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PubMed1991

The effects of alpha-amanitin and cycloheximide on nuclear progression, protein synthesis, and phosphorylation during bovine oocyte maturation in vitro.

Bovine cumulus oocyte complexes were cultured for various periods and either denuded and orcein stained or radiolabeled with 35S-methionine or 32P-orthophosphate. Specific inhibitors were added to the culture medium to investigate mRNA and protein synthesis requirements for both nuclear and cytoplasmic changes during maturation in vitro. Inhibition of mRNA synthesis by alpha-amanitin during the first 2 h of culture prevented the phosphorylation of some specific proteins preceding GVBD and decreased the occurrence of GVBD from 97% to 27%. In addition, in oocytes that had undergone GVBD, only part of the changes in protein synthesis after GVBD were observed. Addition of alpha-amanitin after 3 h of culture had no effect on meiotic maturation. When cumulus oocyte complexes were cultured in the presence of cycloheximide, the phosphorylation of specific proteins was also blocked and only 5% of the oocytes underwent GVBD. Addition of cycloheximide after 4, 6, or 8 h of culture resulted in an increasing percentage of GVBD, but the oocytes became arrested in metaphase I. When cycloheximide was added from 12 h of culture onwards, nuclear progression to metaphase II was increasingly restored. It is concluded that after the onset of culture, both mRNA and protein synthesis are necessary for the phosphorylation of specific proteins and for GVBD. Furthermore, transcription during the first hours of culture is needed for the synthesis of new proteins after GVBD.

Amanitins↗
PubMed1994

Nuclear dynamics of parthenogenesis of bovine oocytes matured in vitro for 20 and 40 hours and activated with combined ethanol and cycloheximide treatment.

Bovine oocytes matured in vitro (IVM) for 20 hr vs. 40 hr were treated for activation with 7% ethanol in Dulbecco's phosphate-buffered saline for 5 min followed by incubation in M199 + 7.5% fetal calf serum containing cycloheximide (10 micrograms/ml). Treated IVM oocytes and the controls (no ethanol and cycloheximide exposures) were fixed after 0, 1, 2, 3, 4, 5, 7, 10, and 20 hr of incubation and stained 24 hr later with 1% acetoorcein to examine nuclear events. Different stages of nuclear development of the activated oocytes were identified on the basis of nuclear and chromosomal morphology. Pronuclear development was classified into four stages (PN I, II, III, and IV) according to pronuclear progression in chromatin decondensation, nucleoplasm appearance, and nuclear size. The results demonstrated that the combined activation treatment effectively drove the IVM oocytes, both young (20 hr) and aging (40 hr), out of metaphase arrest. The activation rates for young oocytes examined immediately after 0, 1, 2, 3, 4, 5, 7, 10, and 20 hr of incubation with cycloheximide were, respectively, 7%, 24%, 77%, 96%, 92%, 97%, 98%, 93%, and 98%. For aging oocytes (40 hr) the corresponding activation values at the same time intervals were 6%, 84%, 100%, 100%, 100%, 100%, 98%, 100%, and 100%, respectively. These values were significantly higher than those for the corresponding controls. The activated aging oocytes achieved peak activation response more rapidly than did young oocytes. In addition, nuclear events in aging oocytes proceeded faster than those in young ones. Spontaneous activation rates of the aging oocytes were also higher (6-57%) than those of the young ones (0-14%).

Anaphase↗
PubMed1982

Interactions between cycloheximide and T-locus alleles during mouse embryogenesis.

