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Biomedical subjects

A Nanci

Publications and source records attributed to A Nanci.

At least 91 records · Page 5Linked to original sources
PubMed1988

Backscattered electron imaging of lectin binding sites in tissues following freeze-fracture cytochemistry.

Recently, cytochemical techniques have been applied for localizing membrane components; however, transmission electron microscopy only provides two-dimensional information about their distribution. Scanning electron microscopy, on the other hand, offers the possibility of examining the three-dimensional architecture of biological samples. The fracture-label cytochemical technique was combined with the backscattered electron imaging (BEI) mode of the scanning electron microscope to visualize the in vivo distribution of lectin binding sites on freeze-fractured biological membranes in tissues and cells. Pancreatic and testicular tissues, fixed with glutaraldehyde, were freeze-fractured and labeled with Helix pomatia lectin-gold or Ricinus communis I-gold complexes. The labeled specimens were then critical-point dried and replicated with platinum-carbon for routine transmission electron microscopy or with carbon alone for BEI. Lectin-gold labeling of fractured plasma and intracellular membranes observed with BEI showed a labeling pattern similar to that seen by the replica method. However, BEI-fracture-label provided additional information about the distribution of the labeling with respect to three-dimensional organization of tissues and cells. Large sample areas could be examined, making this technique particularly useful as a survey method for specimens that are either differentially labeled or composed of heterogenous cell populations.

Animals↗
PubMed1988

Demonstration by staining and radioautography of cyclical distributions of protein at the enamel surface in rat incisors.

Staining patterns in the enamel during the maturation stage of amelogenesis reflect the banded distribution of ruffle-ended and smooth-ended ameloblasts. This study investigated the possibility that proteins at the enamel surface may be distributed cyclically according to cyclical changes in ameloblast morphology. Dissected lower rat incisors were wiped free of their enamel organs and immediately immersed in fixative containing one of the following heavy metal and histological stains: uranyl acetate, lead citrate, Coomassie blue, alcian blue and ruthenium red. Other animals were injected with [35S]methionine to label newly-formed enamel proteins. Their incisors were dissected, the enamel organs were wiped from the enamel surface, and the teeth were processed as whole mounts for radioautography. Teeth stained by heavy metals were also viewed by back-scattered electron imaging. The in-situ staining revealed that proteins were distributed in bands and stripes across maturing enamel. Radioautography revealed that the proteins in the stripes were newly-synthesized and secreted into the enamel by certain maturation ameloblasts. We conclude that the enamel organ expresses cyclical activity in part through secretion of proteins.

Ameloblasts↗
PubMed1988

Sequential expression and differential function of multiple enamel proteins during fetal, neonatal, and early postnatal stages of mouse molar organogenesis.

We have established the time and position of expression for multiple enamel proteins during the development of the mouse molar tooth organ. Using high-resolution two-dimensional gel electrophoresis coupled with immunoblotting and immunocytochemistry, a 46-kDa enamel protein (pI, 5.5) was detected during late cap stage (18-days gestation, E18d) within differentiation-zone-II inner enamel epithelia associated with an intact basal lamina. At E19d a second enamel polypeptide of 72 kDa (pI, 5.8) was identified at the time and position of initial biomineralization in differentiation zone V. At 20 days, differentiation-zone-VI ameloblasts without basal lamina (late bell stage) expressed 46- and 72-kDa enamel proteins and, in addition, expressed a relatively more basic 26-kDa enamel protein (pI, 6.5-6.7); detected after initial formation of calcium hydroxyapatite crystals. Antibodies raised against chemically synthesized enamel peptides cross-reacted with both the 72-kDa and 26-kDa polypeptides, but did not cross-react with the 46-kDa enamel polypeptide. The sequential expression of multiple enamel proteins suggests several functions: (a) the anionic enamel proteins may provide an instructive template for calcium hydroxyapatite crystal formation; (b) the more neutral proteins possibly serve to regulate size, shape and rates of enamel crystal formation. We suggest that initial expression of enamel gene products during mouse tooth development possibly recapitulates ancestral features of amelogenesis documented in prereptilian vertebrates. These results imply that multiple instructive signals may be responsible for mammalian enamel protein induction and that the sequential expression of a family of enamel proteins reflects the evolutionary acquisition of a more complex genetic program for amelogenesis.

