• 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.18 no.1
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• pp.69-73
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• 2003

This study was undertaken to investigate the effect of three different porcine IVM media, TCM-199, mSOF and NCSU-23 on early development of porcine spontaneous parthenogenotes. Spontaneous parthenogenotes were induced by a prolonged culture of porcine oocytes in each medium. In Experiment 1, oocytes derived from gilts were matured in three IVM media and maturation of oocytes was evaluated by the status of chromatin configuration. Oocytes matured in mSOF, NCSU-23 and TCM-199 showed no significant difference (P>0.05) in maturation. Maturation rates at 48h after IVM were 83.1$\pm$2% (mSOF), 78.0$\pm$3% (NCSU-23) and 83.5$\pm$2% (mSOF), 78.0$\pm$3% (NCSU-23) and 83.5$\pm$2% (TCM-199). In Experiment 2, pronucleus formation and development to 6~8 cell stage of pig oocytes activated spontaneously. Pronucleus formation, cleavage rate and development to 6~8 cell embryos of porcine spontaneous parthenogenotes were assessed at 55~58 h, 96 h and 168h after IVM, respectively. Pronucleus formation (5.4$\pm$2% and 3.7 $\pm$ 1% vs 1.7 $\pm$ 3%) and development to the 6$\pm$8 cell (3.2$\pm$3% and 4.0$\pm$1% vs 1.4$\pm$3%) was significantly (P<0.05) higher in mSOF or NCSU-23 than TCM-199. In conclusion, the present study showed that oocytes matured in mSOF and NCSU-23 were spontaneously activated with higher frequency.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.265-271
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• 2014

Implementation of smart embryo technologies in cattle e.g. ovum pick-up followed by in vitro embryo production (OPU-IVP). Seasonal variation is important factor for follicular growth, oocytes quality, quantity and developmental competence. Therefore the aim of present study was carried out to investigated whether the seasons (hot and cool) effect on follicular development, oocyte recovery and subsequent embryo development. Follicular oocytes were aspirated from Korean native cows (Hanwoo) by the ovum pick-up (OPU) method, which was performed 24 times during two different seasons, the hot (July to September) and cool (October to December), from OPU donors. The recovered oocytes were classified according to morphological categories and used for in vitro embryo production (IVEP). The mean number of total follicles was significantly higher (p<0.05) during the hot season ($18.32{\pm}2.26$) compared to cool season ($15.41{\pm}3.34$). Furthermore, seasons did not significantly effect on the number of oocytes recovered (hot season: 41.16% vs. cool season: 46.14%). However, the average number of Grade A oocytes was significantly greater during hot ($1.75{\pm}1.86$) season compared to the cool season ($1.00{\pm}1.46$), but there was no significant difference of other grades oocytes. The cleavage rate (hot: 66.67% vs. cool: 63.3%) and embryo development (hot: 58.95% vs. cool: 56.97%) did not differ significantly between the seasons. In conclusion, the results of present study suggest that the season (hot and cool) does not have effects on the oocyte recovery and embryo developmental competence of in vitro cultured embryos.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.131-138
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• 2017

In most mammals, metaphase II (MII) oocytes having high maturation promoting factor (MPF) activity have been considered as good oocytes and then used for assisted reproductive technologies including somatic cell nuclear transfer (SCNT). Caffeine increases MPF activity in mammalian oocytes by inhibiting p34cdc2 phosphorylation. The objective of this study was to investigate the effects of caffeine treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after SCNT in pigs. To this end, morphologically good (MGCOCs) and poor oocytes (MPCOCs) based on the thickness of cumulus cell layer were untreated or treated with 2.5 mM caffeine during 22-42, 34-42, or 38-42 h of IVM according to the experimental design. Caffeine treatment for 20 h during 22-42 h of IVM significantly inhibited nuclear maturation compared to no treatment. Blastocyst formation of SCNT embryos was not influenced by the caffeine treatment during 38-42 h of IVM in MGCOCs (41.1-42.1%) but was significantly improved in MPCOCs compared to no treatment (43.4 vs. 30.1%, P<0.05). No significant effects of caffeine treatment was observed in embryo cleavage (78.7-88.0%) and mean cell number in blastocyst (38.7-43.5 cells). The MPF activity of MII oocytes in terms of p34cdc2 kinase activity was not influenced by the caffeine treatment in MGCOCs (160.4 vs. 194.3 pg/ml) but significantly increased in MPCOCs (133.9 vs. 204.8 pg/ml). Our results demonstrate that caffeine treatment during 38-42 h of IVM improves developmental competence of SCNT embryos derived from MPCOCs by influencing cytoplasmic maturation including increased MPF activity in IVM oocytes in pigs.