Female CD-1 mice were mated with CD-1 X T/ + F1 males that were heterozygous for the brachyury (T') semidominant lethal gene or were +/+. Fetuses from CD-1 X +/+ matings were normal when observed on gestation Day 17 (plug day = Day 0). Those from the CD-1 X T/+ cross exhibited the expected 1:1 ratio of short:normal tail lengths, but 10% of these fetuses were tailless, apparently due to factors in the CD-1 genotype that increased the expressivity of the T-gene with regard to reduction of tail length. Additional CD-1 females were mated with CD-1 X tw18/+ F1 males. Fetuses from the CD-1 X tw18/+ matings were normal. CD-1 females carrying CD-1 X +/+, CD-1 X T/+, or CD-1 X tw18/+ litters were injected ip on gestation Day 9 with 30 mg/kg cycloheximide or were untreated. Cycloheximide was teratogenic for litters from all three crosses. Polydactyly, oligodactyly, and a variety of skeletal abnormalities were observed. Gross malformations and total skeletal malformations were increased in treated CD-1 X T/ + or tw18/+ litters in comparison with CD-1 X +/+ litters, as were nonvertebral skeletal defects in CD-1 X tw18/+ litters. Prenatal mortality was also greater in treated mutant-containing litters than in +/+ litters, and fetal weights were similarly decreased in treated CD-1 X tw18/+ litters. The incidence of taillessness was also higher in treated (26%) than in control (10%) CD-1 X T/+ litters. Thus both the T and tw18 alleles appear to have enhanced the teratogenicity of cycloheximide, and the inhibitor may have increased the expressivity of T.

Abnormalities, Drug-Induced↗
PubMed1993

Superinduction by cycloheximide of cytochrome P4502H1 and 5-aminolevulinate synthase gene transcription in chick embryo liver.

The present study examines the effect of inhibiting protein synthesis with cycloheximide on the induction of the genes for cytochrome P4502H1 (CYP2H1) and 5-aminolevulinate synthase (ALAS) in phenobarbital-treated chick embryo livers. Phenobarbital administration caused a 10- to 15-fold increase in the levels of mRNAs for both CYP2H1 and ALAS. Cycloheximide treatment alone also induced the levels of mRNA for CYP2H1 and ALAS by 7- and 3-fold, respectively, but in combination, cycloheximide and phenobarbital elicited an additional effect resulting in a 33- and 40-fold increase, respectively. To investigate whether these effects were due to transcriptional activation or a post-transcriptional mechanism, nuclear transcription run-on experiments were conducted. The observed changes in mRNA levels for CYP2H1 and ALAS were shown to be predominantly due to changes in the rate of transcription of the respective genes. These findings establish that drug induction of the CYP2H1 and ALAS genes can proceed in the almost complete absence of protein synthesis and also imply that a labile repressor protein may be involved in modulating expression of these genes. In addition, these results indicate that drug induction of ALAS does not require concomitant synthesis of P450 apoprotein.

5-Aminolevulinate Synthetase↗
PubMed1997

Protein synthesis inhibitors cycloheximide and anisomycin induce interleukin-6 gene expression and activate transcription factor NF-kappaB.

In two human cell lines, MDA-MB-231 and HeLa, the inducible expression of the interleukin-6 (IL-6) gene by two protein synthesis inhibitors, cycloheximide and anisomycin, was compared with the induction by the most potent physiological inducer of IL-6 described to date, interleukin-1beta (IL-1beta). In cycloheximide or anisomycin treated cells, the accumulation of the IL-6 message and the activation of transcription factors required for IL-6 gene expression occurs at an extent similar to that obtained with IL-1beta. Furthermore, IL-6 mRNA accumulation stimulated by cycloheximide or anisomycin is almost completely inhibited in the presence of actinomycin D, indicating that this effect occurs mainly through the activation of the transcriptional machinery. These data indicate that transcriptional induction of the IL-6 gene by inhibitors of protein synthesis is triggered by the same nuclear signals as other inducers.

Anisomycin↗
PubMed1996

Effect of anthracycline antitumor antibiotics (adriamycin and nogalamycin) and cycloheximide on the biosynthesis and processing of major UsnRNAs.

