• Title/Summary/Keyword: T7 expression system

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Antioxidant capacity and Raw 264.7 macrophage anti-inflammatory effect of the Tenebrio Molitor (갈색거저리(Tenebrio Molitor)의 항산화능과 Raw 264.7 대식세포의 항염증 효과)

  • Yu, Jae-Myo;Jang, Jae-Yoon;Kim, Hyeon-Jeong;Cho, Yong-Hun;Kim, Dong-in;Kwon, O-jun;Cho, Yeong-Je;An, Bong-Jeun
    • Food Science and Preservation
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    • v.23 no.6
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    • pp.890-898
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    • 2016
  • The purpose of this paper is to investigate potential anti-inflammatory and anti-oxidant effects of Tenebrio molitor. Macrophage cell response by outside stimulation leads expression of pro-inflammatory cytokines, such as tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$), interleukin-6 (IL-6), $interleukin-1{\beta}$ ($IL-1{\beta}$), and trigger expression of genes which are affected by inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), resulting in formation of inflammatory factors like nitric oxide (NO) and Prostaglandin $E_2$ (PGE2). Cell viability was determined by MTT assay. In order to investigate anti-inflammatory agents, the inhibitory effects on the production of lipopolysaccharide (LPS)-induced NO in RAW 264.7 cells were examined. T. Molitor significantly decreased the production of NO in a dose-dependent manner, and also reduced the expression of iNOS, a COX-2 protein. As a result, the levels of protein such as $PGE_2$, iNOS, COX-2 and MARKs were significantly reduced compared to non-treated group in T. Molitor water extract (TDW) treated group. Also, antioxidant effect of T. Molitor were investigated using DPPH, ABTS+ and superoxide anion radical scavenging activity tests in cell-free system. Antioxidant activity of T. molitor was found low in the DPPH radical scavenging test while high in the ABTS+ and superoxide anion radical scavenging activity tests. These results show that TDW could be an effective anti-pro-inflammatory and anti-oxidant agent.

The Effects of Liriopis Tuber Water Extract on Innate Immune Activation and Anti-Inflammation (맥문동 물 추출물의 선천면역 활성과 염증억제 효과)

  • Kang, Nu-Ri;Hwang, Deok-Sang;Lee, Jin-Moo;Lee, Chang-Hoon;Jang, Jun-Bock
    • The Journal of Korean Obstetrics and Gynecology
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    • v.34 no.3
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    • pp.15-28
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    • 2021
  • Objectives: This study was designed to examine the anti-cancer activity by innate immunomodulating and anti-inflammatory effects of liriopis tuber water extract (LPE). Methods: Cell cytotoxicity was tested with 4T1 mouse mammary carcinoma cells, spleen cells, macrophage, and RAW264.7 cells. To investigate innate immunomodulating effects of LPE on macrophage, we measured tumor necrosis factor-alpha (TNF-α), interleukin-12 (IL-12), and interleukin-10 (IL-10). To investigate innate immunomodulating effects of LPE on RAW264.7 cell, we measured TNF-α, interleukin-6 (IL-6). In addition, TNF-α and nitric oxide (NO) induced by lipopolysaccharide (LPS) were measured after treating with LPE to observe innate immunomodulating effect of LPE on RAW264.7 cell. Also, mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) were examined by western blot analysis. Results: In an in vitro cytotoxicity analysis, LPE affected tumor cell growth above specific concentration. As compared with the control group, the production of TNF-α, IL-12, and IL-10 were increased in macrophage. As compared with the control group, the production of TNF-α and IL-6 were increased in RAW 264.7 cell. The expression of TNF-α and NO induced by LPS after treating LPE was decreased. In addition, treatment of RAW 264.7 cell with LPE increased the phosphorylation levels of p-extracellular signal-regulated kinase (p-ERK), p-Jun N-terminal kinase (p-JNK), and p-p38. Conclusions: LPE might have impact on the anti-cancer effect by activation of innate immune system and inflammation control.

