• Journal of Plant Biotechnology
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• 제35권4호
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• pp.265-274
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• 2008

Glutathione S-transferases (GSTs) are multifunctional proteins encoded by a large gene family divided into Phi, Tau, Theta, Zeta, Lambda and DHAR classes on the basis of sequence identity. The Phi(F) and Tau(U) classes are plant-specific and ubiquitous. Their roles have been defined as herbicide detoxification and responses to biotic and abiotic stresses. Fifty-two members of the GST super-family were identified in the Arabidopsis thaliana genome, 13 members of which belong to the Phi class of GSTs (AtGSTFs). Based on the sequence similarities of AtGSTFs, 11 BAC clones were identified from Brassica rapa. Seven unique sequences of ORFs designated the Phi class candidates of GST derived from B. rapa (BrGSTFs) were detected from these 11 BAC clones by blast search and sequence alignment. Some of BrGSTFs were present in the same BAC clones indicating that BrGSTFs could also be clustered as usual in plant. They were mapped on B. rapa linkage group 2, 3, 9 and 10 and their nucleotide and amino acid sequences were highly similar to those of AtGSTFs. In addition, in silico analysis of BrGSTFs using Korea Brassica Genome Project 24K oligochip and microarray database for cold, salt and drought stresses revealed 15 unigenes to be highly similar to AtGSTFs and six of these were identical to one of BrGSTFs identified in the BAC clones indicating their expression. The sequences of BrGSTFs and unigenes identified in this study will facilitate further studies to apply GST genes to medical and agriculture purposes.

• IMMUNE NETWORK
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• 제12권5호
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• pp.207-212
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• 2012

T cell immunoglobulin mucin domain (TIM)-3 is an immunomodulatory molecule and upregulated in T cells by several cytokines. TIM-3 also influences mast cell function but its transcriptional regulation in mast cells has not been clarified. Therefore, we examined the transcript level and the promoter activity of TIM-3 in mast cells. The TIM-3 transcript level was assessed by real-time RT-PCR and promoter activity by luciferase reporter assay. TIM-3 mRNA levels were increased in HMC-1, a human mast cell line by TGF-${\beta}1$ stimulation but not by stimulation with interferon (IFN)-${\alpha}$, IFN-${\lambda}$, TNF-${\alpha}$, or IL-10. TIM-3 promoter -349~+144 bp region relative to the transcription start site was crucial for the basal and TGF-${\beta}1$-induced TIM-3 promoter activities in HMC-1 cells. TIM-3 promoter activity was increased by over-expression of Smad2 and Smad4, downstream molecules of TGF-${\beta}1$ signaling. Our results localize TIM-3 promoter activity to the region spanning -349 to ＋144 bp in resting and TGF-${\beta}1$ stimulated mast cells.

• Steel and Composite Structures
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• 제18권6호
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• pp.1569-1582
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• 2015

This study explores the lateral deflections of diagonal braces in concentrically-braced earthquake-resisting frames. The performance of this widely-used system is often compromised by the flexural buckling of slender braces in compression. In addition to reducing the compressive resistance, buckling may also cause these members to undergo sizeable lateral deflections which could damage surrounding structural components. Different approaches have been used in the past to predict the mid-length lateral deflections of cyclically loaded steel braces based on their theoretical deformed geometry or by using experimental data. Expressions have been proposed relating the mid-length lateral deflection to the axial displacement ductility of the member. Recent experiments were conducted on hollow and concrete-filled circular hollow section (CHS) braces of different lengths under cyclic loading. Very slender, concrete-filled tubular braces exhibited a highly ductile response, undergoing large axial displacements prior to failure. The presence of concrete infill did not influence the magnitude of lateral deflection in relation to the axial displacement, but did increase the number of cycles endured and the maximum axial displacement achieved. The corresponding lateral deflections exceeded the deflections observed in the majority of the previous experiments that were considered. Consequently, predictive expressions from previous research did not accurately predict the mid-height lateral deflections of these CHS members. Mid-length lateral deflections were found to be influenced by the member non-dimensional slenderness (${\bar{\lambda}}$) and hence a new expression was proposed for the lateral deflection in terms of member slenderness and axial displacement ductility.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.1015-1025
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• 2015

The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the L-leucine-specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A_6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A_6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A_6-D-Leu-domain with the A_6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

• Parasites, Hosts and Diseases
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• 제49권1호
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• pp.79-83
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• 2011

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage ${\lambda}$ Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, ${\alpha$-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.

