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J V Ruch

Publications and source records attributed to J V Ruch.

At least 19 recordsLinked to original sources
PubMed1992

Structural variations of different oral basement membranes revealed by cationic dyes and detergent added to aldehyde fixative solution.

The ultrastructural appearance of different types of basement membrane was studied using histochemical methods for visualizing glycosaminoglycans. Samples of rat gingiva and mouse molar germ tissue were fixed either with glutaraldehyde, glutaraldehyde-ruthenium hexammine trichloride (RHT), glutaraldehyde-Cuprolinic Blue (CB) or cetylpyridinium chloride-glutaraldehyde (CPC). Ultrathin sections were stained with uranyl acetate and lead citrate. The results showed that the conventional trilaminar structure of the basement membrane was observed after glutaraldehyde and CB fixation. In contrast, after CPC or RHT fixation, the appearance of the basement membrane was homogeneous without any evidence of a lamina lucida. Furthermore, after single fixation with CPC, the ultrastructure of different basement membranes from oral tissues showed some differences in appearance which were related to their localizations, functions, or both.

Animals↗
PubMed1992

The carboxy-terminal extension of the collagen binding domain of fibronectin mediates interaction with a 165 kDa membrane protein involved in odontoblast differentiation.

Terminal differentiation of the odontoblast is characterized by an elongation and a polarization of the cell. The change in the cell shape and the reorganization of the cytoplasm involve the microfilament system. An immunological approach has previously implicated a transmembrane interaction between fibronectin and vinculin in the control of odontoblast differentiation. A 165 kDa protein localized on the cell-surface of odontoblasts mediated this interaction. In order to define the nature of the interaction of the 165 kDa protein with fibronectin, peptides were prepared by proteolytic cleavage of fibronectin with alpha-chymotrypsin. The results indicate that the 165 kDa protein interacted with a 62 kDa peptide located towards the amino-terminal extremity of fibronectin, but not with a 47 kDa related fragment. Both these 62 kDa and 47 kDa peptides included the collagen-binding domain and were retarded on a heparin-Ultrogel column. Microsequences demonstrated that the 62 kDa and 47 kDa fragments had the same amino-terminal extremity and that the larger fragment was extended in the carboxy-terminal direction. This carboxy-terminal extension of the collagen binding domain of fibronectin is implicated in the interaction of this molecule with the 165 kDa protein. On the other hand, odontoblasts differentiated normally when tooth germs were cultured in the presence of GRGDS synthetic peptide, suggesting that RGD-dependent integrins were not involved in odontoblast differentiation. Staining of dental mesenchymal cells in primary culture and of differentiated odontoblasts in situ with antibodies directed against the beta 1-subunit of integrins confirmed previous observations and showed that although beta 1 integrins are involved in the attachment of cultured dental cells, they are not implicated in the process of odontoblast differentiation.

Actin Cytoskeleton↗
PubMed1992

Characterization of a monoclonal antibody that specifically binds to choline phospholipids and its use in immunocytochemistry.

A monoclonal IgM (MC22-33F), raised in response to mouse embryonic dental papilla cells, was selected for further analysis on the basis of the unusual resistance of its epitope to detergent extractions and protease treatments of cell cultures. Binding of MC22-33F to cultured cells was abolished after either pre-treatment of the cells with phospholypase C or pre-incubation of the hybridoma culture supernatant with multilamellar phosphatidylcholine-containing vesicles. MC22-33F reacted with phosphatidylcholine, with the phosphatidylcholine analogue dimethylphosphatidylethanolamine, and with sphingomyelin immobilized on polystyrene surfaces or in thin-layer chromatograms. Crossreaction with other phospholipids was not observed. The surface of cultured epithelial cells was labeled by MC22-33F at sites of bleb formation. Combining immunostaining by MC22-33F and histochemical staining of cultured cells revealed codistribution of phospholipid-containing inclusions with either lysosomes or neutral fat droplets, and inhibition of lipid degradation by kanamycin resulted in a parallel accumulation of these inclusions and of neutral fats in the cytoplasm. Immunolabeling by MC22-33F of frozen mouse tissues was maximal in fat-storing and steroid-producing cells. Extracellular phospholipids present in calcifying cartilage septa strongly reacted with MC22-33F. This monoclonal antibody offers an interesting alternative to histochemical lipid stains for investigating fatty metamorphosis and extracellular lipid deposition under physiological and pathological conditions.

