• Proceedings of the KSAR Conference
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.75-75
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• 2003

Since the establishment of embryonic stem cell, pluripotency of the cells was known to allow differentiation of the cells into various cell types consisting whole body. Several protocols have been developed to induce expression of specific genes.. However, no precise protocol that will generate a single type of the cells from stem cells has been reported. In order to produce cells suitable for transplantion into brain of PD animal model, which arouse due to a progressive degeneration of dopaminergic neurons in midbrain, human embryonic stem cell (hESC, MB03) was transfected with cDNAs cording for tyrosine hydroxylase (TH). Successful transfection was confirmed by western immunoblotting. Newly transfected cell line (TH#2/MB03) was induced to differentiate by the two neurogenic factors retinoic acid (RA) and b-FGF. Exp. I) Upon differentiation using RA/ascorbic acid (AA), embryoid bodies (EB, for 4days) derived from hES cells were exposed to RA (10$^{-6}$ M)/AA (50 mM) for 4 days, and were allowed to differentiate in N2 medium for 7, 14, 21, or 28 days. Exp. II) When bFGF was used, neuronal precursor cells were selected for 8 days in N2 medium after EB formation. After selection, cells were expanded at the presence of bFGF (20 ng/ml) for another 6 days followed by a final differentiation in N2 medium for 7, 14, 21 or 28 days. By indirect immunocytochemical studies, proportion of cells expressing NF200 increased rapidly from 20% at 7 days to 70 % at 28 days in RA/AA-treated group, while those cells expressing NF160 decreased from 80% at 7 days to 10% at 28 days upon differentiation in N2 medium. However, in differentiation by RA/AA treatment system, there was a significant increase in proportion of neuron maturity (73%) at day 14 after N2 medium. TH#2/MB03 cells expressing TH are >90% when matured at the absence of either bDNF or TGF-$\alpha$. These results suggested that TH#2/MB03 cells could be differentiated in vitro into mature neurons by RA/AA.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.103-103
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• 2003

The purpose of this study is to evaluate an efficacy of in vitro differentiated human embryonic stem (hES, MB03) cells expressing Nurr1 in relief of symptomatic motor behavior of Parkinson's disease (PD) animal models MB03 was genetically modified to express Nurr1 protein and was induced to differentiate according to 2-/4+ protocol using retinoic acid and ascorbic acid. The differentiation-induced cells were selected for 10 to 20 days thereafter in N2 medium. Upon selection, cells expressing GFAP, TH, or NF200 were 38.8%, 11%, and 20.5%, respectively. in order to examine therapeutic effects of the differentiated cells in PD animal model, rats were unilaterally lesioned by administration of 6-kydroxydopamine HCI (6-OHDA) into medial forebrain region (MFB, AP -4.4 mm, ML 1.2 mm, DV 78 mm with incision bar set at -2.4 mm), as a reference to bregma and the surface of the skull. Confirmation of successful lesion by apomorphine-induced rotational behavior, differentiated cells were transplanted into the striatum (AP 1.0, ML 3.5, DV -5.0; AP 0.6, ML 2.5, DV -4.5). Improvements of asymmetric motor behavior by the transplantation were examined every two weeks after the surgery. In two weeks, numbers of rotation by the experimental rats were $-14.8 \pm 33.9%$ (P<0.05) of the number before transplantation, however, the ratio increased slightly to $13.6 \pm 56.3%$ in six weeks. In contrast, the ratio of sham-grafted animals ranged from 112.3+8.5% to 139.2+28.9% during the examination. Immunohistochemical studies further confirmed the presence, survival, migration, and expression of TH of the transplanted human cells.

