Histochemical and immunohistochemical examination of odontoblasts (petroblasts) in petrodentine formation of lungfish.

OBJECTIVE: Petrodentine, the core of the lungfish tooth plate, is a well-mineralized tissue similar to mammalian enamel and analogous to enameloid in fish teeth. Petrodentine is formed solely by petroblasts, which are specialized odontoblasts, whereas enameloid is a composite tissue produced by both odontoblasts and dental epithelial cells. To clarify the details of petrodentine formation, petroblasts were investigated using histochemical and immunohistochemical techniques. METHODS: Extant lungfish (Lepidosiren paradoxa) were used in this study. Tooth plates during the stage of petrodentine formation were observed by means of histochemistry and immunohistochemistry. Commercial kits were used to detect enzyme activity. Correlative sections were immunostained using antibodies against selected peptides. Routine staining such as periodic acid-Schiff (PAS) reaction to identify glycogen and Elastica van Gieson staining for the detection of elastic fibers in histological sections were performed. In addition, conventional transmission electron microscopy was used for observing the fine structure. RESULTS: Petroblasts showed marked acid and alkaline phosphatase activities, and positive immunoreactivities against anti-nestin, anti-V-ATPase, and anti-Ca2+-ATPase, during the maturation stage, but in the matrix formation stage, reactions were much weaker than that of the maturation stage. During the maturation stage, petroblasts showed intense PAS reactivity, and glycogen particles were observed in petroblasts by transmission electron microscopy. Glucose transporter 1-immunoreactivity was observed in petroblasts in the matrix formation stage and the initial to mid part of the maturation stage. CONCLUSIONS: The results in this study suggested that petroblasts have two functional stages, matrix formation and maturation, and glycogen plays an important role in the modulation of petroblasts.

Laminin γ2 knockout mice rescued with the human protein exhibit enamel maturation defects.

The epithelial ameloblasts are separated from the maturing enamel by an atypical basement membrane (BM) that is enriched in laminin 332 (LM-332). This heterotrimeric protein (α3, ß3 and γ2 chains) provides structural integrity to BMs and influences various epithelial cell processes including cell adhesion and differentiation. Mouse models that lack expression of individual LM-332 chains die shortly after birth. The lethal phenotype of laminin γ2 knockout mice can be rescued by human laminin γ2 (LAMC2) expressed using a doxycycline-inducible (Tet-on) cytokeratin 14 promoter-rtTA. These otherwise normal-looking rescued mice exhibit white spot lesions on incisors. We therefore investigated the effect of rescue with human LAMC2 on enamel maturation and structuring of the atypical BM. The maturation stage enamel organ in transgenic mice was severely altered as compared to wild type controls, a structured BM was no longer discernible, dystrophic matrix appeared in the maturing enamel layer, and there was residual enamel matrix late into the maturation stage. Microtomographic scans revealed excessive wear of occlusal surfaces on molars, chipping of enamel on incisor tips, and hypomineralization of the enamel layer. No structural alterations were observed at other epithelial sites, such as skin, palate and tongue. These results indicate that while this humanized mouse model is capable of rescue in various epithelial tissues, it is unable to sustain structuring of a proper BM at the interface between ameloblasts and maturing enamel. This failure may be related to the atypical composition of the BM in the maturation stage and reaffirms that the atypical BM is essential for enamel maturation.

Amelogenins as potential buffers during secretory-stage amelogenesis.

