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Molecular Cell Nov 2021Rapid protein degradation enables cells to quickly modulate protein abundance. Dysregulation of short-lived proteins plays essential roles in disease pathogenesis. A...
Rapid protein degradation enables cells to quickly modulate protein abundance. Dysregulation of short-lived proteins plays essential roles in disease pathogenesis. A focused map of short-lived proteins remains understudied. Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we identified 1,017 short-lived proteins (half-lives ≤ 8 h). These short-lived proteins are less abundant, evolutionarily younger, and less thermally stable than other proteins. We quantified 103 proteins with different stabilities among cell lines. We showed that U2OS and HCT116 cells express truncated forms of ATRX and GMDS, respectively, which have lower stability than their full-length counterparts. This study provides a large-scale resource of human short-lived proteins under translational arrest, leading to untapped avenues of protein regulation for therapeutic interventions.
Topics: Alanine; Cell Line; Cell Line, Tumor; Cycloheximide; Fucose; Geminin; HCT116 Cells; HEK293 Cells; Humans; Peptides; Principal Component Analysis; Protein Biosynthesis; Proteins; Proteome; Proteomics; Quality Control; RNA, Small Interfering; Telomere
PubMed: 34626566
DOI: 10.1016/j.molcel.2021.09.015 -
Viruses Aug 2022Enterovirus infections affect people around the world, causing a range of illnesses, from mild fevers to severe, potentially fatal conditions. There are no approved...
Enterovirus infections affect people around the world, causing a range of illnesses, from mild fevers to severe, potentially fatal conditions. There are no approved treatments for enterovirus infections. We have tested our library of broad-spectrum antiviral agents (BSAs) against echovirus 1 (EV1) in human adenocarcinoma alveolar basal epithelial A549 cells. We also tested combinations of the most active compounds against EV1 in A549 and human immortalized retinal pigment epithelium RPE cells. We confirmed anti-enteroviral activities of pleconaril, rupintrivir, cycloheximide, vemurafenib, remdesivir, emetine, and anisomycin and identified novel synergistic rupintrivir-vemurafenib, vemurafenib-pleconaril and rupintrivir-pleconaril combinations against EV1 infection. Because rupintrivir, vemurafenib, and pleconaril require lower concentrations to inhibit enterovirus replication in vitro when combined, their cocktails may have fewer side effects in vivo and, therefore, should be further explored in preclinical and clinical trials against EV1 and other enterovirus infections.
Topics: Anisomycin; Antiviral Agents; Cycloheximide; Drug Combinations; Emetine; Enterovirus Infections; Humans; Picornaviridae; Vemurafenib
PubMed: 36146673
DOI: 10.3390/v14091866 -
Drug Design, Development and Therapy 2021The incidence of fungal infection after corneal transplant has increased significantly in recent years, especially spp. This study aimed to evaluate the efficacy and...
PURPOSE
The incidence of fungal infection after corneal transplant has increased significantly in recent years, especially spp. This study aimed to evaluate the efficacy and safety of the addition of cycloheximide in Optisol-GS media in decreasing the growth of spp. strains.
METHODS
This in vitro laboratory efficacy study measured fungal colony growth in 24 vials of Optisol-GS that were divided into 6 groups of 4 vials each, as follows: (1) MIC/2 cycloheximide, (2) MIC cycloheximide, (3) MICx5 cycloheximide, (4) MICx10 cycloheximide, from MIC values obtained for each strain, (5) unsupplemented optisol-GS as a positive control (added inoculum), and (6) unsupplemented optisol-GS as a negative control (no inoculum). In each group was added and , except in the negative control. The evaluated variables were fungal colony growth from the Optisol-GS vials, corneal endothelial cell density and endothelial cell viability at different concentrations of cycloheximide.
RESULTS
In the efficacy study, all strains showed a reduction in fungal cell growth from the second day at all evaluated concentrations of optisol-GS supplemented with cycloheximide, even at subinhibitory concentrations (MIC/2). For , the colony count was reduced to 99%. No evidence of corneal endothelial toxicity was found at any concentration, in the safety study, compared with the paired control.
CONCLUSION
The addition of cycloheximide to optisol-GS decreased the fungal growth, demonstrating fungicide action against and fungistatic action against and . This drug did not demonstrate toxicity to the corneal endothelium at different concentrations.
