• Tuberculosis and Respiratory Diseases
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• v.50 no.1
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• pp.52-66
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• 2001

Background : NF-${\kappa}B$ is the most important transcriptional factor in IL-8 gene expression. Triptolide is a new compound that recently has been shown to inhibit NF-${\kappa}B$ activation. The purpose of this study is to investigate how triptolide inhibits NF-${\kappa}B$-dependent IL-8 gene transcription in lung epithelial cells and to pilot the potential for the clinical application of triptolide in inflammatory lung diseases. Methods : A549 cells were used and triptolide was provided from Pharmagenesis Company (Palo Alto, CA). In order to examine NF-${\kappa}B$-dependent IL-8 transcriptional activity, we established stable A549 IL-8-NF-${\kappa}B$-luc. cells and performed luciferase assays. IL-8 gene expression was measured by RT-PCR and ELISA. A Western blot was done for the study of $I{\kappa}B{\alpha}$ degradation and an electromobility shift assay was done to analyze NF-${\kappa}B$ DNA binding. p65 specific transactivation was analyzed by a cotransfection study using a Gal4-p65 fusion protein expression system. To investigate the involvement of transcriptional coactivators, we perfomed a transfection study with CBP and SRC-1 expression vectors. Results : We observed that triptolide significantly suppresses NF-${\kappa}B$-dependent IL-8 transcriptional activity induced by IL-$1{\beta}$ and PMA. RT-PCR showed that triptolide represses both IL-$1{\beta}$ and PMA-induced IL-8 mRNA expression and ELISA confirmed this triptolide-mediated IL-8 suppression at the protein level. However, triptolide did not affect $I{\kappa}B{\alpha}$ degradation and NF-$_{\kappa}B$ DNA binding. In a p65-specific transactivation study, triptolide significantly suppressed Gal4-p65T Al and Gal4-p65T A2 activity suggesting that triptolide inhibits NF-${\kappa}B$ activation by inhibiting p65 transactivation. However, this triptolide-mediated inhibition of p65 transactivation was not rescued by the overexpression of CBP or SRC-1, thereby excluding the role of transcriptional coactivators. Conclusions : Triptolide is a new compound that inhibits NF-${\kappa}B$-dependent IL-8 transcriptional activation by inhibiting p65 transactivation, but not by an $I{\kappa}B{\alpha}$-dependent mechanism. This suggests that triptolide may have a therapeutic potential for inflammatory lung diseases.

• Korean journal of applied entomology
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• v.54 no.1
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• pp.7-15
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• 2015

CpBV (Cotesia plutellae bracovirus) is a polydnavirus and encodes a cystatin (CpBV-CST1) gene. Its overexpression suppresses insect immunity and alters insect developmental processes. This study aimed to construct a genetically modified (GM) tobacco to further explore the physiological function of the viral cystatin and to apply to control insect pests. To this end, the transgenic tobacco lines were screened in expression of the target gene and assessed in insecticidal activity. A recombinant vector (pBI121-CST) was prepared and used to transform a bacterium, Agrobacterium tumefasciens. The transformed bacteria were used to generate transgenic tobacco lines, which were induced to grow callus and resulted in about 92% of shoot regeneration. The regenerated plants were screened by PCR analysis to confirm the insertion of the target gene in the plant genome. In addition, the expression of the target gene was assessed in the regenerated plants by quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis showed that the transgenic line plant expressed the target gene about 17 times more than the control tobacco, indicating a stable insertion and expression of the target gene in the transgenic tobacco line. The insecticidal activity was then analyzed using the screened transgenic tobacco lines against the teneral 1st instar larvae of the oriental tobacco budworm, Helicoverpa assulta. Though there was a variation in the insecticidal efficacy among transgenic lines, T9 and T12 lines exhibited more than 95% mortality at 7 days after feeding treatment. These results suggest that CpBV-CST1 is a useful genetic resource to be used to generate GM crop against insect pests.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.994-1004
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• 2005

