• Archives of Craniofacial Surgery
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• v.19 no.3
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• pp.181-189
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• 2018

Background: Autogenous bone grafts have several limitations including donor-site problems and insufficient bone volume. To address these limitations, research on bone regeneration is being conducted actively. In this study, we investigate the effects of a three-dimensionally (3D) printed polycaprolactone (PCL)/tricalcium phosphate (TCP) scaffold on the osteogenic differentiation potential of adipose tissue-derived stem cells (ADSCs) and bone marrow-derived stem cells (BMSCs). Methods: We investigated the extent of osteogenic differentiation on the first and tenth day and fourth week after cell culture. Cytotoxicity of the 3D printed $PCL/{\beta}-TCP$ scaffold was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, prior to osteogenic differentiation analysis. ADSCs and BMSCs were divided into three groups: C, only cultured cells; M, cells cultured in the 3D printed $PCL/{\beta}-TCP$ scaffold; D, cells cultured in the 3D printed $PCL/{\beta}-TCP$ scaffold with a bone differentiation medium. Alkaline phosphatase (ALP) activity assay, von Kossa staining, reverse transcription-polymerase chain reaction (RT-PCR), and Western blotting were performed for comparative analysis. Results: ALP assay and von Kossa staining revealed that group M had higher levels of osteogenic differentiation compared to group C. RT-PCR showed that gene expression was higher in group M than in group C, indicating that, compared to group C, osteogenic differentiation was more extensive in group M. Expression levels of proteins involved in ossification were higher in group M, as per the Western blotting results. Conclusion: Osteogenic differentiation was increased in mesenchymal stromal cells (MSCs) cultured in the 3D printed PCL/TCP scaffold compared to the control group. Osteogenic differentiation activity of MSCs cultured in the 3D printed PCL/TCP scaffold was lower than that of cells cultured on the scaffold in bone differentiation medium. Collectively, these results indicate that the 3D printed PCL/TCP scaffold promoted osteogenic differentiation of MSCs and may be widely used for bone tissue engineering.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.1
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• pp.1-7
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• 2007

Altered environmental gravity, including both hypo- and hypergravity, may result in space adaptation syndrome. To explore the characteristics of this adaptive plasticity, the expression of immediate early gene c-fos mRNA in the vestibular related tissues following an exposure to hypergravity stimulus was determined in rats. The animals were subjected to a force of 2 g (twice earth's gravity) for 1, 3, or 12 h, and were examined poststimulus at 0, 2, 6, 12, and 24 h. RT-PCR (reverse transcription polymerase chain reaction) and real-time quantitative RT-PCR were adopted to analyze temporal changes in the expression of c-fos mRNA. The hypergravity stimulus increased the expression of c-fos mRNA in the vestibular ganglion, medial vestibular nucleus, inferior vestibular nucleus, hippocampus, cerebellum, and cortex. The peak expression occurred at 0 h poststimulation in animals stimulated with hypergravity for 1 h, and at 6 h poststimulus in those stimulated for 3 h. In contrast, those stimulated for 12 h exhibited dual peaks at 0 and 12 h poststimulus. Bilateral labyrinthectomy markedly attenuated the degree of c-fos mRNA expression. Glutamate receptor antagonist also dramatically attenuated the degree of c-fos mRNA expression. These results indicate that expression of c-fos mRNA in response to hypergravity occurs in the vestibular related tissues of the central nervous system, in which peripheral vestibular receptors and glutamate receptors play an important role. The temporal pattern of c-fos mRNA expression depended on the duration of the hypergravity stimulus.

• Applied Biological Chemistry
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• v.39 no.5
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• pp.355-360
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• 1996

In vitro-tested ribozyme against synthesized cucumber mosaic virus (CMV) RNA (Agric. Chem. & Biotech. 37:56-63(1994)) was expressed in tobacco plant to develop virus resistant plants. The ribozyme sequence was linked to cauliflower mosaic virus 35S promoter and nopaline synthase(nos) terminator and this chimeric 35S-ribozyme-nos gene was sequenced. The sequenced chimeric gene was transferred to Agrobacterium tumefaciens LBA4404 using tri-parental mating system. The E. coli HB101 containing chimeric gene was incubated with E. coli HB101(pRK2073) as a helper and Agrobacterium tumefaciens LBA4404. Then Agrobacterium cells containing the ribozyme construct was cocultivated with tobacco leaf pieces. Ten different plants were regenerated from kanamycin containing MS medium. The presence of the ribozyme construct in the transgenic tobacco plants was confirmed by polymerase chain reaction (PCR). Seven different transgenic plants in ten different kanamycin resistant plants showed the expected size (570 base pairs) of 35S-ribozyme-nos gene fragment. Total RNAs were isolated from four different transgenic plants and separated on a 1% agarose gel containing formamide. Northern hybridization with 35S-ribozyme-nos gene fragment as a probe indicated that ribozyme transcripts may be degraded tv nuclease. Therefore, nuclease-resistant ribozymes are needed for the development of virus-resistant transgenic plants using ribozymes.

