The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to meet a multi-parellel process. We have developed a novel recombinant bacmid, bEasyBm that enabling easy and fast generation of pure recombinant virus without any purification step. In the bEasyBm, attR recombination sites were introduced to facilitate the generation of recombinant viral genome by in vitro transposition. Moreover, extracellular RNase gene from bacillus amyloliquefaciens, barnase, was expressed under the control of Cotesia plutellae bracovirus early promoter. Therefore, only when the barnase gene was replaced to gene of interest, the bEasyBm could replicate in host insect cells. When the bEasyBm was transposed with pDualBac-EGFP and pDualBac-LUC respectively, there were no non-recombinant backgrounds were detected from unpurified BmEasy-EGFP or BmEasy-LUC stocks. In addition, the resulting recombinant virus, BmEasy-EGFP, showed comparable level of EGFP expression efficiency with the plaque-purified recombinant virus, BmEGFP, which was constructed using bBmGOZA system. Based on these results, high-throughput condition for generation of multiple recombinant viruses in a time was established.

Varieties of Bacillus thuringiensis (Bt) crystal proteins, Cry proteins, have so far been found as one of the most successful biological control agents which are safe to natural environments for a long time. Recently, cry genes encoding these Cry proteins have been widely applied for construction of transgenic crops resistant to pest insects. In this study, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Spodoptera exigua and Ostrinia furnacalis were identified. To construct novel cry genes with improved insecticidal activity, we randomly mutated these 23 amino acid sequences by in vitro muti site-directed mutagenesis, resulting in totally 24 mutant cry genes. For further characterization, these mutant cry genes were expressed as a fusion protein with polyhedrin using baculovirus expression system. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. In the further study, we plan to investigate their insecticidal activity against Plutella xylostella, S. exigua and O. furnacalis larvae.

Adipose tissue-derived mesenchymal stem cells (ASCs) are very interesting in several laboratory animals and humans because they are easy to harvest and expand to generate millions of cells from a small quantity of fat. ASCs are known as useful materials for clinical applications in human cell therapy and as a donor cell in somatic cell nuclear transfer (SCNT). Here, we investigated if 1) minipig ASCs can be isolated, self-renewed and differentiated into multiple tissue lineages, 2) ASCs can be a suitable donor cell type for generation of cloned pig. In order to isolate ASC, adipose tissues were collected from inguinal region of a 6-year-old female minipig. The ASCs were attached to the culture dish with a fibroblast-like morphology. They expressed cell-surface marker characteristics of stem cell, underwent osteogenic, adipogenic, myogenic, neurogenic and chondrogenic differentiation when exposed to specific differentiation-inducing conditions. To investigate its potential as donor cell for cloning, we respectively carried out SCNT using ASC, adult skin fibroblast (ASF) and fetal fibroblast (FF) derived from same minipig. The ratio of blastocysts to 2-cell embryos and total cell number of blastocysts were monitored as experimental parameters. In results, cleavage and developmental competence to blastocysts rate showed no significant difference among the three groups. On the other hand, total cell numbers of blastocysts derived from ASC and FF were significantly higher than in ASF (89±7.9 and 105±5.5 vs. 57.5±5.2, respectively). Our results demonstrated that ASC have potential compared to ASF and FF in terms of the in vitro development and blastocyst formation ability. In further study, we will investigate the in vivo developmental ability of ASC as donor cell for pig cloning. * This study was supported by IPET (#311011-05-1-SB010), RNL Bio (#550-20120006), Institute for Veterinary Science, the BK21 program, TS Corporation and Optifarm Solution.

ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene whose homologs are found in all lepidoteran Group I NPV, but its function is unknown so far. To determine the role of ac11 in baculovirus life cycle, ac11 knock-out mutant, Ac11KO, was constructed using the plasmid capture system (PCS). Real-Time PCR analysis showed that ac11 transcript was first detected at 6 h post-infection (p.i.) and accumulated to maximum at 48 h p.i., indicating that ac11 is belong to late gene. When the genomic DNA of Ac11KO was transfected into Sf9 cells, viral replication was restricted to a cell transfected originally. While viral transmission of the Ac11KO was not observed in Sf9 cells, production of budded virus (BV) in Sf9 cells transfected with Ac11KO was observed by transmission electron microscopy (TEM). These results suggest that the ac11 is essential for AcMNPV to produce infective BV.

