RNA interference (RNAi) is an universal gene-knockdown mechanism in eukaryotic organisms including insects. RNAi has been considered as an alternative strategy to control agricultural pests whereby double-stranded RNA triggers a potent and specific inhibition of its homologous mRNA. Bacillus thuringiensis (Bt) is a spore-forming bacterium that produces a copious amount of crystal proteins δ-endotoxins under the control of sporulation-dependent promoter. In order to develop dsRNA mass-production platform utilizing recombinant Bt, the pHT1K-EGFP which expresses dsRNA against EGFP under the control of Cyt1-Aa sporulation-dependent promoter was constructed and the expression level of transgene (EGFP) was confirmed by qPCR analysis. These results suggested that Bt’s potential of becoming a new platform in dsRNA production.

Streptomyces is the largest genus of Actinobacteria that forms fungus-like branched networks of hyphae. Streptomyces has been clinically important because they produce various secondary metabolites with antibacterial, antifungal, and nematocidal activities. In order to explore novel insecticidal compounds, extracts from 363 strains of Actinobacteria were screened for their juvenoid and anti-juvenoid activities using yeast-two hybrid system. Among them, extract of Streptomyces spp. showed high anti-juvenoid activity. This extract also showed high level of insecticidal activities against larvae of Aedes albopictus, Laodelphax striatellus, and Ostrinia furnacalis. These results suggested that the secondary metabolites of Streptomyces could be natural sources of novel insecticidal compounds.

Medical mushroom, Phellinus linteus and Phellinus baumii called as “Sanghwang” have cultivated in Korea. PL has been studied extensively for its extraordinary capacity of suppressing cancer or enhancing body immunity. The mycelial materials of PL have mainly been used as research samples worldwide because fruiting bodies was difficult to be artificially cultivated. Alternatively, P. baumii (variety, ‘Jangsu’) have been cultivated in Korea. However, fruiting body morphology of P. baumii is clearly different to that of PL. Generally, Phellinus spp. including P. linteus slowly grow on artificial medium such as Potato Dextrose Agar (PDA). In contrast, P. baumii strains were rapidly grown on the artificial media when compared to other Phellinus spp. and thus it was considerable that its mycelial growing ability can be acted as a factor for producing fruiting bodies. This study aimed to find Phellinus isolates having high mycelial growth rate. Five Phellinus isolates that show rapid growth rate on YGM medium were selected from 36 Phellinus isolates collected in Korea. They were identified on nucleotide sequences of rDNA-ITS region. Phellinus linteus strain and Phellinus spp. showing mycelial growth rate comparing to P. baumii were characterized on cultural and bioactive characteristics (antioxidant activity and immune activation).

Bean bug, Riptortus pedestris is an agriculturally serious pest in north eastern Asian countries, damaging to several legumes and fruit trees. Chemical pesticides have been largely used to control the pest but it encounters insecticide-resistance and environmental toxicity issues. Alternatively different mode of action and environmentally sound pest management system can be found in entomopathogenic fungal insecticides. Herein we developed a platform to optimize the fungal production to express their maximum virulence against bean bug, by focusing on solid culture system for thermotolerance, formulation to select effective surfactants to carry the fungal conidia on the cuticles, and relationship between environmental abiotic factors and fungal mortality. First to produce highly thermotolerance fungal conidia, Beauveria bassiana and Metarhizium anisopliae isolates were cultured on several granular cereal substrates, which could be subjected to formulation process. Among the tested media, four media (millet, non-glutinous italian foxtail millet, glutinous italian foxtail millet, brown rice) were superior to the other grains in the spore production and thermotolerance. Next to efficiently deliver the fungal conidia on the cuticles of bean bug, total of six surfactant (CO-2.5, CO-12, LE-7, PE-61, TED-3 and Siloxane) was used to experiment. CO-12 was superior to the other surfactant in mortality of 100 ppm consistence. This work suggests that solid culture system and formulation and application should be seriously considered to reach an optimal level of mortality by inducing their maximum virulence.

The green pale plant bug, Apolygus spinolae was one of the main insect pests that damaged leaves and fruit in grapes and its damage status was firstly reported in 2000 in grape orchards. This research was conducted to evaluate the distribution and difference in damage rate depending in management type of grapevine orchards (domestic sale farm vs export farm) in the export complex area of Korea (Hwangsung in Gyeonggii, Sangju and Yeongcheon in Gyeongbuk, Namwon in Junbuk and Yeongdong in Chungbuk) from 2010 to 2012. Damage by A. spinolae occurred in all 62 survey farms and damage rate differed depending on locality and individual farms in the same area. Damage rate was lower in export farms than in domestic sale farms, and damage rate of leaves was highly correlated with damage rate of new shoots. 15 species of hemipteran insect were attracted to sticky traps and A. spinolae was the dominant species. The attracted number of A. spinolae in the sticky traps differed depending on locality, and more occurred in domestic sale farms than expert farms. A. spinolae was continually attracted to sticky traps in the harvest period in grapevine orchards.