Amelogenins are the most abundant protein species in forming dental enamel, taken to regulate crystal shape and crystal growth. Unprotonated amelogenins can bind protons, suggesting that amelogenins could regulate the pH in enamel in situ. We hypothesized that without amelogenins the enamel would acidify unless ameloblasts were buffered by alternative ways. To investigate this, we measured the mineral and chloride content in incisor enamel of amelogenin-knockout (AmelX(-/-)) mice and determined the pH of enamel by staining with methyl-red. Ameloblasts were immunostained for anion exchanger-2 (Ae2), a transmembrane pH regulator sensitive for acid that secretes bicarbonate in exchange for chloride. The enamel of AmelX(-/-) mice was 10-fold thinner, mineralized in the secretory stage 1.8-fold more than wild-type enamel and containing less chloride (suggesting more bicarbonate secretion). Enamel of AmelX(-/-) mice stained with methyl-red contained no acidic bands in the maturation stage as seen in wild-type enamel. Secretory ameloblasts of AmelX(-/-) mice, but not wild-type mice, were immunopositive for Ae2, and stained more intensely in the maturation stage compared with wild-type mice. Exposure of AmelX(-/-) mice to fluoride enhanced the mineral content in the secretory stage, lowered chloride, and intensified Ae2 immunostaining in the enamel organ in comparison with non-fluorotic mutant teeth. The results suggest that unprotonated amelogenins may regulate the pH of forming enamel in situ. Without amelogenins, Ae2 could compensate for the pH drop associated with crystal formation.

Analysis of the proliferative potential of odontogenic epithelial cells of pericoronal follicles.

AIM: To evaluate the proliferative potential and the cell proliferation rate of odontogenic epithelial cells. MATERIALS AND METHODS: Forty-two cases of pericoronal follicles of impacted third molars were submitted to silver impregnation technique for quantification of argyrophilic nucleolar organizer regions (AgNOR) and immunohistochemical staining for EGFR and Ki-67. For AgNOR quantification, the mean number of active nucleolar organizer regions per nucleus (mAgNOR) and the percentage of cells with 1, 2, 3 and 4 or more AgNORs per nucleus (pAgNOR) were quantified. Ki-67 immunolabeling was quantified, whereas for EGFR, a descriptive analysis of staining patterns (membrane, cytoplasm or membrane + cytoplasm positivity) was performed. We evaluated the reduced epithelium of the enamel organ and/or islands of odontogenic epithelium present in the entire connective tissue. RESULTS: mAgNOR were 1.43 (1.0-2.42) and were significantly different among pericoronary follicles from upper and lower teeth (p = 0.041). Immunostaining of Ki-67 was negative in all cases. EGFR immunolabeling was found mainly in the cytoplasm and was more intense in islands and cords when compared to reduced epithelium of the enamel organ. CONCLUSION: Odontogenic epithelial cells of some pericoronal follicles have proliferative potential, suggesting their association with the development of odontogenic lesions. CLINICAL SIGNIFICANCE: The authors suggest that nonerupted, especially of the lower teeth, should be monitored and if necessary removed.

Consequences for enamel development and mineralization resulting from loss of function of ameloblastin or enamelin.

Although the nonamelogenin proteins, ameloblastin and enamelin, are both low-abundance and rapidly degrading components of forming enamel, they seem to serve essential developmental functions, as suggested by findings that an enamel layer fails to appear on teeth of mice genetically engineered to produce either a truncated form of ameloblastin (exons 5 and 6 deleted) or no enamelin at all (null). The purpose of this study was to characterize, by direct micro weighing, changes in enamel mineralization occurring on maxillary and mandibular incisors of mice bred for these alterations in nonamelogenin function (Ambn(+/+, +/-5,6, -5,6/-5,6), Enam(+/+, +/- ,-/-)). The results indicated similar changes to enamel-mineralization patterns within the altered genotypes, including significant decreases by as much as 50% in the mineral content of maturing enamel from heterozygous mice and the formation of a thin, crusty, and disorganized mineralized layer, rather than true enamel, on the labial (occlusal) surfaces of incisors and molars along with ectopic calcifications within enamel organ cells in Ambn(-5,6/-5,6) and Enam(-/-) homozygous mice. These findings confirm that both ameloblastin and enamelin are required by ameloblasts to create an enamel layer by appositional growth as well as to assist in achieving its unique high level of mineralization.

A developmental comparison of matrix metalloproteinase-20 and amelogenin null mouse enamel.

