• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.50-50
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• 1993

최근의 암치료용 제제는 암세포의 분화를 재개시켜서 정상적인 세포로 분화 유도하는 약재개발에 촛점을 맞추고 있으므로 본 연구에서는 F9 EC세포주에 의한 분화인자 스크리닝 시스템을 확립하고 나아가 새로운 분화 유도인자를 스크리닝 하는데 그 목표를 두고 연구를 수행하였다. 즉 F9 EC세포의 세포 배양조건을 먼저 확립하였으며 retinoic acid에 의한 F9 EC세포의 분화유도 시스템을 다음과 같이 확립하였다. 1 단계: F9 EC세포에 스크리닝할 물질의 단독처리 2 단계: 스크리닝할 물질에 dibutyryl cyclic AMP(dbc-AMP)와 theophylline을 첨가하여 처리 3 단계: 분화 marker유전자의 발현조사 그리고 이 시스템을 이용하여 천연 식물 및 해양동식물에서 추출한 성분을 대상으로 하여 새로운 분화인자의 검색을 시도한 결과, saponin, steroid 및 glycolipid계열의 물질이 분화인자로서 효과가 있음이 나타났다.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.33-33
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• 2003

This study was conduct to compare the efficacy to produce male and female somatic cloned piglets. Maturation of porcine COCs was accomplished by incubation in NCSU-23 medium supplemented with 0.6 mM cysteine, 10% porcine follicular fluid, 1mM dibutyryl cyclic adenosine monophosphate (dbc-AMP, Sigma, USA), and 0.1 IU/ml human menopausal gonadotrophin (hMG, Teikokuzoki, Japan) for 20h and then cultured without dbcAMP and hMG for another 18 to 24 h. Female and male fetal cells were isolated from each fetus, cultured in ES-DMEM medium containing 10% FCS. Enucleated oocytes were fused with fetal fibroblasts (passage 4 to 15). Reconstructed embryos were cultured in NCSU-23 with 4 mg/ml BSA under mineral oil at 39$^{\circ}C$ in 5% $CO_2$ in air. A total of 12,328 nuclear-transferred embryos (1- to 4-cell stage) were surgically transferred into 69 surrogate gilts. Three recipients aborted during the period of conception. Three gilts delivered eleven female piglets, and five recipients gave rise to birth 22 male piglets. The average birth weigh of the cloned piglets was 1.52 kg (1.38~1.83 kg) in female piglets and 0.84 kg (0.45~1.25 kg) in male piglets. Alive cloned pigs was seven in female piglets (63.6%) and four in male piglets (18.2%). The other two recipients is ongoing. This study suggests that female-derived fetal cell as a nuclear donor has more capability on production of cloned piglets than male.

• Journal of Ginseng Research
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• v.10 no.2
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• pp.123-132
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• 1986

The pepsinogen secretion was stimulated by the cholecystokinin, caerulein, isoproterenol, and carbachol, respectively. But it was increased slightly and returned to control level by the combiantions of total saponin with each above the agents. Even though the atropine had the inhibition effect, the pepsinogen secretion was recovered to normal level from depressed condition by the combination of the atropine with total saponin. Propranolol showed the same pattern as atropine, too. On the other hand, the pepsinogen secretion was stimulated by the DBcAMP alone, but decreased to control level by the combination with the total saponin. In the case of DBcGMP, the pepsinogen secretion was decreased by itself, but stimulated the above control level by the combination with total saponin. Histamine alone had little effect on the pepsinogen secretion, but when combinated with total saponin, the pepsinogen secretion was increased. Serotonin alone and with total saponin, had no effect respectively, From the above results, the total saponin may have the normalization action stimulating or decreasing the pepsinogen secretion to the control level.

• Journal of Life Science
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• v.8 no.2
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• pp.162-166
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• 1998

The acute myelogeous leukemia cell line, HL-60 is good model to examine leukemia differentiation with nitro blue terazolium reduction assay. We investigated that effect of triterpene acids and ginseng saponin on differentiation of HL-60 cells. Differentiation of HL-60 cells was induced in proportion to ,olar concentration by dibutylyl cAMP, ginseng saponin, lithocholic acid, ginsenoside RH2, and ginsenoside RH3.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.87-94
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• 1996

