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Cycloheximide (CyX), a toxic antibiotic with a unique chemical structure generated by the actinomycete, Streptomyces griseus, has emerged as a primary focus of studies on mammalian bitter taste. Rats and mice avoid it at concentrations well below the thresholds for most bitter stimuli and T2R G-protein-coupled receptors specific for CyX with appropriate sensitivity are identified for those species. Like mouse and rat, golden hamsters, Mesocricetus auratus, also detected and rejected micromolar levels of CyX, although 1mM CyX failed to activate the hamster chorda tympani nerve. Hamsters showed an initial tolerance for 500microM CyX, but after that, avoidance of CyX dramatically increased, plasticity not reported for rat or mouse. As the hamster lineage branches well before division of the mouse-rat lineage in evolutionary time, differences between hamster and mouse-rat reactions to CyX are not surprising. Furthermore, unlike hamster LiCl-induced learned aversions, the induced CyX aversion neither specifically nor robustly generalized to other non-ionic bitter stimuli; and unlike adverse reactions to other chemosensory stimuli, aversions to CyX were not mollified by adding a sweetener. Thus, CyX is unlike other bitter stimuli. The gene for the high-affinity CyX receptor is a member of a cluster of five orthologous T2R genes that are likely rodent-specific; this "CyX clade" is found in the mouse, rat and probably hamster, but not in the human or rabbit genome. The rodent CyX-T2R interaction may be one of multiple lineage-specific stimulus-receptor interactions reflecting a response to a particular environmental toxin. The combination of T2R multiplicity, species divergence and gene duplication results in diverse ligands for multiple species-specific T2R receptors, which confounds definition of 'bitter' stimuli across species.
Protein synthesis inhibitors such as cycloheximide (CHX) are known to suppress protein degradation including autophagy. The fact that CHX inhibits autophagy has been generally interpreted to indicate that newly synthesized protein is indispensable for autophagy. However, CHX is also known to increase the intracellular level of amino acids and activate mTORC1 activity, a master negative regulator of autophagy. Accordingly, CHX can affect autophagic activity through inhibition of de novo protein synthesis and/or modulation of mTORC1 signaling. In this study, we investigated the effects of CHX on autophagy using specific autophagy markers. We found that CHX inhibited starvation-induced autophagy but not Torin1-induced autophagy. CHX also suppressed starvation-induced puncta formation of GFP-ULK1, an early-step marker of the autophagic process which is regulated by mTORC1. CHX activated mTORC1 even under autophagy-inducible starvation conditions. Finally, the inhibitory effect of CHX on starvation-induced autophagy was cancelled by the mTOR inhibitor Torin1. These results suggest that CHX inhibits starvation-induced autophagy through mTORC1 activation and also that autophagy does not require new protein synthesis at least in the acute phase of starvation.
Cycloheximide (CHX) is a small molecule derived from Streptomyces griseus that acts as fungicide. As a ribosome inhibitor, CHX can restrict the translation elongation of eukaryotic protein synthesis. Once protein synthesis is inhibited by CHX, the level of intracellular proteins decreases by degradation through the proteasome or lysosome system. Thus, the CHX chase assay is widely recognized and used to observe intracellular protein degradation and to determine the half-life of a given protein in eukaryotes. Here, we present a complete experimental procedure of the CHX chase assay. Graphical overview.
The ability of the protein synthesis inhibitor cycloheximide (CHX) to prevent neuronal death in different paradigms has been interpreted to indicate that the cell death process requires synthesis of "killer" proteins. On the other hand, data indicate that neurotrophic factors protect neurons in the same death paradigms by inducing expression of neuroprotective gene products. We now provide evidence that in embryonic rat hippocampal cell cultures, CHX protects neurons against oxidative insults by a mechanism involving induction of neuroprotective gene products including the antiapoptotic gene bcl-2 and antioxidant enzymes. Neuronal survival after exposure to glutamate, FeSO4, and amyloid beta-peptide was increased in cultures pretreated with CHX at concentrations of 50-500 nM; higher and lower concentrations were ineffective. Neuroprotective concentrations of CHX caused only a moderate (20-40%) reduction in overall protein synthesis, and induced an increase in c-fos, c-jun, and bcl-2 mRNAs and protein levels as determined by reverse transcription-PCR analysis and immunocytochemistry, respectively. At neuroprotective CHX concentrations, levels of c-fos heteronuclear RNA increased in parallel with c-fos mRNA, indicating that CHX acts by inducing transcription. Neuroprotective concentrations of CHX suppressed accumulation of H2O2 induced by FeSO4, suggesting activation of antioxidant pathways. Treatment of cultures with an antisense oligodeoxynucleotide directed against bcl-2 mRNA decreased Bcl-2 protein levels and significantly reduced the neuroprotective action of CHX, suggesting that induction of Bcl-2 expression was mechanistically involved in the neuroprotective actions of CHX. In addition, activity levels of the antioxidant enzymes Cu/Zn-superoxide dismutase, Mn-superoxide dismutase, and catalase were significantly increased in cultures exposed to neuroprotective levels of CHX. Our data suggest that low concentrations of CHX can promote neuron survival by inducing increased levels of gene products that function in antioxidant pathways, a neuroprotective mechanism similar to that used by neurotrophic factors.
