돼지 卵子의 透明帶에 대한 單一클론抗體生産과 그 特性에 關한 硏究

金鐘培,劉永春,金昌圭,權五中,鄭盛元,鄭吉生 건국대학교 동물자원연구센터 1991 動物資源硏究誌 Vol.16 No.-

本 試驗은 單一클론抗體의 강한 特異性과 抗體性質의 不變性을 이용하여 發生學的 側面에서 哺乳動物 卵子의 透明帶의 機能과 構造를 이해하고, 또한 種特異的인 精子 受容體의 存在 및 生化學的 構造를 규명하기 위한 기초연구로서, 돼지 卵子의 透明帶를 免疫原으로 하여 BABL/c 생쥐로부터 單一클론抗體를 생산하고 그 특성을 구명하였던 바 다음과 같은 결과를 얻었다. 1) 3마리의 BABL/c 생쥐(YⅠ, YⅡ, ZI)에 돼지卵子의 透明帶를 免疫化하고, 複合抗體 生成을 확인한 후 생쥐의 脾臟細胞와 Myeloma(SP2/O-Ag14)를 polyethylene glycol를 融合을 실시한 결과 각각 25.8%, 54.5% 그리고 59.7%의 融合效率을 나타내었으며, ELISQ에 의해 陽性反應을 조사한 결과 각각 17.3%, 32.6% 그리고 6.2% 陽性反應 效率을 나타내었다. 2) YI에서 강한 陽性反應을 보인 6개의 well에 대한 cloning을 실시하고 抗體檢證을 행한 결과 20.8% ∼ 48.4%의 Cloning效率과 54.6% ∼ 82%의 陽性反應 效率을 나타내었다. 3) 강한 陽性反應을 나타낸 항체에 대해 sandwich ELISA法에 의해 isotype을 決定하였던바 2E93C(YⅠ), 3E83E7(YⅠ), 4E3(YⅡ)각각 IgG₂b, IgG₂a, IgM으로 확인되었다. 4) Isotype이 決定된 2E93C9(YⅠ), 3E84E7(YⅠ), 4E3(YⅡ)의 세포를 생쥐의 腹腔에 주사하여 얻은 腹水를 indirect ELISA法에 의해 titer를 決定한 결과 모두 1:400,000 이상의 높은 titer를 나타내었다. 5) 處理區로서 單一클론抗體의 腹水와 對照區로서 normal mouse serum이 각각 2%씩 함유된 배양액속에서 난자를 배양한 후 顯徵鏡下에서 관찰했을 때 對照區에서 배양된 난자의 표면은 정상적인 형태를 나타냈으나 處理區에서 배양된 卵子는 표면에 뚜렷한 沈澱層을 형성하였다. 6) 處理區와 對照區 卵子를 Rabbit anti-mouse IgG-FITC가 1% 함유된 배양액속에서 배양하고 洗滌한 후 螢光顯徵鏡下에서 관찰한 바 處理區의 卵子는 透明帶 주위에서 螢光이 나타났으나, 對照區에서는 나타나지 않았다. This study was carried out ot produce and characterize monoclonal antibodies against porcine zona pellucida, and undertaken as a basic study to develop immunocontraceptive vaccine and to investigate the function of zana pellucida in early fertilization process. The results obtained were summarized as follows: 1. Spleen cells of three BALB/C mice(YⅠ, YⅡ and ZI) which were immunized with porcine zona pelucida were fused with myeloma cells(SP2/O-Ag14) by polyethylene glycol. At the result of fusion, fusion efficiency was 25.8 , 54.5% and 59.7% and positive efficiency 17.3%, 32.6% and 6.2%, respectively. 2. Cloning efficiency was shown to be from 20.8% to 48.4% and positive efficiency of them were 54.6% to 82%. 3. Sub-isotypes of three strong positive antibodies, 2E93C(YⅠ), 3E83E7(YⅠ) and 4E3(YⅡ) were determined by sandwich ELISA method and shown to be as IgG2b, IgH2a and IgM, respectively. 4. The titers of three ascitic fluids containing antibodies, 2E93C9(YⅠ), 3E84E7(YⅠ) and 4E3(YⅡ) were determined by indirect ELISA and all of them showed above 1:400,000. 5. The layer of precipitate was observed on the zona incubated with medium containing 2% ascitic fluid of monoclonal antibody while the eggs treated with 2% normal mouse serum did not. 6. Porcine eggs incubated with medium containing 2% ascitic fluid of monoclonal antibody and followed by subsequent incubation with Rabbit anti-mouse IgG-FITC conjugate showed strong fluorescent light on the zona surface while the zona of normal mouse serum-treated eggs did not show fluorescence.

