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Find video protocols related to scientific articles indexed in Pubmed.
The Rates and Clinical Characteristics of Pneumolabyrinth in Temporal Bone Fracture.
Otol. Neurotol.
PUBLISHED: 11-20-2014
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Pneumolabyrinth is a rare inner ear clinical manifestation. To date, only about 50 cases have been reported-all as case reports. Consequently, the rate and clinical characteristics of pneumolabyrinth have not been evaluated.
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Melanopsin mediates light-dependent relaxation in blood vessels.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-19-2014
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Melanopsin (opsin4; Opn4), a non-image-forming opsin, has been linked to a number of behavioral responses to light, including circadian photo-entrainment, light suppression of activity in nocturnal animals, and alertness in diurnal animals. We report a physiological role for Opn4 in regulating blood vessel function, particularly in the context of photorelaxation. Using PCR, we demonstrate that Opn4 (a classic G protein-coupled receptor) is expressed in blood vessels. Force-tension myography demonstrates that vessels from Opn4(-/-) mice fail to display photorelaxation, which is also inhibited by an Opn4-specific small-molecule inhibitor. The vasorelaxation is wavelength-specific, with a maximal response at ?430-460 nm. Photorelaxation does not involve endothelial-, nitric oxide-, carbon monoxide-, or cytochrome p450-derived vasoactive prostanoid signaling but is associated with vascular hyperpolarization, as shown by intracellular membrane potential measurements. Signaling is both soluble guanylyl cyclase- and phosphodiesterase 6-dependent but protein kinase G-independent. ?-Adrenergic receptor kinase 1 (?ARK 1 or GRK2) mediates desensitization of photorelaxation, which is greatly reduced by GRK2 inhibitors. Blue light (455 nM) regulates tail artery vasoreactivity ex vivo and tail blood blood flow in vivo, supporting a potential physiological role for this signaling system. This endogenous opsin-mediated, light-activated molecular switch for vasorelaxation might be harnessed for therapy in diseases in which altered vasoreactivity is a significant pathophysiologic contributor.
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Actin-capping proteins play essential roles in asymmetric division of maturing mouse oocytes.
J. Cell. Sci.
PUBLISHED: 11-15-2014
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Actin polymerization is essential for various stages of mammalian oocyte maturation, including spindle migration, actin cap formation, polar body extrusion, and cytokinesis. The heterodimeric actin-capping protein (CP) is an essential element of the actin cytoskeleton. It binds to the fast-growing (barbed) ends of actin filaments and plays essential roles in various actin-mediated cellular processes. However, the roles of CP in mammalian oocyte maturation are poorly understood. We investigated the roles of CP in mouse oocytes and found that CP is essential for correct asymmetric spindle migration and polar body extrusion. CP mainly localized in the cytoplasm during maturation. By knockdown or ectopically overexpression of CP revealed that CP is critical for efficient spindle migration and maintenance of the cytoplasmic actin mesh density. Expression of the CP inhibiting protein CARMIL impaired spindle migration and polar body extrusion during oocyte maturation and decreased the cytoplasmic actin mesh density. Taken together, these findings show that CP is an essential component of the actin cytoskeleton machinery that plays crucial roles in oocyte maturation, presumably by controlling the cytoplasmic actin mesh density.
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Variation Between Residents and Attending Staff Interpreting Radiographs to Verify Placement of Nutrition Access Devices in the Neonatal Intensive Care Unit.
Nutr Clin Pract
PUBLISHED: 11-12-2014
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Introduction: Although radiography is considered the standard for confirming the position of nutrition access devices, it is sometimes difficult to visualize their tips. The purpose of this study was to evaluate how well pediatric residents could confirm placement via radiography of feeding tubes and intravenous (IV) nutrition catheter support in a neonatal intensive care unit (NICU). Methods: Seventy radiographs in a NICU during May 2013 were retrospectively evaluated. Eight pediatric residents (mean NICU experience, 5 months; range, 0-12 months) recorded the location of feeding tubes and IV nutrition catheters and marked their tips on computerized radiographs. Consensus review of radiographs by a radiologist and a NICU expert using a picture archiving communication system monitor in a reading room served as the reference standard. Detection rates and correct tip localization percentages were evaluated. Results: Of the 70 neonates, 38 had nutrition access devices: orogastric tube (n = 36), oroduodenal tube (n = 4), or central venous catheter (CVC) (n = 8). Detection rates were 89.6% for orogastric tubes (range, 75.0%-100%), 90.6% for oroduodenal tubes (range, 50.0%-100%), and 46.9% for CVCs (range, 12.5%-75.0%). Percentage of correct tip localizations was 85.7% for orogastric tubes (range, 74.1%-100%), 86.2% for oroduodenal tubes (range, 25.0%-100%), and 70% for CVCs (range, 50.0%-100%). Conclusion: It is not easy for pediatrician residents to confirm the position of nutrition access devices in neonates by using radiographs. Reinforcement of radiology teaching, second opinions from radiologists or NICU experts, and other methods for verifying the positions of nutrition access devices are needed to minimize complications.
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A New Class of Peptidomimetics Targeting the Polo-Box Domain of Polo-Like Kinase 1.
J. Med. Chem.
PUBLISHED: 10-28-2014
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Recent progress in the development of peptide-derived Polo-like kinase (Plk1) polo-box domain (PBD) inhibitors has led to the synthesis of multiple peptide ligands with high binding affinity and selectivity. However, few systematic analyses have been conducted to identify key Plk1 residues and characterize their interactions with potent Plk1 peptide inhibitors. We performed systematic deletion analysis using the most potent 4j peptide and studied N-terminal capping of the minimal peptide with diverse organic moieties, leading to the identification of the peptidomimetic 8 (AB-103) series with high binding affinity and selectivity. To evaluate the bioavailability of short peptidomimetic ligands, PEGylated 8 series were synthesized and incubated with HeLa cells to test for cellular uptake, antiproliferative activity, and Plk1 kinase inhibition. Finally, crystallographic studies of the Plk1 PBD in complex with peptidomimetics 8 and 22 (AB-103-5) revealed the presence of two hydrogen bond interactions responsible for their high binding affinity and selectivity.
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Risk Assessment of Volatile Organic Compounds Benzene, Toluene, Ethylbenzene, and Xylene (BTEX) in Consumer Products.
J. Toxicol. Environ. Health Part A
PUBLISHED: 10-25-2014
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Exposure and risk assessment was performed by evaluating levels of volatile organic compounds (VOC) benzene, toluene, ethylbenzene, and xylene (BTEX) in 207 consumer products. The products were categorized into 30 different items, consisting of products of different brands. Samples were analyzed for BTEX by headspace-gas chromatography/mass spectrometry (headspace-GC/MS) with limit of detection (LOD) of 1 ppm. BTEX were detected in 59 consumer products from 18 item types. Benzene was detected in whiteout (ranging from not detected [ND] to 3170 ppm), glue (1486 ppm), oil-based ballpoint pens (47 ppm), and permanent (marking) pens (2 ppm). Toluene was detected in a leather cleaning product (6071 ppm), glue (5078 ppm), whiteout (1130 ppm), self-adhesive wallpaper (15-1012 ppm), shoe polish (806 ppm), permanent pen (609 ppm), wig adhesive (372 ppm), tapes (2-360 ppm), oil-based ballpoint pen (201 ppm), duplex wallpaper (12-52 ppm), shoes (27 ppm), and air freshener (13 ppm). High levels of ethylbenzene were detected in permanent pen (ND-345,065 ppm), shoe polish (ND-277,928 ppm), leather cleaner (42,223 ppm), whiteout (ND-2,770 ppm), and glue (ND-792 ppm). Xylene was detected in permanent pen (ND-285,132 ppm), shoe polish (ND-87,298 ppm), leather cleaner (12,266 ppm), glue (ND-3,124 ppm), and whiteout (ND-1,400 ppm). Exposure assessment showed that the exposure to ethylbenzene from permanent pens ranged from 0 to 3.11 mg/kg/d (men) and 0 to 3.75 mg/kg/d (women), while for xylene, the exposure ranges were 0-2.57 mg/kg/d and 0-3.1 mg/kg/d in men and women, respectively. The exposure of women to benzene from whiteout ranged from 0 to 0.00059 mg/kg/d. Hazard index (HI), defined as a ratio of exposure to reference dose (RfD), for ethylbenzene was 31.1 (3.11 mg/kg/d/0.1 mg/kg/d) and for xylene (2.57 mg/kg/d/0.2 mg/kg/d) was 12.85, exceeding 1 for both compounds. Cancer risk for benzene was calculated to be 3.2 × 10(-5) based on (0.00059 mg/kg/d × 0.055 mg/kg-d(-1), cancer potency factor), assuming that 100% of detected levels in some products such as permanent pens and whiteouts were exposed in a worst-case scenario. These data suggest that exposure to VOC via some consumer products exceeded the safe limits and needs to be reduced.
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Potential Application of Benzo(a)Pyrene-Associated Adducts (Globin or Lipid) as Blood Biomarkers for Target Organ Exposure and Human Risk Assessment.