Cycloheximide (CHX) is one of the most interesting protein synthesis inhibitors. For this reason, fluorescent derivatives of CHX could find useful applications in cell biology. We report the successful synthesis of a set of novel fluorescent derivatives of CHX. The effect of different functional groups on the biological activity of CHX was studied upon their modification through suitable strategies, i.e., acetylation of the hydroxyl group and reductive amination of the ketone group. The first route induced a complete loss of biological activity, while the second approach allowed a retained inhibition of protein synthesis, as demonstrated by in vitro translation assays. Various fluorescent dyes for reductive amination were tested (i.e., ANTS, APTS, and Rhodamine-123), and the success of the syntheses was demonstrated by diverse analytical techniques. Cycloheximide labeling with fluorescent dyes is a promising approach for developing fluorescence reporters for various applications, both in vitro (fluorescence spectroscopy) and in vivo (live imaging).
Regulation of the efficiency with which an mRNA is translated into proteins represents a key mechanism for controlling gene expression. Such regulation impacts the number of actively translating ribosomes per mRNA molecule, referred to as translation efficiency (TE), which can be monitored using ribosome profiling and RNA-seq, or by evaluating the position of an mRNA in a polysome gradient. Here we show that in budding yeast, under nutrient limiting conditions, the commonly used translation inhibitor cycloheximide induces rapid transcriptional upregulation of hundreds of genes involved in ribosome biogenesis. Cycloheximide also prevents translation of these newly transcribed messages, leading to an apparent drop in TE of these genes under conditions that include key transitions during the yeast metabolic cycle, meiosis, and amino acid starvation; however, this effect is abolished when cycloheximide pretreatment is omitted. This response requires TORC1 signaling, and is modulated by the genetic background as well as the vehicle used to deliver the drug. The present work highlights an important caveat to the use of translation inhibitors when measuring TE or mRNA levels, and will hopefully aid in future experimental design as well as interpretation of prior results.
Cycloheximide is the most common protein synthesis inhibitor, and is believed to specifically inhibit the cytoplasmic protein synthesis. Here we demonstrate that cycloheximide induces internalization and redistribution of EGF receptor to early endosomes in HeLa cells independent of receptor tyrosine phosphorylation, but dependent on p38 MAPK activity. Degradation of EGF receptor or its downstream effectors was not observed. EGF-induced activation of ERK1/2 was inhibited upon pre-treatment with cycloheximide, but did not activate JNK. The observed effects of treatment with cycloheximide alone are significant and therefore results involving the use of cycloheximide for inhibition of protein synthesis must be interpreted with caution.
Symbiotic microbes help a myriad of insects acquire nutrients. Recent work suggests that insects also frequently associate with actinobacterial symbionts that produce molecules to help defend against parasites and predators. Here we explore a potential association between Actinobacteria and two species of fungus-farming ambrosia beetles, Xyleborinus saxesenii and Xyleborus affinis. We isolated and identified actinobacterial and fungal symbionts from laboratory reared nests, and characterized small molecules produced by the putative actinobacterial symbionts. One 16S rRNA phylotype of Streptomyces (XylebKG-1) was abundantly and consistently isolated from the galleries and adults of X. saxesenii and X. affinis nests. In addition to Raffaelea sulphurea, the symbiont that X. saxesenii cultivates, we also repeatedly isolated a strain of Nectria sp. that is an antagonist of this mutualism. Inhibition bioassays between Streptomyces griseus XylebKG-1 and the fungal symbionts from X. saxesenii revealed strong inhibitory activity of the actinobacterium toward the fungal antagonist Nectria sp. but not the fungal mutualist R. sulphurea. Bioassay guided HPLC fractionation of S. griseus XylebKG-1 culture extracts, followed by NMR and mass spectrometry, identified cycloheximide as the compound responsible for the observed growth inhibition. A biosynthetic gene cluster putatively encoding cycloheximide was also identified in S. griseus XylebKG-1. The consistent isolation of a single 16S phylotype of Streptomyces from two species of ambrosia beetles, and our finding that a representative isolate of this phylotype produces cycloheximide, which inhibits a parasite of the system but not the cultivated fungus, suggests that these actinobacteria may play defensive roles within these systems.
