• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.584-592
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• 2004

A thermostable $\beta$-glycosidase gene, tfi $\beta$-gly, was cloned from the genomic library of Thermus filiformis Wai33 A1. ifi $\beta$-gly consists of 1,296 bp nucleotide sequence and encodes a polypeptide of 431 amino acids. It shares a strong amino acid sequence similarity with the $\beta$-glycosidases from other Thermus spp. belonging to the glycosyl hydrolase family 1. In the present study, the enzyme was overexpressed in Escherichia coli BL21 (DE3) using the pET21b(+) vector system. The recombinant enzyme was purified to homogeneity by heat treatment and a $Ni^{2+}$-affinity chromatography. Polyacrylamide gel electrophoresis (PAGE) showed that the recombinant Tfi $\beta$-glycosidase was a monomeric form with molecular mass of 49 kDa. The temperature and pH range for optimal activity of the purified enzyme were 80- $90^{\circ}C$ and 5.0-6.0, respectively. Ninety-three percent of the enzyme activity was remained at $70^{\circ}C$ after 12 h, and its half-life at $80^{\circ}C$ was 6 h, indicating that Tfi $\beta$-glycosidase is highly thermostable. Based on its K_m$, or $K_{cat}K_m$, ratio, Tfi $\beta$-glycosidase appeared to have higher affinity for $\beta$-D-glucoside than for $\beta$-D-galactoside, however, $K_{cat} for \beta$-D-galactoside was much higher than that for $\beta$-D-glucoside. The activity for lactose hydrolysis was proportionally increased at $70^{\circ}C$ and pH 7.0 without substrate inhibition until reaching 250 mM lactose concentration. The specific activity of Tfi TEX>$\beta$-glycosidase on 138 mM lactose at $70{^\circ}C$ and pH 7.0 was 134.9 U/mg. Consequently, this newly cloned enzyme appears to have a valuable advantage of conducting biotechnological processes at elevated temperature during milk pasteurization in the production of low-lactose milk.

• Korean Journal of Clinical Pharmacy
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• v.26 no.4
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• pp.298-305
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• 2016

Background: This study is to investigate the prescription patterns and factors related to the number of medications treating chronic obstructive pulmonary disease (COPD) in patients under 65 years old according to GOLD guidelines. Methods: We retrospectively analyzed the medical records of patients aged 40-64 years with a diagnosis of COPD from January to March 2016. Patients were classified by combined assessment of COPD (grades A, B, C, D) using spirometry, exacerbation history, mMRC, and/or CAT results. We analyzed prescribed medications, treatment options and factors related to the numbers of COPD medications. Results: The total number of prescriptions were 251. About 35.5% of patients were classified as GOLD A, 34.2% as GOLD B, 17.1% as GOLD C and 13.2% as GOLD D. Inhaled bronchodilator was prescribed for 86.9% of patients and the most frequent COPD medication was long-acting muscarinic antagonist (LAMA) followed by inhaled corticosteroids/long acting beta agonist (ICS/LABA). The majority of low risk patients (GOLD A/B) were prescribed a monotherapy with LAMA or LABA. For high risk patients (GOLD C/D), combination treatment with ICS+LAMA+LABA was mostly prescribed. The 21.2% of patients in GOLD D received systemic corticosteroid. The average number of medications per prescription was 3.7, and this number increased with increasing COPD grade, COPD duration and lung function reduction ($FEV_1$, $FEV_1/FVC$). Conclusion: Generally high adherence to GOLD guideline recommendations was reported. Given the progressive nature of the disease, results suggest that closer attention to respiratory symptoms for early detection, diagnosis, and appropriate treatment of COPD is warranted.

• Journal of Life Science
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• v.20 no.2
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• pp.281-291
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• 2010

We investigated the antioxidant effects of hederagenin 3-O-b-D-glucopyranosyl($1{\rightarrow}3$)-a-L-rhamnopyranosyl($1{\rightarrow}2$)-a-L-arabinopyranoside (HDL) isolated from root bark of Ulmus davidiana on the activity of enzymes related to reactive oxygen species (ROS) in human osteosarcoma U2OS cells. Cobalt chloride ($CoCl_2$), a transition metal, was used as an inducer of oxidative stress, generating hydrogen peroxide ($H_2O_2$) via increasing xanthine oxidase (XO) activity. The increased levels of $H_2O_2$, XO, ferritin, and ferritin iron by $CoCl_2$ were diminished effectively by co-treatment with HDL in U2OS cells. Furthermore, decreased levels of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) by $CoCl_2$ were highly increased by co-treatment with HDL in U2OS cells; however, the levels of glutathione peroxidase (GPx) did not change. The increased contents of TBARS related to lipid peroxidation were significantly reduced by HDL in U2OS cells. The concentration of GSH changed in a pattern that went against regulated TBARS by $CoCl_2$ and HDL. We examined the expression of p53, $p21^{CIP1/WAF1}$, and $p27^{KIP1}$ proteins related to oxidative stress and cell cycle regulation. As a result, the expression of $p27^{KIP1}$ modulated by $CoCl_2$ was not changed by HDL. However, the expression of p53 and $p21^{CIP1/WAF}$ increased by $CoCl_2$ was reduced by HDL in U2OS cells. Together with alteration of p53 and $p21^{CIP1/WAF1}$ proteins, the accumulated cells at G1 phase by $CoCl_2$ was decreased by HDL in U2OS cells. Our data suggests that HDL inhibits $CoCl_2$-generated ROS in U2OS cells, providing potentially new antioxidant compounds that are isolated from natural products.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.238-243
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• 2000