• Korean Journal of Microbiology
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• v.36 no.3
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• pp.216-220
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• 2000

Eighty-eight strain3 o~methicillin resistant Stopllylococcus awecis were Isolated from pus (64.7%); spuhm (26.2%), blood, fluid, andurine of 83 patients at Dong-A Hospital in P~~san to invesligate theil-coagulase typ- Ing, and multi-drug resistaut ppattems. The presence of niec A gene confe~~ing melhicillin resistance was tested by polymerase chain reaction (PCR) with uwo mec A gene specific primers using purified clromosonlal DNA as templates. DNA fragments of expected size wel-e detected frorn 86 strains, but not from two strains. !i coagulase typmg, the 86 isolates were assigned to 5 coagulase lypes, I, 11, lll. 1V, VI, VII, VIlI, but there was no isolate helong lo type V. The most abundant coagulase type was type TI(50 %), lollowed by type IV Rest ofthe coagulase types were ininor; ranging fmm 4.5 to 12.5 '% Most of the type I1 ~netlucillin resistant Stapl\ulcorneryiococcus nwem (MRSA) strams were isolated from the generd sulzely ward, but major strains of type IV were Isolated from the otorhinolq~ngology of the hospital's outpatient clinic center. All of the 88 st~nins were sensitive to vancomycin and teicoplanin, but 71 (81%) strains showed multi-drug resitant to penicillin, cephalotl~n, eiythroinycin, gentan~ycin, imipenem, clindamycin, ciprofloxacin and ooxacillin. Yo relationship was found between the antibiotic resistance pattems aud the coagulase typing patterns.

• Journal of Life Science
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• v.17 no.3 s.83
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• pp.402-406
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• 2007

This study was performed to develop the molecular marker for sex determination of hare (Lepus coreanus) distributed in Korea which focused on sexual dimorphism between X and Y chromosomal homologous genes, zinc finger-X (ZFX) and -Y (ZFY). The intron 7 regions of ZFX and ZFY genes exhibited differential amplification patterns between male and female hares. The lengths of intron 7 region of ZFX and ZFY genes were 538 and 233-bp, respectively. Especially, the ZFX intron 7 contained a repetitive sequence identified as member of RNA-mediated transposable elements which was similar to CSINE2 commonly found in the rabbit genome. However, it was not present in intron 7 of ZFY gene. The molecular sex typing by polymerase chain reaction (PCR) was also carried out to determine the sex of hare based on difference in lengths between the intron 7 regions of ZFX and ZFY genes. All DNA samples tested had common band amplified from ZFX. However, the male hare DNAs had two distinct bands which amplified from ZFX and ZFY genes, respectively. The results from ZFX-ZFY PCR sex typing were identical to those from phenotypic investigation and from amplification patterns using male-specific sex determining region Y (SRY) gene as well. Finally, this study suggested that the sexual dimorphism between intron 7 regions of ZFX and ZFY could be useful genetic marker to determine sex of hare.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.3
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• pp.269-277
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• 2005

Objectives: Previously, we sought to compile a list of genes expressed during early folliculogenesis by using cDNA microarray to investigate follicular gene expression and changes during primordialprimary follicle transition and development of secondary follicles (Yoon et al., 2005). Among those genes, a group of genes related to the cell size growth was characterized during the ovarian development in the present study. Methods: We determined ovarian expression pattern of six genes related to the cell size growth (cyr61, emp1, fhl1, socs2, wig1 and wisp1) and extended into CCN family (${\underline{c}}onnective$ tissue growth factor/${\underline{c}}ysteine$-rich 61/${\underline{n}}ephroblastoma$-overexpressed), ctgf, nov, wisp2, wisp3, including cyr61 and wisp1 genes. Expression of mRNA and protein according to the ovarian developmental stage was evaluated by in situ hybridization, and/or semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemistry, respectively. Results: Among 6 genes related to the cell size growth, cyr61 and wisp1 mRNA was detected only in oocytes in the postnatal day5 mouse ovaries. cyr61 mRNA expression was limited to the nucleolus of oocytes, while wisp1 was expressed in the cytoplasm and nucleolus of oocytes, except nucleus. cyr61 mRNA expression, however, was found in granulosa cells from secondary follicles. The rest 4 genes in the cell size growth group were detected in oocytes, granulosa and theca cells. Cyr61 and Wisp1 proteins were expressed in the oocyte cytoplasm from primordial follicle stage. Especially, Cyr61 protein was detected in pre-granulosa cells, Wisp1 protein was not. By using RT-PCR, we evaluated and decided that Cyr61 protein is produced by their own mRNA in pre-granulosa cells that was not detected by in situ hybridization. cyr61 and wisp1 genes are happen to be the CCN family members. The other members of CCN family were also studied, but their expression was detected in oocytes, granulose and theca cells. Conclusions: We firstly characterized the ovarian expression of genes related to the cell size growth and CCN family according to the early folliculogenesis. Cyr61 protein expression in the pre-granulosa cells is profound in meaning. Further functional analysis for cyr61 in early folliculogenesis is under investigation.

