scholarly journals PHOSPHATASES OF EPIPHYSEAL CARTILAGE STUDIED BY ELECTRON MICROSCOPIC CYTOCHEMICAL METHODS

1971 ◽  
Vol 19 (12) ◽  
pp. 801-808 ◽  
Author(s):  
TATSUJI MATSUZAWA ◽  
H. CLARKE ANDERSON
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Plasma Membranes ◽  
Matrix Vesicles ◽  
Electron Microscopic ◽  
Previous Suggestion ◽  
Hypertrophic Zone ◽  
Dense Bodies ◽  
The Matrix ◽  
Epiphyseal Plates

The presence and distribution of alkaline phosphatase, adenosine triphosphatase (ATPase) and acid phosphatase activities in the epiphyseal plates of young mice were studied by electron microscopic cytochemical methods. Alkaline phosphatase and ATPase activities were associated with the plasma membranes of chondrocytes and with the investing membranes of matrix vesicles. These vesicles contain the earliest recognized deposits of hydroxyapatite and may promote calcification through an active process. Alkaline phosphatase and ATPase activities were greatest in the hypertrophic zone of the epiphysis, which is an area of beginning calcification. Acid phosphatase activity was demonstrable in the cytoplasm of chondrocytes in association with dense bodies which were larger than matrix vesicles and were devoid of alkaline phosphatase and ATPase activities. These cytoplasmic lysosome-like bodies were slightly more numerous in the hypertrophic zone but disappeared in the underlying zone of chondrocyte degeneration and matrix calcification. Our observations do not support a previous suggestion by others that matrix vesicles represent lysosomes. The presence of ATPase and alkaline phosphatase is compatible with an enzymatic calcium- and/or phosphate-concentrating mechanism in the matrix vesicles.

scholarly journals Demonstration of lysosomal and extralysosomal sites for acid phosphatase in mouse kidney tubule cells with p-nitrophenylphosphate lead-salt technique.

1975 ◽  
Vol 23 (6) ◽  
pp. 439-451 ◽  
Author(s):  
H Miyayama ◽  
R Solomon ◽  
M Sasaki ◽  
C W Lin ◽  
W H Fishman
Keyword(s):  
Endoplasmic Reticulum ◽  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Epithelial Cells ◽  
Normal Mouse ◽  
Plasma Membranes ◽  
Mouse Kidney ◽  
Lead Salt ◽  
Acetate Buffer ◽  
Electron Microscopic

Dual localization of acid phosphatase in lysosomal and extralysosomal sites of the tubule epithelial cells of normal mouse kidney was observed at the light and electron microscope level using a modified Gomori lead-salt method with p-nitrophenylphosphate (pNPP) as substrate. Based on previous biochemical and cytochemical findings, we developed optimal conditions for the enzyme activity in extralysosomal sites. The conditions used for the light microscopic level consisted of 1.5 mM PNPP, 2.0 MM Pb(NO3)2 and 0.05 M acetate buffer (pH 5.8). Those for the electron microscopic study required 3.0 mM PNPP, 3.6 MM Pb(NO3)2 and 0.1 M acetate buffer (pH 5.8). This modified lead-salt technique was highly specific and provided a suitable method for the demonstration of nonlysosomal as well as lysosomal sites of acid phosphatase activity in the tubule epithelial cells of normal mouse kidney. As expected, the enzyme activity appeared in the lysosomes, but the prominent reaction in the brush border, the rough endoplasmic reticulum and basal infolding plasma membranes was not anticipated. We were able to demonstrate in situ organelle precursors of microsomal acid phosphatase such as endoplasmic reticulum, plasma membrane and basal infolding membranes showing the same substrate preference, which had been observed previously in biochemical studies in our laboratory. Since the possible participation of alkaline phosphatases, K+-pNPPase or Na+-K+-adenosine triphosphatase was ruled out by use of appropriate inhibitors, the enzyme-reactive sites can be interpreted as reflecting nonspecific acid phosphatase.

scholarly journals ELECTRON MICROSCOPIC LOCALIZATION OF ACID PHOSPHATASE AND THIAMINE PYROPHOSPHATASE ACTIVITY IN HYPOTHALAMIC NEUROSECRETORY CELLS OF THE RAT

