• Korean Journal of Pharmacognosy
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• v.48 no.3
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• pp.179-186
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• 2017

Pyrrosia lingua which belongs to Polypodiaceae has been used as a traditional medicine for the treatment of urinary system inflammation, urination disorder, and bronchitis. However, there are not enough phytochemical and pharmacological studies of P. lingua up to now. Here in this study, the protective effect of MeOH extract of whole plant of Pyrrosia lingua (MPL) against 2% glucose-induced toxicity was investigated using Caenorhabditis elegans (C. elegans) model system. We found that MPL significantly extended the lifespan of wild-type nematode under normal culture condition. MPL also effectively recovered the decreased lifespan caused by 2% glucose-toxicity. In addition, MPL efficiently attenuated the increased glucose concentration inside of nematode. Further studies evaluating diabetes-related factors revealed that MPL reduced both intracellular ROS and lipid accumulation which were up-regulated under 2% glucose supplement condition. Our data also showed that MPL improved the 2% glucose-induced shortened body movement of nematode. Lastly, we carried out genetic studies using several single gene knockout mutants to establish the possible target of MPL. Our results demonstrated that genes such as daf-2 and daf-16 were responsible for the protective activity of MPL against 2% glucose-induced toxicity. These results indicate that MPL exerts protective action against 2% glucose via regulation of insulin/IGF-1 sinaling pathway and FOXO activation.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.267-273
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• 2005

To develop effective and powerful probiotic, Saccharomyces cerevisiae strains producing cellulolytic enzymes were genetically brooded. For the production of exoglucanase, the plasmid pVT-TExo (8.8 kb) was constructed, in which Thermomonosporafusca exoglucanase gene (E3) was under the control of ADHl promoter, and introduced into S. cerevisiae SEY2102. When the transformant, S. cerevisiae SEY2102/pVT-TExo, was cultivated on YPD medium, the total expression level of avicelase reached about 190 unit/l. The secretion efficiency and plasmid stability were about $50\%\;and\;91\%$, respectively. Recombination exoglucanase enzyme bound to avicel better than Clostridium endoglucanase (CelA) and Trichoderma endoglucanase (C4) enzymes. The mixing ratio of E3 and CelA displaying the best synergistic hydrolysis for avicel was observed at 4:1. The mixture of endoglucanase (CelA) and exoglucanase (E3) resulted in 3.2-fold increase of avicelase activity and 2.5-fold enhanced production of sugar production from avicel, compared to the single enzyme treatment.

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.436-439
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• 1989

The chromosomal DNA fragments of thermophilic alkalophilic Bacillus sp, K-17, a potent xylanhydrolyzing bacterium, were ligated to a vector plasmid pBR322 and transformed into Escherichia coli HB101. The plasmid pAX278, isolated from a transformant forming yellow color on the LB agar plate containing 1 mM p-nitrophenyl- $\beta$-xylopyranoside, was found to enable the transformants to produce p-xylosidase. The 5.0 kilobase insert of pAX278 had single sites for EcoRI, PstI, XbaI, and PvuII, and 2 sites for BglII. Biotinylated pAX218 was hybridized to 0.9 kb as well as 5.0 kb fragment from Bacillus sp. K-17 DNA on nitrocellulose filter. pGX718 was constructed by inserting the 5.0 kb HindIII fragment of pGX278 at the HindIII site of pGR71, E. coli and B. subtilis shuttle vector. The enzymatic properties of $\beta$-xylosidase from E. coli HB101 carrying recombinant plasmid were the same those of $\beta$-xylosidase from Bacillus sp. K-17.

• Journal of Microbiology and Biotechnology
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• v.26 no.10
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• pp.1708-1716
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• 2016

Glucose dehydrogenase (GDH) is an oxidoreductase enzyme and is used as a biocatalyst to regenerate NAD(P)H in reductase-mediated chiral synthesis reactions. In this study, the glucose 1-dehydrogenase B gene (gdhB) was cloned from Bacillus thuringiensis subsp. kurstaki, and wild-type (GDH-BT^WT) and His-tagged (GDH-BT^N-His, GDH-BT^C-His) enzymes were produced in Escherichia coli BL21 (DE3). All enzymes were produced in the soluble forms from E. coli. GDH-BT^WT and GDH-BT^N-His showed high specific enzymatic activities of 6.6 U/mg and 5.5 U/mg, respectively, whereas GDH-BT^C-His showed a very low specific enzymatic activity of 0.020 U/mg. These results suggest that the intact C-terminal carboxyl group is important for GDH-BT activity. GDH-BT^WT was stable up to 65℃, whereas GDH-BT^N-His and GDH-BT^C-His were stable up to 45℃. Gel permeation chromatography showed that GDH-BT^WT is a dimer, whereas GDH-BT^N-His and GDH-BT^C-His are monomeric. These results suggest that the intact N- and C-termini are required for GDH-BT to maintain thermostability and to form its dimer structure. The homology model of the GDH-BT^WT single subunit was constructed based on the crystal structure of Bacillus megaterium GDH (PDB ID 3AY6), showing that GDH-BT^WT has a Rossmann fold structure with its N- and C-termini located on the subunit surface, which suggests that His-tagging affected the native dimer structure. GDH-BT^WT and GDH-BT^N-His regenerated NADPH in a yeast reductase-mediated chiral synthesis reaction, suggesting that these enzymes can be used as catalysts in fine-chemical and pharmaceutical industries.

