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H C Slavkin

Publications and source records attributed to H C Slavkin.

At least 127 records · Page 7Linked to original sources
PubMed1994

Isolation and characterization of a mouse amelogenin expressed in Escherichia coli.

A mouse cDNA encoding a 180 amino acid amelogenin was subcloned into the pET expression plasmid (Novagen, Madison, WI) for production in Escherichia coli. A simple growth and purification protocol yields 20-50 mg of 95-99% pure recombinant amelogenin from a 4.5-liter culture. This is the first heterologous expression of an enamel protein. The expressed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption mass spectrometry, and hydroxyapatite binding. The recombinant amelogenin is 179 amino acids in length, has a molecular weight of 20,162 daltons, and hydroxyapatite binding properties similar to the porcine 173 residue amelogenin. Solubility analyses showed that the bacterially expressed protein is only sparingly soluble in the pH range of 6.4-8.0 or in solutions 20% saturated with ammonium sulfate. The purified protein was used to generate rabbit polyclonal anti-amelogenin antibodies which show specific reaction to amelogenins in both Western blot analyses of enamel extracts and in immunostaining of developing mouse molars.

Amelogenin↗
PubMed1994

Alternative splicing of the mouse amelogenin primary RNA transcript.

A heterogeneous mixture of amelogenins can be extracted from developing tooth enamel matrix. In an attempt to discover the extent to which alternative splicing of the amelogenin primary RNA transcript can generate unique isoforms, we have conducted a thorough search for cDNAs amplified by reverse transcription-polymerase chain reaction (RT-PCR). Over 2400 colonies were screened by colony hybridization. Seven different alternatively spliced amelogenin mRNAs were isolated. The predicted translation products of the messages are 194, 180, 156, 141, 74, 59, and 44 amino acids in length. RT-PCR amplification products not predicted by these seven amelogenin cDNAs were characterized. The intron separating exons 5 and 6 was cloned and sequenced. Using rapid amplification of cDNA ends (RACE) techniques, the 5' ends of the amelogenin mRNAs were cloned and characterized. The finding that the same exon 1 is common to all of the cloned mRNAs indicates that mouse amelogenin is transcribed from a single promoter. The mouse amelogenin transcription and translation initiation sites, the 5' untranslated leader, and the segment encoding the signal peptide were determined. The distinctly nonamelogenin-like exon 4, first observed in human amelogenin cDNAs, has also been found in mice. Antibodies were raised to synthetic exon 4-encoded polypeptides and used to immunostain Western transfers and histologic tooth sections.

Alternative Splicing↗
PubMed1993

Rieger syndrome revisited: experimental approaches using pharmacologic and antisense strategies to abrogate EGF and TGF-alpha functions resulting in dysmorphogenesis during embryonic mouse craniofacial morphogenesis.

The major manifestations of Rieger syndrome (RS), an autosomal dominant disorder, include absent maxillary incisor teeth, malformations of the anterior chamber of the eye, and umbilical anomalies [Aarskog et al., 1983: Am J Med Genet 15:29-38; Gorlin et al., 1990: "Syndromes of the Head and Neck" 3rd ed.]. Linkage of RS to human chromosome 4q markers has been identified with tight linkage to epidermal growth factor (EGF) [Murray et al., 1992: Nat Genet 2:46-48]. Mutations associated with genes of the EGF superfamily are implicated in malformations arising from abnormal development of the first branchial arch [Ardinger et al., 1989: Am J Hum Genet 45:348-353; Sassani et al., 1993: Am J Med Genet 45:565-569]. Down-regulation of EGF during early mouse development results in ablation of tooth formation [Kronmiller et al., 1991: Dev Biol 147:485-488]. Since EGF, TGF-alpha, and EGF receptor (EGFr) transcripts are expressed in the mouse first branchial arch and derivatives, experimental strategies were employed to investigate the consequences of down-regulation of EGF translation and inhibition of EGF receptor during embryonic mandibular morphogenesis. Antisense inhibition of EGF expression produces mandibular dysmorphogenesis with decreased tooth bud size; these effects are reversed by the addition of exogenous EGF to the culture medium [Shum et al., 1993: Development 118:903-917]. Tyrphostin RG 50864, which inhibits EGF receptor kinase activity, inhibits EGF or TGF-alpha stimulation of tyrosine phosphorylation in a concentration-dependent manner and severely retards mandibular development [Shum et al., 1993: Development 118:903-917].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Role of epidermal growth factor expression in early mouse embryo lung branching morphogenesis in culture: antisense oligodeoxynucleotide inhibitory strategy.

