PURPOSE: Reduction-oxidation reaction homeostasis is vital for regulating inflammatory conditions and its dysregulation may affect the pathogenesis of chronic airway inflammatory diseases such as asthma. Peroxiredoxin-6, an important intracellular anti-oxidant molecule, is reported to be highly expressed in the airways and lungs. The aim of this study was to analyze the expression pattern of peroxiredoxin-6 in the peripheral blood mononuclear cells (PBMCs) of asthmatic patients and in bronchial epithelial cells (BECs).METHODS: The expression levels and modifications of peroxiredoxin-6 were evaluated in PBMCs from 22 asthmatic patients. Phosphorylated and acetylated peroxiredoxin-6 in hydrogen peroxide-treated human BECs was detected using immunoprecipitation analysis. The expression level of peroxiredoxin-6 was also investigated in BECs treated with hydrogen peroxide. Cycloheximide and proteasome inhibitors were used to determine whether peroxiredoxin-6 is degraded by proteasomes.RESULTS: Peroxiredoxin-6 expression was significantly reduced in the PBMCs of asthmatic patients compared to control subjects. Distinct modification patterns for peroxiredoxin-6 were observed in the PBMCs of asthmatic patients using 2-dimensional-electrophoresis. The levels of phosphorylated serine and acetylated lysine in peroxiredoxin-6 were significantly increased in the BECs following hydrogen peroxide treatment. The level of peroxiredoxin-6 expression was reduced in hydrogen peroxide-stimulated BECs, presumably due to proteasomes.CONCLUSIONS: The expression of peroxiredoxin-6, which is down-regulated in the immune cells of asthmatic patients and BECs, can be modified by oxidative stress. This phenomenon may have an effect on asthmatic airway inflammation.
Asthma ; Cycloheximide ; Epithelial Cells ; Homeostasis ; Humans ; Hydrogen ; Hydrogen Peroxide ; Immunoprecipitation ; Inflammation ; Lung ; Lysine ; Oxidative Stress ; Proteasome Inhibitors ; Protein Processing, Post-Translational ; Serine

T-cell internal antigen-1 (TIA-1) has roles in regulating alternative pre-mRNA splicing, mRNA translation, and stress granule (SG) formation in human cells. As an evolutionarily conserved response to environmental stress, SGs have been reported in various species. However, SG formation in chicken cells and the role of chicken TIA-1 (cTIA-1) in SG assembly has not been elucidated. In the present study, we cloned cTIA-1 and showed that it facilitates the assembly of canonical SGs in both human and chicken cells. Overexpression of the chicken prion-related domain (cPRD) of cTIA-1 that bore an N-terminal green fluorescent protein (GFP) tag (pntGFP-cPRD) or Flag tag (pFlag-cPRD) induced the production of typical SGs. However, C-terminal GFP-tagged cPRD induced notably large cytoplasmic granules that were devoid of endogenous G3BP1 and remained stable when exposed to cycloheximide, indicating that these were not typical SGs, and that the pntGFP tag influences cPRD localization. Finally, endogenous cTIA-1 was recruited to SGs in chicken cells and tissues under environmental stress. Taken together, our study provide evidence that cTIA-1 has a role in canonical SG formation in chicken cells and tissues. Our results also indicate that cPRD is necessary for SG aggregation.
Chickens ; Clone Cells ; Cycloheximide ; Cytoplasmic Granules ; Humans ; Protein Biosynthesis ; RNA Precursors ; RNA-Binding Proteins ; T-Lymphocytes

β-Amyloid peptide (Aβ) is the main component of senile plaques in patients with Alzheimer's disease, and is known to be a main pathogenic factor of the disease. Recent evidence indicates that activation of NADPH oxidase (NOX) in microglia or astrocytes may be a source of Aβ-induced reactive oxygen species (ROS). We investigated the role of neuronal NOX in Aβ-induced neuronal death in mouse mixed cortical cultures. Cell death was assessed by measuring lactate dehydrogenase efflux to bathing media 24 or 48 hr after exposure to Aβ₂₅₋₃₅, a fragment of Aβ with an equivalent neurotoxic effect. Aβ₂₅₋₃₅ induced neuronal death in concentration- and time- dependent manners with apoptotic features. Neuronal death was significantly attenuated, not only by anti-apoptotic drugs, such as z-VAD-fmk and cycloheximide, but also by antioxidants, such as trolox, ascorbic acid, and epigallocatethin gallate. We also demonstrated that treatment with 20 µM Aβ₂₅₋₃₅ increased fluorescent signals in mixed cortical cultures, but produced only weak signals in pure astrocyte cultures in the presence of 2',7'-dichlorofluorescin diacetate (DCF-DA), an indicator for intracellular ROS. Increased DCF-DA fluorescence was markedly inhibited, not only by trolox, but also by selective NOX inhibitors, such as apocynin and AEBSF. Western blot analyses revealed that Aβ₂₅₋₃₅ increased the expression of gp91phox, a main subunit of NOX in cells. The above antioxidants, apocynin, and AEBSF significantly attenuated neuronal death induced by Aβ₂₅₋₃₅. Furthermore, the gp91phox-specific siRNA-based knockdown of NOX significantly inhibited neuronal death. These results suggest that activation of neuronal NOX is involved in Aβ25-35-induced neuronal death.
Alzheimer Disease ; Amyloid beta-Peptides ; Animals ; Antioxidants ; Ascorbic Acid ; Astrocytes ; Baths ; Blotting, Western ; Cell Death ; Cycloheximide ; Fluorescence ; Humans ; L-Lactate Dehydrogenase ; Mice ; Microglia ; NADP ; NADPH Oxidase ; Neurons ; Plaque, Amyloid ; Reactive Oxygen Species