Stable Maintenance of Recombinant Plasmid Containing trp $^+$ Operon in E. coli Cultures by the phe W$^+$ -pheS$^{t8}$ System (대장균 배양 중 phe W$^+$-pheS-$^{-ts}$ System에 의한 재조합 trp$^+$ 플라스미드의 안정적 유지)

  • 강충민;최장원;이세영
    • Microbiology and Biotechnology Letters
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    • v.18 no.1
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    • pp.89-93
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    • 1990
  • To improve the stability of recombinant pBR322-trip$^+$ plasmid (pLTW24) in E. coli culture, a positive selection system was devised. A DNA fragment containing pheW$^+$ gene (a structural gene for tRNA$^{phe}$ was isolated and inserted into the pBR322-trip$^+$ plasmid(pLTP24). A temperature sensitive host strain. LC901-pheS$^{-ts}$, was constructed for this plasmid by transducing pheS$^{-ts}$ allele (phenylalanyl-tRNA synthetase) to E. coli LC901 using P1kc bacteriophage. The LC901-pheS$^{-ts}$ cells were unable to grow at a restrictive temperature when they had lost the pBR322 :: pheW$^+$ (pLTP24) plasmid. The effects of pheW$^+$ gene on the plasmid stability and the expression level of trip$^+$ gene in LC901-pheS$^{-ts}$ strain were investigated. The proportion of Trip$^+$ colonies among LC901-pheS$^{-ts}$ strain carrying plasmid pLTP24 was 99%, whereas that of LC901 strain carrying plasmid pLTW24 was 7% at the end of 20 generations. After 100 generations of growth, the strain LC901-pheS$^{-ts}$ carrying plasmid pLTP24 showed little loss of plasmids. While the majority of plasmid pLTW24 in LC901 strain were lost in the same period. The activities of tryptophan synthetase (T. Sase) and anthranilate synthetase (A. Sase) in LC901 strain carrying pLTW24 were about 1.2 times and 1.8 times respectively of those in LC901-pheS$^{-ts}$ strain carrying pLTP24.

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Studies on Genetice of Blast Resistance in Rice L Inheritance of Resistance to Specific Races of Blast Fungus and Relationship between Their Resistance and II, VIII, XI and XII Linkage Groups in Some Rice Varieties (수도품종의 도열병 저항성 유전분석 제1보 특정 도열병 균계에 대한 저항성 품종들의 저항성 유전분리와 II, VIII, XI 및 XII번 연관과의 관계)

  • Chae, Y.A.;Park, S.Z.;Ha, S.B.
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.26 no.1
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    • pp.32-39
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    • 1981
  • In order to study the genetic system of the blast resistant varieties, the conidial suspension of mutant races of T-2$^{+t}$, N-2$^{+t}$, C-8$^{+t}$ was inoculated at 4-5 leaf stage by injector for F_2 seedlings from the crosses between seven resistant varieties and four maker lines easily detectable at seedling stage. The results are summarized as follows; 1. The fertility of cross between Semi-dwaf testers and Indica resistant varieties except Carreon was about 74 percents. 2. The segregation modes of resistance varied with varieties and blast races. However, the resistance was expressed as dominance in all cases. Tetep, Tadukan and Carreon showed more complicated segregation for resistance than that of the bred lines. 3. For blast races used, four segregation ratios such as 3:1, 9:7, 13:3 and 37:27 were found in the Tatukan, Tetep, and IR747, and three segregation ratios such as 3:1, 13:3 and 15:1 in the Carreon, and two segregation ratios of 3:1 and 13:3 with Suweon 287, Suweon 288, and Iri342. 4. In the segregation of the resistance to the each races, the ratios of 3:1, 13:3, 15:1 were fitted to T-2$^{+t}$, and the ratios of 3:1, 13:3, 9:7 and 37:27 to N-2$^{+t}$ and C-8$^{+t}$. 5. Suweon 287, Suweon 288 and Iri342 carried one simple dominant gene and inhibitor gene was considered in some cross combinations. Meanwhile Tadukan, Tetep and IR747 seemed to carry one to three resistant genes, and in some cross combinations, the expression of these genes were simple dominant, inhibiting, duplicating and complimentary action. 6. Resistance genes to blast races, T-2$^{+t}$, N-2$^{+t}$ and C-8$^{+t}$ in the Tadukan, Tetep, Carreon, Suweon 287, Suweon 288 and Iri342 were found to be independent with the linkage group of II(lg), VIII(la), XI(bc), and XII(gl).bc), and XII(gl).

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Evaluation of primary hepatocyte function using 2D or 3D culture method for primary rat hepatocytes (Rat Primary Hepatocyte의 2차원 배양과 3차원 배양에 따른 생리 활성능과 대사능에 관한 연구)