Mutations in both the human amelogenin and human matrix metalloproteinase-20 (MMP20, enamelysin) genes cause amelogenesis imperfecta. Both genes have also been individually deleted from the mouse and each deletion results in defective dental enamel. Here, we compare the stage-specific progression of enamel development in continuously erupting mouse incisors from amelogenin null and MMP-20 null mice. Our goal was to closely examine differences in enamel and enamel organ structure between these mice that would allow a better understanding of each protein's function. The predominant feature of the amelogenin null incisors was the late onset of mineral deposition, with little or no protein present within the forming mineral. Conversely, the developing MMP-20 null incisors had a layer of protein between the apical surface of the ameloblasts and the forming enamel. Furthermore, the protein present within the enamel matrix was disorganized. An analysis of crystal structure demonstrated that the thin amelogenin null enamel was plate-like, while the MMP-20 null enamel had a disrupted prism pattern. These results suggest that amelogenin is essential for appositional crystal growth during the early to mid-secretory stage and for the maintenance of the crystal ribbon structure. They also suggest that MMP-20 is responsible for enamel matrix organization and for subsequent efficient reabsorption of enamel matrix proteins. Both genes are essential for the generation of full-thickness enamel containing the characteristic decussating prism pattern.

Expression and characterization of a Rana pipiens amelogenin protein.

Amelogenin, the major protein of developing enamel matrix, controls enamel crystal growth via unique supermolecular features. While much has been contributed to our understanding of mammalian amelogenin function, little is known about how amelogenin and its unique physico-chemical features have evolved among vertebrates. Here we report, for the first time, amphibian amelogenin recombinant protein expression and characterization in Rana pipiens. In order to characterize R. pipiens amelogenin, the newly discovered amelogenin coding sequence was amplified, subcloned, and expressed in Eshcerichia coli. Our newly generated R. pipiens amelogenin-specific antisera resolved a major 19-kDa band on western blots of frog tooth extracts and revealed an enamel organ tissue-specific localization pattern using immunohistochemistry. Using mass spectroscopy, a single major compound with a molecular weight of 21.6 kDa was detected, which corresponded to the amino acid sequence-based molecular weight prediction of the His fusion recombinant protein. Dynamic light scattering studies resolved 41-nm radius subunits compared with 14-nm radius subunits from mouse recombinant amelogenin controls. Transmission electron microscopy revealed defined spherical subunits in R. pipiens matrix self-assembly in contrast with a homogeneous 'stippled' matrix in mouse amelogenin matrix self-assembly. Our data suggest that R. pipiens amelogenin is distinguished from mammalian amelogenins by a number of unique physico-chemical properties which may be related to specific modes of crystal formation in frog enamel.

Structural features of forming and developing blood capillaries of the enamel organ of rat molar tooth germs observed by light and electron microscopy.

The process of vascularization of the enamel organ, a unique epithelial structure, occurs when the tooth germ is fully developed, i.e., at the onset of dentinogenesis. Although the three-dimensional organization of the capillaries has been previously investigated, the structural features underlying the formation of the new capillaries remains poorly understood. Thus, in the hope of better understanding the mechanism of formation of the stellate reticulum capillaries, upper first molar tooth germs of newborn and 3-day-old rats were fixed in glutaraldehyde-formaldehyde and processed for light and electron microscopy. Our results showed that blood capillaries are initially in close proximity to the outer enamel epithelium. Between and intercalated with the capillaries are round/ovoid clusters of cells, some of which are vacuolated, closely apposed to the outer enamel epithelium. The outer enamel epithelium is not a continuous layer, but exhibits gaps between the cells. This suggests that the capillaries penetrate the enamel organ through these gaps, since no invagination of the epithelium was observed. The presence of a cluster of cells containing vacuoles suggests that vasculogenesis is taking place. Images showing loss of the basal lamina, proliferation of endothelial cells, presence of filopodia and lateral sprouting suggests that angiogenesis is also occurring. Thus, neoformation of capillaries of the molar enamel organ of rat seems to occur simultaneously by mechanisms of vasculogenesis and angiogenesis.

Fine structural and cytochemical mapping of enamel organ during the enameloid formation stages in gars, Lepisosteus oculatus, Actinopterygii.

