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.     

Distribution of extracutaneous melanin pigment in Sparus auratus, Mugil cephalus, and Dicertranchus labrax (Pisces, Teleostei)

The morphological and biochemical characteristics of pigment accumulations found in the kidney, liver, spleen, and mesentery of three different species of teleost fishes have been studied. There are significant differences in number, distribution, and morphology of pigment accumulations in different organs of the three species. Biochemical studies have shown the existence of tyrosinase activity in the mesentery of Mugil cephalus and in the kidney and mesentery of Sparus auratus. No tyrosinase activity was found in any internal organs of Dicertranchus labrax. That activity was assayed using three methods: tyrosine hidroxylation, dopa oxidation, and melanin formation. The morphological and biochemical observations are in agreement. In those organs in which we have demonstrated melanin synthetic activity, the pigment cells are morphologically and like melanophores, while in the organs that show no melanin synthetic activity, the pigment cells resemble macrophages.     

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.     

Hyperpigmentation of Chinchilla Stria Vascularis Following Acoustic Trauma

This report describes morphological alterations of the chinchilla stria vascularis seen 30 days after exposure to impulse noise. The observed changes included a dramatic increase in strial melanin content which occurred in 7 of 36 animals exposed to electronically synthesized impulses presented in various temporal patterns at either 135 or 150 dB peak SPL. In these animals, densely pigmented areas of stria 1.5 to 3 mm in length were found in the basal cochlear turn. Light and electron microscopic study revealed that these areas contained large numbers of melanin granules situated primarily in pale-staining cells of the middle layer of the stria. Unlike the pigment granules present in normal chinchilla stria, the melanosomes found in the noise-exposed material clearly showed ultrastructural features characteristic of eumelanin. Melanin granules were also observed in marginal and basal cells of the noise-exposed stria. In some cases, pigment granules which had apparently been expelled from the marginal cells were present in the endolymphatic space beneath Reissner's membrane and on the strial surface. These findings support the view that the melanin-bearing cells of the inner ear are capable of markedly increased activity in response to stressful conditions.     

Hyperpigmentation of chinchilla stria vascularis following acoustic trauma.

This report describes morphological alterations of the chinchilla stria vascularis seen 30 days after exposure to impulse noise. The observed changes included a dramatic increase in strial melanin content which occurred in 7 of 36 animals exposed to electronically synthesized impluses presented in various temporal patterns at either 135 or 150 dB peak SPL. In these animals, densely pigmented areas of stria 1.5 to 3 mm in length were found in the basal cochlear turn. Light and electron microscopic study revealed that these areas contained large numbers of melanin granules situated primarily in pale-staining cells of the middle layer of the stria. Unlike the pigment granules present in normal chinchilla stria, the melanosomes found in the noise-exposed material clearly showed ultrastructural features characteristic of eumelanin. Melanin granules were also observed in marginal and basal cells of the noise-exposed stria. In some cases, pigment granules which had apparently been expelled from the marginal cells were present in the endolymphatic space beneath Reissner's membrane and on the strial surface. These findings support the view that the melanin-bearing cells of the inner ear are capable of markedly increased activity in response to stressful conditions.     

Intestinal Ultrastructure of Nerita picea (Mollusca: Gastropoda), an Intertidal Marine Snail of Hawaii

The neritid snail Nerita picea is a marine prosobranch mollusc which resides high in the intertidal zone on the Hawaiian Islands. Since other studies have shown considerable variations in molluscan gut histology and the relatively few recent ultrastructural reports have revealed novel cellular structures in the molluscan gastrointestinal tract, this investigation was directed toward ultrastructural clarification of the neritid intestine. Seven principal cell types constituted the intestinal architecture, including absorptive cells, zymogen cells, neural and endocrine cells, myocytes, pigment and gland cells. The intestinal epithelium was composed mainly of tall ciliated (9 plus 2 complement of microtubules) columnar absorptive cells which also possessed microvilli, extensive deposits of non-membrane-bound lipid-like droplets, and large reservoirs of glycogen-like granules. Less frequent, columnar zymogen cells contained numerous large zymogen secretory granules and possessed microvilli but not cilia. Small endocrine-like cells with secretory granules were observed basolaterally between some absorptive cells, resembling mammalian gut endocrine cells. Nerve fibers were prevalent in close association with the epithelial cells. A thin layer of non-striated muscle was present, as well as a serosally located gland composed of storage cells with a granular matrix and large granules.     