  • Lim, Malgum;Kim, Yeongji;Shin, Yurianna;Oh, Keon Bong;Hwang, Seongsoo;Kim, Youngim;Hur, Tai-Young;Ock, Sun A
    • Journal of Embryo Transfer
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    • v.31 no.3
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    • pp.169-177
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    • 2016
  • There is a growing interest in the application of primary hepatocytes for treatment of liver diseases in humans and for drug development. Several studies have focused on long-term survival and di-differentiation blocking of primary hepatocytes in an in vitro culture system. Therefore, the present study also aimed to optimize an in vitro culture system using primary rat hepatocytes. Primary rat hepatocytes from 6-week-old male Crl:CD rats were isolated using a modified two-step collagenase perfusion. Healthy $3.5{\times}10^6$ primary rat hepatocytes were seeded into a 2 dimensional (2D) culture in a 25T culture flask coated with collagen type I or into a 3D culture in a 125-ml spinner flask for 7 days. Production of plasma protein (ALB and TF), apoptosis (BAX and BCL2), and CYP (CYP3A1) related genes were compared between the 2D and 3D culture systems. The 3D culture system had an advantage over the 2D system because of the relatively high expression of ALB and low expression of BAX in the 3D system. However, the level of CYP3A1 did not improve in the 3D culture with and without the presence of a dexamethasone inducer. Therefore, 3D culture has an advantage for albumin production and primary rat hepatocyte survivability, but a low expression of CYP3A1 indicated that primary rat hepatocytes require a high-density culture for stress reduction by continuous flow.

Molecular Cloning of a Gene Encoding a Putative Antibacterial Peptide from Bombyx mori (누에에서 새로운 항세균성 펩타이드 유사 유전자의 분리와 염기서열 결정)

  • 김상현;제연호;윤은영;강석우;김근영;강석권
    • Korean journal of applied entomology
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    • v.35 no.4
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    • pp.321-325
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    • 1996
  • To isolate a novel gene for antibacterial peptide, an inducible clone(BmInc8) was selected by differential screening strategy from Bombyx mori cDNA library prepared from lavae injected with Escherichia coli. This clone contained a cDNA insert of 564 nucleotides and encoded 59 amino acids with an apparent molecular mass of 6.3 kDa. The cDNA sequence of BmInc8 had 61.2% identity compared to that of the bactericidin from Manduca sexta and also the deduced amino acids sequences from this insert had 65% identity compared to that of the cecropin D peptide Hyalophora cecropia. The transient expression assay of this insert using prokaryotic expression vector system revealed that the expressed peptide displayed the antibacterial activity. The cDNA sequence was deposited in GenBank under the accession number U30289.

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Innate Immunity Activation and Anti-Inflammation Effects of Evodia Rutaecarpine Water Extract (오수유 물 추출물의 선천 면역 활성과 염증 억제 효과)

  • Jeong, So-Mi;Lee, Jin-Moo;Lee, Chang-Hoon;Hwang, Deok-Sang;Jang, Jun-Bock
    • The Journal of Korean Obstetrics and Gynecology
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    • v.34 no.2
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    • pp.1-15
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    • 2021
  • Objectives: This study was designed to examine immuno-modulatory effects of Evodia Rutaecarpine by activating innate immune system and inhibiting inflammation. Methods: First, Cell cytotoxicity was examined with 4T1 breast carcinoma and TG-induced macrophage. To investigate activating innate immune system of Evodiamine Rutacarpine Extract (ERE) on macrophage, we tested tumor necrosis factor-alpha (TNF-α), interleukin-12 (IL-12), and interleukin-6 (IL-6). In addition, TNF-α and nitric oxide (NO) induced by lipopolysaccharide (LPS) were measured after treating with ERE to observe innate immune modulating effect of ERE on RAW 264.7 cell. Also, mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) were examined by western blot analysis. Results: In cytotoxicity analysis, ERE significantly affected tumor cell growth above specific concentration. Also, ERE significantly affected macrophage growth above specific concetration. As compared with the control group, the production of TNF-α, IL-12 and IL-6 were increased in TG-induced macrophage. As compared with the control group, TNF-α and IL-6 were significantly up-regulated in RAW 264.7 cell. The expression of TNF-α and NO induced by LPS after treating ERE was significantly decreased compared with control group. In addition, We observed ERE inhibited the phosphorylation levels of p-extracellular signal-regulated kinase (p-ERK), p-Jun N-terminal kinase (p-JNK), and p-p38 in western blotting by treating ERE on RAW 264.7 cell. Conclusions: ERE seems to have considerable impact on the anti-cancer effect by activation of innate immune system and inflammation control.