BACKGROUND: Pediatric onychomycosis has been previously investigated; however, the specific causative agents of onychomycosis in Korean children have not been reported. OBJECTIVE: This study aimed to determine the most common causative agents of onychomycosis in Korean children. METHODS: We reviewed the medical records of 149 pediatric patients (<18 years of age) referred for fungal cultures because of a clinical suspicion of onychomycosis between 2005 and 2014 at our clinic. Patient specimens were cultured on Sabouraud's dextrose agar with and without cycloheximide. RESULTS: Onychomycosis was clinically suspected in 149 children. Of the 44 patients with onychomycosis, confirmed by culture, 72.7% had toenail onychomycosis, 22.7% had fingernail onychomycosis, and 4.5% had toenail and fingernail onychomycosis. The male-to-female patient ratio was 1.93:1. Fourteen (31.8%) children had concomitant tinea pedis, and 12 (27.2%) had family members with tinea pedis or onychomycosis. Distal and lateral subungual onychomycosis were the most common (68%) clinical types. Trichophyton rubrum was the most frequently isolated pathogen (66.7%), followed by Candida albicans (14.8%), Microsporum canis (11.1%), Candida parapsilosis (3.7%), and Candida tropicalis (3.7%). Candida albicans was the most commonly isolated pathogen (50.0%) in fingernail onychomycosis. CONCLUSION: Pediatric onychomycosis is more common than most people think. Thus, we suggest the need for a careful mycological examination of children with suspected onychomycosis.
Agar ; Candida ; Candida albicans ; Candida tropicalis ; Child* ; Clinical Study* ; Cycloheximide ; Glucose ; Humans ; Medical Records ; Microsporum ; Nails ; Onychomycosis* ; Tinea Pedis ; Trichophyton

In the previous study, we found that flavonoids and ginsenosides exhibited high eliminate rates of human immunodeficiency virus type 1 (HIV-1) D3-transfected macrophages. Based on these findings, here we synthesized the derivatives of gallic acid, including methyl gallate, methyl 4-O-methyl gallate, methyl 3,4-O-dimethyl gallate, and methyl 3,4,5-O-trimethyl gallate and measured their cellular toxic effects against HIV-1-infected macrophages. Of these, treatment with methyl 4-O-methyl gallate in the presence of lipopolysaccharide (LPS) and cycloheximide (CHX) most effectively eliminated HIV-1-transfected cytoprotective human microglial CHME5 cells and HIV-1-D3-infected human primary macrophages. Furthermore, these strongly inhibited LPS/CHX-induced phosphorylation of phosphoinositide 3-kinase (PI3K), pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK1), Akt, and glycogen synthase kinase-3β (GSK-3β) in the Tat-transfected cells and HIV-1-D3-infected human primary macrophages. These findings suggest that methyl 4-O-methyl gallate may be a promising candidate for eliminating HIV-1 infected macrophages by blocking PI3K/Akt signaling pathway.
Cycloheximide ; Flavonoids ; Gallic Acid* ; Ginsenosides ; Glycogen Synthase ; HIV-1 ; Humans ; Macrophages ; Microglia* ; Oxidoreductases ; Phosphorylation ; Phosphotransferases ; Pyruvic Acid

Reducing [Mg2+]o to 0.1 mM can evoke repetitive [Ca2+]i spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM [Mg2+]o are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether Ca2+ spikes are involved in this process. Preconditioning by an exposure to 0.1 mM [Mg2+]o for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type Ca2+ channel antagonist nimodipine, which blocked 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the [Ca2+]i spikes. The intracellular Ca2+ chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the [Ca2+]i spikes. While Go6976, a specific inhibitor of PKCalpha had no effect on the tolerance, both the PKCepsilon translocation inhibitor and the PKCzeta pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the [Ca2+]i spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low [Mg2+]o preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the [Ca2+]i spike-induced activation of PKCepsilon and PKCxi, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.
Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Cell Death ; Cycloheximide ; N-Methylaspartate ; Neurons* ; Nimodipine ; Phosphatidylinositol 3-Kinases ; Phosphotransferases ; Rats* ; Receptors, AMPA ; Seizures

