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Biomedical subjects

R M Pratt

Publications and source records attributed to R M Pratt.

At least 55 records · Page 3Linked to original sources
PubMed1983

Growth and differentiation of embryonic mouse palatal epithelial cells in primary culture.

The study of both normal and abnormal mammalian palatal development would be greatly enhanced by the advent of a cell culture system for the palatal epithelium in the absence of its mesenchyme. We have developed such a method for the primary culture of the secondary palatal epithelium from the embryonic mouse which allows for both epithelial cell proliferation and differentiation into the three cell types normally found in vivo. These include the terminally differentiated medial epithelial cells and the appearance of the nasal epithelial cell phenotype (ciliated pseudostratified), as well as the oral epithelial cell phenotype (stratified squamous). The most successful culture medium tested consisted of a 1:1 mixture of DMEM/F-12 basal medium supplemented with fetal bovine serum and epidermal growth factor (EGF). Epithelial cell attachment, DNA synthesis and differentiation are greatly stimulated by the presence of EGF and by an extracellular matrix (ECM) substratum. Our results have demonstrated for the first time the feasibility of culturing embryonic palatal epithelial cells in primary culture in the absence of any mesenchymal tissue.

Animals↗
PubMed1982

Prescreening for environmental teratogens using cultured mesenchymal cells from the human embryonic palate.

Mesenchymal cells from the prefusion human embryonic palate have been established in culture and can be grown in either a serum-free hormone-supplemented medium or a serum-containing medium. The growth of these cells is quite rapid in culture and inhibited in a dose-dependent manner by most teratogens thus far tested, such as dexamethasone. These cells are highly sensitive to a variety of DNA synthetic and mitotic inhibitors. The responses of these cells are complementary to the ovarian tumor cell attachment assay of Braun et al [1, and in this volume]. When used in conjunction with the tumor cells, the overall reliability is greater than 90% with only one false-negative, allopurinol.

Cells, Cultured↗
PubMed1982

Vitamin B6 reduces cortisone-induced cleft palate in the mouse.

Administration of Vitamin B6 during gestation to mice on a Vitamin B6-containing diet resulted in a substantial reduction in cortisone-induced cleft palate. Mice maintained on a Vitamin B6-deficient diet demonstrated an increase in the frequency of cortisone-induced cleft palate; this effect was prevented by administration of Vitamin B6. Vitamin B6 inhibited the specific binding of a labeled glucocorticoid to cytosolic receptors from cultured palatal mesenchyme cells. These results indicate that Vitamin B6 reduces the incidence of cortisone-induced cleft palate by altering the binding of glucocorticoids to their cytoplasmic receptors and subsequently nuclear acceptors.

Abnormalities, Drug-Induced↗
PubMed1982

Glucocorticoid receptors in palatal mesenchymal cells from the human embryo: relevance to human cleft palate formation.

Glucocorticoids are potent inducers of cleft palate (CP) in experimental animals. The present study was performed in order to examine whether human embryonic palatal mesenchymal (HEPM) cells contain glucocorticoid receptors and are responsive to the growth inhibitory effects of glucocorticoids. We have found that there is a single population of specific, high affinity, saturable glucocorticoid receptors in HEPM cells. Scatchard analysis of the binding data revealed that the apparent dissociation constant (Kd) was 26 nM and the number of receptors was 1.4 x 10(5) per cell. Dexamethasone (DEX), a potent synthetic glucocorticoid analog, inhibited growth of HEPM cells cultured in serum-free chemically defined medium by 20-40%. Dose-response experiments showed that DEX inhibition of HEPM cell growth was closely correlated with specific binding of [3H]DEX to the cells, suggesting that the suppression of HEPM cell growth may be receptor-mediated. DEX also inhibited collagen synthesis in HEPM cells. These results indicate that HEPM cells are capable of responding to both physiological and pharmacological levels of glucocorticoids and suggest that either endogenous or exogenous glucocorticoids may have the potential to interfere with human palate formation by perturbing the growth and differentiation of palatal mesenchymal cells.

