• Asian-Australasian Journal of Animal Sciences
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• v.14 no.2
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• pp.163-169
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• 2001

Despite the high potency of the retrovirus vector system in gene transfer, one of the main drawbacks of has been difficulty in preparing highly concentrated virus stock. Numerous efforts to boost the virus titer have ended in unsatisfactory results mainly due to fragile property of retrovirus envelope protein. In this study, to overcome this problem, we constructed our own retrovirus vector system producing vector viruses encapsulated with VSV-G (vesicular stomatitis virus G glycoprotein). Concentration process of the virus stock by ultracentrifuge did not sacrifice the virus infectivity, resulting in more than 108 to 109 CFU (colony forming unit) per ml on most of the target cell lines tested. Application of this high-titer retrovirus vector system was tested on chicken embryos. Injection of virus stock beneath the blastoderms of pre-incubated fertilized eggs resulted in chick embryos expressing E. coli LacZ gene with 100% efficiency. Therefore, our results suggest that it is possible to transfer the foreign gene into chicken embryo using our high-titer retrovirus vector.

• Korean Journal of Veterinary Research
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• v.46 no.2
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• pp.127-133
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• 2006

Pasteurella multocida is a terrible veterinary pathogen that causes widespread infections in husbandry. To induce homologous and/or heterologous immunity against the infections, outer membrane protein Hs (OmpH) in the envelope of different strains of P. multocida are thought to be attractive vaccine candidates. Previously we cloned and characterized a gene for OmpH from pathogenic P. multocida (A : 3) (In Press, Korean J. Microbiol. Biotechnol. 2005, 33, December). The gene is composed of 1,047 nucleotides (nt) coding 348 amino acids (aa) with signal peptide of 20 aa. The truncated ompH, a gene without nt coding for the signal peptide, was generated using pRSET A to name "pRSET A/OmpH-F2". This truncated ompH was well expressed in Escherichia coli BL21 (DE3). Truncated OmpH was purified for induction of immunity against live pathogen of fowl cholera (P. multocida A : 3) in mice. Some $50{\mu}g$ of the purified polypeptide was intraperitoneally injected into mice two times with 10 day interval. Lethal dose ($25{\mu}l$) of live P. multocida A : 3 was determined by directly injecting the pathogen into wild mice (n = 25). To demonstrate the vaccine candidate of the truncated OmpH, the live pathogen ($25{\mu}l$) was challenged with the OmpH-immunized mouse group as well as positive & negative controls (n = 80). The results show that the truncated OmpH can be used for an effective vaccine production to prevent fowl cholera caused by pathogenic P. multocida (A : 3).

• International Journal of Industrial Entomology and Biomaterials
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• v.29 no.2
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• pp.207-213
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• 2014

The structural proteins of classical swine fever virus (CSFV) consist of nucleocapsid protein C and envelope glycoprotein $E^{rns}$ (E0), E1 and E2. Among them, E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. In this study, to determine the optimal expression conditions of glycoprotein E2 using baculovirus system, we investigated the influence of insect cells and media to the expression of recombinant E2. Recombinant virus containing glycoprotein E2 coding gene was constructed with bApGOZA DNA. Expression of the glycoprotein E2 was analyzed by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. Expression of glycoprotein E2 in Sf21 cells was first observed after 3 days and reached a maximum on the 5th day after infection. Furthermore, the highest levels of glycoprotein E2 expression were observed at multiplicity of infection (MOI) of 5. When three different insect cell lines (Sf21, High-Five and Se301) were tested, High-Five cells showed the highest production. In addition, four different serum-free and serum-supplemented media, respectively, were tested for the expression of glycoprotein E2 and the budded virus (BV) titers. As a result, serum-supplemented medium provided the best conditions for protein production and the BV yield.

