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PubMed1978

High-pressure liquid chromatographic determination of cycloheximide in ointment and suspension formulations.

A high-pressure liquid chromatographic procedure is described for the assay of the antibiotic cycloheximidein bulk drug and two experimental formulations. The method utilizes a reversed-phase C18 chromatographic column and refractive index detection. Possible impurities or degradation products, isocycloheximide, anhydrocycloheximide, and dimethylcyclohexone, are well separated from cycloheximide by this procedure. Complete extraction of cycloheximide from the formulations was obtained. The assay has a relative standard deviation of approximately 1%.

Chromatography, High Pressure Liquid↗
PubMed1991

Effects of actinomycin D and cycloheximide on transcript levels of IGF-I, actin, and albumin in hepatocyte primary cultures treated with growth hormone and insulin.

The stability of several RNA transcripts in cultured hepatocytes is known to increase when serum is omitted from the culture medium. In order to investigate possible mechanisms for this phenomenon, we examined the effects of actinomycin D and cycloheximide on the levels of actin, albumin, and insulin-like growth factor I transcripts in primary cultures incubated in serum-free medium. The levels of IGF-I and albumin transcripts per culture increased for the first 4 hours following addition of actinomycin D and then declined. The levels of actin transcripts and total RNA per culture declined immediately following actinomycin D addition in a manner consistent with exponential decay. IGF-I and albumin transcript levels were relatively unaffected by cycloheximide, while actin transcript levels increased 7-fold over 7 hours. The half-lives of actin transcripts and total RNA were calculated to be 4.6 to 7.7 hours and 11 to 19 hours, respectively, with no statistically significant correlation with hormone treatment. The data suggest that the stability of albumin and IGF-I transcripts, but not actin transcripts, is controlled in part by an actinomycin D-sensitive process.

Actins↗
PubMed1994

Inhibitory effects of actinomycin D and cycloheximide on neuronal death in adult Manduca sexta.

A decline in circulating 20-hydroxyecdysone permits the emergence of the adult Manduca sexta moth; this endocrine signal also triggers the death of approximately half of the neurons in the unfused abdominal ganglia of the moth central nervous system. This programmed death of neurons was markedly reduced by treatment with either actinomycin D (an RNA synthesis inhibitor) or cycloheximide (a protein synthesis inhibitor). Similar results were found after addition of these agents to ventral nerve cord cultures. The effectiveness of these treatments in delaying or blocking neuronal death depended upon their time of administration relative to the normal time of post-emergence death in the particular motoneuron under study: late-dying neurons, for example, could still be saved by these treatments even after early-dying neurons had already initiated degeneration. In both intact moths and cultured ventral nerve cords, the ability of actinomycin D to prevent neuronal death waned at the same time at which replacement of the steroid hormone could no longer block neuronal death. This suggests that the steroid commitment point represents the time at which the genes that mediate cell death are transcribed. Cycloheximide remained effective in delaying or blocking neuronal death until shortly before the onset of degeneration, suggesting that ongoing protein synthesis is essential for the initiation of the degeneration response.

Animals↗
PubMed2005

Proteome analysis of cortical neuronal cultures following cycloheximide, heat stress and MK801 preconditioning.

Studying endogenous neuroprotective mechanisms induced by preconditioning may provide drug leads to reduce ischemic neuronal death. In this study, we used 2-DE to examine protein expression following cycloheximide, heat stress, and MK801 preconditioning in rat cortical neuronal cultures. Of 150 differentially expressed protein spots selected for identification the protein or tentative protein(s) were identified in 84 cases, representing 50 different proteins. Different protein spots representing the same protein or closely related protein(s) occurred for 21 of the identified proteins and are likely to represent PTMs or proteolytic fragments of the protein. Six protein spots (actin, elongation factor 1-alpha 1, peptidyl-prolyl cis-transisomerase A, Cu/Zn superoxide dismutase, stathmin, tropomyosin) were differentially expressed in all three preconditioning treatments. Twenty-seven protein spots were differentially expressed in two preconditioning treatments, while 51 spots were differentially expressed in one treatment. Three proteins heterogeneous nuclear ribonucleoproteins A2/B1, mitochondrial stress-70 protein, and tropomyosin were detected in control neuronal cultures, but not following one or more preconditioning treatments, while a posttranslational modified form of the voltage dependent anion channel 1 was only detected following cycloheximide preconditioning. In summary, this study has revealed multiple protein changes potentially involved in neuroprotective and neurodamaging pathways, which require further characterization.

Animals↗
PubMed2002

Use of a YAP1 overexpression cassette conferring specific resistance to cerulenin and cycloheximide as an efficient selectable marker in the yeast Saccharomyces cerevisiae.

