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Cycloheximide inhibition of ovulation, prostaglandin biosynthesis and steroidogenesis in rabbit ovarian follicles.

Cycloheximide (5 mg/kg, i.v.) significantly inhibited ovulation in the rabbit when it was administered as early as 20 h before the ovulation process was initiated by hCG, and as late as 1 h after hCG. The ovulation rate was significantly reduced, but follicular biosynthesis of prostaglandins E and F was only partly inhibited. The biosynthesis of progesterone and oestradiol in follicles during the early stages of the ovulation process was also inhibited. Cycloheximide may therefore inhibit ovulation by a mechanism which is different from the action of indomethacin, and this mechanism may involve the suppression of ovarian steroidogenesis.

Animals↗

Effects of actinomycin D and cycloheximide on the differentiation of epidermal melanocytes of newborn mice.

The increase in the number of epidermal melanocytes positive to the dopa reaction after birth was suppressed in C57BL/10J strain mice when injected with actinomycin D or cycloheximide. On the other hand, no change was observed in the melanoblast-melanocyte population positive to the combined dopa-premelanin reaction. The initiation of melanogenesis in the skin of newborn mice, therefore, seems to require de novo transcription and translation. The increase in the number of melanocytes after treatment with alpha-MSH or DBc-AMP was also completely suppressed when mice were injected with actinomycin D or cycloheximide. The results suggest that normal and hormone-induced differentiation of epidermal melanocytes requires de novo transcription and translation.

Age Factors↗

Protection with cycloheximide or emetine against pulmonary edema induced by ozone or nitrogen dioxide.

Pretreatment with cycloheximide or emetine provided significant protection against pulmonary edema in rats exposed to ozone or nitrogen dioxide. Other inhibitors of protein-synthesis, actinomycin D or puromycin, failed to show such effects. Possible actions of these agents as well as the doses and times that afforded the significant protection were investigated. These agents, by themselves, did not alter the water content of the lungs. In vitro study revealed that both cycloheximide and emetine hardly acted as scavengers of oxidant. Pretreatment with either agent was associated with a significant increase in the activity of glucose 6-phosphate dehydrogenase of the lungs, but the increase did not necessarily coincide with the protection. Activity levels of non-protein SH, glutathione-peroxidase and -reductase in the lungs of rats treated with either agent were scarcely altered. The effect of these agents administered in vivo or in vitro on the in vitro lipid peroxidation by air was also investigated. Other possible mechanisms of these agents responsible for the protective effect against pulmonary edema induced by oxidants were also discussed.

Animals↗

Effects of growth hormone-releasing factor (1-44) on growth hormone release from human somatotrophinomas in vitro: interaction with somatostatin, dopamine, vasoactive intestinal peptide and cycloheximide.

The effect of GH-releasing factor(1-44)(GRF) alone, or together with somatostatin (SRIF), dopamine (DA), vasoactive intestinal peptide (VIP) or cycloheximide was studied in a total of ten human somatotrophinomas using a static cell culture system. Growth hormone-releasing factor (2.0 X 10(-8) mol/l) significantly (P less than 0.05) stimulated GH release from nine out of ten tumours over 4-h incubations, and a dose-related effect (2.0 X 10(-10) -2.0 X 10(-8) mol/l) was observed in five tumours thus studied. Repeated GRF (2.0 X 10(-8) mol/l)-mediated GH release was seen during 96% (n = 25) of experiments performed on six tumours over 4 h and up to 27 days in culture. Growth hormone-releasing factor (2.0 X 10(-8) mol/l) also stimulated GH release from five out of seven somatotrophinomas during 60-min incubations. Somatostatin (6.1 X 10(-9) mol/l) completely inhibited GRF-induced GH secretion from four tumours studied over 4 h, but in each case there was significant (P less than 0.05) 'rebound' of GH release from cultures exposed to both GRF and SRIF during a subsequent recovery period. Dopamine suppressed basal GH release from two out of four tumours, but in each case had a greater inhibitory effect on GRF-mediated GH release. Vasoactive intestinal peptide directly stimulated GH release from two out of three tumours, and the effects were additive to maximal stimulatory doses of GRF. Cycloheximide significantly (P less than 0.01) enhanced GRF-stimulated release of GH during a 60-min incubation, but inhibited both basal and GRF-stimulated release over 4 and 8 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗

Combined cycloheximide and 8-hydroxyquinoline pretreatment for study of plant chromosomes.

