and affinity constant of low affinity binding sites of insulin and glucagon to isolated hepatocytes decreased when the cells were incubated with Escherichia coli 0111:B4 lipopolysaccharide. This effect agrees with a non-specific binding of lipopolysaccharide to hepatocytes, similar to the well-recognized non-specific binding of albumin. Also, binding of different lectins to their glycoprotein receptors did not affect the [¹⁴C]lipopolysaccharide interaction with the cell membrane surface. Endotoxin depresses gluconeogenesis from lactate when the precursor was incubated with the cells for short time intervals. The longer the preincubation interval with lipopolysaccharide, the higher the inhibition of gluconeogenesis in the absence and in the presence of glucagon.The effect of endotoxin was also studied on the glucagon-induced synthesis of cyclic AMP and the glucagon binding. Levels of cyclic AMP and hormone binding decreased with increasing both endotoxin concentrations and preincubation intervals at which cells were in contact with endotoxin.     

Pyruvic acid is attached through its central carbon atom to the amino terminus of the recombinant DNA-derived DNA-binding protein Ner of bacteriophage Mu.

Ner protein of bacteriophage Mu, produced by recombinant DNA techniques in Escherichia coli, has been found to possess a molecule of pyruvic acid attached covalently through carbon-2 to the amino-terminal cysteine residue. The intact protein and the amino-terminal chymotryptic peptide were found by mass spectrometry to be 70 mass units heavier than expected. The modified peptide was unstable under mildly acid or mildly basic conditions. Two-dimensional nuclear magnetic resonance spectroscopy of the modified and unmodified forms of the amino-terminal chymotryptic peptide was consistent with the presence of pyruvate linked through carbon-2 to the amino-terminal Cys residue. Treatment of the modified form with 2,4-dinitrophenylhydrazine in acid medium led to the expected hydrazone of pyruvic acid, which was identified by high pressure liquid chromatography. Of the two proteins known to be modified by pyruvate through its central carbon (the other being human adult hemoglobin, in which the modified form represents only a very minor fraction), Ner is the first protein found to be modified quantitatively. Given the instability of the modification, it may be more prevalent than recognized hitherto. Incubation with 2,4-dinitrophenylhydrazine may offer a useful means of detecting the presence of pyruvate linked to proteins in this way.     

Determination of picotamide in human plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of picotamide in human plasma and urine is described. After addition of an internal standard (bamifylline), the plasma and urine samples were subjected to liquid—liquid extraction and clean-up procedures. The final extracts were evaporated to dryness and the resulting residues were reconstituted in 100 μl of methanol—water (50:50, v/v) and chromatographed on a LiChrosorb RP-SELECT B reversed-phase column coupled to an ultraviolet detector monitored at 230 nm. Chromatographic analysis takes about 10 min per sample. The assay was linear over a wide range and has a limit of detection of 0.005 and 0.1 μg/ml in plasma and urine, respectively. It was selective for picotamide, accurate and robust and thus suitable for routine assays after therapeutic doses of picotamide.     

Synthesis,semipreparative HPLC separation,biological evaluation,and 3D-QSAR of hydrazothiazole derivatives as human monoamine oxidase B inhibitors

The present study reports on synthesis in high yields (70–99%), HPLC enantioseparation, inhibitory activity against human monoamino oxidases, and molecular modeling including 3D-QSAR studies, of a large series of (4-aryl-thiazol-2-yl)hydrazones (1–45). Most of the synthesized compounds proved to be potent and selective inhibitors of hMAO-B isoform in the micromolar or nanomolar range, thus demonstrating that hydrazothiazole could be considered a good pharmacophore to design new hMAO-B inhibitors. Due to the presence in some derivatives of a chiral center, we also performed a semipreparative chromatographic enantioseparation of these compounds obtained by a stereoconservative pattern. The separated enantiomers were submitted to in vitro biological evaluation to point out the stereorecognition of the active site of the enzyme towards these structures. Finally, a 3D-QSAR study was carried out using Comparative Molecular Field Analysis (CoMFA), aiming to deduce rational guidelines for the further structural modification of these lead compounds.     

Adsorption of chitosan on PET films monitored by quartz crystal microbalance

The adsorption behavior of chitosan on poly(ethylene terephthalate) (PET) model film surface was studied using the quartz crystal microbalance (QCM) technique. QCM with a dissipation unit (QCM-D) represents a very sensitive technique for adsorption studies at the solid/liquid interface in situ, with capability of detecting a submonolayer of adsorbate on the quartz crystal surface. Chitosan as well as PET were chosen for this study due to their promising biocompatible properties and numerous possibilities to be used in biomedical applications. As a first step, PET foils were activated by alkaline hydrolysis in order to increase their hydrophilicity. Model thin films were prepared from PET foils by the spin coating technique. The chemical composition of the obtained model PET films was analyzed using X-ray photoelectron spectroscopy (XPS) and their morphology was characterized by atomic force microscopy (AFM). Furthermore, the adsorption behavior of chitosan on these activated PET films and the influence of adsorption parameters (pH, ionic strength and chitosan solution concentration) were investigated in detail. Additionally, the surface chemistry and morphology of the PET films and the chitosan coated PET films were analyzed with XPS and AFM.     

In situ analysis of native microbial communities in complex samples with high particulate loads

In the present study a procedure combining a cell extraction method and Fluorescence In Situ Hybridization (FISH) for molecular monitoring and quantification of bacteria in soil and aquifer samples is presented. FISH was applied to bacterial cells extracted from the matrix by density gradient centrifugation. This separation method was applied to soil and aquifer samples and produced high cell recovery of 76.5%+/-4.4 and 78.0%+/-3.2, respectively. FISH, performed on the harvested cells, permitted a perfect visualization and quantification of bacteria. This approach is therefore promising for in situ detection of indigenous bacterial communities in complex samples.