• Biomolecules & Therapeutics
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• v.26 no.6
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• pp.576-583
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• 2018

Human rhinoviruses (HRV) are one of the major causes of common cold in humans and are also associated with acute asthma and bronchial illness. Heat-shock protein 90 (Hsp90), a molecular chaperone, is an important host factor for the replication of single-strand RNA viruses. In the current study, we examined the effect of the Hsp90 inhibitor pochonin D, in vitro and in vivo, using a murine model of human rhinovirus type 1B (HRV1B) infection. Our data suggested that Hsp90 inhibition significantly reduced the inflammatory cytokine production and lung damage caused by HRV1B infection. The viral titer was significantly lowered in HRV1B-infected lungs and in Hela cells upon treatment with pochonin D. Infiltration of innate immune cells including granulocytes and monocytes was also reduced in the bronchoalveolar lavage (BAL) by pochonin D treatment after HRV1B infection. Histological analysis of the lung and respiratory tract showed that pochonin D protected the mice from HRV1B infection. Collectively, our results suggest that the Hsp90 inhibitor, pochonin D, could be an attractive antiviral therapeutic for treating HRV infection.

• Bulletin of the Korean Chemical Society
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• v.33 no.11
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• pp.3676-3680
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• 2012

An Epstein-Barr virus (EBV)-based plasmid contains the EBV nuclear antigen 1 (EBNA1) gene and EBV replication origin (oriP) sequence. Since EBNA1 (the only EBV-encoded protein) is combined with oriP, it is replicated simultaneously with chromosomal DNA in human, primate, and canine cells and is faithfully segregated at a stable copy number upon cell division. Consequently, it can be used to stably express gene inserts over a prolonged time in target cells. We have previously shown that the polyamidoamine (PAMAM) dendrimer can be surface-modified with L-arginine. Arginine is present at a high frequency in the transactivator of transcription (Tat) sequences of human immunodeficiency virus (HIV). It presents high membrane permeability and permits effective transfer of DNA inside the cells. In this study, we constructed two kinds of recombinant DNA by inserting the luciferase gene and enhanced green fluorescence protein (eGFP) gene as reporter genes into the pCEP4 plasmid vector. We measured dynamic light scattering (DLS) and zeta potential after preparing PAMAM-based cationic polymer/EBV-based plasmid complexes. We performed transfection of HEK 293 cell lines with the polyplexes, and monitored luciferase activity and green fluorescence protein (GFP) expression. Our results show that PAMAM-based cationic polymer/EBV plasmid complexes provide enhanced and sustained gene expression.

• Molecules and Cells
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• v.41 no.2
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• pp.127-133
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• 2018

Chromatin remodeling factors are involved in many cellular processes such as transcription, replication, and DNA damage response by regulating chromatin structure. As one of chromatin remodeling factors, remodeling and spacing factor 1 (RSF1) is recruited at double strand break (DSB) sites and regulates ataxia telangiectasia mutated (ATM) -dependent checkpoint pathway upon DNA damage for the efficient repair. RSF1 is overexpressed in a variety of cancers, but regulation of RSF1 levels remains largely unknown. Here, we showed that protein levels of RSF1 chromatin remodeler are temporally upregulated in response to different DNA damage agents without changing the RSF1 mRNA level. In the absence of SNF2h, a binding partner of RSF1, the RSF1 protein level was significantly diminished. Intriguingly, the level of RSF1-3SA mutant lacking ATM-mediated phosphorylation sites significantly increased, and upregulation of RSF1 levels under DNA damage was not observed in cells overexpressing ATM kinase. Furthermore, failure in the regulation of RSF1 level caused a significant reduction in DNA repair, whereas reconstitution of RSF1, but not of RSF1-3SA mutants, restored DSB repair. Our findings reveal that temporal regulation of RSF1 levels at its post-translational modification by SNF2h and ATM is essential for efficient DNA repair.

• Journal of the Korea Organic Resources Recycling Association
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• v.12 no.2
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• pp.61-71
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• 2004

