Cycloheximide In Combination Research Articles

TNF Apoptosis Protection Fraction (TAPF) prevents apoptosis induced by TNF, but not by Fas or TRAIL, via NF-κB-induced increase in cFLIP

Tumor Necrosis Factor α (TNFα) induces both the apoptotic pathway and anti-apoptotic factors. Incubation of human dermal fibroblasts with TAPF (TNF Apoptosis Protection Fraction) protects them from apoptosis induced by the subsequent addition of TNF and cycloheximide (CHX). TAPF does not protect against apoptosis induced by CHX in combination with either TRAIL (TNF related apoptosis inducing ligand) or an agonistic Fas antibody, or against apoptosis induced by the chemotherapeutic agent doxorubicin. Incubation with TAPF does not affect the quantity of TNF that binds to the cell. TAPF prevents TNF-induced cleavage of caspases 8, 9, 3 and 7 and the apoptotic substrate PARP (poly-ADP ribose polymerase), but has no effect when these molecules are induced by an agonistic Fas antibody. TAPF induces rapid phosphorylation of the NF-κB/p65 (nuclear factor-κB) transcription factor at serine 536 which is indicative of its activation. TAPF increases the expression of cFLIP (cellular FLICE-inhibitory protein) which is a potent inhibitor of apoptosis that acts by preventing the cleavage of caspase 8. This increase in cFLIP is coincident with protection from TNF-induced apoptosis. Decreasing cFLIP levels using shRNA (short hairpin RNA) decreases protection by TAPF. TAPF also induced the anti-apoptotic A20 protein. These data indicate that TAPF protects human dermal fibroblasts from TNF-induced apoptosis by induction of cFLIP and subsequent inhibition of caspase 8 cleavage.

Long-term phase reorganization of conditioned food aversion memory in edible snail.

The specific features of memory reconsolidation in edible snails were studied over 30 days after learning of conditioned food aversion. Injections of a NMDA glutamate receptor antagonist MK-801 or protein synthesis inhibitor cycloheximide in combination with the conditioned food stimulus (reminder) on day 2 after learning were followed by the development of amnesia. Repeated training on day 10 after the induction of amnesia did not result in skill formation. Injections of MK-801 or cycloheximide and reminder by the 10th day after training had no effect on memory retention. Injections of MK-801 or cycloheximide and reminder by the 30th day after training were followed by the development of amnesia. In these experiments, memory was recovered after repeated training. Our results indicate that a complex phase reorganization of memory occurs over 30 days after learning. This process includes memory consolidation over the first days after training, stabilization and resistance to adverse factors after 10 days, and newly acquired ability for reconsolidation by the 30th day after training.

Artificial Meiotic Arrest and Parthenogenetic Activation of Buffalo Oocytes Using Cycloheximide

Maintaining oocytes at germinal vesicle (GV) stage without damaging their quality would allow synchronization of maturation and homogenization of the oocyte population. Activation of the oocyte is very important for a number of oocyte or embryo related technologies including intracytoplasmic sperm injection and cloning by nuclear transfer. This work will focus on induction of meiotic inhibition and oocyte activation by cycloheximide (CHX), in buffalo. For this purpose 2 experiments were conducted. In experiment I, buffalo cumulus-oocyte complexes (COCs) were cultured in 100 μl droplets of tissue culture medium 199 enriched with 10% v/v fetal calf serum, FSH and LH (0, 05 iu/ml, PERGOVET 75, Serono, Rome, Italy) and 50 μg/ml gentamycin for 24–26 h at 38.5°C in 5% CO2 and humidified air for 24 h in the presence of 2 μg/ml CHX. COCs were significantly blocked at the GV stage (20.85% vs. 82.72% in control). Reversibility of the CHX effect was assessed by culturing COCs an additional 24 h in CHX-free culture medium. About 65.0% of treated oocytes (control 80.1%) resumed meiosis and progressed to the MII stage. In experiment II, buffalo oocytes were activated by 10 μM calcium ionophore A23187 for 5 min followed by 10 μg/ml CHX for 3 h. Control oocytes were exposed to conventional in vitro fertilization (IVF) in BO medium. After 48 h of culture, significantly more oocytes (p<0.05) were cleaved in the IVF group than in treated group and spontaneous activated one. In conclusion, CHX can be used to block spontaneous resumption of meiosis in buffalo oocytes and its effect is reversible. Also, CHX in combination with calcium ionophore have the ability to induce parthenogenetic activation in buffalo oocytes.

