Mechanism of Apparent Transcription Inhibition by Methyl Mercury in Cerebellar Neurons

Abstract: We have investigated the mechanism of inhibition of RNA synthesis by methyl mercury (MeHg) in isolated neonatal rat cerebellar cells. Each of the three component steps involved in the incorporation of exogenous [³H]uridine into cellular RNA was examined separately in whole-cell and/or subcellular preparations. Nuclear RNA polymerase activity was measured in preparations containing both free nuclei and whole cells. Incorporation of [³H]UTP into nuclear RNA was found to be unimpaired at concentrations of MeHg that inhibited whole-cell incorporation of [³H]uridine by > 75%. Cellular uptake of [³H]uridine was assayed in cerebellar cells treated with KCN to deplete ATP levels and block subsequent phosphorylation reactions of transported uridine. Uptake activity under these conditions was unaffected by MeHg. Measurement of intracellular phosphorylation of [³H]uridine indicated that inhibition of this activity closely paralleled that of RNA synthesis. Quantitation of individual uridine nucleotides by polyethyleneimine-cellulose TLC revealed reduced levels of UTP and UDP whereas levels of UMP were elevated, suggesting that impairment of phosphorylation was not the result of cellular ATP depletion but, more likely, a direct effect on phosphouridine kinase enzymes. This mechanism of MeHg-induced inhibition of RNA synthesis was confirmed by assays of uridine phosphorylation using cell-free extracts in which exogenous ATP was supplied.     

Stimulation of uridine phosphorylation in primary cultures of Xenopus laevis hepatocytes by estradiol-17 beta

The effects of estrogen on the uridine uptake into cells were examined in primary cultures of liver parenchymal cells from Xenopus laevis. The total uptake of [3H]uridine into the estrogen-treated cells and its incorporation into RNA were about 1.5 times higher than the values for control cells. The uptake of [3H]adenosine and its incorporation into RNA were not affected by estrogen. An experiment in which liver parenchymal cells were double labeled with [3H]uridine and [3H]adenosine showed that estrogen elevated the specific radioactivity of the UTP pool 1.4-fold the value found for the control cells, but that of the ATP pool was not altered by estrogen. Short term labeling revealed that estrogen did not significantly alter the rate of the initial uptake of [3H]uridine into the cells, but it did stimulate [3H]uridine phosphorylation about 1.7-fold. Uridine kinase activity measured in cell-free extracts of hepatocytes treated with estrogen had a value 1.6 times that of the control cells. These data indicate that the stimulation of [3H]uridine uptake and phosphorylation in Xenopus laevis hepatocytes in the presence of estrogen is caused by the enhancement of uridine kinase activity.     

Suppression of temperature-sensitive sporulation of a Bacillus subtilis elongation factor G mutant by RNA polymerase mutations.

A class of rifampin-resistant (rfm) mutations of Bacillus subtilis suppresses the temperature-sensitive sporulation of a fusidic acid-resistant mutant. FUS426, which has an altered elongation factor G. The rfm mutation suppressed only the sporulation defect caused by the elongation factor G mutation, but could not suppress other types of induced sporulation defects. Genetic and biochemical analyses showed that the sporulation suppression by the rfm mutation was caused by a single mutation in RNA polymerase. After the early sporulation phase, the apparent rate of RNA synthesis of FUS426, measured by [3H]uracil or [3H]uridine incorporation into RNA, became lower than that of the wild-type strain, and this decrease was reversed by the rfm mutation. However, when the total rate of RNA synthesis of FUS426 was calculated by measuring the specific activity of [3H]UTP and [3H]CTP, it was higher than that of the rfm mutant, RIF122FUS426. The possible mechanism of the functional interaction between elongation factor G and RNA polymerase during sporulation is discussed.     

Effect of streptovaricin on the incorporation of uridine into cellular and viral RNA.

Poxvirus replication is inhibited by streptovaricin. The most readily observed effect is the inhibition of incorporation of [3H]uridine into viral mRNA, suggesting an inhibition of RNA synthesis. Streptovaricin also inhibits the incorporation of [3H]uridine into cellular RNA but not as severely as viral RNA. On the other hand, [3H]uridine incorporation into the RNA of Semliki Forest virus (SFV), which contains a positive strand RNA genome, does not seem to be inhibited by streptovaricin. The inhibitory effect of streptovaricin is completely reversible after removal of the inhibitor. In addition to inhibiting RNA synthesis, streptovaricin also may inhibit the methylation of cellular RNA. Viral RNA is stable in the presence of streptovaricin.     

Inhibition of cell division in amoebae: the incorporation of tritiated precursors into Amoeba proteus after the injection of non-homologous cytoplasm.

