Electron-dense Cytoplasmic Granules Research Articles

Ultrastructure of enterochromaffin cells and associated neural and vascular elements in the mouse duodenum.

Enterochromaffin cells of adult mouse duodenum were studied with light- and electron-microscopical techniques. They were distinguished from other enteroendocrine cells by their pleomorphic, electron-dense secretory granules in the basal cytoplasm. At the apices of enterochromaffin cells, tufts of short microvilli bordered the gut lumen. At their bases, irregular cytoplasmic extensions were either in contact with or passed through the basal lamina. The presence of cytoplasmic extensions in close proximity to fenestrated capillaries and subepithelial nerves suggested an endocrine or paracrine function. Electron micrographs of serial thin sections were used to reconstruct an enterochromaffin cell from the crypt epithelium in three dimensions and to determine its relationship with the underlying neural plexus. Although extensions from the serially sectioned and reconstructed cell and other enterochromaffin cells studied in crypt epithelia protruded through the basal lamina, no synaptic contacts were seen. Evidence of a synaptic contact between a neurite and another type of enteroendocrine cell (possibly an intestinal A cell), suggested a neurocrine role for some of the basally-granulated cells. Possible functions of enterochromaffin cells are discussed in the light of recent literature on the system of enteroendocrine cells, also known as APUD (amine precursor uptake and decarboxylation) cells and/or paraneurons.

Generation of natural killer cell lines from murine long-term bone marrow cultures.

Functionally active natural killer (NK) cells with the ability to lyse 51Cr-labeled YAC-1 lymphoma target cells are no longer detectable by 1 wk of culture in cultured marrow cells harvested from Dexter-type long-term marrow cultures (LMC). Interferon, which enhances NK cell-mediated target cell lysis, fails to induce NK activity from LMC cells even at high effector to target cell ratios. However, such LMC cells, when placed in secondary cultures in the presence of Con A-splenic leukocyte-conditioned medium (spleen-CM) generated a population of cells with NK activity within 1 wk. Kinetic studies showed that the generation of NK activity was not due simply to proliferation of a few surviving NK cells, but suggested derivation from NK precursors through clonal expansion and functional maturation. This NK activity was further shown to be associated with a subpopulation of cells bearing surface Thy-1, Ly-5, and NK-1 as well as asialo-GM1 antigens but lacking Ly-1 antigen. The expression of Ly-2 antigen, however, was variable. Electron microscopy studies of isolated asialo-GM1-positive cells showed a uniform lymphoblastoid morphology with large cytoplasmic to nuclear ratios and prominent electron dense cytoplasmic granules characteristic of large granular lymphocytes. In support of the NK nature of such cultured cells was the ability of anti-asialo-GM1 and complement to abrogate, and of interferon to augment, target cell lysis. Isolated cell lines also showed target selectivity similar to NK cells. The implications of the studies on further analysis of the nature of NK precursors is discussed.

Growth and differentiation of hamster tracheal epithelial cells in culture.

The purpose of these studies was to define culture conditions that support growth and differentiation of normal epithelial cells obtained from hamster tracheas. Epithelial cells from tracheas of adult hamsters were collected using enzymatic procedures and cultured under various conditions. The medium used consisted of a 1:1 mixture of medium 199 and Dulbecco's modified Eagle's medium with 2% fetal bovine serum, which was conditioned by mouse 3T3 cells before use. Insulin, transferrin, hydrocortisone, epidermal growth factor, and an extract from bovine hypothalamus were used as supplements. When seeded on uncoated or collagen-coated tissue culture dishes, the hamster cells grew only poorly. When the cells were seeded on collagen gels, however, rapid and prolonged growth ensued. The cultures had a population doubling time of 20 hr and a colony-forming efficiency of 7-10%, and they could be grown for up to three passages. Growth was dependent on the presence of transferrin, insulin, epidermal growth factor, and 3T3 conditioning factors in the medium. The latter could be omitted if the concentration of serum was increased. Less important for growth was the presence of hydrocortisone and bovine hypothalamus extract. In contrast to results with tracheal epithelial cells from adult rabbits, rats, and mice, differentiation into ciliated cells regularly occurred in cultures of cells derived from hamster tracheas. The appearance of ciliated cells in the cultures was dependent on the presence of collagen gel as a substratum and of 3T3 conditioning factors in the medium. In addition, there were numerous cells that contained electron-dense cytoplasmic granules. The granules were not stained by dialyzed iron, which stains acidic glycoproteins, but were stained positively by periodic acid-Schiff reagents and the periodic acid-thiocarbohydrazide-silver proteinate method, suggesting the presence of secretory granules containing neutral glycoproteins. A similar staining pattern was observed for the secretory granules of intact hamster tracheas. The culture system described supports growth and cellular differentiation of normal tracheal epithelial cells of hamsters. We believe therefore that it will be a useful model for studying the regulation of tracheal cell function on the cellular and biochemical level.

