Takuji Takeuchi

Induction of melanogenesis in the epidermal melanoblasts of newborn mouse skin by MSH

SummaryThe number of epidermal melanocytes positive to the dopa reaction increased when skin explants from newborn mice were cultured with MSH or dbc-AMP. These agents seem to induce melanogenesis in the pre-existing melanoblasts. This hormone-induced melanogenesis is suppressed by actinomycin D or cycloheximide, suggesting that the initiation of melanogenesis in the epidermal melanoblasts requires de novo transcription and translation.

Ultrastructural change of melanosomes associated with agouti pattern formation in mouse hair

Abstract We have studied the structural alteration of melanosomes in the melanocytes of agouti mice whose genetic characteristic is to produce eumelanin and phaeomelanin alternately in a single hair bulb. Melanocytes of hair bulbs from 1 to 2 day old mice of the black phase were observed to contain rod-shaped melanosomes of the eumelanin type (eumelanosome). In the melanocytes of the hair bulbs from 4 to 6-day old skin, which exclusively contain phaeomelanin, spherical melanosomes (phaeomelanosomes) were seen. On the other hand, the mice of the transitional phase from black to yellow possessed melanocytes that contained both eumelanosomes and phaeomelanosomes within a single cell. This result indicates that the shift from the eumelanin formation to the phaeomelanin formation or vice versa in agouti hair occurs within a single melanocyte. We observed multivesicular bodies in both the agouti melanocytes of the yellow phase and the genotypically yellow melanocytes. These bodies are considered to be the precursor of the phaeomelanin-containing melanosome. They are sometimes observed to have continuity with E. R. suggesting that the melanosomes are derived from E. R. in the phaeomelanin-forming melanocytes.

Dysgenesis of melanocytes and cochlear dysfunction in mutant microphthalmia (mi) mice

In order to evaluate the cytological homology of intermediate cells and melanocytes, and to investigate the function of melanocytes in the inner ear, hearing acuity and cochlear pathology were studied in three strains of mice, namely, wild type mice (+/+), albino mice without melanin (c2J/c2J), and microphthalmia mice with no melanocytes (mibw/mibw). Our histochemical data indicated that intermediate cells showed cytological characteristics almost identical to those of melanocytes and that disorders of melanin and/or melanocytes were reflected in the stria vascularis of each mouse. While c2J/c2J presented the same normal hearing acuity and normal structure of the stria vascularis as +/+, the hearing acuity of mibw/mibw mutants was severely impaired. Their stria vascularis was abnormally thin, lacking intermediate cells. According to these results, lack of melanin has little influence on hearing acuity; however, the absence of intermediate cells or melanocytes causes severe hearing loss, presumably due to a strial dysfunction.

Immunoelectron Microscopic Localization of Tyrosinase in the Mouse Melanocyte

In the melanocyte, tyrosinase is known as the dey enzyme for melanin formation. Purified tyrosinase protein was prepared that was capable of oxidizing tyrosine. The localization of tyrosinase antigen in the melanocyte was studied using antiserum against tyrosinase. DOPA (L-3,4-dihydroxyphenylalanine)-reaction product and tyrosinase antigen were found on the same organelles i.e., premelanosomes, melanosomes, GERL, and Golgi vesicles. This result seems to suggest that it is cytochemically appropriate to use DOPA as the substrate of tyrosinase. It appeared that tyrosinase antigen was present as granule-like structures inside GERL cisterna and associated with its membrane.

Expression of tyrosinase gene in amelanotic mutant mice

In order to study the molecular aspects of albinism, we analyzed the expression of tyrosinase gene in some amelanotic mutant mice by Northern blotting using mouse tyrosinase cDNA as a probe. It hybridized with a species of 2.1 kilobase RNA prepared from the skin of wild-type mice and two albino strains in the same quantity. In black-eyed white mouse, however, no RNA transcript encoding tyrosinase was detected. Our results suggest that these albinism is due to a point mutation in the structural region of the tyrosinase gene, not to a deficiency of tyrosinase gene expression and that the black-eyed white mouse has a deficiency in the gene expression possibly related to melanocyte differentiation.

