Acid-phosphatase activity of reticular cells and macrophages in the lymph node of the rat after ingestion of mast-cell granules

Summary Acid phosphatase (ACPase) was ultracytochemically demonstrated in the lymph-node sinus reticular cells and macrophages of rats. After the uptake of horseradish peroxidase (HRP), marked ACPase activities were seen in both reticular cells and macrophages, although only sparse ACPase activity was detected in the reticular cells of the control. After the injection of HRP into the footpad, the mast cells in the regional lymph node became degranulated, and the released granules were taken up by reticular cells and macrophages. In macrophages, these taken-up mast-cell granules exhibited ACPase reaction products, whereas none of the granules taken up by reticular cells showed ACPase activity. The heparin-protamine complex was also engulfed by reticular cells and macrophages, and ACPase activity was demonstrable in the complex taken up by both types of cell. It is probable that, as is the case in macro-phages, reticular cells in the lymph-node sinuses take up and digest foreign substances through the formation of phagolysosomes, but they do not digest granules originating from the mast cells in the lymph node of the same animal.     

Acid-phosphatase activity of reticular cells and macrophages in the lymph node of the rat after ingestion of mast-cell granules

Acid phosphatase (ACPase) was ultracytochemically demonstrated in the lymph-node sinus reticular cells and macrophages of rats. After the uptake of horseradish peroxidase (HRP), marked ACPase activities were seen in both reticular cells and macrophages, although only sparse ACPase activity was detected in the reticular cells of the control. After the injection of HRP into the footpad, the mast cells in the regional lymph node became degranulated, and the released granules were taken up by reticular cells and macrophages. In macrophages, these taken-up mast-cell granules exhibited ACPase reaction products, whereas none of the granules taken up by reticular cells showed ACPase activity. The heparin-protamine complex was also engulfed by reticular cells and macrophages, and ACPase activity was demonstrable in the complex taken up by both types of cell. It is probable that, as is the case in macrophages, reticular cells in the lymph-node sinuses take up and digest foreign substances through the formation of phagolysosomes, but they do not digest granules originating from the mast cells in the lymph node of the same animal.     

Histochemical changes in the midgut of two ixodid tick speciesBoophilus microplus andRhipicephalus appendiculatus during digestion of the blood meal

The changes in the midgut epithelia of two ixodid tick species,Boophilus microplus andRhipicephalus appendiculatus, have been studied using several histochemical techniques. It was revealed that there is an accumulation of RNA at the time of tick attachment to the host and prior to the arrival of the blood meal, indicating that the midgut digest cell is furnished with the machinery characteristic of a synthetic cell. There appears to be a synchrony in the appearance of granules with peroxidase activity and the uptake of haemoglobin into the midgut digest cells. Alkaline phosphatase activity was observed in the midgut epithelia of all ticks except in a few of the long-starved ticks, and was concentrated in the apical plasma membrane regions of those digest cells involved in absorption and the intracellular digestion of haemoglobin. The presence of these enzymes suggests that the midgut digest cell is a multifunctional cell capable of both secretory and digestive activities. The colloidal material in the midgut lumen was found to result from the accretion of several products both secreted and excreted by the midgut epithelial cells and exhibited different staining reactions depending on which component dominated. The nature of the material suggests that in addition to its digestive function it may serve as a sink to bind all the by-products of digestion and thereby facilitate their excretion.     

An ultrastructural study on gastric endocrine cells in the stomach gland patch of the koala Phascolarctos cinereus

The endocrine cells in the stomach gland patch of the koala (Phascolarctos cinereus) were studied ultrastructurally. They were classified into 3 types based on the ultrastructural profiles of their endocrine granules and tentatively categorized as type I, II, and III endocrine cells. Type I cells contained round granules that were for the most part larger than those observed in the other 2 cell types. The granules ranged from moderate to relatively high in electron density. Type II cells were angular in shape and characterized by the presence of granules that were polymorphous in profile. Contents of the endocrine granules in type II cells also showed a range of high to moderate electron density. Type III cells were oval or pyramidal in shape. They contained highly polymorphous granules that were round, oval, dumbbell-like or comma in shape and characterized by the presence of a clear space or halo separating the high to low electron-dense core from the limiting membrane of granules. Type III cells were observed most often whereas type I and II cells were a less frequent observation.     