Modulation of CCK mRNA in cell lines in response to isoproterenol and retinoic acid.

Regulation of cholecystokinin (CCK) expression was studied in the human neuroepithelioma cell line SK-N-MCIXC and the rat medullary thyroid carcinoma cell line WE 4/2. The cells were treated with the beta-adrenergic agonist isoproterenol and retinoic acid, a natural derivative of vitamin A, which plays a role in cell growth and proliferation. Levels of CCK mRNA were determined after 6, 12 and 24 h drug treatments, with Northern blot analysis using human CCK riboprobes. In WE 4/2 cells no differences were observed in CCK mRNA levels, between control and isoproterenol treated cells, after 6, 12 or 24 h treatments. In SK-N-MCIXC cells isoproterenol increased CCK mRNA levels at all time points examined, the beta-adrenergic antagonist propranolol blocked this effect. SK-N-MCIXC cells were also treated with actinomycin D or cycloheximide in combination with isoproterenol. Actinomycin D decreased CCK mRNA levels. Cycloheximide increased CCK mRNA levels when compared to isoproterenol acting alone. Retinoic acid did not affect CCK mRNA levels in WE 4/2 cells. In SK-N-MCIXC cells, retinoic acid consistently decreased CCK mRNA level. CCK mRNA levels in SK-N-MCIXC cells treated with retinoic acid combined with either isoproterenol or phorbol-12-myristate-13 acetate, were not significantly different from cells treated with retinoic acid alone.

Regulation of Type 1 Plasminogen Activator Inhibitor Gene Expression in Cultured Bovine Aortic Endothelial Cells: Induction by transforming growth factor-β, lipopolysaccharide, and tumor necrosis factor-α

Cultured bovine aortic endothelial cells (BAEs) synthesize and secrete type 1 plasminogen activator inhibitor (PAI-1), an Mr 50,000 glycoprotein which inhibits both urokinase and tissue-type plasminogen activators. The synthesis of PAI-1 in BAEs is positively regulated by a variety of agents. To elucidate the mechanisms which govern expression of the PAI-1 gene, total cytoplasmic RNA was prepared from BAEs and analyzed by Northern blotting using a 1.3-kilobase (kb) human PAI-1 cDNA probe. Hybridization under conditions of high stringency revealed two bovine PAI-1 RNA species, 3.0 and 1.6 kb in length. The ratio of the two species was approximately 4:1. The 3.0-kb mRNA was bound by oligo(dT)-cellulose, whereas the 1.6-kb form was not, suggesting that the latter form lacked a poly(A) terminus. Treatment of BAEs with transforming growth factor beta (TGF-beta), bacterial lipopolysaccharide (LPS), or tumor necrosis factor alpha (TNF-alpha) markedly enhanced the steady-state levels of both RNA species. In each case, increases were detectable within 1 h, and maximal effects (i.e. greater than 30-fold increase) were observed between 6 and 18 h of treatment, followed by a decline to near-basal levels by 48 h. The response to each of these agents was dose-dependent, with maximal induction observed at concentrations of 10 ng/ml TGF-beta, 10 ng/ml LPS, and 25 ng/ml TNF-alpha. Induction of PAI-1 mRNA by these agents was not blocked by the protein synthesis inhibitor cycloheximide, suggesting that de novo protein synthesis was not required. In fact, treatment with cycloheximide (2 micrograms/ml) alone also increased PAI-1 mRNA levels. Treatment with cycloheximide in combination with TGF-beta, LPS, or TNF-alpha further enhanced the accumulation of PAI-1 mRNA. Nuclear transcription run-on experiments indicated that these agents elevated the rate of PAI-1 gene transcription 20-30-fold and that gene template activity was temporally correlated with the accumulation of PAI-1 mRNA. These data are consistent with the conclusion that the observed increases in PAI-1 steady-state mRNA levels result from primary effects of these agents on the rate of PAI-1 gene transcription.

