• Journal of Life Science
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• v.26 no.2
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• pp.226-233
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• 2016

Vascular smooth muscle cell (VSMC) apoptosis has been identified in various vascular diseases, including atherosclerosis and restenosis after angioplasty, and has been known to precipitate atherosclerotic plaque instability and rupture. Oxysterols are known as inducers of apoptosis in VSMC, and 7-ketocholesterol (7KC) is the major nonenzymically formed oxysterol in atherosclerotic lesions. The precise mechanism underlying VSMC apoptosis is still poorly understood. In this study, we investigated whether 7KC causes apoptosis, and characterized its apoptotic mechanisms in primary cultured rat aortic VSMC. Cell viability was assessed by MTT assay and trypan blue assay. Apoptosis was assessed by flow cytometry, immunofluorescence, immunoprecipitation, and Western blot analyses. 7KC markedly decreased the VSMC viability in a time- and concentration-dependent manner, and increased the production of 4-hydroxynonenal (HNE), a major end-product of lipid peroxidation, which also decreased the VSMC viability. Pretreatment with 2,4-dinitrophenylhydrazine, a well-known reagent of lipid peroxidation-derived aldehydes, significantly restored the 7KC-decreased viability of VSMC. Furthermore, HNE, as well as 7KC, reduced the level of total Akt, a major mediator of cell survival. The 7KC-decreased level of total Akt was significantly restored by pretreatments with 2,4-dinitrophenylhydrazine and N-acetylcysteine. Lactacystin, a proteasome inhibitor, protected VSMC against apoptosis and Akt degradation, but did not inhibit HNE production. In the immunoprecipitation assay, 7KC increased HNE-modified Akt. From the results, it seems that, in atherosclerotic lesions, 7KC induces HNE production in VSMC, and this HNE binds to Akt, proceeding to proteasomal degradation of Akt, through which mechanism the atherosclerotic plaque instability may be facilitated.

• Journal of Life Science
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• v.26 no.2
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• pp.147-154
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• 2016

Osteosarcoma (OS) is the most common and malignant bone tumors. Although many types of resection surgery and experimental agents were developed, median survival and clinical prognosis are poorly investigated. Recently, several researches have reported that Eucheuma cottonii has potent as protective effects of coal dust-induced lung damage via inhibition of malondialdehyde (MDA) and oxidative stress in bronchoalveolar lavage fluids (BALF). However, anti-cancer effects and specific molecular mechanism of extract from Eucheuma cottonii (EE) has not been clearly studied yet. This study evaluated that anti-cancer potential of EE in human osteosarcoma Saos-2 cells. EE indicated cytotoxicity on Saos-2 cells in a dose-dependent manner. Morphological degradation and nucleic condensation were also observed under the EE treatment. However, it did not significantly affect on non-cancerous kidney HEK-293 cells under the same concentration which is shown cytotoxicity on Saos-2 cells. The phosphorylation of Fas-Associated Death Domain (FADD) and expression of cleaved caspase-8, -7 and -3 were upregulated in a dose-dependent manner. In immunofluorescence staining, expression level of Fas and cleaved PARP were upregulated by EE treatment. Furthermore, treatment of EE induces upregulation of sub G1 phase by flow cytometry analysis. The results demonstrated that EE has a therapeutic potential against osteosarcoma via FADD mediated caspase cascade apoptosis signal pathway.

• Investigative Magnetic Resonance Imaging
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• v.16 no.1
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• pp.47-54
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• 2012

Purpose : This study was performed to evaluate the characteristics of rat mesenchymal stem cells (RMSCs) transduced with human ferritin gene and investigate $in$ $vitro$ MRI detectability of ferritin-transduced RMSCs. Materials and Methods: The RMSCs expressing both myc-tagged human ferritin heavy chain subunit (myc-FTH) and green fluorescence protein (GFP) were transduced with lentiviurs. Transduced cells were sorted by GFP expression using a fluorescence-activated cell sorter. Myc-FTH and GFP expression in transduced cells were detected by immunofluorescence staining. The cell proliferative ability and viability were assessed by MTT assay. The RMSC surface markers (CD29+/CD45-) were analyzed by flow cytometry. The intracellular iron amount was measured spectrophotometically and the presence of ferritin-iron accumulation was detected by Prussian blue staining. $In$ $vitro$ magnetic resonance imaging (MRI) study of cell phantoms was done on 9.4 T MR scanner to evaluate the feasibility of imaging the ferritin-transduced RMSCs. Results: The myc-FTH and GFP genes were stably transduced into RMSCs. No significant differences were observed in terms of biologic properties in transduced RMSCs compared with non-transduced RMSCs. Ferritin-transduced RMSCs exhibited increased iron accumulation ability and showed significantly lower $T_2$ relaxation time than non-transduced RMSCs. Conclusion: Ferritin gene as MR reporter gene could be used for non-invasive tracking and visualization of therapeutic mesenchymal stem cells by MRI.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.129-137
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• 2018

