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M Zeichner-David

Publications and source records attributed to M Zeichner-David.

At least 37 records · Page 2Linked to original sources
PubMed1992

Characteristics of phosphorylated and non-phosphorylated dentine phosphoprotein.

Heterogeneity among the odontoblast-specific, highly phosphorylated acidic protein dentine phosphoprotein (DPP) obtained from different species has been reported by several investigators. In the present study, the apparent molecular-mass variations in rabbit and mouse DPP were investigated. Extracellular matrix (ECM) DPPs were isolated and characterized. Primary gene products, before post-translational phosphorylation, were analysed based upon translation products produced in a rabbit reticulocyte lysate cell-free system using a polyclonal mouse anti-DPP antibody. Nascent non-phosphorylated DPPs were also identified from intracellular protein extracts. Mouse and rabbit ECM phosphoproteins exhibited a 10 kDa difference in size. However, nascent intracellular or translation products from both species showed the same lower molecular mass (approx. 45 kDa). Furthermore, Northern-blot analysis showed a single mRNA of the same size in both species (approx. 1.6 kb) which contains information for a protein no larger than 50 kDa. Our results indicate that the difference in molecular mass (or electrophoretic behaviour) among DPPs from different species is due to post-translational modifications, in this case phosphorylation.

Amino Acid Sequence↗
PubMed1992

Protein kinases in dentinogenesis.

Protein modifications such as phosphorylation and dephosphorylation are known to control several cell functions including regulation of the cell cycle, signal transduction and enzyme activation/inactivation. Bone and dentin contain highly phosphorylated anionic proteins that appear to be involved in the regulation of mineralization. This study was designed to identify and characterize the enzyme(s) responsible for phosphorylation (kinases) of dentin phosphoprotein (DPP) during dentinogenesis. DPP-protein kinase activity was demonstrated in a crude homogenate of dental pulp and odontoblast cells. In parallel studies, oligonucleotides to conserved amino acid sequences present in the active site of kinases were constructed and used to screen a lambda-gt11 tooth organ cDNA library. Several cDNA clones were isolated, the size of the insert determined by PCR (polymerase chain reaction) amplification, and in situ hybridization was used to determine cellular localization during tooth organ development. Preliminary evidence provides additional molecular determinants involved with candidate kinases responsible for DPP phosphorylation and dentinogenesis.

Animals↗
PubMed1992

Dentin phosphoprotein gene locus is not associated with dentinogenesis imperfecta types II and III.

Dentinogenesis imperfecta (DGI) is an autosomal dominant inherited dental disease which affects dentin production and mineralization. Genetic linkage studies have been performed on several multigeneration informative kindreds. These studies determined linkage between DGI type II and III and group-specific component (vitamin D-binding protein). This gene locus has been localized to the long arm of human chromosome 4 in the region 4q11-q21. Although this disease has been mapped to chromosome 4, the defective gene product is yet to be determined. Biochemical studies have suggested abnormal levels of dentin phosphoprotein (DPP) associated with DGI type II. This highly acidic protein is the major noncollagenous component of dentin, being solely expressed by the ectomesenchymal derived odontoblast cells of the tooth. The purpose of the present study was to establish whether DPP is associated with DGI types II and III, by using molecular biology techniques. The strategy was to use a synthetic degenerative DPP oligonucleotide probe to map this sequence to the long arm of human chromosome 4, 4q13-q21, by using somatic cell hybrids. Our results indicated that DPP is not localized to any region of human chromosome 4, thus suggesting that the DPP gene is not directly associated with DGI type II or DGI type III. Our data do not exclude the possibility that other proteins associated with DPP posttranslational modifications might be responsible for this genetic disease.

Adult↗
PubMed1989

Human and mouse cementum proteins immunologically related to enamel proteins.

SDS-polyacrylamide gel electrophoresis, immunoblot and amino acid composition analyses were applied to human and mouse acellular cementum proteins immunologically related to enamelins and amelogenins. In this analysis, anti-mouse amelogenin, anti-human enamelin and synthetic peptide (e.g., -LPPHPGHPGYIC-) antibodies were shown to cross-react with tooth crown-derived enamelin with a molecular mass of 72,000 Da (72 kDa), amelogenins (26 kDa), and also to four human cementum proteins (72, 58, 50 and 26 kDa) and two mouse cementum proteins (72 and 26 kDa). Each of the antibodies recognized tooth root-derived cementum polypeptides which share one or more epitopes with tooth crown-derived enamel proteins. The molecular mass and isoelectric points for crown-derived and root-derived enamel-related proteins were similar. Analysis of human and mouse cementum proteins revealed a characteristic amino acid composition enriched in glutamyl, serine, glycine, alanine, proline, valine and leucine residues; compared to the major enamel protein amelogenin, cementum proteins were low in proline, histidine and methionine. The human and mouse putative intermediate cementum proteins appear to represent a distinct class of enamel-related proteins. Moreover, these results support the hypothesis that epithelial root sheath epithelia express several cementum proteins immunologically related to canonical enamel proteins.

