• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.847-852
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• 2007

This study investigated an efficient method for the extraction of astaxanthin from the red yeast Xanthophyllomyces dendrorhous. The extraction process comprised three steps: 1) cultivating the yeast; 2) treating the yeast culture suspension with microwaves to destroy the cell walls and microbodies; and 3) drying the yeast and extracting the astaxanthin pigment using ethanol, methanol, acetone, or a mixture of the three as the extraction solvent. Ultimately, various treatment tests were performed to determine the conditions for optimal pigment extraction, and the total carotenoid and astaxanthin contents were quantified. A frequency of 2,450 MHz, an output of 500 watts, and irradiation time of 60 s were the most optimum conditions for yeast cell wall destruction. Furthermore, optimal pigment extraction occurred when using a cell density of 10g/l at $30^{\circ}C$ over 24 h, with a 10% volume of ethanol.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.235-244
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• 1995

The present study was designed to demonstrate that ES cell lines efficiently could be isolated from explanted blastocysts of hybrid BCF1 mouse when grown on STO feeder layer derived from mouse fibroblasts in culture medium supplemented with leukemia inhibitory factor (LIF). The expanded blastocysts were attached to mitomycin C-inactivated STO feeder layer and were cultured for 4 days. Four days later the ICM was disaggregated by a short term trypsin treatment (0.25% trypsin / 0.04% EDT A for 2-3 min). The resulting cell suspension was seeded on a new STO feeder layer and covered with DMEM supplemented with 10% FCS, 0.1 mM nonessential amino acid, 0.1 mM sodium pyruvate, 0.1 mM mercaptoethanol and 1,000 U/ml LIF. Colonies of ES-like cells were observed after the second passage. These colonies were repeatedly passaged at approximately 5 day intervals. In this study, five ES-like celllines were isolated by directly explanting blastocysts, but three lines were lost after the 5th passage, possibly due to toxic effects of a new FCS batch. The characterization of developmental potential of isolated cell lines was performed with respect to in vitro differentiation and specific activity of alkaline phosphatase (AP). When cells were cultured in suspension, the aggregates of cell lines were capable of forming simple embryoid bodies (EB), and showed the capacity for forming cystic multilayer EBs. In addition, the cell lines were positive for AP staining, a biochemical marker characteristic of mouse ES cells.

• Journal of Plant Biotechnology
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• v.30 no.2
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• pp.135-141
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• 2003

Peroxiredoxin(Pix) are large family of peroxidases that reduce alkyl hydroperoxides and hydrogen peroxide. A cDNA clone (referred to as swPrxl) encoding Pix was from a sweetpotato cDNA library constructed from suspension-sultured cells, and its expression was investigated in terms of stress. The swPrxl contained an open reading frame (ORF) encoding mature protein of 193 amino acids with calculated molecular mass of 20.8kDa. The predicted amino acid sequence of swPrxl has two conserved cysteines that are essential resicues for the reduction of peroxides. It showed high amino acid sequence homology ot PixIIF of Arabidopsis (77%) and putative Prx of rice(72%). RNA gel-blot analysis showed that swPrxl gene was expressed dominantly in leave among intact tissues, and also highly detect in suspension-cultured cells. Interestingly, the level of swPrxl transcripts was almost the same regardless of the growth stage in suspension culture. Furthermore, the transcription level of swPrxl gene was not significantly changed in response to various stress treatments such as wounding, extreme temperature and stress-related chemicals RT-PCR analyses.

• Journal of Ginseng Research
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• v.43 no.2
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• pp.261-271
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• 2019

Background: Protopanaxatriol (PPT) is an aglycone of ginsenosides, which has high medicinal values. Production of PPT from natural ginseng plants requires artificial deglycosylation procedures of ginsenosides via enzymatic or physicochemical treatments. Metabolic engineering could be an efficient technology for production of ginsenoside sapogenin. For PPT biosynthesis in Panax ginseng, damarenediol-II synthase (PgDDS) and two cytochrome P450 enzymes (CYP716A47 and CYP716A53v2) are essentially required. Methods: Transgenic tobacco co-overexpressing P. ginseng PgDDS, CYP716A47, and CYP716A53v2 was constructed via Agrobacterium-mediated transformation. Results: Expression of the three introduced genes in transgenic tobacco lines was confirmed by Reverse transcription-polymerase chain reaction (RT-PCR). Analysis of liquid chromatography showed three new peaks, dammarenediol-II (DD), protopanaxadiol (PPD), and PPT, in leaves of transgenic tobacco. Transgenic tobacco (line 6) contained $2.8{\mu}g/g$ dry weight (DW), $7.3{\mu}g/g$ DW, and $11.6{\mu}g/g$ DW of PPT, PPD, and DD in leaves, respectively. Production of PPT was achieved via cell suspension culture and was highly affected by auxin treatment. The content of PPT in cell suspension was increased 37.25-fold compared with that of leaves of the transgenic tobacco. Transgenic tobacco was not able to set seeds because of microspore degeneration in anthers. Transmission electron microscopy analysis revealed that cells of phloem tissue situated in the center of the anther showed an abnormally condensed nuclei and degenerated mitochondria. Conclusion: We successfully achieved the production of PPT in transgenic tobacco. The possible factors deriving male sterility in transgenic tobacco are discussed.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.3
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• pp.272-278
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• 2006