The actions of cycloheximide and 8-hydroxyquinoline on dividing cells of root meristems of Zea mays L. have been studied during the development of a new cytological technique for sugar cane (Saccharum) root tips. The determination of mitotic phase indices revealed that combined treatment with cycloheximide (70 ppm) plus 8-hydroxyquinoline (250 ppm) was superior to treatments with either chemical separately. After the combined treatment, the preparations contained nearly ten times more cells in prophase and metaphase that were suitable for chromosome counting than those given a single pretreatment with 8-hydroxyquinoline. This new pretreatment has been developed especially for chromosome studies in tropical grasses with a large number of small chromosomes. However, both chemicals are active in a wide range of plant species.

Chromosomes↗

Effect of cycloheximide and actinomycin D on thymidylate synthase and thymidine kinase in regenerating rat liver after partial hepatectomy.

Thymidylate synthase and thymidine kinase, which catalyze the formation of thymidylate via the de novo and salvage pathways, respectively, are rate-determining enzymes in DNA synthesis. The increases in the activities of hepatic thymidylate synthase and thymidine kinase were significantly suppressed at 24 h after 70% partial hepatectomy in rats that had been administered cycloheximide. Concomitantly, other regenerative parameters such as the liver weight and contents of protein, RNA, and DNA were also significantly reduced in 24-h regenerating liver of cycloheximide-treated rats. When actinomycin D was injected, the activity of thymidine kinase, the liver weight, and contents of protein, RNA, and DNA were completely depressed in 24-h regenerating liver. However, the activity of thymidylate synthase in actinomycin D-administered rats rose to the level similar to the control (70% partially hepatectomized). The immunoblotting assay showed that thymidylate synthase is newly synthesized during liver regeneration after partial hepatectomy without being affected with actinomycin D.

Animals↗

Effects of cycloheximide and streptovitacin A on protein synthesis and gastric secretion in rats.

Cycloheximide and streptovitacin A administered in vivo to rats display a similar dual effect on the labelling of soluble liver proteins by valine-14C, and result in a similar enhancement of liver uridine kinase activity. On the other hand, in pylorus-ligated rats, both antibiotics markedly depress gastric secretion, acid output, and the level of mucoproteins and proteolytic activity in secreted juice. Streptovitacin A on a molar basis was in all cases 5 approximately 8 times more effective than cycloheximide.

Animals↗

Effect of cycloheximide on pregnenolone-16alpha-carbonitrile (PCN)-induced ultrastructural changes in the rat liver.

Cycloheximide (10 mg/100 g body weight i.v. twice), an inhibitor of protein synthesis, caused smooth endoplasmic reticulum (SER) accumulation in the hepatocytes of female rats. Pregnenolone-16alpha-carbonitrile (PCN), given p.o., had a similar effect. When cycloheximide was administered to PCN-treated rats, SER proliferation as well as myelin figure formation were noted.

Animals↗

On the regulation of rat testicular steroidogenesis. Short term effect of luteinizing hormone and cycloheximide in vivo and Ca2+ in vitro on steroid production in cell-free systems.

