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Formation of melanin-tyrosinase complex and its possible significance as a model for control of melanin synthesis.

Previous reports have shown that the tyrosinase activities of melanosomes and soluble tyrosinase isolated from melanoma were diminished when these preparations were incubated with 3,4-dihydroxyphenylalanine (dopa). It was concluded from these results that the tyrosinase activities of these preparations decreased when melanin was synthesized in these systems. The present investigation has revealed that melanin synthesized in vitro from dopa formed a complex with purified mushroom tyrosinase. The addition of melanin diminished the tyrosinase activity of the sample. These results show that the formation of the melanin-tyrosinase complex results in a decrease in the activity of the tyrosinase solution. The tyrosinase activity of the melanin-tyrosinase complex could not be increased by certain procedures which were previously found to enhance the tyrosinase activity of isolated melanosomes.

Catechol Oxidase

Melanin containing neurofibroma: case report with evidence of Schwann cell origin of melanin.

This case report describes a melanin-containing neurofibroma involving a spinal nerve root. Electron microscopy of the tumor shows that neoplastic Schwann cells are capable of melanogenesis. Although this capability is suggested in the literature, few reports provide ultrastructural confirmation. The likely identity between reported "cellular blue nevi of spinal nerve roots" and nerve sheath tumors is discussed. It is possible that pigmented nerve sheath tumors behave more aggressively than nonpigmented ones, although it is debatable.

Adult

Melanin in the rat brain.

Melanin was measured in various parts of the rat brain by a spectrophotofluorometric assay. This method could detect natural, Sepia melanin as well as melanin synthesized from L-DOPA. Contrary to published expectations of other investigators, measurable amounts of melanin were found in the brain of albino as well as pigmented rats. The highest concentrations of melanin occurred in the pons-medulla and midbrain, but all regions within the blood-brain barrier contained greater concentrations than samples from many other tissues in the body. No significant change in the melanin content was found after various endocrine manipulations such as removal of the pituitary, pineal, adrenals, thyroid, testes, or ovaries, exposure to constant illumination or darkness, and daily injection for 5 weeks of alpha-MSH, Pro-Leu-Gly-NH2 (MIF-I) or melatonin. As expected, retinal tissue from black-hooded rats contained extremely high levels of melanin whereas that from albino rats contained no melanin. It is thought that the presence of melanin in the brain of albino and pigmented rats may have a function which is still unknown.

Albinism

Use of mutants to establish (+)-scytalone as an intermediate in melanin biosynthesis by Verticillium dahliae.

Melanin biosynthesis in Verticillium dahliae Kleb, was studied with mutants deficient for normal black melanin or for production of microsclerotia. Seven genetically different mutants had apparent blocks in melanin biosynthesis. Four mutants (brm-1 to -4) produced brown microsclerotia and extruded pigments into media; three (alm-1 to -3) produced albino microsclerotia. Other mutants produced no microsclerotia (nms) or had greatly reduced numbers of microsclerotia (rms). Mutation alm-1 was due to a single recessive gene; the other melanin-deficient characters were recessive but their genetic bases were not determined. Cultures of the brown mutants brm-1 and -3 extruded and accumulated a metabolite that blackened the albino microslerotia of alm-1 to -3. The metabolite was identified as (+)-scytalone (3,4-dihydro-3,6,8-trihydroxy-1(2H)naphthalenone). Pigment formed by alm-1 microsclerotia from (+)-scytalone had chemical and physical properties identical with those of melanin in the wild-type fungus. (+)-Scytalone was produced and converted to melanin by microsclerotia but not by conidia or hyphae. Conversion of (+)-scytalone to melanin appeared to involve two or more enzymes and probably involved conversions to 1,3,8,-trihydroxynaphthalene and 1,8-dihydroxynaphthalene. Albino mutants of Thielaviopsis basicola, Drechslera sorokiniana, Pleospora infectoria (Alternaria), Ulocladium sp., and Curvularia sp. also converted scytalone to pigments indistinguishable from the melanins found in their respective wild types. Scytalone melanin may be common in fungi with dark brown or black pigments.

Catechols

Elemental analysis of melanins from bovine hair, iris, choroid, and retinal pigment epithelium.

According to recent theory on the structure of melanin, the sulfur content of melanin is a reflection of the amount of cysteine used in its manufacture. We compared the sulfur contents of melanins from hair, choroid, iris, and retinal pigment epithelium from black-haired cattle and separately from red-haired cattle. In black-haired cattle, sulfur contents of hair and ocular melanins were all between 0.60% and 0.94%, however, in red-haired cattle, hair melanin had a sulfur content (8.66%) different from those of choroid (0.94%) or RPE (1.72%) MELANIN. One may conclude that in an individual animal, ocular melanins can have different chemical structures from hair melanins. The clinical importance of this finding is discussed with regard to sympathetic ophthalmia.