Purpose: This study is designed to evaluate biocompatibility of three types of absorbable collagen GBR membrane in vitro. Material and Method: The human PDL fibroblasts culture was obtained through typical way and the cells used in the experiment was forth passage. The membranes examined were Experimental group A, B, C. All the 3-experimental groups were made of bovine pericardium and the membranes were excised into 5$\times$5mm respectively. The samples of the membranes were fixed on the 24-well plate with the double-sided adhesive tape. Then, 2ml of cell suspension which included $2{\times}10^4$cells was inoculated into the 24-well plate, and the cells were cultured for 1 week. Cellular viability and the alkaline phosphatase activity were measured with ELISA. The membranes in the culture were processed to examine with SEM. Results: The survival rate was highest in control and Experimental group A is the next, group B and group C in order of the value. The values are analyzed for statistical difference using Wilcoxon test. All the values of experimental groups are significantly lower than those of control, and the vaules among the experimental groups significantly differ from each other. Alkaline phosphatase level was identical order with the viable cell rate. SEM examination revealed that the PDL fibroblasts adherent on culture dish (control) and group A were spindle-shaped, but on group B and C, the cells were round-shaped without processes.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.4
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• pp.209-215
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• 2004

Objective: Embryonic stem (ES) cells could be differentiated into the specific cell types by alternation of culture condition and modification of gene expression. This study was performed to evaluate the differentiation protocol for mouse and human ES cells to insulin secreting cells. Methods: Undifferentiated mouse (JH-I) and human (Miz-hESI) ES cells were cultured on STO feeder layer, and embryoid bodies (EBs) were formed by suspension culture. For the differentiation, EBs were cultured by sequential system with three stage protocol. The differentiating ES cells were collected and marker gene expressions were analyzed by seIni-quantitative RT-PCR in each stage. Amount of secreted insulin levels in culture media of human ES cells were measured by human insulin specific RIA kit. Results: During the differentiation process of human ES cells, GATA-4, a-fetoprotein, glucose transporter-2 and Ngn-3 expression were increased whereas OctA was decreased progressively. Insulin and albuInin mRNAs were expressed from stage IT in mouse ES cells and from stage III in human ES cells. We detected 3.0~7.9 IlU/rnl secretion of insulin from differentiated human ES cells by in vitro culture for 36 days. Conclusion: The sequential culture system could induce the differentiation of mouse and human ES cells into insulin secreting cells. This is the fIrst report of differentiation of human ES cells into insulin secreting cells by in vitro culture with serum and insulin free medium.

• KSBB Journal
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• v.10 no.1
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• pp.23-29
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• 1995

Calli were induced from leaf base region of germinated rice(Oryza sativa L. cv. Nakdong) with high frequency of up to 65% on LS medium supplemented with $2.5mg/{\ell}2$, 4-D in the dark at $27^{\circ}C$. Embryogenic calli of pale yellow, globular type were selected and used for the initiation of cell suspension cultures in AA2 liquid medium with $2mg/\ell$ 2,4-D, 0.2mg/$\ell$ kinetin arid $0.1mg/\ell$ GA3. Protoplasts were isolated from the embryogenic cell suspensions after 4 months of culture and then were electroporated with 400V/cm for 1 msec. Electroporated protoplasts divided with plating efficiency of 1.1% on PCM liquid medium supplemented with $2.5mg/\ell$ 2, 4-D, $0.1mg/\ell$ kinetin and 10mM proline. The protoplasts-derived microcalli were cultured on $0.2{\mu}m$ membrane fitter placed onto LS2.5 solid medium containing fine suspension cells as a feeder cells, for 2 weeks in the dark at $27^{\circ}C$. After an additional 2 weeks of culture under fluorescent light of $30{\pm}/3{\mu}E$.m^{-2}S^{-1}, yellow calli of 2mm diameter were transferred to regeneration medium. Shoots were produced from the green spot of protoplasts-derived calli and plants were regenerated form protoplast-derived green calli with frequencies of 11∼33%.

