Actinomycin D and cycloheximide

McLachlin, J.R., Goyer, R.A. and Cherian, M.G. (1980). Formation of lead-induced inclusion bodies in primary rat kidney epithelial cell cultures effect of actinomycin D and cycloheximide. Toxicol. Appl. Pharmacol. 56 418-431. 

Figure 5. Effect of actinomycin D and cycloheximide on the induction of MnP activity. Mn-deficient cultures were grown for 4 days after which MnSO (180 //M) was added alone (triangles), simultaneously with actinomycin D (50 //g/mL) (solid circles), or with cycloheximide (50 //g/mL) (open circles). Extracellular enzyme activity was assayed at the indicated intervals after the additions, by the ABTS method 13,32). (Reproduced with permission from Ref. 32. Copyright 1990, mencan Society for Microbiology.)...

Morphological changes were prevented in butyrate-treated HeLa cells by actinomycin D and cycloheximide (2,8,13). After removal of butyrate, the cells reverted to a normal morphology over a 24 h time course (2,8,12,13). When butyrate-treated cells were detached from the cuTture dishes with trypsin, they assumed a spherical shape and, when replated in the absence of butyrate, their neurite-like processes transiently re-extended (13). This re-extension was blocked when cycloheximide but not the calcium ionophore was included during the initial exposure of the cells to butyrate (13). Process formation, however, did resume in the presence of cycloheximide (1 3). These results were interpreted as indicating that the fatty acid induces a protein(s) required for process formation which can accumulate in the absence of processing and promote processing in the absence of inducer (13). 

In normal and regenerating rat liver tissue, neuraminidase has been shown to be localized primarily in the fraction of light mitochondria, enriched with lysosomes. Actinomycin D and cycloheximide did not lower the neuraminidase activity of lysosomes of the regenerating liver, indicating absence of de novo biosynthesis of the enzyme in the process of tissue regeneration. 

The development of amylase activity in extracts of embryo-free and of GA3-treated, embryo-free maize kernels has been determined. The increase in amylase activity was accompanied by the appearance of several starch-degrading enzymes. Actinomycin D and cycloheximide prevented the amylolytic activity from developing. Other results indicated that the development of a-amylase activity in embryo-free maize kernels does not depend on gibberellic acid, but involves the de novo synthesis of protein. 

Fig. 3. The effects of chloramphenicol and puromycin on protochlorophyllide regeneration by etiolated bean leaves. Panel A Absorption spectra of 7-day-old dark-grown red kidney bean (Phaseolus vulgaris) leaves taken 4 hours after illumination for 1 minute. Panel B Absorption spectra of the same leaf sets as shown in panel A, but spectra were deteimined after leaves had again been irradiated for 1 minute. Panel C Difference spectra obtained by subtracting an absorption spectrum in panel A from the corresponding one in panel B. Curves a control bi leaves incubated with 5 X 10" M chloramphenicol for 4 hours before initial illumination c leaves incubated with 10" M puromycin for 4 hours prior to initial illumination d control leaves (no inhibitors) incubated with 10 M ALA in darkness during the 4-hour period after the initial illumination e as in b, but incubated with 10 M ALA in the same manner as d. After Gassman and Bogorad (1967a). Data for actinomycin D and cycloheximide are not shown.

The use of antibiotics for the control of plant virus diseases( ) is of interest. Several antibiotics have been tested for inhibition of replication of viral nucleic acid and/or protein synthesis within the host cell. Chloramphenicol, cycloheximide, actinomycin D and others are the most used antibiotics and the disease caused by tobacco mosaic... 

Butyrate appears to induce sialyl transferase activity as addition of actinomycin D or cycloheximide to the medium along with butyrate blocked the increase in activity (4,8). Specific cell cycle inhibitors such as thymidine and colcemid did not cause an increase in activity in control cells or prevent induction in butyrate-treated cells (8). Induction of sialyl transferase activity also occurred in serum-free medium (8). When homogenates of control and butyrate-treated cells were admixed and assayed for sialyl transferase activity, there was no evidence of an inhibitor in the former or activator in the latter cells (8). 

