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Biomedical subjects

A Nanci

Publications and source records attributed to A Nanci.

At least 55 records · Page 3Linked to original sources
PubMed1995

Osteopontin and the bone remodeling sequence. Colloidal-gold immunocytochemistry of an interfacial extracellular matrix protein.

Relative to other noncollagenous, extracellular matrix proteins in mineralized tissues, colloidal-gold immunocytochemistry has demonstrated that the ultrastructural distribution of osteopontin (OPN) is unique in that this protein preferentially accumulates at mineralized tissue interfaces. In bone, this protein is present as a major component of cell-matrix and matrix-matrix interfacial structures called cement lines and laminae limitantes. In the present article, the implications of this distinct tissue distribution are discussed in terms of the bone remodeling sequence, and a detailed account of the secretion, accumulation and potential role of OPN is presented and related to current theory on the cellular and extracellular matrix events associated with basic multicellular unit (BMU)-based bone remodeling. In this context, a proposal is made describing the production of this protein as one of the earliest, and latest, secretory activities of the osteoblastic lineage, and that this activity manifests itself morphologically as a cement line ('plane') and a lamina limitans, respectively, at bone matrix interfaces. When integrated with other, known functional characteristics of this protein, the present morphological and compositional data indicate that OPN in cement lines and laminae limitantes may participate in initial and late extracellular matrix organization and mineralization, matrix-matrix/mineral adhesion and/or cell adhesion at bone interfaces.

Animals↗
PubMed1995

Ultrastructural immunodetection of osteopontin and osteocalcin as major matrix components of renal calculi.

The organic matrix of renal calculi has long been considered to influence the crystal growth that occurs in these pathological mineral deposits. Recent advances in characterizing individual organic moieties from mineralized tissues in general and the combined use of antibodies raised against these molecules with different immunocytochemical approaches have allowed their precise distribution to be visualized in a variety of normal and pathological mineralized tissues. The present ultrastructural study reports on the epithelial expression and extracellular localization of several noncollagenous proteins in rat and human kidney stones using high-resolution colloidal-gold immunocytochemistry. To this end, we have examined in an ethylene glycol-induced calcium oxalate model of urolithiasis in the rat, and in human kidney stones, the distribution of certain noncollagenous and plasma proteins known to accumulate in bone and other mineralized tissues that include osteopontin, osteocalcin, bone sialoprotein, albumin, and alpha 2HS-glycoprotein. Of these proteins, osteopontin (uropontin) and osteocalcin (or osteocalcin-related gene/protein) were prominent constituents of the calcium oxalate-associated crystal "ghosts" found in the nuclei, lamellae, and striations of the organic matrix of lumenal renal calculi in the rat and of small crystal ghosts found within epithelial cells. Immunocytochemical labeling for both proteins of the content of secretory granules in tubular epithelial cells from treated rats, together with labeling of a similarly textured organic material in the tubular lumen, provides evidence for cosecretion of osteopontin and osteocalcin by epithelial cells, their transit through the urinary filtrate, and ultimately their incorporation into growing renal calculi. In normal rat kidney, osteopontin was localized to the Golgi apparatus of thin loop of Henle cells. In human calcium oxalate monohydrate stones, osteopontin was similarly detected in the lamellae and striations of the organic matrix. Based on these data, it is proposed that during urolithiasis, secretion of osteopontin (uropontin) and osteocalcin (or osteocalcin-related gene/protein), and the subsequent incorporation of these proteins into kidney stone matrix, may influence the nucleation, growth processes, aggregation, and/or tubular adhesion of renal calculi in mammalian kidneys.

Animals↗
PubMed1995

Immunoblotting studies on artifactual contamination of enamel homogenates by albumin and other proteins.