• Journal of Korean Society of Environmental Engineers
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• v.29 no.8
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• pp.904-908
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• 2007

The protozoan pathogen Giardia lamblia has been major cause of waterborne enteric disease. In this study, we tried to identify G. lamlbia of human infectious species and to detect viable C. lamblia in river water samples including three sites of Han River mainstream and an its creek using PCR and RT-PCR technique. The PCR/RT-PCR methods were performed by using giardin primer based on the giardin gene targeting ventral disk of Giardia. Sensitivity testing in the DNA/RNA extraction and PCR/RT-PCR amplification steps showed that it was possible to detect a single cyst of G. lamblia and viable G. lamblia. The PCR/RT-PCR methods were compared with immunofluorescence(IF) assay by analyzing 48 samples collected from the mainstream water and the creek water. The mean concentration of the total cysts were 6.3 cysts/10 L(arithmetic mean, n = 48) and the positive detection rate were 62.5%(30/48). And the mean concentration of the cysts excluding empty cysts were 4.5 cysts/10 L and the positive detection rate were 52.1%(25/48). It resulted that 24 of 48 samples included Giardia lamblia by PCR assay and 10 of 48 samples included viable G. lamblia by RT-PCR assay. It resulted that the PCR/RT-PCR technique would be available to river water samples with low concentration of Giardia cysts. And it could support the Korean protozoan standard method, which provides useful information for species and viability.

• Applied Biological Chemistry
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• v.48 no.4
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• pp.345-351
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• 2005

To access the natural products of uncultured microorganisms, we constructed and screened the metagenomic DNA libraries by using a cosmid vector and DNA inserts isolated directly from soil. Initial screening of the libraries in Escherichia coli resulted in the isolation of several clones that produce a dark brown color when grown in LB medium. One of the positive clones, designed pYS85C, was transposon mutagenized and the DNA surrounding the transposon insertions in cosmids that no longer conferred the production of brown pigment to E. coli was sequenced. Annotation of the pYS85C sequence obtained from the transposon mutagenesis experiment indicated a single 393 amino acid open reading frame (ORF) with a molecular mass of about 44.5 kDa, predicted to be a 4-hydroxyphenylpyruvate dioxygenases (HPPDs), was responsible for the observed brown pigment. In a BLAST search against deposited sequence, the translated protein from this ORF showed moderate-level identity (>60%) to the other known HPPDs and was most conserved in the C-terminal region of the protein. These results show that genes involved in natural product synthesis can be cloned directly from soil DNA and expressed in a heterologous host, supporting the idea that this technology has the potential to provide novel natural products from the wealth of environmental microbial diversity and is a potentially important new tool for drug discovery.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.9
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• pp.1236-1243
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• 2010

The aim of this study was to screen lactic acid bacteria for the fermentation of garlic and to assess the increase in inhibitory activity of garlic fermented against antibiotic-resistant pathogens for use as an animal feed supplement. We screened 45 strains of lactobacillus for the fermentation of garlic. Of these strains, 23 showed similar growth rates with or without allicin. Cultures of the 23 strains were mixed with an equivalent amount of garlic juice and incubated overnight at $37^{\circ}C$. The three strains with the lowest pH values were Lactobacillus paracasei KCTC 3169, L5 strain, and L. reuteri SW. Garlic juice fermented by the L5 strain more strongly inhibited antibiotic-resistant pathogenic bacteria than L. paracasei KCTC 3169, L. reuteri SW, or garlic juice itself. By examining carbohydrate utilization, morphologic properties and 16S rRNA gene sequences, we identified the L5 strain as Pediococcus pentosaceus and deposited it in the name of P. pentosaceus KACC 91419 into the Korea Agricultural Culture Collection. To identify the antimicrobial compound from the garlic filtrate fermented by P. pentosaceus KACC 91419, we fractionated P. pentosaceus KACC 91419 culture on a C18 column and checked the antimicrobial activity of fractions A6 to A10. Only fraction A9 showed inhibitory activity on Staphylococcus aureus. Comparing the mass spectra of the fractions with and without antimicrobial activity, we observed a single dominant product ion (m/z 157.99) from the fraction showing antimicrobial activity. Its molecular mass (157.99) was 2 atomic mass units less than that of allicin (162.02). This suggests that allicin might be converted to its derivative, which has antimicrobial activity, during fermentation by P. pentosaceus KACC 91419.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.61-61
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• 2001

