• Clinical and Experimental Reproductive Medicine
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• v.31 no.4
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• pp.261-271
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• 2004

Objective: This study was performed to evaluate the possibility of prolonged culture of human embryonic stem cells (hESC; SNUhES2) on human amniotic fluid cells (hAFC), which had been storaged after karyotyping. Method: The hAFC was prepared for feeder layer in the presence of Chang's medium and STO medium (90% DMEM, 10% FBS) at $37^{circ}C$ in a 5% $CO_2$ in air atmosphere. Prior to use as a feeder layer, hAFC was mitotically inactivated by mitomycin C. The hESCs on hAFC were passaged mechanically every seven days with ES culture medium (80% DMEM/F12, 20% SR, bFGF). Results: The hAFC feeder layer support the growth of undifferentiated state of SNUhES2 for at least 59 passages thus far. SNUhES2 colonies on hAFC feeder appeared slightly angular and flatter shape as compared with circular and thicker colonies observed with STO feeder layer and showed higher level with complete undifferentiation in seven days. Like hESC cultured on STO feeders, SNUhES2 grown on hAFC expressed normal karyotype, positive for alkaline phosphatase activity, high telomerase activity, Oct-4, SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81 and formed embryoid bodies (EBs). Conclusion: The hAFC supports undifferentiated growth of hESC. Therefore, these results may help to provide a clinically practicable method for expansion of hESC for cell therapies.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.243-248
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• 2003

Sperrnatogenesis, the process by which the male germ-line stem cells(GSCs; type A spermatogonia) divide and differentiate to produce the mature spermatozoa, occurs in the seminiferous tubules of the testis. The GSCs proliferate actively to produce two types of cells: other GSCs and differentiating spermatogonia. GSCs have unipotentcy, devoted solely to the generation of sperm. The function of GSCs has broad implications for development, disease, and evolution. Spermatogenesis is fundamental for propagation of species and the defects of this system can result in infertility or disease. The ability to identify, isolate, culture, and alter GSCs will allow powerful new approaches in animal transgenesis and human gene therapy relating to infertility. Until recently, research on stem cells in the testis has been limited because of technical difficulties in isolating and identifying these cell populations. Here, we were trying to find out optimal conditions for in vitro culture of GSCs for identifying and isolating GSCs. We collected mouse GSCs from 3-days old mouse by two-step enzyme digestion method. GSCs were plated and grown on mouse embryonic fibroblasts in Dulbecco's modified Eagle's medium (DMEM) containing 15％ fatal bovine serum, 10 mM 2-mercaptoethanol, 1％ non-essential amino acids, 1 ng/$m\ell$ bFGF, 10 $\mu$M forskolin, 1500 U/$m\ell$ human recombinant leukemia inhibitory factor (LIF). Over a period 3∼5 days, GSCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells. After 5th passages, cells within the colonies continued to be alkaline phosphatase and Oct-4 positive and tested positive against a panel of two immunological markers(Integrin $\alpha$ 6 and Integrin $\beta$ 1) that have been recognized generally to characterize GSCs. SSEA-1, SSEA-3, and SSEA-4 also showed positive signals. Based on our data, these GSCs-derived cultures meet the criteria for GSCs itself and even other pluripotent stem cells. We reported here the establishment of in vitro cultures from mouse male GSCs.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.5
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• pp.2291-2295
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• 2014

Although nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis and expression has been observed in some types of human cancer and stem cells, the molecular mechanisms involved in mediation of cell proliferation and cell cycling remains largely elusive. The aim of the present study was to evaluate NS as a potential target for gene therapy of human breast carcinoma by investigating NS gene expression and its effects on SKBR-3 cell proliferation and apoptosis. NS mRNA and protein were both found to be highly expressed in all detected cancer cell lines. The apoptotic rate of the pcDNA3.1-NS-Silencer group ($12.1-15.4{\pm}3.8%$) was significantly higher than those of pcDNA3.1-NS ($7.2-12.0{\pm}1.7%$) and non-transfection groups ($4.1-6.5{\pm}1.8%$, P<0.01). MTT assays showed the knockdown of NS expression reduced the proliferation rate of SKBR-3 cells significantly. Matrigel invasion and wound healing assays indicated that the number of invading cells was significantly decreased in the pcDNA3.1-NS-siRNA group (P<0.01), but there were no significant difference between non-transfected and over-expression groups (P>0.05). Moreover, RNAi-mediated NS down-regulation induced SKBR-3 cell G1 phase arrest, inhibited cell proliferation, and promoted p53 pathway-mediated cell apoptosis in SKBR-3 cells. NS might thus be an important regulator in the G2/M check point of cell cycle, blocking SKBR-3 cell progression through the G1/S phase. On the whole, these results suggest NS might be a tumor suppressor and important therapeutic target in human cancers.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.4
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• pp.247-265
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• 2008

