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Biomedical subjects

R M Greene

Publications and source records attributed to R M Greene.

At least 91 records · Page 5Linked to original sources
PubMed1981

Glutamine synthetase activity in the developing secondary palate and induction by dexamethasone.

Glutamine synthetase (EC 6.3.1.2) (GS) and glutamyltransferase (EC 2.3.2.1) (GT) specific activity were examined in developing A/Jax and C57BL/6J (C57) mouse fetal secondary palates. In addition, the induction of palatal GS was also examined after maternal injection of dexamethasone. Palatal GT activity was uniformly higher in A/J than C57 palates with both strains showing highest activity late on day 13 of gestation and a drop in activity by early day 14. In contrast, A/J palatal GS activity peaked transiently late on day 13, dropped by early day 14 and remained lower throughout the remaining period of palatal development. Palatal GS activity in C57 mouse fetuses, although failing to show a discrete transient peak of activity, remained at a constant elevated level from early day 13 to late day 14 and did not decrease until day 15 of gestation. These elevated levels of palatal GS and GT activity correspond to the gestation period of maximal palatal glycoconjugate biosynthesis. Thus, palatal GS activity may play an important regulatory role in the synthesis of these macromolecules. A/J and C57BL/6J mice exhibit different susceptibilities to glucocorticoid-induced cleft palate. However, maternal administration of a non-teratogenic dose of dexamethasone on either late day 12 or late day 13 resulted in a dramatic stimulation of both A/J and C57 fetal palatal GS but not GT activity when assay 18 h later. A/J palatal tissue responded to dexamethasone with greater induction of palatal GS activity than enzyme activity in C57 palates. Palatal GS, sensitive to glucocorticoid stimulation, may thus be an important link in expressing hormonal control of normal palatal differentiation.

Animals↗
PubMed1980

Collagen synthesis in cultures of differentiating neural crest cells.

Neural crest cells from the cranial region of chick embryos were cultured in the presence of fetal calf serum in which they differentiate into melanocytes or in the presence of horse serum in which they differentiate into neuron-like cells. Undifferentiated cultures, as well as pigmented and neuronal cultures, incorporated [14C]proline into types I and III collagen and into non-collagenous proteins. When cells differentiated into melanocytes, the ratio of collagen to non-collagenous protein did not change. In contrast, in cultures where a portion of the cells differentiated into neuron-like cells, the ratio of collagen to non-collagenous protein was reduced. Indirect immunofluorescence studies using specific antibodies against collagen or procollagen types I, II, III, and IV demonstrated that undifferentiated neural crest cells, melanocytes and neuron-like cells stained only with antibody to type III procollagen, while fibroblastic cells present in some cultures stained only with antibody to type I procollagen. Our results demonstrate that although the types of collagen synthesized by neural crest cells do not change with time in culture, the relative amounts of collagen may reflect the pathway of differentiation of the cells.

Animals↗
PubMed1980

Immunohistochemical localization of cyclic AMP in the developing rodent secondary palate.

During development of the mammalian secondary palate, medial-edge epithelia (MEE) from apposing palatal shelves adhere and undergo autolysis allowing palatal mesenchymal regions to unite. In a prior study (Greene & Pratt, 1979), we reported a transient increase in levels of cyclic AMP (cAMP) in the mouse and rat palate during epithelial adhesion and cell death. The objective of this study was to examine the distribution of cyclic AMP in the developing rodent secondary palate using immunohistochemistry to localize cyclic AMP. Staining for cAMP was observed in the epithelium just prior to and during epithelial fusion (day 16 in the rat; day 14 in the mouse). Cyclic AMP was distributed throughout the epithelial cytoplasm whereas no staining was seen in nuclei. Epithelial staining for cAMP was faint or absent 24 and 48 h prior to epithelial contact. Mesenchymal staining for cAMP was minimal and associated with the plasma membrane at all stages studied. These results demonstrate that elevated levels of cAMP in the rat and mouse palate during epithelial adhesion and cell death are mainly due to increases of the nucleotide in palatal epithelium. This observation suggests that a transient increase in epithelial cAMP may play a role in palatal epithelial differentiation.