Topics: Antifungal Agents; Candida; Chondroitin Sulfates; Complex Mixtures; Cycloheximide; Dextrans; Gentamicins; Microbial Sensitivity Tests
PubMed: 34040347
DOI: 10.2147/DDDT.S298059 -
Scientific Reports Jan 2022This study was initiated following the serendipitous discovery of a unialgal culture of a Stichococcus-like green alga (Chlorophyta) newly isolated from soil collected...
This study was initiated following the serendipitous discovery of a unialgal culture of a Stichococcus-like green alga (Chlorophyta) newly isolated from soil collected on Signy Island (maritime Antarctica) in growth medium supplemented with 100 µg/mL cycloheximide (CHX, a widely used antibiotic active against most eukaryotes). In order to test the generality of CHX resistance in taxa originally identified as members of Stichococcus (the detailed taxonomic relationships within this group of algae have been updated since our study took place), six strains were studied: two strains isolated from recent substrate collections from Signy Island (maritime Antarctica) ("Antarctica" 1 and "Antarctica" 2), one isolated from this island about 50 years ago ("Antarctica" 3) and single Arctic ("Arctic"), temperate ("Temperate") and tropical ("Tropical") strains. The sensitivity of each strain towards CHX was compared by determining the minimum inhibitory concentration (MIC), and growth rate and lag time when exposed to different CHX concentrations. All strains except "Temperate" were highly resistant to CHX (MIC > 1000 µg/mL), while "Temperate" was resistant to 62.5 µg/mL (a concentration still considerably greater than any previously reported for algae). All highly resistant strains showed no significant differences in growth rate between control and treatment (1000 µg/mL CHX) conditions. Morphological examination suggested that four strains were consistent with the description of the species Stichococcus bacillaris while the remaining two conformed to S. mirabilis. However, based on sequence analyses and the recently available phylogeny, only one strain, "Temperate", was confirmed to be S. bacillaris, while "Tropical" represents the newly erected genus Tetratostichococcus, "Antarctica 1" Tritostichococcus, and "Antarctica 2", "Antarctica 3" and "Arctic" Deuterostichococcus. Both phylogenetic and CHX sensitivity analyses suggest that CHX resistance is potentially widespread within this group of algae.
Topics: Antarctic Regions; Chlorophyta; Cycloheximide; DNA, Algal; Drug Resistance; Eukaryota; Eukaryotic Cells; Microbial Sensitivity Tests; Phylogeny; Soil; Soil Microbiology
PubMed: 35058560
DOI: 10.1038/s41598-022-05116-y -
British Journal of Pharmacology Sep 2022Limonin, a naturally occurring tetracyclic triterpenoid, has extensive pharmacological effects. Its role in cardiac hypertrophy remains to be elucidated. We investigated...
BACKGROUND AND PURPOSE
Limonin, a naturally occurring tetracyclic triterpenoid, has extensive pharmacological effects. Its role in cardiac hypertrophy remains to be elucidated. We investigated its effects on cardiac hypertrophy along with the potential mechanisms involved.
EXPERIMENTAL APPROACH
The effects of limonin on cardiac hypertrophy in C57/BL6 mice caused by aortic banding, plus neonatal rat cardiac myocytes (NRCMs) stimulated with phenylephrine to induce cardiomyocyte hypertrophy in vitro were investigated.
KEY RESULTS
Limonin markedly improved the cardiac function and heart weight in aortic banded mice. Limonin-treated mice and NRCMs also produced fewer cardiac hypertrophy markers than those treated with the vehicle in the hypertrophic groups. Sustained aortic banding- or phenylephrine-stimulation impaired cardiac sirtuin 6 (SIRT6) protein levels, which were partially reversed by limonin associated with enhanced activity of PPARα. Sirt6 siRNA inhibited the anti-hypertrophic effects of limonin in vitro. Interestingly, limonin did not influence Sirt6 mRNA levels, but regulated ubiquitin levels. Thus, the protein biosynthesis inhibitor, cycloheximide and proteasome inhibitor, MG-132, were used to determine SIRT6 protein expression levels. Under phenylephrine stimulation, limonin increased SIRT6 protein levels in the presence of cycloheximide, but it did not influence SIRT6 expression in the presence of MG-132, suggesting that limonin promotes SIRT6 levels by inhibiting its ubiquitination degradation. Furthermore, limonin inhibited the degradation of SIRT6 by activating ubiquitin-specific peptidase 10 (USP10), while Usp10 siRNA prevented the beneficial effects of limonin.
