Although embryonic stem cells (ESCs) or ES-like cells are reported from many mammalian species other than the mouse, the culture system for murine ESCs may not be suitable to the other species. Previously many other research groups have modified either human or mouse ESC culture systems for bovine ESC culture. In this study, we compared three different culture mediums consisting of DMEM, -MEM or KnockOut-DMEM (KO), which are modified from human or mouse ESC culture system, for the generation of bovine ESCs. In this study, some pre-requisite events which are important for establishment and long-term propagation of ESCs such as inner cell mass (ICM) attachment on feeder cells, primary colony formation and sustainability after passaging. Once the ICM clumps attached on feeder cells, this was designated as passage 0. In regards to the rate of ICM attachment, -MEM was superior to the other systems. For primary colony formation, there was no difference between DMEM and -MEM whereas KO showed lower formation rate than the other groups. For passaging, the colonies were split into 2~4 pieces and passed every 5~6 days. From passage 1 to passage 3, DMEM system seemed to be appropriate for maintaining putative bovine ESCs. On the other hand, -MEM tended to be more suitable after passage 6. Although -MEM support to maintain a ES-like cell progenies to passage 15, all three culture systems which are modified from human or mouse ESC culture media failed to retain the propagation and long-term culture of putative bovine ESCs. Our findings imply that more optimized alternative culture system is required for establishing bovine ESC lines.

The 3-isobutyl-1-methylxanthine (IBMX) is non-selective phosphodiesterase and is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte. The present study was conducted to analyze: (1) nuclear maturation (examined by the Hoechst staining), (2) whether cytoplasmic maturation (examined by the intracellular glutathione (GSH) concentration) of porcine oocytes is improved during meiotic arrest after prematuration (22 h) with IBMX. Before in vitro maturation (IVM), oocytes were treated with 1 mM IBMX for 22 h. After 22 h of pre-maturation, the higher rate of IBMX treated group oocytes were arrested at the germinal vesicle (GV) stage (42.3%) than control IVM oocytes (10.1%). It appears that the effect of IBMX on the resumption of meiosis has shown clearly. In the end of IVM, the reversibility of the IBMX effect on the nuclear maturation has been corroborated in this study by the high proportions of MII stage oocytes (72.5%) reached after 44 h of IVM following the 22 h of inhibition. However, intracellular GSH concentrations were lower in the oocytes treated with IBMX than the control oocytes (6.78 and 12.94 pmol/oocyte, respectively). These results demonstrate that cytoplasmic maturation in porcine oocytes pre-treated with IBMX for 22 h did not equal that of control oocytes in the current IVM system. These results indicate that pre-maturation with IBMX for 22 h may not be beneficial in porcine IVM system.

The present study was conducted to develop a simple method for porcine oocyte maturation without CO2 regulation. In experiment 1, we evaluated that the effect of CO2 non-supplement on porcine oocyte maturation. Cumulusoocyte complexes (COCs) were collected from 2～6 mm follicles and divided into three groups (Control, tube-CO2, and tube-non-CO2). For control, COCs were cultured in 4-well multidish in a CO2 incubator. For tube-CO2, COCs were cultured in a round-bottom tube in a CO2 incubator, and for tube-non-CO2, COCs were cultured in a round-bottom tube sealed tightly without CO2 supplement in a dry incubator. The proportion of oocytes reached to metaphase II (M-II) was not significantly different among three groups (87.9% to 91.4%). In experiment 2, we evaluated the effect of CO2 non-supplement during oocyte maturation on development of embryos. Oocytes with a polar body were divided into two groups (Control and tube-non-CO2) and applied 1.1 kV/cm or 1.2 kV/cm voltages for parthenogenetic activation. After activation, embryos were cultured for 6 days and examined the development. The proportion of embryos cleaved was not significantly different among treatment (86.3% to 91.5%). The proportion of embryo reached to blastocyst stage was not significantly different among treatment (13.9% to 25.2%). The cell number of blastocysts was not significantly different among treatment (29.0 to 32.4). In conclusion, oocytes cultured in a dry incubator without CO2 supplement have enough competence to development after parthenogenetic activation. These results would be useful for transporting oocytes or embryos a long distance.