Ultrastructure of the granule cells in the small intestine of the rainbow trout (Salmo gairdneri) before and after stratum granulosum formation

The ultrastructure of the small intestinal granule cells (GC) of the rainbow trout (Salmo gairdneri) was studied in fry and fingerlings before the cells organized to form the stratum granulosum (SG) and in 12-month-old juveniles which possess a well-developed SG several cell diameters in thickness. The GC of juvenile trout exhibit a striking reduction in nucleolar numbers and in the quantity of rough endoplasmic reticulum relative to the GC of younger fish, but contain even greater numbers of large, electron-dense cytoplasmic granules. The final stages of cellular maturation therefore take place in the GC after their organization to form a layer, and the cells appear to lose their capacity to synthesize protein(s) for the cytoplasmic granules. Since the latter structures are, nevertheless, maintained in large numbers in mature GC, it is suggested that they function intact within the cell. No precursor to the granulated form of the GC was identified in the intestine, a result which suggests that the earliest developmental stages of the GC locate within another organ.

In vitro generation of mast cell-like cells from human peripheral mononuclear phagocytes.

An in vitro culture method, previously shown to induce the transformation of rat macrophages to mast cells, has been adapted to humans. Adherent peripheral human blood mononuclear cells were cultured in a medium supplemented with 30% L-cell supernatant and 30% horse serum. During the first week of culture, the cells were phagocytic (80%), lacked histamine, were not metachromatic and stained faintly for naphthol-AS-D-chloroacetate esterase, primarily in a paranuclear location. During the first 2 weeks of culture, the cells grew in size, synthesized DNA, became nonadherent and developed cytoplasmic chloroacetate esterase staining which was strongly positive in 50-60% of the cells during the third week. Intracellular histamine levels increased steadily during culture, and electron-dense metachromatic cytoplasmic granules were present during the third week. Human peripheral blood mononuclear cells contain therefore a subpopulation of cells that can differentiate into mast cells-like cells under specific in vitro culture conditions.

A cytotoxic T-lymphocyte line propagated from a rabbit infected with sheep associated malignant catarrhal fever

The propagation of a cell line from a rabbit affected with the sheep associated form of malignant catarrhal fever is described. Immunological and morphological characteristics of the cell indicated that it was a T-lymphocyte and the presence of electron dense cytoplasmic granules suggested that the cell could be further classified as a large granular lymphocyte. The cell line required a feeder layer and was cytotoxic to both primary cell cultures and cell lines, a characteristic of large granular lymphocytes. No evidence of the nature of the agent could be detected but as few as 10(2) cells transmitted the disease. These findings are discussed and the possibility that infection and subsequent dysfunction of large granular lymphocytes may have a central role in the pathogenesis of malignant catarrhal fever is considered. That cells with similar characteristics have been derived from Herpesvirus saimiri and H ateles infected marmoset lymphocytes suggests that the lymphoproliferation associated with infection by these two simian herpes-viruses and malignant catarrhal fever may have a similar pathogenesis.

Mast cell clones: a model for the analysis of cellular maturation.

