George P. Tryfiates

Effect of benzene on rat liver polyribosomes

Abstract The effect(s) of benzene on rat liver polyribosomes was studied. The data show that administration of benzene to rats intraperitoneally resulted in disaggregation of liver polyribosomes, accumulation of ribosomal monomer-dimers and appearance of an intermediate, previously absent, ribosomal peak. The protein synthetic capacity of disaggregated liver polyribosomes as measured by the incorporation of L -[ 3 H]-phenylalanine in vitro was significantly impaired (>50 per cent) but the poly U-directed polymerization of L -[ 3 H]-phenylalanine was not affected. Protein synthetic tests with polyribosomes and pH 5·1 enzyme fractions from benzene-treated and untreated animals showed that treatment with benzene in vivo did not affect the pH 5·1 enzyme fraction. Further, other experiments showed that the incorporation of labeled ribonucleic acid precursors into liver polyribosomes during treatment with benzene was considerably impaired while incorporation into total liver ribonucleic acid and the size of the acidsoluble fraction radioactive label pool were not affected. These results are discussed in terms of benzene action at the molecular level.

Hormonal and cofactor induction of tyrosine transaminase in vitamin B6 deficiency

Abstract The activity of liver tyrosine transaminase of vitamin B 6 deficient rats is approximately half that of normal animals. Administration of hydrocortisone to deficient animals raises the level of liver transaminase activity to a value 6 times greater than that achieved by the administration of pyridoxine. Actinomycin D or puromycin given with the vitamin or the hormone at zero time do not prevent the rise in enzymatic activity but further enhance it to values above the induced level. The results suggest that, in vitamin B 6 deficiency, cofactor and hormonal enzyme induction reflect stabilization of enzyme in vivo rather than de novo enzyme synthesis.

Vitamin B6 Effects on the Growth of Morris Hepatomas and the Development of Enzymatic Activity

Vitamin B6 is not only required for normal growth and development, in general, but also for the growth of neoplasms. In attempts to decipher the effects of the vitamin on tumor growth and metabolic function, i.e., enzyme activity, studies were carried out in our laboratory using Morris hepatomas of increasing degree of differentiation grown in animals fed ad libitum a pyridoxine free diet and in pair-fed or ad libitum (controls) given the same diet supplemented with pyridoxine2. The effects of the absence of pyridoxine from the diet on (a) hepatoma growth, (b) enzyme activity, (c) enzyme induction and (d) enzyme expression in liver and hepatomas were investigated using, in addition to conventional and chromatographic procedures, (a) immunoprecipitation in agar-gel and (b) in situ, ‘on the gel’, histochemical staining for detection of resolved, enzymatically active protein bands. Further, the effects of progressive ‘cofactor depletion’ (vitamin deficiency) and hepatoma growth on hormonal enzyme induction and vitamin B6 were also investigated.

Enzyme Activities in Vitamin B6-Deficient, Normal, and Tumor-Bearing Animals: Effect of Hydrocortisone

Summary The effect of vitamin B6, deficiency on the activity of two vitamin B6-requiring enzymes and the induction of these enzymes by hydrocortisone was studied in normal and hepatoma #7794A-bearing Buffalo rats. Enzyme activities were generally lower in vitamin B6-deficient animals. Serine dehydratase was absent in this hepatoma. Administration of hydrocortisone resulted in increased transaminase activity of normal, host liver, and hepatoma. The increase in activity was greater in animals fed the pyridoxine-supplemented basal diet. Cycloheximide blocked the hormonally induced rise in enzyme activity. Hormonal induction of serine dehydratase was not seen in the host liver or tumor of animals fed the basal diet. However, a small increase in activity did occur in the host liver of animals fed the pyridoxine-supplemented basal diet. Administration of hydrocortisone with or without cycloheximide resulted in lower activity values of this enzyme in the host liver of vitamin B6-deficient animals.

Isolation of Rat Liver Mitochondria and of Their Ribonucleic Acid

Abstract Mitochondria prepared from normal or regenerating rat liver appeared homogeneous on examination by electron microscopy. Ribonucleic acid (RNA) isolated from such mitochondria by phenol extraction or by deproteinization without phenol was resolved on sucrose density gradients into 18S, 12S and 4S optical density peaks. Administration of 5-[3H]-uridine to normal or partially hepatectomixed animals for 16 hours resulted in the labeling of A IS, 36S, 28–29S, 14S, 9–10S and 4S RNA species. Labeling of 18S RNA from regenerating liver but not from normal liver was also observed.

Effect of Vitamin A Deficiency on the Protein Synthetic Activity of Rat Liver Ribosomes

Summary Vitamin A deficiency enhances the in vitro capacity of rat liver ribosomes to make protein. This stimulatory effect of vitamin A deficiency on protein synthesis is due to the pH 5.1 enzyme fraction.