Fed-batch Fermentations of Recombinant Escherichia coli to Produce Bacillus macerans CGTase

  • Park, Yong-Cheol;Kim, Chang-Sup;Kim, Chung-Im;Choi, Kyu-Hwan;Seo, Jin-Ho
    • Journal of Microbiology and Biotechnology
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    • v.7 no.5
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    • pp.323-328
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    • 1997
  • The recombinant Escherichia coli BL21(DE3)pLysE : pTCGT1 was grown to overproduce Bacillus macerans cyclodextrin glucanotransferase (CGTase) able to synthesize ${\alpha}$-cyclodextrin (CD) with a selectivity of 67%. A number of batch fermentations were performed to test the possibility of using lactose as an inducer of the E. coli T7 promoter system. A mixture of isopropyl ${\beta}$-D-thiogalactoside (IPTG) and lactose (1 : 1) gave a maximum CGTase activity of 2.4 U/ml, which was higher than the value obtained with induction by IPTG alone. Fed-batch fermentations involving a glucose-controlled growth period followed by a gene-expression phase with mixtures of IPTG and lactose were employed to achieve high cell density and thereby increase total CGTase activity. Optimized fed-batch fermentation using the modified inducer (IPTG : lactose=1 : 3) and 100 g/l yeast extract solution in the gene-expression phase resulted in a maximum CGTase activity of 62.9 U/ml and a final cell mass of 53.5 g/l, corresponding to a 31-fold increase in CGTase activity and a 29-fold increase in cell mass compared with the control batch fermentation.

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The emerging role of lncRNAs in inflammatory bowel disease

  • Yarani, Reza;Mirza, Aashiq H.;Kaur, Simranjeet;Pociot, Flemming
    • Experimental and Molecular Medicine
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    • v.50 no.12
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    • pp.7.1-7.14
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    • 2018
  • Dysregulation of long noncoding RNA (lncRNA) expression is linked to the development of various diseases. Recently, an emerging body of evidence has indicated that lncRNAs play important roles in the pathogenesis of inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative Colitis (UC). In IBD, lncRNAs have been shown to be involved in diverse processes, including the regulation of intestinal epithelial cell apoptosis, association with lipid metabolism, and cell-cell interactions, thereby enhancing inflammation and the functional regulation of regulatory T cells. In this review, we aim to summarize the current knowledge regarding the role of lncRNAs in IBD and highlight potential avenues for future investigation. We also collate potentially immune-relevant, IBD-associated lncRNAs identified through a built-by association analysis with respect to their neighboring protein-coding genes within IBD-susceptible loci. We further underscore their importance by highlighting their enrichment for various aspects of immune system regulation, including antigen processing/presentation, immune cell proliferation and differentiation, and chronic inflammatory responses. Finally, we summarize the potential of lncRNAs as diagnostic biomarkers in IBD.

Comparison of immune cell populations in bronchoalveolar lavage cells and PBMC cytokine expressions in porcine reproductive and respiratory syndrome and porcine respiratory disease complex

  • Yang, Myeon-Sik;Jeong, Chang-Gi;Nazki, Salik;Mattoo, Sameer ul Salam;Lee, Sang-Myeong;Kim, Won-Il;Kim, Bumseok
    • Korean Journal of Veterinary Service
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    • v.42 no.4
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    • pp.201-216
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    • 2019
  • Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failure in sows and respiratory distress in all age pigs. Porcine respiratory disease complex (PRDC) is a disease caused by opportunistic bacterial infection secondary to a weakened immune system by a preceding respiratory infection. In this study, we tried to compare the immune responses in PRRS and PRDC groups to clearly characterize the disease severity. Eighty-five pigs were infected with various Korean field PRRS virus strains. Infected animals were classified into PRRS (n=32) and PRDC (n=53) groups based on lung lesions such as interstitial pneumonia, suppurative pneumonia, and pleuropneumonia. The immune cell population of bronchoalveolar lavage cells (BALc) was evaluated on 14 and 28 days post infection (dpi) and PMBC cytokine expression was measured on 0, 3, 7, 14 dpi to investigate early inflammatory reactions. Pulmonary lesion severity was negatively correlated with alveolar macrophage (AM) in both PRRS and PRDC groups on 14 and 28 dpi. AM in BALc was less populated in PRDC group on 28 dpi compared to PRRS group. AM in BALc was significantly less populated in PRDC group on 28 dpi compared to 14 dpi. In addition, cytotoxic T lymphocyte (CTL) in BALc was higher populated in PRDC group on 14 dpi and 28 dpi compared to PRRS group. In the case of PBMC cytokine TNF-α, IFN-α, IL-1β, IFN-γ, FoxP3, and IL-2, the PRRS group showed higher expression than the PRDC group on 7 dpi, 14 dpi, 7 dpi, 14 dpi, 14 dpi, and 14 dpi, respectively. On the other hand, in the case of IFN-β, IL-6, IL-8, IL-4, and IL-17, the PRDC group showed higher PBMC cytokine expression at 14 dpi, 7 dpi, 14 dpi, 3 dpi, and 3 dpi, respectively, than the PRRS group. Based on these results, our study could characterize differential immune responses in pigs with PRRS or PRDC.