Bladder cancer is a common cancer in smoking men and may correlate with mechanosensitive potassium channels because the urinary bladder is a stretch sensing organ. Two-pore K+ channels (K2P), such as TASK3 and TREK1, have recently been shown to play a critical role in both cell apoptosis and tumorigenesis. Of the channels, TREK1 can be activated by many physiological stimuli, including polyunsaturated fatty acids, and intracellular pH, hypoxia, and neurotransmitters. Here we attempted to determine whether TREK1 is functionally expressed in bladder cancer 253J cells. K2P channels, including TREK1, TREK2, TASK1, TASK3, and TWIK1, were quantified in cultured human bladder cancer 253J cells using real time quantitative RT-PCR (qRT-PCR) analysis. Among them, TREK1-like channel was recorded at a single channel level using the patch-clamp technique. The TREKl-like channel, with single-channel conductance of ~90 pS at −80 mV, was recorded in symmetrical 150 mM KCl using an excised inside-out patch configuration. The current- voltage relationships were linear and were insensitive to tetraethylammonium. The channel was activated by membrane stretch, free fatty acids, and intracellular acidosis. These results with electrophysiological properties resemble to those of K2P channel, for instance, TREK1. Therefore, we conclude that TREK1 channel is functionally present in bladder cancer 253J cells.

Hipparchia autonoe belongs to the family Nymphalidae (Lepidoptera) and is designated as an endangered insect and national monument in Korea. It only inhabits a very restricted area on Mt. Halla but is widely distributed in several Asian countries including Mongolia. A previous study conducted to understand the genetic relationship between Mt. Halla and Mongolian H. autonoe for conservation purposes suffered from a limited number of samples. Therefore, we sequenced the DNA barcode region of an additional 36 H. autonoe individuals, combined them with previous data from 19 individuals, and performed phylogenetic and population genetic analyses. Furthermore, the internal transcribed spacer 2 (ITS2) region was also sequenced from the 36 samples as a nuclear DNA marker. The existence of independent haplotypes, sequence types, and significant FST estimates (p < 0.05) between Mt. Halla and Mongolian populations indicated hampered gene flow between the populations. Nevertheless, an absence of a reciprocal monophyletic group in Mt. Halla and Mongolian populations by cytochrome oxidase subunit I gene- and ITS2-based phylogeny suggests that the genetic isolation of the Mt. Halla population from the Mongolian populations seemed not large enough to consider them independent genetic entities.

Lepidoptera is one of the largest insect orders, but the phylogenetic relationships within this order, have yet to be completely described. One of the unresolved relationships includes the monophyly of Papilionoidea in relationship with the monotypic superfamily Hesperioidea. We newly sequenced five hesperid mitochondrial genomes (mitogenomes), representing four subfamilies: Pyrginae (Daimio tethys and Lobocla bifasciatus), Coeliadinae (Choaspes benjaminii), and Hesperiinae (Potanthus flavus), and Heteropterinae (Carterocephalus silvicola). Along with these newly sequenced hesperid genomes phylogenetic analysis was conducted with all available lepidopteran mitogenomes including three reported species of Hesperiidae that consisted of ~70 species in ten lepidopteran superfamilies. The test for the effect of optimization schemes, such as exclusion and inclusion of third codon position of 13 PCGs, other genes (22 tRNAs and two rRNAs), and with and without partitions also was performed. Majority of datasets consistently placed the monophyletic Hesperiidae the sister to ((Pieridae + Lycaenidae) + Nymphalidae), placing another true butterfly family Papilionidae as the basal lineage of this group, presenting the relationships (Papilionidae + (Hesperiidae + ((Pieridae + Lycaenidae) + Nymphalidae))). Consistent to previous result, Pyraloidea was placed as the sister to ((Bombycoidea + Geometroidea) + Noctuoidea), placing the Macrolepidoptera as non-monophyletic group.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is widely utilized for greenhouse pollination in South Korea. Understanding the magnitude of genetic diversity and geographic relationships is of fundamental importance for long term preservation and utilization. As a first step, we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to the “DNA barcode” region and the complete internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA from 88 individuals collected in nine South Korean localities. The complete ITS2 sequences were longest among known insects, ranging in size from 2,034 bp ~ 2,052 bp, harboring two duplicated 112-bp long repeats. The 658-bp long mtDNA sequences provided only six haplotypes with a maximum sequence divergence of 0.61% (4 bp), whereas the ITS sequences provided 84 sequence types with a maximum sequence divergence of 1.02% (21 sites). The combination of the current COI data with those of published data suggest that the B. ignitus in South Korea and China are genetically a large group, but those in Japan can be roughly separated into another group. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population were found to exist among the South Korean populations of B. ignitus, which suggests panmixia. This finding is consistent with our understanding of the dispersal capability of the species.