J. Toxicol. Environ. Health Part A
PUBLISHED: 10-25-2014
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In order to investigate the potential application of blood biomarkers as surrogate indicators of carcinogen-adduct formation in target-specific tissues, temporal formation of benzo[a]pyrene (BaP)-associated DNA adducts, protein adducts, or lipid damage in target tissues such as lung, liver, and kidney was compared with globin adduct formation or plasma lipid damage in blood after continuous intraperitoneal (ip) injection of [(3)H]BaP into female ICR mice for 7 d. Following treatment with [(3)H]BaP, formation of [(3)H]BaP-DNA or -protein adducts in lung, liver, and kidney increased linearly, and persisted thereafter. This finding was similar to the observed effects on globin adduct formation and plasma lipid damage in blood. The lungs contained a higher level of DNA adducts than liver or kidneys during the treatment period. Further, the rate of cumulative adduct formation in lung was markedly greater than that in liver. Treatment with a single dose of [(3)H]BaP indicated that BaP-globin adduct formation and BaP-lipid damage in blood reached a peak 48 h after treatment. Overall, globin adduct formation and lipid damage in blood were significantly correlated with DNA adduct formation in the target tissues. These data suggest that peripheral blood biomarkers, such as BaP-globin adduct formation or BaP-lipid damage, may be useful for prediction of target tissue-specific DNA adduct formation, and for risk assessment after exposure.
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Comparative efficacy and bioequivalence of novel h1-antihistamine bepotastine salts (nicotinate and salicylate).
J. Toxicol. Environ. Health Part A
PUBLISHED: 10-25-2014
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Bepotastine salts (nicotinate and salicylate) were investigated for their physicochemical properties to develop novel salt forms of bepotastine, bioequivalent to the bepotastine besilate-loaded tablet (Talion). These bepotastine salts of either nicotinate- or salicylate-loaded tablets were prepared by conventional wet granulation method, and dissolution profiles and pharmacokinetics in beagle dogs were compared to those of Talion. A novel bepotastine nicotinate has a higher solubility at varying pH levels (1.2, 4, or 6.8) than salicylate-loaded or besilate-loaded salt. In addition, those bepostastine salt forms (nicotinate and salicylate) are stable in heat, light, and water. Further, the novel nicotinate- and salicylate-loaded tablets showed similar dissolution rates to Talion in several selected dissolution media and were bioequivalent to Talion in beagle dogs in terms of area under the concentration-time curve (AUC) and maximum observed concentration (Cmax). A pharmacokinetic study performed in beagle dogs demonstrated that test and reference products were found to be bioequivalent in terms of safety, efficacy, and pharmacokinetic properties. These results suggest that bepostastine nicotinate and salicylate formulations are considered applicable candidates and are well tolerated versus the conventional bepostastine besilate formulation.
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A One-Generation Reproductive Toxicity Study of 3,4-Methylenedioxy-N-Methamphetamine (MDMA, Ecstasy), an Amphetamine Derivative, in C57BL/6 Mice.
J. Toxicol. Environ. Health Part A
PUBLISHED: 10-25-2014
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3,4-Methylenedioxy-N-methamphetamine (MDMA, ecstasy) is an amphetamine derivative and is a popular type of drug that is abused due to its effects on the central nervous system (CNS), including alertness and euphoria. However, life-threatening (brain edema, heart failure, and coma) and fatal hyperthermia sometimes occur in some individuals taking MDMA. In a one-generation reproductive toxicity study, the potential toxicity of chronic exposure of MDMA was investigated on the reproductive capabilities of parental mice (F0), as well as the survival/development of their subsequent offspring (F1). Male and female C57BL/6 mice were administered orally MDMA at 0, 1.25, 5 or 20 mg/kg body weight (b.w.) throughout the study, beginning at the premating period, through mating, gestation, and lactation periods. MDMA did not produce any apparent clinical signs in F0 or F1 mice, and produced no significant changes in body weight, feed/water intake, or organ weights. In contrast, administration of MDMA produced external abnormalities in fetuses, stillbirth and labored delivery, and diminished viability and weaning indices in offspring, but these data were not significant. In addition, physical development of F1 mice was not markedly influenced by MDMA treatment. Nonetheless, serum biochemistry markers showed that levels of alkaline phosphatase (ALP), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) were markedly elevated in a dose-dependent manner from 5 mg and higher MDMA/kg b.w., whereas levels of triglycerides (TG), potassium (K), and uric acid (UA) were reduced. Data suggest that MDMA may exert a weak reproductive and developmental toxicity, and the no-observed-adverse-effect level (NOAEL) of MDMA is estimated to be 1.25 mg/kg b.w./d.
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Evaluation of cadmium-induced nephrotoxicity using urinary metabolomic profiles in sprague-dawley male rats.
J. Toxicol. Environ. Health Part A
PUBLISHED: 10-25-2014
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The aim of this study was to investigate urinary metabolomic profiles associated with cadmium (Cd)-induced nephrotoxicity and their potential mechanisms. Metabolomic profiles were measured by high-resolution (1)H-nuclear magnetic resonance (NMR) spectroscopy in the urine of rats after oral exposure to CdCl2 (1, 5, or 25 mg/kg) for 6 wk. The spectral data were further analyzed by a multivariate analysis to identify specific urinary metabolites. Urinary excretion levels of protein biomarkers were also measured and CdCl2 accumulated dose-dependently in the kidney. High-dose (25 mg/kg) CdCl2 exposure significantly increased serum blood urea nitrogen (BUN), but serum creatinine (sCr) levels were unchanged. High-dose CdCl2 (25 mg/kg) exposure also significantly elevated protein-based urinary biomarkers including osteopontin, monocyte chemoattractant protein-1 (MCP-1), kidney injury molecules-1 (Kim-1), and selenium-binding protein 1 (SBP1) in rat urine. Under these conditions, six urinary metabolites (citrate, serine, 3-hydroxyisovalerate, 4-hydroxyphenyllactate, dimethylamine, and betaine) were involved in mitochondrial energy metabolism. In addition, a few number of amino acids such as glycine, glutamate, tyrosine, proline, or phenylalanine and carbohydrate (glucose) were altered in urine after CdCl2 exposure. In particular, the metabolites involved in the glutathione biosynthesis pathway, including cysteine, serine, methionine, and glutamate, were markedly decreased compared to the control. Thus, these metabolites are potential biomarkers for detection of Cd-induced nephrotoxicity. Our results further indicate that redox metabolomics pathways may be associated with Cd-mediated chronic kidney injury. These findings provide a biochemical pathway for better understanding of cellular mechanism underlying Cd-induced renal injury in humans.
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Nephrotoxic potential and toxicokinetics of melamine combined with cyanuric Acid in rats.
J. Toxicol. Environ. Health Part A
PUBLISHED: 10-25-2014
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To investigate the nephrotoxic potential of melamine (MEL) and cyanuric acid (CA) in male Sprague-Dawley rats, 7-d repeated-dose studies were performed. The experimental groups of MEL100 and CA100 were orally administered with MEL and CA at 100 mg/kg/d for 7 d, respectively. In groups dosed with MEL-CA mixtures, melamine and cyanuric acid (1:1) were simultaneously administered at 4, 20, or 100 mg/kg/d for 7 d (i.e., MEL-CA4, MEL-CA20, or MEL-CA100, respectively). Body weights were not markedly affected in MEL100, CA100, and MEL-CA4 groups, but significantly reduced in MEL-CA 20 and 100 rats. Most parameters determined in sera and tissues were not markedly altered in MEL100, CA100, and MEL-CA4-treated rodents. However, BUN, creatinine, total protein, and kidney weights were significantly increased in MEL-CA20- and MEL-CA100-treated animals. Renal histopathologic findings also revealed signs of toxicity, including tubular dilatation, crystal deposition, granulomatous tubulo-interstitial inflammation, and tubular necrosis with regeneration. Data suggested that the combination of MEL and CA might be responsible for observed nephrotoxicity that was not seen following individual exposure to either MEL or CA alone. Subsequently, the concentrations of MEL and CA were determined in serum, urine, and kidney tissues by using liquid chromatography-mass spectrometry. Toxicokinetic studies indicated that MEL or CA alone might be eliminated almost completely within 24 h after dosing showing no accumulation in kidney. However, the combined MEL-CA dose produced marked accumulation of chemicals in blood and kidneys. These results suggested that combined MEL and CA might produce renal toxicity due to significant chemical accumulation in kidney accompanied by low excretion.
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The No-Observed-Adverse-Effect Level (NOAEL) of Baby Aloe Powder (BAP) for Nutraceutical Application Based Upon Toxicological Evaluation.
J. Toxicol. Environ. Health Part A
PUBLISHED: 10-25-2014
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Aloe has been used in versatile herbal medications and nutraceuticals throughout history. Aloe is widely considered to be generally safe for humans and used globally. The effectiveness and pharmacological properties of aloe are dependent upon when the plant is collected. However, little is known about the toxicology of whole-body aloe collected within less than 1 yr. Based upon widespread exposure to aloe, it is important to determine a daily intake level of this chemical to ensure its safety for humans. To determine the no-observed-adverse-effect level (NOAEL) of baby aloe powder (BAP) for clinical application, Sprague-Dawley (SD) rats were treated orally for 4 wk with 4 different concentrations: 0, 0.125, 0.5, and 2 g/kg body weight (bw). In this study, no significant or dose-dependent toxicological effects of BAP were observed in biochemical or hematological parameters, urinalysis, clinical signs, body weight, and food and water consumption. There were changes in some biomarkers in certain treated groups compared to controls; however, all values were within their reference ranges and not dose-dependent. Based on these results, the NOAEL of BAP was estimated to be greater than 2 g/kg bw in male and 2 g/kg bw in female SD rats. Collectively, these data suggest that BAP used in this study did not produce any marked subacute toxic effects up to a maximum concentration of 2 g/kg bw, and thus use in nutraceuticals and in pharmaceutical and cosmetic applications at a concentration of >2 g/kg is warranted.
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Autocrine stimulation of IL-10 is critical to the enrichment of IL-10-producing CD40hiCD5+ regulatory B cells in vitro and in vivo.
