Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cell (iPS) experiments have generally demonstrated that a differentiated cell directly converts into a undifferentiated or pluripotent state. In SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor cell nuclei to the recipient cytoplasm of matured oocytes. Although nuclear reprogramming of cells by the ex-ovo methods is not always consistent or efficient, it has been suggested that a combination of nuclear reprogramming technique may improve the efficiency or frequency of normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from GV stage sturgeon's oocytes prior to their use as nuclear donors for SCNT will improve subsequent development. We reported a reversible permeabilization protocol with digitonin to deliver sturgeon oocyte exteact (SOE) to porcine fetal fibroblast cell nuclei ex ovo. Porcine fibroblasts were permeabilized by 4 μg/ml of digitonin for 2 min at 4℃ and then incubated in SOE for 7h at 15 18℃ followed by resealing of cell membrane. As results, no difference was observed in the number of fused couplets or the number of fused couplets that cleaved between the extract treated or control group. However, there was a significantly decrease in the percentage of fused couplets that developed to the blastocyst stage in the SOE treated group (p<0.05). Histone acetylation status was determined using an antibody to acetylation at lysine 9 on histone 3 (H3K9Ac). The intensity of H3K9Ac staining in 1-cell stage NT embryos was significantly increased when treated with the SOE (p<0.05), similar to that in 1-cell stage IVF embryos. In addition, porcine NT embryos reconstructed by using donor cell exposed to SOE prior to cell fusion significantly decreased developmental competence to the blastocyst stage but increased pluripotent gene expressions (Sox2, Nanog and Oct3/4) when compared with those in normal NT embryos (p<0.05).

Urokinas type plasminogen activator (uPA) has been used as a therapeutic agent for treating human diseases such as thrombosis. Attempts to transgenically overexpress the uPA in animal bioreactors have been hampered due to side effects associated with this functional protein hormone on homeostasis. Recently, chicken has been emerged as a potential candidate for use as bioreactor to produce proteins of pharmaceutical importance. Since this species has low homology uPA sequence with mammals, we hypothesized that chicken could be used as a potential bioreactor for production of human uPA. In this study, using replication‐defective Murine Leukemia Virus (MLV)‐based retrovirus vectors encapsidated with Vesicular Stomatitis Virus G Glycoprotein (VSV‐G), we attempted to make transgenic chicken expressing human uPA (huPA). The recombinant retrovirus was injected beneath the blastoderm of non‐incubated chicken embryos (stage X, at laying). After 21 days of incubation (at hatching), all of the 38 living chicks that assayed, were found to express the vector‐encoded huPA gene in various organs and tissues, which was under the control of the Rous Sarcoma Virus (RSV) or Cytomegalovirus (CMV) promoter. Using specific primer set for huPA, PCR and RTPCR analyses of gDNA isolated from these samples demonstrated these chickens were transgenic for huPA. Furthermore, successful germ line transmission of huPA transgene was confirmed and next generation whole body huPA transgenic chickens were also produced. We also assayed huPA protein titer in blood (17.1 IU/ml) and eggs (4.4 IU/ml) of whole body huPA transgenic chicken. Thus, our results demonstrated that chicken could be used as bioreactors to produce huPA.

Testes‐derived unipotent male germ‐line stem (GS) cells can acquire multipotency under appropriate culture conditions to become mGS cells which can contribute to all three germ‐layers. This study was designed to investigate the epigenetic characteristics of mGS cells derived from adult mouse testes (maGS cells). The GS cells were isolated from 4 6 week DBA mouse and were cultured in Dulbecco’s modified Eagle Medium supplemented with 15% (v/v) fetal bovine serum, 1,000 U/ml LIF, 4 ng/ml GDNF at 37℃ in an humidified atmosphere of 5% CO2 in air to derive the maGS cells. The multipotency of maGS cells were verified by morphological and gene expression analyses, teratoma formation upon transplantation into nude mouse and in vitro differentiation ability. Bisulfite genomic sequencing revealed that GS cells had androgenetic DNA methylation pattern at the Igf2‐H19, Gnas‐Nespas , and Dlk1‐Dio3 imprinted gene clusters which changed to hemi‐zygotic embryonic stem (ES)‐cell like pattern in the maGS cells. Western blot analysis, using modification‐ and residue‐specific antibodies, revealed that both maGS and ES cells had similar level of histone di‐methylation at 4th and 27th lysine residue of histone 3 (H3K4me2 and H3K27me2) which represent “bivalent domain” for regulating self‐renewal and differentiation of mouse ES cells. Both maGS and ES cells also shared similar hisone modification for H3K9me2, H3K79me2, H3K9ac and H3K18ac. However, maGS cells had higher level of H3K- 36me2 and H3S10p. These data suggest that maGS and ES cells share several epigenetic characteristics but they also have their own unique epigenetic marks that may be useful as a molecular marker for their identification.