Objectives: Many types of liver diseases can damage regenerative potential of mature hepatocytes, hepatic progenitor cells or oval cells. In such cases, a stem cell-based therapy can be an alternative therapeutic option. We examined whether human amnion-derived mesenchymal stem cells (HAM) and human umbilical cord-derived stem cells (HUC) could differentiate into hepatocyte-like cells as therapeutic cells for the liver diseases. Methods: HAM and HUC were isolated from the amnion and umbilical cord of the volunteers after a caesarean section with informed consent. In order to differentiate these cells into hepatocyte-like cells, cells were cultivated in hepatogenic medium using culture plates coated with fibronectin. Effects of hepatocyte growth factor, L-ascorbic acid 2-phosphate, insulin premixture fibroblast growth gactor 4, dimethylsulfoxide, oncostatin M and/or dexamethasone were examined on the hepatic differentiation. After differentiation, the cells were analyzed by RT-PCR, immunocytochemistry, immunoblotting, albumin ELISA, urea assay and periodic acid-schiffs staining. Results: Initial fibroblast-like appearance of HAM and HUC changed to a round shape during culture in the hepatogenic medium. However, in all hepatogenic conditions examined, HUC secreted more amounts of albumin or urea into medium than HAM. Expression of some of hepatocyte-specific genes increased and expression of new genes were observed in HUC following cultivation in hepatogenic medium. Results of immunocytochemistry and immunoblotting analyses demonstrated that HUC secreted albumin into the culture medium. PAS staining further demonstrated that HUC could store glycogen inside of the cells. Conclusions: Both HUC and HAM could differentiate into albumin-secreting, hepatocyte-like cells. Under the same hepatogenic conditions examined, HUC more efficiently differentiated into hepatocyte-like cells compared with the HAM. The results suggest that HUC and HAM could be used as sources of stem cells for the cell-based therapeutics such as in liver diseases.

• Journal of Applied Biological Chemistry
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• v.43 no.2
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• pp.69-73
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• 2000

In order to understand the biological role of calmodulin in plants, transgenic plants expressing a mutant calmodulin (VU-4, Iys to ile-115) have been analyzed. We found that tobacco plants expressing VU-4 calmodulin have approximately twofold higher $\gamma$-aminobutyric acid (GABA) levels than the control plants. Cell suspension cultures established from the stem explants of the transgenic tobacco seedlings also have higher levels of GABA than the control cell cultures. Specific activity of glutamate decarboxylase (GAD), which catalyzes the decarboxylation of glutamate to $CO_2$ and GABA, of the transgenic tobacco cell extracts was about twofold higher than the activity of the control cell extracts. Western-blot analysis showed that the GAD is highly expressed in the transgenic tobacco plants. GAD partially purified from tobacco cell extracts showed approximately threefold $Ca^{2+}$/calmodulin-dependent activation. These data suggest that GABA synthesis in the transgenic tobacco plants is elevated, possibly due to higher levels of the calmodulin-dependent GAD enzyme and/or as a result of enhanced activation due to increased levels of the foreign calmodulin.

• Korean Journal of Veterinary Pathology
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• v.2 no.1
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• pp.37-46
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• 1998

It has been known that $\gamma$-irradiation usually induces cell death in regenerating stem cell in normal tissues like skin, intestine and hematopoietic organ. The experiment were carried out to evaluate the early response of radiation injury in radiosensitive and intermediate radiosensitive tissues in feeding and starving rats with the doses of 3.5 and 7.0 Gy. The results of the study showed that the histological phenomenon was apoptosis in the doses of the radiation as the early response of tissue injury. Apoptosis were showed organ-specific and cellular specific responses suggesting that the selection of apoptosis be exactly focused on highly renewal organs and cells. It was interesting that the rats starved for 72 hours prior to irradiation induced less apoptosis in liver than fed rats. As for cellular responses it appeared that apoptotic cells were mostly distributed in ductal or periportal cells in liver of feeding rats unlikely in liver of Starving rots which showed no difference in zonal distribution. In salivary gland apoptotic cells in fed rats were highly induced in intercalating and ductal cell population than in acinar cell population although unlikely in starved rats. This study showed the value of apoptosis using the detection system of TUNEL for evaluating cellular damage after radiation injury and the diminished effect of starvation on cell damage after ionizing irradiation.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.101-101
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• 2003

