by Dr. Jiro Matsumoto

This meeting was organized by Toho University with Professor Noriko Oshima (Department of Biomolecular Science, Faculty of Science) as chairperson.

The science programs consisted of a presidential address, an invited lecture, 2 special lectures, a Symposium, 4 research seminars and 28 oral presentations, with a special emphasis on the symposium.

The symposium entitled with "Phylogenic aspects of tyrosinase genes" was organized with an expectation to provide up-to-date, unified concepts on the evolution of genes playing a central role in pigmentation.

Four speakers reported their own recent works on tyrosinase or its associated genes deriving from different phyletic levels of organisms and their biological significance.

Melanogenesis and prokaryotes: Dr. Y. Nishimura reviewed recent findings on the wide distribution of homologues of eukaryotic tyrosinase in prokaryotes and pointed out that melanin-like pigments in bacteria are formed mostly by the tyrosinase catabolic pathway forming homogentisic acid as observed in human alkaptonuria. It was emphasized that bacteria with melanins or alike indicate a high survival rate against UV-irradiation, and that melanogenic Escherichia coli carrying plasmid tyrosinase genes from actinomycete (S. antibioticus) is available for screening of inhibitors for melanogenesis.

Insect pro-phenol oxidase and its activation mechanism: Dr. M. Ashida reported the homology of pro-phenol oxidase and hemocyanin present in the hemolymph of arthropods based on the similarity in the base sequence of their cloned cDNA, and presumed possible evolution of these two molecules from the common ancestral binuclear copper protein. He mentioned that dimeric phenol oxidase in insect hemolymph is activated by breakage of the peptide-bond at Arg (50)-Phe (51) under the presence of zymogen proteinase and then is released into tissues, and that quinones thus produced by activated phenol oxidase cause hardening of exoskeleton, attributing the protection against bacterial infections.

Expression of the ascidian tyrosinase gene in the developing brain: Dr. H. Yamamoto and his associate cloned tyrosinase gene from ascidian species, Halocynthia roretzi, and found that its open reading frame has 36 - 39% homology with its counterpart of vertebrate origin in terms of amino acid sequence. He also reported that this gene is expressed in the neural plate of early neurulae, even though the neural crest of this species shows no further differentiation, yielding only two sensory cells having different functions in the brain. Based on the presence of melanin granules, these two sensory cells were considered to be an ancestry type of pigment cells. Albinism in fish: insertion of an

Ac-like transportable element in the tyrosinase gene: Dr. H. Hori reported that albinism in the i4-mutant of Medaka fish is caused by insertion of a 4.6 kb sequence into the exon 5 of the tyrosinase gene, and that the inserts, Tol-2 in their designation, are homologous in its basic structure and mobility inside the genome to a maize transposable element, Ac. It was also shown that the gene for tyrosinase, TRP1 and TRP2 in this fish are similar in their basic structure to those of mammalian origin, but are different with regard to the size of their introns which are about 10 kb smaller than those of mammals.

MITF isoform multiplicity and a new aspect in melanin research: Dr. S. Shibahara reported that microphthalmia-associated transcription factor (MITF) plays an important role in cell type-specific transcription of the tyrosinase and the TRP-1 gene, and that MITF fails to transactivate the TRP-2 promoter, despite the presence of an MITF-binding site in the promoter of this gene. He pointed out that transgenic insertional mutation and/or mutation at the mi locus are closely associated with various dysfunctional phenotypes such as small non-pigmented eyes, a lack of melanocytes in the inner ear associated with deafness, a deficiency in mast cell and osteopetrosis, suggesting an intimate correlation of such disorders with the multiplicity of MITF isoforms.