육성돈에 미생물제제 급여시 분뇨 특성에 미치는 효과 연구

곽정훈,최동윤,박치호,김재환,정광화,양창범,유용희,라창식,Kwag, J.H.,Choi, D.Y.,Park, Ch.H.,Kim, J.H.,Jeong, K.H.,Yang, Ch.B.,Yoo, Y.H.,La, C.S. 한국축산환경학회 2007 축산시설환경학회지 Vol.13 No.1

본 시험은 육성돈 사료에 미생물제제를 사료에 미생물제제 A 및 B 0.1 미생물제제 C를 0.2% 혼합 급여할 경우 사료섭취량 및 돈분의 오염물질 배설농도에 미치는 영향을 분석하기 위하여 4처리$\times$반복당 5두로서 총 20두를 공시하여 실시하였는데 그 결과를 요약하면 다음과 같다. 1. 육성비육돈의 일일 평균사료섭취량은 대조구 2.06kg/일.두였고 미생물 A, B, C 구는 각각 2.13kg/일.두, 2.17, 2.34로 미생물제제 C구에서 일일 사료섭취량이 가장 높게 조사되었으며(p<0.05), 2. 일일평균 음수량은 사료섭취량이 높았던 미생물 C구에서 2.89kg/일/두로 가장 높게 조사되었다(p<0.05). 3. 미생물제제 처리구별로 분뇨 배설량은 사료섭취량이 높았던 미생물제제 C 구에서 가장 많이 배설되는 것으로 조사되었으며 (0<0.05), 돈뇨의 배설량도 미생물제제 C구에서 2.31kg/일/두에서 높았다(p<0.05). 4. 육성돈의 성장단계별 돈분뇨의 수분 함량은 및 비료성분인 T-N, $P_2O_5,\;K_2O$ 성분도 처리간에 큰 차이를 보이지 않았다(p<0.05). 5. 육성돈 분뇨의 평균 BOD 농도는 돈분의 경우 미생물제제 A, B제제 급여구가 유의적으로 높게 조사되었다(p<0.05). 그리고 돈뇨의 BOD의 경우에도 미생물제제 A급 여구에서 $6,537mg/\ell$로 가장 높은 것으로 조사되었다(p<0.05). 6. COD의 경우에도 미생물 C급여구에서 가장 높게 조사되었으며(p<0.05). 돈뇨의 경우에는 미생물제제 A급여구에서 평균 $8,566mg\ell$로 가장 높았다(p<0.05). 7. SS 농도는 대조구에서 가장 높게 조사 되었으며(p<0.05), 그 다음이 미생물 B> 미생물 C> 대조구 순으로 조사되었다. 8. 돈분뇨중의 T-N 농도는 처리구간에 유의적인 차이가 나타나지 않았다(p<0.05). 9. 돈분중의 T-P 농도는 미생물제제 처리 구간별로 미생물제제 A, C 급여구에서 유의적인 차이가 나는 것으로 조사되었다(p<0.05). 이상의 결과를 요약해보면 육성돈에 미생물제제 혼합급여는 사료섭취량을 증가시키는데 효과가 있으나 비료성분 배설량에는 큰 차이를 보이지 않는 것으로 조사되었으며, 오염물질 배설량의 경우에는 사료섭취량이 높은 미생물제제 C 급여구에서 높은 것으로 조사되었다. The effects of microbial feedstuff additives on feed conversion rate and physical and chemical characteristics of excreta in growing pigs were investigated. Three different products (A, B and C) were compared. Microbial population tests showed B contained higher numbers of total bacteria, Lactobacillus spp. and yeasts. The amylase activity of B was also higher than that of A and C. The daily feed intake rates fer control, A, B and C were 2.06, 2.13, 2.17 and 2.34 kg, respectively. Pigs feed product C had the highest liveweight gain(2.89 kg). However, the results of feed conversion rate were not significantly different between treatments. Amount of faces excreted for control, A, B and C was 1.18, 1,19, 1.23 and 1.32 kg, respectively. Urine volume for control, A, B, and C was 1.91, 1.80, 2.19 and 2.31 kg respectively. Moisture content, T-N, $P_2O_5$ and $K_2O$ in pig manure were not significantly different between treatments. The range of BOD values was 63,453 to $73,758mg/\ell$ for faeces, and 5,678 to $7,428mg/\ell$, for urine. SS values of solid and liquid excreta ranged from 142,200 to 176,000 and from 710 to $1,025mg/\ell$, respectively.

