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PubMed2001

Antiapoptotic effect of dipyrone on HL-60, Jurkat and Raji cell lines submitted to UV irradiation, arachidonic acid and cycloheximide treatments.

The effect of dipyrone (metamizol) on cell viability was evaluated in human leukocyte cell lines upon different apoptotic treatments: arachidonic acid (AA), cycloheximide (CHX), tumor necrosis factor (TNF) and ultraviolet (UV) irradiation. Dipyrone had a dual effect: at high concentrations (beyond 300 microM), it was cytotoxic, leading to apoptosis, whereas at lower concentrations (37.5-300 microM), it was cytoprotective, delaying the loss of membrane integrity triggered by arachidonic acid (100-200 microM) and UV irradiation and the cytotoxicity of cycloheximide (25-50 microM). No effect of dipyrone was found on TNF-induced cytotoxicity (250 ng/ml). The cytoprotective effect of dipyrone is associated with a decrease in DNA fragmentation, as assessed by electrophoresis of genomic DNA and by flow cytometry; a reduction in the percentage of condensed nuclei, as evaluated by DNA staining with Hoescht 33342 and a decrease in poly(ADP)-ribose polymerase (PARP) cleavage, as assessed by Western blotting. The cytoprotective effect of dipyrone on leukocyte apoptosis occurs at concentrations usually found for the main active metabolite of the drug and may have implications on the therapeutic and side effects caused by this agent.

Analgesics↗
PubMed2001

Yeast ribosomal protein deletion mutants possess altered peptidyltransferase activity and different sensitivity to cycloheximide.

The major function of the ribosome is its ability to catalyze formation of peptide bonds, and it is carried out by the ribosomal peptidyltransferase. Recent evidence suggests that the catalyst of peptide bond formation is the 23S rRNA of the large ribosomal subunit. We have developed an in vitro system for the determination of peptidyltransferase activity in yeast ribosomes. Using this system, a kinetic analysis of a model reaction for peptidyltransferase is described with Ac-Phe-tRNA as the peptidyl donor and puromycin as the acceptor. The Ac-Phe-tRNA-poly(U)-80S ribosome complex (complex C) was isolated and then reacted with excess puromycin to give Ac-Phe-puromycin. This reaction (puromycin reaction) followed first-order kinetics. At saturating concentrations of puromycin, the first-order rate constant (k(3)) is identical to the catalytic rate constant (k(cat)) of peptidyltransferase. This k(cat) from wild-type yeast strains was equal to 2.18 min(-1) at 30 degrees C. We now present for the first time kinetic evidence that yeast ribosomes lacking a particular protein of the 60S subunit may possess significantly altered peptide bond-forming ability. The k(cat) of peptidyltransferase from mutants lacking ribosomal protein L24 was decreased 3-fold to 0.69 min(-1), whereas the k(cat) from mutants lacking L39 was slightly increased to 3.05 min(-1) and that from mutants lacking both proteins was 1.07 min(-1). These results suggest that the presence of ribosomal proteins L24 and, to a lesser extent, L39 is required for exhibition of the normal catalytic activity of the ribosome. Finally, the L24 or L39 mutants did not affect the rate or the extent of the translocation phase of protein synthesis. However, the absence of L24 caused increased resistance to cycloheximide, a translocation inhibitor. Translocation of Ac-Phe-tRNA from the A- to P-site was inhibited by 50% at 1.4 microM cycloheximide for the L24 mutant compared to 0.7 microM for the wild type.

Cycloheximide↗
PubMed1976

Effects of alpha-amanitin, cycloheximide, and thioacetamide on low molecular weight nuclear RNA.

