• 한국수정란이식학회지
• /
• 제10권1호
• /
• pp.65-72
• /
• 1995

The purposes of this study were to produce cloned rabbit embryos and offsprings by nuclear transplantation(NT) using in vitro matured oocytes as nuclear recipient cytoplasm and to determine the effect of frozen nuclei donor embryos on the production efficiency of cloned embryos. The 8cell embryos were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline containing 10% fetal calf serum(FCS) at 40 hours after hGG injection. A portion of collected embryos were preserved at 4$^{\circ}C$ for 24 hours and a portion of them were frozen by vitrification method. The embryos used for donor nuclei were synchronized in the phase of Gi /S transition. The in vitro matured oocytes were used as recipient cytoplasm following removing the nucleus and the first polar body. The synchronized blastomeres from fresh, cooled or frozen embryos were injected into the enucleated oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.0 W /cm in 0.28 M mannitol 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 NT embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The results obtained were summarized as follows: 1. The fusion rates of the blastomeres from fresh, cooled and frozen embryos with the in vitro matured and enucleated oocytes were 100, 95.8 and 64, 3%, respectively. 2. Development in vitro to blastocyst was significantly(p<0.05) different between the cloned embryos with the blastomeres from fresh, cooled or frozen embryos as 39.0, 20. 9 and 15.7%, respectively. 3. The mean numbers of cell cycle per day during in vitro culture of cloned embryos blastomeres from fresh, cooled or frozen embryos was 1.31, 1.29 and 1.16, respectively. 4. A total of 77 nuclear transplant embryos were transferred into 6 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer.

• 한국수정란이식학회지
• /
• 제10권1호
• /
• pp.73-82
• /
• 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
• /
• 제8권1호
• /
• pp.15-22
• /
• 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
• /
• 제16권1호
• /
• pp.125-131
• /
• 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.

• 한국식품영양과학회지
• /
• 제34권9호
• /
• pp.1443-1449
• /
• 2005

본 연구는 품질 특성과 기능성을 가진 우리밀 국수를 개발하기 위하여 적 채 분말을 물과 $70\%$ 에탄올로 추출하여 추출물의 전자공여능 및 아질산염 소거능 등의 기능성을 검토하고 적채를 첨가한 우리밀 국수의 품질특성을 검토하였다. 추출물의 전자공여능은 물 및 에탄을 추출물 모두에서 농도가 증가함에 따라 전자공여능도 증가하였으며 1000 ppm에서 물 추출물은 $64\%$, 에탄을 추출물은 $76\%$로 에탄올 추출물의 전자공여능이 물 추출물보다 높았다. 추출물의 아질산염소거능은 물 및 에탄을 추출물 모두에서 pH가 낮아짐에 따라 증가하여 PH 1.2에서 가장 높았고 동일 pH에서는 농도가 높아짐에 따라 증가하였다. 적채를 첨가한 우리밀 국수 생면을 $20^{\circ}C$에서 일주일간 저장하였을 때 적채첨가량이 증가할수록 총균수 및 진균수는 대조구의 최대균수를 나타낸 저장일의 균수에 비해 유의적으로 낮았다. 우리밀 적채국수의 색도 측정 결과 명도(L)는 생면과 건면의 경우 적채첨가량이 증가할수록 낮아졌으며 적색도(a)는 생면, 건면, 삶은면 모두에서 적채 첨가량이 증가할수록 높아져 $7\%$ 첨가구가 가장 높았고 황색도(b)는 생면과 건면의 경우 적채 첨가량이 증가할수록 낮았다. 삶은면에서는 적색도 감소가 가장 큰 $5\%$ 첨가구에서 황색도 및 명도가 가장 높았다. 적채를 첨가한 우리밀 건면과 삶은면의 관능검사 결과 색을 제외한 모든 기호도에서 $3\%$ 첨가구가 가장 높아 유의적인 차이를 보였다(p<0.05).

