Altered hepatic and cerebral protein synthetic activity following diethylstilbestrol treatment.
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Biomedical subjects
Publications and source records attributed to S Tewari.
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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.
Decreased protein synthesis in a cell free system of brain has been reported for male adult rats following chronic ethanol ingestion. To assess the developmental and maturational changes occurring in the neonatal brain, the effects of pre- and postnatal maternal ingestion of ethanol were determined. For these studies young female rats were given a 10% w/v ethanol/water solution for varying periods after impregnation and seven days post pregnancy. Data showed that maternal ethanol ingestion produced a large deficit in the in vitro incorporation of (14C) leucine into the hot TCA extractable residue of ribosomes of neonatal brain. Maximum inhibition was obtained when ethanol was given postnatally. To determine the molecular sites of ethanol's action, ribosomes and pH 5 enzymes from the adult and neonatal brain were examined. Data showed that the highest activity was obtained with control neonatal brain enzymes and ribosomal fractions.
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.
Previous observations have demonstrated decreased in vivo and in vitro protein synthesis by brain ribosomal systems following long-term ethanol ingestion. For further investigation of the properties of brain ribosomes, the 40S and 60S ribosomal subunits were successfully isolated from control and chronic 10% ethanol-drinking rats. For a successful dissociation of ribosomes into subunits NH4Cl, puromycin and a high-salt treatment at 10 degrees C were essential with a critical concentration of Mg2+ since ribosomes could not be resolved at less than 7 mM Mg2+. Analysis of the A260 profile of the subunits on the sucrose gradients showed no significant differences between the control and ethanol-ingesting groups. Studies on 3H-labeled ribosomes following in vivo RNA labeling showed correspondence of the radioactive profiles from the incorporation of [5(-3) H) orotic acid into RNA with the sucrose gradient absorbance profile of 60S and 40S ribosomal subunits. Furthermore, active reassociation of both subunits occurred at 37 degrees C as demonstrated by the increased [14 C]-phenylalanine incorporation in the presence of poly(U). Results further showed that the poly(U)-dependent [14C]phenylalanine incorporation was significantly reduced by the subunits from the ethanol-ingesting animals. These findings suggest that long-term ingestion of ethanol caused functional changes in the properties of brain ribosomes, specifically on the reassociation process of the two subunits.
Recent laboratory endeavors have been primarily concerned with determining the effects of chronic ethanol ingestion on cerebral RNA metabolism within the inner mitochondrial membrane (IMM). Toward this goal, a series of experiments were carried out by determining the RNA and protein synthetic activity of the organelle. Brain mitochondria actively incorporated [5-3H]orotic acid into RNA under in vivo conditions. In addition, data showed that a major portion of the radioactivity present in the cold TCA insoluble residue from labeled intact mitochondria could be recovered in the digitonin treated purified IMM fraction. Maximum incorporation of [5-3H]orotic acid into RNA took place within 24 hrs followed by a rapid decline in activity on the 5th day of the pulse in both 'control' and 'ethanol' ingesting rats. Data revealed that ethanol ingestion affected the degree of incorporation of labeled RNA precursor into the mitochondrial RNA fraction. The observed effect was in the form of inhibition and was dependent on the duration of the initial pulse. Thus, while at earlier time points a decreased incorporation of the precursor into mitochondrial RNA was obtained in the ethanol imbibing rats, the inhibition was not observed when the pulse time was extended to 5 days. Such results indicate the possibility of a rapid turnover rate for the brain mitochondrial RNA with the 'ethanol' group having a faster rate of turnover when compared to the 'control' group.
Prior investigation of the protein synthesizing properties of mitochondria involved the whole organelle. In order to better characterize these properties, the present study was concerned more specifically with the activity of the inner mitochondrial membranes (IMM) which recent investigation has implicated as the primary location of mitochondrial ribosomes. To further define mitochondrial protein synthesis simultaneous experimentation was also conducted utilizing cytoplasmic ribosomes thus enabling both qualitative and quantitative comparison between the two systems. Results from this series of investigations reveal a dramatic amino acid incorporating ability by the IMM fraction of the brain mitochondria. This activity, in turn, was shown to be highly independent of exogenous sources of ATP, GTP, pH 5 enzymes, and cytoplasmic ribosomes. Furthermore, the addition of an exogenous source of messenger RNA, polyuridylic acid or (poly (U)) which resulted in an increased incorporation of [14C]phenylalanine into polypeptide in the cytoplasmic system was found to have no effect on the IMM system. Upon comparison of the in vitro protein synthesizing properties of the IMM fraction with those of the cytoplasmic ribosomal system, it became evident that obvious differences existed in the degree of amino acid incorporation and in the sensitivity of this process to the various protein synthesizing inhibitors. Cytoplasmic ribosomes demonstrated a much greater [14C]leucine and [14C]phenylalanine incorporating activity than the IMM fraction. In addition, RNase and cyclohexamide had their greatest effect on the cytoplasmic system while the action of chloramphenicol was most potent on the IMM system. Although puromycin inhibited both protein synthesizing systems, this effect was greatest in the presence of cytoplasmic ribosomes.
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The effects of ethanol withdrawal were determined on cell free brain protein synthesis in physically dependent rats. Following the development of physical dependence, ethanol abstinence for 24 h resulted in decreased protein synthesis in cerebral tissue. The observed inhibition of [14C]leucine incorporation into protein was found to be reversible after 7 days of ethanol withdrawal. Although the ribosomes from control, ethanol-treated and ethanol-withdrawn animals were highly responsive to polyuridylic acid stimulation, the ribosomes from the control group consistently exhibited higher activity. The determination of protein content of the ribosomal fraction showed a significant increase following ethanol administration and was further enhanced by ethanol abstinence. The results suggest that ethanol-induced changes at the ribosomal level may result in defective association of mRNA causing depression of brain protein synthesis.
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The effects of chronic ethanol intake by mice and rats have been determined on brain ribosomes. Under conditions of ethanol administration, when physical dependence on ethanol either does or does not develop in rats, significant inhibition of polypeptide synthesis was observed in comparison with animals not receiving ethanol. In both rats and mice drinking a 10% ethanol solution, in vitro protein synthesis was found to be diminished more in the free than membrane-bound polyribosomes (polysomes) when compared to similar fractions obtained from control animals. Addition of exogenous amino acids stimulated protein synthesis of free polysomes to a greater extent than that of bound polysomes obtained from both groups of micemin vivo incorporation of [5-3H]-orotic acid into RNA was more inhibited in polysomes than ribosomes of ethanol-drinking mice, suggesting that ethanol affects messenger RNA. In addition, chronic ethanol ingestion produced a decreased in vivo incorporation in ribosomal RNA of a mixed population of ribosomes and polysomes. Chronic ethanol ingestion by mice led also to a differential effect on the polysomal population of the brain. It decreased the amount and the incorporation of precursor label into RNA of free polysomes while exerting an opposite effect on bound polysomes. The significance of these findings relative to brain metabolism is discussed.
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