This study was carried out to identify how a self-stretching exercise program affects pain for each body area, pain relief and job satisfaction for care workers. 20 of 40 care workers with musculoskeletal symptom were randomly selected and participated a self-stretching exercise program consisting of 15 motions. The intervention was done five times or more per weeks for 8 weeks and 1 session lasted within 15 minutes. 'Musculoskeletal symptom survey table' of the Korea Occupational Safety and Health Agency(KOSHA) and JDI(Job Descriptive Index) was used for pain on the musculoskeletal symptom and job satisfaction. Survey were done twice before and after the program. The result of this study showed that self-stretching exercise program group(SSPG) relieved from pain significantly in the shoulders(p<.01) and lumbar(p<.05), comparing to the non selfstretching exercise program group(NSPG). Although no significant difference on variations in the JDI appeared in SSPG, the significant reduction appeared from the colleague relationship and organization in NSPG(p<.05). SSPG showed the significant increase on variations in JDI from the job and organization comparing to NSPG. Especially, the improvement on satisfaction for the organization was shown(p<.05). Accordingly, the self-stretching exercise program for care workers can be said to positively affect the overall pain relief and increase on the JDI.

고무화합물 형태로 구성된 조영제의 병에 Syringe Connector의 Spike를 연결 시 고무의 찢김 정도를 알아보고 찢김 및 분쇄로 인한 합성고무의 혼입 유무와 분쇄된 합성고무가 검출 시 분쇄물의 크기를 실험을 통해 알아보고자 하였다. 그 결과 찢김 정도의 경우 Syringe Connector의 끝과 최초 접촉하는 앞면이 약 3.14±0.04 ㎜로 뒷면 보다 많이 찢겼으며, 실험 대상인 10 병의 조영제에서 평균 7 개에서 15 개로 모두 분쇄물이 검출되었다. 검출된 분쇄물을 이용하여 크기를 측정한 결과 평균크기는 약 7.89±0.31 ㎛이었다. 향 후 다양한 실험 및 분석방법을 통한 추가실험과 더불어 흡인된 분쇄물 차단을 위한 미세 필터타입 자동주입장치의 개발이 필요하며, 분쇄물 유입 시 치명적 사고를 대비하여 관련기관의 관심 또한 필요할 것으로 사료된다.

봄수확 수삼의 항공 및 선박의 모의수출 유통 중 손실유형을 조사하기 위해 수삼을 기능성 LDPE 필름에 5 kg 대포장 또는 500 g 소포장으로 포장하고 각기 다른 모의수출 유통 온도 및 모의수출 기간 즉 항공 모의수출 유통은 15℃와 25℃로 설정된 저장고에 4일 동안 저장하고, 선박모의수출 유통은 1℃와 5℃로 설정된 저장고에 14일 동안 저장한 뒤 유통실험을 위해 최종 10℃로 설정된 저장고에 수삼을 옮기고 수삼의 품질 변화를 조사하였다. 수삼의 품질 조사는 포장단계부터 10℃유통기간까지 총 기간 수행하였다. 조사결과 수삼의 유통 중 손실률에 가장 크게 영향을 미치는 것은 물러짐과 곰팡이 발생에 의한 부패로 나타났다. 물러짐과 곰팡이에 의한 부패를 합한 종합 손실률에서 손실률 20%를 유통한계 기간으로 정했을 때 항공 모의수출유통 조건인 25℃에서 5 kg 대포장은 10일, 500 g 소포장은 6-7일에 20%에 도달했으며, 15℃는 5 kg 대포장은 17일, 500 g 소포장은 12-13일에 20%에 도달했다. 선박 모의유통 조건인 5℃는 5 kg 대포장은 21일, 500 g 소포장은 28일경에 20%에 도달했고, 1℃는 5 kg 대포장은 24일, 500 g 소포장은 29일 경에 20%에 도달하는 것으로 나타나 수삼의 유통은 모의수출 유통 방법뿐 아니라 포장용량도 손실률에 상당한 영향을 미치는 것으로 나타났다. 수삼의 부패에 관여하는 병원균 중 곰팡이 균은 대부분Fusarium sp., Botrytis sp., Penicillium sp. 균들로 동정되었고, 25℃에서는 Fusarium sp. 균이 우세종이고, 그 이하의 온도에서는 Botrytis sp. 균이 우세종으로 나타났다.

