José Imbenotte

Persistance d'une synthèse de D-RNA dans le foie de rat traité par l'actinomycine

Abstract Actinomycin D-treated rats were injected with 32 P-labelled orthophosphate, 3 h later the animals were sacrificed and both RNA and DNA were extracted from their livers using phenol plus 0.5% or 1.0% sodium dodecylsulfate. RNA was fractionated by centrifugation in sucrose gradients. Separation of ribosomal and DNA-like RNA from s-RNA was achieved using 1.5 M NaCl or 0.2 M MgCl 2 . Complete inhibition of the synthesis of ribosomal RNA and s-RNA was found in the livers of actinomycin-treated rats, since, whether or not the RNA was treated with snake-venom phosphodiesterase (EC 3.14.1) before analysing the labelled 2′,3′-nucleotides, residual labelling of s-RNA could be attributed exclusively to the end groups pC-pC-pA. Synthesis of another fraction of RNA (8–10 S) was only partially inhibited (50–70%) by actinomycin. The (A+U)/(C+G) ratio of this fraction was identical to the (A+T)/(C+G) ratio of DNA, but generally the proportion of A was much larger (32–33%) than the proportion of U (20–22%). This DNA-like RNA could be recovered in a form closely associated with DNA itself. A similar DNA-like RNA was found in normal rat liver. These findings suggest that synthesis of all cellular RNA fractions is DNA dependent, but synthesis of DNA-like RNA is much less sensitive to actinomycin than synthesis of ribosomal and s-RNA. These facts pose certain questions about the manner in which ‘messenger’ RNA is replicated in mammalian cells.

FRACTIONS WITH DIFFERING BASE COMPOSITION IN RNA FROM MALIGNANT CELLS OF MOUSE.

RNA labeled with 32 P was extracted from mouse ascites tumor cells, and centri-fuged in a linear sucrose gradient. After a pulse of 30 minutes most of the label was in a “heavy” (>30 s) and a “light” (4 to 10 s) fractions. The base composition of the fractions differed depending on whether or not sodium dodecyl-sulfate was included in the phenol extraction procedure. Without sodium dodecylsulfate the radioactivity of the “light” fraction predominated. This fraction contained a high proportion of cytosine which disappeared after treatment with phosphodiesterase. After the enzymic treatment, the base-ratios resembled those of transfer RNA. The “heavy” fraction differed from ribosomal RNA by a low proportion of adenine and a high proportion of uracil. More radioactivity was recovered after a short pulse when sodium dodecylsulfate and phenol were used. The “heavy” fraction differed from ribosomal RNA (28 8) only by slightly higher adenine and uracil contents. A DNA-like RNA was separated from transfer RNA contained in the light fraction (4 to 10 s). DNA-like RNA was also directly separated from DNA fibers. After labeling for 4·5 to 7 hr RNA was uniformly labeled, but there were still differences in the base ratios of the fractions. Ribosomal 26 to 28 s RNA had lower adenine and uracil contents than ribosomal 16 to 18 s RNA.

Stimulation by TGFβ of chick embryo fibroblasts—Inhibition by an IGFBP-3

The multiple effects of TGF beta on cell proliferation are not well understood. Our results show that TGF beta was a good but transient mitogen for chick embryo fibroblasts. DNA synthesis was three- to fourfold increased, even at high concentrations of TGF beta. We did not show a bimodal effect. An inhibitor of cell growth, that inhibits 100% of stimulation induced by serum in CEF, was purified to homogeneity from medium conditioned by mouse 3T3 cells. This inhibitor has been shown to be an IGF-binding protein (mIGFBP-3). In the present work, this mIGFBP-3 inhibited the TGF beta stimulation by about 50%, while the stimulation induced by PDGF or insulin was not inhibited by mIGFBP-3. Furthermore, TGF beta stimulation, in the presence of a high concentration of insulin in conditions which would saturate IGF receptors, was not significantly inhibited by mIGFBP-3. All together these results suggest that a part of the mitogenic effect of TGF beta may be through increasing IGF secretion and eventually other growth factors such as PDGF (as suggested previously).

Nature of the iron requirement for chick embryo cells cultured in the presence of horse serum

Chick or bovine transferrin induces proliferation of chick embryo cells cultured in the presence of horse serum, which cannot itself assure their multiplication. Cell growth can also be induced by iron salts and iron complexes such as hemoglobin or hemin, but also by biliverdin which has no iron atom in its molecule.