• 대한수의학회지
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• 제39권2호
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• pp.327-337
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• 1999

The transposon TnphoA was used to generate avirulent mutants from a type A Pasteurella multocida. A suicide vector plasmid pRT733 carrying TnphoA, having the kanamycin resistant gene and harbored in Escherichia coli K-12 strain SM10(${\lambda}pir$), was mated with streptomycin resistant P. multocida P-1059 strain as recipient. This resulted in the generation of two TnphoA insertion mutants (transconjugants, tc95-a and tc95-b) which were resistant both to kanamycin ($Km^{R}$) and streptomycin ($Sm^{R}$), secreted alkaline phosphatase, and were avirulent to turkeys. Southern blot hybridization using two probes derived from internal fragments of TnphoA, confirmed the insertion of TnphoA into 12.9kb or 13.7kb DNA fragment from the EcoRV digested genomic fragments of transconjugants. The two transconjugants, tc95-a and tc95-b, were distinguishable from their parent strains by differences in ribotypes, and outer membrane protein profiles. TnphoA insertion in both transconjugants also resulted in constitutive expression of a 33Kd iron regulated outer membrane protein (IROMP). The gene encoding $Sm^{R}$ was also located within the same 12.9kb EcoRV genomic fragment from both transconjugants. Furthermore, our finding that the recipient P. multocida P-1059 $Sm^{R}$ strain and both transconjugants were avirulent to turkeys suggest that the either 12.9kb or 13.7kb genomic DNA contains the virulence gene and speculate that the presence of $Sm^{R}$ gene or TnphoA insertion may be responsible for regulating and inactivating the gene(s) encoding virulence in P. multocida.

• 한국수산과학회지
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• 제30권6호
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• pp.984-991
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• 1997

New tumor suppressor gene, snm23, homologous to human nm23/NDP kinase (human nucleoside diphosphate kinase) gene whose product has a tumor metastasis inhibitory activity, was first cloned from Korean tiger shark (Scyliorhinus forazame) skin cDNA library constructed by using a $\lambda$ ZAP-II cDNA synthesis kit. About $1\times10^5$ plaques were screened and several positive plaques were isolated and confirmed by second screening. The phagemid containing a positive clone from the Uni-Zap XR vector was excised in vivo and the gene containing the tumor metastasis suppressor protein was named as snm23. Cloned gene, snm23, was sequenced with ABI-PRISM 310 Genetic Analyzer. The nucleotide and deduced amino acid sequences of snm23 have shown an open reading frame consisting of 450 base pairs that correspond to a protein of 150 amino acid residues, with a calculated molecular mass of 16.8 kDa. Sequence comparison of snm23 with human nm23/NDP kinase was performed by using Blast protein data base of National Center for Biotechnology Information. In order to determine tissue specificity, reverse transcription-polymerase chain reaction (RT-PCR) was used. Good expression level of snm23/NDP kinase was detected at the tissues from skin, cartilage, and liver of Korean tiger shark.

• Journal of Microbiology and Biotechnology
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• 제30권4호
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• pp.515-525
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• 2020

Interferon (IFN)-λ plays an essential role in mucosal cells which exhibit strong antiviral activity. Lactobacillus plantarum (L. plantarum) has substantial application potential in the food and medical industries because of its probiotic properties. Alphacoronaviruses, especially porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV), cause high morbidity and mortality in piglets resulting in economic loss. Co-infection by these two viruses is becoming increasingly frequent. Therefore, it is particularly important to develop a new drug to prevent diarrhea infected with mixed viruses in piglets. In this study, we first constructed an anchored expression vector with CWA (C-terminal cell wall anchor) on L. plantarum. Second, we constructed two recombinant L. plantarum strains that anchored IFN-λ3 via pgsA (N-terminal transmembrane anchor) and CWA. Third, we demonstrated that both recombinant strains possess strong antiviral effects against coronavirus infection in the intestinal porcine epithelial cell line J2 (IPEC-J2). However, recombinant L. plantarum with the CWA anchor exhibited a more powerful antiviral effect than recombinant L. plantarum with pgsA. Consistent with this finding, Lb.plantarum-pSIP-409-IFN-λ3-CWA enhanced the expression levels of IFN-stimulated genes (ISGs) (ISG15, OASL, and Mx1) in IPEC-J2 cells more than did recombinant Lb.plantarum-pSIP-409-pgsA'-IFN-λ3. Our study verifies that recombinant L. plantarum inhibits PEDV and TGEV infection in IPEC-J2 cells, which may offer great potential for use as a novel oral antiviral agent in therapeutic applications for combating porcine epidemic diarrhea and transmissible gastroenteritis. This study is the first to show that recombinant L. plantarum suppresses PEDV and TGEV infection of IPEC-J2 cells.

• Journal of Veterinary Science
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• 제22권3호
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• pp.39.18-39.18
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• 2021