Animals↗
PubMed1979

Calcification of differentiating skeletal mesenchyme in vitro.

Embryonic limb-bud mesenchyme was induced to calcify in culture by the addition of 3 mM inorganic phosphate to the medium. Phosphate enhanced calcification of the matrix produced by mesenchymal or fibroblast-like cells, whereas no calcification was evident in areas where cartilage had developed. However, calcification was induced throughout the cell layer by altering the cartilage matrix properties with certain enzymes or by changing the phenotypic expression of the cells with vitamin A.

Animals↗
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 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↗
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↗
PubMed1975

Some aspects of corticosteroid-induced cleft palate: a review.

Since the discovery 25 years ago that cortisone can produce cleft palate in mouse embryos investigations into possible mechanisms of this corticosteroid-induced defect have been many and varied. However, the teratogenic mode of action remains not fully clarified. It is with this thought in mind that we have reflected upon what is known concerning corticosteroids and cleft palate. The major metabolic pathways upon which glucocorticoids act as well as their intracellular mode of action are well known. Differential sensitivity of various mouse strains to cortisone treatment as well as recent results from interstrain blastocyst transfer experiments demonstrate that corticosteroid action is influenced by both the fetal and maternal genomes. Labeling experiments indicate that corticosteroid-induced cleft palate is the result of direct action of the steroid molecule on the fetus, whose own sensitivity to insult, perhaps owing to differences in binding of corticosteroids to tissue proteins, determines the final effect. Possible mechanisms that have been proposed by which corticoids may produce cleft palate include: disruption of glycosaminoglycan or collagen synthesis or both, intracellular lysosomal membrane stabilization, myopathy, weakened midline fusion, and loss of amniotic fluid. Also discussed is the role of stress and stress-induced corticosteroids and their possible role in the production of cleft palate.

Adrenal Cortex Hormones↗
PubMed1975

Limb development in mouse embryos: protection against teratogenic effects of 6-diazo-5-ox-L-norleucine (DON) in vivo and in vitro.

The glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON), has been shown to inhibit biosynthesis of purines and glycosaminoglycans, presumably by blocking the glutamine-dependent steps in the biosynthetic pathways. The teratogenic potential of DON on the developing mouse limb-bud in vivo and in vitro was studies in an attempt to discriminate whether DON is exerting its teratogenic effect by interfering with glycosaminoglycan or purine metabolism. A single intramuscular injection of DON (0-5 mg/kg) to ICR/DUB mice on day 10 of gestation resulted in 76% resorption, while fetuses surviving to day 17 exhibited growth retardation, median cleft lip, and limb malformation. Concurrent administration of L-glutamine (250 mg/kg) provided no protection against resorption of malformations, while 5-aminoimidazolecarboxamide (AIC, 250 mg/kg) decreased the resorption rate to 34% without significantly altering the incidence of malformations. Injection of DON alone on day 11 resulted in 87% of fetuses exhibiting limb formations, with only 2% resorption. Concurrent injection of AIC decreased the frequency of limb malformations to 32% L-Glutamine, D-glucosamine, or inosinic acid were without any protective effect in vivo. DON (5mug/ml medium) added in vitro to organ cultures of day 11 mouse limb-buds caused all limbs to evidence cartilage abnormalities. In this system, either L-glutamine ro D-glucosamine (0-5 mg/ml medium) provided protection against DON effects while AIC (0-5 mg/ml medium) offered no protection in vitro. These data suggest that DON exerts its effects in vivo by interfering with purine metabolism while in vitro its teratogenic action may be interruption of glycosaminoglycan biosynthesis. This may reflect upon the relative importance of growth and differentiation to limb development in vivo and in vitro. These data infer that limb development in vitro relies more on the differentiative process (differentiation of cartilage) than on growth, whereas limb development in vivo is dependent, at this stage, to a greater extent on growth for normal phenotypic expression.

Abnormalities, Drug-Induced↗