The dihydrofolate reductase (dhfr) promoter contains cis-acting element for the transcription factors Spl and E2F. Transcription of dhfr gene shows maximal activity during the Gl/S phase of cell cycle. The member of the Spl transcriptional factor family can act as both negative and positive regulators of gene expression. There was a report that Spl-Rb and E2F4-pl30 complexes cooperate to establish stable repression of dhfr gene expression in CHOC400 cells. Here, we examined the role of HDAC in dhfr, cyclin E, and cyclin A gene regulation using the histone deacetylation inhibitor, trichostatin A (TSA) in U2OS and C33A cells, a Rb-positive human osteosarcoma cell line, and a Rb-negative cervical carcinoma cell line, respectively. When the dhfr promoter constructs were applied in U2OS cells, TSA markedly stimulated over 14-fold of dhfr promoter activity through dhfr-Spl sites by the deletion of an E2F element. In contrast, the deletion of dhfr-Spl binding sites completely abolished promoter stimulation by TSA. The dhfr promoter activity including dhfr-Spl sites increased only 2-fold in C33A cells. Promoter activity containing only dhfr-E2F site did not have much effect by the treatment of TSA in both U2OS and C33A cells. On the other hand, treatment with TSA induced significantly mRNA expression of dhfr and cyclin E, whereas levels of cyclin A decreased in U2OS cells, but had no effect in C33A cells. These results indicate that TSA have contradictory effect, activation of dhfr and cyclin E genes on Gl phase, and down-regulation of cyclin A on G2 phase through transcriptional regulation in U2OS cells.

• Korean Journal of Heritage: History & Science
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• v.53 no.3
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• pp.62-79
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• 2020

The rock-carved seated Avalokiteśvara statue at Ganghwa Bomunsa Temple is a giant rock-carved Buddhist statue that was built in 1928 during the Japanese colonial era. Although it is a year-recorded Buddhist statue that occupies a prominent place in modern Korean Buddhist sculpture history, it has not been the subject of in-depth discussion due to weak research on modern Buddhist sculptures. In this study, to examine the various significant aspects of the rock-carved Seated Avalokiteśvara statue at Bomunsa Temple as a modern Buddhist sculpture, I have managed to determine its construction year, artificers, and patrons by deciphering the inscription around the rock-carved statue; in addition I have researched the effects of the rock shapes and landforms on the formation of the Buddhist statue by comparing and analyzing the points of view of both artificers and worshipers. I have also identified the specific circumstances of the time of construction from interviews with the descendants of artificers. A monk from Geumgangsan Mountain, Lee Hwaeung, took the role of sponsor and chief painter to construct the rock-carved seated Avalokiteśvara statue at Bomunsa temple. In the beginning of its construction in 1928, more than 100 donators jointly sponsored the construction of the statue. Gansong Jeon Hyoungphil sponsored alone at the time of the place of worship's expansion in 1938. Bomunsa Temple has been regarded as one of the top-three sacred places of Avalokiteśvara Bodhisattva together with Naksansa Temple in Yang Yang and Boriam in Nam Hae, due to the construction of the rock-carved statue. It took about three months to construct the statue. Lee Hwaeung drew a rough sketch and then Un Songhag and five masons from Ganghwa Island took part in the carving process. We can observe the line drawing technique around the rock-carved statue because the statue was carved based on the rough sketch of the monk painter. The aspect of Lee Hwaeung as a painter is revealed; therefore, we can identify the clue of painting pattern leading to Seogongchulyou- Hwaunghyoungjin- Ilonghyegag. The rock-carved seated Avalokiteśvara statue at Bomunsa Temple is a typical Avalokiteśvara that wears a jeweled crown and holds Kundica. It makes a strong impression as it has a big square-shaped face and a short neck and is unsophisticated in general. The artificers solved the issue of visual distortion of the rock-carved statue caused by carving on a 10-meter high and 40-degree sloping rock by controlling motion to its maximum, omitting detailed expression by emphasizing symmetry, and adjusting the head-to-body proportion to be almost one-to-one. In this study, especially, I presume the unified form of sacred sculptures and Buddhist altars, without making a Buddhist altar like the rock-carved seated Avalokiteśvara statue at Bomunsa Temple, to be a key characteristic of modern Buddhist sculptures. Furthermore, I make newly clear that the six letters of Sanskrit carved on nimbus, which had been interpreted as a Six-Syllable Mantra, are a combination with Jeongbeopgye and Sabang Mantras. In addition, three iron rings driven on eaves rock were used as a reference point, and after construction they were used as a decoration for the Bodhisattva with hanging wind chimes.

• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.312-320
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• 2000

Abstract The full sequence of the plasmid pKJ36, which was derived from Bifidobacterium longum KJ, was determined and analyzed to construct shuttle vectors between E. coli and Bifidobacterium. The plasmid pKJ36 was composed of 3,625 base pairs with a 65.1% G+C content. The structural organization of pKJ36 was highly similar to that of pKJ50, and the three major ORFs on pKJ36 showed high amino acid sequence homologies with those of pKJ50. The putative proteins coded by these three ORFs were designated as RepB (32.0 kDa, pI=9.25), MembB (29.0 kDa, pI=12.25), and MobB (39.0 kDa, pI=IO.66), respectively. The amino acid sequence of RepB showed a 57% identity and 70% similarity with that of the RepA protein of pKJ50. Upstream of the repB gene, the so-called iteron sequence was directly repeated four-and-ahalf times and a conserved dnaA box was identified. An amino acid sequence comparison between the MobB and MobA of pKJ50 revealed a 48% identity and 61 % similarity. A conserved oriT sequence with an inverted repeat identical to that of pKJ50 was also found upstream of the mobB gene. A hydropathy analysis of MembB revealed four possible transmembrane regions. The expressions of the repB and membB genes were confirmed by RT-PCR. The in vitro translation reaction of pKJ36 showed protein bands with anticipated sizes with respect to each putative gene product. S 1 endonuclease treatment and Southern hybridization suggested that pKJ36 replicates by a rolling circle mechanism via a single-stranded DNA (ssDNA) intermediate. A shuttle vector between E. coli and Bifidobacterium sp. was constructed using the pKJ36, pBR322, and staphylococcal chloramphenicol acetyl transferase (CAT) gene. The successful transformation of the Bifidobacterium strains was shown by Southern hybridization and PCR. The transformation efficiency differed from strain to strain and, depending on the electroporation conditions, with a range between $1.2{\times}10^1-2.6{\times}10^2{\;}cfu/\mu\textrm{g}$ DNA.X> DNA.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.3
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• pp.192-197
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• 2007

Suppression subtractive hybridization (SSH) was performed to construct an cDNA library of olive flounder Paralichthys olivaceus peripheral blood leukocytes (PBL) stimulated with lipopolysaccharide (LPS). Total 470 clones were randomly selected and sequenced, 214 out of 470 sequences showed identities with known genes and 252 sequences were unknown genes. Among the 218 known genes, 34 sequences were found to be homologous to IL-1RII gene, and 14 sequences were identified as IL-$1{\beta}$. RT-PCR analysis showed that IL-$1{\beta}$ mRNA was induced 1h after LPS-stimulation, and IL-1RII was increased from 3h after stimulation, indicating that most of SSH clones are associated with inflammatory responses in fish, and this SSH cDNA library would be useful to identify bio-defense and immuno-related genes in fish.

• Journal of Life Science
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• v.16 no.3 s.76
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• pp.454-458
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• 2006

HSF1 is the heat shock transcription factor in Saccharomyces cerevisiae. S. cerevisiae KNU5377 can ferment at high temperature such as $40^{\b{o}}C$. We have been the subjects of intense study because Hsf1p mediates gene expression not only to heat shock, but to a variety of cellular and environmental stress challenges. Basing these facts, we firstly tried to construct the hsf1 gene-deleted mutant. PCR-method for fast production of gene disruption cassette was introduced in a thermotolerant yeast S. cerevisiae KNU5377, which allowed the addition of short flanking homology region as short as 45 bp suffice to mediate homologous recombination to kanMX module. Such a cassette is composed of linking genomic DNA of target gene to the selectable marker kanMX4 that confers geneticin (G418) resistance in yeast. That module is extensively used for PCR-based gene replacement of target gene in the laboratory strains. We describe here the generation of hsf1 gene disruption construction using PCR product of selectable marker with primers that provide homology to the hsf1 gene following separation of haploid strain in wild type yeast S. cerevisiae KNU5377. Yeast deletion overview containing replace cassette module, deletion mutant construction and strain confirmation in this study used Saccharomyces Genome Deletion Project (http:://www-sequence.standard.edu/group/yeast_deletion_project). This mutant by genetic manipulation of wild type yeast KNU5377 strain will provide a good system for analyzing the research of the molecular biology underlying their physiology and metabolic process under fermentation and improvement of their fermentative properties.