Cancer is the second leading cause of death worldwide. Current treatment strategies based on multi-agent chemotherapy and/or radiation regimens have improved overall survival in some cases. However, resistance to apoptosis often develops in cancer cells, and its occurrence is thought to contribute to treatment failure. Non-apoptotic cell death mechanisms have become of great interest, therefore, in hopes that they would bypass tumor cell resistance. Glioblastoma multiforme (GBM), a grade IV astrocytic tumor is the most frequent brain tumor in adults, and has a high rate of mortality. We report that NIM811, a small molecule cyclophilin-binding inhibitor, induces catastrophic vacuolization and cell death in GBM cells. These unique features are distinct from many known cell death pathways, and are associated with an incompletely defined cell death mechanism known as paraptosis. We found that NIM811-induced paraptosis is due to unresolved ER stress. The abnormal upregulation of protein translation was responsible for the build-up of misfolded or unfolded proteins in ER, whereas pro-survival autophagy and UPR signals were shutdown during prolonged treatment with NIM811. Although cycloheximide has been claimed to suppress paraptosis, instead we find that it only temporarily delayed vacuole formation, but actually enhanced paraptotic cell death in the long term. On the other hand, mTOR inhibitors rescued cells from NIM811-induced paraptosis by sustaining autophagy and the UPR, while specifically restraining cap-dependent translation. These findings not only provide new insights into the mechanisms underlying paraptosis, but also shed light on a potential approach to enhance GBM treatment.
This study was done to determine the neuroprotective effect of cycloheximide on neonatal hypoxic-ischemic brain injury. Seven day-old newborn rat pups were subjected to 90 min of 8% oxygen following a unilateral carotid artery ligation. The extent of cerebral infarction was evaluated at 1 and 4 week of recovery. Apoptosis was identified by performing terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and flow cytometry with a combination of fluoresceinated annexin V and propidium iodide. Brain infarction area was significantly increased at 4 week compared to 1 week after hypoxia-ischemia in the control group. With cycloheximide treatment, the number of TUNEL positive cells in the ipsilateral cerebral cortex at 48 hr and peri-infarct area at 1 and 4 week of recovery was significantly reduced, both apoptotic and necrotic cells by flow cytometry 48 hr after the injury were significantly reduced, and the extent of cerebral infarction at 1 and 4 week of recovery was also significantly attenuated compared to the hypoxia-ischemia control group. In summary, our data suggest that apoptosis plays an important role in the development of delayed infarction, and inhibition of apoptosis with cycloheximide significantly reduces the ensuing cerebral infarction in a newborn rat pup model of cerebral hypoxia-ischemia.
Toll-like receptor 3 (TLR3) is a critical component of the innate immune response to dsRNA viruses, which was considered to be mainly expressed in immune cells and some endothelial cells. In this study, we investigated the expression and proapoptotic activity of TLR3 in human and murine tumor cell lines.
Stress conditions lead to global and gene-specific changes in RNA translation. Ribosome profiling experiments have identified genome-wide alterations in the distribution of ribosomes along mRNAs. However, it is contentious whether these changes reflect real responses, or whether they are artefacts caused by the use of inhibitors of translation (notably cycloheximide). To address this issue we performed ribosome profiling with the fission yeast Schizosaccharomyces pombe under conditions of exponential growth (unstressed) and nitrogen starvation (nutritional stress), and both in the presence and absence of cycloheximide. We examined several aspects of the translational response, including density of ribosomal footprints on coding sequences, 5' leader ribosomal densities, distribution of ribosomes along coding sequences, and ribosome codon occupancies. Cycloheximide had minor effects on overall ribosome density, which affected mostly mRNAs encoding ribosomal proteins. Nitrogen starvation caused an accumulation of ribosomes on 5' leaders in both cycloheximide-treated and untreated cells. By contrast, stress-induced ribosome accumulation on the 5' side of coding sequences was cycloheximide-dependent. Finally, codon occupancy showed strong positive correlations in cycloheximide-treated and untreated cells. Our results demonstrate that cycloheximide does influence some of the results of ribosome profiling experiments, although it is not clear if this effect is always artefactual.
The adaptive increase in bile flow was studied up to 24 h after selective biliary obstruction (SBO) in the rat. Between 6 and 12 h after SBO bile flow increased significantly, such that 12 and 24 h after SBO bile flow did not differ significantly from control. The prior administration of cycloheximide (300 micrograms/100 g) resulted in a significant impairment of the increase in bile flow normally occurring between 6 and 12 h after SBO. These data indicate that the adaptive response in bile flow is dependent on de novo protein synthesis and suggest that the protein composition of the plasma membrane may determine the adaptive increase in bile flow that occurs after SBO in the rat.
