Cycloheximide Research Articles

Circular RNA‑MTO1 suppresses breast cancer cell viability and reverses monastrol resistance through regulating the TRAF4/Eg5 axis.

Circular RNAs (circRNAs), a class of endogenous RNAs, have emerged as an enigmatic class of genes. However, little is known about their value in the progression and chemoresistance of cancers. The present study sought to determine the expression profiles and potential modulatory role of circRNAs on breast cancer cell viability and monastrol resistance. Monastrol-resistant cell lines were established by exposing breast cancer cells to increasing concentrations of monastrol. A human circRNA microarray was used to search for dysregulated circRNAs in monastrol-resistant cells, then circRNA‑MTO1 (hsa‑circRNA-007874) was validated as a circRNA that exhibited elevated expression levels in monastrol-resistant cells. Mechanistic investigations suggested that upregulation of circRNA‑MTO1 suppressed cell viability, promoted monastrol-induced cell cytotoxicity and reversed monastrol resistance. Subsequently, Eg5 was identified as the functional target of circRNA‑MTO1, and MTO1 inhibited Eg5 protein level but not mRNA level. By treating with protein synthesis inhibitor cycloheximide (CHX), it was revealed that MTO1 did not affect the protein stability of Eg5. RNA-pull down experiments followed by mass spectrometry revealed that MTO1 interacted with tumor necrosis factor receptor associated factor4 (TRAF4), and sequester TRAF4 from activating Eg5 translation, thereby inhibiting the Eg5 protein level. Taken together, the data reveal a regulatory mechanism by circRNA‑MTO1 to control cell viability and monastrol resistance in breast cancer cells.

Green Fluorescent Protein-Based Viability Assay in a Multiparametric Configuration.

Green fluorescent protein (GFP) is considered to be suitable for cell viability testing. In our study, GFP transfected A549 lung carcinoma cell line was treated with sodium fluoride (NaF), cycloheximide (CHX) and ochratoxin A (OTA). GFP fluorescence, intracellular ATP, nucleic acid and protein contents were quantified by a luminescence microplate assay developed in our laboratory. Flow cytometry was used to confirm the findings and to assess the intensity of GFP during different types of cell death. A 24 h NaF and CHX exposure caused a dramatic decrease in ATP contents (p < 0.05) compared with those of the controls. GFP fluorescence of the cells was in close correlation with total protein; however, GFP/ATP increased at NaF and decreased at CHX treatments (p < 0.05). ATP/protein and ATP/propidium iodide (PI) were largely decreased at NaF exposure in a dose-dependent manner (p < 0.05), while CHX and OTA showed markedly fewer effects. Both treatments caused apoptosis/necrosis at different rates. NaF induced mainly late apoptosis while OTA, mainly apoptosis. CHX effects varied by the incubation time with 100-fold elevation in late apoptotic cells at 24 h treatment. GFP intensity did not show a significant difference between live and apoptotic populations. Our results suggest when using GFP, a multiparametric assay is necessary for more precise interpretation of cell viability.

Mutations of G38R and D40G cause amyotrophic lateral sclerosis by reducing Annexin A11 protein stability

To explore the role of the mutations G38R and D40G of Annexin A11 (ANXA11) in the onset of amyotrophic lateral sclerosis (ALS). Methods: The plasmids expressing ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein were constructed, respectively. The recombinant plasmids were then transfected into HEK293 cells respectively followed by cycloheximide (CHX) treatment for 0, 2, 4 and 8 h. The protein expressions of ANXA11 wild type, ANXA11 G38R and ANXA11 D40G mutations were determined by Western blot. Gray analysis by Image J was performed to compare the half-life of each protein. The NSC-34 cell lines constantly expressing ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein were established. The cells were treated with NP-40 lysis buffer to examine the protein solubility by Western blot. Results: Both ANXA11 G38R protein and ANXA11 D40G protein showed a shorter half-life than ANXA11 wild type protein (P<0.05), while there was no difference between ANXA11 G38R protein and ANXA11 D40G protein (P>0.05). There was no visible insoluble substance in the NP-40 lysates for ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein. Conclusion: G38R and D40G mutations reduce the stability of ANXA11 protein. G38R and D40G mutations do not alter ANXA11 solubility.

Alteration of Androgen Receptor Protein Stability by Triptolide in LNCaP Cells.