1964 ◽  
Vol 21 (1) ◽  
pp. 35-47 ◽  
Author(s):  
Joseph Osinchak
Keyword(s):  
Acid Phosphatase ◽  
Osmium Tetroxide ◽  
Neurosecretory Cells ◽  
Electron Microscopic ◽  
Vascular Perfusion ◽  
Dense Bodies ◽  
Thiamine Pyrophosphatase ◽  
Pyrophosphatase Activity ◽  
Supraoptic Nuclei ◽  
Electron Microscopic Localization

Supraoptic nuclei in the hypothalamus of rats were fixed for the electron microscope by vascular perfusion with solutions of glutaraldehyde followed by post fixation with osmium tetroxide. Cytochemical methods for detection of acid phosphatase and thiamine pyrophosphatase activity have been applied to glutaraldehyde-fixed frozen sections containing the neurosecretory cells. The enzyme activities have been localized to certain Golgi cisternae. Acid phosphatase activity is present in the large (0.4 µ to 1.0 µ) granules or dense bodies which are surrounded by a single limiting membrane; both features characterize these structures as lysosomes. Smaller (0.1 µ) granules also present in the perikarya are generally unreactive towards enzyme activity and resemble in form the neurosecretory granules in the neurohypophysis.

scholarly journals Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca2+-binding glycoprotein.

1986 ◽  
Vol 103 (4) ◽  
pp. 1615-1623 ◽  
Author(s):  
B de Bernard ◽  
P Bianco ◽  
E Bonucci ◽  
M Costantini ◽  
G C Lunazzi ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Protein A ◽  
Matrix Vesicles ◽  
Immune Reactivity ◽  
The Matrix ◽  
Immunohistochemical Evidence

A glycoprotein that exhibits alkaline phosphatase activity and binds Ca2+ with high affinity has been extracted and purified from cartilage matrix vesicles by fast protein liquid chromatography. Antibodies against this glycoprotein were used to analyze its distribution in chondrocytes and in the matrix of calcifying cartilage. Under the light microscope, using immunoperoxidase or immunofluorescence techniques, the glycoprotein is localized in chondrocytes of the resting zone. At this level, the extracellular matrix does not show any reaction. In the cartilage plate, between the proliferating and the hypertrophic region, a weak immune reactivity is seen in the cytoplasm, whereas in the intercolumnar matrix the collagen fibers appear clearly stained. Stained granular structures, distributed with a pattern similar to that of matrix vesicles, are also visible. Calcified matrix is the most stained area. These results were confirmed under the electron microscope using both immunoperoxidase and protein A-gold techniques. In parallel studies, enzyme activity was also analyzed by histochemical methods. Whereas resting cartilage, the intercellular matrix of the resting zone, and calcified matrix do not exhibit any enzyme activity, the zones of maturing and hypertrophic chondrocytes are highly reactive. Some weak reactivity is also shown by chondrocytes of the resting zone. The observation that this glycoprotein (which binds Ca2+ and has alkaline phosphatase activity) is synthesized in chondrocytes and is exported to the extracellular matrix at the time when calcification begins, suggests that it plays a specific role in the process of calcification.

scholarly journals FINE STRUCTURAL LOCALIZATION OF ACID AND ALKALINE PHOSPHATASES IN CELLS OF RABBIT BLOOD AND BONE MARROW

1967 ◽  
Vol 15 (6) ◽  
pp. 311-334 ◽  
Author(s):  
B. K. WETZEL ◽  
S. S. SPICER ◽  
R. G. HORN
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Acid Phosphatase Activity ◽  
Mononuclear Cells ◽  
Nuclear Staining ◽  
Alkaline Phosphatases ◽  
Structural Localization ◽  
Dense Granules ◽  
Dense Bodies

In rabbit heterophils, acid phosphatase activity occurs in primary (azurophil) granules which predominate in early cells and persist in mature cells and in tertiary granules which are seen only in mature cells. Alkaline phosphatase activity occurs in secondary granules which appear in intermediate heterophils and later predominate in mature cells. Acid phosphatase activity in heterophil Golgi zones coincides developmentally with the genesis of primary and, later, tertiary granules, whereas alkaline phosphatase in the Golgi complex coincides with secondary granulogenesis. In developing eosinophils, acid phosphatase reaction product occurs in Golgi elements, rims the spherical precursors of angular, mature granules and appears inconsistently within mature granules. Basophil myelocytes show acid phosphatase in Golgi elements but not in specific granules. Additional acid phosphatase reactive structures include: granules of mononuclear cells; phagocytic vacuoles in macrophages; autophagic vacuoles in maturing erythroid cells; small dense granules of platelets; dense bodies in lipocytes; and Golgi elements of mononuclear cells, macrophages, nucleated red cells, megakaryocytes and lipocytes. Localized deposits were absent in control specimens except for enzyme-independent nuclear staining in alkaline phosphatase preparations.

