MELANIN AND URATE ACT TO PREVENT ULTRAVIOLET DAMAGE IN THE INTEGUMENT OF THE SILKWORM,BOMBYX mori

The phenomenon that epidermal cells under the white stripes rather than black stripes contain many uric acid granules was found in larvae of several Lepidopteran species. However, the biological mechanism of this phenomenon is still unknown. In the present study, we take advantage of several silkworm (Bombyx mori) body color mutant strains to investigate the deposition patterns and biological mechanism of urate and melanin in the integuments of these mutant larvae. By imaging with transmission electron microscope, we found that there were some melanin granules in the larval cuticle in black body color mutant plain Black (p^B), but not in background strain plain (p) with white larval body color. In contrast, the larval epidermal cell of background strain had much more urate granules than that of black one. Furthermore, the uric acid content under the black stripes was significantly lower than that under the white stripes in a single individual of mottled stripe (p^S) with black and white stripes in each segment. Ultraviolet A (UVA) exposure experiments showed that the distinct oily (od) mutant individuals with translucent larval integument were more sensitive to the UVA damage than black body color mutant and background strain without any pigmentation in the larval cuticle. This is likely due to the absence of melanin granules and few urate granules in the integument of od mutant. Thus, both the deposited melanin granules in the cuticle and the abundant urate granules in the epidermis cells constitute effective barriers for the silkworm to resist UVA‐induced damage.     

Loss of melanin by eye retinal pigment epithelium cells is associated with its oxidative destruction in melanolipofuscin granules

The effect of superoxide radicals on melanin destruction and degradation of melanosomes isolated from cells of retinal pigment epithelium (RPE) of the human eye was studied. We found that potassium superoxide causes destruction of melanin in melanosomes of human and bovine RPE, as well as destruction of melanin from the ink bag of squid, with the formation of fluorescent decay products having an emission maximum at 520-525 nm. The initial kinetics of the accumulation of the fluorescent decay products is linear. Superoxide radicals lead simultaneously to a decrease in the number of melanosomes and to a decrease in concentration of paramagnetic centers in them. Complete degradation of melanosomes leads to the formation of a transparent solution containing dissolved proteins and melanin degradation products that do not exhibit paramagnetic properties. To completely degrade one melanosome of human RPE, 650 ± 100 fmol of superoxide are sufficient. The concentration of paramagnetic centers in a melanolipofuscin granule of human RPE is on average 32.5 ± 10.4% (p < 0.05, 150 eyes) lower than in a melanosome, which indicates melanin undergoing a destruction process in these granules. RPE cells also contain intermediate granules that have an EPR signal with a lower intensity than that of melanolipofuscin granules, but higher than that of lipofuscin granules. This signal is due to the presence of residual melanin in these granules. Irradiation of a mixture of melanosomes with lipofuscin granules with blue light (450 nm), in contrast to irradiation of only melanosomes, results in the appearance of fluorescent melanin degradation products. We suggest that one of the main mechanisms of age-related decrease in melanin concentration in human RPE cells is its destruction in melanolipofuscin granules under the action of superoxide radicals formed during photoinduced oxygen reduction by lipofuscin fluorophores.     

The formation of glandular secretion in larval Austramphilina elongata (Amphilinidea)

The ultrastructure of three types of gland cells of embryos and free-swimming larvae of Austramphilina elongata is described. Type I gland cells contain large, more or less round electron-dense granules which are formed by numerous Golgi complexes. Type II gland cells contain thread-like, membrane-bound secretory granules with longitudinally arranged microtubules inside the granules; secretory droplets are produced by Golgi complexes and the microtubules apparently condense in the cytoplasm or in the droplets. Type III gland cells contain irregular-ovoid membrane-bound granules with coiled up microtubules which have an electron-dense core; the granules are formed by secretionderived from Golgi complexes and the microtubules aggregate around and migrate into the secretion; microtubules are at first hollow and the early secretory granules have a central electron-dense region.     

