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Find video protocols related to scientific articles indexed in Pubmed.
Mesenchymal Stem Cell Implantation in Osteoarthritic Knees: Is Fibrin Glue Effective as a Scaffold?
Am J Sports Med
PUBLISHED: 10-29-2014
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The cell-based tissue engineering approach that uses mesenchymal stem cells (MSCs) has addressed the issue of articular cartilage repair in osteoarthritic (OA) knees. However, to improve outcomes, an advanced surgical procedure with tissue-engineered scaffolds may be needed to treat patients with large cartilage lesions.
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Negligible pharmacokinetic interaction of red ginseng and antihypertensive agent amlodipine in sprague-dawley rats.
J. Toxicol. Environ. Health Part A
PUBLISHED: 10-25-2014
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Red ginseng (RG) is the top-selling functional food in Korea, but is not recommended for use in hypertensive patients. This study was performed to determine the pharmacokinetic (PK) interaction between RG and amlodipine, an antihypertensive drug. RG (0, 0.5, 1, or 2 g/kg/d) was administered orally for 2 wk, and then amlodipine (10 mg/kg) was given orally, to Sprague-Dawley (SD) rats. Blood was collected at 0.08, 0.25, 1, 1.5, 2, 3, 6, 12, and 24 h after amlodipine administration. In intravenous (iv) study, RG (0, 1, or 2 g/kg/d) was administered orally to SD rats for 2 wk, followed by amlodipine (2 mg/kg) intravenously (iv). Plasma concentrations of amlodipine were analyzed using a high-pressure liquid chromatography-tandem mass system (LC-MS/MS). Oral administration of amlodipine produced an increase of time to maximum plasma concentration (tmax: 2.6, 4.1, 8.3, and 8.9 h at 0, 0.5, 1, and 2 g/kg/d, respectively), and a decrease of maximum plasma concentration (Cmax: 278.5, 212.4, 232.1, and 238.7 ng/ml at 0, 0.5, 1, and 2 g/kg/d, respectively.). However, the area under the concentration-time curve from time 0 to 24 h measurable concentration (AUC0-24 h was 3487.4, 2895.4, 3158.2, and 3495 ng/h/ml at 0, 0.5, 1, and 2 g/kg/d respectively) was not significantly changed among the different dose groups. Administration of amlodipine iv produced no significant changes in the apparent terminal half-life, volume of distribution, and AUC0-24 hr among the different dose groups. These results suggest that RG induced negligible influence on amlodipine pharmacokinetically in rats.
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Aptamer-based single-molecule imaging of insulin receptors in living cells.
J Biomed Opt
PUBLISHED: 08-05-2014
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We present a single-molecule imaging platform that quantitatively explores the spatiotemporal dynamics of individual insulin receptors in living cells. Modified DNA aptamers that specifically recognize insulin receptors (IRs) with a high affinity were selected through the SELEX process. Using quantum dot-labeled aptamers, we successfully imaged and analyzed the diffusive motions of individual IRs in the plasma membranes of a variety of cell lines (HIR, HEK293, HepG2). We further explored the cholesterol-dependent movement of IRs to address whether cholesterol depletion interferes with IRs and found that cholesterol depletion of the plasma membrane by methyl-?-cyclodextrin reduces the mobility of IRs. The aptamer-based single-molecule imaging of IRs will provide better understanding of insulin signal transduction through the dynamics study of IRs in the plasma membrane.
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Aptamer-conjugated gold nanorod for photothermal ablation of epidermal growth factor receptor-overexpressed epithelial cancer.
J Biomed Opt
PUBLISHED: 08-05-2014
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Biomarker-specific photothermal nanoparticles that can efficiently sense markers that are overexpressed in distinguished adenocarcinomas have attracted much interest in an aspect of efficacy increase of cancer treatment. We demonstrated a promising prospect of a smart photothermal therapy agent employing anti-epidermal growth factor receptor aptamer (AptEGFR)-conjugated polyethylene glycol (PEG) layted gold nanorods (AptEGFR-PGNRs). The cetyltrimethylammonium bromide bilayer on GNRs was replaced with heterobifunctional PEG (COOH-PEG-SH) not only to serve as a biocompatible stabilizer and but also to conjugate AptEGFR. Subsequently, to direct photothermal therapy agent toward epithelial cancer cells, the carboxylated PEGylated GNRs (PGNRs) were further functionalized with AptEGFR using carbodiimide chemistry. Then, to assess the potential as biomarker-specific photothermal therapy agent of synthesized AptEGFR-PGNRs, the optical properties, biocompatibility, colloidal stability, binding affinity, and epicellial cancer cell killing efficacy in vitro/in vivo under near-infrared laser irradiation were investigated. As a result, AptEGFR-PGNRs exhibit excellent tumor targeting ability and feasibility of effective photothermal ablation cancer therapy.
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Overexpression of TGF-?1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells.
Biochem. Biophys. Res. Commun.
PUBLISHED: 07-04-2014
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Transforming growth factor-beta (TGF-?) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-? up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuous TGF-?1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-?1. The results revealed that continuous overexpression of TGF-?1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-?1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-?1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs.
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Endothelial deletion of phospholipase D2 reduces hypoxic response and pathological angiogenesis.
Arterioscler. Thromb. Vasc. Biol.
PUBLISHED: 06-19-2014
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Aberrant regulation of the proliferation, survival, and migration of endothelial cells (ECs) is closely related to the abnormal angiogenesis that occurs in hypoxia-induced pathological situations, such as cancer and vascular retinopathy. Hypoxic conditions and the subsequent upregulation of hypoxia-inducible factor-1? and target genes are important for the angiogenic functions of ECs. Phospholipase D2 (PLD2) is a crucial signaling mediator that stimulates the production of the second messenger phosphatidic acid. PLD2 is involved in various cellular functions; however, its specific roles in ECs under hypoxia and in vivo angiogenesis remain unclear. In the present study, we investigated the potential roles of PLD2 in ECs under hypoxia and in hypoxia-induced pathological angiogenesis in vivo.
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Heterozygous mutations in cyclic AMP phosphodiesterase-4D (PDE4D) and protein kinase A (PKA) provide new insights into the molecular pathology of acrodysostosis.
Cell. Signal.
PUBLISHED: 06-14-2014
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Acrodysostosis without hormone resistance is a rare skeletal disorder characterized by brachydactyly, nasal hypoplasia, mental retardation and occasionally developmental delay. Recently, loss-of-function mutations in the gene encoding cAMP-hydrolyzing phosphodiesterase-4D (PDE4D) have been reported to cause this rare condition but the pathomechanism has not been fully elucidated. To understand the pathogenetic mechanism of PDE4D mutations, we conducted 3D modeling studies to predict changes in the binding efficacy of cAMP to the catalytic pocket in PDE4D mutants. Our results indicated diminished enzyme activity in the two mutants we analyzed (Gly673Asp and Ile678Thr; based on PDE4D4 residue numbering). Ectopic expression of PDE4D mutants in HEK293 cells demonstrated this reduction in activity, which was identified by increased cAMP levels. However, the cells from an acrodysostosis patient showed low cAMP accumulation, which resulted in a decrease in the phosphorylated cAMP Response Element-Binding Protein (pCREB)/CREB ratio. The reason for this discrepancy was due to a compensatory increase in expression levels of PDE4A and PDE4B isoforms, which accounted for the paradoxical decrease in cAMP levels in the patient cells expressing mutant isoforms with a lowered PDE4D activity. Skeletal radiographs of 10-week-old knockout (KO) rats showed that the distal part of the forelimb was shorter than in wild-type (WT) rats and that all the metacarpals and phalanges were also shorter in KO, as the name acrodysostosis implies. Like the G-protein ?-stimulatory subunit and PRKAR1A, PDE4D critically regulates the cAMP signal transduction pathway and influences bone formation in a way that activity-compromising PDE4D mutations can result in skeletal dysplasia. We propose that specific inhibitory PDE4D mutations can lead to the molecular pathology of acrodysostosis without hormone resistance but that the pathological phenotype may well be dependent on an over-compensatory induction of other PDE4 isoforms that can be expected to be targeted to different signaling complexes and exert distinct effects on compartmentalized cAMP signaling.
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Regulation of C1-Ten protein tyrosine phosphatase by p62/SQSTM1-mediated sequestration and degradation.
Cell. Signal.
PUBLISHED: 05-21-2014
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C1-Ten is a member of the tensin family of focal adhesion molecules but recent studies suggest it plays a more active role in many biological processes because of its potential association with diabetes and cancers. However, relatively little is known about the regulation of C1-Ten, such as changes in its protein level or cellular localization. The cellular localization of C1-Ten is unique because it is expressed in cytoplasmic puncta but nothing is known about these puncta. Here, we show that p62 sequestrates C1-Ten into puncta, making C1-Ten diffuse into the cytoplasm upon p62 depletion. More importantly, p62-mediated C1-Ten sequestration promoted C1-Ten ubiquitination and proteasomal degradation. p62-mediated protein reduction was specific to C1-Ten, and not other tensins such as tensin1 and tensin3. Thus, our results link cellular localization of C1-Ten to an off-switch site for C1-Ten. Additionally, p62 expression increased but C1-Ten protein decreased during muscle differentiation, supporting a role for p62 as a physiological regulator of C1-Ten.
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Clinically useful diagnostic tool of contrast enhanced ultrasonography for focal liver masses: comparison to computed tomography and magnetic resonance imaging.
Gut Liver
PUBLISHED: 05-16-2014
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To evaluate the diagnostic value of contrast (SonoVue(®)) enhancement ultrasonography (CEUS) and to compare this method with computed tomography (CT) and magnetic resonance imaging (MRI) in evaluating liver masses.
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Design of pH-responsive alginate raft formulation of risedronate for reduced esophageal irritation.