Transcriptional and posttranscriptional regulation of tumor necrosis factor gene expression in human monocytes.

Regulation of tumor necrosis factor (TNF) gene expression was investigated in resting human monocytes and in 12-O-tetradecanoylphorbol-13-acetate (TPA) activated monocytes. TNF transcripts were undetectable in resting monocytes. However, in TPA-activated monocytes, TNF mRNA was first detectable by 3 h and reached maximal levels by 12 h of drug exposure. Using run-on transcription assays, the TNF gene was transcriptionally inactive in resting monocytes, but was rapidly activated after TPA exposure. The protein synthesis inhibitor, cycloheximide (CHX), had no detectable effect on levels of TNF transcripts in resting monocytes, while this agent superinduced the level of TNF mRNA by 50-fold in TPA-activated cells. TPA activated monocytes were also exposed to actinomycin D and/or CHX to determine whether transcriptional or posttranscriptional control of TNF gene expression was responsible for the induction of TNF transcripts. After 1 h of actinomycin D treatment, the amount of TNF transcripts was reduced by 75%. In contrast, no difference in TNF mRNA levels was observed in TPA-activated monocytes exposed to CHX alone or CHX in combination with actinomycin D. These findings indicated that CHX prevented the degradation of TNF mRNA by inhibiting the synthesis of a labile protein. Run-on transcription assays performed on cells exposed to either TPA or the combination of TPA and CHX further indicated that CHX treatment increased transcription of the TNF gene. Thus, TNF gene expression is controlled at the transcriptional level in resting human monocytes, while both transcriptional and posttranscriptional events regulate the level of TNF transcripts in TPA-activated cells.

Translational sites of thylakoid membrane proteins. Studies based on selective inhibition of cytoplasmic and chloroplast ribosomes by cycloheximide and chloramphenicol

To inhibit chloroplast and cytoplasmic protein synthesis, respectively, excised shoots of 19-day-old Vicia faba plants and 10-day-old Hordeum vulgare plants were incubated with d-threochloramphenicol or cycloheximide in combination with l-[ 35S]methionine. Incubation was performed for 22 h under light subsequent to a 30 min inhibitor pre-incubation without methionine. In order to analyse the translational sites of thylakoid membrane polypeptides, isolated thylakoids were dispersed by sodium dodecyl sulfate and separated by means of two electrophoretic systems. Based on results obtained with electrophoretic system III, 19 of the Vicia faba thylakoid membrane polypeptides sufficiently labeled with l-[ 35S]methionine were inhibited by chloramphenicol and 17 were inhibited by cycloheximide. Nine polypeptides were found to be sufficiently stained but very weakly labeled, indicating low metabolic activity or low methionine content. These polypeptides of low specific radioactivity did not allow analysis of inhibitor effects. Chloramphenicol inhibition included three chlorophyll a-apoproteins. In contrast, the synthesis of three chlorophyll a b- apoproteins , besides some unidentified polypeptides found in different light-harvesting complex preparations of various composition, was severely inhibited by cycloheximide. Comparison of V. faba and H. vulgare thylakoids revealed significant differences in the polypeptide patterns. Both plant species, however, are characterized by three rapidly labeled polypeptides located closely together on the polyacrylamide gels. The identity of these rapidly labeled polypeptide(s) with a herbicide binding protein is discussed.

Effects of cycloheximide administered in conjunction with nicotine on retention of a passive avoidance task in mice.

Retention of a passive avoidance task was studied in mice given either cycloheximide or cycloheximide in combination with nicotine. Two similar experiments were conducted: In the first experiment, the effects, of these drugs were studied in inbred C57Bl/6J mice. In the second experiment, the effects of these drugs were studied in genetically heterogenous mice. Cycloheximide was found to have a deleterious effect on retention of the passive avoidance task. Larger doses of cycloheximide were found to be necessary to disrupt memory in heterogenous mice than in C57Bl/6J animals. Nicotine, when administered in conjunction with cycloheximide, abolished the memory disruptive effects of cycloheximide. The results of these experiments are discussed in terms of a time-dependent consolidation model of memory storage.