Animals↗
PubMed1987

Immunocytochemical and radioautographic evidence for secretion and intracellular degradation of enamel proteins by ameloblasts during the maturation stage of amelogenesis in rat incisors.

In the continuously erupting rat incisor the ameloblasts progress through distinct stages associated with the secretion and maturation of enamel. We have examined the possibility that the so-called "postsecretory" ameloblasts of the maturation stage of amelogenesis remain biosynthetically active and are engaged in the synthesis, secretion, and degradation of enamel gene products. The ultrastructural distribution of antigenic sites for enamel proteins was studied within enamel organ cells during the early maturation stage of amelogenesis in rat incisors by using the protein A-gold immunocytochemical technique and rabbit polyclonal antibodies developed against mouse amelogenins. All regions of amelogenesis from late secretion through the first complete modulation from ruffle-ended to smooth-ended ameloblasts were examined. Specific immunolabelling was found within the rough endoplasmic reticulum, Golgi saccules, secretory granules, and lysosomes of ameloblasts throughout these regions. The heaviest intracellular immunolabelling was found within secretory granules and lysosomes (multivesicular type). Quantitative analyses showed that the Golgi saccules and the multivesicular lysosomes of modulating ameloblasts were generally less immunoreactive compared to similar organelles in ameloblasts secreting the inner enamel layer. Radioautographic studies confirmed that ameloblasts of the maturation stage incorporated 3H-leucine and 3H-methionine and secreted labelled proteins into the enamel layer. Grain counts indicated that ameloblasts from the first ruffle-ended band incorporated about two-fold less 3H-methionine and secreted about tenfold less labelled proteins into the enamel compared to ameloblasts secreting the inner enamel layer. The results of this study confirm that ameloblasts do not terminate biosynthesis and secretion of enamel proteins once the final layer has been deposited on the surface of the developing enamel. They continue to form and release new proteins during the maturation stage which intermix with older proteins laid down initially during the secretory stage of amelogenesis. Secretory activity for enamel proteins has been detected in ameloblasts up to at least the second ruffle-ended phase of maturation, at which point the enamel matrix is partially soluble in EDTA.

Ameloblasts↗
PubMed1987

Analysis of crystallite shape in rat incisor enamel.

The hydroxyapatite crystallites of mammalian enamel appear as hexagons when seen in cross-sections examined with the transmission electron microscope. Using goniometric transmission electron microscopy, stereo-pair electron micrographs and freeze-fracture replicas, two models have been proposed to explain the hexagonal crystallite profile. The "hexagonal ribbon" model proposes that hexagonal profiles are true cross-sections of elongated hexagonal ribbons. The "rectangular ribbon"model proposes that crystallite profiles are three-dimensional rectangular segments (parallelepipeds), which are contained in the Epon sections and project as opaque hexagons in routine transmission electron micrographs. Morphological observations together with predictions from models indicate that the crystallites in rat incisor enamel are flat ribbons with rectangular cross-sectional profiles. The hexagonal images seen in electron micrographs of thin sections of enamel result from viewing parallelepiped-shaped segments of these crystallites as two-dimensional shadows.

Animals↗
PubMed1987

The effects of vinblastine on the secretory ameloblasts: an ultrastructural, cytochemical, and immunocytochemical study in the rat incisor.

