The action of alpha-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids

alpha-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of alpha-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of alpha-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10-20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by alpha-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.     

Triiodothyronine regulation of multiple rat hepatic genes: requirement for ongoing protein synthesis

We have examined the role of rapidly turning over proteins in the T3 regulation of multiple rat hepatic genes. T3 induction of the rapidly responsive mRNA-S14 was markedly inhibited by cycloheximide (1 mg/100 g BW) or emetine (3 mg/100 g) injected ip 30 min before T3 (mRNA-S14 concentration was only 35% of that in T3-treated controls 8.5 h after administration of either protein synthesis inhibitor, P less than 0.01). Cycloheximide exhibited a similar effect on each of five other more slowly responsive T3 regulated genes. When cycloheximide was given 10 h after T3, the expected T3-induced rise of mRNA-S7 activity was completely prevented, and for mRNA-S4 activity the anticipated rise was blunted to 40% of T3-treated control (P less than 0.05). Cycloheximide caused sharp declines in the activity of two other mRNAs, S6 and S8, which because of shorter lag times of response to T3, had already risen when the drug was given. Values for both these mRNAs returned to the baseline hypothyroid level within 6 h of injection of the drug and remained low for a further 8 h (P less than 0.05). The expected deinduction of mRNA-S10 by T3 was also markedly modified. T3 lowered this mRNA to 11% of the hypothyroid control after 8 h, whereas cycloheximide given 30 min before the hormone blunted this fall to only 72% of control (P less than 0.01). Thus there appeared to be a 70% reduction in the rate of T3 induced fall of mRNA-S10. We did not find that cycloheximide caused a generalized decrease in poly (A)+ RNA mass.(ABSTRACT TRUNCATED AT 250 WORDS)     

The action of α-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids

α-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of α-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of α-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10–20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by α-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.     

The effect of inhibitors of protein and RNA synthesis on 1 alpha,25-dihydroxyvitamin D3-dependent calcium uptake in cultured embryonic chick duodenum

To determine whether 1 alpha, 25-dihydroxyvitamin D3-dependent increases in intestinal calcium uptake require de novo protein and RNA synthesis, the effects of several inhibitors of these processes have been re-examined in vitro using cultured embryonic chick duodenum. To minimize the contributions of antibiotic toxicity to the interpretation of results, care was taken to examine inhibitor effects at early times after the onset of the 1 alpha, 25-dihydroxyvitamin D3 response. Cycloheximide at a concentration of 5 microM blocked hormone-dependent calcium uptake at all times examined (6 to 24 h). Actinomycin D was similarly effective at 6 to 12 h. The effects of cycloheximide were totally reversible while actinomycin D inhibition was only partially reversible. These compounds inhibited protein or RNA synthesis by 68.4 +/- 1.4 and 51.4 +/- 1.1%, respectively. Anisomycin, another inhibitor of polypeptide chain elongation and alpha-amanitin, an inhibitor of RNA polymerase I, also blocked 1 alpha, 25-dihydroxyvitamin D3-dependent calcium uptake after 12 h in culture. These results further strengthen the hypothesis that 1 alpha, 25-dihydroxyvitamin D3 stimulates intestinal calcium transport via a nuclear mechanism involving new gene expression.     

Effect of protein synthesis inhibitors on DNA replication. I. Effect of the short-term action of cycloheximide on cell permeability for thymidine in vitro]

The effect of protein synthesis inhibitors on DNA replication was studied on L cells. After a 10 minutes' action of the inhibitors, protein synthesis was seen to be completely blocked, and DNA synthesis decreased by 85%. Four hours after a 20-minutes' cycloheximide treatment, the cells completely restored their ability to protein synthesis and DNA replication and even surpass the control level, due, probably, to a partial cell synchronization in S period. The short action of cycloheximide did not interfere with thymidine uptake by the cells. The rate of the exogenous precursor uptake was even higher than that in the control, apparently, because of its much reduced utilization in the inhibited DNA synthesis.     

Selective Inhibition of Reovirus Ribonucleic Acid Synthesis by Cycloheximide

Cycloheximide, at a concentration of 10 mug/ml, rapidly blocked protein synthesis in L cells infected with reovirus. When the drug was added before 5 hr postinfection, synthesis of both single- and double-stranded varieties of virus-specific ribonucleic acid (RNA), which normally commences between 6 and 7 hr after infection, was blocked. When the cycloheximide was added at 9 hr after infection, uptake of uridine-H(3) into RNA, for the succeeding 6 hr at least, was similar to that of an infected culture without the drug. This latter uptake of uridine-H(3) in the presence of cycloheximide was largely into single-stranded RNA, since double-stranded RNA synthesis was rapidly and markedly inhibited by the cycloheximide. Single-stranded RNA formed in the presence of cycloheximide was found not to be a precursor of viral progeny, double-stranded RNA. Synthesis of double-stranded RNA in the infected cell probably requires prior synthesis of a new protein, which has a rapid rate of turnover. The possibility that formation of single-stranded RNA is preceded by synthesis of a second new protein is discussed.     

