Biomedical subjects

S Lécolle

Publications and source records attributed to S Lécolle.

14 recordsLinked to original sources

PubMed2000

Inositol hexasulphate, a casein kinase inhibitor, alters enamel formation in cultured embryonic mouse tooth germs.

Post-translational modification of enamel proteins is regulated by casein kinases (CK) and results in binding sites for calcium ions that subsequently play a key role during the initial stages of mineralization. Phosphorylation may also influence the secretion and extracellular organization of enamel proteins. Previous studies indicated that inositol hexasulphate inhibited the activity of CK-I and/or CK-II in mouse tooth germs (Torres-Quintana et al., 1998). We hypothesized that inositol hexasulphate would also inhibit the activity of the specific casein kinase(s) identified in secretory ameloblasts, and would prove useful for determination of the extent to which phosphorylation might influence the organization of enamel proteins at early stages of enamel formation. To test this hypothesis, we dissected mandibular first molars from 18-day-old mouse embryos and cultured them for 11 days in the presence of 0-0.1 mM inositol hexasulphate. Ultastructural analysis revealed that the formation of enamel was largely impaired at an inhibitor concentration > or = 0.08 mM. Quantitative radioautographic analysis of [33P]phosphate incorporation indicated that radiolabeled phosphate normally secreted into forming enamel was retained within ameloblasts. In contrast, no significant difference was observed between control and inositol-hexasulphate-treated tooth germs when cultures were labeled with [3H]serine and [3H]proline. SDS-PAGE and Western blot analysis confirmed that while inositol hexasulphate inhibited CK-mediated phosphorylation, it did not significantly alter protein synthesis. We conclude that impairment of phosphorylation leads to intracellular accumulation of [3H]phosphate-containing material by ameloblasts. We also conclude that when non-phosphorylated enamel matrix proteins are secreted, they are either unable to form an enamel matrix that supports mineralization, or they diffuse throughout a poorly mineralized dentin.

Ameloblasts↗

PubMed1999

[3H]choline uptake and turnover into membrane and extracellular matrix phospholipids, visualized by radioautography in rat incisor dentin and enamel.

In order to study the uptake and fate of [3H]choline into cellular and extracellular phospholipids in the forming part of mandibular rat incisors, radioautography was carried out after treatment with the iodoplatinate reaction which retains phospholipids. Thirty minutes and 1 hour after the intravenous injection of the radiolabeled precursor, grain density in secretory odontoblasts and ameloblasts was not significantly above background labeling whereas dentin was actually labeled. Therefore, at this early period, odontoblasts cannot be responsible for the secretion of phospholipids incorporated into dentin, and intercellular diffusion of components originating from blood could explain this early dentin labeling. After 2 hours, odontoblasts and ameloblasts were labeled. In cells, grain density reached a maximum at 4 hours, reduced at 24 hours, and strongly decreased at 4 days. In predentin and enamel, grain density peaked at 24 hours and diminished at 4 days. However, in the forming enamel 4 days after the injection, labeling was twice as high as in any other compartment. Altogether, the results highlighted two distinct pathways for phospholipids in dental mineralized dental tissues: a first one shows evidence of early incorporation of [3H]choline into dentin resulting from intercellular diffusion independently from odontoblasts secretion, whereas inside the forming enamel, higher labeling and longer retention of choline-containing membrane components were detected between 4 hours and 4 days. This suggests an accumulation of membranes that are not subjected to rapid turnover in contrast with other dental compartments.

Ameloblasts↗

PubMed1998

Effects of inositol hexasulphate, a casein kinase inhibitor, on dentine phosphorylated proteins in organ culture of mouse tooth germs.