Background and Objective: Although triptolide was effective for prostate cancer (PCa), the mechanism is still unclear. Androgen receptor (AR) plays a large role in the development and progression of PCa, even after castration. The present study aimed at investigating the effects of triptolide on AR protein stability and the possible mechanism. Materials and Methods: By blocking protein synthesis with cycloheximide (CHX), the effect of triptolide on AR protein stability was investigated with western blot assay. The potential role of calpains in triptolide reduced AR protein stability was investigated with calpain inhibitor and Ca2+ chelator. Results: Triptolide down-regulated AR protein level when protein synthesis was blocked by CHX, demonstrating the decrease of AR protein stability. The AR protein level was restored when the cells were co-treated with triptolide and calpain inhibitor or Ca2+ chelator, indicating the important role of calpains. Conclusions: The results indicate that triptolide can activate calpain via promoting intracellular Ca2+ accumulation, and thus decrease the stability of AR protein, subsequently resulting in the breakdown of the AR protein in LNCaP cells. This work provides an experimental basis and evidence to elucidate the anti-PCa mechanisms of triptolide.

Scutellaria baicalensis targets the hypoxia-inducible factor-1α and enhances cisplatin efficacy in ovarian cancer.

Hypoxia-inducible factor-1alpha (HIF-1α) is aberrantly upregulated in tumors and implicated in angiogenesis, metastasis, and drug resistance. Therefore, developing treatments that target HIF-1α may be a viable therapeutic approach. In Traditional Chinese Medicine (TCM), Scutellaria baicalensis (SB) is used for the treatment of cancer but the anti-cancer mechanisms are not known. We examined the effects of SB on HIF-1α expression in ovarian cancer (OC) cell lines grown under normoxic and hypoxic conditions. SB treatment attenuated HIF-1α expression in cancer cell lines. Treatment of cells with cycloheximide (CHX) reduced HIF-1α levels similar to cells treated with SB. Furthermore, SB-induced HIF-1α inhibition was abrogated by the proteasomal inhibitor MG132 and a lysosome inhibitor, chloroquine. Activation of PI3K/AKT and MAPK/ERK seen in OC cells was reduced with SB. Pretreatment of cells with LY294002 (phosphoinositide 3-kinase inhibitor) and PD98059 (mitogen-activated protein kinase inhibitor) reduced HIF-1α expression comparable to SB-treated cells. SB potentiated the anti-growth effects of cisplatin on OC cells by attenuating the expression of HIF-1α, ABCG1, and ABCG2. Taken together, the findings suggest that targeting HIF-1α with SB could be an effective treatment strategy for cancer and SB can improve the sensitivity of cancer cells to cisplatin, which is a major challenge in therapy for ovarian tumors.

Mitoxantrone is More Toxic than Doxorubicin in SH-SY5Y Human Cells: A 'Chemobrain' In Vitro Study.

The potential neurotoxic effects of anticancer drugs, like doxorubicin (DOX) and mitoxantrone (MTX; also used in multiple sclerosis), are presently important reasons for concern, following epidemiological data indicating that cancer survivors submitted to chemotherapy may suffer cognitive deficits. We evaluated the in vitro neurotoxicity of two commonly used chemotherapeutic drugs, DOX and MTX, and study their underlying mechanisms in the SH-SY5Y human neuronal cell model. Undifferentiated human SH-SY5Y cells were exposed to DOX or MTX (0.13, 0.2 and 0.5 μM) for 48 h and two cytotoxicity assays were performed, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction and the neutral red (NR) incorporation assays. Phase contrast microphotographs, Hoechst, and acridine orange/ethidium bromide stains were performed. Mitochondrial membrane potential was also assessed. Moreover, putative protective drugs, namely the antioxidants N-acetyl-l-cysteine (NAC; 1 mM) and 100 μM tiron, the inhibitor of caspase-3/7, Ac-DEVD-CHO (100 μM), and a protein synthesis inhibitor, cycloheximide (CHX; 10 nM), were tested to prevent DOX- or MTX-induced toxicity. The MTT reduction assay was also done in differentiated SH-SY5Y cells following exposure to 0.2 μM DOX or MTX. MTX was more toxic than DOX in both cytotoxicity assays and according to the morphological analyses. MTX also evoked a higher number of apoptotic nuclei than DOX. Both drugs, at the 0.13 μM concentration, caused mitochondrial membrane potential depolarization after a 48-h exposure. Regarding the putative neuroprotectors, 1 mM NAC was not able to prevent the cytotoxicity caused by either drug. Notwithstanding, 100 μM tiron was capable of partially reverting MTX-induced cytotoxicity in the NR uptake assay. One hundred μM Ac-DEVD-CHO and 10 nM cycloheximide (CHX) also partially prevented the toxicity induced by DOX in the NR uptake assay. MTX was more toxic than DOX in differentiated SH-SY5Y cells, while MTX had similar toxicity in differentiated and undifferentiated SH-SY5Y cells. In fact, MTX was the most neurotoxic drug tested and the mechanisms involved seem dissimilar among drugs. Thus, its toxicity mechanisms need to be further investigated as to determine the putative neurotoxicity for multiple sclerosis and cancer patients.