The reason for the presence of albumin and other serum, cytoskeletal, cytosolic, and extracellular matrix proteins in enamel fractions was investigated by immunoblotting using homogenates prepared from freeze-dried and freshly dissected rat incisors, and antibodies capable of resolving at least 1 ng of the primary antigen. The data indicated that most of the 16 antibodies examined in this study reacted with antigens present only within "cell" homogenates (enamel organ cells + adhering labial connective tissue and blood vessels). One exception was rat serum albumin which was detected routinely in enamel homogenates prepared from freshly dissected, wiped incisors but rarely within enamel homogenates prepared from freeze-dried incisors. Another exception was calbindin-D 28 kDa which was consistently found within secretory stage enamel homogenates irrespective of preparative technique. A third exception was enamel proteins (amelogenins) which were enriched in secretory and early maturation stage enamel homogenates compared with cell homogenates and distributed as multiple molecular weight, antigenic bands in enamel homogenates (14-30 kDa), but mostly as a single antigenic band in cell homogenates (near 27 kDa). Overall, the results of this study suggest that developing rat incisor enamel naturally contains few exogenous proteins such as albumin. High concentrations of albumin (or other serum proteins) in crude homogenates, or purified fractions, derive mostly from blood and/or tissue fluids soaking into the enamel during sample preparation. This type of artifact can be avoided by using freeze-dried teeth for biochemical analyses.

Amelogenin↗
PubMed1995

Spatial distribution of enamel proteins and fibronectin at early stages of rat incisor tooth formation.

Enamel proteins are secreted very early during amelogenesis, that is prior to mantle dentine formation, raising the possibility that they may participate in epithelial-mesenchymal interactions taking place during tooth development. These first enamel proteins associate with elements of the basement membrane interposed between the differentiating ameloblasts and odontoblasts. Fibronectin, a component of the basement membrane, is redistributed and accumulates along the apical portion of odontoblasts during their terminal differentiation. In order to determine whether any correlation exists between the redistribution of fibronectin and the secretion of the first enamel proteins, the spatial distribution of these two extracellular matrix proteins was examined during the presecretory stage of amelogenesis. Male Wistar rats were perfused with a formaldehyde-based fixative, and undemineralized and EDTA demineralized incisors were dehydrated in methanol and embedded in Lowicryl K4M resin. Ultrathin tissue sections were then processed for post-embedding, colloidal-gold immunocytochemistry with antibodies to enamel proteins, fibronectin or type III collagen. In the region of ameloblasts facing pulp, labelling for fibronectin was weak and mostly associated with the lamina fibroreticularis of the basement membrane separating differentiating ameloblasts and odontoblasts. As the mantle predentine formed the immunoreaction for fibronectin increased, particularly in the region of the basement membrane. Enamel proteins were also immunodetected in association with the lamina fibroreticularis and gradually accumulated as patches within mantle dentine and at its interface with ameloblasts. Von Korff collagen bundles, present between odontoblasts and in dentine, were immunolabelled for fibronectin and for type III collagen. Patches of granular material, immunoreactive for fibronectin and/or enamel proteins, were found along the odontoblastic processes and cell bodies. Although no evidence was obtained indicating a precise colocalization of fibronectin and enamel proteins, the results confirm that these two proteins can be found within similar extracellular compartments during mantle predentine-dentine formation. These data suggest that enamel proteins, by themselves or synergistically with other proteins, may play a part in the differentiation and/or formative events taking place at the ameloblast-odontoblast interface during the early stages of tooth development.

Ameloblasts↗
PubMed1995

Overview of morphological changes in enamel organ cells associated with major events in amelogenesis.

The formation and mineralization of enamel is controlled by epithelial cells of the enamel organ which undergo marked, and in some cases repetitive, alterations in cellular morphology as part of the developmental process. The most dramatic changes are seen in ameloblasts which reverse their secretory polarity during differentiation to allow for extracellular release of large amounts of proteins from plasma membrane surfaces that were originally the embryonic bases of the cells. Secreted enamel proteins at first do not accumulate in a layer but, in part, percolate into the developing predentin and subjacent odontoblast layer. Appositional growth of an enamel layer begins with mineralization of the dentin, and ameloblasts develop a complicated functional apex (Tome's processes) to direct release of matrix proteins, and perhaps proteinases, at interrod and rod growth sites. Once the full thickness of enamel is produced, some ameloblasts degenerate, and the surviving cells shorten in height and spread out at the enamel surface. They reform a basal lamina to cover the immature enamel, and continue producing small amounts of enamel proteins that pass through the basal lamina into the enamel. Ameloblasts also undergo cycles of modulation where apical invaginations enriched in Ca-ATPases and other enzymes are formed and shed on a repetitive basis (ruffle-ended/smooth-ended transitions). As this happens, apatetic crystals seeded earlier expand in volume by gradual layering of new mineral at the surfaces of the preformed crystals. Ameloblasts stop modulating when the crystals almost fill existing volume formerly occupied by protein and water.(ABSTRACT TRUNCATED AT 250 WORDS)

Ameloblasts↗
PubMed1994

The epithelial attachment and the dental junctional epithelium: ultrastructural features in porcine molars.