Int. J. Biol. Macromol.
PUBLISHED: 05-08-2014
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Risedronate sodium (RA), a pyridinyl bisphosphonate, is widely used in the treatment of osteoporosis. However, the free acid form of the bisphosphonate below pH 3.5 has the potential to produce severe impatience of the upper gastrointestinal tract, particularly esophagitis. A pH-responsive raft-forming tablet (PRR-T) was designed to prevent the esophageal irritation, mainly consisting of low-molecular-weight alginate (LFR 5/60, 300 mg) as raft-forming polymer, sodium bicarbonate (1000 mg) as gas-generating agent and citrate and sodium citrate (600 and 200 mg, respectively) as buffer system. A PRR-T was rapidly liquefied in water within 80 s with a low viscosity 8.0 mPa s, offering ease of swallowing in patients. A formulation profoundly neutralized simulated gastric fluid over pH 5.5, leading to an ionization of the bisphosphonate, without raft formation. On the other hand, the raft was rapidly formed on the top layer preventing the reflux of RA, if the contact with acidic medium is much higher than 0.5 N of hydrochloric acid. Nevertheless, the release rate of the drug was equivalent, providing over 95% release within 5 min. Our study demonstrated the potential usefulness of alginate-based PRR-T for an oral therapy with bisphosphonates for reduced esophageal adverse experiences.
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Parkin ubiquitinates mTOR to regulate mTORC1 activity under mitochondrial stress.
Cell. Signal.
PUBLISHED: 04-21-2014
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mTORC1, a kinase complex that is considered a master regulator of cellular growth and proliferation, is regulated by many extra- and intracellular signals. Among these signals, mitochondrial status is known to have an impact on the effects of mTORC1 on cell growth and survival. However, how mitochondrial status affects mTORC1 activity, notably the molecular link, is not fully elucidated. Here, we found that Parkin can interact with and ubiquitinate mTOR. We also identified K2066 and K2306 as Parkin-dependent and mitochondrial stress-induced mTOR ubiquitination residues. This ubiquitination by Parkin is required for maintenance of mTORC1 activity under mitochondrial stress. With regard to the physiological meaning of mTORC1 activity under mitochondrial stress, we suggest that mTORC1 plays a pro-survival role.
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DJ-1 contributes to adipogenesis and obesity-induced inflammation.
Sci Rep
PUBLISHED: 04-09-2014
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Adipose tissue functions as an endocrine organ, and the development of systemic inflammation in adipose tissue is closely associated with metabolic diseases, such as obesity and insulin resistance. Accordingly, the fine regulation of the inflammatory response caused by obesity has therapeutic potential for the treatment of metabolic syndrome. In this study, we analyzed the role of DJ-1 (PARK7) in adipogenesis and inflammation related to obesity in vitro and in vivo. Many intracellular functions of DJ-1, including oxidative stress regulation, are known. However, the possibility of DJ-1 involvement in metabolic disease is largely unknown. Our results suggest that DJ-1 deficiency results in reduced adipogenesis and the down-regulation of pro-inflammatory cytokines in vitro. Furthermore, DJ-1-deficient mice show a low-level inflammatory response in the high-fat diet-induced obesity model. These results indicate previously unknown functions of DJ-1 in metabolism and therefore suggest that precise regulation of DJ-1 in adipose tissue might have a therapeutic advantage for metabolic disease treatment.
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A novel feeder-free culture system for expansion of mouse spermatogonial stem cells.
Mol. Cells
PUBLISHED: 04-04-2014
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Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost- effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.
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Co-culture with human synovium-derived mesenchymal stem cells inhibits inflammatory activity and increases cell proliferation of sodium nitroprusside-stimulated chondrocytes.
Biochem. Biophys. Res. Commun.
PUBLISHED: 04-04-2014
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Rheumatoid arthritis (RA) and osteoarthritis (OA) are primarily chronic inflammatory diseases. Mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the mesodermal lineage, and to regulate immunomodulatory activity. Specifically, MSCs have been shown to secrete insulin-like growth factor 1 (IGF-1). The purpose of the present study was to examine the inhibitory effects on inflammatory activity from a co-culture of human synovium-derived mesenchymal stem cells (hSDMSCs) and sodium nitroprusside (SNP)-stimulated chondrocytes. First, chondrocytes were treated with SNP to generate an in vitro model of RA or OA. Next, the co-culture of hSDMSCs with SNP-stimulated chondrocytes reduced inflammatory cytokine secretion, inhibited expression of inflammation activity-related genes, generated IGF-1 secretion, and increased the chondrocyte proliferation rate. To evaluate the effect of IGF-1 on inhibition of inflammation, chondrocytes pre-treated with IGF-1 were treated with SNP, and then the production of inflammatory cytokines was analyzed. Treatment with IGF-1 was shown to significantly reduce inflammatory cytokine secretion in SNP-stimulated chondrocytes. Our results suggest that hSDMSCs offer a new strategy to promote cell-based cartilage regeneration in RA or OA.
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Proteomic analysis of hypoxia-induced U373MG glioma secretome reveals novel hypoxia-dependent migration factors.
Proteomics
PUBLISHED: 03-25-2014
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High-grade gliomas are one of the most common brain tumors and notorious for poor prognosis due to their malignant nature. Gliomas have an extensive area of hypoxia, which is critical for glioma progression by inducing aggressiveness and activating the angiogenesis process in the tumor microenvironment. To resolve the factors responsible for the highly malignant nature of gliomas, we comprehensively profiled the U373MG glioma cell secretome-exosome and soluble fraction under hypoxic and normoxic conditions. A total of 239 proteins were identified from the exosome and soluble fractions. Vascular endothelial growth factor, stanniocalcin 1 (STC1) and stanniocalcin 2, and insulin-like growth factor binding protein 3 and 6, enriched in the soluble fraction, and lysyl oxidase homolog 2 enriched in the exosomal fraction were identified as upregulated proteins by hypoxia based on a label-free quantitative analysis. STCs and insulin-like growth factor binding proteins, which were identified as secretory proteins under hypoxic conditions, were highly correlated with glioma grade in human patients by microarray analysis. An in vitro scratch wound assay revealed that STC1 and 2 have important functions in the induction of cell migration in a hypoxia-dependent manner, suggesting that they are hypoxia-dependent migration factors.
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Development of an aptamer-conjugated fluorescent nanoprobe for MMP2.
Nanoscale Res Lett
PUBLISHED: 02-24-2014
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Matrix metalloproteinase 2 (MMP2) plays critical roles in various diseases, such as atherosclerosis and cancer, and has been suggested to contribute to the instability of atherosclerotic plaque. To visualize MMP2 in pathologic tissues, we developed an aptamer targeting MMP2 protein by performing eight rounds of modified DNA systematic evolution of ligands by exponential enrichment (SELEX). The aptamer showed high affinity for MMP2 (Kd?=?5.59 nM), precipitated MMP2, and detected MMP2 protein in pathological tissues such as atherosclerotic plaque and gastric cancer tissues. Furthermore, a MMP2 aptamer-conjugated fluorescent nanoprobe successfully visualized atherosclerotic plaques in apolipoprotein E (ApoE) knockout mice. These results suggest that the devised MMP2 aptamer could be useful for the development of various diagnostic tools.
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CXCL12 secreted from adipose tissue recruits macrophages and induces insulin resistance in mice.
Diabetologia
PUBLISHED: 02-15-2014
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Obesity-induced inflammation is initiated by the recruitment of macrophages into adipose tissue. The recruited macrophages, called adipose tissue macrophages, secrete several proinflammatory cytokines that cause low-grade systemic inflammation and insulin resistance. The aim of this study was to find macrophage-recruiting factors that are thought to provide a crucial connection between obesity and insulin resistance.
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Serum and ultrastructure responses of common carp (Cyprinus carpio L.) during long-term exposure to zinc oxide nanoparticles.
Ecotoxicol. Environ. Saf.
PUBLISHED: 01-30-2014
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The uptake of nanoparticles by aquatic organisms such as fish has raised concerns about the possible adverse effects of nanoparticles (NPs). In this study, we aimed to evaluate the toxicological effects in juvenile common carp exposed to zinc oxide nanoparticles (ZnO-NPs) for 12 weeks. The carp were exposed to 0 (control), 0.1, 0.3, 0.8, and 2.4mg/L of ZnO-NPs under a flow-through exposure system. Fish were sampled at 0, 4, 8, and 12 weeks to test for zinc in the test water and blood, and biochemistry analysis; further, they were sampled at 12 weeks to observe ultrastructural changes in the liver, kidney, and gill. In the organic serum, changes in the glutamic pyruvic transaminase/alanine aminotransferase (GPT/ALT) and glutamic oxaloacetic transaminase/aspartate aminotransferase (GOT/AST) levels were significant, but changes in the lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) levels were not significantly different across all exposure periods. In the inorganic serum, the magnesium (Mg), inorganic phosphorus (IP), sodium (Na(+)), and chloride (Cl(-)) levels were significantly different in the exposure group and across exposure periods. However, calcium (Ca) and potassium (K(+)) levels were not significantly different. In the enzyme serum, the glucose (GLU) level significantly increased for the highest exposure group, but the total cholesterol (TCHO), triglyceride (Tg), and total protein (TP) levels were not significantly different during the exposure period. Ultrastructural changes in the liver induced changes in the black granules (of various sizes) in the lysosomes, indistinct nucleus membrane, and non-spherical nucleus. In the kidney, some mild changes were observed in the size and number of the lysosomes in the renal tubule. Desquamation and hypertrophy of pavement epithelial cells and vacuolation in the cytoplasm of the chloride cells were observed in the gill. Nanoparticles were also observed in the red blood cells, cytoplasm of all tissues, and glomerulus of the kidney. The observed changes in the serum and tissues may provide useful information regarding environmental conditions and risk assessments of aquatic organisms.