To study the effects of impaired protein phosphorylation on dentine formation and mineralization, inositol hexasulphate, an intracellular type I and type II casein kinase inhibitor, was used in an in vitro organotypic culture system. Mandibular first molar tooth germs were dissected from 18-day-old mouse embryos and cultured for 11 days with and without inositol hexasulphate at different concentrations. At 0.04-0.08 mM inhibitor, cellular alterations were not detected. Dentine displayed the characteristic purple-blue colour when Stains all, a specific stain for extracellular phosphoproteins, was used. At 0.1 mM, dentine failed to stain and mineralization did not occur, as seen from the von Kossa method. The presence of numerous lysosome-like vesicles inside cells indicated that the experiment was at the limits of cytotoxicity; higher concentrations induced severe cellular alterations. Therefore, quantitative radioautography was carried out on germs treated or not with the inhibitor at 0.1 mM. [33P]-phosphate incorporation showed that grain density in inhibited germs compared with that in control germs was about double in odontoblasts and half in the predentine/dentine compartment. In the presence of inositol hexasulphate the incorporation of [3H]serine into odontoblast cell bodies was unchanged between 2 and 24 h while in predentine/dentine, grain density was higher between 1 and 4 h, and reduced at 24 h. Both with [33P]phosphate and [3H]serine, labelling was seen throughout the porous dentine formed in vitro and not as a band located at the predentine/dentine junction, as is the case in vivo. With [3H]proline, in the presence of the inhibitor, a small reduction of grain density occurred in cell bodies, no significant difference was seen between 1 and 4 h in predentine/dentine, and more silver grains were present after 24 h both in cells and in the matrix. The radioautographic data support the view that the inhibitor interacts mostly with post-transductional phosphorylation and does not alter significantly other cell synthetic pathways and functions. Finally, the experiments presented here confirm that phophorylated proteins have a key role in dentine mineralization.

Analysis of Variance↗

PubMed1998

Fragmentation of the distal portion of Tomes' processes of secretory ameloblasts in the forming enamel of rat incisors.

In order to investigate enamel and dentin phospholipid metabolic pathways, two separate experiments were carried out. Firstly, rats were given chloroquine, a drug which induces a lipidosis-like disease. Extensive accumulation inside lysosomes was seen in all the groups of cells in the forming part of the rat incisor, except secretory ameloblasts which were unaffected by the drug. Secondly, the uptake and fate of 3H-choline were studied by radioautography on rats fed either normally or on an essential fatty acid deficient diet (EFAD). Four hours after the injection of the precursor, incorporation reached a maximum then decreased gradually. At 4 days the forming enamel displayed higher silver grain density than any other compartment. In EFAD rats 3H-choline incorporation was decreased drastically in each compartment except in the forming enamel which was not affected by the deficiency. The longer retention of the labeling in the forming enamel and the lack of lysosomal accumulation in chloroquine-treated secretory ameloblasts support the hypothesis that fragments of the distal Tomes' process are released during enamel formation. Disconnected from the cells, membrane remnants are neither reinternalized nor subjected to further degradation inside lysosomes.

Ameloblasts↗

PubMed1996

Dual incorporation of (35S)sulfate into dentin proteoglycans acting as mineralization promotors in rat molars and predentin proteoglycans.

Autoradiographic investigations were carried out 0.5, 1, 2, 4, 24, 48, 72, and 120 hours after the injection of a single dose of [35S]-sulfate on undemineralized molars of 7-15-day-old rats. In predentin, labeling was detected at 0.5 hours. Silver grain density reached a plateau value between 1 and 24 hours, then decreased and disappeared 120 hours after injection. In dentin, the mineralization front started to be labeled as early as 0.5 hours after injection. Labeling increased at the dentin edge between 1 and 2 hours, reached a maxima at 4 hours, then started to decrease, the labeled band seen 24 hours after injection being further incorporated into dentin. This band stood at constant distance from the dentin-enamel junction with stable grain density, even at 120 hours. This investigation proves the existence of two distinct groups of [35S]-labeled proteoglycans, one exclusively related to predentin and disappearing with time, and the second one located in dentin behaves as a stable component. The fact that an early labeling appeared at the mineralization front which was further incorporated into dentin, confirms that dentin proteoglycans constitute an individual group of molecules that are not derived from predentin proteoglycans, and act as mineralization promotors.

Animals↗

PubMed1995

Lipids in predentine and dentine.

Using two histochemical methods, malachite green-aldehyde and iodoplatinate, phospholipids were visualized in the predentine of rat incisors in the spaces located between collagen fibers and in dentine as needle-like structures located along individual or groups of mineralizing collagen fibers. The same staining pattern was seen with phospholipase A2-gold. Autoradiographic investigation using 3H choline as labelled precursor, visualized the incorporation of membrane-associated and extracellular choline-containing phosphatidyl choline and sphingomyelin. The cell and membrane-associated labelling decreased gradually between 24 and 4 days, whereas incorporation of the labelled precursor as stable extracellular matrix component was seen in dentine. In addition to these investigations, pharmacologically induced (suramine) and genetically (Krabbe's disease) lysosomal storage pathology was investigated. Defects due to lipid metabolism alterations were seen in predentine and/or in dentine. The major differences visualized here between the non-mineralized and mineralized compartments and interactions between phospholipids and proteoglycans, support the view that phospholipids as matrix components play an important role in the mechanisms of dentine formation and mineralization.