银鲳消化道形态与组织学结构特征及其消化酶活性

为了解银鲳(Pampus argenteus)消化道结构特点与其功能及食性的相关性, 采用解剖、石蜡切片、AB-PAS染色及酶活性检测技术对银鲳消化道的形态、组织结构、黏液细胞分布及消化酶活性进行研究。结果显示, 银鲳的消化道由口咽腔(舌)、食道侧囊、食道、胃及肠构成, 胃肠交界处有很多幽门盲囊。食道侧囊呈椭球形, 食道粗短, 胃呈U型, 肠有多个盘曲, 肠指数为2.03。舌上皮内有少量味蕾及较多黏液细胞。食道侧囊、食道、胃及肠均由黏膜层、黏膜下层、肌层及浆膜组成。食道侧囊内皱襞较发达, 被覆复层扁平上皮, 内含较多黏液细胞, 且以Ⅳ型为主, 皱襞顶端及侧面有内含角质刺的次级突起; 黏膜下层及肌层中有固定皱襞的骨质脚根; 侧囊内胃蛋白酶活性较高。食道内皱襞较高, 被覆复层扁平上皮, 内含较多黏液细胞, 且以Ⅳ型为主。胃内皱襞发达, 被覆单层柱状上皮, 未见黏液细胞分布; 胃腺发达, 胃内蛋白酶活性较高。肠道内褶襞多, 高度呈先下降后上升趋势, 黏液细胞密度前、中肠较高, 后肠较低, 且均以Ⅰ型为主; 肠道内胰蛋白酶、脂肪酶、淀粉酶及碱性磷酸酶活性较高。幽门盲囊组织结构与肠相似。银鲳的消化道结构特点、黏液细胞分布及消化酶活性与其功能及偏肉食的杂食性相适应。     

Ultrastructure and functional versatility of hirudinean botryoidal tissue

In leeches, the botryoidal tissue is composed of two different cell types--granular botryoidal cells and flattened endothelial-like cells--localized in the loose connective tissue between the gut and the body wall sac. We have observed that the botryoidal tissue undergoes functional and structural modifications in response to the different needs arising during the life-cycle of the animal. In healthy, untreated leeches, botryoidal cells are organized in cords or clusters, sometimes surrounding few, small lacunae. Conversely, in wounded animals we have observed the transition of the botryoidal tissue from cluster/cord-like structures to a hollow/tubular architecture, typical of pre-vascular structures. We have documented in botryoidal cell cytoplasm the presence of large calcium storage. Moreover, the cytoplasm of botryoidal cells was filled with granules of different form and size, containing iron or melanin, as tested by classic histochemical methods. The presence of elements like iron and calcium was confirmed by the well-established EDS analysis. In response to a surgical wound, botryoidal tissue cells changed their shape and formed new capillary vessels. Concurrently, botryoidal cells secreted iron from cytoplasmic granules into the new cavity: this secretory activity appeared to be related to intracellular calcium fluctuations. At the end of the angiogenic process, botryoidal cells lost their contact with the basal lamina and moved freely in the circulating fluid towards the lesioned area. Interestingly, circulating botryoidal cells were found to carry melanin in the wounded area. This function is probably involved in defense processes. Thus, we have shown that stimulated botryoidal tissue displays a variety of striking structural, secretory and defensive activities.     

Epithelial and neuronal calbindin in avian intestine An immunohistochemical study

Summary It is well known that calbindin immunoreactivity is highly concentrated in the duodenal absorptive cells of young birds. We have shown that in the adult intestine of three avian species, calbindin content is much more variable. In addition to absorptive cells, we have detected throughout the gut of both sexes of the domestic fowl and in the large intestine of the Japanese quail a second type of calbindin-positive epithelial cell which has the shape of a typical endocrine cell. These cells were particularly abundant in the large intestine, in contrast to the usual distribution of endocrine cells along the gut. Calbindin was also detected in the nervous system of the intestine. Calbindinpositive nerve fibres were rare in the duodenum and ileum, numerous in plexuses and nerve processes in both muscular layers and lamina propria of the large intestine in domestic fowl and Japanese quail. In the mallard, nerve fibres were rarely calbindin positive while definitively positive for VIP. Calbindin of the peripheral nervous system of the domestic fowl and Japanese quail comigrates with the duodenal calbindin (27000 dalton) in SDS gel electrophoresis.     

Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall

Melanin pigments protect against both ionizing radiation and free radicals and have potential soil remediation capabilities. Eumelanins produced by pathogenic Cryptococcus neoformans fungi are virulence factors that render the fungal cells resistant to host defenses and certain antifungal drugs. Because of their insoluble and amorphous characteristics, neither the pigment bonding framework nor the cellular interactions underlying melanization of C. neoformans have yielded to comprehensive molecular-scale investigation. This study used the C. neoformans requirement of exogenous obligatory catecholamine precursors for melanization to produce isotopically enriched pigment “ghosts” and applied 2D ¹³C-¹³C correlation solid-state NMR to reveal the carbon-based architecture of intact natural eumelanin assemblies in fungal cells. We demonstrated that the aliphatic moieties of solid C. neoformans melanin ghosts include cell-wall components derived from polysaccharides and/or chitin that are associated proximally with lipid membrane constituents. Prior to development of the mature aromatic fungal pigment, these aliphatic moieties form a chemically resistant framework that could serve as the scaffold for melanin synthesis. The indole-based core aromatic moieties show interconnections that are consistent with proposed melanin structures consisting of stacked planar assemblies, which are associated spatially with the aliphatic scaffold. The pyrrole aromatic carbons of the pigments bind covalently to the aliphatic framework via glycoside or glyceride functional groups. These findings establish that the structure of the pigment assembly changes with time and provide the first biophysical information on the mechanism by which melanin is assembled in the fungal cell wall, offering vital insights that can advance the design of bioinspired conductive nanomaterials and novel therapeutics.     

Relations between pigmentation of the cornea and the number of epidermal melanophores in Rana temporaria L. larvae

The dynamics of the external cornea pigmentation in Rana temporaria L. larvae at the 22d developmental stage have been studied under conditions favourable for various course of certain morphological reactions in the pigment system. The cornea together with the surrounding skin is transferred on the dorsal surface of the larva body, and the piece of the dorsal surface skin is put instead of the cornea removed. When using the reciprocal transplantation method and preserving the organism's integrity (without disturbing melanocyte-stimulating source--namely, the hypophysis, and melatonine sources--namely, the pineal gland and the lateral eyes) the corneal pigmentation is observed on the background of perfect morphological reactions in the pigment system, while the larvae are maintained on the dark and light substrates, that is at various density of the pigment cells (120 larvae have been used). The pigmentation dynamics have been studied from the 6th up to the 20th day in total preparations. The epidermal melanophores density is estimated in 4 areas of each preparation. The melanin amount is estimated by means of the electron paramagnetic resonance-spectrometry according to the contents of free radicals expressed in relative units. A direct proportional dependence between the significantly higher melanin contents (1.5-fold) and a significantly quicker (1.5-fold) process of the corneal pigmentation is revealed, that agrees with an increasing number of the pigment cells per one unit of the body surface in the larvae maintained on the dark substrate. In the larvae maintained on the light substrate, the dependence is of a reverse character. It is probable that the factors forcing the pigmented cells, at cultivation the neural crest cells in vitro to reject from each other, affect the pigmentation of the larval cornea in vivo. If it is the case, the processes specific for the embryonal period, transgress during the cornea pigmentation at the larval stages of development.     