During cap enameloid formation in gars (Lepisosteus oculatus), the dental epithelial cells that constitute the enamel organ were observed by means of transmission electron microscopy and enzyme cytochemistry to detect the hydrolytic enzyme activities, alkaline phosphatase (ALPase), acid phosphatase (ACPase), calcium-dependent adenosine triphosphatase (Ca-ATPase) and potassium-dependent p-nitrophenylphosphatase (K-NPPase) (sodium, potassium-activated adenoshine triphosphatase (Na-K-ATPase)). The enameloid formation process in gars was divided into three stages: matrix formation, mineralisation and maturation. The enamel organ consisted of the outer dental epithelial (ODE) cells, stellate reticulum (SR), stratum intermedium (SI) and the inner dental epithelial (IDE) cells during the whole of the cap enameloid formation stages. During the matrix formation stage, many cisternae of rough endoplasmic reticulum and widely distributed Golgi apparatus, in which the procollagen granules containing cross-striations were often found, were remarkable elements in the IDE cells. During the stage of mineralisation, the IDE cells were tall columnar, and infoldings of distal plasma membrane of the IDE cells became marked. The most developed Golgi apparatus was visible at this stage, and large secretory granules containing fine granular or tubular materials were found in the distal cytoplasm that was close to the infoldings of the distal end. Many lysosomes that were ACPase positive were seen near the Golgi apparatus and in the distal cytoplasm of the IDE cells. ACPase positive granules often contained the cross-striation structure resembling procollagen, suggesting that the procollagen is degenerated in the IDE cells. During the maturation stage, the distal infoldings became unclear, and there were no large granules containing tubular materials, but many ACPase positive lysosomes were still present in the IDE cells. Non-specific ALPase was detected at the plasma membrane of the IDE cells at the mineralisation and maturation stages. K-NPPase was markedly detected at the plasma membrane of the IDE cells at the maturation stage. These results demonstrate that the IDE cells might be mainly involved in the removal of degenerated organic matrix from enameloid during the later formation stages. Strong Ca-ATPase activity was observed at the entire plasma membrane of the stratum intermedium cells, and there was slightly weak activity at the plasma membrane of the IDE cells during the mineralisation and maturation stages, implying that these cells are related to the active Ca transport to the maturing enameloid. It is likely that although the structure of the enamel organ is different, the function, especially at the mineralisation and maturation stages, is similar to other actinopterygians having well-mineralized cap enameloid.

Explainable and critical periods during human dental morphogenesis and their control.

OBJECTIVE: To what extent is the current knowledge about regulatory and patterning processes gained from research on animal models (predominantly mouse) applicable to describe certain aspect of human prenatal dental development? METHODS: 3D-reconstructions were produced from serial sections of human dental primordia (Radlanski collection, Berlin) and scanning electron microscopic visualisation techniques were applied. RESULTS AND CONCLUSION: There are several examples, where present knowledge of regulatory processes allows the understanding of changes in outline and form. However, many other examples show that much more complex regulatory mechanisms should be expected to explain the details of human prenatal dental development.

Hertwig's epithelial root sheath, enamel matrix proteins, and initiation of cementogenesis in porcine teeth.

OBJECTIVES: The aim of this study was to analyze the association between Hertwig's epithelial root sheath (HERS) cells, enamel matrix proteins (EMPs), and cementogenesis. MATERIAL AND METHODS: Porcine teeth were examined at the beginning of root formation by light and transmission electron microscopy. Colloidal gold immunocytochemistry was used to analyze the protein expression of amelogenin and ameloblastin. RESULTS: Before and during disintegration of HERS, its cells displayed the cytologic features of protein synthesis and secretion. While some cells assumed an ameloblast-like phenotype, others extended their territory away from the root surface. A collagenous matrix filled the widening intercellular spaces, and tonofilaments and desmosomes were still present in cells featuring the morphologic characteristics of cementoblasts. Labeling for amelogenin was observed but ameloblastin was not immunodetected. Labeling was associated with organic matrix deposits that were sporadically and randomly distributed both along the root surface and away from it among the dissipated epithelial cells. CONCLUSIONS: These findings suggest that HERS' cells occasionally assume a lingering ameloblastic activity at the beginning of root formation in the pig. While the results do not support the hypothesis of a causal relationship between EMPs and cementogenesis, they lend support to the concept of an epithelial origin of cementoblasts.