In the present study, anthracycline antitumor antibiotics (e.g. adriamycin and nogalamycin), the potent RNA synthesis inhibitors and cycloheximide, the protein synthesis inhibitor, have been used to understand the events of biosynthesis and processing of major UsnRNAs (U1-U6). The anthracyclines inhibit the UsnRNAs biosynthesis (in terms of labelling) differentially in a dose dependent manner. The inhibitory effect of adriamycin and nogalamycin reached plateau at a concentration of 2.5 micrograms/ 10(6) cells/ml and 0.1 microgram/10(6) cells/ml respectively and indicates that nogalamycin is more inhibitory than adriamycin. The inhibition of the UsnRNAs synthesis (in terms of labelling) became maximum within 30 min of incubation and remained unaltered even after 2 h. Thus, it shows that the anthracyclines preferentially inhibit the initiation of the UsnRNA genes' transcription as it has been seen in cases of other large RNAs' synthesis by some other laboratories. The higher inhibitory effect of the anthracyclines on the biosynthesis of U5 and U6 compared to other UsnRNAs indicates the presence of more binding sites on the U5 and U6 snRNA genes. In presence of the anthracyclines, there was high retention of cytoplasmic major pre-UsnRNAs/ UsnRNAs which indicates that the elongation of the UsnRNA synthesis is probably impaired along with initiation; because for the proper processing of the pre-UsnRNAs, formation of the correct secondary structure of that pre-UsnRNA is necessary. Cycloheximide showed some differential effect on the pol II transcribed UsnRNAs (U1-U5) biosynthesis (in terms of labelling) however it has no effect on the pol III transcribed U6 snRNA. It implies that in the pol II transcribed UsnRNAs, some transacting labile factors, either activator or inhibitor, are involved. Whereas, the processing of the UsnRNAs (either pol II or pol III transcribed) was affected more or less in a similar fashion in presence of cycloheximide, indicating the involvement of some transacting labile factors in this event.

Animals↗
PubMed1978

Genetical studies on revertants to sensitivity from a cycloheximide resistant strain of Schizosaccharomyces pombe.

Six UV induced cycloheximide-sensitive revertants were isolated from the cyh1-C7 strain of Schizosaccharomyces pombe which is resistant to cycloheximide. In all cases reversion to sensitivity was due to a forward mutation in a second suppressor gene. Genetical analysis showed that at least two genes, designated scr1 and scr2 (scr=suppression of cycloheximide resistance) were involved. Both scr1 and scr2 suppressed the resistance of six independently isolated alleles at the cyh1 locus. They had no effect on two known nonsense mutations in the ade7 locus. The cyh1-C7 strain has an altered 60S ribosomal protein which can be detected by two-dimensional polyacrylamide gel electrophoresis. In two suppressed strains, cyh1-C7 scr1 and cyh1-C7 scr2, the original altered protein was present. However no further ribosomal protein differences were observed which could be correlated with the presence of the scr genes. Both scr mutations conferred cold sensitivity on the organism indicating that they were of the missense type. Hence it seems certain that scr1 and scr2 are not mutations in tRNA genes leading to either nonsense or missense suppression. There is however no direct evidence that they code for ribosomal proteins and exert their effect on cyh1-C7 at the ribosomal level.

Cycloheximide↗
PubMed1994

Transcriptional activation of a cycloheximide-inducible gene encoding laccase is mediated by cpc-1, the cross-pathway control gene, in Neurospora crassa.

Expression of the laccase gene (lacc) of Neurospora crassa is transcriptionally inducible by the protein synthesis inhibitor cycloheximide. A lni-1 mutation, conferring the laccase non-inducible phenotype, was found to be a cpc-1 allele. Northern blots probed with plasmid pLA1, which carries the lacc gene revealed that the cpc-1 mutation abolishes the induced transcription of the lacc gene, indicating requirement of the cpc-1 gene for transcriptional activation of the lacc gene. In Northern blots probed with plasmid pAB1, which bears arg-2 a gene whose transcription is under the control of CPC1, the level of the arg-2 transcript was shown to increase several-fold in wild-type mycelia but remained low in cpc-1 mycelia, after treatment with cycloheximide. This suggests that inhibition of protein synthesis with cycloheximide, as well as amino acid limitation, elicits the CPC1-mediated cross-pathway control. Characterization of the lacc upstream region using a series of 5'-deletion plasmids led to the identification of a 170 bp DNA region required for the induced lacc expression. Sequence analysis of this DNA region demonstrated that it includes a 9 bp sequence with dyad symmetry, ATGAATCAT, which differs only by a central base pair from ATGA(C/G)TCAT, the recognition sequence characteristic of CPC1 and GCN4 binding sites. Possible mechanisms by which CPC1 mediates transcriptional activation of the lacc gene are discussed.

