Protein purification, gene cloning and sequencing of an acidic endoprotease from Myxococcus xanthus DK101.

An acidic endoprotease (MAEP) secreted during vegetative growth by Myxococcus xanthus DK101 was purified to homogeneity by a series of chromatographic procedures. The endoprotease cleaved the Phe-Met bond of kappa-casein under acidic conditions (pH 5.9). Its apparent molecular mass and its isoelectric point have been estimated to be 12 kDa and 4.5, respectively. From the N-terminal amino acid sequence, a set of two primers for polymerase chain reaction have been designed. Amplification of the corresponding DNA fragment (84 bp) generated a probe, then used to screen an expression DNA library of M. xanthus and to isolate a recombinant plasmid which contained a 2127-bp insert. The nucleotide sequence included an open reading frame (ORF) of 585 nucleotides, encoding 195 amino acids, that exhibited a high degree of similarity with the N-terminal amino acid sequence of the purified MAEP. The polypeptide sequence inferred from this ORF revealed that the mature enzyme should contain 131 amino acids arising from a 195-amino-acid precursor protein.

Effects of retinoic acid on the transformation and metabolic cooperation of rat liver cells in vitro, and on the growth of hepatoma cells in vivo.

By different experimental approaches in culture, we obtained convergent responses to 13 cis-retinoic acid (RA) in rat liver epithelial cell lines. We showed that the degree of transformation of the cells which had already been spontaneously transformed in culture, could be enhanced, since the capacity of these cells both to grow in soft agar and to express gamma-glutamyl transpeptidase increased markedly. We also showed that RA acted synergistically with a promoter, 12-tetradecanoyl-phorbol-13-acetate (TPA), as regards the promoter's property of blocking the metabolic cooperation between cells. RA did not, however, trigger cell transformation in the lines which had not yet been transformed, and unlike TPA, did not by itself inhibit intercellular communications. On the other hand, in our in vivo experiments, it appeared that RA, by slowing down the growth of a transplanted rat hepatoma, might have a slightly protective effect against tumour development.

The effects of N-methyl-N'-nitro-N-nitrosoguanidine on KB cells. II. Cell sensitivity to the lethal effect of the drug as a function of the cell age and of the nature of the culture medium.

We have tested the sensitivity of KB cells to the lethal effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), measured as cell loss within the 20-h period following a 1-h drug treatment, as a function of culture age and of the medium in which treated cells were incubated after elimination of MNNG. We showed that KB cell sensitivity to the lethal effect of the drug decreased with time after seeding when the treated cells were post-incubated in drug-free medium conditioned by untreated cells of the same age as treated ones but not when they were post-incubated in fresh drug-free medium. This difference was due in part to the fact that the conditioned medium had acquired with time a protective activity for treated cells and in part to an increased competence of aging cells to be protected by this medium. By post-incubating treated stationary cells sequentially in both media, we showed that a brief (15 min) post-incubation of the cells in fresh medium was sufficient to trigger cell death even if the cells were afterwards transferred to conditioned medium. In contrast, long post-incubation in fresh medium did not cause cell death if the cells were first post-incubated in conditioned medium for about 3 h. We conclude that: the medium acted on cell sensitivity to the lethal effect of MNNG through its growth regulatory ability; quiescent cells were less sensitive to the drug than growing cells; the sensitive phase of the cell was located before S; cell hypersensitivity might be due to deficient repair of cellular lesions rather than to increased lesion formation.

The effects of N-methyl-N'-nitro-N-nitrosoguanidine on KB cells. I. Growth inhibition and cell killing in relation to inhibition of macromolecular synthesis.