In the present study, the 17,694-bp long complete mitochondrial genome (mitogenome) of the dwarf honey bee, Apis florea (Hymenoptera: Apidae), is described with an emphasis on the noteworthy triplicated tRNAser(AGN) region and an extraordinary long A+T-rich region with repeat regions. The gene arrangement of A. florea mitogenome is identical to that of A. mellifera, but has triplicated tRNASer(AGN), each of which contains the precedent 44 bp-long and following another 64 bp-long repeats plus one complete first repeat abutting to tRNAMet. A total of 1,610-bp long two repeat regions in 1,987 bp-long A+T-rich region is composed of nearly identical 141 ~ 219-bp long five tandem repeats and 50 ~ 52-bp long 12 tandem repeats that are encompassed by three non-repeat sequences. One of the common interpretations for such repeat sequence is slipped-strand mispairing and unequal crossing-over events during DNA replication.

We previously reported Pear Pest Forecasting Management System (PPFMS) for the Improvement of pass ratio of Korean exporting pears. It is consisted of regular field forecasting by pear farmers, meteorological data obtained by automatic weather station (AWS), an internet web page (http://pearpest.jnu.ac.kr/) as information collecting and providing ground, and information providing service. Currently, we are expanding this system to the area, Cheonan and Ansung, where pear orchards are organized into exportation-specific group. Further, the information obtained from field forecasting and AWS were up-loaded to under-constructing upgraded webpage (http://www.kpear.kr), with several pest/disease-related information. We hope this pest forecasting management system increases the pass ratio of Korean exporting pears throughout establishment of farmer-oriented forecasting, inspiring farmers’ effort for the prevention and forecasting of diseases and pests occurring at pear orchards.

This study was conducted to estimate the control thresholds (CTs) at different larval densities of Oides decempunctatus Billberg (Coleoptera: Chrysomelidae) of Campbell early in the vineyard and investigated life cycle. Each stage of O. decempunctatus was sampled 18 times from May to September in 2010~2012. The seasonal occurrence of O. decempunctatus showed the highest peak in mid-late June and mid-late August. Overwintered O. decempunctatus's eggs were hatched from late May to early June. Larva period was from late May to mid July and adults appeared in mid July. The percentage of leaf damage (Y) of Campbell early inoculated by different densities of O. decempunctatus (X, no. of larvae/fruiting mother branch) for six weeks was estimated by Y= 0.498X+2.041 (R2=0.988) during vegetation period. The decreasing rate of soluble solid (Y) after grape harvest of Campbell early damaged by different densities of O. decempunctatus (X) was estimated by Y= - 0.046X+15.3 (R2=0.8543). Based of the relationships between the densities of O. decempunctatus larvae and the index of reducing soluble solid of Campbell early, the number of larvae (2nd to 3rd instar) which decreased less than 15°Bx loss of soluble solid was determined as the injury level of 7/fruiting mother branch.

휴면이 타파된 무늬둥굴레의 촉성재배와 억제재배시 생육 적정 온도 범위를 알아보았다. 촉성재배를 염두한 낮과 밤의 온도가 15/5, 20/10, 25/15, 30/20oC의 처리, 억제재배를 염두한 28/ 18, 32/22, 36/26, 40/30oC 처리에서 무늬둥굴레를 생육시켰다. 촉성재배을 위한 생육온도 실험에서 온도가 올라갈수록 맹아소요 일수, 개화소요일수, 낙화소요일수가 줄어들었다. 그러나 맹아율, 개화율, 초장, 엽수에서는 온도에 따른 유의적인 차이를 보이지 않았다. 다만 초장이 15/5oC 처리에서 상대적으로 작았다. 이 실험의 결과로 촉성재배시 20/10oC 이상의 온도를 유지하는 것 이 권장할 수 있다. 억제재배를 위한 생육온도 실험에서는 재배 온도가 높아질수록 낙화소요일수, 초장, 엽수에서 감소하였다. 그 러나 맹아소요일수, 개화소요일수, 맹아율, 개화율에서는 온도간 유의적 차이가 없었다. 초장이 40/30oC 처리에서 상대적으로 줄 어드는 현상이 나타났다. 그러므로 무늬둥굴레의 억제재배시 36/ 26oC 이하의 온도로 재배할 것을 권할 수 있다.

