Abstract. The majority of malignant melanoma cell types are able to produce melanin and the degree of melanin synthesis in various types of cultured cell line differs. In this study, we evaluated three types of cultured cell line, MNT-1, HM3KO and G-361, with differing melanin production levels. The level was greatest in the MNT-1 cells, lower in the HM3KO cells and lowest in the G-361 cells. In addition, a positive correlation between melanin production and tyrosinase activity was observed. The molecular masses of tyrosinases from HM3KO and G-361 cells were marginally lower than those from MNT-1 cells. Glycosylation inhibitor treatment on MNT-1 cells caused decreases in the molecular mass of tyrosinase, its activity and melanin production. An immunoprecipitation assay using anti-tyrosinase indicated that the immature glycosylated tyrosinases were associated with a type of chaperone, Hsp70. The interaction between tyrosinase and Hsp70 was also detected in HM3KO and G-361 cells. The results indicated that the immature glycosylation of tyrosinase has a critical effect on the melanin-producing ability of melanoma cells. IntroductionMelanin is important in protecting the skin from the harmful effects of ultraviolet irradiation and absorbing toxic drugs and chemicals. Melanin is synthesized in two predominant forms, black-brown pigments (eumelanin) and red-yellow pigments (pheomelanin), within the melanosomes of melanocytes by at least three melanogenic enzymes. Tyrosinase (EC1.14.18.1) is one of the predominant enzymes mediated by the melanin production process in melanosomes (1). The enzyme catalyzes the rate-limiting step of the hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA) and its further oxidation to DOPA-quinone (2). Melanin synthesis is maintained by a number of regulatory processes that act at various steps during the synthesis of the protein in the endoplasmic reticulum (ER), Golgi body and melanosomes, and disorders in any of these processes lead to abnormal pigmentation.Malignant melanoma (MM) cells are derived from epidermal melanocytes and nevi (3), and numerous cultured melanoma cell lines have been established from human and mouse MMs. The majority of MMs produce melanin, and the degree of melanin synthesis differs for each type of cultured cell line (4-6). For example, MNT-1 cells were demonstrated to be highly pigmented MMs (4); however, SK-Mel-28 cells were not able to produce melanin due to a mutation in the endocytic pathway that affected the endosomal trafficking of tyrosinase (5). In addition, B16F10 cells were pigmented; whereas amelanotic melanoma was not pigmented, due to the increased expression of proteasome subunit p27 (6).In this study, three cultured cell lines, MNT-1, HM3KO and G-361, were observed. These cell lines differed in their degrees of melanin synthesis, and thus, the determinants of the degree of melanin synthesis were investigated. Materials and methodsCell culture. The MNT-1 (from Dr VJ Hearing, Laboratory of Cell Biology, National Cancer Institute, National Ins...

We report the occurrence of neuronal cytoplasmic inclusions (NCIs) in the external cuneate nucleus of humans. The NCIs appeared as accumulations of eosinophilic rod-like structures in the neuronal somata in 20 (9.5%) of 211 consecutive autopsy cases. Histochemically, the NCIs were stained bright red with Gomori trichrome, Azan-Mallory and methyl green-pyronin, indicating that they contain protein and RNA. Immunohistochemically, the NCIs were positive for stress granule marker proteins, including Hu-antigen R, eukaryotic translation initiation factor 3 and poly(A)-binding protein 1, but negative for ubiquitin- and autophagy-related proteins. Ultrastructurally, the NCIs were composed of randomly oriented arrays of parallel fibrillar crystalline material with a well-defined substructure consisting of longitudinal striations, and were often associated with ribosome-like granules. These NCIs are morphologically, immunohistochemically and topographically distinct from any other inclusions previously described. Their incidence was found to increase with age. A high incidence was also observed in individuals with noninfectious inflammatory disease. These findings suggest that eosinophilic NCIs in the external cuneate nucleus are novel inclusions and might be formed under stress conditions.