본 연구는 막걸리 제조 시 아로니아의 첨가 방법을 달리하 여 제조한 후, 발효하면서 항산화 활성 및 이화학적 품질특성 을 분석하였다. 아로니아는 생과와 마쇄한 형태로 첨가하였 고, 쌀 함량의 10%, 30% 그리고 50%로 하였다. 막걸리의 pH 는 발효 초기에 생과를 첨가한 시료가 3.9~4.2로, 마쇄하여 첨 가한 시료(3.6~3.9)보다 높았고, 발효가 진행되면서 생과로 첨가한 경우는 pH가 낮아지다가 다시 증가하는 경향성을 보 였다. 총산의 경우는 초기 발효에는 생과보다 마쇄한 시료에 서 더 높게 나타났고, 발효가 진행될수록 총산의 함량이 증가 하였다. 환원당 함량은 발효가 진행되며 대부분의 시료에서 감소하였다. 에탄올 함량은 1단 담금 시 11.4%로 나타났고, 2단 담금 6일 후에는 10~15%였으며, 특히, 아로니아를 50% 첨가한 실험구는 1단 담금 시 보다 낮은 에탄올 함량을 보였 으며, 마쇄하여 첨가할수록 더 낮은 알코올함량을 보였다. 색 도 중 a값(적색도)을 비교했을 때 생과 사용 시 천천히 a값이 높아져 마지막 날에는 생과 처리구 간 유의적으로 차이가 났 지만, 마쇄한 경우는 처음부터 마지막 발효까지 높은 a값을 가져 생과를 처리한 경우보다 높게 나타났다. 총 폴리페놀함 량 측정과 DPPH radical 소거능 측정 결과는 아로니아 첨가량 이 증가할수록 함께 증가하였는데, 생과첨가군의 경우 초기 에는 낮은 값을 가지다가 발효가 진행하며 급속히 증가하였 고, 마쇄첨가군에서는 초기에서부터 높은 값을 가졌다. 또한 아로니아를 30% 첨가하여도 50%를 첨가한 것과 같이 뛰어난 항산화 활성을 나타내었다. 관능검사는 생과 30%를 첨가 한 시료가 색, 쓴맛, 전체적 기호도에서 높아 관능적 만족도 가 우수한 것으로 나타났다. 이상의 결과로 아로니아를 첨가 한 기능성 막걸리의 특성을 보았을 때 아로니아를 마쇄한 형 태보다 생과 형태로 30%를 첨가하는 것이 관능적 특성과 항 산화 활성을 고려하였을 때 가장 적합한 것으로 나타났다.

Among hemipteran insects which is the most important insect vector of plant viruses, small brown planthopper, Laodelphax striatellus, transmits the rice stripe virus (RSV) causing rice stripe disease. For effective control of RSV, it is important to understand interaction between RSV and L. striatellus. Therefore, in this study, expressed sequence tag (EST) databases were generated based on 454 GS-FLX pyrosequencing for comparative transcriptome analysis between nonviruliferous and RSV-viruliferous L. striatellus. By comparing the two EST libraries, we showed that 108 host genes were significantly up-regulated and 28 host genes were significantly down-regulated in viruliferous insects. Interestingly, genes encoding ribosomal proteins were mainly up-regulated in viruliferous L. striatellus, whereas genes related to translation were concentrated in the downregulated cohort. These RSV-dependently regulated genes may have important function in the behavior of planthopper or the transmission of RSV.

Bacillus thuringiensis serovar mogi of a novel serogroup (H3a3b3d), which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis serovar mogi contained only megaplasmid (> 30 MDa) on which the toxin genes were occasionally located. Sequence analysis using 454-pyrosequencing revealed that the megaplasmid harbored at least seven putative cry genes, showing about 84%, 75%, 73%, 58%, 84%, 39% and 75% similarities in amino acid sequences with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2, respectively. These cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K, and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. To investigate the role of these genes in crystal production, the expression profiles of these toxin genes were analyzed by quantitative real-time PCR (qrtPCR) from the wild type strain as well as transformant strains. The results clearly indicate that the cry39orf2 was the dominant ingredient in the crystal. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, could be used as a good resource for studying unknown mosquitocidal cry genes.

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.

In the transcriptome surveys of Laodelphax striatellus, several cDNA sequences showed a high level of similarities to the insect picorna-like virus genomes. Interestingly, there was no sequence similarity between picorna-like virus sequences from the RSV-viruliferous and those from the non-viruliferous L. striatellus. Picorna-like virus from the non-viruliferous L. striatellus was a geographical isolate of Himetobi P virus (HiPV). The genome of the HiPV was 9,272 nt in length excluding the poly(A) tail and contained two open reading frames (ORFs), which were separated by a 176 nt intergenic region that functions as an internal ribosome entry site (IRES). The 5' ORF encodes the non-structural proteins and the 3' ORF encodes the capsid proteins. The partial genomic RNA of the picorna-like virus from the RSV-viruliferous L. striatellus, LsPV-2, was 8,769 nt in length excluding the poly(A) tail and contained a single, large open reading frame (nt 1–8,535) encoding a 2,845 aa polyprotein. In terms of sequence similarity, identity, and genome organization, LsPV-2 resembled insect picornalike viruses belonging to the family Iflaviridae. A phylogenetic analysis based on RNA-dependent RNA polymerase (RdRp) sequence showed that LsPV-2 was most closely related to the deformed wing virus (DWV). The HiPV and LsPV-2 were incompatible each other in L. striatellus, suggesting that these two picorna-like viruses may have important functions in transmission of the RSV.

