Tacaribe virus infection may induce inhibition of the activity of the host cell Ca2+ and Na+/K+ pumps.

Infection of Vero cells with Tacaribe virus stocks containing a high ratio of standard (plaque-forming) viruses to defective interfering particles (DIP) induced inhibition of the host cell Ca2+ ATPase (Ca2+ pump) and the ouabain-sensitive Na+/K+ ATPase (Na+/K+ pump). The Mg2+ ATPase which is not involved in cation transport was not affected. The presence of DIP in the inocula protected the cells from alteration of the transport-associated ATPases induced by standard viruses.

Transcription of liver ribosomal RNA: differential effect of 5-fluorouracil depending on the nutritional state of mice.

The effect of 5-fluorouracil (5-FUra) on RNA transcription in mice liver cells was studied using animals exposed to different nutritional conditions as a model of normal, nondividing cells. There are two levels of rRNA transcription in mouse liver: a basal level found in mice fed on a complete diet (control mice) and a higher level, two- to three-fold increased over the basal, found in mice fed on a protein-depleting diet for about 3 days and refed on a complete diet for at least 5 hr (refed mice). The rRNA transcription was measured as the activity of RNA polymerase I in isolated liver nuclei. It was found that the intraperitoneal administration of 5-FUra (30 mg/kg body wt) to refed mice rapidly decreases the higher level of rRNA transcription towards the basal level. This is accomplished through a decrease of the initiation frequency of rRNA chains by RNA polymerase I. 5-FUra however, does not affect the basal level of rRNA transcription in liver from mice fed on a complete diet. Under this condition the drug does not affect the initiation frequency of rRNA chains. The effect of 5-FUra on rRNA transcription in refed mice is not mediated by an inhibition of protein synthesis.

Increased formation and keeping of ribosomes during dietary recovery of mouse kidney.

In the kidney of 5-day protein-depleted mice there is a decrease of 23% in the rRNA mass. When these animals are fed with a complete diet, rRNA content is restored to its normal value after 24 h of refeeding. The mechanisms that underlie this phenomenon were studied. It was found that the activity of rRNA polymerase I in the nuclei of kidneys from refed mice showed an increase of about twofold compared with the activity in normal and protein-depleted nuclei. The in vivo incorporation of a large dose (nontrace) of [14C]orotic acid into rRNA was also twofold enhanced in kidneys from refed mice. Ribosome degradation (measured by the disappearance of radioactivity from either ribosomal proteins or rRNA previously labeled by the injection of NaH14CO3 and [14C]orotic acid to the mice, respectively) stopped during the 1st day after refeeding. The estimation of the difference between the rRNA synthesis rate and the net rRNA increase also demonstrated a decrease in the rRNA degradation rate in refed mice.

Control of rRNA transcription in liver of mice exposed to nutritional changes: initiation frequency may be a regulatory mechanism.

Shortly after feeding protein-depleted mice on a meal containing proteins, the RNA polymerase I activity in isolated liver nuclei shows a two fold to threefold activation over the basal value in nuclei of either normal or protein-depleted mice. This activation can be accounted for by the increase in the number of growing rRNA chains. Moreover, the template-bound RNA polymerase I fraction in nuclei from re-fed mice is about three times that from protein-depleted animals. An excess of template- unbound enzyme was found in liver nuclei from animals under either nutritional condition. Shortly after inhibition of protein synthesis by pactamycin administration to re-fed mice, the number of transcribing RNA polymerase I molecules in liver nuclei decreases to the basal level found in nuclei from protein-depleted mice, while in the latter, protein synthesis inhibition has no effect. These results support the suggestion that short-lived proteins may enhance the initiation frequency by RNA polymerase I after re-feeding.

Transcription of ribosomal RNA is differentially controlled in resting and growing BALB/c 3T3 cells.

Shortly after serum-deprived BALB/c 3T3 fibroblasts are stimulated to grow in medium containing 10% calf serum, the RNA polymerase I activity in permeabilized cells shows a two-fold increase over the values observed in either serum-deprived or density-inhibited resting cells. Inhibition of protein synthesis by pactamycin or cycloheximide specifically reduces the enhanced RNA polymerase I activity in serum-stimulated cultures without affecting the values in resting cells. On the other hand, inhibition of rRNA processing by the nucleoside analogs 5-fluoruridine and toyocamycin decreases the rate of 45S rRNA transcription in serum-stimulated cells but has no effect on the values found in resting cultures. These data suggest that the regulation of rRNA transcription occurs by two different mechanisms, depending on the growth state of the cell. One mechanism, in serum-stimulated cells, is dependent on a continuous protein synthesis and a correct 45S rRNA processing; the other, in resting cells, is independent of these two parameters.

Lack of correlation between liver RNA polymerase I and ornithine decarboxylase activities after re-feeding of protein-depleted mice.

When mice fed on a protein-depleted diet are restored to the normal diet (re-feeding), there is a 2-fold increase in liver RNA polymerase I activity. The results obtained with pactamycin; an inhibitor of protein synthesis, suggest the presence of short-lived proteins which are required for inducing an activated state of transcription. To gain an insight on whether ornithine decarboxylase (ODC)--the first enzyme in polyamine biosynthesis--is the labile protein that regulates rRNA synthesis, we have investigated the correlation between liver ODC and RNA polymerase I activities under different nutritional conditions. We have also studied the effects of alpha-difluormethylornithine (alpha-DFMO)--a specific ODC inactivator--on rRNA transcription. The results indicate that, after re-feeding, there is an abrupt increase in ODC activity that rapidly declines, while RNA polymerase I is still increasing. On the other hand, alpha-DFMO--which inhibits the elevated activity of ODC--has not effect on rRNA transcription.