The region of epithelial apposition with a tooth surface is the site of an unusual stratified integument, the junctional epithelium, which combines tight attachment to the tooth, cell turnover, tissue permeability, and epithelial versatility into the first line of defense against periodontal destruction by oral pathogens. To better understand the structure and function of the junctional epithelium we have reviewed its developmental and cell biology, and undertaken a multidisciplinary analysis of its composition in the pig, an omnivore whose dietary and dental development and occlusion patterns are similar to the human condition, and which, because of its size, is more readily amenable to experimental manipulation. The porcine junctional epithelium was also compared with this well-described epithelium in the rat. Morphological analyses by light microscopy and scanning and transmission electron microscopy showed the porcine junctional epithelium and epithelial attachment were similar to that in the rat except that apically, extracellular matrix lamellae associated with the internal basal lamina were more complex, and more coronally there was extensive layering of a dental cuticle-like material. Biochemical analysis of the porcine junctional epithelium by dissociative extraction and SDS-PAGE revealed the presence of some proteins not present in gingival epithelium. Together, these studies show that the porcine junctional epithelium has predictable morphological and biochemical features which establish the pig as an advantageous model to study the basic and clinical biology of this unique epithelium.

Animals↗
PubMed1994

Translocation of enamel proteins from inner enamel epithelia to odontoblasts during mouse tooth development.

The developmental problem of how dental epithelia and/or dental papilla ectomesenchyme induce and/or up- or down-regulate tooth formation are as yet unresolved issues. We have designed studies to map the synthesis and fate pathways of secreted amelogenin proteins from Kallenbach differentiation zones II-IV during in vivo and in vitro mouse mandibular first molar tooth development (M1). Tooth organs from cap, bell, and crown stages were processed for reverse transcriptase/polymerase chain reaction (RT-PCR) and high resolution Protein A immunocytochemistry using anti-amelogenin and anti-peptide antibodies. Cap stage M1 were cultured for periods ranging from 10-21 days in vitro using either serum-less, or 15% fetal calf sera-supplemented, chemically-defined medium. Amelogenin transcripts are expressed in the mouse embryonic molar from E15 through early postnatal development. Amelogenin antigens were first detected in Kallenbach's differentiation zone II. Amelogenin proteins secreted from preameloblasts were identified along cell processes and cell surfaces of odontoblasts adjacent to forming mantle dentine extracellular matrix (ECM) prior to biomineralization. Amelogenin proteins were restricted to forming endocytotic vesicles, clathrin-coated vesicles, and lysosomes within odontoblasts. At later stages (e.g. 2 days postnatal development), enamel proteins were not identified in odontoblasts or predentine matrix following mineralization. Comparable observations for stages of development were noted for in vitro cultured tooth explants. Preameloblasts synthesize and secrete amelogenin proteins which bind to odontoblast cell surfaces possibly through the process of receptor-mediated endocytosis. We conclude that amelogenin proteins secreted from preameloblasts, prior to the initiation of biomineralization, were translocated to odontoblasts to serve as yet unknown biological functions.

Amelogenin↗
PubMed1994

Ultrastructural studies and immunolocalization of enamel proteins in rodent secretory stage ameloblasts processed by various cryofixation methods.