• Microbiology and Biotechnology Letters
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• v.40 no.1
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• pp.1-9
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• 2012

This study's aim was to isolate microorganisms producing agarase with a high activity, with possible applications in improving the performance of the pretreatment processes for bioethanol production. Marine algaes were collected from the south coast of Korea, from which three kinds of microorganisms were isolated. After a 4-day culture of these strains at $25^{\circ}C$, crude enzymes were obtained from culture supernatant or cell-free extract by ammonium sulfate precipitation and membrane dialysis. Agarase activity was observed in these crude enzymes. Notably higher specific activity was observed in the crude enzyme obtained from the culture supernatant rather than that from the cell-free extract. This indicates that a secreted enzyme has a much greater activity than a cellular enzyme. Crude enzymes from the GNUM08122 strain were inferred to have ${\alpha}$-agarase activity because release of p-nitrophenol was observed, possibly due to the cleavage of p-nitrophenyl-${\alpha}$-D-galactopyranoside. The 16S rRNA sequence of GNUM08122 showed a close relationship to Pseudoalteromonas issachenkonii KMM 3549 (99.8%) and Pseudoalteromonas tetraodonis IMA 14160 (99.7%), which led us to assign it to the genus Pseudoalteromonas. Biochemical and physiological study revealed that this strain can grow well at $40^{\circ}C$ under a wide range of pH (pH 4~8) in high-salt conditions (10% NaCl).

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.119-126
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• 2004

The present study was carried out to examine the effect of four different media (NCSU (North Carolina State University)-23, PZM (Porcine Zygotes Medium)-3, PZM-4 and TCM (Tissue Culture Medium)-l99) and two oxygen concentrations (39 , 5% $O_2$, 5% $CO_2$ and 90% $N_2$, 5% $CO_2$ in air) on in vitro production of porcine IVM/IVF embryos. The results were summarized as follows: The rates of GVBD and nuclear maturations were not significantly different (p>0.05) for 44 hours of culture with four media in two oxygen concentrations. The rates of polyspermy, penetrated sperm(s) and male and female prouclei formation were not significantly different (p>0.05). among four media in two oxygen concentrations. The cleavage rates were not significantly different (p>0.05) among four media in two oxygen concentrations. At day 7 under gas atmosphere of 5% $O_2$, 5% $CO_2$ and 90% $N_2$, the blastocyst formation was significantly higher (p<0.05) in PZM-3 (19.9$\pm$2.4) than other media. Also, NCSU-23 medium gave high rate of blastocyst formation at day 7 under gas atmosphere of 5% $CO_2$ in air (p<0.05). Based on the result of differential staining of porcine blastocyst at dat 7, inner cell mass cell and total cell numbers were not significantly different (p>0.05) among four media in two oxygen concentrations. However, the observed total cell number was higher in PZM-3 medium (36.8$\pm$6.5) than other madia. In conclusion, these results suggested that in vitro production of porcine embryos in PZM-3 medium under a gas atmosphere of 5% $O_2$, 5% $CO_2$ and 90% $N_2$ was effective on the blastocyst formation rate and total blastocyst cell number.

