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.     

Hydrogen Peroxide-dependent Synthesis of Flavonols in Mesophyll Cells of Vicia faba L.

Mesophyll cells of Vicia faba contain kaempferol and quercetinglycosides. When isolated mesophyll cells were treated with0.1 mM H₂O₂ for 2 h, the levels of these flavonols increasedby 10–70% of the control values (mean values, 19.6% and34.4% for kaempferol and quercetin glycosides, respectively).Such increases in levels of flavonols were also observed inisolated vacuoles of mesophyll cells. However, when mesophyllcells and vacuoles were treated with 10 mM H₂O₂₎degradationof flavonols was observed. These data suggest that H₂O₂ hastwo effects on the metabolism of flavonols: induction of theirsynthesis and stimulation of their oxidation. (Received March 6, 1989; Accepted July 10, 1989)     

Taq I-generated HLA-DQ αpolymorphism in Japanese patients with narcolepsy

Taq I-generated HLA-DQrestriction fragment length polymorphism was examined in Japanese patients with narcolepsy. All patients were DR2 positive and shared a 6.0 kb fragment, although this fragment was found only in 54 % of the healthy DR2-positive Japanese. This finding added the DQ gene to the list of candidates for the possible narcolepsy-susceptibility gene. In contrast, there was no complete association between narcolepsy and DXrestriction fragment length polymorphism. These findings suggest that a narcolepsy-susceptibility gene is located closer to the DQ locus than to the DX locus.     

Pigment types in sheep, goats, and llamas

Pigment types in various colors of fiber from sheep, goats, and llamas were assayed by a method using high performance liquid chromatography. In these three species the black/gray group is due to eumelanin, which is fully intense in all three species. Red phenotypes are due to pheomelanin and fade considerably with age in fiber from sheep and goats, but not in llamas. This phenomenon has implications on the genetic mechanisms used in generating white fiber. Brown phenotypes in sheep are due to eumelanin, in goats these phenotypes are equivocal, and they were not observed in llamas.     

On the glutamate transport through cell envelope vesicles of Halobacterium halobium

Glutamate uptake by envelope vesicles of Halobacterium halobium was measured. Previous authors showed that the glutamate uptake needs the illumination as well as Na+ gradient across the membrane. The latter is considered to be the driving force for the uptake. No satisfactory explanation for the necessity of the illumination has not been given. We found that in the absence of Cl- in the medium, only Na+ gradient was enough to induce the glutamate uptake, i.e. no illumination was needed. Glutamate uptake was measured with various strains of H. halobium. We found that the envelope vesicles prepared from strains containing no bacteriorhodopsin showed the glutamate uptake in the dark and in the presence of Cl- in the medium provided only that Na+ gradient is imposed.     

Production of transgenic fish: introduction and expression of chicken delta-crystallin gene in medaka embryos

To produce a model of transgenic fish, recombinant plasmids containing chicken delta-crystallin gene were microinjected into the oocyte nucleus of a small teleost, medaka (Oryzias latipes). About 50% of the microinjected oocytes developed to 7-day-old embryos. By Southern blotting delta-crystallin gene was detected in 4 of 8 embryos, and, by Western blotting, delta-crystallin polypeptides in 5 of 16. In 1 of 6 examined histologically, delta-crystallin DNA was detected in all the tissues, and delta-crystallin polypeptides, in many of the tissues including the lens. Thus, the exogenous gene and/or its products were detected in 10 of 30 embryos examined. This is the first report of successful production of transgenic fish.     

Differential subcellular localization of ACTH and α-MSH in corticotropes of the rat anterior pituitary

Summary Specific antisera to -melanotropin (-MSH) and corticotropin (ACTH 1-39) were used to obtain immunocytochemical evidence for the differential localization of -MSH and ACTH in the secretory granules of corticotropes of rat anterior pituitary. The specificity of the antisera was established by binding ¹³¹I-labeled -MSH and ACTH 1-39 to their respective antisera. Double-labeling immunocytochemistry (for -MSH, ferritin; for ACTH, colloidal gold) was performed. Some secretory granules were labeled with ferritin particles (-MSH), whereas others contained gold particles (ACTH). Only a few granules showed both ACTH and -MSH. In typical corticotropes (stellate in form with a small number of secretory granules aligned along the cell periphery) only some of the secretory granules that were labeled with anti-ACTH serum were also immunoreactive to anti--MSH. In atypical corticotropes (polygonal in shape and containing a large number of secretory granules) almost all of the immunoreactive ACTH secretory granules were also positive to anti--MSH serum. An intermediate type of corticotrope was observed containing a small number of secretory granules, almost all of which were labeled with anti--MSH. Thus, rat anterior pituitary corticotropes may be classified into three types according to the distribution and content of -MSH. The light-microscopic immuncytochemistry provided similar results.     

Immunotherapy with bestatin for acute nonlymphocytic leukemia in adults

Summary In a cooperative randomized control study of immunotherapy with bestatin in combination with chemotherapy in adults with acute nonlymphocytic leukemia (ANLL), 101 patients (48 in the bestatin group and 53 in the control group) out of 115 patients registered were evaluated as eligible. The bestatin group achieved a statistically significant prolongation of survival compared with the control group in overall ANLL and acute myelogenous leukemia. In the analysis of patient age, the bestatin group achieved a statistically significant prolongation of both the remission duration and survival in patients aged 50 to 65 years, while the differences were not significant in the 15 to 49 age group. The bestatin group tended to achieve a higher rate of reinduction of remission in patients who had recurrence of leukemia. Side effects developed in only 5 (9.6%) of 52 patients treated with bestatin. None of these side effects were particularly serious in nature. It is concluded that bestatin is useful for prolongation of survival of adult patients with ANLL, making for a longer remission duration especially in elderly patients and with few side effects.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.