• Journal of Microbiology and Biotechnology
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• v.27 no.9
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• pp.1701-1710
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• 2017

Classical swine fever virus (CSFV) is the etiologic agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. The lapinized C-strain, a widely used vaccine strain against CSFV, has low growth efficiency in cell culture, which limits the productivity in the vaccine industry. In this study, a recombinant virus derived from C-strain was constructed and subjected to continuous passaging in PK-15 cells with the goal of acquiring a high progeny virus yield. A cell-adapted virus variant, RecCpp80, had nearly 1,000-fold higher titer than its parent C-strain but lost the ability to induce fever in rabbits. Sequence analysis of cell-adapted RecC variants indicated that at least six nucleotide changes were fixed in RecCpp80. Further adaption of RecCpp80 variant in swine testicle cells led to a higher virus yield without additional mutations. Introduction of each of these residues into the wild-type RecC backbone showed that one mutation, M979R (T3310G), located in the C-terminal region of E2 might be closely related to the cell-adapted phenotype. Rabbit inoculation revealed that $RecCpp40_{+10}$ failed to induce fever in rabbits, whereas $RecCpp80_{+10}$ caused a fever response similar to the commercial C-strain vaccine. In conclusion, the C-strain can be adapted to cell culture by introducing specific mutations in its E2 protein. The mutations in RecCpp80 that led to the loss of fever response in rabbits require further investigation. Continuous passaging of the C-strain-based recombinant viruses in PK-15 cells could enhance its in vitro adaption. The non-synonymous mutations at 3310 and 3531 might play major roles in the enhanced capacity of general virus reproduction. Such findings may help design a modified C-strain for improved productivity of commercial vaccines at reduced production cost.

• Tuberculosis and Respiratory Diseases
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• v.48 no.6
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• pp.853-869
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• 2000

Background : Following several decades of decline, the incidence of tuberculosis has recent1y begun to increase in many countries of this the control of this disease has been impeded by the emergence of multi-drug resistant tuberculosis (MDR-TB). The development of rapid diagnostic methods and effective new drugs are needed to control MDR-TB. One of the new drugs for MDR-TB is rifabutin (RBU) which has been known to be effective in some patients with MDR-TB. A few reports showed that some types of mutations of the rpoB gene, which were known to be present in 96-98% of rifampicin-resistant M. tuberculosis, were associated with the rifampicin-resistant but RBU-susceptible phenotype. This study was performed to investigate the correlation between RBU susceptibility and the patterns of rpoB gene mutations in Korean MDR-TB. Methods : Sixty-five clinical isolates of multi-drug resistant Mycobacterium tuberculosis, gathered from patients who visited the Asan Medical Center from July 1997 to June 1999, were investigated. Clinical responses to rifabutin-containing regimen were evaluated. An RBU susceptibility test and sequencing analysis of rpoB gene were performed, and the results were analyzed to confirm which mutations correlated with RBU-susceptible MDR-TB. Results : Fifty-three of 56 (95%) clinical isolates of MDR-TB had 60 mutations of the rpoB gene. The most frequent mutations were found at codon 531 (43%), and two mutations were combined in seven clinical isolates. Five of 53 (10%) clinical isolates showed the RBU-susceptible phenotype, and in them the characteristic patterns of point mutations were found at codon 509, 516, and 526. Conclusion : The frequency and pattern of mutations of the rpoB gene of Korean MDR-TB isolates were similar to those in western countries, where the prevalence of tuberculosis is low, but some show RBU-susceptible phenotypes. RBU-susceptible MDR-TB isolates showed the characteristic pattern of mutations of the rpoB gene which could be used to rapidly diagnose RBU susceptibility.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.62 no.1
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• pp.1-8
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• 2017