Studies were made on the effects of alpha-amanitin, cycloheximide, and thioacetamide on synthesis and content of low molecular weight nuclear RNA. Cycloheximide, an inhibitor of protein synthesis and the synthesis of 45S pre-rRNA and 5S RNA, also inhibited synthesis of nuclear U1 and U3 RNAs. alpha-Amanitin, an inhibited the synthesis of U1 and U2 low molecular weight nuclear RNA. Thioacetamide, which induces nucleolar hypertrophy and increased nucleolar RNA polymerase activity, markedly increased synthesis of 5.8S RNA and U3 RNA. These results show that syntheses of individual low molecular weight nuclear (LMWN) RNAs are controlled by different regulatory mechanisms. In particular, there appears to be a specific relationship between U3 RNA and functional states of the nucleolus.

Acetamides↗
PubMed2002

Cycloheximide prevents inhibition of expression of immediate early gene c-fos in paraventricular nuclei of rat hypothalamus produced by delta sleep-inducing peptide.

We studied expression of the immediate early gene c-fos in hypothalamic paraventricular nuclei in rats with different prognostic resistance to emotional stress receiving delta-sleep-inducing peptide after intracerebroventricular administration of cycloheximide. Delta-sleep-inducing peptide inhibited expression of the c-fos gene in rats receiving intracerebroventricular injection of physiological saline. Pretreatment with the protein synthesis blocker cycloheximide abolished changes produced by delta-sleep-inducing peptide.

Animals↗
PubMed1978

The effect of cycloheximide on cholecystokinin-evoked pancreatic juice of the anaesthetized rat.

Cholecystokinin (CCK) is a major stimulant for enzyme secretion by pancreatic acinar cells. In most animals this enzyme secretion is accompanied by a small volume of chloride-rich "carrier" fluid in which the enzymes are dissolved. In order to determine whether this carrier fluid is secreted independently of the pancreatic digestive enzymes, and how its composition is altered by interaction with them, we have studied CCK-evoked juice collected from anaesthetized rats before and after administration of cycloheximide, a powerful inhibitor of protein synthesis. Following treatment with cycloheximide, fluid secretion continued unchanged for 30--40 min but protein excretion declined without appreciable delay towards zero. As protein content declined, the juice bicarbonate content rose from 30 to more than 60 mmole 1(-1) so that the juice resembled that evoked in the rat by secretin. It is concluded that secretion of CCK-evoked fluid is independent of the secretion of digestive enzymes and that the carrier fluid is actually alkaline, like secretin-evoked fluid, and only becomes neutral and chloride-rich by interaction with the acidic contents of zymogen granules.

Anesthesia↗
PubMed1970

Secretion of cell-wall glycoproteins by yeast protoplasts. Effect of 2-deoxy-D-glucose and cycloheximide.

The effect of 2-deoxy-d-glucose and cycloheximide on the synthesis and secretion of the cell-wall constituents protein and mannan in yeast protoplasts was examined in detail. Although the 2-deoxy-d-glucose hardly influenced protein synthesis, a significant parallel inhibition of carbohydrate and protein secretion into the medium was observed. The mechanism of this inhibition is considered as an interference of metabolites of 2-deoxy-d-glucose with the synthesis of yeast mannan. Cycloheximide, which is an effective inhibitor of protein synthesis in yeast (Kerridge, 1958), inhibited the secretion of non-diffusible carbohydrate in yeast protoplasts, but on the other hand had no effect on the activity of particulate yeast mannan synthetase. Our results clearly show that blocking the synthesis of either part of the mannan-protein complex prevents the extracellular appearance of the other component. The nature of this phenomenon is discussed.

Carbon Isotopes↗
PubMed1980

Effect of tunicamycin and cycloheximide on the secretion of acid hydrolases from I-cell cultured fibroblasts.

I-cell cultures fibroblasts secrete excessive amounts of N-acetyl-beta-D-hexosaminidase and alpha-L-fucosidase into the culture media as compared with normal fibroblasts. Addition of tunicamycin or cyd [14C]leucine (40--50%) into trichloroacetic acid-precipitable material decreased the secretion of these I-cell hydrolases to normal values within 24 h, but had no effect on the secretion of acid hydrolases from normal fibroblasts. These results indicate that I-cell cultured fibroblasts secrete at least two types of acid hydrolases: one is tunicamycin- and cycloheximide-sensitive and constitutes the greater proportion of the secreted hydrolases, and a smaller proportion is insensitive to tunicamycin and cycloheximide, similar t9 the acid hydrolases secreted by normal cultured fibroblasts.

