Emmanuel Farber

Cycloheximide, an inhibitor of peptide chain termination or release in liver in vivo and in vitro☆

Abstract It has been found that cycloheximide at low dosage has a selective inhibitory effect on peptide chain termination or release in the liver in vivo and in vitro , In vivo , cycloheximide in a dosage that inhibits total liver synthesis by over 90% has little or no effect on labeling of nascent protein on polysomes by radioactive leucine but a marked effect on the labeling of sap protein. In vitro , cycloheximide in a dosage range of 0.018 to 0.89 mM shows a progressive inhibitory effect on the labeling of soluble protein and no effect on the labeling of nascent protein on polysomes. Only at a much higher concentration range (above 1.88 mM) is there an increasing effect on incorporation of radioactive amino acids into nascent protein.

Highly selective in vivo ethylation of rat liver nuclear protein by ethionine

Abstract Ethionine ethylates rat liver nuclear proteins in a highly selective manner. The proteins most ethylated are those insoluble in 20–40% (NH 4 ) 2 SO 4 in the saline soluble fraction. With L-ethionine-ethyl-1- 14 C, only one peak of radioactivity was found. This peak has been tentatively identified as an ethyl analogue of methylguanidino arginine, recently reported to occur in nuclear proteins. No radioactive ethionine was found in the proteins after giving ethyl-labeled or 35 S-labeled ethionine. The pattern of labeling with methionine-methyl- 14 C is more complex and is presented.

CORRESPONDENCE BETWEEN RIBOSOME AGGREGATION PATTERNS IN RAT LIVER HOMOGENATES AND IN ELECTRON MICROGRAPHS FOLLOWING ADMINISTRATION OF ETHIONINE.

Electron micrographs from livers of rats treated four hours previously with ethionine show a loss of the characteristic aggregation and organization of the ribo -somes. Within one and one-half hours after the administration of adenine or adenine plus methionine to the ethionine-treated animal, the ribosomes show a remarkable reassembly and reorganization into their normal configuration. These changes coincide in time with changes in the patterns of aggregation of isolated ribosome and with alterations in protein synthesis. These results indicate that, under some experimental conditions, a correspondence exists between the ribosome aggregates in the isolated cell fraction and in tissue sections examined with the electron microscope.

Resistance of liver polysomes to ATP deficiency in the male rat

Abstract 1. 1. Mature male and female rats respond in a similar fashion to a single dose of ethionine by the following changes in the liver: (a) marked depression of ATP concentration, (b) large accumulation of S- adenosylethionine and (c) severe inhibition of radioactive orotate incorporation into nuclear and microsomal RNA. 2. 2. However, a divergence in the response between the sexes is seen in hepatic protein synthesis. In the female rat, there is a rapid and marked inhibition of protein synthesis both in vivo and in vitro accompanied by a breakup of polysomes into monomers. In contrast, in male rats there is much less inhibition of protein synthesis in vivo , no inhibition in vitro and only a slight loss of polysomes. The unusual resistance of the polysomes from the male to the ATP deficiency lasts for at least 24 h. At that time, the male shows less depression of ATP concentration and a decrease in S- adenosylethionine content of the liver when compared to the female. The possible usefulness of this model for the analysis of some aspects of polysome metabolism in the intact cell is briefly discussed.

Reformation of functional liver polyribosomes from ribosome monomers in the absence of RNA synthesis.

The administration to rats of the ethyl analog of methionine, ethionine, results in the rapid decrease in the hepatic concentration of adenosine triphosphate followed by an extensive disaggregation of polysomes to ribosome monomers and a concomitant inhibition of protein synthesis. These effects are readily reversed by the injection of methionine or precursors of adenine nucleotides such as adenine. The reformation of liver polyribosomes in such animals following the administration of adenine plus methionine was found to occur under conditions in which new RNA synthesis was markedly inhibited. Free messenger RNA without attached ribosomes must be capable of remaining functionally active in the liver cytoplasm for many hours.

