Melanin content and hydroperoxide metabolism in human melanoma cells

Human melanoma cells were grown to exponential and stationary phases showing melanin contents of 4.2 +/- 0.3 and 11.3 +/- 0.6 micrograms/10(6) cells, respectively. The cells were separated in four subpopulations by a Percoll gradient; the subpopulation of density 1.07 (g/ml) was the most enriched in pigmented cells and produced 28 and 58% of the cells in exponential and stationary phases, respectively. Melanoma cells had similar superoxide dismutase and glutathione peroxidase activities in exponential and stationary phases. Moreover melanoma cells exhibited a higher catalase activity in the stationary phase: whole homogenate and cytosol activities were 7.0 +/- 0.3 and 10.8 +/- 0.6 U/mg protein, whereas in exponential phase the activities were 4.9 +/- 0.1 and 7.6 +/- 0.3 U/mg protein for whole homogenate and cytosol, respectively. The intracellular H2O2 steady-state concentration was 3.3 +/- 0.2 and 2.1 +/- 0.2 microM H2O2 for exponential and stationary phases, respectively. The spontaneous chemiluminescence of the two culture phases was 169 +/- 27 cps/10(6) cells (exponential) and 78 +/- 24 cps/10(6) cells (stationary). The cytotoxicity of H2O2 generated extracellularly by glucose oxidase was determined after 60 min of exposure. IC50 values for exponential and stationary cell cultures were 0.9 and 2.4 mU/ml of glucose oxidase, respectively. The increased catalase activities in the stationary phase as compared with the exponential phase are consistent with the decreased intracellular H2O2, with the decreased spontaneous chemiluminescence, and with the increased resistance to exogenous H2O2.     

The satellite cells of normal anuran skeletal muscle

Population counts and size measurements of satellite cell nuclei and myonuclei were carried out on the normal gastrocnemius muscles of adult Rana pipiens and Rana clamitans. Satellite cell profiles occurred with an observed frequency of about 1.3% in the muscles of the R. pipiens, and with an observed frequency of about 1.6% in the muscles of the R. clamitans. These frequencies were not found to be significantly different. The observed frequencies were corrected for the sampling bias introduced by the difference in the mean size of the satellite cell nuclei and myonuclei. This correction suggests that R. pipiens and R. clamitans both have a true satellite cell frequency of approx 2.7%. Analysis of these data indicates that the satellite cells of the normal anuran gastrocnemius occur in sufficient numbers to account for the regeneration seen after injury to this muscle.     

Best practices for storing and shipping cryopreserved cells

Successfully recovering stored cryopreserved cells requires attention to two major factors, i.e., storage temperature and proper handling of the frozen samples. These key considerations are in turn impacted by how the inventory system is constructed, the frequency and process for retrieving specimens, as well as how the material is secured. In addition, shipping biological materials requires attention to the type of material transported, adherence to regulatory requirements, packaging materials and proper assembly, labeling, and engaging reputable carriers. This paper provides an overview discussion of the current best practices for storing, handling, and shipping of cryopreserved cells and provides references to more detailed information on specific topics.     

Ultrastructural identification of cells storing pancreatic-type glucagon in dog stomach

Summary The oxyntic mucosa of the dog stomach is rich in cells storing pancreatic-type glucagon. The cells were identified by immunocytochemistry and found to be indistinguishable from pancreatic A cells in the electron microscope. Ultrastructural identification of the immunoreactive cells was accomplished by the use of consecutive semithin-ultrathin sections, a technique that permitted the use of optimally fixed material.     

Melanin and dopa-positive cells in the skin of tropical cattle.