This study was carried out to investigate effective condition for producing somatic cell nuclear transfer (SCNT) embryos of Jeju native cattle. As donor cells for SCNT, ear skin cells from Jeju native cattle were used. In experiment 1, the effect of recipient oocyte sources on the development of Jeju native cattle SCNT embryos were examined. Fusion rate of recipient oocyte and donor cell was not different between the Hanwoo and Holstein recipient oocytes (86.0% vs 89.9%). The rate of embryos developing to the blastocyst stage was significantly (p<0.05) higher in Hanwoo recipient oocytes than in Holstein recipient ones (28.2% vs 14.7%). Blastocysts derived from Hanwoo recipient oocytes contained higher numbers of total cells than those derived from Holstein ones ( vs ), although there were no significant difference. The mean proportion of apoptotic cells in blastocyst was not different between the sources of recipient oocytes. In experiment 2, the development of Jeju native cattle and Hanwoo SCNT embryos were compared. Hanwoo oocytes were used as the recipient oocytes. Fusion rate was not different between the Jeju native cattle and Hanwoo SCNT embryos (92.1% vs 92.9%). The blastocyst rate of SCNT embryos was significantly (p<0.05) lower in Jeju native cattle than in Hanwoo (16.9% vs 31.0%). Blastocysts derived from Jeju native cattle SCNT embryos contained smaller numbers of total cells than those derived from Hanwoo ones ( vs ), but there were no significant difference. The mean proportion of apoptotic cells in blastocyst was not different between the Jeju native cattle and Hanwoo SCNT embryos. The present study demonstrated that Hanwoo recipient oocytes were more effective in supporting production of Jeju native cattle SCNT embryos, although Jeju native cattle SCNT embryos showed reduced developmental capacity when compared to Hanwoo SCNT embryos.

Peri-implantitis (PI) is bacteria-induced inflammatory condition which affects the alveolar bone and soft tissue around implants and may result in the loss of supporting bone. Attenuation of the P. gingivalis lipopolysaccharide (LPS)-induced inflammatory response can be a new therapeutic approach in the treatment of PI. This study was conducted to evaluate the anti-inflammatory effect of 635-nm light-emitting diode (LED) irradiation over MG63 osteoblast-like cell. Scratch was made on MG63 cells with or without LPS, then 635-nm irradiated. The expression of the cyclooxygenase-2 (COX-2) proteins was evaluated with western blot. The production of the prostaglandin E2 (PGE2) and expression of the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) was measured with enzyme-linked immunoassay, and the cytokine profile was evaluated with the human inflammation antibody array. Wound closure effect presented in the cells treated with LPS was observed more significantly in the cells with 635-nm irradiation than the cells without irradiation. The 635-nm irradiaiton reduced LPS-induced expression of the COX-2 and production of the PGE2. Also, 635-nm irradiation affect the expression of RANKL, OPG, and proinflammatory cytokines. These results indicate that 635-nm irradiation could reduce the alveolar bone resorption induced by LPS stimulation through the inhibition of COX-2 expression and PGE2 production, the suppression of proinflammatory cytokine, and the modulation of RANKL/OPG balance in MG63 cells.

To evaluate the antioxidant and potential of Humulus japonicus, total polyphenol and flavonoid content, radical scavenging activities, and antitumor activities were measured. The total polyphenol and flavonoid contents of the methanol extracts from Humulus japonicus were 30.13±1.13 and 13.61±0.49 ㎎ gallic acid equivalent/g extract, respectively. DPPH radical and hydroxyl radical scavenging activities of methanol extracts of Humulus japonicus were 60% and 35%, respectively. The Humulus japonicus higher activities of anticancer activities on liver cancer cell lines compared to other cancer cell lines.

본 연구는 반추동물의 조사료 자원으로서 거대억새를 개발하기 위한 목적으로 수행되었다. 우리나라에서 새롭게 개발된 품종인 거대억새 1호를 완숙기 이후에 채취하여 in vitro 반추위 발효를 이용해반추위내 pH, 암모니아태 질소, 가스발생량, 휘발성 지방산 생성량 및 건물소화율을 조사하였으며,볏짚과 비교하여 평가하였다. 거대억새는 볏짚에 비하여 유의적으로 높은 반추위내 pH를 나타내었다(p<0.01). 암모니아태 질소의 경우 배양 12시간 이후에는 두 처리구간의 유의적인 차이를 나타내지 않았다 (p>0.05). 배양 6시간 이후 부터는 거대억새의 가스발생량이 볏짚에 비하여 유의적으로 낮게 나타났다 (p<0.05). 휘발성 지방산 생성량에 있어 acetate, propionate, butyrate, valerate 및 총생상량에서 볏짚이 거대억새보다 높게 나타났다. 그러나 iso-butyrate와 iso-valerate에서는 두 조사료원별 차이는 발견되지 않았다. 건물소화율에 있어 배양 12~24시간 사이의 거대억새 소화율이 볏짚에 비하여 유의적으로 나타났다. 결론적으로 거대억새의 이용성은 볏짚의 약 80% 수준인 것으로 나타났다.