Melanin monomer genesis investigations leading to therapeutic control of malignant melanoma and melanin pigmentation -molecular to clinical level : Dr. Y. Mishima reviewed a recent successful development of the selective boron neutron capture therapy by application of 10B-p-boronphenylalanine (10B-BPA) which had accentuated polymer forming ability within melanoma cells. He emphasized that 1) the accumulation of 10B-BPA becomes possible by formation of complexes with DHICA and DHI which are abundant in these cells, 2) this therapy becomes applicable even to amelanotic melanoma upon transfection of the genes for tyrosinase or its related proteins, and 3) complex forming ability of BPA with DHICA and DHI in vitro led to the idea of using this compound as an inhibitor for melanogenesis in hyperpigmented human skin. At the end of this presentation, he emphasized the necessity of conceptual designs in the research.

Structural and enzymatic components of mammalian melanosomes: Dr. V. J. Hearing, an invited speaker, addressed current understanding of mammalian melanosomes with a particular emphasis on the role of two intercellular signaling molecules, melanocyte stimulating hormone (MSH) and agouti signal protein (ASP), which switch between eumelanin and pheomelanin synthesis. He indicated based on his recent findings that ASP elicits down-regulation in transcription of several pigment specific loci such as albino, brown, slaty, silver and pink eyed-dilution, all of which encode proteins associated with catalytic and structural components of melanosomes. He set forth a view that melanosomes emerge as organelles common to lysosomes, which then differentiate into two distinguishable forms with spherical or elongated fibrillar phenotypes known as pheomelanosomes and eumelanosomes, and that selection of such differential courses of maturation can be defined by combination of specific proteins that are synthesized and deposited under the guidance of signaling molecules.

Signals and molecules regulating melanosome biosynthesis and transport of tyrosine-related protein: Dr. K. Jimbow reported his group's recent findings regarding the fate of tyrosinase-related protein (TRP-1) in the course of melanosome formation, with particular interest in biosynthesis and structural integration of this protein in the ER and Golgi and its transport via the trans Golgi network to the melanosomes under guidance of signal molecules. He pointed out that (1) calnexin plays an important role in folding of tyrosinase and TRP-1 in the ER, suggesting its correlation with melanosomal proteins, (2) newly synthesized TRP-1 is fully glycosylated in the trans Golgi network and then transported to late endosomes installed with the mannose-6-phosphate receptors, possibly by binding with its tyrosine-dileucine residues, (3) several small GTP-binding proteins present in melanosomes, particularly rab 7, are implicated in trapping of TRP-1, together with PI-3-kinase.

Pigment cells and gene transfer in Medaka: Dr. K. Ozato reviewed recent progress in gene-transfer studies using Medaka fish and reported his own success in production of transgenic Medaka carrying the gene for salmon melanin concentrating hormone (MCH) with CMV promoter. He addressed that the transgenes of salmon origin are expressed in a variety of tissues of homozygous progenies, indicating the presence of a detectable level of MCH in their serum and thereupon causing a distinguishable paler body coloration over 10 generations. He also reported his current success in establishing embryonic stem cell lines from Medaka embryos, suggesting the usefulness of the nuclear transplantation technique in production of chimeric animals.

At the research seminars, four topics were dealt with: the chemistry of melanins (Dr. K. Wakamatsu), the effects of ACF (stem cell factor) on the development of cultured neural crest cells into melanocytes (Dr. H. Ono), the role of c-KIT expression in melanoma (Dr. Y. Funasaka), and the expression of SCF and c-KIT in cutaneous mastocytosis (mastocytoma) (Dr. Y. Kubota). In this seminar, Ono reported clear evidence indicating the necessity of stem cell factor for melanocyte differentiation which is considered to be inducible solely by endothelin-3, based on in vitro neural crest culture using homozygous sl mutant mice. Dr. Funasaka reported a marked decrease of c-kit expression in terms of mRNA levels in dysplastic nevus and malignant melanoma cells. She predicted, based on the inhibitory effects of SCF on the growth of melanoma cells, that apoptosis would be inducible by application of phosphorylation to c-kit proteins.

In the regular presentation, Dr. T. Hirobe reported that the differentiation-inducing potential of ACTH for murine melanocytes exists in the site corresponding to 1 to 13 amino acid residues, showing a higher activity with shortening of accessory residues and reaching to its maximal with the sequence similar to MSH.