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Chlormadinone acetate promotes osteoblast differentiation of human mesenchymal stem cells through the ERK signaling pathway.
Eur. J. Pharmacol.
PUBLISHED: 01-08-2014
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Bone is continuously remodeled throughout life, and this remodeling is regulated by osteoclasts and osteoblasts. Bone-forming osteoblasts are derived from mesenchymal stem cells in bone marrow. Here, we have identified a new function of chlormadinone acetate (CMA) as an osteogenic activator in human bone marrow-derived mesenchymal stem cells (hBMSCs). To date, CMA has been used as an oral contraceptive and is known to have antiandrogenic activity. Our results show that CMA promotes osteoblast differentiation and calcium deposition in hBMSCs, whereas CMA treatment suppresses adipogenesis of hBMSCs. CMA activates and potentiates the phosphorylation of extracellular signal-regulated kinases (ERK1/2) in an osteogenic differentiation conditions. In addition, CMA-stimulated osteoblast differentiation is suppressed by inhibiting the ERK pathway, suggesting that CMA promotes the osteogenic differentiation program of hBMSCs through the ERK activation. Taken together, these results suggest a novel function of CMA as an osteogenic activator and intracellular signaling pathway mediated by CMA in osteoblast differentiation.
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Association of inflammation, myocardial fibrosis and cardiac remodelling in patients with mild aortic stenosis as assessed by biomarkers and echocardiography.
Clin. Exp. Pharmacol. Physiol.
PUBLISHED: 01-07-2014
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1. The aim of the present study was to investigate the relationships among inflammation, myocardial fibrosis and cardiac remodelling in patients with mild aortic stenosis (AS), as assessed by biomarkers and echocardiography. 2. We evaluated 32 consecutive patients with mild AS, as well as 30 age- and gender-matched healthy individuals with normal aortic valves as control subjects. 3. Baseline echocardiography showed that the left ventricular (LV) mass index (111.3 ± 26.9 vs 94.5 ± 18.2 g/m(2); P = 0.006) and left atrial (LA) volume index (LAVI 27.5 ± 9.0 vs xx.x ± 5.2 mm(3)/mm(2); P = 0.005) were significantly higher in patients with mild AS. 4. Furthermore, LA enlargement (LAVI > 33 mm(3)/mm(2); 32.4% vs 3.3%; P = 0.003) and elevated LV filling pressure (E/e' > 15; 50.0% vs 23.3%; P = 0.036) were higher in patients with mild AS. 5. In patients with mild AS, stepwise, multivariate linear regression analysis revealed that the LV end-diastolic volume index was independently associated with matrix metalloproteinase (MMP)-1 (? = 0.371; P = 0.015), that the aortic valve mean pressure gradient was independently associated with MMP-2 (? = 0.19; P = 0.019), that MMP-2 was independently associated with transforming growth factor-? (? = 0.95; P < 0.001) and interleukin (IL)-1 (? = 0.17; P = 0.019) and that IL-1 was independently associated with tissue inhibitor of matrix metalloproteinase-1 (? = 0.68; P = 0.001). 6. Myocardial fibrosis in mild AS is independently associated with three factors: LV volume overload, aortic valve pressure overload and inflammation.
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Enhancement of ?-Glucan Content in the Cultivation of Cauliflower Mushroom (Sparassis latifolia) by Elicitation.
Mycobiology
PUBLISHED: 01-06-2014
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The effectiveness of three kinds of enzymes (chitinase, ?-glucuronidase, and lysing enzyme complex), employed as elicitors to enhance the ?-glucan content in the sawdust-based cultivation of cauliflower mushroom (Sparassis latifolia), was examined. The elicitors were applied to the cauliflower mushroom after primordium formation, by spraying the enzyme solutions at three different levels on the sawdust-based medium. Mycelial growth was fully accomplished by the treatments, but the metabolic process during the growth of fruiting bodies was affected. The application of a lysing enzyme resulted in an increase in the ?-glucan concentration by up to 31% compared to that of the control. However, the treatment resulted in a decrease in mushroom yield, which necessitated the need to evaluate its economic efficiency. Although we still need to develop a more efficient way for using elicitors to enhance functional metabolites in mushroom cultivation, the results indicate that the elicitation technique can be applied in the cultivation of medicinal/edible mushrooms.
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Xanthene derivatives increase glucose utilization through activation of LKB1-dependent AMP-activated protein kinase.
PLoS ONE
PUBLISHED: 01-01-2014
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5' AMP-activated protein kinase (AMPK) is a highly conserved serine-threonine kinase that regulates energy expenditure by activating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. Therefore AMPK activators are considered to be drug targets for treatment of metabolic diseases such as diabetes mellitus. To identify novel AMPK activators, we screened xanthene derivatives. We determined that the AMPK activators 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-nitro-phenyl)-thioureido]-ethyl}-amide (Xn) and 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-cyano-phenyl)-thioureido]-ethyl}-amide (Xc) elevated glucose uptake in L6 myotubes by stimulating translocation of glucose transporter type 4 (GLUT4). Treatment with the chemical AMPK inhibitor compound C and infection with dominant-negative AMPKa2-virus inhibited AMPK phosphorylation and glucose uptake in myotubes induced by either Xn or Xc. Of the two major upstream kinases of AMPK, we found that Xn and Xc showed LKB1 dependency by knockdown of STK11, an ortholog of human LKB1. Single intravenous administration of Xn and Xc to high-fat diet-induced diabetic mice stimulated AMPK phosphorylation of skeletal muscle and improved glucose tolerance. Taken together, these results suggest that Xn and Xc regulate glucose homeostasis through LKB1-dependent AMPK activation and that the compounds are potential candidate drugs for the treatment of type 2 diabetes mellitus.
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Computational design of binding proteins to EGFR domain II.
PLoS ONE
PUBLISHED: 01-01-2014
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We developed a process to produce novel interactions between two previously unrelated proteins. This process selects protein scaffolds and designs protein interfaces that bind to a surface patch of interest on a target protein. Scaffolds with shapes complementary to the target surface patch were screened using an exhaustive computational search of the human proteome and optimized by directed evolution using phage display. This method was applied to successfully design scaffolds that bind to epidermal growth factor receptor (EGFR) domain II, the interface of EGFR dimerization, with high reactivity toward the target surface patch of EGFR domain II. One potential application of these tailor-made protein interactions is the development of therapeutic agents against specific protein targets.
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Pattern Recognition Analysis for Hepatotoxicity Induced by Acetaminophen Using Plasma and Urinary (1)H NMR-Based Metabolomics in Humans.
Anal. Chem.
PUBLISHED: 11-14-2013
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Drug-induced liver injury (DILI) is currently an increasingly relevant health issue. However, available biomarkers do not reliably detect or quantify DILI risk. Therefore, the purpose of this study was to comparatively evaluate plasma and urinary biomarkers obtained from humans treated with acetaminophen (APAP) using a metabolomics approach and a proton nuclear magnetic resonance (NMR) platform. APAP (3 g/day, two 500 mg tablets every 8 h) was administered to 20 healthy Korean males (age, 20-29 years) for 7 days. Urine was collected daily before and during dosing and 6 days after the final dose. NMR spectra of these urine samples were analyzed using principal component analysis (PCA) and partial least-squares-discrimination analysis. Although the activities of aspartate aminotransferase and lactate dehydrogenase were significantly increased 7 days post-APAP treatment, serum biochemical parameters of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, ?-glutamyl transpeptidase, and lactate dehydrogenase were within normal range of hepatic function. However, urine and plasma (1)H NMR spectroscopy revealed different clustering between predosing and after APAP treatment for global metabolomic profiling through PCA. Urinary endogenous metabolites of trimethylamine-N-oxide, citrate, 3-chlorotyrosine, phenylalanine, glycine, hippurate, and glutarate as well as plasma endogenous metabolites such as lactate, glucose, 3-hydroxyisovalerate, isoleucine, acetylglycine, acetone, acetate, glutamine, ethanol, and isobutyrate responded significantly to APAP dosing in humans. Urinary and plasma endogenous metabolites were more sensitive than serum biochemical parameters. These results might be applied to predict or screen potential hepatotoxicity caused by other drugs using urinary and plasma (1)H NMR analyses.
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Clinical implications and risk factors of acute pancreatitis after cardiac valve surgery.
Yonsei Med. J.
PUBLISHED: 10-30-2013
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Acute pancreatitis is one of the potentially lethal complications that occurs after cardiac surgery. We tried to identify risk factors for and the prognosis of acute pancreatitis after cardiac valve surgery with cardiopulmonary bypass.
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The impact of high sensitivity C-reactive protein level on coronary artery spasm as assessed by intracoronary acetylcholine provocation test.
Yonsei Med. J.
PUBLISHED: 10-22-2013
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High sensitive C-reactive protein (hs CRP) is well known as a strong risk factor of cardiovascular disease (CVD). The aim of this study is to evaluate the impact of elevated hs CRP on coronary artery spasm (CAS) as assessed by intracoronary acetylcholine (ACh) provocation test.
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Mutagenic assessment of olmesartan cilexetil by bacterial mutation assay.
Toxicol Res
PUBLISHED: 09-26-2013
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Hypertension is a serious health problem due to high frequency and concomitant other diseases including cardiovascular and renal dysfunction. Olmesartan cilexetil is a new antihypertensive drug associated with angiotensin II receptor antagonist. This study was conducted to evaluate the mutagenicity of olmesartan cilexetil by bacterial reverse mutation test using Salmonella typhimurium (TA100, TA1535, TA98, and TA1537) and Escherichia coli (WP2 uvrA). At the concentrations of 0, 62, 185, 556, 1667, and 5000 ?g/ plate, olmesartan cilexetil was negative in both Salmonella typhimurium and Escherichia coli regardless of presence or absence of metabolic activation system (S9 mix). These results demonstrate that olmesartan cilexetil does not induce bacterial reverse mutation.
