한국 내 전국적으로 5곳의 담수에서 2010년 4월부터 2011년 6월에 걸쳐 담수시료를 채취한 후, 각 담수시료에서 Botryococcus braunii의 출현을 확인하고 microcapillary-pipetting method를 이용하여 5주를 분리하였다. 분리된 B. braunii의 18S rRNA sequencing 후, BLAST search를 통한 분석 결과는 B. braunii와 가장 높은 상동성을 나타낸 것으로 확인되어 형태적 동정결과와 일치하였다. 진화, 계통분류학적 분석결과, 분리된 5주의 B. braunii는 단일 계통인 Trebouxiophyceae으로 분류되었다. 18S rRNA sequence의 계통 분류학적 분석에서 B. braunii KNU6는 다른 분리된 B. braunii와 같은 분류군 내에서 거리의 차이가 있었고, 또한 세포크기, 점액질의 양 등의 형태적 특징도 차이를 보였다. 한국에서 분리된 5주의 B. braunii는 모두 race A type의 분류군에 포함되며, 탄소수 C23-C33의 지방산에서 유래된 alkadienes과 alkatrienes를 생산하는 것으로 사료되며, B. braunii를 이용한 바이오디젤을 생산하기 위한 최적의 배양조건을 찾는 연구가 필요하다.

The objective of this study was to compare of different isolation method of mouse preantral follicles, and to examine in vitro development of mouse preantral follicles isolated by different method. Preantral follicles were mechanically or enzymatically extracted from mouse ovaries. Mechanical isolation method used fine gauge needles and enzymatic method of isolating follicles used collagenase. The recovered preantral follicles were cultured for 10 days in alpha-minimal essential medium (-MEM) + 5% FBS + Insulin-Transferrin-Selenium (ITS) + 100 mIU/ml FSH. The collected primary follicles by enzymatic treatment were higher than mechanical method. Others stage preantral follicle by mechanical isolation were higher than enzymatic method. After 10 days of culture, no statistical differences were shown in survival rates of preantral follicle among the 2 culture groups. The metaphase II rates of the oocytes were significantly higher (p<0.05) in mechanical method (17.8%) than in enzymatic method (5.1%). These results suggest that the isolation method of choice depends on the target stage preantral follicles and mechanical isolation is an optimal method of preantral folliclesin a culture of mouse preantral follicle.

버섯 생산 후 발생되는 부산물인 새송이버섯 수확 후 배지로부터 surfactin을 생성하는 4종의 균주를 분리하였으며 이 중 곰팡이 독소를 생성하는 A. flavus와 A. ochraceous 에 대한 항균활성이 우수한 균주를 최종 선발하여 YJ07로 명명하였다. Bacillus ID kit와 VITEK 2 system를 이용하여 분리균 YJ07의 생리적·생화학적 특성을 조사한 결과 분리균은 B. subtilis, B. amyloliquefaciens와 유사한 생리적·생화학적 특성을 나타내었으며 16S rDNA 염기서열 분석을 통한 계통학적 유연관계에서도 B. amyloliquefaciens와 99.5%의 상동성을 나타내었다. 이와 같은 결과를 종합하여 분리균 YJ07은 B. amyloliquefaciens YJ07로 동정되었으며 분리균 YJ07이 생성하는 항균물질은 TLC와 HPLC 분석에서 reference 물질로 사용한 srfactin과 유사한 특성을 나타내었다.

현재까지 스마트 면진시스템은 일본이냐 미국 같은 강진지역에서 개발되고 적용되어 왔다. 이렇게 강진지역에 있는 건축물을 지진하중으로부터 보호하기 위하여 개발된 스마트 면진시스템은 우리니라와 같은 중약진 지역에 있는 건축물에 그대로 적용되기에는 많은 한계점이 있다. 따라서 본 연구에서는 강진지역에 건설되는 건축물을 위한 스마트 면진시스템을 중약진 지역에 건설되는 건축물에 작용하였을 때 발생하는 문제점음 검토해보았다. 이를 위하여 예제구조물로 대공간 아치구조물을 선택하였고 스마트 면전시스템은 MR 감쇠기와 저감쇠 탄성베어링을 사용하여 구성하였다. 강진지역과 중약진 지역에서 발생하는 지진하중으로는 기존에 발생한 역사지진을 바탕으로 인공지진을 생성하였다. 수치해석결과 강진지역에 건설되는 대공간구조물을 위하여 개발된 스마트 면진시스템을 그대로 중약진 지역에 적용하면 면진효과가 상당히 줄어들므로 스마트 제어장지의 용량이 중약진 지역에 맞추어 주의 깊게 설계되어야 함을 알 수 있었다.

