Gisèle Beck

Action of Ochratoxin a on cultured hepatoma cells—reversion of inhibition by phenylalanine

Ochratoxin A (OT-A), a mycotoxin composed of a phenylalanine moiety bound by an a-amide bond to a chlorinated dihydroisocoumarin is highly toxic for some ~croorg~isms [ I] as well as for higher animals [Z] essentially causing liver and kidney damage. It inhibits protein synthesis by competition with phenylalanine in the phenyl~~yl-tea synthetase catalyzed reaction as has been shown either with bacterial or with yeast systems [ 1,3,4]; but on animal cells neither this effect nor the effect on nucleic acid synthesis has yet been tested. In this paper we have determined the amount of OT-A necessary to completely inhibit protein synthesis in hepatoma tissue culture cells (HTC) and have also tested its activity on DNA and RNA synthesis. Finally, since OT-A is a competitive inhibitor with respect to phenylal~~e, we have tested the capability of this amino acid for reversion of the mycotoxin action.

Complete complementary DNA of rat tyrosine aminotransferase messenger RNA. Deduction of the primary structure of the enzyme.

The primary structure of rat tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase; EC 2.6.1.5), a liver-specific enzyme involved in gluconeogenesis, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 2362 nucleotides long (excluding the poly(A) tail) and codes for a polypeptide of 454 amino acids with a molecular weight of 50634. Unambiguous identification was obtained by comparison of this sequence with the amino acid sequences of several peptides obtained from the purified enzyme.

Action des sels d'ammonium quaternaire sur les acides nucléiques I. Solubilité des sels d'ammonium quaternaire des acides nucléiques dans les solvants organiques

Abstract Deoxyribonucleic acid and ribonucleic acid can be precipitated by a number of quaternary ammonium salts. The products obtained are quaternary ammonium nucleates, soluble in numerous polar organic solvents. Sodium salts can be regenerated from organic solutions by addition of sodium chloride.

Purification and characterization of rat liver tyrosine aminotransferase

Several studies have been published describing the purification and properties of L-tyrosine-2-oxoglutarate aminotransferase (EC 2.6.1 S.). The purification was first attempted from the livers of L-tyrosine treated rats [ 1,2] . Later, a more extensive purification from the livers of rats previously treated with glucocorticoid hormones was reported 13-51. In this paper, we describe a purification method for the soluble tyrosine aminotransferase (TAT) from livers of rats treated with the synthetic glucocorticoid: dexamethasone. The enzyme was purified by fractionation techniques utilizing heat treatment, chromatography on DEAE-cellulose and hydroxyapatite columns, gel-filtration on Sephadex G-200 and sedimentation in a sucrose gradient. During the whole purification procedure, the enzyme remained soluble since steps like precipitation by ammonium sulfate or concentration by freeze-drying were avoided. The overall purification factor was in the range of 30004000 with a recovery of 30-40% of the activity originally present in the crude extract. Several properties of the enzyme were studied.

ACTIVATION OF EPSTEIN-BARR VIRUS PROMOTERS BY A GROWTH-FACTOR AND A GLUCOCORTICOID

Transforming growth factor‐β (TGF‐β) and a glucocorticosteroid, Dexamethasone (DXM), both cause transcriptional induction of Epstein‐Barr virus (EBV) early antigens (EA) in Daudi lymphoma cells. The viral induction occurs through the viral promoter DR overlapping an origin of replication which is active during the lytic cycle. Each hormone requires specific regions on the DR promoter. Since these regions also mediate the action of two viral transcription factors, EBI and R, it may be emphasized that EBI and/or R are involved in the EA induction process by TGF‐β and by DXM.

Inhibition of steroid inducible tyrosine aminotransferase by mouse and rat interferon in hepatoma tissue culture cells.

Interferons are cell-derived glycoproteins induced by viral infection and by a variety of non-viral agents. They are characterized principally by their capacity to establish an antiviral resistance in sensitive cells PI. Recent reports from several laboratories [2-41 have shown that the translation of messenger RNAs, from viral and cellular origin, was impaired in cellfree systems derived from interferon treated cells. The synthesis of total cellular proteins, measured either in whole cells or in cell-free systems, was inhibited by the interferon treatment, but to a lesser extent [5]. In the present work, the effect of interferon on the synthesis of a specific enzymic protein has been studied in vivo. We utilised the particularity of the outstanding property of hepatoma tissue culture (HTC) cells which respond to adrenal steroids by a 5-15-fold increase of the synthesis of tyrosine aminotransferase (TAT) [6]. Our results show that partially purified mouse interferon, which has antiviral activity in rat cells, and crude rat interferon act on HTC cells by an inhibition of steroid-induced TAT.