New use for CETSA: monitoring innate immune receptor stability via post-translational modification by OGT.

O-GlcNAcylation is a dynamic and functionally diverse post-translational modification shown to affect thousands of proteins, including the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (Nod2). Mutations of Nod2 (R702W, G908R and 1007 fs) are associated with Crohn's disease and have lower stabilities compared to wild type. Cycloheximide (CHX)-chase half-life assays have been used to show that O-GlcNAcylation increases the stability and response of both wild type and Crohn's variant Nod2, R702W. A more rapid method to assess stability afforded by post-translational modifications is necessary to fully comprehend the correlation between NLR stability and O-GlcNAcylation. Here, a recently developed cellular thermal shift assay (CETSA) that is typically used to demonstrate protein-ligand binding was adapted to detect shifts in protein stabilization upon increasing O-GlcNAcylation levels in Nod2. This assay was used as a method to predict if other Crohn's associated Nod2 variants were O-GlcNAcylated, and also identified the modification on another NLR, Nod1. Classical immunoprecipitations and NF-κB transcriptional assays were used to confirm the presence and effect of this modification on these proteins. The results presented here demonstrate that CETSA is a convenient method that can be used to detect the stability effect of O-GlcNAcylation on O-GlcNAc-transferase (OGT) client proteins and will be a powerful tool in studying post-translational modification.

Central Angiotensin II regulates Protein Inhibitor of Neuronal Nitric Oxide Synthase through post‐translational mechanisms in the Paraventricular Nucleus resulting in increased Sympathetic outflow

Increased activation of Renin‐Angiotensin System in the paraventricular nucleus (PVN) of the hypothalamus has been associated with the increased sympathetic outflow observed in hypertension and chronic heart failure (CHF). Previously we have shown increased protein expression of the PIN (a protein inhibitor of nNOS: neuronal nitric oxide synthase, known to dissociate nNOS dimers into monomers) with concomitantly reduced levels of catalytically active dimers of nNOS in the PVN of rats with CHF. However, PIN transcripts levels remain unchanged. To elucidate the molecular mechanism of increased PIN expression post‐transcriptionally, we used Sprague‐Dawley rats (250–300 g) subjected to intracerebroventricular infusion of Ang II (20 ng/min, 14 days, 0.5 μl/h) through osmotic mini‐pumps and NG108‐15 hybrid neuronal cell line treated with Ang II as an in vitro model. Ang II infusion significantly increased baseline mean arterial pressure (126 ± 9 vs. 84 ± 4 mmHg) and baseline renal sympathetic nerve activity (20.5 ± 2.3 vs. 6.4 ± 1.9 % of Max activity). Ang II infusion increased the expression of the PIN (1.36 ± 0.04* Ang II vs. 0.81 ± 0.02 Veh) a with concomitant decrease in PIN‐Ub conjugates (0.73 ± 0.04* Ang II vs. 1 ± 0.03 Veh) in the PVN. Further, PIN translation was inhibited in vitro using protein synthesis inhibitor cycloheximide (CHX) for 0–4h after 20h of pretreatment with Ang II. Ang II‐mediated increase in PIN expression(0.67±0.06* CHX AngII vs. 0.40±0.08 CHX 0h) was independent of CHX mediated decrease in PIN expression (0.41±0.06* CHX AngII vs. 0.19±0.04 CHX 4h) suggesting Ang II‐mediated post‐translational stabilization of PIN. Ubiquitination assay in cells transfected with pCMV‐(HA‐Ub) 8 vector revealed a reduction of HA‐Ub‐PIN conjugates after Ang II and proteasome inhibitor lactacystin (LC) treatment (4.5 ± 0.6* LC Ang II vs. 9.2 ± 2.2 LC). TUBE (Tandem Ubiquitin‐Binding Entities) assay showed decrease PIN‐Ub conjugates in Ang II‐treated cells (0.82 ± 0.12* LC Ang II vs. 1.23 ± 0.05 LC) while AT1R blocker Losartan(Los) treatment diminishes the Ang II‐mediated stabilization of PIN (1.21 ± 0.07 LC Los vs. 1.14 ± 0.04* LC AngII Los). These results suggest that PIN is targeted for rapid degradation by the ubiquitin‐proteasome and Ang II via AT1R delays the rate of degradation resulting in accumulation of PIN. It of interest to note that previously we observed that there was a decreased accumulation of PIN‐Ub conjugates in the PVN of CHF rats compared to Sham (0.63 ± 0.05* CHF vs. 1.00 ± 0.10 Sham). Taken together, our studies suggest that increased central levels of Ang II contribute to the upregulation of PIN expression in the PVN via decreasing the ubiquitination which may be involved in reducing the expression of dimeric nNOS of pre‐autonomic neurons in the PVN resulting in the increased sympathetic outflow.Support or Funding InformationSupported by AHA ‐ 14SDG19980007 and NIH grants HL124104 &amp; HL62222.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Site saturation mutagenesis of ribosomal protein L42 at 56th residue and application as a consecutive selection marker for cycloheximide resistance in yeast.

