• Molecules and Cells
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• v.42 no.11
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• pp.783-793
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• 2019

When endoplasmic reticulum (ER) functions are perturbed, the ER induces several signaling pathways called unfolded protein response to reestablish ER homeostasis through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Although it is important to measure the activity of ATF6 that can indicate the status of the ER, no specific cell-based reporter assay is currently available. Here, we report a new cell-based method for monitoring ER stress based on the cleavage of $ATF6{\alpha}$ by sequential actions of proteases at the Golgi apparatus during ER stress. A new expressing vector was constructed by using fusion gene of GAL4 DNA binding domain (GAL4DBD) and activation domain derived from herpes simplex virus VP16 protein (VP16AD) followed by a human $ATF6{\alpha}$ N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-$hATF6{\alpha}$ deletion variant was cleaved to liberate active transcription activator encompassing GV-$hATF6{\alpha}$ fragment which could translocate into the nucleus. The translocated GV-$hATF6{\alpha}$ fragment strongly induced the expression of firefly luciferase in HeLa Luciferase Reporter cell line containing a stably integrated 5X GAL4 site-luciferase gene. The established double stable reporter cell line HLR-GV-$hATF6{\alpha}$(333) represents an innovative tool to investigate regulated intramembrane proteolysis of $ATF6{\alpha}$. It can substitute active pATF6(N) binding motif-based reporter cell lines.

• Animal Bioscience
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• v.34 no.5
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• pp.811-823
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• 2021

Objective: Eggshell color is an important indicator of egg quality for consumers, especially for brown eggs. Various factors related to laying hens and their environment affect brown eggshell coloration. However, there have been no studies investigating hepatic functions of laying hens with variable intensity of brown eggshell color. Therefore, this study was aimed to identify potential factors affecting brown eggshell coloration in aged laying hens at the hepatic transcriptomic level. Methods: Five hundred 92-wk-old Hy-line Brown laying hens were screened to select laying hens with different intensity of brown eggshell color based on eggshell color fans. Based on eggshell color scores, hens with dark brown eggshells (DBE; eggshell color fan score = 14.8) and hens with light brown eggshells (LBE; eggshell color fan score = 9.7) were finally selected for the liver sampling. We performed RNA-seq analysis using the liver samples through the paired-end sequencing libraries. Differentially expressed genes (DEGs) profiling was carried out to identify their biological meaning by bioinformatics. Results: A total of 290 DEGs were identified with 196 being up-regulated and 94 being down-regulated in DBE groups as compared to LBE groups. The Kyoto encyclopedia of genes and genomes (KEGG) analysis revealed that these DEGs belong to several biological pathways including herpes simplex infection (toll-like receptor 3 [TLR3], cyclin-dependent kinase 1, etc.) and influenza A (TLR3, radical S-adenosyl methionine domain containing 2, myxovirus [influenza virus] resistance 1, etc.). Genes related to stress response (ceremide kinase like) and nutrient metabolism (phosphoenolpyruvate carboxy-kinase 1, methylmalonic aciduria [cobalamin deficiency] cblB type, glycine receptor alpha 2, solute carrier family 7 member 11, etc.) were also identified to be differentially expressed. Conclusion: The current results provide new insights regarding hepatic molecular functions related to different intensity of brown eggshell color in aged laying hens. These insights will contribute to future studies aiming to optimize brown eggshell coloration in aged laying hens.

• Molecules and Cells
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• v.38 no.9
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• pp.773-780
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• 2015

3D8 single chain variable fragment (scFv) is a recombinant monoclonal antibody with nuclease activity that was originally isolated from autoimmune-prone MRL mice. In a previous study, we analyzed the nuclease activity of 3D8 scFv and determined that a HeLa cell line expressing 3D8 scFv conferred resistance to herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). In this study, we demonstrate that 3D8 scFv could be delivered to target tissues and cells where it exerted a therapeutic effect against PRV. PRV was inoculated via intramuscular injection, and 3D8 scFv was injected intraperitoneally. The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays. Intraperitoneal injection of 5 and $10{\mu}g$ 3D8 scFv resulted in no detectable toxicity. The survival rate in C57BL/6 mice was 9% after intramuscular injection of 10 $LD_{50}$ PRV. In contrast, the 3D8 scFv-injected C57BL/6 mice showed survival rates of 57% ($5{\mu}g$) and 47% ($10{\mu}g$). The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.

• Pediatric Infection and Vaccine
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• v.28 no.2
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• pp.92-100
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• 2021

Purpose: Rapid detection of etiologic organisms is crucial for initiating appropriate therapy in patients with central nervous system (CNS) infection. This study aimed to evaluate the diagnostic value of the BioFire^® Meningitis/Encephalitis (ME) panel in detecting etiologic organisms in cerebrospinal fluid (CSF) samples from febrile infants. Methods: CSF samples from infants aged <90 days who were evaluated for fever were collected between January 2016 and July 2019 at the Seoul National University Children's Hospital. We performed BioFire^® ME panel testing of CSF samples that had been used for CSF analysis and conventional tests (bacterial culture, Xpert^® enterovirus assay, and herpes simplex virus-1 and -2 polymerase chain reaction) and stored at -70℃ until further use. Results: In total, 72 (24 pathogen-identified and 48 pathogen-unidentified) CSF samples were included. Using BioFire^® ME panel testing, 41 (85.4%) of the 48 pathogen-unidentified CSF samples yielded negative results and 22 (91.7%) of the 24 pathogen-identified CSF samples yielded the same results (enterovirus in 19, Streptococcus agalactiae in 2, and Streptococcus pneumoniae in 1) as those obtained using the conventional tests, thereby resulting in an overall agreement of 87.5% (63/72). Six of the 7 pathogen-unidentified samples were positive for human parechovirus (HPeV) via BioFire^® ME panel testing. Conclusions: Compared with the currently available etiologic tests for CNS infection, BioFire^® ME panel testing demonstrated a high agreement score for pathogen-identified samples and enabled HPeV detection in young infants. The clinical utility and cost-effectiveness of BioFire^® ME panel testing in children must be evaluated for its wider application.

