• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.365-373
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• 2000

The purpose of this study was to investigate the effects of the fusion pulses and fusion media on fusion rate and the development of embryos produced by somatic cell nuclear transfer in Hanwoo (Korean cattle). Nuclear donor cumulus and fetal fibroblast cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at 38.5$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. The cumulus cell and cytoplast were fused using one pulse of 70 volts for 40$mutextrm{s}$, two pulses of 70 volts for 40$mutextrm{s}$ and one pulse of 180 volts for 15$mutextrm{s}$. The fetal fibroblast cell and cytoplast were fused using one pulse of 180 volts for 15$mutextrm{s}$ or 30$mutextrm{s}$. The cumulus cell and cytoplast were fused using mannitol and Zimmerman cell fusion medium (ZCFM) as a fusion medium. The fused embryos were activated after the fusion with 10 $\mu$M calcium ionophore for 5 min and 2 mM 6-dimethyl- aminopurine for 3 h. The nuclear transfer embryos were cultured in 500 ${mu}ell$ well of modified CR1aa supplemented with 3 mg/$m\ell$ BSA in th $\varepsilon$ four well dish cove red with mineral oil. After 3 days culture, culture medium was changed into modified CRlaa medium containing 1.5 mg/$m\ell$ BSA and 5% FBS for 4 days. The incubation environment was 5% $CO_2$, 5% $O_2$, 90% $N_2$ at 38.5$^{\circ}C$. When the cumulus cells were fused with enucleated oocytes by three different fusion pulses, one pulse of 180 volts for 15 $mutextrm{s}$ yielded the highest fusion rate and developmental rate to blastocyst among the pulses (P＜0.05). When the fetal fibroblast cells were fused with enucleated oocytes, one pulse of 180 volts for 30$mutextrm{s}$ yielded significantly higher fusion rate compared with that for 15 $mutextrm{s}$(P＜0.05). The present result indicates that the fusion rate between karyoplast and cytoplast was affected by the cell type and the optimal fusion condition was different according to cell type or size. When the fusion was conducted by the use of mannitol and ZCFM, the fusion rate was 71.2% and 65.8%, respectively. The developmental rates to blastocyst were 37.8% and 39.8%, respectively. There was no significant difference between two fusion media in the developmental rate of cumulus cell nuclear transfer embryos. These results indicate that optimal electric current should be selected according to cell type.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.1
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• pp.49-60
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• 2008

Objective: This study was carried out to investigate the effects of 3-dimensional co-culture of human endometrial cells decidualized with progesterone and TGF-${\beta}1$ on the development of 2-cell mouse embryos. Methods: Stromal and epithelial cells isolated from human endometrial tissue were immunostained for cytokeratin and vimentin. Expression of TGF-${\beta}1$, its receptor-1, -2, integrin-${\beta}3$ and prolactin in mono or co-culture according to three different hormone conditions was investigated by RT-PCR. Differential staining was used to investigate the number of ICM and trophectoderm of hatched mouse blastocysts in different three conditions. Results: The immunohistochemical study was positive for cytokeratin or vimentin and confirmed that epithelial and stromal cells were isolated from endometrial tissue successfully. In co-culture, TGF-${\beta}1$, its receptor-1, integrin-${\beta}3$ and prolactin except TGF-${\beta}1$-r2 were expressed in progesterone dominant condition. The hatching and attaching rate were higher in the co-culture with decidualized cells (p<0.05). However, we observed that lots of the incomplete hatched blactocysts attached on non-decidualized cells. The ICM number of hatched mouse blastocysts was higher in co-culture with decidualized and non decidualized cells than media only culture (p<0.05). The trophectoderm number of hatched blastocyst was higher in the co-culture with decidualized cells than non-decidualized cells or media only culture (p<0.05). Conclusion: The administration of progesterone, estrogen and TGF-$\beta$ could induce decidualization of stromal and epithelial cells isolated from human endometrial tissue using 3-dimensional co-culture, and the decidualization of human endometrial cells could increase the hatching and attaching rate of 2-cell mouse embryos.