Amelogenin↗
PubMed1989

Hertwig's epithelial root sheath differentiation and initial cementum and bone formation during long-term organ culture of mouse mandibular first molars using serumless, chemically-defined medium.

Studies were designed to test the hypothesis that Hertwig's epithelial root sheath (HERS) synthesizes and secretes enamel-related proteins that participate in the process of acellular cementum formation. Our experimental strategy was to examine sequential root development of the mouse mandibular first molar in vivo and in long-term organ culture in vitro using serumless, chemically-defined medium. Using anti-amelogenin, anti-enamelin and anti-peptide antibodies, enamel-related antigens were localized within intermediate cementum during HERS differentiation and root formation in vivo. Cap stage molars maintained for periods of up to 31 days in organ culture expressed morphogenesis and cytodifferentiation as identified by tooth crown and initial root, cementum and bone formation. Metabolically-labeled HERS products were analyzed by immunodetection using enamel-related antibodies and one- and two-dimensional SDS gel electrophoresis. A 72 kDa and 26 kDa polypeptide were identified in forming mouse cementum. Both of these root putative cementum proteins yield similar (identical) amino acid compositions; however, both proteins differed from the compositions of either mouse crown enamelin or amelogenin proteins. This approach provides a new and novel in vitro model towards understanding HERS differentiation and functions related to root and bone formation. The data support the hypothesis that HERS cells synthesize polypeptides related to but also different from canonical crown enamel proteins.

Animals↗
PubMed1989

Enamelins and amelogenins share the same amino-terminal sequence.

Previous results from our laboratory indicated that rabbit enamel high molecular weight proteins have the same amino-terminal sequence that amelogenins, thus suggesting the possibility that this domain is shared by both, enamelins and amelogenins. To determine if this is true for other species, enamel proteins and mRNA were extracted from rabbit and hamster developing teeth and analyzed using probes targeted towards the N-terminal sequence of the amelogenins. Our results strongly suggest that both, enamelins and amelogenins share the same amino-terminal amino acid sequence.

Amelogenin↗
PubMed1989

Characterization of extracellular and nascent dentin phosphoproteins.

Experiments were designed to compare extracellular dentin phosphoprotein (DPP) and nascent DPP prior to post-translational modifications from several vertebrate species. Dental matrix proteins were extracted with acetic acid, followed by GuHCl-EDTA, and precipitated with CaCl2. Cross-reactivity of the DPPs with a mouse DPP antibody was determined by a dot-immunobinding assay. To analyze nascent DPP, mRNA was isolated from developing tooth organs and the mRNA-directed translation products were immunoprecipitated with the DPP specific antibody. All DPP components identified in the species which contained a DPP were shown to cross-react with the polyclonal mouse DPP antibody. The extracellular matrix DPPs were found to exhibit as much as a 30 kDa size difference using the same SDS PAGE system. In contrast, nascent DPPs were found to be the same size for all species examined. Our results indicate that differences in the molecular weight size of DPPs between species may be due to the degree of post-translational modifications such as phosphorylation.

Animals↗
PubMed1989

Of mice and men: anatomy of the amelogenin gene.

Mammalian enamel matrix is composed of two principal proteins, the enamelins and amelogenin. Recombinant complementary DNA (cDNA) molecules for the predominant mouse amelogenin have been identified, characterized by direct determination of the DNA sequence, and used as a specific hybridization probe. The spatial- and temporal-restricted pattern for amelogenin gene expression within developing mouse molars has been traced at the level of a single cell using in situ hybridization. The mouse genome has been shown to contain only one copy of the amelogenin (AMEL) gene which is not amplified or rearranged during ameloblast determination. In contrast, the human genome contains two copies of the AMEL gene, one residing on the X chromosome and one upon the Y chromosome. These observations, the availability of specific enamel gene probes coupled with the application of new techniques in molecular biology now afford unique opportunities for the analysis of the molecular basis of inherited defects of human enamel such as amelogenesis imperfecta. Recent advances towards obtaining a physical map and the complete nucleotide sequence for the human genome, as well as the documented developmental biology, defined genetics and transgenic capability of the mouse, suggest that mouse and man are the most relevant and potentially informative models for analysis of normal and abnormal enamel biomineralization.

