Effects of lithium on pigmentation in the embryonic zebrafish (Brachydanio rerio).
Pigment cell precursors of the embryonic zebrafish give rise to melanophores, xanthophores and/or iridophores. Cell signaling mechanisms related to the development of pigmentation remain obscure. In order to examine the mechanisms involved in pigment cell signaling, we treated zebrafish embryos with various activators and inhibitors of signaling pathways. Among those chemicals tested, LiCl and LiCl/forskolin had a stimulatory effect on pigmentation, most notable in the melanophore population. We propose that the inositol phosphate (IP) pathway, is involved in pigment pattern formation in zebrafish through its involvement in the: (1) differentiation/proliferation of melanophores; (2) dispersion of melanosomes; and/or (3) synthesis/deposition of melanin. To discern at what level pigmentation was being effected we: (1) counted the number of melanophores in control and experimental animals 5 days after treatment; (2) measured tyrosinase activity and melanin content; and (3) employed immunoblotting techniques with anti-tyrosine-related protein-2 and anti-melanocyte-specific gene-1 as melanophore-specific markers. Although gross pigmentation increased dramatically in LiCl- and LiCl/forskolin treated embryos, the effect on pigmentation was not due to an increase in the proliferation of melanophores, but was possibly through an increase in melanin synthesis and/or deposition. Collectively, results from these studies suggest the involvement of an IP-signaling pathway in the stimulation of pigmentation in embryonic zebrafish through the synthesis/deposition of melanin within the neural crest-derived melanophores. (+info)
Isolation and characterization of a Golgi-rich fraction from the Harding-Passey mouse melanoma.
Golgi-rich fraction was isolated from Harding-Passey mouse melanoma by centrifugation through the discontinuous sucrose density gradient and its properties were compared with those of the same fraction isolated from rat liver. The specific activity of UDP-galactose: N-acetylglucosamine galactosyltransferase was 35 times higher in the melanoma Golgi fraction than in the melanoma homogenate and was a half that in the rat liver Golgi fraction. The specific activities of marker enzymes for other subcellular components such as 5'-nucleotidase, acid phosphatase and glucose-6-phosphatase in the melanoma Golgi fraction were all one-third those in the melanoma homogenate. Electron micrographs of the negatively-stained Golgi fractions of melanoma and liver revealed the presence of a system of tubules, vesicles and plate-like center regions which are known as components of Golgi apparatus. Tyrosinase activity was found to be present in this fraction of mouse melanoma, but its specific activity was lower than that in the rough or smooth surface membrane fraction or in the melanosome fraction. (+info)
Tumor necrosis factor alpha-mediated inhibition of melanogenesis is dependent on nuclear factor kappa B activation.
Melanogenesis is a physiological process resulting in the synthesis of melanin pigments which play a crucial protective role against skin photocarcinogenesis. In vivo, solar ultraviolet light triggers the secretion of numerous keratinocyte-derived factors that are implicated in the regulation of melanogenesis. Among these, tumor necrosis factor alpha (TNFalpha), a cytokine implicated in the pro-inflammatory response, down-regulates pigment synthesis in vitro. In this report, we aimed to determine the molecular mechanisms by which this cytokine inhibits melanogenesis in B16 melanoma cells. First, we show that TNFalpha inhibits the activity and protein expression of tyrosinase which is the key enzyme of melanogenesis. Further, we demonstrate that this effect is subsequent to a down-regulation of the tyrosinase promoter activity in both basal and cAMP-induced melanogenesis. Finally, we present evidence indicating that the inhibitory effect of TNFalpha on melanogenesis is dependent on nuclear factor kappa B (NFkappaB) activation. Indeed, overexpression of this transcription factor in B16 cells is sufficient to inhibit tyrosinase promoter activity. Furthermore, a mutant of inhibitory kappa B (IkappaB), that prevents NFkappaB activation, is able to revert the effect of TNFalpha on the tyrosinase promoter activity. Taken together, our results clarify the mechanisms by which TNFalpha inhibits pigmentation and point out the key role of NFkappaB in the regulation of melanogenesis. (+info)
A cytoplasmic sequence in human tyrosinase defines a second class of di-leucine-based sorting signals for late endosomal and lysosomal delivery.
Distinct cytoplasmic sorting signals target integral membrane proteins to late endosomal compartments, but it is not known whether different signals direct targeting by different pathways. The availability of multiple pathways may permit some cell types to divert proteins to specialized compartments, such as the melanosome of pigmented cells. To address this issue, we characterized sorting determinants of tyrosinase, a tissue-specific resident protein of the melanosome. The cytoplasmic domain of tyrosinase was both necessary and sufficient for internalization and steady state localization to late endosomes and lysosomes in HeLa cells. Mutagenesis of two leucine residues within a conventional di-leucine motif ablated late endosomal localization. However, the properties of this di-leucine-based signal were distinguished from that of CD3gamma by overexpression studies; overexpression of the tyrosinase signal, but not the well characterized CD3gamma signal, induced a 4-fold enlargement of late endosomes and lysosomes and interfered with endosomal sorting mediated by both tyrosine- and other di-leucine-based signals. These properties suggest that the tyrosinase and CD3gamma di-leucine signals are distinctly recognized and sorted by distinct pathways to late endosomes in non-pigmented cells. We speculate that melanocytic cells utilize the second pathway to divert proteins to the melanosome. (+info)
Catecholamine synthesis is mediated by tyrosinase in the absence of tyrosine hydroxylase.