Animals↗
PubMed1992

Effects of ascorbate-deficiency on collagen secretion and resorption in cultured mouse incisor germs.

The effects of ascorbic acid deficiency on mouse incisors, grown in vitro, has been investigated at the histological and cytological levels. In this model, continuously growing mouse incisors are characterized by the existence of different type of predentin-dentin matrix on its lingual (root-analogue) and labial (crown-analogue) surface and the absence of enamel on the lingual surface. Our observations indicated that ascorbate-deficiency affected the behavior of mouse tooth germs in vitro: odontoblast differentiation was disturbed and morphological evidence for odontoblast-mediated collagen resorption were observed. An abnormal amorphous predentin-dentin matrix existed and the basement membrane was prematurely disrupted. The dentin mineralization, as well as functional differentiation of ameloblasts were strongly hampered. Chronic deficiency led to disorganization of the dental tissues.

Absorption↗
PubMed1992

Immunolocalization of acidic and basic fibroblast growth factors during mouse odontogenesis.

Acidic and basic fibroblast growth factors (aFGF and bFGF), are both known to bind to extracellular matrix components, particularly proteoheparin sulfates, and to regulate in vitro proliferation, differentiation and morphology of cells of neuroectodermal and mesodermal origins. Their patterns of distribution were studied during mouse odontogenesis by means of indirect immunofluorescence and immunoperoxidase histochemistry on frozen fixed sections and after Bouin's fixative and paraffin embedding. Localization of aFGF on frozen fixed sections was observed in the oral epithelium, dental lamina and oral mesenchyme (day-12 of gestation), the stellate reticulum and oral epithelium (day-14), the stratum intermedium and at the basal and apical poles of preameloblasts at bell stage. After birth aFGF epitopes were localized within the predentin-dentin area, the stratum intermedium and at the secretory pole of ameloblasts. There was no staining with anti-aFGF antibodies after Bouin's fixative and paraffin embedding. In contrast, using this protocol, intense stainings were found with anti-bFGF antibodies predominantly within dental and peridental basement membranes and mesenchyme: staining of the dental basement membranes was transient (bud and cap stage) and discontinuous; a preferential concentration of bFGF epitopes in the condensed dental mesenchyme of incisors (cap stage) and the dental papillae mesenchymal cells of molars (bell stage) was observed in the posterior and the cervical part of tooth germs. An intense immunostaining of the stellate reticulum with anti-bFGF antibodies was also found on paraffin sections from bud to bell stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Effects of dentin proteins, transforming growth factor beta 1 (TGF beta 1) and bone morphogenetic protein 2 (BMP2) on the differentiation of odontoblast in vitro.

We have studied the effects of dentin proteins, of Transforming Growth Factor beta 1 (TGF beta 1) and Bone Morphogenetic Protein (BMP2) on the differentiation of odontoblasts in vitro. The total EDTA-soluble fraction of dentin proteins, prepared from rabbit incisors was further separated by chromatography on DEAE-Cellulose and heparin-agarose columns. While the total EDTA-soluble fraction of dentin had no effect on cultured dental papillae, fractions retained on both columns were able to initiate functional differentiation of preodontoblasts of isolated day-17 first lower mouse molar dental papillae cultured in vitro. TGF beta 1 and BMP2, both stimulated the matrix secretion by dental papillae cells. TGF beta 1 and BMP2, combined with the inactive total EDTA-soluble fraction, stimulated odontoblast differentiation. An active fraction retained on DEAE-Cellulose completely lost the inductive activity after incubation with a neutralizing anti-TGF beta antibody. These results demonstrate that a TGF beta-like molecule present in dentin could interact with some component which acts as a modulator of its activity on the initiation of the cytological and functional differentiation of odontoblasts.

Animals↗
PubMed1992

Effects of retinoids on tooth morphogenesis and cytodifferentiations, in vitro.

