소 體外受精卵의 超急速凍結에 관한 硏究 : Ⅱ. 소 體外受精卵의 超急速凍結 融解後의 生存性에 관한 硏究 Ⅱ. Studies on the Survival Rates after Rapid Frozen-Thawing of In Vitro Fertilized Bovine Embryos

金相根,李晩徽 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

This study was carried out in order to investigate the effects of cryoprotective concentration and equilibration time on survival rate of ultrarapidly frozen in vitro fertilized bovine embryos. In vitro fertilized bovine embryos, following dehydration by cryoprotective agents and sucrose were directly plunged into liquid nitrogen and thawed in 38℃ water. Survival rate was defined by development rate to the morula and blastocyst stage after in vitro culture of by FDA test. The results are summarized as follows. 1. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0M, 2.5M. 3.0M, 3.5M, 4.0M glycerol were 75.0%, 72.0%, 67.6%, 44.8% and 18.3% respectively. 2. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0M, 2.5M. 3.0M, 3.5M, 4.0M DMSO were 64.0%, 66.7%, 70.8%, 52.7% and 18.6, respectively. 3. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0M, 2.5M. 3.0M, 3.5M, 4.0M propanediol were 68.4%, 64.9%, 63.2%, 62.2% and 34.7%, respectively. 4. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing medium of 2.50M glycerol added 0.1M, 0.25M, 0.5M, 0.75M, sucrose were 60.5%, 72.2%, 70.1% and 54.9%, respectively. The survival rates of in vitro fertilized embryos after ultrarapid frozen-thawing in the freezing medium of 2.5M glycerol added 0.25M sucrose were higher than concentration of 0.10M, 0.50M and 0.75M sucrose. 5. The equilibration time on the survival rate of in vitro fertilized bovine embryos was attained after short period of time(2.5∼5min.) in the freezing medium added 0.25M sucrose and 3.0M DMSO higher tham long periodef time(1∼20min.).

Effect of Antimuscarinic Autoantibodies in Primary Sjögren’s Syndrome

Kim, N.,Shin, Y.,Choi, S.,Namkoong, E.,Kim, M.,Lee, J.,Song, Y.,Park, K. SAGE Publications 2015 Journal of dental research Vol.94 No.5

<P>The presence of functional autoantibodies against the muscarinic type 3 receptor (M3R) has been reported in primary Sjogren's syndrome (pSS). However, the pathogenic role of these autoantibodies in pSS development remains to be elucidated. In this experiment, we investigated a pathologic role of pSS autoantibodies (pSS IgG) associated with downregulation of the major histocompatibility complex I (MHC I) molecule with M3R through internalization. Anti-M3R autoantibodies in purified control and pSS IgG were detected using 4 synthesized cyclic M3R peptides by enzyme-linked immunosorbent assay. The binding reactivity of pSS IgG to M3R in situ was analyzed by a dual immunostaining method. Surface expression, interaction, and internalization of M3R with MHC I were analyzed by immunofluorescence confocal microscopy and biochemical assays. Synthetic cyclic peptides M3RP(205-221) and M3RP(520-527) showed significantly high reactivity with pSS IgG compared to the control IgG or the other 3 peptides (P < 0.05). Significantly high reactivity of pSS IgG to M3R in situ was observed. PSS IgG increased the interaction of membrane M3R with MHC I and induced their internalization in primary human submandibular gland cells. The pSS IgG-induced internalization of M3R with MHC I was significantly inhibited by the cholesterol-sequestering drug filipin. Our novel findingnamely, strong downregulation of the membrane MHC I with M3R through internalization of the cholesterol-rich microdomain associating with anti-M3R autoantibodiescould be an important mechanism contributing to the impaired salivation seen in pSS and linking secretory hypofunction to autoimmune pathogenesis.</P>

Multiple heterologous M2 extracellular domains presented on virus-like particles confer broader and stronger M2 immunity than live influenza A virus infection

Kim, M.C.,Lee, J.S.,Kwon, Y.M.,O, E.,Lee, Y.J.,Choi, J.G.,Wang, B.Z.,Compans, R.W.,Kang, S.M. Elsevier/North-Holland 2013 Antiviral research Vol.99 No.3

