The aim of this study was to examine the effect of acteoside (the cyclin-dependent kinase inhibitor) on the SCNT efficiency with adult fibroblasts in dog. Canine adult fibroblasts were obtained from muscle and cell cycle of fibroblasts was synchronized by culturing to confluency, serum starvation and treating with 30 μM acteoside for 48 h. Cell cycle stages, cell cytotoxicity (apoptosis) and, prduction of reactive oxygen species (ROS) were analyzed using flow cytometry. The canine cells, prepared by confluent-cell culture or treating with 30 μM acteoside for 48 h, were injected into enucleated in vivo matured oocytes, the couplets were electrical fused and activated by calcium ionomycin. SCNT embryos using acteoside-treated fibroblasts were surgically transferred into oviducts of estrus cycle synchronized recipient dogs. In cell cycle synchronization (G0/G1), there was no significant difference between serum starvations (83.9%) and acteoside treated groups (81.3%) that were higher than confluent group (78.5%). In production of apoptosis, confluent and acteoside treated groups (4.3 and 4.5%, respectively) were generated less than serum starvation group (21.8%). In case of ROS, serum starvation group was induced a significantly higher than other groups. After synchronization of the donor cell cycle, either confluent or acteoside treated, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into naturally estrus-synchronized surrogates. Fusion and cleavage rate of acteoside treated group were 64.1 and 41.5%, which were higher than those of confluent group (53.9 and 20.6%, respectively). The reconstructed embryo development rates to 4-cell and 8-cell in acteoside treated group were 29.5 and 14.8%, respectively, while confluent group showed 11.1 and 3.2%, respectively. Total 54 SCNT embryos using acteoside-treated fibroblasts were transferred into oviducts of 2 recipient dogs and one recipient finally delivered one puppy, whereas din`t detected pregnancy on transfer of cloned embryos reconstructed with confluent cells in 6 surrogate dogs. In conclusion, the results of the current study demonstrated that canine fibroblasts could be successfully arrested at the G0/G1 stage with reduced the formation of ROS and apoptosis after acteoside treatment. This results may contribute to improve the effi-ciency of canine SCNT. * This research was supported by iPET (Grants 110056-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Acteoside acts as an anti-oxidative activity and anti-apoptosis in the cells. But, it has been not studied on maturation and development of porcine oocytes. The aims of the present study were to examine the effects of acteoside on the morphological progress of meiosis, developmental competence, and ROS in porcine oocytes. Oocytes were matured in tissue culture medium-199, supplemented with acteoside at various concentrations: 0 (control), 10, 30 and 50 μM. The oocytes maturation rates of groups supplemented with acteoside were no significantly different (81.13, 85.96, 82.95 and 83.68%, respectively). Level of ROS was significantly decreased in acteoside treated group. Furthermore, the parthenogenetic blastocyst rate was significantly improved in 10 μM acteoside treated group compared with control group (44.83 vs. 27.75%). And we investigated effect of acteoside on the oocytes condition represented by cytoplasmic maturation by homogeneous distribution and formation of cytoplasmic organelles and regulation of apoptosis-related genes. In the results. during IVM, 10 μM acteoside treated oocytes showed that the mitochondria and lipid droplet were smaller and homogeneous distribution in cytoplasm compare with control oocytes. And reverse transcription polymerase chain reaction (RTPCR) of parthenogenetic blstocysts revealed that acteoside increased the anti-apoptotic genes (Mcl-1, Bcl-2 and Bcl-xL), whereas reduced the expression of pro-apoptotic genes (Bax and Bak). In conclusion, based on the results, the effect of acteoside on IVM was not attractive. However, in acteoside treated group, cytoplasmic maturation seemed to be improved with morphologically uniform distribution of cytoplasmic organelles. Furthermore, embryonic development in acteoside treated group was significantly highly increased than that of non-treated group. Our results represents that addition of acteoside to the IVM medium has a beneficial effect in physiology of porcine oocytes, providing a improved method for porcine oocytes in vitro. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.

