The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas9) system can be applied to produce transgenic pigs. Therefore, we applied CRISPR/Cas9 system to generate FoxN1-targeted pig parthenogenetic embryos. Using single guided RNA targeted to pig FoxN1 genes was injected into cytoplasm of in vitro matured oocyte before electrical activation. In results, regardless of the concentrations of vector, the cleavage rate were significantly (p<0.05) decreased (4 ng/μl, 51.24%; 8 ng/μl, 40.88%; and 16 ng/μl; 45.22%) compared to no injection group (70.44%). The blastocyst formation rates were also decreased in vector injected 3 groups (4 ng/μl, 7.96%; 8 ng/μl, 6.4%; and 16 ng/μl; 9.04%) compared to no injection group (29.07%). In addition, the blastocyst formation rates between sham injected group (13.51%) and no injection group (29.07%) also showed significant difference (p<0.05). The mutation rates were comparable between groups (4 ng/μl, 18.4%; 8 ng/μl, 12.5%; and 16 ng/μl; 20.0%). The sequencing analysis showed that blastocysts derived from each group were successfully mutated in FoxN1 loci regardless of the vector concentrations. However, the deletion patterns were higher than the patterns of point mutation and insertion regardless of the vector concentrations. In conclusion, we described that cytoplasmic microinjection of FoxN1-targeted CRISPR/Cas9 vector could efficiently generate transgenic pig parthenogenetic embryos in one-step.

Previously, we performed de novo RNA sequencing of Scolopendra subpinipes mutilans using high-throughput sequencing technology and identified several AMP candidates. Among them, a synthetic peptide (CP112) was designed based on the physicochemical properties of antimicrobial peptide such as length, charge, isoelectric point. Here, we have assessed the antimicrobial activities of CP112 against various microbes and the antioxidative effects. The results showed that CP112 had antimicrobial activities in radial diffusion assay and colony count assay. In addition, we found that CP112 bound to the surface of microorganisms via a specific interaction with lipoteichoic acid, lipopolysaccharide and peptidoglycan, which is one of bacteria cell wall components. Furthermore, CP112 has shown significant DPPH radical scavenging activity. Taken together, the results would be provided the basis for developing of peptide antibiotics and antioxidants.

The purpose of this study was performed to determine the whitening effect of organic solvent extracts from the centipede, Scolopendra subspinipes mutilans. We prepared different concentrations (50%, 70% and 100%) of ethanol, methanol, 100% ethyl acetate and water extracts. We tested melanin inhibitory effect and tyrosinase activity using B16/F10 melanoma cell. As a result, treatment of organic solvent extracts is decreased the biosynthesis of melanin and tyrosinase activity to 30~60%. Especially the 70% ethanol extracts was the most effective in B16/F10 melanoma cells. In the study on melanogenic protein expression, 70% ethanol extracts of Scolopendra subspinipes mutilans blocked glycosylation of tyrosinase. Therefore this result suggests that 70% ethanol extracts could be developed as a skin whitening agents.

Previously, we have performed de novo RNA sequencing of Scolpendra subpinipes mutilans using next generation sequencing technology and identified several AMP candidates. Among them, a synthetic peptide (scolopendrasin I) was designed based on SVM algorithm. In this study, we reported that the synthetic peptide scolopendrasin I had an antimicrobial and anticancer activity. As a result, scolopendrasin I showed antibacterial activities against Gram positive and Gram negative bacteria strains in radial diffusion assay and colony count assay without hemolytic activity. In addition, we confirmed that scolopendrasin I bound to the surface of bacteria via a specific interaction with lipoteichoic acid and lipopolysaccharide, which is one of bacteria cell membrane components. In addition, we found that scolopendrasin I had anticancer activities in the human leukemic T lymphocyte cell line Jurkat using MTS assay. In conclusion, our results suggested that scolopendrasin I could be useful for developing peptide antibiotics and anticancer agents.