Conversion of C2C12 Myoblast into Adipoblast with Thiazolidinediones - A Possible Basis for Intramuscular Fat Generation in Meat Animals

Singh, N.K.,Chae, H.S.,Hwang, I.H.,Yoo, Y.M.,Ahn, C.N.,Lee, H.J.,Park, H.J.,Chung, H.Y. Asian Australasian Association of Animal Productio 2007 Animal Bioscience Vol.20 No.3

Thiazolidinediones (TZDs) act as potent activators of the adipose differentiation program in established preadipose cell lines. TZD's have also been investigated in diabetic patients and reported to act as PPAR-${\gamma}$ ligands. In this report, the effects of TZDs on the differentiation pathway of myoblasts have been investigated. C2C12 mouse myoblasts were grown in Dulbecco's Modified Eagles medium for 4-5 days until they reached almost 100% confluency. Post-confluent cells (day 0) were further exposed to adipogenic induction medium along with TZDs for 48 hours. Thereafter, cells were exposed only to TZDs every 48 h until day 10. The control was provided with differentiation medium without any treatment. Alterations in the cells during the differentiation programme were analyzed on the basis of fusion index, oil-red-o staining, adipocyte index, adipocyte stain uptake measurement, immuno-histochemistry and western blotting. Exposure of C2C12 mouse myoblasts to TZDs prevented the expression of myosin heavy chain with parallel increase in the expression of C/EBP-${\alpha}$ and PPAR-${\gamma}$ and acquisition of adipocyte morphology, thus abolishing the formation of multinucleated myotubes. TZDs exert their adipogenic effects only in non-terminally differentiated myoblasts; myotubes were insensitive to the compound. Continuous exposure (at least 4-5 doses) to inducers after the growth arrest was essential to provide a sustained environment to the cells converting to fully matured adipoctyes. The results indicate that TZDs specifically converted the differentiation pathway of myoblasts into that of adipoblasts.

2, 4-Thiazolidindion Induced Plasticity of Myoblast (C2C12) and Satellite Cells (Porcine) - A Comparative Study

Singh, N.K.,Chae, H.S.,Hwang, I.H.,Yoo, Y.M.,Ahn, C.N.,Lee, H.J.,Park, H.J.,Chung, H.Y. Asian Australasian Association of Animal Productio 2007 Animal Bioscience Vol.20 No.7

This study was conducted to determine the difference between satellite cells (porcine) and myoblasts (C2C12) in their differentiation under the influence of 2, 4-thiazolidindion. C2C12 myoblast cells and porcine satellite cells (isolated from 10 d old $Landrace{\times}Duroc$ piglets) were grown to absolute confluency. Post confluent cells (day 0) were further exposed to adipogenic induction medium along with 2, 4-thiazolidindion ($8{\mu}M$) for 2 d. Thereafter, cells were exposed to 2, 4-thiazolidindion alone every 2 d till day 10 and analysed. The control was cultured in differentiation medium without any treatment. Increased (p<0.05) expression of transcriptional factors i.e. C/EBP-${\alpha}$ and PPAR-${\gamma}$ and transition of cells to adipocyte morphology was noticed from 2 d and 4 d onwards in satellite cells (Porcine) and myoblasts (C2C12) respectively. Myogenesis was observed to be suppressed completely in case of satellite cells compared to myoblasts in response to 2, 4-thiazolidindion. Pax-7 (transcriptional factor) appeared as a sole entity to satellite cells only, as it was not identified in case of myoblasts. Although both the cells were converting to adipoblasts, the degree of their conversion was different in response to 2, 4-thiazolidindion. Therefore, the hypothesis that satellite cells contribute various domains to the growing myoblasts appeared obscured and found to be dependent on the proliferative energy/or degree of fusion. However, it revealed satellite cells as currency to myoblasts/muscle.

