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Biomedical subjects

R M Pratt

Publications and source records attributed to R M Pratt.

At least 19 recordsLinked to original sources
PubMed1991

Effects of retinoic acid on embryonic development of mice in culture.

The effects of all-trans-retinoic acid (RA) (tretinoin) on the craniofacial development of mouse embryos were examined using whole embryo culture. In day 8 embryos cultured for 48 h, embryonic growth was inhibited concentration-dependently by all-trans-RA treatment. Most of the treated embryos exhibited hypoplasia of the primary palatal processes and a reduction in the development of the first visceral arches. In day 10 embryos cultured for 48 h, although embryonic growth was not inhibited at any concentrations of all-trans-RA, median cleft lip (93%), hypoplasia of the primary palatal processes (37%) and limb reduction deformities (48%) occurred commonly. Furthermore, RA treatment greatly reduced the size of the secondary palatal processes. The incorporation of 3H-thymidine in the treated maxillary processes was decreased to 65% of the control value at 1.0 x 10(-7) M all-trans-RA. These findings indicate that all-trans-RA is teratogenic in mouse whole embryo culture.

Animals↗
PubMed1991

Influence of retinoids on sister chromatid exchanges and chromosomes in cultured human embryonic palatal mesenchymal cells.

The cytogenetic effects of retinoids and their metabolites on human embryonic palatal mesenchymal (HEPM) cells were investigated in cell culture. Treatment with 13-cis, 4-oxo-13-cis-, and all-trans-retinoic acids did not induce chromosomal structural aberrations even at high concentrations in the presence or absence of metabolic activation system. The frequencies of sister chromatid exchanges (SCE) tended to decrease in HEPM cells treated with retinoids in the absence of S-9. 4-Oxo-13-cis-retinoic acid, which is one metabolite of 13-cis-retinoic acid, significantly decreased the SCE frequencies. The mitotic index decreased with increasing concentration of retinoic acids, indicating that retinoids inhibit cell proliferation in HEPM cells. We concluded that retinoids inhibit cell proliferation in HEPM cells in culture without causing DNA or chromosome damage.

Cells, Cultured↗
PubMed1990

Effect of retinoids on proliferation of human embryonic palatal mesenchymal cells in culture.

The cytokinetics and growth effects of retionic acid (RA) (13-cis-RA, and all-trans-RA) were determined in human embryonic palatal mesenchymal (HEPM) cells using cell culture and sister chromatid differential staining. The lowest concentration tested that showed growth inhibition was 1.0 X 10(-4)M for 13-cis-RA, and 6.8 X 10(-5)M for all-trans-RA after 40 h of treatment. When HEPM cells were grown in the presence of BrdU (5-bromodeoxyuridine) for 44 h, approximately 70% of the control cells were in metaphase in the 3rd replication cycle (M3). The percentage of cells in M3 decreased in the presence of RA with or without a metabolic activation system (S-9), thus indicating a longer cell proliferation time for the RA-treated cells. The estimate of cell proliferation time in 13-cis-RA-treated cells (1.0 X 10(-4)M) was approximately 25 h at the 2nd cell cycle compared with 20 h in the control cells. When HEPM cells were exposed for 16 h to [3H]-thymidine, labeling indices in 13-cis-RA-treated cells were significantly reduced even at 1.0 X 10(-6)M. Both 13-cis-RA and all-trans-RA caused concentration-dependent decreases in [3H]-thymidine incorporation into HEPM cells. These results suggest that retinoic acids or their major metabolites interfere with DNA synthesis and decrease the proliferation of HEPM cells.

Cell Count↗
PubMed1988

Teratogenicity of benzoic acid derivatives of retinoic acid in cultured mouse embryos.

