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A cell-free protein-synthesizing system active in initiation of translation of both endogenous mRNA and exogenous mRNA has been obtained from postmitochondrial supernatant (S-12) of the liver of ethionine-treated rats by adding reticulocyte ribosomal extract as a source of initiation factor. Formation of polysomes in the course of protein synthesis in vitro has also been demonstrated. Homogenization of the liver in the presence of 50 microM hemin stabilizes the initiation activity of S-12 fraction, which otherwise decays rapidly even at 0 degrees C. The mechanism of inhibition of protein synthesis by ethionine is discussed in view of these results.

The effect of ethionine, an amino acid analog of methionine, has been studied in Saccharomyces cerevisiae in relation to cell growth, oxygen consumption, in vitro protein synthesis of mitochondrial translation products (MTPs) and the degradation of those mitoribosomally made proteins by an ATP-dependent process present within the organelle. Ethionine was found to increase the generation time of those cells already committed to cell division and to abolish the initiation of new cell cycles. Oxygen consumption of cultures grown in the presence of the analog was drastically reduced. Ethionine was also found to impair the incorporation of methionine and leucine into mitochondrial translation products, however the synthesis of proteins was not totally blocked and, apparently, mitochondria utilized ethionine as a precursor amino acid. MTPs synthesized by isolated mitochondria in the presence of ethionine were rapidly degraded inside the organelle at a faster rate compared with the normal proteins synthesized under identical conditions in the mitochondria. It is also shown that these in vitro synthesized proteins are degraded by an ATP-stimulated proteolytic system, as has been previously established.

S-Adenosyl methionine (AdoMet) and several structurally related compounds were added to in vitro systems for the synthesis of single-stranded RNA by cytoplasmic polyhedrosis virus (CPV) types 1 and 2. The effects of these compounds were examined on the level of transcription and methylation of the RNA products. Of the compounds tested, five increased the polymerase activity in both viruses, the most effective being the D- and L-stereoisomers of S-adenosyl homocysteine (AdoHcy), and the least effective, adenosine. L-AdoHcy, unlike D-AdoHcy, was also a competitive inhibitor of RNA methylation in the presence of [3H]AdoMet. The different response of both viruses to D- and L-AdoHcy suggests that CPV virions contain at least two functionally distinct sites to which AdoMet, or its analogues, bind. One of these is the transcription control site, while the other is the active site(s) for RNA methylation. CPV RNA synthesised in the presence of the methyl donor AdoMet was more efficiently translated in vitro in a wheat-germ translation system than RNA synthesised in the presence of methylation inhibitors. Type 2 CPV-RNA transcripts had a greater degree of methylation than type 1 CPV transcripts and were more effective in stimulating protein synthesis in the translation system. It seems likely that the allosteric control of CPV polymerase by AdoMet and its analogues, and the methylation of the transcripts, ensures the effective transcription and translation of the CPV genome and the stability of the viral messenger RNA.

The activity of poly(adenosine diphosphoribose) polymerase was measured in isolated rat pancreatic nuclei and was found to increase during pancreatic nuclei which follows an ethionine treatment, although a possible relationship of enzyme activity to the initial degenerative phase may also be considered. There is a 2-fold increase in the enzyme activity during the destruction process which remains high during the regeneration period. This increase of activity observed during regeneration is not related to a decrease of the polymer degradation. We have studied the adenosine diphosphate ribosylation of proteins during pancreatic regeneration, and we have found increases in the level of adenosine diphosphate ribosylations of these proteins just before regeneration and during the regeneration period. The in vivo adenosine diphosphate ribosylation of nuclear proteins does not correlate with synthetase activity measured in nuclei during the degeneration period but does correlate during the regeneration period and thereafter with the relative amount of enzymatic activity found in nuclei. Furthermore, as verified by autoradiography, labeling of the nuclei by polyadenosine diphosphoribose polymer shows a marked increase during regeneration.