Various histochemical and histological techniques were used to study the melanin and dopa-positive cell distribution in the skin of some tropical and temperate breeds of cattle in Nigeria. Melanin pigments were concentrated in the basal and lower spinous layers of the epidermis and in the hair cortex, follicle sheaths and papillae of the various breeds. In the White Fulani and N'Dama breeds, melanin pigments were however found in all layers of the epidermis. Dopa-positive cells (melanocytes) were observed in the epidermis, dermis and hair follicles; the distribution pattern varied among breeds, being copiously disposed in the basal epidermis and papillary dermis in the White Fulani and Muturu and, except in areas of thick epidermal ridges, scanty in the epidermis and dermis of the Friesian and N'Dama. Mast cell distribution pattern in the various breeds was similar to that of the dopa-positive cells. Peroxidase-positive cells were present in the basal epidermis and upper dermis of the Muturu, widespread in the subepidermal layer of the N'Dama and very scanty in the dermis of the White Fulani and Friesian. Acid phosphatase activity was intense in the granular layer of the Muturu and N'Dama breeds and also in the papillary dermis and hair follicles, whereas alkaline phosphatase-positive dendritic cells, and 'clear' cells were also observed in the basal and upper epidermis.     

Stellate cells as phagocytes of the anuran pars distalis

When the stellate cells (nongranulated cells) from dissociated-cell preparations of the anuran pars distalis were examined, they were seen to contain debris within phagocytic vacuoles (phagosomes). These phagosomes were variable; some contained granules from secretory cells while others were similar to lipid-like bodies and myelin figures. In situ partes distales from frogs were examined at the breeding season. The tissues were divided into lobules that were bounded by processes of stellate cells located between the secretory cells. Processes of stellate cells in the interior of a lobule interdigitate with processes extending inward from the stellate cells forming the border of the lobule. When these processes come together, a small cavity is formed. In many of the intact frogs the spaces between the stellate and secretory cells were greatly enlarged. At this particular time the processes of the stellate cells were attenuated and enclosed secretory granules that were also present as debris in these dilated, intercellular spaces. Within the cytoplasm of these stellate cells were not only phagosomes containing secretory granules but also organelles that appeared to be lipid bodies and lysosomes. Thus, the stellate cells of the pars distalis function in vivo, as well as in vitro, as phagocytes. In addition, macrophage-like cells moving from the blood may form another component of this system of phagocytes.     

Ultrastructure of neuroendocrine cells in the lungs of three anuran species

Neuroendocrine cells in the lungs of three species of anurans, Bombina variegata, Bufo bufo and Bu. viridis, occur both as single cells and in the form of neuroepithelial bodies. Neuroendocrine cells are covered by ciliated cells or pneumocytes, which separate them from the lumen of the lung. Neuroepithelial bodies are dispersed in the apical part of the main septa of lungs of Bo. variegata and Bu. bufo or are situated on special protrusions of septa in the lungs of Bu. viridis. Neuroepithelial bodies are innervated by intraepithelial nerve endings of afferent and efferent types.     

Extra-Cutaneous Melanin

Extra-cutaneous melanocytes derive from either the neural crest, the outer wall of the optic cup, or the cranial neural tube. Those of neural crest origin reach most bodily regions, and may give rise to primary melanoma in various tissues. The Kupffer cell produces a form of melanin, but is hardly a melanocyte. Melanocytes of the internal ear may be concerned with secretion of endolymph, trans-epithelial ion transport, and with protection against ototoxic drugs and high-intensity noise damage. There is evidence from albino animals that retinal pigment epithelium determines co-ordinates of the neural retina, and its decussation pattern during development. Neuromelanin derives from Dopamine, and is found in dopaminergic neurons widely distributed throughout the brain-stem and hypothalamus, and which project to the striatum and limbic system. Parkinsonism is due to degeneration of melanin-containing dopaminergic neurons of locus coeruleus and substantia nigra, and MPTP provides an investigative probe for studying the causes of Parkinsonism. Neuromelanin should not be regarded as a waste-product, but as something which can affect the firing properties of neurons with specific functional effect.     

Dendritic cells in oral tolerance in the gut

Oral tolerance is a process that allows generation of systemic unresponsiveness to food antigens. Hence if the same antigen is introduced systemically even under immunogenic conditions it does not induce immune responsiveness. Dendritic cells (DCs) have been identified as essential players in this process. DCs in the gut are located in a strategic position as they can interact directly with luminal antigens or indirectly after their transcytosis across epithelial cells. DCs can then migrate to associated lymphoid tissues to induce tolerance. Antigen presenting cells in the gut are specialized in function and have divided their labour so that there are cells capable to migrate to the draining mesenteric lymph node for induction of T regulatory cells, while other subsets are resident and are required to enforce tolerance locally in the gut after food antigen exposure. In this review, I shall summarize the characteristics of antigen presenting cells in the gut and their involvement in oral tolerance induction. In addition, I will also emphasize that tolerance to food allergens may be contributed by plasmacytoid DCs in the liver that participate to the elimination or anergy of allergen-specific CD8 T cells. Hence specialized functions are associated to different subsets of antigen presenting cells and different organs.     