CONCLUSION AND IMPLICATIONS
Limonin mediates the ubiquitination and degradation of SIRT6 by activating USP10, providing an attractive therapeutic target for cardiac hypertrophy.
Topics: Animals; Cardiomegaly; Cycloheximide; Limonins; Mice; Myocytes, Cardiac; Phenylephrine; RNA, Small Interfering; Rats; Sirtuins; Ubiquitin Thiolesterase; Ubiquitin-Specific Proteases
PubMed: 35727596
DOI: 10.1111/bph.15899 -
British Journal of Pharmacology Dec 2021Lymphatic transport of drugs after oral administration is an important mechanism for absorption of highly lipophilic compounds. Direct measurement in lymph duct...
BACKGROUND AND PURPOSE
Lymphatic transport of drugs after oral administration is an important mechanism for absorption of highly lipophilic compounds. Direct measurement in lymph duct cannulated animals is the gold standard method, but non-invasive cycloheximide chylomicron flow blocking method has gained popularity recently. However, concerns about its reliability have been raised. The aim of this work was to investigate the validity of cycloheximide chylomicron flow blocking method for the evaluation of lymphatic transport using model compounds with high to very high lipophilicity, that is, abiraterone and cinacalcet.
EXPERIMENTAL APPROACH
Series of pharmacokinetic studies were conducted with abiraterone acetate and cinacalcet hydrochloride after enteral/intravenous administration to intact, lymph duct cannulated and/or cycloheximide pre-treated rats.
KEY RESULTS
Mean total absolute oral bioavailability of abiraterone and cinacalcet was 7.0% and 28.7%, respectively. There was a large and significant overestimation of the lymphatic transport extent by the cycloheximide method. Mean relative lymphatic bioavailability of abiraterone and cinacalcet in cycloheximide method was 28-fold and 3-fold higher than in cannulation method, respectively.
CONCLUSION AND IMPLICATIONS
Cycloheximide chylomicron flow blocking method did not provide reliable results on lymphatic absorption and substantially overestimated lymphatic transport for both molecules, that is, abiraterone and cinacalcet. This non-invasive method should not be used for the assessment of lymphatic transport and previously obtained data should be critically revised.
Topics: Administration, Oral; Animals; Biological Availability; Biological Transport; Chylomicrons; Cycloheximide; Intestinal Absorption; Pharmaceutical Preparations; Rats; Reproducibility of Results
PubMed: 34365639
DOI: 10.1111/bph.15644 -
Behavioural Brain Research Aug 2012This experiment examined the effects on memory of interactions of cycloheximide dose and training foot shock intensity. Mice received injections of cycloheximide (120...
This experiment examined the effects on memory of interactions of cycloheximide dose and training foot shock intensity. Mice received injections of cycloheximide (120 mg/kg, s.c.) or saline 30 min prior to inhibitory avoidance training with shock intensities of 100, 150, 250 or 300 μA (1 s duration). Memory was tested 48 h later. The saline control mice showed increasing memory latencies as a function of shock intensity. The ability of cycloheximide to impair memory increased as the training shock intensity increased. In a second experiment, mice were trained with a 200 μA (1 s duration) shock and received injections of saline or cycloheximide at one of several doses (30, 60 or 120 mg/kg). Under these training conditions, cycloheximide enhanced memory in an inverted-U dose-response manner. These findings are consistent with prior findings suggesting that protein synthesis inhibitors act on memory by altering modulators of memory formation as a secondary consequence of the inhibition of protein synthesis rather than by interfering with training-initiated synthesis of proteins required for memory formation.
Topics: Animals; Avoidance Learning; Biophysics; Cycloheximide; Disease Models, Animal; Electroshock; Male; Memory Disorders; Mice; Mice, Inbred ICR; Protein Synthesis Inhibitors; Reaction Time
PubMed: 22610049
DOI: 10.1016/j.bbr.2012.05.010 -
American Journal of Physiology.... Dec 2019Compounds described by humans as "bitter" are sensed by a family of type 2 taste receptors (T2Rs). Previous work suggested that diverse bitter stimuli activate distinct...