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Aptamer-modified magnetic nanoprobe for molecular MR imaging of VEGFR2 on angiogenic vasculature.
Nanoscale Res Lett
PUBLISHED: 06-25-2013
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Nucleic acid-based aptamers have been developed for the specific delivery of diagnostic nanoprobes. Here, we introduce a new class of smart imaging nanoprobe, which is based on hybridization of a magnetic nanocrystal with a specific aptamer for specific detection of the angiogenic vasculature of glioblastoma via magnetic resonance (MR) imaging. The magnetic nanocrystal imaging core was synthesized using the thermal decomposition method and enveloped by carboxyl polysorbate 80 for water solubilization and conjugation of the targeting moiety. Subsequently, the surface of the carboxylated magnetic nanocrystal was modified with amine-functionalized aptamers that specifically bind to the vascular growth factor receptor 2 (VEGFR2) that is overexpressed on angiogenic vessels. To assess the targeted imaging potential of the aptamer-conjugated magnetic nanocrystal for VEGFR2 markers, the magnetic properties and MR imaging sensitivity were investigated using the orthotopic glioblastoma mouse model. In in vivo tests, the aptamer-conjugated magnetic nanocrystal effectively targeted VEGFR2 and demonstrated excellent MR imaging sensitivity with no cytotoxicity.
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Magnifying endoscopy for the diagnosis of specialized intestinal metaplasia in short-segment Barretts esophagus.
World J. Gastroenterol.
PUBLISHED: 05-12-2013
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To determine whether magnified observation of short-segment Barretts esophagus (BE) is useful for the detection of specialized intestinal metaplasia (SIM).
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Morphological and genetic characteristics of newly crossbred cauliflower mushroom (Sparassis latifolia).
J. Microbiol.
PUBLISHED: 04-30-2013
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Cauliflower mushroom (Sparassis latifolia or S. crispa) is popular for food and medicine. Importance of new varieties of Sparassis was raised and studied widely by protection system of UPOV. In this study, 10 crossbred strains of Sparassis latifolia that specifically expressed distinctive features during basidiocarp formation and mycelium growth were applied to sawdust medium inoculated with S. latifolia mycelia. The 10 crossbred strains were divided into 3 groups on the basis of morphological (size of marginal wave and basidiocarp color) and genetic characteristics. Each phenotype of the parent and crossbred strains represented 3 marginal wave-sizes (large, medium, and small) and 3 color notations (NN155D, 163C, and 8D). Our result suggests that morphological characteristics of cauliflower mushroom can be affected by various environmental and genetic stimuli under artificial conditions such as crossbreed. Also this research showed genetic differences among breeding isolates and their morphological characteristics were correlated with the molecular data within parent and crossed strain.
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Plant-derived mAbs have effective anti-cancer activities by increasing ganglioside expression in colon cancers.
Biotechnol. Lett.
PUBLISHED: 04-29-2013
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An epithelial cell adhesion molecule (EpCAM) was selectively expressed in human colorectal carcinoma. Treatment with plant-derived anti-EpCAM mAb (mAbP CO17-1A) and RAW264.7 cells inhibited cell growth in the human colorectal cancer cell line SW620. In SW620 treated with mAbP CO17-1A and RAW264.7 cells, expression of p53 and p21 increased, whereas the expression of G1 phase-related proteins, cyclin D1, CDK4, cyclin E, and CDK2, decreased, similar to mammalian-derived mAb (mAbM) CO17-1A. Similar to mAbM CO17-1A, treatment with mAbP CO17-1A and RAW264.7 cell decreased the expression of anti-apoptotic protein, Bcl-2, but the expression of pro-apoptotic proteins Bax, TNF-?, caspase-3, caspase-6, caspase-8 and caspase-9, increased. Cells treated with mAbP CO17-1A and RAW264.7 cells expressed metastasis-related gangliosides, GM1 and GD1a, similar to mAbM CO17-1A. These results suggest that mAbP CO17-1A is as effective on anti-cancer activity as mAbM CO17-1A.
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A Randomized Controlled Trial about the Levels of Radiation Exposure Depends on the Use of Collimation C-arm Fluoroscopic-guided Medial Branch Block.
Korean J Pain
PUBLISHED: 04-03-2013
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C-arm fluoroscope has been widely used to promote more effective pain management; however, unwanted radiation exposure for operators is inevitable. We prospectively investigated the differences in radiation exposure related to collimation in Medial Branch Block (MBB).
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Periostin-binding DNA aptamer inhibits breast cancer growth and metastasis.
Mol. Ther.
PUBLISHED: 03-19-2013
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Periostin is an extracellular matrix (ECM) protein that is overexpressed in a variety of human cancers, and its functions appear to be linked to tumor growth, metastasis, and angiogenesis. Recent clinical evidence suggests that aberrant periostin expression is correlated with poor outcome in patients with breast cancer. To identify novel tools to regulate the functional role of periostin, we generated benzyl-d(U)TP-modified DNA aptamers that were directed against human periostin (PNDAs) and characterized their functional roles in breast cancer progression. PNDA-3 selectively bound to the FAS-1 domain of periostin with nanomolar affinity and disrupted the interaction between periostin and its cell surface receptors, ?v?3 and ?v?5 integrins. PNDA-3 markedly antagonized the periostin-induced adhesion, migration, and invasion of breast cancer cells and blocked the activation of various components of the ?v?3 and ?v?5 integrin signal transduction pathways. In a 4T1 orthotopic mouse model, PNDA-3 administration significantly reduced primary tumor growth and distant metastasis. Thus, our results demonstrated that periostin-integrin signaling regulates breast cancer progression at multiple levels in tumor cells and the tumor microenvironment. DNA aptamers targeting periostin may potentially be used to inhibit breast cancer progression.
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Cytological Evaluation and REBA HPV-ID HPV Testing of Newly Developed Liquid-Based Cytology, EASYPREP: Comparison with SurePath.
Korean J Pathol
PUBLISHED: 03-18-2013
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The objective of this study was to evaluate a newly-developed EASYPREP liquid-based cytology method in cervicovaginal specimens and compare it with SurePath.
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Gender differences in the effect of comorbid insomnia symptom on depression, anxiety, fatigue, and daytime sleepiness in patients with obstructive sleep apnea.
Sleep Breath
PUBLISHED: 02-22-2013
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PURPOSE: This study investigated gender differences in the effect of comorbid insomnia symptom on depression, anxiety, fatigue, daytime sleepiness, and quality of life in patients with obstructive sleep apnea. There are gender differences in the presentation of obstructive sleep apnea. However, the influence of gender on the presentation of comorbid insomnia symptom and obstructive sleep apnea is not known. METHODS: Allparticipantsperformed overnightpolysomnography and completed a battery of questionnaires including Beck Depression Inventory, State-Trait Anxiety Inventory, Multidimensional Fatigue Inventory, Epworth Sleepiness Scale, and Short Form-36 Health Survey. Insomnia symptom was defined as present if a patient had any insomnia complaints longer than 1 month and at least one time per week. RESULTS: Six hundred fifty-five adult patients with obstructive sleep apnea were enrolled; 233 (35.5 %) reported comorbid insomnia symptom with obstructive sleep apnea. The severity of obstructive sleep apnea was not related to comorbid insomnia symptom. Based on linear regression, women had higher depression, fatigue, and daytime sleepiness and lower health-related quality of life than men (all, p?
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An activator of the cAMP/PKA/CREB pathway promotes osteogenesis from human mesenchymal stem cells.
J. Cell. Physiol.
PUBLISHED: 02-15-2013
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Mesenchymal stem cells (MSCs) are multipotent adult stem cells capable of differentiating along the osteoblast, adipocyte, and chondrocyte lineages. Regulation of MSCs differentiation may be a useful tool for regenerative medicine and cell-based therapy. The discovery of small molecule that activates the osteogenic differentiation of MSCs could aid in the development of a new anabolic drug for osteoporosis treatment. We identified CW008, a derivative of pyrazole-pyridine, that stimulates osteoblast differentiation of human MSCs and increases bone formation in ovariectomized mice. CW008 promotes osteogenesis by activating cAMP/PKA/CREB signaling pathway and inhibiting leptin secretion. These results suggest that CW008 is an agonist of cAMP/PKA/CREB pathway in osteogenic differentiation and that application of CW008 may be useful for the treatment of bone-related diseases and for the study of bone biology.
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Functional interplay between Aurora B kinase and Ssu72 phosphatase regulates sister chromatid cohesion.
Nat Commun
PUBLISHED: 02-13-2013
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Cohesins establish cohesion between replicated sister chromatids and are maintained as a multiprotein complex on chromosome arms until they are phosphorylated by mitotic kinases, such as Aurora B and Plk1. However, the mechanics of how the phosphorylation and dephosphorylation of cohesin subunits by kinases and phosphatases, respectively, leads to the dissociation of the cohesin complex from chromosomes remain unclear. Here we report that Aurora B kinase directly interacts with and phosphorylates Ssu72, a new cohesin-binding phosphatase, at Ser 19 in vitro and in vivo. The Aurora B-mediated phosphorylation of Ssu72 causes the structural modification of Ssu72 protein, downregulates phosphatase activity and triggers the ubiquitin-dependent degradation of Ssu72. Overexpression of the Aurora B-mediated phosphomimetic mutant of Ssu72 prevents maintainance chromosome arm cohesion. These results provide evidence that Aurora B kinase directly targets Ssu72 phosphatase for regulation of sister chromatid cohesion during early mitosis.
