본 연구는 신생아 검사 중 포수클로랄(chloral hydrate)을 투여 후 진행되는 신생아 진정 검사 대비 진정 대체 방식 중 하나인 피드 및 랩(feed and wrap) 방식의 유용성을 평가한 연구이다. 본 연구에선 진정으로 진행한 신생아의 두뇌 T2 축면 영상과 피드 및 랩 방식으로 진행한 같은 영상 각 30개의 운동 허상(motion artifact)과 백질과 회백질의 구분 정도를 두 명의 영상의학과 전문의가 정성적으로 평가하였고, 운동 허상을 측정하기 위해서 위상부호화(phase encoding) 방향의 배경 영역(background area)의 평균 신호 강도(mean signal intensity)를 구하여서 정량적 방식으로 평가하였다. 또한 총검사 시간을 정리한 뒤 정량적 방식으로 평가하였고 투약 기록의 여부와 간호일지를 토대로 피드 및 랩 방식의 총 39건의 검사 건수 대비 성공률을 측정하였다. 운동 허상의 정량적 평가와 영상 품질의 정성적 평가 모두에서 두 집단은 유의미한 차이가 없었으나, 검사 시간의 정량적 평가에선 p값이 0.001로 유의한 차이가 있었다. 피드 및 랩 방식의 총검사 건수 대비 성공률은 100%였다. 결론적으로 본 논문에선 피드 및 랩 방식과 진정 방식의 영상 품질이 유의한 차이가 없고 성공률이 높기에 유용하다고 판단하였으나, 검사 시간이 더 지연되는 한계가 있다는 사실을 확인하였다.

Three acetylcholinesterases (ACEs) were identified from the pinewood nematode, Bursaphelenchus xylophilus, and named BxACE-1, BxACE-2, and BxACE-3. Sequence comparison with known ACEs in conjunction with three-dimensional structure analysis suggested that all BxACEs share typical characteristics of ACE but show some differences in the peripheral anionic site. BgACE-3 was most predominantly transcribed, followed by ACE-1 and ACE-2. Immunohistochemistry using anti-BxACEs antibodies revealed that BxACE-1 is most widely distributed whereas BxACE-2 exhibits more localized distribution in neuronal tissues. BxACE-3 was detected from entire body together with some limited tissues, and determined to be soluble. Kinetic analysis of in vitro expressed BxACEs revealed that BxACE-1 has the highest substrate specificity whereas BxACE-2 has the highest catalytic efficiency with BxACE-3 having the lowest catalytic efficiency. Interestingly, presence of BxACE-3 in the pool of BxACEs significantly reduced the inhibition of BxACE-1 and BxACE-2 by inhibitors. Knockout of BxACE-3 by RNAi significantly increased the toxicity of nematicides, supporting the protective role of BxACE-3 against these toxicants. Taken together, BxACE-1 appears to be the major ACE with the function of postsynaptic transmission whereas BxACE-3 has been evolved to acquire the function of chemical defense. BxACE-2 appears to play a role in post-synaptic transmission in specialized neurons.

Injection of nematicides such as emamectin benzonate and milbemectin is the most common practice to control the pinewood nematode, Bursaphelenchus xylophilus, in Korea. These macrocyclolactone nematicides, however, are expensive, limiting their practicability despite of high efficacy. In an attempt to screen affordable alternative organophosphate (OP) and carbamate (CB) nematicides, we identified and characterized three acetylcholinesterases (ACE, EC 3.1.1.7) from B. xylophilus and functionally expressed them using baculovirus system. In inhibition assay using 11 OPs and 3 CBs, all the three ACEs were highly inhibited by paraoxon, DDVP, chlorpyrifos-oxon and mevinphos of OPs and carbofuran and carbaryl of CBs but not inhibited well by the others. Interestingly, inhibition assay revealed that BxACE-3 is less sensitive to all insecticides tested than other two ACEs. In additional bioassay, chlorpyrifos, DDVP and parathion showed a high LC50 but all CBs tested did a very low mortality. The inhibition kinetic data and bioassay data obtained in this study should provide essential information for the development of OP-based nematicidal agents against B. xylophilus. Availability of expressed ACE will also facilitate the development of in vitro screening system to develop potential OP nematicides.