The 56th residue of ribosomal protein L42 (Rpl42) determines the sensitivity of yeast cells to the antibiotic cycloheximide (CYH). In this study, we identified the relationship between the 56th residue of Rpl42 and the function of the ribosome by site saturation mutagenesis. The resulting 20 RPL42 mutants harbouring one of 20 amino acids at the 56th residue were classified into five groups: sensitive to CYH (RPL42aP); weak resistance (RPL42aA, RPL42aM, RPL42aC, RPL42aN, RPL42aD, RPL42aS and RPL42aT), moderate resistance (RPL42aL, RPL42aI, RPL42aV, RPL42aG and RPL42aH), and strong resistance (RPL42aQ, RPL42aE, RPL42aR and RPL42aK) to CYH; and non-functional (RPL42aF, RPL42aY and RPL42aW). Three RPL42a mutants from each group, RPL42aA, RPL42aL and RPL42aQ, were used as CYH-resistant selection marker genes for the sequential transformation of CYH-sensitive yeast. A series of RPL42 mutants conferring different levels of resistance to CYH should be useful for the dose-dependent multiple selection of prototrophic industrial yeasts.

RNA helicase DDX5 participates in oxLDL-induced macrophage scavenger receptor 1 expression by suppressing mRNA degradation

The DEAD box protein DDX5, an ATP-dependent RNA helicase, plays an important role in transcriptional regulation and is associated with solid tumors and leukemia. However, its role in oxLDL-induced lipid uptake in macrophages remains unclear. In this study, we detected the expression of DDX5 mRNA and protein in oxidized low-density lipoprotein (oxLDL)-treated human primary macrophages that were induced from monocytes isolated from human peripheral blood with or without several chemical inhibitors using quantitative real-time PCR (qRT-PCR) or Western blotting. We found that oxLDL induced DDX5 expression to be independent of both the MAPK and NF-κB pathways. We also found that DDX5 promoted macrophage lipid uptake by evaluating the fluorescence intensity of engulfed dil-oxLDL. Various scavenger receptors that participate in lipid uptake were detected in siR-DDX5 transfected macrophages using qRT-PCR and Western blotting. Macrophage scavenger receptor A (MSR1) was found to be involved the upregulation of DDX5-mediated lipid uptake. Through the use of a dual luciferase reporter assay system and incubation with cycloheximide (CHX) MG132 and actidione (ActD), we found that DDX5 promoted MSR1 protein expression by stabilizing MSR1 mRNA. Moreover, the mechanism involved in DDX5 regulation of MSR1 mRNA was also explored using mass spectrum analysis; Immunoprecipitations (IPs) and RNA- Immunoprecipitations (R-IPs) revealed that mettl3 was involved in DDX5-mediated MSR1 mRNA stabilization. In addition, we also demonstrated that DDX5 inhibited mettl3 to catalyze m6a methylation in MSR1 mRNA, which contributed to the maintenance of MSR1 mRNA stability. In conclusion, ox-LDL promotes DDX5 expression in macrophages, which interacts with mettl3 to stabilize MSR1 mRNA by decreasing the m6a modification of MSR1 mRNA, ultimately promoting lipid uptake in macrophages.

