Epithelial-mesenchymal interactions: effects of a dental biomatrix on odontoblasts

The functional differentiation of odontoblasts requires specific interactions between these cells and the extracellular matrix. To further analyze these phenomena we studied the effects of a "dental papillae biomatrix" on isolated dental papillae cultured in vitro. The dental papillae biomatrix was extracted from EDTA-dissociated day-18 mouse dental papillae by homogenization, NaCl and enzymatic treatments, and deposited on Millipore filters. This biomatrix was studied by means of transmission electron microscopy and indirect immunofluorescence: it contained collagen fibrils, type IV collagen, fibronectin and laminin; cellular residues were also observed. The dental papillae were isolated by trypsin treatment of homologous tooth germs and cultured on uncoated (control) and coated filters. As shown by histological and cytological data, odontoblast-like cells never differentiated in control cultures. In presence of biomatrix and serum, polarized functional cells were observed. The functional state of these cells was enhanced by the addition of ascorbic acid to the culture media. Study of the incorporation of 3H-proline in cultured dental papillae and in macromolecules secreted into the culture media corroborated the morphological findings.     

Secretion of a major phosphorylated glycoprotein by hepatocytes. Characterization of specific antibodies and investigations of the processing, excretion kinetics, and phosphorylation

Isolated rat hepatocytes secreted a major phosphorylated glycoprotein (PP63) with apparent Mr = 63,000 and isoelectric point ranging from 4.8 to 5.3. Specific antibodies were raised in a rabbit using material obtained from plasma as an antigen. The biosynthesis of PP63 was studied in vitro in a cell-free system and in intact hepatocytes incubated with or without tunicamycin. The mRNA translation product had a Mr = 43,000 and was of the same size as the major unglycosylated precursor found in intact cells. This precursor was rapidly processed into two major intracellular forms of Mr = 53,000 and 56,000. These species were insensitive to neuraminidase but susceptible to endoglycosidase H, indicating that they contained oligosaccharide side chains of the high mannose-type. Terminal glycosylation gave rise to the mature Mr = 63,000 protein that contained sialic acid and fucose. This species represented the exportable form of the protein and was the only one to be phosphorylated. The charge heterogeneity observed for the mature protein already existed in all the precursors, indicating that it could not be ascribed to sialylation or to phosphorylation. However, these covalent modifications were mainly responsible for the acidic character of PP63. PP63 secretion was altered by tunicamycin. Pulse-chase experiments showed that the phosphorylated glycoprotein was secreted according to kinetics similar to that described for other liver glycoprotein, with slower kinetics than albumin. Permanent phosphorylation did not appear mandatory for excretion since the dephosphorylated PP63 was excreted with an efficacy comparable to that of the phosphorylated protein. Phosphorylation of PP63 was shown to occur on a single tryptic peptide, at a serine residue.     

Control of cell division in Escherichia coli. DNA sequence of dicA and of a second gene complementing mutation dicA1, dicC.

A mutation in a gene dicA of Escherichia coli leads to temperature-sensitive cell division, by allowing expression of a nearby division inhibition gene dicB (1). We have now established the sequence of the DicA region and identified DicA as a 15.5 KD protein. A second gene dicC transcribed divergently from dicA and coding for an 8.5 KD protein can also complement mutation dicA1 when provided on a multicopy plasmid.     

Bidirectional transport of glutamine across the cell membrane in rat liver.

Hepatocytes isolated from fed rats were used to investigate glutamine transport. Glutamine transport appears as a composite process involving at least two saturable components. The Na+-dependent component probably represents the entry through the N system. The Na+-independent component was also inhibited by histidine and exhibited trans-stimulation, suggestive of a facilitated diffusion process. Kinetic parameters for both systems suggest that facilitated diffusion only plays a minor role in glutamine influx. In contrast, the Km for glutamine efflux was consistent with a physiological role of the facilitated-diffusion component in glutamine release. In Na+ medium, relatively constant distribution ratios (about 8) between intra- and extra-cellular concentrations were observed, with external glutamine ranging from 0.5 to 5 mM. The present observations suggest that glutamine influx might largely be mediated by the N system, whereas facilitated diffusion allows hepatocytes to release glutamine when intracellular concentrations are elevated. The physiological consequences of this bidirectional transfer of glutamine across the liver cell membrane is discussed.     

Study of some enzyme activities in cultured chick embryo brain nerve cells treated by chick embryo brain extracts

Brain extracts from 8-day-old chick embryos have been shown to influence morphological development of dissociated brain cells from 7-day-old chick embryos in culture. Stimulatory, effects on size of the neuronal somas and on growth of long processes were observed by adding the cytosol of the brain extract or the dialysate of the cytosol. These morphological changes parallel modifications of various enzyme activities according to the age of the cultures. Adenyl cyclase, (Na⁺, K⁺)- and Mg²⁺-ATPase, 5-nucleotidase, choline acetyltransferase, and acetylcholinesterase activities were studied between 5 and 14 days of culture. Adenyl cyclase activity was strongly stimulated at 8 days by both extracts. (Na⁺, K⁺)-and Mg²⁺-ATPase activities were stimulated in 8-day-old cultures only by the dialysate. 5-Nucleotidase activity was stimulated in 8-day-old cultures by the dialysate and in 11-day-old cultures by both extracts. Choline acetyltransferase activity was stimulated by the cytosol in 8-day-old cultures and by the dialysate in 11-day-old cultures. The total acetylcholinesterase activity was higher in 8-, 11-, and 14-day-old cultures treated with the cytosol. When the cells were treated with the dialysate, the activity was only higher in 14-day-old cultures. We also found that following the addition of brain extracts, the specific activity of the enzymes we studied was enhanced and became close to the values found in vivo during embryogenesis. Thus in parallel to the morphological modifications observed in nerve cell cultures treated by embryo brain extracts, biochemical variations especially involved in synaptogenesis and membrane development could be measured.     

Ultrastructural and biochemical studies of isolated adult rat hepatocytes prepared under hypoxic conditions : Cryopreservation of hepatocytes

Isolated adult rat hepatocytes were prepared under hypoxic conditions, following a two-step procedure as described by Seglen. A simple recirculating apparatus was used and some modifications were made. Electron microscope studies revealed that the fine structure of the hepatocytes was not extensively damaged during cell preparation. Isolated hepatocytes prepared under hypoxic conditions are functional as demonstrated by their ability to carry out gluconeogenesis under normal and stimulated conditions, by the selectivity of the cell membrane towards various substrates and by the preservation of insulin receptors on the cell membrane. As previously observed for other cell types, survival of hepatocytes after freezing was dependent on the cooling rate. About 80% of cells appeared to survive when cooled at 7-2 °C/min; this apparent optimum cooling rate was also found suitable to preserve insulin-binding sites; on the other hand, the gluconeogenic capacity of hepatocytes frozen under these conditions was consistently decreased and the fine structure of organelles was damaged.     

Synthesis of the growth hormone-regulated rat liver anti-protease GHR-P63 is inhibited by acute inflammation

Synthesis of the growth hormone-regulated anti-protease GHR-P63 by rat hepatocytes was strongly reduced during acute inflammation. This decrease was detected 8 h after the onset of inflammation and reached a maximum after 24 h. A decrease in the GHR-P63 mRNA level measured by in vitro translation and by hybridization mainly accounted for the alteration of GHR-P63 synthesis. Besides this major pretranslational mechanism, inflammation also interfered with GHR-P63 synthesis at a posttranslational level. This was indicated by the production of abnormal immunoprecipitable species at early stages of the acute-phase response.