For evaluating the suitability of human follicular fluid(HFF) as protein supplement in ART, this preliminary study was performed to examine the maturation promoting activity of HFF on mouse follicular oocytes in vitro. Mouse follicular oocytes were collected from ovaries of 21-28 day old ICR mice by puncturing the antral follicles with fine needle at 48 hours after PMSG injection. The oocytes were rinsed and cultured in modified Whittingham's $T_6$ medium containing purines or dbcAMP to maintain meiotic arrest, and different concentrations of HFF were added into the culture medium to examine the effect of HFF on releasing the oocytes from the suppressive influence of the meiotic inhibitors. As a control for HFF, the maturation promoting activity of human fetal cord serum(HFCS) was investigated and compared with the activity of HFF. While HFF was separated, by molecular weight(M.W), into high M.W. fraction(M.W>30,000) and low M.W. fraction(M.W<30,000) and the effects of the fractions on meiotic resumption were investigated in the presence of the meiotic inhibitors. Also hormone analysis was performed to compare the content of hormones in HFF with that in HFCS. Same concentrations of HFF and HFCS induced similar germinal vesicle break down(GVBD) rates of the oocytes meiotic arrested by purines(4mM hypoxanthine+0.75mM adenosine), but the extrusion rate of 1st polar body(PB) of the oocytes cultured in HFF(65.0%, P<0.05) was significantly higher than that(51.6%) in HFCS. While, in the presence of 200 M dbcAMP, the maturation promoting activity of HFF (GVBD: 70.5%, $p<10^{-6}$; 1st PB extrusion: 67.1%, $p<10^{-3}$) was significantly greater than that of HFCS(GVBD: 35.2%; 1st PB extrusion: 41.1%). The oocytes cultured in the fraction of HFF containing high M.W. components showed higher meiotic maturation rates than the oocytes cultured in the low M.W. fraction of HFF. Gonadotropins and $E_2$ were known to improve the completion of maturation changes, and the levels of these hormones were higher in HFF than in HFCS. Therefore, HFF was more effective than HFCS to use for promoting meiotic resumption of mouse oocytes in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.1
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• pp.99-110
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• 1994

One consequence of fertilization in mammals is an increased resistance of the zona pellucida (ZP) to proteases and various chemical reagents. This phenomenon has been called 'zona pellucida hardening' (ZPH), and it is generally accepted that it is caused by the secretory products of cortical granules released by the egg at fertilization. ZP of mouse oocytes maturing in vitro in a chemically defined medium becomes progressively more resistant to solubilization by chymotrypsin ("Spontaneous" ZP hardening). In the present study, it was aimed to find the specificity of spontaneous ZPH in relation to its possible relevance to the cortical reaction and the physiological block to polyspermy. When a maturation inhibitors, cAMP analog(dbcAMP) and phosphodiesterase inhibitor (IBMX) was added to culture medium, it prevent spontaneous ZPH of mouse oocyte during in vitro culture. Thus spontaneous ZPH requires GVBD, since it is prevented by those agents, which inhibit GVBD in vitro. However, culture for 3 hours in the presence of PMA(lOng/ml), a protein kinase C activator, resulted in ZPH without GVBD, thus suggesting that ZPH may be regulated independently apart from the event of GVBD. Pretreatment of mouse oocyte with FBS result in partially inhibitory effect on subsequent spontaneous ZPH. Induction of GVBD in vivo had a inhibitory effect on the spontaneous ZPH, but subsequent spontaneous ZPH. Induction of GVBD in vivo had a inhinbitory effect on the spontaneous ZPII, but had no inhibitory effect on PMA-induced ZPH. Treatment with a microfilament formation blocker(cytochalasin-B) at 1${\mu}g$/ml concentration, resulted in the excellent inhibitory effect on spontaneous ZPH. However cytochalasin-B did not inhibit PMA-induced ZPH. Thus this suggesting that spontaneuse ZPH had a different mechanism from PMA-induced ZPH.

• The Korean Journal of Zoology
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• v.36 no.2
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• pp.277-284
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• 1993

A possible involvement of maturation promoting factor (nfPF) in the two-cell block phenomenon was studied by fusion experiments. Germinal vesicle (GlF) ooeyte was fused with a blastomore from late or blocked 2-cell mouse embryos. and germinal vesicle breakdoum (GVBD) of fused GV oocvtes in the presence of dbcAMP (100$\mu$g/ml) was scored as an index of MPF aniviD. GnD was induced approximately 30% by fusion of a blastomere derived from late 2-cell embryos, but not from blocked 2-cell embryos. The rate of GVBD was changed when GV oocyte was fused with a blastomere from late 2-cell embryos which were treated with u-amanitin, puromvcin or colcemid before and after hsion: Treatment of late 2-cell embryos with puromycin (50 Is/mll but not with u-amanitin (100 Is/ml) clearly inhibited GVBD, indicating that do novo protein synthesis maw be required for the appearance of MPF activity in late 2-cell embryos. Treatment of late 2-cell embryos w기h colcemid (0.1 Is/mll doubled GVBD, presumably due to the maintenance of metaphase or mitotic phase. SDS-PAGE and twoiimensional electrophoresis revealed that there was no difference in protein synthetic pattern in late and blocked 2-cell embryos, but three phosphoproteins with 27, 35 and 46 M)a, presumsblv M-phase components were phosphorylated in late 2-cell embryos but not in blocked 2-cell embryos. It seems then that MPF activity is closely related to phosphorylstion of M-phase components in late 2-cell embryos.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.427-432
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• 1997