Molecular mechanisms of cutis laxa– and distal renal tubular acidosis–causing mutations in V-ATPase a subunits, ATP6V0A2 and ATP6V0A4

The a subunit is the largest of 15 different subunits that make up the vacuolar H+-ATPase (V-ATPase) complex, where it functions in proton translocation. In mammals, this subunit has four paralogous isoforms, a1-a4, which may encode signals for targeting assembled V-ATPases to specific intracellular locations. Despite the functional importance of the a subunit, its structure remains controversial. By studying molecular mechanisms of human disease-causing missense mutations within a subunit isoforms, we may identify domains critical for V-ATPase targeting, activity and/or regulation. cDNA-encoded FLAG-tagged human wildtype ATP6V0A2 (a2) and ATP6V0A4 (a4) subunits and their mutants, a2P405L (causing cutis laxa), and a4R449H and a4G820R (causing renal tubular acidosis, dRTA), were transiently expressed in HEK 293 cells. N-Glycosylation was assessed using endoglycosidases, revealing that a2P405L, a4R449H, and a4G820R were fully N-glycosylated. Cycloheximide (CHX) chase assays revealed that a2P405L and a4R449H were unstable relative to wildtype. a4R449H was degraded predominantly in the proteasomal pathway, whereas a2P405L was degraded in both proteasomal and lysosomal pathways. Immunofluorescence studies disclosed retention in the endoplasmic reticulum and defective cell-surface expression of a4R449H and defective Golgi trafficking of a2P405L Co-immunoprecipitation studies revealed an increase in association of a4R449H with the V0 assembly factor VMA21, and a reduced association with the V1 sector subunit, ATP6V1B1 (B1). For a4G820R, where stability, degradation, and trafficking were relatively unaffected, 3D molecular modeling suggested that the mutation causes dRTA by blocking the proton pathway. This study provides critical information that may assist rational drug design to manage dRTA and cutis laxa.

Delayed Degradation and Impaired Dendritic Delivery of Intron-Lacking EGFP-Arc/Arg3.1 mRNA in EGFP-Arc Transgenic Mice.

Arc is a unique immediate early gene (IEG) whose expression is induced as synapses are modified during learning. Newly-synthesized Arc mRNA is rapidly transported throughout dendrites and localizes near recently activated synapses. Arc mRNA levels are regulated by rapid degradation, which is accelerated by synaptic activity in a translation-dependent process. One possible mechanism is nonsense-mediated mRNA decay (NMD), which depends on the presence of a splice junction in the 3′UTR. Here, we test this hypothesis using transgenic mice that express EGFP-Arc. Because the transgene was constructed from Arc cDNA, it lacks intron structures in the 3′UTR that are present in the endogenous Arc gene. NMD depends on the presence of proteins of the exon junction complex (EJC) downstream of a stop codon, so EGFP-Arc mRNA should not undergo NMD. Assessment of Arc mRNA rundown in the presence of the transcription inhibitor actinomycin-D confirmed delayed degradation of EGFP-Arc mRNA. EGFP-Arc mRNA and protein are expressed at much higher levels in transgenic mice under basal and activated conditions but EGFP-Arc mRNA does not enter dendrites efficiently. In a physiological assay in which cycloheximide (CHX) was infused after induction of Arc by seizures, there were increases in endogenous Arc mRNA levels consistent with translation-dependent Arc mRNA decay but this was not seen with EGFP-Arc mRNA. Taken together, our results indicate: (1) Arc mRNA degradation occurs via a mechanism with characteristics of NMD; (2) rapid dendritic delivery of newly synthesized Arc mRNA after induction may depend in part on prior splicing of the 3′UTR.

Long Non-Coding RNA CYP4B1-PS1-001 Inhibits Proliferation and Fibrosis in Diabetic Nephropathy by Interacting with Nucleolin

