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.

The generation and application of porcine iPSCs (piPSCs) as a large animal model may enable the test for the efficacy and safety of the therapy in the field of human regenerative medicine. Here, we report the generation of piPSC from wild (a 10-day-old Massachusetts General Hospital miniature pig; MGH minipig) and genetically modified pig, alpha1,3-Galactosyltransferase knock-out (—/—) (GalT KO homo) and human CD46 (membrane cofactor protein) knock-in (hCD46 KI) MGH minipig (a 10-day-old). Fibroblasts were isolated from the ear skin of wild and MGH minipigs, respectively. After 2 passages, each of fibroblasts was transduced with cocktail of 6 human factors (POU5F1, NANOG, SOX2, C-MYC, KLF4, and LIN28) and cultured on a mitotically inactive mouse embryonic fibroblast (MEF) monolayer. Both of reprogrammed somatic cells expressed the classical pluripotency markers (POU5F1, NANOG, and SOX2) and surface marker (SSEA1). Similar to mouse ESCs, both piPSCs from wild and transgenic minipigs were negative for SSEA3, Tra-1-60, and Tra-1-81. Further these cells could form embryoid body (EB) and differentiate into 3 germ layers in vitro (ectoderm: FOXJ3 and PAX6, mesoderm: HAND2, and endoderm: SOX17 and GATA6). Our piPSCs may provide useful source as a large animal model for studying approaches that can reduce an immune- rejection of cell or organ transplantation.

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 average life expectancy has continuously increased with the development of medicine. However, Some of men have been suffered from the insufficient secretion of testosterone causing by physical factors, social and psychological factors. Testosterone is an essential steroid hormone controlling male reproductive function. Alternative medicines in plants, fungi, and insects have been studied to enhance sexuality. In the present study, we determined the effect of an acorn pollen and Cordyceps militaris fruiting body on testosterone concentration in Sprague-Dawley rats. Eighteen rats per group were housed to regular diet or diet supplemented with acorn pollen powder or fruiting body power for 4 weeks. Serum was collected from 6 rats per group. Results showed that changes of the body weight, food and water intake of the rats were not observed in this study. However, acorn pollen powder and Cordyceps militaris fruiting body significantly stimulated testosterone production (p < 0.05) compared to the control, respectively. Hence, it suggests that acorn pollen or Cordyceps militaris fruiting body might be developed as a complementary medicine to improve sexual hormones.

Mushrooms has been used for food ingredients since early times. Recently it is being used as a good pharmaceutical material or functional bio-material. Especially Ganoderma lucidum, Phellinus and Inonotus are used for medicinal purposes. Ganoderma is a basidiomycete white rot fungus which has been used for medicinal purposes for centuries particularly in China, Japan and Korea. A great deal of work has been carried out on Ganoderma lucidum. The metabolites consist of mainly polysaccharides and terpenoids. At present, Ganoderma is a health food supplement to support cancer patients, yet the evidence supporting the potential of direct in vivo anticancer effects should not be underestimated. the aim of this study was to investigate the anti-cancer effects and anti-inflammatory of Ganoderma lucidum in some kinds of human cell lines (Gastric:AGS, liver:HepG2, breast:MCF-7, lung:A549, uterine:Hela, Raw 264.7) we are examined the cell viability by MTT assay and effects of anti-inflammatory by NO assay. Anticancer and anti-inflammatory effect in the 10strains were selected.

This study was carried out to investigate anticancer activities fruiting body extracts and fractions of Cordyceps militaris. Fruiting body of this mushroom was extracted using by 80% MeOH. Fractionations of these extracts were performed by n-hexane, methylene chloride, ethyl acetate, n-BuOH. AGS(human gastric cancer line) was cultured in media conditions (10% FBS, 1% Penicillin-Streptomycin in RPMI). Anticancer activities of each fractions of Cordyceps militaris were examined by using MTT, Cell titer Glo.