The influenza M2 ectodomain (M2e) is poorly immunogenic and has some amino acid changes among isolates from different host species. We expressed a tandem repeat construct of heterologous M2e sequences (M2e5x) derived from human, swine, and avian origin influenza A viruses on virus-like particles (M2e5x VLPs) in a membrane-anchored form. Immunization of mice with M2e5x VLPs induced protective antibodies cross-reactive to antigenically different influenza A viruses and conferred cross protection. Anti-M2e antibodies induced by heterologous M2e5x VLPs showed a wider range of cross reactivity to influenza A viruses at higher levels than those by live virus infection, homologous M2e VLPs, or M2e monoclonal antibody 14C2. Fc receptors were found to be important for mediating protection by immune sera from M2e5x VLP vaccination. The present study provides evidence that heterologous recombinant M2e5x VLPs can be more effective in inducing protective M2e immunity than natural virus infection and further supports an approach for developing an effective universal influenza vaccine.

Microneedle patch delivery to the skin of virus-like particles containing heterologous M2e extracellular domains of influenza virus induces broad heterosubtypic cross-protection

Kim, M.C.,Lee, J.W.,Choi, H.J.,Lee, Y.N.,Hwang, H.S.,Lee, J.,Kim, C.,Lee, J.S.,Montemagno, C.,Prausnitz, M.R.,Kang, S.M. Elsevier Science Publishers 2015 Journal of controlled release Vol.210 No.-

A broadly cross-protective influenza vaccine that can be administrated by a painless self-immunization method would be a value as a potential universal mass vaccination strategy. This study developed a minimally-invasive microneedle (MN) patch for skin vaccination with virus-like particles containing influenza virus heterologous M2 extracellular (M2e) domains (M2e5x VLPs) as a universal vaccine candidate without adjuvants. The stability of M2e5x VLP-coated microneedles was maintained for 8weeks at room temperature without losing M2e antigenicity and immunogenicity. MN skin immunization induced strong humoral and mucosal M2e antibody responses and conferred cross-protection against heterosubtypic H1N1, H3N2, and H5N1 influenza virus challenges. In addition, M2e5x VLP MN skin vaccination induced T-helper type 1 responses such as IgG2a isotype antibodies and IFN-γ producing cells at higher levels than those by conventional intramuscular injection. These potential immunological and logistic advantages for skin delivery of M2e5x VLP MN vaccines could offer a promising approach to develop an easy-to-administer universal influenza vaccine.

Supplemented vaccination with tandem repeat M2e virus-like particles enhances protection against homologous and heterologous HPAI H5 viruses in chickens

Song, B.M.,Kang, H.M.,Lee, E.K.,Jung, S.C.,Kim, M.C.,Lee, Y.N.,Kang, S.M.,Lee, Y.J. Butterworths ; Elsevier Science Ltd 2016 Vaccine Vol.34 No.5

Highly pathogenic avian influenza (HPAI) H5 viruses derived from A/Goose/Guangdong/1/96 have been continuously circulating globally, severely affecting the public health and poultry industries. The matrix 2 protein ectodomain (M2e) is considered a promising candidate for a universal cross-protective influenza vaccine that provides more effective control over HPAI H5 viruses harboring variant hemagglutinin (HA)-antigens. Here, we evaluated the protective efficacy of a tandem repeat construct of heterologous M2e presented on virus-like particles (M2e5x VLPs) either alone or as a supplement against HPAI H5 viruses in a chicken model. Chickens immunized with M2e5x VLPs alone induced M2e-specific antibodies but were not protected against HPAI H5. The homo- and cross-protective efficacy of M2e5x VLP-supplemented vaccination of chickens was also examined. Importantly, supplementation with M2e5x VLPs induced significantly higher levels of antibodies specific for M2e and different viruses as well as provided improved protection against homologous and heterologous HPAI H5 viruses. Considering the limited efficacy of inactivated vaccines, supplement vaccination with M2e5x VLPs may be an effective measure for preventing outbreaks of HPAI viruses that have the ability to constantly change their antigenic properties in poultry.

