To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of polyhedrin fragments were investigated by fusion expression them with the enhanced green fluorescence protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the minimal region for self-assembly and the KRKK nuclear localization signal (NLS). The increase of EGFP production by fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production of EGFP was shown by the mutation of the NLS within the fused polyhedrin fragment. Among the fusion expressed protein in cytoplasm, the most hyper-expression was observed in the fusion of amino acids 32 to 59 of polyhedrin. Polyhedrin fragment fusion expression with classical swine fever virus E2 protein also resulted hyper-enhanced expression of E2 protein. However, the fusion expression of porcine circovirus ORF2 with polyhedrin fragment did not show significant enhance of ORF2 production. These results suggested that the enhancement of foreign protein production when fused with polyhedrin is caused by the enhanced stability of expressed protein.

Interferon induced transmembrane protein-1 (Ifitm-1) has been reported to have an important role in primordial germ cell formation, and it has expressed in female reproductive organ. In the present study, Ifitm-1 gene expression was identified in testes and all part of epididymis using western immunoblot and immunohistochemistry. Interestingly, Ifitm-1 expression was observed on the head of spermatozoa. To investigate the role of Ifitm-1 gene expression in behavior of spermatozoa after acrosome reaction, fresh sperm was incubated with calcium ionophore to induce acrosome reaction, whereas the expression of Ifitm-1 was not altered after the acrosome reaction. Then to identify the effect of Ifitm-1 in sperm motility and other seminal parameters, different concentration of Ifitm-1 antibody was incubated with spermatozoa, and seminal parameters were assessed using computer-assisted semen analysis (CASA). Interestingly, motility, progressive, and VAP were increased in the sperm with Ifitm-1 antibody treated compared to rabbit serum, however other parameters such as straightness were not changed. In order to identify the functional significance of Ifitm-1 in fertilization, capacitated spermatozoa were pre-incubated with anti- Ifitm-1 antibody and subsequently examined the ability to adhere to mouse oocytes. However, any defection or alteration in sperm-egg fusion was not found, Ifitm-1 antibody treated or non-treated spermatozoa showed a normal penetration. Although the precise role of Ifitm-1 in sperm motility and following fertilization need to be elucidated, this study suggests that the activation of Ifitm-1 on the sperm may enhance the motility of spermatozoa in mice.

True hermaphrodites are animals of equivocal sex in which both male and female gonads develop simultaneously. The frequency of true hermaphroditism is higher in pigs than in other domestic animals. Two Korean pigs were diagnosed with true hermaphroditism showing ovotestes, epididymes, penes, and uteri. Histomorphologically, the testicular tissues consisted of Sertoli cells that were devoid of spermatogenic germ cells and showed proliferation of interstitial cells. However, the uteri were of normal architecture and had well-developed uterine endometrial glands. The samples were 38, XX female karyotype without the sex-determining region Y (SRY) gene. The findings of this study could contribute to the understanding of true hermaphroditism in the Korean pig industry. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.

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.

The purpose of this study is to examine the effects of gait training using functional electrical stimulation on the improvement of hemiplegic patients' functions for balance and gait velocity. The subjects of the experiment were determined to be 10 each hemiplegic patients who had been diagnosed with stroke or brain damage six months or longer earlier assigned to an experimental group and a control group respectively. The subjects were evaluated before the experiment using Tetrax and 10M gait tests, received gait training five times a week for four weeks using functional electrical stimulation and were evaluated after the experiment in the same method as used in the evaluation before the experiment. In order to examine differences between the experimental group that received gait training using functional electrical stimulation and the control group that was treated by functional electrical stimulation and received gait training thereafter, differences between before and after the experiment were analyzed using paired sample t-tests and differences in changes after the experiment between the experimental group and the control group were analyzed using independent sample t-tests in order to compare the two groups with each other. Experimental results showed significant differences in weight bearing, balance and gait velocity between before and after the experiment in the experimental group(p<.05). In the control group, whereas weight bearing and gait velocity did not show any significant difference between before and after the experiment(p>.05), balance showed significant differences(p<.05). Weight bearing, balance and gait velocity change rates showed significant differences between the experimental group and the control group(p<.05). In conclusion, it was indicated that gait training using functional electrical stimulation is effective for enhancing stroke patients' weight bearing rates, balance abilities and gait velocity.

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.

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.

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.

Three species of the genus Neolygus Knight - N. hakusanensis (Yasunaga, 1991), N. roseus (Yasunaga, 1991) and N. zhugei (Yasunaga, 1991) - are recognized for the first time in Korea. An identification key to the eleven Korean Neolygus species is presented. Some illustrations of male genitalic structures are also provided.

The purpose of this study was to analyze and compare the effect of resistance exercise and balance exercise on proprioception and WOMAC index of patients with degenerative knee osteoarthritis. A total of 40 subjects participated in this study. The subjects were diagnosed with degenerative knee osteoarthritis and all were more than 60 years old. They were divided into three groups. Group Ⅰ(n=8) was trained with resistance exercise, Group Ⅱ(n=6) was trained with balance exercise and GroupⅢ(n=6) was trained with range of motion as a control. The results of this study were as follows. It was significantly indicated that the resistance exercise group and balance exercise group elicited error-reduction on proprioception goal-angle (p<.05). There was a statistically significant difference on proprioception between resistance exercise group and control(range of motion) group. There was a statistically significant reduction on WOMAC index between resistance exercise group and balance exercise group (p<.05) and on the WOMAC index between resistance exercise group and range of motion group(p<.05). In conclusion, resistance exercise and balance exercise are effective on degenerative knee osteoarthritis and resistance exercise is the most effective for improving proprioception and WOMAC index. More research on the intervention according to the degree of degenerative knee osteoarthritis is needed.