• Journal of Life Science
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• v.24 no.7
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• pp.757-761
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• 2014

Olive flounder (Paralichthys olivaceus) is an important aquaculture fish species in Jeju Island, South Korea. Due to the intensification of flounder fish farming, huge amounts of chemical antibiotics are used against several fish diseases. This has many harmful side effects on fish, as well as human consumers. Hence, an alternative to chemical antibiotic agents is needed for disease control. In this study, three strains of rhizobacteria (BRH433-2, TRH415-2, and THJ609-3) were isolated from the rhizosphere of plants. Assays of their antibacterial activity against fish pathogens, such as S. iniae, S. parauberis, V. anguillarum, and E. tarda, were performed with untreated broth culture (without cell separation), supernatant, and precipitated pellets separated by centrifugation. Among these, the cell suspension prepared from the precipitated pellet showed significant antimicrobial activity when compared with that of the untreated broth culture and centrifugal supernatant. These results indicate that the three isolated rhizobacterial strains exhibit antibacterial activity. Analysis of the 16S rDNA sequences of the BRH-433-2, THJ609-3, and TRH415-2 strains showed the highest similarity to Burkholderia gladioli (99.5%), Pseudomonas baetica (97.7%), and P. koreensis and P. baetica (98.4%), respectively. We suggest that the strains hold promise in disease management of fish.

• Journal of Plant Biotechnology
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• v.32 no.2
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• pp.105-109
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• 2005

To enhance salidroside productivity from a cell suspension cultures of Rhodiola sachalinensis, various combinations of auxin (NAA) and cytokinins (BA, Kinetin) concentration and addition of $GA_3$, TDZ, zeatin, spermine and spermidine at a hormone combination to be established were examined. NAA/BA combination is superior to NAA/kinetin combination in biomass and salidroside content. Maximum salidroside production ($64.6{\pm}8.9\;mg/L$ medium) was obtained from $2B_5$ medium with 1 mg/L NAA and 5 mg/L BA. The adding of $GA_3$ ($0.01\;mg/L{\sim}5\;mg/L$) was beneficial for salidroside accumulation and the highest productivity of salidroside, $90.3{\pm}8.34\;mg/L$, was obtained from $2B_5$ medium supplemented with 1 mg/L NAA, 5 mg/L BA and 0.1 mg/L $GA_3$. On TDZ, zeatin, spermine and spermidine, expectant results were not obtained except a little affirmative effect of spermidine ($69{\pm}2.88$ at 1 mg/L).

• Development and Reproduction
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• v.12 no.1
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• pp.87-95
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• 2008

Neural stem/precursor derived from pluripotent human embryonic stem cells (hESCs) has considerable therapeutic potential due to their ability to generate various neural cells which can be used in cell-replacement therapies for neurodegenerative diseases. However, production of neural cells from hESCs remains technically very difficult. Understanding neural-tube like rosette characteristic neural precursor cells from hESCs may provide useful information to increase the efficiency of hESC neural differentiation. Generally, neural rosettes were derived from differentiating hEBs in attached culture system, however this is time-consuming and complicated. Here, we examined if neural rosettes could be formed in suspension culture system by bypassing attachment requirement. First, we tested whether the size of hESC clumps affected the formation of human embryonic bodies (hEBs) and neural differentiation. We confirmed that hEBs derived from $500{\times}500\;{\mu}m$ square sized hESC clumps were effectively differentiated into neural lineage than those of the other sizes. To induce the rosette formation, regular size hEBs were derived by incubation of hESC clumps($500{\times}500\;{\mu}m$) in EB medium for 1 wk in a suspended condition on low attachment culture dish and further incubated for additional $1{\sim}2$ wks in neuroectodermal sphere(NES)-culture medium. We observed the neural tube-like rosette structure from hEBs after $7{\sim}10$ days of differentiation. Their identity as a neural precursor cells was assessed by measuring their expressions of neural precursor markers(Vimentin, Nestin, MSI1, MSI2, Prominin-1, Pax6, Sox1, N-cadherin, Otx2, and Tuj1) by RT-PCR and immunofluorescence staining. We also confirmed that neural rosettes could be terminally differentiated into mature neural cell types by additional incubation for $2{\sim}6$ wks with NES medium without growth factors. Neuronal(Tuj1, MAP2, GABA) and glial($S100{\beta}$ and GFAP) markers were highly expressed after $2{\sim}3$ and 4 wks of incubation, respectively. Expression of oligodendrocyte markers O1 and CNPase was significantly increased after $5{\sim}6$ wks of incubation. Our results demonstrate that rosette forming neural precursor cells could be successfully derived from suspension culture system and that will not only help us understand the neural differentiation process of hESCs but also simplify the derivation process of neural precursors from hESCs.