Actinomycin D and puromycin (B12, B13, R3) block thyroxine inhibition of TSH release after TRF stimulation but do not block the release phenomenon. This observation suggests that the ability of thyroxine to block TRF depends on protein synthesis. There are conflicting reports about the ability of cycloheximide to block the T4 effect (B13, W2). Ouabain had no effect in one study, and oligomycin and... 

The increased rate of ABA biosynthesis in dehydrated leaves can be blocked by inhibitors of transcription, such as actinomycin D and cordycepin [163-165], as well as by cycloheximide, an inhibitor of cytoplasmic protein synthesis [163,165-167]. These results indicate that nuclear gene transcription and cytosolic protein synthesis are required before an increase in ABA biosynthesis can take place. These processes probably account for the lag period prior to ABA accumulation following the onset of stress [94,163]. Water stress and cycloheximide had no effect on the conversion of XAN to ABA [54]. This indicates that the enzymes catalyzing these conversions are constitutively expressed. The most likely step stimulated by dehydration is, therefore, at the level of xanthophyll cleavage, although isomerization of xanthophylls cannot be ruled out. 

Effect of temperature on protein Synthesis by isolated Chloroplasts Intact chloroplasts of V. Sinensis mediated protein synthesis in a light-dependent reaction without the requirement for ATP. No protein synthesis was seen in the dark. Fig.l shows the fluorographic profile of the proteins synthesized at various temperatures. The general profile of the polypeptides synthesized was not altered very much by the change in incubation temperature. However, at 35 C and above, there was a distinct increase in the synthesis and/or accumulation of four polypeptides in the molecular sizes of 85,70, 60 and 23 kDa. All four polypeptides could be observed, at least as faint bands, even at 25 C. The synthesis of HSPs at 40 C in the isolated chloroplasts were completely inihibited by chloramphenicol but not by cycloheximide. The transcriptional inihibitors like actinomycin-D and rifamycin were also inihibitory to the HSP synthesis (Fig 2). 

In a mouse lung epithelial cell Une, dexamethasone decreased retinoic acid receptor-P and surfactant protein C expression to 75 and 70 % of the control values, respectively, with greatest effects at 48 h and at 10" M (Grummer and Zachman 1998). There was no effect of dexamethasone on either retinoic acid receptor-P or surfactant protein C mRNA disappearance with actinomycin D. However, cycloheximide prevented the effect of dexamethasone. Despite dexamethasone, retinoic acid increased both retinoic acid receptor-P and surfactant protein C mRNA. 

Our own work (3) and that of others (2) with E. coll have shown that the de novo purine biosynthetic pathway is regulated by both a repressor molecule (pur R gene product) and by feedback inhibition. However, Chinese hamster cells are much more sensitive to feedback inhibition by adenine than E, coli and, unlike the situation in E. coli, no repression of PRPP amidotransferase or formyglycinamide biosynthesis could be detected. If repression did occur, it would have to be by a mechanism not normally associated with the purine biosynthetic pathways or at a site late in the purine bios3mthetic pathway. Moreover, the nucleotide pools of cells treated for 2 h with with actinomycin D or cycloheximide showed a substantial increase in nucleotide levels. This Increase in nucleotide concentration is probably sufficient in itself to inhibit de novo purine biosynthesis by feedback inhibition without recourse to a repression mechanism, Snyder and Henderson (10) have also reported an effect of actinomycin D on purine metabolism in Ehrlich ascites cells. In this case, there was no large effect (11% inhibition) on de novo purine biosynthesis, Snyder and Henderson (10) proposed that this decrease was due to a 29% reduction in PRPP levels as a result of increased (1,3-fold increase in ATP and 2,8-fold Increase in GTP) nucleotide pools. These observations are consistent with our data in which a 58% decrease in PRPP level is found over a 2-h period in Chinese hamster cells grown in actinomycin D, The extent of inhibition in Chinese hamster cells is much greater than that reported for Ehrlich ascites cells and may reflect a difference between cells,... 

Transcription is the first viral metabolic event in cells penetrated by infectious VSV nucleocapsids. The viral transcriptase functions in the presence of actinomycin D and inhibitors of protein synthesis, such as cycloheximide this has been called primary transcription. 

