Morphological heterogeneity among fractionated alveolar macrophages in their release of lysosomal enzymes

When coupled with separation of alveolar macrophages (AM) into four different density fractions (I, II, III and IV) by discontinuous Percoll gradient centrifugation, ultrastructural heterogeneity was evident in secreting process of lysosomal enzymes. Lower dense AM (I and II) released high levels of acid phosphatase and cathepsin B, whereas higher dense ones (III and IV) did not. Ultrastructurally, there were multiple ruffling and active extension of long cytoplasmic processes from one pole or around the cell surface of AM obtained from the higher density fractions. In contrast, AM from lower dense fractions had much less cytoplasmic processes and contained more cytoplasmic vacuoles showing positive reactions of acid phosphatase. These cells featured more frequently round or ovoid knobs with acid phosphatase activity along and from the tips of the cytoplasmic processes, suggestive of exocytosis. It was suggested that these ultrastructural changes linked to the maturation process and release of lysosomal enzymes from differentiated AM.     

Histochemical and biochemical studies on the localization and activity of lysosomal enzymes in fracture callus

Summary Quantitative biochemical studies on the activities of four lysosomal hydrolases during different stages of fracture healing in the rat were performed, and the results obtained were integrated with those of histochemical observations relating to changes in the localization of acid phosphatase in the same tissue.The findings showed presence of all the four lysosomal enzymes assayed in the callus; during early callus formation the enzyme activities calculated on a DNA basis increased up to about 12 days after the fracture. The enzyme activities appeared to be roughly reflected histochemically by the acid phosphatase staining. The increasing activity during early callus formation seemed to depend on the presence of numerous macrophage-like cells in the tissue containing many large lysosomes. A decrease in enzyme activity was found after day 12. Comparison with the histochemical and ultrastructural findings suggested that this decrease was due to a reduction in the number of macrophage-like cells and a concomitant increase in osteogenic cells with a lower enzyme content.     

Histological localization of hydrolases in the epididymis of the dog]

Localization and activity of five hydrolases (alkaline phosphatase, adenosine triphosphatase, acid phosphatase, nonspecific esterase and leucylamino-peptidase) were evaluated histochemically in the epididymides of mature dogs. In the ductuli efferentes, cilia and apical parts of the epithelial cells displayed high activity of alkaline phosphatase and adenosine triphosphatase. Strong activity of acid phosphatase, nonspecific esterase and leucylamino-peptidase was present in the basal and supranuclear zones of the epithelium of the ductuli efferentes. Stereocilia of all three segments of the ductus epididymidis showed a high activity of alkaline phosphatase. Positive adenosine triphosphatase reaction was confined to the stereocilia of the initial segment. A complex pattern of acid phosphatase activity was observed in the middle segment. The subdivision of the middle segment in four subsegments was therefore suggested. In the epithelium of the initial segment only a few nonspecific esterase-positive cells were seen. The infranuclear and basal areas of the epithelium in the middle segment and the supranuclear zone of the terminal segment displayed distinct nonspecific esterase activity. The possible contribution of the hydrolases to the function of the epididymis is discussed.     

Localization of acid phosphatase in protoplasts from Saccharomyces cerevisiae.

The localization of acid phosphatase (EC 3.1.3.2) in secreting protoplasts prepared from Saccharomyces cerevisiae is reported for the first time. Using a Gomori technique we were able to show acid phosphatase at those organelles in the protoplasts which are generally involved in the processes of biosynthesis and secretion of glycoproteins in eukaryotic cells.     

Prolactin secreting cells in the hypophysis of the brown spiny mouseMus platythrix (Bennett)

Prolactin secreting cells are identified in thepars distalis of Mus platythrix by conventional methods of light and electron microscopy. Two types of prolactin secreting cells are recognised. These types are estrone-sensitive, mammotrophic type I, and luteotrophic type II, respectively. Histochemical analysis revealed that the cells are rich in RNA, basic proteins, alkaline phosphatase and are resistant to extraction with 0·5% trichloroacetic acid. Quantitative data showed that the prolactin secreting cells increase during pregnancy, lactation and estrone treatment. Estrone at low dose levels caused immense hyperplasia whereas at higher levels there was no corresponding increase in the percentage of type I cells. Ultrastructurally, prolactin secreting cells are characterised by the presence of stacked endoplasmic reticulum, oval or irregular secretory granules. The Golgi apparatus is seen rich in vacuolar system.     