Osmoregulatory-like mitochondria-rich cells in the developing pancreatic ducts of young anuran tadpoles

Pancreatic ducts of young posthatching Rana temporaria tadpoles are the main component of the developing pancreas. At this stage (free-swimming tadpoles with internal gills), duct cells display a high degree of development of basal and lateral outfoldings of the cell membrane with extensive interdigitation, and numerous mitochondria are present throughout the cytoplasm. Wide intercellular spaces also exist, sometimes forming canaliculi-like structures. Since these traits are characteristic of cells engaged in osmotic regulation, we suggest the possibility that this temporary duct system participates in such control. Duct cells in tadpoles with well-developed hindlegs have diminished interdigitation, and mitochondria are localized apically. © 1993 Wiley-Liss, Inc.     

Melanin inhibits cytotoxic effects of doxorubicin and daunorubicin in MOLT 4 cells

In the present study we have developed a simple method to elucidate the melanin binding ability of different chemotherapeutic agents. The anthracyclines, doxorubicin and daunorubicin, or the alkylating agent cisplatin were preincubated with melanin (Sepia). Melanin and free drug was then separated through centrifugation and the cytotoxic effects of corresponding drug were evaluated in a MTT (3-(4,5-dimetyltiazol-2-yl)-2,5-difenyl-tetrazoliumbromide) assay using MOLT-4 cells. Our results show that melanin pretreatment shifted the IC50 value for doxorubicin from 0.06 to 0.97 microM and for daunorubicin from 0.04 to 0.80 microM. In contrast, the IC50 values of cisplatin was not influenced by melanin pre-treatment indicating that cisplatin does not bind to melanin. By comparing equi-active concentrations from concentration-response curves with or without melanin pretreatment an approximate binding capacity of melanin could be estimated. Our results show that melanin binds about 900 nmol/mg doxorubicin and 760 nmol/mg daunorubicin. Chloroquine, which is known to bind to melanin with high affinity, was found to inhibit melanin binding of both daunorubicin and doxorubicin, thereby leading to an increased sensitivity of the anthracyclines. The clinical implications of melanin binding regarding unwanted accumulation of anthracyclines in the skin as well as chemoprotective effects against chemotherapy are discussed.     

Autolysis and heterolysis of the epidermal cells in anuran tadpole tail regression

Autolysis and heterolysis of the degenerating epidermis of the tail fin of Rana japonica tadpoles during spontaneous metamorphosis were observed by transmission and scanning electron microscopy. In the early climactic stages of metamorphosis (st. 19–20), the outermost epidermal cells developed vacuoles that were acid phosphatase positive and showed apparent breakdown of the cell membrane. The cells shrunk, perhaps due to the rupture of the cell membrane, and sloughed off without typical cornification. As tail resorption proceeded, autolysis of the epidermal cells spread towards the inner layers, in which some epidermal cells lost desmosomal junctions. They also displayed atrophic figures with condensed cytoplasm, breakdown of the cell membrane, and pycnotic nuclei. Lymphocytes, neutrophils and macrophages were already present in the basal layers of the premetamorphic epidermis (st. 10). Based on ultrastructural observation, blood cells could be distinguished from autolysing epidermal cells. Only a few blood cells were found in the early climactic stages of metamorphosis (st. 19–20), but the number of the blood cells, especially macrophages, greatly increased during the final stages of metamorphosis (st. 23–24). During the final stages, many macrophages were observed to phagocytose the autolysing epidermal cells by projecting slender pseudopodia into the inner epidermis. Macrophages also were observed to pass through the degraded basal lamella. These results suggest that not only autophagy but also heterophagy of the epidermal cells by the macrophages is a major process in the regression of the tail fin epidermis.     

