Induced pluripotent stem cells (iPSCs), generated by the overexpression of transcription factors Oct4, Sox2, Klf4 and c‐Myc in somatic cells, are pluripotent. iPSCs reprogrammed from differentiated cells get through a epigenetic modification during reprogramming and finally have the similar epigenetic state to embryonic stem cells (ESCs). In this study, these epigenetic changes were observed in reprogramming of uni‐parental parthenogenetic somatic cells. Furthermore, we have shown that parthenogenetic pattern of imprinted genes were changed during pluripotential reprogramming. Parthenogenetic neural stem cells (pNSCs) containing only maternal alleles regain the biparental imprinting patterns after reprogramming. However, we have yet to define whether the changed imprinted genes are maintained or reverted to the parthenogenetic state when the reprogrammed cells are differentiated into specialized cell types. To address this question, we compared genome‐wide expression profiles of biparental female neural stem cells (fNSCs), parthenogenetic neural stem cells (pNSCs), and NSCs differentiated from parthenogenetic maternal iPSC (miPS‐NSCs). Furthermore, this study establishes the correlation between the alteration of genome methylation and activation of imprinting genes in the parthenogenetic cells and reports for the first time that the silenced PWS‐related imprinted genes are activated in miPS‐NSCs. Our data demonstrated that pluripotential reprogramming of parthenogenetic somatic cells were able to reset the parthenogenetic imprinting patterns; reprogrammed miPSCs showed erasure of maternal methylation imprints and acquisition of methylation in paternally imprinted genes. Furthermore, the changed imprinting patterns were maintained when the reprogrammed cells are differentiated into specialized cell type. * This work was supported by the Next‐Generation BioGreen 21 program (Grant PJ008- 009) funded by the Rural Development Administration, Republic of Korea.

Pluripotent stem cells can be derived from both pre- and post-implantation embryos. Embryonic stem cells (ES cells), derived from inner cell mass (ICM) of blastocyst are naïve pluripotent and epiblast stem cells (EpiSCs) derived from post-implantation epiblast are primed pluripotent. The phenotypes and gene expression patterns of the two pluripotent stem cells are different each other and EpiSCs thought to be in a more advanced pluripotent (primed pluripotent state) than mouse ES cells (naïve pluripotent state). Therefore, we questioned whether EpiSCs are less potential to be differentiated into specialized cell types in vitro. EpiSCs were isolated from 5.5～6.5 day post coitum mouse embryos of the post-implantation epiblast. The EpiSCs could differentiate into all tree germ layers in vivo, and expressed pluripotency markers (Oct4, Nanog). Interestingly, EpiSCs also were able to efficiently differentiate into neural stem cells (NSCs). The NSCs differentiated from EpiSCs (EpiSC-NSCs) expressed NSC markers (Nestin, Sox2, and Musasi), self-renewed over passage 20, and could differentiate into two neural subtypes, neurons, astrocytes and oligodendrocytes. Next, we compared global gene expression patterns of EpiSC-NSCs with that of NSCs differentiated from ES cells and brain tissue. Gene expression pattern of brain tissue derived NSCs were closer to ES cell-derived NSCs than EpiSC-NSCs, indicating that the pluripotent stem cell-derived somatic cells could have different characteristics depending on the origin of pluripotent stem cell types. * This work was supported by the Next Generation Bio-Green 21 Program funded by the Rural Development Administration (Grant PJ 008009).

Recent advances in stem cell biology have shown that terminally differentiated somatic cells can be directly converted to the different types of somatic cells such as neurons and cardiomyocytes with defined sets of transcription factors without going through a pluripotent state. Recently, it was demonstrated that the hepatocyte-specific transcription factors, Hnf4α plus Foxa1, Foxa2 or Foxa3 could erase somatic memory and reset hepatocyte program on the differentiated somatic genome. Here, we show that Foxa3 together with Hnf4α could efficiently reprogram fibroblasts into hepatocytes. However, the direct conversion into hepatocytes is not observed with Hnf4α plus Foxa1. After two weeks of retroviral transduction of Hnf4α and Foxa3, we observed epithelial colonies emerged from starting fibroblasts and were able to establish stable hepatocyte cell lines, namely induced hepatocytes (iHep cells). The iHep cells closely resemble primary hepatocytes in a number of characteristics such as their polygonal shapes, the hepatic gene expression patterns and the presence of E-cadherin signals as determined by immunocytochemistry. In addition, iHep cells show the storage of glycogen as revealed by Periodic acid-Schiff (PAS) staining, indicating that iHep cells are functionally similar to their in vivo counterparts. Taken together, our findings suggest that the combination of hepatic transcription factors, Hnf4α with Foxa3 but not Foxa1 could induce hepatocyte fate on the differentiated somatic cells.

