Effect of hydrogen peroxide on cytoskeletal proteins of Drosophila cells: comparison with heat shock and other stresses.

Hydrogen peroxide, which was shown to trigger the heat-shock response by activating the immediate binding of the heat-shock factor to DNA heat shock regulatory elements in the promoter of heat-shock genes of Drosophila cells, has also been reported to enhance the synthesis of actin. We show here that very short and transient H2O2 treatments, from 1 s to 2 min, are sufficient to induce an increase of actin synthesis. This increase becomes apparent 2 to 3 h after the short H2O2 treatment. It is inhibited if actinomycin D is present during the short H2O2 treatment. An increase of actin synthesis was also observed during the recovery period after two other stresses: reoxygenation after anoxia and ethanol treatment. The synthesis of two cytoskeletal proteins, tubulin and a 46-kDa insoluble protein of the intermediate filament fraction, was also slightly increased by H2O2 in Drosophila cells, but this increase was not actinomycin D-dependent. H2O2 does not provoke the translocation of the 46-kDa protein to the nuclear fraction as does heat shock. The very rapid stimulation of actin synthesis by H2O2 and the involvement of cytoskeletal elements in many stress situations suggest that actin may play a key role in the response to external stimuli.

The response of the centrosome to heat shock and related stresses in a Drosophila cell line.

The centrosome of Drosophila melanogaster cells cultured in vitro has been followed by immunofluorescence techniques with the Bx63 antibody of Frasch and Saumweber. After a heat shock, the centrosome labelling becomes very small and finally disappears after 30 min. Other heat-shock protein (hsp) inducers such as ethanol, arsenite and ecdysterone lead to the same disappearance. Moreover, the functional ability of centrosomes to nucleate microtubule assembly is inhibited by these treatments, particularly by heat shock, ethanol and ecdysterone. Two other hsp inducers, cadmium chloride and hydrogen peroxide, do not affect the centrosome seriously. With the exception of cadmium, the rapidity and the intensity of hsp induction are in good agreement with the kinetics of alteration of the organelle. We propose that a close link exists between the heat-shock response and the centrosome and that the physiological induction of hsps could be reinterpreted in terms of cell division control.

Early activation of heat shock genes in H2O2-treated Drosophila cells.

Drosophila cells of a diploid clone derived from line Kc were treated with 1 mM H2O2 for 1 to 20 minutes. Dot blot and Northern blot analysis of RNAs extracted from control and treated cells showed that the transcriptional activation of the 6 heat-shock genes tested was early, and maximal within 5 minutes of H2O2 treatment. Analysis of the kinetics of induction of the heat-shock proteins (hsps) after an exposure to H2O2 of 2 or 5 minutes, followed by removal, suggests that this brief treatment was sufficient to trigger the synthesis of all the hsps, which was maximal 1.5 to 3h after this short H2O2 treatment.

[Isolation by binding to ultrafiltration membranes of a testicular factor limiting the number of germ cells in fetal ovary of rats in vitro]. / Isolement par liaison à des membranes d'ultrafiltration, d'un facteur testiculaire limitant le nombre de cellules germinales dans l'ovaire foetal de rat in vitro.

The medium used for culturing in vitro fetal or neonatal testes, when used subsequently to culture for 4 days ovaries from 13.5 day old rat fetuses, has the property of severely limiting the number of ovarian germ cells. The non-dialysable factor(s) responsible for the observed effect binds to ultrafiltration membranes (Diaflo, Amicon) and can be eluted from these membranes with fresh medium added 1 M NaCl.

Effect of serum on organogenesis of the rat testis in vitro.

It was observed previously that primordia of fetal rat testes when explanted in vitro in a synthetic medium at the outset of sexual differentiation differentiate seminiferous cords during the following days, but that the addition of 15% fetal bovine serum prevents this morphogenesis. In the present study, human, horse, bovine calf, and rat sera were shown to exert the same effect. Very low concentrations of human or fetal bovine serum (0.5 or 1%) were sufficient to produce the serum effect, which was only slightly reduced when the serum was heated. The serum activity was not removed by dialysis (membrane cut-off 15 000), but it disappeared after treatment with trichloroacetic or perchloric acids or after trypsin digestion. Partial purification of the active factor(s) from human serum was achieved by successive gel filtration, affinity chromatography, and ion exchange chromatography. Analysis of the active fractions by electrofocusing and immunoelectrophoresis placed the activity within the alpha globulin group. Among a series of purified serum proteins tested, alpha 2-HS-glycoprotein was found to exhibit the serum effect, though this activity was heat labile.