Anti-neurodegenerative activities of aqueous extract from propolis were investigated. The aqueous extracts showed strong antioxidant activities in malondialdehyde (MDA) assay, and it effectively inhibited lipid peroxidation. In addition, the aqueous extract presented protective effects against H2O2-induced neurotoxicity in a dose-dependent manner. Our study verified that the aqueous extract has strong antioxidant activity and neuronal cell protective effect which is correlated with its total phenolics (290.75 mg GAE/g) including p-coumaric acid, vanillic acid and gallic acid. These phenolics of propolis may reduce the risk of neurodegenerative disorders, and can be utilized as effective and safe resources of functional food.

This study was conducted to examine the effect of dietary n-3/n-6 fatty acid (FA) ratio on in vitro dry matter digestibility (IVDMD), fermentation indices and FA profile. Rice bran was mixed with oil sources (cotton seed oil and linseed oil) to make the diets at 0.02, 0.29 and 0.61 of dietary n-3/n-6 FA ratio. These diets (0.5g) were placed into the incubation bottles with 40 ml of anaerobic culture medium, which contained rumen fluid and Van Soest medium at 1:2 ratio. Five replicates of each diet and two blanks were incubated at 39℃ for 48 hours. After incubation, the incubated contents were centrifuged. The residues were freeze-dried for DMD and FA analyses. The supernatant was used for pH, NH3-N and volatile fatty acid analyses. The concentrations of lactate (p<0.001) and iso-valerate (p<0.001) decreased linearly with increasing dietary n-3/n-6 FA ratio, but acetate concentration (p=0.056) and the ratio of acetate to propionate (p=0.005) was increased linearly. The concentrations of n-3, n-6 FA and the ratio of n-3/n-6 FA in residues increased (p<0.001) linearly with increasing dietary n-3/n-6 FA ratio, but C18:1n-9 FA concentration was decreased (p<0.001) linearly. With these results, it could affect fermentation characteristics and FA profile of rumen content by dietary n-3/n-6 FA ratio.

친환경 유기농자재를 이용하여 잿빛곰팡이병균의 포자 발아, 균사생장 억제효과를 기내에서 조사하였다. 그 결과 잿빛곰팡이병의 균사생장 억제 효과는 Bacillus subtilis를 주성분으로 하는 제제가 100%의 억제효과를 보였으며, 그 외의 제제는 20% 이하의 범위에서 억제효과를 나타내었다. 포자발아 억제효과는 유기황 수화제 2종('BTB', '황스타')이 각각 97.7%, 92.3%으로 나타났다. 균사생장 억제와 포자 발아억제에 모두 효과를 보이는 제제는 없었다. 따라서 잿빛곰팡이병 방제를 위해서는 포자발아 억제를 위한 방제와 균사생장억제를 위한 제제를 각각 처리하여야 할 것으로 사료되었다. 아울러 사물 기생성을 가진 잿빛곰팡이병의 방제를 위해서는 잔재물에서 생장을 막는 것이 중요하며 이를 위해 예방을 전제로 한 방제의 경우 유기황을 주성분으로 하는 제제가 적용 가능할 것으로 생각된다.

친환경 유기농자재를 이용하여 고추 탄저병균의 포자 발아, 균사생장 억제효과를 기내에서 조사하였다. 그 결과 균사생장 억제효과 기내검정. 탄저병의 균사생장 억제 효과는 Bacillus subtilis를 주성분으로 하는 제제가 100%의 억제효과를 보였으며, 그 외의 제제는 20~40%의 범위에서 억제효과를 나타내었다. 포자발아억제효과는 유황 수화제 2종('BTB', '황스타')이 각각 100%, 95.1%, '고려역'(95.0%), '보르스타'(99.0%), '지하부대 KM'(96.1%) 순으로 나타났다. 균사생장 억제와 포자 발아억제, 부착기 형성억제 결과를 고려하여 고추 탄저병 방제에 적용 가능한 친환경 방제제는 Bacillus subtilis, Panibacillus polymyxa 혼용제('고려역')가 가장 효과적인 것으로 판단되었으며, 예방을 전제로 한 방제의 경우 유황 수화제, 보르도액을 주성분으로 하는 제제가 적용 가능할 것으로 생각된다. 본 연구에서 선택된 제제는 고추 포장에서 탄저병 방제효과가 최종적으로 인정되면 차후 고추 탄저병 방제에 이용될 것으로 사료된다.