Immunohistochemical localization of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the forming rat incisor.

Western blots analyses and gelatin zymography established the presence of matrix metalloproteinase (MMP)-2 and -9 in the forming zone of rat incisor. Light microscope immunohistochemistry carried out on undemineralized material provided evidence for strong MMP-2 staining in the secretory ameloblasts, odontoblasts, in the enamel organ, and in the pulp. A weaker staining was observed in predentin and in the outer part of the forming enamel. Using MMP-9 antibodies, the staining was generally weak, except for the secretory ameloblasts that were positively stained. Electron microscopic immunohistochemistry of undemineralized sections revealed a close association between gold-antibodies complexes and cytoskeletal microfilaments in the cytosol of secretory ameloblasts and odontoblasts, within the rough endoplasmic reticulum and along the plasma membrane. The striking feature of MMP-2 and -9 electron immunostaining was the particularly high labeling in the mantle dentin. By contrast, staining of tissue inhibitors of metalloproteinases (TIMP-1 and -2) was lowest in this region. We suggest that this uneven distribution may have some functional implications.

Transmission and scanning electron microscopic observations of epithelial-mesenchymal interface of rat tooth germs using dithiothreitol separation.

Aperiodic fibrils (AF) project from the interstitial side of the lamina densa of the basement membrane (BM) of the inner enamel epithelium (IE), and show remarkable changes in their morphology during development. The three-dimensional morphology of aperiodic fibrils during development has not been observed, because of the difficulty of exposing the interstitial surface of the BM of the inner enamel epithelium. In the present study, the dithiothreitol separation method was applied to expose the interstitial side of the inner enamel epithelial BM of rat tooth germs for the purpose of observing the exposed aperiodic fibrils by transmission and scanning electron microscopy (TEM and SEM, respectively). After dithiothreitol treatment, the enamel organ (EO) was mechanically separated from the dental papilla (DP). In the region with poorly-developed aperiodic fibrils, the separation occurred at the junction between the inner enamel epithelial BM and the dental papilla, and the aperiodic fibrils were exposed, showing the typical picture of dithiothreitol separation. SEM observation of this region revealed that the aperiodic fibrils were connected to each other and they formed networks. These networks resembled those formed by the anchoring fibrils of epidermal and mucosal epithelial BMs. TEM and SEM observations revealed that there were sidechain-like structures on the surface of the aperiodic fibrils. In the region with well-developed aperiodic fibrils, dithiothreitol treatment was not entirely effective, and some mesenchymal tissues remained on the BM. In this region, TEM observation revealed that the aperiodic fibrils were arranged in parallel with each other, and were connected by the sidechains. Several thin collagen fibrils, which were thought to be immature collagen fibrils (CF) of the predentine, were also connected to the aperiodic fibrils with these sidechains and arranged in parallel with them. Based on SEM and TEM observations, the aperiodic fibrils may be regarded as a kind of anchoring fibrils and they may play a role in connecting the BM with the mesenchymal tissue below. They are also thought to guide the arrangement of collagen fibrils in the surface layer of the predentin.

Fine structure and Ca-ATPase activity of the stratum intermedium cells during odontogenesis in gars, Lepisosteus, Actinopterygii.

This is the first report on the stratum intermedium in vertebrates other than mammals. The aim of this study is to elucidate the fine structure and cytochemical features of the stratum intermedium during the stages of enameloid formation in Lepisosteus. Inner dental epithelium, stratum intermedium, stellate reticulum, and outer dental epithelium are consistently present in the tooth germs of Lepisosteus. The stratum intermedium cells are oval in shape, contain elliptical nuclei, and extend many small processes. It is implied that the structure of the enamel organ is different among actinopterygians, and that constitution of the enamel organ in Lepisosteus resembles that in higher vertebrates. Marked Ca-ATPase activity is observed at the cell membrane of the stratum intermedium cells, suggesting that the cells are involved in calcium transport during the stages of enameloid formation.

