Staphylococcus (S.) aureus is commonly found on the skin and mucous membranes of animals. Moreover, some isolates producing staphylococcal enterotoxins (SE) are also responsible for food poisoning. This study was conducted to explore the prevalence of S. aureus enterotoxin from slaughtered pigs and cattle. A total of 202 carcass swabs were collected from slaughterhouses: 102 samples were taken from slaughtered pigs and 100 were taken from cattle, respectively. Among them, 16 (7.9%) from slaughtered pigs were found to contain S. aureus, while S. aureus was not isolated from any of the slaughtered cattle samples. Additionally, six (37.5%) of the S. aureus isolates contained genes that encode staphylococcal enterotoxin type A. Therefore, it is necessary to investigate the management of food-borne pathogens based on differences in the process by which pigs and cattle are slaughtered.

가스 생산용 해양플랜트 설비의 경우 폭발의 위험에 노출되어 있으며, 폭발사고는 구조물의 안전성에 치명적인 영향을 미칠 수 있다. 따라서, 이러한 폭발사고에 의한 피해를 최소화하기 위해서는, 폭발하중에 의한 구조부재의 동적응답 특성을 명확히 파악할 필요가 있다. 폭발하중의 경우 매우 짧은 시간 동안에 구조물에 가격되었다가 소멸되기 때문에 구조부재의 고유주기 및 폭발하중의 지속시간을 고려한 동적응답 평가가 필수적으로 요구된다. 일반적으로 가스 폭발하중의 경우, 부 압력단계가 전체 하중 이력에서 상당 부분 존재하며, 본 연구에서는 이러한 부 압력단계의 형상에 따라 총 하중 지속시간을 결정하는 하중 모델을 제안하였다. 방화벽은 폭발사고 시 장비 및 인명 피해를 방지하고자 FPSO 탑사이드 모듈 사이에 배치되는 구조부재이므로 폭발하중에 의한 응답이력 특성 분석이 반드시 필요하다. 때문에 무 감쇠 단 자유도 모델에 가스 폭발하중을 적용하여 변위응답 특성을 분석하였으며, 평판으로 구성된 방화벽의 FE 모델을 이용한 하중 지속시간과 구조부재들의 고유주기를 고려한 응답 특성을 분석하였다. LS-DYNA를 이용한 선형/비선형 구조해석 분석결과, 부 압력단계의 지속시간이 구조물의 동적응답에 큰 영향을 주는 것을 보였다.

가스 생산용 해양플랜트 설비에서 발생할 수 있는 폭발사고의 경우, 구조 시스템의 기하학적 특성이나, 바람, 가스 누출율 등과 같은 환경적 조건에 의해 피해 규모의 범위가 상당하다. 따라서 폭발파에 의한 구조 부재의 응답을 분석하기 위해서는 이러한 조건들을 고려한 가스폭발 수치해석 과정이 반드시 필요하다. 본 연구에서는 FPSO 탑사이드의 형상 및 장비 배치와 같은 세부적인 부분까지 고려하여 폭발해석을 수행하였으며, 이를 바탕으로 획득한 하중 이력들의 특성을 분석하였다. 또한 다양한 형태로 나타나는 폭발하중 이력들 중 구조물 손상에 직접적으로 영향을 미칠 수 있는 최대 압력과 지속시간들을 고려하여 유한요소해석 시 하중조건으로 적용한 후, 부재의 응답특성에 관한 분석을 수행하였다. 유한요소해석 모델은 실제 구조물에 적용이 가능하고, 복잡한 형상을 이상화한 단 자유도 및 다 자유도 모델을 사용하였다. 정 압력 및 부 압력단계의 최대 압력이 증가함에 따라 구조 부재의 최대 응답이 증가하였고, 부 압단계에서 하중 지속시간이 증가함에 따라 구조물의 최대 변위가 증가는 경향을 보였다.