The injection of non-homologous cytoplasm into any strain of large free-living amoebae leads to a 60% inhibition of division amongst recipient cells. When the post-microsomal supernatant fraction of Amoeba discoides was injected into A. proteus, this inhibition of division was as high as 95%. The incorporation of tritiated precursors, either [3H]uridine or 3H-amino acids, into these inhibited amoebae was studied at various times after the injection of the inhibitory material using autoradiography. When cells were grown in [3H]uridine, autoradiographs indicated that RNA synthesis had ceased 2 days after the injection of non-homologous material. However, if [3H]uridine was injected into the inhibited cells, some synthesis of RNA could be detected up to 4 days after the injection of inhibitor. These results suggested that uptake of [3H]uridine was impaired and that one site of action of the inhibitory molecules was RNA synthesis for membrane components. Experiments with a variety of 3H-amino acids suggested that protein synthesis continued for at least 9 days after the injection of non-homologous cytoplasm, and that in these cells some informational RNA molecules were long-lived. There seemed to be accumulation of material containing [3H]lysine in the nuclei of control cells taken at random from cultures, and this was seen in the nuclei of inhibited cells 1 day after injection. However, 2 days after the injection of inhibitor, no accumulation of [3H]lysine-containing material was found in the nuclei.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of (3H] uridine incorporation into RNA and [3H] leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10-21 M). Insulin stimulated the rate of [3H] thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100-1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H] thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of 3H- uridine, [3H] thymidine and [3H] leucine into their respective precursor pools is not responsible for the apparent stimulation of RNA, DNA and protein synthesis.     

Electron microscope analysis of amplifying ribosomal DNA from Xenopus laevis.

The inhibition of human prostatic epithelial cell (MA-160) replication by cAMP and certain analogs was explored in tissue cultures. When untreated fetal bovine serum was used to supplement the culture medium, cyclic AMP (cAMP) markedly inhibited cell growth. The inhibition was reversed by equimolar concentrations of uridine. Inhibition by 8-methyl-thio-cAMP (MES) was somewhat less effective and was not reversed by uridine. After heat treatment of the fetal bovine serum, which inactivated the cAMP phosphodiesterases, cAMP became less effective in cell growth inhibition, whereas the activity of MES remained unaltered. Dibutyryl cAMP (db-cAMP) had no effect on cell growth, however, when combined with the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), significant retardation of cell replication was observed. Cells treated for 24 h with 0.5 mM MES took up and incorporated significantly less [³H]TdR and [³H]uridine than control cells. Treatment of cells with 0.5 mM cAMP for 24 h, on the other hand, resulted in both substantially increased [³H]TdR uptake and increased [³H]uridine incorporation into RNA. The effects of similar treatment with db-cAMP plus MIX closely paralleled those of MES with marked inhibition of the uptake and incorporation of both thymidine and uridine.     

Effect of cycloheximide on protein and ribonucleic acid synthesis in cultured human lymphocytes

1. Phytohaemagglutinin stimulates the transformation into blast cells of human lymphocytes incubated in vitro. This transformation is accompanied by an increase in the incorporation of [(14)C]leucine into protein and [(3)H]uridine into RNA. 2. The incorporation of [(14)C]leucine by cultures grown in the presence or absence of phytohaemagglutinin is inhibited to the same extent by cycloheximide, a known inhibitor of protein synthesis. 3. Lymphocytes grown without phytohaemagglutin synthesize mainly non-ribosomal RNA. [(3)H]Uridine incorporation by these cells was increased by cycloheximide. 4. Lymphocytes incubated with phytohaemagglutinin begin to synthesize substantial quantities of ribosomal RNA. Under these conditions [(3)H]uridine incorporation was partially inhibited by cycloheximide. This inhibition is shown to be largely a result of inhibition of the synthesis of ribosomal RNA.     

Cartilage-derived factor (CDF) : II. Somatomedin-like action on cultured chondrocytes

Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [³⁵S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [³⁵S]sulfate incorporation but also [³H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [³H]uridine incorporation into rabbit chondrocytes and significantly enhanced [³H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit chondrocytes.     

Effect of SV40 infection on [3H]uridine incorporation.

More [3H]uridine was incorporated into RNA of SV40-infected than into uninfected cells 31 h after infection. When the specific activity of the uridine triphosphate pools in infected and uninfected cells was equated by the addition of appropriate amounts of exogenous unlabelled uridine, no difference in the rate of [3H]uridine incorporation into RNA was observed. Although no difference in [3H]uridine entry or phosphorylation was demonstrable, the apparently smaller pools of endogenous RNA precursors in infected cells resulted in less isotope dilution and thus to synthesis of uridine triphosphate and RNA of higher specific activity.     

