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Biomedical subjects

F R Denys

Publications and source records attributed to F R Denys.

At least 55 records · Page 3Linked to original sources
PubMed1983

Ultrastructural visualization of complex carbohydrates in epiphyseal cartilage with the tannic acid-metal salt methods.

The present study has ultrastructurally applied the tannic acid-ferric chloride (TA-Fe) and the TA-uranyl acetate (TA-UA) methods to thin sections of glutaraldehyde-fixed, unosmicated embedded epiphyseal cartilage from rat tibiae to demonstrate complex carbohydrates. The strongest TA-Fe and TA-UA staining was observed after fixation of the specimens in glutaraldehyde containing TA. TA-Fe (pH 1.5) strongly stained matrix granules presumed to be proteoglycan monomers and chondrocyte secretory granules at various maturational stages but did not stain collagen fibrils and glycogen. TA-UA (pH 4.2) strongly stained matrix granules, intracellular glycogen, and chondrocyte secretory granules, and moderately stained collagen fibrils in the cartilage matrix. Ribosomes and nuclei were not stained above background staining with UA alone. In alpha-amylase-digested specimens, all TA-UA-reactive cytoplasmic glycogen was selectively removed. Testicular hyaluronidase digestion of specimens selectively removed TA-UA staining in matrix granules and all TA-Fe staining. When the pH of the UA solution was reduced to 1.5, TA-UA staining of glycogen and collagen was markedly decreased or absent, whereas staining of anionic sites was unaltered and significantly greater than with UA staining alone. Thus the TA-metal salt methods are pH dependent and allow differential intracellular and extracellular localization of complex carbohydrates in cartilage tissues at the electron microscope level.

Animals↗
PubMed1983

Ultrastructural cytochemistry and immunocytochemistry of proteoglycans associated with epiphyseal cartilage calcification.

Proteoglycans (PGs) are closely associated with cartilage calcification. We have examined the hypertrophic zone of rat epiphyseal cartilage, in which calcification is occurring, using the high-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycosaminoglycans, an immunoferritin method specific for chondroitin sulfate A, and the tannic acid-ferric chloride (TA-Fe) method to stain cartilage matrix granules (MGs) presumed to be PG monomers. HID-TCH-SP produced stain deposits with a diameter of 11.2 +/- 3.2 nm (mean +/- SD; n = 200) in the MGs. However, HID-TCH-SP staining was not discernible in membrane-limited matrix vesicles (MVs). In areas of advanced calcification, partially disrupted MVs and globular bodies (GBs), derived in part from disrupted and/or degenerated MVs, contained a few too many small HID-TCH-SP stain deposits. Further down the epiphyseal cartilage, intact MVs markedly decreased and the GBs, containing many small HID-TCH-SP stain deposits, significantly increased in number. These GBs were found exclusively in the longitudinal septa rather than in the transverse septa. After enzyme digestion with testicular hyaluronidase, small (7.2 +/- 1.2 nm in diameter) stain deposits remained in the MGs and GBs, presumably localized to keratan sulfate. Immunoferritin localizing chondroitin sulfate strongly stained MGs, whereas MVs and GBs lacked staining. TA-Fe staining of glycoconjugates in the GBs demonstrated a striking decrease in the diameter of MGs associated with calcification in the GBs as compared with those in the noncalcifying area around the GBs. These results indicate that the GBs containing needle-like apatite crystals in morphologic preparations represent sites of chondroitin sulfate degradation. Testicular hyaluronidase-resistant sulfated glycosaminoglycans presumed to be keratan sulfate and partially degraded PGs selectively remain within the GBs as a probable requisite for expansion of the initial calcification in MVs.

Animals↗
PubMed1982

Ultrastructural visualization of complex carbohydrates in eosinophilic leukocytes.