Ganoderma lucidum, a popular medicinal mushroom, has been used in China for longevity and health promotion since ancient times. Recently it is being used as a good pharmaceutical material or functional bio-material. Total 106 strains was cultured at temperature(25 ℃, 30 ℃ ,35 ℃) to study cultural properties of Ganoderma lucidum. Then the mycelium growth was digitized in the average diameter. With the result, Ganoderma lucidum strain has the optimum growing temperature at 25 ℃ with 25 strains, 30 ℃ with 70 strains and 35 ℃ with 11 strains. The 84strains were cultivated in wood for investigating the cultivation properties of Ganoderma lucidum. With the result, 70mm~90mm of the pileus diameter (major axis, mm) and 70mm-80mm of pileus diameter (minor axis, mm) was measured after one year cultivation. And 1,000-2,000 ㎟ of strain took the most the area (major axis x minor axis). The 30-40mm of stipe length,1time of harvesting and volume was 200g~400g took the most. 10cm-15cm of the pileus diameter (major axis, mm), 5cm~7.5cm of pileus diameter (minor axis, mm) and 15-20mm of thickness was measured after 2 year cultivation. And 50~100 ㎟ of strain took the most the area (major axis x minor axis). The 5-10mm of stipe length, 2 times of harvesting and volume was 200g~400g took the most. we are selected a middle model through this result. phylogenetic relationships was drawn based on the ITS sequence analysis in Ganoderma lucidum.

This study was carried out to know whether the host plant feeding have an influence to subsequent prey consumption of a zoophytophagous mirid, Nesidiocoris tenuis, with biological control potential. Piece of leave of Paprika, Sesame, and cotton ball soaked with water were respectively provided to the test insects, adult mirid starved about a day, and frozen eggs of Ephestia kuehniella, were presented to them for a day and counted the number of the eggs consumed by them. The mirid fed by Sesame leave took significantly less prey than both the Paprika and the water fed one.

Spider silks hold great potential as biomaterials with extraordinary properties. Here we report cloning and characterization of the major ampullate silk protein gene from the spider Araneus ventricosus. A cDNA coding for the partial major ampullate silk protein (AvMaSp) was cloned from A. ventricosus. Analysis of the cDNA sequence shows that AvMaSp consists of 240 amino acids of a repetitive region and 99 amino acids of a C-terminal non-repetitive domain. The peptide motifs found in spider major ampullate silk proteins, (A)n, (GA)n, and (GGX)n, were conserved in the repetitive region of AvMaSp. Phylogenetic analysis further confirmed that AvMaSp belongs to the spider major ampullate spidroin proteins. The AvMaSp-R cDNA, which contains sequences encoding for 240 amino acids of a repetitive domain, was expressed as a 22 kDa polypeptide of soluble form in baculovirus-infected insect cells. Recombinant AvMaSp-R was degraded abruptly by trypsin. However, AvMaSp-R was stable at 100 °C for at least 30 min. Additionally, the AvMaSp-R was stable at various pH values from 2 to 12 for at least 1 h. Taken together, our findings provide the molecular structure and biochemical property for A. ventricosus major ampullate silk protein as a biomaterial.

In the transcriptome surveys of Laodelphax striatellus, several cDNA sequences showed a high level of similarities to the insect picorna-like virus genomes. Interestingly, there was no sequence similarity between picorna-like virus sequences from the RSV-viruliferous and those from the non-viruliferous L. striatellus. Picorna-like virus from the non-viruliferous L. striatellus was a geographical isolate of Himetobi P virus (HiPV). The genome of the HiPV was 9,272 nt in length excluding the poly(A) tail and contained two open reading frames (ORFs), which were separated by a 176 nt intergenic region that functions as an internal ribosome entry site (IRES). The 5' ORF encodes the non-structural proteins and the 3' ORF encodes the capsid proteins. The partial genomic RNA of the picorna-like virus from the RSV-viruliferous L. striatellus, LsPV-2, was 8,769 nt in length excluding the poly(A) tail and contained a single, large open reading frame (nt 1–8,535) encoding a 2,845 aa polyprotein. In terms of sequence similarity, identity, and genome organization, LsPV-2 resembled insect picornalike viruses belonging to the family Iflaviridae. A phylogenetic analysis based on RNA-dependent RNA polymerase (RdRp) sequence showed that LsPV-2 was most closely related to the deformed wing virus (DWV). The HiPV and LsPV-2 were incompatible each other in L. striatellus, suggesting that these two picorna-like viruses may have important functions in transmission of the RSV.

Plasmids from Bacillus thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases in mammals and insects. B. thuringiensis serovar mogi of a novel serogroup (H3a3b3d), which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis serovar mogi contained only megaplasmid (> 30 MDa) on which the toxin genes were occasionally located. Sequence analysis using 454-pyrosequencing revealed that the megaplasmid harbored at least seven putative cry genes, showing about 84%, 75%, 73%, 58%, 84%, 39% and 75% similarities in amino acid sequences with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2, respectively. These cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K, and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, could be used as a good resource for studying unknown mosquitocidal cry genes.

ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene whose homologs are found in all lepidoteran Group I NPV, but its function is unknown so far. To determine the role of ac11 in baculovirus life cycle, ac11 knock-out mutant, Ac11KO, was constructed using the plasmid capture system (PCS). Real-Time PCR analysis showed that ac11 transcript was first detected at 6 h post-infection (p.i.) and accumulated to maximum at 48 h p.i., indicating that ac11 is belong to late gene. When the genomic DNA of Ac11KO was transfected into Sf9 cells, viral replication was restricted to a cell transfected originally. While viral transmission of the Ac11KO was not observed in Sf9 cells, production of budded virus (BV) in Sf9 cells transfected with Ac11KO was observed by transmission electron microscopy (TEM). These results suggest that the ac11 is essential for AcMNPV to produce infective BV.

The toxicity of red pine needle hydrodistillate (RPN-HD), 19 RPN-HD constituents and 12 structurally related compounds and control efficacy of four experimental spray formulations containing RPN-HD (0.5. 1, 2 and 3% sprays) to adult Dermatophagoides farinae was evaluated. RPN-HD (24 h LC50,68.33 μg cm–2) was toxic. Menthol was the most toxic compound (12.69 μg cm–2) and the toxicity of this compound and benzyl benzoate did not differ significantly from each other. High toxicity was also produced by α-terpineol, bornyl acetate, geranyl acetate, thymol, linalyl acetate, terpinyl acetate, citral, linalool and camphor (18.79–36.51 μg cm–2). These compounds were more toxic than either deet or dibutyl phthalate. In vapour-phase mortality tests, these compounds were consistently more toxic in closed versus open containers, indicating that their mode of delivery was largely a result of vapour action. RPN-HD 3% experimental spray provided 95% mortality against adult D. farinae, whereas permethrin (cis:trans,25:75)2.5gL–1 spray treatment resulted in 0% mortality. In the light of global efforts to reduce the level of highly toxic synthetic acaricides in indoor environments, RPN-HD and the compounds described merit further study as potential biocides for the control of Dermatophagoides populations as fumigants with contact action.

Substrate affinity and insecticide sensitivity of acetylcholinesterase (AChE) from Daphnia magna S., Bombyx mori L., Musca domestica L., Myzus persicae S., Anguilla anguilla L., Cyprinus carpio L., Oryzias latipes T.&S., Homo sapiens L., Bos taurus L. were tested. The Km values of M. domestica AChE to acetylthiocholine (ATCh), propionylthiocholine (PTCh), butyrylthiocholine (BTCh) were 57.3 μM, 13.4 μM and 85.9 μM respectively, which were lower than those of A. anguilla, C. carpio, O. latipes, H. sapiens and B. taurus. In nontarget organisms, the I50 values of AChE to fenitroxon and DDVP were 1.5×10-6 M~7.8×10-5 M and 2.4×10-6 M~1.1×10-4 M respectively, thus they have lower sensitivity compared with M. domestica. The I50 value of M. persicae AChE to pirimicarb was 1.3×10-8 M, which was higher sensitivity compared with other test animals except D. magna. The I50 values of D. magna AChE to fenitroxon, DDVP, carbaryl, eserine, pirimicarb were 5.2×10-10 M~2.1×10-8 M, which were higher sensitivity compared with the other test animals used for this study. cDNA of Daphnia magna AChE precursor was sequenced and compared with those of Musca domestica, Drosophila melanogaster and Torpedo californica.

The wood-boring and bark beetle (Cerambycidae, Curculionidae, Scolytinae) community in Korean white pine, Pinus koraiensis Sieb. & Zucc., forests was surveyed using Malaise traps in 2007. A total of 1,669 wood-boring and bark beetles were collected, including 193 cerambycids from 16 species, 221 curculionids from 21 species, and 1,255 scolydid beetles from 6 species, of which the dominant species was the ambrosia beetle Xyleborus mutilatus Blandford. Ranked by order of population size, the wood-boring and bark beetle community in Korean white pine showed high dominance by one species of Scolytinae, suggesting the community was unstable and had low biological diversity. Thinning in Korean white pine forests influenced the abundance of bark and ambrosia beetles, whose populations in particular stands increased 1 year after thinning, and then decreased the following year.