Background/Aims: Our previous studies demonstrated that a novel long non-coding RNA, CYP4B1-PS1-001, was significantly downregulated in early diabetic nephropathy in vivo and in vitro, and CYP4B1-PS1-001 overexpression could inhibit the proliferation and fibrosis of mouse mesangial cells (MMCs). However, the underlying mechanism of the CYP4B1-PS1-001-mediated regulation of proliferation and fibrosis in diabetic nephropathy remains undetermined. Methods: RNA-protein pull-down assay, RNA-binding protein immunoprecipitation, and mass spectrometry were used to investigate CYP4B1-PS1-001 interacted with the upregulated protein nucleolin (NCL). siRNA method was applied to knockdown NCL in MMCs, the interaction between CYP4B1-PS1-001 and NCL were determined by Western blot analysis and RT-qPCR. The effect of CYP4B1-PS1-001 in the regulation of NCL was detected by cycloheximide (CHX) and ubiquitination assays. Results: We found that CYP4B1-PS1-001 interacts with NCL, and CYP4B1-PS1-001 inhibits the proliferation and fibrosis of MMCs depending on interaction with NCL. Furthermore, degradation of CYP4B1-PS1-001-associated NCL was mediated by a ubiquitin proteasome-dependent pathway. Conclusion: Our study provides evidence that CYP4B1-PS1-001 regulates the ubiquitination and degradation of NCL and thereby plays a critical role in the proliferation and fibrosis of MMCs, indicating that CYP4B1-PS1-001 and NCL may be promising prognostic biomarkers and molecular targets for the treatment of diabetic nephropathy.

Characterization of Non-amyloidogenic G101S Transthyretin.

Transthyretin (TTR) is a tetrameric beta-sheet-rich protein that is important in the plasma transport of thyroxine and retinol. Mutations in the TTR gene cause TTR tetramer protein to dissociate to monomer, which is the rate-limiting step in familial amyloid polyneuropathy. Amyloidogenicity of individual TTR variants depends on the types of mutation that induce significant changes in biophysical, biochemical and/or biological properties. G101S TTR variant was previously identified in a Japanese male without amyloidotic symptom, and was considered as a non-amyloidogenic TTR variant. However, little is known about G101S TTR. Here, we found slight but possibly important biophysical differences between wild-type (WT) and G101S TTR. G101S TTR had slower rate of tetramer dissociation and lower propensity for amyloid fibril formation, especially at mild low pH (4.2 and 4.5), and was likely to have strong hydrophobic interaction among TTR monomers, suggesting relatively higher stability of G101S TTR compared with WT TTR. Cycloheximide (CHX)-based assay in HEK293 cells revealed that intracellular G101S TTR expression level was lower, but extracellular expression was higher than WT TTR, implying enhanced secretion efficiency of G101S TTR protein compared with WT TTR. Moreover, we found that STT3B-dependent posttranslational N-glycosylation at N98 residue occurred in G101S TTR but not in other TTR variants, possibly due to amino acid alterations that increase N-glycosylation preference or accelerate rigid structure formation susceptible to N-glycosylation. Taken together, our study characterizes G101S TTR as a stable and N-glycosylable TTR, which may be linked to its non-amyloidogenic characteristic.

Novel Mutations in PRPF31 Causing Retinitis Pigmentosa Identified Using Whole-Exome Sequencing.

The purpose of this study was to investigate the disease-causing mutations for retinitis pigmentosa (RP) patients and function of mutations. We recruited RP families and sporadic RP patients, and performed whole-exome sequencing (WES) to screen for sequence variations. Subsequently, we investigated the expression of green fluorescent protein (GFP) merged expression vectors containing PRPF31 wild type (WT) and its variants. We determined protein stability by cycloheximide (CHX) treatment. Two frameshift variants, c.547delG (p.E183fs) and c.804delG (p.L268fs), and one stopgain variant, c.1060C>T (p.R354X), in the pre-mRNA processing factor 31 gene (PRPF31) were identified in three RP families. In comparison with WT, the expressions of GFP-fused PRPF31 (GFP-PRPF31) protein with the mutation c.547delG or c.804delG in HEK293 cells were significantly reduced. However, the expression of GFP-PRPF31 containing the stopgain mutation (GFP-PRPF31sg) was increased. CHX treatment of HEK293 showed the GFP-PRPF31sg protein was more stable than GFP-PRPF31 WT. The WT protein expression was localized in the nuclei, and the mutants in both nuclei and cytoplasm. We screened for PRPF31 mutations in 131 sporadic RP patients by WES and successfully identified three novel mutations: c.G781C (p.G261R), c.A1373T (p.Q458L), and c.C1222T (p.R408W). Our study revealed novel mutations of PRPF31 in RP. Our results also showed that the two mutations (c.547delG or c.804delG) affect gene expression and GFP-PRPF31sg has increased protein stability.

Relative Contribution of nirK- and nirS- Bacterial Denitrifiers as Well as Fungal Denitrifiers to Nitrous Oxide Production from Dairy Manure Compost.

