• Journal of Microbiology and Biotechnology
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• v.31 no.11
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• pp.1591-1600
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• 2021

Streptomyces coelicolor is a filamentous soil bacterium producing several kinds of antibiotics. S. coelicolor abs8752 is an abs (antibiotic synthesis deficient)-type mutation at the absR locus; it is characterized by an incapacity to produce any of the four antibiotics synthesized by its parental strain J1501. A chromosomal DNA fragment from S. coelicolor J1501, capable of complementing the abs- phenotype of the abs8752 mutant, was cloned and analyzed. DNA sequencing revealed that two complete ORFs (SCO6992 and SCO6993) were present in opposite directions in the clone. Introduction of SCO6992 in the mutant strain resulted in a remarkable increase in the production of two pigmented antibiotics, actinorhodin and undecylprodigiosin, in S. coelicolor J1501 and abs8752. However, introduction of SCO6993 did not show any significant difference compared to the control, suggesting that SCO6992 is primarily involved in stimulating the biosynthesis of antibiotics in S. coelicolor. In silico analysis of SCO6992 (359 aa, 39.5 kDa) revealed that sequences homologous to SCO6992 were all annotated as hypothetical proteins. Although a metalloprotease domain with a conserved metal-binding motif was found in SCO6992, the recombinant rSCO6992 did not show any protease activity. Instead, it showed very strong β-glucuronidase activity in an API ZYM assay and toward two artificial substrates, p-nitrophenyl-β-D-glucuronide and AS-BI-β-D-glucuronide. The binding between rSCO6992 and Zn²⁺ was confirmed by circular dichroism spectroscopy. We report for the first time that SCO6992 is a novel protein with β-glucuronidase activity, that has a distinct primary structure and physiological role from those of previously reported β-glucuronidases.

• Journal of Veterinary Science
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• v.21 no.2
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• pp.22.1-22.9
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• 2020

Rabid raccoon dogs (Nyctereutes procyonoides koreensis) have been responsible for animal rabies in South Korea since the 1990s. A recombinant rabies vaccine strain, designated as ERAGS, was constructed for use as a bait vaccine. Therefore, new means of differentiating ERAGS from other rabies virus (RABV) strains will be required in biological manufacturing and diagnostic service centers. In this study, we designed two specific primer sets for differentiation between ERAGS and other RABVs based on mutation in the RABV glycoprotein gene. Polymerase chain reaction analysis of the glycoprotein gene revealed two DNA bands of 383 bp and 583 bp in the ERAGS strain but a single DNA band of 383 bp in the field strains. The detection limits of multiplex reverse transcription polymerase chain reaction (RT-PCR) were 80 and 8 FAID₅₀/reaction for the ERAGS and Evelyn-Rokitnicki-Abelseth strains, respectively. No cross-reactions were detected in the non-RABV reference viruses, including canine distemper virus, parvovirus, canine adenovirus type 1 and 2, and parainfluenza virus. The results of multiplex RT-PCR were 100% consistent with those of the fluorescent antibody test. Therefore, one-step multiplex RT-PCR is likely useful for differentiation between RABVs with and those without mutation at position 333 of the RABV glycoprotein gene.

• Fisheries and Aquatic Sciences
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• v.25 no.11
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• pp.559-571
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• 2022

Galectins, a family of ß-galactoside-binding lectins, have emerged as soluble mediators in infected cells and pattern recognition receptors (PRRs) responsible for evoking and regulating innate immunity. The present study aimed to evaluate the role of galectin-1 in the host immune response of redlip mullet (Liza haematocheilia). We established a cDNA database for redlip mullet, and the cDNA sequence of galectin-1 (LhGal-1) was characterized. In silico analysis was performed, and the spatial and temporal expression patterns in gills and blood in response to lipopolysaccharide polyinosinic:polycytidylic acid, and Lactococcus garvieae were estimated via quantitative real-time PCR. Functional assays were conducted using recombinant protein to investigate carbohydrate binding, bacterial binding, and bacterial agglutination activity. LhGal-1 was composed of 135 amino acids. Conserved motifs (H-NPR, -N- and -W-E-R) within the carbohydrate recognition domain were found in LhGal-1. The tissue distribution revealed that the healthy stomach expressed high levels of LhGal-1. The temporal monitoring of LhGal-1 mRNA expression in the gill and blood showed its significant upregulation in response to immune challenges with different stimulants. rLhGal-1 exhibited binding activity in response to carbohydrates and bacteria. Moreover, the agglutination of rLhGal-1 against Escherichia coli was observed. Collectively, our findings suggest that LhGal-1 may function as a PRR in redlip mullet. Furthermore, LhGal-1 can be considered a significant gene to play a protective role in redlip mullet immune system.