The objective of the current study was to describe in vitro embryo production in Hanwoo, analyzing oocytes yield and embryo production. The effects of oocytes production and the number of OPU procedures per animal on embryo production were also evaluated. OPU was done every 3～4 days during experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, we compared the recovery rate of oocytes based on OPU session (Experiment 1). The average of collected oocytes was calculated from every 10 session. The average number of total oocytes recovered per animalonsessionwas 5.16 (mean). Second, we compared the recovery rate base on collection period of OPU (Experiment 2). The following results show the difference of the number of recovered oocytes in every month during the procedure between the months of session. Every animal shows the constant number of recovered oocytes for the first 5 months. However, the recovery rate of oocytes was decreased from month 6 to 8. Third, we compared the developmental rate to blastocyst in two groups (Experiment 3). Oocytes by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24～48 h after in vitro fertilization (IVF) was 75.8% and blastocyst development rate was 18.8% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (61.1%), blastocyst development rate was higher compared with needle puncture group (28.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio- technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being.

This study examines how students and instructors perceived online instructors’ roles in asynchronous courses designed for language teachers. Five instructor interviews, 46 student questionnaires, and ten course evaluation summaries presenting the opinions of 69 out of 120 students in total were analyzed. Based upon Berge’s (1995) framework, asynchronous online course instructors’ roles were divided into four categories: pedagogical, social, managerial, and technical. The results highlight and give suggestions to what is characteristic and likely problematic in online teaching—using the tools and pedagogical techniques to make learning more social than conventional classroom exhibit, orchestrating discussion not only technically but also by instructing and modeling effective postings, assessing the various ways that students participate in the class, and managing time, the students’ as well as the instructor’s. This study provides an opportunity for language educators and researchers to think about the unique characteristics of web-based distance education in general and the changing roles of instructors in that new form of education in more critical way.

For reconstituting genetic resource(Korean Native Chicken: KNC) with grem-line chimeric chicken made with cryopreserved biastdermal cells, the experiments were carried out to optimize cryopreservating conditions. Stage X biastdemal cells were collected from KNC embryos and dissociated. Cells were susupended in medium containing cyopretectant and fetal bovine serum(FBS), and distributed into plastic ampules. Cell susupensions were seeded to induce ice formation at —7 ℃ to —35 ℃ at in the experiments, the effect of modification of dissociation way, concentration of FBS and cell density on the vaibility of frezen-thawed cells were investigated by trypan blue exclusion. Then change the way of cell dissociation from pipetting to short time vortexing, viability of frozen-thawed cell tended to be increaced from 29 % to 52 %. Increase concentraition of FBS in frozen medium from 20 % to 80 % made viability of thawed cell from 28 % to 35 %. The viability of thawed cells were 33.9% frozen at 2 embryos/ 0.5ml, and 43.6 % frozen at 20 embryos/0.5 ml. Furthermore, combination of three modifications make big improvement. The viability of frozen-thawed cell was 60 % for combinated method, and 41 % for general method. This result means the advance to practical cryoreservation of blastdermal cell of the KNC(Ogolgye breed).

Cytokines are known to function as regulatory molecules that can be produced by virtually every nucleated cell type in the body, including lymphocytes, monocytes/macrophages, epithelial cells, fibroblasts, and many others. Cytokines include lymphocyte-derived factors (lymphokines), monocyte-derived factors (monokines), hematopoietic factors (colony-stimulating factors), connective tissue/ growth factors, and chemotactic chemokines. Cytokines released in response to infection can affect tumor development in different ways. When exposed to infectious agents, cytokines are secreted by sentinel cells, such as macrophages and dendritic cells. These cytokines include interleukin 1 (IL-1) and tumor necrosis factor-α, as well as others, such as IL-6, IL-12, and IL-18. When released in sufficient quantities, these molecules can cause inflammation. Chronic inflammation is highly associated with tumor initiation, promotion, and progression. In this article, we review the roles and mechanisms of cytokines in tumor development.