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Ruptured right sinus of valsalva aneurysm to the right atrium mimicking acute myocardial infarction.
J Cardiovasc Ultrasound
PUBLISHED: 02-13-2013
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We present a rare case involving a ruptured sinus of Valsalva aneurysm (SVA) and acute myocardial infarction in a 39-year-old male patient. Coronary angiography showed normal findings; however, the patient showed remarkably elevated levels of cardiac enzymes and decreased left ventricular function with apical akinesia on transthoracic echocardiography. Transesophageal echocardiography revealed shunt flow from the SVA to the right atrium without significant aortic regurgitation. Preoperative cardiac arrest was managed by cardiopulmonary resuscitation, and surgical repair was performed by closing the entrance of the aneurysm. However, the compromised hemodynamic status was not reversed by surgery.
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C1-Ten is a protein tyrosine phosphatase of insulin receptor substrate 1 (IRS-1), regulating IRS-1 stability and muscle atrophy.
Mol. Cell. Biol.
PUBLISHED: 02-11-2013
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Muscle atrophy occurs under various catabolic conditions, including insulin deficiency, insulin resistance, or increased levels of glucocorticoids. This results from reduced levels of insulin receptor substrate 1 (IRS-1), leading to decreased phosphatidylinositol 3-kinase activity and thereby activation of FoxO transcription factors. However, the precise mechanism of reduced IRS-1 under a catabolic condition is unknown. Here, we report that C1-Ten is a novel protein tyrosine phosphatase (PTPase) of IRS-1 that acts as a mediator to reduce IRS-1 under a catabolic condition, resulting in muscle atrophy. C1-Ten preferentially dephosphorylated Y612 of IRS-1, which accelerated IRS-1 degradation. These findings suggest a novel type of IRS-1 degradation mechanism which is dependent on C1-Ten and extends our understanding of the molecular mechanism of muscle atrophy under catabolic conditions. C1-Ten expression is increased by catabolic glucocorticoid and decreased by anabolic insulin. Reflecting these hormonal regulations, the muscle C1-Ten is upregulated in atrophy but downregulated in hypertrophy. This reveals a previously unidentified role of C1-Ten as a relevant PTPase contributing to skeletal muscle atrophy.
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Synthesis, characterization, and surface wettability properties of amine functionalized graphene oxide films with varying amine chain lengths.
J Colloid Interface Sci
PUBLISHED: 02-06-2013
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Surface functionalization of graphene oxide (GO) an important graphene precursor using alkylamines of varying chain lengths followed by thermal treatment resulted in the formation of superhydrophobic surfaces. Alkylamines consisting of hydrophobic long chain alkyl groups and hydrophilic amine groups were chemically reacted to the GO surface via two types of reactions viz. (i) amidation reaction between amine groups and carboxylic acid sites of GO and (ii) nucleophilic substitution reactions between amine and epoxy groups on GO surface. Successful grafting of alkylamines was confirmed using Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance ((1)H NMR), and thermogravimetric analysis (TGA). Alkylamine-modified GO surfaces showed enhanced roughness, and this effect was more pronounced with increasing amine chain length. Water contact angle measurements revealed that the hydrophobic nature of graphene depended on the chain length of the grafted alkylamines, and this fact may be corroborated to the decrease in the surface energy values. Our results indicate that superhydrophobic graphene films can be produced by thermal treatment of hexadecylamine- and octadecylamine-grafted GO films. These results will provide valuable guidance for the design and manufacture of graphene-based biomaterials, medical instruments, structural composites, electronics, and renewable energy devices.
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Aptamer-conjugated magnetic nanoparticles enable efficient targeted detection of integrin ?v?3 via magnetic resonance imaging.
J Biomed Mater Res A
PUBLISHED: 01-30-2013
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An understanding of neovascularization and/or angiogenesis in cancer is acutely required for effective cancer therapy due to concerns about tumor growth and metastasis. In particular, integrin ?v?3 is closely associated with cell migration and invasion during angiogenesis. Hence, we developed aptamer?v ? 3 -conjugated magnetic nanoparticles (Apt?v ? 3 -MNPs) to enable precise detection of integrin-expressing cancer cells using magnetic resonance imaging. Apt?v ? 3 -MNPs exhibited not only cytocompatibility, but also an efficient targeting ability with high magnetic sensitivity through in vitro/in vivo studies. The results of this study demonstrate that Apt?v ? 3 -MNPs have the potential to be used for accurate tumor diagnosis and therapy. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.
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Chromosomal gain promotes formation of a steep RanGTP gradient that drives mitosis in aneuploid cells.
J. Cell Biol.
PUBLISHED: 01-14-2013
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Many mitotic factors were shown to be activated by Ran guanosine triphosphatase. Previous studies in Xenopus laevis egg extracts and in highly proliferative cells showed that mitotic chromosomes were surrounded by steep Ran guanosine triphosphate (GTP) concentration gradients, indicating that RanGTP-activated factors promote spindle assembly around chromosomes. However, the mitotic role of Ran in normal differentiated cells is not known. In this paper, we show that although the steep mitotic RanGTP gradients were present in rapidly growing cell lines and were required for chromosome congression in mitotic HeLa cells, the gradients were strongly reduced in slow-growing primary cells, such as HFF-1 fibroblasts. The overexpression of RCC1, the guanine nucleotide exchange factor for Ran, induced steeper mitotic RanGTP gradients in HFF-1 cells, showing the critical role of RCC1 levels in the regulation of mitosis by Ran. Remarkably, in vitro fusion of HFF-1 cells produced cells with steep mitotic RanGTP gradients comparable to HeLa cells, indicating that chromosomal gain can promote mitosis in aneuploid cancer cells via Ran.
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Emodin regulates glucose utilization by activating AMP-activated protein kinase.
J. Biol. Chem.
PUBLISHED: 01-09-2013
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AMP-activated protein kinase has been described as a key signaling protein that can regulate energy homeostasis. Here, we aimed to characterize novel AMP-activated kinase (AMPK)-activating compounds that have a much lower effective concentration than metformin. As a result, emodin, a natural anthraquinone derivative, was shown to stimulate AMPK activity in skeletal muscle and liver cells. Emodin enhanced GLUT4 translocation and [(14)C]glucose uptake into the myotube in an AMPK-dependent manner. Also, emodin inhibited glucose production by suppressing the expression of key gluconeogenic genes, such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, in hepatocytes. Furthermore, we found that emodin can activate AMPK by inhibiting mitochondrial respiratory complex I activity, leading to increased reactive oxygen species and Ca(2+)/calmodulin-dependent protein kinase kinase activity. Finally, we confirmed that a single dose administration of emodin significantly decreased the fasting plasma glucose levels and improved glucose tolerance in C57Bl/6J mice. Increased insulin sensitivity was also confirmed after daily injection of emodin for 8 days using an insulin tolerance test and insulin-stimulated PI3K phosphorylation in wild type and high fat diet-induced diabetic mouse models. Our study suggests that emodin regulates glucose homeostasis in vivo by AMPK activation and that this may represent a novel therapeutic principle in the treatment of type 2 diabetic models.
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The Survey about the Degree of Damage of Radiation-Protective Shields in Operation Room.
Korean J Pain
PUBLISHED: 01-08-2013
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Medical doctors who perform C-arm fluoroscopy-guided procedures are exposed to X-ray radiation. Therefore, radiation-protective shields are recommended to protect these doctors from radiation. For the past several years, these protective shields have sometimes been used without regular inspection. The aim of this study was to investigate the degree of damage to radiation-protective shields in the operating room.
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Radiation Exposure of the Hand and Chest during C-arm Fluoroscopy-Guided Procedures.
Korean J Pain
PUBLISHED: 01-04-2013
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The C-arm fluoroscope is an essential tool for the intervention of pain. The aim of this study was to investigate the radiation exposure experienced by the hand and chest of pain physicians during C-arm fluoroscopy-guided procedures.
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Inhibitory effect on NO production of triterpenes from the fruiting bodies of Ganoderma lucidum.
Bioorg. Med. Chem. Lett.
PUBLISHED: 01-03-2013
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Four new lanostane triterpenes, butyl lucidenate P (1), butyl lucidenate D(2) (2), butyl lucidenate E(2) (3) and butyl lucidenate Q (4) along with 11 known compounds (5-15) were isolated from the fruiting bodies of Ganoderma lucidum. Their chemical structures were established mainly by 1D and 2D NMR techniques and mass spectrometry. Their anti-inflammatory activity was evaluated against LPS-induced NO production in macrophage RAW 264.7 cells. Compounds 1, 3, 4, 9, 10 and 15 showed inhibitory potency with IC(50) values of 7.4, 6.4, 4.3, 9.4, 9.2 and 4.5 ?M, respectively. Compounds 1, 3 and 15 dose-dependently reduced the LPS-induced iNOS expressions. Preincubation of cell with 1, 3 and 15 significantly suppressed LPS-induced expression of COX-2 protein.
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Intracellular reprogramming of expression, glycosylation, and function of a plant-derived antiviral therapeutic monoclonal antibody.
PLoS ONE
PUBLISHED: 01-01-2013
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Plant genetic engineering, which has led to the production of plant-derived monoclonal antibodies (mAb(P)s), provides a safe and economically effective alternative to conventional antibody expression methods. In this study, the expression levels and biological properties of the anti-rabies virus mAb(P) SO57 with or without an endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) in transgenic tobacco plants (Nicotiana tabacum) were analyzed. The expression levels of mAb(P) SO57 with KDEL (mAb(P)K) were significantly higher than those of mAb(P) SO57 without KDEL (mAb(P)) regardless of the transcription level. The Fc domains of both purified mAb(P) and mAb(P)K and hybridoma-derived mAb (mAb(H)) had similar levels of binding activity to the Fc?RI receptor (CD64). The mAb(P)K had glycan profiles of both oligomannose (OM) type (91.7%) and Golgi type (8.3%), whereas the mAb(P) had mainly Golgi type glycans (96.8%) similar to those seen with mAb(H). Confocal analysis showed that the mAb(P)K was co-localized to ER-tracker signal and cellular areas surrounding the nucleus indicating accumulation of the mAb(P) with KDEL in the ER. Both mAb(P) and mAb(P)K disappeared with similar trends to mAb(H) in BALB/c mice. In addition, mAb(P)K was as effective as mAb(H) at neutralizing the activity of the rabies virus CVS-11. These results suggest that the ER localization of the recombinant mAb(P) by KDEL reprograms OM glycosylation and enhances the production of the functional antivirus therapeutic antibody in the plant.