• Journal of Ginseng Research
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• v.46 no.1
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• pp.115-125
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• 2022

Background: Ginsenosides (GS) have potential value as cosmetic additives for prevention of skin photoaging. However, their protective mechanisms against skin barrier damage and their active monomeric constituents are unknown. Methods: GS monomer types and their relative proportions were identified. A UVB-irradiated BALB/c hairless mouse model was used to assess protective effects of GS components on skin epidermal thickness and transepidermal water loss (TEWL). Skin barrier function, reflected by filaggrin (FLG), involucrin (IVL), claudin-1 (Cldn-1), and aquaporin 3 (AQP3) levels and MAPK phosphorylation patterns, were analyzed in UVB-irradiated hairless mice or HaCaT cells. Results: Total GS monomeric content detected by UPLC was 85.45% and was largely attributed to 17 main monomers that included Re (16.73%), Rd (13.36%), and Rg1 (13.38%). In hairless mice, GS ameliorated UVB-induced epidermal barrier dysfunction manifesting as increased epidermal thickness, increased TEWL, and decreased stratum corneum water content without weight change. Furthermore, GS treatment of UVB-irradiated mice restored protein expression levels and epidermal tissue distributions of FLG, IVL, Cldn-1, and AQP3, with consistent mRNA and protein expression results obtained in UVB-irradiated HaCaT cells (except for unchanging Cldn-1 expression). Mechanistically, GS inhibited JNK, p38, and ERK phosphorylation in UVB-irradiated HaCaT cells, with a mixture of Rg2, Rg3, Rk3, F2, Rd, and Rb3 providing the same protective MAPK pathway inhibition-associated upregulation of IVL and AQP3 expression as provided by intact GS treatment. Conclusion: GS protection against UVB-irradiated skin barrier damage depends on activities of six ginsenoside monomeric constituents that inhibit the MAPK signaling pathway.

• Journal of Microbiology
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• v.44 no.1
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• pp.83-91
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• 2006

The sudden appearance and potential lethality of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) in humans has resulted in a focusing of new attention on the determination of both its origins and evolution. The relationship existing between SARS-CoV and other groups of coronaviruses was determined via analyses of phylogenetic trees and comparative genomic analyses of the coronavirus genes: polymerase (Orflab), spike (S), envelope (E), membrane (M) and nucleocapsid (N). Although the coronaviruses are traditionally classed into 3 groups, with SARS-CoV forming a $4^{th}$ group, the phylogenetic position and origins of SARS-CoV remain a matter of some controversy. Thus, we conducted extensive phylogeneitc analyses of the genes common to all coronavirus groups, using the Neighbor-joining, Maximum-likelihood, and Bayesian methods. Our data evidenced largely identical topology for all of the obtained phylogenetic trees, thus supporting the hypothesis that the relationship existing between SARS-CoV and group 2 coronavirus is a monophyletic one. Additional comparative genomic studies, including sequence similarity and protein secondary structure analyses, suggested that SARS-Co V may bear a closer relationship with group 2 than with the other coronavirus groups. Although our data strongly suggest that group 2 coronaviruses are most closely related with SARS-CoV, further and more detailed analyses may provide us with an increased amount of information regarding the origins and evolution of the coronaviruses, most notably SARS-CoV.

• ALGAE
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• v.31 no.4
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• pp.379-390
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• 2016

When a multinucleate cell of Bryopsis plumosa was collapsed by a physical wounding, the extruded protoplasm aggregated into numerous protoplasmic masses in sea water. A polysaccharide envelope which initially covered the protoplasmic mass was peeled off when a cell membrane developed on the surface of protoplast in 12 h after the wounding. Transmission electron microscopy showed that the protoplasmic mass began to form a continuous cell membrane at 6 h after the wounding. The newly generated cell membrane repeated collapse and rebuilding process several times until cell wall developed on the surface. Golgi bodies with numerous vesicles accumulated at the peripheral region of the rebuilding cell at 24 h after the wounding when the cell wall began to develop. Several layers of cell wall with distinctive electron density developed within 48-72 h after the wounding. Proteome profile changed dramatically at each stage of cell rebuilding process. Most proteins, which were up-regulated during the early stage of cell rebuilding disappeared or reduced significantly by 24-48 h. About 70-80% of protein spots detected at 48 h after the wounding were newly appeared ones. The expression pattern of 29 representative proteins was analyzed and the internal amino acid sequences were obtained using mass spectrometry. Our results showed that a massive shift of gene expression occurs during the cell-rebuilding process of B. plumosa.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.121-127
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• 2004

