Masataka Obika

Association of pteridines with amphibian larval pigmentation and their biosynthesis in developing chromatophores

Abstract In order to ascertain the interrelation between chromatophores and pteridine derivatives in urodeles, qualitative changes in these substances were observed during development. In all species employed here, young embryos having no apparent chromatophores were free of fluorescent pteridines. A small quantity of pteridines appeared simultaneously with the melanophore pigmentation. When xanthophores appeared, yellow fluorescent pteridines became distinguishable. The amount of pteridines increased thereafter with the augmentation of chromatophore population. A very small quantity of pteridines was found in skin containing melanophores, but no specific association of pteridines with melanophores was observed. The greatest quantity of pteridines was found in skins having xanthophores containing water-soluble yellow granules. These are assumed to be pteridines. Red chromatophores of Triturus adults have carotenoid pigments, but lack pteridines. Isolated posterior trunk neural crest cells of neurulae, cultured with or without an epidermal envelope, formed pteridines comparable to those of intact larvae. The presence of epidermis or of humoral factors appear to have little to do with the quality of pteridine synthesis. The pteridine pattern is intrinsic to neural crest cells.

The effects of melanophore-stimulating hormone and cyclic nucleotides on teleost fish chromatophores

Abstract The effects of melanophore-stimulating hormone (MSH) and cyclic nucleotides on pigment translocation within the isolated scale chromatophores of the medaka, Oryzias latipes , were examined in relation to the second-messenger hypothesis. In leucophores, MSH, cyclic AMP, dibutyryl cyclic AMP (DBcAMP), and methylxanthines brought about a rapid pigment dispersal. β-Adrenergic blockers completely abolished methylxanthine-mediated dispersion without inhibiting the response induced by MSH or DBcAMP. In melanophores and xanthophores, MSH did not accelerate pigment dispersion. Dibutyryl cyclic GMP (DBcGMP) was more effective than cyclic AMP or DBcAMP in producing pigment dispersal. In the presence of DBcGMP, epinephrine was incapable of stimulating pigment aggregation in melanophores whereas the presence of DBcAMP did not interfere with the epinephrine-induced aggregation. The results implicate the involvement of the adenylate cyclase-cyclic AMP system in the regulation of leucophore response while in melanophores and xanthophores, cyclic GMP appears to mediate pigment displacement.

Epinephrine-induced changes in the cyclic nucleotide content of fish melanoma cells

Abstract Intracellular levels of adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) in melanoma tissues developed in platyfish and swordtail hybrids were determined by radioimmunoassay. The melanoma tissues were composed mostly of pigment cells, many of which responded to epinephrine with pigment aggregation. The aggregated melanosomes redispersed gradually by washing the cells with physiological saline, and rapidly by the perfusion with theophylline. Determination of intracellular levels of cyclic nucleotides revealed that epinephrine produced a small but significant decrease in cyclic AMP level, whereas theophylline elicited a remarkable increase in the intracellular concentration of cyclic AMP. On the other hand, the change in the levels of cyclic GMP was not significant among the tissues subjected to the different experimental conditions. The present results suggest that the centrifugal and centripetal translocations of melanosomes within melanophores are regulated directly by the intracellular concentration of cyclic AMP.

Fish Hereditary Melanoma Cell Lines of Different Degrees of Cell Differentiation

Four cell lines including two sublines were established from hereditary melanomas in interspecific hybrids between platyfish (Xiphophorus maculatus) carrying the Sp gene and swordtails (X. helleri) and maintained in vitro for more than 34 months. Cells in each cell line grew randomly across each other with an apparent lack of contact inhibition of growth and at a population doubling time of 50 to 72 hr. They retained the characteristics of young pigment cells in regard to ultrastructure, tyrosinase activity, the DOPA and combined DOPA‐premelanin reactions. In the degree of differentiation, the cells of the three cell lines seemed comparable to early melanocytes close to melanoblasts, and those of the remaining one cell line seemed comparable to young melanocytes but were in a more differentiated state than the early melanocytes. Colony forming ability on plastic plates was at a level of 10% in the three cell lines but only 1% in the one cell line. All four cell lines failed to form colonies in soft agar. Chromosome analysis revealed that these four cell lines were heteroploid with many abnormal figures of chromosomes and double minute chromosomes. None of the cell lines showed transplantability to fish.