Background: Interferon lambda receptor 1 (IFNLR1) is a type II cytokine receptor that clings to interleukins IL-28A, IL29B, and IL-29 referred to as type III IFNs (IFN-λs). IFN-λs act through the JAK-STAT signaling pathway to exert antiviral effects related to preventing and curing an infection. Although the immune function of IFN-λs in virus invasion has been described, the molecular mechanism of IFNLR1 in that process is unclear. Objectives: The purpose of this study was to elucidate the role of IFNLR1 in the pathogenesis and treatment of porcine reproductive and respiratory syndrome virus (PRRSV). Methods: The effects of IFNLR1 on the proliferation of porcine alveolar macrophages (PAMs) during PRRSV infection were investigated using interference and overexpression methods. Results: In this study, the expressions of the IFNLR1 gene in the liver, large intestine, small intestine, kidney, and lung tissues of Dapulian pigs were significantly higher than those in Landrace pigs. It was determined that porcine IFNLR1 overexpression suppresses PRRSV replication. The qRT-PCR results revealed that overexpression of IFNLR1 upregulated antiviral and IFN-stimulated genes. IFNLR1 overexpression inhibits the proliferation of PAMs and upregulation of p-STAT1. By contrast, knockdown of IFNLR1 expression promotes PAMs proliferation. The G0/G1 phase proportion in IFNLR1-overexpressing cells increased, and the opposite change was observed in IFNLR1-underexpressing cells. After inhibition of the JAK/STAT signaling pathway, the G2/M phase proportion in the IFNLR1-overexpressing cells showed a significant increasing trend. In conclusion, overexpression of IFNLR1 induces activation of the JAK/STAT pathway, thereby inhibiting the proliferation of PAMs infected with PRRSV. Conclusion: Expression of the IFNLR1 gene has an important regulatory role in PRRSV-infected PAMs, indicating it has potential as a molecular target in developing a new strategy for the treatment of PRRSV.

• 한국수산과학회지
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• 제28권2호
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• pp.183-193
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• 1995

본 연구에서는 그물을 그것의 영역권 내로 물을 유입한 후 영역권 밖으로 투과시키는 하나의 유공성 구조물로 간주하고, 벽 면적이 S되는 그물이 유속 v에서 받는 저항 R을 $R=kSv^2$으로 취하여, 레이놀즈수를 $R_e$, 그물 입구의 단면적을 $S_m$, 흐름에 수직인 평면에 대한 그물의 총 투영면적을 $S_m$이라 할 때 저항계수 k를 $$k=c\;Re^{-m}(\frac{S_n}{S_m})n(\frac{S_n}{S})$$으로 표시한 후, 지금까지 행해진 평면 그물감에 대한 저항 실험 결과들을 이용하여 이 식의 타당성과 각계수 값을 함께 조사하였다. 조사 결과, 발의 지름이 d, 그물코의 크기가 21, 전개각이 $2\varphi$ 그물감의 $R_e$에 관한 대표치수를 그물코의 면적에 대한 발의 체적의 비 $\lambda$, 즉 $$\lambda={\frac{\pi\;d^2}{21\;sin\;2\varphi}$$로 택하였을 때, c와 m의 값은 각각 $240(kg\;\cdot\;sec^2/m^4)$ 및 0.1로 일정해졌고, n의 합은 1.2로서 1.0보다 컸기 때문에 매듭과 발에서 생기는 반류가 그물코 속으로의 물의 투과를 나쁘게 하여 저항을 증대시킨다는 것을 알 수 있었다. 반면, $R_e$가 커서 그 영향이 무시되는 경우는 $cR_e\;^{-m}$의 값이 상수가 되는데, 그 값은 흐름에 대한 그물감의 영각 $\theta$가 $ 45^{\circ}<\theta\leq90^{\circ}$의 구간에 있을 때 100$(kg\cdot sec^2/m^4)$으로 주어졌고, $ 0^{\circ}<\theta\leq45^{\circ}$의 구간에 있을 때는 후류의 영향 때문에 $100(S_m/S)^{0.6}\;(kg\cdot\;sec^2/m^4)$으로 주어 졌다. 그런데, 평면 그물감에 대 한 $S_m$ 및 $S_n$의 값은 각각 $$S_m=S\;sin\theta$$ 및 $$S_n=\frac{d}{I}\;\cdot\;\frac{\sqrt{1-cos^2\varphi cos^2\theta}} {sin\varphi\;cos\varphi} \cdot S$$로 주어지므로, 이들과 상기 c, m 및 n 값을 이용하면 평면 그물감의 저항계수 k가 구해지는데, $\theta=0^{\circ}$인 경우는 저항 특성 자체가 변하여 k가 그물감 표면의 조도에 따라 달라졌으므로 $$k=9(\frac{d}{I\;cos\varphi})^{0.8}$$으로 주어졌다. 그러나, 이상의 결과를 실제 그물에 적용할 때는 $\theta=0^{\circ}$ 때의 것은 고려하지 않아도 되고, 전기한 c 및 m 값도 불충분한 자료에 의한 것들이기 때문에 $R_e$의 영향이 무시되는 경우의 것만을 이용하면, 그물 각부의 $\theta$가 $45^{\circ}<\theta\leq90^{\circ}$의 구간 또는 $0^{\circ}<\theta\leq45^{\circ}$의 구간에 들어오는 그물의 저항계수 $k(kg\cdot sec^2/m^4)$는 $$k=100(\frac{S_n}{S_m})^{1.2}\;(\frac{S_m}{S})$$ 또는 $$k=100(\frac{S_n}{S_m})^{1.2}\;(\frac{S_m}{S})^{1.6}$$으로 주어진다는 것을 알 수 있었다.