• Journal of Microbiology and Biotechnology
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• v.33 no.6
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• pp.788-796
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• 2023

Canine parvovirus type 2 (CPV-2) has high morbidity and mortality rates in canines. Nonstructural protein 1 (NS1) of CPV-2 has endonuclease activity, initiates viral DNA replication, and is highly conserved. Thus, it is a promising target for antiviral inhibitor development. We overexpressed a 41.9 kDa active recombinant endonuclease in Escherichia coli and designed a nicking assay using carboxyfluorescein and quencher-linked ssDNA as substrates. The optimal temperature and pH of the endonuclease were 37℃ and pH 7, respectively. Curcumin, bisdemethoxycurcumin, demethoxycurcumin, linoleic acid, tannic acid, and α-tocopherol inhibited CPV-2 NS1 endonuclease with IC₅₀ values of 0.29 to 8.03 µM. The extracted turmeric, yerba mate, and sesame cake suppressed CPV-2 NS1 endonuclease with IC₅₀ values of 1.48, 7.09, and 52.67 ㎍/ml, respectively. The binding affinity between curcumin, the strongest inhibitor, and CPV-2 NS1 endonuclease by molecular docking was -6.4 kcal/mol. Curcumin inhibited CPV-2 NS1 endonuclease via numerous hydrophobic interactions and two hydrogen bonds with Lys97 and Pro111 in the allosteric site. These results suggest that adding curcuminoids, linoleic acid, tannic acid, α-tocopherol, extracted turmeric, sesame cake, and yerba to the diet could prevent CPV-2 infection.

• Journal of Microbiology and Biotechnology
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• v.33 no.9
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• pp.1228-1237
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• 2023

The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/㎍ RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.2 no.2
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• pp.169-177
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• 1999

Purpose: To evaluate the efficacy of interferon alpha therapy with or without prednisolone in children with chronic hepatitis B. Methods: Twenty-eight children (22 boys, 6 girls, mean age 130 months) had seropositive results for HBsAg, HBeAg and HBV DNA; 11 had chronic persistent hepatitis and 17 had chronic active hepatitis. The patients were divided into two groups depending upon their inflammatory activity on liver biopsy, pretreatment serum ALT levels and HBV DNA levels. Fourteen children (group 1: chronic active hepatitis, ALT ${\geq}$ 100 IU/L and HBV DNA ${\leq}$ 100 pg/$300\;{\mu}L$) received interferon alpha 2a 5 $MU/m^2$ of body surface three times weekly for 6 months. Fourteen children (group 2: chronic persistent hepatitis or chronic active hepatitis with ALT < 100 IU/L or HBV DNA > 100 pg/$300\;{\mu}L$) received prednisolone in decreasing daily doses of 60 mg/$m^2$, 40 mg/$m^2$, and 20 mg/$m^2$, each for 2 weeks, followed after 2 weeks by interferon alpha 2a on the same schedule. At the end of therapy, 3 end points were analyzed: HBeAg seroconversion, serum ALT normalization rate and clearance of serum HBV DNA. Results: At the end of treatment, HBe antigen-to antibody seroconversion was higher but not more significant in group 1 than group 2 (71.4% vs. 50.0%). Only one patient in group 2 who lost HBeAg, also cleared HBsAg. ALT normalization was similar in both groups (64.3% in group 1 vs. 55.6% in group 2). Clearance of serum HBV DNA was observed in 78.6% of patients in group 1 and 64.3% in group 2, but no significant differences. Complete response was similarly achieved in both groups (57.1% in group 1 vs. 50.0% in group 2). Interferon alpha therapy with prednisolone priming was well tolerated and all children finished therapy. Conclusion: The combined therapy with prednisolone followed by interferon alpha may be safe and effective in inducing a serological and biochemical remission of the disease in approximately 50% of children with chronic hepatitis B and with a high level of viral replication and less active disease. However, a controlled study should be performed to confirm these results.