• Natural Product Sciences
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• v.11 no.4
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• pp.220-224
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• 2005

Extensive chemical studies on the stem bark extracts of two Guttifereous plants namely Mesua daphnifolia and Garcinia nitida have led to the isolation of eight xanthones. Mesua daphnifolia gave cudraxanthone G (1), ananixanthone (2), 1,3,5-trihydroxy-4-methoxyxanthone (3) and euxanthone (4) while Garcinia nitida gave inophyllin B (5), 1,3,7-trihydroxy-2,4-bis (3-methylbut-2-enyl)xanthone (6), 3-isomangostin (7) and rubraxanthone (8). All these compounds were assayed against the MDA-MB-231 (human estrogen receptor negative breast cancer) cells. A structure-activity relationship study showed that structurally, all the 1, 3-oxygenated xanthones which carried unsaturated prenyl side chains (either 3-methylbut-2-enyl or 1,1-dimethyl-2-propenyl) at carbones C-2 and C-4 in the xanthone ring A are essential for the outstanding activities in the assay.

• Journal of Life Science
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• v.27 no.3
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• pp.370-381
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• 2017

Protein dephosphorylation is important for cellular regulation, which is catalyzed by protein phosphatases. Among protein phosphatases, carboxy-terminal domain (CTD) phosphatases are recently emerging and new functional roles of them have been revealed. There are 7 CTD phosphatases in human genome, which are composed of CTD phosphatase 1 (CTDP1), CTD small phosphatase 1 (CTDSP1), CTD small phosphatase 2 (CTDSP2), CTD small phosphatase-like (CTDSPL), CTD small phosphatase-like 2 (CTDSPL2), CTD nuclear envelope phosphatase (CTDNEP1), and ubiquitin-like domain containing CTD phosphatase 1 (UBLCP1). CTDP1 dephosphorylates the second phosphor-serine of CTD of RNA polymerase II (RNAPII), while CTDSP1, STDSP2, and CTDSPL dephosphorylate the fifth phosphor-serine of CTD of RNAPII. In addition, CTDSP1 dephosphorylates new substrates such as mothers against decapentaplegic homologs (SMADs), cell division cycle-associated protein 3 (CDCA3), Twist1, tumor-suppressor protein promyelocytic leukemia (PML), and c-Myc. CTDP1 is related to RNA polymerase II complex recycling, mitosis regulation and cancer cell growth. CTDSP1, CTDSP2 and CTDSPL are related to transcription factor recruitment, tumor suppressor function and stem cell differentiation. CTDNEP1 dephosphorylates LIPIN1 and is related to neural tube formation and nuclear envelope formation. CTDSPL2 is related to hematopoietic stem cell differentiation. UBLCP1 dephosphorylates 26S proteasome and is related to nuclear proteasome regulation. In conclusion, noble roles of CTD phosphatases are emerging through recent researches and this review is intended to summarize emerging roles of CTD phosphatases.

• Biomolecules & Therapeutics
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• v.18 no.2
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• pp.178-183
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• 2010

Costunolide is an active compound isolated from the stem bark of Magnolia sieboldii, and is considered a potential therapeutic for the treatment of various cancers. In this study, we investigated the underlying mechanism whereby costunolide induces the apoptosis of human leukemia cells. Using apoptosis analysis and quantitative reverse transcription-polymerase chain reaction (RT-PCR) results obtained during this study show that costunolide is a potent inducer of apoptosis and that it is triggered due to the premature activation of Cdc2. $G_1$-synchronized cells, which cannot undergo mitosis, were found to be more sensitive to costunolide, and Cdc2 mRNA levels were increased by costunolide treatment. Furthermore, the Cdk inhibitors, olomucine and butyrolactone I, were found to suppress costunolide-induced apoptosis. In addition, the PKC activator TPA rescued cells from cell death by costunolide, and this was prevented by the PKC inhibitor staurosporin. The present study suggests that costunolide induces the apoptosis of HL-60 leukemic cells by modulating cyclin-dependent kinase Cdc2.