많은 춘계단명식물들의 종자들은 모식물체에서 미숙 배를 가지고 탈리되는 것으로 보고되고 있다. 이러한 종자들은 형태학적 또는 형태생리학적 휴면을 가지고 있다. 따라서 본 연구는 몇 가지 자생 관상용 춘계단 명식물들의 형태학적인 종자휴면 연구를 위한 기초자 료를 제공하고자 수행하였다. 깽깽이풀, 한계령풀, 복수 초, 매발톱꽃, 동강할미꽃, 바위미나리아재비, 얼레지 및 큰연영초의 종자를 5월부터 6월 사이에 채종하여 배의 형태와 발아율을 조사하였다. 연구대상 8종 모두 미숙배 종자였다. 한계령풀의 종자는 심장형 배를, 깽 깽이풀, 복수초, 매발톱, 바위미나리아재비 및 동강할미 꽃의 종자는 어뢰형 배를 가지고 있었다. 백합과의 얼 레지와 큰연영초는 구형과 심장형의 중간형태 정도였다. 동강할미꽃과 바위미나리아재비는 배의 길이가 종 자 길이의 15% 이상이었고, 나머지는 10% 미만이었다. 매발톱과 동강할미꽃은 30일 동안 각각 92%, 84%가 발아하였으나, 나머지 종자들은 전혀 발아하지 않았다. 따라서 매발톱과 동강할미꽃은 형태적 휴면으로, 나머 지 종자들은 형태생리적 휴면으로 분류할 수 있었다. 이 결과들은 추후 형태적 종자휴면을 연구하는데 유용 한 자료가 될 것이다.

To identify subspecies and stocks of minke whale meats purchased from Korean markets during 2005-2007, we first obtained their complete sequences of mitochondrial DNA cytochrome b and control region sequences, and compared these sequences to the corresponding sequences of the common minke whale (Balaenoptera acutorostrata), obtained from GenBank. From analyses with partial cytochrome b sequences (383 bp) and non-coding, partial control region sequences (463 bp), Korean mink whale meats are identified as products from the North Pacific minke whale (B. a. scammoni). In addition, the sequences of the partial control region from these meats showed G at site no. 298 and G or A at site no. 463, and the meats appeared to originate from the J stock within this subspecies. Thus, because the J stock has been protected since 1986, implementation of strict regulation measures to reduce their accidental fisheries by catch seems urgent. In addition, B. a. scammoni is distinct from B. a. acutorostrata, with an average Jukes-Cantor distance of 2.21% in the complete control region sequence analysis (935 bp) and 1.31% in the complete cytochrome b gene sequence analysis; the current results support the current subspecies classification, although further sequencing analyses with nuclear genes are necessary.