These studies were performed to approach the precise pathway inducing the meiotic inhibitory action of hypoxanthine on mouse follicular oocytes and to identify the cause of detrimental effect of hypoxanthine on viability of the oocyte in vitro. In addition, a correlation between the meiotic inhibitory effect and the detrimental effect of hypoxanthine was investigated. Mouse follicular oocytes at germinal vesicle(GV) stage were collected from the ovaries of ICR mice by puncturing the antral follicles with a fine needle, at 48 hours after PMSG injection. Oocytes were cultured in Modified Whittingham's T6 media containing hypoxanthine and several materials that involved in metabolism of hypoxanthine, and the effects of the materials on the actions of hypoxanthine were investigated by observing germinal vesicle breake down (GVBD), 1st polar body (PB) extrusion and viability of the oocytes. Phophodiesterase significantly reduced the meiotic inhibitory effect of dbcAMP but did not influence on the inhibitory effect of hypoxanthine. Allopurinol and 6-MP significantly enhanced the meiotic inhibitory effect of hypoxanthine, but the materials themselves also showed the meiotic inhibitory action like hypoxanthine. Hypoxanthine-guanine phosphoribosyltransferase significantly enhanced the meiotic inhibitory effect of hypoxanthine, on the contrary HGPRT itself promoted meiotic resumption of the oocytes. Catalase did not induce any change in the meiotic inhibitory effect of hypoxanthine, but SOD increased the GVBD rate suppressed by hypoxanthine. The detrimental effect of hypoxanthine on viability of the oocytes was significantly reduced by allopurinol and catalase, but SOD increased the GVBD rate suppressed by hypoxanthine. The detrimental effect of hypoxanthine on viability of the oocytes was significantly reduced by allopurinol and catalase, but SOD did not reduce the deterimental effect of hypoxanthine. In conclusion, the meiotic inhibtory effect of hypoxanthine may be caused by guanyl dervartives converted from hypoxanthine via salvage pathway, and superoxide anion may partially participate in the inhibitory effect of hypoxanthine. The detrimental effect of hypoxanthine on viability of the oocytes be cused by hydrogen peroxide produced during the metabolism of hypoxanthine.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.1
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• pp.1-12
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• 2001

Objective: The present study was done to clarify the effects of nicotine and nicotine tartrate on the mouse oocyte maturation in vitro. Methods: GV (germinal vesicle) oocytes were isolated from Graafian follicle of ovaries with sharp needles under a stereomicroscope from female mouse of ICR strain (4 weeks old). Collected oocytes were cultured for 17 hours at $37^{\circ}C$, 5% $CO_2$ in air and 100% humidified condition in incubator. New MHBS was the basic medium used in which nicotine, nicotine tartrate, and mecamylamine (antagonist of nicotinic acetylcholine receptor) were added depending on the experimental group. GV oocytes were cultured in one of these media. Results: Nicotine ($300{\mu}M{\sim}5mM$) had no effects on GVBD (germinal vesicle breakdown) compared to the control, but increasing concentration of nicotine led to an decrease in the first polar body formation. However, nicotine ($10{\sim}500{\mu}M$) induced GVBD in a dose-dependent manner of GV oocytes in a medium containing dbcAMP. Nicotine tartrate ($50{\mu}M{\sim}5mM$) had no effects on GVBD compared to the control but, increasing concentration of nicotine tartrate led to an decrease in the first polar body formation. Mecamylamine $10{\mu}M$ added to the medium containing nicotine ($300{\mu}M{\sim}5mM$) showed higher percentage of the first polar body formation compared to the nicotine ($300{\mu}M{\sim}5mM$) treatment group. Mecamylamine $10{\mu}M$ added to the medium containing nicotine tartrate ($50{\mu}M{\sim}5mM$) showed higher percentage of the first polar body formation compared to the nicotine tartrate ($50{\mu}M{\sim}5mM$) treatment group. Conclusion: The present study suggest that nicotine and nicotine tartrate have the harmful effects on the meiotic maturation of the mouse oocytes in vitro. However, mecamylamine block harmful effects of nicotine and nictine tartrate.

• Journal of Periodontal and Implant Science
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• v.26 no.2
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• pp.376-395
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• 1996

The neuropeptide substance P(SP) has been recognized to modulate immune systems, with close proximity between peptidergic sensory nerve endings and immune cells. These include the macrophage and neutrophil activation, IL-2 production in T cell, augmentation of Ig synthesis, mast cell degranulation, $PGE_2$ and collagenase secretion in synoviocytes. In this study I examined SP-induced various biological activities such as antimicrobial action, cytokine production, and mast cell degranulation in the presence or absence of other inflammatory cell activators. Antimicrobial studies showed that undifferentiated HL-60 cells were not affected by SP. However, SP significantly enhanced antimicrobial action of TPA-treated or dbcAMP-treated HL-60 cells which had been differentiated into PMN or macrophage/monocyte. I could not find synergistic relationship between SP and LPS in parallel experiments of the above. SP did not induce IL-l production from murine macrophage cell line RAW264.7 whether costimulated with LPS or not. Mast cell degranulation was occured only when stimulated with high dose ($10^{-5}M$) of SP and the degree of this activation was slightly reduced by simultaneous application of $MIP-1{\alpha}$. In addition, CGRP which is known to be a common coexisting neuropeptide with SP within specific fibers did not augment the function of SP on mast cell degranulation. These results suggest that immunoregulatory activities of SP could be mediated through direct upregulation of various functions of immune cells and also upregulation of responsiveness of immune cells to other immune activators.