Ultrastructure of salivary glands of Ornithodoros (Ornithodoros) moubata (Ixodoidea: Argasidae)

The paired salivary glands of unfed adult Ornithodoros (Ornithodoros) moubata are composed of type I (agranular) and type II (granular) alveoli. Type I alveoli consis of one large central cell surrounded by peripheral cells having the morphology of fluid-transporting epithelia. Type II alveoli contain granular and agranular cells; the former are comprised of morphologically distinct types of cells (a, b, and c) containing granules of different structures and chemical composition with respect to polysaccharide and protein. The agranular cells are the interstitial and cap cells. Golgi bodies and rough endoplasmic reticulum (RER) are found in all granular cells and apparently are involved in granule formation. No appreciable structural changes were observed in type I alveoli during or after feeding. Type c cell granules are released before granules from types a and b cells and may contain anticoagulant substances that promote the blood flow of the host during the tick feeding. Although the cap cells are not structurally affected by feeding, interstitial cells are developed into transporting epithelia.     

A Neoplastic Connective Tissue Mast Cell Capable of Continuous Growth In Tissue Culture

A neoplastic connective tissue mast cell from a dog mast cell sarcoma has been grown in tissue culture for 50 passages over a period of 2 years. The cells were grown as monolayer cultures in glass bottles, using Eagle's basal medium fortified with calf serum. The cultures were contaminated with an Alkaligenes sp. for 10 months but finally were sterilized bacteriologically by treatment with specific antiserum combined with antibiotics. The cells grow in a fibroblastic pattern, and contain mitochondria, mast cell granules, and lipid granules or droplets. The mast cell granules stain basophilic with Giemsa's stain and metachromatically with azure A or toluidine blue. They also stain with Sudan black B and with periodic acid-Schiff stain. The interphase nuclei are vesicular, contain from 1 to 20 nucleoli, and frequently show bizarre outlines. Multinucleate cells are often seen, as are mitotic figures. Extracellular fibrous material occurs in all cultures and apparently originates from the cell surface. This material does not have the structure of connective tissue fibers and has not been identified. The cells develop an increased number of metachromatic granules when grown in medium containing heparin and an increased number of sudanophilic granules when grown in medium containing stearic acid. Only small amounts of histamine were present in the tumor from which this cell line was derived and in the cells grown in tissue culture.     

Identification of two types of melanocyte within the stria vascularis of the mouse inner ear

We have distinguished two types of melanocyte within the intermediate layer of the stria vascularis in the cochlea of normally pigmented mice: light and dark intermediate cells. The light intermediate cells are present in the stria from birth and have the typical appearance of a melanocyte. They are large and dendritic with electron-lucent cytoplasm containing numerous vesicles that show tyrosinase activity, and pigment granules in various stages of development. These granules have the ultrastructural and histochemical characteristics of premelanosomes and melanosomes. The light intermediate cells persist throughout life, but less frequently contain pigment in older animals. The dark intermediate cells, present only in adult mice, vary considerably in number and distribution between animals. Pigment granules, bound within an electron-dense acid phosphatase-rich matrix, form the main component of the dark intermediate cells. The intermediate cells may comprise either two distinct cell populations or different developmental stages of the same cell type; ultrastructural observations suggest the latter. In young mice, light intermediate cells contain the electron-dense matrices, which at later stages of development are found almost exclusively in dark cells. The dark intermediate cells contain few cell organelles other than pigment granules accumulated within lysosomal bodies and they often have pycnotic nuclei. These observations suggest that the dark intermediate cells are a degenerate form of the light intermediate cells. Clusters of melanosomes also occur in the basal cells, and to a much lesser extent in the marginal cells. These cells do not stain after incubation in DOPA, suggesting that they are not capable of melanin synthesis, and therefore probably acquire melanin by donation from adjacent melanocytes. Pigment clusters are also found within the spiral ligament at all stages of development.     

Biogenesis of Melanosomes in Kupffer Cells of Proteus anguinus (Urodela,Amphibia)

The ultrastructural characteristics of melanosomes and premelanosomes observed during the biogenesis of melanosomes in liver pigment cells of the neotenic cave salamander Proteus anguinus (Proteidae) are described. It is well known that amphibian liver pigment cells, also known as Kupffer cells (KC), contain melanosomes and are able to synthesize melanin. Liver pigment cells of P. anguinus contain numerous siderosomes and melanosomes. The melanosomes are grouped together within single‐membrane‐bounded bodies, named as ‘clusters of melanosomes’ or ‘melanosomogenesis centers’. Inside such clusters, different structures are present: (1) filament‐like structures, characteristic of the initial stage of melanosome biogenesis, (2) medium electron‐dense melanosomes in different stages of melanization, (3) melanosomes with an electron‐dense cortical area and a less electron‐dense medullar area, and (4) uniformly highly electron‐dense mature melanosomes or melanin granules. Histochemical and cytochemical dihydroxyphenylalanine (DOPA) oxidase reactions in pigment cells were positive. Our results confirm the ability of amphibian KC to synthesize melanin and contribute to this little known subject.     