An attempt has been made to correlate the rapid effect of luteinizing hormone on testicular steroid production in vivo with testicular steroid concentrations and in vitro steroid production rates in testis tissue preparations. Within 20 min after intravenous administration of 25 mug luteinizing hormone, increases were observed in testosterone concentrations in testicular venous plasma and in whole testis tissue and in pregnenlone concentrations isolated testis mitochondrial fractions. Testosterone production by whole testis homogenates and pregnenolone production by isolated mitochondrial fractions were significantly increased within 5 min after in vivo administration of luteinizing hormone. Injection of cycloheximide 10 min prior to luteinizing hormone prevented the stimulating effect of luteinizing hormone to steroid levels in testicular venous plasma and testis tissue and on steroid production rates by preparations of rat testis tissue. Cycloheximide treatment of control animals did not significantly alter testosterone concentrations and testosterone production rates vitro, although mitochondrial pregnenolone concentrations and production rates were decreased. Testosterone production by whole testis homogenates as well as the pregnenolone production by isolated mitochondrial fractions obtained from luteinizing hormone treated testes and control glands showed a biphasic time curve A period (5-10 min) of high steroid production was followed by a period lower steroid production. Addition of 25 mug luteinizing hormone or 10(-8)--10(-5) M adenosine 3':5'-monophosphate (cyclic AMP) to the incubation medium had no effect pregnenolone production by isolated mitochondrial fractions. Administration of leuteinizing hormone in vivo markedly enhance the stimulating effect of Ca2+ on testosterone production by whole testis homogenates and on pregnenolone production by isolated mitochondrial fractions.

Animals↗

[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.

Cell Membrane Permeability↗

Cycloheximide blocks TGF-beta1-induced apoptosis in murine hepatocytes.

AIM: To study the mechanism of transforming growth factor beta1-induced apoptosis in cultured hepatocytes. METHODS: DNA fragmentation and fluorescent microscopy were used to characterize cell apoptosis. Crystal violet staining was used to assess cell viability. Immunoblotting was used to detect Tak1, p53, and Bax. Dual luciferase assay was used to determine TGF-beta1-induced gene expression. Thin layer chromatography was used to examine ceramide level in AML12 cells. RESULTS: In response to TGF-beta1 treatment, AML12 cells exhibited typical chang es, which was characteristic of apoptosis, such as condensation of chromatin, disintegration of nuclei, and DNA fragmentation. TGF-beta1-induced apoptosis in AML12 cells was completely blocked in the presence of cycloheximide. The inhibitory effect of cycloheximide was accompanied with down-regulation of Tak1 expression and TGF-beta1-induced PAI-1 expression. TGF-beta1 induced p53 expression but not Bax. No increase of ceramide was observed in TGF-beta1-induced apoptosis. CONCLUSION: TGF-beta1-induced apoptosis requires TGF-beta1-induced gene expression.

Animals↗

[The effect of cycloheximide on incorporation of newly formed mRNA into membrane-bound polyribosomes in rat liver cells].

Incorporation kinetics of new synthesized mRNA into free and endoplasmic membrane-bound polyribosomes in the absence of normal translation (when protein synthesis in inhibited by 98% with cycloheximide) is studied. mRNA is found to incorporate into both free and bound polyribosomes. Relative content of new synthesized membrane-bound polyribosomes in the presence of cycloheximide within 2.5-4.5 hours is by 30-40% lower as compared with the control. This fact can be explained either by the absence of a growing peptide of a sufficient length, which is necessary for the formation of a part of membrane-bound polyribosomes, or by a restricted number of attachment sites on membranes as a result of delayed translation of mRNA in pre-existed polyribosomes. It is suggested that 1) the growing peptide in liver cells is responsible for the recognition of a membrane only under the formation of only one type of membrane-bound polyribosomes, or 2) the formation of all bound polyribosomes has a single mechanism and the growing peptide does not participates in the membrane recognition.

Animals↗

[Ataxia telangiectasia: effects of cycloheximide on G2 repair of chromosome damage in lymphocytes cultured in vitro].