• Proceedings of the Korean Journal of Food and Nutrition Conference
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• 한국식품영양학회 2001년도 동계 학술심포지움
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• pp.126-126
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• 2001

The Rhus verniciflua contains alkly(en)-catechol type allergens with a saiurated or unsaturated alkly chain of 15 or 17 carbon atoms. It has been recognized as an extremely active allergen causing skin reactions similar In poison ivy. The allergic contact dermatitis induced by the urushiol is known to be mediated be T lymphocytes whicht specifically recognize the hepten urushiol. Therefore. direct use of this plant as a medicinal purpose might imply a considerable hazard in Korea. In this study, using the established method for the detoxification from the stem bark of Rhus verniciflua, an strong antioxidant substance was isolated and characterized DPPH (diphenypricryl hydrazyl) assay measures hydrogen atom-donating activity and hence provides a measure of free radical scavenging antioxidant activity. DPPH, a purple-colored stable free radical, is reduced to yellow-colored diphenylpicryl hydrazine by antioxidants to deducing agents. Antioxidative effects of the water extract from RV were measured by DPPH assay. Twenty microliters of the extract was added to 1ml of 100mM DPPH solution in ethanol The mixture was shaken and left to stand for 10min at room temperature. The crude water extracts was purified by using HPLC method with a DEAE (anionic type), CN, ODS column. The purified compound remained stable at pH 3.0-6,0, but unstable above pH 6.5. It was stable heat at 10$0^{\circ}C$ for 4 hours, but still had about 80% of residual activity after treatment at 10$0^{\circ}C$ for 5 hours. The elemental composition of the HR-EI mass spectrum at m/z 170.02 was estimated the empirical formula as $C_{7}$ $H_{6}$ $O_{5}$. $C_{10}$ $H_4$ $O_2$N$_1$, $C_{5}$ $H_4$ $O_4$N$_3$, $C_{8}$$H_2O$$_1$N$_4$. In antimicrobial test, no inhibition was observed against Gram-positive and negative bacteria. This compound was stronger than that of commercial antioxidant by DPPH test, such as BHT, BHC at the same concentration (20$\mu$g/ml).ml).

• IMMUNE NETWORK
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• 제10권1호
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• pp.15-25
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• 2010

Background: Rat mast cells were regarded as a good model for mast cell function in immune response. Methods: Rat bone marrow mast cells (BMMC) were prepared both by recombinant rat IL-3 (rrIL-3) and by recombinant mouse stem cell factor (rmSCF), and investigated for both proliferation and differentiation in time course. Rat BMMC was induced by culture of rat bone marrow cells (BMCs) in the presence of both rrIL-3 (5 ng/ml) and rmSCF (5 ng/ml). Culture media were changed 2 times per week with the cell number condition of $5{\times}10^4/ml$ in 6 well plate. Proliferation was analyzed by cell number and cell counting kit-8 (CCK-8) and differentiation was by rat mast cell protease (RMCP) II and histamine. Results: Cell proliferation rates reached a maximum at 8 or 11 days of culture and decreased thereafter. However, both RMCP II production and histamine synthesis peaked after 11 days of culture. By real time RT-PCR, the level of histidine decarboxylase mRNA was more than 500 times higher on culture day 11 than on culture day 5. By transmission electron microscopy, the cells were heterogeneous in size and contained cytoplasmic granules. Using gated flow cytometry, we showed that cultured BMCs expressed high levels of $Fc{\varepsilon}RI$ and the mast cell antigen, ganglioside, on culture day 11. Conclusion: These results indicate that rat BMMCs were generated by culturing BMCs in the presence of rrII-3 and rmSCF and that the BMMCs have the characteristics of mucosal mast cells.

• Korean Journal of Clinical Pharmacy
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• 제12권1호
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• pp.7-12
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• 2002