The first embryonic lower mouse molar was used as a model system to investigate the effects of two retinoids, retinoic acid (RA) and a synthetic analogue, Ch55, on morphogenesis and cytodifferentiations in vitro. Exogenous retinoids were indispensable for morphogenesis of bud, cap and bell-stage molars in serum-free, chemically-defined, culture media. Transferrin and RA or transferrin and Ch55 acted synergistically in promoting morphogenesis from bud and cap-stage explants. Transferrin, per se, had no morphogenetic effect. Epithelial histogenesis, odontoblast functional differentiation and ameloblast polarization always occurred in RA-depleted explants. Comparison of the distributions of bromodeoxyuridine (BrdU) incorporation between explants cultured in the absence or presence of RA revealed that RA could modify the patterns of cell proliferation in the inner dental epithelium and dental mesenchyme. Inner dental epithelium cell proliferation is regulated by the dental mesenchyme through basement membrane-mediated interactions, and tooth morphogenesis is controlled by the dental mesenchyme. Laminin is a target molecule of retinoid action. Using a monospecific antibody, we immunolocalized laminin and/or structurally-related molecules sharing the laminin B chain in the embryonic dental mesenchyme and in the dental basement membrane and showed that RA could promote the synthesis or secretion of these molecules. Based on previous in situ hybridization data, it was speculated that CRABPs might regulate the effects of RA on embryonic dental cell proliferation. The fact that Ch55, a retinoid which does not bind to CRABPs, is 100 times more potent than RA in promoting tooth morphogenesis in vitro seems to rule out this hypothesis. On the other hand, the stage-specific inhibition of tooth morphogenesis by excess RA is consistent with the hypothesis that CRABPs might protect embryonic tissues against potentially teratogenic concentrations of free retinoids.

Animals↗
PubMed1992

[Cell-matrix interactions and odontoblast differentiation].

The terminal differentiation of odontoblasts requires the integrity of the cytoskeleton and is controlled by cell-matrix interactions. These interactions implicate both matrix molecules and matrix-associated growth factors. On the one hand, predentin-dentin constituents were found to initiate odontoblast differentiation and to allow the maintenance of this state; TGF-beta or related molecules are implicated. Fibronectin on the other hand can induce the differentiation of second generation odontoblasts and interacts with three high molecular weight proteins present in membrane prepared from dental mesenchymal cells. One of these proteins (165 kDa) was localized on the surface of odontoblasts and is involved in the organization of microfilaments. Two main axes of research will have to be developed in the future in order to understand how matrix molecules and growth factors interactions can be modulated in time and space by epithelial and mesenchymal cells, and how such modulations can affect the phenotype of these cells.

Cell Differentiation↗
PubMed1992

Patterned distributions of chondroitin sulfate isoforms, retinoic acid receptor gamma and cellular retinoic acid binding proteins in the embryonic mouse incisor.

Odontogenesis is an example of developmental process whereby pluripotential embryonic ectodermal and ectomesenchymal cells give rise to specific, highly ordered, spatial arrangements of overtly differentiated tissues. This process implies the sequential and coordinated expression of several molecules of defined biological activities. The data presented here demonstrate the occurrence, in the embryonic mouse incisor, of a striking correspondence between gradients of mitoses, chondroitin 6-sulfate distribution, retinoic acid receptor gamma and cellular retinoic acid binding protein I transcripts. Although purely phenomenological, these observations provide an important guide to the design of experiments for understanding the mechanisms of pattern formation during tooth development.

Animals↗
PubMed1991

Mosaic 46,XY/92,XXYY,del(5)(q13 q34) in an adult lymphoblastic leukemia.

Usually the chromosome anomalies encountered in ALL are modal number abnormalities (hyperdiploidy or hypodiploidy) and structural anomalies such as t(8;14), t(11;14), t(9;22), t(1;19) and del(6p). The 5q- syndrome is mainly associated with myelodysplastic syndromes and with ANLL (M1, M2, M3). We report the case of a patient presenting with a mosaic karyotype 46,XY/92,XXYY,del(5)(q13 q34) in the following proportion 1/3 normal mitoses and 2/3 tetraploid mitoses.

Aged↗
PubMed1991

Involvement of cellular retinoic acid-binding proteins I and II (CRABPI and CRABPII) and of the cellular retinol-binding protein I (CRBPI) in odontogenesis in the mouse.

