Evolution of ornithine decarboxylase activity during the cell cycle of Euglena gracilis Z in synchronous culture. Influence of vitamin B-12.

Ornithine decarboxylase activity in Euglena gracilis Z was studied during the normal cell cycle and in vitamin B-12 deficiency. The cells were synchronized by means of alternating periods of light and dark. During the normal cell cycle, ornithine decarboxylase activity was very weak in the dark period, while three peaks of activity were recognized in the light period. The first peak, in the G1 phase, occurred when luminous stimulation started; the second preceded the S phase and the third was found in G2. In B-12-deficient cells, ornithine decarboxylase activity was greatly decreased and only the first peak remained. Elimination of the deficiency by addition of vitamin B-12 to the medium induced a very fast and significant increase in ornithine decarboxylase activity.

Interleukin 1 beta modulates hepatic synthesis of alpha 1-acid glycoprotein in the fetal rat.

The ability of the fetal rat to respond to interleukin 1 beta (IL1 beta) by expressing alpha 1-acid glycoprotein (AGP) was investigated. Eight and 20 h after injection of 7 ng IL1 beta into 19-day fetuses, liver AGP mRNA increased by a factor of 66 and 82 respectively, while serum AGP levels increased by a factor of 3 and 5. Similar treatment of the mothers altered in the fetuses neither AGP serum levels nor the amount of liver AGP mRNA. The induction of AGP gene expression in the fetal liver in response to IL1 beta was similar to that observed in the adult liver. These results demonstrate that at day 19 the fetal rat liver has acquired a mature acute-phase system.

The complete cDNA sequence encoding dog gastric lipase.

Oligodeoxyribonucleotide ligation to single-stranded cDNA (SLIC) and polymerase chain reaction (PCR) techniques were used to clone an entire dog gastric lipase (DGL) cDNA. The size of the cDNA is confirmed by Northern blot analysis. The DGL is synthesized as a 379-amino acid mature polypeptide with a molecular mass of 43176 Da which is preceded by a 19-amino acid signal sequence located at the NH2-terminus. Comparison of the signal sequences reveals a high degree of similitude between the DGL, the human gastric lipase (HGL), the rabbit gastric lipase (RGL) and the rat lingual lipase (RLL).

Nucleic acids methylation of synchronized BHK 21 HS 5 fibroblasts during the mitotic phase.

The methylation of nucleic acids has been investigated during the cell cycle of an asparagine dependent strain of transformed fibroblasts (BHK 21 HS 5). The synchrony was carried out by a partial asparagine starvation of cells for 24 hours. The amino acid supply induced all cells to enter synchronously the G1 phase. Methylation and DNA synthesis were respectively measured by pulsed [methyl-14C] methionine and [methyl-3H] thymidine incorporation. DNA methylation followed a biphasic pattern with maximal methyl incorporations during both S phase and mitosis. A partial desynchronisation induced the S phase of the second cycle to proceed before all the cells have achieved their division. Hydroxyurea was used in order to inhibit the DNA synthesis of cells entering the second cell cycle, which might interfer with the mitosis of the first one. The inhibitor was added either at the first beginning of cell division or during all the G1 phase. In both conditions it suppressed 3H thymidine incorporation of the second cycle. However, mitosis took place and methylations occurred as in previous experiments. The DNA methylation of the mitotic phase in the first cell cycle could thus be dissociated from the classical post-synthetic DNA maturation and did not correspond to any DNA methylation appearing in the course of the second cell cycle.

Amplification of ribosomal DNA in the nucleolus of vitamin B12-deficient Euglena cells.

In the nucleoli of vitamin B12-deprived Euglena cells there is a 10-fold amplification of rDNA which correlates with increased numbers of fibrillar centers in the hypertrophic nucleoli. This rDNA amplification was demonstrated by means of molecular hybridization using a specific cytoplasmic rRNA probe. Most of the amplified sequences appear as extrachromosomal copies which were observed without digestion of total cellular or nuclear Euglena DNA. Five different hybridizing sequences, three of high molecular weight and two of 19.0 and 11.5 kb, were identified. The rDNA repeat unit which contains the 11.5-kb transcriptible sequence is 19.0 kb in length and was always more abundant in the undigested total cellular DNA. The possible mechanisms involved in the induction of rDNA amplification in vitamin B12-deficient cells are discussed.