The chromosomal sensitivity to the spontaneous and induced DNA damage detected in lymphocytes from patients affected with ataxia telangiectasia (AT) might be related with disturbances in DNA repair mechanisms. We have studied the effect of caffeine, an inhibitor of G2 repair and the enlargement of G2 by 0.5 ug/ml cycloheximide on chromosomal aberration frequencies in AT lymphocytes, both in control and X-ray irradiated conditions. The increase of spontaneous and X-ray induced chromosomal aberrations by caffeine treatments during G2 in AT lymphocytes was higher than in control cells. The number of spontaneous and X-ray induced lesions repaired during G2 in AT cells was higher than in normal cells. The enlargement of G2 duration by 0.5 ug/ml cycloheximide decreased the spontaneous and X-ray induced chromosomal aberrations in AT cells, whereas no such effect was observed in control cells. We postulate that disturbances in the mechanism that control G2 duration might be involved in the high frequency of spontaneous and induced chromosomal aberrations detected in AT lymphocytes.

Ataxia Telangiectasia↗

Cycloheximide enhances maintenance of methamphetamine-induced conditioned place preference.

Accrued evidence demonstrated the necessity of protein synthesis at acquisition, consolidation and expression stages in conditioning/learning tasks, while the underlying mechanisms of the maintenance of memory remained less explored. This study was designed to characterize the maintenance of methamphetamine-induced conditioned place preference, a drug-induced learning and memory. In addition, cycloheximide, a protein synthesis inhibitor, was used to examine the involvement of protein synthesis in the maintenance of such place preference memory. We found that the maintenance of the rapidly-established methamphetamine (2 mg/kg, i.p.) -induced conditioned place preference could be long-lasting and even over fifty days under the present protocol of extinction. Moreover, it was of interest to note the undulating expression of this conditioned place preference throughout the extinction protocol. Most importantly, as the methamphetamine-induced conditioned place preference was acquired and expressed by mice, the saline-pretreated control mice underwent numbers of intermittent extinction across a long-term retention test period, while cycloheximide-pretreated mice exhibited unaltered methamphetamine-induced conditioned place preference throughout the same retention test period. Taken together, we conclude that [1] methamphetamine-induced conditioned place preference could last for a long period of time, and such place preference memory is reluctant to extinguish even animals' repeated exposure to the previous conditioned environment at a drug-free status, and [2] blockade of protein synthesis may enhance the maintenance of the methamphetamine-induced conditioned place preference.

Animals↗

Cycloheximide-mediated sensitization to TNF-alpha-induced apoptosis in human colorectal cancer cell line COLO 205; role of FLIP and metabolic inhibitors.

Current efforts are focused on revealing the cellular factors that determine the "immune escape" of cancer cells. As an example in our study human colorectal cancer cell line COLO 205 was resistant to TNF-alpha - a death inducing ligand. Nonetheless, co-incubation TNF-alpha (10 ng/ml) with cycloheximide (5 micro g/ml, CHX) caused time-dependent (6, 12, 24 hours) cell death even though, at that concentration cycloheximide did not exert cytotoxic effect unless 24 hour of treatment. After additional pretreatment it is concluded that CHX sensitizes cells to TNF-alpha-induced apoptosis. The aim of this study was to investigate the role and properties of cellular proteins that granted COLO 205 cells survival as well as to assess the effect of several metabolic inhibitors on cell viability at the above-mentioned time-points. PKCs inhibitor staurosporine (5 microM) did not influence, whereas cPKC activator PMA (100 microM) prevented TNF-alpha-induced apoptosis in the presence of CHX. Although EDTA (2 mM) and to a lesser degree EGTA (5 mM) were individually cytotoxic, they exerted protective effect at 6 and partially at 12 hour of combined TNF-alpha and CHX treatment. Ionophore A23187 (1 microM) protected cells against cell death at 12 and 24 but only partially at 6 hour of treatment. On the other hand, AD (10 ng/ml) acted synergistically with TNF-alpha and CHX at 6 and 12 hour. It appears that resistance of COLO 205 cells is determined on genomic level by a few reaction steps in which both Ca(2+)-dependent and antiapoptotic proteins are involved. Further studies revealed that CHX treatment reduces the level of FLIP but not other members of TNF-alpha-dependent death signalosome.