가스폭발은 해양플랜트 산업에서 발생할 수 있는 치명적인 사고 중 하나이며, 탑사이드 플랫폼은 폭발압력에 따른 구조건전성을 확보해야만 한다. 따라서, 해양플랜트 분야에서는 이러한 폭발사고에 대비한 방폭설계에 관한 많은 연구가 수행되었지만, 여전히 추가적으로 세밀한 분석이 더 필요한 실정이다. 폭발 설계하중 계산과정에서 도출된 충격량은 CFD 해석결과로 계측된 폭발 압력 응답에서의 곡선 아래 면적의 절대 값에 의해 결정되어 진다. 하지만 가스폭발에서의 부압구간은 TNT 폭발이나 가스폭발과는 달리 상당부분 존재한다. 본 연구의 목표는 이러한 부압구간이 구조물의 거동에 미치는 영향에 대해서 분석하는 것이다. 따라서 방폭설계가 필수적으로 요구되어지는 FPSO 탑사이드의 방화벽을 폭발하중에 따른 구조응답을 분석하기 위한 대상물로 선정하였다. 폭발 하중-시간이력 데이터는 FLACS를 이용한 폭발 시뮬레이션 과정을 통해 획득하였으며, LS-DYNA는 비선형 과도 응답해석을 위해 사용되었다.

This peptide has antibacterial activity against several Gram-positive and Gram-negative bacteria. BmCecB1 is antimicrobial peptides from Bombyx mori and belongs to cecropin family. Antimicrobial peptides are important components of the innate immune systems in all living organism. To produce the BmCecB1 antimicrobial peptide, we constructed transgenic silkworm that expressed BmCecB1 gene under the control BmA3 promoter using piggyBac vector. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor vector and helper vector were micro-injected into 600 eggs of bivoltin silkworms, Baegokjam. In total, 49 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 22 broods, and we selected 2 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 11%. We identified 9 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a BmCecB1 as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that BmCecB1 possesses high antibacterial activities against Gram-negative bacteria.

Silkworm transgenesis is now a routine method leading to a satisfactory yield of transformed animals and the reliable expression of transgenes during multiple successive generations. However, the screening of G1 transgenic individuals from numerous progeny has proved to be difficult and time-consuming work. Previously, we characterized the promoter of heat shock protein 70 from Bombyx mori (bHsp70), which is ubiquitously expressed in all tissues and developmental stages. To investigate the utilization of the bHsp70 promoter to screen transgenic individuals, the EGFP marker gene was inserted into the piggyBac vector under the control of the bHsp70 promoter. Mixtures of the donor and helper vectors were micro-injected into 3,060 eggs of bivoltine silkworms (Keomokjam). EGFP fluorescence was observed in 17 broods of transgenic silkworms under a florescence stereomicroscope. Interestingly, this fluorescent marker protein was detected not only in parts of the embryo segments on the seventh day of the G1 embryonic developmental stage but it was also detected in a part of the body of G1 hatched larvae, in the middle silk gland of G1 fifth instar larvae, and in the wings of seven-day-old G1 pupae and G1 moths. Therefore, we suggest that the bHsp70 promoter can be used for the rapid and simple screening of transgenic silkworms.

To product the blue fluorescent protein (AmCyan) expressed cocoon, we were fused AmCyan cDNA to the heavy chain gene and injected the gene into a silkworm. AmCyan was one of the existing violet fluorochromes and originally derived from the fluorescent protein amFP486. AmFP486 was cloned from the sea anemone Anemonia majano (GenBank accession number AF168421), and belongs to the family of fluorescent proteins (FPs) isolated from coral reef organisms. The AmCyan fusion protein, each with N- and C- terminal sequences or the fibroin H-chain, were designed to be secreted into the lumen of the posterior silk glands. The expression of the AmCyan/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3 EGFP as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor and helper vectors were micro-injected into 300 eggs of bivoltine silkworms (Baegokjam). EGFP fluorescence was observed in 3 broods of transgenic silkworms under a florescence stereomicroscope. The cocoon was displayed strong blue fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the AmCyan gene expressed cocoon will be enable the production of the novel biomaterials based on the transgenic silk.

