• Korean Journal of Veterinary Service
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• v.39 no.2
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• pp.107-116
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• 2016

In the study, we developed and evaluated a uracil N-glycosylase (UNG)-supplemented single-tube nested reverse transcription-polymerase chain reaction (UsnRT-PCR) assay that can carried out first-round RT-PCR and second-round nested PCR in a reaction tube without reaction tube opening and can simultaneously detect EU- and NA-PRRSV. The UsnRT-PCR confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PRRSV DNA from previous UsnRT-PCR. Primer specificities were evaluated with RNAs extracted from 8 viral strains and our results revealed that the primers had a high specificity for both genotypes of PRRSV. The sensitivity of the UsnRT-PCR was 0.1 $TCID_{50}$/0.1 mL for EU- or NA-PRRSV, respectively, which is comparable to that of previously reported real time RT-PCR (RRT-PCR). Clinical evaluation on 110 field samples (60 sera and 50 lung tissues) by the UsnRT-PCR and the RRT-PCR showed that detection rates of the UsnRT-PCR was 70% (77/110), and was relatively higher than that of the RRT-PCR (69.1%, 76/110). The percent positive or negative agreement of the UsnRT-PCR compared to RRT-PCR was 96.1% (73/76) or 90.9% (30/33), showing that the test results of both assays may be different for some clinical samples. Therefore, it is recommend that diagnostic laboratory workers use the two diagnostic assays for the correct diagnosis for the relevant samples in the swine disease diagnostic laboratories. In conclusion, the UsnRT-PCR assay can be applied for the rapid, and reliable diagnosis of PRRSV without concerns about preamplified DNA carryover contamination that can occurred in PCR process in the swine disease diagnostic laboratories.

• Journal of Life Science
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• v.15 no.3 s.70
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• pp.317-322
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• 2005

The cycloinulooligosaccharide fructanotransferase (CFTase) gene (cft) from Paenibacillus macerans was subcloned into an E. coli-yeast shuttle vector, pYES2.0, resulting in pYGECFTN. The plasmid pYGECFTN (8.6 kb) was introduced into Saccharomyces cerevisiae SEY2102 cells and then the transformants were selected on the synthetic defined media lacking uracil. The cft gene expression in yeast transformant was demonstrated by the analyses cyclofructan (CF) spots on thin-layer chromatogram. The recombinant CFTase was not secreted into the medium and localized in the periplasmic space. The production of CF was observed after 5 min of the enzymatic reaction with inulin. The optimun pH and temperature for CF production were found to be at pH 8.0 and $45^{\circ}C$, respectively. Enzyme activity was stably maintained up to $55^{\circ}C$. The CF was produced from all inulin sources and was most efficiently produced from dahlia tubers and Jerusalem artichokes.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.241-247
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• 2007

The cycloinulooligosaccharide fructanotransferase (CFTase) gene (cft) from Paenibacillus macerans was subcloned into the surface display vector, pCTcon (GAL1 promoter). The constructed plasmid, pCTECFTN (9.0 kb) was introduced to S. cerevisiae EBY100 cell and then east transformants were selected on the synthetic defined medium lacking uracil and on the inulin containing medium. The surface display of CFTase was confirmed by immunofluorescence microscopy and its enzymatic ability to form cycloinulooligosaccharides(cyclofructans, CFs) from inulin. The total activity of the CFTase was reached about 5.52 unit/1 by cultivation of yeast transformant on YPDG medium. The optimized conditions determined were as follows; pH, 8.0; temperature, $50^{\circ}C$ ; substrate concentration, 5%; inulin source, Jerusalem artichoke. By the reaction with inulin, CFs consisting of cycloinulohexaose (CF6), cycloinuloheptaose (CF7), and cycloinulooctaose (CF8) were produced and CF6 was the major product.