Cloned mouse mast cells resemble, by ultrastructure, immature mast cells observed in vivo. These mast cell clones can be grown in the absence of any other cells, facilitating direct investigations of their biochemistry and function. We find that cloned mast cells express plasma membrane receptors (Fc epsilon R) that bind mouse IgE with an equilibrium constant (KA) similar to that of normal mouse peritoneal mast cells. In addition, cloned mast cells do not display detectable la antigens and cannot enhance lg secretion when added to lymphocyte cultures or mediate natural killer lysis. In the presence of 1 mM sodium butyrate, cloned mast cells stop dividing and acquire abundant electron-dense cytoplasmic granules similar to those of mature mast cells. Their histamine content increases concomitant with cytoplasmic granule maturation and may exceed that of untreated mast cells by 50-fold. Unlike peritoneal mast cells, cloned mast cells incorporate 35SO4 into chondroitin sulfates rather than heparin. These findings demonstrate that, unlike fully differentiated mouse peritoneal mast cells, cloned immature mouse mast cells contain no heparin and low levels of histamine. In addition, they establish that high-affinity Fc epsilon R are expressed early in mast cell maturation, well before completion of cytoplasmic granule synthesis and mediator storage.

In vitro differentiation of rat peritoneal macrophages into mast cells: an enzymecytochemical study.

Under special culture conditions, rat peritoneal mononuclear phagocytes have previously been shown to transform into typical mast cells, based on their histamine contents, the increase of their membrane IgE-receptors, and the development of metachromatic, electron-dense cytoplasmic granules. In the present investigation, the enzyme-cytochemical characteristics of these cells were examined prior to and during culture. The peritoneal macrophages were found to have strong paranuclear activity of nonspecific esterases and acid hydrolases, while they were negative for peroxidase and chloroacetate esterase. During culture, two main changes in enzyme activity were observed within all the cells: (1) Paranuclear activity was lost in favour of granular enzyme activity throughout the cytoplasm, and (2) chloroacetate esterase appeared in cultured cells and developed a strong granular activity. These findings are consistent with the macrophage nature of the seeded cells and their development into mast cells during culture. They give support to the theory that mast cells originate from the monocyte-macrophage lineage.

Messenger RNA dependent synthesis of a protein containing relaxin related sequences.

Serum relaxin levels in pregnant rats, sows, and guinea pigs are very low during most of gestation (Sherwood et al. 1975); mean relaxin concentrations in the sow range between 0.1 and 2.0 ng/ml throughout the first 100 days of pregnancy. During this period small electron dense cytoplasmic granules begin to appear in luteal cells. This granule population continued to increase in late gestation, reaching a maximum several days before parturition. About 24–48 hours before delivery a rapid disintegration of the cytoplasmic granules occurs. Serum levels of relaxin rise rapidly and then drop precipitously during parturition and lactation (Belt et al.3 1971). Studies in rat ovarian homogenates also suggest a steady, gradual rise in relaxin production throughout pregnancy (Sherwood and Crnekovic, 1979). The stimuli for the initial production of the hormone, the factors controlling its secretion and storage during pregnancy, and the reasons for its explosive release at term remain obscure. Relaxin has traditionally been assigned to the class of pregnancy-related hormones, but at least one study suggests that in humans the protein is also present in non-pregnant sera (Bryant-Greenwood et al.3 1977).

Detoxication of zinc and cadmium by the freshwater protozoan Tetrahymena pyriformis: II. Growth experiments and ultrastructural studies on sequestration of heavy metals

Sublethal concentrations of zinc and cadmium limited the growth of batch cultures of the ciliate Tetrahymena pyriformis. Electron microscopy of cultured populations revealed many electron-dense cytoplasmic granules in cells exposed to zinc (60 ppm), but fewer in cells grown with less zinc (6 ppm) or cadmium (2 ppm). Ultrastructural abnormalities were observed only in cells grown with cadmium. These contained autophagic vacuoles and had damaged nuclei. Zinc was shown to exert a protective effect against cadmium toxicity both in terms of growth and ultrastructural appearance. Electron probe X-ray microanalysis revealed both zinc and cadmium within the cytoplasmic granules of cells exposed to these metals. Since the granules are normally composed predominantly of calcium and magnesium, these ions are implicated in the intracellular detoxication of zinc and cadmium. Zinc was detected only in the granules, while cadmium was also found in autophagic vacuoles and damaged nuclei.