Isotretinoin (13-cis-RA) is a human teratogen and mouse embryos exposed to 13-cis-RA in vivo exhibit many of the same defects as humans. Early postimplantation mouse embryos exposed to 13-cis-RA in culture exhibit developmental alterations of the visceral arches, similar to those seen after in vivo exposure. Certain benzoic acid derivatives of retinoic acid have been shown to possess activity equal to or greater than retinoic acid in several in vitro systems. This study examines the teratogenic effects of some of these retinoids on mouse embryos in vitro. Day 8 CD-1 mouse embryos were cultured for 48 hours in the presence of these benzoic acid derivatives. With the exception of Ro-15-0778, all compounds produced visceral arch malformations similar to those seen in embryos exposed to 13-cis-RA, but at dramatically different effective concentrations. Extremely low concentrations of the retinoic acid-related compounds tested appear to have detrimental effects on embryonic development and these compounds may be poor candidates for therapeutic use.

Animals↗
PubMed1986

Karyotype, growth, and cell cycle analysis of human embryonic palatal mesenchymal cells: relevance to the use of these cells in an in vitro teratogenicity screening assay.

Human embryonic palatal mesenchyme (HEPM) is an established cell line that is presently under investigation as an in vitro prescreening assay used to determine the teratogenic potential of chemicals. We describe here general growth characteristics, karyotype, and cell cycle analysis of these cells. HEPM cells had plating efficiencies of less than 95% and displayed notable contact growth inhibition following an exponential growth phase that lasted for approximately 6 days. These cells had the diploid karyotype of a female human embryo. The chromosomal complement showed no dramatic change between passage 5 and 14. Flow cytofluorometry analysis using bromodeoxyuridine (BrdU) pulse labeling and a direct immunofluorescence anti-BrdU FITC probe revealed that the total cell cycle transit time was approximately 22 hr: the duration of G1 was 12.2 hr, S was 6.1 hr, and G2-M lasted for 3.7 hr. The results indicate that HEPM cells met the criteria regarding karyotype stability that were assumed by the National Toxicology Program of the USA.

Bromodeoxyuridine↗
PubMed1985

Receptor-dependent mechanisms of glucocorticoid and dioxin-induced cleft palate.

Glucocorticoids (triamcinolone) and dioxins (TCDD) are highly specific teratogens in the mouse, in that cleft palate is the major malformation observed. Glucocorticoids and TCDD both readily cross the yolk sac and placenta and appear in the developing secondary palate. Structure-activity relationships for glucocorticoid- and TCDD-induced cleft palate suggest a receptor involvement. Receptors for glucocorticoids and TCDD are present in the palate and their levels in various mouse strains are highly correlated with their sensitivity to cleft palate induction. Receptors for glucocorticoids appear to be more prevalent in the palatal mesenchymal cells whereas those for TCDD are probably located in the palatal epithelial cells. Glucocorticoids exert their teratogenic effect on the palate by inhibiting the growth of the palatal mesenchymal cells whereas TCDD alters the terminal cell differentiation of the medial palatal epithelial cells.

Animals↗
PubMed1979

Selective inhibition of mandibular growth and induction of cleft palate by diazo-oxo-norleucine (DON) in the rat.

A high percentage of cleft palates can be induced in rat fetuses by a single injection of the glutamine analog diazo-oxo-norleucine (DON) on day 15 of gestation. The purpose of this study was to evaluate the effects of DON in vivo on craniofacial growth and spatial relations in order to identify factors that may contribute to the palatal defects. Sprague-Dawley rats in the experimental groups were given a single IP injection of 2.0 mg DON (6 mg/kg maternal body weight) on day 15 and were killed on day 16 or 17. Control fetuses were collected on days 15, 16 and 17. Fetal heads were fixed in Bouin's solution, embedded in Paraplast and serially-sectioned. Midsagittal and coronal sections were projected at 30 X and a series of linear and angular measurements were made. DON had limited effect on growth of the cranial base, nasomaxillary complex, and palatine processes, but dramatically reduced the length of Meckel's cartilage. Treatment with DON delayed shelf elevation approximately 24 hours, and tongue position remained high in the oronasal cavity. Growth retardation in Meckel's cartilage therefore may contribute to delayed shelf movement by retarding downward and forward positioning of the tongue-mandibular complex.