Although somatic cell nuclear transfer (SCNT) has successfully been produced cloned animals in several species, the cloning efficiency is extremely low. It is generally believed that the low cloning efficiency is mainly attributed to faulty epigenetic modifications underlying the aberrant reprogramming of donor cell nuclei in recipient cytoplasm after SCNT. The nuclear reprogramming process involves epigenetic modifications, such as DNA demethylation and histone acetylation, which may be a key factor in improving the cloning efficiency. Recently, the histone deacetylase inhibitors (HDACi), such as trichosatin A (TSA) and m-carboxycinnamic acid bishydroxamide (CBHA), to increase histone acetylation have been used to improve the developmental competence of SCNT embryos. Therefore, we compared the effects of TSA with CBHA on the in vitro developmental competence and pluripotency-related gene expressions (Nanog, Oct3/4 and Sox2) in porcine cloned blastocysts. The porcine cloned embryos were treated with a 50 nM concentration of TSA or a 100 μm concentration of CBHA during the in vitro early culture (10h) after cell fusion and then were assessed to cleavage rate, development to the blastocyst stage and pluripotency-related gene expressions in NT blastocysts. All data was analyzed by chi-square. Following 4-5 replicates (245, 200 and 222 for NT, TSA and CBHA treated NT embryos respectively) there was no difference between normal NT and CBHA treated NT embryos, whereas TSA treated NT embryos was significantly decreased for cleavage rate (p<0.05). The developmental competence to the blastocyst stage in CBHA treated NT embryos (18.9%) significantly increased than that of normal NT and TSA treated NT embryos (9.4% and 11.5%) (p<0.05). In addition, all of pluripotent transcription factors (Nanog, Oct3/4 and Sox2) were highly expressed in the CBHA treated NT embryos, however, Sox2 and Oct3/4 were expressed in TSA treated NT embryos and Sox2 was only expressed in normal NT embryos (p<0.05). In conclusion, the treatment of CBHA as a histone deacetylase inhibitor significantly increased the developmental competence of porcine NT embryos and pluripotency- related gene expressions (Nanog, Oct3/4 and Sox2) in NT blastocysts.

Preservation of sperm is essential for long-term storage of valuable animal genetic resources and for the conservation of threatened mammalian species undergoing progressive extinction. In this study, using pig as a model system, we evaluated the feasibility of germ-plasm preservation via sperm cell lyophilization. We show that, pig sperm can be successfully lyophilized and stored in a liquid nitrogen-free condition for at least 6 months. Intracytoplasmic injection of lyophilized sperm (ICSI), stored at 4℃ for four months, into in vitro matured pig oocytes could successfully develop up to blastocyst stage (13.0±3.0%). Lyophilized sperm could also be stored at room temperature for at least three weeks without further compromising their in vitro development up to the blastocyst stage (14.6±3.2 vs. 16.6±5.1%; p>0.05). Blastocysts produced from ICSI of lyophilized sperm stored at 4℃ or room temperature contained similar number of cells per blastocyst (44.9±3.2 vs. 44.0±4.3; p>0.05) but was significantly lower than those produced from non-lyophilized fresh sperm (52.1±5.8 p>0.05). Interestingly, use of a custom-designed HEPES-buffered, calcium-free, defined medium for the lyophilization resulted in normal post-ICSI embryonic development up to blastula stage (23.4±2.8 vs. 24.0±2.9%) and, the resultant blastocysts contained similar number of cells per blastocyst (47.9±4.3 vs. 50.6±7.0) compared to those generated from non-lyophilized fresh sperm (p>0.05). These lyophilized sperm could also be stored at room temperature for at least three weeks with slight reduction in post-ICSI embryonic development (19.6±1.4%). Therefore, these results suggest that, pig sperm could be successfully and efficiently lyophilized for their long-term storage at 4℃. Lyophilization of sperm could be a practical option for long-term storage of mammalian germ-plasm.

