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.

Nicotine, a major teratogen of cigarettes smoke induces embryonic abnormalities during the early stages of organogenesis. In this study, the protective effect of β-carotene against nicotine–induced embryos was evaluated by morphologic scoring, nile blue staining, lipid peroxidation, SOD activity assay and real-time PCR. The embryos exposed to nicotine (1 μM) revealed remarkable morphological anomalies compared to normal control group (p<0.05), but when β-carotene (1×10‒4 μM or 5×10‒4 μM) was added concurrently to the embryos exposed to nicotine, morphological parameters were significantly improved (p<0.05). Nicotine induced oxidative stress by increased lipid peroxidation, expression of proinflammatory cytokines (TNF-α and IL-1β), caspases-3 and decreased SOD activity. However, administration of β-carotene (1×10‒4 μM or 5×10‒4 μM) restored the SOD level and decreased oxidative damage in the embryos. These results indicate that β-carotene effectively counteracts the deleterious effects of nicotine on embryos and attenuates oxidative damage possibly through its antioxidant effects.

Nicotine, a major toxic component in tobacco smoke, leads to severe embryonic damages on organogenesis. We investigated if resveratrol can inhibit the nicotine–induced teratogenesis in the cultured mouse embryos (embryonic day 8.5) for 48 hours using a whole embryo culture system. The embryos exposed to nicotine (1 μM) revealed severe morphological anomalies, the increased levels of caspase-3 mRNA and lipid peroxidation, and further the lowered levels of mitochondrial manganese superoxide dismutase (SOD), cytosolic glutathione peroxidase (GPx), phospholipid hydroperoxide GPx, hypoxia-inducible factor 1α, and sirtuin mRNAs and SOD activity significantly compared to normal control group (p<0.05). However, whenre sveratrol(1×10‒5 μMor1 ×10‒4 μM) was added concurrently to the embryos exposed to nicotine, these all parameters were significantly improved (p<0.05).These findings indicate that resveratrol has a protective effect against nicotine-induced teratogenesis in mouse embryos throughout antioxidative and anti-apoptotic activities.

Zinc oxide nanoparticles (nZnO) are used in a various range, including ceramic manufacture, photocatalysis, UV filters, and the food industry. However, little is known about the effects of micro- and nano-particles during mouse embryo organogenesis. To determine whether ZnO affects size-dependent anomalies during embryonic organogenesis, mouse embryos were cultured for two days with 300 ug/ml micro ZnO (mZnO;80±25 μm) and nZnO (< 100 nm) and the developmental changes were then investigated. Quantity of Zn by inductively coupled plasma mass spectrometry analysis, and expression patterns of various antioxidant enzymes in the embryos were investigated. Embryos exposed to mZnO or nZnO exhibited severe retardation of growth and development. In embryos exposed to mZnO and nZnO, yolk sac diameter, crown-rump length, and head length were significantly diminished. The morphological parameters, including yolk sac circulation, allantois, flexion, heart, hindbrain, midbrain, forebrain, otic system, optic system, branchial bars, maxillary process, mandibular process, olfactory system, caudal neural tube, forelimb, hindlimb, and somites in mZnO and nZnO-treated groups were significantly decreased. Zn absorption of the nZnO-treated group was significantly higher than that of the mZnO-treated group. Significantly decreased levels of CuZn-SOD, Mn-SOD, cGPx, and PHGPx mRNA were observed in the ZnO-treated group. In addition, antioxidant enzyme mRNA expressions of the nZnO group were significantly diminished, less than those of the mZnO treated group. These findings indicate that 300 ug/ml ZnO showed abnormality and nZnO may have a more severe effect than mZnO in developing embryos.

Porcine Circovirus Type2 (PCV2), a single-stranded DNA virus associated with Postweaning multisystemic wasting syndrome(PMWS) of swine, has two major open reading frames, ORF1 and ORF2. The genomic size and molecular weight of ORF2 is respectively 699bp, 28kDa. ORF2 encodes the capsid protein (structural protein) that has type-specific epitopes and is very immunogenic and associated with the induction of neutralizing antibodies, suggesting its potential use in diagnostic assays as well as vaccine development. For efficient production of the capsid proteins, we expressed the PCV2 ORF2 gene with baculovirus in the insect cells. In this study, PCV2 ORF2 was appropriately ligated into the baculovirus transfer vector, pBacPAK9 and pB9-Acpol19-110-EK. Sf21 cells were transfected with a mixture of the purified recombinant transfer vector and bAcGOZA. We generated and purified recombinant viruses containing PCV2 ORF2, and named rAc-B9-PCV2ORF2 and rAc-B9-19-110-EK-PCV2ORF2, respectively. Expression levels of capsid fusion proteins with a partial polyhedrin region of AcNPV more increased than recombinant proteins from non-fusion expressed. Also, expression efficiency increased over time and differed at MOI. As a results, fusion expression of porcine circovirus type2 ORF2 using baculovirus could be utilized as an alternative expression method to produce recombinant antigen against PCV2 infection and is worthy of further investigation.