CONTENT AND ULTRASTRUCTURAL LOCALIZATION OF TRANSITIONAL METALS IN ASCIDIAN OVARY.

The content of some transitional metals (Ti, V, Cr. Mn. Fe, Cu) in the ovary of Ascidia malaca and Ciona intestinalis and in the gonads ofPolycarpa gracilis has been reported. The metals were localized histospectrographically and ultrastructurally. In the ovaries of C. intestinalis. the quantitative analysis shows a variable content of metals related to the ovary maturative cycle. The iron is localized in the electron dense cytoplasmic granules ofA. malaca oocytcs and in the cytoplasmic dense formation of P. gracilis test cells. Cl, S, Si and P are present in the test, follicles and inierovarian cells of all examined species. The results are discussed in relation to the zoological position of these species. The presence of metals and Ascidiacee phylogenesis might be correlated.

Ultrastructure of the gut neurotensin cell

In mammals, neurotensin cells occur scattered in the epithelium of the jejunum-ileum. In chicken, neurotensin cells are abundant in the region of the gizzard-duodenal junction (antrum) where they occur intermingled with numerous somatostatin and gastrin cells. The neurotensin cells in chicken, dog and man were identified at the electron microscopic level by immunocytochemistry, using the consecutive semithin/ultrathin section technique. They contain numerous electron dense cytoplasmic granules, pre-dominantly in the basal portion of the cell. It was shown that these granules are the storage site for neurotensin. The neurotensin granules are round, highly electron dense and of about the same size in the different species examined (mean diameter 260--290 nm). In dog and man the granules have a tightly applied surrounding membrane while in the chicken a relatively electron lucent zone separates the electron dense core from the granule membrane. The ultrastructure of the neurotensin granules in chicken is somewhat reminiscent of that of the gastrin granules. The mean diameter of the gastrin granules in chicken antrum is 230 nm; for the somatostatin granules the mean diameter is 305 nm.

Studies on the 14C-histamine release induced by noradrenaline in mouse neoplastic mast cells.

Murine neoplastic mast cells incubated with labelled histamine released 14C-histamine when exposed to increasing concentrations of noradrenaline. This process was blocked by low temperature, by inhibiting oxidative metabolism and glycolysis, by calcium deprivation and by cytochalasin B. Electron microscope observations of cells exposed to noradrenaline revealed an increase of the Juxtanuclear Golgi Apparatus, and the appearance of a pronounced peripheral vacuolization. Cells exposed simultaneously to noradrenaline and cytochalasin B were characterized by a further increase of the Golgi apparatus, and by the appearance of many electron dense cytoplasmic granules. The possible mechanisms of the noradrenaline induced histamine release were discussed.

Structure and ultrastructure of the pituitary gland in the sea lamprey, Petromyzon marinus at different stages in its life cycle.

The structure and fine structure of the pituitary gland in the sea lamprey, Petromyzon marinus L., are described at all major stages in the life cycle. Synthetically active adenohypophysial cells are evident in even the smallest ammocoetes examined (body length 2.5 cm). An attempt is made to correlate changes in the activity of pituitary cell types with biological events. In the neurohypophysis a single fibre type was discriminated in electron micrographs. This contained electron-dense cytoplasmic granules 800 to 1600 A in diameter.

Fluorescence and electron microscopy of amine-storing enterochromaffin-like cells in tracheal epithelium of mouse.