Autophagy, the process of bulk degradation and recycling of long-lived proteins, macromolecular aggregates, and damaged intracellular organelles, has recently been shown to be important for pre-implantation development and cavitation in mouse embryos. This study investigated the occurrence of autophagy and its importance in determining the in vitro development of pig embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). Western blot analysis for autophagy marker, microtubule associated protein light chain 3 (MAP-LC3), revealed the temporal pattern of LC3-conversion with intense changes during 10 20 h post-insemination and at morula-blastocyst transition in pig embryos. Specific inhibition of autophagy in 2 4 cell stage pig embryos, by treatment with 3-methyladenine (3MA), did not affect their embryonic development up to morula stage (p>0.05) but completely blocked their progression to the blastocyst stage (0.0±0.0 vs. 28.5±1.7% p<0.05). On the other hand, autophagy-inhibition in morula stage embryos significantly inhibited the formation of blastocoel (14.9±3.6 vs. 37.5±7.2%) and reduced the proportion of expanded blastocysts (5.6±2.6 vs. 29.6± 4.6% p<0.05). TUNEL assay revealed that autophagy-inhibited embryos had significantly increased indices of apoptosis (10.2±0.4 vs. 2.3±0.2) and DNA fragmentation (0.8± 0.1 vs. 0.3±0.1) than those of controls (p<0.05). Interestingly, while anti-oxidants reduced (p<0.05) the apoptosis and improved the blastocyst formation rate in pig embryos, it had no influence (p>0.05) on the expression of MAP-LC3. These data therefore, suggest that autophagy may have essential role during blastocyst formation in pig embryos.

The nematicidal activity of Phellodendron amurense rhizome-derived materials (methanol extract) toward Meloidogyne Spp. second-stage juveniles (J2) and these effects on Cucumis sativus and Cucumis melo. Results were compared with these of fosthiazate. J2 was examined using 24-well plate tests, pot bioassays (C. sativa and C. melo) and Field trials (C. melo). In 24-well plate test with J2, methanol extract of P. amurense exhibited 98.7% and 69.8% mortality at 0.25 and 0.125 mg/ml toward J2, respectively, whereas Fosthiazate showed 100% mortality at 1 mg/ml. In pot bioassays with J2, P. amurense rhizome methanol extract gave 79.5% and 57.4% mortality at 2ℓ/m2(1,000x) and 2ℓ/m2(2,000x)/3kg soil from C. sativa and 73.7% and 53.3% mortality at 2ℓ/m2(1,000x) and 2ℓ/m2(2,000x)/3kg soil from C. melo, respectively. In Field test at C. melo in greenhouse showed 55.1% and 26.9% mortality at 2ℓ/m2(1,000x) and 2ℓ/m2(2,000x) applied soil. P. amurense rhizome-derived materials, merit further study as potential root-knot nematode control agents because of their nematicidal activity.

