• Journal of Embryo Transfer
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• v.10 no.1
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• pp.73-82
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• 1995

large scale production of cloned embryos requires the technology of multiple generation nuclear transplantation(NT) using NT embryos as the subsequent donor nuclei. The purposes of this study were producing the second generation cloned rabbit embryos, and also to determine the electrofusion rate and in vitro developmental potential comparatively in the cloned embryos of the first and second NT generation. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gi /S transition of 32-cell stage. The first generation NT embryos which were developed to 8-cell were synchronized in Gi /S transition phase of the following 16-cell stage and used as donor nuclei for second generation Synchronization of the cell cycle of blastomeres was induced, first, using an inhibitor of microtuble polymerization, colcemid for 10 hours to arrest blastomeres in M phase, and secondly, using a DNA synthesis inhibitor, aphidicolin for 1.5 to 2 hours to arrest them in Gi /S transition boundary. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 14 hours after hCG injection. The separated donor blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.25 kV /cm in 0.28 M rnannitol solution The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. Following in vitro culture of the first and second generation cloned embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The results obtained were summarized as follows: 1. The electrofusion rate was found to be similar as 79.4 and 91.5% in the first and second generation NT rabbit embryos, respectively. 2. The in vitro developmental potential to blastocyst stage of the second generation NT embryos (23.3%) was found significantly(p<0.05) lower, compared with that of the first generation NT embryos (56.8%). 3. The mean blastomeres counts of embryos developed to blastosyst stage following in vitro culture for 120 hours and also their daily cell cycles during the culture period were decreased significantly (p<0.05) to 104.3 cells and 1.33 cylces in the second NT generation, compoared with 210.4 cells and 1.54 cycles in the first NT generation, respectively.

• The Korean Journal of Physiology
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• v.8 no.1
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• pp.15-22
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• 1974

The influence, upon male rat's mating behavior, of Korean Panax Ginseng administered for 3 and 5 days was investigated by direct behavioral observation and also by counting the number of copulation plugs the animals deposited. Four쇼-four male albino rats were used. Part of the animals received ginseng for 3 days (ginseng-3 day group, N= 12) or for 5 days(ginseng-5 day group, H=10), while the remaining animals received saline for 3 days (saline-3 day group, N=12) or for 5 days (saline-S day group, N=10). Each animal belonging to the 2 ginseng groups received subcutaneously 0.5 ml/100 g body weight of ginseng alcohol extract solution (4 mg of the ethyl alcohol extract in 1 ml of physiological saline), and each rat belonging to the 2 saline groups received the same amount of saline per day. During the dark period of the light-dark cycle on the next day following the last drug administration, a female rat in the artificial estrus was introduced to each male and the mating behavior was observed for 45 minutes. The observation session was divided into two parts and, in the early part which terminated with the first ejaculation and succeeding intromission, following behavioral measures were taken: mounting latency, intromission latency, inter-intromission period, ejaculatory latency(time from the first intromission until the first ejaculation), occurrence of mounting with intromission, occurrence of mounting without intromission, and postejaculatory interval. Behavioral measures taken in the later part of the session after the first ejaculation were: occurrence of mounting with intromission, occurrence of mounting without intromission, and occurrence of ejaculation. Immediately after the behavioral observation session the experiment turned to measure, for 10 days, the number of copulation plug which each pair of rats deposited. Following results were obtained: 1. After several mountings mounting with intromission, males of the 2 ginseng groups finished the first ejaculation significantly earlier than the corresponding 2 saline groups did. 2. The postejaculatory latency was significantly reduced in the ginseng-5 day group compare with the value of the saline-5 day group and also compared with the value of the ginseng-3 day group. 3. The 2 ginseng groups ejaculated significantly more often in 45 minutes' observation session than the corresponding 2 saline groups did. 4. The number of copulation plug deposited in 10 days by the animals of the 2 ginseng groups. significantly exceeded the number deposited by the corresponding 2 saline group animals. The animals of the ginseng-5 day group deposited copulation plugs significantly more than the animals of the ginseng-3 day group did. It is inferred from the above results that the ginseng facilitates mating behavior of male rats, and that the degree of facilitation may be influenced by the duration of drug administration.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.1
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• pp.125-131
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• 2015