In order to isolate thermophilic compost-promoting bacteria with high activity of cellulase and xylanase, spent mushroom substrates with sawdust were collected from mushroom cultivation farm, Jinju, Gyeongnam in Korea. Among of the isolates, one strain, designated HJ01 was selected by agar diffusion method. The strain HJ01 was identified as members of the Bacillus subtilis by biochemical characteristics using Bacillus ID kit and VITEK 2 system. Comparative 16S rDNA gene sequence analysis showed that strain HJ01 formed a distinct phylogenetic tree within the genus Bacillus and was most closely related to Bacillus subtilis with 16S rDNA gene sequence similarity of 99.3%. On the basis of its physiological properties, biochemical characteristics and phylogenetic distinctiveness, strain HJ01 was classified within the genus Bacillus, for which the name Bacillus subtilis HJ0 is proposed. The cellulase and xylanase activity of B. subtilis HJ0 was slightly increased according to bacterial population from exponential phase to stationary phase in growth curve for B. subtilis HJ01.

Spent mushroom substrate(SMS) is a by-product remaining after a crop of mushrooms. About 9 thermophilic strains were isolated from SMS(Flammulina velvtipes). Among of them, one isolate, designated UJ09, showed the antifungal activity against Aspergillus flavus and Aspergillus ochraceous producing mycotoxin on PDA medium, potentially. The strain UJ09 was produced cellulase and xylanase as hydrolase. The strain UJ21 was identified as members of the genus Bacillus by biochemical characteristics using Bacillus ID kit and VITEK 2 system. Comparative 16S rDNA gene sequence analysis showed that strain UJ09 formed a distinct phylogenetic tree within the genus Bacillus and was most closely related to Bacillus amyloliquefaciens with 16S rDNA gene sequence similarity of 99.2%. On the basis of its physiological properties, biochemical characteristics and phylogenetic distinctiveness, strain UJ09 was classified within the genus Bacillus, for which the name Bacillus amyloliquefaciens UJ09 is proposed.

Culture of preantral follicles has important biotechnological implications through its potential to produce large quantities of oocytes for embryo production and transfer. The objective of this study was to determine the comparison of different isolation method of mouse preantral follicles, and to examine in vitro development of mouse preantral follicles isolated by different method. Preantral follicles were mechanically or enzymatically extracted from mouse ovaries. Mechanical isolation method used fine gauge needles and enzymatic method of isolating follicles used 1 mg/ml collagenage (Type IA) and 0.2 mg/ml DNase Ⅰ in Leibovitz L-15 medium. The solution containing Leibovitz L-15 medium, enzyme and ovary fragments was incubated at 37℃ for 30 min. The selection criteria are as follows: primary follicle of 75 to 99 μm, early secondary follicle of 100 to 125 μm and late secondary follicle of 126 to 150 μm in diameter. The recovered preantral follicles were cultured for 10 days in alpha-minimal essential medium (α-MEM) + 5% FBS + ITS + 100 mIU/ ml FSH. The collected primary follicles by enzymatic treatment were higher than mechanical method. Others stage preantral follicle by mechanical isolation were higher than enzymatic method. After 10 days of culture, no statistical differences were shown in survival rates of preantral follicle among the 2 culture groups. The metaphase Ⅱ rates of the oocytes were significantly higher (p<0.05) in mechanical method (17.8%) than in enzymatic method (5.1%). These results suggest that the isolation method of choice depends on the target stage preantral follicles and mechanical isolation is an optimal method of preantral follicles in a culture of mouse preantral follicle.

A bacterium was isolated from feces of Allomyrina dichotoma larva that feeds on well-rotted sawdust of woods. The isolate was identified as a Gram-positive and spore-forming stain. We named the isolate as Bacillus amyloliquefaciens on the basis of morphological and biochemical properties as well as 16S rRNA gene sequences. The culture of bacteria beneficially increased root and shoot growth of tomato, pepper and cucumber plants compared to the distilled water control. In addition, the bacterial culture strongly inhibited the mycelial growth of several fungal phytopathogens. The drop collapse assay with this culture showed a surfactant activity that is a major indicator for the selection of biocontrol agent. Also, a bacterium has ability of wastewater treatment. These data demonstrate the potential application of B. amyloliquefaciens as a biocontrol agent.