Angelica tenuissima, also known as Ligusticum tenuissimum, is classified as a food-related plant and has been used as traditional medicines treating headache and anemia in Asia. However, its anti-melanogenic effect has not been reported in detail. When the extract of Angelica tenuissima (ATE) was prepared by the extraction with 70% EtOH at 80°C (final yield = 22%), the contents of decursin and Z-ligustilide in ATE were determined 0.06% and 8.43%, respectively. Total flavonoid and phenolic content in mg ATE were 5.52±0.07 ㎍ quercetin equivalents and 237.27±13.24 ㎍ gallic acid equivalents, respectively. Antioxidant capacity of ATE determined by DPPH and ABTS assay was increased with a dose dependent manner up to 1000 ㎍/㎖. The amount of melanin synthesis followed by α-melanocyte stimulating hormone on B16F10 cells were significantly reduced in the presence of ATE (250 to 1000 ㎍/㎖, p<0.05). ATE (125 to 1000 ㎍/㎖, p<0.05) suppressed the tyrosinase activity but did not show any significant effect on α-glucosidase activity at the same condition. Taken together, ATE possesses tyrosinase inhibitory potential with significant antioxidant capacities. These effects of ATE might be involved in suppression of melanin synthesis, at least, in B16F10 cells. The anti-melanogenic potential of ATE will provide an insight into developing a new skin whitening product.

Spatiotemporal expressions of microRNAs (miRNAs) are altered by the physiological states of cells which could be influenced by microenvironment. Function of miRNAs has been focused as a new regulator of gene expressions and cell differentiation in human health and diseases. We found and identified the several miRNAs, which were related to developmental competence of preimplantation and implantation process of mouse blastocysts and outgrowth embryos by microarray-based bioinformatical studies. In this study, we evaluated three miRNAs expressions related to third cleavage event in conditioned media (CM) and blastocysts. Mouse 2-cell stage embryos were collected and monitored for 9 hours. The embryos were divided two groups as early third cleavage before 9 hours of collection and late third cleavage after 9 hours of collection. They were cultured to blastocyst stage up to day-5 after hCG injection. The total number of cells and the number of cells with fragmented DNA were assessed in blastocysts by terminal dUTP nick-end labelling (TUNEL) staining and DAPI staining. Mean cell number of early third cleavage group was significantly higher than that of late third cleavage group (105.3±8.0 vs 81.8±7.0, p<0.05), but apoptotic index was not different. The miRNAs of CM and blastocysts from early and late group were prepared, and quantified by qRT-PCR with TaqMan probes. The expression levels of three miRNAs (mmu-let-7b, mmu-miR-183, and mmu-miR-429) in CM and blastocysts were slightly upregulated in late third cleavage group. Our study suggested that the expression level of miRNAs could be altered with embryo quality, and miRNAs in CM may be used to predict miRNAs expression of embryos and developmental competence.

EP was obtained through 20% ethanol extraction of Pueraria lobata root, and the fermented form of EP, FEP, was prepared from the EP after incubating with Lactobacillus rhamnosus vitaP1. There was no significant toxicity by EP and FEP up to 1000 ㎍/㎖ in NIH-3T3, HaCaT, and B16F10 cells. In addition to antioxidant potentials of EP and FEP determined by DPPH and ABST assays, we confirmed increase of procollagen type I and elastin synthesis by supplementation of the EP and FEP at the concentration of 50 ㎍/㎖ using ELISA kits. The protein expression levels of matrix metalloprotease (MMP)-1, -3, and -9, those are involved in the degradation of collagen or other skin matrix proteins, were remarkably suppressed while their inhibitory protein metallopeptidase inhibitor 1 (TIMP-1) was greatly up-regulated by supplementation of the EP and FEP at a concentration of 50 ㎍/㎖. Taken together, both EP and FEP supplementation could be involved in the suppression of the skin wrinkle formation through inhibiting degradation of collagen and stimulating the synthesis of collagen and elastin. The results showed that the anti-wrinkle potential of the EP and FEP will be a promising candidate for developing cosmeceutical compounds or products.