A Novel Histochemical Method for a Simultaneous Staining of Melanin and Collagen Fibers

For the histopathologic diagnosis of melanocytic lesions, it could be necessary to identify the melanin pigment because its visualization is unspecific with hematoxylin-eosin (HE). The Fontana-Masson (FM) technique is used in histopathology in this type of lesion, which allows the identification of the pigment, but it loses all the morphologic parameters. The authors describe a modification to the FM method, for the evaluation of the morphology, the argentaffin reaction of the melanin, and collagens fibers of the extracellular matrix simultaneously, for which they have developed the Fontana-Masson picrosirius (FMPS) method. Biopsies of different melanocytic lesions were used for the performance of the HE, FM, and FMPS methods. The pixel intensity of the reaction for melanin, collagen, and epithelium was determined with ImageJ software. The FMPS method allows the evaluation of morphological characteristics, identifying the melanin pigment and collagen fibers with high intensity simultaneously. This method does not differ significantly from FM in the identification of melanin, maintaining its sensitivity and specificity. In addition, it does not differ in the demonstration of the morphology with HE. However, FMPS is significantly superior in the identification of collagen fibers. The FMPS method combines morphological and histochemical parameters that could be useful in the study of pigmented lesions of melanocytic origin.     

Gut morphology and morphometry in the epauletted Wahlberg's fruit bat (Epomophorus wahlbergi, Sundevall, 1846)

The morphological adaptations of the fruit bat small intestine to which the high functional efficiency could be related and the possible landmarks delineating the various parts of the gut were examined. The stomach was the carnivorous type with large rugae spanning the entire luminal aspect down to the pyloric sphincter, which was reflected internally as a prominent fold. Externally, the intestine was a continuous tube uninterrupted by any structures. The cranial fifth of the small gut had long, branching and anastomosing villi, which caudally turned to finger-like discrete structures that became rather short and stumpy and diminished at the beginning of the colon. The colon had longitudinal folds that were macroscopically discernible from the mucosal aspect of the opened intestine and that continued into the rectum. The small gut formed 94% of the whole intestinal length, the colon and the rectum taking 4 and 2%, respectively. Ultrastructurally, the enterocyte showed a prominent brush border and the lateral membranes were modified into numerous tortuous interdigitating processes. Adjacent enterocytes were joined by these processes through desmosomes. The processes also participated in pinocytotic fluid uptake from the intercellular spaces with resultant numerous intracellular vacuoles of varied sizes. Solutes absorbed into the cells were probably first passed into the intercellular compartment to create a concentration gradient thus enhancing further absorption into the cell. We conclude that the uniquely elaborate ultrastructure of the enteric epithelium coupled with the vast microvillous surface areas reported elsewhere are partly responsible for the very high absorption rates reported in the fruit bat small intestine.     

Spontaneous lesions in cynomolgus monkeys used in toxicity studies.

Spontaneous lesions in wild-caught, laboratory-maintained cynomolgus monkeys used in drug-toxicity studies were examined histopathologically in an effort to better distinguish toxic changes from spontaneous lesions and assess the toxicity of drugs more exactly. In the liver and kidney, where toxic changes are observed frequently, many spontaneous lesions were observed. Infiltration of mononuclear cells, vacuolization of the hepatocytes, dilatation of the renal tubules, and vacuolization of the renal epithelia were observed at a relatively high frequency. It is considered important to examine these changes carefully, because they closely resemble the changes recognized as toxic. Deposition of brownish pigment was observed in various organs such as the liver, kidney, spleen, intestinal tract, lung and brain, however the type of pigment differed among the organs, and histochemical examination revealed anthracosis or accumulation of hemosiderin, or melanin. Since the monkeys were caught in the wild, many parasitic lesions were observed especially in the large intestine and liver. Helminthous worms were frequently observed in the granulomas in the large intestine, however, no parasites were observed in the granulomas in the liver. Such lesions in the liver may be misinterpreted as toxic changes, when only scars or inflammatory lesions are observed.     

Color and morphological characteristics of the skin in people of different racial groups

A complex colorimetric, spectrometric and morphological research of race-dependent differences in skin colour and structure has been carried out. Certain regularities in the quantity, distribution and morphological composition of melanin-containing structures and Hb pigment have been revealed in the Russians, Vietnamese, Angolans and Mulattoes. The study has shown that sex differences in skin color depend on hemoglobin concentration--in people of the Caucasian race; both on melanin and hemoglobin concentration--in people of the Mongolian race and only on melanin concentration--in people of the Negroid race.     