Beauveria bassiana is one of universal insect pathogenic fungi that have been used for biocontrol agent against insect pests. This fungus has also been studied for medicinal use. To meet for commercial use, the artificial production of the fruit body of this fungus has been established by the Mushtech Co in Korea. This study was carried out to define the morphological features of the fruit body of B. bassiana developed through artificial cultivation. For the observation of mycelia growth, B. bassiana was cultured on the Sabouraud Dextrose agar plus Yeast Extract(SDAY), nut-supplemented medium, and Fe ion-supplemented SDAY at 25℃ for 15 days. The variation of colony color was observed between the different media. Strong pigmentation was observed on Fe ion-supplemented SDAY. To investigate morphological characteristics of fruit body, geminating ascospores and vegetative hyphae were observed though light microscopy and scanning microscope. During seven weeks of cultivation period, the development process of apical fertile part of stromata can be separated by the development stage of perithecia. To understand the developing process of fruit body at the transcript level, investigating process of distinct gene expression according to cultural condition and developmental stage was discussed.

The purpose of this study is to examine the effect of stabilization, resistance exercise, and combined exercise for lumbar on balance and walking ability of elderly. This study selected 22 subjects of over 65 years old elderly women who satisfy the study condition from A, B and C Senior Citizen's Center, in Daegu. The subjects were divided into groups; 7 for resistance exercise group, 8 for stabilization exercise group, and 7 for combined exercise group(resistance and stabilization exercise). The exercises were conducted for 60min a day, three times a week for 12 weeks. Balance and walking ability were checked before the exercise, 6 weeks later, and 12 weeks later. First, all lumbar muscle strengthening exercises were effective for static balance which changed according to exercising period. In dynamic balance, the resistance exercise group showed significant improvement in sit to stand. Stabilization exercise group showed significant improvement in all factors. The combined exercise group showed significant improvement in sit to stand and timed up and go. There was no difference between the exercise types. Second, the resistance exercise group showed significant change in Cadence which changed according to exercising period. The combined exercise group showed significant improvement in all factors. Between the exercise types, combined exercise was a bit more effective than resistance exercise. According to the result of 12 weeks of lumbar muscle exercise, combined exercise is considered to be the most effective exercise to prevent fall as it helps balance and walking ability slightly more than other exercises. Therefore, this study can understand the risk factors for fall accidents that frequently occur among elderly and adopt the combined exercise to prevent fall which in turn will prevent secondary problems occur from fall accidents and improve quality of life of elderly.

The objective of this study was to evaluate in vitro production of bovine embryos in Hanwoo. Oocytes were collected by ovum pick up (OPU) from ovaries of genetically high-value Hanwoo or by needle puncture from ovaries of slaughtered cattle. OPU was done every 3 4 days duing experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, We compared the in vitro maturation rate in two groups (Experiment 1). 545 oocytes were recoverd from 4 females by 32 trials of OPU and then 433 oocytes were shown MⅡ stage after in vitro maturation (79.4%). In case of needle puncture group, 1905 oocytes were collected and then 1420 oocytes were matured to MⅡ stage during in vitro culture(74.5%). Second, we compared the developmental rate to blastocyst in two groups (Experiment 2). 1420 oocyte by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24 48 h after in vitro fertilization (IVF) was 88.6% and blastocyst development rate was 20.5% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (84.8%), blastocyst development rate was higher compared with needle puncture group (26.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio-technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being. This research was suppoted by Imsil-gun agricultural technology service center.