Blocking of the induction and expression of immunologically functional T lymphocytes by rat antiactivated T-cell serum

A hybridoma, F133, that produces macrophage activation factor (MAF) after mitogen stimulation was developed by fusing the AKR-derived BW5147 thymoma with alloantigen-stimulated C3H/HeJ splenocytes. F133 supernatants were shown to contain MAF, migration inhibition factor, and a factor capable of suppressing the plaque-forming response to sheep erythrocytes but not lymphotoxin, interleukin II, or interferon. Both concanavalin A (Con A) and phytohemagglutinin (PHA) induced MAF production by F133. Time course and dose-response experiments showed that maximal concentrations of MAF were present 48 hr after stimulation with either 1.5 μg/ml Con A or 6 μg/ml PHA. F133 and normal splenocyte MAF preparations shared physicochemical properties in that heating at 100 °C for 30 min abolished MAF activity while 56 °C for 30 min or 100 °C for 2 min had little effect. In addition, both MAF preparations were dependent on the presence of lipopolysaccharide for macrophage activation and each was inactivated by pH 4.0 or pH 10 treatment while pH 6.0 and pH 8.0 had little effect. Also, pretreatment of both MAF preparations with either trypsin or chymotrypsin inactivated MAF activity.     

In vitro DNA and RNA synthesis by human platelets.

In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria.     

Effect of puromycin on synthesis, processing, and nucleocytoplasmic translocation of rRNA in Tetrahymena pyriformis.

1. Treatment of Tetrahymena pyriformis with various concentrations of puromycin results in a more pronounced inhibition of [3H]uridine accumulation in stable RNA than of protein synthesis. 2. At a concentration of 500 micrograms/ml, which is almost completely inhibitory to [3H]uridine incorporation in vivo, puromycin has no influence on the incorporation of [3H]UTP into RNA in isolated macronuclei. Pretreatment of the cells with the antibiotic, however, reduces the activity of RNA polymerases in isolated nuclei to less than 30%. 3. In puromycin-treated cells a small amount of pre-rRNA is synthesized but not processed into cytoplasmic rRNAs. 4. Puromycin reduces the nucleocytoplasmic translocation of pre-existing RNA to about 25% of the control rate within 5 min, resulting in an accumulation of relatively stable rRNA precursor molecules in the macronucleus.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

The pattern of methylation of RNA in peripheral nerve of the chick during development

The pattern of the methylation of RNA was investigated in organ cultures of the sciatic nerve of the chicken. Nerve tissue from 14-day embryos, 17-day embryos and 3-day- old chicks was incubated with [methyl-³H]methionine or with [2-¹⁴C]uridine and [methyl-³H]methionine simultaneously for various periods of time. Subsequently, RNA was extracted from the tissues and the purified preparations were fractionated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the rapidly labelled RNA changed during the three developmental stages. The incorporation of both uridine and the methyl groups from methionine was highest in the‘heavy’RNA species of the 14-day embryonic nerve during the 0.5 and 1.0 h incubation periods. In contrast, in the nerves of 3-day-old chicks during a 0.5 h pulse with both precursors, methylation was almost entirely limited to the transfer RNA species. Furthermore, the incorporation of uridine in the nerves from 3-day-old animals revealed the presence of a heterogeneous population of rapidlylabelled, unmethylated species of RNA, most of which migrated between the smaller ribosomal RNA and transfer RNA components of the bulk RNA. The pattern of uridine incorporation and the methylation of the rapidly-labelled RNA of the 17-day embryonic nerve represented a transitional state between that of the 14-day embryos and that of the 3-day-old chicks. The 17-day embryonic stage of development corresponded to the phase of the onset of rapid deposition of myelin lipids in the sciatic nerve. Pulse-chase experiments on the embryonic nerves indicated that a number of methylated precursors of ribosomal RNA and labile, heterogeneous, probably DNA-like RNA were synthesized.     

Comparison of the effects of zinc deprivation and actinomycin D on ribonucleic acid synthesis by stimulated lymphocytes.