2단계 급속동결 및 초자화 동결한 토끼상실배의 체외생존성에 관한 연구

정구민,이창규,임경순,김수헌 서울대학교농과대학농업개발연구소 1990 서울대농학연구지 Vol.15 No.1

본 시험은 수정란 급속동결보존기술의 개발을 위한 기초자료를 얻기 위하여 2단계 급속동결 및 초자화동결이 토끼 상실배의 체외발생등에 미치는 영향을 검토하고자 실시하였으며, 얻어진 결과는 다음과 같다. 1. 토끼 상실배를 1.5, 2.0, 2.5 및 3.0 M glycerol과 0.5 M sucrose가 포함된 동결액에 실온에서 10분간 노출후 -30℃에서 30∼40분간 정치하여 급속동결하였을 때 발생율은 각각 36.4, 83.3, 92.3 및 84.2%로 2.5 M glycerol에서 가장 높았다. 2. 토끼 상실배를 1.5, 2.0, 2.5 및 3.0 M 1,2-propanediol과 0.5 M sucrose가 포함된 동결액에 실온에서 10분간 노출 후 -30℃에서 30∼40분간 정치하여 급속동결하였을 때 발생율은 각각 26.6, 55.6, 65.0 및 52.9%로 2.5 M 1,2-propanediol에서 가장 높았으나, glycerol을 사용했을 때 보다 낮았다. 3. 토끼 상실배를 2.5, 3.0 및 3.5 M glycerol과 0.5 M trehalose가 포함된 동결액에 실온에서 10분간 노출후 액체질소에 침지하여 초급속동결한 결과 회수율은 각각 87.5, 92.5 및 92.5%, 형태적으로 정상인 수정란의 비율은 각각 37.5, 55.5 및 60.0%, 그리고 발생율은 각각 13.3, 36.4 및 37.5%로 3.5 M glycerol에서 가장 높았으나 초자화동결법보다 현저하게 낮았다. 4. 토끼 상실배를 25% glycerol과 25% 1,2-propanediol을 함유한 동결액에 실온에서 10분간 노출후 초자화동결했을 때 발생율은 75.0%로 실온에서 형평한 후 초자화동결이 가능하였다. This experiment was carried out to investigate on in vitro development of rabbit monla frozen by 2-step feezing and vitrification. The results obtained from this experiment are as follows; 1. When rabbit morula in m-PBS containing 1.5, 2.0, 2.5 or 3.0 M glyceral and 0.5 M sucrose for 10 min at room temperature were cooled at -30℃ for 30 to 40 min and plunged into liquid nitrogen, the proportion of embryo developed to expanded blastoyst was 36.4, 83.3, 92.3 and 84.2%, respectively. Glycerol 2.5 M showed higher survival than others. 2. When rabbit morula in m-PBS containing 1.5, 2.0, 2.5 or 3.0 M 1, 2-propanediol and 0.5 M sucrose for 10 min at room temperature were cooled at -30℃ for 30 to 40 min and plunged into liquid nitrogen, the proportion of embrye developed to expanded blastocyst was 26.6, 55.6, 65.0 and 52.9%, respectively. 1, 2-propanediol was less effective than glycerol. 3. When rabbit morula in m-PBS containing 2.5, 3.0 or 3.5 M glycerol and 0.5 M trehalose for 10 min at room temperature were plunged into liquid nitrogen and thawed rapidly, the recovery rate of embryo was 87.5, 92.5 and 92.5%, the proportion of morphologically normal embryo was 37.5, 55.5 and 60.0%, and the proportion of embryo developed to expanded blastocyst was 13.3. 36.4 and 37.5%, respectively. The proportion of embryo developed to expanded blastocyst was higher in vitrification than in plunging into liquid nitrogen. 4. When rabbit morula were frozen by vitrification in m-PBS containing 25% glycerol. 25% 1, 2-propanediol, the proportion of embryo developed to expanded blastocyst was 75.0%, the result suggested that rabbit embryos could be frozen by vitrification after equilibration at room temperature.

