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Biomedical subjects

R M Pratt

Publications and source records attributed to R M Pratt.

At least 91 records · Page 5Linked to original sources
PubMed1978

Autoradiographic analysis of altered glycosaminoglycan synthesis in the epiphyseal cartilage of neonatal brachymorphic mice.

Brachymorphic (bm/bm) mice are disproportionately short in stature. Past biochemical studies on neonatal mice (Orkin et al., '76) demonstrated that epiphyseal cartilage from these mutants synthesizes glycosaminoglycans (GAG) that are undersulfated. In this study, synthesis of GAG, as determined autoradiographically with Na2 35SO4 and 3H-glucosamine was reduced in all areas of bm/bm epiphyses both in vivo and in vitro as compared to normal C57BL/6J mice. Incorporation of both isotopic precursors into GAG of the brachymorphic proliferative zone was reduced to a greater extent than in the reserve zone. In addition, incorporation of these precursors into GAG of epiphyseal cartilages in vitro, as determined biochemically, was reduced by 40%. In contrast, the incorporation of 3H-leucine and 3H-proline into protein did not show differences between mutant and normal epiphyses. These results suggest that alterations in GAG synthesis in bm/bm epiphyseal growth plates are not exclusive to any one zone, but do appear to be most pronounced in the proliferative zone.

Animals↗
PubMed1978

Inhibition of growth in vitro by glucocorticoids in mouse embryonic facial mesenchyme cells.

The growth of primary embryonic facial mesenchyme cells established from cleft palate sensitive A/J and resistant C57BL/6J (C57) mice is inhibited by glucocorticoid treatment. A reduction in cell number in both A/J and C57 culture is accompanied by a significant decrease in [3H] thymidine incorporation into both acid soluble and insoluble material. No significant changes in total cellular protein or [14C] leucine incorporation were observed in either cell type. A greater reduction in [3H] thymidine incorporation occurs in cells undergoing exponential growth following steroid exposure than in cells approaching stationary growth. In both A/J and C57 cultures the reduction in cell number exhibits a dose-dependent response to dexamethasone; is specific for glucocorticoids; and is dependent upon the concentration of serum in which the cells are maintained. A/J cells show a greater sensitivity to the inhibitory effect of dexamethasone on cell number and thymidine incorporation than comparably treated C57 cells. Specific, high affinity, saturable cytoplasmic receptors for [3H] dexamethasone are present in the maxillary cytosols from which the primary cultures were established. These receptors exhibit binding specificity for glucocorticoids, and have properties which are similar to glucocorticoid receptors identified in other systems. In both cell types, a correlation exists between the degree of growth inhibition or reduction of [3H] thymidine incorporation and the level of glucocorticoid receptors. These results provide evidence for a receptor-mediated set of responses to glucocorticoids in these cells.

Animals↗
PubMed1978

Enhancement of chlorcyclizine teratogenicity in the rat by coadministration of calcium chelating agents.

Chlorcyclizine and structurally related drugs induce a high incidence of cleft palate and skeletal malformations in fetal rats. We have shown previously that these teratogens bind tightly and reversibly to chondroitin sulfate of cartilage and compete with calcium for binding. Experiments reported here demonstrate that co-administration of calcium chelating agents with chlorcyclizine significantly increases both the frequency of malformations and retention of [14C] chlorcyclizine by embryos. Retention of radioactive teratogen by embryos is inverse to retention of [45Ca]calcium. These findings suggest that drug binding to embryonic glycosaminoglycans is involved in the pathogenesis of malformations produced by chlorcyclizine.

Abnormalities, Drug-Induced↗
PubMed1978

Chlorcyclizine induction of cleft palate in the rat: degradation of palatal glycosaminoglycans.

Administration of the cleft palate teratogen chlorcyclizine or norchlorcyclizine to pregnant rats causes an alteration in glycosaminoglycans (GAGs) in embryonic palatal shelves. Pulse-chase experiments in vitro indicate that norchlorcyclizine enhances the degradation of hyaluronic acid and chondroitin sulfate but has little or no effect on their synthesis. These changes in GAGs are caused by concentrations of norchlorcyclizine that have no appreciable effect on DNA or protein synthesis. These findings suggest that degradation of palatal GAGs may be the primary biochemical defect responsible for the inhibition of palatal shelf elevation by norchlorcyclizine.

Abnormalities, Drug-Induced↗
PubMed1978

Role of carbohydrates in protein secretion and turnover: effects of tunicamycin on the major cell surface glycoprotein of chick embryo fibroblasts.

Using tunicamycin, we have investigated the role of glycosylation in the biosynthesis, processing and turnover of CSP, the major cell surface glycoprotein of chick embryo fibroblasts (CEF). This antibiotic specifically inhibits glycosylation mediated by dolichol pyrophosphate and consequently inhibits the glycosylation of asparaginyl residues of glycoproteins. Tunicamycin inhibited the incorporation of 3H-mannose into CSP by 92--98% and 14C-glucosamine by 84--96%, whereas total protein synthesis was decreased by only 15--45%. Tunicamycin treatment decreased total amounts of CSP by approximately 50--65%, with equal decreases in CSP occurring on the cell surface and in culture medium, whereas intracellular pools of CSP were not substantially affected. In contrast to CSP, three other membrane-associated proteins of apparent molecular weights 75,000, 95,000 and 150,000 daltons were found in increased amounts. Procollagen secretion was not inhibited by tunicamycin. Both procollagen and CSP secretion into culture medium were also not increased in AD6, a glycosylation-deficient, mutant mouse 3T3 cell line compared to wild-type cells. We examined the mechanism of the decrease in CSP after tunicamycin treatment. The rate of CSP biosynthesis as measured by pulse-labeling with 14C-leucine was not altered. Tunicamycin had only a slight effect on the initial times and rates of CSP appearance on the cell surface; some apparent intracellular redistribution of CSP was detected by immunofluorescence. The major effect of tunicamycin treatment was to accelerate the rate of degradation of CSP 2--3 fold. This increase is sufficient to account for the observed decreases after tunicamycin treatment. Our results suggest that carbohydrates may not be essential for CSP or procollagen synthesis, intracellular processing and secretion, but that carbohydrates may help stabilize CSP against proteolytic degradation.