Morphological phenotypes and functional capabilities of submandibular parenchymal cells of the ferret investigated by protein, mucosubstance and enzyme histochemistry

Submandibular glands obtained post-mortem from mature ferrets of both sexes were examined with the use of light microscopical histochemical methods for proteins, mucosubstances and enzymes associated with cell functions or organelles. Demilunar cells showed carboxylated mucosubstances that were mainly non-sulphated, and diffuse activity for peroxidase, E600-sensitive esterase and acid phosphatase. Thiol groups were also detected in these cells. Central acinar cells showed sulphated mucosubstances, disulphides and reticular staining for thiamine pyrophosphatase. Intercalary ducts showed diffuse activity for NADH and NAD(P)H dehydrogenases. Striated ducts contained protein, tryptophan, disulphides, neutral mucosubstances and E600-sensitive esterase periluminally. Basally, the striated ductal cells showed variable activity for peroxidase, cytochrome oxidase, succinate dehydrogenase and acid phosphatase. Basolateral plasma membranes of these cells exhibited ouabain-sensitive Na,K-ATPase activity. The collecting ducts were characterized by variable periluminal staining for acid phosphatase, -glucuronidase, acid -galactosidase and E600-resistant esterase. The results suggest that the histological appearances of the acini of the submandibular gland of the ferret are dependent on the synthesis of secretory acid glycoproteins, that the striated ducts are involved with the secretion of tryptophan-rich product comprising neutral glycoproteins and showing esterase activity and with marked transport of ions and that the collecting ducts are involved with absorption.     

Histochemical localization of phosphomonoesterases in Avitellina lahorea Woodland, 1927 (Cestoda: Anoplocephalida)

Non-specific and specific phosphatases have been histochemically localized in the tissues of Avitellina lahorea, an intestinal parasite of sheep and goats. Large quantities of acid phosphatase, alkaline phosphatase and adenosine triphosphatase were observed in almost all organs except the parenchyma where there were moderate amounts of acid phosphatase and no alkaline phosphatase; the reproductive ducts contained moderate amounts of alkaline phosphatase. 5-nucleotidase was observed only in the uterus, egg pouches and eggs and glucose-6-phosphatase activity was restricted to the tegument. The probable functions of these moieties at different sites are discussed.     

Salivary glands of the cat: A histochemical study

Synopsis The submandibular, sublingual and parotid glands of the cat have been studied. Mucosubstance histochemistry demonstrated acidic mucosubstances with varying properties in the acini. Thiamine pyrophosphatase and nucleoside diphosphatase reaction products were seen with a Golgi-like appearance in acinar cells. Granules of acid phosphatase, -glucuronidase and E600-resistant esterase reaction products, presumably representing lysosomal enzyme activities, were seen in acinar and ductal cells. Diffuse acid phosphatase and -glucuronidase reaction products were seen in central cells of the submandibular acini, and diffuse non-specific esterase reaction product was seen in acinar and ductal cells. Arylamidase reaction product was associated with some acinar cells. Reaction product from a peroxidase technique was seen in demilunar cells of the submandibular acini, in parts of the sublingual acini, in parotid acini, and in ductal cells. Cytochrome oxidase and succinate dehydrogenase reaction products were seen most strongly in striated ducts, whereas NADH- and NADPH-diaphorase reaction products were seen at a high level throughout the ducts.     

Morphocytochemical and electron microscopic research on murine hybridoma cells producing and not producing monoclonal antibodies

Cytochemical and ultrastructural features of mouse hybridomas and also of the parental cells--myeloma P3-X63-Ag8.653 and spleen cells of the Balb/c mice immunized with cell line RPMI-1788 have been studied. Differences in cytomorphological signs and activity of acid phosphatase, acid nonspecific esterase, nonspecific-alpha-naphthyl acetate esterase were shown in hybrid cell lines secreting and not secreting monoclonal antibodies.     