The experimental approach in most of the systems studied so far was to follow the greening process at different levels (ultrastructure, composition of membranes, development of activity) and try to establish a correlation between the different parameters observed which will lead to the formulation of explanatory hypotheses. The validity of the hypotheses can then be assessed by inducing alteration in the process at one level and studying the effect at the other levels. The use of protein and nucleic acid synthesis inhibitors has so far been the method of choice. Cycloheximide was extensively used to block protein synthesis by cytoplasmic 80 S type ribosomes, while chloramphenicol, spectinomycin, and lyncomycin were used to inhibit protein synthesis by the chloroplast ribosomes. Actinomycin D and, more recently, rifampicin have been used as inhibitors of RNA synthesis. Initially, rifampicin was considered to be specific for the DNA-dependent RNA polymerase of the chloroplast. Although it appears that in some algae, such as Chlamydomonas and Acetabularia, rifampicin has a reasonable degree of specificity, its action against RNA polymerase of the chloroplast partially purified from plants was not found to be specific. ... 

Those herbicides that block mitotic entry decrease or prevent the formation of mitotic figures in meristems. Amino acid, protein, RNA, DNA, and ATP synthesis and/or utilization can all attest cell growth (163,166). Although not registered as herbicides, cycloheximide [66-81-9] inhibits mitotic entry by inhibiting protein synthesis (167) hydroxyurea/727-(97-/7 inhibits DNA synthesis (168) and actinomycin D [50-76-0] nh2oix.s RNA synthesis (167). 

Actinomycin D, Antibiotic 205-2B, Blasticidin, Cycloheximide(actidione), Daunomycin DPB, Mithramycin, Mitomycin C, Pentaene G8, and Tubercidin. 

Recently it has been demonstrated by Rayton and Harris (30) that the role of copper, in addition to its presumed role as a cofactor, is related to the induction of lysyl oxidase. Cycloheximide, but not actinomycin D, completely inhibits the incorporation of 64cuII into lysyl oxidase. They suggest that the mechanism may be similar to the induction of ferritin by iron. 

Induction of de novo synthesis of a-amylase by GA in isolated aleurone layers is evident after a lag period of approximately 8 hr following administration of the hormone. In keeping with hormone responses generally, GA must be present continuously if the de novo synthesis of hydrolases is to be sustained. Synthesis of new RNA is essential to the GA-induction of de novo synthesis of hydrolases. Actinomycin D, an inhibitor of RNA synthesis, inhibits the synthesis and release of a-amylase if the inhibitor is presented during the first 7 to 8 hr after treatment. Inhibitors of protein synthesis, such as cycloheximide, also inhibit GA-induction of hydrolases. And, interestingly, abscisic acid, a growth-inhibiting hormone, inhibits GA-induced a-amylase synthesis as well. 

Indeed, inhibitors of protein synthesis have been used in the in vivo rabbit [60] and in the rat LangendorfT models [61]. Cycloheximide or actinomycin D did not reverse the PC effect [60]. This is probably due to non-complete inhibition of protein synthesis. The latter study [61] showed that the PC effect was abolished only with cycloheximide, but not with actinomycin D, indicating that PC protection is regulated at the post-transcriptional level. Recent findings [62] have reconfirmed the translational control of protein synthesis in ischemic (but not pharmacological) PC. These are in accord with our findings concerning de novo synthesis of ferritin. 

McKenzie, J. M., and Williamson, A., Experience with the bioassay of the long-acting thyroid stimulator. J. Clin. Endocrinol. Metab. 26, 518-526 (1966). McKenzie, J. M., Adiga, P. R., and Murthy, P. V. N., Effects of actinomycin-D, cycloheximide and puromycin on thyroid stimulation. Endocrinology 83, 1132-1139 (1968). 

Selye H, Tuchweber B, Bertok L (1966) Effect of lead acetate on the susceptibility of rats to bacterial endotoxins. J Bacteriol 91 884-890 Seyberth HW, Schmidt-Gayk H, Hackental E (1972) Toxicity, clearance and distribution of endotoxin in mice as influenced by actinomycin D, cycloheximide, a-amanitin and lead acetate. Toxicon 10 491-500... 

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