BACKGROUND: Cryofixation rapidly immobilizes cell and tissue components in their native state, thereby resulting in an ultrastructural preservation very close to the living situation. We have applied this approach to examine the morphology of secretory stage ameloblasts and the distribution of enamel proteins in these cells. METHODS: Molar and incisor tooth germs from newborn mice and/or rats were quickly dissected and divided into segments. The segments were then rapidly frozen using slam, plunge or pressure freezing, freeze-substituted and embedded in Epon. In addition, incisors from older rats were chemically fixed by vascular perfusion and also dehydrated by freeze-substitution. RESULTS: Well-preserved ameloblasts were obtained with all four tissue processing methods. However, slam freezing often showed mechanical damage to the ameloblasts, particularly at the level of the distal portion of Tomes' processes which appeared severed or distorted. Plunging into liquid nitrogen-cooled liquid propane resulted in comparatively less tissue distortion. High pressure freezing gave a relatively higher yield of well-preserved specimens, although displacement of organelles in ameloblasts was sometimes observed, probably resulting from hydrostatic pressure. Minimal ice crystal and mechanical damage was observed in chemically fixed tooth samples processed by freeze-substitution since such specimens are cryoprotected and their examination is not restricted to a surface layer. With all of the above cryopreparation methods, the ultrastructure of well-preserved ameloblasts was, in general, similar to that obtained following conventional chemical fixation, and immunocytochemistry with an anti-amelogenin antibody indicated no profound differences in the distribution of enamel proteins. CONCLUSIONS: These results indicate that, despite some limitations, it is possible to adequately cryofix tooth organs while preserving the architecture of ameloblasts and permitting immunolocalization of enamel proteins. Furthermore, they confirm the general morphology of secretory stage ameloblasts as currently derived from conventional chemical tissue processing.

Ameloblasts↗
PubMed1994

Homogeneity in the distribution of matrix components in the hamster zona pellucida as revealed by backscattered electron imaging fracture-label.

BACKGROUND: Backscattered electron imaging fracture-label (BEI-FL), an adaptation of the fracture-label method for scanning electron microscopy, offers the advantage of providing information about the distribution of antigenic and receptor sites with respect to the three-dimensional organization of tissues and cells over relatively large surfaces. Recently, using post-embedding cytochemistry on thin-sections of Lowicryl-embedded oocytes, a homogenous distribution of glycoproteins in the zona pellucida (ZP) was demonstrated (Kan et al., 1989. Biol. Reprod., 40:585-598, Anat. Rec., 226:37-47; Roux and Kan, 1991. Anat. Rec., 230:347-360). However, it can be argued that the chemical nature of resins and the physical conditions of tissue processing required for post-embedding cytochemistry may introduce changes in the tissue components and result in altered distribution of components. On the other hand, freeze-fracture exposes constituents in a minimally denaturing manner and, since no embedding media are used, binding sites are sterically available to the probe. We have, therefore, applied BEI-FL to examine the distribution of matrix glycoproteins in the ZP of hamster oocytes. METHODS: Ovaries and cumulus masses obtained from superovulated female golden hamsters were fixed by immersion in 2.5% glutaraldehyde and processed for fracture-label. Tissues were labeled, respectively, with Wheat germ agglutinin (WGA) followed by ovomucoid-colloidal gold, Ricinus communis agglutinin I (RCA I)-colloidal gold or a monoclonal antibody against Hamster Oviductin-1 followed by protein A-gold, and then examined in the scanning electron microscope. RESULTS: Backscattered electron imaging revealed a homogenous distribution of WGA and RCA I binding sites throughout the cross-fractured matrix of the ZP of ovarian and postovulatory oocytes. Hamster Oviductin-1, an oviductal glycoprotein which is transferred to the ZP of oocytes during oviductal transit, was also found to be uniformly distributed throughout the ZP of postovulatory oocytes. CONCLUSIONS: Our results indicate that BEI-FL can be advantageously used to examine extracellular matrices and are consistent with the concept that glycoproteins are uniformly distributed throughout the ZP of the hamster oocyte.

Animals↗
PubMed1994

Bone sialoprotein mRNA expression and ultrastructural localization in fetal porcine calvarial bone: comparisons with osteopontin.