F. occidentalis causes to decrease the yield of chrysanthemum flower economically because it is the major crop pest injuring the flower directly at flowering period in chrysanthemum artificial houses. The control of it with chemicals is very difficult because the resistance of it to chemicals is so high. F. occidentalis began to occur at early-May and increased rapidly mid-May after in standard chrysanthemum artificial houses, Yesan, Chungcheongnam-do. In case of a farm, the density of it increased continuously in spite of chemical control . The times of chemical control to depress the density of it was 6 or 7th for cultivation period of chrysanthemum. We put the flowering yellow pot-mum already (trap plant attracting F. occidentalis) in standard chrysanthemum PVC-houses and set the yellow sticky cards up at an interval of 5m from trap plant(TP). The more yellow sticky trap near to trap plant, the more F. occidentalis was attracted to yellow sticky trap. TP was more attractive to adult F. occidentalis up to distances of 10m. The numbers of it attracted to yellow sticky traps at different distance from TP was not different significantly when standard chrysanthemum bloomed. We put the TP in standard chrysanthumum PVC-house at an distance of 20m and released the natural enemies (Orius laevigatus) on TP at 3 times, an interval of 7 days. And then we set yellow sticky traps up at an distance of 5m from TP and compared the plot with TP+natural enemy(NE) to the plot without them. The density of F. occidentalis was rather lower at TP+NE plot than the plot without at the formative period of flower bud of standard chrysanthemum. Therefore, The yellow flowering chrysanthemum possesses great value of TP attracting F. occidentalis in chrysanthumum PVC-house up to flowering period of cultivating chrysanthemum, and if it is used with natural enemy and chemicals simultaneously, farmers can minimize the quantity of chemicals used.

복숭아혹진딧물에 기생하는 목화면충좀벌의 발육기간, 머미율, 번데기 사망률, 성비를 20, 25, 30, 25℃ 인큐베이터에서 수행하였다. 실험을 위하여 피망잎이 놓인 콤팩트샤레를 사용하였다. 각각의 4개 온도에서 발육단계별로 복숭아혹진딧물을 사육하였고 기생을 유도하기 위하여 12시간 동안 목화면충좀벌에 제공하였다. 목화면충좀벌의 알에서 성충까지 발육기간은 20℃ 18.3일, 25℃ 14.7일, 30℃ 10.4일, 35℃ 9.3일 이었다. 복숭아혹진딧물에 기생한 목화면충좀벌의 머미율은 20℃와 25℃, 1령과 2령에서 가장 높았으나 30℃와 25℃에서는 3령충에서 84.4~85.0%로 가장 높았다. 목화면충좀벌의 기주섭식률은 20~35℃까지 각각 12.5%, 17.3%, 16.0%, 10.9%였다. 목화면충좀벌 암컷의 성비는 온도별로 각각 49%, 46%, 69%, 60% 였다.

The purpose of this study was to assess the feasibility of task-oriented arm training for chronic hemiparetic stroke patients. The experimental design in this study was the pre-test and post-test with control group for 4-week intervention. Thirty patients with chronic hemiparetic stroke were recruited from 2 rehabilitation units. The subjects were divided randomly into experimental and control groups. The experimental group conducted task-oriented approach, involving 3 subparts of upper extremity activities, and the control group involved in the general upper extremity exercises. Functional movements of the upper extremities were assessed using clinical measures, including the Fugl-Meyer Assessment-Upper Extremity Section, Box and Block Test, and Action Research Arm Test. The score of Fugl-Meyer Assessment showed greater increases in the experimental group than in the control group after training. The improvement in Box and Block Test between pre-test and post-test measurements was significantly greater after task-oriented arm training compared to general upper extremity exercises. Action Research Arm Test scores also improved after task-oriented arm training compared to exercises in the control group. The task-oriented arm training improves the gross and fine motor activities and encouraging the use of the paretic arm through activity dependent intervention expedites the recovery of functional activities in the upper extremities for chronic hemiparetic stroke.

꼬마배나무이 (Cacopsylla pyricola)에 저항성 품종육성을 위한 육종재료를 선발코자 월동형 성충수, 단과지내의 산란수, 과총엽내의약충수 및 수관 전체적인 그을음 발생정도를 15개 종 및 종간잡종133개 유전자원을 대상으로 조사하였다. 월동형 성충수는 Pyrus.calleryana 4.6마리, 단과지내 산란수는 P. calleryana 0.3개, 과총엽내약충수는 P. calleryana 0마리, 그리고 그을음 발생정도는 P.betulaefolia, P. calleryana, P. communis, P. hybrid (P.pyrifolia × P.communis), P. lindleyi 0으로 발생 및 피해정도가 가장 낮았으며 종간의 차이가 뚜렷하였다. 전체적으로 대목으로 이용하고 있는 콩배인P. calleryana와 P. betulaefolia가 조사 대상 유전자원 중 가장 높은 항객성(antixenosis)을 보였으나 품질개량에 많은 기간이 요구됨에 따라 실용적으로는 서양배(P. communis) 중 ‘Conference', 'Cascade','Bosc', 'Winter Nelis' 가 가장 낮은 발생 및 피해정도를 보여 꼬마배나무이 저항성 품종 육성을 위한 좋은 육종재료로 판단되었다.