Pharmacokinetic parameters and dosage of aminoglycosides (AGs) were studied retrospectively in 36 patients with neutropenic fever after stem cell transplantation in Seoul National University Hospital from July 1996 to June 2001. AGs pharmacokinetic parameters were calculated with steady-state peak and trough serum drug concentrations by the method of Sawchuk and Zaske et at. The calculated aminoglycosides volume of distribution and clearance were greater than population value $(0.36\pm0.06\;L/kg,\;116\pm32\;ml/min/1.73\;m^2,\;respectively)$. The average dosage of aminoglycosides required to maintain optimal serum AGs concentration was also greater than recommended dose in insert paper. The average dosage of amikacin was $11\pm2.1$ mg/kg every 12 hours (In case of tobramycin, $2.09\pm0.37$ mg/kg every 8 hours or $2.59\pm0.20$ mg/kg every 12 hours). The relationship between AGs volume of distribution and sex, serum albumin (g/dl), body mass index $(kg/m^2)$, body weight change $(\%)$, the amount of fluid inpu (ml/kg/day), the degree of hematocrit decrease $(\%)$ were studied respectively. Univariate anlysis revealed that body mass index $(kg/m^2)$, the amount of fluid input (ml/kg/day) and the degree of hematocrit decrease $(\%)$ had significant correlation with aminoglycosides volume of distribution. But sex, serum albumin, body weight change $(\%)$ had no significant correlation with aminoglycosides volume of distribution.

• Journal of Microbiology and Biotechnology
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• 제30권3호
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• pp.325-332
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• 2020

Methyl linderone (ML), a cyclo-pentenedione, was isolated from the fruit of Lindera erythrocarpa Makino (family Lauraceae). This plant has well-known anti-inflammatory effects; however, the anti-cancer effects of ML have not yet been reported. Thus, in the present study we investigated the effects of ML on the metastasis of human breast cancer cells. We used 12-O-tetradecanoyl phorbol-13-acetate (TPA)-stimulated MCF-7 cells as the cell model to study the effects of ML on invasion and migration. ML was found to reduce the invasion and migration rate of TPA-stimulated MCF-7 cells. Moreover, it inhibited two metastasis-related factors, matrix metalloproteinase-9 (MMP-9) and interleukin-8 (IL-8), at the mRNA and protein expression levels, in TPA-treated MCF-7 cells. The mechanism by which ML exerted these effects was through the inhibition of translocation of activator protein-1 (AP-1) and signal transducer and activator of transcription-3 (STAT3), mediated via phosphorylation of extracellular signal-regulated kinase (ERK). Taken together, our findings indicated that ML attenuated the TPA-stimulated invasion and migration of MCF-7 cells by suppressing the phosphorylation of ERK and its downstream factors, AP-1 and STAT3. Therefore, ML is a potential agent for the treatment of breast cancer metastasis.

• The Korean Journal of Physiology and Pharmacology
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• 제16권1호
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• pp.11-16
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• 2012

Cancer stem cells (CSCs) are often characterized by the elevated expression of drug-resistance related stem-cell surface markers, such as CD133 and ABCG2. Recently, we reported that CSCs have a high level of expression of the IL-6 receptor (IL-6R). The purpose of this study was to investigate the effect of anticancer drugs on the expression of the drug resistance-related cancer stem cell markers, ABCG2, IL-6R, and CD133 in non-small cell lung cancer (NSCLC) cell lines. A549, H460, and H23 NSCLC cell lines were treated with the anticancer drugs 5-fluorouracil (5-FU; $25{\mu}g/ml$) and methotrexate (MTX; $50{\mu}g/ml$), and the expression of putative CSC markers was analyzed by fluorescent activated cell sorter (FACS) and the gene expression level of abcg2, il-6r and cd133 by reverse transcriptase-polymerase chain reaction (RT-PCR). We found that the fraction of ABCG2-positive(+) cells was significantly increased by treatment with both 5-FU and MTX in NSCLC cells, and the elevation of abcg2, il-6r and cd133 expressions in response to these drugs was also confirmed using RT-PCR. Also, the number of IL-6R(+) cells was increased by MTX in the 3 cell lines mentioned and increased by 5-FU in the H460 cell line. The number of CD133(+) cells was also significantly increased by both 5-FU and MTX treatment in all of the cell lines tested. These results indicate that 5-FU and MTX considerably enhance the expression of drug-resistance related CSC markers in NSCLC cell lines. Thus, we suggest that antimetabolite cancer drugs, such as 5-FU and MTX, can lead to the propagation of CSCs through altering the expression of CSC markers.