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/ 0.5 ml, and 43.6 % frozen at 20embryos/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).

Neural stem cells (NSCs) are self-renewing tripotent cell populations and have capacity of neuronal (neurons) and glial (astrocytes and oligodendrocytes) differentiation. Many researchers have reported that NSCs have therapeutic effects in neurological disease by transplantation. However, it is not easy to obtain NSCs in vitro. Recently, Yamanaka and colleagues showed that somatic cells could be reprogrammed into pluripotent state by enforcing reprogramming factors. Induced pluripotent stem (iPS) cells undergo unlimited self-renewal and have differentiation potential into various types of cells like embryonic stem cells. Direct differentiation into a specialized cell types from iPS cells hold considerable promise for regenerative medicine as well as basic research. Here, we induced differentiation of iPS cells into NSCs in vitro and in vivo, which were compared with embryonic stem (ES) cell-derived NSCs and brain derived NSCs. NSCs from ES and iPS cells were morphologically indistinguishable from brain derived NSCs and stained positive for NSCs markers Nestin and Sox2. ES cells derived NSCs were transcriptionally distinguishable from brain derived NSCs. However, global gene expression pattern were similar but distinct between iPS derived NSCs and brain derived NSCs. Moreover, iPS derived NSCs were spontaneously aggregated upon passaging, formed ES cell like colonies, and finally reactivated Oct4-GFP. The spontaneously reverted GFP-positive cells (iPS-NSC-iPS) expressed similar levels of pluripotency markers (Oct4,Nanog) to ES and iPS cells, and could form germ line chimera. One possible explanation for this phenomenon is that spontaneously re-reprogramming was associated with transgene re-activation when iPS cells were differentiated into NSCs. However, NSCs from dox-inducible iPScells could not be reprogrammed into pluripotent state without doxycycline. Taken together, iPS derived NSCs were morphologically and similar to brain derived NSCs, but differ in gene expression pattern and maintenance. * This work was supported by the Next Generation Bio-Green21 Program funded by the Rural Development Administration (Grant PJ008009).

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).

This study compared the stability of the cervical spine according to the presence of neck pain and deep neck flexor performance. Thirty subjects with neck pain, and thirty subjects without neck pain were recruited for this study. The Cranio-cervical flexion (CCF) test was applied using a pressure biofeedback unit to classify the subjects into four subgroups; no cervical pain and good deep neck flexor performance (NG group), no cervical pain and poor deep neck flexor performance (NP group), cervical pain and good deep neck flexor performance (PG group), and cervical pain and poor deep neck flexor performance (PP group). The head sway angle was measured using a three-dimensional motion analysis system. A 3-kg weight was used for external perturbation with the subject sitting in a chair in the resting and erect head positions with voluntary contraction of the deep neck flexors. A one-way analysis of variance (ANOVA) was performed with a Bonferroni post hoc test. The deep neck flexor performance differed significantly among the four groups (p<.05). The NG group had significantly greater deep neck flexor performance than NP and PP groups. The stability of the cervical spine also differed significantly among the four groups in the resting head position (p<.05). The head sway angle was significantly smaller in NG group as compared with the other groups. The PP group had the greatest head sway angle in the resting head position. However, there was no significant difference in the stability of the cervical spine among the groups in the erect head position with voluntary contraction of deep neck flexors (p=.57). The results of this study suggest that the deep neck flexor performance is important for maintaining the stability of cervical spine from external perturbation.

The green peach aphid (Myzus persicae) is a cosmopolitan pest of agricultural and horticultural crops and causes serious economic damages. M. persica has rapidly developed resistance to a wide variety of insecticides, including pyrethroids. Target site insensitivity mechanism mediated by two mutations (L1014F and M918T) on the para-type voltage-sensitive sodium channel (vssc) is mainly responsible for pyrethroid resistance. To predict the vssc resistance allele frequency, quantitative sequencing (QS) protocol was established. Frequency prediction equations generated from the plots of signal ratios and amplification critical time showed a high correlation coefficient (r2>0.993), indicating its high accuracy in prediction. QS results revealed that the kdr-type L1014F mutation is only present in Pyeongchang strain. No field strains of M. persicae possessed the super-kdr type M918T mutation. However, a novel M918L mutation was found by genotyping approach. The allele frequencies of M918L and L1014F were 0% to 53% in populations examined, and the level of M918L mutation frequency was closely related with pyrethroid resistance. Therefore, QS-based detection of M918L mutation frequency should faciltate the monitoring of pyrethroid resistance in the field.