Genetic control of LDH isozymes in the house fly, Musca domestica

Electrophoretic variations in lactate dehydrogenase from adult whole body homogenates are described for three laboratory strains of house fly, Musca domestica. Several crosses between different electrophoretic forms provided evidence that the observed variations are due to segregation of alleles at two distinct loci (designated as A and B loci) and that the LDH isozymes of house flies are dimers formed by a random association of subunits controlled by the two loci.

Cell type conversion in a mouse melanoma cell clone.

SummaryA melanoma cell clone was isolated from cultured B16 mouse melanoma cells. This clone,conv, which was characterized by rounded and spindle-shaped cell morphology, was not highly melanotic under the usual culture condition but had high tyrosinase (dopa oxidase) activity.When the cells were seeded to form colonies on a plastic culture dish in Eagle’s minimum essential medium supplemented with 10% bovine calf serum, two kinds of cell types always appeared. One was cytochemically dopa-positive and spindle-shaped (S type cell) with the same phenotypes as those of the parental cells. The other was dopa-negative and fibroblastlike (F type cell) containing no melanosomes. It was observed that the conversion from S type to F type occurred with a high frequency. The conversion from F type to S type also occurred but with a low frequency.

Differentiation of reptilian neural crest cells in Vitro

An attempt was made to culture neural crest cells of the turtle embryo in vitro. Trunk neural tubes from the St. 9/10 embryos were explanted in culture dishes. The developmental potency of the turtle neural crest cells in vitro was shown to be essentially similar to that of avian neural crest cells, although they seem to be more sensitive to melanocyte-stimulating hormone (MSH) stimulation. We describe conditions under which explanted neural tube gives rise to neural crest cells that differentiate into neuronal cells and melanocytes. The potency of melanocyte differentiation was, found to vary according to the concentration of fetal bovine serum (FBS, from 5 to 20%). Melanization of neural crest cells cultured in the medium containing FBS and α-MSH was more extensive than those cultured with FBS alone, combinations of FBS and chick embryo extract, or turtle embryo extract. These culture conditions seem to be useful for the study of the developmental potency of the neural crest cells as well as for investigating local environmental factors.

A simple organ culture method for differentiation of melanocytes in mouse embryonic skin using “origami” membrane filter

A simple organ culture method for culturing embryonic skin was developed. A piece of skin with a part of the neural tube from mouse embryo (11 to 12 d) was placed on a 25 mm d membrane filter. The filter was folded to wrap the explant and inserted into glass tubing. The explant and filter in the glass tubing were placed in a rotating tissue culture tube containing 5 ml culture medium (Hams F12 supplemented with 15 to 20% fetal bovine serum) and filled with a mixture of 95% air:5% CO2. In explants cultured for 6 d fully differentiated melanocytes were observed in the epidermis.

Anti-Bovine Rhodopsin Monoclonal Antibody Recognizing Light-Dependent Structural Change

Abstract The antigenic structure of the bovine rhodopsin molecule was investigated by using a bovine rhodopsin-specific monoclonal antibody designated Rh29. Competition assay with sealed intact disks and broken disks indicated that the antibody-binding region was localized in the intradiscal surface. An antigenic peptide obtained by a cyanogene bromide cleavage of rhodopsin was purified and determined as residues 2–39 in the amino acid sequence. Further analysis suggested that the antigenic determinant included at least residues 21–25. These results were consistent with the structural model for membrane topology of rhodopsin. The antigenicity of the rhodopsin was compared among several states. The antibody bound to both ammonyx LO-solubilized unbleached and bleached rhodopsin. In contrast, upon membrane-embedded rhodopsin, unbleached one was 100-times less antigenic than bleached one. The results suggested that the segment around the determinant of membrane-embedded rhodopsin should undergo a structural change upon absorption of light. Rh29 detected a band corresponding to bovine, porcine and octopus opsins in immunoblotting. Protein blot of crayfish rhabdome did not show any reactive band. These bands except for crayfish reacted with concanavalin A as well. The N-terminal structure may, therefore, conserved between mammal and erthropoda and diverge between them and cepharopoda.