Epidermal growth factor (EGF) expression and branching morphogenesis were inhibited using a 5' 15-mer antisense oligodeoxynucleotide (ODN) directed against EGF precursor mRNA in embryonic mouse lung in culture under chemically defined, serumless conditions. Antisense EGF ODN resulted in > 90% inhibition of EGF immunoreactive peptide synthesis, 75% reduction in branching morphogenesis, 73% decrease in DNA content, 64% decrease in RNA content, 73% decrease in protein synthesis, and 65% decrease in [3H]thymidine incorporation into DNA compared to Embryonic Day 11 controls in culture for 4 days. Sense ODN results were similar to control. Supplementing antisense ODN with EGF partially reversed antisense effects. The results further support a role for EGF in pulmonary organogenesis.

Animals↗
PubMed1993

Endogenous epidermal growth factor regulates limb development.

Mutations associated with genes of the EGF superfamily are implicated in limb malformations. To evaluate the potential role of EGF-mediated signal transduction in the control of early mammalian limb development, we developed a simple in vitro system which is permissive for morphogenesis and cytodifferentiation in serumless, chemically defined medium. Our experimental strategy was to ascertain if the EGF precursor gene was transcribed and translated into potentially bioactive growth factor. EGF mRNA transcripts are expressed in Swiss Webster mouse embryonic (42-44 somite pairs) forelimbs as determined by mRNA phenotyping. EGF transcripts are translated into precursor EGF polypeptides which were localized to limb covering epithelium and the chondrogenic mesenchymal cell lineages. EGF immunostaining patterns suggested a paracrine type of regulation for the cartilage blastema associated with forelimb development. To test whether EGF effects the timing and positional information required for limb-specific cartilage morphogenesis, we employed tyrphostin (RG 50864) which inhibits EGF receptor kinase activity in a concentration-dependent manner and severely retards limb development. These findings support our hypothesis that endogenous EGF or EGF-like proteins provide signaling for the size and shape of discrete forelimb cartilage formations during mouse embryonic morphogenesis.

Animals↗
PubMed1993

Messenger RNA phenotyping for semi-quantitative comparison of glucocorticoid receptor transcript levels in the developing embryonic mouse palate.

A specific complex of glucocorticoid and receptor interacts with responsive genes in target tissues of developing palate and controls its morphogenesis. In order to examine developmental changes in glucocorticoid receptor gene expression during embryonic palate formation, variations in transcript levels have been determined using a simple semi-quantitative reverse transcription-polymerase chain reaction (PCR) procedure. Oligo (dT)16 was used as the primer for reverse transcription, and the amount of PCR product for glucocorticoid receptor was normalized against those for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin. We found that either GAPDH or beta-actin were suitable internal standards, and that the mRNA levels of glucocorticoid receptor undergoes a significant decrease of 30% at the developmental stage of embryonic mouse palatal shelf re-orientation. Immunolocalization of the receptor demonstrated a significant change in the distribution pattern of glucocorticoid receptor during the re-orientation stage of secondary palate formation. Our present data suggest that developmental variations in glucocorticoid receptor level control morphogenetic events of secondary palate formation.

Animals↗
PubMed1993

Antisense inhibition of AMEL translation demonstrates supramolecular controls for enamel HAP crystal growth during embryonic mouse molar development.

During tooth development, enamel organ epithelial cells express a tissue-specific gene product (amelogenin) which presumably functions to control calcium hydroxyapatite crystal growth patterns during enamel biomineralization. The present studies were designed to test the hypothesis that amelogenin as a supramolecular aggregate regulates crystal growth during enamel biomineralization. Antisense oligodeoxynucleotide strategy was used in a simple organ culture system to inhibit amelogenin translation. Under these experimental conditions, antisense treatment prior to and during amelogenin expression resulted in inhibition of amelogenin translation products within immunoprecipitated [35S]methionine metabolically labeled proteins. To determine the efficiency of antisense treatment in this model system, digoxigenin-labeled oligodeoxynucleotides were observed to diffuse throughout the tooth explants including the target ameloblast cells within 24 hours. Ultrastructural analyses of amelogenin supramolecular assembly as electron-dense stippled materials in antisense treated cultures demonstrated dysmorphology of the extracellular enamel matrix with a significant reduction in crystal length and width. We conclude that secreted extracellular proteins form a supramolecular aggregate, which controls both the orientation and dimensions of enamel crystal formation during tooth development.

Amelogenesis↗
PubMed1992

Alternative splicing of the mouse amelogenin primary RNA transcript contributes to amelogenin heterogeneity.

A heterogeneous population of amelogenin proteins is derived from a single copy of the mouse amelogenin gene. To investigate the one gene--multiple protein enigma, we designed a study to distinguish between alternative splicing and proteolytic cleavage models. A pulse of [35S]methionine labeling demonstrated that multiple amelogenins are synthesized concurrently, a result consistent with an alternative splicing mechanism. Using reverse transcription and polymerase chain reaction we cloned a segment from the 5' end of a mouse amelogenin mRNA and connected it to a previously isolated abbreviated cDNA clone. Four additional cDNAs derived from alternatively spliced amelogenin mRNAs have been cloned and characterized. The five transcripts encode amelogenins 180, 156, 141, 74, and 59 amino acids in length.