Periodic,multimodal distribution of granule volumes in mast cells

The areas of 2327 mast cell granules in transmission electron micrographs of sections of peritoneal mast cells from adult rats were measured by digitized planimetry. A histogram constructed using equivalent volumes calculated from the measured areas assuming approximation of the granules to spheres showed a periodic multimodal distribution in which the modes fell at volumes that were successively larger integral multiples of the volume at the first mode. Application of a moving-bin technique to the data confirmed the presence of the modes. We propose a mechanism of fusion of unit sized granules to account for the multimodal distribution. The presence of pear- and dumbbell-shaped granules in mast cells is consistent with this mechanism.     

The significance of paravacuolar granules of the thyroid. A histologic, cytologic and ultrastructural study

The presence of the so-called "paravacuolar granules" in thyroid follicular cells has been associated with increased metabolic activity of the gland, regressive changes, degeneration, phagocytic activity and benign papillary hyperplasia. During the course of a review of the intraoperative cytologic preparations and corresponding histologic sections from 73 thyroid cases, the presence of granules within follicular cells was noted in 25 cases (18 adenomatous or colloid goiters, 3 follicular adenomas, 2 papillary carcinomas, 1 follicular carcinoma and in thyroid tissue surrounding a follicular adenoma in 1 case). Histochemical and ultrastructural studies showed the granules to consist of lysosomes containing hemosiderin or lipofuscin pigments. These findings indicate that the presence of paravacuolar granules in thyroid cells is a common nonspecific finding that simply reflects: (1) the erythrophagocytic capability of the follicular epithelial cells, which results in the accumulation of iron within lysosomes, and (2) the accumulation of lipofuscin pigments within lysosomes as a result of degradation of endogenous cellular material.     

The effects of heparin and protamine on resorption of bone particles

Implantation of bone particles into rats initiates rapid, reproducible resorption that can be quantitated by histomorphometric analysis. Mast cells are found with the mononuclear and multinucleated cells that digest the bone. One of the constituents of mast cell secretory granules, heparin, is known to regulate collagenase activity. This study shows that 1) exogenous heparin stimulated resorption of implanted bone to 41% of control values and that 2) protamine, a heparin antagonist, reduced resorption to rates 50% of controls.     

Horny cell formation in the epidermis of Rana pipiens

Well preserved transitional cells were found between differentiated cells and horny cells of the frog epidermis, thus facilitating the study of the sequential events involved in horny cell formation. Autolysosomes appear to play an important role in the formation of horny cells. These structures preferentially digest those cytoplasmic components which are not necessary constituents of the terminal horny cell. The release of the contents of the small mucous granules into the intercellular spaces is one of the initial events in horny cell formation. Filaments and large mucous granules seem to be resistant to the lytic digestion and contribute to the bulk of the horny cell. Loss of fluids through the plasma membrane and consolidation of the remaining constituents, results in a flattened horny cell. The appearance of a thickened membrane around the horny cell signifies the completion of the transformation process.     

Morphological and functional characterization of beige mouse adrenomedullary secretory vesicles

We tested whether the giant secretory granules observed in the mast cells of the naturally occurring mutant beige mouse (BM) (C57BL/6N-bg) were also present in the adrenal chromaffin cells. The presence of large chromaffin granules (CG) would be a valuable tool for the study of exocytosis in neuronal tissues. Conversely, the observation of large vesicles within chromaffin cells that are different from CG could indicate that CG are of a different origin than granules of mast cells. Ultrastructural analysis demonstrated the presence of large lysososmal-like vesicles in the BM, and also a discrete increase in the number of CG with diameters larger than 240 nm but not of giant CG. In addition, amperometric measurements of single-event exocytosis, using carbon fiber microelectrodes, showed no differences between the quantal size of secretory events from BM and wildtype or bovine chromaffin cells. Minor but significant differences were found between the kinetics of exocytosis in BM cells andwild-type mouse cells. We conclude that CG, but not the abnormal-sized vesicles found in BM chromaffin cells contribute to the catecholamine secretion and that abnormal secretory granules are not present in adrenergic cell lineage.     

The presence and localization of heat shock protein 70 in rat mast cells

Heat shock protein 70 (Hsp70) is considered not only as a cytosolic stress protein, but also as an extracellular molecule with immunomodulatory and signaling functions that play a role in adaptation to stress on cellular and systemic levels. The active involvement of mast cells in adaptation to stress may be associated with the presence of Hsp70 in secretory granules. Using immunoelectron microscopy, we showed that Hsp70 localized in secretory granules of rat pericardial and peritoneal mast cells. Localization of Hsp70 in rat perinoneal mast cells isolated by centrifugation on Percoll was confirmed by immunoblotting. The proposed involvement of mast cells in production of extracellular Hsp70 and possible functions of Hsp70 inside the mast cells granules are discussed.     