• Journal of Food Hygiene and Safety
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• v.13 no.2
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• pp.106-111
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• 1998

This study has been focused on both estrogenic and proliferating activity of genistein (GEN) and bisphenol A (BPA). GEN and BPA enhance the proliferation of estrogen-dependent MCF-7 human breast cancer cells at concentrations as low as 100 nM of GEN and 8 ng/ml of BP A achieving similar effect to that of estradiol at 1 nM. Expression of the estrogen responsive gene, pS2 was also induced in MCF-7 cells by treatment with genistein at dose as low as 1 nM and BPA at dose as low as 4 ng/ml. Using 21 day-old ovariectomized nude mice, we examined end-bud formation and mammary gland development after treatment with bisphenol A or genistein. Compared with untreated control, mammary gland development and end-bud formation were significantly increased in mice fed genistein or bisphenol A (p<0.05). Taken together, it is concluded that GEN and BP A can act as an estrogen agonist resulting in cell proliferation and induction of the estrogen responsive pS2 gene in MCF-7 cells in vitro and in athymic mice in vivo, respectively. Therefore, it is suggested that GEN and BP A might modulate human endocrine system and these compounds might be considered as a endocrine modulator at the low levels of doses.

• Journal of Radiopharmaceuticals and Molecular Probes
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• v.9 no.1
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• pp.9-16
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• 2023

Liposomes as drug delivery system have proved useful carrier for various disease, including cancer. In addition, perfluorocarbon cored microbubbles are utilized in conjunction with high-intensity focused-ultrasound (HIFU) to enable simultaneous diagnosis and treatment. However, microbubbles generally exhibit lower drug loading efficiency, so the need for the development of a novel liposome-based drug delivery material that can efficiently load and deliver drugs to targeted areas via HIFU. This study aims to develop a liposome-based drug delivery material by introducing a substance that can burst liposomes using ultrasound energy and confirm the ability to target tumors using PET imaging. Liposomes (Lipo-DOX, Lipo-DOX-Au, Lipo-DOX-Au-RGD) were synthesized with gold nanoparticles using an avidin-biotin bond, and doxorubicin was mounted inside by pH gradient method. The size distribution was measured by DLS, and encapsulation efficiency of doxorubicin was analyzed by UV-vis spectrometer. The target specificity and cytotoxicity of liposomes were assessed in vitro by glioblastoma U87mg cells to HIFU treatment and analyzed using CCK-8 assay, and fluorescence microscopy at 6-hour intervals for up to 24 hours. For the in vivo study, U87mg model mouse were injected intravenously with 1.48 MBq of ⁶⁴Cu-labeled Lipo-DOX-Au and Lipo-DOX-Au-RGD, and PET images were taken at 0, 2, 4, 8, and 24 hours. As a result, the size of liposomes was 108.3 ± 5.0 nm at Lipo-DOX-Au and 94.1 ± 12.2 nm at Lipo-DOX-Au-RGD, and it was observed that doxorubicin was mounted inside the liposome up to 52%. After 6 hours of HIFU treatment, the viability of U87mg cells treated with Lipo-DOX-Au decreased by around 20% compared to Lipo-DOX, and Lipo-DOX-Au-RGD had a higher uptake rate than Lipo-DOX. In vivo study using PET images, it was confirmed that ⁶⁴Cu-Lipo-DOX-Au-RGD was taken up into the tumor immediately after injection and maintained for up to 4 hours. In this study, drugs released from liposomes-gold nanoparticles via ultrasound and RGD targeting were confirmed by non-invasive imaging. In cell-level experiments, HIFU treatment of gold nanoparticle-coupled liposomes significantly decreased tumor survival, while RGD-liposomes exhibited high tumor targeting and rapid release in vivo imaging. It is expected that the combination of these models with ultrasound is served as an effective drug delivery material with therapeutic outcomes.