Alternative Splicing↗
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

Distribution of type I collagen, type II collagen and PNA binding glycoconjugates during chondrogenesis of three distinct embryonic cartilages.

Previous studies of chondrogenesis have been focused on limb bud cartilage, whereas little is known about chondrogenic processes of other cartilages with different developmental fates. We hypothesize that cartilages with various developmental fates might show identical characteristics of chondrogenesis. The chondrogenic processes in the nasal septum, the mandible, and the limb bud of the mouse were examined by means of PNA-binding glycoconjugate, and types I and II collagen expression. Swiss-Webster mouse embryos of 11 days (E11) to 14 days (E14) gestation were fixed and processed for immuno- and lectin histochemistry. The blastema of mesenchymal cell aggregates stained positively with anti-type I collagen, but very weakly with anti-type II collagen in all three models at E12, whereas PNA bound to the blastema in the limb bud but not in nasal septum or mandible. Types I and II collagens coexisted in cartilages at E13. Type II collagen was predominant in E14; type I collagen was confined to the peripheral region. The synchronized transitional expression of the collagen phenotypes in all three embryonic cartilages may be systemically regulated. The presence or absence of the PNA-binding glycoconjugates may be involved in characterizing the nature of the cartilages.

Animals↗
PubMed1992

Epigenetic role of epidermal growth factor expression and signalling in embryonic mouse lung morphogenesis.

A major unsolved problem in developmental biology is to determine when and how time- and position-restricted instructions are signaled and received during secondary embryonic inductions such as branching morphogenesis. The mouse embryonic lung rudiment was used to test the hypothesis that endogenous peptide growth factors, specifically epidermal growth factor (EGF), serve as instructive epigenetic signals for morphogenesis. The presence of EGF precursor mRNA transcripts was detected using the reverse-transcriptase-coupled polymerase chain reaction both in E11-E17-day mouse embryo lung tissues in vivo and in E11-day lung cultured for up to 7 days in vitro under chemically defined, serum-free conditions. Immunolocalization identified a position-restricted distribution of EGF in and around the primitive airways both during in vivo lung morphogenesis and in culture. EGF receptors (EGFR) coimmunolocalized with EGF in the primitive airways. Addition of exogenous EGF to lungs in culture resulted in significant concentration-dependent stimulation of branching morphogenesis, DNA, RNA, and protein content, and in [3H]thymidine incorporation into DNA. Conversely, the addition of tyrphostin (specific EGF receptor kinase antagonist) to lungs in culture resulted in concentration-dependent inhibition of branching morphogenesis, DNA, RNA, and protein content, and in [3H]thymidine incorporation into DNA without apparent cytotoxicity. The inhibition of the EGF signal by tyrphostin was confirmed by immunoprecipitation of tyrosine phosphoproteins. We conclude that early mouse embryo lungs express EGF transcripts and corresponding EGF peptides in a specific position-restricted distribution which coimmunolocalizes with EGFR in the primitive airways, while stimulatory and inhibitory studies indicate a functional role for the transduced EGF signal in the epigenetic regulation of lung branching morphogenesis. We speculate that the peptide growth factor EGF serves a function in secondary embryonic morphogenetic inductions, which may be modulated by interaction with other growth factors.

Animals↗
PubMed1992

Polyoma virus-induced murine odontogenic tumors.

Neonatal mouse pups were injected subcutaneously with polyoma virus to induce odontogenic tumors. This treatment resulted in a spectrum of tumors that arose in organs dependent upon epithelial-mesenchymal interactions for their organogenesis, which included the teeth, salivary glands, thymus, and lacrimal glands. In addition, several odontogenic tumors with a histologic resemblance to ameloblastoma were identified and analyzed with respect to the presence of markers specific for various stages of ameloblast differentiation. Immunodetection analyses of the odontogenic tumors identified fibronectin and laminin, typical of basement membrane organization during early tooth organogenesis. These same tumors failed to express amelogenin, a gene whose expression is limited to differentiated ameloblasts. In contrast, a 47 kDa enamelin-like polypeptide was identified with the use of an antienamelin antibody. These data were interpreted to suggest that the polyoma virus truncated the differentiation pathway for these odontogenic tissues at an early stage of their development and retained the expression of basement membrane components and the enamelin-like polypeptides, yet excluded expression of amelogenin gene products. This observation suggests that polyoma viral transformation may dysregulate odontogenic tissue interactions and produce tumors composed of cells arrested at a specific stage in their development.

Ameloblastoma↗
PubMed1992

Distribution of glycoconjugates localized by peanut and Maclura pomifera agglutinins during mouse molar root development.