Aberrant deposition of collagen is associated with cancer development and tissue fibrosis. Proline hydroxylation, catalyzed by collagen prolyl 4-hydroxylases (C-P4Hs), is necessary for collagen maturation and secretion. Here, we try to evaluate the mechanism of the regulation of CHX on collagen maturation. Using pepsin digestion, liquid chromatograph mass spectrometry and gene knockout, we find that treatment of mouse embryonic fibroblasts with cycloheximide (CHX) increases type I collagen proline hydroxylation partially via P4HA1 and mainly via P4HA2. Western blot analysis results show that CHX treatment reduces type I collagen but does not obviously impact the level of P4HA1/2 protein in the endoplasmic reticulum, which enhances the molar ratio of P4HA1/2 to type I collagen, and coimmunoprecipitation results confirm that more P4HA1/2 can bind to each type I collagen. Since C-P4Hs possess the capability to hydroxylate proline independent of ascorbate for a few cycles, this enhanced binding between P4HA1/2 and type I collagen can partially explain how CHX stimulates type I collagen maturation.
We have previously shown that cycloheximide significantly inhibited apoptosis, and reduced ensuing cerebral infarction in a newborn rat model of cerebral hypoxiaischemia. This study was performed to determine the therapeutic window for cycloheximide therapy. Seven day-old newborn rat pups were subjected to 100 min of 8% oxygen following a unilateral carotid artery ligation, and cycloheximide was given at 0, 6, 12 and 24 hr after hypoxia-ischemia (HI). Apoptosis or necrosis was identified by performing flow cytometry with a combination of fluorescinated annexin V and propidium iodide, and the extent of cerebral infarction was evaluated with triphenyl tetrazolium chloride (TTC) at 48 hr and 72 hr after HI, respectively. With cycloheximide treatment at 0 hr after HI, both apoptotic and necrotic cells by flow cytometry were significantly reduced, only necrotic cells were significantly reduced at 6 and 12 hr, and no protective effect was seen if administration was delayed until 24 hr after HI compared to the HI control group. Infarct volume, measured by TTC, was significantly reduced by 92% and 61% when cycloheximide was given at 0 or 6 hr after HI respectively; however, there was an insignificant trend in infarct reduction if cycloheximide was administered 12 hr after HI, and no protective effect was observed when administration was delayed until 24 hr after HI. In summary, cycloheximide was neuroprotective when given within 6 hr after HI in the developing newborn rat brain.
Here, we report for the first time the successful use of cycloheximide (CHX) as an inhibitor to block de novo viral protein synthesis during WSSV (white spot syndrome virus) infection. Sixty candidate IE (immediate-early) genes were identified using a global analysis microarray technique. RT-PCR showed that the genes corresponding to ORF126, ORF242 and ORF418 in the Taiwan isolate were consistently CHX-insensitive, and these genes were designated ie1, ie2 and ie3, respectively. The sequences for these IE genes also appear in the two other WSSV isolates that have been sequenced. Three corresponding ORFs were identified in the China WSSV isolate, but only an ORF corresponding to ie1 was predicted in the Thailand isolate. In a promoter activity assay in Sf9 insect cells using EGFP (enhanced green fluorescence protein) as a reporter, ie1 showed very strong promoter activity, producing higher EGFP signals than the insect Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) ie2 promoter.
Macrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L. pneumophila, the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung-epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180 nM and 1.7 μM, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30-40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach, we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple targets in L. pneumophila.
Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax-/-Bak-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.
Pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neurite outgrowth, reduces proliferation and inhibits apoptosis of PC12 cells. We have partially characterized the transcriptome changes induced by PACAP after 6 h of treatment, when commitment to differentiation has occurred. Here, we have investigated the effects of a 6-h treatment with PACAP (10(-7) m) in the presence of cycloheximide (5 microm) to identify, via superinduction, components of the transitional transcriptome initially induced by PACAP and potentially participating in the regulation of late-response genes required for differentiation. Approximately 100 new transcripts were identified in this screen, i.e. as many individual genes as make up the 6-h PACAP differentiation transcriptome itself. Six known transcripts in this cohort were then measured at several time points between 0 and 6 h by real-time PCR to determine whether these transcripts are induced early following PACAP treatment in the absence of cycloheximide, and therefore may be of functional importance in differentiation. Five out of the six transcripts were indeed induced by PACAP alone soon (between 30 min and 3 h) after cell treatment. beta-Cell translocation gene 2, antiproliferative (Btg2), serum/glucocorticoid-regulated kinase (Sgk), nuclear factor for the kappa chain of B-cells (NFkappaB), seven in absentia homologue 2 (Siah2) and FBJ osteosarcoma related oncogene (Fos) showed a 2.5-200-fold induction by PACAP between 15 min and 3 h, and mRNA levels returned either to baseline or near baseline after 6 h. This work provides new information concerning genes whose transient regulation early after PACAP exposure may contribute to the expression of the differentiated transcriptome in PC12 cells, and should help to elucidate the molecular mechanisms involved in the control of nerve cell survival and differentiation.
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