This study was conducted to see the initial increasing time of population of citrus leafminer(CLM) in spring and early summer seasons in 2007-2011. CLM adults were monitored using sex pheromone trap which is mainly composed of Z,Z-7, 11-hexadecadienal in the 4, 8 and 5 orchards of the east, south and north part in jeju island, respectively during 2007-2011 seasons. The traps and lures were changed at 10 days and 1 month interval, respectively. Though CLMs were captured on traps from March or April, it was 17, 28 and 28 May in 2007, 16, 26 May and 5 June in 2008, 8, 26 and 26 May in 2009, 27 May, 7 and 7 June in 2010 and 25 May, 2, 2 June in the south, east and north part of Jeju Island, respectively that the CLM population rapidly began to increase toward peaking. The accumulated temperature except less than developmental zero, 12.1 Ujiye T 1990). from January was 248.0, 270.5 and 289.7 in 2007, 193.2, 203.8 and 261.4 in 2008, 202.0, 245.5 and 259.9 in 2009, 261.4, 192.2 and 268.3 in 2010 and 243.4, 246.3 and 252.4 in 2011 at that time. After May overwintered CLM adults are little possible to be counted to the number of captured CLM because adult longevity is about 100 in degree days (Lim 2006). CLM populations in the south part of Jeju Island in first CLM adults increasing periods during 2007-2011 is higher than other parts.

Cicadas that produce loud calling songs for mate attraction can be a nuisance to city dwellers in Korea. The exuviae of final instars can be used for estimation of population density. To provide a key for species identification, we investigated morphological characteristics of final instar exuviae for four cicada species that are abundant in urban areas of central Korea: Cryptotympana atrata, Meimuna opalifera, Oncotympana fuscata, and Graptopsaltria nigrofuscata. Eight morphological characters were used for statistical analyses. The results of a general linear model showed that species and sex were significant for morphological characters and that mean values of most of the morphological characters measured were significantly different among four cicada species. C. atrata and M. opalifera were separated from the other species in most characters measured. Although O. fuscata and G. nigrofuscata were not distinguishable based on size-related characters, these two species differed in femoral tooth angle. Therefore, the exuviae of all four cicada species can be easily distinguished based on the morphological characters used in this study.

Maize weevil (Sittophilus zeamais) and Indian meal moth (Plodia interpunctella) are the dominant species among the stored grain insect pests of the rice grain and bran. This experiment was conducted to investigate developmental characteristic and damages of S. zeamais and P. interpunctella on the rice. Under five constant temperatures, 15, 20, 25, 30 and 35℃, developmental periods from egg to adult of Sittophilus zeamais were 43.0, 36.4, 29.2, 20.8 and 16.3 days, respectively. With egg periods being 9.6, 7.3, 5.2, 3.2 and 2.6 days, and larval periods being 25.2, 22.6, 19.8, 14.5 and 11.3 days, and adult periods being 129.3, 116.1, 108.6, 89.2 and 73.3 days, respectively. Damages of S. zeamais adult at 15, 20, 25 and 30℃ were 67.2, 96.2, 134.0 and 174.5 for 24hr on the rice. Damages of P. interpunctella larval 15, 20, 25 and 30℃ were 56.2, 78.3, 109.4 and 138.7 for 24hr on the rice. The duration of maximum occurrence were June to August for S. zeamais, late May to early August for P. interpunctella.

This study was conducted to identify an insect species in Genus Ostrinia (Lepidoptera: Crambidae) that gave serious damage to the red bean, Vigna angularis. The species has ever been described as O. zaguliaevi in the previous presentation (Jung et al., 2010). Because, however, inconsistent information has been recognized for the species, we reviewed characteristics in morphological, molecular and sex pheromone levels, and host-range. Male genitalia had 3-lobed uncus and tibia of midleg showed massive type. The morphology indicated that the species might be one of O. zaguliaevi, O. scapulalis and O. zealis. Partial nucleotide sequences of cytochrome oxidase subunit Ⅰ(COⅠ) and Ⅱ genes were not identical with those of the 3 species in GenBank, respectively. The deduced amino acid sequence of COⅠ was not identical with that of O. zealis. In the 23 analyses that sex pheromones were checked, (Z)-9-tetradecenyl acetate, which was reported in the sex pheromone components of both O. zaguliaevi and O. zealis, was not detected at all. An intensive study in Japan has reported that the feeding habit of O. scapulalis is polyphagous, while that of O. zaguliaevi is monophagous (only in Petasites japonicus) (Ishikawa et al., 1999). After considering all these information, we concluded that it is reasonable to decide that the insect species in the red bean in Korea is O. scapulalis.