We investigated the effect of silicate coating of rice seeds on bakanae disease incidence and the quality of seedlings raised in seedling boxes and transplanted into pots. The silicate-coated rice seed (SCS) was prepared as follows. Naturally infested rice seeds not previously subjected to any fungicidal treatment were dressed with a mixture of 25% silicic acid at pH 11 and 300-mesh zeolite powder at a ratio of 50 g dry seed - 9 mL silicic acid - 25 g zeolite powder. The following nursery conditions were provided : Early sowing, dense seeding in a glass house with mulching overnight and no artificial heating, which were the ideal conditions for determining the effect on the seed. The nursery plants were evaluated for Gibberella. fujikuroi infection or to determine the recovery to normal growth of infected nursery plants in the Wagner pot. Seedlings emerged 2-3 days earlier for the SCS than they did for the non-SCS control, while damping-off and bakanae disease incidence were remarkably reduced. Specifically, bakanae disease incidence in the SCS was limited to only 7.8% for 80 days after sowing, as compared to 91.6% of the non-SCS control. For the 45-days-old SCS nursery seedlings, the fresh weight was increased by 11% and was two times heavier, with only mild damage compared to that observed for non-SCS. Even after transplanting, SCS treatment contributed to a lower incidence of further infections and possibly to recovery of the seedlings to normal growth as compared to that observed in symptomatic plants in the pot. The active pathogenic macro-conidia and micro-conidia were considerably lower in the soil, root, and seedling sheath base of the SCS. In particular, the underdeveloped macro-conidia with straight oblong shape without intact septum were isolated in the SCS ; this phenotype is likely to be at a comparative etiological disadvantage when compared to that of typical active macro-conidia, which are slightly sickle-shaped with 3-7 intact septa. A active intact conidia with high inoculum potential were rarely observed in the tissue of the seedlings treated only in the SCS. We propose that promising result was likely achieved via inhibition of the development of intact pathogenic conidia, in concert with the aerobic, acidic conditions induced by the physiochemical characteristics associated with the air porosity of zeolite, alkalinity of silicate and the seed husk as a carbon source. In addition, the resistance of the healthy plants to pathogenic conidia was also important factor.

• Tuberculosis and Respiratory Diseases
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• v.65 no.6
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• pp.476-486
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• 2008

Background: TRAIL (TNF-related apoptosis inducing ligand) is a newly identified member of the TNF gene family which appears to have tumor-selective cytotoxicity due to the distinct decoy receptor system. TRAIL has direct access to caspase machinery and induces apoptosis regardless of p53 phenotype. Therefore, TRAIL has a therapeutic potential in lung cancer which frequently harbors p53 mutation in more than 50% of cases. However, it was shown that TRAIL also could activates $NF-{\kappa}B$ in some cell lines which might inhibit TRAIL-induced apoptosis. This study was designed to investigate whether TRAIL can activate $NF-{\kappa}B$ in lung cancer cell lines relatively resistant to TRAIL-induced apoptosis and inhibition of $NF-{\kappa}B$ activation using proteasome inhibitor MG132 which blocks $I{\kappa}B{\alpha}$ degradation can sensitize lung cancer cells to TRAIL-induced apoptosis. Methods: A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells were used and cell viability test was done by MTT assay. Apoptosis was confirmed with Annexin V assay followed by FACS analysis. To study $NF-{\kappa}B$-dependent transcriptional activation, a luciferase reporter gene assay was used after making A549 and NCI-H1299 cells stably transfected with IgG ${\kappa}-NF-{\kappa}B$ luciferase construct. To investigate DNA binding of $NF-{\kappa}B$ activated by TRAIL, electromobility shift assay was used and supershift assay was done using anti-p65 antibody. Western blot was done for the study of $I{\kappa}B{\alpha}$ degradation. Results: A549 and NCI-H1299 cells were relatively resistant to TRAIL-induced apoptosis showing only 20~30% cell death even at the concentration 100 ng/ml, but MG132 ($3{\mu}M$) pre-treatment 1 hour prior to TRAIL addition greatly increased cell death more than 80%. Luciferase assay showed TRAIL-induced $NF-{\kappa}B$ transcriptional activity in both cell lines. Electromobility shift assay demonstrated DNA binding complex of $NF-{\kappa}B$ activated by TRAIL and supershift with p65 antibody. $I{\kappa}B{\alpha}$ degradation was proven by western blot. MG132 completely blocked both TRAIL-induced $NF-{\kappa}B$ dependent luciferase activity and DNA binding of $NF-{\kappa}B$. Conclusion: This results suggest that inhibition of $NF-{\kappa}B$ can be a potentially useful strategy to enhance TRAIL-induced tumor cell killing in lung cancer.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.65 no.4
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• pp.406-415
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• 2020

Maize (Zea mays L.) is one of the most valuable agricultural crops and is grown under a wide spectrum of environmental conditions. However, maize is moderately sensitive to salt stress, and soil salinity is a serious threat to its production worldwide. In this study, we used ethyl methane sulfonate (EMS) to generate salt-tolerant silage maize mutants. We screened salt-tolerant lines from 203 M₃ mutant populations by evaluating the morphological phenotype after salt stress treatment and selected the 140ES91 line. The 140ES91 mutant showed improved plant growth as well as higher proline content and leaf photosynthetic capacity compared with those of wild-type plants under salt stress conditions. Using whole-genome re-sequencing analysis, 1,103 single nucleotide polymorphisms and 71 insertions or deletions were identified as common variants between KS140 and 140ES91 in comparison with the reference genome B73. Furthermore, the expression patterns of three genes, which are involved in salt stress responses, were increased in the 140ES91 mutant under salt stress. Taken together, the mutant line identified in our study could be used as an improved breeding material for transferring salt tolerance traits in maize varieties.