BMB Rep
PUBLISHED: 10-25-2014
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IL-10-producing B (Breg) cells regulate various immune responses. However, their phenotype remains unclear. CD40 expression was significantly increased in B cells by LPS, and the Breg cells were also enriched in CD40hiCD5+ B cells. Furthermore, CD40 expression on Breg cells was increased by IL-10, CD40 ligand, and B-cell activating factor, suggesting that CD40hi is a common phenotype of Breg cells. LPS-induced CD40 expression was largely suppressed by an anti-IL-10 receptor antibody and in IL-10-/-CD5+CD19+ B cells. The autocrine effect of IL-10 on the CD40 expression was largely suppressed by an inhibitor of JAK/STAT3. In vivo, the LPS treatment increased the population of CD40hiCD5+ Breg cells in mice. However, the population of CD40hiCD5+ B cells was minimal in IL-10-/- mice by LPS. Altogether, our findings show that Breg cells are largely enriched in CD40hiCD5+ B cells and the autocrine effect of IL-10 is critical to the formation of CD40hiCD5+ Breg cells.
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Ru(II)-catalyzed selective C-H amination of xanthones and chromones with sulfonyl azides: synthesis and anticancer evaluation.
J. Org. Chem.
PUBLISHED: 09-24-2014
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A ketone-assisted ruthenium-catalyzed selective amination of xanthones and chromones C-H bonds with sulfonyl azides is described. The reactions proceed efficiently with a broad range of substrates with excellent functional group compatibility. This protocol provides direct access to 1-aminoxanthones, 5-aminochromones, and 5-aminoflavonoid derivatives known to exhibit potent anticancer activity.
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CRISPR/Cas9 Nuclease-Mediated Gene Knock-In in Bovine-Induced Pluripotent Cells.
Stem Cells Dev.
PUBLISHED: 09-12-2014
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Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3? and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.
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Amino Acid supplementation affects imprinted gene transcription patterns in parthenogenetic porcine blastocysts.
PLoS ONE
PUBLISHED: 09-02-2014
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To determine whether exogenous amino acids affect gene transcription patterns in parthenogenetic porcine embryos, we investigated the effects of amino acid mixtures in culture medium. Parthenogenetic embryos were cultured in PZM3 medium under four experimental conditions: 1) control (no amino acids except L-glutamine and taurine); 2) nonessential amino acids (NEAA); 3) essential amino acids (EAA); and 4) NEAA and EAA. The rate of development of embryos to the four-cell stage was not affected by treatment. However, fewer (P<0.05) embryos cultured with EAA (12.8%) reached the blastocyst stage as compared with the control group (25.6%) and NEAA group (30.3%). Based on these findings, we identified genes with altered expression in parthenogenetic embryos exposed to medium with or without EAAs. The results indicated that EAA influenced gene expression patterns, particularly those of imprinted genes (e.g., H19, IGF2R, PEG1, XIST). However, NEAAs did not affect impaired imprinted gene expressions induced by EAA. The results also showed that mechanistic target of rapamycin (MTOR) mRNA expression was significantly increased by EAA alone as compared with control cultures, and that the combined treatment with NEAA and EAA did not differ significantly from those of control cultures. Our results revealed that gene transcription levels in porcine embryos changed differentially depending on the presence of EAA or NEAA. However, the changes in the H19 mRNA observed in the parthenogenetic blastocysts expression level was not related to the DNA methylation status in the IGF2/H19 domain. The addition of exogenous amino acid mixtures affected not only early embryonic development, but also gene transcription levels, particularly those of imprinted genes. However, this study did not reveal how amino acids affect expression of imprinted genes under the culture conditions used. Further studies are thus required to fully evaluate how amino acids affect transcriptional regulation in porcine embryos.
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Dinuclear [{(p-cym)RuCl}2(?-phpy)](PF6)2 and heterodinuclear [(ppy)2Ir(?-phpy)Ru(p-cym)Cl](PF6)2 complexes: synthesis, structure and anticancer activity.
Dalton Trans
PUBLISHED: 08-28-2014
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Phpy bridged homodinuclear Ru-Ru () and heterodinuclear Ir-Ru complexes () have been developed. Complex induces autophagy towards the cisplatin resistant human breast cancer (MCF7) cell line, whereas is inactive.
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Zearalenone exposure affects epigenetic modifications of mouse eggs.
Mutagenesis
PUBLISHED: 08-25-2014
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Zearalenone (ZEA) is a mycotoxin produced by various Fusarium fungi, which has been shown to cause several cases of mycotoxicosis in farm animals and humans. However, there is no evidence regarding the effect of ZEA on mouse egg developmental competence. In this study, we found that the activation rate of maturated oocytes was affected in mice by ZEA treatment, indicating that ZEA affects egg developmental competence. And we explored possible mechanisms of low mouse maturated oocyte developmental competence after ZEA treatment from an epigenetic modification perspective. The fluorescence intensity analysis showed that 5-methyl cytosine level increased after ZEA treatment, indicating that the general DNA methylation level increased in the treated eggs. Moreover, histone methylations were also altered: H3K4me2 as well as H3K9me3 and H4K20me1, me2, me3 levels decreased in eggs that were cultured in high-dose ZEA medium. Thus, our results indicated that ZEA decreased egg developmental competence by affecting the epigenetic modifications.
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Histone deacetylase inhibitor trichostatin A affects porcine oocyte maturation in vitro.
Reprod. Fertil. Dev.
PUBLISHED: 08-22-2014
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Previous studies show that porcine oocyte aging resulting from asynchronised IVM impairs embryo developmental competence. In the present study we investigated whether trichostatin A (TSA; an inhibitor of histone deacetylation) prolongs the maturation time and prevents the aging of oocytes. Porcine oocytes were cultured in medium containing increasing concentrations of TSA (300nM) for 24, 44 or 64h. The percentage of oocytes that underwent germinal vesicle breakdown was significantly lower in the TSA-treated group (300nM) than in the control group. TSA did not affect oocyte quality at MII based on levels of maturation-promoting factor, the phosphorylation status of mitogen-activated protein kinase or histone H3K9 acetylation analysis. We also compared the preimplantation developmental competence and the viability of pathenogenetic embryos treated with 100nM TSA for 24h and then continuously cultured for another 24h in TSA free condition. No significant differences were observed for either parameter between the TSA-treated and control groups. These results indicate that TSA prolongs the IVM of porcine oocytes but that oocyte quality and aging are not affected. These findings provide a feasible option by which to adjust the initiation time of downstream experiments based on porcine matured oocytes.
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Zinc depletion activates porcine metaphase II oocytes independently of the protein kinase C pathway.
In Vitro Cell. Dev. Biol. Anim.
PUBLISHED: 08-09-2014
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Zinc is an important trace element that regulates several biological functions. This study investigated the role of zinc in the metaphase (M) II arrest of porcine oocytes. N, N, N', N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a Zn(2+) chelator, was used to deplete free zinc from porcine MII stage oocytes. TPEN treatment significantly (P?
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Changes in the gray matter volume during compensation after vestibular neuritis: A longitudinal VBM study.
Restor. Neurol. Neurosci.
PUBLISHED: 08-07-2014
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Peripheral vestibular dysfunction following vestibular neuritis (VN) often persists but functional recovery of balance can be variable. The authors compared structural changes in the brain before and after post-VN compensation and related it to the functional recovery.
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A polymorphism in AGT and AGTR1 gene is associated with lead-related high blood pressure.
J Renin Angiotensin Aldosterone Syst
PUBLISHED: 07-18-2014
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We investigated the association of polymorphisms in two renin-angiotensin system-related genes, expressed as angiotensinogen (AGT) and angiotensin II type 1 receptor (AGTR1), with blood lead levels and lead-related blood pressure in lead-exposed male workers in Korea. A cross-sectional study involving 808 lead-exposed male workers in Korea was conducted using a restriction fragment length polymorphism-based strategy to differentiate the various genotypes of polymorphisms in the AGT and AGTR1 genes. The association of clinical characteristics with genotypes as modifiers was estimated after adjustment for age, smoking status, drinking status, body mass index and job duration of each subject. Genotype and allele frequencies of the M235T polymorphism in AGT were associated with lead-related high blood pressure status. Moreover, blood lead levels were associated with allele frequencies of the AGT M235T polymorphism. These results suggested that the M/M genotype and M allele of AGT are risk factors for lead-related high blood pressure.
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Viriditoxin regulates apoptosis and autophagy via mitotic catastrophe and microtubule formation in human prostate cancer cells.
Int. J. Oncol.
PUBLISHED: 06-26-2014
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Microtubule targeting chemicals are considered excellent antitumor drugs through their binding to tubulin, which affects the instability of microtubules resulting in arrest of cancer cells. The present study was designed to investigate the antitumor effects of viriditoxin (VDT) against human prostate cancer cells. VDT, isolated from Paecilomyces variotii fungus, which was derived from the jellyfish Nemopilema nomurai, offers a new approach for controlling resistant bacterial infections by blocking bacterial cell division proteins. VDT produced dose-dependent cytotoxicity against human prostate cancer cells. Treatment with VDT promoted both apoptosis and autophagy in LNCaP cells. Annexin V/FITC staining indicated that apoptosis occurred in VDT-treated LNCaP cells. DAPI staining revealed morphological changes in the cell nuclei indicative of mitotic catastrophe in LNCaP cells. VDT caused cell growth inhibition via G2/M phase arrest. Moreover, VDT also increased autophagic cell death in LNCaP cells by induction of several autophagy-related proteins such as LC3  II, Atg5, Atg7 and beclin-1 protein, which are essential for autophagy induction. These results were also confirmed by acridine orange staining. This study indicates that VDT could potentially be effective against prostate cancer by promoting multiple modes of growth arrest and cell death coupled with apoptosis and autophagy.