Cytotoxic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on KB cells were analyzed taking into account cell killing and growth inhibition. Attempts to correlate these effects with macromolecular synthesis inhibition were monitored. At low doses (1 X 10(-6) at 1 X 10(-5) M), MNNG acting for 1 h only inhibited cell proliferation in a dose dependent manner and specifically inhibited DNA synthesis. At intermediate doses (1.6 X 10(-5) at 3.2 X 10(-5) M), treatment resulted in detachment and death of the cells during a 20-h post-incubation period. This effect was related to a dose-dependent inhibition of RNA and protein synthesis. At high doses (greater than 3.2 X 10(-5) M) MNNG treatment resulted in cell killing which was distinct from that produced by lower doses, since the cells did not detach from the glass. At these high doses strong inhibition of RNA and protein synthesis was observed early. When cells prelabelled with radioactive uridine or amino acids were treated with MNNG, the release of labelling into the culture medium increased, especially with uridine prelabelled cells. This increase was very high with the intermediate doses of the drug, but more modest with the strong doses. Furthermore, with the moderate doses, cell size showed greater reduction compared to the control than with the high doses. Thus, cell size reduction and loss of cellular material varied parallel to cell detachment. We hypothesize that moderate doses of MNNG may induce cellular degradation, whereas high doses may prevent it. The significance of this finding is discussed.

Hydroxyurea: protection of KB cells against the toxic effect of N-methyl-N'-nitro-N-nitrosoguanidine.

This report shows that hydroxyurea (HU) protected KB cells specifically against the toxic effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). It appears unlikely that this action was due to stimulation of DNA repair since HU neither protected the cells when added after removal of MNNG, nor shielded them against the toxicity of other DNA damaging agents. Similarly, it seems difficult to account for HU protection by its specific effect as an inhibitor of DNA synthesis, because cell protection was found to be maximal when the proliferative activity of the cultures was minimal, and because much higher doses of HU were required to ensure cell protection than to inhibit DNA synthesis. In fact, the shift of the MNNG dose-response curves in the presence of increasing HU concentrations suggests that HU might interfere with the MNNG-molecule. This was confirmed by our finding that HU enhanced the decomposition rate of MNNG, as did other substances like cysteine or ascorbic acid, which also appeared able to protect the cells against the toxic effect of MNNG.

Induction of cross-links between DNA and protein by PR toxin, a mycotoxin from Penicillium roqueforti.

PR toxin, a mycotoxin from Penicillium roqueforti, induces DNA--protein cross-links in chromatin of both cultured cells and isolated rat-liver nuclei. The presence of the aldehyde group in the PRT molecule is required for the induction of cross-linking; methylene bridges between nucleic acid and protein are presumably involved in the complex formation. The role of other functional groups of PR toxin is discussed.

Effects of PR toxin on liver cells in culture.

We studied the effects on liver cells in culture of PR toxin, a substance produced from Penicillium roqueforti. PR toxin displayed cytotoxicity which increased as a function of its concentration but the form of such toxicity differed, depending on the toxin's concentration. Thus, cells only underwent quick retraction and intensive vacuolization when treated with low drug concentrations, and they came away from the substrate easily under these conditions. By contrast, the major events observed in the case of high concentrations were loss of structure of the nuclei and strong adhesiveness of dead cells to the support. PR toxin already inhibited cell multiplication at low concentrations and became toxic when the concentration was raised; growth inhibition decreased but the toxic effect increased when cells passed from the exponential growth phase to a phase of slower growth. PR toxin inhibited tritiated precursor incorporation into DNA, RNA and proteins in a similar time and concentration-dependent manner. Inhibition of DNA synthesis persisted even after removal of the drug from the medium.

[Relationship between the growth rate of the liver of young rats and the inhibitory effect of extracts of their liver on the multiplication of cultured cells]. / Relation entre la vitesse de croissance du foie de jeunes Rats et laction inhibitrice d'extraits de leur foir sur la multiplication de cellules en cultures

We have shown that extracts of liver from young Rats are less active, than extracts of liver from adult Rats, in inhibiting the multiplication of cells in culture. This inhibitory activity is at a minimum in livers taken from 10 to 15 days old Rats, which corresponds to the time of maximum increase in weight of the liver. The existence of an inverse relationship between the inhibitory activity of these extracts and the state of proliferation of the liver suggests that the inhibitory substance contained in the liver extracts may act as a regulator of growth of the organ.