Complex carbohydrates in eosinophils from human, rabbit, and rat marrow were identified and localized by cytochemical and radioautographic methods. The high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP), low iron diamine (LID)-TCH-SP, and periodate (PA)-TCH-SP methods were used for the localization of sulfate, sulfate and carboxyl, and vicinal glycol- containing complex carbohydrates, respectively. Golgi vesicles and small precursor granules (0.2-0.4 micrometer in diameter) demonstrated strong HID-TCH-SP staining and labeled intensely after a 10-minute pulse with 35SO4(2). Crystalloid-free or immature specific granules (0.5-0.9 micrometers in diameter) labeled heavily after a 60-minute incubation and 60-minute chase with 35SO4(2). Immature granules were graded according to their HID-TCH-SP staining. Type I granules demonstrated strong rim staining and similar or somewhat less central staining, whereas type 2 granules only demonstrated rim staining, and type 3 granules lacked staining. Fully mature crystalloid-containing granules lacked staining. LID-TCH-SP similarly stained the HID-positive sulfated material in cytoplasmic granules. PA-TCH-SP stained some Golgi vesicles and diffusely stained all precursor granules and type 1 granules. Weaker staining was observed in type 2 granules and staining was very weak or absent in type 3 and crystalloid-containing granules. In early eosinophils, tubulovesicular structures (TVS) were observed rosetting and contacting precursor and type 1 granules. These TVS contained material with strong PA-TCH-SP staining but lacked HID-TCH-SP or LID-TCH-SP-reactive acidic glycoconjugates. Flattened Golgi saccules or early eosinophils stained weakly or not at all with the PA-TCH-SP method. Small granules and TVS in late (bilobed) eosinophils displayed PA-TCH-SP reactivity and lacked HID-TCH-SP staining but differed from TVS in early eosinophils in that they were not associated as rosettes with specific granules. These results indicate that sulfated and vicinal glycol-containing complex carbohydrates are differently disturbed in immature specific granules of eosinophils and presumably become masked to staining as the granule matures.

Animals↗
PubMed1982

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method.

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.

Animals↗
PubMed1982

Extracellular and intracellular digestion of complex carbohydrates by osteoclasts.

The role of osteoclasts or chondroclasts in degradation and synthesis of complex carbohydrates was investigated using the high iron diamine-thiocarbohydrazide-silver proteinate method (HID-TCH-SP) for sulfated glycoconjugates and the periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HID-TCH-SP stained the calcified cartilage matrix, the osteoclast ruffled border, vacuoles and heterophagosomes but not the Golgi apparatus and primary lysosomes. The size and number of HID-TCH-SP stain deposits progressively decreased from the calcified cartilage matrix to the ruffled border (p less than 0.001). Enzyme digestion with testicular hyaluronidase removed most HID-TCH-SP stain deposits averaging 13 nm. in diameter in the extracellular matrix, cytoplasmic vacuoles, and heterophagosomes of osteoclasts. Only sparse stain deposits averaging 8 nm. in diameter and presumed to be keratan sulfate remained in these sites after enzyme digestion. Osteoclast heterophagosomes contained the highest concentration of hyaluronidase-resistant material suggesting delayed degradation of keratan sulfate at this site. PA-TCH-SP strongly stained collagen fibrils in the calcified cartilage matrix. Reactive collagen fibrils were also observed in extracellular channels but only rarely were identifiable collagen fibrils observed in cytoplasmic vacuoles. A progressive decrease in the diameter of PA-TCH-SP reactive collagen fibrils was observed between the calcified cartilage matrix and the ruffled border region (p less than 0.001). PA-TCH-SP stained cisternae of rough endoplasmic reticulum, Golgi saccules, and primary lysosomes consistent with the synthesis and packaging of glycoprotein enzymes at these sites. These results indicate that the dissolution of sulfated glycoconjugates requires osteoclastic engulfment of degraded material and subsequent intracellular digestion, whereas the dissolution of collagen fibrils appears to be completed extracellularly.

Animals↗
PubMed1981

Heparan sulfate of AH-130 ascites hepatoma cells: a cell-surface glycosaminoglycan not displaced by heparin.

This study reports on the ultrastructural location and biophysical properties of cell-associated glycosaminoglycans of AH-130 cells, an azo dye-induced ascites hepatoma. Earlier studies have shown that a low-sulfated heparan sulfate, which comprises 93% of their total glycosaminoglycan (GAG) content, is associated with these cells. High-iron diamine, an ultrastructural stain for sulfated glycoconjugates, stained the hepatoma cell surfaces heavily. With the exception of occasional light staining in a few cytoplasmic granules, intracellular organelles did not stain with this method. The lack of an extensive pool of intracellular GAG was confirmed by quantitative fluorescence microscopy of cells vitally stained with acridine orange. The nature of the binding of the cell-surface heparan sulfate was explored by competitive binding studies with exogenous heparin. When cells were incubated with exogenous heparin, release of heparan sulfate into the medium was not detected, although heparin was bound. We conclude that low-sulfated heparan sulfate is an integral component of the AH-130 hepatoma cell surface and is bound at a site different than heparin.