Comparative effects of TSH and of insulin on fetal rat thyroid gland maintained in organ culture.

Summary In organ culture of fetal rat thyroid glands, both insulin and TSH enhance significantly the 24 hr incorporation of 125I into protein-bound iodotyrosines and iodothyronines; however, TSH is much more active than insulin. Under the same experimental conditions, although insulin is not so effective as TSH, these two hormones stimulate, in a similar fashion, the iodination of soluble proteins. Both TSH and insulin enhance also the biosynthesis of ribosomal RNAs and of nuclear DNA-like RNAs. The technical assistance of Mrs. Jennequin-Gerbod is gratefully acknowledged.

Stimulatory effects of protein synthesis inhibitors on the spreading rate of 3T3 cells

Three protein synthesis inhibitors, puromycin, cycloheximide and anisomycin were found to increase the rate of spreading of Swiss-3T3 cells after 1 h incubation in the presence of low concentrations of serum. Data from experiments with anisomycin suggest that this effect is roughly proportional to the extent of inhibition of protein synthesis.

Stimulatory and inhibitory effects of serum and conditioned medium on cell spreading

This work deals with the effects of coating culture dishes with chick embryo fibroblast conditioned medium (CM) and 1% fetal calf serum (FCS) on the rates of attachment and spreading of chick embryo fibroblasts. It appeared that a FCS coating over a CM precoating exerted a remarkable promoting cooperative effect on cell spreading whereas a CM coating over a FCS precoating had a very marked inhibitory effect. Precoating with cobalt-protoporphyrin IX (CoPP) was also performed to serve as a substrate for FCS or CM coating. This CoPP precoating was found to exert a promoting effect for FCS coating only.

The inhibitory effect of serum on cell attachment can be prevented by cobalt-protoporphyrin IX

The rate of attachment of chick embryo fibroblasts (CEF) incubated for 1 h at pH 6 in the absence of serum, was equal to about 90% with 2% of spreading cells. The addition of 1% horse serum (HS) decreased to about 30% the rate of attached cells but increased to about 30% the proportion of spreading cells. We found that 3 microM cobalt-protoporphyrin IX (CoPP) added for 3 min before the addition of 1% HS increased both the rate of cell attachment (about 60%) and the proportion of cell spreading (about 50%).

Infection par le virus de la myéloblastose aviaire, biosynthèse de RNA lourd polysomal rapidement marqué et différenciations cellulaires

Summary Secundary cultures of normal chick embryos were treated with the incubation medium of avian leukemic cells (AMV transformed myeloblasts kept at 4°C for 1 h or 24 h in Eagles medium) which in all cases contains numerous virions bound to macromolecular complexes (apparently deoxyribonucleoproteins). On the first day the treatment very markedly increases the radioactivity of pulse labelled RNA ( 3 H Uridine, 15 min) associated with the polysomes with a peak of radioactivity between 30 and 40S. This increase (up to 16 times the level of control cultures) is not due to stimulation of the cellular uptake of uridine. It cannot be attributed to heavy molecular weight RNA or ribonucleoproteins of nuclear origin, as demonstrated by zonal centrifugation of polysomes after treating with EDTA, or equilibrium sedimentation in Cs Cl gradient after formaldehyde fixation. The early stimulation of polysomal RNA synthesis is followed by abundant virus production on the 2 d day and cell differentiation, both muscular and epithelial, start appearing on the 3 d day. The early stimulation of RNA biosynthesis may, to a great extent be dissociated from both the later virus production or cell differentiations. The supernate of the 1 h incubation medium made virus free by filtering through Millipore membranes, retains the capacity to induce cell differentiation, without virus production, although the effect on polysomal RNA synthesis is small or negligible. Besides in embryonic cells infected by purified AMV, no marked effect is observed on the labelling of polysomal RNA, but virus is subsequently produced in the absence of noticeable cell differentiation. In contrast a mixture of purified virus and 55 000 g supernate of the 1 h incubation medium has the same enhancing effect on RNA synthesis, as the total incubation medium of leukemic cells. Finally no similar effect was observed in serially passaged cells consisting of fibroblasts. Different interpretations of these results are discussed.