Presence of anaerobic bacteroides in aerobically grown microbial granules

Microbial granules were grown in a column-type sequential aerobic sludge blanket reactor inoculated with activated sludge flocs taken from a wastewater treatment plant and containing a medium with glucose as the main carbon source. The reactor selected for granules that could settle rapidly by employing a short settling time of 2 min. Matured granules with diameters between 2 and 3 mm were examined for anaerobic bacteria as their presence can signal the onset of diffusion limitation problems that can potentially diminish granule stability due to the bacterial production of fermentation gases and organic acids under anaerobic conditions. To detect the anaerobes in the granules, clones were constructed from 16S rRNA PCR amplicons. Two sequence types associated with a strict anaerobe Bacteroides spp. were identified from these clones. Fluorescence in situ hybridization (FISH) followed by confocal laser scanning microscopy (CLSM) demonstrated that cells of Bacteroides spp. were concentrated at a depth of approximately 800 mm below the surface of the granule. Cell enumeration using flow cytometry showed that the percentage of labeled cells of Bacteroides spp. compared to total bacterial cells in the granules was 0.56%. This is the first study to use a suite of culture-independent techniques to report the presence of a defined species of anaerobic bacteria in aerobically grown microbial granules.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cysteine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Structural changes occurring in interrenal tissue of the duck (Anas platyrhynchos) following adenohypophysectomy and treatment in vivo and in vitro with corticotropin

Adrenal glands from ACTH-treated intact ducks and chronically adenohypophysectomized ducks showed clear zonation into a subcapsular zone (SCZ) and an inner zone (IZ). Adenohypophysectomy caused ultrastructural changes in the IZ but not in the SCZ cells. These included increases in lipid droplets, changes in mitochondrial cristae from tubular to shelf-like, and changes in the shape of the nuclei from spherical to crenated. These changes were reversed by treatment with ACTH. Also, cells of the IZ, but not the SCZ, of adrenals from intact birds given ACTH showed more SER, more dense bodies, fewer lipid droplets and more prominent Golgi complexes. IZ cells incubated in buffer containing no ACTH developed mitochondria with shelf-like cristae and numerous opaque granules in the matrix. Exposure to buffer containing ACTH caused the mitochondrial cristae to become tubular and the matrix granules either decreased in number or disappeared. The granules could be extracted by incubating sections with chelating agents. The mitochondria in SCZ cells did not respond structurally to the presence of ACTH in the incubation medium but the matrix granules, like those in IZ cells, responded to the presence of chelating agents.     

Intergranular bridges in the anterior pituitary cell and their possible involvement in Ca2+-induced granule-granule fusion

Quick-freeze deep-etch electron microscopy showed the presence of bridge-like structures between adjacent secretory granules in rat anterior pituitary secretory cells. These intergranular bridges were variable in length and thickness. The finest bridges were 7–8 nm in length, while the longest ones were as long as 80 nm. Annexin II, one of the Ca²⁺-dependent phospholipid-binding proteins, is known to interlink between two membranes and induce aggregation of liposomes and chromaffin granules under the presence of Ca²⁺. In anterior pituitary cells, annexin II was detected by immunoelectron microscopy at the contact sites of secretory granules with other granules. The anterior pituitary cells treated under the presence of extracellular Ca²⁺ with Clostridium perfringens enterotoxin which induces Ca²⁺ influx showed multigranular exocytosis, i.e., multiple fusions of secretory granules with each other and with the plasma membrane. The granule-granule fusion in progress could be captured by the quick-freeze deep-etch technique. The membranes of adjacent secretory granules were partially fused at their contact sites where intergranular strands were no longer seen, while there existed intergranular strands between unfused portions of the granule membranes. From these results, we consider that the intergranular bridges, some of which may be composed of annexin II, are involved in Ca²⁺-induced granule-granule fusion in anterior pituitary cells.     