Bone sialoprotein (BSP) and osteopontin (OPN) are two major non-collagenous proteins in bone that have similar biochemical properties and can mediate cell attachment through an RGD (Arg-Gly-Asp) motif that recognizes the vitronectin receptor. To facilitate evaluations of the biological functions of BSP and OPN in bone formation, affinity-purified rabbit polyclonal antibodies against porcine BSP and OPN were used, together with a high-resolution protein A-gold immunocytochemical technique to reveal the ultrastructural localization of these proteins in undermineralized sections of 50-day fetal porcine calvarial bone. In addition, 35S-labelled antisense riboprobes were prepared to demonstrate the cellular expression of BSP and OPN in the same tissues using in situ hybridization. Immunolocalization for both BSP and OPN revealed the highest density of gold particles associated with electron-dense organic material found at the mineralization front and in 'cement lines'. Labelling was also observed in the mineralized matrix over electron-dense material between collagen fibrils. In the osteoid of newly-formed bone, immunogold labelling for BSP and OPN was associated with loci of mineralization, which were often characterized by feathery clusters of fine needle-like crystals. Results of in situ hybridization on the same tissues demonstrated that BSP mRNA expression was restricted to differentiated osteoblasts with particularly strong signals evident at sites of de novo bone formation. More moderate expression of BSP was observed in 'older' osteoblasts and in some of the newly-entrapped osteocytes. Although expression of OPN mRNA was also observed in osteoblasts and osteocytes, the level of hybridization was similar for most bone cells and not markedly stronger than the signal observed in some stromal cells. While it is evident from these and other studies that both BSP and OPN are associated with bone formation, the differences observed in cellular expression indicate distinct roles for these proteins in bone formation.

Amino Acid Sequence↗
PubMed1993

Effects of chronic fluoride exposure on morphometric parameters defining the stages of amelogenesis and ameloblast modulation in rat incisors.

The response of ameloblasts to long-term (6 weeks) exposure to 100 ppm fluoride was examined in continuously erupting mandibular incisors of female Sprague-Dawley rats as compared to control rats receiving a similar diet (Teklad L-356) but no sodium fluoride in their drinking water. After treatment, animals from both groups were perfused intravascularly with glutaraldehyde, and the incisors were removed and processed for light microscope morphometric analyses directly from 1 microns thick Epon sections. Other animals were injected intravenously with calcein (green fluorescence) followed 4 hours later by xylenol orange (red fluorescence) in order to reveal smooth-ended ameloblast modulation bands and thereby allow quantification of parameters related to the creation and movement of modulation waves within the maturation zone of these teeth. The results indicated that rat incisors expressed four major changes in normal amelogenesis which could be attributed to the chronic fluoride treatment. First, ameloblasts produced a thinner than normal enamel layer by the time they completed the secretory stage and entered the maturation stage of amelogenesis. Second, enamel organ cells within the maturation zone, especially those from the papillary layer, were shorter in height than normal. Third, ameloblasts related to maturing enamel in areas where it was partially soluble and/or fully soluble in EDTA modulated at a rate that was much slower than normal. In some locations ameloblasts remained ruffle-ended for as much as 30% longer than normal per cycle. This upset the usual pattern such that fewer total modulation cycles were completed per unit time by these ameloblasts. Fourth, enamel proteins were lost from the maturing enamel layer at a rate that was about 40% slower than normal. The data suggested that ameloblasts detected the delay in the extracellular breakdown and/or loss of enamel proteins and they responded by remaining ruffle-ended for longer intervals than usual (positive feedback).

Ameloblasts↗
PubMed1993

Ultrastructural immunolocalization of noncollagenous (osteopontin and osteocalcin) and plasma (albumin and alpha 2HS-glycoprotein) proteins in rat bone.

