OBJECTIVETo search for an optimal activation protocol by comparing the chemical activation effects of single-activator and combined activation protocols on mouse oocytes following injection of round spermatids (ROSI) from spermatogenic cells cultured in vitro.

METHODSUsing different concentrations of ethanol, ionomycin (Ion), calcium ionophore A23187 (CIA), strontium chloride (SrCl2), cycloheximide (CHX), and 6-dimethylaminopurine (6-DMAP) , we activated post-ROSI oocytes for different times, and activated them by combined protocols at optimal concentrations and action times according to different activation channels. We compared the activation effects of single-activator and combined activation protocols by comparing the rates of fertilization, cleavages, and morulas and blastocysts.

RESULTSWith a single activator, the optimal protocols of different activators were as follows: 7% ethanol for 6 min, 5 micromol/L CIA for 5 min, 5 micromol/L Ion for 5 min, 2 mmol/L 6-DMAP for 2 h, 10 mmol/L SrCl2 for 1.5 h, and 10 microg/ml CHX for 1.5 h, among which 10 mmol/L SrCl2 for 1.5 h achieved the highest rate of morulas and blastocysts, significantly better than CHX (P < 0.05) but with no remarkable difference from other activators. The ethanol + 6-DMAP group showed a significantly higher rate of morulas and blastocysts (29.63%) than all other combined activation groups and single-activator groups except SrCl2 (P < 0.05), and it also exhibited higher rates of normal fertilization, cleavages and morula than the SrCl2 group, but with no significant difference.

CONCLUSIONThe single-activator 10 mmol/L SrCl2 for 1.5 h and the combined activation of 7% ethanol for 6 min + 2 mmol/L 6-DMAP for 2 h are the optimal protocols for chemical activation of mouse oocytes following ROSI, and the combined activation of ethanol + 6-DMAP is even superior to the single-activator protocol.

Animals ; Cycloheximide ; pharmacology ; Female ; Fertilization in Vitro ; Ionomycin ; pharmacology ; Male ; Mice ; Mice, Inbred Strains ; Oocytes ; cytology ; drug effects ; Spermatids ; cytology ; drug effects

This study was performed to determine the accuracy of proton magnetic spectroscopy (1H-MRS) lipid peak as a noninvasive tool for quantitative in vivo detection of brain cell death. Seven day-old Sprague Dawley rats were subjected to 8% oxygen following a unilateral carotid artery ligation. For treatment, cycloheximide was given immediately after hypoxic ischemia (HI). Lipid peak was measured using 1H-MRS at 24 hr after HI, and then brains were harvested for fluorocytometric analyses with annexin V/propidium iodide (PI) and fluorescent probe JC-1, and for adenosine-5'-triphosphate (ATP) and lactate. Increased lipid peak at 1.3 ppm measured with 1H-MRS, apoptotic and necrotic cells, and loss of mitochondrial membrane potential (DeltaPsi) at 24 hr after HI were significantly improved with cycloheximide treatment. Significantly reduced brain ATP and increased lactate levels observed at 24 hr after HI showed a tendency to improve without statistical significance with cycloheximide treatment. Lipid peak at 1.3 ppm showed significant positive correlation with both apoptotic and necrotic cells and loss of DeltaPsi, and negative correlation with normal live cells. Lipid peak at 1.3 ppm measured by 1H-MRS might be a sensitive and reliable diagnostic tool for quantitative in vivo detection of brain cell death after HI.
Adenosine Triphosphate/*analysis ; Animals ; Animals, Newborn ; *Apoptosis ; Brain/metabolism/pathology ; Cycloheximide/pharmacology ; Hypoxia-Ischemia, Brain/*metabolism/*pathology ; Lactic Acid/*analysis ; Lipids/*analysis ; Magnetic Resonance Spectroscopy ; Membrane Potential, Mitochondrial ; Rats ; Rats, Sprague-Dawley