• Korean Journal of Veterinary Service
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• v.37 no.4
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• pp.263-271
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• 2014

In this study, we developed a cost and time saving one-step multiplex RT-PCR for the simultaneous detection and differentiation of swine influenza viruses (SIV) and 2009 pandemic influenza H1N1 virus (pH1N1). The one-step multiplex RT-PCR using four sets of primer was confirmed to be capable of detection of all SIV subtypes and differential diagnosis of major SIV subtype H1, H3 and pH1N1 on individual or mixed viral culture samples. The sensitivity of the multiplex RT-PCR was determined to be at least $2^{-6}$ $HA/25{\mu}L$ of the presented SIVs, providing sufficient efficacy for a routine SIV monitoring in diagnostic laboratories. In addition, compared with the conventional RT-PCR methods that cannot avoid the carryover DNA contamination, the developed RT-PCR applied with the uracil DNA glycosylase (UNG) system was proven to prevent a false positive reaction by carryover contamination of the pre-amplified DNA. In conclusion, the one-step RT-PCR with UNG system could be applicable to detect and differentiate of SIV from the viral cultures without worry of carryover DNA contamination in clinical laboratories.

• Journal of Life Science
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• v.29 no.3
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• pp.376-381
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• 2019

To construct useful yeast strain for bioethanol production, we improved yeast harboring various phenotypes by using yeast protoplast fusion method. In this study, S. cerevisiae BYK-F11 strain which have ethanol tolerance, thermotolerance and ${\beta}-glucanase$ activity and P. $stipitis{\Delta}ura$ strain which has xylose metabolism pathway were fused by genome shuffling. P. $stipitis{\Delta}ura$ strain was constructed for protoplast fusion by URA3 gene disruption, resulting in uracil auxotroph. By protoplast fusion, several fused cells were selected and BYKPS-F8 strain (fused cell) showing both karyotypes from two parent strains (S. cerevisiae BYK-F11 and P. $stipitis{\Delta}ura$ strain) among 22 fused cells was finally selected. Sequentially, various phenotypes such as ${\beta}-glucanase$ activity, xylose utility, ethanol tolerance, thermotolerance and ethanol productivity were analyzed. The BYKPS-F8 strain obtained ${\beta}-glucanase$ activity from BYK-F11 strain and 1.2 fold increased xylose utility from P. $stipitis{\Delta}ura$ strain. Also, the BYKPS-F8 strain showed thermotolerance at $40^{\circ}C$ and increased ethanol tolerance in medium containing 8% ethanol. In this fused cell, 7.5 g/l ethanol from 20 g/l xylose was produced and the multiple phenotypes were stably remained during long term cultivation (260 hr). It was proved that novel biological system (yeast strains) is easily and efficiently bred by protoplast fusion among yeasts having different genus.

• Journal of Life Science
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• v.11 no.6
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• pp.517-522
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• 2001

To Confirm the repression of off-odor and improvement of food quality in small black bean chungkugjang (SBC), some taste compounds of SBC added with kiwi and radish homogenate and fermented fro 3 days at 42$^{\circ}C$ were investigated. Contents of free amino acids in SBC were lower than that of soybean chungkugjang (SC) and were increased by adding kiwi and radish homogenate to black bean, indicating that two materials were effective to the enzymatic digestibility of soy protein. Of organic acids, citric acid was the most abundant, followed by acetic acid and lactic acid. Fatty acid composition was high in the order of linoleic acid (50.82~54.51%), oleic acid (17.76%~22.10%) and plamitic acid(12.13~13.79%). There were no significant difference in compositions of organic acids and fatty acids of chungkugjang. Major volatile compounds of SBC were indole , 2-5-dimethyl pyrazine and trimethyl pyrazine. Contents of alkyl pyrazines that contribute the characteristic aroma and flavor of SBC added and fermented with kiwi and radish homogenate were 3 times lower than those of SC. Uracil and UMP were major nucleic acie-drlated compounds in all four chungkugjangs. Contents of the other nucleic acid-related compounds were a similar trend in all chungkugjangs.