Cells, Cultured↗
PubMed1985

Inhibition of protein synthesis and intestinal absorptive cell ultrastructure in cycloheximide or puromycin-treated rats.

Inhibitors of protein synthesis are useful for the investigation of some aspects of intestinal fatty acid absorption. The aim of the present study was to investigate both the biochemical and morphological effects of two inhibitors of protein synthesis on the intestinal mucosa of adult rats. Our results showed that 4 or 4 1/2 h after a single dose of cycloheximide or multiple doses of puromycin, the inhibition of protein synthesis in the jejunal mucosa, measured by the incorporation of 14C-leucine, stabilized at 60% of the controls. At the same time, these two drugs had different effects on absorptive cell morphology: cycloheximide definitely altered the apical surface of these cells, disorganizing their cytoplasm; puromycin did not modify brush border morphology but remodeled the mitochondria and the Golgi complexes.

Animals↗
PubMed1995

Microtubule rearrangement during in vitro maturation of pig oocytes. Effect of cycloheximide.

In freshly isolated fully grown pig oocytes at the germinal vesicle (GV) stage, the cytoplasmic microtubules are arranged in a meshwork. This microtubule arrangement is also maintained during the initial phases of meiotic maturation in vitro. A perinuclear array of microtubules is formed immediately before germinal vesicle breakdown (GVBD). Short-term treatment of oocytes with taxol when oocytes are at metaphase I stage induced formation of cytoplasmic asters. The oocyte cytoplasm is unable to respond to the taxol treatment at the earlier stages of meiotic maturation. In oocytes cultured with the proteosynthesis inhibitor cycloheximide, meiotic maturation is blocked. Condensation of chromatin occurs but the nuclear envelope is preserved and the microtubule arrangement is unchanged. A perinuclear array of microtubules does not appear and oocyte cytoplasm does not respond to short-term taxol treatment by the formation of cytoplasmic asters. We can conclude that the microtubule rearrangement and the acquisition of competence for tubulin assembly are blocked by cycloheximide and are thus dependent on de novo proteosynthesis.

Animals↗
PubMed2002

Synchronization of porcine oocyte meiosis using cycloheximide and its application to the study of regulation by cumulus cells.

This paper describes the use of the protein synthesis inhibitor cycloheximide (CHX) to synchronize nuclear progression during meiotic maturation in porcine oocytes, and also the time-dependence of nuclear maturation on exposure of the oocyte to cumulus cells. Prior to culture, the majority of oocytes were at the germinal vesicle (GV) stage (95-100%), but distributed from GVI to GVIV (GVI 56.1 +/- 9.1%, GVII 15.3 +/- 1.4%, GVIII 21.5 +/- 7.1%, GVIV 7.1 +/- 3.5%). During culture of cumulus-enclosed oocytes (COCs) from 12 h to 48 h in a conventional culture system, all meiotic stages were represented at any time point examined, with 63.6 +/- 4.2% of oocytes maturing to metaphase II (MII). Cycloheximide blocked the progression of nuclear development in a dose-dependent manner. Treatment for 12 h with CHX at 1-25 microg mL(-1) resulted in 95-100% oocytes being arrested and synchronized at GVII. With >5 microg mL(-1) CHX, all oocytes were arrested before germinal vesicle breakdown (GVBD) (mostly at GVIII) by 24 h. A 12 h preincubation with 5 microg mL(-1) CHX followed by 24 h of further culture without CHX resulted in >80% of oocytes maturing to MII. The profile of nuclear progression during maturation revealed discrete peaks of occurrence of different meiotic stages, with GVBD at 6-12 h, metaphase I (MI) at 10-18 h and anaphase I/telophase I at 16-20 h. After 12 h preincubation with 5 microg mL(-1) CHX, denuded oocytes (DOs) matured to MI as COCs. However, DOs matured to MII as normal when denuded at MI. In conclusion, CHX not only efficiently blocks and synchronizes the meiotic progression of porcine oocytes at a specific GV stage, but it also effectively synchronizes subsequent meiotic progression to MII, resulting in discrete peaks of occurrence of different meiotic stages. Using this technique, the study showed that cumulus cells are essential for oocytes to mature from MI to MII but exposure to cumulus cells must occur before MI.

