• International Journal of Oral Biology
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• v.33 no.4
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• pp.179-186
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• 2008

Several lines of evidence suggest that osteocytes play a critical role in bone remodeling. Both healthy and apoptotic osteocytes can send signals to other bone surface cells such as osteoblasts, osteoclasts, osteoclast precursors, and bone lining cells through canalicular networks. Osteocytes responding to mechanical strain may also send signals to other cells. To determine the role for osteocytes an mechanical strain in bone remodeling, we examined the effects of fluid flow shear stress on osteoclast precursor cell and osteoblast proliferation and recruitment induced by osteocytes. In addition, the effects of fluid flow shear stress on osteocyte M-CSF, RANKL, and OPG mRNA expression were also examined. MLO-Y4 cells were used as an in vitro model for osteocytes, RAW 264.7 cells and MOCP-5 cells as osteoclast precursors, and 2T3 cells as osteoblasts. MLO-Y4 cells conditioned medium (Y4-CM) was collected after 24h culture. For fluid flow experiments, MLO-Y4 cells were exposed to 2h of pulsatile fluid flow (PFF) at 2, 4, 8, $16{\pm}0.6\;dynes/cm^2$ using the Flexcell $Streamer^{TM}$ system. For proliferation assays, MOCP-5, RAW 264.7, and 2T3 cells were cultured with control media or 10-100% Y4 CM. Cells were cultured for 3d, and then cells were counted. RAW 264.7 and 2T3 cell migration was assayed using transwells with control media or 10-100% Y4-CM. M-CSF, RANKL and OPG in MLO-Y4 mRNA expression was determined by semiquantitative RT-PCR. Y4-CM increased osteoclast precursor proliferation and migration, but decreased 2T3 cell proliferation and migration. CM from MLO-Y4 cells exposed to PFF caused decreased RAW 267.4 cell proliferation and migration and 2T3 migration compared to control Y4-CM. However, Y4-CM from cells exposed to PFF had no effect on 2T3 osteoblastic cell proliferation. PFF decreased RNAKL mRNA and increased OPG mRNA in MLO-Y4 cells compared to control(without PFF). PFF had no effect on M-CSF mRNA expression in MLO-Y4 cells. These results suggest that osteocytes can regulate bone remodeling by communication with osteoclast precursors and osteoblasts and that osteocytes can communicate mechanical signals to other cells.

• Horticultural Science & Technology
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• v.29 no.2
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• pp.87-94
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• 2011

This study was carried out to investigate the effects of hot solution soaking and chilling treatment on sprouting and enlargement of bulblets obtained through in vitro culture in Korean native lilies with ornamental values. In vitro bulblets of Lilium cernuum, L. hansonii, L. hansonii for. mutatum, L. leichtlinii, and L. tsingtauense were soaked in distilled water or 100 $mg{\cdot}L^{-1}$ $GA_3$ and $GA_{4+7}$ solution maintained at $35^{\circ}C$ for two hours (hot water treatment) and/or exposed at $4^{\circ}C$ for 0, 4, or 8 weeks (chilling treatment) and then planted in plastic trays filled with media and grown in a greenhouse at $20^{\circ}C$ and under 16 h photoperiod. In all species, no bulblet propagated by tissue culture sprouted without chilling or hot water treatment due to dormancy. For dormancy breaking, $GA_{4+7}$ hot solution treatment increasing sprouting by 55-96%, whereas distilled water or GA was not effective in sprouting. Chilling treatment for 4 weeks induced sprouting by 50-70% in L. cernuum and L. leichtlinii, whereas 8 weeks was needed for sprouting of L. hansonii and L. hansonii for. mutatum. Combined treatment of hot water and chilling treatments synergistically promoted sprouting. Especially, in L. cernuum and L. hansonii, $GA_{4+7}$ hot solution soaking prior to chilling for 4 weeks promoted sprouting by 35-45% compared with the reverse order. Enlargement of bulblets resulted from increase in fresh weight and diameter was promoted by the treatments that increased the sprouting percentage of bulblets. Only in L. cernuum, shoots emerged from bulblets soaked in hot $GA_{4+7}$ solution or chilled at $4^{\circ}C$ and shoot emergence rate was highest in bulblets soaked in hot $GA_{4+7}$ solution and then chilled for 8 weeks. From these results, the most effective method for bulblet sprouting and enlargement was to soak in hot $GA_{4+7}$ solution and then chill for 4 weeks in L. hansonii, hansonii for. mutatum, and leichtlinii, and to soak in hot $GA_{4+7}$ solution and then chill for 4 weeks in L. cernuum and tsingtauense.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.04a
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• pp.43-43
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• 2019