The main goal of this study was to produce transgenic piglets by the method of injection of sperm-mediated exogenous DNA. Spermatozoa (1$\times$106 sperm of final concentration) obtained from caudal epididymis were mixed with pBC1-hEPO (20 ng/${mu}ell$) or pcDNA3 LAC Z (20 ng/${mu}ell$), and followed by electroporation (500 V, 25 ㎌). Matured oocytes having the first polar body and dense cytoplasm were selected and centrifuged at 12,000g for 6 min. After sperm injection, the oocytes were activated electrically (1.7 ㎸/cm, 30 $\mu$ sec, single pulse) in 0.3 M mannitol solution. Eggs injected sperm were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air for 192 h. This study were comprised 3 experiments. Experiment 1 compared the developmental efficiencies between the sperm-injected oocytes (Group 1) and further activated electrically (Group 2). Experiment 2 compared the expression of pcDNA3 LAC Z in the embryos produced by Group 1 and Group 2. Finally, experiment 3 carried out transfer of embryos (1-8 cell stage) transfected with pBC1 -hEPO into surrogate recipients synchronized by injection of combination of PG600 with hCG. The rates of cleavage and development into blastocyst stage in Group 2 were significantly higher than those of Group 1 (71.3% and 28.1% vs. 43.3% and 10.3%, respectively, p<0.05). Thirty (24.2%) out of 124 embryos analyzed in Group 2 were positive by X-gal. Similarly, in Group 1, 16.3% (8/49) were positive. After transfer of 789 embryos to 7 recipient gilts, three out of them examined by ultrasound became pregnant. One recipient is in day 50 pregnancy. On day 54 of gestation, two were carried out uterotomy in order to confirm the pregnancy One had 7 and another had 2 fetuses. We conclude that injection of sperm-mediated gene transfer will be used as a valuable tool for the production of transgenic piglets.

• Fisheries and Aquatic Sciences
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• v.22 no.10
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• pp.21.1-21.7
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• 2019

Background: Lactococcus garvieae is one of the most important risk factors in the rainbow trout culture. Therefore, the purpose of this study was to identify and detect strains isolated from rainbow trout suspected of having Lactococcus garvieae using biochemical characteristics and PCR and determination of the degree of severity of isolated strains. Methods: In this study, the cause of lactococcosis in selected rainbow trout farms in Kohkilooieh and Boyerahmad province was assayed. Gram-positive and catalase-negative bacterial isolates were first obtained from selected trout fish farms using conventional biochemical tests and PCR assay. The 10-day LD₅₀ method (concentration causing 50% mortality in 10 days) was used to determine the severity of the isolated bacteria. Results: One bacterial isolate was detected from all sampled fish which confirmed as Lactococcus garvieae using a specific PCR assay based on the 16S rDNA gene by producing a single band of 1107 bp. Analysis of the rate of mortality showed that the 10-day LD₅₀ was 4.6 × 10⁵ CFU/fish. The results of this study showed that isolated bacteria had high severity for rainbow trout. The presence of bacteria in internal organs of suspected fish showed a severe systemic infection in challenged fish. Antibiogram assay also indicated that the isolated Lactococcus garvieae were resistant to some mostly used antibiotics in rainbow trout. Conclusions: According to current research, it can be concluded that the condition of lactococcosis in the studied area is not suitable, and despite the presence of disease, there is no proper action to control and prevent the disease. Unfortunately, isolated bacteria from the studied area have a very high severity compared to bacteria isolated from other regions of the country or other countries. Therefore, further investigation is needed to determine the cause of this difference and possibly in the design of the vaccine.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.627-632
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• 2007

FoxO1, FoxO3a and FoxO4 belong to the FoxO gene family, which play important roles in the PI3K/PKB pathway. In this study, we cloned the porcine FoxO1, FoxO3a and FoxO4 sequences and assigned them to SSC11p11-15, SSC1p13 and SSC xq13 using somatic cell hybrid panel (SCHP) and radiation hybrid panel (IMpRH). RT-PCR results showed that these three genes are expressed in multiple tissues. Sequencing of PCR products from different breeds identified a synonymous T/C polymorphism in exon 2 of FoxO3a. This FoxO3a single nucleotide polymorphism (SNP) can be detected by AvaII restriction enzyme. The allele frequencies of this SNP were investigated in Dahuabai, Meishan, Tongcheng, Yushan, Large White, and Duroc pigs. Association of the genotypes with growth and carcass traits showed that different genotypes of FoxO3a were associated with carcass length and backfat thickness between 6th and 7th ribs (BTR) and drip loss (p<0.05).