Apoptosis↗

[The effect of p38 on the cycloheximide-induced HL-60 cell death through mitochondria pathway].

OBJECTIVE: To study the effect of p38 on the cycloheximide (CHX)-induced HL-60 cell death through mitochondria pathway. METHODS: Inhibition of p38 pathway was by SB203580 (SB). Four groups were set up: control, SB only, CHX only and SB + CHX. Sub-diploid cell ratio was detected by PI staining flow cytometry at 6, 9, 12, 18, 24 h time points, and apoptotic cell ratio by Annexin V-FITC/PI double staining flow cytometry at 6 h and 18 h time points. High J-aggregate cells were evaluated by the J-aggregate contents, measurement of the J-aggregate (FL2) and J-monomer (FL1) by JC-1 flow cytometry, calculation of the delta psi m by FL2/FL1 and analysis of the delta psi m changes at 18 h time points. RESULTS: The sub-diploid cell ratio in CHX group was significantly higher than that in control group at 6 h time point, and the ratio in SB + CHX group was significantly higher than that in CHX group at 9 h time point. At 18 h time point the apoptotic cell ratios in both CHX and SB + CHX groups were significantly higher than those in control group (P < 0.01). There was no significant difference of apoptotic cell ratio between CHX group and SB + CHX group (P > 0.05). At 18 h time point the necrotic cell ratios in both CHX and SB + CHX groups were significantly higher than that in control group (P < 0.01); and that in SB + CHX group was significantly higher than that in CHX group (P < 0.01). The high J-aggregate cell ratios in CHX and SB + CHX groups were significantly lower than that in control group (P < 0.05), and that was signficantly lower in SB + CHX group than in CHX group (P < 0.01). For the FL2/FL1 value (delta psi m) CHX group (0.17 +/- 0.01) and SB + CHX group (0.05 +/- 0.003) were significantly higher than control group (0.38 +/- 0.02) (P < 0.01), and SB + CHX group was significantly lower than CHX group (P < 0.01). CONCLUSION: CHX can induce HL-60 cell apoptosis and the cell mitochondria depolarization, and the latter was intensified by inhibition of the p38 pathway. p38 pathway may related to the cell necrosis in the cycloheximide-induced HL-60 cell apoptosis model. s

Apoptosis↗

Use of cycloheximide on intracellular growth of Mycobacterium leprae in cultured murine macrophages.

Mycobacterium leprae is an obligate intracellular parasite and grows within mononuclear phagocytes. When murine macrophages derived from the peritoneal cavities of CBA mice were infected in vitro with M. leprae (Thai-53 strain), intracellular multiplication was observed three weeks after infection. On the other hand, there was no increase in the number of heat-killed M. leprae at the same times after infection. Morphological studies showed that the growth rate of the bacteria increased by about 20-30% in medium supplemented with cycloheximide. With the addition of cycloheximide to the culture medium, metabolic activity of macrophages was decreased but infected cells were maintained in good condition and seldom floated off from the culture flask.

Animals↗

[The effect of cycloheximide on the entry into the S period of the nuclei in heterodikaryons].

Serum-deprived (0.2%) resting NIH 3T3 mouse fibroblasts were fused with serum-stimulated (10%) proliferating cells to elucidate mechanisms of entering into S-period operating in the nuclei of the heterokaryons under the effect of cycloheximide--an inhibitor of protein synthesis. Using radioautography DNA synthesis was investigated in mono-, homo- and heterodikaryons. After short (0.5-3.0 h) depressing of protein synthesis, the nuclei of stimulated cells in heterokaryons were found to enter into S-period. Under these conditions no induction of DNA synthesis was found in the nuclei of resting cells in heterodikaryons. In other experiments, resting cells were under the effect of cycloheximide during 2-4 h before the fusion, that led to a great induction of DNA synthesis in the nuclei of these cells in heterodikaryons. The data obtained are consistent with the idea of fibroblast transition to the rest under the action of labile proteins-repressors.

3T3 Cells↗