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.     

Role of actin in the cAMP-dependent activation of sodium/glucose cotransporter in renal epithelial cells

We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin-Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [¹⁴C]-α-methyl glucopyranoside ([¹⁴C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [¹⁴C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments.     

Structural studies of a human gamma 3 myeloma protein (Jir) bearing the allotypic marker Gm(st)

The authors established the amino acid substitutions determining G3m(s) and G3m(t) specificities, which characterize Mongoloid populations, by sequence analysis of the Fc region of a myeloma protein (Jir). By comparing the amino acid sequences of the IgG3 (Jir) and the other IgG subclasses analyzed to date, it was found that G3m(s) was an isoallotype specified by an amino acid substitution at position 435; i.e., whereas the subclasses IgG1, IgG2, and IgG4 had histidine in common, G3m(s-) had arginine in this position. This was also confirmed by the observation that the Fc fragment in question bound to protein A. It was also established that the amino acid at position 379 of G3m(t-) IgG3 and the other subclasses was valine, whereas methionine in this position was specific for G3m(t+). In addition, the amino acids at position 339 of G3m(u-) IgG3 Jir was threonine, and at position 296 of G3m(g-) IgG3 Jir was tyrosine. These findings are not in accord with the hitherto postulated relations of alanine and phenylalanine to G3m(u-) and G3m(g-), respectively. Finally, this study showed that a large number of substitutions occurred at positions 384 through 389, which suggests that many specificities of the G3m(b) group occur on IgG3 proteins.     

Immunochemical subfractionation of a microsomal fraction of rat liver with antibody-coated Staphylococcus aureus cells

The procedure for immunochemical adsorption of vesicles with specific antigen on their outer surfaces was improved. When microsomal vesicles were mixed with Staphylococcus aureus cells coated with the antibody against NADPH-cytochrome c reductase, more than 90% of the enzyme activity was adsorbed on the cell, whereas, only about 10% of the activity was adsorbed on cells coated with the same amount of anti-ovalbumin antibody. NADH-cytochrome c reductase and aldehyde dehydrogenase activities were adsorbed on the cell to the same extent as was NADPH-cytochrome c reductase activity. Under this condition, there was no adsorption of the activities of the marker enzymes of lysosomes and Golgi apparatus, whereas large amounts of the activities of the plasma membrane enzymes were adsorbed. The specific activity of NADPH-cytochrome c reductase in the adsorbed vesicles from the microsomal fractions increased considerably. In contrast, marker enzymes of the Golgi or of the plasma membranes could be enriched in unadsorbed vesicles from the Golgi fractions.     

Effects of delayed mating on preimplantation embryos in spontaneously ovulated mice

The effects of delayed mating on mouse preimplantation embryos (78 ± 1 hours) were studied by setting up different mating periods in relation to the estimated time of spontaneous ovulation. Copulation occurred even in the late morning and early afternoon after the night of spontaneous ovulation. However, females mated in the early afternoon had no viable embryos at the time of laparotomy. Although embryonic development was not affected in the groups mated 6 or 10 hours after estimated ovulation, the percentage of degenerated embryos was increased in these groups. These results suggest that prolonged intervals between the estimated time of ovulation and mating have some deleterious effects on preimplantation embryos.     

Oxidative degradation of thymine with O2 promoted by L-ascorbic acid and Cu(II) ion

Oxidation of thymine with O2 was promoted by copper(I) ion generated from reaction of L-ascorbic acid (AA) with copper (II) ion. The main oxidation products were thymine glycol (TG) and N-formyl-N'-pyruvylurea (FPU). At higher concentration of O2, formation of FPU was favored, while TG was dominant at higher Cu(II) ion and lower O2 concentrations. Reaction mechanism involving hydroxy thyminyl radical was proposed.     

Hyperthermic treatment of chromomycosis with disposable chemical pocket warmers

A case of chromomycosis in which hyperthermia proved effective is reported. The patient was a 56-year-old male bean curd maker who, without any previous history of minor trauma, developed on the extensor side of the left upper arm an eczematous lesion that underwent gradual radial expansion. The lesion showed a well-defined, 7×10 cm infiltrated erythematous plaque with the central area healed and, at the upper and lower borders, adherent scales and crusts on the surface. Histological examination revealed granulomatous changes in the dermis, as well as sclerotic cells within giant cells and microabscesses. On culturing,Fonsecaea pedrosoi was isolated. The patient was treated with disposable chemical pocket warmers, which were secured over the lesion with a rather tight elastic bandage, so that they kept the affected area warm for 24 hours a day. After a month of such hyperthermic treatment, the erythema and infiltration had decreased considerably, and microscopic examination and culture of the crusts both yielded negative results. Examination of biopsy specimens of the lesion after the third month showed that it had cicatrized. The treatment was stopped after 4 months, and no relapse occurred. We also summarize the published results of local hyperthermic treatment of chromomycosis in Japan.