BACKGROUND: As the life expectancy has risen globally because of the advance of medicine, onychomycosis in the elderly has been increasing with higher concerns over nails. Onychomycosis has been studied quite extensively, however, few reports on onychomycosis in a geriatric Korean population have been available. OBJECTIVE: The purpose of this study was to investigate the clinical features of onychomycosis in the elderly compared with other age groups and to identify the etiological agents during 10-year period. METHODS: A total of 629 patients over 65 years of age had been diagnosed with onychomycosis during a 10-year period (2001-2010). The etiological agents were identified by cultures on Sabouraud's dextrose agar with and without cycloheximide. Nondermatophytic molds and yeasts were considered as pathogens, if the identical fungal elements were observed at the initial direct microscopy and repeatedly in specimen-yielding cultures at a follow-up visit. RESULTS: The 629 elderly patients represented 22.1% of all onychomycosis patients. Toenails were involved in 567 (90.1%) patients; fingernails in 39 (6.2%); both toenails and fingernails in 23 (3.7%). The ratio of male to female was 1.01:1. Associated systemic diseases were found in 327 (52.0%) cases. Distal and lateral subungual onychomycosis (80.2%) was the most common clinical type of onychomycosis, followed by TDO (10.7%), SWO (6.2%) and PSO (2.9%). TDO was increasing significantly in the elderly. Organisms causing onychomycosis were dermatophytes (76.5%), yeasts (14.3%) and nondermatophytic molds (9.2%). The most common cause of onychomycosis in the elderly was Trichophyton rubrum. Nondermatophytic molds were more frequently responsible for onychomycosis in the elderly. CONCLUSION: Onychomycosis has been increased in the elderly and there are many differences from other age groups in aspects of clinical features, associated diseases and etiologic agents. Therefore, we suggest the need of a careful mycological examination in the elderly patients with onychomycosis.
Agar ; Aged* ; Arthrodermataceae ; Cycloheximide ; Female ; Follow-Up Studies ; Fungi ; Glucose ; Humans ; Life Expectancy ; Male ; Microscopy ; Nails ; Onychomycosis* ; Trichophyton ; Yeasts

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

BACKGROUND: Reperfusion in ischemia is believed to generate cytotoxic oxidative stress, which mediates reperfusion injury. These stress conditions can initiate lipid peroxidation and damage to proteins, as well as promote DNA strand breaks. As biliverdin and bilirubin produced by heme oxygenase isoform 1 (HO-1) have antioxidant properties, the production of both antioxidants by HO-1 may help increase the resistance of the ischemic brain to oxidative stress. In the present study, the survival effect of HO-1 was confirmed using hemin. METHODS: To confirm the roles of HO-1, carbon monoxide, and cyclic guanosine monophosphate further in the antioxidant effect of HO-1 and bilirubin, cells were treated with cycloheximide, desferoxamine, and zinc deuteroporphyrin IX 2,4 bis glycol, respectively. RESULTS: HO-1 itself acted as an antioxidant. Furthermore, iron, rather than carbon monoxide, was involved in the HO-1-mediated survival effect. HO-1 activity was also important in providing bilirubin as an antioxidant. CONCLUSION: Our results suggested that HO-1 helped to increase the resistance of the ischemic brain to oxidative stress.
Animals ; Antioxidants ; Bilirubin ; Biliverdine ; Brain* ; Carbon Monoxide ; Cycloheximide ; DNA ; Guanosine Monophosphate ; Heme ; Heme Oxygenase (Decyclizing) ; Hemin ; Iron ; Ischemia ; Lipid Peroxidation ; Microvessels* ; Oxidative Stress ; Oxygen* ; Oxygenases ; Rats* ; Reperfusion ; Reperfusion Injury ; Zinc

Onychomycosis is usually caused by dermatophytes, but some nondermatophytic molds and yeasts are also associated with invasion of nails. The genus Chaetomium is a dematiaceous nondermatophytic mold found in soil and plant debris as a saprophytic fungus. We report the first Korean case of onychomycosis caused by Chaetomium globosum in a 35-year-old male. The patient showed brownish-yellow discoloration and subungual hyperkeratosis on the right toenails (1st and 5th) and left toenails (1st and 4th). Direct microscopic examination of scraping on the potassium hydroxide preparation revealed septate hyphae and repeated cultures on Sabouraud's dextrose agar (SDA) without cycloheximide slants showed the same fast-growing colonies, which were initially velvety white then turned to dark gray to brown. However, there was no growth of colony on SDA with cycloheximide slants. Brown-colored septated hyphae, perithecia and ascospores were shown in the slide culture. The DNA sequence of internal transcribed spacer region of the clinical sample was a 100% match to that of C. globosum strain ATCC 6205 (GenBank accession number EF524036.1). We confirmed C. globosum by KOH mount, colony, and light microscopic morphology and DNA sequence analysis. The patient was treated with 250 mg oral terbinafine daily and topical amorolfine 5% nail lacquer for 3 months.
Agar ; Arthrodermataceae ; Base Sequence ; Chaetomium ; Cycloheximide ; Fungi ; Glucose ; Humans ; Hydroxides ; Hyphae ; Lacquer ; Light ; Male ; Morpholines ; Nails ; Naphthalenes ; Onychomycosis ; Plants ; Potassium ; Potassium Compounds ; Sequence Analysis, DNA ; Soil ; Sprains and Strains ; Yeasts