• Journal of Embryo Transfer
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• v.22 no.2
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• pp.121-126
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• 2007

The objective of this study was carried out to examine the selection effects of in vitro matured porcine follicular oocytes with polar body extrusion and early cleavage as non-invasive marker to know the developmental competence in advance. The porcine oocytes matured for 48 h were examined the polar body extrusion. The examined oocytes were matured for additional $16{\sim}18h$ and activated with 7% ethanol and cultured in $5{\mu}g/ml$ cytochalasin B for 5 h for diploid formation. The treated oocytes were cultured and examined the cleavage after 48 h and continued culturing for 5 days. The oocytes of 21.9% were discarded in morphological selection and 32.1% oocytes were discarded by failure of first polar body extrusion. The selected oocytes were matured and activated and then after 48 h the cleavage rates were examined. In morphologically selected oocytes, 15.8% oocytes were not cleaved and 52.6% oocytes were normally cleaved and 31.6% oocytes were hyper-cleaved over 8-cell stage. However in the first polar body extruded oocytes, 7.1% oocytes were not cleaved and 73.1% oocytes were normally cleaved and 19.8% oocytes were hyper-cleaved. The morphologically selected embryos that not cleavage-selected were developed in 16.7% up to blastocyst and the morphologically selected and cleavage-selected embryos were developed in 31.7%. The polar body extruded oocytes that were not carried out cleavage selection were developed in 39.0% and the polar body extruded and cleavage-selected embryos were developed 49.0%. The first cleavage timing was examined with 12 h interval after activation. In $0{\sim}12,\;12{\sim}24,\;24{\sim}36,\;and\;36{\sim}48h$ intervals, 4.1%, 68.6%, 19.1%, and 2.3% oocytes were cleaved and 5.9% oocytes were not cleaved until 48 after activation. The cleaved oocytes in each interval were cultured and developed upto blastocyst with 0, 39.1, 9.5, and 0%, respectively. This results suggests that polar body extruded and cleaved at $12{\sim}36h$ embryo has higher developmental potential than the others.

• Journal of Life Science
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• v.27 no.12
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• pp.1445-1451
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• 2017

Mitochondria play a central role in energy generation by using electron transport coupled with oxidative phosphorylation. They also participate in other important cellular functions including metabolism, apoptosis, signaling, and reactive oxygen species production. Therefore, mitochondrial dysfunction is known to contribute to a variety of human diseases. Furthermore, there are various inherited diseases of energy metabolism due to mitochondrial DNA (mtDNA) mutations. Unfortunately, therapeutic options for these inherited mtDNA diseases are extremely limited. In this regard, mitochondrial replacement techniques are taking on increased importance in developing a clinical approach to inherited mtDNA diseases. In this study, green fluorescence protein (GFP)-tagged mitochondria were isolated by differential centrifugation from a mammalian cell line. Using microinjection technique, the isolated GFP-tagged mitochondria were then transferred to bovine oocytes that were triggered for early development. During the early developmental period from bovine oocytes to blastocysts, the transferred mitochondria were observed using fluorescent microscopy. The microinjected mitochondria were dispersed rapidly into the cytoplasm of oocytes and were passed down to subsequent cells of 2-cell, 4-cell, 8-cell, morula, and blastocyst stages. Together, these results demonstrate a successful in vitro transfer of isolated mitochondria to oocytes and provide a model for mitochondrial replacement implicated in inherited mtDNA diseases and animal cloning.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.171-178
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• 2003