Retention of the cyanobacterial neurotoxin β‐N‐methylamino‐l‐alanine in melanin and neuromelanin‐containing cells – a possible link between Parkinson‐dementia complex and pigmentary retinopathy

β‐N‐methylamino‐l ‐alanine (BMAA), a neurotoxic amino acid produced by cyanobacteria, has been suggested to be involved in the etiology of a neurodegenerative disease complex which includes Parkinson‐dementia complex (PDC). In PDC, neuromelanin‐containing neurons in substantia nigra are degenerated. Many PDC patients also have an uncommon pigmentary retinopathy. The aim of this study was to investigate the distribution of ³H‐BMAA in mice and frogs, with emphasis on pigment‐containing tissues. Using autoradiography, a distinct retention of ³H‐BMAA was observed in melanin‐containing tissues such as the eye and neuromelanin‐containing neurons in frog brain. Analysis of the binding of ³H‐BMAA to Sepia melanin in vitro demonstrated two apparent binding sites. In vitro‐studies with synthetic melanin revealed a stronger interaction of ³H‐BMAA with melanin during synthesis than the binding to preformed melanin. Long‐term exposure to BMAA may lead to bioaccumulation in melanin‐ and neuromelanin‐containing cells causing high intracellular levels, and potentially changed melanin characteristics via incorporation of BMAA into the melanin polymer. Interaction of BMAA with melanin may be a possible link between PDC and pigmentary retinopathy.     

Alteration of Racial Differences in Melanosome Distribution in Human Epidermis after Exposure to Ultraviolet Light

CAREFUL studies of human epidermis have revealed that there is no significant difference between the number of melanocytes in the various racial groups, although there are regional differences in the population density of DOPA-positive melanocytes in various areas of the body (for example, on the forehead, 2,310 mm^?2; abdomen, 800 mm^?2; and back, 1,100 mm^?2)¹. These differences in the number of melanocytes may explain the colour differences in various areas of the body, but the colour of skin in the various races is apparently due to variations in the number of melanin granules, or melanosomes, in the melanocytes and in the epidermal keratinocytes, as well as to the degree of melanization of the individual melanosomes.     

Intergenic complementation in chick melanocyte heterokaryons

Melanocytes from chick embryos of the pinkeye (pk/pk) and recessive white (c/c) genotypes do not produce melanin in cell culture. However, aberrant melanogenic organelles are evident when these cells are examined with the electron microscope. Melanocytes of each genotype, previously grown for 5 days in cell culture, were co-cultured for 24 h and then fused with inactivated Sendai virus. Twenty-four hours after fusion faintly pigmented cells could be seen in the culture dishes. These cells were invariably multinucleated. At 48 h post-fusion many darkly pigmented, multinucleated cells could be seen. Pigment-producing cells were found in four separate experiments and occurred at a frequency of approx. 1 per 40 000 cells treated. Co-culturing of the melanocytes without virus treatment failed to elicit pigment production. When one genotype was labeled with [³H]thymidine prior to fusion, autoradiograms showed that the pigmented cells contained at least one labeled and one unlabeled nucleus. Electron micrographs of the pigmented cells confirmed that cell fusion was complete and showed normal pigment granules with welldefined matrices and deposited melanin. The results show that recessive white and pinkeye can complement as heterokaryons. This indicates that each mutation affects a different melanogenic function and that the expression of the normal function of each does not require nuclear integration. The simplest hypothesis is that the two mutations affect structural genes and that the complementing cytoplasms contain functional gene products. The hypothesis that one or both mutants have altered control functions cannot be ruled out, however.     