The coordination of the activities of individual cells during development is regulated in part by epigenetic signals either encoded in the insoluble extracellular matrix or provided by small diffusible factors such as growth factors peptides and retinoids. Odontogenesis offers a suitable model to correlate the temporospatial distributions of such molecules, and of their cell receptors and ligands, with particular developmental processes. We have analyzed, by in situ hybridization, the distribution patterns of CRABPI, CRABPII and CRBPI transcripts during odontogenesis in the mouse. CRABPI transcripts were restricted to the mitogenic regions of the dental mesenchyme during late bell stages and were absent from post-mitotic odontoblasts. The only epithelial site of CRABPI transcription was the labial epithelial loop of the continuously growing incisor. CRABPII transcription was preponderant in the mitogenic zones of the dental epithelium: differential labeling of the dental epithelium occurred as early as the dental bud stage and during subsequent molar morphogenesis, this labeling became confined in the epithelial loops. The graded distribution of CRABPII transcripts along the anteroposterior axis of the continuously growing incisor was superimposed with the gradient of mitoses. CRABPII transcripts were absent from post-mitotic ameloblasts. It is concluded that during odontogenesis the expressions of the CRABPI and CRABPII genes are confined to regions exhibiting the highest rate of cell proliferation whenever differential mitotic activity is required. Moreover, the putative effects of retinoic acid on the regulation of cell proliferation kinetics in the dental epithelium and in the dental mesenchyme imply distinct CRABPs. CRBPI transcripts were restricted to the dental mesenchyme prior to the onset of CRABPI transcription. This observation supports the hypothesis that the two proteins might perform antagonistic functions in some retinoic acid-mediated developmental processes.

Animals↗
PubMed1990

Establishment and characterization of somatic hybrids between human differentiated macrophages and mouse myeloma NS1 cells.

Human macrophages obtained from circulating monocytes matured in vitro by culture for seven days in hydrophobic flexible teflon bags were successfully fused with murine myeloma NS1 cells. Six of 20 clones, selected for their adherence properties, were further studied. All possessed human chromosomes (mean number ranging from 4 to 14 depending on the clones studied), exhibited non-specific esterases (but no peroxidase activity) and expressed CD14 antigen and C3 receptors (but no MAX-1 antigen). Moreover, the hybridomas retained phagocytic activity and high interferon plus lipopolysaccharide-activable cytolytic activity against tumor cells.

Animals↗
PubMed1990

Immunolocalization of transforming growth factor beta 1 and epidermal growth factor receptor epitopes in mouse incisors and molars with a demonstration of in vitro production of transforming activity.

Day-14 lower incisors and day-18 first lower molars of mouse embryos produced in vitro transforming activities for non-confluent NRK cells co-cultured in agar, and mitogenic activities for exponentially growing NRK and BHK cells. The patterns of distribution of TGF beta 1 and EGF receptor, both known to regulate cell proliferation, differentiation and transformation in vitro and suspected to play important roles in developmental processes, were studied during mouse odontogenesis by means of indirect immunofluorescence on fixed or frozen fixed sections. TGF beta 1 epitopes were detected in the stellate reticulum of day-13 to day-16 incisors and of molars from day-17 onwards. Dental mesenchyme of day-14 incisors and postnatal molars, and peridental mesenchyme of bud and cap stage molars and incisors were also stained by TGF beta 1 antibodies. EGF receptor was localized in the enamel organs of incisors and molars; the inner dental epithelium and later the outer dental epithelium rapidly became negative while the stellate reticulum remained stained. Incisor apical mesenchyme showed an intense reaction with EGF receptor antibodies after birth.

Animals↗
PubMed1990

Effects of cerulenin, an inhibitor of fatty acid synthesis on reconstitution of the dental basement membrane.

When trypsin-dissociated enamel organs and dental papillae were recombined in the presence of cerulenin--an antibiotic which is a potent inhibitor of fatty acid synthesis--the newly synthesized basement membrane seemed defective in a dose-dependent manner. The three-dimensional relationship between the basement membrane components and the plasma membrane appears to be regulated in part by lipids.

Animals↗
PubMed1990

Effects of beta-D-xyloside on morphogenesis and cytodifferentiation in cultured embryonic mouse molars.