Glycolipids, glycoproteins, glycosaminoglycans and sialoglycoproteins have all been implicated in a number of developmentally significant processes related to complex interactions between cell surfaces and the extracellular matrix. The present study was designed to localize glycoconjugates recognized by peanut agglutinin (PNA) and Maclura pomifera (MPA) lectins during mouse molar root development. Postnatal ICR mice at 10, 15, 21, 28 and 42 days were used. Lower jaws were dissected, fixed in 4% paraformaldehyde, decalcified in 5% EDTA and embedded in paraffin. Serial sections were made and stained with FITC-conjugated PNA or MPA. beta-Lactose was used as an inhibitory sugar for PNA, and alpha-D-melibiose for MPA. PNA specifically stained Hertwig's epithelial root sheath (HERS), whereas MPA stained a number of tissues. The outermost layer of root dentin, forming cellular cementum, alveolar bone and HERS showed positive reactions with MPA. Glycoconjugates localized by the lectins may be functionally related to molecules which contribute to root formation and cemento-genesis.

Animals↗
PubMed1992

Incidence of cleft lips, palates rising.

About 1:700 live human births are affected by cleft lip and palate. Socio-economic factors--teenage pregnancies, pregnancies in women older than 35, increased consumption of teratogens during early months of pregnancy--have resulted in increased numbers of congenital malformations in the United States.

Adolescent↗
PubMed1992

Endogenous epidermal growth factor regulates the timing and pattern of embryonic mouse molar tooth morphogenesis.

The tooth organ provides a model for discrete patterns of morphogenesis over short periods of developmental time. Studies were designed to test the hypothesis that endogenous epidermal growth factor (EGF) functions to regulate multiple cusp molar tooth morphogenesis during embryonic mouse development. The relative levels of endogenous EGF and EGF receptor (EGFR) transcripts were determined in both enamel organ epithelia and dental ectomesenchyme by reverse transcription-polymerase chain reaction (RT-PCR) assays. EGF and EGFR were localized by immunohistochemistry; both antigenic determinants were demonstrated on the same odontogenic cells in cultured tooth explants. To examine EGF-mediated signal transduction, cap stage mouse molar tooth organs (E16) were cultured in serumless, chemically-defined medium as either (i) controls, or supplemented with (ii) tryphostin (an EGF receptor kinase inhibitor), (iii) tyrphostin plus exogenous EGF, and (iv) exogenous EGF. Antisense oligodeoxynucleotide (ODN) strategy was used to investigate the functions of endogenous EGF employing (i) non-treated control, (ii) sense ODN control, (iii) antisense ODN, (iv) exogenous EGF, (v) sense ODN with exogenous EGF, and (vi) antisense ODN with exogenous EGF. Tyrphostin inhibited DNA synthesis and produced a significant decrease in the volume of the explants. These effects were recovered by addition of exogenous EGF. Antisense ODN inhibition resulted in abnormal cusp formations, decreased DNA synthesis, total DNA, RNA and protein content, and decreased stellate reticulum and tooth explant volumes. The decreased tooth size was not uniform, the most pronounced effect was in the stellate reticulum. This pattern of changes was not seen when antisense ODN treatment was supplemented with exogenous EGF. These results suggest that during cap stage of odontogenesis endogenous EGF acts to stimulate DNA synthesis, which increases the cell number of specific phenotypes within the enamel organ epithelia, and thereby regulates molar tooth morphogenesis.

Animals↗
PubMed1992

Gene expression, signal transduction and tissue-specific biomineralization during mammalian tooth development.

Tooth development provides a paradigm for intrinsic molecular controls for cell- and extracellular matrix (ECM)-mediated biomineralization. The intent of this review is to evaluate the sequential timing and positional information prerequisite for tissue-specific biomineralization. Recent investigations suggest that 1,25-dihydroxyvitamin D3 functions to up-regulate VDR (vitamin D receptor) that in turn could induce structural gene products, including calcium-binding proteins and several ECM proteins (e.g., enamelins, amelogenins, dentine sialoglycoproteins (DSP) and dentine phosphoproteins (DPP)), resulting in dentine and enamel formation. Inhibition of regulatory gene products and/or their receptors likely results in hypoplastic and/or hypomineralized ECM as a direct consequence of down-regulated (1) transcription and/or translation of structural and regulatory genes, (2) posttranslational modifications, (3) and/or decreased calcium transport to the forming dentine and enamel matrices. Advances in serumless in vitro culture methodology; computer-assisted access to nucleic acid sequences for probes to define when, where, and how many specific regulatory and structural gene products are expressed; antisense oligodeoxynucleotides to inhibit specific translation; and microtechniques to analyze biomineralization all provide additional avenues to investigate tissue-specific biomineralization.

Animals↗
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↗