Catecholamine neurotransmitters are synthesized by hydroxylation of tyrosine to L-dihydroxyphenylalanine (L-Dopa) by tyrosine hydroxylase (TH). The elimination of TH in both pigmented and albino mice described here, like pigmented TH-null mice reported previously (Kobayashi et al., 1995; Zhou et al., 1995), demonstrates the unequivocal requirement for catecholamines during embryonic development. Although the lack of TH is fatal, TH-null embryos can be rescued by administration of catecholamine precursors to pregnant dams. Once born, TH-null pups can survive without further treatment until weaning. Given the relatively rapid half-life of catecholamines, we expected to find none in postnatal TH-null pups. Despite the fact that the TH-null pups lack TH and have not been supplemented with catecholamine precursers, catecholamines are readily detected in our pigmented line of TH-null mice by glyoxylic acid-induced histofluorescence at postnatal day 7 (P7) and P15 and quantitatively at P15 in sympathetically innervated peripheral organs, in sympathetic ganglia, in adrenal glands, and in brains. Between 2 and 22% of wild-type catecholamine concentrations are found in these tissues in mutant pigmented mice. To ascertain the source of the catecholamine, we examined postnatal TH-null albino mice that lack tyrosinase, another enzyme that converts tyrosine to L-Dopa but does so during melanin synthesis. In contrast to the pigmented TH-null mice, catecholamine histofluorescence is undetectable in postnatal albino mutants, and the catecholamine content of TH-null pups lacking tyrosinase is 18% or less than that of TH-null mice with tyrosinase. Thus, these extraordinary circumstances reveal that tyrosinase serves as an alternative pathway to supply catecholamines. (+info)
The reactions of copper proteins with nitric oxide.
Nitric oxide (NO) can act as a ligand for copper atoms and may also engage in redox chemistry with the metal once bound. Furthermore NO posses an unpaired electron which can couple with the unpaired electron on Cu2+. These properties have been exploited to probe the active sites of copper-containing enzymes and proteins. We review these studies. In addition to the use as a spectroscopic probe for the active site we draw attention to the rapid reactions of NO at the copper sites in Cytochrome c oxidase (CcO) and laccase. These reactions in CcO occur in the ms time range, at low NO concentrations and in the presence of oxygen and may therefore be of physiological relevance to the control of respiration. Finally we speculate on the wider role that NO may play in regulation of an important group of Type 2 copper containing enzymes. (+info)
A system for rapid generation of coat color-tagged knockouts and defined chromosomal rearrangements in mice.
Gene targeting in mouse embryonic stem (ES) cells can be used to generate single gene mutations or defined multi-megabase chromosomal rearrangements when applied with the Cre- loxP recombination system. While single knockouts are essential for uncovering functions of cloned genes, chromosomal rearrangements are great genetic tools for mapping, mutagenesis screens and functional genomics. The conventional approach to generate mice with targeted alterations of the genome requires extensive molecular cloning to build targeting vectors and DNA-based genotyping for stock maintenance. Here we describe the design and construction of a two-library system to facilitate high throughput gene targeting and chromo-somal engineering. The unique feature of these libraries is that once a clone is isolated, it is essentially ready to be used for insertional targeting in ES cells. The two libraries each bear a complementary set of genetic markers tailored so that the vector can be used for Cre- loxP -based chromosome engineering as well as single knockouts. By incorporating mouse coat color markers into the vectors, we illustrate a widely applicable method for stock maintenance of ES cell-derived mice with single gene knockouts or more extensive chromosomal rearrangements. (+info)
Protein kinase C-beta activates tyrosinase by phosphorylating serine residues in its cytoplasmic domain.
We have previously shown that protein kinase C-beta (PKC-beta) is required for activation of tyrosinase (Park, H. Y., Russakovsky, V., Ohno, S., and Gilchrest, B. A. (1993) J. Biol. Chem. 268, 11742-11749), the rate-limiting enzyme in melanogenesis. We now examine its mechanism of activation in human melanocytes. In vivo phosphorylation experiments revealed that tyrosinase is phosphorylated through the PKC-dependent pathway and that introduction of PKC-beta into nonpigmented human melanoma cells lacking PKC-beta lead to the phosphorylation and activation of tyrosinase. Preincubation of intact melanosomes with purified active PKC-beta in vitro increased tyrosinase activity 3-fold. By immunoelectron microscopy, PKC-beta but not PKC-alpha was closely associated with tyrosinase on the outer surface of melanosomes. Western blot analysis confirmed the association of PKC-beta with melanosomes. Only the cytoplasmic (extra-melanosomal) domain of tyrosinase, which contains two serines but no threonines, was phosphorylated by the serine/threonine kinase PKC-beta. These two serines at positions 505 and 509 both are present in the C-terminal peptide generated by trypsin digestion of tyrosinase. Co-migration experiments comparing synthetic peptide standards of all three possible phosphorylated tryptic peptides, a diphosphopeptide and two monophosphopeptides, to tyrosinase-phosphorylated in intact melanocytes by PKC-beta and then subjected to trypsin digestion revealed that both serine residues are phosphorylated by PKC-beta. We conclude that PKC-beta activates tyrosinase directly by phosphorylating serine residues at positions 505 and 509 in the cytoplasmic domain of this melanosome-associated protein. (+info)