This study was conducted to examine the community structure of ground beetles between coniferous and deciduous forest in rural landscapes along latitudinal gradient, such as Miryang-si, Yeongdong-gun, Icheon-si, and Cheolwon-gun from April to October in 2009. A total of 19 species belonging to 15 genera of 9 subfamilies were identified from 6,253 collected ground beetles. Between forest stands, 14 species belonging to 10 genera of 7 subfamilies were identified from 2,874 ground beetles in coniferous forests and 14 species belonging to 11 genera of 7 subfamilies were identified from 3,379 ground beetles in deciduous forests. Dominant species from 4 regions were Synuchus nitidus (59.4% of total) and Synuchus cyloderus (28.5%) respectively. Analysis of similarity (ANOSIM) showed no difference between forest stands, but it was significantly different among regions. Non-metric multidimensional scaling showed that 8 study sites were divergence to 2 major groups between Miryang-si and the others. Overall, community structure of ground beetles showed no difference between forest stands. In conclusion, rural forests is being discussed about important role in biodiversity conservation such as refuge from human activity, and thus, studies on the community structure of ground beetles in the rural forests will be important to manage the biodiversity in agricultural landscapes, because ground beetles are well known for general predator groups in the ecosystems.

Endothelial cells are a vital constituent of most mammalian organs and are required to maintain the integrity of these tissues. These cells also play a major role in angiogenesis, inflammatory reactions, and in the regulation of thrombosis. Angiogenesis facilitates pulp formation and produces the vessels which are essential for the maintenance of tooth homeostasis. These vessels can also be used in bone and tissue regeneration, and in surgical procedures to place implants or to remove cancerous tissue. Furthermore, endothelial cell regeneration is the most critical component of the tooth generation process. The aim of the present study was to stimulate endothelial regeneration at a site of acute cyclophosphamide (CP)-induced endothelial injury by treatment with human umbilical cord-derived endothelial/mesenchymal stem cells (hEPCs). We randomly assigned 16 to 20-week-old female NOD/SCID mice into three separate groups, a hEPC (1 × 105 cells) transplanted, 300mg/kg CP treated and saline (control) group. The mice were sacrificed on days 5 and 10 and blood was collected via the abdominal aorta for analysis. The alanine transaminase (ALT), aspartate aminotransferase (AST), serum alkaline phosphatase (s-ALP), and albumin (ALB) levels were then evaluated. Tissue sections from the livers and kidneys were stained with hematoxylin and eosin (HE) for microscopic analysis and were subjected to immunohistochemistry to evaluate any changes in the endothelial layer. CP treatment caused a weight reduction after one day. The kidney/body weight ratio increased in the hEPC treated animals compared with the CP only group at 10 days. Moreover, hEPC treatment resulted in reduced s-ALP, AST, ALT levels compared with the CP only group at 10 days. The CP only animals further showed endothelial injuries at five days which were recovered by hEPC treatment at 10 days. The number of CD31-positive cells was increased by hEPC treatment at both 5 and 10 days. In conclusion, the CP-induced disruption of endothelial cells is recovered by hEPC treatment, indicating that hEPC transplantation has potential benefits in the treatment of endothelial damage.

We present the effect of a coupling agent on the optoelectrical properties of few-walled carbon nanotube (FWCNT)/epoxy resin hybrid films fabricated on glass substrates. The FWCNT/epoxy resin mixture solution was successfully prepared by the direct mixing of a HNO3-treated FWCNT solution and epoxy resin. FWCNT/binder hybrid films containing different amounts of the coupling agent were then fabricated on UV-ozone-treated glass substrates. To determine the critical binder content (Xc), the effects of varying the binder content in the FWCNT/silane hybrid films on their optoelectrical properties were investigated. In this system, the Xc value was approximately 75 wt%. It was found that above Xc, the coupling agent effectively decreased the sheet resistance of the films. From microscopy images, it was observed that by adding the coupling agent, more uniform FWCNT/binder films were formed.

The genomic structure and phylogenetic relationships of HSP88 genes from P. tenuipes Jocheon-1, P. tenuipes, C. militaris and C. pruinosa are described. The HSP88 genomic DNA from P. tenuipes Jocheon-1, P. tenuipes and C. militaris all contain 5 introns and 6 exons with the length of 13, 62, 32, 1438, 306, 288 bp, encoding 713 amino acid residues. C. pruinosa HSP88 genomic DNA contains 4 introns and 5 exons encoding 713 amino acids. The length of each exon of C. pruinosa HSP88 is 13, 62, 32, 1744, 288 bp and the length of exon 4 is identical to the total length of exon 4 and exon 5 of HSP88 of P. tenuipes Jochoen-1, P. tenuipes, and C. militaris. The deduced amino acid sequence of P. tenuipes Jocheon-1 HSP88 showed 99% identity with the P. tenuipes, 97% identity with the Cordyceps militaris, and 98% identity with the C. pruinosa. Phylogenetic analysis confirmed that the P. tenuipes Jocheon-1, P. tenuipes, C. militaris and C. pruinosa HSP88 are placed together within the ascomycetes group of fungal clade.

