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Characterization of a rabbit enamelin cDNA clone
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316 CHARACTERIZATION OF A RABBIT ENAMELIN cDNA CLONE. C. Gniewkowski-Taylor, M. Zeichner-David and H. Slavkin. University of Southern California, Los Angeles, CA 90089-0191 Developing enamel proteins are divided into two main classes: amelogenins and enamelins. Fetal rabbit tooth organs are highly enriched for enamelins and repr~sent an excellent model system for the study of enamelin gene expression. The objective of this study is the construction of a cDNA library for rabbit fetal enamel proteins. These probes will be used to study enamel gene expression and to elucidate the structure and organization of the enamelin genes. Total poly(A) RNA from rabbit molar tooth organs is used to produce double stranded cDNA for transformation. Enamel cDNA clones are screened by antibiotic resistance and colony hybridization. The identity of the enamelin cDNA clone is established by hybrid selection of enamelin mRNA followed by translation in a cell-free system and immunoprecipitation with an anti-enamelin antibody. Enamelin cDNA probes will be used to detect the onset of enamelin mRNA transcription during tooth organ development.
PROLACTIN A C T I V I T Y - WITH AND WITHOUT GONADOTROPINS - ON THE OVARYt. H. Gitay, E.S. Lindenbaum and Z. Kraiem*. Dept. of Morphological Sciences, Technion Faculty of Medicine, and *Endocrine Research Unit, Carmel Hospital, Haifa, Israel. It is well established that the gonadotropins are the main activators of steroidogenesis in the ovary. Prolactin, as a modulator of gonadotropic action, also influences steroidogenesis directly at an ovarian site. Reports on the nature and mechanism of prolactin activity are conflicting due to the various systems used. In this study, prolactin action was examined with and without gonadotropins, using a well-defined in vitro culture system. In order to analyze cytodifferentiation o f ovarian granulosa cells in the immature rat, biochemical and histological parameters were tested simultaneously. Data on cyclic AMP and steroid hormone concentrations were correlated with light and electron microscopic observations, in the presence and absence of prolactin and follicle-stimulating and luteinizing hormones. t Supported by Technion V.P.R. F u n d - D. Rose Fund for Pregnancy Research.
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EXOCYTOSIS AND ENDOCYTOSIS IN RAT INCISOR ODONTOBLASTS. M. Goldberg, D.Septier and F.Escaig. Lab.Histol. Fac. Chir.Dent. I rue M.Arnoux 92120 Montrouge, France. Various technics were used to study the location of exocytic areas in rat incisor odontoblasts. Using labelled precursors, the first localization of silver grains was observed along odontoblast processes as well as in the proximal predentine.We never obtained any evidence of distal secretion. Three classes of vesicles were observed inside processes using electron histochemistry and/or cryotechnics:1) 20-35 nm secretory vesicles,2)75-100 nm coa~ed and uncoated vesicles-related to endocytic events,3)200-350 nm elongated or rounded vesicles, likely lysosome-like structures. These facts do not support earlier assumption on the occurence of s ~ cretory process in the distal cell body membrane or the existence of two (proximal and distal) zones of secretion. Exocytosis, observed all along the plasma membrane of odontoblast processes implies a transport pathway through cell process. This does not preclude diffusion throughout the ground substance in predentine. Reabsorptions and membrane retrieval also take place along this plasmaless portion.
CALMODULIN STIMULATION OF PROSTAGLANDIN SYNTHESIS IN EMBRYONIC PALATE MESENCHYMAL CELLS IN VITRO. R. Greene and M. Lloyd, Daniel Baugh Institute, Thomas Jefferson Univ., Phila., PA 19107 USA. Prostaglandin (PG) synthesis by embryonic palate mesenchymal cells has been suggested as playing a key role in orofacial growth and differentiation. Factors regulating palatal PG synthesis are largely unknown. The present study examined wb~ther calmodulin plays a role in Ca *z stimulated palatal PGE 2 synthesis. Endogenous ealmodulin levels were determined by RIA to be 0.21 ng/ug DNA. The calcium ionophore, A23187, induced a dose dependent stimulation of palatal PGE 2 synthesis. Sensitivity to A23187 was eliminated by pretreatment with 2~M EGTA or 10uM trifluoroperazine (TFP), an inactivator of calmodulin. Exogenous arachidonic acid was able to stimulate PG synthesis despite TFP pretreatment. These results are consistant with a role for calmodulin in Ca+2 mediated arachidonate mobilization requisite for palatal PG synthesis. Supported by DE05550 and DE06397.