Animals↗
PubMed1976

Induction and decay of glucagon-induced amino acid transport in primary cultures of adult rat liver cells: paradoxical effects of cycloheximide and puromycin.

Liver parenchymal cells were isolated from adult rats and cultured in collagen-coated plastic petri dishes in serum-free medium. Glucagon induced 4- to 5-fold increases in alpha-aminoisobutyric acid (AIB) transport within 6 hr. Dexaemthasone had no direct effect on AIB transport but greatly potentiated the induction by glucagon ("permissive effect"). Levels of 3':5'-cyclic AMP increased 30- to 100-fold within 30 min after glucagon addition to cultures that had been treated with dexamethasone, and dibutyryl cyclic AMP mimicked the glucagon induction of AIB transport. Additionally, dexamethasone exerted a "permissive" effect on induction of AIB transport by dibutyryl cyclic AMP, whereas the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine induced AIB transport only in cultures that had been treated with dexamethasone. Induction of AIB transport was not dependent upon the continued presence of glucagon, but induced AIB transport activity decayed to uninduced levels within 3-4 hr after glucagon removal. The protein syntesis inhibitors puromycin and cycloheximide inhibited both induction and decay of glucagon induced AIB transport, but had a stabilizing effect if added once induction or decay had commenced. Unlike cycloheximide, the inhibitory effect of puromycin on the glucagon induction of AIB transport was reversible.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1976

Temperature dependence of cycloheximide-sensitive phase of circadian cycle in Acetabularia mediterranea.

The biochemical nature of the circadian regulatory system that controls many cellular activities is still unclear. Recent results obtained from the application of protein synthesis inhibitors to individual Acetabularia cells expressing circadian rhythms of photosynthesis indicate that some protein(s) must be synthesized on 80S ribosomes during a discrete part of each cycle to insure correct time-keeping. A comparative study of the effects of brief cycloheximide treatments on cells investigated at different temperature has revealed that the phase of cycloheximide sensitivity is 4-6 hr longer and occurs about 8 hr later in the cycle when cells are kept at 20 degrees rather than 25 degrees. Temperature is known to influence the function of the circadian regulatory system in Acetabularia, but the effect on frequency is small (Q10 approximately equal to 0.8) due to the existence of a temperature-compensating feature. The large effects of temperature observed here thus favor the interpretation that protein synthesis on 80S ribosomes, while providing an essential component of the circadian timing mechanism, does not itself generate the period of the photosynthesis rhythm.

Acetabularia↗
PubMed1979

Conservative segregation of parental histones during replication in the presence of cycloheximide.

Long stretches of protein-free, nonbeaded DNA were observed electron microscopically in nuclear spreads prepared from cells that had replicated their DNA in the absence of protein synthesis. The amount of this DNA increased with increasing time of replication in the presence of cycloheximide and was greatly decreased when replication was inhibited with 1-beta-D-arabinofuranosylcytosine (cytosine arabinoside). This DNA is considered to be "free" DNA because it has the same diameter as marker PM2 DNA and it is preferentially sensitive to DNase I digestion. Reversal of the cycloheximide block resulted in a burst of histone synthesis and repair of the depleted chromatin within 5 min. In addition, 26 presumptive replication forks were observed with beaded chromatin on two arms and free DNA on the third. These results suggest that new histones are usually deposited onto new DNA, that the cellular histone pool is very small, that histone migration is minimal in vivo for at least 18 hr, that for most fibers nuclesome assembly and segregation is conservative for stretches of DNA as long as 100 kbases, and that some part of the octameric histone core may remain bound to DNA during the replication process. The regularity we have observed for the assembly and segregation of nucleosomes is likely to be important for our understanding of how chromosomal information is segregated during development.