In general, zona pellucida (ZP) of the blastocyst has to be removed first, then either isolated the inner cell mass (ICM) or ZP-removed whole blastocyst, which is then cultured on the feeder layer to induce ICM outgrowth for the generation of embryonic stem cells (ESC). However, it is unclear whether ICM isolation before seeding on feeder layer is beneficial or not because the interaction between ICM and trophoblasts may affect cellular growth and/or pluripotency during the culture on the feeder. In the present study, two ZP removal methods (mechanically by splitting with a 28-gauge needle versus chemically by the treatment of acid-Tyrode's solution) and two ICM isolation methods (ZP-free whole blastocyst seeding versus mechanical isolation of ICM) were evaluated for the efficient isolation and culture of putative parthenogenetic bovine ESC. The number of maintained outgrown colonies was counted in each experimental group. As the result, mechanical removal of ZP with a needle and followed by whole ZP-free blastocyst seeding on feeder cells tended to attach more on the feeder layer and resulted in more outgrown colonies with its simple and less time-costing benefits. Currently we are generating ESC lines in HanWoo cattle by using this method for initial outgrowth of the parthenogenetic bovine blastocysts.

Erysipelothrix rhusiopathiae is a causative agent of erysipelas in avian and mammalian species globally. Eight dead 5-week-old Ross broiler chickens were submitted for diagnostic tests in September 2005. Typical signs including white necrotic foci on the liver, focal congestion of the pancreas, severe atrophy and hardening of the bursa of Fabricius were detected. E. rhusiopathiae was isolated from haemorrhagic lung samples. However, no specific PCR product for the viral diseases tested was detected in these chicken samples. To our best knowledge, this is the first report of the isolation of E. rhusiopathiae in broiler chickens in Korea.

본 연구에서는 지진하중을 받는 대공간구조물의 동적응답을 저감시키기 위하여 스마트 면전시스템을 제안하였다. MR 감쇠기와 저감쇠 탄성베어링을 사용하여 스마트 면진시스템을 구성하였으며 최적설계된 LRB 면진시스템과 비교하여 진동 제어성능을 검토하였다. 스마트 면진시스템은 제어알고리즘에 따라서 제어성능이 크게 좌우된다. 본 연구에서는 스마트 면진시스템이 설치된 대공간 구조물을 효과적으로 제어하기 위하여 퍼지제어기를 사용하였다. 면전시스템이 적용된 대공간 구조물의 동적응답과 면진층 변위는 서로 상충관계가 있으므로 퍼지제어기를 최적화하기 위하여 두 응답을 목적함수로 하는 다목적 유전자알고리즘을 사용하여하였다. 수치해석결과 본 연구에서 제안한 스마트 면진시스템을 적용하면 최적설계된 LRB 시스템에 비하여 면진층 변위 및 대공간 구조물의 동적응답을 대폭 줄일 수 있는 것을 확인하였다.

xylanolytic gut bacterium isolated from Eisenia fetida, Cellulosimicrobium sp. strain HY-13, produced an extracellular glycoside hydrolase capable of efficiently degrading mannose-based substrates such as locust bean gum (LBG), guar gum, mannotetraose, and mannopentaose. The purified mannan-degrading enzyme (ManS, 34,926 Da) from strain HY-13 was found to have an N-terminal amino acid sequence of DEATTDGLHVVDD, which has not yet been identified. Under the optimized reaction conditions of 50℃ and pH 7.0, ManK exhibited extraordinary high specific activities of 7,109 IU/mg and 5,158 IU/mg toward LBG and guar gum, respectively, while the enzyme showed no effect on sugars substituted with p-nitrophenol and various non-mannose carbohydrates. ManK strongly attached to Avicel, lignin, β-cyclodextrin, and poly(3-hydroxybutyrate) granules, but not bound to chitin, chitosan, curdlan, or insoluble oat spelt xylan. The aforementioned characteristics of ManS suggest that it is a unique endo-β -1,4-mannanase with out additional carbohydrolase activities, which differentiates it from other well-known carbohydrolases.