Animals↗
PubMed1979

Enhanced cellular fibronectin accumulation in chondrocytes treated with vitamin A.

Chick sternal chondrocytes cultured at high cell density lack fibronectin as a surface protein, while vitamin A-treated chondrocytes contain it as the major cell surface protein. We investigated the mechanism of fibronectin accumulation under these conditions. Control chondrocytes synthesized nearly as much fibronectin as vitamin A-treated chondrocytes, but it was secreted primarily into culture medium. Althought the fibronectin of control chondrocytes was of a slightly lower apparent molecular weight than the fibronectin synthesized by the treated cells, it bound as effectively to the cell layer of both normal and treated cells. In contrast, the vitamin A-treated cultures were 2.7 fold more effective in binding fibronectin synthesized by either control or treated cells. Thus in chondrocytes, vitamin A appears to regulate the cellular accumulation of fibronectin by increasing the ability of the cell layer to bind fibronectin rather than by altering its synthesis or its adhesivity for the cell layer.

Animals↗
PubMed1979

Evidence for role of glycoprotein carbohydrates in membrane transport: specific inhibition by tunicamycin.

Using tunicamycin, we have investigated the role of glycoproteins in membrane transport. Tunicamycin is a glucosamine-containing antibiotic that specifically inhibits dolichol pyrophosphate-mediated glycosylation of asparaginyl residues of glycoproteins. Inhibition of protein glycosylation in chick embryo fibroblasts by tunicamycin or other inhibitors of glycosylation resulted in defective transport of glucose, uridine, and amino acid analogs (alpha-aminoisobutyrate and cycloleucine). The defect in glucose transport is accompanied by decreased glucose metabolism, as determined by rates of CO2 and lactate production. In contrast, tunicamycin treatment did not affect other membrane-associated processes, such as secretion of fibronectin and procollagen, uptake of glucose by passive diffusion, Na+/K+ ATPase and adenylate cyclase activities, or stimulation of adenylate cyclase by prostaglandin and cholera toxin. Two glucose/glycosylation-regulated membrane proteins with apparent subunit molecular weights of 95,000 and 75,000 were induced by tunicamycin treatment. Our results indicate that glycoprotein glycosylation is required for membrane transport.

Amino Acids↗
PubMed1979

Role of carbohydrate in biological function of the adhesive glycoprotein fibronectin.

We have investigated the role of the carbohydrate moiety in the biological activity of fibronectin in vitro by using tunicamycin to inhibit the glycosylation of this glycoprotein. Tunicamycin is a glucosamine-containing antibiotic that specifically inhibits glycosylation of protein asparaginyl residues mediated by dolichol pyrophosphate. Fibronectin synthesized in the presence of 0.5 microgram of tunicamycin per ml was not glycosylated, as determined by amino sugar analysis, lack of incorporation of [14C]glucosamine and [3H]mannose, and concanavalin A binding studies. Nonglycosylated fibronectin that was isolated from chicken embryo fibroblasts and added to transformed cells in vitro was as effective as the glycosylated protein in promoting a more normal fibroblastic phenotype, including cell flattening, elongation of cell processes, and parallel alignment of cells. The nonglycosylated protein was also as effective as the glycosylated species in mediating cell attachment to collagen and spreading on plastic, as well as in agglutination of formalin-fixed sheep erythrocytes. The nonglycosylated protein was twice as sensitive as the glycosylated protein to proteolytic hydrolysis in vitro as had been suggested by previous studies with intact cells [Olden, K., Pratt, R.M. & Yamada, K.M. (1978) Cell 13, 461-473]. We conclude that the carbohydrate moiety of fibronectin is not required for the mediation of a number of biological activities characteristic of this glycoprotein.

Animals↗
PubMed1979

Increase in levels of cyclic AMP during avian limb chondrogenesis in vitro.