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Transcriptional activation of OCT4 by the ETS transcription factor PEA3 in NCCIT human embryonic carcinoma cells.
FEBS Lett.
PUBLISHED: 06-17-2014
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We examined the molecular mechanism of OCT4 gene regulation by polyomavirus enhancer activator 3 (PEA3) in NCCIT cells. Endogenous PEA3 and OCT4 were significantly elevated in undifferentiated cells and reduced upon differentiation. PEA3 knockdown led to a reduction in OCT4 levels. OCT4 promoter activity was significantly up-regulated by dose-dependent PEA3 overexpression. Deletion and site-directed mutagenesis of the OCT4 promoter revealed a putative binding site within the conserved region 2 (CR2). PEA3 interacted with the binding element within CR2 in NCCIT cells. This study reveals the molecular details of the mechanism by which the oncogenic factor PEA3 regulates OCT4 gene expression as a transcriptional activator.
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Non-muscle tropomyosin (Tpm3) is crucial for asymmetric cell division and maintenance of cortical integrity in mouse oocytes.
Cell Cycle
PUBLISHED: 06-13-2014
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Tropomyosins are actin-binding cytoskeletal proteins that play a pivotal role in regulating the function of actin filaments in muscle and non-muscle cells; however, the roles of non-muscle tropomyosins in mouse oocytes are unknown. This study investigated the expression and functions of non-muscle tropomyosin (Tpm3) during meiotic maturation of mouse oocytes. Tpm3 mRNA was detected at all developmental stages in mouse oocytes. Tpm3 protein was localized at the cortex during the germinal vesicle and germinal vesicle breakdown stages. However, the overall fluorescence intensity of Tpm3 immunostaining was markedly decreased in metaphase II oocytes. Knockdown of Tpm3 impaired asymmetric division of oocytes and spindle migration, considerably reduced the amount of cortical actin, and caused membrane blebbing during cytokinesis. Expression of a constitutively active cofilin mutant and Tpm3 overexpression confirmed that Tpm3 protects cortical actin from depolymerization by cofilin. The data indicate that Tpm3 plays crucial roles in maintaining cortical actin integrity and asymmetric cell division during oocyte maturation, and that dynamic regulation of cortical actin by Tpm3 is critical to ensure proper polar body protrusion.
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Intrinsic relationship between enhanced oxygen reduction reaction activity and nanoscale work function of doped carbons.
J. Am. Chem. Soc.
PUBLISHED: 06-13-2014
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Nanostructured carbon materials doped with a variety of heteroatoms have shown promising electrocatalytic activity in the oxygen reduction reaction (ORR). However, understanding of the working principles that underpin the superior ORR activity observed with doped nanocarbons is still limited to predictions based on theoretical calculations. Herein, we demonstrate, for the first time, that the enhanced ORR activity in doped nanocarbons can be correlated with the variation in their nanoscale work function. A series of doped ordered mesoporous carbons (OMCs) were prepared using N, S, and O as dopants; the triple-doped, N,S,O-OMC displayed superior ORR activity and four-electron selectivity compared to the dual-doped (N,O-OMC and S,O-OMC) and the monodoped (O-OMC) OMCs. Significantly, the work functions of these heteroatom-doped OMCs, measured by Kelvin probe force microscopy, display a strong correlation with the activity and reaction kinetics for the ORR. This unprecedented experimental insight can be used to provide an explanation for the enhanced ORR activity of heteroatom-doped carbon materials.
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Analysis of the prevalence of and risk factors for tinnitus in a young population.
Otol. Neurotol.
PUBLISHED: 06-11-2014
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Tinnitus in children and adolescents is known to be as common as in adults. However, tinnitus in this young population is often overlooked, and a large population-based study designed to adjust for various risk factors for tinnitus is lacking.
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Empirical treatment of highly suspected nontuberculous mycobacteria infections following aesthetic procedures.
Arch Plast Surg
PUBLISHED: 06-09-2014
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Infection caused by nontuberculous mycobacteria (NTM) has been increasing. Awareness of this infection is crucial yet problematic. Delayed management may lead to destructive results. We empirically treated a series of patients with clinical suspicion of NTM infection prior to the identification of the pathogen.
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OxLDL triggers retrograde translocation of arginase2 in aortic endothelial cells via ROCK and mitochondrial processing peptidase.
Circ. Res.
PUBLISHED: 06-05-2014
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Increased arginase activity contributes to endothelial dysfunction by competition for l-arginine substrate and reciprocal regulation of nitric oxide synthase (NOS). The rapid increase in arginase activity in human aortic endothelial cells exposed to oxidized low-density lipoprotein (OxLDL) is consistent with post-translational modification or subcellular trafficking.
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Transcriptional regulation of OCT4 by the ETS transcription factor ESE-1 in NCCIT human embryonic carcinoma cells.
Biochem. Biophys. Res. Commun.
PUBLISHED: 06-02-2014
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The epithelium-specific ETS transcription factor-1 (ESE-1) is physiologically important in the pathogenesis of various diseases. Recently, OCT4, a transcription factor involved in stem cell pluripotency, has been implicated in tumorigenesis. In this study, we invested the molecular mechanism by which ESE-1 regulates transcription of OCT4 in NCCIT human embryonic carcinoma cells. Real-time PCR analysis revealed that OCT4 levels were high in undifferentiated NCCIT cells but significantly decreased upon retinoic acid-mediated differentiation, concomitant with up-regulation of ESE-1 expression. OCT4 mRNA level rose following shRNA-mediated knockdown of ESE-1, but declined when ESE-1 was overexpressed, suggesting that the expression levels of OCT4 and ESE-1 may be coordinated in an opposite manner. Promoter-reporter assays revealed that induced OCT4 promoter activity in NCCIT cells was significantly down-regulated by ESE-1 overexpression in a dose-dependent manner. The inhibitory effect of ESE-1 on OCT4 promoter activity was relieved by co-expression of an ESE-1 mutant lacking the transactivation domain, but not by mutants lacking other domains. Serial deletion and site-directed mutagenesis of the OCT4 promoter revealed that a potential ETS binding site (EBS) is present in the conserved region 2 (CR2). ESE-1 interacted with the EBS element in CR2 and enrichment of CR2 significantly increased upon RA-mediated differentiation of NCCIT cells, suggesting that this binding is likely to be involved in ESE-1-mediated repression of OCT4 promoter activity upon differentiation. Taken together, the results of this study reveal the molecular details of the mechanism by which the oncogenic factor ESE-1 regulates expression of the stem cell transcription factor OCT4 in pluripotent NCCIT cells.
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Development of a method of vitrification, thawing, and transfer of mammalian blastocysts using a single closed cryo-straw.
Cryo Letters
PUBLISHED: 05-30-2014
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There are different methods for cryopreservation of mammalian embryos with variable degrees of success. These methods require specific vessels for embryo vitrification, thawing, and transfer.
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Influence of Panax ginseng on the offspring of adult rats exposed to prenatal stress.
Int. J. Mol. Med.
PUBLISHED: 05-28-2014
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The exposure of pregnant females to stress during a critical period of fetal brain development is an environmental risk factor for the development of schizophrenia in adult offspring. Schizophrenia is a group of common mental disorders of unclear origin, affecting approximately 1% of the global population, showing a generally young age at onset. In the present study, a repeated variable stress paradigm was applied to pregnant rats during the final week of gestation. The effects of an extract of Panax ginseng C.A. Meyer (PG) on rats exposed to prenatal stress (PNS) were investigated in terms of behavioral activity and protein expression analyses. In the behavioral tests, grooming behavior in a social interaction test, line-crossing behavior in an open-field test and swimming activity in a forced-swim test were decreased in the rats exposed to PNS compared with the non-stressed offspring; the changes in behavioral activity were reversed upon oral treatment with PG (300 mg/kg). Subsequently, western blot analysis and immunohistochemical analyses of the prefrontal cortex and hippocampus revealed that the downregulation of several neurodevelopmental genes which occurred following exposure to PNS was reversed upon treatment with PG. The current findings demonstrate that the downregulation of several genes following exposure to PNS may affect subsequent behavioral changes, and that these phenomena are reversed following treatment with PG during pregnancy. Our results suggest that oral treatment with PG reduces the incidence of psychiatric disorders, such as schizophrenia.
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Transcriptional regulation of endothelial arginase 2 by histone deacetylase 2.
Arterioscler. Thromb. Vasc. Biol.
PUBLISHED: 05-15-2014
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Arginase 2 (Arg2) is a critical target in atherosclerosis because it controls endothelial nitric oxide, proliferation, fibrosis, and inflammation. Regulators of Arg2 transcription in the endothelium have not been characterized. The goal of the current study is to determine the role of specific histone deacetylases (HDACs) in the regulation of endothelial Arg2 transcription and endothelial function.
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Isolated horizontal positional nystagmus from a posterior fossa lesion.
Ann. Neurol.
PUBLISHED: 05-09-2014
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Isolated vertigo with horizontal positional nystagmus as an impending sign of a central lesion has rarely been reported. Here we present neuro-otologic findings of patients with these clinical signs. Lesion overlays from 6 patients with ageotropic positional nystagmus revealed that the nodulus and vermis are common areas of injury. In contrast, 2 patients with geotropic positional nystagmus had cerebellar peduncle and lateral medullary lesions. These clinical findings suggest that vertigo with horizontal positional nystagmus, even in the absence of other initial neurological signs, may indicate a posterior fossa lesion, including that in the nodulus, vermis, and deep cerebellar structures. Ann Neurol 2014.