Schizophyllum commune, a basidiomycetous fungus, rarely causes disease in humans. We report a rare case of allergic fungal sinusitis caused by S. commune in a 14-yr-old girl. The patient presented with nasal obstruction and a purulent nasal discharge. Materials obtained during endoscopic surgery of the frontal recess revealed allergic mucin and a few fungal hyphae. A potato dextrose agar (PDA) culture from the allergic mucin yielded a rapidly growing white woolly mold. Although no distinctive features including hyphae bearing spicules or a clamp connection were present, the case isolate disclosed compatible mycological features including growth at 37degrees C, susceptibility to cycloheximide, and production of a tart and disagreeable smell. S. commune was confirmed by sequence analysis of the internal transcribed spacer region and D1/D2 regions of the 26S ribosomal DNA. We believe this is the first report of allergic fungal sinusitis caused by S. commune in Korea. Moreover, this report highlights the value of gene sequencing as an identification tool for non-sporulating isolates of S. commune.
Adolescent ; Antifungal Agents/pharmacology ; Cycloheximide/pharmacology ; DNA, Ribosomal/chemistry/genetics ; Female ; Humans ; Hypersensitivity/*diagnosis ; Schizophyllum/drug effects/genetics/*isolation & purification ; Sequence Analysis, DNA ; Sinusitis/*diagnosis/microbiology ; Tomography, X-Ray Computed

Cycloheximide (CHX) inhibits protein synthesis in most eukaryotic cells and it is a well-known tool commonly used in biochemical research. In this paper, the antiviral spectrum of CHX against several DNA and RNA viruses have been evaluated. CHX showed strong inhibitory activities against several RNA viruses such as HIV-1, influenza viruses, coxsackie B virus, enterovirus (EV71) and several DNA viruses such as HSV and HCMV. Especially the strong inhibitory activities of CHX against coxsackie B virus and enterovirus caught our attention, since effective drugs available in clinic are limited. The SAR of CHX derivatives also has been discussed in the paper. The hydroxyl group at C-2' and carbonyl group at C-2" of CHX are essential for its antiviral activity. And modification to these groups results its derivatives' antiviral activities reduced or lost.
Antiviral Agents ; chemical synthesis ; chemistry ; pharmacology ; Cell Line ; Cycloheximide ; analogs & derivatives ; chemical synthesis ; chemistry ; pharmacology ; DNA Viruses ; drug effects ; Enterovirus ; drug effects ; Enterovirus B, Human ; drug effects ; Humans ; RNA Viruses ; drug effects

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, induces the expression of heat shock proteins (Hsp) in a variety of cells. In this study, we investigated the role of CD36, an OxLDL receptor, and peroxisome proliferator-activated receptor gamma (PPAR gamma) in OxLDL-induced Hsp70 expression. Overexpression of dominant-negative forms of CD36 or knockdown of CD36 by siRNA transfection increased OxLDL-induced Hsp70 protein expression in human monocytic U937 cells, suggesting that CD36 signaling inhibits Hsp70 expression. Similar results were obtained by the inhibition of PPAR gamma activity or knockdown of PPAR gamma expression. In contrast, overexpression of CD36, which is induced by treatment of MCF-7 cells with troglitazone, decreased Hsp70 protein expression induced by OxLDL. Interestingly, activation of PPAR gamma through a synthetic ligand, ciglitazone or troglitazone, decreased the expression levels of Hsp70 protein in OxLDL-treated U937 cells. However, major changes in Hsp70 mRNA levels were not observed. Cycloheximide studies demonstrate that troglitazone attenuates Hsp70 translation but not Hsp70 protein stability. PPAR gamma siRNA transfection reversed the inhibitory effects of troglitazone on Hsp70 translation. These results suggest that CD36 signaling may inhibit stress- induced gene expression by suppressing translation via activation of PPAR gamma in monocytes. These findings reveal a new molecular basis for the anti-inflammatory effects of PPAR gamma.
Antigens, CD36/*physiology ; Cell Line, Tumor ; Chromans/pharmacology ; Cycloheximide/pharmacology ; HSP70 Heat-Shock Proteins/*biosynthesis ; Humans ; Lipoproteins, LDL/pharmacology/*physiology ; Monocytes/drug effects/metabolism ; PPAR gamma/agonists/antagonists & inhibitors/*physiology ; Protein Synthesis Inhibitors/pharmacology ; Signal Transduction ; Thiazolidinediones/pharmacology