Cell Line↗
PubMed1981

Cortisone-induced cleft palate in A/J mice: failure of palatal shelf contact.

Although cortisone treatment for induction of cleft palate in mice has been shown to delay the time of palatal shelf elevation, the effects of delayed elevation of shelf contact have not been critically evaluated in a cortisone-sensitive mouse strain. The objective of this study was to evaluated palatal development in cortisone-treated A/J mice in order to determine whether the shelves make contact upon elevation. Morphometric analysis of frozen sections revealed that cortisone-treated shelves were smaller than control shelves with apparent reductions in both the content of extracellular matrix and the number of cells. At a light microscopic level, thinning of medial epithelium in cortisone-treated palates appeared similar to that in untreated palates with spontaneous cleft lip and palate. Shelf elevation was delayed by approximately 12 hours and only half of the cortisone-treated palates achieved complete horizontal positioning of the shelves in all regions of the palate. Immediately after elevation, all control palates had extensive vertical contact along the complete length of the palate. In contrast, approximately 20% of the cortisone-treated fetuses had contact between the shelves in the middle palate region only, with the mean area of contact only 20% as large as in control fetuses. As result, the net shelf contact in all the cortisone-treated fetuses was only 4% of the potential contact shown in control fetuses. Therefore, failure of the palatal shelves to elevate and make extensive contact appeared to be the major factor contributing to cortisone-induced cleft palate in A/J mice.

Animals↗
PubMed1981

Distribution of fibronectin and collagen during mouse limb and palate development.

Indirect immunofluorescence has been used to study the distribution of fibronectin and collagen types I, II, and III in the developing primary and secondary palatal processes and forelimb buds of the Swiss Webster (NIH) mouse. In the palatal processes fibronectin and types I and III collagen are distributed throughout the mesenchyme. Fibronectin is present in the basement membrane, while types I and III collagen are localized in a linear, discontinuous fashion beneath the basement membrane. Fibronectin is not observed in the epithelium, including the presumptive fusion areas. In the forelimb bud these components show a similar distribution prior to chondrogenesis (early day 11). When chondrogenesis commences (late day 11 or early day 12) fibronectin and, to a lesser degree, types I and III collagen are apparently concentrated in the core mesenchyme, suggesting that fibronectin has a role in initiating chondrogenesis, perhaps by increasing cellular aggregation. Type II collagen is observed only in chondrogenic regions. The codistribution of fibronectin and types I and III collagen supports in vitro studies which indicate that cells use fibronectin to bind to collagen in the matrix. The developing chondrogenic regions appear to lose fibronectin gradually, concomitant with the appearance of type II collagen, suggesting that fibronectin is not involved in the maintenance of functional chondrocytes in their matrices.

Animals↗
PubMed1981

Interaction between glucocorticoids and epidermal growth factor in vitro in the growth of palatal mesenchymal cells from the human embryo.

Previous studies of ours have shown that palatal mesenchymal cells from the human embryo (HEPM cells) are responsive both to the glucocorticoid dexamethasone (DEX) and epidermal growth factor (EGF) through mechanisms associated with cytoplasmic and cell surface receptors, respectively. HEPM cell growth was inhibited by DEX and was stimulated by EGF. In the present study, the interactions between DEX and EGF were investigated. DEX (10(-6) M) enhanced EGF-stimulated HEPM cell growth as assessed by an increase in cell number and ornithine decarboxylase activity under serum-free cell culture conditions. DEX also enhanced the specific binding of 125I-EGF to these cells, which was reflected in an increase in both the number and the affinity of EGF receptors. EGF (1 ng/ml), on the other hand, decreased the number of sites per cell which specifically bind 3H-DEX. EGF completely prevented the inhibition by DEX of HEPM cell growth. These results indicated that DEX and EGF interact with each other in the process(es) regulating HEPM cell growth. This interaction may be partially influenced by direct modulation of existing receptors for DEX and EGF present in the cells.