• Korean Journal of Microbiology
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• v.54 no.1
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• pp.60-68
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• 2018

A cold-active and alkaline serine protease (Pro21717) was partially purified from the Antarctic marine bacterium Pseudoalteromonas arctica PAMC 21717. On a zymogram gel containing skim milk, Pro21717 produced two distinct clear-zones of approximately 37 kDa (low intensity) and 74 kDa (high intensity). These were found to have identical N-terminal sequences, suggesting they arose from an identical precursor and that the 37 kDa protease might homodimerize to the more active 74 kDa form of the protein. Pro21717 displayed proteolytic activity at $0-40^{\circ}C$ (optimal temperature of $40^{\circ}C$) and maintained this activity at pH 5.0-10.0 (optimal pH of 9.0). Notably, relative activities of 30% and 45% were observed at $0^{\circ}C$ and $10^{\circ}C$, respectively, in comparison to the 100% activity observed at $40^{\circ}C$, and this enzyme showed a broad substrate range against synthetic peptides with a preference for proline in the cleavage reaction. Pro21717 activity was enhanced by $Cu^{2+}$ and remained stable in the presence of detergent surfactants (linear alkylbenzene sulfonate and sodium dodecyl sulfate) and other chemical components ($Na_2SO_4$ and metal ions, such as $Ba^{2+}$, $Mg^{2+}$, $Ca^{2+}$, $Zn^{2+}$, $Fe^{2+}$, $K^+$, and $Na^{2+}$), which are often included in commercial detergent formulations. These data indicate that the psychrophilic Pro21717 has properties comparable to the well-characterized mesophilic subtilisin Carlsberg, which is commercially produced by Novozymes as the trademark Alcalase. Thus it has the potential to be used as a new additive enzyme in laundry detergents that must work well in cold tap water below $15^{\circ}C$.

• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.1
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• pp.163-170
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• 2006

Nardostachyos Rhizoma (N. Rhizoma) belonging to the family Valerianaceae has been anti-arrhythmic effect, and sedation to the central nerve and a smooth muscle. We reported that the water extract of N. Rhizoma induced apoptotic cell death and differentiation in human promyelocytic leukemia (HL-60) cells. Cytotoxicity of N. Rhizoma was detected only in HL-60 cells (IC50 is about 200 ${\mu}g/ml$). The cytotoxic activity of N. Rhizoma in HL-60 cells was increased in a dose-dependent manner. We used several measures of apoptosis to determine whether these processes were involved in N. Rhizoma-induced apoptotic cell death. The high-dose (200 ${\mu}g/ml$) treatment of N. Rhizoma to HL-60 cells showed cell shrinkage, cell membrane blobbing, apoptotic bodies, and the fragmentation of DNA, suggesting that these cells underwent apoptosis. Treatment of HL-60 cells with N. Rhizoma time-dependently induced activation of caspase-3, caspase-8, and caspase-9 and proteolytic cleavage of poly(ADP-ribose) polymerase. Also, we investigated the effect of N. Rhizoma on cellular differentiation and proliferation in HL-60 cells. Differentiation and proliferation of HL-60 cells was determined through expression of CD11b and CD14 surface antigens using flow cytometry and nitroblue tetrazolium (NBT) assay, and through analysis of cell cycle using propidium iodide assay, respectively. N. Rhizoma induced the differentiation of HL-60 at the low-dose (100 ${\mu}g/ml$) treatment, as shown by increased expression of differentiation surface antigen CD11b, but not CDl4 and increased reducing activity of NBT. When HL-60 cells were treated with N. Rhizoma at concentration of $50{\mu}g/ml\;and\;100{\mu}g/ml$, NBT-reducing activities induced approximately 1.5-fold and 20.0-fold as compared with the control. In contrast, HL-60 cells treated with the N. Rhizoma-ATRA combination showed markedly elevated levels of 26.3-fold at $50{\mu}g/ml$ N. Rhizoma-0.1 ${\mu}M$ ATRA combination and 27.5-fold at 50 ${\mu}g/ml$ N. Rhizoma-0.2 ${\mu}M$ ATRA combination than when treated with N. Rhizoma alone or ATRA alone. It may be that N. Rhizoma plays important roles in synergy with ATRA during differentiation of HL-60 cells. DNA flow-cytometry indicated that N. Rhizoma markedly induced a G1 phase arrest of HL-60 cells. N. Rhizoma-treated HL-60 cells increased the cell population in G1 phase from 32.71% to 42.26%, whereas cell population in G2/M and S phases decreased from 23.61% to 10.33% and from 37.78% to 33.98%, respectively. We examined the change in the $p21^{WAF1/Cip1}\;and\;p27^{Kip1}$ proteins, which are the CKIs related with the G1 phase arrest. The expression of the CDK inhibitor $p27^{Kip1},\;but\;not\;p21^{WAF1/Cip1}$ were markedly increased by N. Rhizoma. Taken together, these results demonstrated that N. Rhizoma induces apoptotic cell death through activation of caspase-3, and potently inhibits the proliferation of HL-60 cells via the G1 phase cell cycle arrest in association with $p27^{Kip1}$ and granulocytic differentiation induction .