Despite of the absence of hyperacute rejection and acute humoral xenograft rejection, the organ graft of the a1,3-galactosyltransferase (GalT) gene knockouted (KO) and complement regulatory protein (CRP) expressing pig into a nonhuman primate is rejected by development of a thrombotic microangiopathy and/or a consumptive coagulopathy. Thus further introduction of genes to overcome the coagulation incompatibilities between pig and primate under GalT KO/CRP genetic background has been strongly suggested. CD73 (ecto-5'-nucelotidase) is an enzyme attached via a glycosyl phosphoinositol anchor to the extracellular membrane of endothelial cells, which catalyses the hydrolysis of adenosine triphosphate to adenosine. Loss of activity of CD73 results in activation and aggregation of platelets by a reduced capacity to convert nucleotides to adenosine. In previous study, we reported generation of GalT KO fibroblasts concurrently expressing membrane cofactor protein and produced cloned pigs by nuclear transfer of the fibroblast cells (1). In this study, we constructed a vector for expression of human CD73 under control of promoter of pig Icam2 gene expressed specifically at endothelial cells. This vector was introduced into porcine fibroblasts using the nucleofection technology, by which we had forty three fibroblasts clones carrying pIcam2- CD73 vector. Somatic cell nuclear transfer resulted in generation of two transgenic piglets survived.

To identify genes that are differentially expressed, we compared the mRNA expression profile of Harmonia axyridis larvae untreated and treated with LPS. We extracted mRNAs from the larvae with or without LPS treatment, and subjected them to ACP RT-PCR analysis using a combination of 120 arbitrary primers (ACP1-ACP120)and oligo (dT) primer (dT-ACP2). After synthesized cloning DNA from 37 DEGs, it practiced the sequencing homology analysis using BLAST search. Among the 37 DEGs differentially expressed, we identified a cDNA showing homology with previously reported antimicrobial peptide. A cDNA encoding a 82-mer propeptide was identified and its predicted molecular mass and pI was 9.25 kDa and 7.54, respectively. A 35-mer mature peptide was also selected and named herein as Hamoniasin. The antimicrobial activity of chemically synthesized peptide (Mou def 1~8) against human bacterial pathogens was investigate. the result showed all bacteria strains were susceptible to Mou def 2,8 with MIC values in the 32 uM range. And biological changes of the respective cells according to peptide (Mou def 8) treatment were compared. MTT assay was tested that treatment of Mou def 8 decreased cell viability in AML-2, Jurkat, U937 (maximum 200ug/ml, 24hours). That is, fragmentation of DNA, typical characteristics observed in the process of apoptosis, was confirmed in the nucleus of cells dying owing to Mou def 8 treatment.

The present study was carried out to establish an animal model, displaying long-term learning and memory dysfunction, since single intracerebroventricular (icv) injection of amyloid β peptide (Aβ) causes a short-term memory impairment. Male ICR mice were fed a high-cholesterol diet (HCD) containing 3% cholesterol, 1% corn oil and 0.5% cholic acid, and 1 week later, icv injected with Aβ_1-42 (5 μg/head). Learning/ memory function was assessed via passive avoidance performances 1 day and 2, 4, and 6 weeks after Aβ_1-42 injection, in addition to blood biochemical analyses for lipid profiles and hepatic function. Total cholesterol, lowdensity lipoproteins and hepatic dysfunction parameters markedly increased, while high-density lipoproteins were reduced following HCD feeding. Whereas single injection of Aβ induced temporary memory loss 1 day after administration, exhibiting full recovery after 2 weeks, Aβ treatment in combination with HCD feeding lasted the learning/memory impairment up to 6 weeks. Therefore, it is suggested that hypercholesterolemia augments Aβ-induced memory loss, and that Aβ injection plus HCD feeding could be a long-term memorydeficit model suitable for long-term treatment with drugs or stem cells.