Essais de mise en evidence d'une regulation hormonale au niveau des RNA de transfert: I. Etude comparative des RNA de transfert de foie de poules immatures et de poules en ponte

Abstract Hormonal regulation of transfer RNAs in the liver of hens A comparative study of tRNAs prepared by cold phenolic extraction from the “post-nucleic” supernatant and from that of the 105 000× g supernatant from livers of immature hens and laying hens has shown an increase in the serine-accepting capacity of the tRNA from the liver of laying hens. We have observed that this phenomenon is due to an increase in the ratio of tRNA Ser in the total tRNA from the liver of laying hens. It did not appear to be a new tRNA Ser , but a significant difference could be seen in the proportion of certain isoaccepting tRNA Ser s. We have likewise observed an increase in the ratio of seryl-tRNA synthetase in the enzymatic preparation from the liver of laying hens. These results suggest that the oestradiol induces a regulation of the serine tRNAs and seryl-tRNA synthetase in the liver of the hens.

Antagonistic action of RU38486 on the activity of transforming growth factor-β in fibroblasts and lymphoma cells☆

Transforming growth factor-beta (TGF-beta) is a multifunctional protein involved in the control of proliferation, differentiation and other functions in many cell types. The anchorage-independent growth of some established lines of untransformed fibroblasts in soft agar is induced by TGF-beta and requires in addition exogenous EGF for certain target cells, notably rat NRK-49 cells. The formation of colonies of NRK-49F cells is completely inhibited by the synthetic 11-beta substituted nor-steroid RU38486 added at a final concentration of 1.3 X 10(-5) M. We also explored the effect of TGF-beta on Daudi and Raji lymphoma cells by measuring the production of Epstein-Barr Virus (EBV) early antigens (EA). In Daudi cells an induction capacity giving rise to 10-16% positive EA-cells was observed; in Raji cells the induction only reached between 6 and 8%. The induction was partially inhibited by the anti-steroid RU38486 in both systems. Thus, RU38486 not only antagonizes the glucocorticoid hormone action but also interferes with the effects of TGF-beta in fibroblasts and in lymphoma cells. The molecular basis of the interactions observed was investigated by considering (1) the binding to specific receptors, (2) transfection experiments, in order to examine if the interference of the anti-steroid with TGF-beta activities occurs at the transcriptional level as in the case of glucocorticoid induction. The results suggest that the blocking by antiglucocorticoids of the effects of TGF-beta and glucocorticoids, in fibroblasts and lymphoma cells, occurs by different mechanisms.

Synthesis of ferritin in cultured hepatoma cells

Ferritin is the major intracellular site for iron storage [ 11. It has many useful properties and advantages, despite the fact that it is not an enzyme, for studying the regulation of protein synthesis and turnover in mammalian tissues [ 2-41. In the present report the synthesis of ferritin has been studied in a cell line of a hepatoma tissue culture called ‘HTC cells’. These cells were derived from a malignant tumor of rat liver parenchymal cells (Morris hepatoma 7288 C) and adapted to growth in cell culture by Thompson et al. [S] . The outstanding characteristic of HTC cells is their response to the adrenal steroids. The synthesis of ferritin has been studied in suspension cultures of HTC cells as well as in cell free extracts incubated in vitro by measuring the incorporation of [3 H] leucine into proteins. The labelled ferritin was isolated by antibody precipitation after a preliminary purification in the presence of carrier rat liver ferritin. Apoferritin was synthesized to the same rate by the whole cells and by the cell free extracts but only a slight increase in ferritin protein concentration following iron treatment was observed. No measurable effect of glucocorticoids was observed on ferritin synthesis. Thus, it becomes interesting to retain ferritin as an internal control protein in the study of hormonal induction of tyrosine aminotransferase (TAT) in HTC cells. 2. Material and methods

Antagonism of glucocorticoid induction of Epstein-Barr virus early antigens by different steroids in Daudi lymphoma cells

Four antiglucocorticoids, RU38486, RU5020, RU25055 and progesterone were found to antagonize the induction of latent Epstein-Barr virus (EBV) information by dexamethasone. The dose response studies show that the antagonization was more prominent with the synthetic steroids than with the natural hormone. Specific binding characteristics of dexamethasone measured in whole cells indicate the presence of glucocorticoid receptors. Total cellular receptor contents deduced from binding data give values similar to those reported for B-lymphoblasts. Competition experiments between dexamethasone and RU38436 strongly suggest that RU38486 binds to two distinct sites in the whole cell; one is the glucocorticoid receptor but the nature of the other site is unknown. Inhibition by antiglucocorticoids differs from antagonism by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) since the latter does not compete for any sites interacting with RU38486.