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Effect of mycotoxin-containing diets on epigenetic modifications of mouse oocytes by fluorescence microscopy analysis.
Microsc. Microanal.
PUBLISHED: 05-09-2014
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Mycotoxins, such as aflatoxin (AF), fumonisin B1, zearalenone (ZEA), and deoxynivalenol (DON), are commonly found in many food commodities. Mycotoxins have been shown to increase DNA methylation levels in a human intestinal cell line. We previously showed that the developmental competence of oocytes was affected in mice that had been fed a mycotoxin-containing diet. In this study, we explored possible mechanisms of low mouse oocyte developmental competence after mycotoxin treatment in an epigenetic modification perspective. Mycotoxin-contaminated maize (DON at 3,875 ?g/kg, ZEA at 1,897 ?g/kg, and AF at 806 ?g/kg) was included in diets at three different doses (mass percentage: 0, 15, and 30%) and fed to mice for 4 weeks. The fluorescence intensity analysis showed that the general DNA methylation levels increased in oocytes from high dose mycotoxin-fed mice. Mouse oocyte histone methylation was also altered. H3K9me3 and H4K20me3 level increased in oocytes from mycotoxin-fed mice, whereas H3K27me3 and H4K20me2 level decreased in oocytes from mycotoxin-fed mice. Thus, our results indicate that naturally occurring mycotoxins have effects on epigenetic modifications in mouse oocytes, which may be one of the reasons for reduced oocyte developmental competence.
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WASH complex regulates Arp2/3 complex for actin-based polar body extrusion in mouse oocytes.
Sci Rep
PUBLISHED: 05-06-2014
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Prior to their fertilization, oocytes undergo asymmetric division, which is regulated by actin filaments. Recently, WASH complex were identified as actin nucleation promoting factors (NPF) that activated Arp2/3 complex. However, the roles of WASH complex remain uncertain, particularly for oocyte polarization and asymmetric division. Here, we examined the functions of two important subunits of a WASH complex, WASH1 and Strumpellin, during mouse oocyte meiosis. Depleting WASH1 or disrupting Strumpellin activity by WASH1 morpholino (MO) injection or Strumpellin antibody injection decreased polar body extrusion and caused oocyte symmetric division, and this may have been due to spindle formation and migration defects. Time lapse microscopy showed that actin filaments distribution and relative amount at the membrane and in the cytoplasm of oocytes was significantly decreased after disrupting WASH complex. In addition, Arp2/3 complex expression was reduced after WASH1 depletion. Thus, our data indicated that WASH complex regulated Arp2/3 complex and were required for cytokinesis and following polar body extrusion during mouse oocyte meiotic maturation.
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Poly-lysine peptidomimetics having potent antimicrobial activity without hemolytic activity.
Amino Acids
PUBLISHED: 04-29-2014
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Diversity of sequence and structure in naturally occurring antimicrobial peptides (AMPs) limits their intensive structure-activity relationship (SAR) study. In contrast, peptidomimetics have several advantages compared to naturally occurring peptide in terms of simple structure, convenient to analog synthesis, rapid elucidation of optimal physiochemical properties and low-cost synthesis. In search of short antimicrobial peptides using peptidomimetics, which provide facile access to identify the key factors involving in the destruction of pathogens through SAR study, a series of simple and short peptidomimetics consisting of multi-Lys residues and lipophilic moiety have been prepared and found to be active against several Gram-negative and Gram-positive bacteria containing methicillin-resistant Staphylococcus aureus (MRSA) without hemolytic activity. Based on the SAR studies, we found that hydrophobicity, +5 charges of multiple Lys residues, hydrocarbon tail lengths and cyclohexyl group were crucial for antimicrobial activity. Furthermore, membrane depolarization, dye leakage, inner membrane permeability and time-killing kinetics revealed that bacterial-killing mechanism of our peptidomimetics is different from the membrane-targeting AMPs (e. g. melittin and SMAP-29) and implied our peptidomimetics might kill bacteria via the intracellular-targeting mechanism as done by buforin-2.
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Involvement of Dynamin 2 in actin-based polar-body extrusion during porcine oocyte maturation.
Mol. Reprod. Dev.
PUBLISHED: 04-15-2014
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Mammalian oocyte meiotic maturation involves a unique asymmetric division, but the regulatory mechanisms and signaling pathways involved are poorly understood. Dynamins are ubiquitous eukaryotic GTPases involved in membrane trafficking and actin dynamics, whose roles in mammalian oocyte maturation have not been determined. In this study, we used porcine oocytes to show that Dynamin 2 accumulated at the meiotic spindle and in the cortex of oocytes, with a distribution similar to that of actin. Inhibiting Dynamin 2 activity in porcine oocytes with the specific inhibitor dynasore resulted in failed polar-body extrusion. This phenotype may have been due to aberrant actin distribution and/or spindle positioning as inhibitor treatment disrupted the formation of the actin cap and cortical granule-free domain, which negatively impacted spindle positioning. Moreover, the distribution of ARP2, a key actin-nucleation factor, was severely reduced in the cortex after dynasore treatment. Thus, our results suggest that Dynamin 2 possibly regulates porcine oocyte maturation through its effects on actin-mediated spindle positioning and cytokinesis, and that this may depend on regulating ARP2 localization.
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Molecular basis for unidirectional scaffold switching of human Plk4 in centriole biogenesis.
Nat. Struct. Mol. Biol.
PUBLISHED: 04-14-2014
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Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. We show that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal-structure analyses revealed that Cep192- and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152 peptide bound to the CPB markedly better than did the Cep192 peptide and effectively 'snatched' the CPB away from a preformed CPB-Cep192 peptide complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation. Thus, Plk4 is intricately regulated in time and space through ordered interactions with two distinct scaffolds, Cep192 and Cep152, and a failure in this process may lead to human cancer.
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Polymerase subunit gamma 2 affects porcine oocyte maturation and subsequent embryonic development.
Theriogenology
PUBLISHED: 04-09-2014
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Deoxyribonucleic acid polymerase subunit gamma (POLG) is an enzyme encoded by the mitochondrial Polg gene. Polymerase (DNA directed), gamma 2, accessory subunit, also known as POLG2, is involved in mitochondrial replication. In the present study, we examined the role of Polg2 in the maturation of porcine oocytes. After Polg2 knockdown, the mitochondrial DNA copy number was significantly (P < 0.05) lower than that in the control group. However, there was no decrease in mitochondrial membrane potential. The decrease in mitochondrial DNA copy number led to reductions in adenosine-5'-triphosphate content (P < 0.05) and the maturation rate (P < 0.05) of oocytes. Furthermore, in the Polg2-knockdown group, maturation-promoting factor activity was decreased (P < 0.05) and the percentage of oocytes displaying abnormal actin filaments and microtubules was significantly increased (P < 0.05). This likely led to the reduced development rate and number of cells per blastocyst in this group (P < 0.05). In conclusion, Polg2 seems to be critical for mitochondrial replication and regulation of adenosine-5'-triphosphate content and affects porcine oocyte maturation and subsequent embryonic development.
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Aberrant methylation of the VIM promoter in uterine cervical squamous cell carcinoma.
Oncology
PUBLISHED: 04-03-2014
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To identify prognosis-associated methylation markers of uterine cervical squamous cell carcinoma (SCC) and to verify potential clinical correlations.
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Detection of blebs and bullae in patients with primary spontaneous pneumothorax by multi-detector CT reconstruction using different slice thicknesses.
J Med Imaging Radiat Oncol
PUBLISHED: 03-31-2014
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The aim of this study was to compare the diagnostic performances of multi-detector computed tomography (MDCT) reconstruction at two different slice thicknesses (1?mm, 'high resolution' vs. 5?mm, 'routine') with respect to the detection of blebs and bullae (BBs) in patients with primary spontaneous pneumothorax (PSP).
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Evaluation of renal toxicity by combination exposure to melamine and cyanuric Acid in male sprague-dawley rats.
Toxicol Res
PUBLISHED: 03-29-2014
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Melamine-induced nephrotoxicity is closely associated with crystal formation in the kidney caused by combined exposure to melamine (Mel) and cyanuric acid (CA). However, there are few dosage-finding studies for toxicological evaluation of chronic co-exposure to Mel and CA. The objective of this study was to investigate the possible mechanism by which a Mel and CA mixture lead to renal toxicity in rats. Mel and CA were co-administered to rats via oral gavage for 50 days. Nephrotoxicity was determined by measuring blood urea nitrogen (BUN) and serum creatinine (sCr) levels. Relative kidney weights were significantly increased in rats after co-exposure to Mel+CA (63/6.3 or 630/6.3 mg/kg) mixtures. BUN and sCr levels were significantly increased after Mel and CA co-exposure. Taken together, significant increase in KIM-1, NGAL, and calbindin levels were observed in the urine of rats exposed to Mel+CA (63/6.3 or 630/6.3 mg/kg) compared with the corresponding control group. Histological analysis revealed epithelial degeneration and necrotic cell death in the proximal tubules of the kidney after co-exposure to Mel+CA (63/6.3 or 630/6.3 mg/kg). Our data suggest that Mel-mediated renal toxicity may be influenced by CA concentrations in Mel-contaminated milk or foods.
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Transcriptional coactivator undifferentiated embryonic cell transcription factor 1 expressed in spermatogonial stem cells: A putative marker of boar spermatogonia.
Anim. Reprod. Sci.