• Journal of Veterinary Science
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• v.22 no.4
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• pp.43.1-43.10
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• 2021

Background: The H5 avian influenza viruses (AIVs) of clade 2.3.4.4 circulate in wild and domestic birds worldwide. In 2017, nine strains of H5N6 AIVs were isolated from aquatic poultry in Xinjiang, Northwest China. Objectives: This study aimed to analyze the origin, reassortment, and mutations of the AIV isolates. Methods: AIVs were isolated from oropharyngeal and cloacal swabs of poultry. Identification was accomplished by inoculating isolates into embryonated chicken eggs and performing hemagglutination tests and reverse transcription polymerase chain reaction (RT-PCR). The viral genomes were amplified with RT-PCR and then sequenced. The sequence alignment, phylogenetic, and molecular characteristic analyses were performed by using bioinformatic software. Results: Nine isolates originated from the same ancestor. The viral HA gene belonged to clade 2.3.4.4B, while the NA gene had a close phylogenetic relationship with the 2.3.4.4C H5N6 highly pathogenic avian influenza viruses (HPAIVs) isolated from shoveler ducks in Ningxia in 2015. The NP gene was grouped into an independent subcluster within the 2.3.4.4B H5N8 AIVs, and the remaining six genes all had close phylogenetic relationships with the 2.3.4.4B H5N8 HPAIVs isolated from the wild birds in China, Egypt, Uganda, Cameroon, and India in 2016-2017, Multiple basic amino acid residues associated with HPAIVs were located adjacent to the cleavage site of the HA protein. The nine isolates comprised reassortant 2.3.4.4B HPAIVs originating from 2.3.4.4B H5N8 and 2.3.4.4C H5N6 viruses in wild birds. Conclusions: These results suggest that the Northern Tianshan Mountain wetlands in Xinjiang may have a key role in AIVs disseminating from Central China to the Eurasian continent and East African.

• Journal of Veterinary Science
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• v.22 no.5
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• pp.63.1-63.14
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• 2021

Background: Recently, mesenchymal stem cells therapy has been performed in dogs, although the outcome is not always favorable. Objectives: To investigate the therapeutic efficacy of mesenchymal stem cells (MSCs) using dog leukocyte antigen (DLA) matching between the donor and recipient in vitro. Methods: Canine adipose-derived MSCs (cA-MSCs) isolated from the subcutaneous tissue of Dog 1 underwent characterization. For major DLA genotyping (DQA1, DQB1, and DRB1), peripheral blood mononuclear cells (PBMCs) from two dogs (Dogs 1 and 2) were analyzed by direct sequencing of polymerase chain reaction (PCR) products. The cA-MSCs were co-cultured at a 1:10 ratio with activated PBMCs (DLA matching or mismatching) for 3 days and analyzed for immunosuppressive (IDO, PTGS2, and PTGES), inflammatory (IL6 and IL10), and apoptotic genes (CASP8, BAX, TP53, and BCL2) by quantitative real-time reverse transcriptase-PCR. Results: cA-MSCs were expressed cell surface markers such as CD90⁺/44⁺/29⁺/45^- and differentiated into osteocytes, chondrocytes, and adipocytes in vitro. According to the Immuno Polymorphism Database, DLA genotyping comparisons of Dogs 1 and 2 revealed complete differences in genes DQA1, DQB1, and DRB1. In the co-culturing of cA-MSCs and PBMCs, DLA mismatch between the two cell types induced a significant increase in the expression of immunosuppressive (IDO/PTGS2) and apoptotic (CASP8/BAX) genes. Conclusions: The administration of cA-MSCs matching the recipient DLA type can alleviate the need to regulate excessive immunosuppressive responses associated with genes, such as IDO and PTGES. Furthermore, easy and reliable DLA genotyping technology is required because of the high degree of genetic polymorphisms of DQA1, DQB1, and DRB1 and the low readability of DLA 88.