Deparia pycnosora (Christ) M. Kato is a fern used as ornamental plant. In addition, it is called "Teol-go-sa-ri" in Korean name. The aim of this study was to develop a practical propagation method of D. pycnosora using tissue culture technique. Prothallus obtained from spore germination was the used as experiment materials. The prothalli (300 mg) used in all experiments were sub-cultured for 8-week intervals. The most suitable media for prothallus propagation were identified by culturing 300 mg of prothalli in $1/4{\times}$, $1/2{\times}$, $1{\times}$, $2{\times}$ MS medium and in Knop medium for 8 weeks. Also, the prothalli were cultured by chopping with a scalpel. In addition, sucrose, activated charcoal, and total nitrogen source were added in different concentrations based on the culture medium selected. Cultures were maintained at a temperature of $25{\pm}1^{\circ}C$, light intensity of $30{\times}1.0{\mu}mol-m-2{\cdot}s-1$, and a photoperiod of 16/8 h (light/dark) in in vitro. The results showed that optimum was achieved prothallus fresh weight and development in $1{\times}$ MS medium. When other components were added to the basic $1{\times}$ MS medium, prothallus propagation was maximized in $1{\times}$ MS medium supplemented with 2% sucrose, 0.2% activated charcoal, and 60 mM total nitrogen. To select a suitable soil mixture for sporophyte formation, 1.0 g of prothallus was blended with distilled water, spread on five combinations of different soil substrates (decomposed granite, horticultural substrates, peat moss, and perlite), and cultivated for 12 weeks. The sporophyte cultures were maintained at a temperature of $25{\pm}1^{\circ}C$, light intensity of $43{\pm}2.0{\mu}mol-m-2{\cdot}s-1$, humidity of $84{\pm}1.4%$, and a photoperiod of 16/8 h (light/dark). As a results, horticultural substrate alone, 2:1 (v:v) mixtures of horticultural substrate and perlite, and 2:1 mixtures of horticultural substrate and decomposed granite induced 208.0, 201.3 and 248.8 sporophytes per pot, respectively. Therefore, this result could provide a practical mass propagation method of D. pycnosora

• Korean Journal of Plant Resources
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• v.35 no.4
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• pp.502-507
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• 2022

In the present study, plant regeneration through adventitious shoot formation from hypocotyl segments of in vitro seedlings of groundcherry (Physalis angulata L.) was investigated to determine the optimum culture conditions for highly efficient regeneration of the species. Adventitious shoots in hypocotyl segments were efficiently induced on MS media with low concentrations of BAP, specifically, with 0.5-1.0 mg/L BAP singly or in combination with 0.1-0.5 mg/L NAA. The 1.0 mg/L BAP single treatment was most effective for forming multiple adventitious shoots. When the induced shoots were transferred to the root induction media, low concentrations of NAA, IBA, and IAA enhanced the development of adventitious roots from adventitious shoots, suggesting that low concentrations of auxins were optimal for producing regenerated plantlets. The number of roots per shoot was large (> 2.0), and the root length exceeded 8.0 cm. In particular, the development and the overall shape of the roots were ideal. Furthermore, the number and length of shoots exceeded 2 and 6.0 cm, respectively. When the regenerated plantlets were transferred to compost soil, the root and shoot systems had developed well to the point that all of the regenerated plantlets acclimated successfully, resulting in normal morphology and growth characteristics, similar to those of the mother plant. Therefore, plant regeneration via adventitious shoot formation is expected to be one of the main methods for producing groundcherry on a large scale for a stable supply of the raw materials.

• The korean journal of orthodontics
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• v.24 no.4 s.47
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• pp.871-883
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• 1994