PUBLISHED: 03-28-2014
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Spermatogenesis is initiated from spermatogonial stem cells (SSCs), which are derived from gonocytes. Although some rodent SSC markers have been investigated, other species- and developmental stage-specific markers of spermatogonia have not been identified. The objective of this study was to characterize the expression of undifferentiated embryonic cell transcription factor 1 (UTF1) gene as a potential marker for spermatogonia and SSCs in the boar testis. In boar testis tissue at pre-pubertal stages (tissues collected at 5, 30, and 60 days of age), UTF1 gene expression was detected in almost all spermatogonia cells that expressed a protein gene product 9.5 (PGP9.5), and immunocytochemical analysis of isolated total testicular cells showed that 91.14% of cells staining for PGP9.5 also stained for UTF1. However, in boar testis tissue at pubertal and post-pubertal stages (tissues collected at 90, 120, 150, and 180 days of age), UTF1 was not detected in all PGP9.5-positive cells in the basement membrane. While some PGP9.5-positive cells stained for UTF1, other cells stained only for PGP9.5 or UTF1. PGP9.5, UTF1, and NANOG was assessed in in vitro cultures of pig SSCs (pSSCs) from testes collected at 5 days of age. The relative amounts of PGP9.5, NANOG, and UTF1 mRNA were greater in pSSC colonies than in testis and muscle tissue. Thus, the UTF1 gene is expressed in PGP9.5-positive spermatogonia cells of pigs at 5 days of age, and its expression is maintained in cultured pSSC colonies, suggesting that UTF1 is a putative marker for early-stage spermatogonia in the pre-pubertal pig testis. These findings will facilitate the study of spermatogenesis and applications in germ cell research.
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Zearalenone exposure affects mouse oocyte meiotic maturation and granulosa cell proliferation.
Environ. Toxicol.
PUBLISHED: 03-28-2014
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Zearalenone (ZEN) is a metabolite of Fusarium and is a common contaminant of grains and foodstuffs. ZEN acts as a xenoestrogen and is considered to be cytotoxic, tissue toxic, and genotoxic, which causes abortions and stillbirths in humans and animals. Since estrogens affect oocyte maturation during meiosis, in this study we investigated the effects of ZEN on mouse oocyte meiotic maturation and granulosa cell proliferation. Our results showed that ZEN-treated oocyte maturation rates were decreased, which might be due to the disrupted cytoskeletons: (1) ZEN treatment resulted in significantly more oocytes with abnormal spindle morphologies; (2) actin filament expression and distribution were also disrupted after ZEN treatment, which was confirmed by the aberrant distribution of actin regulatory proteins. In addition, cortical granule-free domains (CGFDs) were disrupted after ZEN treatment, which indicated that ZEN may affect mouse oocyte fertilization capability. ZEN reduced mouse granulosa cell proliferation in a dose-dependent manner as determined by MTT assay and TUNEL apoptosis analysis, which may be another cause for the decreased oocyte maturation. Thus, our results demonstrated that exposure to zearalenone affected oocyte meiotic maturation and granulosa cell proliferation in mouse. © 2014 Wiley Periodicals, Inc. Environ Toxicol, 2014.
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Association of ultraviolet radiation resistance-associated gene polymorphisms with rheumatoid arthritis.
Biomed Rep
PUBLISHED: 03-21-2014
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The ultraviolet radiation resistance-associated gene (UVRAG) protein binds to the Beclin 1/PI3-kinase III complex and promotes autophagy. Autophagy may be upregulated by endoplasmic reticulum (ER) stress. Persistent and excessive ER stress may alter synovial fibroblast apoptosis and this alteration may affect the pathogenesis of rheumatoid arthritis (RA). In this study, we investigated whether UVRAG genetic polymorphisms are associated with RA. To determine the association between UVRAG polymorphisms and RA, we genotyped five UVRAG single-nucleotide polymorphisms (SNPs; rs7111334, intron C/T; rs7933235, intron A/G; rs1380075, intron T/A; rs1458836, near the 5' gene terminal G/A; and rs636420, exon 15 C/T) using a direct sequencing method in 243 RA patients and 417 control subjects. Among these, one SNP (rs7111334) exhibited significant genotypic/allelic differences between RA patients and the control group. Therefore, this study suggested a possible association between UVRAG polymorphisms and RA susceptibility.
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Role of the epithelial-mesenchymal transition and its effects on embryonic stem cells.
Exp. Mol. Med.
PUBLISHED: 03-04-2014
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The epithelial-mesenchymal transition (EMT) is important for embryonic development and the formation of various tissues or organs. However, EMT dysfunction in normal cells leads to diseases, such as cancer or fibrosis. During the EMT, epithelial cells are converted into more invasive and active mesenchymal cells. E-box-binding proteins, including Snail, ZEB and helix-loop-helix family members, serve as EMT-activating transcription factors. These transcription factors repress the expression of epithelial markers, for example, E-cadherin, rearrange the cytoskeleton and promote the expression of mesenchymal markers, such as vimentin, fibronectin and other EMT-activating transcription factors. Signaling pathways that induce EMT, including transforming growth factor-?, Wnt/glycogen synthase kinase-3?, Notch and receptor tyrosine kinase signaling pathways, interact with each other for the regulation of this process. Although the mechanism(s) underlying EMT in cancer or embryonic development have been identified, the mechanism(s) in embryonic stem cells (ESCs) remain unclear. In this review, we describe the underlying mechanisms of important EMT factors, indicating a precise role for EMT in ESCs, and characterize the relationship between EMT and ESCs.
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Congenital cholesteatoma localized to the tip of the mastoid bone: a case report and possible etiology.
Korean J Audiol
PUBLISHED: 02-26-2014
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Congenital cholesteatomas of mastoid origin are extremely rare. We reported one in 2007 and experienced an additional case. A male presented with a 5-month history of right-sided ear discharge. Computed tomography of the temporal bone showed a soft tissue density occupying the mastoid tip. At surgery, the cholesteatoma sac was completely isolated from the mastoid antrum and lateral air cell in the mastoid tip area. We now doubt the rarity of this entity. With a brief literature review, we consider how the cholesteatoma localizes to the tip of the mastoid bone.
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Ethanol production from galactose by a newly isolated Saccharomyces cerevisiae KL17.
Bioprocess Biosyst Eng
PUBLISHED: 02-19-2014
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A wild-type yeast strain with a good galactose-utilization efficiency was newly isolated from the soil, and identified and named Saccharomyces cerevisiae KL17 by 18s RNA sequencing. Its performance of producing ethanol from galactose was investigated in flask cultures with media containing various combination and concentrations of galactose and glucose. When the initial galactose concentration was 20 g/L, it showed 2.2 g/L/h of substrate consumption rate and 0.63 g/L/h of ethanol productivity. Although they were about 70 % of those with glucose, such performance of S. cerevisiae KL17 with galactose was considered to be quite high compared with other strains reported to date. Its additional merit was that its galactose metabolism was not repressed by the existence of glucose. Its capability of ethanol production under a high ethanol concentration was demonstrated by fed-batch fermentation in a bioreactor. A high ethanol productivity of 3.03 g/L/h was obtained with an ethanol concentration and yield of 95 and 0.39 g/L, respectively, when the cells were pre-cultured on glucose. When the cells were pre-cultured on galactose instead of glucose, fermentation time could be reduced significantly, resulting in an improved ethanol productivity of 3.46 g/L/h. The inhibitory effects of two major impurities in a crude galactose solution obtained from acid hydrolysis of galactan were assessed. Only 5-Hydroxymethylfurfural (5-HMF) significantly inhibited ethanol fermentation, while levulinic acid (LA) was benign in the range up to 10 g/L.
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Chk2 regulates cell cycle progression during mouse oocyte maturation and early embryo development.
Mol. Cells
PUBLISHED: 02-19-2014
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As a tumor suppressor homologue during mitosis, Chk2 is involved in replication checkpoints, DNA repair, and cell cycle arrest, although its functions during mouse oocyte meiosis and early embryo development remain uncertain. We investigated the functions of Chk2 during mouse oocyte maturation and early embryo development. Chk2 exhibited a dynamic localization pattern; Chk2 expression was restricted to germinal vesicles at the germinal vesicle (GV) stage, was associated with centromeres at pro-metaphase I (Pro-MI), and localized to spindle poles at metaphase I (MI). Disrupting Chk2 activity resulted in cell cycle progression defects. First, inhibitor-treated oocytes were arrested at the GV stage and failed to undergo germinal vesicle breakdown (GVBD); this could be rescued after Chk2 inhibition release. Second, Chk2 inhibition after oocyte GVBD caused MI arrest. Third, the first cleavage of early embryo development was disrupted by Chk2 inhibition. Additionally, in inhibitor-treated oocytes, checkpoint protein Bub3 expression was consistently localized at centromeres at the MI stage, which indicated that the spindle assembly checkpoint (SAC) was activated. Moreover, disrupting Chk2 activity in oocytes caused severe chromosome misalignments and spindle disruption. In inhibitor-treated oocytes, centrosome protein ?-tubulin and Polo-like kinase 1 (Plk1) were dissociated from spindle poles. These results indicated that Chk2 regulated cell cycle progression and spindle assembly during mouse oocyte maturation and early embryo development.
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Highly reactive nonheme iron(III) iodosylarene complexes in alkane hydroxylation and sulfoxidation reactions.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 02-18-2014
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High-spin iron(III) iodosylarene complexes bearing an N-methylated cyclam ligand are synthesized and characterized using various spectroscopic methods. The nonheme high-spin iron(III) iodosylarene intermediates are highly reactive oxidants capable of activating strong C-H bonds of alkanes; the reactivity of the iron(III) iodosylarene intermediates is much greater than that of the corresponding iron(IV) oxo complex. The electrophilic character of the iron(III) iodosylarene complexes is demonstrated in sulfoxidation reactions.
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ROCK inhibition prevents early mouse embryo development.