Cell Division↗
PubMed1981

Induction of cleft lip in cultured rat embryos by localized administration of tunicamycin.

Whole-embryo culture techniques have advanced to the point where the study of normal and abnormal primary palate development in vitro is possible. The purpose of this study was to ascertain whether localized administration of tunicamycin (TM) an inhibitor of protein glycosylation, into the region of the developing primary palate would induce cleft lip in culture. Rat embryos were explanted on late day 11 of gestation and cultured with open yolk sacs for 40 h. TM was administered by implanting a sterile eyelash soaked in TM into the nasal placode region. The contralateral placode was used as the control by implanting an eyelash lacking TM. Under these conditions, TM-treated placodes were found to develop cleft lip in 14 out of 15 embryos compared to 0 for the controls. These experiments demonstrate that localized administration of TM results in cleft lip formation in whole embryo culture. The technique of localized administration of drugs and teratogens in whole embryo culture should prove useful for similar studies on embryonic development.

Animals↗
PubMed1981

Decreased number of glucocorticoid receptors in dermal fibroblasts from individuals with facial clefting: a preliminary report.

Glucocorticoids are potent inducers of cleft palate in various laboratory animals. Recent work has demonstrated that the susceptibility of mice to the teratogenic action of glucocorticoids is strongly correlated with the number of fetal receptors for glucocorticoids. In the present study we have investigated whether there is a difference in levels of glucocorticoid receptors between individuals with or without cleft lip +/- cleft palate or isolated cleft palate. We have found that dermal fibroblasts from the affected individuals have significantly fewer glucocorticoid receptors than do fibroblasts from unaffected persons. This decrease in receptors may reflect a basic hormonal defect in these individuals, which presumably could play a role in the etiology of facial clefting.

Adolescent↗
PubMed1980

Characterization of fibronectin interactions with glycosaminoglycans and identification of active proteolytic fragments.

Fibronectin is a major cell-surface glycoprotein which has been reported to interact with glycosaminoglycans. A nitrocellulose filter-binding assay was developed to quantitate these interactions at physiological pH and ionic strength. Fibronectin isolated from chick embryo fibroblasts binds both hyaluronic acid and heparin; heparan sulfate is bound less efficiently, and chondroitin sulfate and glycopeptides are bound minimally. The binding of hyaluronic acid and heparin to fibronectin is saturable and reversible and occurs at separate binding sites. The binding of both molecules to fibronectin is not blocked by EDTA or by other glycosaminoglycans, and is only moderately inhibited by elevated ionic strength. Scatchard analyses revealed nonlinear, high affinity binding to fibronectin with a KD of approximately 10(-7) to 10(-8) M for these glycosaminoglycans. The affinity for heparin was utilized for the isolation of heparin-binding domains of fibronectin on heparin-agarose affinity columns. Heparin-binding proteolytic fragments with apparent molecular weights of 160,000 and 50,000 were isolated following hydrolysis of fibronectin by chymotrypsin or pronase, respectively. The possible involvement of such high affinity binding sites of fibronectin in the binding of glycosaminoglycans to the cell surface or in the organization of extracellular matrices is discussed.

Animals↗
PubMed1980

Cortisone-induced cleft palate in the brachymorphic mouse.