Amelogenesis Imperfecta↗
PubMed1988

Biosynthesis and characterization of rabbit tooth enamel extracellular-matrix proteins.

Tooth enamel biomineralization is mediated by enamel proteins synthesized by ameloblast cells. Two classes of proteins have been described: enamelins and amelogenins. In lower vertebrates the absence of amelogenins is believed to give rise to aprismatic enamel; however, rabbit teeth, which apparently do not synthesize amelogenins, form prismatic enamel. The present study was designed to characterize the enamel proteins present in rabbit tooth organs and to gain an insight into the process of biomineralization. Rabbit enamel extracellular-matrix proteins were isolated and characterized during sequential stages of rabbit tooth organogenesis. The biosynthesis of enamel proteins was analysed by metabolic 'pulse-chase' experiments as well as mRNA-translation studies in cell-free systems. Our results indicated that rabbit enamel extracellular matrix contains 'amelogenin-like' proteins. However, these proteins are not synthesized as typical amelogenins, as in other mammalian species, thus suggesting that they are the processing products of higher-molecular-mass precursors. An N-terminal amino acid sequence of 29 residues, considered characteristic of mammalian amelogenins, was present in the rabbit 'amelogenin-like' proteins. By using anti-peptide antibodies to this region, similar epitopes were detected in all nascent enamel proteins, including enamelins. These studies suggest that the N-terminal sequence might be characteristic of all enamel proteins, not only amelogenins.

Amino Acid Sequence↗
PubMed1988

Factors influencing the expression of dental extracellular matrix biomineralization.

The forming tooth organ provides a number of opportunities to investigate the cellular and molecular biology of cell-mediated extracellular matrix (ECM) biomineralization. Regulatory processes associated with tooth formation are being investigated by identifying when and where cell adhesion molecules (CAMs), substrate adhesion molecules (SAMs), dentine phosphoprotein and enamel gene products are expressed during sequential developmental stages. In vitro organotypic culture studies in serumless, chemically-defined medium, have shown that instructive and permissive signalling are required for both morphogenesis and cytodifferentiation. Intrinsic developmental instructions (autocrine and paracrine factors) act independently of long-range hormonal or exogenous growth factors and mediate morphogenesis from the initiation of the dental lamina to the crown stages of tooth development. This review summarizes the results of studies using experimental embryology, recombinant DNA technology and immunocytology to elucidate mechanisms responsive to instructive epithelial-mesenchymal interactions associated with ameloblast differentiation, odontoblast differentiation, and dentine and enamel ECM biomineralization.

Animals↗
PubMed1988

Molecular determinants of cranial neural crest-derived odontogenic ectomesenchyme during dentinogenesis.

Positional information on tooth morphogenesis is investigated by the identification of when and where phenotypic markers are expressed during odontogenesis. This temporal and positional information is correlated with the instructive and permissive signaling required for both dentinogenesis and amelogenesis. Of particular interest is the establishment of a map for the cranial neural crest-derived dental papilla ectomesenchyme and the odontoblast cell lineages. The expression of ectomesenchyme-derived cytotactin, dentin phosphoprotein, and epithelial-derived enamel proteins was studied in mice using embryonic, fetal, and postnatal mandibular first molar tooth organ development. This review summarizes the observations in the context of instructive epithelial-mesenchymal interactions and suggests that amelogenesis imperfecta and dentinogenesis imperfecta may in part be explained by alterations in these differentiation markers. Recombinant DNA methods should facilitate future investigations of these inherited dental disorders.

Animals↗
PubMed1988

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

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

Animals↗
PubMed1987

Amelogenin antigenic domain defined by clonal epitope selection.

To experimentally examine the participation of amelogenins in controlled mineral-phase maturation of mammalian enamel, the identification of the individual proteins and their corresponding gene(s) is required. For this purpose, cDNAs were constructed from polyadenylated RNA from 2-day postnatal murine teeth, molecularly cloned into lambda-gt11 expression vectors and transfected into E. coli. The cDNA library was screened for amelogenin gene(s) by using either antibody or nucleic acid probes. An amelogenin cDNA clone encoding 79 carboxy-terminal amino acid residues and 100 nucleotides of the 3' noncoding sequence was demonstrated to contain a major antigenic site for amelogenin protein by immunostaining of specific amelogenin proteins from total extracted enamel protein blots using clonal epitope selected antibody. This is the first report linking amelogenin epitope(s) to a defined DNA sequence, and consequently a defined portion of the amino acid sequence for amelogenins. Secondary structure analysis, based on the relative average linear hydropathy of the amino acid sequence of amelogenin, predicted epitopes in the amino terminus of the molecule rather than the carboxy terminus. Our present data suggest that the carboxy terminus of the amelogenins is sufficiently externalized to be an antigenic domain. These data may be useful in subsequent structural analysis of amelogenin proteins and enhancing our understanding of their physicochemical participation in biomineralization.