Alleles↗
PubMed1992

High-level resistance to cycloheximide resulting from an interaction of the mutated pdr3 and cyh genes in yeast.

In addition to pdr3-1, the S. cerevisiae nuclear pleiotropic drug resistance mutant 2D was found to contain another recessive nuclear mutation, cyh, conferring specific resistance to cycloheximide only. The cycloheximide resistance level due to either the pdr3-1 or the cyh mutation alone was low and was not altered by the ogd1 mutation which increased the physiological acidification of the culture. When pdr3-1 and cyh mutations occurred simultaneously in the haploid yeast strain their interaction was synergistic and resulted in high-level resistance to cycloheximide.

Cycloheximide↗
PubMed1976

Effect of 5-fluorouracil and cycloheximide on the early development of Phycomyces blakesleeanus spores and the activity of N-acetylglucosamine synthesizing enzymes.

The development of germinating Phycomyces spores was not inhibited by 5-fluorouracil (1mM) until the emergence of the germination tube. Fluorouracil was incorporated into RNA as efficiently as uracil; it did not inhibit the synthesis of proteins and the increase in respiratory activity during early development. Cycloheximide inhibited development as well as the increase in respiration and protein synthesis. This suggested that protein synthesis or some other cycloheximide dependent process, but no mRNA synthesis, was needed for the first developmental stages. The activity of two enzymes involved in the synthesis of N-acetylglucosamine increased markedly during germination. THis increase was inhibited by both 5-fluorouracil and cycloheximide; this suggested that those enzymes were synthesized on mRNA formed during germination.

Acetylglucosamine↗
PubMed1979

Resistance against cycloheximide in cell lines from Chinese hamster and human cells is conferred by the large subunit of cytoplasmic ribosomes.

Cell lines from Chinese hamster ovary [CHO-K1-D3] and human fibroblast cells [46, XX, 18p-] were mutagenized with N-nitrosomethylurea followed by a selection for cycloheximide resistance. Two mutants resistant against the drug were selected from either wildtype. 80S ribosomes and their ribosomal subunits were isolated from all mutant and wildtype cells. 80S ribosomes reassociated from the isolated subunits were as active as isolated 80S couples in the poly (U) dependent poly (Phe) synthesis. Hybrid 80S ribosomes constructed from subunits of the various cell lines of the same species were fully active, whereas the interspecies 80S hybrids were not active at all in poly (Phe) synthesis. Hybrid 80S ribosomes from subunits of mutant and the corresponding wildtype cells were tested in the poly (U) assay in the presence and absence of cycloheximide. The results strikingly indicate that in all four mutant cell lines the resistance against cycloheximide is conferred by the large subunit of cytoplasmic ribosomes.

Animals↗
PubMed1975

The effect of puromycin and cycloheximide on vacuole formation and exocytosis in Tetrahymena pyriformis GL-9.