128S
316 CHARACTERIZATION OF A RABBIT ENAMELIN cDNA CLONE. C. Gniewkowski-Taylor, M. Zeichner-David and H. Slavkin. University of Southern California, Los Angeles, CA 90089-0191 Developing enamel proteins are divided into two main classes: amelogenins and enamelins. Fetal rabbit tooth organs are highly enriched for enamelins and repr~sent an excellent model system for the study of enamelin gene expression. The objective of this study is the construction of a cDNA library for rabbit fetal enamel proteins. These probes will be used to study enamel gene expression and to elucidate the structure and organization of the enamelin genes. Total poly(A) RNA from rabbit molar tooth organs is used to produce double stranded cDNA for transformation. Enamel cDNA clones are screened by antibiotic resistance and colony hybridization. The identity of the enamelin cDNA clone is established by hybrid selection of enamelin mRNA followed by translation in a cell-free system and immunoprecipitation with an anti-enamelin antibody. Enamelin cDNA probes will be used to detect the onset of enamelin mRNA transcription during tooth organ development.
PROLACTIN A C T I V I T Y - WITH AND WITHOUT GONADOTROPINS - ON THE OVARYt. H. Gitay, E.S. Lindenbaum and Z. Kraiem*. Dept. of Morphological Sciences, Technion Faculty of Medicine, and *Endocrine Research Unit, Carmel Hospital, Haifa, Israel. It is well established that the gonadotropins are the main activators of steroidogenesis in the ovary. Prolactin, as a modulator of gonadotropic action, also influences steroidogenesis directly at an ovarian site. Reports on the nature and mechanism of prolactin activity are conflicting due to the various systems used. In this study, prolactin action was examined with and without gonadotropins, using a well-defined in vitro culture system. In order to analyze cytodifferentiation o f ovarian granulosa cells in the immature rat, biochemical and histological parameters were tested simultaneously. Data on cyclic AMP and steroid hormone concentrations were correlated with light and electron microscopic observations, in the presence and absence of prolactin and follicle-stimulating and luteinizing hormones. t Supported by Technion V.P.R. F u n d - D. Rose Fund for Pregnancy Research.
317
318
EXOCYTOSIS AND ENDOCYTOSIS IN RAT INCISOR ODONTOBLASTS. M. Goldberg, D.Septier and F.Escaig. Lab.Histol. Fac. Chir.Dent. I rue M.Arnoux 92120 Montrouge, France. Various technics were used to study the location of exocytic areas in rat incisor odontoblasts. Using labelled precursors, the first localization of silver grains was observed along odontoblast processes as well as in the proximal predentine.We never obtained any evidence of distal secretion. Three classes of vesicles were observed inside processes using electron histochemistry and/or cryotechnics:1) 20-35 nm secretory vesicles,2)75-100 nm coa~ed and uncoated vesicles-related to endocytic events,3)200-350 nm elongated or rounded vesicles, likely lysosome-like structures. These facts do not support earlier assumption on the occurence of s ~ cretory process in the distal cell body membrane or the existence of two (proximal and distal) zones of secretion. Exocytosis, observed all along the plasma membrane of odontoblast processes implies a transport pathway through cell process. This does not preclude diffusion throughout the ground substance in predentine. Reabsorptions and membrane retrieval also take place along this plasmaless portion.
CALMODULIN STIMULATION OF PROSTAGLANDIN SYNTHESIS IN EMBRYONIC PALATE MESENCHYMAL CELLS IN VITRO. R. Greene and M. Lloyd, Daniel Baugh Institute, Thomas Jefferson Univ., Phila., PA 19107 USA. Prostaglandin (PG) synthesis by embryonic palate mesenchymal cells has been suggested as playing a key role in orofacial growth and differentiation. Factors regulating palatal PG synthesis are largely unknown. The present study examined wb~ther calmodulin plays a role in Ca *z stimulated palatal PGE 2 synthesis. Endogenous ealmodulin levels were determined by RIA to be 0.21 ng/ug DNA. The calcium ionophore, A23187, induced a dose dependent stimulation of palatal PGE 2 synthesis. Sensitivity to A23187 was eliminated by pretreatment with 2~M EGTA or 10uM trifluoroperazine (TFP), an inactivator of calmodulin. Exogenous arachidonic acid was able to stimulate PG synthesis despite TFP pretreatment. These results are consistant with a role for calmodulin in Ca+2 mediated arachidonate mobilization requisite for palatal PG synthesis. Supported by DE05550 and DE06397.
128S