The objective of this study was to determine the effects of E. coli isolated from porcine semen on sperm viability, motility, and semen pH. Semen samples were prepared using commercial extender, SeminarkPro (Noahbio Tech, Korea) that did not contain antibiotics. And 4 different levels of E. coli were artificially innoculated to semen with following concentrations; 4,000 of sperms with 1 of E. coli (T1), 400 with 1 (T2), 40 with 1 (T3), and 4 with 1 (T4). Semen samples were preserved at 17℃ for 5 days in semen storage box until analyzed by flowcytometer. Aliquots were subjected to measure the sperm viability (Live/Dead® stain), motility (mitochondrial function), and semen acidity (pH) from day 0 (day of semen collection) to day 5. Sperm motility and viability were significantly decreased (p<0.05) on day 0 (4 hrs after preservation at 17℃) in T3 and T4 compared to control groups and were significantly decreased (p<0.05) in all groups from day 3. Sample pH was acidic in T3 (6.90～6.86) and T4 (6.86～6.65) from day 3 to day 5 (p<0.05). On the other hand, sample pH was maintained 7.0～7.1 in control, T1, and T2 during the experimental period. Sperm motility and viability were significantly decreased from day 0 to day 5 compared to control in samples contaminated with E. coli above a value of 40:1 (20×106 sperm cells/ml : 5×105 cfu/ml). Even on day 1 in T4 and on day 3 in T3, semen pH was acidic probably due to the acidification of dead spermatozoa. These results suggest that E. coli contamination has a concentration-dependent detrimental effect on extended porcine semen quality.

Oleanolic acid is a natural triterpenoid that exists widely in foods and some medicinal herbs. The purpose of this study was to determine the antimicrobial activity of oleanolic acid against Streptococcus mutans strains isolated from a Korean population. Antimicrobial activity against these bacteria was evaluated by minimal inhibitory concentration (MIC) and time kill curves. The tolerance of human gingival fibroblasts and human periodontal ligaments to oleanolic acid was tested using a methyl thiazolyl tetrazolium (MTT) assay. The MIC90 value of oleanolic acid for both S. mutans and S. sobrinus isolated from Koreans was 8μg/mℓ. Oleanolic acid showed bactericidal effects against S. mutans ATCC 25175T and S. sobrinus ATCC 33478T at 1 × MIC(8μg/mℓ) and had no cytotoxic effects against KB cells at this dose. The results suggest that oleanolic acid could be useful in the future development of oral hygiene products for the prevention of dental caries.

This study was to investigate the effects of upper and lower limb composing patterns of PNF(proprioceptive neuromuscular facilitation) on the static balance ability by 20 subjects for 6 weeks. This study was measured left one leg standing and right one leg standing with closed eyes on Good Balance system. These results led us to the conclusion that the mean speed of X, Y direction, COP(center of Pressure) velocity moment showed a statistical decrease when applying post-exercise. The above results from this study indicated that upper and lower limb composing patterns of PNF exercise has improved the static balance ability. As a result, this study showed that upper and lower limb composing patterns exercise improve the ability of balance in young adults. Based on this study, it may be applied to old people.

Pyrifluquinazon, as a quinazinalone chemical group, based on a new mode of biological activity. It is reported that mode of action is modifies insect behavior, rapidly stopping feeding such that insects starve to death. Time-release feature and mortality effect on M. persicae using different pyrifluquinazon nano type and non-nano type were compared. Pyrifluquinazon nano type was formulated with different molecular weight and density of used chitosan (CS 30000 0.1% and CS 3000 0.3%). In the CS 30,000 0.1%, the mortality was weakly occurred at early time, but steadily increased after 4days. Finally, we confirmed more than 70% mortality as a peak at 16days. In CS 3000 0.3%, the mortality showed about 70% until 18days as a effective controlled release. Also, We examine time-release feature and mortality effect on M. persicae according to the different pyrifluquinazon nano type(CS 30000 0.1% and CS 3000 0.3%) of concentrations. The CS 30000 0.1% bioassay results of different concentration were showed that the highest concentration(100ppm) was measured better mortality than other concentration at 0 day, but cannot confirm different effect about dissimilar concentration. However, increasing rates of M. persicae were low as treatment concentrate was high. In CS 3000 0.3% 100ppm concentration bioassay result, aphid mortality reached peak at 24 days and increasing rate also low. Additionally, for the comparing of bioassay and feeding behavior of M. persicae against pyrifluquinazon nano types and non-nano type, EPG technique was carried out. In case of non nano type, feeding inhibition efficacy was showed during 4 days after treatment, but appeared similar level with control after 10days. In CS 3000 0.3% 50ppm, residual efficacy was specially showed until 28days after treatment whereas treatments with CS 30000 0.1% were similar to the control after 22days. These result show that the change of feedinng behavior and motrality of M. persicae is correlated with the change of nano type or non nano type of pyrifluquinazon.