Ultrastructural phosphatase histochemistry of submandibular and parotid salivary glands of man

Summary Thiamine pyrophosphatase was demonstrated in the Golgi complex and acid phosphatase in the GERL of acinar cells of submandibular and parotid glands and were previously demonstrated in cells of intercalary ducts. Thiamine pyrophosphatase was also demonstrated in the Golgi complex of cells of striated and excretory ducts and myoepithelial cells. Acid phosphatase was also demonstrated in lysosomes. Alkaline phosphatase was rarely demonstrated light microscopically at luminal surfaces of striated and excretory ducts and electron microscopically in luminal vesicles in cells of striated ducts. The demonstration of the phosphatases in Golgi complexes and GERLs indicates that investigations on these structures in experimental animals are relevant to human salivary glands and supports the opinion that ductal cells as well as acinar cells secrete organic material. The presence of alkaline phosphatase at luminal surfaces of striated and excretory ducts suggests that resorption as well as secretion may occur in them.     

Alkaline phosphatase and myoepithelial cells in the parotid gland of the rat

Synopsis Alkaline phosphatase activity has been studied in the parotid glands of rats at the light and electron microscopical levels. Reaction product was found to outline the plasma membranes of myoepithelial cells. It was also found in the walls of many capillaries and on the luminal surface and between apposing cells in some intercallary ducts.The distribution of myoepithelial cells in the rat parotid is unusual. The cells run longitudinally around intercalary ducts and send processes on to the bases of adjoining acini but do not embrace the acini. The possible functions of these myoepithelial cells are discussed.     

Ascorbic acid induces alkaline phosphatase, type X collagen, and calcium deposition in cultured chick chondrocytes

During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic chondrocytes, which then deposit a mineralized matrix to form calcified cartilage. Chondrocyte hypertrophy and matrix mineralization are associated with expression of type X collagen and the induction of high levels of the bone/liver/kidney isozyme of alkaline phosphatase. To determine what role vitamin C plays in these processes, chondrocytes derived from the cephalic portion of 14-day chick embryo sternae were grown in the absence or presence of exogenous ascorbic acid. Control untreated cells displayed low levels of type X collagen and alkaline phosphatase activity throughout the culture period. However, cells grown in the presence of ascorbic acid produced increasing levels of alkaline phosphatase activity and type X collagen mRNA and protein. Both alkaline phosphatase activity and type X collagen mRNA levels began to increase within 24 h of ascorbate treatment; by 9 days, the levels of both alkaline phosphatase activity and type X collagen mRNA were 15-20-fold higher than in non-ascorbate-treated cells. Ascorbate treatment also increased calcium deposition in the cell layer and decreased the levels of types II and IX collagen mRNAs; these effects lagged significantly behind the elevation of alkaline phosphatase and type X collagen. Addition of beta-glycerophosphate to the medium increased calcium deposition in the presence of ascorbate but had no effect on levels of collagen mRNAs or alkaline phosphatase. The results suggest that vitamin C may play an important role in endochondral bone formation by modulating gene expression in hypertrophic chondrocytes.     

Development of an autophagic system in differentiating cells of the cellular slime moldDictyostelium discoideum

Summary Changes in an autophagic system during differentiation of cells ofDictyostelium discoideum, NC-4 were studied under light and electron microscopes, and it was demonstrated cytochemically that acid phosphatase was almost exclusively localized in food and autophagic vacuoles. Autophagic vacuoles first appeared during formation of loose aggregates, coupled with the defecation of food vacuoles. Autophagic vacuoles seem to originate from flat sacs which segregate parts of the cytoplasm. No acid phosphatase was detected in the vacuoles when first formed, but activity appeared later probably due to fusion with Golgi-like vesicles. When starved cells were not allowed to aggregate due to a low cell density, they formed no autophagic vacuoles but retained many food vacuoles. This indicates that the formation of autophagic vacuoles is not simply due to starvation, but to cell interaction mediated by cell contact. Autophagic vacuoles containing acid phosphatase rapidly increased in number in all cells in the early stage of aggregation. After papillae formed, however, they selectively decreased in the prespore cells, but developed further and grew larger in the prestalk cells.     