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig ($GalT^{-MCP/-MCP}$). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain $GalT^{-MCP/-MCP}/CD39/CD73$ pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from $GalT^{-MCP/-MCP}$ pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate $GalT^{-MCP/-MCP}/CD39/CD73$ pig expressing CD39 and CD73 at endothelial cells.

• Korean Journal of Animal Reproduction
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• v.13 no.2
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• pp.113-120
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• 1989

In order to determine the sex of preimplantation embryos prior to transfer in cattle, a series of experiments were carried out using 45 Holstein donor cows to examine the ovarian response on the gonadotropin and PGF2${\alpha}$, and the morphology of fresh embryos or frozen/thawed embryos after deep freezing at -196$^{\circ}C$. The sexing of embryos treated with the medium containing H-Y antiserum(10%, v/v) and FITC anti-mouse IgG(10%, v/v) were analysed by chromosomal analysis, and the sex of the embryos which survived were ascertain after delivering the pups. The results obtained were summarized as follows ; 1. The average number of developed follicle and corpus luteum per cow were 13.5 and 8.1, and the ovalation rate was 60.1%. 2. Of 220-ova recovered, 75(34.1%) were morula and 91(41.4%) were blastocyst, and the morphological normal and abnormal rate of ova recovered were 75.5% and 24.5%, respectively. 3. Of 39 frozen/thawed embryos, the scores of normal morula and blastocyst, after thawing were 79.2%(19/24) and 73.3%(11/15). The average rate of frozen/thawed embryos which appeared morphologically normal post thawing was 76.9%(30/39). 4. The sex ratio was measured using the embryos treated with immunofluorescence assay to examine the relationship between embryo developmental stage, sex ratio of morula stage embryo was 42.2%(19/45) fluorescing and 57.8%(26/45) non-fluorescing, on the other hand, the ratio switched to 46.8%(29/62) fluorescing and 53.2%(33/62) non-fluorescing embryo in blastocyst stage. The sex ratio was also measured between fresh and frozen/thawed embryos, fresh and frozen/thawed treated embryos were indicated 45.8%(38/83) fluorescing, 54.2%(45/83) non-fluorescing and 41.7%(10/24) fluorescing, 58.3%(14/24) non-fluorescing. This trend indicated the approximal sex ratio was 1 : 1. 5. The result of karyotype test showed the successful rate of sexing embryo is fluorescing and non-fluorescing was 21.2%(7/33) and 29.6%(8/27). The female to male ratio within 33 fluorescing was 28.6 : 71.4, and the ratio of 27 non-fluorescing embryos was 87.7 : 12.5. 6. Of the embryo transferred after assignment of H-Y phenotype, five of the fluorescing embryos survived to term, all was males. Whereas six non-fluorescing embryos also survived to term and the sexes of the calves were 1 male 5 female.

• Journal of the Korean Academy of Child and Adolescent Psychiatry
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• v.17 no.1
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• pp.27-31
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• 2006

Objectives : We examined whether D8/17 expression in Tourette syndrome children who suggested PANDAS were higher than comparison group, and there was my clinical difference by D8/l7 expression. Methods : Nine Tourette's syndrome children suggested PANDAS and two ADHD children without tic disorder were evaluated far percentage of D8/17 expression-positive B cells by immunofluorescence flow cytometric assay and anti-streptolysin O titer. Results : The frequency of D8/17 positive B lymphocyte rate was significantly higher in Tourette's syndrome than ADHD, whose average rate were 77.9 and 24.8, respectively. Among 9 TD patients,4 patients showed above 90% D8/l7 expression. There was high concordance expression rate between mother (98.4%) and daughter (99.0%) The significant relation between percentage of D8/17 expression and tic severity were not detected. The significant relation between percentage of D8/17 expression and anti-streptolysin O titer were not detected, however in 66.7% TD patients showed above 100IU/ml. Conclusion : We concluded that subgroup of TD children are streptococcal infected tic disorder, so called PANDAS.