Dopamine melanin‐loaded PC12 cells: a model for studies on pigmented neurons

The most conspicuous feature in idiopathic parkinsonism is the degeneration of pigmented neurons in the substantia nigra. A major problem for the study of the significance of neuromelanin for the development of parkinsonism is that common experimental animals lack neuromelanin in substantia nigra. The aim of this study was to develop an in vitro model that could be used to study the role of neuromelanin in chemically induced toxicity in dopaminergic cells. Cultured neuron‐like PC12 cells were exposed to synthetic dopamine melanin (0–1.0 mg/ml) for 48 h, resulting in uptake of dopamine melanin particles into the cells. The intracellular distribution of dopamine melanin granules was similar to that found in neuromelanin‐containing neurons. Dopamine melanin, up to 0.5 mg/ml, had negligible effects on ultrastructure, induction of the endoplasmic reticulum‐stress protein glucose regulating protein 78, activation of caspase‐3 and cell viability. The decreased cell viability in response to the cytotoxic peptide amyloid‐β_25?35 was similar in melanin‐loaded cells and in control cells without melanin. The results of the studies suggest that melanin‐loaded PC12 cells can serve as an in vitro model for studies on the role of neuromelanin for the toxicity of chemicals, in particular neurotoxicants with melanin affinity, in pigmented neurons.     

Fine Structure of Melanogenesis in the Ink Sac of Sepia offidnalis

The ink sac epithelium of the cuttlefish Sepia officinalis was investigated by electron microscopy. Melanogenesis in a simplified view seems to follow the general scheme of melanin formation in vertebrates. First, a membrane-bound protein matrix is formed, which is called an early stage melanosome. The early stage melanosomes are more or less irregular in shape with a size up to 1.5 μm and contain membranous, granular, or vesicular material. They seem to originate from Golgi bodies and/or endoplasmic reticulum. Membranes that frequently are present in the early stage melanosomes may originate from fusion of vesicles or from incorporation of Golgi membranes into early stage melanosomes. Free cytoplasmic material or mitochondria probably are also incorporated into the early stage melanosomes or melanosomes. Therefore, the origin of the early stage melanosomes seems to be similar to that of autophagosomes. The early stage melanosomes mature to melanosomes in which several dozen melanin granules are formed. These melanosomes, at last, release the melanin granules together with other cellular material, including early stage melanosomes, into the lumen of the ink gland. This finding confirms the earlier postulated holocrine character of the release. Active tyrosinase was localized in the lumen of the ink sac as already shown by biochemical methods. There was also additional evidence that most of the material of broken down cells inside the lumen of the ink sac seems to be converted into melanin granules.     

Ultrastructure of the adenohypophysis in the larval anadromous sea lamprey,Petromyzon marinus L.

The ultrastructure of the adenohypophysis (AH) in the larval anadromous sea lamprey, Petromyzon marinus L., was examined. The AH is subdivided into three regions, the pro-, meso-, and meta-AH. Cells of the nasopharyngeal stalk extend directly beneath the pro- and meso-AH to form the ventral surface of the gland. Some cells in the pro- and meso-AH are arranged into small follicles. Each region of the AH is characterized by a single granulated (secretory) cell type. Granulated cells constitute 80–90% of the pro-AH and contain secretory granules that range from 800 to 2400 Å in diameter. Only 10–20% of the cells in the meso-AH are granulated and they contain much smaller secretory granules (400 to 1250 Å diameter) than those in the pro-AH. Granulated cells constitute 80–90% of the meta-AH and contain only a few secretory granules, ranging from 1000 to 2500 Å in diameter, and many vesicles containing either a loose flocculent or dense granular material. Nongranulated (stellate) cells are found in all regions. They are characterized by their long cell processes, abundant cytoplasmic filaments, and variable electron density. The appearance of organelles in these cells suggests they are nonsecretory. They may play a role in maintaining the structural integrity of the gland and the regulation of granule release in the pro-AH. Two types of nongranulated cells make up 80–90% of the meso-AH. Type I are stellate cells, type II may be undifferentiated cells. The functional significance of the secretory cells in the larval AH is discussed.     