Plasmids from Bacillus thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases in mammals and insects. B. thuringiensis serovar mogi of a novel serogroup (H3a3b3d), which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis serovar mogi contained only megaplasmid (> 30 MDa) on which the toxin genes were occasionally located. Sequence analysis using 454-pyrosequencing revealed that the megaplasmid harbored at least seven putative cry genes, showing about 84%, 75%, 73%, 58%, 84%, 39% and 75% similarities in amino acid sequences with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2, respectively. These cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K, and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, could be used as a good resource for studying unknown mosquitocidal cry genes.

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.

Stem cell therapy is undoubtedly the most promising therapeutic approach for neurological disorders. Adipose tissue is ubiquitous and it can be easily harvested in large quantities under local anesthesia with little patient discomfort, making adipose tissue into the ideal large-scale source for research on clinical applications. In this study we monitored the neuronal cell differentiation potential of human adipocyte in the following condition; i) N2 medium containing 200 uM ascorbic acid (AA) and/or 10 uM flavonoid (F) and ⅱ) N2 medium containing AA and/or 10 ng/ml brain derived neurotrophic factor (BDNF) and/or, 200 ng/ml sonic hedgehog (SHH) plus 100 ng/ml fibroblast growth factor (FGF) 8. Adipose stem cells were cultured in above described differentiation condition for three weeks. RT-PCR analysis demonstrated that the mRNA levels of neuronal cell markers in differentiated adipose stem cells. Under the culture condition using N2 medium containing AA, the expression level of nestin (neural progenitor marker) m- RNA was high in all groups, while those of Neuro D, and LEP and FABP4 (adipocyte marker) mRNA were significantly decreased. Also, the addition of BDNF or SHH+FGF8 in N2 medium containing AA enhanced the neural cell differentiation from adipose stem cells, the expression level of Map2 (mature neuron) mRNA was increased, and that of TH (dopaminergic neuron marker) mRNA was high. In addition, we confirmed that the flavonoid addition has effect on the increase of Map2 expression. These results demonstrate that our designed culture condition has effect on the neural cell differentiation of adipose stem cells and this stimulatory effect may be further enhanced by transplantation.

It is known that oocytes can be activated without male contribution in vitro and develop to blastocysts which are used to isolate parthenogenetic embryonic stem (ES) cells. Differentiation capacity of the parthenogentic ES cells was rather lower than that of fertilized embryos derived ES cells, which might be the result of the absence of male genome. However, parthenogenetic ES cells might be useful research tool for genetic engineering and generating SCNT embryo derived ES cells. In our previous study, we reported that establishment of several bovine ES cell lines from in vitro fertilized (IVF) embryos named JNU-ibES. Based on this data, the objective of this study is to generate parthenogenetic ES cells and to examine their stem cell characteristics. Total 107 parthenogenetic embryos produced at day 8 or 9 were classified into their developmental stages (full expanded x 40, hatched x 67). For producing ES cells, ICM and trophetoderm-rich clumps were mechanically dissociated and were cultured on mitomycin- C treated mouse embryonic fibroblast feeder cell drop and covered with mineral oil in DMEM medium containing 20% FBS, 5 ng/ml basic FGF, 1% nonessential amino acids, and 0.55 mM b-mercaptoethanol. We obtained 20 primary parthenogenetic bovine ES (pbES)-like cell colonies. And pbES colony formation was higher in hatched blastocyst (25.4%, 16/67) than expanded blastocysts (10%, 4/40). Among those colonies, 5 pbES cell lines were successfully established and they were named as a series of JNU-pbES. These pbES cells were positively expresssed pluripotency markers such as Oct4, Nanog, TRA-1-81, SSEA-1 and alkaline phosphatase. This result demonstrated that the establishment efficiency and characteristics of pbES cell line was very similar to those of ibES cell line.

The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. This study was to examine the in vitro neuron cell differentiation characteristics of our established human (h) iPS cells (IMR90-iPS-1~2) derived from human somatic cells. For the neuron differentiation, well grown hiPS colonies were recovered by collagenase treatment and then suspended cultured in a non-adherent bacteriological culture dish using human embryonic stem (hES) cell culture medium for 4 days. Embryoid bodies were plated and cultured in serum-free ITSFN (insulin/transferrin/selenium/fibronectin) medium for 8 days to select neural precursor cells. Then selected neuronal cells were dissociated, plated onto poly-L-ornithin/laminin coated dish at a concentration of 2 x 105 cells/cm2 and expanded in N2 medium containing 20 ng/ml bFGF, 200 ng/ml SHH and 100 ng/ml FGF-8 for 7 days. For the final differentiation step involved removing agents and culturing for 14 days in 20 ng/ml BDNF added N2 medium. In the neural precursor stage, >90% of nestin positive cells and >50% NCAM positive cells were obtained. Also, in final differentiation step, we confirmed the high percent (>80%) of mature neuron tubulin-β positive cells and approximately >20% of tyrosine hydroxylase positive cells. Also, these results were confirmed by RT-PCR. These results indicated that hiPS cells have potential to generate specific neuron differentiation and especially TH+ neuron was also can be obtained, and thus hiPS-derived neural cells might be an usable source for the study of neuro-degenerative disease.