[Ultrastructural changes in the nucleus and the nucleolus of Euglena gracilis Z during the cell cycle]. / Evolution ultrastructurale du noyau et du nuclêole de Euglena gracilis Z au cours du cycle cellulaire

The chromosomes and nucleolus of the Euglenineae remain visible in electron microscopy throughout the cell cycle. This peculiarity allowed to show cyclic variations of the nucleus structure in synchronized cells of Euglena gracilis Z: I. different stages of chromosome condensation was observed; 2. ultrastructural modifications in the organization of the nucleolus were evidenced, characterized by its apparent continuity ("open appearence") or discontinuity ("closed appearance") with the nucleoplasm.

Separation of naturally occurring or induced methylated nucleobases of DNA by reversed-phase high-performance liquid chromatography.

Separation of the major bases of DNA along with seven minor methylated ones was obtained by reversed-phase high-performance liquid chromatography using an isocratic elution system. Application of this procedure to DNA treated in vitro by N-methyl-N-nitrosourea has allowed identification of two induced minor bases; a third one was resolved using a slightly modified mobile phase. Baseline resolution of 3-methyl- and 5-methylcytosine, detected in Euglena DNA hydrolyzates, was also achieved.

Modifications of hepatic alpha-1-acid glycoprotein and albumin gene expression in rats treated with phenobarbital.

The serum level of alpha 1-acid glycoprotein (alpha 1-AGP) is significantly increased in various animal species by treatment with cytokines, glucocorticoids and phenobarbital. The mechanisms responsible for the cytokine-induced and glucocorticoid-induced increases are now well documented, but not so in the case of phenobarbital. The main purpose of this study was to assess whether phenobarbital acts on alpha 1-AGP synthesis in the liver at the transcriptional or translational level. Male Dark Agouti rats received 70 mg phenobarbital/kg daily for 7 days. The analysis of total hepatic RNA showed that a single injection of phenobarbital induced an 11-fold increase in phenobarbital-dependent cytochrome P450IIB mRNA, whereas seven injections of phenobarbital were required to induce a maximum 5.5-fold increase in alpha 1-AGP mRNA. Concurrently, the transcription rate of the alpha 1-AGP gene rose 3.5-fold. Hepatocytes isolated after the seventh injection of phenobarbital showed a threefold increased capacity to secrete alpha 1-AGP, corresponding to a 3.2-fold increased alpha 1-AGP mRNA content in the liver. In conditions in which its effect on the induction of alpha 1-AGP synthesis was maximum, phenobarbital caused a 30% reduction in liver albumin mRNA and in albumin secretion by isolated hepatocytes, resulting from a 60-70% reduction in the rate of transcription of the albumin gene measured in isolated nuclei. We conclude that the effect of phenobarbital on alpha 1-AGP and albumin gene expression occurs at the transcriptional rather than the translational level.

Evaluation of the hepatotoxicological effects of a drug in an in vivo/in vitro model.

Both in vivo and in vitro models have certain disadvantages for the study of the chronic hepatotoxicity of drugs. The aim of this work was to evaluate a new approach based on an in vivo/in vitro model. After chronic in vivo treatment of rats with Vincamine and Vindeburnol (an eburnamenine derivative which exhibits hepatotoxic properties in man) liver cells were isolated, and functional and metabolic disorders (metabolic utilization of fructose and protein biosynthesis) were studied to determine injury. The results showed no modification of blood parameters, but a direct relationship between the dose of Vindeburnol administered in vivo and the metabolic disorders observed in vitro, evidencing the high sensitivity and reliability of this model.

Separation and characterization of the main methylated nucleobases from nuclear, cytoplasmic and poly (A)+ RNA by high-performance liquid chromatography and mass spectrometry.

We were able to detect nine methylated nucleobases (3-methyluracil, 1-, 2-, 3- and 7-methylguanine, 1-, 2-, 3- and 6-methyladenine) in RNA from rat and calf liver, baker's yeast, Torula and Euglena cells by using reversed-phase high-performance liquid chromatography and thermospray mass spectrometry. Total cellular, nuclear, cytoplasmic and poly (A)+ RNA from rat liver showed marked methylation, mainly of 1- and 3- methylguanine, and 3- and 2-methyladenine. These bases were especially abundant in nuclear RNA and, to a lesser extent, in poly (A)+ RNA. In contrast, 7-methylguanine and 6-methyladenine were poorly represented in poly (A)+ RNA.