Translational regulation in growing clonal human astrocytoma cells in culture.

The in vivo and in vitro protein synthesis by polysomes prepared from Cox astrocytoma cells grown in the presence of 100 mM ethanol were examined during transition from exponential to stationary growth phase. A sharp decline of translational activities of Cox poly (A) + messenger RNAs (mRNAs) occurred during this transition. This decline was accentuated when cells were grown in the presence of ethanol. The observed decline in mRNA translational activity was investigated in vitro in a micrococcal nuclease treated, mRNA depleted postmitochondrial supernatant (PMS) fraction containing [35S]methionine. The formation of the 35S-labeled 40S ternary complex in the absence of mRNA and of the 35S-labeled 80S initiation complex in the presence of Cox or brain poly (A) + mRNAs were reduced substantially when the source of PMS was from stationary phase or ethanol exposed cells. The sedimentation of peaks containing 40S ternary and 80S initiation complexes following sucrose density gradient analysis showed marked reductions in [35S]methionine labeling during the transition to stationary phase and also following ethanol exposure. The reduced formation of initiation complexes suggests possible functional modifications of eukaryotic initiation factor-2 (eIF-2) present in the PMS fraction and of mRNAs under these conditions. Data suggest that cells initiate adaptive or protective mechanisms by reducing the rate of the initiation reaction following environmental alterations produced by ethanol.

Properties of cerebral polyribosomes and membranes of the rough endoplasmic reticulum after ethanol dependence in rats.

Effects of physical dependence upon ethanol on the polyribosomal properties and the reconstitution of the rough endoplasmic reticulum (RER) of the brain has been examined. The purified free polyribosomes (polysomes), membrane-bound polysomes, and a fraction of RER membrane that has been stripped of polysomes were isolated from rat brain. RNA yields, amino acid incorporation activities, and electron micrographs established the purity of stripped membranes, but no differences were detected following the ethanol treatment. For the polyribosomal fractions, the stability of the mRNA/ribosomal complex was decreased after ethanol dependence as was the in vitro translation of endogenous mRNA. In the reconstitution reaction, the incubation of membranes from ethanol-treated animals with either source of purified bound polysomes resulted in higher yields of protein than when control membranes were used. The above results suggest that ethanol dependence affects the properties of both the RNA and membrane components of the RER of brain.

Ethanol-induced changes in properties of rat brian ribosomes.

Altered in vivo and in vitro brain protein metabolism have been demonstrated in rodents following long-term ethanol ingestion. In the present study, ethanol effects were examined on properties of brain ribosomes of male Sprague-Dawley rats ingesting a specially formulated Lieber-DeCarli liquid diet. The development of physical dependence was demonstrated by the presence of withdrawal reactions within 24 hr of ethanol abstinence. Data showed significant inhibition of in vitro protein synthesis by ribosomes from the "ethanol" and "1-day-withdrawn" groups. Partial reversal of inhibition occurred by using a control brain pH 5 enzymes source instead of the matched source. The observed [14C]leucine-incorporating activity was temperature dependent, with the optimum temperature being 37 degrees C. The determination of the state of ribosomal aggregation showed an increased monosomes--disomes ratio in the "ethanol" group. The ratio was even more increased in the "1-day-withdrawn" group. Data suggest that reduced ribosomal binding to stable mRNA may be a contributing factor in the ethanol-induced effects on protein synthesis.