Feeding mechanisms in anuran larvae

Larvae of the neotropical frogs Phyllomedusa are distinctive in that they feed normally in mid-water on phytoplankton, maintaining neutral buoyancy by means of an independently beating tail filament. The feeding mechanism of larval Phyllomedusa trinitatis was studied morphologically and experimentally. The primary feeding mechanism involves a buccal rasp which may in some circumstances render food into small particles, a pumping mechanism which forces water through the buccal cavity and the gill filters, an entanglement system which traps particles in mucous strings produced in special organs, and the formation of mucous cords which transport particles to the oesophagus. In mid-water feeding and surface feeding the buccal rasp serves only its other function in preventing backflow of the respiratory stream. The primary feeding mechanism is discussed and compared with schemes proposed for Rana temporaria and R. agilis. Little agreement exists between these schemes and that which is here proposed. It is concluded that the primary feeding mechanism is the same in the three forms but that there are behavioural differences in feeding generally. Some comment is made on the primary feeding mechanism in the Microhylidae.     

Neuromast topography in anuran amphibians

Generalized anuran tadpoles across families exhibit a similar neuromast morphology on their heads, as follows: (1) all neuromast lines known for anurans are present; (2) within these lines total neuromast number ranges from about 250 to 320; (3) neuromasts form linear stitches composed of two to three, but sometimes up to five, neuromasts; (4) neuromast linear dimensions are ? 10 μm; and (5) neuromasts contain ? 15 hair cells. Compared with generalized forms, stream, arboreal, carnivorous, and desert-pond forms have fewer neuromasts but they contain more hair cells. They do not, however, form stitches. Obligate midwater suspension-feeding forms, including Xenopus (Pipidae), Rhinophrynus (Rhinophyrnidae), and Phrynomerus (Microhylidae), form stitches that contain > six, but potentially up to 18 or more, loosely aggregated neuromasts. Xenopus and Rhinophrynus have large neuromasts (up to 40 μm across). Chiasmocleis (Microhylidae) tadpoles form stiches that are linearly arranged with up to ten neuromasts. Whereas urodeles can have more than one neuromast row per line and may form both linear and transverse stitches, anurans have only one row of neuromasts per line and form only transverse stitches. Neuromasts in anurans tend to be smaller and more circular than in urodeles and positioned flush with the epidermal surface. A greater percentage of anurans form stitches, and anurans have greater intrafamilial variation in stitch formation than do urodeles.     

Melanin Biosynthesis in Cryptococcus neoformans

Pigment production by Cryptococcus neoformans is virulence associated. Dopamine- and 3,4-dihydroxyphenylalanine–melanin products were identified after acidic permanganate oxidation, alkaline hydrogen peroxide oxidation, or hydrolysis with hydriodic acid. These data provide direct chemical evidence for the formation of eumelanin polymers by catecholamine oxidation by laccase alone followed by oxidative coupling of dihydroxyindole.     

Stellate cells storing retinol in the liver of adult lamprey,Lampetra japonica

Summary Distribution, localization and fine structure of the stellate cells in the liver of lamprey, Lampetra japonica, were studied during the spawning migration by use of Kupffer's gold-chloride method, fluorescence microscopy for vitamin A (retinol) and electron microscopy. The stellate cells in the lamprey liver differ in some of their properties from those in mammalian livers. Stellate cells which store abundant retinol in lipid droplets, occur not only in the hepatic parenchyma, but also in the dense perivascular and capsular connective tissue of the liver and in the interstitium of pancreatic tissue. In the hepatic parenchyma these cells are located perisinusoidally or along thick bundles of collagen fibrils. The stellate cells display a number of large retinol-containing lipid droplets, granular endoplasmic reticulum, tubular structures, dense bodies, Golgi complex, microtubules, and microfilaments. In the space of Disse, the stellate cells and extracellular fibrilar components such as collagen fibrils and microfibrils (11–12 nm in diameter) are intervened between the two layers of basal laminae. Differentiation and possible functions of the stellate cells in the lamprey liver are discussed.