This experiment was designed to study possible roles of $interleukin-1\beta$, interleukin-6 and tumor necrosis $factor-\alpha$ in bone remodeling by measuring their effects on $PGE_2,\; LTB_4$ and collagenase production when they were administered to human periodontal ligament fibroblasts. Human periodontal ligament fibroblasts were collected from first premolars extracted for orthodontic treatment. They were incubated in the environment of $37^{\circ}C,\;5\%\;Co^2,\;and\;100\%$ humidity. They were treated with $0.25\%$ trypsin-EDTA solution and centrifuged. PDL cells in the fifth to seventh passage were used for the experiment. Cells were seeded onto the culture dishes and when they were successfully attached, human recombinant $interleukin-1\beta$, interleukin-6, and tumor necrosis $factor-\alpha$ were administered, alone or in combination. They were incubated for 4, 8 and 24 hours and the levels of $PGE_2,\;LTB_4$ and collagenase released into the culture media were assessed by enzymeimmunoassay and collagenase activity assay. The conclusions are as follows: 1. $IL-1\beta\;and\;TNF-\alpha$ were very active in stimulating the production of $PGE_2$ and collagenase by human periodontal ligament fibroblasts, while IL-6 increased $LTB_4$ production. 2. $IL-1\beta$ significantly increased $PGE_2$, but $LTB_4$ Production was not increased. $IL-1\beta$ is thought to act mainly via the cyclooxygenase pathway of arachidonic acid metabolism. 3. IL-6 tended to inhibit $IL-1\beta$ in the production of $PGE_2$ and collagense whereas IL-6 and $TNF-\alpha$ showed auditive effect in the level of $PGE_2$. The above cytokines increased the release of at least one of $PGE_2,\;LTB_4$ and collagenase. It suggests that cytokines are involved in bone remodeling process by stimulating PDL fibroblasts to produce various bone-resorptive agents. The roles of cytokines in bone remodeling as a whole would need further study.

• Journal of Life Science
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• v.18 no.12
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• pp.1631-1636
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• 2008

Rhei Rhizoma (RR; 大黃) consists of the underground parts (rhizome and root) of Rheum officinale Baill. and Rheum palmatum L. (Polygonaceae), and is widely used in Southeast Asian folk medicine to alleviate liver and kidney damages. In this study, we investigated into the efficacy and mechanism of RR water extract in supporting neuronal survival in a hypoxia model of cultured rat hippocampal neurons. RR exhibited no cytotoxicity up to 10 ${\mu}g$/ml and exhibited neurosupportive effects at 2.5 ${\mu}g$/ml in normoxia. When RR was added to the culture media on 10 days in vitro (DIV10) and given a hypoxic shock (2% $O_2$/5% $CO_2$, 3 hr, $37^{\circ}C$) on DIV13, RR exhibited neuroprotective effects on 5 days post-shock. $H_2DCF$ stainings indicated that RR effectively prevents ROS production in both normoxia and hypoxia. JC-1 stainings showed that RR prevents dissipation of MMP in hypoxia. These results indicate that RR protects neurons by suppressing ROS production and MMP loss.

• Development and Reproduction
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• v.5 no.1
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• pp.17-21
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• 2001

This study was conducted to investigate the expression pattern of Trypsin-like enzyme and the effect of a trypsin inhibitor(benzimidine) on hatching process during in-vitro culture of mouse preimplantation embryos. The Trypsin-like enzyme was identified by rhodamine-conjugated Trypsin substrate probe. The expression of trypsin-like enzyme was firstly detected at the late morula stage, and the enzyme was uniformly localized in the trophectoderm of late blastocysts. Especially, intense fluorescence was observed in the blebbing area of hatching blastocysts. Bisbenzamidine, contained in culture media, did not alter embryonic development from 4-cell stage to the expanded blastocyst but decrease the hatching rate in ImM concentration (15.8% vs 89.7%, p<0.02). In the treatment of bisbenzimidine (5mM) for 12 hours according to the embryonic stage of mouse, the hatching rate of control (83.0%) and treatment in late blastocysts (8.7%) were significantly (p<0.01) different. From these results, we suggested that the hatching enzyme having trypsin-like activity was localized from the late morula stage, and the hatching process by this enzyme was activated in the late blastocyst stage of mouse embryos.

• Journal of Periodontal and Implant Science
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• v.35 no.2
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• pp.345-357
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• 2005