To determine differential gene expression profiles in the salivary gland of a water stick-insect, Ranatra chinensis Mayrt, a subtractive cDNA library was constructed by suppression subtractive hybridization. The salivary gland was determined among three salivary gland-like tissues by investigating transcription levels of five trypsin genes isolated from R. chinensis. The major transcripts encoding trypsins (64.4% of the total ESTs) were eliminated from the library and then remaining salivary gland-specific genes were searched. A total of 643 expressed sequence tags (ESTs) were clustered and assembled into 148 contigs (49 multiple sequences and 99 singletons), among which 35 contigs had matched BLASTx hits (E ≤ 1.00E-4). Salivary apyrase occupied 5.6% (36 ESTs) of the library. Apyrase is known to be released by female mosquitoes or blood-sucking assassin bugs to prevent blood clots during blood sucking. Therefore, apyrase in the salivary of R. chinensis might allow R. chinensis to facilitate feeding. Several contigs encoding acid phosphatase, hyaluronidase, prophenoloxidase, and dipeptidylpeptidase IV, commonly found in venoms of Hymenoptera, were also identified from the salivary gland-specific library. Discovery of salivary glandspecific genes should promote further studies on biologically active components in the saliva of R. chinensis.

The genomic structure and phylogenetic relationships of HSP88 genes from P. tenuipes Jocheon-1, P. tenuipes, C. militaris and C. pruinosa are described. The HSP88 genomic DNA from P. tenuipes Jocheon-1, P. tenuipes and C. militaris all contain 5 introns and 6 exons with the length of 13, 62, 32, 1438, 306, 288 bp, encoding 713 amino acid residues. C. pruinosa HSP88 genomic DNA contains 4 introns and 5 exons encoding 713 amino acids. The length of each exon of C. pruinosa HSP88 is 13, 62, 32, 1744, 288 bp and the length of exon 4 is identical to the total length of exon 4 and exon 5 of HSP88 of P. tenuipes Jochoen-1, P. tenuipes, and C. militaris. The deduced amino acid sequence of P. tenuipes Jocheon-1 HSP88 showed 99% identity with the P. tenuipes, 97% identity with the Cordyceps militaris, and 98% identity with the C. pruinosa. Phylogenetic analysis confirmed that the P. tenuipes Jocheon-1, P. tenuipes, C. militaris and C. pruinosa HSP88 are placed together within the ascomycetes group of fungal clade.

In this study, a full-length heat shock protein88 complementary DNA (cDNA) of Paecilomyces tenuipes Jocheon-1 was obtained by screening of P. tenuipesJocheon-1 Uni-Zap cDNA library and 5' RACE polymerase chain reaction. The Paecilomyces tenuipes Jocheon-1 heat shock protein88 cDNA contains an open reading frame of 2,139 bp encoding 713 amino acid residues. The deduced amino acid sequence of the P. tenuipes Jocheon-1 HSP88 cDNA showed 77% identity to N. haematococca HSP88 and 45-76% identity to other fungi HSP88. Phylogenetic analysis and BLAST program analysis confirmed that the deduced amino acid sequences of the P. tenuipes Jocheon-1 HSP88 gene belonged to the ascomycetes group within the fungal clade and P. tenuipes Jocheon-1 HSP88 also contains the conserved ATPase domain at the N-terminal. The cDNA encoding P. tenuipes Jocheon-1 HSP88 was expressed as a 88 kDa polypeptide in baculovirus-infected insect Sf9 cells. Under different stress conditions, mRNA expression of P. tenuipes Jocheon-1 HSP88 were quantified by real-time PCR and the result showed that heat shock stress affected the mRNA expression levels of P. tenuipes Jocheon-1 HSP88.