• Korean Journal of Veterinary Research
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• v.48 no.4
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• pp.441-449
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• 2008

The genus Lactobacillus is the largest of the genera included in lactic acid bacteria and is associated with mucosal membranes of human and animal. Only a few Lactobacillus plasmid-encoded functions have been discovered and used. In this study, a novel plasmid (pILR091) was isolated from a wild L. reuteri isolated from pig and described the characteristics of its replicons, genetic organization, and relationship with other plasmids. After digestion of the plasmid, pILR091, with SalI, plasmid DNA was cloned into the pQE-30Xa vector and sequenced. The complete sequence was confirmed by the sequencing of PCR products and analyzed with the Genbank database. The isolate copy number and stability were determined by quantitative-PCR. The complete sequence of L. reuteri contained 7,185 nucleotides with 39% G-C content and one cut site by two enzymes, SalI and HindIII. The similar ori sequence of the pC194- rolling circle replication family (TTTATATTGAT) was located 63 bp upstream of the protein replication sequence, ORF 1. Total of five ORFs was identified and the coding sequence represented 4,966 nucleotides (70.4%). ORF1 of pILR091 had a low similarity with the sequence of pTE44. Other ORFs also showed low homology and E-values. The average G-C content of pILR091 was 39%, similar with that of genomic DNA. The copy number of pILR091 was determined at approximately 24 to 25 molecules per genomic DNA. These results suggested that pILR091 might be a good candidate to construct a new vector, which could be used for cloning and expression of foreign genes in lactobacilli.

• Korean Journal of Microbiology
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• v.33 no.1
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• pp.1-6
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• 1997

5-Enolpyruvylshikimate 3-phosphate(EPSP) synthase is the sixth enzyme of the shikimate pathway that synthesizes aromatic amino acids. The enzyme is a primary target for the glyphos'lte which is a broad-spectrum and environmetally safe herbicide. As a first step toward development of glyphpsate-resistant EPSP synthase, the EPSP synthase gene(aroA) was amplified by polymerase chain reaction and cluned into pET-25b vector. In this construct. designated pET-aro, the aroA gene is expressed under control of strong T7 promoter. and the EPSP synthase is produced as a fusion protein with pelB leader at N-terminus and HSV-tag and His-tag at C-terminus. When the pET-aro clone was induced to produce the enzyme, it was found that the EPSP synthase was successfully exported to peri plasmic space. The periplasmic transport was greatly dependent on the induction temperatures. Among the induction temperatures examined($25^{\circ}C$, $30^{\circ}C$, $34^{\circ}C$ and $37^{\circ}C$). induction at $34^{\circ}C$ gave rise to maximal periplasmic transport. The recomhinant EPSP synthase could have been purified hy $Ni^{2+}$ -affinity chromatography using the His-tag. and detected hy anti-HSV -tag antibody. The recombinant EPSP synthase also hound to phosphocellulose resin and was eluted hy shikimate 3-phosphate and phosphoenolpyruvate. as expected. The recombinant EPSP synthase purified from phosphocellulose resin showed typical EPSP synthase activity.

• Journal of Educational Research in Mathematics
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• v.20 no.1
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• pp.85-102
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• 2010

Linear programming(LP) is useful for finding the best way in a given condition for some list of requirements represented as linear equations. This study analysed LP in mathematics contexts and LP in school mathematics contexts, considered learning process of LP from an epistemological point of view, and explored a hypothetical learning trajectory of LP. The differences between mathematics contexts and school mathematics contexts are whether they considered that the convex polytope $\Omega$ is feasible/infeasible or bounded/unbounded or not, and whether they prove the theorem that the optimum is always attained at a vertex of the polyhedronor not. And there is a possibility that students could not understand what is maximum and minimum of a linear function when the domain of the function is limited. By considering these three aspects, we constructed hypothetical learning trajectory consisted of 4 steps. The first step is to see a given linear expression as linear function, the second step is to partition a given domain by straight lines, the third step is to construct the conception of y-intercept by relating lines and the range of k, and the forth step is to identify whether there exists the optimum in a given domain or not.