The sodium bicarbonate cotransporter (NBC) protein is functionally expressed in salivary glands. In this experiment, we examined the role of NBC in HCO₃-formation in human parotid gland acinar cells. Intracellular pH (pHi) was measured in 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded cells. Acetazolamide (0.1 mM) and 4,4'-diisothio cyanatostilbene-2,2'-disulphonic acid (DIDS, 0.5 mM) were used as specific inhibitors of carbonic anhydrase and NBC, respectively. The degree of inhibition was assessed by measuring the pHi recovery rate (△pHi/min) after cell acidification using an ammonium prepulse technique. In control experiments, △pHi/min was 1.40±0.06. Treatment of cells with 0.5 mM DIDS or 0.1 mM acetazolamide significantly reduced △pHi/min to 1.14±0.14 and 0.74±0.15, respectively. Simultaneous application of DIDS and acetazolamide further reduced △pHi/min to 0.47±0.10. Therefore, DIDS and acetazolamide reduced △pHi/min by 19% and 47%, respectively, while simultaneous application of both DIDS and acetazolamide caused a reduction in △pHi/min of 67%. These results suggest that in addition to carbonic anhydrase, NBC also partially contributes to HCO₃- formation in human parotid gland acinar cells.

The morecular cloning, gene structure, expression and enzyme activity of a serine-like proteas frome Laccotrephes Japonensis were examined. In this study, RT-PCR was used to amplify cDNA fragments for serine-like proteases from total RNA the hole body of Laccotrephes japonensis. The flanking sequences of the 5'- and 3'- end of the this gene were characterized by RACE-PCR. Sequence analysis showed that this gene contained an 963bp ORF encoding 321 amino acids. The deduced amino acid sequence of this protease showed 62% identity to the serine protease of Creontiades dilutus, 58% to Lygus loneolaris trypsin precuror LlsgP4, 54% to Triatonatoma infestans salivary trypsin. To generate Laccotrephes japonensis serine-like protease, the DNA fragment coding for serine protease is cloning into suttle vector pBACⅠ, named pBAC1-JG and infected to Spodoptera frugiperda (sf9) insect cell. The cDNA encoding JG was expressed as a 32-kDa polypeptide in baculovirus infected insect cells and the recombinant JG showed activity in the protease enzyme assay using gelatin as a substrate.

We have previously shown that the larvae of swallowtail butterfly, Papilio xuthus, exhibit substantial antibacterial activity in the hemolymph, upon challenging with bacterial lipopolysaccharide (LPS). Here we report the isolation and molecular characterization of several immune inducible genes that are specifically expressed by employing annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) from P. xuthus the larva. Using 120 arbitrary ACPs, we identified 24 differentially expressed genes (DEGs) that are up-regulated in response to injected LPS. Sequence analysis showed that 18 DEGs revelaed a high sequence similarity to the previously characterized genes of other insects, although 6 DEGs showed no significant similarity to any known genes. Among these inducible transcripts we found 8 putative immune-related genes including cecropin and attacin. Finally, we analysed the expression profiles of potential immune-related genes by RT-PCR and found all of them were considerably increased in the mRNA levels by LPS injection.

Protease from various sources have been studied biotecnologically. For biotechnological applications, one highly preferred enzyme is protease. There have been no reports of cloned genes encoding digestive proteases in the Laccotrephes japonenis, Ranatra unicolor, Muljarus japonicus. These insects are considered to be a predator of aquatic insects. RT-PCR was used to amplify cDNA fragments for digestive proteases from total RNA the hole body of the insects. The flanking sequences of the 5'- and 3'- end of the these genes were characterized by RACE-PCR. Sequence analysis showed that these genes contained complete ORF. The deduced amino acid sequences of these protease showed 62% identity to the serine protease of Creontiades dilutus, 58% to Lygus loneolaris trypsin-like serine proteinase , 54% to Triatonatoma infestans salivary trypsin. In the further study, to generate digestive protease, the DNA fragment coding for serine protease, trypsin-like serine protease were cloning into suttle vector pBACⅠ, and infected to Spodoptera frugiperda (sf9) insect cell. After that, we expect to carry out the proteolytic activity of these recombinant proteases. This is intended as a basis for future studies on the digestive protease in the insects.