In the present study the level of cAMP was measured during in vitro chondrogenesis of wing mesenchyme of stage 24 chick embryos and was found to increase significantly from 6.3 pmol/mg protein at the end of the first day of culture to 9.7 pmol/mg protein on the second day, when chondrogenic expression is first detected by the appearance of an Alcian blue staining extracellular matrix. Nonchondrogenic cultures derived from wings of stage 19 embryos had a lower level of cAMP (4.4 +/- 0.07 pmol/mg protein). The level of cAMP in intact wings was 4.5 +/- 0.4 pmol/mg protein and did not change between stages 19 through 25. The correlatin between increased levels of cAMP and the onset of chondrogenesis is consistent with a role of cAMP in the expression of differentiated functions in chondrocytes, as well as in some other cell types.

Animals↗
PubMed1979

Involvement of glucocorticoids in the development of the secondary palate.

Genetic differences between various inbred strains of mice in the levels of glucocorticoid receptors embryonic in maxillary mesenchyme cells appear to be reflected in the magnitude of the responses to steroids in these cells. High levels of glucocorticoids cause significant growth inhibition in maxillary mesenchyme cells with subsequent alterations in the production of extracellular matrix components. The presence of higher levels of cytoplasmic glucocorticoid receptor proteins may be one factor which could predispose those strains such as A/J to a greater inhibition of craniofacial growth in vivo by glucocorticoids and therefore increase the frequency of cleft palate production. Furthermore, women with infertility treated with glucocorticoids to support pregnancy give birth to infants with a marked decrease in birth weight [98]. Pharmacologic doses of glucocorticoids can also cause a dramatic reduction in the growth of a number of fetal tissues in mice and humans. In fact, there is evidence that glucocorticoids may be a causative factor in the production of cleft palate in primates [52]. The nature of the molecular elements which determine the biochemical and physiologic responses to glucocorticoids in the palate still remains largely unknown. Although in the mouse there is some evidence to suggest that the major histocompatibility locus (H-2) might be involved, the level(s) at which this control is exerted is unknown. It is possible that this locus may regulate in some manner the level of glucocorticoid receptors and the response to glucocorticoids in the secondary palate. Moreover, there is evidence to suggest that other genes distinct from, but closely linked to the H-2 locus may be important in determining both the strain-dependent differences in susceptibility to glucocorticoid-induced cleft palate and the intracellular levels of cyclic AMP in the secondary palate. It is also apparent that glucocorticoids in conjunction with other hormones or growth factors such as epidermal growth factor and agents which regulate cyclic nucleotide metabolism are essential for the normal development of the secondary palate. Excesses or deficiencies in either the level of these growth regulators and/or in their receptors in specific fetal tissues at defined periods in development are likely to lead to certain fetal malformations. Definition and integration of the genetic, biochemical, and endocrine factors which are involved in the control of cellular growth as influenced by alterations in the composition of cell surface and extracellular matrix components should provide some insights into the events associated with normal palatogenesis.

Animals↗
PubMed1979

Corticosterone levels during midgestation in the maternal plasma and fetus of cleft palate-sensitive and -resistant mice.

Corticosterone levels in the maternal plasma and fetal extracts during days 11--15 of gestation were measured by RIA in cleft palate-sensitive A/J and -resistant C57BL/6J mice. In both strains, the maternal plasma corticosterone increased from 10 micrograms/100 ml in nonpregnant animals to 100 microgram/100 ml between days 12--13. In A/J mice, the corticosterone level declined to 60 micrograms/100 ml by day 15, whereas in C57BL/6J mice, the corticosterone concentration remained elevated during this period. In contrast, fetal corticosterone levels in both strains remained unchanged between days 11-13 (0.5--1.0 ng/embryo) and increased on days 14 and 15 to 4--7 ng/embryo. The absence of significant differences in the concentration of maternal or fetal corticosterone between A/J and C57BL/6J mice suggests that the in vivo strain differences in the glucocorticoid susceptibility to cleft palate production are not related to any intrinsic differences in the endogenous levels of corticosterone between these two strains during midgestation.

Animals↗