Neurotoxicity and oxidative injury induced by glutamate cause neuronal degeneration related to various central nervous system diseases. Resveratrol, a polyphenolic compound, is known to have antioxidative and anti-inflammatory effects. The aim of this study was to investigate the question of whether resveratrol has a neuroprotective effect against glutamate-induced toxicity in cultured cortical neurons. Following exposure to glutamate for 15 min, cortical neurons originating from ICR mouse fetuses on embryonic days 15-16 were then treated with resveratrol for 24 h in the post-treatment paradigm. Glutamate induced a significant reduction in cell viability; however, resveratrol induced a significant increase in cell viability. Glutamate induced generation of ROS and apoptotic neuronal death; however, these were decreased by exposure to resveratrol. mRNA expression in antioxidant enzymes, cytoplasmic glutathione peroxidase, copper/zinc superoxide dismutase (SOD), and manganese SOD, and anti-apoptotic regulator Bcl-xL were decreased by exposure to glutamate, however, exposure to resveratrol resulted in a significant increase in their mRNA levels. In addition, mRNA expression of pro-inflammatory cytokines, interleukin-1β and tumor necosis factor-α, was increased by glutamate insult, but significantly reduced by resveratrol. These findings indicate that resveratrol is neuroprotective against glutamate-induced toxicity, suggesting a useful therapeutic application in treatment of neurodegenerative disorders.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a unique antioxidant enzyme involved in reduction of peroxidized phospholipids within biomembranes. To investigate the expression pattern of the PHGPx gene during fetal development, in situ hybridization analyses were performed using mouse FITC-labeled PHGPx cRNA probes in fetal tissues on embryonic days (Ed) 13.5-18.5. During these periods, PHGPx mRNA appeared in the developing telencephalon, diencephalon, spinal cord, and spinal ganglion. In particular, PHGPx mRNA was strongly expressed in pyramidal cells of the cerebral cortex. On Eds 17.5-18.5, PHGPx mRNA was detected in various tissues including liver, intestinal villi and crypt, pancreas, lung, and olfactory epithelium of the nasal cavity. In addition, PHGPx mRNA was highly expressed in the inner ear on Eds 14.5-18.5, brown fat on Ed 17.5, and adrenal gland on Ed 18.5. It is conceivable that PHGPx may act as an important antioxidant against fetal oxidative stress during mouse organogenesis.

Animal crude drugs (natural medicines derived from animal organs) have been widely used in various Chinese medicine for the therapeutic effects and for enhancement of immunologic functions. We found the specific identification methods using DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses for mitochondrial DNA (mt DNA) in order to discriminate between the animal species and organs as well as the placenta of humans. Species-specific PCR bands of D-loop mt DNA for equine, bovine, porcine, and human were 133 bp, 137 bp, 231 bp, and 240 bp, respectively. Porcine organs were identified using restriction enzyme, HphI cut into two subfragments, 36 bp and 195 bp bands in the heart, spleen, and liver, except for kidney. The heart and liver of porcine were identified using restriction enzyme, SpeI cut into two subfragments, 84 bp and 147 bp bands, except for kidney and spleen. Bovine organs were cut into 68 bp and 69 bp bands in the liver, kidney, and spleen using NalIV, except heart and placenta. Placentas of bovine and humans were easily identified using each primer. Our results suggest that sequencing of mt DNA and its PCR-RFLP methods are useful for identification and discrimination of inter- and intra-specific variations in equine, bovine, porcine, and human by routine analysis.

This study was to investigate pregnancy rate of IVM/IVF/IVC Korean cattle (registered in government) embryos according to transport time course. For the production of embryos, oocytes recovered from slaughtered excellent grade cow and highly motile frozen‐thawed bull semen (purchased from LIMC, KPN#497) was used. In vitro produced embryos were cultured in CR1aa medium for 8 days and some of them were frozen. The rate of average cleavage (>2‐cell) was 83.0% (308/371) and blastocyst rate at day 8 was 34.7% (107/308). Among in vitro produced blastocyst embryos at day 8, most healthy embryos were freshly transferred on production day and some frozen embryos were direct transferred on appropriate day. These embryos were produced in a laboratory, embryo transfer (ET) was planned in 10 areas of the remote island (Jeju) from the laboratory by airplane. Thus, we examined the pregnancy rate in recipient cow according to embryo of transport time course before ET. From embryo transferred 44 recipient cows, overall pregnancy was 40.9% (18/44), these 18 cows were all calved [single, 94% (17/18); twin, 6% (1/18)] and total embryo implantation rate was 26% (19/66). Comparing transport time in the base of 6 hr, pregnancy rate in ET group required less 4 hr (60%, 9/15) was significantly higher than that required more 6 hr (26.3%, 5/19). In direct ET of freezing embryos, the pregnancy rate was 40% (4/10). However, it was difficult to find the meaning of temperature, pH and corpus luteum quality of recipients on comparison of pregnancy rate. When the cell death level of embryos according to storage time in thermos (straw container) before ET was measured by TUNEL staining, apoptotic index was increased with storage time‐dependent. These results demonstrated that long distance transfer of IVM/IVF/IVC embryos is possible and the time of embryo transport is very important for the pregnancy rate on field trial.