Amphotericin B (AmB), an amphipathic polyene macrolide, is an antifungal drug produced by Streptomyces nodosus. Recently, AmB has been shown to exert antiviral activity against rubella virus and human immunodeficiency virus by different mechanisms. In this study, we evaluated the antiviral effect of AmB against Japanese encephalitis virus (JEV) and investigated which step of the viral life cycle was inhibited by AmB to understand the mechanism of antiviral action of AmB. AmB reduced both plaque size and number in the infected cells in a dose-dependent manner. In addition, a 200-fold reduction of infectious virus titer was observed by treatment of infected cells with $5\mug/ml$ of AmB. AmB acted at the post virus-infection step, but not during adsorption of virus to host cells. Western blot analysis revealed that the accumulated level of JEV envelope protein dramatically decreased in the infected cells by treatment with $5-10\mug/ml$ of AmB. Our results indicate that AmB inhibits the replication of JEV at the postinfection step by interfering with viral replication and/or by inhibiting the synthesis of viral proteins.

• International Journal of Industrial Entomology and Biomaterials
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• v.23 no.1
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• pp.147-153
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• 2011

The Pseudorabies (PR), also called Aujeszky's disease (AD), is an infectious viral disease caused by an alpha herpes virus and has domestic and wild pigs, as well as a wide range of domestic and wild animals, as the natural host. Pseudorabies virus (PRV) virions contain several envelope glycoproteins. Among them, gB, gC and gD are regarded as the major immunogenic proteins. We expressed these glycoproteins using the bacterial expression system and analyzed recombinant proteins. Expression of glycoproteins gC and gD were observed on SDS-PAGE or Western blot analysis, but gB was not. Optimal concentration of IPTG and inducing time were determined as 1.0 mM and 4 h, respectively, for the expression of both gC and gD in E. coli. A sodium dodecyl sulfate (SDS) was the most efficient detergent in solubilizing insoluble recombinant protein.

• Bulletin of the Korean Chemical Society
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• v.34 no.8
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• pp.2466-2472
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• 2013

Human immunodeficiency virus type-1 (HIV-1) is a causative agent of Acquired immunodeficiency syndrome (AIDS), which has affected a large population of the world. Viral envelope glycoprotein (gp120) is an intrinsic protein for HIV-1 to enter into human host cells. Molecular docking guided molecular dynamics (MD) simulation was performed to explore the interaction mechanism of heterobiaryl derivative with gp120. MD simulation result of inhibitor-gp120 complex demonstrated stability. Our MD simulation results are consistent with most of the previous mutational and modeling studies. Inhibitor has an interaction with the CD4 binding region. Van der Waals interaction between inhibitor and Val255, Thr257, Asn425, Met426 and Trp427 were important. This preliminary MD model could be useful in exploiting heterobiaryl-gp120 interaction in greater detail, and will likely to shed lights for further utilization in the development of more potent inhibitors.

• Archives of Pharmacal Research
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• v.18 no.3
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• pp.173-178
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• 1995

Ciprofloxacin resistance mechanisms were studied by investigating the inhibitory effect of ciprofloxacin on the gyrase-mediated DNA supercoiling and the intracellular accumulation of ciprofloxacin in clinical isolates of Pseudomonas aeruginosa. A higher amount of ciprofloxacin was required to inhibit the gyrases purified from the ciprofloxacin-resistant strains than that from the sensitive strain. Reconstitution of heterologous gyrase subunits from different strains revealed alterations in the A and/or the B subunits of gyrase in these strains. In addition, the resistant strains accumulated approximately a half amount of ciprofloxacin inside the cells, compared to the sensitive strain. However, when the active efflux was blocked by carbonyl cyanide m-chlorophenyl hydrazone treatment, intracellular concentration of ciprofloxacin was elevated about 4-7 fold in these strains, while the sensitive strain was not significantly affected by this treatment, indicating that the ciprofloxacin-resistant strains developed a drug efflux system. Interestingly, these resistant strains expressed an envelope protein of approximately 51 kD. These studies suggest that alterations in the gyrase as well as the active drug-efflux system conferred dual ciprofloxacin resistance mechanisms to these clinical isolates of P. aeruginosa.