The objective of this study was to investigate the effects of NEAA and leptin supplemented to in vitro culture medium on the developmental competence of porcine embryos after intracytoplasmic sperm injection (ICSI), and to modify the culture condition to improve the quality and the development of ICSI-derived porcine embryos in vitro. After ICSI, the putative zygotes were then cultured in PZM-3 medium with/without NEAA or leptin. The proportion of embryos that developed to the blastocyst stage significantly increased when 1% NEAA (24.62%) was added to the medium compared with 2% NEAA and no NEAA group (17.24% and 20.24%, respectively, p<0.05). The effect of different concentration of leptin (0, 10, 100, 500 ng/ml) was evaluated on the development of porcine ICSI embryos cultured in vitro. In case of blastocyst formation, 100 ng/ml group (27.05%) showed significantly higher rate than 10, 500 ng/ml, and control group (23.45%, 17.99%, and 19.68%, respectively, p<0.05). We also evaluated the effects of different NEAA and leptin treatment time on the development of porcine embryos after ICSI. Among groups of embryos cultured in the presence of NEAA or leptin for whole 7 days (D 1-7), first 4 days (D 1-4), the subsequent 3 days (D 5-7), both NEAA (27.13%, 21.17 %, and 17.56%, respectively, p<0.05) and leptin (25.60%, 20.61%, and 16.53%, respectively, p<0.05) showed that supplementation for whole 7 days significantly increased the blastocyst formation rate compared with the other groups of D1-4 and D5-7. We further evaluated the combination effect of 1% NEAA and 100 ng/ml leptin compared with the effect of each supplementation with 1% NEAA or 100 ng/ml leptin or no supplementation on development of embryos. For blastocyst formation, combination group of NEAA and leptin (24.78%) showed significantly higher rate than other three groups (18.37%, 20.44 %, and 13.27%, respectively, p<0.05). We further evaluated the expression of proapoptosis genes such as BAX and BAK and anti-apoptosis genes, BCL-XL and BCL-2 in blastocysts cultured in the presence of 100 ng/ml leptin. RT-PCR analysis revealed that leptin supplementation significantly decreased the expression of pro-apoptosis genes as well as increased the expression of anti-apoptosis genes. These results of present study demonstrate that NEAA and leptin could improve the in vitro development of ICSI- derived porcine embryos with optimal concentration of each reagent. Furthermore, the optimal culture condition could increase the quality of ICSI-derived embryos in vitro.

Chicken Insulin-like Growth Factor-1 (cIGF-1), one of the most important hormone for regulating physiological function includes body growth, muscle volume, bone density, chicken cell development and metabolism. In order to find in vitro Knokdown expression of cIGF-1, this study introduced tetracycline inducible RNA interference expression system (TetRNAi system). Tet system can inductively control high expression of extrinsic genes and expression of intrinsic genes. So it has advantages such as minimized physiological side-effects any cell and low cytotoxicity. RNAi system is proving to be a powerful experimental tool for inhibition of gene expression and post-transcriptional mechanism of gene silencing. RNAi is mediated by small interfering RNA (siRNA) consisting of 19- to 23- nucleotide double-stranded RNA duplexes that promote specific endonucleolytic cleavage of mRNA targets through an RNA-induced silencing. Then, this study RNAi-based gene knockdown can be achieved by retroviral-based expression systems. Stable integration of our inducible siRNA vector allowed the production of siRNA on doxycycline induction, followed by specific down regulation of chicken IGF-1 gene. Analyses of Real-time PCR to determine expression of the cIGF-1 gene showed successful from chicken embronic fibroblast (CEF) cells with the reduced rate of an approximately 92%. Our results demonstrate the successful regulation of cIGF-1 knockdown expression in CEF cells and support the application of an tetracycline inducible RNAi expression system in transgenic Mini chicken production. This research was supported by Bio-industry Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