Cell Line↗
PubMed1979

Cycloheximide inhibition of hormonal induction of alpha 2u-globulin mRNA.

The induction of hepatic alpha 2u-globulin synthesis by glucocorticoids in isolated hepatocytes occurs via an increase in the level of its mRNA as measured by cell-free translation and by hydbridization to an alpha 2u-globulin cDNA probe. To explore whether induction of this mRNA is a direct or an indirect consequence of the interaction of the dexamethasone-receptor complex with the alpha 2u-globulin genome, the requirement for ongoing protein synthesis was examined. Concentrations of cycloheximide too low to prevent precursor incorporation into total poly(A)-containing RNA do prevent the hormonal induction of alpha 2u-globulin mRNA. Furthermore, incorporation of 3H-labeled amino acids into total protein was decreased by only 40-50%, and the appearance of the dexamethasone-induced glycosylated forms of alpha 2u-globulin was completely prevented in these cycloheximide-treated hepatocytes. The results suggest that the synthesis of a protein mediator(s) may be required for the induction of alpha 2u-globulin mRNA by glucocorticoids and that the steroid-receptor complex may not interact directly with the alpha 2u-globulin genome.

Alpha-Globulins↗
PubMed1980

Kinetic aspects of cycloheximide-induced reversal of adrenocorticotropin effects on steroidogenesis and adrenal phospholipids in vivo.

Intraperitoneal injection of maximally effective doses of corticotropin(1-24) [ACTH(1-24)] provoked maximal increases in rat adrenal phospholipids as follows: phosphatidic acid within 1.5-2 min, phosphatidylinositol and phosphatidylglycerol within 4-6 min, and polyphosphoinositides and corticosterone within 5-15 min. Continued maximal adrenal stimulation by ACTH(1-18) treatment caused sustained increases in adrenal phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, polyphosphoinositides, and corticosterone. Treatment with cycloheximide during this steady-state caused rapid decreases in all of these substances to basal levels. The observed half-lives of adrenal phosphatide acid, phosphatidylinositol, polyphosphoinositides, phosphatidylglycerol, and corticosterone during cycloheximide inhibition were 0.15, 1.0, 1.7, 3.3, and 3.5 min, respectively. Calculated production rates during maximal ACTH stimulation were 1060, 991, 90, 34, and 41 nmol/g of tissue per min, respectively. These findings suggest that (i) an initial effect of ACTH on de novo synthesis of phosphatidic acid can account for all subsequently observed increases in other phospholipid derivatives of CDP-diacylglycerol, (ii) a labile protein is required for the ACTH-induced increase in phosphatidic acid, (iii) the phosphatidate leads to polyphosphoinositide-polyglycerophospholipid pathway is rapidly and dramatically responsive to hormonal stimulation, (iv) changes in steroidogenesis correlate well with changes in this phospholipid pathway, and (v) stimulation of this pathway is rapidly reversible.

Adrenal Cortex↗
PubMed1984

Specific stimulation of actin gene transcription by epidermal growth factor and cycloheximide.