This study was conducted to investigate the effect of electric fusion methods on cell fusion rate and embryo development by somatic cell nuclear transfer in Korean Native Cattle. The KNC ear cell was cultured in vitro for confluence in serum starvation condition(DMEM＋0.05％ FBS) for cell confluence. The zona pellucida of IVM oocytes were partially dissection using micro pipette. Ear cells were transferred into an enucleated oocyte. The reconstructed embryos were electrically fused with Zimmermann Cell Fusion Medium(ZCFM). Nuclear transfer embryos were activated with a combination of 10${\mu}{\textrm}{m}$ calcium ionophore(5 min) and 2.0mM 6-DMAP(3 hr). The activated embryos were cultured in CR1 -aa medium contains 0.3％ BSA or 10％ FBS at 37$^{\circ}C$, 90％ $N_2$, and 5％ $CO_2$in incubator for 6 days. The fusion rates were 51.6％(chamber) and 68.9％(needle), respectively and there were significantly difference between the fusion method(P<0.05). But, lysis rates were not significantly different(10.7％, 11.5％), respectively. The cleavage rates were significantly different between the chamber method(73.2％) and needle method(80.3％), respectively(P<0.05). The rates of early embryos(2∼4cells) and blastocysts of chamber and needle methods were 54.1％, 61.1％ and 18.4％, 26.3％ respectively, and needle method was significantly higher than chamber method(P<0.05). But, morulae formation rate were not significantly differences between the chamber(6.7％) and needle(6.2) method(P <0.05). These result suggest that electric fusion of needle method was to be profitable for nuclear transfer embryo fusion rate, blastocyst formation rate and reduce of oocyte lysis.

• Journal of Embryo Transfer
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• v.25 no.1
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• pp.15-20
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• 2010

This study was performed to identify the optimal timing for oocyte donor replacement during OPU procedure. OPU was carried out to collect oocytes from every donor at an interval of $3{\sim}4$ days (2 times a week). The collected oocytes were matured in vitro in TCM-199 supplemented with 10% FBS, 10 mg/ml of FSH and 1 mg/ml of estradiol for 24 h. After 24 h of exposure to sperm, the presumptive zygotes were cultured in CR1aa medium supplemented with 4 mg/ml of BSA for 3 days before being changed to CR1aa medium with 10% of FBS for another $3{\sim}4$ days. The mean numbers of retrieved oocytes were remained constantly up to 3 months ($6.0{\pm}0.5$, $6.2{\pm}0.7$, $5.2{\pm}0.6$), but significantly decreased at over 4 to 6 months ($3.7{\pm}0.5$, $2.8{\pm}0.4$, $1.2{\pm}0.2$) (p<0.05). The blastocyst development potential was also very similar rate from 1 to 3 months (37.2%, 40.4% and 44.6%), but significantly decreased from 4 to 6 months (24.8%, 29.3% and 28.6%, respectively) (p<0.05). The production of OPU derived embryos in periods of 1 to 3 months ($2.2{\pm}0.3$, $2.5{\pm}0.3$ and $2.3{\pm}0.4$) were significantly higher than those in 4 to 6 months ($0.9{\pm}0.2$, $0.8{\pm}0.2$ and $0.3{\pm}0.2$, respectively) (p<0.05). In conclusion, the efficient periods for the production of OPU derived embryos was until 4 months, twice per week to produce over 64 transferable embryos and then replace new donor after 3 months use. The best replacement time is 3 months and could be maximized production of OPU derived embryos.

• Journal of the Korean Society of Food Science and Nutrition
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• v.44 no.10
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• pp.1439-1449
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• 2015