Lack of correlation between liver RNA polymerase I and ornithine decarboxylase activities after re-feeding of protein-depleted mice.

When mice fed on a protein-depleted diet are restored to the normal diet (re-feeding), there is a 2-fold increase in liver RNA polymerase I activity. The results obtained with pactamycin; an inhibitor of protein synthesis, suggest the presence of short-lived proteins which are required for inducing an activated state of transcription. To gain an insight on whether ornithine decarboxylase (ODC)--the first enzyme in polyamine biosynthesis--is the labile protein that regulates rRNA synthesis, we have investigated the correlation between liver ODC and RNA polymerase I activities under different nutritional conditions. We have also studied the effects of alpha-difluormethylornithine (alpha-DFMO)--a specific ODC inactivator--on rRNA transcription. The results indicate that, after re-feeding, there is an abrupt increase in ODC activity that rapidly declines, while RNA polymerase I is still increasing. On the other hand, alpha-DFMO--which inhibits the elevated activity of ODC--has not effect on rRNA transcription.

Control of activation of liver RNA polymerase I occurring after re-feeding of protein-depleted mice.

Shortly after feeding protein-depleted mice with a meal containing protein, the RNA polymerase I activity in isolated liver nuclei shows a 2-fold increase over the values in the nuclei of either normal or protein-depleted mice. The activity of the RNA polymerase I solubilized from nuclei of re-fed mice was slightly enhanced, probably reflecting an increase in enzyme amount. However, this increase only accounts for about 30% of the stimulation of transcription in the intact nuclei. Administration of pactamycin, an inhibitor of protein synthesis, to normal or protein-depleted mice has almost no inhibitory effect on the RNA polymerase I activity in the isolated nuclei. On the contrary, within 15 min after treatment with the drug, the stimulated activity in nuclei from re-fed mice declines towards the values in normal or protein-depleted mice and then remains constant. The activity of the solubilized enzyme remains slightly elevated for at least 2 1/2 h after re-fed mice are treated with pactamycin. These observations indicate that the stimulation of the RNA polymerase I activity in the intact nuclei after re-feeding is controlled by mechanisms other than an increase in the enzyme amount and suggest the presence of short-lived proteins required for inducing an activated state of transcription.

Aurintricarboxilic acid as a tool for investigating the template-bound and unbound forms of RNA polymerase I in permeabilized cells.

The presence of template-bound and unbound RNA polymerase I in permeabilized cells was investigated. The two enzyme forms were defined on the basis of their different susceptibilities towards aurintricarboxilic acid (ATA). It was found that addition of ATA to permeabilized cells suppresses initiation of new RNA chains by RNA polymerase I but has no effect on the activity of the enzyme already engaged in transcription. This last activity is not affected even after washing the permeabilized cells for removal of the ATA. The RNA polymerase I activity solubilized from permeabilized cells pre-treated with ATA is 60-70% of that obtained from non-treated controls. The decrease of the solubilized enzyme activity was observed after purification of the enzymes by DEAE-Sephadex columns and cannot be attributed to the presence of inhibitory or activating factors in the enzyme preparations. The simplest interpretation of these findings is that two distinct RNA polymerase I fractions, showing different sensitivities towards ATA are present in permeabilized cells. These fractions should represent the template-bound and unbound RNA polymerase I. The results also show that the amount of ATA-insensitive activity is lower in nuclei than in permeabilized cells, suggesting that detachment of template-bound enzyme occurs during nuclei isolation.

Regulation of rRNA synthesis and processing in animal cells. Effect of nucleoside analogues.

The nucleoside analogues fluorouridine and fluorodeoxyuridine (both at 100 muM) and 8-azaguanine (at 500 muM) inhibit both rRNA transcription and processing in Ehrlich ascites cells. In BHK21 cells fluorodeoxyuridine has no effect on either rRNA maturation or transcription, whereas toyocamycin (at 2 microM) inhibits both processes in BHK21 cells and Ehrlich ascites cells. The drugs inhibit transcription in cells incubated in the complete medium, but have no effect on the decreased transcription in cells incubated in a medium without amino acids. This lack of effect cannot be explained by an altered uptake of the drugs in the amino acid-starved cells, since maturation of the rRNA precursor is affected in cells incubated in media with or without amino acids. The effect of the drugs on rRNA transcription is not the consequence of the inhibition of protein synthesis. The results lend support to the proposal that rRNA processing and transcription are co-ordinately controlled in cells with a high rate of rRNA synthesis.

Regulation of ribosomal RNA synthesis in mammalian cells: effect of toyocamycin.

The present study shows that the antitumor agent toyocamycin (4-amino-5-cyano-7beta-D-ribofuranosylpyrrolo(2-3d)pyrimidine) affects rRNA transcription in Ehrlich ascites cells. This action of the antibiotic is dependent on the amino acid composition of the cell culture medium. In cells incubated in a medium rich in amino acids, the high transcription rate of rRNA is lowered by the addition of 2 X 10(-6) M toyocamycin, while in amino acid starved cells the decreased level of rRNA synthesis remains unaffected. Processing of the 45S rRNA precursor is markedly inhibited by toyocamycin in cells incubated in either medium, indicating that the uptake of the drug is unimpaired by amino acid starvation. Toyocamycin does not affect RNA polymerase I (RNA nucleotidyltransferase EC 2.7.7.6) activity when added to in vitro assay systems derived from cells grown in complete or in amino acid deficient media. The drug prevents the activation of rRNA synthesis following the refeeding of amino acid starved cells without affecting the stimulation of protein synthesis.