scholarly journals Electron Microscopic Studies on Ovarian Oocytes and Unfertilized Tubal Ova in the Rat

1960 ◽  
Vol 7 (3) ◽  
pp. 567-574 ◽  
Author(s):  
D. Louise Odor
Keyword(s):  
Granulosa Cells ◽  
Plasma Membranes ◽  
Developmental Stages ◽  
Structural Characteristics ◽  
Polar Body ◽  
Electron Microscopic ◽  
Space Forms ◽  
First Polar Body ◽  
The Matrix ◽  
Microscopic Studies

Developing rat ova have been studied with the electron microscope. Special attention was paid to relations of ova to the granulosa cells, the developmental stages of ovarian follicles, and the cytology of the unfertilized tubal ova. The relationship of the oocyte to the surrounding granulosa cells was found to change from one of a simple apposition of the plasma membranes to a complex interdigitation of microvilli from the ovular surface and processes from the granulosa cells extending into the matrix of the zona pellucida. This complex interrelation is maintained until the formation of the first polar body is initiated. At this time no microvilli are found and the oolemma presents a gently undulating outline. Also at this time, a perivitelline space forms and the granulosa cell processes retract. In the unfertilized tubal ova no microvilli are present and the processes of the follicular cells are completely withdrawn. The cytoplasmic elements of the oocyte in various stages of development are described in some detail. Of particular interest is the change noted in position and degree of aggregation of the Golgi complex as maturation proceeds. The distribution and structural characteristics of the mitochondria, ergastoplasm, dense particles, and multivesicular bodies are described.

scholarly journals COMBINED CYTOCHEMICAL AND ELECTRON MICROSCOPIC DEMONSTRATION OF ß-GLUCURONIDASE ACTIVITY IN RAT LIVER WITH THE USE OF A SIMULTANEOUS COUPLING AZO DYE TECHNIQUE

1968 ◽  
Vol 36 (2) ◽  
pp. 289-297 ◽  
Author(s):  
Masando Hayashi ◽  
Tsuranobu Shirahama ◽  
Alan S. Cohen
Keyword(s):  
Electron Microscopy ◽  
Rat Liver ◽  
Dense Body ◽  
Electron Microscopic ◽  
Glucuronidase Activity ◽  
Dense Bodies ◽  
The Matrix ◽  
Matrix Control ◽  
Parenchymal Cells ◽  
Cytochemical Technique

Rat liver was fixed in formal-cacodylate-sucrose and frozen sections were incubated in a simultaneous-coupling medium containing naphthol AS-BI glucuronide as substrate and hexazonium pararosanilin as the diazo reagent. By light microscopy, the sections demonstrated ß-glucuronidase activity as red discrete granules in the pericanalicular cytoplasm and as a generalized cytoplasmic stain in the parenchymal cells. Brief treatment of sections in cold ethanol prior to incubation markedly enhanced the staining for the enzyme and made it possible to demonstrate sufficient amounts of the reaction product in sections embedded in epoxy resin following dehydration and propylene oxide treatment. Electron microscopy revealed that the reaction product was moderately electron opaque and deposited in greater amounts in the vacuolated dense bodies and occasionally in the dense bodies which did not show obvious vacuoles. In each dense body, the deposits occurred preferentially at the edge as well as in the area surrounding the vacuoles in the matrix. Control sections incubated in the presence of glucosaccharo-1:4-lactone were devoid of the reaction product. No deposits of the reaction product were found in the nucleus, mitochondria, or microbodies. The limitations of the present cytochemical technique for use in electron microscopy are briefly discussed.

scholarly journals ELECTRON MICROSCOPIC LOCALIZATION OF GLYCOPROTEINS IN PITUITARY CELLS OF DUCK AND QUAIL

1971 ◽  
Vol 19 (12) ◽  
pp. 775-797 ◽  
Author(s):  
ANDRÉE TIXIER-VIDAL ◽  
RENÉE PICART
Keyword(s):  
Endoplasmic Reticulum ◽  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Acid Phosphatase Activity ◽  
Secretory Granules ◽  
Periodic Acid ◽  
Pituitary Cells ◽  
Electron Microscopic ◽  
Gonadotropic Cells ◽  
Dense Bodies