Animals↗

PubMed1995

Dental mineralization.

Extracellular matrix components and cell-derived microstructures are implicated in mineralization processes which occur in dental tissues. The respective role(s) of collagenic and non-collagenic matrix components are reviewed: phosphorylated and non-phosphorylated proteins, proteoglycans and phosphpholipids. Space-filling amphiphilic molecules seem to play an important role in the preorganization and oriented deposition of calcium phosphate on structures serving more or less as passive support in dentine as well as in enamel.

Amelogenesis↗

PubMed1995

Poly-L-lysine-gold complexes used at different pH are probes for differential detection of glycosaminoglycans and phosphoproteins in the predentine and dentine of rat incisor.

At neutral pH, poly-L-lysine-gold complexes labelled the predentine extensively, whereas in dentine the number of gold complexes was reduced by half. Hyaluronidase pretreatment of the section at pH 6.8, prior to labelling, suppressed most of the staining in predentine and did not affect dentine. In contrast, alkaline phosphatase pretreatment at pH 9 enhanced the gold complex labelling in predentine and removed most of the labelling in dentine. This proves that at pH 7.2, the polyanions which are stained include a heterogeneous population of glycosaminoglycans, located in predentine, and phosphoproteins, visualized in dentine. At acidic pH levels (2.9 and 1.1), the number of scored gold complexes decreased, but the ratio between predentine and dentine labelling remained constant. Hyaluronidase pretreatment removed or firmly reduced the gold complex labelling both in predentine and dentine, whereas alkaline phosphatase pretreatment of the sections at pH 9 prior to labelling did not induce any change. This argues in favour of an increased specificity of polylysine-gold complex staining for glycosaminoglycans, stained at low pH in both predentine and dentine. Differential staining of glycosaminoglycans and phosphoproteins according to the pH provides a useful tool for studying the role played respectively by the two matrix components in dentine mineralization.

Animals↗

PubMed1994

Effects of suramin, a polyanionic drug inducing lysosomal storage disorders on tooth germs in vitro.

Suramin, a potent inhibitor of lysosomal enzymes, is commonly employed as a tool for inducing experimental mucopolysaccharidosis and lipidosis. The effects of the drug on embryonic mouse molars were analysed. Presecretory ameloblasts and odontoblasts were loaded with lysosome-like vacuoles. Staining with MC22-33F, an antibody to choline phospholipids and sphingomyelin, was completely reversed in the suramin-treated germs, in that it stained only presecretory ameloblasts (versus odontoblasts and some pulpal cells in the control group), according to a developmentally regulated pattern. The suramin-induced cytoplasmic changes were reminiscent of the features of mucopolysaccharidoses and lipidoses. The basement membrane, separating the enamel organ from the dental papilla, displayed suramin-induced patches, and in predentin collagen fibrillogenesis was found to be disturbed. Furthermore, autoradiography was employed to reveal uptake and distribution of [3H] suramin in the cells and predentin. Finally, a suramin-induced disturbance of the metabolism of sulphated macromolecules was found. The results imply that suramin effects in vitro on tooth germs can be used as a useful experimental model with to study both the action of the drug as well as cell and extracellular matrix perturbations in a mucopolysaccharidosis-like condition.

Animals↗

PubMed1994

Cuprolinic blue visualization of cytosolic and membrane-associated glycosaminoglycans in the rat junctional epithelium and gingival epithelia.

The gingiva of rat molars was studied at the light microscope level using glutaraldehyde as fixative, Cuprolinic Blue for visualizing polyanionic glycosaminoglycans and the autometallographic technique for enhancing the copper signal of the cationic dye. The polyanions were located inside the epithelial cells in the junctional epithelium, whereas a network located along either the plasma membrane or the intercellular spaces, or both, of the gingival oral epithelium and sulcular oral epithelium was evident with autometallography. In these cases, positive staining was limited to the basal and spinous layers, the granular and keratinized layers being unstained. With the transmission electron microscope, electron-dense aggregates were seen in the gingival lamina propria, in the basement membrane and along the plasma membrane of the keratinocytes of the basal and spinous layers of the gingival and sulcular oral epithelia. In the junctional epithelium, Cuprolinic Blue-positive granules, 25 nm in diameter, were seen in the cytoplasm. Together with some vesicles containing electron-dense material, they may account for the staining process noted after autometallography. When the ultra-thin sections were digested with bovine testicular hyaluronidase, the staining was abolished. This indicates that glycosaminoglycans were primarily responsible for the staining pattern visualized with these methods. In the junctional epithelium, the cytosolic location of the 25 nm granules reflects either transcellular transfer between the plasma membrane and the nucleus or accumulation of glycosaminoglycans in this group of keratinocytes. The glycoconjugates located inside vesicles or vacuoles are related to endocytosis and lysosomal degradation. Interstitial glycosaminoglycans seen in the two types of oral epithelium may play a role in the diffusion of water and nutriments.