The high-resolution, postembedding protein A-gold immunocytochemical technique was used to visualize the distribution of two noncollagenous bone proteins, osteopontin (OPN) and osteocalcin (OC), and two plasma proteins, alpha 2HS-glycoprotein (alpha 2HS-GP) and albumin (ALB), in sections of Lowicryl K4M-embedded rat tibial and alveolar bone. In the primary spongiosa of the metaphysis, a seam of organic material (lamina limitans) that labeled intensely with OPN and OC antibodies was observed at the bone/calcified cartilage interface just below the zone of vascular invasion of the growth plate. With deposition of bone matrix proper by osteoblasts in this region and its subsequent mineralization, extensive areas of bone were heavily labeled with anti-OPN, anti-OC, and anti-alpha 2HS-GP antibodies, where the majority of gold particles were associated with amorphous, electron-dense patches of organic material throughout the mineralized bone. In the unmineralized osteoid, substantially less labeling was observed, and where occasional mineralization loci were dispersed throughout the osteoid layer, these sometimes showed a concentration of gold particles. ALB labeling, on the other hand, was moderate and generally diffuse throughout the mineralized bone matrix and the osteoid. In alveolar bone, labeling patterns were generally similar to those found in tibial bone. Particularly striking in alveolar bone, however, was an intense anti-OPN labeling of (1) the lamina limitans at cell-lined bone surfaces, including that surrounding cell processes and osteocytes, (2) cement (reversal, resting) lines, and (3) the perilacumar matrix of some osteocytes. In summary, these data suggest that certain plasma proteins, such as alpha 2HS-GP, interact with bone matrix proteins, such as OPN and OC, at sites of tissue mineralization and that the presence of OPN in mineralized bone and at bone surfaces (lamina limitans) and cement lines has a multifunctional role, including regulation of mineralization and mediation of cell dynamics during endochondral and intramembranous bone modeling and remodeling.

Animals↗
PubMed1993

Cytochemical characterization of basement membranes in the enamel organ of the rat incisor.

Ameloblasts are unique epithelial cells, in that once they have deposited the entire thickness of enamel and the process of maturation begins, they reform a basal lamina-like structure at their apical surface. In order to characterize further this basal lamina, its composition was analysed using (1) lectin-gold cytochemistry for glycoconjugates, (2) high-iron diamine (HID) staining for sulfated glycoconjugates and (3) immunogold labeling for collagen type IV and laminin. The labeling patterns were compared to that of other more "typical" basement membranes found in the enamel organ. Sections of rat incisor enamel organs embedded in Lowicryl K4M were stained with Helix pomatia agglutinin (HPA), Ricinus communis I agglutinin (RCA), wheat germ agglutinin (WGA) and Ulex europaeus I agglutinin (UEA). Samples from the late maturation stage were also reacted en bloc with lectins and embedded in Epon for transmission electron microscopic examination or prepared for scanning electron microscopy. Such samples were also stained with HID and conventionally processed for Epon embedding. Tissue sections were then reacted with thiocarbohydrazide-silver proteinate (TCH-SP). Analysis of the lectin labeling suggested that the region of extracellular matrix immediately adjacent to ameloblasts, where the basal lamina is situated, was intensely reactive with HPA and RCA, moderately reactive with WGA, and weakly reactive with UEA. In general, other basement membranes were mildly reactive with all lectins used. No HID-TCH-SP staining was observed directly over the basal lamina while numerous stain deposits were present over other basement membranes of the enamel organ. Immunolocalization of collagen type IV and laminin yielded a weak and variable labeling over the basal lamina.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Characterization of the wheat germ agglutinin-binding property of Treponema denticola.

Lectins were used to characterize glycoconjugates on the cell surface of Treponema denticola, a suspected periodontopathogen. Bacteria were first screened by light microscopy using fluorescein isothiocyanate-coupled lectins. Wheat germ agglutinin (WGA) showed a high reactivity to T. denticola. While the WGA-binding activity was accentuated following heating or detergent treatments of bacterial cells, the reaction was inhibited by incorporation of competing carbohydrates. Scanning and transmission electron microscope studies were conducted in order to characterize the distribution of the WGA-binding sites on the cell surface of T. denticola. Data from these studies confirmed that heat treatment increases the percentage of labeled profiles and suggest that the WGA-binding sites are concentrated on specific regions on the spirochete surface. Initial biochemical analysis indicated that the high reactivity to WGA resides in a peptidoglycan fraction.

Bacterial Adhesion↗
PubMed1993

High-resolution scanning electron microscopy of rat incisor ameloblasts.