Currently, taxol is mainly extracted from the bark of yews; however, this method can not meet its increasing demand on the market because yews grow very slowly and are a rare and endangered species belonging to first-level conservation plants. Recently, increasing efforts have been made to develop alternative means of taxol production; microbe fermentation would be a very promising method to increase the production scale of taxol. To determine the activities of the taxol extracted from endophytic fungus N. sylviforme HDFS4-26 in inhibiting the growth and causing the apoptosis of cancer cells, on comparison with the taxol extracted from the bark of yew, we used cellular morphology, cell counting kit (CCK-8) assay, staining (HO33258/PI and Giemsa), DNA agarose gel electrophoresis and flow cytometry (FCM) analyses to determine the apoptosis status of breast cancer MCF-7 cells, cervical cancer HeLa cells and ovarian cancer HO8910 cells. Our results showed that the fungal taxol inhibited the growth of MCF-7, HeLa and HO8910 cells in a dose-and time-dependent manner. IC50 values of fungal taxol for HeLa, MCF-7 and HO8910 cells were $0.1-1.0{\mu}g/ml$, $0.001-0.01{\mu}g/ml$ and $0.01-0.1{\mu}g/ml$, respectively. The fungal taxol induced these tumor cells to undergo apoptosis with typical apoptotic characteristics, including morphological changes for chromatin condensation, chromatin crescent formation, nucleus fragmentation, apoptotic body formation and G2/M cell cycle arrest. The fungal taxol at the $0.01-1.0{\mu}g/ml$ had significant effects of inducing apoptosis between 24-48 h, which was the same as that of taxol extracted from yews. This study offers important information and a new resource for the production of an important anticancer drug by endofungus fermentation.

• Journal of the Korean Society of Food Science and Nutrition
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• v.34 no.9
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• pp.1443-1449
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• 2005

Quality characteristics and functional properties of Korean wheat noodle with brassica oleracea L. powder were investigated to develop health promoting and high quality product. Brassica oleracea L. powder was extracted with water and $70\%$ ethanol, and the extracts were tested its electron donating ability (EDA) and nitrite scavenging ability (NSA). Quality characteristics of the noodle were evaluated by its color, flavor, moisture, softness, texture and taste evaluation. Microbiological Quality was also tested counting total viable cells. EDA was highest at 1000 ppm of both water extract ($64\%$ of EDA) and ethanol extract ($76\%$ of EDA). NSA was highest pH 1.2 of both water extract ($42\%$ of NSA) and ethanol extract ($46\%$ of NSA). In antimicrobial activity test, Korean wheat noodle with $3\%$ pine pollen powder displayed $0.5{\~}1$ log cycle of total viable cell counts lower than that of control at 5 days of storage. Sensory evaluation of Korean wheat dried and cooked noodles with $3\%$ Brassica oleracea L. powder showed significantly higher scores in overall.

• Journal of Life Science
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• v.19 no.6
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• pp.799-803
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• 2009

The killing of male germ cells by radiation and other toxicants has recently been attributed to apoptosis, but a critical evaluation of the presence of the different features of apoptosis in each epithelial stage has not been performed. In this study, mouse testes exposed to radiation were examined by light microscopy and terminal transferase-mediated end labeling (TUNEL) with periodic acid-Schiff (PAS) stains to determine whether the cells were apoptotic according to several criteria. Apoptosis was easily recognized by the presence of peroxidase-stained, entirely apoptotic bodies. In the TUNEL-positive cells or bodies, the stained products correlated precisely with the typical morphologic characteristics of apoptosis as seen at the light microscopic level. The changes that occurred from 0 to 24 hours after exposing the mice to 2 Gy of gamma-rays (2 Gy/min) were examined. The numbers of apoptotic cells reached a peak at 12 hours after irradiation and then declined. The mice that received 0-8 Gy of gamma-rays were examined 8 hours after irradiation. Dose-response relationships were generated for each stage of the epithelial cycle by counting TUNEL-positive cells. The dose-response curves were linear- quadratic [y=(-0.014${\pm}$0.009)$D^{2}$+(0.31${\pm}$0.697)D+0.3575. Where y=the number of apoptotic cells per seminiferous tubule, and D=the irradiation dose in Gy, $r^{2}$=0.9] and there was a significant relationship between the frequency of apoptotic cells and the radiation dose. Although the maximum response was produced by 8 Gy, even 0.5 Gy induced marked changes. These changes were most pronounced in B spermatogonia of stage V and the spermatocyte at the mitotic cells of stage XII.