The multicolored Asian ladybird beetle, Harmonia axyridis, is a generalist predator of aphids also, shows a high level of phenotype polymorphism in color pattern of elytra. Although, it is not sure about genetic information of color polymorphism, it has been confirmed that this phenomenon comes from their genetic traits. The color of H. axyridis elytra is mainly composed of black and red pigment. Phenoloxidase (PO) plays an important role in many insect physiological functions, i.e. sclerotization and pigmentation of cuticle and melanization of parasites. Following activation, PO catalyses the hydroxylation of tyrosine and subsequent oxidation of phenolic substance into quinines, which are further converted to melanin. However, the molecular bases of H. axyridis color pattern formation are almost unknown but it may be that the different pro-POs have different expression. In this study, total RNA samples from four each color pattern individuals, for example, succinea 1, succinea 2, conspicua and spectabilis was extracted. A cDNA enconding pro-PO was molecular cloned from each color pattern of H. axyridis and its putative amino acid sequence shared homology with pro-PO of other insects. We are pursuing to elucidate that their pro-PO sequence will be similar with those other insect PPO sequence. There are also regions of high sequence similarity, including putative activation site and two copper binding sites.

Apolipophorin-III (apoLp-III) is a hemolymph protein whose function is to facilitate lipid transport in an aqueous medium in insects. Recently, apolipophorin-III in Galleria mellonella and Hyphantria cunea was shown to play an unexpected role in insect immune activation. We show here a novel possible function/role of the apoLp-III in insects. To investigate the genes which have a relationship with apoLp-III in fall webworm larvae, we reduced endogenous Hc apoLp-III mRNA levels in larvae via RNA interference (RNAi). The RNAi-mediated Hc apoLp-III reduction resulted in the reduction of antioxidants, like MnSOD, catalase, and glutathione S transferase as well as immune proteins. In particular, expression of MnSOD commonly decreased in fat body, midgut, and hemocytes following the knockdown of Hc apoLp-III, which induced an elevated level of superoxide anion in Hyphantria cunea larvae. The observed effect of Hc apoLp-III RNAi suggests that Hc apoLp-III is related to the action/expression of antioxidants, especially MnSOD.

A new insect member of the STAT family of transcription factors (HcSTAT) has been cloned from the lepidopteran, Hyphantria cunea. The domain involved in DNA interaction and the SH2 domain are well conserved. The gene is transcribed at a low level during all stages of development, and transcribed in hemocyte, fat body, midgut, epidermis, and Malpighian tubule. Especially, hemocyte and Malpighian tubule showed transcriptional activation of HcSTAT upon Gram-negative and -positive bacteria challenge. Gram-negative and -positive bacteria challenge specifically results in nuclear translocation of HcSTAT protein and induction of DNA-binding activity that recognizes a STAT target site in H. cunea hemocyte. In vivo treatment with sodium orthovanadatetranslocates HcSTAT to the nucleus in hemocyte cells.

Apolipophorin-Ⅲ (apoLp-Ⅲ) is a hemolymph protein whose function is to facilitate lipid transport in an aqueous medium in insect. Recently, apolipophorin-Ⅲ in Galleria mellonella and Hyphantria cunea was shown to play an unexpected role in insect immune activation. We show here a novel possible function/role of apoLp-Ⅲ in insects. To investigate the genes which have a relationship with apoLp-Ⅲ in fall webworm larvae, we reduction of endogenous Hc apoLp-Ⅲ mRNA levels in larvae via RNA interference (RNAi). The RNAi-mediated Hc apoLp-Ⅲ reduction resulted in the reduction of antioxidants, like MnSOD, catalase, and glutathione S transferase as well as immune proteins. In particular, expression of MnSOD commonly decreased in fat body, midgut, and hemocytes following the knockdown of Hc apoLp-Ⅲ, which induced an elevated level of superoxide anion in H. cunea larvae. The observed effect of Hc apoLp-Ⅲ RNAi suggests that Hc apoLp-Ⅲ is related to the action/expression of antioxidants.