Localization of glutathione-insulin transhydrogenase (protein-disulfide interchange enzyme) in pancreas using immunocytochemistry, immunodiffusion, and enzymatic activity assay techniques

The localization of the protein-disulfide interchange enzyme, glutathione-insulin transhydrogenase (GIT), in rat and mouse pancreas was studied by protein A-gold immunocytochemistry, immunodiffusion, and assay of enzymatic activity. Immunocytochemistry on tissue sections using antibody to GIT and protein A-gold complex indicated the presence of GIT in alpha and beta cells in islets as well as acinar cells. The beta cells in obese (ob/ob) hyperinsulinemic mice showed increased GIT immunoreactivity. In both alpha and beta cells, GIT immunoreactive sites were associated predominantly with secretory granules. In pancreas from rats injected with glibenclamide, the degranulated beta cells contained GIT immunoreactive sites on the cisternal surface of the rough endoplasmic reticulum (RER). In acinar cells, the RER, Golgi elements, condensing vacuoles, and zymogen granules possessed GIT immunoreactive sites as did mitochondria. Immunocytochemistry on sections of isolated subcellular fractions showed that GIT was associated with different membranes. The enzymatic activity of GIT was found in the following order: Golgi elements greater than mitochondria greater than microsomes greater than zymogen granules greater than cytosol. In Ouchterlony immunodiffusion tests, each subcellular fraction showed a precipitin band which was continuous with that of purified GIT, a result indicating the presence of immunologically identical GIT in all fractions.     

Presence of calcium in polychaete cortical granules demonstrated by X-ray microanalysis on ultrathin cryo-sections of oocytes and eggs

Summary Cortical granules from fertilized eggs, oocytes and nurse cells of Ophryotrocha labronica have been analyzed for the presence of calcium using cryo-ultramicrotomy and X-ray microprobe analysis. All cortical granules showed a significant peak for calcium, but yolk granules were without calcium. These results support the hypothesis that the discharge of cortical granules shortly after fertilization is a self-propagating phenomenon involving the diffusion of Ca²⁺ from bursting granules.     

The lipofuscin in neuroglial cells of the optic nerve of Formosan rock-monkey (Macaca cyclopis)

The ultrastructure of lipofuscin granules in neuroglial cells of the optic nerve of the Formosan Rock-Monkey was investigated by electron microscopy. In the cytoplasm of astroglial cells, numerous irregular lipofuscin granules were characterized by the presence of large lipid droplets, small electron-dense pigment granules, and some lamellar structures. The lipofuscin granules of the oligodendroglial cells were composed largely of dense, coarse pigment granules, multilinear structures, and a few small lipid droplets. The lipofuscin granules in microglial cells were characterized by numerous lipid droplets in various sizes, small electron-dense pigment granules, and prominent lamellar structures. It was reported that the lipofuscin granules are wear-and-tear materials and products from the cells in lower functional activity. However, our observations suggest that the presence of lipofuscin granules in the neuroglial cells of the optic nerve is likely a characteristic product of active phagocytosis.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Summary Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cystine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Protein kinase C-dependent supply of secretory granules to the plasma membrane

To elucidate the mechanism for supplying secretory granules to the cell membrane, chromaffin cells isolated from the bovine adrenal medulla were observed by the evanescent wave microscopy after staining their granules with acridine orange. The secretory granules showed only a very small fluctuation, indicating their docking to the plasma membrane. The rate and range of movement increased greatly by application of botulinum toxin A or C. The number of secretory granules docked to the plasma membrane significantly decreased by botulinum toxin C. Conversely, the number increased greatly by activation of protein kinase C with phorbol 12,13-dibutyrate (PDBu). In the presence of an anti-actin reagent cytochalasin D, no increasing effect of PDBu on the number of docked granules was observed. While in the presence of an anti-mitotic reagent, colchicine, a clear increasing effect of PDBu was observed. The final step for supplying granules to the plasma membrane in endocrine cells is concluded to be mediated by a phosphorylation-dependent and actin-based transport system.     

Occurence and cytodifferentiation of mucopolysaccharide secreting cells in the pancreas of children with nesidioblastosis.

Large numbers of mucopolysaccharide secreting cells were found in the pancreatic tissue of children with nesidioblastosis. Ultrastructural studies showed that mucus cells contained secretion granules and a characteristic smooth endoplasmic reticulum composed of an array of anastomosed tubules. The periodic acid - thiocarbohydrazide - silver reaction demonstrated the presence of glycogen in the hyaloplasm and of polysaccharides in secretion granules, the Golgi apparatus and in vesicles. A hypothesis is proposed, according to which mucus cells differentiate from a pancreatic stem cell common to both endocrine and exocrine tissues, through a mitochondria-rich intermediate cell stage.