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Involvement of exercise-induced macrophage migration inhibitory factor in the prevention of fatty liver disease.
J. Endocrinol.
PUBLISHED: 01-01-2013
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Physical inactivity can lead to obesity and fat accumulation in various tissues. Critical complications of obesity include type II diabetes and nonalcoholic fatty liver disease (NAFLD). Exercise has been reported to have ameliorating effects on obesity and NAFLD. However, the underlying mechanism is not fully understood. We showed that liver expression of macrophage migration inhibitory factor (MIF) was increased after 4 weeks of treadmill exercise. Phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase in human hepatocyte cell lines was enhanced after MIF treatment. These responses were accompanied by increases in lipid oxidation. Moreover, inhibition of either AMPK or cluster of differentiation 74 resulted in inhibition of MIF-induced lipid oxidation. Furthermore, the administration of MIF to a human hepatocyte cell line and mice liver reduced liver X receptor agonist-induced lipid accumulation. Taken together, these results indicate that MIF is highly expressed in the liver during physical exercise and may prevent hepatic steatosis by activating the AMPK pathway.
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Comparative secretome analysis of human bone marrow-derived mesenchymal stem cells during osteogenesis.
J. Cell. Physiol.
PUBLISHED: 01-01-2013
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Osteogenesis is a tightly regulated process that involves coordinated extracellular signals from autocrine and paracrine loops. Secretory proteins during osteogenesis can inhibit cell proliferation and activate cell differentiation toward mature osteoblasts, which are characterized by mineralization. In this study, we attempted to identify these secretory proteins during osteogenesis using LC-MS/MS analysis. We compared the secretome between undifferentiated human bone marrow-derived mesenchymal stem cells (hBMSCs) and differentiated osteoblasts. Among 315 proteins that were identified, 177 proteins were present at increased levels in osteoblasts, whereas 88 proteins were present at decreased levels. Among the identified proteins, several were validated by quantitative RT-PCR and immunoblot analysis. Of particular interest, calcium homeostasis-related proteins were upregulated, whereas stem cell proliferation-related proteins and other lineage-related proteins were downregulated during osteogenesis. These findings provide information about the dynamic changes in the expression and secretion of proteins during osteogenesis and suggest the putative role of secretory proteins in osteogenesis.
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Understanding of the roles of phospholipase D and phosphatidic acid through their binding partners.
Prog. Lipid Res.
PUBLISHED: 12-28-2011
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Phospholipase D (PLD) is a phosphatidyl choline (PC)-hydrolyzing enzyme that generates phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling. Through interactions with signaling molecules, both PLD and PA can mediate a variety of cellular functions, such as, growth/proliferation, vesicle trafficking, cytoskeleton modulation, development, and morphogenesis. Therefore, systemic approaches for investigating PLD networks including interrelationship between PLD and PA and theirs binding partners, such as proteins and lipids, can enhance fundamental knowledge of roles of PLD and PA in diverse biological processes. In this review, we summarize previously reported protein-protein and protein-lipid interactions of PLD and PA and their binding partners. In addition, we describe the functional roles played by PLD and PA in these interactions, and provide PLD network that summarizes these interactions. The PLD network suggests that PLD and PA could act as a decision maker and/or as a coordinator of signal dynamics. This viewpoint provides a turning point for understanding the roles of PLD-PA as a dynamic signaling hub.
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Regulatory roles of ganglioside GQ1b in neuronal cell differentiation of mouse embryonic stem cells.
BMB Rep
PUBLISHED: 12-23-2011
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Gangliosides play an important role in neuronal differentiation processes. The regulation of ganglioside levels is related to the induction of neuronal cell differentiation. In this study, the ST8Sia5 gene was transfected into mESCs and then differentiated into neuronal cells. Interestingly, ST8Sia5 gene transfected mESCs expressed GQ1b by HPTLC and immunofluorescence analysis. To investigate the effects of GQ1b over-expression in neurogenesis, neuronal cells were differentiated from GQ1b expressing mESCs in the presence of retinoic acid. In GQ1b expressing mESCs, increased EBs formation was observed. After 4 days, EBs were co-localized with GQ1b and nestin, and GFAP. Moreover, GQ1b co-localized with MAP-2 expressing cells in GQ1b expressing mESCs in 7-day-old EBs. Furthermore, GQ1b expressing mESCs increased the ERK1/2 MAP kinase pathway. These results suggest that the ST8Sia5 gene increases ganglioside GQ1b and improves neuronal differentiation via the ERK1/2 MAP kinase pathway.
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Proteomic analysis of tumor necrosis factor-alpha (TNF-?)-induced L6 myotube secretome reveals novel TNF-?-dependent myokines in diabetic skeletal muscle.
J. Proteome Res.
PUBLISHED: 11-11-2011
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There is a strong possibility that skeletal muscle can respond to irregular metabolic states by secreting specific cytokines. Obesity-related chronic inflammation, mediated by pro-inflammatory cytokines, is believed to be one of the causes of insulin resistance that results in type 2 diabetes. Here, we attempted to identify and characterize the members of the skeletal muscle secretome in response to tumor necrosis factor-alpha (TNF-?)-induced insulin resistance. To conduct this study, we comparatively analyzed the media levels of proteins released from L6 skeletal muscle cells. We found 28 TNF-? modulated secretory proteins by using separate filtering methods: Gene Ontology, SignalP, and SecretomeP, as well as the normalized Spectral Index for label-free quantification. Ten of these secretory proteins were increased and 18 secretory proteins were decreased by TNF-? treatment. Using microarray analysis of Zuker diabetic rat skeletal muscle combined with bioinformatics and Q-PCR, we found a correlation between TNF-?-mediated insulin resistance and type 2 diabetes. This novel approach combining analysis of the conditioned secretome and transcriptome has identified several previously unknown, TNF-?-dependent secretory proteins, which establish a foothold for research on the different causes of insulin resistance and their relationships with each other.
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Four-Week Repeated Oral Toxicity Study of AIP1, a Water-soluble Carbohydrate Fraction from Artemisia iwayomogi in Mice.
Toxicol Res
PUBLISHED: 11-03-2011
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Artemisia iwayomogi, a member of the Compositae, is a perennial herb easily found in Korea and used as a traditional medicine to treat liver disease. AIP1, a water-soluble carbohydrate fraction from Artemisia iwayomogi, showed anti-tumor and immuno-modulating activities in animal studies. A subacute toxicological evaluation of AIP1 was performed for 4 weeks in ICR mice. After administration of AIP1 (0, 20, 100, 500 mg/kg/day), the clinical signs, mortalities, body weight changes, hematology, blood clinical biochemistry, urinalysis, organ histopathology, organ weights and gross finding were examined. The results showed that there were no significant differences in body weight changes, food intakes, water consumptions, or organ weights among different dose groups. Also we observed no death and abnormal clinical signs during the experimental period. Between the groups orally treated with AIP1 and the control group, there was no statistical significance in hematological test or serum biochemical values. Histopathological examination showed no abnormal changes in AIP1 groups. These results suggest that no observed adverse effect level (NOAEL) of the oral administration of AIP1 for 4 weeks was considered to be more than 500 mg/kg/ day in mice under the condition investigated in current study.
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Multivessel versus single vessel spasm, as assessed by the intracoronary acetylcholine provocation test, in Korean patients.
Clin. Exp. Pharmacol. Physiol.
PUBLISHED: 09-22-2011
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1. Coronary artery spasm (CAS) is known to be a major cause of myocardial ischaemia. Multivessel coronary spasm (MVS) in particular is likely to induce more severe and prolonged myocardial ischaemia than single vessel spasm (SVS). 2. In the present study, a total of 1082 consecutive patients without significant coronary artery disease who underwent an acetylcholine (ACh) provocation test between March 2004 and April 2009 were investigated. Patients were divided into three groups: an MVS group (n = 275), an SVS group (n = 376) and a non-CAS group (n = 431). Differences in clinical and angiographic characteristics following the ACh provocation test were evaluated between the MVS, SVS and non-CAS groups. 3. At baseline, patients in the MVS group had the highest prevalence of peripheral artery disease (PAD), hyperlipidaemia, smoking and old age, as well as the highest triglyceride levels. Calcium channel blockers were most frequently prescribed in MVS patients before the ACh test. During the ACh test, the highest prevalence of chest pain, ischaemic electrocardiogram changes, baseline spasms and diffuse and severe spasms were observed in the MVS group. The response rate to lower ACh doses that induce CAS was also higher in the MVS group. Multivariate analysis showed that the presence of PAD (odds ratio (OR) 2.0; P = 0.006) and baseline spasm (OR 1.4; P = 0.045) were independent predictors of ACh-induced MVS. 4. In conclusion, ischaemic symptoms, diffuse and severe spasm and baseline spasm were more frequently associated with MVS patients, suggesting more intensive medical therapies and close clinical follow up would be required for this patient group.
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Residual tumor after the salvage surgery is the major risk factors for primary treatment failure in malignant ovarian germ cell tumors: a retrospective study of single institution.