Morphological and histochemical studies on the parotid gland of Praomys (Mastomys) natalensis

The parotid gland of Praomys (Mastomys) natalensis follows the same anatomical pattern of Rodentia since no peculiar morphological and histochemical features were found in the present study that differentiate it from homologous glands in other rodents. It consists of serous secreting components and intralobular ducts that demonstrate no sexual dimorphism. Histochemical studies reveal that the glandular cells of the Praomys' parotid gland contain no acid mucosubstance, but that material of a neutral glycoprotein nature can be found in the secreting cells of acini, epithelium of the ducts, and the ductal lumina.     

Histochemical study of enzyme patterns in the human submaxillary gland

Summary The histochemical distribution of various enzymes, such as alkaline phosphatase, acid phosphatase, esterase, -glycosidase, aminopeptidase, succinic dehydrogenese and TPN diaphorase, in human submaxillary glands has been determined.Acini and ducts of human submaxillary gland were devoid of alkaline phosphatase activity, but this enzyme was observed in capillaries and somewhat in myoepithelium.Activities of acid phosphatase, esterase, -glucuronidase and -galactosidase were generally observed in the entire cytoplasm of serous acini; but the cytoplasm of mucous acini was either negative or showed only trace amounts.Aminopeptidase reaction of both acini and ducts was generally negative.Succinic dehydrogenase and TPN diaphorase activities were strongly active in intralobuler ducts. Serous acini exhibited less activity with these enzymes; and mucous cells showed still less and were almost negative. In serous acini, there was much greater activity of TPN diaphorase than of succinic dehydrogenase.With 7 Figures in the Text     

Ultrastructural observations on the intraodontoblastic collagen fibrils of the mouse tooth germs

We examined electron-microscopically and histochemically the ultrastructural features of the intraodontoblastic collagen fibrils of the mouse. These collagen fibrils were most common in secreting odontoblasts (pre-odontoblasts) of the maturating stages. In such cells they were most numerous at the peripheral zone of the Golgi apparatus, and were sometimes seen in odontoblastic processes. Intraodontoblastic collagen fibrils also had morphological variations including a banded structure enclosed by limiting membranes of vacuoles, fusion with primary lysosomes, and an electron-dense material covering with a structure that was not banded. Study of acid phosphatase activity showed that these structural changes were caused by the degradation of intraodontoblastic collagen fibrils by lysosomes. The results of studies of the permeation of lanthanum nitrate and the alkaline phosphatase reaction showed that these collagen fibrils were separate from the extracellular matrix and that there was no phagocytosis of the odontoblasts.     

Regional and Subcellular Distribution in Mammalian Brain of the Enzymes Producing Adenosine

The activities of 5'-nucleotidase, 2'-nucleotidase, alkaline phosphatase, and acid phosphatase were measured in rat and autopsied human brains. The four phosphatases in the rat brain showed little change in activity after death. The activities of adenosine-producing enzymes were compared in various parts of rat and human brains. When phosphatase activity was measured at pH 7.5, 5'-nucleotidase showed the highest activity in the most parts of the brain. The activity of 2'-nucleotidase and that of nonspecific phosphatase were almost the same at pH 7.5. However, higher phosphatase activity was observed in all parts of the brain when nonspecific phosphatase activity was measured at pH 10.0 or 5.5. High specific activity of 5'-nucleotidase in the brain was detected in the membranous components, especially in the synaptic membranes. The activity of 2'-nucleotidase was distributed in the soluble and synaptosomal fractions. The highest activity of both alkaline and acid phosphatases was recovered in the crude mitochondrial fraction, with the highest specific activity in the microsomal fraction. Phosphatase activity was distributed widely in the rat brain. The activity of 5'-nucleotidase was high in the medulla oblongata, thalamus, and hippocampus, but low in the peripheral nerve, spinal cord, and occipital lobe. The activity of 2'-nucleotidase was high in the vermis and frontal lobe. The highest acid and alkaline phosphatase activities were detected in the frontal lobe and in the olfactory bulb, respectively. The distribution of the four phosphatases in the autopsied human brain was similar to that in the rat brain. The highest 5'-nucleotidase activity was observed in the temporal lobe and thalamus.(ABSTRACT TRUNCATED AT 250 WORDS)     