Prunus persica Flos (PPF) were investigated for their antioxidant and anti-inflammatory activities to find a natural functional food resource preventing degenerative diseases associated with excessive oxidative stress and chronic inflammation. PPF was extracted using ethanol (EtOH) and then sequentially fractioned by hexane (Hx), dichloromethane (DM), ethyl acetate (EA), n-butanol (BtOH), and water (DW). Contents of total phenolics and flavonoids, as well as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities were measured. Anti-inflammatory effects in terms of nitric oxide (NO), prostaglandin (PG) E2, and pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-${\alpha}$ production were also measured using LPS-treated RAW 264.7 macrophages. EtOH extract showed relatively high antioxidant activity with high total phenolic (78.1 mg tannic acid/g) and flavonoid contents (55.3 mg rutin/g). EA fraction contained the highest total phenolic and flavonoid contents (394.6 mg tannic acid/g, 253.7 mg rutin/g), followed by BtOH (128.3 mg tannic acid/g, 93.1 mg rutin/g). EA and BtOH fractions and EtOH extract showed higher DPPH radical and ABTS radical scavenging activities than the others (P<0.05). In LPS-treated RAW 264.7 macrophages, EtOH extract ($200{\mu}g/mL$) showed significantly reduced (P<0.05) NO, PGE2, and TNF-${\alpha}$ production levels to 38.5%, 32.3%, and 48.9% of the control, respectively, as well as reduced iNOS and COX-2 protein expression. DM fraction ($50{\mu}g/mL$) showed significantly reduced (P<0.05) NO, PGE2, IL-6, and TNF-${\alpha}$ production levels to 43.5%, 13.3%, 38.7%, and 41.3% of the control, respectively, and EA fraction ($50{\mu}g/mL$) showed significantly reduced NO, PGE2, IL-6, and TNF-${\alpha}$ production levels to 44.8%, 22.4%, 45.7%, and 62.0% of the control, respectively. Taken together, EtOH extract of PPF showed potent antioxidant and anti-inflammatory activities, and EA and BtOH fractions showed comparatively stronger antioxidant activities while DM and EA fractions showed stronger anti-inflammatory activities. It can be concluded that EtOH extract of PPF and its fractions are good candidates as natural resources for the development of anti-oxidative and anti-inflammatory functional food products.

• The Korean Journal of Pharmacology
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• v.26 no.2
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• pp.219-226
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• 1990

Enalapril maleate tablets of two different producers were tested for bioequivalence. Enalapril is rapidly metabolized to an active metabolite, enalaprilat which inhibits angiotensin-converting enzyme (ACE). The pharmacokinetics of enalapril maleate and the time course of inhibition of plasma ACE activity after administration of the drugs were studied. Two single doses of 10mg each of enalapril maleate were administered orally to twelve male volunteers in a balanced, randomized, two-way crossover investigation. Plasma enalaprilat concentrations were determined over a 23-hour after the dose by enzyme inhibition assay and enalapril by the same method following in vitro hydrolysis. Urinary recoveries of enalapril and enalaprilat were determined for the calculation of renal clearance. Plasma ACE activity was determined by an enzyme assay. Peak plasma levels of enalapril were observed about 1 hour after the doses, and practically all enalapril had disappeared from plasma within 6 hour. Peak enalapril concentrations of both formulations were almost identical ($Vasotec^{\circledR}$, 61.38 ng/ml; $Beartec^{\circledR}$, 64.27 ng/ml). The values of the pharmacokinetic parameters of enalaprilat computed for $Vasotec^{\circledR}$ and $Beartec^{\circledR}$ tablets are presented in that order; area under the curve=330.63:320.96 $ng{\cdot}hr/ml$; peak concentration=38.63:39.43 ng/ml; time to peak=3.83:4.08 hour; elimination half-life=3.95:3.92 hours. No statistically significant difference was detected when area under the curve and all other parameters were compared. Using criteria of 95% confidence interval for the comparison of these parameters, only the upper limits of area under the curve and time to peak of enalapril were over 120%. All the parameters of enalaprilat were acceptable. Percent inhibition of plasma ACE to plasma enalaprilat concentration showed the sigmoid concentration-inhibition relationship. Time courses of plasma ACE inhibition after the administration of both formulations were quite similar. The formulations were found to be equivalent when compared on the premise that no significant difference was detected when pharmacokientic parameters and inhibition of ACE activity were compared, based on the confidence limits analysis.