The purpose of the present study was to elucidate the metabolic requirements of autophagocytosis. Two model systems were used for this purpose: a) glucagon-induced autophagocytosis in the rat liver, and b) the wave of autophagocytosis which occurs when isolated flounder kidney tubules are incubated in vitro. In the rat liver, protein synthesis was inhibited by the administration of cycloheximide (1.5 mg/kg) to rats 2 hours prior to glucagon injection. In flounder kidney tubules, protein synthesis was inhibited at least 90% by adding cycloheximide, actinomycin D, pactamycin and puromycin to the medium. In both systems the inhibition of protein synthesis failed to inhibit the formation of autophagic vacuoles or their subsequent transformation into autolysosomes, as depicted from electron microscopic histochemical preparations. In flounder kidney tubules no differences were found in the levels of p-nitrophenylphosphates, beta-DL-glycerophosphatase, N-acetyl-beta-D-glucosaminidase, arylsulphatase, beta-D-galactosidase or acid proteinase when tubules were incubated up to 5 hours in the presence or absence of protein synthesis inhibitors. When ethionine was administered to rats 2 hours before glucagon injection, a decrease of approximately 75% in the ATP levels was observed. After ethionine administration, glucagon failed to induce the formation of autophagic vacuoles. The incubation of flounder kidney tubules in the presence of cyanide or in a nitrogen atmosphere decreased the ATP levels to less than 10% of controls and blocked autophagy. On the other hand, cyanide had little effect on acid hydrolase levels at 1 hour of incubation. A wide variety of other inhibitors were also shown to block autophagy. These results further support the hypothesis that, in the formation of antophagic vacuoles, preexisting enzyme and membrane pools are utilized. On the other hand, the esotropy-exotropy membrane conformational changes occurring in the formation of autophagic vacuoles seem to be energy dependent and can therefore be blocked by lowering intracellular ATP levels.

The acute biochemical effects of ethionine have been well studied in rats but not in mice. These results show that both hepatic RNA and protein synthesis in Swiss mice were inhibited by DL-ethionine. Protein synthesis was inhibited 30 to 40 percent 3 hr after 2500 mg/kg DL-ethionine while RNA synthesis was inhibited 80 to 90 percent 3 hr after 625 mg/kg DL-ethionine. Thus ethionine was a far more potent inhibitor of RNA synthesis than of protein synthesis. There was no sex difference in either of these responses. While both stereoisomers were active, the L isomer was a more potent inhibitor of RNA synthesis than was the D isomer. In male mice, 3 hr after 20 mg/kg L-ethionine, RNA synthesis was inhibited 80%, while after D-ethionine it was inhibited only 51%.

An hypothesis of carcinogenesis is derived which suggests that carcinogens initially induce derepressions of structional genes of post-translational modifying enzymes. Although the resulting cascade of changes in genic activity would be measureable because of the large number of differentiated cells present in a tissue, only stem cells are vulnerable to the neoplastic array of genic expression by virtue of their repressors being more vulnerable to conformational modification. Furthermore it is derived that there are at least three major classes of repressors and the repressors that are synthesized during embryogenesis are particularly susceptible to derepression mechanisms.

2--4 mM L-ethionine completely inhibits DNA synthesis in phytohaemagglutinin- or concanavalin A-stimulated lymphocytes even though it does not prevent the morphological changes characteristic of blast formation. Evidence is presented which indicates that complete commitment to DNA synthesis as well as a substantial increase in the rates of RNA and protein synthesis can occur in the presence of ethionine. Ethionine, however, does inhibit methylation of tRNA and prevents mitogen-induced increase in the activity of histone-modifying enzymes. All of these effects of exposure to ethionine are completely reversible. Removal of ethionine after 24 h or more of exposure results in a rapid, synchronous wave of DNA synthesis, an increase in the rate of methylation of RNA and an increase in activity of histone-modifying enzymes.

The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete cell-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsCl isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [14C]- or [3H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 subunits by SDS-polyacrylamide slab gel electrophoresis revealed that eIF-3 subunits and two unidentified protein fractions of molecular weight of 2.3.10(4) and 2.1.10(4) were decreased in ethionine-treated rate liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with N-[3H]ethylmaleimide or N-[14C]ethylmaleimide, and the 3H to 14C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially eIF-3, as the results of conformational changes of 40 S subunits.

The effect of pregnenolone-16 alpha-carbonitrile (PCN) on female Sprague-Dawley rats was studied in conjunction with ethionine and beta-diethylaminoethyl diphenylpropylacetate (DADP). The cyanosteroid virtually abolished in inhibitory action of ethionine on orotic acid and leucine uptake by liver microsomes. When PCN was given conjointly with DADP, it completely overshadowed the latter's effect on leucine uptake. DADP was found to act as an inhibitor of the translational mechanism 5 h after its administration, while it exerted a stimulatory influence on the transcriptional mechanism after the same time period.