To product the blue fluorescent protein (AmCyan) expressed cocoon, we were fused AmCyan cDNA to the heavy chain gene and injected the gene into a silkworm. AmCyan was one of the existing violet fluorochromes and originally derived from the fluorescent protein amFP486. AmFP486 was cloned from the sea anemone Anemonia majano (GenBank accession number AF168421), and belongs to the family of fluorescent proteins (FPs) isolated from coral reef organisms. The AmCyan fusion protein, each with N- and C- terminal sequences or the fibroin H-chain, were designed to be secreted into the lumen of the posterior silk glands. The expression of the AmCyan/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3 EGFP as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor and helper vectors were micro-injected into 300 eggs of bivoltine silkworms (Baegokjam). EGFP fluorescence was observed in 3 broods of transgenic silkworms under a florescence stereomicroscope. The cocoon was displayed strong blue fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the AmCyan gene expressed cocoon will be enable the production of the novel biomaterials based on the transgenic silk.

BmCecB1 are antimicrobial peptides from Bombyx mori and belongs to cecropin family. Antimicrobial peptides are important components of the innate immune systems in all living organism. This peptide has antibacterial activity against several Gram-positive and Gram-negative bacteria. To produce the BmCecB1 antimicrobial peptide, we constructed transgenic silkworm that expressed BmCecB1 gene under the control BmA3 promoter using piggyBac vector. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor vector and helper vector were micro-injected into 600 eggs of bivoltin silkworms, Baegokjam. In total, 49 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 22 broods, and we selected 2 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 11%. We identified 9 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a BmCecB1 as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that BmCecB1 possesses high antibacterial activities against Gram-negative bacteria.

The differences in adult lifetime among various silkworm strains has been suggested that the adult lifetime may be genetically controlled. In this experiment, using J037 and Daizo strains we investigated genetic factors related to the adult lifetime of silkworm. We constructed the full-length cDNA library from the adult male of the J037 strain. A total of 2,688 clones were randomly selected, and we performed a differential display hybridization with cDNA probes generated from J037 and Daizo adult males. In conclusion, 193 clones were identified as differential expressed genes, and 154 unique genes were generated after the assembly of 193 clones. Of the 154 unique genes, the most abundant genes were cytochrome oxidase subunit-1 gene(9 times) and unknown(clone ID; 1-50) gene(5 times). The functional groups of these unique genes with matches in the AmiGo database were constructed according to their putative molecular functions. Among thirteen functional categories, the largest group was unclassified protein(24%). In addition, We analyzed the nucleotide and deduced amino acid sequences of the most highly occurred gene(1-50, EF434397), which consisted of 240 amino acids. However, it is confirmed yet that these genes really have an affected on the silkworms longevity.

Immune-inducible antimicrobial peptides were produced using transgenic silkworms that expressed Rel family transcription factor, truncated BmRelish1 (BmRelish1t) genes under the control of the BmA3 promoter using the piggyBac vector. BmRelish1t gene contains all domains of Bmrelish: a Rel homolog domain (RHD), nuclear localization signal (NLS), acidic and hydrophobic amino acids (AHAA) rich region except the Ankyrin repeat domain (ANK) and the death domain (DD). (1:1) Mixtures of the donor vector (pG-3xP3EGFP-BmA3BmRelish1t) and helper vector were micro-injected into 1,800 eggs of bivoltin silkworms, Baegokjam and EGFP-induced fluorescence was observed for 25 broods of transgenic silkworms under a florescence stereomicroscope. Analysis by real-time PCR indicated that transgenic silkworms expressing BmRelish1t recombinant proteins displayed higher mRNA expression levels of the Bombyx mori antimicrobial peptides such as lebocin, moricin, and nuecin than the normal silkworms. Moreover, transgenic silkworms expressing BmRelish1t showed antibacterial activity against Escherichia coli. We suggest that transgenic expression of BmRelish1t may find useful applications for the production of various antimicrobial peptides at the same time in transgenic silkworms.