Secretory ameloblasts synthesize the organic matrix of enamel and secrete it at two distinct "putative secretory sites" characterized by membrane infoldings (Nanci and Warshawsky, 1984a). The antimicrotubular agent vinblastine sulphate interferes with secretion. We have examined the effect of this drug on the ameloblast secretory sites and re-evaluated the effect on the intracellular organization of the cell by using conditions that optimize fixation, cytochemistry (ZI0), and immunocytochemistry. Associated with the disappearance of secretory granules and Golgi-related structures from Tomes' process was the loss of membrane infoldings at secretory sites. The Golgi apparatus appeared fragmented and numerous granule clusters were found throughout the cell body. These clusters were often seen in relation to extracellular patches of material in which no crystallites were seen. Immunocytochemistry revealed the presence of enamel proteins in the protein synthetic organelles, including various granule types, in lysosomes and in the extracellular patches. These data suggest that ameloblasts under the effect of vinblastine carry on secretory activities, but the product is not routed to the usual sites. It was confirmed that membrane infoldings characterize the sites where enamel proteins are normally secreted.

Ameloblasts↗
PubMed1987

Immunocytochemical localization of gamma-carboxyglutamic acid-containing proteins (osteocalcin) in rat bone and dentin.

Gla-protein or osteocalcin is one of the most abundant noncollagenous matrix proteins found in bone and dentin. The present study describes, with high resolution, the intracellular and extracellular distribution of Gla-protein in alveolar bone and incisor dentin. Sections of tissues embedded in Lowicryl K4M were incubated with rabbit antibodies to rat dentin Gla-protein. The site of the specific antigen-antibody reaction was revealed by the protein A-gold complex. Labeling was detected over bone and dentin while fewer gold particles were present over prebone and predentin. Gold particles were also seen over the protein synthetic organelles (rough endoplasmic reticulum, Golgi apparatus) of osteoblasts and odontoblasts. These findings confirm, with improved resolution, previous light immunohistochemical studies, and offer the possibility to examine the secretory pathway of the protein.

Alveolar Process↗
PubMed1987

Effect of tissue processing on colloidal gold cytochemistry.

The aim of cytochemical techniques is to localize specific biochemical components in particular tissue and cell compartments. However, since preparation of tissues for structural observation results in major alterations of the properties of their components, a major problem is to retain an adequate degree of their biochemical properties as well as adequate structural preservation. In the present study, we describe results obtained using various colloidal gold cytochemical techniques on tissues processed through different approaches. We found that any manipulation of the tissue during its processing can result in modifications of tissue components, leading to problems in cytochemistry. Indeed, washing of the tissue before fixation, the nature of the fixative solution, the chemical basis of the resins, and the physical conditions of embedding can all introduce changes in tissue components which can be cytochemically demonstrated. This has been illustrated with application of the protein A-gold, lectin-gold, and enzyme-gold cytochemical techniques on tissues submitted to different processings: fixation by perfusion or by immersion; glutaraldehyde vs paraformaldehyde fixative solutions; cryo-ultramicrotomy; embedding in epoxy, GMA, Lowicryl, or LR resins. The results obtained have demonstrated that conditions for optimal labeling must be worked out for each class of binding sites, and that no single procedure can be recommended as THE best approach in cytochemistry.

Acrylic Resins↗
PubMed1987

Application of backscattered electron imaging and lectin-gold cytochemistry to visualize the distribution of glycoconjugates in a basal lamina.

Basement membranes generally appear in transmission electron microscopy as thin, cross-sectioned profiles. Face-views are difficult to obtain particularly when their contours are curved or undulated. Such preparations are suited for the study of the distribution of basement membrane constituents through thickness but are limited for examining their spatial distribution. Scanning electron microscopy offers the potential of visualizing surface views. We have applied this approach to examining the three-dimensional distribution of glycoconjugates in a basement membrane. Large, face-views of the basal lamina related to maturation ameloblasts in the region where enamel of the rat incisor is EDTA soluble were exposed. Glycoconjugates were then revealed by incubating the segments in lectin-gold complexes and binding sites were visualized by backscattered electron imaging. This basal lamina was found to contain fucose, mannose and to be extremely rich in N-acetyl-D-galactosamine. The abundance of glycoconjugates containing the latter sugar residue may impart on this basal lamina selective properties which could regulate the movement of material into, and out of, the enamel layer.