The green peach aphid (Myzus persicae) is a serious pest of agricultural and horticultural crops all over the world. M. persica has rapidly developed resistance to a wide variety of insecticides, including carbamates. The E4/FE4 carboxylesterase is known to be involved in carbamate resistance. To compare the E4/FE4 carboxylesterase gene copy number, as a genetic resistance marker, between seven field strains, quantitative real-time PCR (qPCR) was performed. In addition, quantitative sequencing (QS) was employed to predict the frequencies of acetylcholinesterase (AChE) mutations (A301S and S431F) that are associated with target site insensitivity. All M. persica strains examined possessed the S431F mutation in the heterozygous state except for a susceptible strain, implying the possibility of AChE duplication. In contrast, no A301S mutation was found. Frequency prediction equation was generated from the plots of signal ratios and amplification critical time, which showed a high correlation (r2>0.996). QS analysis of M. persicae populations revealed that the allele frequency of S431F ranged 4% to 63%. Taken together, the AChE resistance allele frequencies determined by QS and the E4/FE4 gene copy number by qPCR should facilitate the detection and monitoring of carbamate resistance in M. persicae in the field.

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.

This study is intended to examine the tDCS and Montoya stair task(MST) on sensorimotor recovery and glial scar expression in MCAo induced stroke model of rat. To achieve this goal, this study selected 80 SD rats of 8 weeks. The experiment groups were divided them into four groups, and assigned 20 rats to each group. GroupⅠ was a experimental control group; GroupⅡ was a tDCS application group after MCAo; GroupⅢ was a MST application group after MCAo; GroupⅣ was a tDCS and MST application group after MCAo. In each group, neurological function test measurement, motor behavior test, montoya stair task test, immunohistochemistric finding of GFAP expression finding were analyzed. In motor behavior test, the outcome of groupⅠ was significantly difference than the other group, especially from 14days. In montoya stair task test, the outcome of groupⅠ was significantly lower than the other group especially, groupⅡ were significantly different on 14days and group Ⅳ was most significantly difference than the other group. In immunohistochemistric finding, groupⅡ, Ⅲ, Ⅳ were decrease GFAP expression on depend on time stream. These results throughout the MCAo due to focal ischemic brain injury rat model four weeks tDCS and MST was applied, when the neurobehavioural, upper extremity function and ability, histopathologic data suggest that sensorimotor function recovery and a positive influence on glial scar decrease and confirmed that.

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.

Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles forBi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

This study was estimated for cabbage aphid, Brevicoryne brassicae in Brassica campestris L. var. rapa (L.) Hartm. in order to institute of Economic injury levels(EILs). B. brassicae was innoculated on April 29, in differently 0, 5, 10, 20 and 40 adults per ten plant, respectively. After inoculated of B. brassicae, the density was increased until harvest ing gradually in all plots except non innoculate plot. and Higher inoculation density were increased higher than lower inoculation density. Percentage of damage leaf was higher in plots with higher initial aphid density than in plots with lower initial aphid density. And the leaf weight of commodity were decreased in higher initial aphid density. The decreasing rates of leaf weight of commodity was increased with increasing initial aphid density. The relationship between initial B. brassicae densities and the decreasing rates of leaf weight of commodity was well described by a linear regression, Y=0.8416X-3.5147, R2=0.94. Based on the relationship, the number of adults per 10 plant which can cause 5% loss of yield was estimated to be approximately 10.1. And EILs was estimated to be approximately 1.0 adults/plant in late April.

The word spawn is derived from an old French verb, espandre , meaning to spread out or expand. Spawn is also defined as “the mycelium of fungi, especially of mushrooms grown to be eaten, used for propagation.” The effects of bag spawn to sawdust substrate on the growth of Pleurotus ostreatus were conducted. The duration of mycelial growth and days of pinhead formation of bag spawn(2.5kg) were 18~19 days and 7~8 days, whereas bottle spawn (1,000㎖) was 18 days and 6 days, respectively. The yield of mushroom fruitbody was that bag spawn is 100~118g, bottle spawn was 95~115g. In economical analysis, bag spawn is increased to 50%, compared to bottle spawn in relative income.