• Journal of Veterinary Clinics
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• v.12 no.1
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• pp.877-886
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• 1995

The recycling nuclear transplantation(NT) technique has the powerful potential of producing a large number of genetically identical embryos and offsprings from one embryo. Multiple generational cloning by this technique utilizes the NT embryo itself as the donor for the next generation of cloning. In this experiment, we have produced the third generational cloned embryos by recycling NT. Further we examined comparatively the electrofusion rate and in vitro developmental potential in the cloned embryos of the first second and third generations. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulberco's phosphate buffered saline containing 10 % fetal calf serum(FCS) at 47 hours after hCG injection. In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gl/S transition of 32-cell stage. The first and second generation NT embryos developed to 16-cell were used as donor nuclei for second and third generation. The recipient cytoplasms were utilized the oocytes collected at 14 hours after hCG injection, following revoming the nucleus and the first polar body by micromanipulation. The separated blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were fused by electrical stimulation. The electrofusion rate was seen to be 78.0, 88.0 and 90.3 % in the first second and third generation NT rabbit embryos, respectively. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10 % FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The in vitro developmental potential to blastocyst stage was significantly(P<0.05) decreased in the third(7.2 %) generation NT embryos compared to the first(53.1 %) and second(16.1 %) generation NT embryos. Following in vitro development to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The mean blastomere numbers and cell cycle numbers of NT embryos during the culture period were significantly(p<0.05) decreased in the second(93.9 cells and 6.55 cylces) and third(81.5 cells and 1.35 cylces) generation, compared to the first(189.9 cells and 7.55 cylces) generation.

• Animal Bioscience
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• v.34 no.6
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• pp.957-966
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• 2021

Objective: Ovarian follicular development, which dependent on the proliferation and differentiation of granulosa cells (GCs), is a complex biological process in which miRNA plays an important role. Our previous study showed that miR-458b-5p is associated with ovarian follicular development in chicken. The detailed function and molecular mechanism of miR-458b-5p in GCs is unclear. Methods: The luciferase reporter assay was used to verify the targeting relationship between miR-458b-5p and catenin beta-1 (CTNNB1), which is an important transcriptional regulatory factor of the Wnt/β-catenin pathway. The cell counting kit-8 (CCK-8) assay, flow cytometry with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC) labeling were applied to explore the effect of miR-458b-5p on proliferation, cell cycle and apoptosis of chicken GCs. Quantitative real-time polymerase chain reaction and Western blot were used to detect the mRNA and protein expression levels. Results: We demonstrated that the expression of miR-458b-5p and CTNNB1 showed the opposite relationship in GCs and theca cells of hierarchical follicles. The luciferase reporter assay confirmed that CTNNB1 is the direct target of miR-458b-5p. Using CCK-8 assay and flow cytometry with PI and Annexin V-FITC labeling, we observed that transfection with the miR-458b-5p mimics significantly reduced proliferation and has no effects on apoptosis of chicken GCs. In addition, miR-458b-5p decreased the mRNA and protein expression of CD44 molecule and matrix metallopeptidase 7, which are the downstream effectors of CTNNB1 in Wnt/β-Catenin pathway and play functional roles in cell proliferation. Conclusion: Taken together, the data indicate that miR-458b-5p regulates ovarian GCs proliferation through Wnt/β-catenin signaling pathway by targeting CTNNB1, suggesting that miR-458b-5p and its target gene CTNNB1 may potentially play a role in chicken ovarian follicular development.

• Journal of Nuclear Fuel Cycle and Waste Technology(JNFCWT)
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• v.16 no.4
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• pp.397-410
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• 2018

When considering the long-term safety assessment of spent-nuclear fuel management, americium is one of the most radio-toxic actinides. Although spectroscopic methods are widely used for the study of actinide chemistry, application of those methods to americium chemistry has been limited. Herein, we purified $^{241}Am$ to obtain a highly pure stock solution required for spectroscopic studies. Quantitative and qualitative analyses of purified $^{241}Am$ were carried out using liquid scintillation counting, and gamma and alpha radiation spectrometry. Highly sensitive absorption spectrometry coupled with a liquid waveguide capillary cell and time-resolved laser fluorescence spectroscopy were employed for the study of Am(III) hydrolysis and oxalate (Ox) complexation. $Am^{3+}$ ions under acidic conditions exhibit maximum absorbance at 503 nm, with a molar absorption coefficient of $424{\pm}8cm^{-1}{\cdot}M^{-1}$. $Am(OH)_3(s)$ colloidal particles formed under near neutral pH conditions were identified by monitoring the absorbance at around 506-507 nm. The formation of ${Am(Ox)_3}^{3-}$ was detected by red-shifts of the absorption and luminescence spectra of 4 and 5 nm, respectively. In addition, considerable enhancements of the luminescence intensities were observed. The luminescence lifetime of ${Am(Ox)_3}^{3-}$ increased from 23 to 56 ns, which indicates that approximately six water molecules are replaced by carboxylate ligands in the inner-sphere of the Am(III). These results suggest that ${Am(Ox)_3}^{3-}$ is formed through the bidentate coordination of the oxalate ligands.