An exoinulinase (${\beta}-D-fructofuranosidase$) gene was cloned by chromosome walking along the upstream region of the endoinulinase gene of Pseudomonas mucidolens isolated from soil. the exoinulinase gene consisted of an ORF of 0,506 bp encoding a polypeptide of 501 amino acids with a deduced molecular weight of 55,000. The exoinulinase produced by the recombinant Escherichia coli $DH5{\alpha}$ strain was also purified to homogeneity as determined by SDS-PAGE and a zymogram. The molecular weight of the purified exoinulinase according to both SDS-PAGE and gel filtration matched the deduced molecular weight of the protein described above, thereby indicating that the native form of the exoinulinase was a monomer. The purified enzyme hydrolyzed activity value of 2.0. Furthermore, no inulo-oligomers were liberated from the inulin substrate in the enzymatic reaction mixtures incubated for 90 min at $55^{\circ}C$. Taken together, these results indicate that the purified ${\beta}-D-fructofuranosidase$ was an exoinulinase. The pH and temperature optima of the exoinulinase were pH 6.0 and $55^{\circ}C$, respectively. the enzymehad no apparent requirement for a cofactor, and its activity was completely inactivated by $Ag^{+},{\;}Hg^{2+},{\;}and{\;}Zn^{2+}$. Kinetic experiments gave $K_m,{\;}V_{max},{\;}and{\;}K_{cat}$ values for inulin of 11.5 mM, 18 nM/s, and $72{\;}s^{-1}$, respectively. the exoinulinase was fairly stable in broad pH conditions (pH 5-9), and at pH 6.0 it showed a residual activity of about 70% after 4 h incubation at $55^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.347-355
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• 2016

An exo-β-D-glucosaminidase (AorCsxA) from Aspergillus oryzae FL402 was heterologously expressed and purified. The deduced amino acid sequence indicated that AorCsxA belonged to glycoside hydrolase family 2. AorCsxA digested colloid chitosan into glucosamine but not into chitosan oligosaccharides, demonstrating exo-β-D-glucosaminidase (CsxA) activity. AorCsxA exhibited optimal activity at pH 5.5 and 50℃; however, the enzyme expressed in Pichia pastoris (PpAorCsxA) showed much stronger thermostability at 50℃ than that expressed in Escherichia coli (EcAorCsxA), which may be related to glycosylation. AorCsxA activity was inhibited by EDTA and most of the tested metal ions. A single amino acid mutation (F769W) in AorCsxA significantly enhanced the specific activity and hydrolysis velocity as revealed by comparison of V_max and k_cat values with those of the wild-type enzyme. The three-dimensional structure suggested the tightened pocket at the active site of F769W enabled efficient substrate binding. The AorCsxA gene was heterologously expressed in P. pastoris, and one transformant was found to produce 222 U/ml activity during the high-cell-density fermentation. This AorCsxA-overexpressing P. pastoris strain is feasible for large-scale production of AorCsxA.

• Bulletin of the Korean Chemical Society
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• v.24 no.11
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• pp.1623-1626
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• 2003

The dependency of the rate of $CO_2$ reforming of methane on the catalyst loading and the reactor size was examined at a fixed temperature of $750\;^{\circ}C$ and a fixed GHSV of 18000 mL(STP)/$g_{cat}.h$. The conversion of methane in $CO_2$reforming decreased with increase in the reactor size. The catalyst was severely deactivated with increase in the catalyst amount. The amount of carbonaceous species combustible below $550\;^{\circ}C$, determined by TPO experiments with the used catalyst samples increased with increase in the catalyst amount, which was again confirmed by XRD and TEM experiments. The increase of the carbonaceous species combustible below $550\;^{\circ}C$ may be due to the suppression of the reverse Boudouard reaction, since the $CO_2$ reforming of methane, a highly endothermic reaction, resulted in lowering the reaction temperature.