Acridine Orange↗
PubMed1981

Ultrastructural localization of complex carbohydrates in odontoblasts, predentin, and dentin.

Glycosaminoglycans (GAGs) and glycoproteins (GPs) are essential components for dentinogenesis. We have examined rat odontoblasts, predentin, and dentin decalcified with EDTA and stained with: 1) Spicer's hig-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycoconjugates, and 2) Thiéry's periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HIS-TCH-SP stained distended portions of Golgi saccules and secretory granules. The predentin contained three times the number of HID-TCH-SP stain precipitates when compared to the mineralization front of the dentin matrix. PA-TCH-SP weakly stained membranes of Golgi saccules and cisternae of rough endoplasmic reticulum (RER), whereas stronger staining was observed in secretory granules, lysosomes, and multivesicular bodies (MVBs). Collagen fibrils in predentin demonstrated moderate PA-TCH-SP staining. In contrast, strong PA-TCH-SP staining was observed on and between collagen fibrils in the mineralization front of the dentin matrix. TCH-SP controls of unosmicated specimens lacked significant staining, however, osmicated control specimens did contain some TCH-SP stain deposits in the mineralization front. These results indicate that sulfated and vicinal glycol-containing glycoconjugates are packaged in the same type of secretory granule and released into the extracellular matrix; subsequently vicinal glycol-containing glycoconjugates concentrate in the calcification front, whereas sulfated glycoconjugates accumulate in the predentin and are either removed or masked to staining in the dentin.

Animals↗
PubMed1981

Ultrastructural cytochemistry and radioautography of complex carbohydrates in secretory granules of epiphyseal chondrocytes.

Secretory granules of chondrocytes contain a variety of complex carbohydrates, which include glycosaminoglycans and glycoproteins necessary for the formation of the cartilage matrix. The present study has utilized ultrastructural, cytochemical, and radioautographic methods to determine the subcellular route of incorporation of sulfated and vicinal glycol-containing complex carbohydrates into secretory granules and their subsequent distribution in granules. High iron diamine specifically stained sulfated glycoconjugates in Golgi saccules, Golgi derived vacuoles, and mature secretory granules. Incubation of chondrocytes with 35SO4 resulted in significant radioautographic labeling of Golgi saccules, condensing vacuoles, intermediate granules containing moderately dense amorphous material, and mature granules containing fibrillar material. Periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of unosmicated thin sections localized vicinal glycol-containing glycoconjugates in cisternae of rough endoplasmic reticulum and coated vesicles, some of which appeared to be budding from rough endoplasmic reticulum. Similar coated vesicles appeared to fuse with condensing vacuoles and granules of intermediate maturity which evidenced moderate PA-TCH-SP reactivity. These vesicles were very seldom seen associated with Golgi saccules which contained 35SO4 radiolabels and high iron diamine reactivity but lacked significant PA-TCH-SP staining. The PA-TCH-SP-positive vesicles were not associated with mature secretory granules which demonstrated strong PA-TCH-SP staining. Lysosomes demonstrated moderately heavy PA-TCH-SP staining. Amylase digestion of en bloc specimens removed all PA-TCH-SP reactive cytoplasmic glycogen, however, staining of cytoplasmic organelles was not decreased. These studies indicate that sulfated glycosaminoglycans and vicinal glycol-containing glycoproteins are transported to secretory granules by Golgi saccules and coated vesicles, respectively, and are subsequently distributed differently within the maturing secretory granules.

Animals↗
PubMed1980

Dysgranulopoietic neutropenia and abnormal monocytes in childhood vitamin B12 deficiency.

Neutropenia and/or leukopenia (associated with elevated serum lysozyme levels) in three children with vitamin B12 deficiency were evaluated using soft agar culture and ultrastructural and cytochemical techniques. In two patients a marked increase in peripheral myeloid colony forming cells (CFC) was observed; whereas a marginal increase in CFC was present in the third, less symptomatic, patient. Marrow CFC was normal or slightly increased. Serum colony stimulating activity (CSA) was normal but elaboration of CSA by white blood cells was low. Normal maturation of the progenitors was present in vitro and serum inhibitors of myelopoiesis were absent. Megaloblastic neutrophils and monocytes with nuclear-cytoplasmic asynchrony were observed ultrastructurally in directly sampled marrow specimens. These cells contained autophagic and/or heterophagic vacuoles and an increase in cytoplasmic granules. Both monocytes and neutrophils also contained enlarged-disrupted centrioles. Many marrow macrophages contained phagocytic vacuoles, which enclosed disrupted neutrophils and cellular debris.