The relative contribution of fungi, bacteria, and nirS and nirK denirifiers to nitrous oxide (N2O) emission with unknown isotopic signature from dairy manure compost was examined by selective inhibition techniques. Chloramphenicol (CHP), cycloheximide (CYH), and diethyl dithiocarbamate (DDTC) were used to suppress the activity of bacteria, fungi, and nirK-possessing denitrifiers, respectively. Produced N2O were surveyed to isotopocule analysis, and its 15N site preference (SP) and δ18O values were compared. Bacteria, fungi, nirS, and nirK gene abundances were compared by qPCR. The results showed that N2O production was strongly inhibited by CHP addition in surface pile samples (82.2%) as well as in nitrite-amended core samples (98.4%), while CYH addition did not inhibit the N2O production. N2O with unknown isotopic signature (SP = 15.3-16.2‰), accompanied by δ18O (19.0-26.8‰) values which were close to bacterial denitrification, was also suppressed by CHP and DDTC addition (95.3%) indicating that nirK denitrifiers were responsible for this N2O production despite being less abundant than nirS denitrifiers. Altogether, our results suggest that bacteria are important for N2O production with different SP values both from compost surface and pile core. However, further work is required to decipher whether N2O with unknown isotopic signature is mostly due to nirK denitrifiers that are taxonomically different from the SP-characterized strains and therefore have different SP values rather than also being interwoven with the contribution of the NO-detoxifying pathway and/or of co-denitrification.

24 Evaluation of Latrunculin A for the Activation of Hand-Made Cloning (HMC) Porcine Embryos

Somatic cell nuclear transfer (SCNT) is an important biotechnological tool. However, production rates of viable offspring remain low. One possible cause of this low efficiency is chromosomal losses during early activation process (Liu et al. 2015 Cell. Reprogram. 17, 463–471). The use of actin inhibitors that block second polar body extrusion during activation protocols might be a strategy to avoid such losses. The objective of this work was to compare the efficiency of the use of 2 actin inhibitors during the activation of hand-made cloning (HMC) porcine embryos. One of the compounds used was latrunculin A (LatA), which joins directly to actin monomers, preventing their assembly to the filaments. The other was cytochalasin B (CB), which is commonly used for activation protocols. It binds to the growing actin filaments and prevents their elongation. For this purpose, in vitro-matured cumulus–oocyte compexes were deprived of their cumulus and zonae pellucidae cells by mechanical and enzymatic treatments. Oocytes were randomly distributed in 2 experimental groups (HMC) and 2 parthenogenetic control groups (PA). For HMC groups, oocytes were bisected using a microblade and the resulting hemioocytes were stained with Hoechst 33342 and observed under UV light to identify those that had lost the metaphase II plate. Adult skin fibroblasts from primary cultures were used as nuclear donors. For nuclear transfer, 2 hemicytoplasts were fused to a donor cell by an electric pulse of 1.42 kV/cm for 30 μs. After 2 h of nuclear reprogramming, the reconstituted embryos were activated by an electric pulse of 1.2 kV/cm for 80 μs and incubated with cycloheximide (CHX, 10 μg mL−1 , 3 h) in combination with one of the actin inhibitors: LatA 2 μM (CHX-LatA goup) or CB 2.5 μg mL−1 (CHX-CB group). The PA groups were subjected to the same activation treatments (PA-CHX+LatA and PA-CHX+CB groups). All embryos were cultured in SOFaa medium, using an adaptation of the well-of-the-well (WOW) system (microwells), in a humidified atmosphere with 5% CO2 in air at 39°C. Cleavage, morulae, and blastocysts rates were evaluated at Days 2, 4, and 7-8, respectively. At least 3 replicates were performed per group. Results are presented in Table 1. Our results demonstrate that the production of embryos by HMC activated with CHX-LatA is as efficient as that with CHX-CB, the protocol currently used in SCNT protocols. Further research is needed to study its effect on chromosomal complements and long-term development. Table 1.Effect of activation with cycloheximide (CHX) and latrunculin A (LatA) on in vitro development of hand-made cloning (HMC) porcine embryos (% ± SD in parentheses)

Communication or Toxicity: What Is the Effect of Cycloheximide on Leaf-Cutting Ant Workers?