The soluble fraction from rat liver contains an inhibitor of protein chain initiation when tested in a cell-free protein-synthesizing system derived from rabbit reticulocytes. The administration of ethionine to rats increased the inhibitory activity in the liver. This liver inhibitor displayed properties similar to those of hemin-controlled inhibitor found in rabbit reticulocytes: (i) the liver inhibitor inhibited protein chain initiation in rabbit reticulocyte lysate with characteristic biphasic kinetics; (ii) the liver inhibitor disaggregated the reticulocyte polysomes with a concomitant increase in 80 S ribosomes; (iii) the inhibition was prevented or reversed by eIF-2. The activation of the liver inhibitor by ethionine was rapidly and completely counteracted by the subsequent administration of methionine and adenine to rats. The mechanism of inhibition of protein synthesis by ethionine was discussed in the light of these findings.

Treatment of rats with ethionine was found to cause severe impairment in the aminoacylation capacity of tRNA. This effect was only observed when assayed in injected oocytes, while invitro assays of aminoacylation failed to detect differences between normal tRNA and tRNA from ethionine treated animals. The effect of ethionine on the tRNA population was not uniform and differed for various amino acid specific tRNAs. Thus liver tRNA from ethionine treated rats showed a decreased capacity for phenylalanine aminoacylation, while no change was found in the case of leucine. On the other hand, the level of histidine aminoacylation was higher for tRNA from ethionine treated animals. An even more complex response was observed with methionine aminoacylation where tRNA from ethionine treated animals showed an initially faster rate than control tRNA. With more prolonged incubation periods, the methionyl-tRNA from ethionine treated animals was deacylated at an accelerated rate while the level of normal methionyl-tRNA remained almost constant. In addition to the aminoacylation reaction, the participation of aminoacyl-tRNA in protein synthesis was severely impaired. In this case, both the injected oocyte system and the cell-free wheat germ assay revealed these differences which were manifested with various mRNA and viral RNA preparations.

This report identifies L-ethionine as an inducer of differentiation in murine erythroleukemia cells. When Friend erythroleukemia cells are grown in the presence of 4mM L-ethionine, globin mRNA accumulates and in 4-5 days, 25-30% of the cells in the culture contain hemoglobin. Incubation of the cells with bromodeoxyuridine prevents both ethionine-induced accumulation of globin mRNA and erythroide differentiation. At the concentration where L-ethionine acts as an inducer of FL cell differentiation it inhibits methylation of DNA and tRNA in vivo but does not prevent macromolecular synthesis or cell division. To establish whether a link existed between inhibition of a specific methyltransferase and activation of globin synthesis in FL cells, we examined the degree of hypomethylation of DNA and tRNA from FL cells induced to differentiate with dimethylsulfoxide and butyrate. In contrast to the tRNA from ethionine-treated cells, tRNA from cells induced by butyrate or Me2SO cannot be methylated in vitro using homologous enzymes. DNA isolated from cells exposed to any of the three inducers, however, was significantly hypomethylated when compared with DNA from uninduced cells. These data suggest that methylation of DNA may play a role in the regulation of gene expression.

Animals treated with AgNO3 and amyloid-enhancing factor deposit large quantities of splenic amyloid in 48 hours. The kinetics of SAA production was examined in such a model in the presence and absence of an inhibitor of liver protein synthesis (ethionine). Ethionine had little effect on splenic protein synthesis but inhibited both the production of SAA and liver protein synthesis. When experiments were performed with fed mice, ethionine induced an inhibition of liver protein synthesis lasting 16 hours. Thereafter, liver protein synthesis began to recover, accompanied by a rise in plasma SAA levels. These results are consistent with hepatic but not splenic synthesis of SAA.

One of the earliest events following the administration of estradiol to ovariectomized rats is an increase in transfer RNA (tRNA) methyltransferase activity activity of the uterus. Ovariectomy diminishes by about one-half the capacity of extracts of uteri to introduce methyl groups in vitro into heterologous tRNA. Administration of estradiol to ovariectomized animals restores the tRNA methyltransferase capacity to normal. Alterations in the population of uterine serine tRNA were also observed following ovariectomy. These findings represent reversible qualitative changes produced by a hormone in the target tissue. Whether the tRNA's are involved in regulating hormone-induced protein synthesis was investigated by studying the estrogen-induced ovalbumin synthesis in immature chick oviducts. tRNA's from oviducts of estrogen-stimulated chicks or from oviducts of laying hens produced an enhanced stimulation of ovalbumin synthesis in vitro compared with tRNA's from oviducts of immature chicks that were withdrawn from estrogen stimulation (these chicks do not synthesize ovalbumin). Some of these hormone-induced alterations may be of importance in understanding the molecular mechanisms of estrogen action on gene expression. During these studies, we have observed a novel effect of the administration of the carcinogen, ethionine, in the immature chicks. Ethionine induces ovalbumin and conalbumin synthesis in immature chick oviducts that were withdrawn from estrogen for 3 to 4 weeks following primary estrogen stimulation.