Drug-resistance markers for yeast transformation are useful because they can be applied to strains without auxotrophic mutations. However, they are susceptible to technical difficulties, namely lower transformation efficiency and the appearance of drug-resistant mutants without the marker. To avoid these problems, we have constructed a phosphoglycerate kinase (PGK) promoter-driven YAP1 expression cassette, called PGKp-YAP1. Yeast cells containing PGKp-YAP1 were resistant to cycloheximide, a protein synthesis inhibitor, and also to cerulenin, a fatty acid synthesis inhibitor, but not to other drugs tested. The transformation efficiency of PGKp-YAP1 using cerulenin selection was comparable to that using a URA3 auxotrophic marker when low concentrations of cerulenin were used. Non-transformed drug-resistant colonies did appear on the low-concentration cerulenin plates. However, these non-transformed colonies could easily be identified, based on their cycloheximide sensitivity and/or their resistance to aureobasidin A to which the transformants were sensitive. Therefore, the dual drug resistance of PGKp-YAP1 could be used as an effective selection for PGKp-YAP1 recipient cells. The PGKp-YAP1 marker was used to disrupt the LYS2 gene and to transform an industrial yeast strain, indicating that this marker can be used for efficient and reliable gene manipulations in any Saccharomyces cerevisiae strain.

Antifungal Agents↗
PubMed2000

E-73, an acetoxyl analogue of cycloheximide, blocks the tumor necrosis factor-induced NF-kappaB signaling pathway.

Proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-1 activate the NF-kappaB signaling pathway which induces the expression of a variety of genes such as the encoding intercellular adhesion molecule (ICAM)-1. We have found that E-73, an acetoxyl analogue of cycloheximide, specifically blocks TNF-induced ICAM-1 expression even at concentrations unable to affect protein synthesis. By contrast, cycloheximide inhibited both TNF- and IL-1-induced ICAM-1 expression primarily due to the blockage of protein synthesis. The nuclear translocation of NF-kappaB as well as the IkappaB degradation induced by TNF, but not by IL-1, was significantly prevented by E-73. These observations suggest that E-73 blocks the TNF-induced NF-kappaB signaling pathway upstream of IkappaB degradation.

Blotting, Western↗
PubMed1994

Nucleolar organization as revealed in cycloheximide treated cells.

The nucleolar organisation has been studied in cycloheximide treated rat kangaroo cells. Within 1 h of treatment most nucleoli become loose and reveal a nucleolonemal network. With longer treatment the granules are scarce and the nucleolonemal network becomes more prominent. The network reveals units comparable to the rDNA transcriptional units in length. The nucleolonemal units consists of tufts of fibrils. When cycloheximide is withdrawn, granules reappear and obliterate the nucleolonemal network.

Animals↗
PubMed1999

Cryopreservation of cattle oocytes: effects of meiotic stage, cycloheximide treatment, and vitrification procedure.

Different parameters likely to influence the survival of bovine oocytes after a vitrification procedure were evaluated: oocyte meiotic stage, cycloheximide treatment at the beginning or the end of maturation, and three vitrification procedures using conventional straws, open pulled straws (OPS), or microdrops. For each procedure a mixture of cryoprotectants (25% ethylene glycol and 25% glycerol) was used. After the oocytes were warmed and subjected to in vitro maturation and fertilization, the number that developed into blastocysts was determined. Results show that cryoprotectant exposure reduced embryo development and that cycloheximide treatment had no beneficial effect on oocytes vitrified in conventional straws. Among the three vitrification procedures, only the OPS method yielded blastocysts (approximately 3% of vitrified oocytes) irrespective of their initial meiotic stage. This result highlights the major influence of the cooling rate in an oocyte vitrification protocol.

Animals↗
PubMed1993

Okadaic acid accelerates germinal vesicle breakdown and overcomes cycloheximide- and 6-dimethylaminopurine block in cattle and pig oocytes.

Pig and cattle oocytes, when released from the follicle, spontaneously resume first meiotic division within 20 or 8 hr, respectively. In oocytes of both species, the activity of histone H1 kinase increases during maturation, exhibiting a maximum in metaphase I. Treatment of these oocytes with okadaic acid results in acceleration of germinal vesicle breakdown (GVBD) and of histone H1 kinase activation. This effect is more important in pig oocytes, in which the acceleration rises for 6 hr, as compared to 2 hr in cattle. Moreover, under these conditions, H1 kinase activity measured after 12 hr of culture appears higher than that observed in control metaphase I oocytes. When added to prophase oocytes, both cycloheximide and 6-DMAP (6-dimethylaminopurine) block GVBD and histone H1 kinase activation. Okadaic acid, at a concentration of 2.5 microM, is able to release the inhibitory effect exerted by cycloheximide on histone H1 kinase activity; however, GVBD occurred only in two-thirds of pig and one-quarter of cattle oocytes after 20 hr of culture. In addition, okadaic acid fully reverses the effect of 6-DMAP on H1 kinase activity and on GVBD in both species. The opposite effects of 6-DMAP and okadaic acid on MPF activation are discussed, as well as the nature of the protein, which has to be synthesized during the first meiotic division and may be involved in the MPF activation cascade.