CHARACTERIZATION OF DOMINANT RESISTANCE TO COBALT CHLORIDE IN DICTYOSTELIUM DISCOIDEUM AND ITS USE IN PARASEXUAL GENETIC ANALYSIS

Strains of Dictyostelium discoideum resistant to cobaltous chloride have been isolated at a frequency of approximately 10(-6). The resistant strains have one of three phenotypes, recessive to wild type, dominant to wild type and dominant to wild type but requiring the presence of cobaltous chloride to maintain resistance. Strains carrying a dominant cobaltous chloride resistance mutation and a recessive growth temperature-sensitive mutation can be mixed with wild-type haploid lines and then subjected to selection so that only diploid lines survive. Differential sensitivity to cycloheximide has also been observed. Hypersensitivity to cycloheximide in combination with dominant cobaltous chloride resistance provides a means of selecting diploids without the use of temperature-sensitive mutations.

The Effect of Four Abscission Chemicals on Orange Fruit and Leaf Ethylene Production1

Abstract Four abscission materials were evaluated to determine their effects on ethylene production and abscission of fruit and leaves of orange (Citrus sinensis (L.) Osbeck cvs. Hamlin and Valencia): 5-chloro-3-methyl-4-nitro-1H-pyrazole (Release) glyoxal dioxime (Pik-Off), cycloheximide (3-[2-(3,5-dimethyl-2-oxocyclohexyl-2-hydroxyethyl]-glutarimide or Acti-Aid) and cycloheximide in combination with chlorothalonil (DS-27914). All chemicals enhanced internal fruit ethylene levels and subsequent fruit loosening. However, the magnitude and timing of the fruit ethylene response varied with each chemical and correlated with fruit loosening and subsequent retightening. The pattern of ethylene production may indicate the optimum fruit harvest period and the degree of leaf abscission for each chemical.

Combined electroconvulsive shock and cycloheximide inhibition of brain protein synthesis in Vivo

Levels of mouse brain synthesis in vivo following combined cycloheximide and electroconvulsive shock treatments were compared to levels following each treatment given separately. The duration of brain protein synthesis inhibition was examined for all treatment conditions. At appropriate times after treatment mice were injected intracerebrally with labeled amino acids. The amount of radioactivity recovered from brain proteins after 5 min of labeling was used to estimate levels of synthesis. Although combined cycloheximide and electroconvulsive shock treatments have been shown to produce more complete behavioral amnesia than either treatment alone, such combined treatments do not produce more protein synthesis inhibition than cycloheximide alone. Hypotheses of memory storage based primarily on brain protein synthesis mechanisms may have to be reexamined.

Factors influencing the production of interferon from L cells

The production of interferon from Newcastle disease virus-infected mouse L929 cells was investigated in relation to superinduction procedures, cell density, cellular cyclic adenosine 3',5'-monophosphate (cAMP) levels, and rate of incorporation of 14C-labeled protein hydrolysate into trichloroacetic acid-precipitable material. Densely populated cultures did not have their interferon production enhanced through "superinduction" using cycloheximide, actinomycin D, or the two antimetabolites in combination. These dense cultures produced more interferon per cell than less dense cultures, even though the interferon production from the latter cells could be enhanced two- to threefold by cycloheximide or combined cycloheximide and actinomycin D. Cells in densely populated cultures relative to those from sparsely populated cultures were smaller in volume, had a correspondingly reduced protein content and a lower concentration of cAMP, and were less able to concentrate 14C-labeled protein hydrolysate, although proportionally they were just as efficient in incorporating labeled precursors into trichloroacetic acid-precipitable polypeptides.

Time variables affecting the permanence of amnesia produced by combined cycloheximide and electroconvulsive shock treatments

Mice pretreated with saline or cycloheximide were trained in a multiple-trial avoidance apparatus. Electroconvulsive shock (ECS) was administered at various intervals following training. The animals were retrained either one day or about one week later. Both transient and permanent memory deficits were observed. Presence and type of memory deficit were dependent upon time of ECS treatment, time of retraining and whether the animals received saline of cycloheximide prior to original training. A possible relationship between the results observed and reported effects of training on macromolecular synthesis in the brain is discussed.