Anti-Bacterial Agents↗
PubMed1978

Inhibition of epithelial cell death in the secondary palate in vitro by alteration of lysosome function.

The secondary palate in vivo and in vitro exhibits selective cell death at its medialedge epithelium (MEE) at a precise developmental age. This epithelial degeneration is mediated, in part, by MEE lysosomes. Previous studies in vitro (27) showed that the glutamine analogue, diazo-oxo-norleucine (DON), prevented MEE cell death by inhibiting glucosamine synthesis and thereby the glycosylation of proteins without affecting either the synthesis or activity of palatal lysosomal enzymes. In the present study, histochemical examination of MEE from DON treated day-15 rat palates demonstrated that acid phosphatase activity was restricted to Golgi saccules and associated vesicles as well as to lysosomes. Control MEE had reaction product in these structures and distributed diffusely throughout the cytoplasm of degenerating cells. DON treatment therefore appears to alter the intracellular distribution of lysosomal enzymes. Since DON treatment appears to have prevented MEE cell death by inhibiting glycosylation of proteins, glycosylation of lysosomal membranes or lysosomal enzymes may be essential for its role in programmed cell death.

Acid Phosphatase↗
PubMed1977

Ultrastructure of the expected fusion zone in rat fetuses with diazo-oxo-norleucine (DON)-induced cleft palate.

The aim of this study was to determine whether the glutamine analog, 6-diazo-5-oxo-L-norleucine (DON), which is known to inhibit cell degeneration in rat palatal processes in vitro, exerted a similar effect on cleft palatal processes in vivo. Sprague-Dawley rats were injected intraperitoneally with 5.0 mg/kg body wt of DON on Day 15 of gestation and fetuses were removed over Days 16, 17 and 18. A high frequency (80-90%) of cleft palate was obtained. Fetal heads were removed on each of the three days and transverse sections of the palatal processes were examined by light and electron microscopy. Results showed that treatment with DON failed to inhibit degenerative changes in the palatal epithelium. The same sequence and timing of ultrastructural changes occurred in the expected fusion zone (EFZ) as have been described previously in cleft palatal processes from Meclozine-treated and amniotic sac punctured rat fetuses; namely, autophagic cytoplasmic degeneration, loss of basal lamina and the subepithelial accumulation of macrophages all of which were most prominent in that part of the EFZ immediately next to the developing oral epithelium. The results therefore suggest that a teratogenic dose of DON does not interfere with the ultrastructurally observable changes in the EFZ.

Abnormalities, Drug-Induced↗
PubMed1977

Glycosaminoglycan and glycoprotein synthesis by cranial neural crest cells in vitro.

In the developing chick embryo, cranial neural crest cells, that will subsequently give rise to facial mesenchyme tissues, migrate beneath the surface ectoderm in a cell-free and hyaluronate-rich matrix. To determine how the crest cells could contribute to this matrix, we cultured crest cells from stage 9 embryos for 2 days and then labeled them for 18--20 h with various precursors of glycosaminoglycans (GAG) and glycoproteins. [3H]fucose and [3H]glucosamine were incorporated into Pronase-sensitive macromolecules associated with the cell layer, but little labeled glycoprotein was released into the medium. Hyaluronate was the major GAG synthesized and was distributed between the cells and medium. Less chondroitin-sulfate was synthesized. In comparison, older cultures as well as fibroblasts produced different proportions of GAG. Our results confirm the autoradiographic findings of Pratt et al. (1975) and suggest that crest cells may contribute GAG to the matrix during migration.

Animals↗
PubMed1976

Developmental aspects of secondary palate formation.

Research on development of the secondary palate has, in the past, dealt primarily with morphological aspects of shelf elevation and fusion. The many factors thought to be involved in palatal elevation, such as fetal neuromuscular activity and growth of the cranial base and mandible, as well as production of extracellular matrix and contractile elements in the palate, are mostly based on gross, light microscopic, morphometric or histochemical observations. Recently, more biochemical procedures have been utilized to described palatal shelf elevation. Although these studies strongly suggest that palatal extracellular matrix plays a major role in shelf movement, interpretation of these data remains difficult owing to the complexity of tissue interactions involved in craniofacial development. Shelf elevation does not appear to involve a single motive factor, but rather a coordinated interaction of all of the abovementioned developmental events. Further analysis of mechanisms of shelf elevation requires development of new, and refinement of existing, in vitro procedures. A system that enables one to examine shelf elevation in vitro would allow more meaningful analysis of the relative importance of the various components in shelf movement. Much more is known about fusion of the palatal shelves, owing in large part to in vitro studies. Fusion of the apposing shelves, both in vivo and in vitro, is dependent upon adhesion and cell dealth of the midline epithelial cells. Adhesion betweeen apposing epithelial surfaces appears to involve epithelial cell surface macromolecules. Further analysis of palatal epithelial adhesion should be directed towards characterization of those cell surface components responsible for this adhesive interaction. Midline epithelial cells cease DNA synthesis 24-36 h before shelf elevation and contact, become active in the synthesis of cell surface glycoproteins, and subsequently manifest morphological signs of necrosis. Death of the midline epithelial cells is thought to involve a programmed, lysosomal-mediated autolysis...

Animals↗