Compounds described by humans as "bitter" are sensed by a family of type 2 taste receptors (T2Rs). Previous work suggested that diverse bitter stimuli activate distinct receptors, which might allow for perceptually distinct tastes. Alternatively, it has been shown that multiple T2Rs are expressed on the same taste cell, leading to the contrary suggestion that these stimuli produce a unitary perception. Behavioral work done to address this in rodent models is limited to Spector and Kopka (Spector AC, Kopka SL. 22: 1937-1941, 2002), who demonstrated that rats cannot discriminate quinine from denatonium. Supporting this finding, it has been shown that quinine and denatonium activate overlapping T2Rs and neurons in both the mouse and rat nucleus of the solitary tract (NTS). However, cycloheximide and 6-n-propylthiouracil (PROP) do not appear to overlap with quinine in the NTS, suggesting that these stimuli may be discriminable from quinine and the denatonium/quinine comparison is not generalizable. Using the same procedure as Spector and Kopka, we tasked animals with discriminating a range of stimuli (denatonium, cycloheximide, PROP, and sucrose octaacetate) from quinine. We replicated and expanded the findings of Spector and Kopka; rats could not discriminate quinine from denatonium, cycloheximide, or PROP. Rats showed a very weak ability to discriminate between quinine and sucrose octaacetate. All animals succeeded in discriminating quinine from KCl, demonstrating they were capable of the task. These data suggest that rats cannot discriminate this suite of stimuli, although they appear distinct by physiological measures.
Topics: Animals; Cycloheximide; Dose-Response Relationship, Drug; Male; Propylthiouracil; Quaternary Ammonium Compounds; Quinine; Random Allocation; Rats; Rats, Long-Evans; Stimulation, Chemical; Sucrose; Taste
PubMed: 31596113
DOI: 10.1152/ajpregu.00213.2019 -
The Journal of Cell Biology May 1969Flagella can be removed from the biflagellate Chlamydomonas and the cells begin to regenerate flagella almost immediately by deceleratory kinetics. Under usual...
Flagella can be removed from the biflagellate Chlamydomonas and the cells begin to regenerate flagella almost immediately by deceleratory kinetics. Under usual conditions of deflagellation, more than 98% of all flagella are removed. Under less drastic conditions, cells can be selected in which one flagellum is removed and the other left intact. When only one of the two flagella is amputated, the intact flagellum shortens by linear kinetics while the amputated one regenerates. The two flagella attain an equal intermediate length and then approach their initial length at the same rate. A concentration of cycloheximide which inhibits protein synthesis permits less than one-third of each flagellum to form when both flagella are amputated. When only one is amputated in cycloheximide, shortening proceeds normally and the degree of elongation in the amputated flagellum is greater than if both were amputated in the presence of cycloheximide. The shortening process is therefore independent of protein synthesis, and the protein from the shortening flagellum probably enters the pool of precursors available for flagellar formation. Partial regeneration of flagella occurs in concentrations of cycloheximide inhibitory to protein synthesis suggesting that some flagellar precursors are present. Cycloheximide and flagellar pulse-labeling studies indicate that precursor is used during the first part of elongation, is resynthesized at mid-elongation, and approaches its original level as the flagella reach their initial length. Colchicine completely blocks regeneration without affecting protein synthesis, and extended exposure of deflagellated cells to colchicine increases the amount of flagellar growth upon transfer to cycloheximide. When colchicine is applied to cells with only one flagellum removed, shortening continues normally but regeneration is blocked. Therefore, colchicine can be used to separate the processes of shortening and elongation. Radioautographic studies of the growth zone of Chlamydomonas flagella corroborate previous findings that assembly is occurring at the distal end (tip growth) of the organelle.
Topics: Arginine; Autoradiography; Carbon Isotopes; Colchicine; Cycloheximide; Eukaryota; Flagella; Microscopy; Microscopy, Electron; Protein Biosynthesis; Regeneration
PubMed: 5783876
DOI: 10.1083/jcb.41.2.600 -
Scientific Reports Sep 2017Stress conditions lead to global and gene-specific changes in RNA translation. Ribosome profiling experiments have identified genome-wide alterations in the distribution...
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.
Topics: Antifungal Agents; Codon; Cycloheximide; Open Reading Frames; Protein Biosynthesis; Protein Synthesis Inhibitors; Ribosomes; Saccharomyces cerevisiae; Schizosaccharomyces
PubMed: 28871121
DOI: 10.1038/s41598-017-10650-1