Preparation of eumelanin-related metabolites 5,6-dihydroxyindole, 5,6-dihydroxyindole-2-carboxylic acid, and their O-methyl derivatives

5,6-Dihydroxyindole (5,6DHI) and 5,6-dihydroxyindole-2-carboxylic acid (5,6DHI2C) are ultimate precursors of the black melanin, eumelanin. These indolic metabolites and their O-methyl derivatives are excreted in urine of melanoma patients at high levels and of healthy persons at low levels. We describe here a simplified procedure for preparing milligram to subgram quantities of 5,6DHI and 5,6DHI2C and their O-methyl derivatives. Dopachrome generated in situ by ferricyanide oxidation of dopa at pH 6.5 underwent spontaneous decarboxylation to give 5,6DHI in 40% isolation yield, while treatment of dopachrome with alkali at pH 13 afforded 5,6DHI2C in 38% isolation yield. Two isomeric O-methyl derivatives of 5,6DHI were prepared by treatment with diazomethane, while those of 5,6DHI2C were prepared by treatment with diazomethane followed by alkaline hydrolysis of the methyl esters. 5,6DHI and 6-hydroxy-5-methoxyindole were also obtained by heating the corresponding carboxylic acids in decalin. 5-Hydroxy-6-methoxyindole and 6-hydroxy-5-methoxyindole-2-carboxylic acid could also be prepared by debenzylation of the commercially available O-benzyl derivatives.     

Clinical adaptation of a high-performance liquid chromatographic method for the assay of pyridoxal 5′-phosphate in human plasma

Vitamin B6, measured as pyridoxal 5′-phosphate (PLP), is a co-enzyme in the transsulfuration pathway of homocysteine metabolism. Since depletion of PLP has been suggested as an independent risk factor for coronary artery disease, PLP is frequently measured to guide patient care. By a change and utilization of an Aquasil C18 column and the addition of an acetonitrile clean-up gradient to the potassium phosphate, with sodium perchlorate and bisulfite buffer between samples we report the modification of a previously described method for analysis of PLP. The result is a more practical, efficient, reliable and robust method for daily clinical use. We also determined and report that it is critical to protect freshly prepared standard PLP samples from light exposure during assay preparation.     

The participation of the striatum in the central regulation of gonadal hormonal function]

Intrastriatal corticotrophin-releasing hormone (CRH) was shown to induce a decrease in the plasma tectosterone concentration. Besides, the 6-OHDA pre-treatment completely prevented the suppression of plasma testosterone in response to intrastriatal CRH administration. The findings suggest that the striatum is involved in the extrahypothalamic regulation of the gonadal endocrine function.     

Expression of a lipocalin in prokaryote and eukaryote cells: quantification and structural characterization of recombinant bovine beta-lactoglobulin.

In this paper we quantify and characterize the expression of recombinant beta-lactoglobulin (rBLG) in prokaryote and eukaryote cells. In Escherichia coli we used the pET26 vector, which permits the secretion of rBLG in periplasm. We studied the expression of rBLG in COS-7 cells and in vivo in mouse tibialis muscle. The expression of rBLG was measured by two immunoassays specific, respectively, for BLG in its native and denatured conformation. We observed that rBLG was essentially expressed in a denatured form in E. coli even in the periplasm, whereas rBLG in eukaryote cells was found in its native conformation.     

Human acetylcholinesterase. Immunochemical studies with monoclonal antibodies

Monoclonal antibodies were used to investigate the immunochemistry of human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). A series of experiments on the sedimentation velocity and Stokes radius of acetylcholinesterase and its immune complexes indicated that each antibody recognized a single high-affinity binding site (epitope) on the monomeric enzyme. Further analysis suggested that the antibody-binding sites were replicated on multimeric enzyme forms but were subject to steric hindrance between nearby IgG molecules or adjacent enzyme subunits. The cellular localization of the epitopes was studied by measuring the binding of monoclonal antibodies to the cholinesterase of intact erythrocytes. The results implied that most of the epitopes are exposed to the external media. However, one antibody failed to bind to intact cells, despite a relatively high affinity for detergent-solubilized antigen, possibly because its epitope is buried in the lipid bilayer.     

Cathepsin B from human renal cortex.

Cysteine-proteinase activity was observed in homogenates of human-cadaver renal cortex. This activity co-purified with renin enzymic activity until separation by aminohexyl-Sepharose--pepstatin affinity chromatography. The cysteine proteinase was purified 1780-fold after the following successive chromatographic procedures: Sephadex G-75, DEAE-cellulose DE-52, and an organomercurial affinity resin. The proteinase activity was dependent upon activation by thiol-containing compounds such as dithiothreitol, as well as by EDTA, and was inhibited by the thiol-group-specific alkylating reagents iodoacetic acid and N-ethylmaleimide. DE-52 cellulose chromatography resolved the cysteine proteinase into two components. On the basis of molecular size (26 000 daltons), activity as a function of pH, stability as a function of pH, substrate specificity and thermal lability, the major component (95%) has been identified as cathepsin B. The DE-52 cellulose elution pattern of the minor component (5%) is suggestive of cathepsin H [Schwartz & Barrett (1980) Biochem. J. 191, 487-497] Enzymic activity was determined with synthetic substrates, in particular alpha-N-benzoyl-DL-arginine 2-naphthylamide (Bz-Arg-NNap), thus precluding the detection of cathepsin L [Kirschke, Langner, Wiederanders, Ansorge, Bohley & Broghammer (1976) Acta Biol. Med. Germ. 35, 285-299]. Inhibition by dimethyl sulphoxide was observed in the determination of Km = 7.0 +/- 0.4 mM for the substrate Bz-Arg-NNap, and care must therefore be taken in the preparation of substrate solutions.