The tracheo-bronchial mucosa of the mouse has been found to contain an extensive system of argyrophilic epithelial cells. In the trachea the cells morphologically resemble enterochromaffin cells. Normally, these enterochromaffin-like cells contain no fluorogenic amine, as revealed by the Falck-Hillarp formaldehyde technique. On the other hand the cells have the capacity to take up and decarboxylate 3,4-dihydroxyphenylalanine (DOPA) or 5-hydroxytryptophan (5-HTP); the amine formed is stored in the cytoplasm in a reserpine-sensitive store. This capacity to produce and store amines under experimental conditions may reflect the presence in the tracheal enterochromaffin-like cells of an amine which can not be demonstrated with available fluorescence histochemical techniques. In the electron microscope the tracheal enterochromaffin-like cells were identified by a positive argyrophil reaction and by their capacity to accumulate radioactivity after administration of 3H-DOPA or 3H-5-HTP as revealed by autoradiography. The radioactive labelling was associated with cytoplasmic electron-dense granules (800–1000 A), suggesting that the amine formed was stored in these granules. Accordingly, the granules stained argentaffin after DOPA-pre-treatment of the animal. It is suggested that, like similar cells in the gastric mucosa, these argyrophilic enterochromaffin-like cells constitute an endocrine system in which amines are of cytophysiological importance.

A comparative ultrastructural study on ultimobranchial glands of some Israeli anurans (Bufo viridis, Rana ridibunda and Hyla arborea).

The comparative fine structure of ultimobranchial (UB) glands of adult Israeli anurans (Bufo viridis, Rana ridibunda, Hyla arborea) taken in the wild during the breeding season is presented. Common aspects of the UB secretory cells are considered with especial reference to secretory granules, lipid droplets and tonofilaments. In B. viridis a second cell type with large electron-dense cytoplasmic granules is found in UB follicles. R. ridibunda and H. arborea UB follicles have a second cell type similar to goblet cells in appearance and these appear to be discharging their mucoid contents into the lumina of the follicles. The possible significance of these various cell types is considered.

Ultrastructural search for homologues of pancreatic β-cells in the intestinal mucosa of the mollusc Buccinum undatum

In the intestinal mucosa of the gastropod mollusc Buccinum undatum epithelial cells have been found, containing small round electron-dense cytoplasmic granules appearing like the secretion granules of endocrine cells of higher vertebrates. These cells are interpreted as corresponding to the granular cells with some tinctorial and histochemical features of mammalian β-cells observed previously in tissues where insulin production was found. As a working hypothesis for future studies it is proposed that these cells are endocrine cells, possibly representing the site of insulin production in Buccinum undatum.

Occurrence of selenium in poisonous plants

A bacterial strain (SeITE02), related to the species Stenotrophomonas maltophilia and resistant to selenite (SeIV) up to 50 mM in the growth medium, was isolated from rhizospheric soil of a selenium hyperaccumulator plant, the legume Astragalus bisulcatus. The influence of SeIV on the active growth of this Se-tolerant bacterial strain has been investigated in oxic conditions, along with the isolate's ability to reduce selenite to elemental selenium (Se0). Interestingly, concentrations of 0.5 mM SeIV were wholly reduced by strain SeITE02 in liquid culture within 52 h. Moreover, 87% of SeIV added to the growth medium at the initial concentration of 2.0 mM underwent again reduction in 120 h. Actually, a selenite-mediated induction of a sort of adaptive response to detrimental SeIV effects magnified the efficiency of SeITE02 in reducing this toxic oxyanion. Furthermore, the SeIV influence on cell morphology of strain SeITE02 was evidenced by phase-contrast and electron microscopy analyses. In particular, transmission electron microscopy (TEM)–energy-dispersive X-ray (EDX) analysis of S. maltophilia strain SeITE02, grown in presence of SeIV, showed electron-dense Se0 granules either in the cell cytoplasm or in the extracellular space. Therefore, the capability of strain SeITE02 to quickly reduce soluble and harmful SeIV to insoluble and unavailable Se0 may be looked at as a promising exploitable option for the setup of low-cost biological treatments tailored to manage contamination in selenium-laden effluents.