• Korean Journal of Acupuncture
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• v.38 no.3
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• pp.140-150
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• 2021

Objectives : The increase of osteoclasts could cause osteoporosis and bone-related diseases. Also, the inhibition of osteoclast differentiation is important in treating bone-related diseases. Traditionally, Psoraleae Semen has been used for geriatric diseases, aging and musculoskeletal diseases. The purpose of this study is to investigate the effect of Psoraleae Semen ethanol extract (PS) on osteoclast differentiation and its function. Methods : To confirm the effect of PS on osteoclastogenesis and bone resorption activity, various levels of concentrations of PS (5, 10, 20 and 40 ㎍/ml) were tested on RAW 264.7 cells cultured with RANKL. We measured tartarate-resistant acid phosphatase (TRAP)-positive cells, TRAP activity, pit formation and F-actin ring formation. The expressions of nuclear factor of activated T-cells (NFATc1) and c-Fos were confirmed through western blot and reverse transcription- polymerase chain reaction (RT-PCR). Also, the expression of bone resorption and fusion-related genes in osteoclast was confirmed by RT-PCR. Results : PS decreased the number of TRAP-positive cells and the TRAP activity. In addition, PS significantly inhibited the formation of pit and F-actin ring. Furthermore, PS decreased the expression of osteoclast related genes. Conclusions : PS inhibits osteoclast differentiation and bone resorption ability through inhibition of the expression of osteoclast-related genes. This indicates that PS may be a potential therapeutic agent to osteoporosis by suppressing osteoclastogenesis.

• Animal Bioscience
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• v.34 no.10
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• pp.1590-1599
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• 2021

Objective: This study investigates the expression patterns of toll-like receptors (TLRs) and intracellular mediators in horse muscle cells after exercise, and the relationship between TLRS expression in stressed horse muscle cells and immune cell migration toward them. Methods: The expression patterns of the TLRs (TLR2, TLR4, and TLR8) and downstream signaling pathway-related genes (myeloid differentiation primary response 88 [MYD88]; activating transcription factor 3 [ATF3]) are examined in horse tissues, and horse peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs) and muscles in response to exercise, using the quantitative reverse transcription-polymerase chain reaction (qPCR). Expressions of chemokine receptor genes, i.e., C-X-C motif chemokine receptor 2 (CXCR2) and C-C motif chemokine receptor 5 (CCR5), are studied in PBMCs and PMNs. A horse muscle cell line is developed by transfecting SV-T antigen into fetal muscle cells, followed by examination of muscle-specific genes. Horse muscle cells are treated with stressors, i.e., cortisol, hydrogen peroxide (H₂O₂), and heat, to mimic stress conditions in vitro, and the expression of TLR4 and TLR8 are examined in stressed muscle cells, in addition to migration activity of PBMCs toward stressed muscle cells. Results: The qPCR revealed that TLR4 message was expressed in cerebrum, cerebellum, thymus, lung, liver, kidney, and muscle, whereas TLR8 expressed in thymus, lung, and kidney, while TLR2 expressed in thymus, lung, and kidney. Expressions of TLRs, i.e., TLR4 and TLR8, and mediators, i.e., MYD88 and ATF3, were upregulated in muscle, PBMCs and PMNs in response to exercise. Expressions of CXCR2 and CCR5 were also upregulated in PBMCs and PMNs after exercise. In the muscle cell line, TLR4 and TLR8 expressions were upregulated when cells were treated with stressors such as cortisol, H₂O₂, and heat. Migration of PBMCs toward stressed muscle cells was increased by exercise and oxidative stresses, and combinations of these. Treatment with methylsulfonylmethane (MSM), an antioxidant on stressed muscle cells, reduced migration of PBMCs toward stressed muscle cells. Conclusion: In this study, we have successfully cultured horse skeletal muscle cells, isolated horse PBMCs, and established an in vitro system for studying stress-related gene expressions and function. Expression of TLR4, TLR8, CXCR2, and CCR5 in horse muscle cells was higher in response to stressors such as cortisol, H₂O₂, and heat, or combinations of these. In addition, migration of PBMCs toward muscle cells was increased when muscle cells were under stress, but inhibition of reactive oxygen species by MSM modulated migratory activity of PBMCs to stressed muscle cells. Further study is necessary to investigate the biological function(s) of the TLR gene family in horse muscle cells.