Leaf-cutting ants are insects that use plant material to grow fungus from which they feed. These fungus-growing ants perform various behavioral activities to establish an environment conducive to the fungus. Among these behaviors are activities that can serve to detect materials harmful to the colony, such as licking, scraping, chopping, etc. However, there are substances that may not be detected as harmful to the fungus on first contact. Cycloheximide (CHX) is one such substance, described as a fungicide that inhibits the synthesis of proteins in eukaryotes, although its mechanism of action remains unclear. The present study aimed at evaluating the behavioral changes of worker ants, time carrying orange pellets, waste deposition and mortality, when subjected to seven days of CHX-incorporated pellets and another seven days of CHX-free pellets. The fungicide adversely and structurally affected the leaf-cutter ant colonies. Their behavior went through changes, such as an increase in pellet-licking frequencies and cleaning among the ants from the third day onward. Moreover, there was an increase in time carrying orange pellets, as well as in the mortality of workers during CHX incorporation.

A novel puromycin decorporation method to quantify skeletal muscle protein breakdown: A proof-of-concept study

The precise roles that the major proteolytic pathways play in the regulation of skeletal muscle mass remain incompletely understood, in part due to technical limitations associated with current techniques used to quantify muscle protein breakdown (MPB). We aimed to develop a method to assess MPB in cells, based on loss of puromycin labelling of translated polypeptide chains. Following an initial 24 h incubation period with puromycin (1 μM), loss of puromycin labelling from murine C2C12 myotubes was assessed over 48 h, both in the presence or absence of protein synthesis inhibitor cycloheximide (CHX). To validate the method, loss of puromycin labelling was determined from cells treated with selected compounds known to influence MPB (e.g. serum starvation, Dexamethasone (Dex), tumour necrosis factor alpha (TNF-α) and MG-132)). Reported established (static) markers of MPB were measured following each treatment. Loss of puromycin labelling from cells pre-incubated with puromycin was evident over a 48 h period, both with and without CHX. Treatment with Dex (−14 ± 2% vs. Ctl; P < 0.01), TNF-α (−20 ± 4% vs. Ctl; P < 0.001) and serum starvation (−14 ± 4% vs. Ctl; P < 0.01) caused a greater loss of puromycin labelling than untreated controls, while the proteasome inhibitor MG-132 caused a relatively lower loss of puromycin labelling (+15 ± 8% vs. Ctl; P < 0.05). Thus, we have developed a novel decorporation method for measuring global changes in MPB, validated in vitro using an established muscle cell line. It is anticipated this non isotopic-tracer alternative to measuring MPB will facilitate insight into the mechanisms that regulate muscle mass/MPB both in vitro, and perhaps, in vivo.

Knockdown of REGγ inhibits the proliferation and migration and promotes the apoptosis of multiple myeloma cells by downregulating NF-κB signal pathway

ABSTRACTObjectives: This study aimed to evaluate the effects of REGγ knockdown on the proliferation, apoptosis and migration of multiple myeloma (MM) cells, and reveal the potential regulatory mechanisms.Methods: The expression of REGγ on myeloma cells of 28 MM patients was detected by Western blot. shRNA-REGγ-1 and shRNA-REGγ-2 were constructed to downregulate REGγ in RPMI-8226 cells. The proliferation, apoptosis and migration of transfected cells were analyzed by Cell Counting Kit 8 (CCK8), flow cytometry and transwell chamber, respectively. The expression of phosphorylated p65 (p-p65), p65, NF-kappa-B inhibitor ε (IkBε), matrix metalloproteinase 2 (MMP2), B-cell lymphoma xL (Bcl-xL) and X-linked inhibitor of apoptosis protein (XIAP) in transfected cells was detected by Western blot. Using cycloheximide (CHX), the half-life period of IkBε was detected by Western blot.Results: The expression of REGγ was positive in myeloma cells. The proliferation and migration of RPMI-8226 cells were significantly inhibited by shRNA-REGγ-1/shRNA-REGγ-2, while the apoptosis rates were significantly increased (p < 0.05). The expression of p-p65 and IkBε was significantly reduced in RPMI-8226 cells transfected with shRNA-REGγ-1/shRNA-REGγ-2. The degradation of IkBε was significantly lower in RPMI-8226 cells transfected with shRNA-REGγ-1 than the control (longer half-life period). Besides, the expression of MMP2, Bcl-xL and XIAP in RPMI-8226 cells was significantly inhibited by shRNA-REGγ-1/shRNA-REGγ-2.Discussion: Knockdown of REGγ may inhibit the proliferation and migration, and promote the apoptosis of RPMI-8226 cells possibly by downregulating NF-κB signal pathway.