From the methanol crude extracts of the tree of heaven (Ailanthus altissima) leaves, the antifeedant substance was isolated and bioassayed with different concentrations against diamondback moth (Plutella xylostella) larvae. The antifeeding activity was evaluated by measuring the feeding area during 24 hr after inoculation. Methanol extracts showing antifeeding activity at 5000 ppm was subsequently fractionated into hexane, chloroform, ethyl acetate and water layer. Third larvae of diamondback moth was tested to each fraction layer. Chloroform layer shows the highest antifeeding activity and the layer was purified by silica gel open column chromatography. The C22 and C23 fractions showed higher antifeeding ratio with 96 and 86%, respectively, and then these two fractions were re-isolated by ODS open column chromatography. As a result, both fractions in methanol 40% (v/v) showed antifeeding ratio over 90%. The C221 fraction showed insecticidal activity in all fraction, however, C231 fraction was showed the antifeeding activity only in C2311 fraction. The C2311 fraction judging to have antifeeding activity was re-isolated and purified by HPLC and recycling, and finally obtained the bioactive substances (C23111) with antifeeding ratio with 88%. The structure of bioactive materials isolated was confirmed by LC-mass and 1H-NMR(500 MHz), 13C-NMR(100 MHz).

Lycorma delicatula (White 1845), which has been recently introduced into Korea, is a notorious pest on grapes. This invasive insect has rapidly spread throughout central and southern Korea. To date, we have no behavioral or population genetics information, such as invasion routes and subsequent dispersal rates in Korea, to help understand and control populations of L. delicatula. Here, we have developed 15 novel microsatellite loci for L. delicatula. The isolated loci were polymorphic, with 2 to 19 alleles in 42 individuals from a single population in Cheonan. The analyses revealed that all 42 individuals had different multilocus genotypes with heterozygosity ranging from 0.214 to 0.866. Eleven of the 15 loci did not deviate significantly from Hardy-Weinberg equilibrium. The isolated markers will facilitate population genetic studies of L. delicatula.

무포자형성 자연돌연변이 균주의 선발 및 특성검정 결과 ASI 2069 균주가 무포자이면서 수량성이 높으나 상품적 가치가 없어 원형질체 재생에 의한 단핵화로 Nh36 (neohaplont 36) 등 4균주를 분리하였다. 원형1호(ASI 2180) 및 무포자느타리간의 단핵교배 (Mon-Mon)를 시도한 결과 128교배조합수를 얻어 이 중 30균주를 특성 검정한 다음 자실체형성 및 포자비산량 조사에 의해 소포자형성 유망 13균주를 선발하였다. 무포자균주의 스크리닝법을 개발하기위해 포자형성 및 감수분열에 관여하는 helicase, DMC1(recombinase) 및 Spo11(topoisomerase II)유전자를 이용하여 PCR 증폭한 결과 단핵화균주 Nh36 및 교배체 2균주 중에서 Spo11유전자가 증폭되지않아 무포자 균주 스크리닝 방법으로 가능하리라 사료된다.

한우 이력추적제에 적용되는 11개의 MS marker (TGLA227, BM2113, TGLA53, ETH10, SPS115, TGLA126, TGLA122, ETH3, ETH225, BM1824 and INRA23)와 성감별을 위한 2개의 sexing primer로 조합된 하나의 Multiplex PCR set를 이용하여 모근에서 추출한 genomic DNA를 이용해 3510두의 대량 시료를 분석한 결과 3.93%의 genotyping 실패율로 성공적인 분석결과를 얻었다. 무작위교배집단으로 가 정 시 동일개체출현확률 (PI)은 1.31×10-23, 반형매교배집단으로 가정 시 동일개체출현확률 (PIhalf-sibs)은 2.52×10-16 그리고 전형매교배집단으로 가정 시 동일개체출현확률 (PIsibs)은 1.09×10-6으로 나타나 현재 사용 중인 11종의 MS marker는 범용적으로 사용하여도 무방할 것으로 재확인 되었다. 또한 생산 및 사 육단계의 생우의 경우 모근을 이용하여 DNA를 추출하는 것은 시료 채취 시 소에게 주어지는 스트레스를 최소화 시킬 수 있을 뿐만 아니라 대립유전자형 분석에 있어서 시간적, 경제적인 효율성을 높일 수 있었 다. 또한 모근 채취 부위 중 등, 배, 꼬리상부와 꼬리하부를 이용하여 검정한 결과 꼬리하부의 모근을 이 용하여 5~13가닥을 사용했을 때 최적의 분석결과를 보였다. 최종적으로 한우의 사육단계 대량 유전자형 분석에 적용 가능한 96 well 단위를 기본으로 하는 모근 DNA분리 체계를 확립하였다.