한국산 겨우살이 렉틴(KML-C)에 대한 단일크론항체의 생산과 특징

윤택준(T. J. Yoon),유영춘(Y. C. Yoo),강태봉(T. B. Kang),김성훈(S-H Kim),김갑수(K. S. Kim),김종배(J. B. Kim) 대한약학회 2001 약학회지 Vol.45 No.2

We have reported that water-extracted Korean mistletoe(KM-110) had various biological activities such as antitumor and immunomodulatory activity and the lectin fraction(KML-C) of the extract was one of the major factors related to its biological functions. In this paper, we produced murine monoclonal antibody(mAb) against KML-C. The mAbs obtained were largely classified into two groups according to specificity to KML-C and ML-I, a lectin from European mistletoe. One group mABs(9H7-D10 and 3C2-1H4) strongly reacted with KML-C, but not ML-I. In contrast, another group mAbs(8B11-2C5, 8E12-3E9 and 5E10-F1) reacted with both KML-C and ML-1. The subisotypes of these mAbs were shown to be IgG1(9H7-1D10, 3C2-1H4 and 8B11-2C5) or IgM(8E12-3E9 and 5E10-F1). To develop an assay system for determination of the amonunt of KML-C, we established the sandwich ELISA(enzyme-linked immunosorbent assay) method using these mAbs and horse radish peroxidase(HRP) labelled mAbs.In various combinations of the mAbs for coated antibody and detection antibody, the sandwich ELISA quantitatively detected KML-C,showing the detection limit ranging from 7-5,000ng/ml. Especially, reproducibility(C.V.) of the sandwich ELISA, in which 8E12-3E9 was used for coating antibody and 8B11-2C5-HRP for detection antibody, was 4.59-5.83 in intra assay, and 3.9-9.4 in inter assay.

Novel dentin phosphoprotein frameshift mutations in dentinogenesis imperfecta type II

Lee, K‐,E,Kang, H‐,Y,Lee, S‐,K,Yoo, S‐,H,Lee, J‐,C,Hwang, Y‐,H,Nam, KH,Kim, J‐,S,Park, J‐,C,Kim, J‐,W Blackwell Publishing Ltd 2011 Clinical genetics Vol.79 No.4

<P>Lee K‐E, Kang H‐Y, Lee S‐K, Yoo S‐H, Lee J‐C, Hwang Y‐H, Nam KH, Kim J‐S, Park J‐C, Kim J‐W. Novel dentin phosphoprotein frameshift mutations in dentinogenesis imperfecta type II.</P><P>The dentin sialophosphoprotein (<I>DSPP</I>) gene encodes the most abundant non‐collagenous protein in tooth dentin and DSPP protein is cleaved into several segments including the highly phosphorylated dentin phosphoprotein (DPP). Mutations in the <I>DSPP</I> gene have been solely related to non‐syndromic form of hereditary dentin defects. We recruited three Korean families with dentinogenesis imperfecta (DGI) type II and sequenced the exons and exon–intron boundaries of the <I>DSPP</I> gene based on the candidate gene approach. Direct sequencing of PCR products and allele‐specific cloning of the highly repetitive exon 5 revealed novel single base pair (bp) deletional mutations (c.2688delT and c.3560delG) introducing hydrophobic amino acids in the hydrophilic repeat domain of the DPP coding region. All affected members of the three families showed exceptionally rapid pulp chambers obliteration, even before tooth eruption. Individuals with the c.3560delG mutation showed only mild, yellowish tooth discoloration, in contrast to the affected individuals from two families with c.2688delT mutation. We believe that these results will help us to understand the molecular pathogenesis of DGI type II as well as the normal process of dentin biomineralization.</P>

Validation of egg yolk antibody based C-ELISA for avian influenza surveillance in breeder duck

Jeong, O.M.,Kim, M.C.,Kang, H.M.,Ha, G.W.,Oh, J.S.,Yoo, J.E.,Park, C.H.,Kwon, J.S.,Pack, M.R.,Kim, H.R.,Kim, Y.J.,Kwon, J.H.,Lee, Y.J. Elsevier Scientific Pub. Co 2010 Veterinary microbiology Vol.144 No.3

Active surveillance for avian influenza virus (AIV) has expanded from chicken to various poultry species including duck. To further effective antibody screening in laying breeder ducks, we validated the egg yolk antibody as alternative source to serum for AIV antibody. Sera and eggs were collected at weekly intervals after two types of AIV vaccination, H5N3 and H9N2. The antibody levels were determined by an agar gel immunodiffusion (AGID) test, haemagglutination inhibition (HI) test and the competitive enzyme-linked immunosorbent assay (C-ELISA). AGID test did not detect antibodies in egg yolk, and the agreement between AGID test and either HI test or C-ELISA in serum was slight and fair based on kappa statistics (kappa value (κ)@?0.19 in H5N3 group and κ@?0.37 in H9N2 groups). However, there was almost perfect agreement between HI test and C-ELISA (κ>0.9 in all group). The C-ELISA was as sensitive and specific as the HI test, and could be used as a pre-screening test for the detection of type A avian influenza virus antibody. Comparison was made between egg yolk and serum antibody titers by a regression analysis. A high correlation was observed between serum and yolk antibody titers (r=0.8762 for H5N3 and 0.8914 for H9N2 in HI test; r=1 for H5N3 and 0.9686 for H9N2 in ELISA test), although egg yolk antibodies were detected later and remained lower levels than serum antibodies. In field trials involving 54 duck flocks, the positive rate of egg yolk and serum samples showed agreement for the detection of AIV antibody. We concluded that as an alternative to serum, antibody monitoring of laying breeder duck using egg yolk with C-ELISA is feasible and is recommended.

