An auxin-auxotrophic mutant of Nicotiana plumbaginifolia

Summary Auxin (indole-3-acetic acid) is considered to be an important signalling molecule in the regulation of plant growth and development but neither auxin synthesis nor its mode of action is clearly understood. To identify genes involved in these processes, mutations were sought that altered the auxin requirement of plant tissues for growth. For the first time mutant plants were obtained that carry a recessive mutation at a single nuclear locus (auxl) which results in an absolute requirement for exogenous auxin for normal growth. In the absence of auxin treatment, mutant plants undergo premature senescence and die.Abbreviations BAP 6-benzylaminopurine - BUdR 5-bromodeoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - FUdR 5-fluorodeoxyuridine - IAA-EE indole-3-acetic acid ethyl ester - IMS indole-3-methanesulfonic acid     

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.     

Trichinosis: Thiabendazole in the Treatment of 11 Cases

We have studied 11 cases of acute human trichinosis; five of these patients were treated with thiabendazole. We have reviewed the laboratory data for these patients and described their course. This study has failed to show any definite effect of thiabendazole on the course of human trichinosis at the doses used. However, thiabendazole may have decreased the infectivity of the larvae which were embedded in the muscles of our patients.     

Influence ofl-thyroxine,l-triiodothyronine,thyroid stimulating hormone,or estradiol on the cell kinetics of cultured mammary cancer cells

Summary We describe the in vitro influence of 3,5,3′-triiodo-l-thyronine (T₃),l-thyroxine (T₄), a thyroid-stimulating hormone (TSH), and/or estradiol (E₂: chosen as the control of the methodology) on the cell kinetics (cell distribution in the S+G₂+M phases) of mouse MXT and human MCF-7 mammary cancer cells. Experiments were performed by means of a cell image processor, analyzing MCF-7 or MXT cells that had been grown on glass cover slips and whose nuclei had been stained by the Feulgen reaction, which is selective and quantitative (stoichiometric) with respect to DNA. We show that T₃, T₄, and TSH at 0.01 μM dramatically stimulate the cell kinetics of the MXT mouse and the MCF-7 human mammary cancer cell lines. Indeed, the three hormones bring about a significant transient increase in the S+G₂+M fraction as does E₂. Furthermore, our data indicate that E₂ and TSH are antagonistic with regards to MXT or MCF-7 cell kinetics. This work is supported by grants awarded by the IRSIA and the Fonds de la Recherche Scientifique Médicale (FRSM, Belgium).     

Analysis of phytochrome- and ABA-deficient mutants suggests that ABA degradation is controlled by light in Nicotiana plumbaginifolia

The phytochrome chromophore-deficient mutant, pew1, of Nicotiana plumbaginifolia exhibited decreased germination and slower dehydration of detached leaves during water stress as compared with the wild-type. These physiological processes are controlled by abscisic acid (ABA) and we examined, therefore, whether phytochrome plays a specific role in the regulation of ABA metabolism using the pew1 mutant. The ABA contents of mature seeds and young leaves were analysed and in both cases mutant material was found to contain higher amounts of ABA as compared with the wild-type. This indicates that the phytochrome activation can lead to a decrease of the ABA level in the wild-type plant. The role of phytochromes was investigated in greater detail using the ABA-deficient mutant aba1 of N. plumbaginifolia exhibiting an early and synchronous germination. This mutant accumulates at very high levels a metabolite derived from a precursor (ABA-aldehyde) in the ABA biosynthetic pathway. The first biochemical characterization of this molecule, which corresponds to the glucose-conjugated ABA-alcohol (ABA-AG) is described. A pew1-aba1 double mutant exhibiting both an etiolated growth and early germination was also obtained. The comparable accumulation of ABA-AG in the pew1-aba1 double mutant as compared with the aba1 mutant allowed the proposition that, in a wild-type plant, the phytochrome-mediated light signal enhances ABA degradation rather than inhibits its biosynthesis.