To identify genes that are differentially expressed, we compared the mRNA expression profile of Harmonia axyridis larvae untreated and treated with LPS. We extracted mRNAs from the larvae with or without LPS treatment, and subjected them to ACP RT-PCR analysis using a combination of 120 arbitrary primers (ACP1-ACP120)and oligo (dT) primer (dT-ACP2). After synthesized cloning DNA from 37 DEGs, it practiced the sequencing homology analysis using BLAST search. Among the 37 DEGs differentially expressed, we identified a cDNA showing homology with previously reported antimicrobial peptide. A cDNA encoding a 82-mer propeptide was identified and its predicted molecular mass and pI was 9.25 kDa and 7.54, respectively. A 35-mer mature peptide was also selected and named herein as Hamoniasin. The antimicrobial activity of chemically synthesized peptide (Mou def 1~8) against human bacterial pathogens was investigate. the result showed all bacteria strains were susceptible to Mou def 2,8 with MIC values in the 32 uM range. And biological changes of the respective cells according to peptide (Mou def 8) treatment were compared. MTT assay was tested that treatment of Mou def 8 decreased cell viability in AML-2, Jurkat, U937 (maximum 200ug/ml, 24hours). That is, fragmentation of DNA, typical characteristics observed in the process of apoptosis, was confirmed in the nucleus of cells dying owing to Mou def 8 treatment.

The two related species in the tribe Archipini, Adoxophyes paraorana and Pandemis heparana (Lepidoptera: Tortricidae), are insect pests of fruit trees in Korea. We investigated differences in pheromone system and seasonal flight of these two species. GC-MS analyses of pheromone gland extracts revealed that females of both species produce blends of Z11-14:OAc, Z9-14:OAc, and Z11-14:OH in similar ratios. The average ratio of three components in extracts was estimated to be 3:100:0.3 for A. paraorana and 3:100:2 for P. heparana. Field tests showed that Z11-14:OAc and Z9-14:OAc were essential for attraction of A. paraorana males and the presence of Z11-14:OH in primary binary blend did not induce any synergistic or inhibitory effect. For the attraction of P. heparana males, however, all three components, Z11-14:OAc, Z9-14:OAc and Z11-14:OH, were indispensable. These results suggest that male A. paraorana do not discriminate between conspecific females and those of P. heparana in the field. Comparison of the flight phenologies in apple and pear orchards showed that the two species are sympatric and overlap in flight periods. This finding eliminates pheromone specificity and seasonal separation as premating reproductive isolation mechanism between A. paraorana and P. heparana.

Embryoid bodies (EBs) generated from human embryonic stem cells (hESCs) include spontaneously induced endodermal lineage cells (ELCs). Activin-A plays important roles in the endoderm differentiation of hESCs. Despite studies on the generation of ELCs from hESCs with treatment of Actvin-A, it was unclear for localization and pattern of ELCs by Activin-A during differentiation of hESCs. Accordingly in this study, we knew that Actvin-A increased the cystic EBs formation, including the highly enriched AFP (endoderm lineage specific marker)-expressing cells in the surface of cystic EBs. To induce the EBs formation from undifferentiated hESCs, cells were transferred onto petri-dish and cultured in suspension condition without bFGF removed hESC media (EB media) for 3 days. Next to investigate the effect of Activin-A, EBs were subsequently cultured in EB media supplement with 100 ng/ml Activin-A for 3 days. After 5～7 days of Activin-A treatment, cystic EBs began to appear which increased in numbers reaching ～60% of initially formed EBs over 5 days. Endoderm lineage marker, AFP were highly expressed and specifically localized at the surface region of cystic EBs comparison with normal EBs. We next attached the cystic EBs onto gelatin-coated plates and cultured for 5 days. In the results of real-time PCR and immunocytochemistry analysis, AFP-expressing cells migrated and localized at the outgrowth region of attached cystic EBs. To obtain the AFP-expressing cells of the outgrowth region, we manually isolated by using micro- dissection and cultured them. These cells strongly express AFP over 70% of isolated cells post re-plating. Here, we first showed an expression pattern of specifically localized ELCs by Activin-A during differentiation of hESCs. From this observation, we could highly purified ELCs from undifferentiated hESCs. Taken together, our system will provide a novel and efficient option to generate ELCs from hESCs.