The fine localization of acid phosphatase activity in the unvacuolated notochordal cells of the early chick embryo

Summary The electron microscopical localization of acid phosphatase activity was investigated in ultra-thin and semi-thin sections of unvacuolated notochordal cells of chick embryos from stages 9 to 14 (as defined by Hamburger & Hamilton). At stage 9, many notochordal cells show a lightly positive reaction for acid phosphatase activity. Thereafter, the acid phosphatase-positive cells of the notochord increase in number and, at stage 14, the reaction products for the enzyme are distributed throughout almost all the cisternae of the nuclear envelope and a well-differentiated endoplasmic reticulum, the parallel cisternal and reticular parts of the Golgi complex, and various lysosomes in nearly all notochordal cells. In the cisternae of the nuclear envelope and endoplasmic reticulum, the acid phosphatase reaction products are in a fine granular form. In the outermost layer of the cisternal parts of the Golgi complex, faint lead deposits similar to those in the endoplasmic reticulum are found, but in other cisternal and reticular regions which may correspond to the GERL, considerable amounts of reaction products are present. Knob-like projections are also seen protruding from the reticular parts of the Golgi complex. These results suggest that, at least up to stage 14, the notochordal cells are actively synthesizing acid phosphatase which is directly transported from the endoplasmic reticulum to the Golgi complex. The enzyme may be accumulated by the Golgi complex from which primary lysosomes are formed. Furthermore, the pattern of the ultrastructural localization of acid phosphatase activity in embryonic notochordal cells of the chick differs from that of adult cells of other animals.     

The multiple functions of protein phosphatase 6

This review presents the accumulating evidence for the roles of protein phosphatase 6 (PP6) in cell cycle, DNA damage repair, inflammatory signaling, lymphocyte development, virus infection, tumor formation/progression, cell/tissue size, and non-coding RNA-mediated regulation. PP6 is an evolutionarily conserved and ubiquitously expressed Ser/Thr protein phosphatase most closely related to protein phosphatase 2A (PP2A) and protein phosphatase 4 (PP4). Although abundantly expressed in cells with multiple roles in cellular signaling, PP6 has received less attention than its close relative PP2A. Many studies used okadaic acid as “PP2A” inhibitor, even though these toxins also inhibit PP6 activity, so effects of the inhibitor could have been due to inhibition of both phosphatases. PP6 has its own dedicated subunits that assemble into heterotrimers that presumably fulfill its discrete functions in cells.     

Relationship between phosphatase activity and cytotoxic effect of two protein phosphatase inhibitors, okadaic acid and pervanadate, on human myeloid leukemia cell line

Protein phosphatases are signalling molecules that regulate a variety of fundamental cellular processes including cell growth, metabolism and apoptosis. The aim of this work was to correlate the cytotoxicity of pervanadate and okadaic acid on HL60 cells and their effect on the phosphatase obtained from these cells. The cytotoxicity of these protein phosphatase inhibitors was evaluated on HL60 cells using phosphatase activity, protein quantification and MTT reduction as indices. The major phosphatase presents in the cellular extract showed high activity (80%) and affinity (Km = 0.08 mM) to tyrosine phosphate in relation to p-nitrophenyl phosphate (pNPP)-(Km = 0.51 mM). Total phosphatase (pNPP) was inhibited in the presence of 10 mM vanadate (98%), 200 microM pervanadate (95%) and 100 microM p-chloromercuribenzoate (80%) but okadaic acid caused a slight increase in enzyme activity (25%). When the HL60 cells were treated with the phosphatase inhibitors (pervanadate and okadaic acid) for 24hours, only 20% residual activity was observed in presence of 200 microM pervanadate, whereas in the presence of okadaic acid this inhibitory effect was not observed. However, in respect to mitochondrial function, cell viability decreased about 80% in the presence of 100 nM okadaic acid. The total protein content was decreased 25% when the cells were treated with 100 nM okadaic acid in combination with 200 microM pervanadate. Our results suggest that both phosphatase inhibitors presented different mechanisms of action on HL60 cells. However, their effect on the cell redox status have to be considered.