Silkworm transgenesis is now a routine method leading to a satisfactory yield of transformed animals and the reliable expression of transgenes during multiple successive generations. However, the screening of G1 transgenic individuals from numerous progeny has proved to be difficult and time-consuming work. Previously, we characterized the promoter of heat shock protein 70 from Bombyx mori (bHsp70), which is ubiquitously expressed in all tissues and developmental stages. To investigate the utilization of the bHsp70 promoter to screen transgenic individuals, the EGFP marker gene was inserted into the piggyBac vector under the control of the bHsp70 promoter. Mixtures of the donor and helper vectors were micro-injected into 3,060 eggs of bivoltine silkworms (Keomokjam). EGFP fluorescence was observed in 17 broods of transgenic silkworms under a florescence stereomicroscope. Interestingly, this fluorescent marker protein was detected not only in parts of the embryo segments on the seventh day of the G1 embryonic developmental stage but it was also detected in a part of the body of G1 hatched larvae, in the middle silk gland of G1 fifth instar larvae, and in the wings of seven-day-old G1 pupae and G1 moths. Therefore, we suggest that the bHsp70 promoter can be used for the rapid and simple screening of transgenic silkworms.

The antimicrobial peptide cecropin was isolated from the larval hemolymph of immune-challenged japanese oak silkworm, Antheraea yamamai. The full-length cDNA of A. yamamai cecropin (Ay-cecA) was cloned by a combination of RT-PCR and 3' RACE based on N-terminal sequence obtained by Edman degradation. The cloned cDNA consists of 419 nucleotides encoding a 64 amino acid precursor containing a 37-residue mature peptide. Like many insect cecropins, Ay-cecA also harbored a glycine residue for C-terminal amidation at the C-end. To understand this peptide better, we successfully expressed bioactive recombinant Ay-cecA in E. coli BL21(DE3) by fusing with ketosteroid isomerase (KSI) to avoid the cell death during induction. The fusion CecA-KSI protein was expressed as inclusion body at high level. Recombinant Ay-cecA was easily released by cleavage of the fusion protein with cyanogen bromide (CNBr), and purified by FPLC chromatography. The purified recombinant Ay-cecA showed considerably antibacterial activity against Gram-negative bacteria, E. coli ML 35, Klebsiella pneumonia and Pseudomonas aeruginosa. The time-kill assay showed that Ay-CecA displayed a time-dependent bactericidal activity, as was also seen after treatment with melittin. our results proved that Ay-cecA can be developed into novel antibacterial agent.

For the purposes of this paper, stress may be defined as any modification of environmental parameters that leads to a response by biological organisms. Stresses that affect biological structures may be non-thermal, such as ultraviolet radiation, pH, or salinity, or thermal. Temperature is one of the major stresses that all living organism face. The major effects of cold shock are decrease of membrane fluidity and the stabilization of secondary structures of RNA and DNA in the cells, which may effect the efficiency of translation, transcription, and DNA replication. In this study in compliance with the cold temperature stress about selection of the useful gene is contents from the silkworm which is been revealed. The survival rate which is caused by with the cold temperature stress until 12 hours was 100% in 0℃, until 2 hours was 100% in -5℃. A total of 960 clones were randomly selected from the subtraction cDNA library, and then performed a differential display hybridization analysis with forward and reverse probes. In conclusion, selected 53 partial clones and novel 2 full-length clones were identified as differential expressed genes. We assumed that the novel gene related with transmembrane.