Histochem. Cell Biol.
PUBLISHED: 02-16-2014
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ROCK is a Rho-GTPase effector that is important for actin assembly and is involved in various cellular functions, including cell contraction, migration, motility, and tumor cell invasion. In this study, we investigated ROCK expression and function during early mouse embryo development. Inhibiting ROCK by Y-27632 treatment at the zygote stage resulted in first cleavage failure, and most embryos failed to develop to the 8-cell stage. When adding Y-27632 at the 8-cell stage, embryos failed to undergo compaction and could not develop into blastocysts. In addition, fluorescence staining intensity analysis indicated that actin expression at blastomere membranes was significantly reduced. After ROCK inhibition, two or more nuclei were observed in a cell, which indicated possible cytokinesis failure. Moreover, after ROCK inhibition with Y-27632, the phosphorylation levels of LIMK1/2, a downstream molecule of ROCK, were decreased at blastomere membranes. Thus, our results showed conserved roles for ROCK in this mammalian embryo model and indicated that a ROCK-LIMK1/2-actin pathway might regulate cleavage and blastocyst formation during early mouse embryo development.
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Dynamin 2 regulates actin-mediated spindle migration in mouse oocytes.
Biol. Cell
PUBLISHED: 02-04-2014
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During meiosis, a bipolar spindle forms in the central cytoplasm of an oocyte and then moves to the cortex to extrude the first polar body. This is dependent on the regulation of actin and actin-related molecules. Dynamin 2, a large guanosine triphosphatases (GTPase) known to regulate clathrin-mediated endocytosis, is involved in actin recruitment and actin-based vesicle mobility. In this study, we investigated the role of Dynamin 2 in oocyte meiosis.
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The association of mitochondrial potential and copy number with pig oocyte maturation and developmental potential.
J. Reprod. Dev.
PUBLISHED: 02-04-2014
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ATP is critical for oocyte maturation, fertilization, and subsequent embryo development. Both mitochondrial membrane potential and copy number expand during oocyte maturation. In order to differentiate the roles of mitochondrial metabolic activity and mtDNA copy number during oocyte maturation, we used two inhibitors, FCCP (carbonyl cyanide p-(tri-fluromethoxy)phenyl-hydrazone) and ddC (2'3-dideoxycytidine), to deplete the mitochondrial membrane potential (??m) and mitochondrial copy number, respectively. FCCP (2000 nM) reduced ATP production by affecting mitochondrial ??m, decreased the mRNA expression of Bmp15 (bone morphogenetic protein 15), and shortened the poly(A) tails of Bmp15, Gdf9 (growth differentiation factor 9), and Cyclin B1 transcripts. FCCP (200 and 2000 nM) also affected p34(cdc2) kinase activity. By contrast, ddC did not alter ATP production. Instead, ddC significantly decreased mtDNA copy number (P < 0.05). FCCP (200 and 2000 nM) also decreased extrusion of the first polar body, whereas ddC at all concentrations did not affect the ability of immature oocytes to reach metaphase II. Both FCCP (200 and 2000 nM) and ddC (200 and 2000 µM) reduced parthenogenetic blastocyst formation compared with untreated oocytes. However, these inhibitors did not affect total cell number and apoptosis. These findings suggest that mitochondrial metabolic activity is critical for oocyte maturation and that both mitochondrial metabolic activity and replication contribute to the developmental competence of porcine oocytes.
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Spontaneous obliteration of right ventricular pseudoaneurysm after blunt chest trauma: diagnosis and follow-up with multidetector CT.
Korean J Radiol
PUBLISHED: 01-24-2014
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Right ventricular (RV) pseudoaneurysm caused by trauma is very rare. We report a case of RV pseudoaneurysm which resolved without surgical treatment in a patient who survived a falling accident. Echocardiography failed to identify the pseudoaneurysm. Electrocardiography-gated CT showed a 17-mm-sized saccular pseudoaneurysm arising from the RV outflow tract with a narrow neck. Follow-up CT after two months showed spontaneous obliteration of the lesion.
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Influence of somatic cell donor breed on reproductive performance and comparison of prenatal growth in cloned canines.
Theriogenology
PUBLISHED: 01-22-2014
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Using in vivo-flushed oocytes from a homogenous dog population and subsequent embryo transfer after nuclear transfer, we studied the effects of donor cells collected from 10 different breeds on cloning efficiency and perinatal development of resulted cloned puppies. The breeds were categorized into four groups according to their body weight: small (?9 kg), medium (>9-20 kg), large (>20-40 kg), and ultra large (>40 kg). A total of 1611 cloned embryos were transferred into 454 surrogate bitches for production of cloned puppies. No statistically significant differences were observed for initial pregnancy rates at Day 30 of embryo transfer for the donor cells originated from different breeds. However, full-term pregnancy rates were 16.5%, 11.0%, 10.0%, and 7.1% for the donor cells originated from ultra-large breed, large, medium, and small breeds, respectively, where pregnancy rate in the ultra-large group was significantly higher compared with the small breeds (P < 0.01). Perinatal mortality until weaning was significantly higher in small breeds (33.3%) compared with medium, large, or ultra-large breeds where no mortality was observed. The mean birth weight of cloned pups significantly increased proportional to breed size. The highest litter size was examined in ultra-large breeds. There was no correlation between the number of embryo transferred and litter size. Taken together, the efficiency of somatic cell cloning and fetal survival after embryo transfer may be affected significantly by selecting the appropriate genotype.
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A canine model of Alzheimer's disease generated by overexpressing a mutated human amyloid precursor protein.
Int. J. Mol. Med.
PUBLISHED: 01-22-2014
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Canines are considered the most authentic model for studying multifactorial human diseases, as these animals typically share a common environment with man. Somatic cell nuclear transfer (SCNT) technology along with genetic engineering of nuclear donor cells provides a unique opportunity for examining human diseases using transgenic canines. In the present study, we generated transgenic canines that overexpressed the human amyloid precursor protein (APP) gene containing well-characterized familial Alzheimer's disease (AD) mutations. We successfully obtained five out of six live puppies by SCNT. This was confirmed by observing the expression of green fluorescence protein in the body as a visual transgenic marker and the overexpression of the mutated APP gene in the brain. The transgenic canines developed AD-like symptoms, such as enlarged ventricles, an atrophied hippocampus, and ?-amyloid plaques in the brain. Thus, the transgenic canines we created can serve as a novel animal model for studying human AD.
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ROCK inhibitor Y-27632 prevents porcine oocyte maturation.
Theriogenology
PUBLISHED: 01-09-2014
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The inhibitor Y-27632 is a specific selective inhibitor of Rho-associated protein kinases (ROCKs), which are downstream effectors of Rho guanosine triphosphatease (GTPases) and regulate Rho-associated cellular functions, including actin cytoskeletal organization. Little is known regarding the effects of Y-27632 on mammalian oocyte maturation. In the present study, we investigated the effects of Y-27632 on porcine oocyte meiosis and possible regulatory mechanisms of ROCK during porcine oocyte maturation. We found that ROCK accumulated not only at spindles, but also at the cortex in porcine oocytes. Y-27632 treatment reduced ROCK expression, and inhibited porcine oocyte meiotic maturation, which might be because of the impairment of actin expression and actin-related spindle positioning. Y-27632 treatment also disrupted the formation of actin cap and cortical granule-free domain, which further confirmed a spindle positioning failure. Thus, Y-27632 has significant effects on the meiotic competence of mammalian oocytes by reducing ROCK expression, and the regulation is related to its effects on actin-mediated spindle positioning.
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Artificial control maturation of porcine oocyte by dibutyryl cyclicAMP.
Animal Cells Syst (Seoul)
PUBLISHED: 01-02-2014
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In this study, we investigated the effects of various durations of dibutyryl cyclic AMP (dbcAMP) treatment on the in vitro maturation (IVM) and subsequent development of parthenogenetically activated embryos. Immature porcine oocytes were cultured with or without 1 mM dbcAMP during the first 20, 28, or 36 h of culture, and then incubated for an additional 24 h without dbcAMP. The expression of Wee1B, Myt, and Cdc25B and the level of maturation promoting factor (MPF) in metaphase II oocytes were analyzed by real-time PCR (qRT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. The distribution of actin microfilaments in oocytes was also assessed. Subsequently, apoptotic cells in blastocysts from each group were visualized by transferase-mediated dUTP nick-end labeling staining. Results showed that oocytes extruded the first polar body between 12 and 18 h after being released from dbcAMP. MPF activity in oocytes at 28 + 24 h and 36 + 24 h after dbcAMP treatment was higher than that in the control group. Significantly more blastocysts were present among embryos in 28 + 24 h (54.28% vs. 39.11%, P < 0.05) and 36 + 24 h (47.24% vs. 32.94%, P < 0.05) groups than among embryos cultured in the absence of dbcAMP. However, the number of total and apoptotic cells was not significantly different between groups. The distribution of actin microfilaments was abnormal in oocytes cultured for 60 h without dbcAMP. In addition, the expression of Wee1B, Myt, and Cdc25B was higher in the control group at 44 h than in the dbcAMP group, but there were no differences in expression at the other time points. In conclusion, dbcAMP treatment delays oocyte maturation and maintains oocyte quality.
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JMY functions as actin nucleation-promoting factor and mediator for p53-mediated DNA damage in porcine oocytes.