These studies were conducted to investigate the effects of feeding dried leftover food (DLF) on growth, feed conversion and carcass characteristics of growing and finishing pigs. In experiment 1, seventy-five three-way cross-hybrids ($Yorkshire{\times}Landrace{\times}Duroc$) pigs weighing approximately 22 kg of body weight on average were assigned to five treatments in a completely randomized design. Each treatment had three replications with five pigs per replication. All pigs were fed experimental diets for 60 days. In experiment 2, seventy-five three-way cross-hybrids pigs weighing approximately 70 kg of body weight were fed experimental diets for 49 days. Each treatment had three replications with five pigs per replication. The treatments included 1) group offered control diet without DLF, 2) group offered diet containing DLF at 25%, 3) group offered diet containing DLF at 50%, 4) group offerred diet containing DLF at 25% with 10% higher protein level and 5) group offerred diet containing DLF at 50% and 20% higher protein level. Average daily gain of growing pigs was highest in control group among all the treatment groups except group offered diet containing DLF at 25% with no significant difference (P>0.05). Feed intake of DLF-offered groups were lower than that of control group while feed intake of groups fed diets containing DLF at 50% with 20% higher protein level was significantly higher (P<0.05) than that of control group. Feed conversion of growing pigs was not significantly different amont treatments although it seemed to be slightly improved in groups fed diets containing DLF at 25%. Average daily gain of finishing pigs fed diets containing DLF was significantly lower than that of control group. However there was no significant differences in average daily gain between groups fed diets containing DLF at 25% with 10% higher protein level and control group (P>0.05). Feed intakes were significantly decreased in DLF-fed groups compared to control group while there was no significant differences in feed intake between groups fed diets containing DLF with 10% and 20% higher protein levels and control group (P>0.05). Feed conversion was lowest in groups fed diets containing DLF at 25% with 10% higher protein level. However, there were no significant differences in feed conversion between groups fed diets containing DLF at 25% with 10% higher protein level and control group. Feed conversion of groups fed diets containing DLF at 50% was significantly higher than that of control group (P<0.05). Carcass weight was decreased with increasing levels of DLF in the diets. There were no significant differences in dressing percentage, backfat thickness and carcass grade among treatments. Feed cost per 1 kg body weight gain of finishing pigs was lowest in groups fed diets containing DLF at 25% with 10% higher protein level.

• Biomedical Science Letters
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• v.7 no.4
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• pp.161-166
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• 2001

Most mammalian cells take up glucose by passive transport proteins in the plasma membranes. The best known of these proteins is the human erythrocyte glucose transporter, GLUT1. High levels of heterologous expression far the transporter are necessary for the investigation of its three-dimensional structure by crystallization. To achieve this, the baculovirus expression system has become popular choice. However, Spodoptera frugiperda Clone 9 (Sf9) cells, which are commonly employed as the host permissive cell line to support baculovirus replication and protein synthesis, grow well on TC-100 medium that contains 0.1% D-glucose as the major carbon source, suggesting the presence of endogenous glucose transporters. Furthermore, very little is known of the endogenous transporters properties of Sf9 cells. Therefore, human GLUT1 antibodies would play an important role for characterization of the GLUT1 expressed in insect cell. However, the successful use of such antibodies for characterization of GLUT1 expression m insect cells relies upon their specificity for the human protein and lack of cross-reaction with endogenous transporters. It is therefore important to determine the potential cross-reactivity of the antibodies with the endogenous insect cell glucose transporters. In the present study, the potential cross-reactivity of the human GLUT1 antibodies with the endogenous insect cell glucose transporters was examined by Western blotting. Neither the antibodies against intact GLUT1 nor those against the C-terminus labelled any band migrating in the region expected fur a protein of M$_r$ comparable to GLUT1, whereas these antibodies specifically recognized the human GLUT1. Specificity of the human GLUT1 antibodies tested was also shown by cross-reaction with the GLUT1 expressed in insect cells. In addition, the insect cell glucose transporter was found to have very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2018.05a
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• pp.588-591
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• 2018

Baculovirus was originally isolated from the alfalfa looper and contains a 134-kbp genome with 154 open reading frames (ORF). The major capsid protein VP39 together with some minor proteins forms the nucleocapsid ($21nm{\times}260nm$) that encloses the DNA with p6.9 protein. They are double-stranded, circular, supercoiled DNA molecules in a rod-shaped capsid. Wild-type baculoviruses exhibit both lytic and occluded life cycles that develop independently throughout the three phases of virus replication. Recombinant baculoviruses can transfer their vectors and express their recombinant proteins in a wide range of mammalian cell types. Especially, inclusion of a dominant selectable marker in these baculoviral vectors can express diverse recombinant genes in many cells. Baculoviral vectors were reconstructed with cytomegalovirus (CMV) promoter,uroplakin II promoter, polyhedron promoter, vesicular stomatitis virus G (VSVG), enhanced green fluorescent protein (EGFP), protein transduction domain (PTD) gene and so on. These reconstructed vectors were infected into various cell and cell lines. We performed transfection and expression of these recombinant vectors comparison with other control vectors. From this study, we knew that transfection and expression of these recombinant vectors have higher efficacy than any control vector. This work was supported by a grant from Mid-Career Researcher Program(NRF-2016R1A2B4016552) through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning(MSIP).