Stimulation of quiescent AKR-2B mouse embryo cells with epidermal growth factor (EGF) results in a rapid and specific induction of actin mRNA sequences. These mRNAs include those coding for both beta- and gamma-cytoskeletal, but not alpha-skeletal muscle, actin isotypes. Elongation of nascent RNA chains in isolated nuclei (run-off transcription) demonstrates that the mRNA accumulation is preceded by an increase in actin gene transcription. This increase is transient, however, and is followed by a rapid attenuation of transcriptional activity. An inhibitor of protein synthesis, cycloheximide, was also found to induce beta- and gamma-actin mRNA accumulation. Furthermore, the simultaneous addition of EGF and cycloheximide produced a synergistic effect on actin sequences in both steady-state nuclear and polysomal RNA. Run-off transcription experiments demonstrate that this synergistic effect results from an increase in the magnitude and duration of actin gene transcription. It is also specific in that alpha-tubulin gene transcription is not similarly affected. These data suggest the existence of a specific labile repressor of actin gene transcription.

Actins↗
PubMed1989

Early estrogen-induced metabolic changes and their inhibition by actinomycin D and cycloheximide in human breast cancer cells: 31P and 13C NMR studies.

Metabolic changes following estrogen stimulation and the inhibition of these changes in the presence of actinomycin D and cycloheximide were monitored continuously in perfused human breast cancer T47D clone 11 cells with 31P and 13C NMR techniques. The experiments were performed by estrogen rescue of tamoxifen-treated cells. Immediately after perfusion with estrogen-containing medium, a continuous enhancement in the rates of glucose consumption, lactate production by glycolysis, and glutamate synthesis by the Krebs cycle occurred with a persistent 2-fold increase at 4 hr. The content of phosphocholine had increased by 10% to 30% within the first hour of estrogen stimulation, but the content of the other observed phosphate metabolites as well as the pH remained unchanged. Pretreatment with either actinomycin D or cycloheximide, at concentrations known to inhibit mRNA and protein synthesis, respectively, and simultaneous treatment with estrogen and each inhibitor prevented the estrogen-induced changes in glucose metabolism. This suggested that the observed estrogen stimulation required synthesis of mRNA and protein. These inhibitors also modulated several metabolic activities that were not related to estrogen stimulation. The observed changes in the in vivo kinetics of glucose metabolism may provide a means for the early detection of the response of human breast cancer cells to estrogen versus tamoxifen treatment.

Breast Neoplasms↗
PubMed1996

Cytosolic and mitochondrial proteins as possible targets of cycloheximide effect on adrenal steroidogenesis.

It is well accepted that protein(s) with a short half-life are required in the pathway leading to steroid synthesis following stimulation by trophic hormones. A correlation between the disappearance of several proteins in different subcellular compartments and the inhibition of steroid synthesis produced by cycloheximide (CHx) has also been shown. In the present report we describe the effect of CHx in the stimulation of steroid synthesis using a cell-free assay. Mitochondrial progesterone (P4) production was studied by recombination of the different subcellular fractions of adrenal zona fasciculata and determined by radioimmunoassay. Soluble factors from ACTH-treated adrenals produced a four-fold stimulation of mitochondrial steroidogenesis (3.0 +/- 0.6 vs. 13.3 +/- 0.5 ng P4/tube for control and ACTH-treated adrenals respectively). Mitochondria obtained from CHx-ACTH-treated adrenals fail to respond to soluble ACTH-dependent factors. A permeable analogue of cholesterol (22(R)-OH cholesterol) could overcome the inhibition imposed by CHx, confirming the role of mitochondrial proteins in intramitochondrial cholesterol transport. The treatment of the adrenals with CHx 10 minutes before ACTH administration abolished also the stimulation induced by the cytosol on control mitochondria (2.6 +/- 0.5 vs. 13.0 +/- 1.0 ng P4/tube for CHx-ACTH-treated cytosol vs. ACTH-treated cytosol). Arachidonic acid (AA) added to CHx-ACTH-treated cytosol subdued this inhibition (10.3 +/- 1.2 ng P4/tube). CHx treatment had no effect on the stimulation by ACTH of the cAMP-dependent protein kinase. These results indicate the involvement of a cycloheximide-sensitive protein in the release of AA in adrenal steroidogenesis.

Animals↗