Cecropin is an antimicrobial peptide that is synthesized in fat body cells and hemocytes of insect in response to a hypodermic injury or bacterial infection. A 503 bp cDNA encoding a cecropin-like antimicrobial peptide was isolated by employing annealing control primer (ACP)-based differential display PCR and 5'-RACE from immunized Papilio xuthus larvae. The open reading frame (ORF) of isolated cDNA encoded a 63 amino acid prepropeptide with a putative 22-residue signal peptide, a 3-residue propeptide and a 38-residue mature peptide with a theoretical mass of 4060.89 Da. The deduced amino acid sequence of peptide showed significant identities with other Lepidopteran cecropins. This peptide was named as papiliocin. RT-PCR revealed that the papiliocin transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). Based on the deduced amino acid sequence of papiliocin, a 38-mer mature peptide was chemically synthesized by Fmoc method, and analyzed antimicrobial activity. The synthetic papiliocin peptide had a broad spectrum of activity against fungi, Gram-positive and negative bacteria, and also showed no hemolytic activity against human red blood cell.

Electroporation is well known today as a powerful transfection technique and is useful for the study of gene expression. The advantage of the electroporation method is that large quantity of silkworm (Bombyx mori) eggs can be transformed in a very short time. However, how to use it for introducing foreign gene into silkworm eggs needs systematical investigation. In our silkworm transgenesis program, we needed an efficient technique to evaluate the functionality of transgenes before their injection into eggs. The goal of this experiment was to find an alternative efficient method of generating transgenic silkworm eggs using a commercially available electroporation device. The Gene Pulser Xcell (Bio-Rad Laboratories, USA) were used. In contrast to other electroporation devices, which are based on a single pulse with exponential decay or square wave technology. We investigated pigmentation-rate and hatching-rate of the silkworm eggs of electroporation. We used foreign gene LacZ, EGFP, Ds-red induced vector system with selection marker for transgenic silkworm. The LacZ gene in 3rd instar larva DNA can be detected by β-galactosidase stain. During these technical studies we found that optimizing parameters such as electrical voltage, number of pulses and their frequency, and conductivity of the buffer was important. These results confirmed that electroporation is available technique for transfecting B. mori egg.

SAGE technique is a sequenced-based approach that identifies which genes are expressed and quantifies their level of expression. The SAGE catalog of gene expression for a given cell or tissue is defined as the 'transcriptome'. With a goal of obtaining a set of quantitative information of expressed genes of posterior silk gland (PSG) of silkworm, we have generated a SAGE tag library from the PSGat day 4 of 5th instars of Bombyx mori. In this study, atotal of 2,406 tags were identified, representing 682 unique transcripts. Of these SAGE tags, 1,982 tags were detected twice or more accounted for 82% of the total tag population, whereas 445 tags were detected only once accounted for 18% of the total tag population. Four percent (27 tags) of the unique tags were detected at least ten times each, which corresponds to a representation of more than 53% of the total tag population. In addition, we have discussed a comparative aspect of the transcript abundance between expressed sequenced tags (ESTs) and the SAGE tags.

역삼투 공정을 농축공정에 이용하기 위해 향류식 역삼투 나권형 모듈을 고안, 제작하여 염수의 농축실험을 수행하였다. 역삼투 분리의 배제도와 농축도의 관계를 고찰한 결과, 배제유량에 대한 투과유량의 비가 양쪽 관계의 중요한 매개 변수이며, 반사계수 값에 따라, 역삼투막의 배제도와 농축도가 Spiegler-Kedem 식의 경향을 따른다. 향류식 역삼투 공정은 일반 역삼투 농축 공정에 비해 농축의 장애 요소인 삼투압차를 효과적으로 저하시키는 효과가 있으며 농축도가 상대적으로 우수하였다. 향류식 나권형 모듈의 수치모사 결과 농축공정의 농축도 증가를 위해서는 모듈의 지름보다 모듈의 길이를 늘리는 것이 유리함을 알 수 있었다.