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.1
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• pp.20-33
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• 2017

Objective: Superstimulatory treatment of one-month-old lambs can achieve synchronous development of numerous growing follicles. However, these growing follicles cannot complete maturation and ovulation. Oocyte maturation and competence are acquired during follicular development, in which granulosa cells play an essential role. Methods: In this study, we applied RNA sequencing to analyze and compare gene expression between prepubertal and adult superstimulated follicle granulosa cells in sheep. Results: There were more than 300 genes that significantly differed in expression. Among these differently expressed genes, many extracellular matrix genes (EGF containing Fibulin Like Extracellular Matrix Protein 1, pentraxin 3, adrenomedullin, and osteopontin) were significantly down-regulated in the superstimulated follicles. Ingenuity pathway and gene ontology analyses revealed that processes of axonal guidance, cell proliferation and DNA replication were expressed at higher levels in the prepubertal follicles. Epidermal growth factor, T-Box protein 2 and beta-estradiol upstream regulator were predicted to be active in prepubertal follicles. By comparison, tumor protein P53 and let-7 were most active in adult follicles. Conclusion: These results may contribute to a better understanding of the mechanisms governing the development of granulosa cells in the growing follicle in prepubertal sheep.

• The Journal of Korean Society of Virology
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• v.28 no.1
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• pp.39-52
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• 1998

HIV-1 tat, a strong transactivator, is essential for the HIV-1 replication and AIDS progression. The Tat function is markedly inhibited by human anti-oncogene p53. This work was initiated to identify the p53-associated inhibitory mechanism on tat-mediated transactivation. Inhibitory function of p53 was confirmed by co-transfection of tat-expressing Jurkat cells with LTR-CAT plasmid, or H3T1 cells (LTR-CAT integrated HeLa cells) with different ratio of pSV-tat/pCDNA-p53 plasmids. Results from the direct protein-protein interaction between soluble p53 and tat, and yeast two-hybrid experiments showed that the co-suppression mechanism is unlikely to be due to the direct interaction. CAT activity was not affected by tat in Jurkat cells which were transfected with p53-promoter-CAT or p53-enhancer-CAT, suggesting that the tat-mediated p53 suppression is not directly associated with p53-promoter. Finally, we have tested protein kinase activity in p53-tranfected Jurkat cells, which might phosphorylate HIV-1 tat, resulting in inhibition of tat function. Some of our data lead us to assume that the p53-mediated tat inhibition is likely to be associated with p53-associated, signaling-mediated phosphorylation of tat, resulting in the dysfunction of tat. This study is now under investigation.

• The Journal of Korean Society of Virology
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• v.29 no.4
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• pp.235-245
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• 1999

HIV-1 Tat, a strong transactivator, is essential for the HIV-1 replication and AIDS progression. The Tat function is markedly inhibited by human p53 anti-oncogene. However, the detail mechanism has not yet been clearly revealed. In our previous report, we have addressed that p53 is unlikely to interact directly with HIV-1 Tat. In the consecutive experiments, Tat-phosphorylation was found to increase in proportional to the amounts of transfected p53. This work was initiated to identify the signaling factor that is involved in the p53-mediated Tat suppression. Several protein kinases were tested for the phosphorylation of Tat, and we found that PKR is likely to be involved in the p53-mediated Tat suppression. PKR was co-immunoprecipitated by anti-Tat antibody in the Tat-expressing Jurkat cell lysates only when the cells were transfected by p53, indicating that PKR-Tat interaction depends on the p53 activity. The interaction seems to result in PKR-mediated Tat-phosphorylation. Tat function was not blocked by p53 when co-transfected trasiently with antisense-PKR. We have generated PKR-knock out Jurkat cell clone. The PKR defective Jurkat cells didn't show the p53-mediated Tat suppression. These data indicate that p53-mediated Tat suppression is strongly associated with PKR. PKR-mediated Tat phosphorylation experiments are now under investigation by kinase assay and co-immunoprecipitation in the presence or absence of p53.

• Journal of Life Science
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• v.22 no.4
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• pp.552-558
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• 2012

Severe acute respiratory syndrome (SARS) is a severe respiratory infectious disease caused by a novel human coronavirus, SARS-CoV. The 3CL protease is a key enzyme in the proteolytic processing of replicase polyprotein precursors, pp1a and pp1ab, which mediate all the functions required for viral genomic replication and transcription. Therefore, this enzyme is a target for the development of chemotherapeutic agents against SARS. A large quantity of active SARS-3CL protease is required for development of anti-SARS agents. Here we have constructed overexpression vector for the production of the SARS-3CL protease. The gene encoding SARS-3CL protease was amplified using polymerase chain reaction and cloned into the pET29a expression vector, resulting in pET29a/SARS-3CLP. Recombinant SARS-3CL protease was successfully synthesized by the dialysis mode of the cell-free protein expression system, and purified by three-step fast protein liquid chromatography using HighQ and MonoP column chromatographies and Sephacryl S-300 gel filtration. In addition, the produced SARS-3CL protease was found to be an active mature form. This study provides efficient methods not only for the development of anti-SARS materials from natural sources, but also for the study of basic properties of the SARS-3CL protease.