Formation of pterinosomes and carotenoid granules in xanthophores of the teleost Oryzias latipes as revealed by the rapid-freezing and freeze-substitution method

SummaryThe rapid-freezing and freeze-substitution method was applied for the ultrastructural study of the dermal chromatophores of a teleost, Oryzias latipes. The method was found to be suitable for preserving fragile membranous structures within melanophores and xanthophores. In addition, relatively high electron density in overall profile indicates that the procedure is effective in reducing the extraction of cytoplasmic ground substances that inevitably occurs during the process of conventional chemical fixation and the following dehydration. The improved ultrastructural images clearly show that the pterinosomes, the characteristic pigmentary organelles of xanthophores, are formed through several distinct developmental stages starting from the loose congregations of vesicles derived from the Golgi complex. The earlier stages of development are similar to those found in melanosome formation. Whereas carotenoid pigments in xanthophores in conventional aldehyde-osmium-fixed materials are found to be electrondense membrane-free particles, they are identified as membrane-bounded organelles in the present study. The envelope of these carotenoid vesicles does not exhibit a typical trilaminar structure but appears to be an extremely thin membrane. Carotenoid vesicles are, in most cases, in direct contact with the outer surface of tubular endoplasmic reticulum.

Dynein 1 from rainbow trout spermatozoa: Immunological similarity between trout and sea urchin dynein 1

Abstract This paper is the first biochemical presentation on dynein 1 from vertebrate spermatozoa. Axoneme of rainbow trout spermatozoon is surrounded by the functionally differentiated flagellar membrane, the undulating membrane. The long axis of the undulating membrane is perpendicular to the axonemal axis. Dynein 1 was obtained in the salt extract of axonemes and Fragment 1A was purified from the tryptic digest of salt-extracted dynein 1. Dynein 1 and Fragment 1A from trout were compared with those from sea urchin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis could not show the difference in the molecular weights of dynein 1, and subunit components and their molecular weights of Fragment 1A between two species. Anti-dynein 1 and anti-Fragment 1A sera raised against sea urchin antigens also reacted with trout dynein 1 and Fragment 1A, and inhibited their ATPase activities. Ouchterlonys double diffusion test indicated the pattern of “partial identity” between trout and sea urchin Fragment 1A. Immunoelectron microscopy using peroxidase-conjugated anti-IgG shows that the decoration was observed on the outer arms when the axonemes from the fresh spermatozoa were employed.

Growth and maturation of melanosomes in the melanophores of a teleost, Oryzias latipes

SummaryFormation of melanosomes in melanophores of a teleost, Oryzias latipes, was studied by means of electron microscopy. Two distinct types of premelanosomes are observed in the same cell: (i) multivesicular premelanosomes, which later develop into melanosomes with electron-lucent “hollows” in the center, appear at early embryonic stages; (ii) premelanosomes with highly organized, fibrous internal structure are formed at later stages of development and give rise to melanosomes with a filamentous center. Melanosomes are generally ellipsoid in shape, and the difference in the dimensions of fibrillar premelanosomes, melanosomes in the cells at younger developmental stages and those developed fully in melanophores of adults indicates that these organelles grow during development. The growth is achieved by fusion of small unmelanized vesicles or fibrillar premelanosomes to preformed melanosome and by fusion of two or more premelanosomes to form a larger organelle. The addition of the matrix of fibrillar premelanosomes around preformed melanosomes, which are derived from either multivesicular or fibrillar premelanosomes, forms a concentric outer deposit, and the fusion of small vesicles produces electron-lucent pits which are scattered irregularly in mature melanosomes.

Reversible binding of riboflavin and pteridines to melanin in vitro.

Abstract 1. 1. Binding of riboflavin, flavin mononucleotide, flavin-adenine dinucleotide, 6,7-dimethyl-8-ribityllumazine, isoxanthopterin and pterin to melanosomes or natural and synthetic melanin has been studied in vitro . 2. 2. Binding of riboflavin to melanosomes or melanin is greater than the binding of the other compounds. 3. 3. The known distribution patterns of the fluorescent substances in the pigmented integuments of cold-blooded vertebrates are explained by the selective binding of these compounds to melanosomes.

Properties and state of particulate tyrosinase of xanthic goldfish

Abstract 1. 1. The properties, subcellular distribution and status of particulate tyrosinase in xanthic goldfish skin were studied using fractionated materials. 2. 2. Tyrosinase occurred in two molecular forms that exhibited different electrophoretic mobilities in acrylamide-gel electrophoresis. 3. 3. Freshly prepared particulate or pterinosome suspension in an isotonic medium had no measurable tyrosinase activity. The enzyme activity was detected after mechanical or chemical breakage of the structural integrity of the particles. 4. 4. No endogenous tyrosinase inhibitor was found in the particulate fractions.

Induction of xanthophores from non-pigmented dermal cells of xanthic goldfish in vitro.

To identify precursor cells of xanthophores (xanthoblasts), non-pigmented cells without any phenotypic traits as pigment cells were isolated from the dermal tissue of xanthic goldfish with an adult color pattern and cultured in a medium containing 1 mM db-cAMP or 0.25 U/ml ACTH and 10% carp serum. These non-pigmented cells differentiated into xanthophores which showed a dendritic morphology and contained a large quantity of fluorescent pteridines and numerous vesicular inclusions. Sepiapterin was the major component, and the vesicles contained fuzzy material in addition to small membranous elements. The fluorescent pattern and the morphological characteristics indicated that the differentiated pigment cells were xanthophores of larval type.