World J Surg Oncol
PUBLISHED: 08-01-2011
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Malignant ovarian germ cell tumors are rare, and knowledge of their prognostic factors is limited, with little available randomized data. This study was conducted to evaluate the clinicopathologic characteristics of malignant ovarian germ cell tumors and to determine the association of their prognostic factors to primary treatment failure.
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The prognostic impact of duration of anemia during chemotherapy in advanced epithelial ovarian cancer.
Oncologist
PUBLISHED: 06-24-2011
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To propose a measure of anemia to be used as a prognostic factor for progression-free survival and overall survival in advanced epithelial ovarian cancer patients.
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Theranostic systems assembled in situ on demand by host-guest chemistry.
Biomaterials
PUBLISHED: 06-15-2011
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Theranostic systems have been explored extensively for a diagnostic therapy in the forms of polymer conjugates, implantable devices, and inorganic nanoparticles. In this work, we report theranostic systems in situ assembled by host-guest chemistry responding to a request. As a model theranostic system on demand, cucurbit[6]uril-conjugated hyaluronate (CB[6]-HA) was synthesized and decorated with FITC-spermidine (spmd) and/or formyl peptide receptor like 1 (FPRL1) specific peptide-spmd by simple mixing in aqueous solution. The resulting (FITC-spmd and/or peptide-spmd)@CB[6]-HA was successfully applied to the bioimaging of its target-specific delivery to B16F1 cells with HA receptors and its therapeutic signal transduction with elevated Ca(2+) and phosphor-extracellular signal-regulated kinase (pERK) levels in FPRL1-expressing human breast adenocarcinoma (FPRL1/MCF-7) cells. Finally, we could confirm in vitro and in vivo stability of the highly specific host-guest interaction. The on-demand theranostic platform technology using host-guest chemistry can be exploited for various bioimaging, biosensing, drug delivery, and tissue engineering applications.
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Defective nuclear translocation of stress-activated signaling in senescent diploid human fibroblasts: a possible explanation for aging-associated apoptosis resistance.
Apoptosis
PUBLISHED: 06-02-2011
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In order to study the nature of aging-dependent apoptosis resistance, we compared the activation pattern of mitogen-activated protein kinases (MAPK) in response to three different stress modalities: hydrogen peroxide (H(2)O(2)), staurosporine, and thapsigargin. We observed the agonist-specific activation pattern of MAP kinases in human diploid fibroblasts (HDFs). When young HDFs were treated with PD98059, a specific inhibitor of extracellular signal-regulated kinase (ERK), H(2)O(2)-induced apoptosis was blocked, whereas staurosporine-induced apoptosis was inhibited by treatment with SB203580, a specific inhibitor of p38. In addition, the levels of anti-apoptotic protein Bcl-2 (B-cell lymphoma protein-2) were restored by PD98059 or SB239063 in cells treated with H(2)O(2) or staurosporine, respectively. We also found that inhibition of the nuclear import of p-Erk and p-p38 using wheat germ agglutinin induced apoptosis resistance in young HDF cells in response to H(2)O(2) or staurosporine. These data indicate a potential role of the nuclear translocation of apoptotic signals in the induction of apoptosis. Moreover, the nuclear translocation of activated ERK1/2 and p38 in response to H(2)O(2) or staurosporine was significantly compromised in senescent HDFs, compared with young cells. Taken together, we propose that the apoptosis resistance of senescent HDFs might be related to the defective nuclear translocation of stress-activated signals in an agonist-specific manner, which would imply the operation of an aging-dependent functional nucleo-cytoplasmic trafficking barrier.
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Activation of AMP-activated protein kinase is essential for lysophosphatidic acid-induced cell migration in ovarian cancer cells.
J. Biol. Chem.
PUBLISHED: 05-20-2011
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Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, and survival, through LPA receptors. Among them, the motility of cancer cells is an especially important activity for invasion and metastasis. Recently, AMP-activated protein kinase (AMPK), an energy-sensing kinase, was shown to regulate cell migration. However, the specific role of AMPK in cancer cell migration is unknown. The present study investigated whether LPA could induce AMPK activation and whether this process was associated with cell migration in ovarian cancer cells. We found that LPA led to a striking increase in AMPK phosphorylation in pathways involving the phospholipase C-?3 (PLC-?3) and calcium/calmodulin-dependent protein kinase kinase ? (CaMKK?) in SKOV3 ovarian cancer cells. siRNA-mediated knockdown of AMPK?1, PLC-?3, or (CaMKK?) impaired the stimulatory effects of LPA on cell migration. Furthermore, we found that knockdown of AMPK?1 abrogated LPA-induced activation of the small GTPase RhoA and ezrin/radixin/moesin proteins regulating membrane dynamics as membrane-cytoskeleton linkers. In ovarian cancer xenograft models, knockdown of AMPK significantly decreased peritoneal dissemination and lung metastasis. Taken together, our results suggest that activation of AMPK by LPA induces cell migration through the signaling pathway to cytoskeletal dynamics and increases tumor metastasis in ovarian cancer.
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Inhibition of ganglioside GD1a synthesis suppresses the differentiation of human mesenchymal stem cells into osteoblasts.
Dev. Growth Differ.
PUBLISHED: 04-16-2011
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In this study, we investigated the regulatory role of ganglioside GD1a in the differentiation of osteoblasts from human mesenchymal stem cells (hMSCs) by using lentivirus-containing short hairpin (sh)RNA to knockdown ST3 ?-galactoside ?-2, 3-sialyltransferase 2 (ST3Gal II) mRNA expression. After hMSCs were infected for 72 h with the lentivirus constructed with ST3Gal II shRNAs, the puromycin-resistant cells were selected and subcultured to produce hMSCs with ST3Gal II mRNA knockdown. The hMSCs established from human dental papilla abundantly expressed CD44 and CD105, but not CD45 and CD117. Osteoblasts that differentiated from normal hMSCs showed a significant increase in alkaline phosphatase (ALP) activity and ganglioside GD1a expression level compared with those in hMSCs. Lentiviral infection of hMSCs successfully induced a marked inhibition of ST3Gal II mRNA expression and caused a significant decrease in ALP activity and ganglioside GD1a expression. During osteoblastic differentiation, the increased ALP activity remarkably reduced by suppression of ganglioside GD1a expression by ST3Gal II shRNA. Ganglioside GD1a and ALP were mainly expressed in the cell body of hMSCs and osteoblasts with colocalization. The phosphorylation of extracellular signal-regulated kinases (ERK) 1/2 mitogen-activated protein (MAP) kinase and epidermal growth factor receptor (EGFR) was significantly reduced in the osteoblasts that had differentiated from the hMSCs with ST3Gal II mRNA knockdown. These results suggest that ganglioside GD1a plays an important role in the regulation of osteoblastic differentiation of hMSCs through the activation of ERK 1/2 MAP kinase and EGFR.
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Inulin induces dendritic cells apoptosis through the caspase-dependent pathway and mitochondrial dysfunction.
Biol. Pharm. Bull.
PUBLISHED: 04-07-2011
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Dendritic cells are professional antigen-presenting cells that are responsible for initiating of the immune response. However, there are no reports on how the polysaccharides in an oral biofilm affect the viability of dendritic cells. Inulin, a fructooligossacharide, is one component of oral biofilm fructan that is used as an energy source by oral bacteria. In this study, we found that murine bone marrow derived dendritic cells were induced to undergo apoptosis after being treated with inulin in a dose-dependent manner, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), annexinV/propidium iodide (PI), and Hoechest staining methods. Inulin activated the apoptotic pathway, including caspase-9 and caspase-3, decreased the level of B-cell lymphoma 2 (Bcl-2) expression, increased the expression of the Bcl-2-associated X protein (Bax) protein and induced poly(ADP-ribose) polymerase (PARP) cleavage. These observations suggest that inulin induces the apoptosis of dendritic cells by altering the Bcl-2/Bax ratio through the caspase dependant pathway. These results indicated that high concentrations of inulin can cause apoptic cell death in murine bone marrow-derived dendritic cells.
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An efficient growth of silver and copper nanoparticles on multiwalled carbon nanotube with enhanced antimicrobial activity.
J. Biomed. Mater. Res. Part B Appl. Biomater.
PUBLISHED: 03-22-2011
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Transition metal nanoparticles (NPs) such as silver (Ag) and copper (Cu) have been grafted onto carbon nanotube surface through wet chemical approach leading to the development of densely packed NP decorated carbon nanotubes. Chemically active surface and high-temperature stability are the basic attributes to use carbon nanotubes as the template for the growth of NPs. Ag NP-grafted carbon nanotubes (Ag-MWCNT) are prepared by complexing Ag ion with acid functionalized carbon nanotubes followed by the reduction method. Alternatively, Cu-grafted carbon nanotubes (Cu-MWCNT) are prepared by simple chemical reduction method. X-ray diffraction results reveal that the Ag or Cu NPs formed on the surface of carbon nanotubes are determined to be face centered cubic crystals. The morphology and chemical structure of NP-grafted carbon nanotubes are investigated using transmission electron spectroscopy, X-ray photoelectron spectroscopy and Raman spectroscopy. The antimicrobial properties of acid-treated MWCNT (MWCNT-COOH), Ag-MWCNT, and Cu-MWCNT are investigated against gram negative Escherichia coli bacteria. Ag-MWCNT and Cu-MWCNT (97% kill vs. 75% kill), whereas MWCNT-COOH only killed 20% of this bacteria. Possible mechanisms are proposed to explain the higher antimicrobial activity by NP-coated MWCNT. These findings suggest that Ag-MWCNT and Cu-MWCNT may be used as effective antimicrobial materials that find applications in biomedical devices and antibacterial controlling system.
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Human papillomavirus 16/18 AS04-adjuvanted cervical cancer vaccine: immunogenicity and safety in 15-25 years old healthy Korean women.