The aim of this study was to examine the effect of acteoside (the cyclin-dependent kinase inhibitor) on the SCNT efficiency with adult fibroblasts in dog. Canine adult fibroblasts were obtained from muscle and cell cycle of fibroblasts was synchronized by culturing to confluency, serum starvation and treating with 30 μM acteoside for 48 h. Cell cycle stages, cell cytotoxicity (apoptosis) and, prduction of reactive oxygen species (ROS) were analyzed using flow cytometry. The canine cells, prepared by confluent-cell culture or treating with 30 μM acteoside for 48 h, were injected into enucleated in vivo matured oocytes, the couplets were electrical fused and activated by calcium ionomycin. SCNT embryos using acteoside-treated fibroblasts were surgically transferred into oviducts of estrus cycle synchronized recipient dogs. In cell cycle synchronization (G0/G1), there was no significant difference between serum starvations (83.9%) and acteoside treated groups (81.3%) that were higher than confluent group (78.5%). In production of apoptosis, confluent and acteoside treated groups (4.3 and 4.5%, respectively) were generated less than serum starvation group (21.8%). In case of ROS, serum starvation group was induced a significantly higher than other groups. After synchronization of the donor cell cycle, either confluent or acteoside treated, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into naturally estrus-synchronized surrogates. Fusion and cleavage rate of acteoside treated group were 64.1 and 41.5%, which were higher than those of confluent group (53.9 and 20.6%, respectively). The reconstructed embryo development rates to 4-cell and 8-cell in acteoside treated group were 29.5 and 14.8%, respectively, while confluent group showed 11.1 and 3.2%, respectively. Total 54 SCNT embryos using acteoside-treated fibroblasts were transferred into oviducts of 2 recipient dogs and one recipient finally delivered one puppy, whereas din`t detected pregnancy on transfer of cloned embryos reconstructed with confluent cells in 6 surrogate dogs. In conclusion, the results of the current study demonstrated that canine fibroblasts could be successfully arrested at the G0/G1 stage with reduced the formation of ROS and apoptosis after acteoside treatment. This results may contribute to improve the effi-ciency of canine SCNT. * This research was supported by iPET (Grants 110056-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Acteoside acts as an anti-oxidative activity and anti-apoptosis in the cells. But, it has been not studied on maturation and development of porcine oocytes. The aims of the present study were to examine the effects of acteoside on the morphological progress of meiosis, developmental competence, and ROS in porcine oocytes. Oocytes were matured in tissue culture medium-199, supplemented with acteoside at various concentrations: 0 (control), 10, 30 and 50 μM. The oocytes maturation rates of groups supplemented with acteoside were no significantly different (81.13, 85.96, 82.95 and 83.68%, respectively). Level of ROS was significantly decreased in acteoside treated group. Furthermore, the parthenogenetic blastocyst rate was significantly improved in 10 μM acteoside treated group compared with control group (44.83 vs. 27.75%). And we investigated effect of acteoside on the oocytes condition represented by cytoplasmic maturation by homogeneous distribution and formation of cytoplasmic organelles and regulation of apoptosis-related genes. In the results. during IVM, 10 μM acteoside treated oocytes showed that the mitochondria and lipid droplet were smaller and homogeneous distribution in cytoplasm compare with control oocytes. And reverse transcription polymerase chain reaction (RTPCR) of parthenogenetic blstocysts revealed that acteoside increased the anti-apoptotic genes (Mcl-1, Bcl-2 and Bcl-xL), whereas reduced the expression of pro-apoptotic genes (Bax and Bak). In conclusion, based on the results, the effect of acteoside on IVM was not attractive. However, in acteoside treated group, cytoplasmic maturation seemed to be improved with morphologically uniform distribution of cytoplasmic organelles. Furthermore, embryonic development in acteoside treated group was significantly highly increased than that of non-treated group. Our results represents that addition of acteoside to the IVM medium has a beneficial effect in physiology of porcine oocytes, providing a improved method for porcine oocytes in vitro. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.

The objective of the current study was to describe in vitro embryo production in Hanwoo, analyzing oocytes yield and embryo production. The effects of oocytes production and the number of OPU procedures per animal on embryo production were also evaluated. OPU was done every 3～4 days during experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, we compared the recovery rate of oocytes based on OPU session (Experiment 1). The average of collected oocytes was calculated from every 10 session. The average number of total oocytes recovered per animalonsessionwas 5.16 (mean). Second, we compared the recovery rate base on collection period of OPU (Experiment 2). The following results show the difference of the number of recovered oocytes in every month during the procedure between the months of session. Every animal shows the constant number of recovered oocytes for the first 5 months. However, the recovery rate of oocytes was decreased from month 6 to 8. Third, we compared the developmental rate to blastocyst in two groups (Experiment 3). Oocytes by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24～48 h after in vitro fertilization (IVF) was 75.8% and blastocyst development rate was 18.8% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (61.1%), blastocyst development rate was higher compared with needle puncture group (28.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio- technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being.

The objective of this study was to evaluate in vitro production of bovine embryos in Hanwoo. Oocytes were collected by ovum pick up (OPU) from ovaries of genetically high-value Hanwoo or by needle puncture from ovaries of slaughtered cattle. OPU was done every 3 4 days duing experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, We compared the in vitro maturation rate in two groups (Experiment 1). 545 oocytes were recoverd from 4 females by 32 trials of OPU and then 433 oocytes were shown MⅡ stage after in vitro maturation (79.4%). In case of needle puncture group, 1905 oocytes were collected and then 1420 oocytes were matured to MⅡ stage during in vitro culture(74.5%). Second, we compared the developmental rate to blastocyst in two groups (Experiment 2). 1420 oocyte by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24 48 h after in vitro fertilization (IVF) was 88.6% and blastocyst development rate was 20.5% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (84.8%), blastocyst development rate was higher compared with needle puncture group (26.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio-technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being. This research was suppoted by Imsil-gun agricultural technology service center.