• Journal of Plant Biotechnology
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• v.6 no.1
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• pp.1-7
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• 2004

Forestry sector has witnessed some unprecedented events in the recent past both in terms of galloping biotechnological developments and heated environmental debates over risks associated with release of transgenic trees. Improvements in the in vitro propagation techniques has made it possible to develop tissue culture based plant regeneration protocols just for about any tree species. And with the inclusion of every new species within the realms of tissue culture technology, it becomes a candidate for genetic improvement through recombinant DNA technology, the so called genetic engineering. Poplars and their hybrids serve as the model tree species on which most of the genetic transformation work as been carried out. A lot of work has also gone in genetic transformation of fruit trees and trees of horticultural interests. Trees have been successfully transformed for traits ranging from reduction of length of juvenile phase to alteration of tree architecture to altering wood quality by lignin and cellulose modification. More-over trees have been genetically engineered successfully to combat various types of insect pests and pathogens causing diseases. But all these developments have ignited controversies over the possible benefits and risks associated with transgenic plantations by various environmental agencies and activists. Solutions to most of these concerns can be found out with more intensive prioritized research.

• Molecules and Cells
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• v.20 no.1
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• pp.43-50
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• 2005

Glutaredoxin (Grx) is a small, heat-stable redox protein acting as a multi-functional glutathione (GSH)-dependent disulfide oxidoreductase. We have cloned the monothiol Grx5 gene from the genomic DNA of the fission yeast Schizosaccharomyces pombe. It has 1,904 bp, with one intron, and encodes a putative protein of 146 amino acids with a molecular mass of 16.5 kDa. Recombinant Grx5 produced functional Grx in S. pombe cells. NO-generating sodium nitroprusside (SNP, 1.0 and 2.0 mM) and potassium chloride (KCl, 0.2 and 0.5 M) increased the synthesis of ${\beta}$-galactosidase from a Grx5-lacZ fusion gene, and transcription of Grx5 was also enhanced by SNP and KCl. Synthesis of ${\beta}$-galactosidase from the Grx5-lacZ fusion was lower in Pap1-negative TP108-3C cells than in wild type KP1 cells, and when Pap1 was overproduced in KP1 cells, the level of ${\beta}$-galactosidase increased. We also found that Pap1 is involved in the induction of Grx5 by SNP and KCl. S. pombe Grx5 may play a crucial role in responses to nitrosative and osmotic stresses.

• Bulletin of the Korean Chemical Society
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• v.33 no.2
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• pp.651-656
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• 2012

Cationic immunoliposomes were prepared by conjugation of Fab' fragments of the recombinant humanized monoclonal antibody (HuCC49) against tumor-associated glycoprotein (TAG)-72 to sterically unilamella liposomes. The cationic immunoliposomes are composed of cationic lipid (O,O'-dimyristyl-N-lysyl aspartate, DMKD), cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)$_{2000}$] (DPPE-PEG-maleimide) with a molar ratio of 0.5:0.47:0.03. Plasmid DNA was effectively condensed by addition of transferrin (Tf) during the formation of anti-TAG-72 PEG-immunolipoplexes (PILPs). These anti-TAG-72 PILPs were able to adhere to the surface of TAG-72-overexepressing LS174T human colon cancer cells more effectively than conventional liposomes, thereby facilitating gene delivery in vitro. Furthermore, intravenous administration of the anti-TAG-72 PILPs into the tumor-carrying mice exhibited efficient localization of the reporter gene in the tumor tissues.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.652-655
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• 2001

The comparative study of enzymes that catalyze a similar reactions but have different substrate spectrum and/or stereospecificity is a powerful approach to understanding the reaction mechanism between the relative enzymes, and it was also an useful tool to cloning the related enzyme, without the typical cloning from DNA library of genomic pools. For this purpose, we conducted an approach that the comparison at the molecular and protein level of esterases, from various sources including a previously identified (S)-stereospecific esterase of Pseudomonas sp. ES1. As expected, we found an esterase family genes that shared a high similarity at the protein and genetic level in the identical genus Pseudomonad. The striking structural and biochemical identity strongly suggested the family genes to be an identical one. We, hence, aligned the family genes and designated a degenerated primer for PCR-cloning using six Pseudomonas strains as templates. As a result, a recombinant esterase from Pseudomonas fluorescens KCTC 1767 was cloned and high-level expressed with high selectivity to (R,S)-ketoprofen ethyl ester. The enzyme exhibited a high ester-hydrolyzing activity to (S)-ketoprofen but did not hydrolyzed the opposite stereoisomer. Further characteristics were discussed in our presentation.