灌漑用水路의 水路損失率 算定에 관한 硏究

李基春,具滋雄,金在英,李宰泳,徐元明 전북대학교 농업과학기술연구소 1982 農大論文集 Vol.13 No.-

This study was carried out in order to estimate water losses in irrigation canals, which may be used to evaluate the water requirement for irrigation projects. The conveyance losses were measured by the inflow-outflow method, the seepage were measured by the ponding method, and the operation losses in the course of irrigation were calculated by comparing the two kinds of losses. The results obtained in this experiment were as follows : Conveyance losses per unit area of wetted perimeter per second by the main irrigation canal, the secondary irrigation canal and the tributary irrigation, canal, were 1.399x10-5m3/m3/sec, 5.154x10-5m3/m3/sec, and 2.670×10-5m3/m3/sec respectively in the Goong-sa area. And they were 1.934x10-5m3/m3/sec, 2.149x10-5m3/m3/sec, and 4.558x10-6m3/m3/sec respectively in the Seong-dug area. Seepage losses per unit area of wetted perimeter per second by the secondary irrigation canal and the tributary irrigation canal, were 2.180×10-6m3/m3/sec and 2.168×10-6m3/m3/sec in the Goons-sa area. 1.150x10-6m3/m3/sec and 1.084x10-6m3 /m3/sec in the Seong-dug area respectively. Operation losses per unit area of wetted perimeter per second by the secondary irrigation canal and the tributary irrigation canal, were 4.936×10-5m3/m3/sec and 2.453×10-5m3/m3/sec in the Goong-sa area, 2.034×10-5m3/m3/sec and 4.450×10-5m3/m3 /sec in the Seong-dug area respectively. Conveyance, seepage and operation losses in the Goong-sa area were 6.7%, 94.6%, and 14.0% more than those in the Seong-dug area. Operation losses amount to about 17 times as much as seepage losses in the Goons-sa area and about 29 times in the Seong-dug area .The seepage losses depend much on the soil texture. ranging from 7.437×10-7m3/m3/sec to 2.430x10-6 m3/m3/sec. Water loss rates in the main irrigation canal, the secondary irrigation canal and the tributary irrigation canal, were estimated as 8.49%. 37.27%, and 9.8l%, respectively in the Goong-sa area. And they were estimated as 15.10%, 32.67% and 13.73% respectively in the Seong-dug area.

Metabolic characterization of (1-(5-fluoropentyl)-1H-indol-3-yl)(4-methyl-1-naphthalenyl)-methanone (MAM-2201) using human liver microsomes and cDNA-overexpressed cytochrome P450 enzymes

Kong, T. Y.,Kim, J. H.,Choi, W. G.,Lee, J. Y.,Kim, H. S.,Kim, J. Y.,In, M. K.,Lee, H. S. Springer Science + Business Media 2017 Analytical and bioanalytical chemistry Vol.409 No.6

<P>MAM-2201 is a synthetic cannabinoid that is increasingly found in recreational drug abusers and cases of severe intoxication. Thus, characterization of the metabolic pathways of MAM-2201 is necessary to predict individual pharmacokinetics and toxicity differences, and to avoid toxic drug-drug interactions. Collectively, 19 phase 1 metabolites of MAM-2201 were identified using liquid chromatography-Orbitrap mass spectrometry following human liver microsomal incubations in the presence of NADPH: 7 hydroxy-MAM-2201 (M1-M7), 4 dihydroxy-MAM-2201 (M8-M11), dihydrodiol-MAM-2201 (M12), N-(5-hydroxypentyl)-MAM-2201 (M13), hydroxy-M13 (M14), N-dealkyl-MAM-2201 (M15), 2 hydroxy-M15 (M16, M17), MAM-2201 N-pentanoic acid (M18), and hydroxy-M18 (M19). On the basis of intrinsic clearance values in human liver microsomes, hydroxy-MAM-2201 (M1), N-(5-hydroxypentyl)-MAM-2201 (M13), and hydroxy-M13 (M14) were the major metabolites. Based on an enzyme kinetics study using human cDNA-expressed cytochrome P450 (CYP) enzymes and an immunoinhibition study using selective CYP antibodies in human liver microsomes, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 enzymes were responsible for MAM-2201 metabolism. The CYP3A4 enzyme played a prominent role in MAM-2201 metabolism, and CYP1A2, CYP2B6, CYP2C8, and CYP2C9 enzymes played major roles in the formation of some metabolites. MAM-2201 is extensively metabolized by multiple CYP enzymes, indicating that MAM-2201 and its metabolites should be used as markers of MAM-2201 abuse and toxicity.</P>