PLoS ONE
PUBLISHED: 01-01-2014
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Junction-mediating and regulatory protein(JMY) is a multifunctional protein with roles in the transcriptional co-activation of p53 and the regulation of actin nucleation promoting factors and, hence, cell migration; however, its role in the maturation of porcine oocytes is unclear. In the current study, we investigated functional roles of JMY in porcine oocytes. Porcine oocytes expressed JMY mRNA and protein, and the mRNA expression level decreased during oocyte maturation. Knockdown of JMY by RNA interference decreased the rate of polar body extrusion, validating its role in the asymmetric division of porcine oocytes. JMY knockdown also down-regulated the mRNA and protein levels of actin and Arp2/3. Furthermore, JMY accumulated in the nucleus in response to DNA damage, and JMY knockdown suppressed DNA damage-mediated p53 activation. In conclusion, our results show that JMY has important roles in oocyte maturation as a regulator of actin nucleation-promoting factors and an activator of p53 during DNA damage during DNA damages in porcine oocytes.
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Design and synthesis of a cell-permeable, drug-like small molecule inhibitor targeting the polo-box domain of polo-like kinase 1.
PLoS ONE
PUBLISHED: 01-01-2014
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Polo-like kinase-1 (Plk1) plays a crucial role in cell proliferation and the inhibition of Plk1 has been considered as a potential target for specific inhibitory drugs in anti-cancer therapy. Several research groups have identified peptide-based inhibitors that target the polo-box domain (PBD) of Plk1 and bind to the protein with high affinity in in vitro assays. However, inadequate proteolytic resistance and cell permeability of the peptides hinder the development of these peptide-based inhibitors into novel therapeutic compounds.
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Zinc regulates meiotic resumption in porcine oocytes via a protein kinase C-related pathway.
PLoS ONE
PUBLISHED: 01-01-2014
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Zinc is an extremely important trace element that plays important roles in several biological processes. However, the function of zinc in meiotic division of porcine oocytes is unknown. In this study, we investigated the role of zinc during meiotic resumption in in vitro matured porcine oocytes. During meiotic division, a massive release of zinc was observed. The level of free zinc in the cytoplasm significantly increased during maturation. Depletion of zinc using N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a Zn2+ chelator, blocked meiotic resumption in a dose dependent manner. The level of phosphorylated mitogen activated protein kinase (MAPK) and p34cdc2 kinase activity were reduced when zinc was depleted. Moreover, zinc depletion reduced the levels of phosphorylated protein kinase C (PKC) substrates in a dose dependent manner. Real-time PCR analysis showed that expression of the MAPK- and maturation promoting factor related genes C-mos, CyclinB1, and Cdc2 was downregulated following zinc depletion. Treatment with the PKC agonist phorbol 12-myristate 13-acetate (PMA) increased phosphorylation of PKC substrates and MAPK and increased p34cdc2 kinase activity. This rescued the meiotic arrest, even in the presence of TPEN. Activation of PKC by PMA increased the level of zinc in the cytoplasm. These data demonstrate that zinc is required for meiotic resumption in porcine oocytes, and this appears to be regulated via a PKC related pathway.
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Reduction in cardiovascular risk using a proactive multifactorial intervention is consistent among patients residing in Pacific Asian and non-Pacific Asian regions: a CRUCIAL trial subanalysis.
Vasc Health Risk Manag
PUBLISHED: 01-01-2014
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Few trials have compared different approaches to cardiovascular disease prevention among Pacific Asian (PA) populations. The Cluster Randomized Usual Care versus Caduet Investigation Assessing Long-term-risk (CRUCIAL) trial demonstrated that a proactive multifactorial intervention (PMI) approach (based on single-pill amlodipine/atorvastatin) resulted in a greater reduction in calculated Framingham 10-year coronary heart disease (CHD) risk compared with usual care (UC) among hypertensive patients with additional risk factors. One-third of CRUCIAL patients resided in the PA region. The aim of this subanalysis was to compare two approaches to cardiovascular risk factor management (PMI versus UC) among patients residing in PA and non-PA regions.
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Arp2/3 complex inhibition prevents meiotic maturation in porcine oocytes.
PLoS ONE
PUBLISHED: 01-01-2014
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The Arp2/3 complex regulates actin nucleation, which is critical for a wide range of cellular processes, such as cell polarity, cell locomotion, and endocytosis. In the present study, we investigated the possible roles of the Arp2/3 complex in porcine oocytes during meiotic maturation. Immunofluorescent staining showed the Arp2/3 complex to localize mainly to the cortex of porcine oocytes, colocalizing with actin. Treatment with an Arp2/3 complex specific inhibitor, CK666, resulted in a decrease in Arp2/3 complex localization at the oocyte cortex. The maturation rate of porcine oocytes decreased significantly after CK666 treatment, concomitant with the failure of cumulus cell expansion and oocyte polar body extrusion. The fluorescence intensity of F-actin decreased in the cytoplasm, and CK666 also disrupted actin cap formation. In summary, our results illustrate that the Arp2/3 complex is required for the meiotic maturation of porcine oocytes and that actin nucleation is critical for meiotic maturation.
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Production of pigs expressing a transgene under the control of a tetracycline-inducible system.
PLoS ONE
PUBLISHED: 01-01-2014
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Pigs are anatomically and physiologically closer to humans than other laboratory animals. Transgenic (TG) pigs are widely used as models of human diseases. The aim of this study was to produce pigs expressing a tetracycline (Tet)-inducible transgene. The Tet-on system was first tested in infected donor cells. Porcine fetal fibroblasts were infected with a universal doxycycline-inducible vector containing the target gene enhanced green fluorescent protein (eGFP). At 1 day after treatment with 1 µg/ml doxycycline, the fluorescence intensity of these cells was increased. Somatic cell nuclear transfer (SCNT) was then performed using these donor cells. The Tet-on system was then tested in the generated porcine SCNT-TG embryos. Of 4,951 porcine SCNT-TG embryos generated, 850 were cultured in the presence of 1 µg/ml doxycycline in vitro. All of these embryos expressed eGFP and 15 embryos developed to blastocyst stage. The remaining 4,101 embryos were transferred to thirty three surrogate pigs from which thirty eight cloned TG piglets were obtained. PCR analysis showed that the transgene was inserted into the genome of each of these piglets. Two TG fibroblast cell lines were established from these TG piglets, and these cells were used as donor cells for re-cloning. The re-cloned SCNT embryos expressed the eGFP transgene under the control of doxycycline. These data show that the expression of transgenes in cloned TG pigs can be regulated by the Tet-on/off systems.
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Rho-GTPase effector ROCK phosphorylates cofilin in actin-meditated cytokinesis during mouse oocyte meiosis.
Biol. Reprod.
PUBLISHED: 01-01-2014
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During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.
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Proper compression landmark and depth for cardiopulmonary resuscitation in patients with pectus excavatum: a study using CT.
Emerg Med J
PUBLISHED: 12-12-2013
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To determine by chest CT the proper compression landmark and depth for cardiopulmonary resuscitation in patients with pectus excavatum (PE).
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Molecular and chemical investigations and comparisons of biomaterials for ocular surface regeneration.
Microsc. Res. Tech.
PUBLISHED: 12-05-2013
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This study investigated and compared the ultrastructural and chemical properties of representative biomaterials for ocular surface regeneration: a human amniotic membrane (AM) in a basal plate, a human AM in reflected chorion, a preserved AM, and a human corneo-scleral tissue. Assessments of the morphological differences in the extracellular matrices were evaluated by hematoxylin-eosin, Massons trichrome (for total collagen), and picrosirius-red (for newly synthesized collagen) staining. Assessments of the changes in the molecular structures and chemical compositions of the biomaterials for ocular surface regeneration were evaluated by Raman spectroscopy. A placental AM (52 %) was a dense and thick collagenous structure compared to a reflected AM (23 %). The spectroscopy did not obtain any structural information for a preserved AM. The cornea group (100 %, control) and sclera group (104 %) showed the collagen lamellae and interfibrillar spacing, and a slight inflammatory reaction with more fibrous and granulomatous tissues. There was a formation of newly synthesized collagen in a placental AM, while there were few collagen components in a reflected AM. Human AM tissues showed consistent Raman spectra and the characteristic collagen bands, similar to the corneal and scleral tissues. Therefore, these findings suggest that human placental AM and reflected AM are structurally suitable for scleral and corneal surface regeneration, respectively, while human placental or preserved AM and reflected AM are molecularly and chemically suitable for corneal and scleral surface regeneration, respectively. Microsc. Res. Tech., 2013. © 2013 Wiley Periodicals, Inc.
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Actin nucleator Arp2/3 complex is essential for mouse preimplantation embryo development.
Reprod. Fertil. Dev.
PUBLISHED: 11-10-2013
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The Arp2/3 complex is a critical actin nucleator, which promotes actin assembly and is widely involved in a diverse range of actin-related processes such as cell locomotion, phagocytosis and the establishment of cell polarity. Previous studies showed that the Arp2/3 complex regulates spindle migration and asymmetric division during mouse oocyte maturation; however, the role of the Arp2/3 complex in early mouse embryo development is still unknown. The results of the present study show that the Arp2/3 complex is critical for cytokinesis during mouse embryo development. The Arp2/3 complex was concentrated at the cortex of each cell at the 2- to 8-cell stage and the peripheral areas of the morula and blastocyst. Inhibition of the Arp2/3 complex by the specific inhibitor CK666 at the zygote stage caused a failure in cell division; mouse embryos failed to undergo compaction and lost apical-basal polarity. The actin level decreased in the CK666-treated group, and two or more nuclei were observed within a single cell, indicating a failure of cell division. Addition of CK666 at the 8-cell stage caused a failure of blastocyst formation, and CDX2 staining confirmed the loss of embryo polarity and the failure of trophectoderm and inner cell mass formation. Taken together, these data suggest that the Arp2/3 complex may regulate mouse embryo development via its effect on cell division.
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