Background : Ginseng widely cultivated as a major medicinal herb in Korea, is economically important crop for farmer. Ginseng root disease caused by soil borne pathogens is main factors restricting the quantity and quality of ginseng. The disease can result in harvest loss of up to 20~70% and limits the replanting of ginseng under same field for long time. The traditional control method of agrochemical use is not recommend to control soil borne disease because of difficulty in use and unstable effect. The objective of this study was to evaluate the efficacy of several antagonistic microbes for developing biological control method of ginseng root rot. Methods and Results : To select biocontrol agents against ginseng soil borne disease, several bacteria were isolated from ginseng root and rhizosphere soil evaluated in vitro screening of antifungal bacterial against ginseng root pathogens. Two antagonistic bacteria, ES17 and CJ4, showed the strongest inhibition effect against ginseng root pathogen. In the pot experiment under greenhouse conditions, ginseng seedling dipped in bacterial suspension at inoculum density of 106 cfu/ml for 1 hour were planted in pot containing inoculum. Control effect was examined depend on disease severity index at 30 days after inoculation. Ginseng root treated with CJ4 and ES17 isolate reduced root rot disease development on the ginseng root with degrees of control efficacy of 85% and 70%, respectively. Conclusion : Two biocontrol agent, Burkholderia ambifaria CJ4 and Paenibacillus strain ES17, had strong antifungal efficacy against ginseng soil borne pathogens. These results obtained from in vitro test and pot experiment suggest the potential applicability of the biocontrol agent to control ginseng root rot caused by various soil borne pathogens.

This study was conducted to determine the stress response [ethological (operculum movement number (OMN)), hematological (hematocrit and hemoglobin), biochemical (glucose, cortisol and glutamic oxaloacetic transaminase (GOT))] in red spotted grouper, Epinephelus akaara during exposure of different water temperature in winter season. This species (Total length, 18.56±0.34 cm) previously maintained in water temperature of 15°C were transferred to 15, 20 and 25°C. During experimental period (7 days), OMN, hematocrit (Ht), glucose and GOT values were significantly high in 15°C when compared to 20 and 25°C. Hemoglobin value was also increased at 15°C, but no significant differences. There was no differences in cortisol levels among the temperature groups. No fish mortality was observed during the experimental period. From these results, 15°C is likely more stressful to red spotted grouper than 20°C and 25°C. These observations confirm that red spotted grouper adapts better to temperatures between 20 and 25°C during the winter season.

Mungbean (Vigna radiata) is a fast-growing, warm-season legume crop that is primarily cultivated in developing countries of Asia. We constructed a draft genome sequence of mungbean to facilitate genome research into the subgenus Ceratotropis and to enable a better understanding of the evolution of leguminous species. The draft genome sequence covers 80% of the estimated genome, of which 50.1% consists of repetitive sequences. In total, 22,427 high confidence protein-coding genes were predicted. Based on the de novo assembly of additional wild mungbean species, the divergence of what was eventually domesticated and the sampled wild mungbean species appears to have predated domestication. Moreover, the de novo assembly of a tetraploid Vigna species (Vigna reflexo-pilosa var. glabra) provided genomic evidence of a recent allopolyploid event. To further study speciation, we compared de novo RNA-seq assemblies of 22 accessions of 18 Vigna species and protein sets of Glycine max and Cajanus cajan. The species tree was constructed by a Bayesian Markov chain Monte Carlo method using highly confident orthologs shared by all 24 accessions. The present assembly of V. radiata var. radiata will facilitate genome research and accelerate molecular breeding of the subgenus Ceratotropis.

We study on the dynamic interaction with a simulated physical-biological coupled model response to nutrient reduction scenario in Jinhae Bay. According to the low relative errors, high regression coefficients of COD and DIN, and realistic distribution in comparison to the observation, our coupled model could be applicable for assessing the marine ecosystem response to nutrient input reduction in Jinhae Bay. Due to the new construction and expansion of sewage treatment plant from our government, we reduce 50% nutrient inputs near Masan Bay and sewage treatment plant. COD achieves Level Ⅱ in Korea standard of the water quality from the middle of the Masan Bay to all around Jinhae Bay except the inner Masan Bay remaining at Level Ⅲ. When our experiment reduces 50% nutrient inputs near Masan Bay and Dukdong sewage treatment plant simultaneously, COD decreases to about 0.1-1.2 mg/L (128°30’~128°40’ E , 35°05’~35°11’ N). The COD from the middle of the Masan Bay to Jinhae Bay achieves Level Ⅱ.