• 생명과학회지
• /
• 제19권6호
• /
• pp.799-803
• /
• 2009

방사선 및 각종 독성물질에 의한 고환 정세관세포의 사멸은 apoptosis와 관련이 있다고 알려져 있으나 정세관상피주기에 따른 apoptosis 발생에 대한 변화연구는 미진하다. 본 연구에서는 감마선을 조사한 ICR 마우스의 고환에서 apoptosis 발생을 transferase-mediated end labeling (TUNEL) 과 periodicacid-Schiff (PAS) 염색을 동시에 실시하여 관찰하였다. Apptosis는 TUNEL 양성으로 나타났으며 특징적 형태변화를 보였다. 2 Gy (분당 2 Gy의 선량률)의 방사선을 조사하고 24시간동안의 변화를 관찰한바 방사선조사 후 12시간에 가장 높은 apoptosis 발생을 보였고 이후 감소하였다. 8 Gy까지의 방사선을 조사하고 8시간에 변화를 관찰한 결과 모든 정세관상피주기에서 방사선 용량에 비례한 apoptosis의 발생이 관찰되었다. 방사선 용량-반응은 linear-quadratic 곡선 [y=(-0.014${\pm}$0.009)$D^{2}$ +(0.31${\pm}$0.697)D+0.3575. Y는 정세관 당 TUNEL 양성세포의 수, D는 방사선 용량(Gy), $r^{2}$=0.9]에 가장 일치 하였다. 최대반응은 8 Gy에서 관찰되었으며, 0.5 Gy조사군에서도 변화가 나타났다. 이러한 변화는 정세관상피주기 V에서 B정조세포와 정세관상피주기 XII의 분열기 정자세포에서 가장 현저하였다.

• 한국임상수의학회지
• /
• 제12권1호
• /
• pp.877-886
• /
• 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
• /
• 제34권6호
• /
• pp.957-966
• /
• 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.

• 방사성폐기물학회지
• /
• 제16권4호
• /
• pp.397-410
• /
• 2018

아메리슘(Am)은 사용후핵연료의 장기 방사성 독성에 크게 영향을 주기 때문에 고준위 방사성 폐기물 처분의 장기 안전성 평가에 필수적으로 고려되어야 할 원소이다. 분광학적 방법을 이용한 일부 악티나이드 원소의 화학반응 연구가 활발히 진행되고 있는 반면, 아메리슘에 대한 연구는 아직까지 미비한 상황이다. 이 연구에서는 고순도의 시료를 필요로 하는 화학반응 연구를 위하여 $^{241}Am$ 시료를 정제한 후, 액체섬광계수기와 감마선 및 알파선 스펙트럼을 이용하여 정량과 정성분석을 하였다. 액체 광도파 모세관 셀을 이용한 고감도의 UV-Vis 흡수 분광학과 시간분해 레이저 형광 분광학을 이용하여 Am(III) 가수분해물과 옥살레이트(oxalate, Ox) 착물반응을 조사하였다. 산성조건에서 $Am^{3+}$은 503 nm에서 최대 흡수봉우리를 보이며, 몰흡광계수는 $424{\pm}8cm^{-1}{\cdot}M^{-1}$임을 확인하였다. 중성 이상의 pH 조건에서 형성되는 $Am(OH)_3(s)$ 콜로이드 입자에서는 506-507 nm 파장에서 최대 흡수봉우리가 관측되었다. ${Am(Ox)_3}^{3-}$ 착물은 $Am^{3+}$에 비교하여 흡수 및 발광스펙트럼이 각각 4와 5 nm정도 장파장으로 이동하였고 몰흡광계수와 발광세기도 크게 증가하였다. ${Am(Ox)_3}^{3-}$의 발광수명은 23에서 56ns으로 증가하였고 이는 Am(III)의 내부권에 결합하고 있던 약 여섯 개의 물분자가 옥살레이트의 카르복실기로 치환되었음을 의미한다. 이 결과로부터 ${Am(Ox)_3}^{3-}$은 각 옥살레이트 리간드가 두 자리 결합(bidentate)을 하고 있다는 것을 제안하였다.

• Molecules and Cells
• /
• 제42권6호
• /
• pp.448-459
• /
• 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.