Population dynamics of greenhouse whitefly, Trialeurodes vaporariorum, were modeled and simulated to compare the temperature effects of air and tomato leaf inside greenhouse using DYMEX model simulator. The population phenology model of T. vaporariorum was developed and simulated. Leaf temperature on reversed side of cherry tomato leafs was monitored according to three tomato plant positions (top, >1.6m above the ground level; middle, 0.9 - 1.2m; bottom, 0.3 - 0.5m) using an infrared temperature gun. Air temperature was monitored at same three positions using a temperature logger. The leaf temperatures from three plant positions were described as a function of the air temperatures. The number of T. vaporariorum immatures was counted by visual inspection in three positions to verify the performance of DYMEX simulation. A significant positive correlation between the observed and the predicted numbers of immature and adults was found when the leaf temperatures were incorporated into DYMEX simulation, but no significant correlation was observed with air temperatures. This study demonstrated that the population dynamics of T. vaporariorum was affected greatly by the leaf temperatures, rather than air temperatures in cherry tomato greenhouses. This work was supported by Development of field-oriented model for forecasting outbreak of diseases or insect pests based on GIS and IT Program (R1003852) from Rural Development Administration, Republic of Korea.

Porcine Circovirus Type2 (PCV2), a single-stranded DNA virus associated with Postweaning multisystemic wasting syndrome(PMWS) of swine, has two major open reading frames, ORF1 and ORF2. The genomic size and molecular weight of ORF2 is respectively 699bp, 28kDa. ORF2 encodes the capsid protein (structural protein) that has type-specific epitopes and is very immunogenic and associated with the induction of neutralizing antibodies, suggesting its potential use in diagnostic assays as well as vaccine development. For efficient production of the capsid proteins, we expressed the PCV2 ORF2 gene with baculovirus in the insect cells. In this study, PCV2 ORF2 was appropriately ligated into the baculovirus transfer vector, pBacPAK9 and pB9-Acpol19-110-EK. Sf21 cells were transfected with a mixture of the purified recombinant transfer vector and bAcGOZA. We generated and purified recombinant viruses containing PCV2 ORF2, and named rAc-B9-PCV2ORF2 and rAc-B9-19-110-EK-PCV2ORF2, respectively. Expression levels of capsid fusion proteins with a partial polyhedrin region of AcNPV more increased than recombinant proteins from non-fusion expressed. Also, expression efficiency increased over time and differed at MOI. As a results, fusion expression of porcine circovirus type2 ORF2 using baculovirus could be utilized as an alternative expression method to produce recombinant antigen against PCV2 infection and is worthy of further investigation.

Aujeszky's disease (AD), also called pseudorabies, is an infectious viral disease caused by an alpha herpes virus and has domestic and wild pigs, as well as a wide range of domestic and wild animals, as the natural host. Aujeszky's disease virus (ADV) virions contain several envelope glycoproteins. Among them, glycoproteins gB, gC and gD are regarded as the major immunogenicity proteins and the antibodies induced by them can neutralize virus in vitro or in vivo. In this study, we investigated expression of these glycoproteins using the bacterial and baculovirus expressionn system. Successful expression of ADV glycoproteins in E. coli was confirmed by SDS-PAGE and Western blot analysis and their optimal expression condition was determined. However, the recombinant proteins generated in the bacterial expression system which lacks glycosylation process frequently lose their biological activity. We tried to express the ADV glycoproteins using the baculovirus expression vector system. The recombinant gB, gC and gD were detected at approximately 100, 60 and 50 kDa on SDS-PAGE and Western blotting, respectively. The optimal expression conditions were determined for MOI(multiplicity of infection) and post-infection days. One MOI and 4 or 5 days post-infection were the best conditions for the expression of the ADV glycoproteins in Sf21 cells. We are currently investigating the antigenicity of recombinant proteins using experimental animals.