The T-cell receptor (TCR) engages with an antigen and initiates a signaling cascade that leads to the activation of transcription factors. Roquin, a protein encoded by the RC- 3H1 gene and characterized as an immune regulator, was recently identified as a novel RING-type ubiquitin ligase family member, but the mechanisms by which Roquin regulates T-cell responses are unclear. We used the EL-4 murine lymphoma cell line to elucidate the role of Roquin in vitro. Roquin-overexpressing EL-4 cells became hyper-responsive after anti-CD3/CD28 stimulation in vitro and were a major source of the cytokines IL-2 and TNF-α. Upon activation, these cells showed particularly enhanced production of IL-2 and TNF-α. To clarify the important role played by Roquin in T-cell responses ex vivo, we generated T-cell-specific Roquin transgenic (Tg) mice. Roquin-Tg CD4+ T-cells showed enhanced production of IL-2 and TNF-α in response to TCR stimulation with anti-CD28 co-stimulation. Further studies are necessary to investigate the role of Roquin in the regulation of primary T-cell activation, survival, and differentiation.

Porcine blastocyst’s quality derived from in vitro is inferior to in vivo derived blastocysts. In this study, to improve in vitro derived blastocyst’s quality and then establish porcine ESCs (pESCs), we treated in vitro fertilized (IVF) embryos and parthenogenetic activated (PA) embryos with three chemicals: porcine granulocyte-macrophage colony stimulating factor (pGM-CSF), resveratrol (RES) and β-mercaptoethanol (β-ME). The control group was produced using M199 media in in vitro maturation (IVM) and porcine zygote medium-3 (PZM3) in in vitro culture (IVC). The treatment group is produced using M199 with 2 μM RES in IVM and PZM5 with 10 ng/mL pGM-CSF, 2 μM RES and 10 μM β-ME in IVC. Data were analyzed with SPSS 17.0 using Duncan’s multiple range test. In total, 1210 embryos in PA and 612 embryos in IVF evaluated. As results, we observed overall blastocyst quality was increased. The blastocyst formation rates were significantly higher (p<0.05) in the treatment groups (54.5%) compared to the control group (43.4%) in PA and hatched blastocysts rates in day 6 and 7 were also increased significantly. Total cell numbers of blastocyst were significantly higher (p<0.05) in the treatment group (55.1) compared to the control group (45.6). In IVF, hatched blastocysts rates in day 7 were increased significantly, too. After seeding porcine blastocyst, the attachment rates were higher in the treatment group (36.2% in IVF and 32.2% in PA) than the control group (26.6% in IVF and 19.5% in PA). Also, colonization rates and cell line derivation rates were higher in treatment group than control group. Colonization rates of control group were 10.8% in IVF and 2.4% in PA, but treatment group were 17.75% in IVF, and 13.1% in PA. And we investigated the correlation between state of blastocysts and attachment rate. The highest attachment rate is in hatched blastocyst (78.35±15.74 %). So, the novel system increased quality of porcine blastocysts produced from in vitro, subsequently increased attachment rates. The cell line derivation rates were 4.2% (IVF) and 2.4% (PA) in control group. In treatment group, they were 10.0% (IVF) and 7.2% (PA). We established 3 cell lines from PA blastocysts (1 cell line in control group and 2 cell lines in treatment group). All cell line has alkaline phosphatase activity and express pluri-potent markers. In conclusion, the novel system of IVM and IVC (the treatment of RES during IVM and RES, β-ME, and pGM-CSF during IVC) increased quality of porcine blastocysts produced from in vitro, subsequently increased derivation rates of porcine putative ESCs.

Althogh Spermatogonial stem cells (SSCs) are widely studied in mice, study of pig SSCs is not sufficient for the isolation, long-term culture, and characterization. To identify the effect of growth factor in cultured pig SSC, newly generated pSSCs like cell from neonatal 5days porcine testis were cultured and investigated for the pSSCs like cell formation. Glial derived neurotrophic gactor (GDNF), fibroblast growth factor (FGF), leukemia inhibitory factor (LIF), and epidermal growth factor (EGF) were applied to culture media to identify the pSSC like cell growth and stem cell formation. The criteria for the determining of stem cell characters, morphology, number of colonies, putative stem cell marker were analysed by microspic, polymerase chain reaction (PCR) and immunocytochemistry (ICC) methods. Most of the pSSCs like cells were formed approximately 100 μm size with sphere shape. Most of the feeder cells were highly dependent on FGF that feeder cells were not stably attached on plate without FGF and colony formation of pSSC was not observed consequently. Immunocyto chemistry data revealed that this cells expressed the ubiquitin-C-terminal hydrolase 1 (UCHL-1, PGP9.5) and Dolichos Biflorus Agglutinin (DBA) in addition of 20 ng/ml EGF, 10 ng/ml FGF, 10 ng/ml GDNF, 10 U3/ml LIF. In addition, Alkaline Phosphatase ()was positive in all period of culture. This study suggest that various growth factorsinp SSC culture system is important to regulate and maintain the pSSC. In conculsion, although the precise role of growth factor in pSSC proliferation need to be clarified, combination of growth factor might be critical in order to derivation and proliferation of neonatal pSSCs and spermatogenesis.