ELECTRON MICROSCOPE STUDIES OF EPIDERMAL MELANOCYTES, AND THE FINE STRUCTURE OF MELANIN GRANULES

Melanocytes and melanin granules have been studied by electron microscopy in normal human and cat skin, and in hyperplastic human skin lesions. The melanocytes have always been found as free cells within the epidermis,i.e., on the epidermal side of the dermal membrane. Melanocytes frequently rest on the dermal membrane or bulge towards the dermis. In such cases the uninterrupted dermal membrane is uniformly thin and smooth in appearance, in contrast with the regions alongside Malpighian cells, where it appears appreciably thicker and seemingly anchored to the basal cell layer. Two types of melanin granules have been distinguished according to their location in the melanocytes and to morphological characteristics which may only express different stages in the maturation of the granules: (a) light melanin granules in which a structure resembling a fine network is apparent; (b) dense melanin granules which, in osmium-fixed preparations, appear as uniformly dense masses surrounded by a coarsely granular, intensely osmiophilic shell. Treatment of sections of osmium-fixed tissues with potassium permanganate has revealed within the dense granules the existence of an organized framework in the form of a regular, crystalline-like lattice. It is suggested that this basic structure is protein in nature and may include the enzymatic system capable of producing melanin. The existence is reported of fine filaments located in the cytoplasm of melanocytes and morphologically distinct from the tonofilaments found in Malpighian cells.     

Cell Division: Pressure-induced reversal of the antimeiotic effects of heavy water in the oocytes of the starfish,Asterias forbesi

In dermal melanocytes of Rana pipiens, colchicine is known to produce a gradual, dosage-dependent dispersion of melanin granules, irreversible over several hours. This effect is potentiated by a number of chemical agents that normally produce a reversible dispersion of granules. In the present study we examined the effect of high hydrostatic pressure on changes induced in melanocytes by colchicine. In Ringer's solution, samples of skin from a single frog were incubated for 30 minutes at room temperature with or without colchicine, 9 × 10^?5 M. Then two samples, one of which had been pretreated with colchicine, were successively subjected to 12,000 psi for one hour at 25 to 26°C. The degree of dispersion of melanin granules in melanocytes was observed before, during and after the period of pressure. In frog skin pretreated with colchicine, the usually gradual, irreversible dispersion of melanin granules in melanocytes was potentiated. Since high pressure is known to produce solational changes in protoplasm, such changes may accompany dispersion of melanin granules in melanocytes. If this be so, then sol-gel equilibria may be important in the action of dispersing and aggregating agents, many of which are hormones and other physiologically active agents. Finally, the present study supports the hypothesis that colchicine shifts protoplasmic sol-gel equilibria toward a less gelated condition.     

PLASTID DEVELOPMENT IN IOJAP- AND CHLOROPLAST MUTATOR-AFFECTED MAIZE PLANTS

Plastids affected by either iojap or chloroplast mutator fail to green, and altered plastids are maternally transmitted to subsequent generations. The ultrastructure of iojap-affected plastids indicates that these plastids contain no ribosomes and are capable of supporting little internal membrane organization in either light or dark-grown plants. Chloroplast mutator-affected plastids of light-grown plants contain some organized internal membrane structures. In dark-grown plants, chloroplast mutator-aftected plastids contain a crystalline prolamellar body, numerous vesicles, and osmiophilic granules. The chloroplast mutator-affecled etioplasts display an abnormal distribution of lamellar membranes; these membranes, rather than radiating in a spokelike pattern from the prolamellar body, are condensed into a portion of the organelle. Light causes disruption of the prolamellar body in chloroplast mutator-affected plastids without promoting the organization of a normal thylakoid membrane system. The effects of iojap and chloroplast mutator are cell autonomous and apparently influence the individual plastid, as evidenced by the persistence of heteroplastidic cells containing normal and affected plastids.     