Adolescent↗
PubMed1979

Ferrocyanide enhancement of concanavalin A-ferritin and cationized ferritin staining blood cell surface glycoconjugates.

Ferrocyanide was used to enhance cationized ferritin and concanavalin A-ferritin (Con A-ferritin) staining of surface glycoconjugates of peripheral blood and bone marrow cells from rabbits and humans. The glutaraldehyde-fixed cells were stained with Con A-ferritin or cationized ferritin and then exposed to a ferrocyanide solution. The resulting cuboidal and irregular stain deposits averaged 50 nm in diameter when viewed with the transmission (TEM) and scanning electron microscope (SEM). Rabbit blood cells demonstrated more Con A binding sites than human blood cells and the decrease in binding sites observed with maturation of human granulocytic and erythrocytic cells was not evident in rabbit cells. Differences in binding of cationized ferritin to rabbit and human cell surfaces were less prominent than that observed for Con A. These results extend previous studies of blood cell surface glycoconjugates and demonstrate that ferrocyanide enhancement significantly facilitates SEM evaluation of Con A-ferritin and cationized ferritin bound to cell surfaces.

Animals↗
PubMed1979

Finishing of enamel surfaces after debonding of orthodontic attachments.

The finishing of enamel surfaces after removal of directly bonded attachments is essential. The following procedures are suggested: 1. The bonding of mesh-backed stainless steel brackets with a lightly filled resin system. 2. Debonding of attachments with a direct bonding bracket remover. 3. Removal of the bulk of the remaining resin with a 12-bladed tungsten carbide bur operated at high speed with adequate air cooling. 4. Finishing of the residual resin and underlying enamel with graded polishing discs or Ceramisté wheels used with light pressure and adequate air cooling. 5. Final finishing with a rubber cup and a water slurry of pumice.

Acid Etching, Dental↗
PubMed1979

Development of annular gap junctions in guinea pig epithelia.

Epithelia from skin, palate, buccal mucosa and dorsal tongue were studied in normal and zinc deficient guinea pigs by transmission electron microscopy. Gap junctions (nexuses) were observed primarily in the basal and prickle cell layers. They were also seen to form bulging and looping configurations which appeared to pinch off into the cytoplasm forming what are recognized as annular gap junctions. These may be attached to the surface or represent separate, intracytoplasmic, nexus-bound inclusions. Their contents varied, but most often they contained glycogen and ribosomal-like particles. Various cell organelles were also seen entrapped, most common of which were membrane coating granules. The development of these annular gap junctions is a prominent feature of many epithelia. It has been suggested that they probably serve to maintain a synchronized differentiation throughout the various epithelial layers.

Animals↗
PubMed1979

Ultrastructure and cytokinetics of leukemic myeloblasts containing giant granules.

Leukemic myeloblasts containing abnormal granules were studied with ultrastructural, cytochemical, and thymidine labeling techniques to evaluate defects in granulogenesis and proliferation. Giant granules (1 to 3 micron in diameter) and Auer rods were observed in leukemic cells from two patients, and only rarely were both abnormal granule types observed in the same cell. The lysosomal origin of these abnormal granules was demonstrated by their content of peroxidase, esterase, and anionic glycoconjugates. Fusion of small dense granules (less than 0.2 micron in diameter) appeared to be increased in cells containing Auer rods and/or giant granules, but fusion of intact primary granules (0.2 to 0.4 micron in diameter) and sequestration of cytoplasmic contents were observed only in giant granules and not in Auer rods. Although the small granules that fused to form giant granules and Auer rods appeared similar, there was no evidence for transformation of giant granules into Auer rods. In one patient, cells with abnormal granules could easily be distinguished from the larger population of cells that lacked abnormal granules. The perturbation of these two distinct populations by chemotherapy was evaluated with thymidine labeling experiments. A high percentage (2- or 3-fold greater) of the abnormally granulated myeloblasts incorporated tritiated thymidine when compared to myeloblasts without abnormal granules in the same specimen. This difference could have resulted from an underlying metabolic defect which affected both granulogenesis and cell division. These results demonstrate that the formation of giant granules in leukemic cells is morphologically similar to that observed in the Chediak-Higashi syndrome and that leukemic cells with abnormal granules may differ cytokinetically from uninvolved leukemic cells.

Cell Division↗