Previous studies have shown that the autosomal recessive gene brachymorphic (bm/bm), which is maintained on a C57BL/6J (C57) background, reduces limb growth and sulfation of cartilage proteoglycans. Hydrocortisone administered on gestational days 11-14 resulted in 20% CP in the C57 mouse, but 95% CP in the bm/bm mouse. The bm/bm mouse had a median effective dose for CP of 45 mg/kg, compared to 325 mg/kg for C57 and 40 mg/kg for A/J. Morphometric analysis indicated that the time of palatal elevation was delayed in the bm/bm relative to the C57 mouse both with and without hydrocortisone treatment. The amount of cytoplasmic glucocorticoid receptor protein present in the bm/bm palate on day 14 was the same as the amount found in the C57 palate, and was not elevated as it is in the A/J palate. The levels of cyclic AMP in the bm/bm palate on day 14 were 30-70% higher than that found in the C57 palate with or without hydrocortisone. These results suggest that both bm/bm and A/J exhibit a delay in palatal shelf rotation and elevated levels of cyclic AMP, which appear to be predisposing factors for cortisone-induced cleft palate. These strains differ in that elevated levels of steroid receptors are present in A/J palate, whereas lower levels are found in the C57 and bm/bm mice.

Animals↗
PubMed1980

Detection of teratogenic compounds using differentiating embryonic cells in culture.

Methods are described for screening for teratogenic compounds using differentiating neural crest and prechondrogenic limb bud mesenchyme cells in culture. Substances to be tested are either added directly to the culture medium or are combined in a dialysis bag with the postmitochondrial fraction from rat liver and certain cofactors. In the latter case, the compound and its metabolites are gradually released into the medium from the dialysis bag. The results obtained with 14 compounds demonstrate a positive relationship between teratogenicity in vivo and alterations in the growth or the differentiation of the cultured cells.

Aminopropionitrile↗
PubMed1980

An improved culture system for secondary palatal elevation.

An organ culture system devised for studying the development of the secondary palate was modified so that it retained high partial pressures of oxygen and supported total anterior and posterior palatal elevation. The cultured tissues appeared healthy as judged by histological examination. Medium was continuously recirculated through the culture system in which Day 13 embryonic mouse heads, with the brain and tongue removed, were totally submerged and suspended. The medium was constantly gassed via hollow fiber devices. A motor-driven stirrer, run at a low rate, agitated the medium so that the boundary layer surrounding the tissue was dispersed. Embryonic mouse heads were cultured in each of four media: Eagle's basal medium, Ham's F-12 medium, Fitton-Jackson's modified BGJb medium, and Waymouth's MB 752/1 medium. Elevation of the palate in both anterior and posterior regions with excellent tissue viability was achieved in all heads grown in BGJb medium.

Animals↗
PubMed1980

Detection of epidermal growth factor-urogastrone and its receptor during fetal mouse development.

Using both a radioimmunoassay and a radioreceptor assay, we have estimated the content of mouse epidermal growth factor-urogastrone (EGF-URO) in fetal mice at 11(1/2) to 17(1/2) days of gestation. Concurrently, the amount of specific EGF-URO receptor binding was determined in crude membrane fractions from the embryos. EGF-URO receptor binding is readily detected in membranes from the youngest embryos (day 11(1/2)) and rises steadily up to parturition (18 days); the rise is more marked in embryo membranes derived from a potential target tissue, such as the maxilla and secondary palate. In the embryonic extracts, EGF-URO proved to be labile, requiring the presence of soybean trypsin inhibitor and sodium azide to stabilize the recovery of added EGF-URO in test samples. Even with added stabilizing agents, immunoreactive EGF-URO was barely detectable before day 14(1/2) (less than 20 fmol per embryo), whereas a substantial increase was observed from day 15(1/2) to 17(1/2) (from 70 to 200 fmol per embryo). In contrast, the radioreceptor assay detected appreciable amounts of an EGF-URO-like substance at 11(1/2) days (50 fmol per embryo); the values estimated by radioreceptor assay (about 10-fold higher than by radioimmunoassay) also increase markedly between days 15(1/2) and 17(1/2) (on average from 500 to 3000 fmol per embryo). We conclude that during fetal mouse development there is an increase both in the receptors for EGF-URO and in a substance (presumably a fetal growth factor) that can occupy the receptor. The differences between the radioreceptor and radioimmunoassay estimates for the EGF-URO content suggest that the fetal form of mouse EGF-URO differs from the adult molecule.

Animals↗