Fine structural and cytochemical observations of dental epithelial cells during the enameloid formation stages in red stingrays Dasyatis akajei.

The fine structure and the localization of nonspecific acid phosphatase (ACPase), nonspecific alkaline phosphatase (ALPase), and calcium-dependent adenosine triphosphatase (Ca-ATPase) activities in the dental epithelial cells in tooth germs of Dasyatis akajei in the later stages of enameloid formation were investigated. Numerous invaginations of the distal cell membrane of the inner dental epithelial (IDE) cells were observed at the early stage of enameloid maturation. The invaginations contain many fine granular and filamentous substances; the lamina densa, which was thicker during the former stages, is obscure. Granules exhibiting defined ACPase activity were usually found in the IDE cells during the stages of enameloid mineralization and maturation. IDE cells are putatively involved in the removal of degenerated enameloid matrix during these stages. Marked ALPase activity was detected at the proximal and the lateral cell membranes of the IDE cells from the late stage of enameloid matrix formation to the early stage of enameloid maturation. Strong activity of Ca-ATPase was localized at the proximal and the lateral cell membranes of the IDE cells during the stages of enameloid mineralization and maturation. ALPase and Ca-ATPase activity is probably related to crystal formation in the enameloid and the removal of degenerated enameloid matrix from the enameloid.

Cell-matrix interactions and cell-cell junctions during epithelial histo-morphogenesis in the developing mouse incisor.

The continuously growing rodent incisor develops mainly along its antero-posterior axis. The labio-lingual asymmetry which characterizes this tooth is initiated at the cap stage and increases further during the cap to bell transition (ED14 to ED16) when histogenesis of the enamel organ proceeds. Histology, transmission electron microscopy (TEM), and immunostaining were used to document the changes in the basement membrane (BM) as well as the modifications of epithelial cell-matrix and cell-cell interactions during this period. The expression of plakoglobin, desmoglein and E-cadherin at ED14 suggested that the main cell-cell junctional complexes were adherens junctions. The expression of desmoglein and TEM observations suggested a progressive antero-posterior stabilization of the enamel organ by means of desmosomes from ED14 to ED18. alpha6 integrin, BP 230 and laminin gamma2 chain were all expressed in the developing incisor but were not always co-distributed. Immunostaining and TEM suggested that only primitive type II hemidesmosomes were present. At ED14, cells of the enamel knot (EK) did not show any specific expression for antigens involved in cell-cell interaction. However, strong staining for the laminin gamma2 chain characterized the BM in contact with EK cells. The BM in the labial part of the cervical loop demonstrated ultrastructural changes: the presence of loops of the lamina densa in this region preceeded the differential expression of the integrin alpha6 subunit and that of the laminin gamma2 chain in the labial/lingual parts of the cervical loop. Apoptosis was transiently observed in the contiguous mesenchyme. This affected osteoblasts and also nerve cells close to the labial part of the cervical loop.

Localization of specific binding sites for 125I-TGF-beta1 to fenestrated endothelium in bone and anastomosing capillary networks in enamel organ suggests a role for TGF-beta1 in angiogenesis.

Previous studies have shown endothelial cells to be a major target for endocrine TGF-beta in several soft tissues in the normal growing rat. The potent effect of TGF-beta1 on bone formation prompted us to analyze in detail the localization of specific binding sites for endocrine TGF-beta in hard tissues. At 2.5 minutes after injection of 125I-TGF-beta1, specific binding, as demonstrated by quantitative radioautography, was localized to fenestrated endothelium participating in angiogenesis in the vascular invasion region of the growth plate in bone as well as to anatomizing capillary networks in the maturation zone of the enamel organ. At 15 minutes after injection, the bound ligand was internalized into endocytic vesicles of endothelial cells. In bone, quantitation revealed significant differences in receptor density between endothelia undergoing proliferation vs those in a state of elongation and anastomosis with neighboring endothelial cells. In the rat incisor, specific binding of 125I-TGF-beta1 to endothelium correlated with increased formation of anastomotic capillary networks. These studies identify differential specific binding sites of 125I-TGF-beta1 in angiogenically active endothelium, providing an important link between TGF-beta1, the endothelium, and hard tissue development.