It has been shown that both puromycin and cycloheximide, at concentrations of 434 and 100 mug/ml respectively, produce a marked inhibition of vacuole formation and exocytosis in Tetrahymena pyriformis GL-9. These effects were analysed in a quantitative manner. At the same time as these inhibitions occurred the incorporation of 1-C14 leucine into trichloroacetic acid precipitable material was inhibited by 90% and 100% respectively over a 40 min period. This inhibition of protein synthesis by cycloheximide occurred almost immediately, whereas the inhibition of vacuole formation and egestion was delayed. The results suggested that the latter processes were dependent upon a continuing supply of proteinaceous material, of which there was only a small store within the cell. Cycloheximide inhibited exocytosis completely -nder the conditions employed (with 100% inhibition of protein synthesis) whereas puromycin (with a 90% inhibition of protein synthesis) only inhibited it by about 50%. This suggested that the amount of newly synthesized protein required for the exocytic egestion process was very small in relation to the total cell requirement for protein synthesis. The entry of both inhibitors into the cell was by means other than vacuole formation. Puromycin appeared to have some effect on vacuole formation which was unconnected with protein synthesis. Microscopic observations of living cells indicated that oral apparatus function and endocytic vacuole formation were probably both affected by the inhibitors. Chloramphenicol, at 200 mug/ml, had little effect on vacuole formation by starved cells with an exposure of an hour. The uptake of 1-C14 leucine from the growth medium was found to be a selective process, giving a concentration of about 2000 times into the cells over a 1 hr period. The results are discussed

Bacterial Proteins↗
PubMed1984

Differential changes of tightly chromatin-bound RNA polymerase II in starved and cycloheximide-treated rat liver nuclei.

In order to investigate the physiological function of loosely and tightly chromatin-bound RNA polymerase II in vivo (1), the changes of these enzyme activities in cycloheximide-treated or starved rat liver nuclei were studied. Total nuclear mRNA synthesis activity in starved rats was considerably decreased, but that of cycloheximide-treated rats was not affected significantly. In starved rats, tightly bound enzyme activity was much more repressed as compared with that of loosely bound enzyme. On the other hand, cycloheximide-treated rats showed the reverse relationship. Thus, mRNA synthesis activity in hepatic nuclei seems to be dependent on the tightly bound RNA polymerase II activity. However, the difference of nuclear mRNA synthesis in both cases can not be explained by the change of chromatin-bound enzyme activity of Yu (2).

Animals↗
PubMed1982

The effects of Cycloheximide and Anisomycin on monoamine synthesis in a brain synaptosome preparation.

Cycloheximide (100 mg/kg) and anisomycin (50 mg/kg) decreased the synthesis of [3H]-dopamine (DA) and [3H]-5-hydroxytryptamine (5-TH) from their labelled amino acid precursors in a brain synaptosomal preparation on hour after drug administration in vivo. Treatment with anisomycin also decreased synthesis of [3H]-norepinephrine (NE). Anisomycin (1mM) decreased the accumulation of newly snythesized monoamines when added to the synaptosomal preparation in vitro, while cycloheximide (1mM) impaired only [3H]-norepinephrine formation. All drug-induced decreases in the accumulation of newly synthesized 5-hydroxytryptamine were associated with concomitant decreases in the accumulation of [3H]-tryptophan (Trp) by the synaptosome fraction, suggesting that the drugs may act in part by decreasing precursor availability. However, the same correlation was not observed between [3H]-catecholamine synthesis and [3H]-tyrosine (try) content of the synaptosomal fraction. Comparison of the results obtained after in vivo and in vitro administration of these drugs indicates that cycloheximide may exert many of its effects on monoamine synthesis via a metabolite, while anisomycin appears to act directly.

Animals↗
PubMed1993

Cycloheximide prevents kainate-induced neuronal death and c-fos expression in adult rat brain.

The present study was directed at evaluating the possible involvement of protein synthesis in excitotoxin-induced neuronal damage and prolonged expression of the proto-oncogene, c-fos. Kainic acid-induced seizure activity elicited varying degrees of neuronal damage and cell loss in selectively vulnerable regions of the adult rat limbic system. Pretreatment with cycloheximide, a protein synthesis inhibitor, did not alter behavioral seizure characteristics, but markedly attenuated damage to susceptible neuronal populations. A prolonged increase in c-fos mRNA was observed by in situ hybridization up to 16 h after the onset of seizures in regions exhibiting neuronal death. Pretreatment with cycloheximide did not affect the transient induction of c-fos observed in numerous structures, but significantly reduced the prolonged expression of c-fos mRNA in kainate-vulnerable regions. Despite producing massive seizure activity, systemic kainic acid administration during the early postnatal period did not induce any neuronal death, and did not result in prolonged c-fos expression in any brain structures. The developmental onset of selective neuronal vulnerability coincided with that of prolonged c-fos expression in susceptible neuronal populations. In adult rats, seizure activity induced by pentylenetetrazole did not produce neuronal damage nor did it produce prolonged c-fos expression. These results not only demonstrate that kainate-induced neurotoxicity and the prolonged expression of c-fos are both prevented by cycloheximide, but also strengthen idea that prolonged c-fos expression is a marker of neuronal death.