• Parasites, Hosts and Diseases
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• v.25 no.2
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• pp.141-148
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• 1987

Circulating antigens in rats experimentally infected with Paragonimus westermani were examined by ELISA. From a total of 22 albino rats, each fed with 25 metacercariae, blood samples were collected until 12 weeks after infection. The specific antibodies against P. westermani in the serum of an infected cat were purified by ammonium sulfate precipitation, DEAE anion-exchange chromatography and affinity chromatography serially. So-called double antibody sandwich ELISA method was used for the detection of circulating antigens. The results were as follows: Mean value of O.D. in control sera was O. 04 (S.D.=0. 04). After infection, mean O.D.(S.D.) values were changed serially: 0.03(0.01) at 0.5 week(3 days), 0.55(0.50) at 1 week, 0.69(0.45) at 1.5 week, O.20 (0.19) at 2 weeks and O.13(0.10) at 2.5 weeks of infection. They returned, thereafter, to the level before infection. When O. 16 (mean+3 S.D.) were considered as cut-off value, those higher than O. 16 were observed only in the sera collected between 1 and 2.5 weeks after infection. Average 8. 4 immature worms (2.2 from the lungs and pleural cavities; 6.2 from muscles) were recovered in a rat at 12 weeks after infection. The fact that circulating antigens were not detected after 3 weeks of infection was considered to the caused by the formation of antigen-antibady complexs.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.1
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• pp.81-87
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• 2013

Quercetin-3-O-${\beta}$-D-glucuronopyranoside (QGC) is a flavonoid glucoside extracted from Rumex Aquaticus Herba. In the present study, anti-oxidative and anti-inflammatory effects of QGC were tested in vitro. Epithelial cells obtained from cat esophagus were cultured. When the cells were exposed to acid for 2 h, cell viability was decreased to 36%. Pretreatment with 50 ${\mu}M$ QGC for 2 h prevented the reduction in cell viability. QGC also inhibited the productions of intracellular ROS by inflammatory inducers such as acid, lipopolysaccharide, indomethacin and ethanol. QGC significantly increased the activities of superoxide dismutase (SOD) and catalase, and also induced the expression of SOD2, while it restored the decrease of catalase expression in cells exposed to acid. QGC inhibited NF-${\kappa}B$ translocation, cyclooxygenase-2 expression and $PGE_2$ secretion in cells exposed to acid, which plays an important role in the pathogenesis of esophagitis. The data suggest that QGC may well be one of the promising substances to attenuate oxidative epithelial cell injury and inflammatory signaling in esophagus inflammation.

• Bulletin of the Korean Chemical Society
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• v.26 no.2
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• pp.260-264
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• 2005

The nature of the active site of Tobacco acetohydroxyacid synthase (AHAS) in the substrate- and cofactorbinding was studied by kinetics and fluorescence spectroscopy. The substrate saturation curve does not follow Michaelis-Menten kinetics at different temperatures (7, 21 and 37 ${^{\circ}C}$), pH (6.5, 7.5 and 8.5) and buffers (Tris-HCl and MOPS). The concentration of one half of the maximum velocity ($S_{0.5}$) decreased in the following order: pyruvate $\gt$ ThDP $\approx$$Mg^{+2}$ $\gt$ FAD. However, the catalytic efficiency (K$_{cat}/S_{0.5}$) inversely decreased in the following order; FAD $\gt$ $Mg^{+2}$ $\approx$ThDP $\gt$ pyruvate, indicating that the cofactors by in decreasing order; FAD, $Mg^{+2}$, ThDP, affect the catalysis of AHAS. The dissociation constant ($K_d$) of the intrinsic tryptophan fluorescence decreased with the same tendency of the concentration of one half of the maximum velocity ($S_{0.5}$) decreasing order. This data provides evidence that the substrate and cofactor binding natures of the active site, as well as its activation characteristics, resemble those of other ThDP-dependent enzymes.

• Parasites, Hosts and Diseases
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• v.52 no.6
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• pp.691-694
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• 2014

The purpose of this study was to conduct a survey of Dirofilaria immitis infection among stray cats in Korea using nested PCR. We included 235 stray cats (121 females and 114 males) and evaluated each for the presence of feline heartworm infection. Blood samples were collected from 135 cats in Daejeon, 50 cats in Seoul, and 50 cats from Gyeonggi-do (Province). Of the 235 DNA samples, 14 (6.0%) were positive for D. immitis. The prevalence of infection in male cats (8/114, 7.0%) tended to be higher than that in female cats (6/121, 5.0%), but the difference was not statistically significant. In each location, 8, 2, and 4 cats were positive for infection, respectively, based on DNA testing. No significant differences in the prevalence were observed among the geographic regions, although the rate of infection was higher in Gyeonggi-do (8.0%) than Daejeon (5.9%) and Seoul (4.0%). We submitted 7 of the 14 D. immitis DNA-positive samples for sequencing analysis. All samples corresponded to partial D. immitis cytochrome c oxidase subunit I gene sequences with 99% homology to the D. immitis sequence deposited in GenBank (accession no. FN391553). To the best of our knowledge, this is the first survey using nested PCR to analyze the prevalence of D. immitis in stray cats in Korea.