Louis M. Nicole

Thermotolerance and heat shock proteins induced by hyperthermia in rat liver cells

Hepatic epithelial cells become thermotolerant when conditioned with a 30 minute heat-treatment at 43 degrees C. The effect reaches a full amplitude after a 4-8 hour period at 37 degrees C and lasts for more than one day at a level corresponding to a 50-fold increase in cellular thermoresistance. During the development period, electrophoretic patterns of proteins from cells incubated in presence of 35S-methionine reveal an increased synthesis of a small set of proteins with molecular weights of 107, 89, 70, 68 and 27KD. The maximal synthesis of the induced proteins occurs concomitantly with the maximal increase of cell thermotolerance and has returned to normal when thermotolerance levels off. The induction of specific protein synthesis is also observed in other liver epithelial cells of normal and cancerous origins and in freshly isolated hepatocytes. It is suggested that the accumulation of these proteins in the cells plays a role in the process leading to a thermotolerant state.

On the Specificity of Antisense RNA to Arrest in vitro Translation of mRNA Coding for Drosophila hsp 23

The specificity of action of antisense RNA for one ofDrosophila low molecular weight heat shock proteins (hsp 23) was tested at the translational level using the rabbit reticulocyte lysate cell-free system. T7 polymerase-driven transcripts of hsp 23 in the antisense orientation were mixed with mRNA from heat-shocked cells under various stringency conditions prior to translationin vitro. Although the four small hsps show considerable sequence homology in their coding sequences, antisense hsp 23 RNA was shown to specifically inhibit hsp 23 mRNA translation under both high (formamide, 45°C and low stringency (37°C conditions. This suggests that the 5′ leader and the ribosome binding region of mRNA are of prime importance in the specificity of action of antisense RNA at the translational level.

Proteins of microdissected polytenic cells

Proteins extracted from unfixed and ethanol-acetic acid fixed salivary glands were compared by SDS-polyacrylamide gel electrophoresis. The electropherograms were nearly identical when care was taken to prevent degradation in the unfixed glands. Steady state proteins from microdissected nuclei and cytoplasms showed approximately the same number of species but displayed fundamentally different electrophoretic distributions. It was calculated that the maximum number of copies for individual polypeptide species ranged from 3.8×108 to 1.8×1010 in large polytene nuclei. A comparison of electropherograms from steady state nuclear proteins and nuclear proteins labeled with 3H-leucine after various periods of in vitro incubation of the glands, may suggest different turnover rates for individual protein species. An unexpected effect of incubation of the explanted salivary glands in a synthetic medium was observed. There are differences, on a quantitative level, between certain labeled proteins which accumulate in the nucleus at the beginning and at the end of a relatively long period of incubation.

Ribosomes of Physarum polycephalum. Subunits and protein composition.

Ribosomes from Physarum polycephalum were purified. Optimal conditions for preparation and stability of subunits were determined. KCl concentration above 200 mM induced protein dissociation from the subunits. It was observed that dissociated ribosomes were more stable in a low ionic strength buffer than in 200 mM KCl, where the 40 S was preferentially degraded by ribonucleases. Ribosomal proteins were analyzed by two-dimensional gel electrophoresis. The first dimension was carried out at pH 8.6 while the second was run at pH 4.6. The monosome contained sixty seven proteins, of which six were acidic. Two proteins were lost after subunit dissociation. Twenty six basic and two acidic proteins were observed in the 40 S subunit while the largest subunit gave thirty nine spots on the basic part of the gel and three additional spots on the acidic side. Five proteins were shared by 40 S and 60 S.