We report for the first time the occurrence of DWV-infected bumble bees (Bombus ignitus). For the present study, the detection of DWV virus from the female and male bumble bee was investigated in the same colony. The Deformed wing virus (DWV) of honeybee (Apis mellifera) is closely associated with characteristic wing deformities, abdominal bloating, paralysis, and rapid mortality of emerging adult bees. Using specific RT-PCR protocols for the detection of DWV followed by sequencing of the PCR products we could demonstrate that the bumble bees were indeed infected with DWV. The virus was detected from Bombus ignitus, and its partial DWV gene was cloned and sequenced. The partial DWV gene encoding the polyprotein is 711-nt of 235 amino acid residues. The deduced nucleotide sequence of the polyprotein partial gene of DWV showed 96.9%, 96.2%, 96.8%, and 96.5% homology to other structure polyprotein partial gene of DWV from insects, respectively. Phylogenetic analysis further conformed that the deduced nucleotide sequence of the polyprotein partial gene of DWV divided to the outside tree. We describe the first time that presence of Deformed wing virus(DWV) from bumble bee(Bombus terrestris) in korea using RT-PCR.

The traditional use of insects as food continues to be widespread in tropical and subtropical countries and to provide significant nutritional, economic and ecological benefits for rural communities. Specially, Bee brood serves as a food source to humans in many countries although limited data exists concerning its nutrient composition. Bee brood (pupa and larvae) were analyzed for Carbohydrate, Saturated fatty acid, Cholesterol, protein, fat, fiber, minerals, and vitamins. Bee brood was high in protein(46.4%～46.73%), fat(18.84%～ 20.75%),carbohydrate(24.66 %～35.79 %), Folic acid(222.30 ㎍/100g), and vitamins. Differentially, folic acid had been contained by high density in pupa of drone. While low in iron, bee brood was a good source of folic acid, and carbohydrate. The fat was composed mostly of saturated and mono-unsaturated fatty acids. The present data suggest bee brood to be an excellent source of many valuable nutrients including energy, amino acids, many essential minerals, and B-vitamins. These data suggest bee brood could be a valuable source of nutrients to various populations.

Wntsignaling is involved in the normal development and tumorigenesis via epithelial- mesenchymal transition (EMT). init iated by down-regul ation of E-cadherin by the transc ription factor Snail. Wnt signaling inhi bits Sna il phosph o rylation t hrough Axin2-dependent pathway that sustains nuclear accumul ation 0 1' Snail by driving CSK3ß nucleocytoplasmic export then consequently increases Snail protein levels and induces an EMT However. the roles of Wnt and Axin expression and their functional implication on Snail dependent EMT program a re not clear du ring the multistep carcinogenic process. We examined that canonical Wnt signaling engagingmul t istep carcinogenic process of uterine cervical cancer through Wnt-Axin2-Snail axis. In nonnal cervi cal mucosa, Wntl. Wnt3a. and Axin2 mRNA expression were locali zed in basal cell layer suggesting that canonical Wnt is required for maintenance of self-renewal program of cervica l epi theli al cells. With progression to cervical int r aepithelial neoplasia (CIN) and carcinoma. Wntl, Wnt3a‘ Axin2, and Snail expression were gradually increased in patient samples suggesting that canonical Wnt pathway is involved in earl y step of carcinogenesis in uterine cervix. LRP6 and Axin2 transfected cells showed the highly increased nuclear Snai l resul ted from dec reased level 0 1' nu clear GSK3ß , indicating that LRP6- Axin2 serves to stabili ze Sna il protein levels and susLains iLs nllclear acc llrnulation by driv ing GSK3ß . RNA interference of Axin2 and Snail on SiHa cells relieved E-cadherin proximal promotel‘ activity and block the in 띠 vo chorioalantoic membra ne ln VaSlOn These results suggest the canon ical Wnt signa ling regul ating Axin2-GSK3ß compartmentalization may important for stabi li zation of E- cad herin repressor Snail during the multistep carcinogen ic process of uteri ne cervix. It may lead to not only tracing the proper biomarker 0 [' ca ncer progression‘ but a lso the development oJ new targets for therapeutic intervention in cancer