Presently, We have constructed an olig-d(T) primed directional cDNA library from the silkworm Dongchunghacho, an entomopathogenic fungus, of which species is belonging to Paecilomyces tenuipes Jocheon-1. To isolate and screen genes in the fungus, 626 expressed sequence tags(ESTs) were generated by a partial sequencing from the cDNA library. Paecilomyces tenuipes Jocheon-1 cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase(Pt-GAPDH) of Paecilomyces tenuipes Jocheon-1 was cloned from the above cDNA library. The complete cDNA sequence of Pt-GAPDHis comprised of 1,014bp encoding 338 amino acid residues. The deduced protein sequence of Pt-GAPDH showed higher homology with Beauberia bassiana-GAPDH(93% amino acid identity). Hydropathy analysis revealed that Pt-GAPDH protein is hydrophilic. The major three amino acids in its composition of amino acid residues were alanine(11.54%), valine(9.47%) and glycine(8.88%). The cDNA encoding Pt-GAPDH was expressed as a 37 kDa polypeptide in baculovirus-infected insect Sf9 cells. The Pt-GAPDH gene of Paecilomyces tenuipes entomopathogenic fungus consisted of three exons and two introns coding for 338 amino acid residues, and the genomic DNA length of the gene spans 1302bp. The accession number of the gene in GenBank are GU997099 for Pt-GAPDH cDNA and GU997102 for Pt-GAPDH genomic DNA.

The differences in the immune response between body lice, Pediculus humanus humanus, and head lice, Pediculus humanus capitis, were regarded as primary factors determining their differential vector competence. To find any differences in genetic components in immune system between body and head lice, whole genome sequences of head lice were determined by both SBS [sequencing by synthesis, Illumina Genome Analyzer (Illumina-GA)] and pyrosequencing (Roche GS FLX), and compared with the reference genome sequences of body lice. The short DNA reads from Illumina-GA (an average mapping depth of 50-fold) were aligned first to the body louse reference genome, to which Roche GS FLX DNA reads (an average depth of 2.5-fold) were subsequently assembled to make up gaps between mapped consensus. Total consensus showed a size of 114 Mb and a coverage of 96% of the published body louse genome sequences. From this head louse genome sequences, a total of 12,651 genes were predicted and used for comparing with the 10,775 genes previously reported from the body louse genome. The homolog analysis identified 873 head louse-specific genes and 422 body lice-specific genes. Comparison of immune response genes between both louse species showed head lice have more number of immune-related genes than body lice. Head lice were determined to possess all of the 107 immune-related genes reported in the previous study (Kim et al., 2011), suggesting that there is no difference in genetic make-up in terms of the 107 immune-related genes between body and head lice.

Venom allergen-like protein 2 (Vap2) was characterized from the pinewood nematode, Bursaphelenchus xylophilus, which is a destructive pathogen in several countries including Japan, China and Korea. Among three vaps of B. xylophilus (Bx-vap)reported in GenBank, Bx-vap2 showed the highest transcript level in both pine-grown propagative stage (PGPS) and media-grown propagative stage (MGPS). Bx-vap2 also was revealed that its transcript level over 10-fold increased in PGPS. In addition, western blot using BxVap2-polyclonal antibody showed that expression level of BxVap2 was significantly increased in PGPS. In immunohistochemistry, moreover, strong signals were detected around putative subventral gland in PGPS, whereas weak signals were observed in MGPS. These experimental results suppose pathogenic function of BxVap2 and migration assay using Bx-vap2 knock-down worms by RNA interference supports this postulation.

Insects or insect remains found in beer are one of major issues in consumer claim. Accurate estimation of inflow time isa critical factor for the settlement of such claims related with beer-contaminating insects but no reliable methods have been developed. In an attempt to establish a molecular marker-based diagnostic method, the degradation rates of 18S rRNA genes in the insectssoaked in 500 ml beer were investigated by quantitative real-time PCR (qPCR) over one month period at room temperature. Among the six insect species tested, the house fly (Musca domestica) and honey bee (Apis mellifera) revealed high correlations (r2=0.974-0.990) between the degradation of 18S rRNA gene and inflow time. In these insects, statistically significant distinction was possible between the samples stored in beer less than 14 days and more than 14 days. Other insects, including the fruit fly, common house mosquito, German cockroach and Indian meal moth, displayed poor correlations, which appeared attributed to the inefficient genomic DNA extraction likely due to small sample size or disintegration of body parts during storage in beer. With proper improvement in DNA extraction, this 18S rRNA-based diagnostic method would be applicable for estimating the inflow time of beer-contaminating insects.