Prostaglandin plays a significant role in the local control of bone metabolism associated with periodontal disease. ${\Delta}^{12}-PGJ_2$ is a natural $PGD_2$ metabolite that is formed in vivo in the presence of plasma. It is known for ${\Delta}^{12}-PGJ_2$ to stimulate calcification in osteoblastic cells. Bone morphogenetic protein(BMP) stimulated osteoblastic differentiation in various types of cells and greatly enhanced healing of bony defects. The purpose of this study was to evaluate the effect of rhEMP-2 on ${\Delta}^{12}-PGJ_2$ induced osteoblastic differentiation and mineralization in vitro. A human osteosarcoma cells line Saos-2 were cultured. In the test groups, 10-7M of ${\Delta}^{12}-PGJ_2$ or mixture of 10-8M of ${\Delta}^{12}-PGJ_2$ and 100ng/ml of rhBMP-2 or 100ng/ml of rhEMP-2 were added to culture media. After 1 day, 2 days and 4 days of culture period, the cell number was measured. Alkaline phosphatase activity was measure at 3 days. Reverse transcription polymerase chain reaction(RT-PCR) was performed to determine the expression of mRNA of bone matrix protein at 8 hours, 1 day and 7 days. The ability to produce mineralized nodules in rat osteoblasts(MC3T3-E1) was evaluated at 21 days. The results were as follows : 1. rhEMP-2 or mixture of rhBMP-2 and ${\Delta}^{12}-PGJ_2$ inhibited cell proliferation of human osteosarcoma cells. 2. rhEMP-2 or mixture of rhBMP-2 and ${\Delta}^{12}-PGJ_2$ stimulated alkaline phosphatase activity significantly higher than ${\Delta}^{12}-PGJ_2$ alone. 3. rhBMP-2 or mixture of rhEMP-2 and ${\Delta}^{12}-PGJ_2$ stimulated mineralization compared to ${\Delta}^{12}-PGJ_2$ alone. 4. mRNA of alkaline phosphatase, BMP-2, cbfa 1, Type I collagen were detected in the group treated with ${\Delta}^{12}-PGJ_2$/rhBMP-2, rhBMP-2 alone, ${\Delta}^{12}-PGJ_2$ alone. These results show that mixture of ${\Delta}^{12}-PGJ_2$ and rhBMP-2 causes more bone formation than ${\Delta}^{12}-PGJ_2$ alone while the bone formation effects of mixture of ${\Delta}^{12}-PGJ_2$ and rhBMP-2 are less than those of rhBMP-2 alone. Further researches would be necessary to clarify the interactions of these agents.

• BMB Reports
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• v.37 no.4
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• pp.454-459
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• 2004

Experimental hyperoxia represents a suitable in vitro model to study some pathogenic mechanisms related to oxidative stress. Moreover, it allows the investigation of the molecular pathophysiology underlying oxygen therapy and toxicity. In this study, a modified experimental set up was adopted to accomplish a model of moderate hyperoxia (50% $O_2$, 96 h culture) to induce oxidative stress in the human leukemia cell line, U-937. Spectrophotometric measurements of mitochondrial respiratory enzyme activities, NMR spectroscopy of culture media, determination of antioxidant enzyme activities, and cell proliferation and differentiation assays were performed. The data showed that moderate hyperoxia in this myeloid cell line causes: i) intriguing alterations in the mitochondrial activities at the levels of succinate dehydrogenase and succinate-cytochrome c reductase; ii) induction of metabolic compensatory adaptations, with significant shift to glycolysis; iii) induction of different antioxidant enzyme activities; iv) significant cell growth inhibition and v) no significant apoptosis. This work will permit better characterization the mitochondrial damage induced by hyperoxia. In particular, the data showed a large increase in the succinate cytochrome c reductase activity, which could be a fundamental pathogenic mechanism at the basis of oxygen toxicity.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.4
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• pp.292-297
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• 2022

Direct injection of CRISPR/Cas9 into zygotes enables the production of genetically modified nonhuman primates (NHPs) essential for modeling specific human diseases, such as Usher syndrome, and for developing novel therapeutic strategies. Usher syndrome is a rare genetic disease that causes loss of hearing, retinal degeneration, and problems with balance, and is attributed to a mutation in MYO7A, a gene that encodes an uncommon myosin motor protein expressed in the inner ear and retinal photoreceptors. To produce an Usher syndrome type 1B (USH1B) rhesus macaque model, we disrupted the MYO7A gene in developing zygotes. Identification of appropriately edited MYO7A embryos for knockout embryo transfer requires sequence analysis of material recovered from a trophectoderm (TE) cell biopsy. However, the TE biopsy procedure is labor intensive and could adversely impact embryo development. Recent studies have reported using cell-free DNA (cfDNA) from embryo culture media to detect aneuploid embryos in human in vitro fertilization (IVF) clinics. The cfDNA is released from the embryo during cell division or cell death, suggesting that cfDNA may be a viable resource for sequence analysis. Moreover, cfDNA collection is not invasive to the embryo and does not require special tools or expertise. We hypothesized that selection of appropriate edited embryos could be performed by analyzing cfDNA for MYO7A editing in embryo culture medium, and that this method would be advantageous for the subsequent generation of genetically modified NHPs. The purpose of this experiment is to determine whether cfDNA can be used to identify the target gene mutation of CRISPR/Cas9 injected embryos. In this study, we were able to obtain and utilize cfDNA to confirm the mutagenesis of MYO7A, but the method will require further optimization to obtain better accuracy before it can replace the TE biopsy approach.