Adenine↗
PubMed2000

Effects of cycloheximide and aminophylline on 5-methoxytryptophol and melatonin contents in the chick pineal gland.

The chick pineal gland rhythmically synthesizes two 5-methoxyindoles, melatonin and 5-methoxytryptophol. These rhythms are circadian in nature and have opposite phases. The aim of this study was to determine the effects of cycloheximide, a protein synthesis inhibitor, and aminophylline, an inhibitor of phosphodiesterase, on 5-methoxytryptophol content in the chick pineal gland and to compare this with the drugs' action on pineal melatonin production. Inhibition of melatonin biosynthesis by cycloheximide (1 mg/kg, i.p. ), revealed by a marked reduction in the nighttime activity of serotonin N-acetyltransferase (AA-NAT; a key regulatory enzyme in melatonin synthesis) and melatonin concentrations, was accompanied by a significant increase in 5-methoxytryptophol content. In contrast, administration of aminophylline (100 mg/kg, i.p.) to light-exposed chicks significantly increased pineal AA-NAT activity and melatonin levels and decreased 5-methoxytryptophol concentrations. It is concluded that in the chick the production of pineal 5-methoxytryptophol and melatonin is inversely correlated.

Acetylserotonin O-Methyltransferase↗
PubMed1992

The effects of insulin, cycloheximide and phalloidin on the content of actin and p35 in extracts prepared from the nuclear fraction of Krebs II ascites cells.

The nuclear fraction isolated from Krebs II ascites cells following cell disruption by nitrogen cavitation was separated into four fractions by salt/detergent extraction: NP-40 soluble fraction, 130 mM KCl extract, DOC/Triton x 100 soluble fraction and salt/detergent treated nuclei. The protein composition of the individual fractions was studied by SDS-PAGE and the relative amounts of actin and a 35 kDa protein (p35) were measured from gel scans. There was a time-dependent shift of actin from the 130 mM KCl extract to the NP-40 soluble fraction upon storage of the nuclear fraction on ice, indicating a progressive depolymerization of microfilaments. Compared with actin there was a slower release of p35 into the NP-40 soluble fraction. The results suggest that p35 is not integrated in the microfilament network. Phalloidin, which stabilizes the microfilaments, enriched the amount of both proteins in the 130 mM KCl extracts, together with a series of other proteins in the range 50-205 kDa. The presence of phalloidin also resulted in a large increase in the actin content in both the DOC/Triton x 100 extract and the fraction containing salt/detergent treated nuclei. Incubation of cells with insulin and/or cycloheximide enriched the amount of actin in the 130 mM KCl fraction. The results show that short term incubation of cells with phalloidin, insulin or cycloheximide increases the actin content of the nuclear fraction and also affects the presence of several other proteins.

Actins↗
PubMed1991

Cycloheximide resistance as a yeast cloning marker.

In CYH2/cyh2 heterozygous diploids of the yeast Saccharomyces cerevisiae resistance is dominant over sensitivity at low (0.5-5 micrograms/ml) cycloheximide (cyh) concentrations. The cyh-resistant haploid strain MMY1 confers relatively high (10 micrograms/ml) cyh-resistance to heterozygous diploids constructed by mating this strain with cyh-sensitive haploid strains. We present here a genetic and biochemical study of strain MMY1. Analysis of tetrads obtained from a MMY1 heterozygous diploid showed that two unlinked nuclear mutations, determining high- and low-cycloheximide resistance, were present in MMY1. From a genomic library of this strain, constructed in vector YCp50, two plasmids (pRC1 and pRC13) have been isolated which, respectively, confer high- and low-resistance phenotypes to cyh-sensitive S. cerevisiae strains. The restriction maps of pRC1 and pRC13 are totally unrelated. This finding suggests that the genes harboring the two mutations encoding cyh-resistance from MMY1 were cloned in plasmids pRC1 and pRC13, respectively. Pulse field gel electrophoresis showed that the DNA insert of pRC1 maps at either chromosome VII or XV, whereas that from pRC13 maps at chromosome XI. This latter gene appears to define a previously unreported locus and has been named cyh5. By restriction and nucleotide sequencing analysis, the cyh gene present in pRC1 has been shown to correspond to cyh2, which maps at chromosome VII. These results suggest that the dominant cyh-resistance phenotype conferred by MMY1 in heterozygous diploids is promoted by the presence of both cyh2 and cyh5.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Mapping↗
PubMed1983

Analysis of mutagenic DNA repair in a thermoconditional repair mutant of Saccharomyces cerevisiae. I. Influence of cycloheximide on UV-irradiated stationary phase rev2ts cells.