Embryonic mouse molars were grown on a semi-solid medium supplemented with 2 mM beta-D-xylopyranoside (beta-xyloside), a specific inhibitor of proteoglycan synthesis. The induced glycosaminoglycan depletion in the extracellular matrix was monitored by immunohistochemistry employing monoclonal antibodies to chondroitin 4- and chondroitin 6-sulfates. beta-Xyloside inhibited formation of the dental bell and delayed the appearance of the first odontoblasts. Odontoblast functional differentiation proceeded in the absence of chondroitin sulfate in the basement membrane. Predentin secreted in the presence of beta-xyloside triggered the polarization of ameloblasts, but did not allow the maintenance of polarized odontoblasts. These results support the hypothesis that, in the tooth germ, chondroitin sulfate proteoglycans participate in the regulation of cell kinetic-dependent morphogenesis (Mark et al., 1990. Differentiation 43, 37-50). On the other hand, the possibility that chondroitin sulfate might play a role in odontoblast terminal differentiation is definitively ruled out.

Ameloblasts↗
PubMed1990

Chondroitin sulfates in developing mouse tooth germs. An immunohistochemical study with monoclonal antibodies against chondroitin-4 and chondroitin-6 sulfates.

The role of glycosaminoglycans and proteoglycans during ontogenesis is not known. The developing tooth offers a potentially important model for studies of structure-function relationships. In this study, we have analysed the temproal and spatial expression of chondroitins of differing sulfation patterns in embryonic molars and incisors. For this purpose, we have used monoclonal antibodies (Mabs) specific for unsulfated, 4-sulfated, and 6-sulfated forms of chondroitin in conjunction with indirect immunofluorescence or immunoperoxidase labeling. Unsulfated chondroitin was not detected in embryonic teeth. Chondroitin 4- and chondroitin 6-sulfates were present in the stellate reticulum but otherwise they were confined to the dental mesenchyme. The 3B3 and MC21C-epitope, which are markers of 6-sulfated chondroitin, were uniformly distributed in the dental mesenchyme during the bud stage; they disappeared from the dental papilla of the cusps and of the anterior region of the incisor as development proceeded. These epitopes were absent from the basement membrane and from the predentin. In the odontoblastic cell lineage, the 3B3 and MC21C-epitopes were detected only between preodontoblasts at an early stage of differentiation. The monoclonal antibody 2B6 served as a probe to localize chondroitin 4-sulfate. This glycosaminoglycan was detected as early as the dental lamina stage but its expression was restricted to the basement membrane of the teeth until the late bell stage. After the onset of cusp formation, strong staining was also observed over the occlusal region of the dental papilla while the cervical region of the dental papilla remained 2B6-negative. Incisors at the bell stage exhibited a decreasing gradient of immunostaining by 2B6 from their anterior region to their posterior end. The extracellular matrix surrounding preodontoblasts reacted with 2B6 and the predentin, produced by the odontoblasts, was also intensely labeled with this antibody. Comparison between immunostaining with 3B3 and 2B6, on consecutive sections revealed a mutually exclusive pattern of distribution of the corresponding epitopes during odontogenesis. Furthermore, in the continuously growing incisor, a striking positive correlation was found between the immunostaining patterns produced by 3B3 and MC21C and the mitotic indices along the anterior-posterior axis of the tooth. Hence, sulfation of chondroitin seems developmentally regulated. We postulate that changes in the sulfation pattern of chondroitin might play a role in ontogenesis by locally altering the functional properties of the extracellular matrix.

Animals↗
PubMed1990

A 165 kDa membrane antigen mediating fibronectin-vinculin interaction is involved in murine odontoblast differentiation.

Membrane-mediated matrix-microfilament interactions are involved in odontoblast differentiation. In this study, we analyzed the interactions of vinculin and fibronectin with plasma membrane proteins separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene-difluoride (PVDF) paper. Vinculin was found to interact with 58, 63 and 165 kDa plasma membrane proteins. Fibronectin interacted with three high molecular weight (145, 165, and 185 kDa) membrane proteins. Attempts were made to characterize the 165 kDa protein which interacted with vinculin and with fibronectin. The interaction of the 165 kDa protein with fibronectin was not competitively inhibited by synthetic peptides such as GRGDS or GRGDSP, suggesting that the protein was not related to integrins. Antibodies directed against the 165 kDa protein allowed the identification of the precise localization and biological role of this membrane antigen. The data presented in this paper and previous observations indicate that the 165 kDa protein, involved in odontoblast elongation and polarization, mediates a fibronectin-vinculin transmembrane interaction.

Actins↗