Morphological Characterization of Three Phenotypes of the Isopod Armadillidium vulgare

The morphological characteristics and ommochrome quantity in the integument of red, white, and wild type (black-grey) Armadillidium vulgare were studied. The red phenotype was found to possess two kinds of immature ommochrome pigment granules within its pigment cells, in addition to mature pigment granules. The immature granules seemed to contain uniformly distributed fibrilles, or to have an electron-dense central region surrounded by an electron-lucent outer edge. Since these immature pigment granules were typically observed to be distributed along with the mature ones, and were also more easily extractable than the wild type's, it is hypothesized that ommochrome granule maturation in the red phenotype may occur slowly due to a defect in the pigment granule internal process which combines pigments with matrix proteins. Regarding the white phenotype, although its pigment cells were undeveloped, several large-sized vesicles containing a small amount of electron-dense material appeared in the pigment cell cytoplasm. The wild and red type males of A. vulgare were found to have an ommochrome content twice as large as that of the corresponding females, with no ommochrome pigment being detected in the white phenotype. The genetic relationship between the white and red phenotypes was discussed using as a basis the observed pigment granule structure.     

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.     

光唇鱼消化道的形态结构特征

采用解剖及石蜡切片显微技术,观察研究了光唇鱼消化道的形态结构特征。消化道由口咽腔、食道、肠构成。口下位、马蹄形,无颌齿,具咽齿,齿式为4/4。舌较小,前端游离,舌粘膜表层为复层鳞状上皮,有较多的杯状细胞和味蕾。食道及肠均由粘膜层、粘膜下层、肌层及外膜构成。食道内皱襞发达,粘膜层有大量杯状细胞。肠道盘曲,由前、中、后肠组成,肠长/体长为1.84±0.24;前肠管腔较大,中、后肠管腔渐变小;前、中肠皱襞及纹状缘比后肠发达;前肠及后肠杯状细胞较少,中肠杯状细胞较多。光唇鱼消化道的形态结构特征与其食性相适应。     

Ultrastructure of the hindgut of Drosophila larvae, with special reference to the domains identified by specific gene expression patterns

The hindgut of Drosophila larvae consists of nine domains that have been distinguished by specific gene expression patterns. In the present study, we examined the ultrastructure of the hindgut of Drosophila larvae, with special reference to the domains, in order to determine whether or not the domains are morphologically distinct functional units. Each domain showed specific ultrastructural features that suggested specific corresponding functions. According to the morphological features, terms are proposed for each domain: the imaginal ring; the "pylorus," which has a thick cuticular layer and well-developed sphincter muscles; the "large intestine," which occupies a major middle portion of the hindgut and has a unique dorsal and ventral subdivision; "border cells," which delineate the anterior and posterior borders of the large intestine and the border between the dorsal and ventral domains of the large intestine; and the "rectum," which is situated at the posterior end of the hindgut and has a thick cuticular layer and sphincter muscles. The morphological features indicate that the large intestine has active absorptive activities. The domains, which have been distinguished by gene expressions, were demonstrated to be functional tissue units of the gut.     

Localization of iodopsin and rod-opsin immunoreactivity in the retina and pineal complex of the river lamprey,Lampetra japonica

Melano-macrophages in the head-kidney, spleen and liver of sea bass and gilthead seabream have been investigated by means of light and electron microscopy, histochemistry and phagocytic assays. The results demonstrate the presence of both free and clustered melano-macrophages (melano-macrophage centres), with similar ultrastructural features. These large cells are PAS-, hemosiderin-and melanin-positive, and contain large, alkaline-and acid phosphatase-positive lysosomes, whose reaction intensity depends on the amount of accumulated pigment. The relationship between the cytochemical features of these lysosomes and the capacity of the melano-macrophages to phagocytose bacteria and latex beads, has been studied. The large melanomacrophage centres have a capsule of flattened cells, whose ultrastructural and cytochemical features are similar to fibroblast-like reticular cells. Melanin is the main accumulated pigment. A subpopulation of head-kidney mononuclear phagocytes engulfs melanin associated with cell debris. The relationship between the origin of the melano-macrophage pigment and the ability of monocytes/macrophages to phagocytise the melanin from melanocytes, is considered. The origin and possible function of melano-macrophage centres are discussed.