Two new mutants are described which are temperature sensitive in protein synthesis. The mutants were obtained by a screening procedure using methionine analogues. The method is based on two findings: a) that in E. coli, and in other members of the Enterobacteriaceae, sensitivity to methionine analogues increases with the growth temperature, and b) that cells which are not synthesizing proteins during treatment with methionine analogues have a shorter lag in resuming growth subsequent to removal of the analogue.

Although the infection of BHK cells with foot-and-mouth disease virus did not cause a marked inhibition of cellular protein synthesis, the proteins synthesized gradually changed from host-specific to virus-specific. The synthesis of at least thirteen virus-induced proteins was demonstrated by polyacrylamide electrophoretic analysis of the infected cells. Only a small proportion of the virus-induced proteins was incorporated into the mature virus particles. The addition of amino acid analogues caused an accumulation of the larger mol. wt. proteins, suggesting that in infected cells, some cleavages occurred, giving rise to the smaller mol. wt. proteins. The operation of the precursor-cleavage mechanism was more clearly shown in pulse-chase experiments, in which it was found that the proteins were degraded from larger to smaller mol. wt.

Pretreatment of rats with cycloheximide protects the liver against ribosome changes but not against alterations in the endoplasmic reticulum membrane induced by the administration of CCl(4), as observed both biochemically and ultrastructurally. Another inhibitor of protein synthesis, tenuazonic acid, has effects similar to cycloheximide. Neither compound has any apparent effect when given after the administration of CCl(4) at a time at which a major shift in ribosome distribution from polysomes to ribosome monomers or subunits has already occurred. Thus, it appears that the ribosomes are not susceptible to damage by CCl(4), or more probably an active metabolic derivative of CCl(4), when they are organized as polysomes but only when they are present as monomers or subunits. Also, unlike the situation with ethionine or puromycin, the damage to the protein synthetic system of the ribosomes induced in the liver by CCl(4) appears to be irreversible. In contrast to cycloheximide or tenuazonic acid, N,N'-diphenyl-p-phenylenediamine (DPPD) protects the liver against CCl(4)-induced changes in both ribosomes and endoplasmic reticulum. These observations suggest that the changes in the ribosome-protein synthetic system induced in the liver by treatment with CCl(4) are not the direct result of damage to the membranes of the endoplasmic reticulum but are either an indirect effect of such damage or are unrelated to it. These possibilities are discussed in terms of three proposed working hypotheses.

Protein metabolism was studied in the regenerating rat pancreas acinar cell by (3)H-leucine autoradiography. Rats were placed for 10 days on a protein-free ethionine (PFE) regimen which caused necrosis of most of the acinar cells. Withdrawing the PFE regimen and instituting a stock diet (SD) on day 11 was followed by good restitution of acinar cell morphology. DNA synthesis peaked at day 15. Intraperitoneal injection of (3)H-leucine and sacrifice at 5, 10 and 30 minutes and at 1, 4 and 24 hours after injection at PFE-SD days 10, 12, 15 and 29 and at the same time periods in control SD animals provided tissue for autoradiography. At PFE-SD days 10, 12 and 15, there were great increases in the concentration of autoradiographic grains over the nucleolus, nucleus and cytoplasm of acinar cells as compared to similar compartments in SD animals. There also were more total grains per nucleus and nucleolus in the PFE-SD animals at these days. The grains per unit area of nucleus and cytoplasm of PFE-SD acinar cells at days 10, 12 and 15 increased to about the same degree, but the increase in concentration of grains per unit area of nucleolus was much higher than in the two other compartments. The increase in total number of grains in the nucleolus was much higher than that per nucleus in PFE-SD animals on days 12 and 15. Some of the disproportionately high concentration and amount of newly synthesized labeled protein in the nucleolus at PFE-SD days 12 and 15 may have been involved in genomic activity, giving rise to the peak of DNA synthesis at day 15.

The rat liver nucleolus, after fragmentation induced by ethionine treatment, has been found to undergo complete reformation by adenine in the presence of a dose of cycloheximide sufficient to cause inhibition of protein synthesis by 90-95%. In contrast, actinomycin D given along with adenine was followed by the appearance of a small compact mass containing only the fibrillar component with no evident granules. This structure resembled pseudonucleoli seen in the anucleolate mutant of Xenopus laevis or in certain early stages of amphibian oocytes. Actinomycin D administered 2 hr after adenine induced a segregation of the fibrillar and granular components of nucleoli similar to that induced in the normal nucleolus. The implications of these findings in relation to nucleolar organization are briefly discussed.