Innate immunity responses are triggered by the immune challenge and therefore involve signaling processes. The cellular response is initiated by hemocytes and mainly involves phagocytosis and encapsulation of intruders by these cells. To address whether Hc-STAT is activated upon bacterial challenge, we examined the subcellular location of STAT protein in hemocyte by immunostaining. A new insect member of the STAT family of transcription factors (Hc-STAT) has been cloned from the lepidopteran, Hyphantria cunea. The domain involved in DNA interaction and the SH2 domain are well conserved. The gene is transcribed at a low level during all stages of development, and the protein is present in hemocytes, fat body, midgut, epidermis, and Malphigian tuble (Mt). Especially, hemocytes and Mt showed transcriptional activation of Hc-STAT upon Gram (-) bacteria and fungal challenge. Gram (-) bacteria and fungal challenge specifically results in nuclear translocation of Hc-STAT protein and induction of DNA-binding activity that recognizes a STAT target site in H. cunea hemocyte. In vitro treatment with pervanadate translocates Hc-STAT to the nucleus in hemocyte cells. Here we report the first evidence for the involvement hemocyte JAK/STAT pathway upon microbial infection in lepidopteran insect.

Human embryonic stem (ES) cells retain the capacity for self‐renewal, are pluripotent and differentiate into the three embryonic germ layer cells. The regulatory transcription factors Oct4, Nanog and Sox2 play an important role in maintaining the pluripotency of human ES cells. The aim of this research was to identify unknown genes upregulated in human ES cells along with Oct4, Nanog, and Sox2. This study characterizes an unknown gene, named chromosome 1 open reading frame 31 (C1orf31) mapping to chromosome 1q42.2. The product of C1orf31 is the hypothetical protein LOC388753 having a cytochrome c oxidase subunit VIb (COX6b) motif. In order to compare expression levels of C1orf‐ 31 in human ES cells, human embryoid body cells, vascular angiogenic progenitor cells (VAPCs), cord‐blood endothelial progenitor cells (CB‐EPCs) and somatic cell lines, we performed RT‐PCR analysis. Interestingly, C1orf31 was highly expressed in human ES cells, cancer cell lines and SV40‐immortalized cells. It has a similar expression pattern to the Oct4 gene in human ES cells and cancer cells. Also, the expression level of C1orf31 was shown to be upregulated in the S phase and early G2 phase of synchronized HeLa cells, leading us to purpose that it may be involved in the S/G2 transition process. For these reasons, we assume that C1orf31 may play a role in on differentiation of human ES cells and carcinogenesis.

Insulin, transferrin and selenium (ITS) complex is reported to improve in vitro development of oocytes and embryos. This study was carried out to investigate the effects of ITS during in vitro culture (IVC) of porcine parthenogenetic and nuclear transfer (NT) embryos on subsequent developmental capacity in vitro. The electrically activated oocytes were cultured in Porcine Zygote Medium (PZM-3) with various concentrations (0, 0.1, 0.5, and 1.0%) of ITS for 7 days. Also, the electrically activated reconstructed embryos were cultured in PZM-3 with various concentrations (0, 0.1, 0.5, and 1.0%) of ITS for 6 days. Addition of ITS to culture medium did not affect development of porcine parthenogenetic embryos in vitro. To test the effect of ITS on the in vitro development of porcine NT embryos, factorial experiments were also performed for in vitro maturation (IVM) medium (TCM-199) with or without 1% ITS and culture medium (PZM-3) with or without 0.5% ITS. Addition of 0.5% ITS to culture medium increased (p<0.05) the proportion of NT blastocysts compared with non-treated group. In contrast, addition of 1% ITS to culture medium was ineffective or had a detrimental effect. Also, addition of ITS only to maturation medium increased (p<0.05) the percentage of NT blastocysts formation compared with the control group. In conclusion, addition of ITS to IVM or IVC medium could improve subsequent blastocyst development of porcine NT embryos.

To examine the differential protein expression pattern in the 11.5 day post-coitus (dpc) and 18.5 dpc placenta of mouse, we have used the global proteomics approach by 2-D gel electrophoresis (2-DE) and MALDI-TOF-MS. The differential protein patterns of 3 placentae at the 11.5 dpc and 18.5 dpc from nature mating mice were analyzed. Proteins within isoelectric point range of 3.0~10.0, separately were analyzed in 2DE with 3 replications of each sample. A total of approximately 1,600 spots were detected in placental 2-D gel stained with Coomassie-blue. In the comparison of 11.5 dpc and 18.5 dpc placentae, a total of 108 spots were identified as differentially expressed proteins, of which 51 spots were up-regulated proteins such as alpha-fetoprotein, mKIAA0635 protein and transferrin, annexin A5, while 48 spots were down-regulated proteins such as Pre-B-cell colony-enhancing factor 1(PBEF), aldolase 1, A isoform, while 4 spots were 11.5 dpc specific proteins such as chaperonin and Acidic ribosomal phosphoprotein P0, while 3 spots were 18.5 dpc specific proteins such as aldo-keto reductase family 1, member B7 and CAST1/ERC2 splicing variant-1. Most identified proteins in this analysis appeared to be related with catabolism, cell growth, metabolism and regulation. Our results revealed composite profiles of key proteins involved in mouse placenta during pregnancy.