New mixture composition of organic solvents for efficient extraction of lipids from Chlorella vulgaris

Jeon, J.m.,Choi, H.W.,Yoo, G.C.,Choi, Y.K.,Choi, K.Y.,Park, H.Y.,Park, S.H.,Kim, Y.G.,Kim, H.J.,Lee, S.H.,Lee, Y.K.,Yang, Y.H. Pergamon ; Elsevier Science Ltd 2013 Biomass & bioenergy Vol.59 No.-

To identify more effective but less toxic compositions of organic solvent mixtures for biodiesel production from Chlorella vulgaris, 15 different organic solvents were examined and compared for their power of lipid extraction from algal biomass. When solvents were individually examined, methanol showed the best efficiency for the extraction of fatty acids, followed by dichloromethane. Although chloroform resulted in yields which were equal to or greater than those of other solvents, it showed a very low fatty acid content and a high level of unknown impurities when analyzed by gas-chromatography (GC). Furthermore, solvent mixtures were applied in order to determine the optimal composition for a high lipid extraction efficiency, using chloroform, methanol and dichloromethane. Through comparison of compositions using the Bligh and Dyers method, the best composition was to be methanol:dichloromethane (1:1). Our findings resulted in a 25% increase of lipid extraction yield, together with C16, C16:2, C18:2 as major components from C. vulgaris.

An exploration of the antioxidant effects of garlic saponins in mouse-derived C2C12 myoblasts

Kang, J. S.,Kim, S. O.,Kim, G.-Y.,Hwang, H. J.,Kim, B. W.,Chang, Y.-C.,Kim, W.-J.,Kim, C. M.,Yoo, Y. H.,Choi, Y. H. Spandidos Publications 2016 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE Vol.37 No.1

<P>In this study, we aimed to confirm the protective effects of garlic saponins against oxidative stress-induced cellular damage and to further elucidate the underlying mechanisms in mouse-derived C2C12 myoblasts. Relative cell viability was determined by 3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bromide assay. Comet assay was used to measure DNA damage and oxidative stress was determined using 2 ',7 '-dichlorofluorescein diacetate to measure intracellular reactive oxygen species (ROS) generation. Western blot analysis and small interfering RNA (siRNA)-based knockdown were used in order to investigate the possible molecular mechanisms. Our results revealed that garlic saponins prevented hydrogen peroxide (H2O2)-induced growth inhibition and exhibited scavenging activity against intracellular ROS. We also observed that garlic saponins prevented H2O2-induced comet tail formation and decreased the phosphorylation levels of gamma H2AX expression, suggesting that they can prevent H2O2-induced DNA damage. In addition, garlic saponins increased the levels of heme oxygenase-1 (HO-1), a potent antioxidant enzyme associated with the induction and phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the translocation of Nrf2 from the cytosol into the nucleus. However, the protective effects of garlic saponins on H2O2-induced ROS generation and growth inhibition were significantly reduced by zinc protoporphyrin IX, an HO-1 competitive inhibitor. In addition, the potential of garlic saponins to mediate HO-1 induction and protect against H2O2-mediated growth inhibition was adversely affected by transient transfection with Nrf2-specific siRNA. Garlic saponins activated extracellular signal-regulated kinase (ERK) signaling, whereas a specific ERK inhibitor was able to inhibit HO-1 upregulation, as well as Nrf2 induction and phosphorylation. Taken together, the findings of our study suggest that garlic saponins activate the Nrf2/HO-1 pathway by enabling ERK to contribute to the induction of phase II antioxidant and detoxifying enzymes, including HO-1 in C2C12 cells.</P>

객담 임상 가검물에서 특이항체를 이용한 레지오넬라 항원검색

이혜경,조경주,박만석,심수경,유천권,박경석,박미연 국립보건연구원 2000 국립보건원보 Vol.37 No.-