The cotton aphid, Aphis gossypii (Glover), is one of the most serious pest in seed potato and various vegetable cultivation. The imidacloprid resistant strain (IR) was over 200 fold more resistant to imidacloprid compared to the susceptible strain (S) as judged by the LC50 values and IR showed cross resistant to acetamiprid, thiamethoxam, thiacloprid, clothianidin. By using the suppression subtractive hybridization method, a imidacloprid resistant associated cDNA library was constructed in adult cotton aphid. In total 115 differentially expressed cDNA clones were obtained. Any point mutation detected in nicotinic acetylcholine receptor alpha 1~5 and beta 1 subunits in the IR. Based on IEF, the IR general esterase isozyme banding patterns were identical with that of S.

Fungi belonging to the Paecilomyces spp. have recently been used as food and herbal medicines in Korea and are greatly popular as commercially available powdered supplement or dried fruiting body. Despite this acceptance and its use, little is known of the genes related to its reactive agents. Presently, We have constructed an olig-d(T) primed directional cDNA library from the silkworm Dongchunghacho, an entomopathogenic fungus, of which species is belonging to Paecilomyces spp. based on the previous identification of ITS1 and ITS2 at the molecular level and collected from Jocheon Miryang, Korea. To isolate and screen genes in the fungus, 626 expressed sequence tags(ESTs) were generated by a partial sequencing from the cDNA library. cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase(Pt-GAPDH) of Paecilomyces tenuipes- Jocheon was cloned from the above cDNA library. The complete cDNA sequence of Pt-GAPDH is comprised of 1,014bp encoding 338 amino acid residues. The deduced protein sequence of Pt-GAPDH showed higher homology with Beauberia bassiana-GAPDH(93% amino acid identity). Hydropathy analysis revealed that Pt-GAPDH protein is hydrophilic. The major three amino acids in its composition of amino acid residues were alanine(11.54%), valine(9.47%) and glycine(8.88%). The Pt-GAPDH gene of Paecilomyces tenuipes entomopathogenic fungus consisted of three exons and two introns coding for 338 amino acid residues, and the genomic DNA length of the gene spans 1302bp. The accession number of the gene in GenBank are GU997099 for Pt-GAPDH cDNA and GU997102 for Pt-GAPDH genomic DNA. More investigation works including gene expression, immunological analysis etc. will be carried continuously without hesitation after this presentation.

Three acetylcholinesterases (AChEs) were identified from the pinewood nematode, Bursaphelenchus xylophilus. Sequence comparison with known AChEs in conjunction with three-dimensional structure analysis suggested that all BxAChEs share typical characteristics of AChE at the major catalytic structures. BgAChE3 was most predominantly transcribed and then followed by AChE1 and AChE2. Immunohistochemistry using anti-BxAChEs antibodies revealed that BxAChE1 is most widely distributed whereas BxAChE2 exhibits more localized distribution in neuronal tissues. BxAChE3 was detected from entire body together with some limited tissues, including mouth parts and alimentary lining, and determined to be the only soluble AChE, suggesting its localization in hemolymph or/and extracellular space. Kinetic analysis of in vitro expressed BxAChEs revealed that BxAChE1 has the highest substrate specificity whereas BxAChE2 has the highest catalytic efficiency with BxAChE3 having the lowest catalytic efficiency. Interestingly, presence of BxAChE3 in the pool of BxAChEs significantly reduced the inhibition of BxAChE1 and BxAChE2 by inhibitors. Knockout of BxAChE3 by RNAi significantly increased the toxicity of nematicides, suggesting the protective role of BxAChE3 against these toxicants. Based on several features, including tissue distribution, expression level, substrate kinetics and inhibition property, it appeared that BxAChE1 is the major AChE with the function of postsynaptic transmission whereas BxAChE3 has been evolved to acquire the function of chemical defense, perhaps intrinsically against secondary toxic compounds from host pine trees, such as α-pinene and limonene. BxAChE2 appears to play a role in post-synaptic transmission in specialized neurons but its detailed physiological function still remains to be elucidated.