• Molecules and Cells
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• v.42 no.6
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• pp.448-459
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• 2019

The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway is a promising target for gastric cancer (GC) treatment; however the efficacy of PI3K/mTOR dual inhibitors in GC has not yet been maximized. Additionally, the effect of autophagy regulation by PI3K/mTOR dual inhibitors has not been clearly elucidated in GC treatment. We aimed to show that our newly developed PI3K/mTOR dual inhibitor, CMG002, when combined with an autophagy inhibitor, chloroquine (CQ), potently induces effective cancer cell death in Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) cells, where both the PI3K/AKT/mTOR and autophagy pathways play important roles in disease pathogenesis. EBV- and mock-infected AGS and NUGC3 GC cell lines were treated with CMG002 +/- CQ. PI3K/AKT/mTOR signaling pathway mediators, cellular apoptosis and autophagy markers were confirmed by Western blot assay. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. CMG002 effectively blocked the PI3K/AKT/mTOR pathway by markedly decreasing phosphorylation of AKT and its downstream mediator S6. CMG002 induced G0/G1 cell cycle arrest and enhanced apoptotic cell death in AGS and NUGC3 cells, particularly EBV-infected cells compared with mock-infected cells, as confirmed by flow cytometric analyses and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays. The combination of CMG002 plus CQ synergistically increased apoptotic cell death in EBV-infected GC cell lines when compared with CMG002 alone (P < 0.05). Our results suggest that the new PI3K/mTOR dual inhibitor, CMG002, when used in combination with the autophagy inhibitor, CQ, provides enhanced therapeutic efficacy against EBVaGC.

• Animal Bioscience
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• v.36 no.9
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• pp.1336-1349
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• 2023

Objective: The study was conducted to screen differentially expressed miRNAs in sows at early pregnancy by high-throughput sequencing and explore its mechanism of action on embryo implantation. Methods: The blood serum of pregnant and non-pregnant Landrace×Yorkshire sows were collected 14 days after artificial insemination, and exosomal miRNAs were purified for high throughput miRNA sequencing. The expression patterns of 10 differentially expressed (DE) miRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR quantified the abundance of serum exosomal miR-192 in pregnant and control sows, and the diagnostic power was assessed by receiver operating characteristic (ROC) analysis. The target genes of DE miRNAs were predicted with bioinformatics software, and the functional and pathway enrichment analysis was performed on gene ontology and the Kyoto encyclopedia of genes and genomes terms. Furthermore, a luciferase reporter system was used to identify the target relation between miR-192 and integrin alpha 4 (ITGA4), a gene influencing embryo implantation in pigs. Finally, the expression levels of miRNAs and the target gene ITGA4 were analyzed by qRT-PCR, and western blot, with the proliferation of BeWo cells detected by cell counting kit-8 (CCK-8). Results: A total of 221 known miRNAs were detected in the libraries of the pregnant and non-pregnant sows, of which 55 were up-regulated and 67 were down-regulated in the pregnant individuals compared with the non-pregnant controls. From these, the expression patterns of 10 DE miRNAs were validated. The qRT-PCR analysis further confirmed a significantly higher expression of miR-192 in the serum exosomes extracted from pregnant sows, when compared to controls. The ROC analysis revealed that miR-192 provided excellent diagnostic accuracy for pregnancy (area under the ROC curve [AUC]=0.843; p>0.001). The dual-luciferase reporter assay indicated that miR-192 directly targeted ITGA4. The protein expression of ITGA4 was reduced in cells that overexpressed miR-192. Overexpression of miR-192 resulted in the decreased proliferation of BeWo cells and regulated the expression of cell cycle-related genes. Conclusion: Serum exosomal miR-192 could serve as a potential biomarker for early pregnancy in pigs. miR-192 targeted ITGA4 gene directly, and miR-192 can regulate cellular proliferation.