Animals

Melanoprotein: absence of a direct melanin-protein relationship in chick embryo melanocytes.

Short-term synthesis of radioactivity labeled melanin (using DL-[2-14C]tyrosine or 2-[2-14C]thiouracil) by chick retinal pigment tissues in vitro was not influenced by inhibitors of protein synthesis, puromycin and cycloheximide. Co-ordinate synthesis of protein is, therefore, unnecessary for melanin synthesis, and melanoproteins must represent secondary interactions between melanin and protein. Melanin was isolated from chick embryo feather germs by extracting the proteins with hot dodecyl sulfate/mercaptoethanol. Melanin isolated from tissues incubated previously in L-[U-14C]valine medium had no associated radioactivity compared to the radioactivity of melanin prepared from tissues incubated in DL-[2-14C]tyrosine or 2-[2-14C]thiouracil. If melanoproteins exist at all, they are non-covalently bonded associations of melanin and melanosomal proteins.

Animals

Lipofuscin and melanin of human retinal pigment epithelium. Fluorescence, enzyme cytochemical, and ultrastructural studies.

The life history of melanin and lipofuscin granules of human retinal pigment epithelium (RPE) was studied in 30 human eyes spanning nine decades of life. Autofluorescent granules in the cytoplasm of eye over 30 years of age were shown, ultrastructurally and through lipid solvent extraction, to be lipofuscin granules. Sparse small fluorescent granules in infant eyes were secondary lysosomes containing small droplets of lipid. Flourescent substances in RPE granules of eyes less than 50 years old were more readily extracted with lipid solvents than those in very old eyes (greater than 70). Lipfuscin granules were positive for acid phosphatase and aryl sulfatase activity. Fusions between primary lysosomes and lipofuscin granules were common in older eyes, suggesting that the over-all degradative process involves repeated injection of lysosomal enzymes, i.e., the initial fusion of lysosomes with phagosomes (phagocytized outer segment disks) is only one of several attempts to hydrolyze the membranous material. Some melanin granules showed hydrolytic enzyme reactions. By use of enzyme cytochemistry, fluorescence microscopy, and lipid extraction two types of melanin-containing complex granules were identified: melanin with a cortex of lipofuscin (melanolipofuscin) and melanin with a cortex of nonlipid, enzyme reactive material (melanolysosomes). These findings indicate that melanin commonly becomes incorporated into the lysosomal system of the RPE cell and suggests that it undergoes modification or degradation there. These studies indicate that a dynamic, complex interrelationship exists between the various components of the phagolysosomal system and the melanin granules in the RPE cytoplasm. Also, the observed variation from one human eye to another in the content and lipid extractability of RPE lipofuscin granules suggests that there may be differences in lipid composition of phagocytized photoreceptor disks and/or differences in the degradation of these lipids in the phagolysosomal system of the RPE cell.

Acid Phosphatase

Rat brain melanin at different ages and after various treatments.

Melanin was measured by a spectrophotofluorometric method in the brains of albino rats from birth to 20 months of age. The concentration of brain melanin increased from Day 1 until adult levels were reached at 1 month. Between 1 and 20 months of age no significant differences were found in brain melanin. Daily injections of alpha-MSH, MIF-I, melatonin, or diluent did not consistently alter the concentration of brain melanin and a high (40%) protein diet did not appear to increase it. After concurrent injections of alpha-MSH and theophylline, an initial elevation of the level of melanin in the brain of newborn rats was found beginning at Day 8 but by age 1 month the values had returned to control levels. The results show that the largest changes in the concentrations of melanin in the brains of rats occur with age during the first month after birth.

Aging

Some aspects of melanin biology: 1950-1975.

Recent advances in the biology of mammalian pigmentation are reviewed. The multicellular epidermal melanin unit (melanocyte and associated pool of keratinocytes) rather than the melanocyte alone forms the focal point for melanin metabolism within mammalian epidermis. Within an epidermal melanin unit, melanosomes are synthesized by melanocytes and transferred to keratinocytes where they are degraded as they ascend to the epidermal surface. During the past 25 years, technical advances in biology and biochemistry have frosted a multidisciplinary approach to research on mammalian pigmentation. Emphasizing this perspective, we have examined the current state of knowledge of the form and function of epidermal melanin units from the levels of biologic organization ranging from the molecules relevant to melanin synthesis through the skin as a totally intergrated system. To an unusual degree, advances in melanin pigmentation have resulted from the integration of clinical medicine and basic science.

Animals

Fluorescence spectrophotometric analysis of melanins in the house mouse.

We extracted the yellow melanin (phaeomelanin), black melanin (eumelanin), and mixed type of melanin from dorsal hair of dominant yellow (Ay/a), non-agouti (a/a), and agouti (A/A) mice, respectively. Spectrophotometric and fluorescence spectrophotometric analysis demonstrated that the yellow melanin was qualitatively distinct from the black melanin and that the agouti hair contained both types of pigment.