Three acetylcholinesterase (ace) genes were identified from Bursaphelenchus xylophilus and named as Bxace1, Bxace2 and Bxace3. Open reading fragments of Bxace1, Bxace2 and Bxace3 were composed of 622, 625 and 588 amino acids, respectively. Sequencing comparison with Torpedo ace gene identified cholinebinding site, catalytic triad functional site, three internal disulfide bonds and aromatic residues for the catalytic gorge. Transcriptional profiling by quantitative real-time PCR revealed that Bxace3 is more actively transcribed than Bxace1 (2-3 times) and Bxace2 (9-18 times) in both propagative and dispersal stages. The three ace genes were functionally expressed using baculovirus system. Kinetic analysis using three choline substrates demonstrated that BxAce2 has higher maximum velocity than BxAce1 (ca. 2 times) and BxAce3 (ca. 100 times) whereas BxAce3 shows lower Michaelis constant than BxAce1 (12.8 times) and BxAce2 (13.6 times). In inhibition assay using five organophosphates (OPs) and three carbamates (CBs), all the three BxAces were highly inhibited by paraoxon, DDVP and chlorpyrifos-oxon but not inhibited well by fenamiphos and fosthiazate. Interestingly, inhibition assay revealed that BxAce3 is less sensitive to all insecticides tested than other two BxAces. The inhibition kinetic data obtained in this study should provide essential information for the development of OP- and CB-based nematicidal agents againt B. xylophilus.

A full genomic DNA microarray technique was employed to investigate the effects of Dongchunghacho on aortal and hepatic gene expression in apolipoprotein E knockout mice fed a high-fat/high-cholesterol diet. Male 8- week - old ApoE-/- mice were randomly divided into two groups, control(high cholesterol group; HC) and supplementation of Dongchunghacho (SD). All of the mice were fed a high-fet/high cholesterol diet with or without Dongchunghacho supplemented by 1% for 6 weeks. At first, lipid profile of the Dongchunghacho was measured by biochemical analysis. No differences were observed in serum triglyceride and total cholesterol levels between the two groups. Antigenotoxic effect of the Dongchunghacho was measured by the single cell gel electrophoresis assay (Comet assay) and quantified as % fluorescence in tail. Dongchunghacho supplementation decreased significantly leukocytic DNA damage and also there was a tendency of reduction in hepatic DNA damage in Dongchunghacho group compared with the control group. In up regulated genes in liver and aorta of the mice, genes with 0 to 2- fold difference in expression level between the two group (HD and SD) was very much more in liver than in aorta, on the contrary, those with 2-fold to 16-flod difference increased greatly rather in aorta than in liver. Also, almost the same results were observed in down regulated genes in liver and aorta between the two groups. These results suggested that supplementation of Dongchunghacho might be helpful in preventing leukocytic DNA damage induced by high fat diet, and has a more crucial roles in aortal gene expression.

The pinewood nematode, Bursaphelenchus xylophilus (Steinner & Buhrer) Nickle, has two different life stages according to several environmental factors: dispersal stage and propagative stages. The dispersal stage is closely related to the migration to other host pines, whereas the propagative stage is coupled to the direct cause of pine wilt. To establish expressed sequence tag (EST) database of two life cycles of B. xylophilus, subtractive EST libraries were constructed using suppressed subtractive hybridization (SSH). From 3,072 and 3,840 sequences of dispersal- and propagative-specific stage cDNA libraries, 1,927 and 2,604 clusters were generated, respectively, which were annotated by BLASTx and Gene ontology (GO). A total of 1,112 (57.7%) and 1,215 (46.7%) clusters from the dispersal- and propagative-specific stage cDNA libraries respectively had the matched BLASTx hits (E≤10-2), among which 913 (47.4%) and 960 (36.9%) were classified into three categories in Gene ontology. From GO database, some respective stage-specific genes were searched and estimated the relative transcripts level according to stages using the quantitative real-time PCR.