Parthenogenetic activation is a procedure that an oocyte at meiosis II stage is activated into mitosis by some chemical or physical stimulation other than a sperm and the embryo is formed in the absence of any contribution from a male gamete. The activation of oocyte is the result of calcium ion oscillations and deactivation of some cytokines such as maturation promoting factor, mitogen-activated protein kinase and cytostatic factor. Parthenogenetic activation is artificially induced by various kinds of physical and/or chemical methods. The main activation method of human oocyte is chemical methods. The rates of activation and cleavage depend on the age, origin,and culture conditions of the oocyte.
Adenine ; analogs & derivatives ; pharmacology ; Animals ; Calcium ; metabolism ; Cycloheximide ; pharmacology ; Cytokines ; metabolism ; Female ; Humans ; Oocytes ; drug effects ; growth & development ; metabolism ; Parthenogenesis ; drug effects

A marine unicellular green alga, Platymonas subcordiformis, was demonstrated to photobiologically produce hydrogen gas from seawater. The objective of this study was to localize and identify the hydrogenase isolated from P. subcordiformis. Adaptation in the presence of inhibitors of protein biosynthesis indicated that the hydrogenase was much more inhibited by cycloheximide than that by chloramphenicol. The result suggested that the hydrogenase isolated from P. subcordiformis is probably synthesized in cytoplasmic ribosomes. Both Western blot analysis and immunogold electron microscopy demonstrate that the P. subcordiformis hydrogenase is mainly located in the chloroplast stroma. The proteins that reacted specifically with the antibodies against the iron hydrogenase isolated from Chlamydomonas reinhardtii were concentrated by immunoprecipitation. The separated protein bands were cut out of the SDS-PAGE gel, in-gel digested by trypsin, and analyzed by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Mascot was employed for analysis of the MALDI data using the public databases NCBInr. The hydrogenase isolated from P. subcordiformis was identified to be the Fe-hydrogenase.
Algal Proteins ; isolation & purification ; metabolism ; Biocatalysis ; drug effects ; Blotting, Western ; Chloramphenicol ; pharmacology ; Chlorophyta ; enzymology ; Cycloheximide ; pharmacology ; Cytoplasm ; enzymology ; ultrastructure ; Electrophoresis, Polyacrylamide Gel ; Hydrogenase ; antagonists & inhibitors ; isolation & purification ; metabolism ; Immunoprecipitation ; methods ; Iron-Sulfur Proteins ; antagonists & inhibitors ; isolation & purification ; metabolism ; Kinetics ; Microscopy, Immunoelectron ; Protein Synthesis Inhibitors ; pharmacology ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; methods

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.
Time Factors ; Rats, Sprague-Dawley ; Rats ; Propidium ; Neuroprotective Agents/*pharmacology ; In Situ Nick-End Labeling ; Hypoxia-Ischemia, Brain/*drug therapy/metabolism/pathology ; Cycloheximide/*pharmacology ; Brain Infarction/pathology/prevention & control ; Apoptosis/drug effects ; Annexin A5/metabolism ; Animals, Newborn ; Animals

OBJECTIVETo study the effect of p38 on the cycloheximide (CHX)-induced HL-60 cell death through mitochondria pathway.

METHODSInhibition of p38 pathway was by SB203580 (SB). Four groups were set up: control, SB only, CHX only and SB + CHX. Sub-diploid cell ratio was detected by PI staining flow cytometry at 6, 9, 12, 18, 24 h time points, and apoptotic cell ratio by Annexin V-FITC/PI double staining flow cytometry at 6 h and 18 h time points. High J-aggregate cells were evaluated by the J-aggregate contents, measurement of the J-aggregate (FL2) and J-monomer (FL1) by JC-1 flow cytometry, calculation of the delta psi m by FL2/FL1 and analysis of the delta psi m changes at 18 h time points.