Using the thermoconditional yeast mutant rev2ts that controls an apparently site-specific step of mutagenic DNA repair it was possible to measure the time course of REV2 dependent UV-induced reversion of the ochre allele his5-2 and recovery of survival for UV-treated stationary phase cells: due to the rev2ts coded protein being active at 23 degrees C, survival and mutation frequencies increased with duration of incubation under permissive conditions in growth medium before the temperature was shifted to 36 degrees C (restrictive temperature). This increase was abolished in the presence of the protein synthesis inhibitor, cycloheximide. Furthermore, the REV2 dependent recovery of survival could be blocked or nearly blocked by cycloheximide added at any time during repair. Therefore, REV2 dependent repair can be characterized as a process requiring concomitant protein synthesis. These findings give further support to the concept that in yeast, mutagenesis involves UV inducible components of DNA repair.

Cycloheximide↗
PubMed1989

Early response and induced tolerance to cycloheximide in Neurospora crassa.

Incubation of Neurospora crassa mycelia with low doses of cycloheximide induces the expression of several genes. After 6 h in the presence of cycloheximide, mycelia become tolerant to further additions of the drug and the rate of protein synthesis exhibits a lower sensitivity to it. The polypeptide pattern is indicative of a stress situation.

Cycloheximide↗
PubMed1979

Cycloheximide-resistance in Chinese hamster ovary cells and human fibroblast cells. Cytogenetic and biochemical characterization.

Cycloheximide(CHM)-resistant mutant Chinese hamster ovary (CHO) and human cells were induced with N-nitrosomethylurea (NMU) and ethyl methanesulfonate (EMS); the mutants were viable and showed unlimited growth in the presence of CHM (7 X 10(-7) M), whereas this concentration inhibits protein synthesis in vivo as well as in vitro. No numerical or structural chromosomal aberrations were found in the mutant cells. In vitro analysis shows that the ribosomes confer resistance against cycloheximide.

Animals↗
PubMed1980

Effect of cycloheximide on urea facilitated transport through toad gallbladder epithelium.

Transepithelial urea outfluxes across toad gallbladder were determined before and after the addition of cycloheximide. The drug inhibits the movement of urea but has no effect on thiourea and antipyrine outfluxes. The inhibition of amide transport is time dependent as also shown in counterflow experiments. These results are consistent with the hypothesis that cycloheximide inhibits the synthesis of membrane proteic sites involved in urea mediated transport.

Animals↗
PubMed1980

Composition of hnRNA-associated proteins in rat liver is specifically altered after cycloheximide treatment.

Rats were treated with non-lethal doses of alpha-amanitin or cycloheximide. Nuclei were prepared and the particles carrying heterogeneous nuclear RNA (30--40 S-particles) isolated by density gradient centrifugation. In the case of alpha-amanitin the yield of particles was reduced to about 45%. Cycloheximide affected the composition of proteins associated with the nuclear RNA. In particular, the concentration of a 110 000 molecular weight protein as determined by sodium dodecylsulfate gel electrophoresis was reduced to 20--30% after 2 h and then increased to 150--180% of the control before it approached the normal level after 30 h. Possible mechanisms underlying these changes are discussed.

Amanitins↗
PubMed1994

Cell death due to ACNU-induced DNA fragmentation: inhibition by cycloheximide.

Several anticancer drugs have recently been shown to induce cell death in a manner similar to programmed cell death or apoptosis. The purpose of this study is to explore the mode of cell death caused by ACNU, a water-soluble nitrosourea. Exposure of rat glioma cell line KEG-1 to ACNU for 2 hours resulted in oligonucleosomal DNA fragmentation, creating a 'ladder' on agarose gel electrophoresis. DNA fragmentation began 18 hours after ACNU treatment, and preceded loss of membrane integrity as evaluated by the trypan blue exclusion test. The extent of DNA fragmentation increased in a dose-dependent manner, and the cell survival rate decreased reciprocally. A translational inhibitor, cycloheximide, suppressed this DNA fragmentation and enhanced cell survival rate with partial inhibition of protein synthesis. However, a transcriptional inhibitor, actinomycin D, failed to inhibit DNA fragmentation or enhance cell survival. Cycloheximide-inhibitable DNA fragmentation was also found in the KEG-1 implanted in vivo rat model following the administration of ACNU. These findings suggest that ACNU induces cell death associates with DNA fragmentation and partially with protein synthesis.

Animals↗