Animals

Electron spin resonance study of melanin treated with reducing agents.

The electron spin resonances (ESR) of several native and modified melanins have been determined. Melanins isolated from black wool and synthesized from 3,4-dihydroxyl-L-phenylalanine (L-DOPA) and tyrosine all show similar ESR signals. Modification of the isolated melanins by treatment with reducing agents causes some lightening in color and slight changes in the ESR spectra. Lithium and liquid ammonia (Birch) reduction applied to melanins from wool and L-DOPA gave very different results, as reflected by ESR spectra, but in both cases the changes were much greater than those produced by other treatments. In general, reductive treatments in nonaqueous media in the presence of metals increase the free radical content and line width, whereas treatment in aqueous media resulted in decreased free radical content. These observations are consistent with a melanin pigment which is an irregular polymer and has unpaired electrons localized on different but similar monomer units.

Ammonia

Ultrastructure of melanin formation in Verticillium dahliae with (+)-scytalone as a biosynthetic intermediate.

Transmission and scanning electron microscopy showed that melanin of wild-type Verticillium dahliae occurred as granules in microsclerotial cell walls and in a fibrillar network encapsulating the walls. An albino microsclerotial mutant and a brown microsclerotial mutant of V. dahliae did not form melanin granules. When albino microsclerotia were treated with (+)-scytalone (a metabolite that the brown mutant accumulates), they formed melanin granules and turned black. These granules were similar in appearance and distribution to those in the wild type. Melanin granules of the wild-type isolate and the scytalone-treated albino mutant were formed at a maximum rate in microsclerotia from 5- to 8-day-old cultures. These observations suggest that scytalone is a natural intermediate of melanin synthesis in V. dahliae.

Cell Wall

Ultrastructural and chemical distinction of melanins formed by Verticillium dahliae from (+)-scytalone, 1,8-dihydroxynaphthalene, catechol, and L-3,4-dihydroxyphenylalanine.

Microsclerotia of three melanin-deficient mutants of Verticillium dahliae formed malanin from (+)-scytalone, 1,8-dihydroxynaphthalene, catechol, and L-3,4-dihydroxyphenylalanine. The melanins formed from (+)-scytalone or 1,8-dihydroxynaphthalene resembled wild-type melanin chemically and ultrastructurally, whereas the melanins formed from catechol and L-3,4-dihydroxyphenlalanine were different. This suggests that scytalone and 1,8-dihydroxynaphthalene but no catechol or L-3,4-dihydroxyphenylalanine are natural intermediates of melanin biosynthesis in V. dahliae.

Catechols

Melanin capacity to accumulate drugs in the internal ear. A study on lidocaine, bupivacaine and chlorpromazine.

The distribution and retention of labelled lidocaine, bupivacaine, and chlorpromazine to melanin in the internal ear after intravenous and intraperitoneal injection were examined by whole-body autoradiography. Both young pigmented hooded rats and albino rats were studied. In the pigmented rats chlorpromazine showed the greatest accumulation, which was more pronounced in the cochlea than in the vestibular portion. The other two substances were evenly distributed in the internal ear. After a single injection of chlorpromazine and of bupivacaine these substances were still bound to the melanin of the internal ear after 14 days, which was the longest survival time. Lidocaine, on the other hand, had disappeared after only 4 days. Strong uptake and retention of the three substances were observed in the eyes of pigmented animals. In albino animals there was very weak, transient uptake in the internal ear of chlorpromazine and bupivacaine, but not of lidocaine. In studies in vitro on isolated bovine eye melanin there was considerably greater adsorption of chlorpromazine than of lidocaine and bupivacaine. An uptake was noted in the human eye in experiments in vitro. Clinical tests revealed no acute or late damage to hearing or sight after large doses of lidocaine. The participation of melanin in different basal labyrinthine functions such as the energy transfer mechanism and the sound protective mechanism is discussed in the light of the results obtained. Further, the theory is put forward that the melanin affinity of certain substances can be of both therapeutic and ototoxic importance.

Animals

[Melanin pigment of Actinomyces umbriferus].

Properties of a dark-brown pigment complex isolated from the spores, mycelium and cultural broth of Actinomyces umbriferus by a conventional technique were compared. The complex consists of three fractions: one is a true melanin while two other fractions differ by several characteristics from melanins and from each other. The fraction which is most similar to melanin prevails in the spores, is contained, in a relatively small amount, in the mycelium, and is absent from the cultural broth. Melanin isolated from the spores has a low relative content of carbon and nitrogen and a higher ash content as compared to melanin isolated from the vegetative mycelium. The pigment complex of the spores is characterized also by differences in properties of the first isolated fraction. The pigment complex from the cultural broth differs from the complex of the mycelium and spores by properties of the studied fractions.

Actinomyces