Ion‐Exchange and Adsorption of Fe(III) by Sepia Melanin

Sepia eumelanin is associated with many metal ions, yet little is known about its metal binding capacity and the chemical nature of the binding site(s). Herein, the natural concentrations of metal ions are presented and the ability to remove metals by exposure of the melanin granules to EDTA is quantified. The results reveal that the binding constants of melanin at pH 5.8 for Mg(II), Ca(II), Sr(II) and Cu(II) are, respectively, 5, 4, 14 and 34 times greater than the corresponding binding constants of these ions with EDTA. By exposing Sepia eumelanin to aqueous solutions of FeCl₃, the content of bound Fe(III) can be increased from a natural concentration of ～180 ppm to a saturation limit of ～80 000 ppm or 1.43 mmol/g of melanin. Similar saturation limits are found for Mg(II) and Ca(II). Exposure of Sepia melanin granules to aqueous solutions containing Ca(II) results in the stoichiometric replacement of the initially bound Mg(II), arguing that these two ions occupy the same binding site(s) in the pigment. The pH‐dependent binding of Mg(II) and Ca(II) suggests coordination of these ions to carboxylic acid groups in the pigment. Mg(II) and Ca(II) can be added to a Fe(III)‐saturated melanin sample without affecting the amount of Fe(III) pre‐adsorbed, clearly establishing Fe(III) and Mg(II)/Ca(II) occupy different binding sites. Taking recent Raman spectroscopic data into account, the binding of Fe(III) is concluded to involve coordination to o‐dihydroxyl groups. The effects of metal ion content on the surface morphology were analyzed. No significant changes were found over the full range of Fe(III) concentration studied, which is supported by the Brunauer–Emmett–Teller surface area analysis. These observations imply the existence of channels within the melanin granules that can serve to transport metal ions.     

Fine structure of melanocytes and macrophages in the Harderian gland of the mouse

The presence of dendritic cells containing melanin granules has been demonstrated employing silver impregnation and electron microscopy in the interstitial tissue of the Harderian gland of the mouse. Two types of melanocytes, either with or without the various developmental stages of melanin granules, were found in the gland. Cells with developing granules were more dendritic and contained a large number of cytoplasmic organelles. The other cells were ellipsoidal or slender in shape and contained few cytoplasmic organelles and a large number of fully melanized granules, but no developing granules. In general, the granules of the Harderian gland melanocytes resembled granules from other organs (particularly the skin of the eyelids). The general size range of the granules was 0.2-0.9 micron. Each granule was enclosed by a membrane. The Harderian gland macrophages contained fully pigmented melanin granules of various sizes. The granules were enclosed by a membrane either singly or in groups. Some of the melanin granules within the phagosomes showed signs of degradation, revealing the underlying matrix.     

Failure of Cytochalasin or Colchicine to inhibit Secretion of Immunoglobulins

SECRETIONS are often packaged in granules which are held within the cells of origin until some specific stimulus brings about release by exocytosis. Granules containing catecholamines are liberated from adrenal medullary cells by acetylcholine; granules containing insulin are released from pancreatic β-cells by high concentrations of glucose; and granules containing histamine, serotonin and slow-reacting substance are discharged from mast cells in the presence of cell-bound antibody and antigen. The release of secretory granules requires calcium ions in the extracellular medium¹ and may follow the entry into the cytoplasm of calcium ions which trigger contraction of an actomyosin-like microfilament system². This interpretation is supported by our recent observation² that induced release from mast cells of granules containing mediators of acute hypersensitivity is strongly inhibited by cytochalasins, a group of fungal products that selectively block the activity of microfilament-related contractile systems in many cells^3,4. Stimulated release of ¹³¹I from previously labelled mouse thyroids and endocytosis of colloid, are also inhibited by cytochalasin⁵. Cytochalasin inhibits cell movement, movement of ruffled membranes, pinocytosis and phagocytosis in macrophages and polymorphonuclear leucocytes^4,6. Release of ¹³¹ I from previously ¹³¹I-labelled mouse thyroid⁷ is also inhibited by colchicine and other agents that disperse labile cytoplasmic microtubules. Thus it seems that a contractile microfilament-related system, acting together with microtubules, brings about the controlled release, when required, of certain secretions.     

THE INDUCTION OF MELANIZATION IN GOLDFISH SCALES WITH ACTH, IN VITRO : Cellular and Subcellular Changes

The induction of melanization in xanthic goldfish scales with ACTH in vitro has been studied by light and electron microscopy utilizing ammoniated silver nitrate staining of premelanin and melanin. The melanized cells (melanophores and melanocytes) and the yellow pigmented cells (lipophores and the newly described lipocytes) were found to possess many similarities at the levels of cellular and subcellular structure. The latter cells contain characteristic cytoplasmic bodies which react positively to the premelanin stain. Changes accompanying ACTH stimulation of goldfish scales in tissue culture suggest that these bodies in the lipocytes and lipophores can become melanized. Electron micrographs illustrate the intermediate staining of newly formed melanin granules in an induced melanocyte and the appearance of a transitional melanolipophore. It is postulated that ACTH can promote the association of the enzyme tyrosinase with the preformed structure of unmelanized granules.