RESULTSThe sub-diploid cell ratio in CHX group was significantly higher than that in control group at 6 h time point, and the ratio in SB + CHX group was significantly higher than that in CHX group at 9 h time point. At 18 h time point the apoptotic cell ratios in both CHX and SB + CHX groups were significantly higher than those in control group (P < 0.01). There was no significant difference of apoptotic cell ratio between CHX group and SB + CHX group (P > 0.05). At 18 h time point the necrotic cell ratios in both CHX and SB + CHX groups were significantly higher than that in control group (P < 0.01); and that in SB + CHX group was significantly higher than that in CHX group (P < 0.01). The high J-aggregate cell ratios in CHX and SB + CHX groups were significantly lower than that in control group (P < 0.05), and that was signficantly lower in SB + CHX group than in CHX group (P < 0.01). For the FL2/FL1 value (delta psi m) CHX group (0.17 +/- 0.01) and SB + CHX group (0.05 +/- 0.003) were significantly higher than control group (0.38 +/- 0.02) (P < 0.01), and SB + CHX group was significantly lower than CHX group (P < 0.01).

CONCLUSIONCHX can induce HL-60 cell apoptosis and the cell mitochondria depolarization, and the latter was intensified by inhibition of the p38 pathway. p38 pathway may related to the cell necrosis in the cycloheximide-induced HL-60 cell apoptosis model. s

Apoptosis ; drug effects ; Cycloheximide ; pharmacology ; HL-60 Cells ; Humans ; Membrane Potentials ; Mitochondria ; physiology ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

BACKGROUNDIslet transplantation represents an ideal therapeutic approach for treatment of type 1 diabetes but islet function and regeneration may be influenced by necrosis or apoptosis induced by oxidative stress and other insults. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in the catabolism of heme into biliverdin, releasing free iron and carbon monoxide. It has also been reported to be an antioxidant enzyme which can improve the function of grafted islets by cytoprotection via free radical scavenging and apoptosis prevention. In the present study, we investigated whether transduction of HO-1 genes into human islets with an adenovirus vector has cytoprotective action on islets cultured in vitro and discuss this method of gene therapy for clinical islet transplantation.

METHODSCadaveric pancreatic islets were isolated and purified in vitro. Transduction efficiency of islets was determined by infecting islets with adenovirus vector containing the enhanced green fluorescent protein gene (Ad-EGFP) at multiplicities of infection (MOI) of 2, 5, 10, or 20. Newly isolated islets were divided into three groups: EGFP group, islets transduced with Ad-EGFP using MOI = 20; HO-1 group, transduced with adenovirus vectors containing the human HO-1 gene using MOI = 20; and control group, mock transduced islets. Insulin release after glucose stimulation of the cell lines was determined by a radioimmunoassay kit and the stimulation index was calculated. Flow cytometry was used to detect apoptotic cells in the HO-1 group and in the control group after induction by recombinant human tumor necrosis factor-alpha (rTNFalpha) and cycloheximide (CHX) for 48 hours.

RESULTSAdenovirus vectors have a high efficiency of gene transduction into adult islet cells. Transduction of islets with the Ad-EGFP was most successful at MOI 20, at which MOI fluorescence was very intense on day 7 after transduction and EGFP was expressed in cultured islet cells for more than four weeks in vitro. The insulin release in the control group was (182.36 +/- 58.96) mIU/L after stimulation by high glucose media (16.7 mmol/L), while insulin release from the HO-1 group and the EGFP group were (270.09 +/- 89.37) mIU/L and (175.95 +/- 75.05) mIU/L respectively. Compared to the control group and the EGFP group, insulin release in the HO-1 group increased significantly (P < 0.05). After treatment with rTNFalpha and CHX the apoptotic ratio of islet cells was (63.09 +/- 10.86)% in the HO-1 group, significantly lower than (90.86 +/- 11.25)% in the control group (P < 0.05).

CONCLUSIONSTransduction of human islets with Ad-HO-1 can protect against TNF-alpha and CHX mediated cytotoxicity. The HO-1 gene also appears to facilitate insulin release from human islets. Transduction of donor islets with the adenovirus vector containing an HO-1 gene might have potential value in clinical islet transplantation.

Adenoviridae ; genetics ; Apoptosis ; drug effects ; Cycloheximide ; pharmacology ; Cytoprotection ; Genetic Therapy ; Heme Oxygenase-1 ; genetics ; physiology ; Humans ; Insulin ; secretion ; Islets of Langerhans ; physiology ; Transduction, Genetic ; Tumor Necrosis Factor-alpha ; pharmacology