Structures demonstrating the presence of glycoproteins, acid phosphatase activity and OsO4 impregnation were localized by means of the electron microscope in duck and in quail pituitary cells. Two methods for the electron microscopic demonstration of glycoproteins were used: a chromic acid-phosphotungstic acid mixture on glycol-methacrylate-embedded tissues, and the periodic acid-thiocarbohydrazide-silver proteinate technique. Both methods showed glycoproteins in the following sites: ( a) the secretory granules in three types of cells (A, B, C) which are part of the seven different cells of the avian pituitary; ( b) the several kinds of dense bodies which are richer in reaction product than the secretory granules. A correlation with previous studies on similar species of birds is helpful in identifying each of the three positive types of cells as thyrotropic cell (A), prolactin cell (B) and gonadotropic cell (C). The presence of glycoproteins within the Golgi saccules (on condensing granules) was found with the periodic acid-thiocarbohydrazide-silver proteinate method in these gonadotropic cells only. In gonadotropic and thyrotropic cells, acid phosphatase activity is weak in the inner Golgi saccules and strong in the "Golgi Endoplasmic Reticulum Lysosomes" system, in the lysosomes, in the dense bodies and in the vacuolated dense bodies. The structures which are richest in glycoproteins are also those which have the most acid phosphatase activity. On the contrary, OsO4-stained structures in duck gonadotropic cells (nuclear pericisterna, rough endoplasmic reticulum, cisternae and outer Golgi saccules) have no glycoproteins or acid phosphatase activity.

Acid and Alkaline Phosphatase Activity in Axenically Grown Physarum Myxamoebae

1980 ◽  
Vol 38 ◽  
pp. 488-489
Author(s):  
Henry C. Aldrich
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Mammalian Cells ◽  
Cellular Localization ◽  
Plasma Membranes ◽  
Cell Fractionation ◽  
Intact Cells ◽  
E Coli ◽  
Food Vacuoles ◽  
Alkaline And Acid Phosphatase

In preparation for biochemical studies of isolated plasma membranes of myxamoebae of the mycetozoan Physarum polycephalum, histochemical studies were undertaken to determine the cellular localization of alkaline and acid phosphatase activities. In a previous paper, Kazama and Aldrich1 showed that myxamoebae of Physarum, when grown with E. coli as the food source, synthesize acid phosphatase in Golgi apparatus and utilize it in food vacuoles in the process of bacterial digestion. There are no data, however, regarding whether myxamoebae grown on an axenic medium secrete and sequester acid phosphatase within the food vacuoles. One purpose of this study was to answer this question. Secondly, alkaline phosphatase is a commonly used marker for plasma membranes in mammalian cells and is often employed in cell fractionation studies. Its presence in plasma membranes of Physarum myxamoebae has not been demonstrated, so the study reported here was also designed to see whether alkaline phosphatase occurs in these intact cells, and if so, where.

scholarly journals Electron microscopic localization of adenosine triphosphate (ATP)-hydrolyzing activity in isolated matrix vesicles and reconstituted vesicles from calf cartilage.

1983 ◽  
Vol 31 (4) ◽  
pp. 462-470 ◽  
Author(s):  
S Kanabe ◽  
H H Hsu ◽  
R N Cecil ◽  
H C Anderson
Keyword(s):  
Atpase Activity ◽  
Phosphatase Activity ◽  
Adenosine Triphosphatase ◽  
Matrix Vesicles ◽  
Matrix Vesicle ◽  
Epiphyseal Growth Plate ◽  
Vesicle Membrane ◽  
Electron Microscopic ◽  
The Matrix ◽  
Electron Microscopic Localization

The presence and distribution of adenosine triphosphatase (ATPase) activity in isolated matrix vesicles and reconstituted vesicles from fetal calf epiphyseal growth plate cartilage was studied by electron microscopic cytochemical methods to determine whether phosphatase activity would be found concentrated on the inside or the outside of matrix vesicle membranes or on both sides, and whether reconstitution of vesicles from deoxycholate-solubilized substituents would lead to the reassembly of membranes with ATPase incorporated. ATPase activity was observed on both the outer and inner surfaces of the investing membranes of isolated matrix vesicles and reconstituted vesicles. A transmembrane location of ATPase could indicate phosphate transfer across the vesicle membrane. Orthophosphate released by phosphatase activity within the protected microenvironment of the matrix vesicle could combine with membrane- or lipid-bound calcium, known to be present in vesicles, to form the first hydroxyapatite mineral during calcification.

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