• Parasites, Hosts and Diseases
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• v.54 no.4
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• pp.385-391
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• 2016

The discovery and understanding of antigenic proteins are essential for development of a vaccine against malaria. In Plasmodium falciparum, Pf92 have been characterized as a merozoite surface protein, and this protein is expressed at the late schizont stage, but no study of Pv92, the orthologue of Pf92 in P. vivax, has been reported. Thus, the protein structure of Pv92 was analyzed, and the gene sequence was aligned with that of other Plasmodium spp. using bioinformatics tools. The recombinant Pv92 protein was expressed and purified using bacterial expression system and used for immunization of mice to gain the polyclonal antibody and for evaluation of antigenicity by protein array. Also, the antibody against Pv92 was used for subcellular analysis by immunofluorescence assay. The Pv92 protein has a signal peptide and a sexual stage s48/45 domain, and the cysteine residues at the N-terminal of Pv92 were completely conserved. The N-terminal of Pv92 was successfully expressed as soluble form using a bacterial expression system. The antibody raised against Pv92 recognized the parasites and completely merged with PvMSP1-19, indicating that Pv92 was localized on the merozoite surface. Evaluation of the human humoral immune response to Pv92 indicated moderate antigenicity, with 65% sensitivity and 95% specificity by protein array. Taken together, the merozoite surface localization and antigenicity of Pv92 implicate that it might be involved in attachment and invasion of a merozoite to a new host cell or immune evasion during invasion process.

• Journal of Yeungnam Medical Science
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• v.15 no.2
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• pp.286-296
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• 1998

Different techniques for culturing respiratory epithelial cells have been developed to overcome the limitations of studies on in vivo and on bioptic material. Traditionally, culture systems are divided into organ cultures, explant cultures and dissociated cell cultures. The first two contain both epithelial and non-epithelial cells. However, in monolayer cultures of dissociated cells only epithelial cells are present, the effects observed are caused by a pure epithelial responses. The purpose of this study is to establish primary culture method of human nasal epithelium (HNEC) by monolayer culture of dissociated cells to evaluate the role of the epithelial cells in the allergic and non-allergic nasal inflammatory reactions. HNEC was prepared by primary culture method of monolayer culture of dissociated cells from human inferior nasal turbinate mucosa of septal deviation patients. Primary cultured cells were characterized by indirect immunofluorescence assay and transmission electron microscopy. The immunoreactivities of cytokeratin-pan and cytokeratin No. 8 were observed in cultured HNEC. However, the immnoreactivities of vimentin and von Willebrand factor were not observed in cultured HNEC. The tonofilaments and desmosome were observed in cultured HNEC. The cultured epithelial cells were identified to be pure nasal epithelial cells. The monolayer culture of dissociated cells could successfully be employed for further study to investigate the role of the epithelial cells in allergic or non-allergic nasal inflammatory diseases.

• Parasites, Hosts and Diseases
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• v.36 no.4
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• pp.269-275
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• 1998

Anti-Neospora caninum antibody was detected in anti-Toxoplasma gondii positive and negative human sera by ELISA, western blot and immunofluorescence assay (IFA). Twelve cases out of 172 (6.7％) Toxoplasma-positive sera cross-reacted with both T. gondii and N. caninum antigens, and one out of 110 Toxoplasma-negative sera reacted with N. caninum antigen by ELISA. By western blot, all 12 sera reacted with T. gondii antigens with various banding patterns but specifically at 30 kDa (SAG 1), and 22 kDa （SAG2) bands. With N. caninum antigen, the number of reactive bands was reduced, however a 43 kDa band reacted in three cases in Toxoplasma-positive sera in addition to one in Toxoplasma-negative control sera. All sera of the Toxoplasma-positive group labeled surface membrane of T. gondii, but reacted differently with N. caninum. Fluorescence was detected in surface membrane, subcellular organelles, or both in N. caninum. And one case in the Toxoplasma-negative group also reacted with N. caninum strongly in subcellular organelles. This suggested that the antibody against N. caninum may be present in human sera although the positive rate was very low in this study. The possibility of human infection with N. caninum remains to be evaluated further.

• Parasites, Hosts and Diseases
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• v.48 no.4
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• pp.319-324
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• 2010

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4-EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in Echerichia coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.