The four genetically distinct isolates have been identified previously from Bombyx mori nucleopolyhedroviruses (BmNPVs) isolated in Korea. To further understand the complex of viruses infecting Bombyx mori, the genome of BmNPV-K1 and K4 strains was completely sequenced and analyzed in comparison with the genome of other sequenced baculoviruses including previously reported BmNPV. BmNPV-K1 consisted of 127,542 bp and 133 open reading frames (ORFs) of 150 nucleotides or longer with minimal overlap have been identified. In contrast, BmNPV-K4 consisted of 128,615 bp and 134 open reading frames (ORFs). Although gene arrangement is virtually identical, the genome of BmNPV-K4 is 1,073 bp longer than BmNPV-K1. This was related to the more existence of bro genes in BmNPV-K4. To investigate the relationship between BmNPV-K1 and K4, phylogenetic analysis with each member of the paired ORFs was performed. The sequence data suggest that BmNPVK1 and BmNPV-K4 are closely related but have diverged and evolved into two separate strains. This was study to identify highly related but separately evolving viruses in the same insect host and geographic location. We are currently comparing the differences of these BmNPV genomes to elucidate characteristics of each virus.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). To enhance the expression level of baculovirus vector system, we constructed several fusion vectors using various fragments of the polyhedrin. The polyhedrin fragments were genetically fused to the enhanced green fluorescent protein (eGFP) under the control of polyhedrin promoter, and their expressions were analyzed in Sf21 insect cells. Expression of the fusion protein was identified by SDS-PAGE and Western blot analysis using anti-GFP and anti-Polyhedrin. The expression level of eGFP was markedly increased by the fusion of partial polyhedrin. Also, the fluorescence intensity of fusion proteins was higher than that of non-fusion protein. Confocal laser scanning microscopy demonstrated that fusion proteins were localized to the cytosol or nucleus of insect cells. In additional, the glycoprotein E2 (gE2) of classical swine fever virus (CSFV) expressed by the these vectors was dramatically increased and its immunogenicity was proofed using experimental animal guinea pigs that were immunized with the partial polyhedrin containing gE2. This study provides a new option for the higher expression of useful foreign recombinant protein by using the partial polyhedrin in BEVS.

Recently, farmers of mushroom farms in Korea express the difficulty of shiitake cultivation due to the damage of oak log beds by unknown reason. To elucidate the cause of the damage, we investigated and isolated fungi from inside wood chip of the damaged shiitake log bed. Four of green fungi were obtained and their morphology and molecular properties examined. Trichoderma atroviride, Trichoderma citrinoviride, Hypocrea lixii (Trichoderma harzianum), and Hypocrea lutea (Gliocladiumviride) were identified. Hypocarea lutea is newly described in Korea. These species were able to produce extracellular enzymes such as cellulase, pectinase, and xylanse that might degrade wood components of oak logs. This study provides strong evidence that Trichoderma fungi are involved in the recent damages of shiitake log beds In Korea.

Mamestra brassicae nucleopolyhedrovirus-K1 (MabrNPV-K1) was isolated from naturally infected Mamestra brassicae (Lepidoptera: Noctuidae) larvae in Korea. Restriction endonuclease fragment analysis using EcoRI, PstI, and BamHI estimated that the total genome size of MabrNPV-K1 is about 150 Kb. The full genome sequences of MabrNPV-K1 were determined, analyzed and compared to those of other baculoviruses. The MabrNPV-K1 genome consisted of 152,471 bp and had an overall G + C contents of 39.90 %. Computer-assisted analysis predicted 159 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. The gene content and arrangement in MabrNPV-K1 were most similar to those of Mamestra configurata nucleopolyhedrovirus-B (MacoNPV-B), including three polh, p10 and lef-8 gene homologues. The MabrNPV-K1 genome contains four homologous repeat regions (hr1,hr2,hr3,hr4) that account for 3.1% of the genome. The genomic positions of MabrNPV-K1 regions hr1– hr4 are conserved with the genomic positions of MacoNPV-B hr1–hr4. This indicates that the position of MabrNPV–K1 hrs is conserved with regard to both the upstream and downstream genes. Given that hrs share higher similarity within a virus strain than any hrs between species, this evidence further indicates that hrs play a fundamental role in viral life cycle and replication process appears to be tightly linked to functional conservation. The dot plot analysis, percent identity of the gene homologues and a phylogenetic analysis suggested that MabrNPV-K1 is a Group II NPV that is closely related to MacoNPV but with a distinct genomic organization.