Recent studies on the pigment patterns of cold-blooded vertebrates have revealed the presence of a protein melanization inhibiting factor (MIF) present in the integument of the ventral surface. This putative MIF is present in the ventral but not the dorsal surface and is presumably responsible for the pigmentary pattern seen widely among vertebrates wherein the dorsal integument is dark due to the presence of melanophores (-cytes) and the ventrum is pale due to the failure of these melanin- containing cells to differentiate. Incubation of pieces of ventral skin from Xenopus or Rana in basic salt solution (BSS) yields a conditioned medium (VCM) which when used in the culture of isolated neural tubes of Xenopus embryos, markedly inhibits on a dose-response basis, the outgrowth of neural crest cells and melanization. Preliminary experiments with S-91 murine melanoma cultures have revealed that VCM inhibits MSH-induced melanogenesis in these cells. It is not surprising that VCM from amphibian sources has an effect on a mammalian pigmentary system because pigment cells from lower and higher vertebrates alike, have much common including a neural crest origin. Because of these commonalities, it might be possible to utilize the putative MIF in human pigmentary pathologies such as vitiligo or melanoma. Accordingly, the major thrust of this research proposal is directed toward extraction and purification of the putative MIF, determination of the N-terminal amino acid sequence, synthesis of a peptide corresponding to the N-terminal acid sequence, and the preparation of polyclonal and monoclonal antibodies to MIF. These antibodies will be used for the development of radioimmunoassays to MIF, affinity purification of MIF, for fluorescent labeling of monoclonal antibodies to be used for establishing tissue localization of MIF, and for monoclonal species variations in MIF's. The effectiveness of pure MIF will be tested on other melanization systems such as mammalian melanocytes and melanoma cells in vitro.