Aging↗
PubMed2001

Characterization of 5-HT2A receptor desensitization and the effect of cycloheximide on it in C6 cells.

Effect of prolonged pretreatment with serotonin (5-HT) on 5-HT2A receptor desensitization was examined by the measurement of intracellular calcium ([Ca2+]i) mobilization in C6 cells. 5-HT-induced desensitization of [Ca2+]i mobilization was in a time and dose dependent manner and reached a plateau after 3 hr. After 1 and 3 hr 5-HT pretreatment, 5-HT concentration in the medium little changed. 5-HT pretreatment with cycloheximide, a protein synthesis inhibitor, produced an enhancement of the desensitization for 3 and 6 hr pretreatment. However, 5-HT pretreatment for 3 and 6 hr caused no marked change in the 5-HT2A receptor mRNA level or Galphaq/11 protein in this study, suggesting that 5-HT may decrease 5-HT-induced [Ca2+]i mobilization independent of 5-HT2A receptor mRNA or G-proteins. Endothelin-1-induced [Ca2+]i mobilization did not alter after 5-HT and/or cycloheximide pretreatment. These results showed that activation of the 5-HT2A receptor induced homologous desensitization and pretreatment with 5-HT and/or cycloheximide did not change the efficacy of the second messenger pathway from Gq to a [Ca2+]i rise.

Animals↗
PubMed1977

Inhibition of DNA synthesis by cycloheximide and blasticidin-S is independent of their effect on protein synthesis.

The effects of cycloheximide and related glutarimide antibiotics on DNA synthesis in Achlya bisexualis Coker and A. Couch were compared to those of other protein synthesis inhibitors, puromycin, p-fluorophenylalanine and blasticidin-S. The inhibitors had no significant effects on intrahyphal pool sizes of dTTP, dCTP, ATP, UTP and CTP, nor on the specific activity of the dTTP pool labelled by [3H] thymidine. DNA was the sole acid-insoluble product of [3H]-thymidine incorporation. Cycloheximide, isocycloheximide, streptimidone and blasticidin-S inhibited DNA synthesis rapidly and completely and anhydrocycloheximide was less effective. Cycloheximide acetate, puromycin and p-fluorophenylalanine did not inhibit DNA synthesis. It is concluded that the effects of the several glutarimide antibiotics and of blasticidin-S on DNA synthesis were independent of their effects on protein synthesis.

Anti-Bacterial Agents↗
PubMed1980

Effect of cycloheximide on the in vivo and in vitro synthesis of ribosomal RNA in rat liver.

The action of low (5 mg/kg body wt;) and high (20 mg/kg body wt.) doses of cycloheximide, both causing a rapid and almost complete inhibition of protein synthesis in rat liver is investigated. Short-term (15 min) [14C]orotate incorporation into nucleolar rRNA in vivo is inhibited only by the high dose acting for periods longer than 1 h. The effect may be correlated with a strongly reduced labelling of the cellular pool of free uridine nucleotides. These results indicate that in vivo transcription of rRNA genes may not be under stringent control. The activity of template-bound RNA polymerase A in nuclei isolated from animals treated with both doses of cycloheximide is reduced within 1 h to about 50% of controls reaching nearly plateau levels at longer times of action of the drug. The differential effect of cycloheximide inhibition of protein synthesis on in vivo and in vitro rRNA synthesis suggests the existence of elongation control protein(s) characterized by a rapid turnover and a loose association with the nucleus.

Animals↗