Ameloblasts↗
PubMed1986

[Ultrastructural characteristics of the regression of a Novikoff hepatoma transplanted into immunized rats].

In order to elucidate ultrastructure of tumoral rejection, we have studied the sequential morphological modifications of Novikoff hepatoma transplanted in striated muscle of normal and immunized rats. Specific immunity was induced by the repeated injection of small doses of tumor cells. One month after immunization, a tumorigenous dose was then injected in striated muscle. The animals were sacrificed at 1, 4, 6 or 7 and 14 or 21 days, and tissue samples of the zone of implantation were taken for morphological study. The transplanted tumor regressed in all immunized rats and the sequential ultrastructural examination of these tumors indicated three major phases in their regression: an initial phase (day 1) during which there was an inflammatory infiltrate rich in neutrophils, a reduction of the mitotic activity of tumoral cells and the necrosis of some of them; an intermediary phase (days 4-7) during which the intratumoral infiltrate was composed mainly of lymphocytes and macrophages. These cells surrounded the tumoral cells and eventually formed microgranulomas. Tumoral cells showed cytostasis and/or cytolysis; a final phase (days 14-21) where the tumor had completely disappeared and had been replaced by fibrous tissue and vacuolated macrophages. The morphologic aspect of the intermediary phase is characteristic of a regressive tumor. Our sequential observations clearly showed the sequestration of tumor cells by macrophages and suggest the contact cytolysis of tumor cells. The sequestration of target cells by macrophages might facilitate the concentration of the cytolytic substances in the area of intercellular contacts, and likewise protect these substances from neutralization by serum or tissue inhibitory factors.

Animals↗
PubMed1985

Enamel protein biosynthesis and secretion in mouse incisor secretory ameloblasts as revealed by high-resolution immunocytochemistry.

Mouse secretory ameloblasts express a number of enamel proteins, which have been divided into amelogenin and enamelin subfamilies. We have used polyclonal antibodies to murine amelogenins to reveal enamel proteins in mouse ameloblasts using the protein A-gold immunocytochemical technique. Specific immunolabeling was detected over the extracellular enamel matrix and over the rough endoplasmic reticulum, the saccules of the Golgi apparatus, and the secretory granules of the ameloblasts. In addition, some lysosome-like granules were also labeled. Only background labeling was obtained over mitochondria, nuclei, cytosol, adjacent odontoblasts, and dentin. Quantitation of the intensity of labeling showed the presence of an increasing gradient along the secretory pathway, which may correspond to the concentration or the maturation of these proteins as they are processed by the cell. These findings indicate that the ameloblast displays an intracellular distribution of its secretory products similar to that of other merocrine secreting cells. The presence of enamel proteins in lysosomes suggests that crinophagy and/or resorption occurs in these cells.

Ameloblasts↗
PubMed1984

Characterization of putative secretory sites on ameloblasts of the rat incisor.

The distribution and structure of the putative sites where enamel matrix is secreted from the ameloblast were studied by correlating the external topography with the distribution of organelles in Tomes' process cut in various planes of section. Both the interrod and rod secretion sites are associated with deep membrane infoldings. It was found that the interrod secretion site completely surrounds each ameloblast, and the marked interdigitation of adjacent cells results in a cooperative growth front for interrod enamel. In contrast, the rod secretion site is present on only one surface of the interdigitating portion of Tomes' process. Numerous granules were observed adjacent to the membrane infoldings associated with both sites, and granules were seen fused to membrane infoldings suggesting that the matrix of enamel is a merocrine secretion product.

Ameloblasts↗
PubMed1984

Relationship between the quality of fixation and the presence of stippled material in newly formed enamel of the rat incisor.