Animals↗

PubMed1994

Galactosylceramide lipidosis (Krabbe's disease) and deciduous dental tissues. A case report.

The deciduous teeth of a 5-year-old girl affected by a protracted type of infantile globoid-cell leukodystrophy or Krabbe's disease, a galactosylceramide lipidosis, were examined with the scanning and transmission electron microscopes. The diagnosis was established on the basis of galactosylceramidase activity (0.04 mu katal/kg proteins versus 0.86 +/- 0.49 in the normal situation) and abnormalities in the white matter and trouble in myelinization assessed by magnetic resonance imaging. In spite of the fact that enamel, dentin and pulp are extraneural tissues, they were also affected by the disease. Ultrastructural observations of dental mineralized tissues revealed enamel hypoplasia as well as abnormal proteinaceous lamina and hypomineralized lines or bands. Inside the mantle dentin, dendritic inclusions of amorphous material were seen. Lysosomal storage inclusions were observed in all the cells of the dental pulp. The myelin sheaths of dental peripheral nerves displayed severe degenerative changes. Proliferation of dentinoclasts contributed to stimulate root resorption.

Child↗

PubMed1993

[Lysosomal storage diseases, genetic or drug-induced? effect of glycosaminoglycan and sphingolipid disorders on dental tissues].

In vivo studies were carried out on dental tissues of rat incisor after a single injection of suramin, a drug which induces mucopolysaccharidosis-like disease. Accumulation of lysosome-like structures was seen in secretory ameloblasts and odontoblasts. In vitro studies on embryonic tooth germ buds showed similar changes when they were cultured in presence of suramin. Anti-phospholipid immunolabelling revealed a developmentally regulated temporo-spatial pattern. Radiolabeling with 3H-suramin indicated cytosolic and nuclear incorporation. The drug acting as polyanion interacted directly with predentine. 35S sulphate incorporation was impaired by the drug. Another lysosomal storage disease, the sphingolipidosis, Krabbe's disease was also investigated in human. Changes were observed in pulp cells and as a consequence in dentin. Enamel also displayed many changes. Pharmacological or genetically acquired diseases constitute models providing insights on the role played by glycosaminoglycans and phospholipids in biomineralization.

Ameloblasts↗

PubMed1989

Autometallography for histochemical visualization of rat incisor polyanions with cuprolinic blue.

Autometallography was applied to semi-thin sections of rat incisors fixed a solution of cuprolinic blue-aldehyde. The resulting reduction of silver ions to metallic silver amplifies the copper sulfide signal of the cationic dye. Silver grains were seen over the cell bodies of ameloblasts and odontoblasts but not over their processes. This was owing to the interaction of cuprolinic blue with the DNA and RNA of these cells. In the extracellular matrix, silver grains were unevenly distributed over the predentin, dentin, and forming enamel. The distal predentin near the mineralization front and a thin band of dentin located near the dentino-enamel junction displayed unexpectedly intense accumulation of silver grains, whereas all other portions of the extracellular matrix exhibited the distribution of glycosaminoglycans expected from previous studies. The present investigation constitutes a new application of autometallography to glycosaminoglycan histochemistry.

Animals↗

PubMedDate not supplied

Apoptosis in human and rat dental pulp.

Apoptotic cells were visualized in human premolar and rat molar pulps using either the TUNEL method, which stains DNA strand breaks by in situ nick end labeling, or labeling sections with an anti-transglutaminase antibody. Apoptotic cells were evident at the periphery of the pulp, mostly in a sub-odontoblastic location, and were more numerous in the crown than in the root region of the pulp. Most odontoblasts were unlabeled. A few apoptotic nuclei in the pulp of rat molars displayed the characteristics of chromatin condensation, as observed by electron microscopy. Many cell debris resembling apoptotic bodies were also observed. The 3 methods support the occurrence of apoptosis in the dental pulp. As this phenomenon was observed in healthy teeth apoptosis is probably involved in the regulation of the pulp cell population.

Animals↗