Rapid increase in poly(A) (+) mRNA content following inhibition of protein synthesis

When resting 3T6 cells undergo a serum-induced transition to the growing state, the cytoplasmic content of ribosomal, transfer and messenger RNA increase as the cells prepare for DNA synthesis. The normal linear increase in mRNA content occurs even when the production of ribosomes is blocked. In this paper we determine the effect of inhibiting protein synthesis on the increase in poly(A) (+) mRNA content. Resting cells were serum stimulated in the presence of cycloheximide or puromycin at levels which inhibit protein synthesis by greater than 95%. Cytoplasmic poly(A) (+) mRNA content was determined at various times thereafter. We found that mRNA content increased five to ten times more rapidly in drug treated cells than in control cells stimulated in the absence of inhibitors. mRNA content increased 50–70% by one hour, and 60–90% by two hours following stimulation in the presence of inhibitor, and remained more or less constant thereafter. In contrast, mRNA content increased linearly in control stimulated cultures and did not double until about 15 hours after stimulation. The rapid increase in mRNA content is most likely the result of inhibition of protein synthesis rather than a secondary effect of the drug since the same observations were made in growth stimulated cells if protein synthesis was blocked with either puromycin or cycloheximide. A similar effect was also observed with resting 3T6, exponentially growing 3T6 and growing HeLa cells following exposure to cycloheximide, although the magnitude of the increase was less than that observed with growth stimulated cells. Puromycin had negligible effect on mRNA content in resting or exponentially growing cells. The rapid increase in cytoplasmic poly(A) (+) mRNA content was not due to rapid unbalanced export of nuclear poly(A) (+) RNA into the cytoplasm since there was no decrease in nuclear poly(A) content following serum stimulation in the presence of cycloheximide.     

Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.     

Induction of deoxyribonucleic Acid synthesis in potato tuber slices: role of protein synthesis

Timing of protein synthesis which is a prerequisite to DNA synthesis induced in potato tuber tissue (Solanum tuberosum L.) by cut injury has been studied using cycloheximide. The induction of DNA synthesis which was measured by incorporation of ³H-thymidine was completely inhibited when the inhibitor was applied to the tuber discs immediately after slicing. When the application of cycloheximide was delayed for 6 hours or more after slicing, DNA synthesis was observed but its rate was reduced to 20% of control. The inhibitory effect of cycloheximide, however, rapidly decreased when the inhibitor was applied at 6 or less hours immediately prior to determination of DNA synthesis. The effect of cycloheximide on the incorporation of ¹⁴C-leucine suggests that the change in the effect of cycloheximide on the induction of DNA synthesis is not due to incomplete inhibition of protein synthesis. Cycloheximide did not have significant effects on either uptake or phosphorylation of ³H-thymidine in the discs. Inhibition of both protein and DNA synthesis by cycloheximide was reversed by washing and further incubation of the discs. Almost no qualitative difference was detected by buoyant density analysis between DNA formed under inhibition of protein synthesis of the later stage and DNA synthesized under normal conditions. These results suggest that DNA synthesis induced in potato tuber tissue by cut injury requires continuous synthesis of new protein molecules in a characteristically programmed sequence.     

Interleukin-3 inhibits cycloheximide of C-Jun mRNA in human monocytes: Possible role for a serine/threonine phosphatase

Cycloheximide is a strong inducer of the c-jun protooncogene mRNA at concentrations (≤50 ng/ml) that do not inhibit protein synthesis in human monocytes. This induction is transient lasting 30–60 min in contrast to the sustained induction obtained with concentrations that inhibit protein synthesis. The pluripotent colony stimulating factor interleukin-3 (IL-3) (10 ng/ml) is also a modest inducer of the c-jun gene in these cells; however, in combination with cycloheximide, IL-3 dramatically reduces the c-jun induction below levels induced by cycloheximide alone. This is a true inhibition and is not due to a change in temporal kinetics of induction because the suppression in the presence of IL-3 is observed at both 30 and 60 min after simultaneous addition of both IL-3 and cycloheximide. Preincubation of monocytes with 12.5 nM okadaic acid (a potent inhibitor of protein phosphatases 1 and 2A) and cycloheximide prior to addition of IL-3 restored the level of c-jun induction to that mediated by cycloheximide alone. This concentration of okadaic acid inhibited almost 70% of the phosphorylase phosphatase activity in monocyte lysates. These observations suggest that activation of protein serine/threonine phosphatase(s) underlies the ability of IL-3 to inhibit cycloheximide induction of c-jun in monocytes. © 1993 Wiley-Liss, Inc.     

Role of protein phosphorylation in activation of interferon-stimulated gene factors.

Possible involvement of protein phosphorylation in interferon (IFN)-mediated activation of IFN-stimulated gene factor 3 (ISGF3) was investigated. For this purpose, in vivo experiments with specific inhibitors of protein kinases and in vitro experiments with protein phosphatases were carried out. In HeLaM cells, 2-aminopurine, an inhibitor of double-stranded RNA-dependent protein kinase, blocked the induction of ISGF3 gamma subunit but not the activation of ISGF3 alpha subunit. A series of experiments using combinations of protein and RNA synthesis inhibitors and 2-aminopurine indicated that the block elicited by 2-aminopurine was at the level of ISGF3 gamma mRNA synthesis. Activation of ISGF3 alpha, although insensitive to 2-aminopurine, was completely blocked by 10 nM staurosporine, an inhibitor of protein kinase C. On the other hand, even 500 nM staurosporine did not block the induction of ISGF3 gamma. Incubation of cytoplasmic or nuclear extracts of IFN-treated HeLaM cells in vitro with alkaline phosphatase completely eliminated their ability to form the ISGF3 complex but not the ISGF1 complex. Treatment with acid phosphatase, on the other hand, changed the electrophoretic mobility of the ISGF3 complex but did not obliterate it. Complementation experiments revealed that ISGF3 alpha was the alkaline phosphatase-sensitive component of the complex. These results suggest that a protein kinase C-mediated phosphorylation step is involved in ISGF3 alpha activation and a 2-aminopurine-sensitive component is involved in ISGF3 gamma mRNA induction.