Extracellular accumulation of a granular material that is presumed to be an organic "precursor" to mineralized enamel has been reported. This material, generally referred to as ""stippled material," was observed mainly after immersion fixation with osmium tetroxide. In studies with perfusion fixation, the presence of stippled material was inconsistent. Therefore, it appeared that the occurrence of stippled material was dependent on the method of fixation. To test this assumption, tissues were fixed by immersion in either osmium tetroxide or glutaraldehyde and by perfusion with either glutaraldehyde or a mixture of acrolein, glutaraldehyde, and formaldehyde. It was found that as the quality of cellular preservation improved, the occurrence of stippled material decreased. Since no stippled material could be found in material judged to be well fixed, it was concluded that stippled material is not an extracellular precursor to mineralized enamel, but is a breakdown product resulting from poor fixation.

Acrolein↗
PubMed1983

The effect of osmium postfixation and uranyl and lead staining on the ultrastructure of young enamel in the rat incisor.

Enamel crystallites are electron opaque without osmium or heavy metal staining and give a crystalline electron diffraction pattern. Since the opacity and diffraction pattern are abolished from ultrathin sections of young enamel by floating on distilled water (Bishop and Warshawsky, 1982), the possibility that aqueous staining may also remove crystallites was tested. In addition, the effect of osmium postfixation on crystallite structure was examined. Rat incisors fixed by perfusion with a mixture of aldehydes were either nonosmicated or osmicated prior to dehydration. Incisor segments in the region of inner enamel secretion were embedded in the same Epon block to ensure reliable comparison. Osmicated enamel was more intensely stained with toluidine blue and more electron opaque than nonosmicated enamel. No other structural differences were seen. However, crystallites in osmicated enamel were more resistant to grid demineralization and electron beam damage. Routine staining was done by floating sections on solutions of uranyl acetate and lead citrate; sections were also floated on similar solutions from which the heavy metals were omitted. These solutions removed the electron opaque crystallites from the youngest enamel. Stained sections showed electron opaque crystallite-like structures similar to unstained enamel. When sections that were extracted by the solutions from which the metals were omitted were restained, they appeared identical to routinely stained enamel. It was concluded that staining of young enamel removes the crystallites and reveals only the organic matrix.

Aging↗
PubMed1983

Selachian tooth development: II. Immunolocalization of amelogenin polypeptides in epithelium during secretory amelogenesis in Squalus acanthias.

We have determined the distribution of amelogenin polypeptides in an order of elasmobranchs using indirect immunofluorescence with rabbit polyclonal antibodies prepared to purified murine amelogenins. We find that amelogenins are definitely present within the inner enamel epithelium prior to the production of the extracellular matrix component termed "enameloid" (row II developing tooth organs). During subsequent stages of selachian tooth development (row III tooth organs), immunofluorescence staining data indicated localization of amelogenin antigens within epithelium as well as the enameloid extracellular matrix. The results from these immunohistochemical studies suggest that the 16-20 kdalton amelogenins, which are characteristic of murine inner enamel epithelial cells undergoing terminal biochemical differentiation into secretory ameloblasts, may also be regarded as molecular markers for amelogenesis in developing teeth in the spiny dogfish, Squalus acanthias.

Amelogenesis↗
PubMed1983

Selachian tooth development: I. Histogenesis, morphogenesis, and anatomical features in Squalus acanthias.

We have determined the general features of epithelial-mesenchymal interactions during fetal and mature adult stages of odontogenesis in the selachian spiny dogfish, Squalus acanthias. The general features of odontogenesis included the formation of an extended dental lamina, bud, cap, bell, and crown stages as identified and described using serial sections from fetal as well as adult specimens and light microscopy. Fetal and adult lengths, the rostrum-caudal fin distance, were correlated with the number of tooth rows and columns present in each specimen. This positional information and terminology was extremely valuable in attempting to acquire reproducible data for analyses. Whereas a number of histologic features of fetal and adult odontogenesis were comparable to those described in Mammalia, we found a number of epithelial-mesenchymal interaction characteristics unique to elasmobranch tooth development, including the persistence of the dental lamina in the adult stages, the coupling of cervical inner enamel epithelia between sequential stages of tooth formation, and the patterns of odontogenesis in the adult dentition.

Animals↗