1. EDTA inhibited incorporation of [3H]uridine into RNA of lymphocytes, but did not decrease uptake into the cold-acid-soluble fraction of the cells. The inhibition by EDTA was largely reversible by simultaneous addition of Zn2+. 2. Low concentrations pf actinomycin D (3 ng/ml) added at the time of stimulation of the cells inhibited [3H]uridine incorporation into RNA, but concentrations of 50-100 ng/ml were required to produce the same degree of inhibition if addition of actinomycin D was delayed until just before the incorporation was measured. This difference in sensitivity did not reg within the cells. 3. When added immediately before phytohaemagglutinin, actinomycin D (3 ng/ml) and EDTA produced similar time-courses of inhibition of uridine incorporation. 4. Uridine incorporation at 32h was inhibited when actinomycin D (3 ng/ml) or EDTA was added just before stimulation of the cells, but was only slightly affected when they were added at 32h. At intermediate times the incorporation of uridine remained sensitive to addition of EDTA for longer than it was sensitive to actinomycin D. 5. Polyacrylamide-gel separation of RNA synthesized in EDTA-treated cultures in the presence or absence of added Zn2+ showed that lower availability of Zn2+ resulted in a decreased rate of transfer of radioactivity from 32S to 28S rRNA and decreased survival of 28S rRNA relative to 18S rRNA. 6. Close similarities have been shown to exist between the effects of EDTA and low concentrations of actinomycin D. Not all the effects of EDTA could be explained by postulating that Zn2+ was a constituent of RNA polymerase I, nor were the effects of actinomycin D readily explained by previously suggested mechanisms of action of this antibiotic.     

Ribonucleic acid synthesis in vitro in primary spermatocytes isolated from rat testis

The incorporation of [(3)H]uridine into RNA was studied quantitatively (by incorporation of [(3)H]uridine into acid-precipitable material) and qualitatively (by phenol extraction and electrophoretic separation of RNA in polyacrylamide gels) in preparations enriched in primary spermatocytes, obtained from testes of rats 26 or 32 days old. The rate of incorporation of [(3)H]uridine into RNA of isolated spermatocytes was constant during the first 8h of incubation, after which it decreased, but the decreased rate of incorporation was not reflected in a marked change in electrophoretic profiles of labelled RNA. In isolated spermatocytes, [(3)H]uridine was incorporated mainly into heterogeneous RNA with a low electrophoretic mobility. Most of this RNA was labile, as shown when further RNA synthesis was inhibited with actinomycin D. Spermatocytes in vivo also synthesized heterogeneous RNA with a low electrophoretic mobility. A low rate of incorporation of [(3)H]uridine into rRNA of isolated spermatocytes was observed. The cleavage of 32S precursor rRNA to 28S rRNA was probably retarded in spermatocytes in vitro as well as in vivo. RNA synthesis by preparations enriched in early spermatids or Sertoli cells was qualitatatively different from RNA synthesis by the spermatocyte preparations. It is concluded that isolated primary spermatocytes maintain a specific pattern of RNA synthesis, which resembles RNA synthesis in spermatocytes in vivo. Therefore isolated spermatocytes of the rat can be used for studying the possible regulation of RNA synthesis during the meiotic prophase.     

Exogenous nucleosides stimulate RNA synthesis in resting cells of a culture of scarlet rose

RNA synthesis in response to exogenous nucleoside precursors was studied in a suspension culture of rose cells. Exponentially growing and resting cells were prelabeled with [3H] uridine, an excess of unlabeled uridine added, and subsequent isotopic incorporation into nuclear and ribosomal fractions measured. The data were compared to control values in cells continuously labeled in the absence of unlabeled uridine. Addition of uridine to the growing culture reduced the further uptake, and incorporation of [3H] uridine into RNA. In contrast, in resting cells, the addition of uridine (or, purine nucleosides) enhanced the apparent utilization of [3H] uridine in RNA synthesis by 2- to 4-fold.     

Macrophage activation factor from EL-4, a murine T-cell line: antigenic characterization by hamster monoclonal antibodies to murine interferon-gamma

A cloned variant of the EL-4 murine T-cell line treated with phorbol myristate acetate (PMA) releases a factor that activates macrophages for nonspecific tumor cytotoxicity. This macrophage activation factor (MAF) is both physicochemically (Mr 25,000; pH 2 stable) and biologically different from interferon-gamma (IFN-gamma). However, EL-4 MAF may represent a breakdown product or otherwise altered fragment of IFN-gamma. We examined this possibility with a unique pair of hamster monoclonal antibodies against different epitopes of murine IFN-gamma. Both antibodies inhibited IFN-gamma-induced fibroblast antiviral activity; H21 but not H1 antibody also inhibited lymphokine (LK)-induced macrophage-mediated tumor cytotoxicity. Neither antibody, however, had any effect on the EL-4 MAF throughout a broad dose response. Moreover, passage through a H21 immunoaffinity chromatography column or addition of staphylococcal protein A and antibody completely inhibited LK-induced macrophage tumoricidal activity but did not affect the activity in EL-4 MAF. Identical effects in both fluid and solid phase were observed with polyclonal rabbit antisera to murine IFN-gamma. Results with all of these antibodies strongly suggest that the EL-4 MAF and murine IFN-gamma are antigenically distinct.