In this study, a full-length heat shock protein88 complementary DNA (cDNA) of Paecilomyces tenuipes Jocheon-1 was obtained by screening of P. tenuipesJocheon-1 Uni-Zap cDNA library and 5' RACE polymerase chain reaction. The Paecilomyces tenuipes Jocheon-1 heat shock protein88 cDNA contains an open reading frame of 2,139 bp encoding 713 amino acid residues. The deduced amino acid sequence of the P. tenuipes Jocheon-1 HSP88 cDNA showed 77% identity to N. haematococca HSP88 and 45-76% identity to other fungi HSP88. Phylogenetic analysis and BLAST program analysis confirmed that the deduced amino acid sequences of the P. tenuipes Jocheon-1 HSP88 gene belonged to the ascomycetes group within the fungal clade and P. tenuipes Jocheon-1 HSP88 also contains the conserved ATPase domain at the N-terminal. The cDNA encoding P. tenuipes Jocheon-1 HSP88 was expressed as a 88 kDa polypeptide in baculovirus-infected insect Sf9 cells. Under different stress conditions, mRNA expression of P. tenuipes Jocheon-1 HSP88 were quantified by real-time PCR and the result showed that heat shock stress affected the mRNA expression levels of P. tenuipes Jocheon-1 HSP88.

Presently, We have constructed an olig-d(T) primed directional cDNA library from the silkworm Dongchunghacho, an entomopathogenic fungus, of which species is belonging to Paecilomyces tenuipes Jocheon-1. To isolate and screen genes in the fungus, 626 expressed sequence tags(ESTs) were generated by a partial sequencing from the cDNA library. Paecilomyces tenuipes Jocheon-1 cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase(Pt-GAPDH) of Paecilomyces tenuipes Jocheon-1 was cloned from the above cDNA library. The complete cDNA sequence of Pt-GAPDHis comprised of 1,014bp encoding 338 amino acid residues. The deduced protein sequence of Pt-GAPDH showed higher homology with Beauberia bassiana-GAPDH(93% amino acid identity). Hydropathy analysis revealed that Pt-GAPDH protein is hydrophilic. The major three amino acids in its composition of amino acid residues were alanine(11.54%), valine(9.47%) and glycine(8.88%). The cDNA encoding Pt-GAPDH was expressed as a 37 kDa polypeptide in baculovirus-infected insect Sf9 cells. The Pt-GAPDH gene of Paecilomyces tenuipes entomopathogenic fungus consisted of three exons and two introns coding for 338 amino acid residues, and the genomic DNA length of the gene spans 1302bp. The accession number of the gene in GenBank are GU997099 for Pt-GAPDH cDNA and GU997102 for Pt-GAPDH genomic DNA.

The impact of transgenic Bt maize plant contained Cry1F was evaluated on the oat aphid Rhopalosiphum padi as a non-target insect species. Slightly reduced rates of survival and alata vivipar production were observed on Bt maize than on the non-Bt maize. In addition, slightly low preference to Bt maize plant was observed. Aphid fecundity, measured as the number of offspring produced for 7 days, was higher on Bt maize than on non-Bt maize but not different significantly. ELISA test using Cry1F-antibody revealed that 26% of Cry1F protein compared to the positive control was detected from the whole body of R. padi when the insects were fed Bt maize for 50 days, showing that R. padi can carry Cry1F protein to the higher trophic level when exposed to Bt maize. Taken together, the Bt maize plant is not likely to cause any negative side impacts on non-target insect R. padi but Bt toxin can be transferred to higher predators via R. padi as it carries the toxin.