• Archives of Pharmacal Research
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• v.25 no.6
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• pp.824-830
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• 2002

Four flavonoid glycosides, flavaprin (7), evodioside B (8), vitexin (11), and hesperidin (12), as well as the coumarins bergapten (1), xanthotoxin (2), and isopimpinellin (3), the lignan simplexoside (10), the steroids ${\beta}-sitosterol$ (4) and daucosterol (5), the limonoids isolimonexic acid (6) and limonin (9), and uracil (13) and myo-inositol (14) have been isolated from Euodia daniellii. The structures of these compounds were established from spectral data. Among the isolates, bergapten showed cyclooxygenase-2 inhibitory activity with an $IC_{50}$ value of $6.2{\;}{\mu\textrm{g}}/ml. Flavonoids isolated from this plant exhibited no cytotoxic activity against the human tumor cell lines, A549, SKOV-3, SKMEL-2, XF498, and HCT15.

• Journal of the Korean Chemical Society
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• v.45 no.4
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• pp.312-317
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• 2001

7'-Aldehyde-nucleosides analogue (2a, 2c) were synthesized from 6-N-benzoyl-2',3'-O-isopropylideneadenosine and uridine. The condensation of 2 with ethoxycarbonylmethylene Wittig reagent produced the adenosine and uridine analogues containing extended conjugated diene and ethoxycarbonyl group, ethyl-1',5',6',7',8'-pentadeoxy-1'-(adenin-9-yl)-$\beta$-D-ribo-nona-5'(E),7'(E)-dienofuranuronate (4b) and ethyl-1'. 5',6',7',8'-pentadeoxy-1'-(uracil-1-yl)-$\beta$-D-ribo-nona-5'(E),7'-(E)-dienofuranuronate (4c). 9-[8',8'-dibromo-5',6',7',8' -tetradeoxy-$\beta$-D-ribo-octa-5'(E),7'(E)-diene]nucleosides (6b, 6c) were also prepared from 2 with similar method.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.3
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• pp.180-185
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• 2005

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. Amino acid residues located in or near the active sites of the intracellular cytosine deaminase from chromobacterium violaceum YK 391 were identified by chemical modification studies. The enzymic activity was completely inhibited by chemical modifiers, such as 1mM NBS, chloramine-T, $\rho-CMB,\;\rho-HMB$ and iodine, and was strongly inhibited by 1mM PMSF and pyridoxal 5'-phosphate. This chemical deactivation of the enzymic activity was reversed by a high concentration of cytosine. Furthermore, the deactivation of the enzymic activity by $\rho-CMB$ was also reversed by 1mM cysteine-HCI, DTT and 2-mercaptoethanol. These results suggested that cysteine, tryptophan and methionine residues might be located in or near the active sites of the enzyme, while serine and lysine were indirectly involved in the enzymic activity. The intracellular cytosine deaminase from C violaceum YK 391 was assumed to be a thiol enzyme.

• Korean Journal of Head & Neck Oncology
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• v.10 no.1
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• pp.74-79
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• 1994

A Phase II study of UFT which is a mixture of Tegafur and Uracil was conducted in two institutions during past two years. Ninty-four patients of head and neck squamous cell carcinoma entered this trial, of which sixty-eight were evaluated. Among those, thirty-six cases were previously untreated and thirty-two cases were recurrent UFT was administrated orally at a daily dose of $400mg/m^2$ for eight weeks. The results were as following: 1) Overall response was 30.88%, but for 38.36% for 36 cases of the untreated cases, 21.88% for 32 cases of recurrent cases. 2) UFT was more effective in early stage and well differentiated squamous cell carcinoma and UFT tended to reduce the tumor size maximally at fourth or fifth week 3) There was no serious side effects except mild gastrointestinal disturbances such as nausea and vomiting, which were recovered immediately after stop or reducing a daily dose. Therefore, UFT therapy is clinically effective for head and neck squamous cell carcinoma and also may be useful for combination or palliative chemotherapy because of mild side effects.