• Korean Journal of Food Science and Technology
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• v.40 no.2
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• pp.207-214
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• 2008

In Asia Saussurea lappa (SL) has been used as a traditional herbal medicine to treat abdominal pain and tenesmus. Recently, in vitro cell culture studies have shown that SL has anti-ulcer, anti-inflammatory, and anti-tumor properties. To explore its potential chemopreventive and chemotherapeutic effects in colon cancer, we examined whether the hexane extract of SL (HESL) could inhibit the growth of HT-29 human colon cancer cells, and investigated the mechanisms for this effect. The cells were cultured with various concentrations (0-5 ${\mu}g/mL$) of HESL. The results indicated that HESL markedly decreased the numbers of viable HT-29 cells; whereas at the concentration of 5 ${\mu}g/mL$, HESL slightly decreased the viable cell numbers of CCD 1108Sk human skin normal fibroblasts at 72 hr. HESL substantially increased the numbers of cells in the sub G1 phase, and dose-dependently increased apoptotic cell numbers. Western blot analysis of the total cell lysates revealed that HESL increased Bax protein levels, but did not affect Bcl-2 levels. HESL induced the cleavage of poly (ADP-ribose) polymerase and caspases 8, 9, 7, and 3. This study demonstrated that HESL inhibits cell growth and induces apoptosis in HT-29 cells, which may be mediated by its ability to increase Bax levels and activate the caspase pathway. These findings may lead to the development of new therapeutic strategies for colon cancer treatment.

• Clean Technology
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• v.25 no.1
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• pp.46-55
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• 2019

CdS and CdZnS/ZnO materials were prepared using precipitation method and used as photocatalysts for the photocatalytic degradation of rhodamine B (RhB) under visible light irradiation. The prepared photocatalysts were also characterized by XRD and UV-vis DRS. The results indicated that the photocatalysts with intended crystalline structures were successfully obtained and both the CdS and CdZnS/ZnO can absorb visible light as well as UV. The photocatalytic activities were examined with the addition of scavenger for various active chemical species and the difference of reaction mechanisms over the catalysts were discussed. The $CH_3OH$, KI and p-benzoquinone were used as scavengers for ${\cdot}OH$ radical, photogenerated positive hole and ${\cdot}O_2{^-}$ radical, respectively. The CdS and CdZnS/ZnO showed different photocatalytic degradation mechanisms of RhB. It can be postulated that ${\cdot}O_2{^-}$ radical is the main active species for the reaction over CdS photocatalyst, while the photogenerated positive hole for CdZnS/ZnO photocatalyst. As a result, the predominant reaction pathways over CdS and CdZnS/ZnO photocatalysts were found to be the dealkylation of chromophore skeleton and the cleavage of the conjugated chromophore structure, respectively. The above results may be mainly ascribed to the difference of band edge potential of conduction and valence bands in CdS, CdZnS and ZnO semiconductors and the redox potentials for formation of active chemical species.