Inositol hexasulphate, a casein kinase inhibitor, alters the distribution of dentin matrix protein 1 in cultured embryonic mouse tooth germs.

Immunohistochemical studies using a polyclonal antibody, raised against the recombinant form of dentin matrix protein 1 (DMP1), show that DMP1 was detected mainly in odontoblasts in cultured mouse embryonic tooth germs. However, in restricted areas, DMP1 staining was also observed in secretory ameloblasts, in the stratum intermedium and stellate reticulum, but only when the odontoblasts located in front of them were unstained. When the embryonic tooth germs were cultured in the presence of inositol hexasulfate, a casein kinase I and II inhibitor, staining of odontoblasts was weak or nil, whereas, in contrast, ameloblasts and enamel organ were strongly immunolabelled, suggesting an enhanced translocation of DMP1 after secretion to the secretory ameloblasts and/or stratum intermedium and stellate reticulum. Moreover, DMP1--was shown to be a good substrate for gelatinase A (MMP-2), but not to gelatinase B (MMP- 9). We hypothesized that DMP1--or the sub-fractions cleaved by the MMP--could behave as diffusible signaling molecule (s) rather than as a true dentin extracellular matrix component.

The ameloblast movement in rat incisor. L.M., S.E.M. and C.L.S.M. study.

The internal epithelium of enamel organ and the below enamel surface during growth of the lower incisor, were examinated in ten Wistar rat 12-27 weeks old and weighing between 150/200 gr, by means of immuno histochemical, light and scanning electron microscopy techniques. Our specimens indicate that during the outer enamel secretion the anti-actin positivity goes from distal terminal web to infra nuclear region of cell body. The results of the present study do not support the active movement hypothesis, conversely they support the Warshawsky (1992) hypothesis, i.e. the distal terminal web permits the maintenance and the assembling of ameloblasts during enamel growth. Hence we do agree with Osborn (1970) who reported that, during secretion, ameloblasts move passively in response to secretory forces.

Calbindin28kDa is specifically associated with extranuclear constituents of the dense particulate fraction.

Recent attempts to understand the function of calbindin28kDa, a widely expressed calcium-binding protein, are confounded by uncertainties over its subcellular location. Using immunoblot analysis of rat brain subregions, we found that the proportion of particulate calbindin28kDa (24-43% of total) was independent of expression level and location. The association of calbindin28kDa with particulate structures appeared to be specific, since it persisted when soluble calbindin28kDa was sequestered by antibodies added before tissue disruption. Moreover, when exogenous calbindin28kDa was added during homogenisation of brain from calbindin28kDa-nullmutant mice, only 10% partitioned to the particulate fraction compared with 33% of endogenous calbindin28kDa in wildtype controls. Confocal microscopy showed that calbindin28kDa was predominantly extranuclear in all tissues analysed (i.e. various brain regions, isolated neurons, and dental enamel epithelium). Dual-label microscopy of neural dense particulate fractions confirmed the extranuclear location of calbindin28kDa and also showed that it partly colocalised with synaptosome and microtubule markers. Using sucrose step gradients, calbindin28kDa was separated from nuclei in parallel with synaptosome and endoplasmic reticulum markers. However, no association with the marker proteins (synaptophysin, ERp29, alpha/beta-tubulin) was detected by calbindin28kDa-immunoprecipitation analysis. Together these findings provide the first consistent picture that calbindin28kDa is located predominantly outside of the nucleus, irrespective of tissue type (neuronal vs. non-neuronal) and experimental approach (biochemical vs morphological). The evidence of a substantial, strong and specific association with insoluble cellular structures challenges the widely held view of calbindin28kDa as a mobile calcium buffer, and supports the existence of important alternative roles that involve target proteins.