Growth in vitro of Cells from Hyperplastic Nodules of Liver Induced by 2-Fluorenylacetamide or Aflatoxin B1

Cell suspensions obtained from hyperplastic nodules induced in rat liver by either of the two hepatic carcinogens, 2-fluorenylacetamide or aflatoxin B1, show growth when cultured in vitro. No growth of cells from liver adjacent to the hyperplastic nodules or from liver of control rats has been obtained so far under comparable conditions. Hepatocarcinoma cells induced by 2-fluorenylacetamide grow readily in vitro but behave differently. These findings suggest that some nonmalignant cells capable of growth in vitro arise during liver carcinogenesis prior to the appearance of unequivocal cancer. Cultures of such cells may offer new avenues for the study of liver carcinogenesis.

Altered and distorted DNA from a premalignant liver lesion induced by 2-fluorenylacetamide

Abstract DNA isolated from a precancerous population of liver cells, weeks after the termination of the dietary exposure to the carcinogen 2-fluorenylacetamide (2-FAA) was found to show obvious alterations in its chemical and physical properties. The DNA had (I) an altered absorption spectrum in the range 350–310 mμ, (2) a small population with an increased buoyant density as observed in equilibrium centrifugation in CsCl, and (3) denatured and distorted fibres as observed with electron microscopy, when compared with DNA from normal control liver, the surrounding non-hyperplastic liver or regenerating liver following partial hepatectomy. These findings indicate the possibility that an observable alteration in DNA may play a role in carcinogenesis and suggest a possible mechanism for the progressive nature of the cellular changes during cancer induction.

Selective effects of adenine and adenosine triphosphate upon the hepatic biochemical lesions induced by ethionine

Abstract The effects of adenine, ATP, or methionine administration upon the ethionine-induced inhibition of labeling of liver phospholipid and liver protein by methionine-methyl-C14 in female rats have been studied. Adenine and ATP completely prevent the inhibition of incorporation of methionine into protein, but are only partially effective in protecting against the inhibition of transmethylation to phospholipid. In contrast, both these effects of ethionine are completely counteracted by methionine administration. These data further support the hypothesis that certain biochemical lesions induced by ethionine are directly due to the primary effect of ethionine on ATP concentration, while others are due to other effects of the analogue upon methionine metabolism.

The biochemical pathology of ethionine-lnduced pancreatic damage: Ethionine incorporation into proteins, and ATP levels in pancreas of rats☆

Abstract Two biochemical mechanisms of possible pathogenetic significance in injury of cells by ethionine are synthesis of abnormal proteins by ethionine incorporation, and induction of ATP deficiency. These hypotheses were evaluated in relation to ethionine-in-duced pancreatic damage in rats. Ethionine incorporation into pancreatic protein was found to conform to the pattern which has been reported for several natural amino acids. Incorporation continued for at least 9 hours following a dose of 0.5 g per kg, apparently because a significant level of free ethionine persisted. The pancreas did not appear to have any unique tendency to incorporate ethionine into structural proteins when compared with liver and kidney, and therefore the results do not suggest that formation of abnormal proteins is of special significance in the pancreas. S-adenosylethionine accumulation and ATP deficit were demonstrated in pancreas 24 hours following single doses of ethionine (1 g per kg). However, at a lower dose which was adequate to produce severe pancreatic damage, there was no decrease in ATP in the pancreas. It therefore appears that the ethionine-induced pancreatic lesion cannot be attributed to ATP deficiency.

The effects of inosine and aminoimidazole carboxamide upon the ethionine fatty liver

Abstract The administration of inosine or 5-amino-4-imidazole carboxamide to female rats is as effective as adenine or ATP in preventing the fatty liver induced by ethionine. Hadacidin, an antibiotic known to prevent the amination of inosinic acid to adenylic acid, counteracts the effect of inosine. Inosine is also very effective in preventing the mortality of female rats after ethionine administration. Aminoimidazole carboxamide is as effective as adenine, ATP, or inosine in returning the hepatic ATP level to normal 2 hr after ethionine administration. The results of this study strengthen further the hypothesis that the ethionine-induced fatty liver is secondary to a more basic interference with ATP metabolism in the rat liver.