The internal three-dimensional organization of secretory and maturation stage ameloblasts was examined using field emission scanning electron microscopy. Particular attention was given to the structure of the Golgi apparatus, the distribution of smooth membrane tubulo-vesicular elements and their relationship with endosomal/lysosomal components. Rat incisors were fixed by intracardiac perfusion with glutaraldehyde, decalcified and divided into segments. The tissues were cryoprotected with glycerol and freeze-fractured. They were then macerated in osmium, and after conductive staining with osmium/tannic acid, the samples were critical-point dried and sputtered with gold. High-resolution scanning electron microscopy showed that ameloblasts contain a well-developed Golgi apparatus, even in the maturation stage where cells are generally believed not to be actively involved in protein secretion. Smooth tubulo-vesicular structures formed a complex network which extended throughout the cell. In secretory stage ameloblasts, this network reached into Tomes' process and consisted of small and large tubules, and distended portions. The smaller tubules radiated from a central core of organelles towards the plasma membrane. Numerous lysosomal/endosomal elements were observed in the Golgi region, and in some cases smooth tubular portions were seen at the surface of multivesicular bodies. These data show that high-resolution scanning electron microscopy can be applied to correlate three-dimensional structural detail with the secretory and resorptive functions of ameloblasts.

Ameloblasts↗
PubMed1992

Correlated biochemical and radioautographic studies of protein turnover in developing rat incisor enamel following pulse-chase labeling with L-[35S]- and L-[methyl-3H]-methionine.

The movement of proteins into and out of enamel was followed over time using a highly sensitive microprecipitation technique to quantify the amount of TCA-insoluble radioactivity present within small pieces of freeze-dried enamel and cells (enamel organ) dissected from the mandibular incisors of rats injected with L-[35S]-methionine. Conventional image processing techniques were also used to estimate the number of silver grains over enamel and cells in radioautographs of mandibular incisors from rats similarly injected with L-[methyl-3H]-methionine. Data from both techniques indicated that the average half-life for labeled proteins secreted into enamel was about 8.9 days. Typically, radioactive proteins accumulated in increasing amounts for 8 hours after which they were lost slowly up to 4 days and more rapidly thereafter when enamel formed during the secretory stage underwent maturation. The half-life for radioactive proteins in cells was only about 20.7 hours. No significant accumulation of radioactivity could be detected in the TCA-soluble or TCA-insoluble fractions of cells as enamel development proceeded. Results from this study suggest that radioautographs provide an accurate estimate of changes occurring to proteins in enamel and cells except at early time intervals (less than 1 hour) when a high percentage of total radioactivity is present within the TCA-soluble fraction of cells.

Animals↗
PubMed1992

Immunolocalization of enamel proteins during amelogenesis in the cat.

Amelogenesis in the cat has been suggested to closely resemble enamel formation in human teeth. In order to further characterize the sequence of events leading to enamel formation in the cat, the expression and distribution of enamel proteins throughout amelogenesis were examined by postembedding immunocytochemistry using an antibody to mouse amelogenins and the high resolution protein A-gold technique. Enamel proteins were first immunodetected in ameloblasts and in the extracellular matrix during the presecretory stage. Secretory stage ameloblasts showed the most intense cellular reactivity. In these cells, protein synthetic organelles, secretory granules, and large lysosome-like structures were all intensely labeled. Extracellularly, numerous gold particles were observed over enamel and over patches of material found at the baso-lateral surfaces of these ameloblasts. During the early maturation stage, the protein synthetic organelles and secretory granules of ameloblasts still showed some immunoreactivity, although the most conspicuous labeling at this later stage was found over enamel and over material present among the extensive apical membrane infoldings of ruffle-ended ameloblasts. Qualitative analysis of lysosome-like elements in ameloblasts suggested that their frequency and immunoreactivity in the maturation stage were relatively lower than in the secretory stage, where some groups of cells often showed numerous large labeled structures. The enamel matrix was intensely labeled at all stages; however, cervical-occlusal and surface-depth gradients were readily apparent by conventional staining and by quantitative analysis of immunolabeling in the late secretory and early maturation stages. These data suggest that the cellular and extracellular distribution of enamel proteins in the cat is generally similar to that reported in other species, although some particularities were observed, perhaps reflecting variation in the timing of developmental parameters.

Amelogenesis↗