J Gynecol Oncol
PUBLISHED: 03-02-2011
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The study assessed the immunogenicity and safety of human papillomavirus (HPV)-16/18 AS04-adjuvanted cervical cancer vaccine in healthy Korean women aged 15-25 years.
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Nanoscale mapping and affinity constant measurement of signal-transducing proteins by atomic force microscopy.
Anal. Chem.
PUBLISHED: 02-07-2011
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Atomic force microscope (AFM) was used to measure the interaction force between two signal-transducing proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Ras homologue enriched in brain (Rheb), and to analyze the binding of glyceraldehyde-3-phosphate (Gly-3-P) to GAPDH. To enhance the recognition efficiency and avoid undesirable multiple interactions, the AFM probe and the substrate were each modified with a dendron, glutathione S-transferase (GST)-fused proteins were employed, and reduced glutathione (GSH) was conjugated at the apex of each immobilized dendron. The resulting median specific force between GAPDH and Rheb was 38 ± 1 pN at a loading rate of 3.7 × 10(3) pN/s. The measurements showed that the GAPDH-Rheb interaction was inhibited by binding of Gly-3-P. An adhesion force map showed individual GADPHs on the surface and that the number density of GAPDH decreased with the concentration of Gly-3-P. Maps obtained in the presence of various Gly-3-P concentrations provided information on the binding behavior, yielding a thermodynamic association constant of 2.7 × 10(5) M(-1).
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Identification of characteristic molecular signature of Müllerian inhibiting substance in human HPV-related cervical cancer cells.
Int. J. Oncol.
PUBLISHED: 02-02-2011
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Müllerian inhibiting substance (MIS), also known as anti-Müllerian hormone (AMH), is a member of the transforming growth factor-? (TGF-?) superfamily that plays an important role in the mesenchymal-epithelial interaction, cell growth and proliferation, extracellular matrix production and tissue remodeling. Previously, we demonstrated that MIS suppressed ovarian cancer cell growth and suggested large-scale genetic elements that could be responsible for anti-neoplastic effects of MIS on ovarian cancer cells. In this study, we demonstrated the expression of MIS type II receptor (MISRII) in the human papillomavirus (HPV)-16-related cervical cancer cell lines CaSki and SiHa, and a non-HPV-related cervical cancer cell line, C33A. We also showed that MIS inhibited growth of cervical cancer cells, and induced cellular apoptosis of C33A. In addition, we identified a characteristic molecular signature of MIS in CaSki cells by using whole genome expression analysis. Of the 1,690 genes that showed significant expression changes by MIS, 21 genes were related to cell cycle; 13 genes to apoptosis; and 52 genes to the cancer pathway. On performing a search for cell cycle pathways in the KEGG pathway database, several gene expressions at the G1/S checkpoint were found. In particular, the expression of p16 and p107 increased and that of E2F2 and E2F3 decreased at an early stage, whereas the expression of E2F4 and E2F5 decreased at a later stage after MIS treatment. These data suggest that MIS produces activity against HPV16-related cervical cancers in vitro, and MIS may also be an effective targeted therapy for HPV16-related cervical cancer. Genetic data obtained here could be useful in determining the treatment strategy of MISR-expressing cervical tumors in the future.
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Phospholipase D2 induces stress fiber formation through mediating nucleotide exchange for RhoA.
Cell. Signal.
PUBLISHED: 01-25-2011
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Phospholipase D (PLD) is involved in diverse cellular processes including cell movement, adhesion, and vesicle trafficking through cytoskeletal rearrangements. However, the mechanism by which PLD induces cytoskeletal reorganization is still not fully understood. Here, we describe a new link to cytoskeletal changes that is mediated by PLD2 through direct nucleotide exchange on RhoA. We found that PLD2 induces RhoA activation independent of its lipase activity. PLD2 directly interacted with RhoA, and the PX domain of PLD2 specifically recognized nucleotide-free RhoA. Finally, we found that the PX domain of PLD2 has guanine nucleotide-exchange factor (GEF) activity for RhoA in vitro. In addition, we verified that overexpression of the PLD2-PX domain induces RhoA activation, thereby provoking stress fiber formation. Together, our findings suggest that PLD2 functions as an upstream regulator of RhoA, which enables us to understand how PLD2 regulates cytoskeletal reorganization in a lipase activity-independent manner.
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Phospholipase C-?1 is activated by intracellular Ca(2+) mobilization and enhances GPCRs/PLC/Ca(2+) signaling.
Cell. Signal.
PUBLISHED: 01-14-2011
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Phospholipase C-?1 (PLC-?1) is the most recently identified PLC isotype and is primarily expressed in nerve tissue. However, its functional role is unclear. In the present study, we report for the first time that PLC-?1 acts as a signal amplifier in G protein-coupled receptor (GPCR)-mediated PLC and Ca(2+) signaling. Short-hairpin RNA (shRNA)-mediated knockdown of endogenous PLC-?1 reduced lysophosphatidic acid (LPA)-, bradykinin (BK)-, and PACAP-induced PLC activity in mouse neuroblastoma Neuro2A (N2A) cells, indicating that PLC-?1 participates in GPCR-mediated PLC activation. Interestingly, ionomycin-induced PLC activity was significantly decreased by PLC-?1, but not PLC-?2, knockdown. In addition, we found that intracellular Ca(2+) source is enough for PLC-?1 activation. Furthermore, the IP(3) receptor inhibitor, 2-APB, inhibited LPA-induced PLC activity in control N2A cells, whereas this effect was not observed in PLC-?1 knockdown N2A cells, suggesting a pivotal role of intracellular Ca(2+) mobilization in PLC-?1 activation. Finally, we found that LPA-induced ERK1/2 phosphorylation and expression of the downstream target gene, krox-24, were significantly decreased by PLC-?1 knockdown, and these knockdown effects were abolished by 2-APB. Taken together, our results strongly suggest that PLC-?1 is activated via intracellular Ca(2+) mobilization from the ER, and therefore amplifies GPCR-mediated signaling.
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Identification of the target proteins of rosiglitazone in 3T3-L1 adipocytes through proteomic analysis of cytosolic and secreted proteins.
Mol. Cells
PUBLISHED: 01-06-2011
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Rosiglitazone, one of the thiazolidinedione (TZD), is an oral antidiabetic drug that activates a gamma isoform of peroxisome proliferator-activated receptor (PPAR?). To identify target proteins induced by rosiglitazone in adipocytes, we first performed simultaneous in-depth proteomic profiling of cytosolic proteins and secreted proteins (secretome) from 3T3-L1 adipocytes using a label-free quantification method with nano-UPLC MS/MS. In total, we identified 646 proteins from 3T3-L1 adipocytes, of which 172 and 162 proteins were upregulated and downregulated >1.5-fold, respectively, in rosiglitazone-treated cells, as compared to controls. Some differentially expressed proteins in particular, including fatty acid translocase (FAT)/CD36, fatty acid binding protein, lipoprotein lipase, acetyl CoA acyltransferase, carnitine O-palmitoyltransferase 2, sterol carrier protein, adiponectin, and phosphoenolpyruvate carboxykinase could explain the current action mechanism of TZDs. Furthermore, this study is the first to report on two potential target proteins of rosiglitazone, such as adenomatosis polyposis coli 2 (APC2), and eukaryotic translation initiation factor 5A-1 (eIF5A) related to apoptosis and cell division. Our data clearly suggest that in-depth proteomic approaches using cytosolic and secreted proteins are important and necessary for identification of drug targets at the protein level.
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Left ventricular hypertrophy determines the severity of diastolic dysfunction in patients with nonvalvular atrial fibrillation and preserved left ventricular systolic function.
Clin. Exp. Hypertens.
PUBLISHED: 11-25-2010
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Regression of left ventricular (LV) hypertrophy (LVH) is known to be related to a lower incidence of stroke in hypertensive patients with nonvalvular atrial fibrillation (NV-AF). However, its mechanism remains controversial. Recently, diastolic dysfunction (DD) was reported to be correlated with ischemic stroke in NV-AF. We hypothesized that hypertension (HTN) and resultant LVH might be associated with the severity of DD in NV-AF. Two hundred and ninety-four patients (204 males, age 66 ± 12 y) with NV-AF with preserved LV systolic function were included. Clinical and echocardiographic data were compared between patients with enlarged left atrial (LA) volume (n = 237) and patients with normal LA. Age (60 ± 12 vs. 67 ± 11 years), sex (male; 81 vs. 62%), duration of NV-AF (4.1 ± 7.8 vs. 45.7 ± 49.0 months), brain natriuretic peptide (108.3 ± 129.3 vs. 236.1 ± 197.0 pg/mL), right ventricular systolic pressure (24.5 ± 5.5 vs. 33.1 ± 11.1 mmHg), mitral inflow velocity (E [77.4 ± 22.2 vs. 88.3 ± 22.0 cm/s]), LV mass index (LVMI [87.6 ± 22.2 vs. 105.1 ± 23.2 g/m(2)]), peak systolic mitral annular velocity (S [7.2 ± 2.0 vs. 5.8 ± 1.8 cm/s]), and mitral inflow velocity to diastolic mitral annular velocity (E/E [9.8 ± 3.4 vs. 12.1 ± 4.4]) were significantly different between the two groups, respectively (P < 0.05). In multivariate analysis, LVMI was independently correlated with increased LA volume (OR: 1.037 [95% CI: 1.011-1.063], P < 0.05), whereas HTN was not. LA enlargement, which reflects the severity and chronicity of DD, is independently associated with LVH in patients with NV-AF. Therefore, regression of LVH with anti-hypertensive treatment may lead to improvement of diastolic function and favorable clinical outcomes in hypertensive patients with NV-AF.
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