Growth of Morris hepatoma No. 7794A with and without vitamin B6. Effect of inoculation time

Abstract The effect of time of inoculation of tumor cells on the growth of the highly differentiated transplantable Morris hepatoma 7794 A under vitamin B 6 deficient and control conditions was studied. Groups of weaning Buffalo strain rats were fed ad libitum a diet lacking pyridoxine or were pair-fed the same diet supplemented with the vitamin. They were then inoculated intramuscularly in both hind legs with 7794 A hepatoma cells at the beginning (to) and after 7, 15 , and 31 days on their respective diets. Animals were sacrificed 39–49 days after inoculation, tumors were excised and after removal of connective tissue they were weighed. The average weight of hepatomas grown in animals fed the vitamin deficient diet for the same length of time was always similar and in all cases was less than the average weight of those hepatomas grown in pair-fed (control) animals. The difference between average tumor weights was not statistically significant (P 0·5 ) except when inoculation was performed after 31 days. The average weight of tumors from pair-fed controls, in this case, was 3 times higher over that of tumors grown in animals fed the vitamin deficient diet. The significance level was P 0·05 . The data are interpreted to show that caloric restriction (pair-feeding) allows hepatoma 7794 A to grow as during ad libitum conditions provided inoculation takes place after 31 days of feeding period.

Hepatic and tumor proteins reactive with tyrosine transaminase—Specific rabbit antiserum☆

Abstract Availability of dietary pyridoxine has been shown to affect tumor growth as well as expression of enzymatic activity. Whether pyridoxine influences the expression of hepatic and tumor tyrosine transaminase (TT) was investigated in the present study by a combination of electrophoretic and immunological procedures. Buffalo strain female weanling rats were fed ad libitum a pyridoxine free diet or pair-fed the same diet with pyridoxine added. After 21 days they were inoculated with No. 7777 Morris hepatoma cells and sacrificed 14 days later 4 hr after the i.p. administration of hydrocortisone hemisuccinate. High speed liver and tumor supernatants were electrophoresed on polyacrylamide gels and TT bands were subsequently immunoprecipitated in agar-gel using rabbit antiserum specific to highly purified rat liver tyrosine transaminase. In addition, Ouchterlony tests were also performed using the same preparations. The results obtained were as follows: Four and 5 gel protein bands were immunoprecipitated from liver supernatants of hormonally induced animals fed the supplemented and pyridoxine free diets, respectively. Hepatomas grown in animals fed the vitamin free and supplemented diets developed 3 and 4 precipitation bands, respectively. The immunoprecipitation pattern of the hepatoma was also distinctly different than that of normal liver. Additional analyses by the technique of double diffusion in agar gel (Ouchterlony) showed three precipitation lines developed with samples from either deficient or control animals. Hepatoma samples from control animals (B 6 plus) showed three precipitation lines while those from deficient animals showed an altered precipitation pattern and only two bands. Our results show that (a) in hydrocortisone treated animals, lack of dietary pyridoxine induced in liver an extra protein species reactive with transaminase antiserum while the appearance of an antitransaminase reactive protein was inhibited in the hepatoma, and (b) an immunoprecipitation pattern peculiar to this tumor tissue.

In vitro RNA synthesis by rat liver and human leukemic nuclei. Isolation of undegraded RNA.

Abstract Incubation of nuclei from rat liver or human leukemic cells in the presence of 3H-UTP2 and other factors results in th incorporation of label into a material precipitable by acid, alcohol or ether. This materials is isolated by phenolsds extraction, is sensititve to ribonuclease digestion and presumed to be RNA. The addition of Cu++ to the incubation system is necessary to inhibit RNA breakdown and allows the isolation of undegraded RNA without interefering with th incorporation of radiosactivity. The time patterns of labl incorporation by the two nuclei preparations are different. Whereas label incorporation by th two nuclei preparations are different. Whereas labelincorporation by liver nuclei continues to increase up to 60 minutes, incorporation by th leukemic nuclei is high during the first 10 minutes and continues at a slower rate up to 45 minutes of incubation. further, th two nuclei preparations also synthesize diferent RNA species. While liver nuclei synthesize RNA sedimenting at 4.5S and...

Methylation of DNA in human chronic granulocytic leukemia cells

Abstract Human leukemic granulocytes actively synthesize and methylate DNA during incubation at 37°C. Maximal DNA synthesis occurred after one hour of incubation while maximal DNA methylation was reached at two hours. The following DNA methylated bases were identified: 1-methyladenine, 5-methylcytosine, 1-methyl and/or 7-methylhypoxanthine, and 6-dimethylamino purine and/or 6-methyl mercaptopurine. It was further found that the main DNA peak banded at ϱ =1.6916 g. cm −3 in the CsC1 gradient and had an approximate guanine-cytosine content of 32.2 per cent.