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.

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.

5-Hydroxy-1,4-naphthoquinone and its derivatives were evaluated for insecticidal effect against Sitophilus oryzae and S. zaemais adults. This study was examined using fumigant method. Mortality was determined after 72 h of treatment. 5-Hydroxy-1,4-naphthoquinone showed strong (+++) activity at 5 mg and the 1,4-naphthoquinone showed strong (+++) and moderate (++) activity at 5 mg, against S. oryzae and S. zaemais, respectively. However, 5-hydroxy-2-methyl-1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone, and 2-methoxy-1,4-naphthoquinone had non-activity (-) at 5 mg. Based on the LD50 values, the compound most highly effect to S. oryzae was 1,4-naphthoquinone (0.012 mg/cm2), followed by 5-hydroxy-1,4-naphthoquinone (0.013 mg/cm2). However, against S. zeamais, 5-hydroxy-1,4-naphthoquinone (0.044 mg/cm2) was the most toxic compound, followed by 1,4-naphthoquinone (0.155 mg/cm2). These results suggest that the introduction of various functional group (hydroxy, methyl and methoxy) into the 1,4-naphthoquinone skeleton contributes to insecticidal activity. Accordingly, 5-hydroxy-1,4-naphthoquinone and 1,4-naphthoquinone could be used highly effective rice weevil control agents.

The rice weevil, Sitophilus oryzae, is the most serious storage pests worldwide of grocery shops, flour mills, and warehouses. The phototactic response of the rice weevil, S. oryzae, to light emitting diodes (LEDs) at five different wavelengths and various light intensities was tested within an LED-equipped Y-maze chamber, and compared with its response to a luring lamp, which is used in commercial traps. Various wavelengths in this study were UV (365 nm), blue (450 nm), green (520 nm), red (660 nm), and infrared (IR) (730 nm). Based on attraction rate (%), blue (84.3%) was the most attractive to S. oryzae, followed by green (74.3%), red (64.3%), UV (63.3%), and IR (48.7%). Moreover, blue and green wavelengths were 1.5 and 1.3 times more attractive than luring lamp (56.7%), whereas the UV wavelength was slightly less attractive to the weevils than luring lamp. These results suggested that blue and green wavelengths could be more useful than those currently used for monitoring and mass trapping of S. oryzae.

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.

The cotton aphid, Aphis gossypii (Glover), is one of the most serious pest in the cultivation of various vegetables. A highly imidacloprid-resistant field population (CA-L) was collected from cucumber at Gangwha island in 4th August 2011. Even though neonicotinoid insecticides especially imidacloprid were sprayed six times during June and July, aphid density was too high to be counted. IEF and 2DE analyses revealed that general esterase isozyme (pI. 5) in CA-L were dramatically overexpressed and more isozyme spots identified in CA-L compared to susceptible (CA-S) strain. To identify differentially expressed genes in CA-L, comparative transcriptome analyses based on GS-FLX were conducted with total RNA extracted from CA-L, which generated ca. 143 Mb reads. Previously reported, comparative transcriptome analyses performed in imidacloprid resistant (CA-IR) and CA-S. The comparative transcriptome analyses re-investigated after all data sets were combined together. As previously reported, seven ATP-binding cassette (ABC) transporter genes were newly identified in A. gossypii, among which only ABCC9 gene was highly expressed in CA-IR and L. These results suggested that ABCC subfamily associated with imidacloprid resistance in A. gossypii.

Larval stages of Callipogon relictus (Semenov) (Coleoptera: Cerambycidae), a gigantic longhorn beetle designated as a natural monument of Korea, has never been studied as it is hardly discovered in nature. The DNA barcoding gene, mt-COI, was used to identify a dead larva found in the Gwangneung forest of the Korea National Arboretum. Based on the result, we provide the morphology of the immature stage, with the illustrations of diagnostic characteristics.

After pea aphid Acyrthosiphon pisum genome project, hologenome concept was applied to pea aphid and symbioant such as Buchnera aphidicola. Here we screened symbiotic microorganism in four lab strains (two genetically different insecticide susceptible strains, host plant: cucumber and two different host adopted imiacloprid resistance strains, host plants: cucumber and potato) and four field populations (Jeju, Goryeong, Gimjae and Muju, host plant: potato) of cotton aphid based on GS-FLX pyrosequencing which were conducted with universal primer amplified partial fragments of 16S rRNA from total DNA which was extracted from each strain and population. B. aphidicola occupied over 90% of all identified prokaryotic microorganisms which all tested samples. It’s interesting that the ratio of B. aphidicola occupied over 99% in all of the tested lab strains. However, specific enterobacteriaceae occupied six to seven percents of all field populations which closely related endosymbiont of Glycaspis brimblecombei. That means B. aphidicola occupied only 91~92% of all identified prokaryotic microorganisms. Futhermore, other actinobacteridae and bacillaceae also were detected in field populations. The results obtained for these ratios suggested that there has some interaction between symbioant and environment NOT in imidacloprid resistance.

This study proposes the compensation method for the mechanical deflection error of a SCARA robot. While most studies on the related subject have dealt with the development of a control algorithm for improvement of robot accuracy, this study presents the control method reflecting the mechanical deflection error which is predicted in advance. The deflection at the end of the gripper of SCARA robot is caused by the self-weights and payloads of Arm 1, Arm 2 and quill. If the deflection is constant even though robot’ posture and payload vary, there may not be a big problem on robot accuracy because repetitive accuracy, that is relative accuracy, is more important than absolute accuracy in robot. The deflection in the end of the gripper varies as robot’ posture and payload change. That’ why the moments ,  and  working on every joint of a robot vary with robot’ posture and payload size. This study suggests the compensation method which predicts the deflection in advance with the variations in robot’ posture and payload using neural network. To do this, I chose the posture of robot and the payloads at random, found the deflections by the FEM analysis, and then on the basis of this data, made compensation possible by predicting deflections in advance successively with the variations in robot’ posture and payload through neural network learning.

The purpose of the current study was bi-fold. First it was to examine how the semantic prosody of eight lexical items that had a specific semantic prosody were presented and explained in the six English-Korean bilingual dictionaries. In addition, it was to investigate how those lexical items were used in university students’ sentence writings in relation to semantic prosody. The result showed that the semantic prosodies of the lexical items were not adequately presented in the dictionaries in general and a number of inappropriate uses of lexical items in relation to semantic prosody were identified in the students’ sentence writings. It was suggested that EFL dictionary publishers should explicitly address the issue of semantic prosody by providing information about the semantic preference and collocational behavior of a lexical item and they should be more cautious when presenting Korean translations/equivalents in the dictionaries. Along the same lines, it was also suggested that EFL/ESL teachers should (a) recognize the value of semantic prosody in L2 communication; (b) avoid the vocabulary teaching practice of explaining the meaning of words by simply providing near synonyms; and (c) make the students more aware of the difference in semantic prosody between English lexical items and their Korean translations/equivalents.

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.