돼지의 선발에 있어서 능력검정 방법에 따른 육종가 및 유전적 개량량의 변화

조규호,김시동,김명직,이일주,전광주 한국동물자원과학회 2001 한국축산학회지 Vol.43 No.6

본 연구에서는 이유시체중(㎏), 90㎏ 도달일령을 대상형질로 하여 폐쇄돈군에서 이유시 체중을 기초로 5가지의 선발방법에 따라 선발된 선발축의 산육 능력을 검정하여 검정이 완료된 개체들에 대해 유전능력 평가를 실시한후 검정대상 선발두수의 크기에 따른 유전적 개량량을 비교하여 이상적인 검정방법을 찾고자 실시하였다. 시뮬레이션 결과 90㎏ 도달일령에 대한 육종가 추정치의 변화는 Method Ⅴ그룹에서 -36.2일의 단축효과를 보였으며 세대당 유전적 개량량 또한 Method Ⅴ그룹에서 -7.5일로 가장 높은 개량을 보였다. 따라서 개량적인 측면에 있어서는 전수검정을 실시하는 것이 가장 좋을 것으로 사료되지만 농장의 검정능력이나 전수검정을 실시할 때 드는 초과비용에 대한 경제적인 분석은 더 연구되어져야 될 것으로 사료된다. This study was carried out to find the ideal testing method by comparing predicted breeding values, and genetic gain by the size of testing group. Tested traits were weaning weight and days to 90㎏. Five methods for selecting testing group were adopted: Method Ⅰ (MⅠ ; 30% selected based on litter weight at weaning for female, and 30% selected based on individual weight at weaning for mail), Method Ⅱ (MⅡ ; 30% selected based on individual weight at weaning for female and male, respectively), Method Ⅲ (MⅢ ; 50% selected based on individual weight at weaning for female and male, respectively), Method Ⅳ (MⅣ ; 70% selected based on individual weight at weaning for female and male, respectively), Method Ⅴ (MⅤ ; all piglets were tested). Genetic ability was evaluated by multiple trait animal model using MTDFREML package. Based on the predicted breeding values for days to 90㎏, the next generation was selected up to 5th generation. The above procedure were repeated and simulated by 4 times. The results obtained in the present study are summarized as follows; Phenotypic mean and predicted breeding values mean of days to 90㎏ was 114.3, -27.8, 113.7, -27.0, 107.5, -32.6, 105.7, -34.4, 103.7 and -36.2 days for MⅠ, MⅡ, MⅢ, MⅣ and MⅤ, respectively. Phenotypic and predicted breeding values variance of days to 90㎏ was 20.9, 4.1, 19.0, 3.4, 15.3, 1.8, 13.8, 1.7, 13.3 and 1.2 days for MⅠ, MⅡ, MⅢ, MⅣ and MⅤ, respectively. Genetic gain of days to 90㎏ was -6.2, -6.0, -7.1, -7.3 and -7.5 days for MⅠ, MⅡ, MⅢ, MⅣ and MⅤ, respectively. In all case, Method Ⅴ group, all piglets be tested, is the most efficient for improving the target economic traits.

Biomarker analysis in stage III–IV (M0) gastric cancer patients who received curative surgery followed by adjuvant 5-fluorouracil and cisplatin chemotherapy: epidermal growth factor receptor ( <i>EGFR</i> ) associated with favourable survival

Kim, J-S,Kim, M-A,Kim, T M,Lee, S-H,Kim, D-W,Im, S-A,Kim, T-Y,Kim, W H,Yang, H-K,Heo, D S,Bang, Y-J,Lee, K-U,Choe, K-J,Kim, N K Nature Publishing Group 2009 The British journal of cancer Vol.100 No.5

<P>The aim of this study was to analyse the impact of epidermal growth factor receptor (<I>EGFR</I>), thymidylate synthase (<I>TS</I>), dihydropyrimidine dehydrogenase (<I>DPD</I>), thymidine phosphorylase (<I>TP</I>), aurora kinase (ARK) A/B, and excision repair cross-complementing gene 1 (<I>ERCC1</I>) on the efficacy of adjuvant chemotherapy with 5-fluorouracil and cisplatin (FP) after curative gastric resection. Normal and cancer tissue were separately obtained from gastrectomy samples of 153 patients with AJCC stage III–IV (M0) who subsequently treated with adjuvant FP chemotherapy. <I>TS</I>, <I>DPD</I>, <I>TP</I>, <I>ERCC1</I>, and ARK proteins were measured by immunohistochemistry (IHC). <I>EGFR</I> expression was investigated using a standardized IHC with the <I>EGFR</I> PharmDx assay. Amplification of <I>EGFR</I> gene was analysed using fluorescent <I>in situ</I> hybridisation (FISH). In multivariate analysis, stage, ratio of positive to removed lymph nodes, and <I>EGFR</I> expression were significant prognostic factors for overall survival. Patients with higher <I>EGFR</I> expression had better overall survival than those with lower expression (relative risk: 0.475 (95% confidence interval, 0.282–0.791, <I>P</I>=0.005). Low <I>EGFR</I> expression might be a predictive marker for relapse in curative resected stage III–IV (M0) gastric cancer patients who received adjuvant FP chemotherapy.</P>