• Advances in Traditional Medicine
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• 제7권2호
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• pp.157-161
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• 2007

We undertook the present study to evaluate different pharmacological as antioxidant, antibacterial and cytotoxic activities of the crude ethanolic extract of the stem bark of Holarhena antidysenterica (Family: Apocynaceae). The antioxidant property of the extract was assessed by 1,1-diphenyl-2-picryl hydrazyl free radical scavenging assay. The extract showed antioxidant activity ($IC_{50}$ about ${\sim}08$ ${\mu}g/ml$), which was comparable to standard drug ascorbic acid ($IC_{50}$ about ${\sim}10$ ${\mu}g/ml$). The extract showed a broad spectrum of antibacterial activity against all tested gram positive and gram negative bacteria most prominent against Enterococci, Staphylococcus pyogenase and Shigella sonnie. And the zones of inhibitions were ranging from 10 - 21 mm for all the tested bacteria. Its cytotoxic property was evaluated by brine shrimp lethality bioassay. The extract showed significant lethality and the $LC_{50}$, $LC_{90}$ values were 80 ${\mu}g/ml$, 320 ${\mu}g/ml$ respectively. The results tend to suggest that the extract might possess chemical constituent(s) that are responsible for antioxidant, antibacterial and cytotoxic activities.

• Advances in Traditional Medicine
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• 제6권2호
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• pp.102-107
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• 2006

The methanol extract and residual methanol fraction of Excoecaria agallocha L. (Euphorbiaceae) stem bark was investigated in this study by wheat rootlet and shoot growth inhibition, and antimicrobial bioassay. The methanol extract and residual methanol fraction showed high inhibitory effect on both the wheat rootlet (82-89%) and shoot growth (85-90%) compared to control. The methanol extract showed a better and dose related inhibition on both the rootlet and shoot growth compared to residual methanol fraction. The $IC_{50}$ value of methanol extract for rootlet and shoot were $2.88\;{\mu}g/ml$ and $2.32\;{\mu}g/ml$, and of residual methanol fraction for rootlet and shoot were $7.91\;{\mu}g/ml$ and $4.45\;{\mu}g/ml$. The methanol extract and residual methanol fraction did not show any antibacterial activity against the tested microorganisms of clinical isolates Escherichia coli, Staphylococcus aureous, Pseudomonas aeruginosa, Proteus vulgaris and Bacillus subtilis. The plant has the potential to be a source of novel cytotoxic compound(s).

• Reproductive and Developmental Biology
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• 제36권1호
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• pp.21-26
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• 2012

Suspension culture is a useful tool for culturing embryonic stem (ES) cells in large-scale, but the stability of pluripotency and karyotype has to be maintained $in$ $vitro$ for clinical application. Therefore, we investigated whether the chromosomal abnormality of ES cells was induced in suspension culture or not. The ES cells were cultured in suspension as a form of aggregate with or without mouse embryonic fibroblasts (MEFs), and 0 or 1,000 U/ml leukemia inhibitory factor (LIF) was treated to suspended ES cells. After culturing ES cells in suspension, their karyotype, DNA content, and properties of pluripotency and differentiation were evaluated. As a result, the formation of tetraploid ES cell population was significantly increased in suspension culture in which ES cells were co-cultured with both MEFs and LIF. Tetraploid ES cell population was also generated when ES cells were cultured alone in suspension regardless of the existence of LIF. On the other hand, the formation of tetraploid ES cell population was not detected in LIF-free condition, in which MEFs were included. The origin of tetraploid ES cell population was turned out to be E14 ES cells and not MEFs by microsatellite analysis and the basic properties of them were still maintained despite ploidy-conversion to tetraploidy. Furthermore, we identified the ploidy shift from tetraploidy to near-triploidy as tetraploid ES cells were differentiated spontaneously. From these results, we demonstrated that suspension culture system could induce ploidy-conversion generating tetraploid ES cell population. Moreover, optimization of suspension culture system may make possible mass-production of ES cells.