Amelogenin↗
PubMed1987

Immunochemical and biochemical studies of human enamel proteins during neonatal development.

The present communication provides descriptions of the developmental, biochemical, and immunological properties of the human enamel extracellular matrix proteins. We report the isolation and partial characterization of the major human enamel proteins, the production of polyclonal antibodies directed against the human enamelins, and a comparison between the immunogenicity of enamelins and amelogenins from human and mouse enamel extracellular matrices. Our results indicate that although enamelins and amelogenins share some epitopes, each one of these proteins appears to invoke a different degree of immunogenicity.

Amelogenin↗
PubMed1985

Production and characterization of antibodies against murine dentine phosphoprotein.

Experiments were designed to produce and characterize a polyclonal antibody directed against mouse dentine phosphoprotein, the major non-collagenous protein of the dentine extracellular matrix. Dental extracellular matrix proteins from 2-day-postnatal Swiss-Webster-mouse tooth organs were extracted with 0.5 M-acetic acid, followed by 4 M-guanidinium chloride/0.5 M-EDTA. Mouse dentine phosphoprotein yields were further increased by precipitation with 1 M-CaCl2. Final purification was achieved by excising and eluting dentine phosphoprotein polypeptide bands from preparative sodium dodecyl sulphate/urea/polyacrylamide gels. Mouse dentine phosphoprotein is a single component of approx. 72 kDa and has a characteristic amino acid composition of 33% aspartic acid and 55% serine/phosphoserine. A polyclonal antibody was raised in rabbits against purified mouse dentine phosphoprotein and was shown to be monospecific by enzyme-linked immunoabsorbent, dot-immunobinding and 'Western transfer' assays. This antibody was used to detect the expression and localization of dentine phosphoprotein in 1-day-postnatal mouse tooth organs. This antigen was localized intracellularly within the monolayer of odontoblasts, which line the perimeter of the dental papilla mesenchyme, and within the odontoblastic cell processes, which traverse the predentine matrix. Newly forming mineralized dentine matrix was also cross-reactive with the dentine phosphoprotein specific antibody. The non-mineralized predentine matrix did not contain any detectable cross-reactive antigens.

Amino Acids↗
PubMed1985

DNA sequence for cloned cDNA for murine amelogenin reveal the amino acid sequence for enamel-specific protein.

Enamel is the unique and highly mineralized extracellular matrix that covers vertebrate teeth. Amelogenin proteins represent the predominate subfamily of gene products found in developing mammalian enamel, and are implicated in the regulation of the formation of the largest hydroxyapatite crystals in the vertebrate body. Previous attempts to isolate, purify and characterize amelogenins extracted from developing matrix have proven difficult. We now have determined the DNA sequence for a cDNA for the 26-kDa class of murine amelogenin and deduced its corresponding amino acid sequence. The murine amino acid sequence is homologous to bovine or porcine amelogenins extracted from developing enamel matrices. However, an additional 10-residues were found at the carboxy terminus of the murine amelogenin. This is the most complete sequence database for amelogenin peptides and the only DNA sequence for enamel specific genes.

Amelogenin↗
PubMed1984

Concepts of epithelial-mesenchymal interactions during development: tooth and lung organogenesis.

One of the major problems in developmental biology concerns how differential gene activity is regionally controlled. One approach to this problem is the use of mesenchyme specification of epithelial-specific gene expression, such as, during tooth morphogenesis or lung morphogenesis. In the example of tooth morphogenesis, dental papilla ectomesenchyme induces de novo gene expression as assayed by detection of amelogenin transcripts, or immunodetection of amelogenin polypeptides within ameloblast cells. This process does not require serum supplementation or exogenous factors during epithelial-mesenchymal interactions in vitro. In contrast, lung morphogenesis requires hormones to mediate mesenchyme-derived influences upon type II epithelial cell differentiation and the production of pulmonary surfactant (eg, neutral and phospholipids, surfactant proteins). Glucocorticoids are required to stimulate the release of fetal pneumonocyte factor (FPF) from fibroblasts which, in turn, enhance the production of pulmonary surfactant. Thyroxin appears to regulate the relative responsiveness of progenitor type II cells to steroid-stimulated release of FPF. This review will highlight key concepts associated with these developing organ systems and emphasize the problem of regional controls which regulate epithelial cell-specific gene activity.

Ameloblasts↗