Background: To enhance the taste and physiological characteristics of Lycii fructus (Gugija) extracts, we investigated the changes in the physiological characteristics of Gugija extracts caused by adding white ginseng (WG) and red ginseng (RG) Methods and Results: Gugija extracts, including 10G10, 10GW-G8 : 2, -G6 : 4, -G4 : 6, -G2 : 8, and -G0 (mixtures made by replacing 0, 20, 40, 60, 80, and 100% of Gugija with WG), as well as 10G10, 10GR-G8 : 2, -G6 : 4, -G4 : 6, -G2 : 8, and -G0 (mixture made by replacing 0, 20, 40, 60, 80, and 100% of Gugija with RG) were extracted with water at 10 times the respective mixture's volume. The antioxidant activities of Gugija extracts were investigated by assessing their 1,1-diphenyl-2-picrydrazyl (DPPH) and 2,2’-azinobis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, ferric reducing antioxidant potential (FRAP) activity, nitrite scavenging activity, and angiotensin converting enzyme (ACE) inhibitory activity. As the amount of WG added increased, the DPPH, and, ABTS radical scavenging activity, and FRAP activity of the Gugija extract decreased. The half maximal inhibitory concentration (IC50) value of 10G10, 10GW-G6 : 4, 10GR-G6 : 4, and 10GR-G0 for DPPH radical scavenging activity were 25.50 ± 1.04, 52.06 ± 1.46, 16.87 ± 1.24, and 9.50 ± 0.16 ㎕/㎖, respectively. On the other hand, the physiological activity of Gugija extract increased with the addition of increasing amounts of RG. However, ACE inhibitory activity was the highest (50.25 ± 2.58%) in the Gugija 10-fold extract without any added RG. Conclusions: From the above results, we suggest that adding RG to Gugija extracts increase their antioxidant, FRAP, and nitrite scavenging activities.

Background : This study examined EC in soil depending on leaking water rate of sun shading facilities and conducted experiments to establish the proper leaking water rate for ginseng depending on rainfall. Methods and Results : For leakage examination, rainfall flowing into a ridge. As sun shading facilities of ginseng to examine leakage, four kinds such as Blue-Pe-sheet, Pe4 (four–layered polyethylene net) + Pe2 (two–layered polyethylene net), Pe4, Pe2 were installed. As for leakage, a plastic box (23 × 30 × 30 ㎝) was installed on the ridge of ginseng field and outside, rainfall into the box during precipitation was examined, and the leakage quantity was calculated as the ratio of the quantity into the ginseng field regarding rainfall outside. The leakage quantity was examined a total of six times on July 2, July 3, August 24, August 30, August 31, and September 4. Regarding EC in soil, WT–1000n (www.rfsenser.co) which is a EC measuring instrument was used, and the average was calculated through 3 repeated examinations. There are little differences in the leakage quantity depending on the sun shading with 0.1% of the Blue-Pe-sheet, 2.3% of Pe4 + Pe2, 4.3% of Pe4, and 30.7% of Pe2. At the leakage rate of 0.1%, EC decreased from 1.52 ds/m on July 3 to 1.04 ds/m on August 8, and increased again to 1.54 ds/m on September 3 At the leakage rate of 2.4%, EC changes were lower than that of the leakage quantity of 0.1%, and similar results depending on periods were found. At the leakage rate of 4.3%, salt concentration was measured at 0.92 ds/m on July 3, decreased since then, increased to 0.90 ds/m on September 3, and overall concentration was less than 1.0 ds/m. At the leakage rate of 30.7%, EC was the lowest at 0.46 ds/m and similar results were found since then. Conclusion : The differences in leakage quantity depending on sun shading facilities of ginsengs affected EC in soil, and EC became lower with more leakage quantity. As for the leakage quantity to maintain the EC in soil proper for ginseng growth and development lower than 1.0 ds/m, it was found to be effective to control the leakage quantity at 30% in May - June, and September - October when there are less rainfall, and at 2 - 5% in July - August when there are heavy rainfall.