• Journal of dental hygiene science
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• v.3 no.2
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• pp.67-70
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• 2003

The biological effects of the crude extracts from medicinal plants, Brachyglottis monroi and Trichocolea hatcheri were investigated. The crude ethanol extract inhibited the growth of the Gram positive bacterium Bacillus subtilis (ATCC 19659, 1 mm inhibition zone at $150{\mu}g/disc$) and the dermatophyte Trichophyton mentagrophytes (ATCC 28185, 6 mm inhibition zone at $150{\mu}g/disc$), and was toxic to P388 murine leukaemia cells ($IC_{50}$ $23.96{\mu}g/mL$ at $75{\mu}g/disc$). B. monroi ethanol extract showed stronger antiviral activity than that of T. hatcheri against Herpes simplex Type I virus (ATCC VR 733) and Polio Type I virus (Pfizer vaccine strain) (50% activity, @ 5 mg/ml at $150{\mu}g/disc$). The crude ethanol extract of T. hatcheri showed stronger antimicrobial activity than that of B. monroi. However, this extract was inactive against P388 murine leukaemia cells.

• Journal of Oral Medicine and Pain
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• v.24 no.2
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• pp.125-135
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• 1999

This study was performed to discover the underlining influences of Herpes Simplex virus (HSV) and Varicella Zoster virus (VZV), to detect the changes of whole protein and mucin level and to observe protein profiles in the saliva when recurrent aphthous ulcer (RAU) was present. Unstimulated whole saliva was collected from 23 patients who for over three years had a clinical history of RAU, in a group of 10 women and 13 men, ranging from 11 to 72 years of age, and 20 healthy subjects, in a group of 8 women and 12 men, who did not have the symptoms nor a past history of RAU. Through the means of Polymerization Chain Reaction, genomic DNA from the HSV and VZV was purified from the saliva samples for identifying precisely the two types of viruses, and the level of whole protein and sialic acids in the saliva and the ratio of sialic acid to whole protein were measured, and SDS-polyacrylamide gel electrophoresis was performed. The results obtained were as follows ; 1. 39.13% of patients showed 224 bp bands of VZV DNA, those were appeared more in patients than in control group (p<0.01), but there was no significant difference between patients and control group in HSV DNA (p>0.05). 2. The concentration of whole protein in men patients was lower than in men control group (p<0.05), but there were no significant differences between patients and control in other groups (p>0.05). 3. The concentration of sialic acids from patients was lower than control group in all groups (p<0.05). 4. The concentration of sialic acids in proportion to that of whole protein was lower in patients than in control group (p<0.05), and in the two women groups (p<0.01), but no noticeable difference was found between the two men groups (p>0.05). 5. There were no consistent differences observed in the protein profiles of patients with control group except that certain protein bands near 50 kDa was lower in patients than in control group. These results suggest that viruses such as HSV and VZV and reduction of salivary whole protein and mucin levels are related to development of RAU.

• Journal of Life Science
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• v.18 no.3
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• pp.374-380
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• 2008

Rapid detection of enterovirus (EVs) is important in the management of aseptic meningitis. We examined the relative efficiency and specificity of the real-time nucleic acid sequence-based amplification (NASBA) comparing with the established reverse transcription polymerase chain reaction (RT-PCR) and viral culture method which were used for the detection of enterovirus RNA in clinical specimens. Of the total 292 samples, 145 were found to be positive to enterovirus RNA by real-time NASBA, 101 were positive by viral culture, and 86 were positive by RT-PCR. 147 samples and 46 samples were determined to be negative and positive by all methods respectively, but 4 samples were positive only by real-time NASBA. To compare the specificity of each method, various clinical samples which were diagnosed for herpes simplex virus (HSV)-1, HSV-2, adenovirus, mumps, and rhinovirus were applied. Except one rhinovirus sample which was false positive to enterovirus RNA by RT-PCR, the other different samples were negative to all three methods. The real-time NASBA procedure can be completed within 5 hours in contrast with 9 hours for the RT-PCR and 3-14 days for the viral culture. From this study, it was suggested that the real-time NASBA assay could be a standardized, rapid, specific, and sensitive procedure for the detection of enterovirus RNA.

• Clinical and Experimental Pediatrics
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• v.46 no.6
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• pp.541-547
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• 2003

Purpose : Gap junction intercellular communication(GJIC) is an important mechanism of the bystander effect in herpes simplex thymidine kinase/ganciclovir(HSVtk/GCV) gene therapy Therefore, we attempted to enhance the bystander effect in vitro by exogenous overexpressing connexin 37(Cx37) in cells to increase GJIC. Methods : NIH3T3 cells were transfected with the Cx37 and HSVtk gene or the HSVtk gene alone by the calcium phosphate method, and we detected their expression from these cells by RT-PCR. GCV-mediated cytotoxicity and the bystander effect of each transfectant was then assessed and compared. Results : Cells transfected with HSVtk became sensitive to low concentration of GCV. We found significantly increased cytotoxicity in HSVtk/GCV gene therapy after introduction of the HSVtk and Cx37 genes together compared with the cytotoxicity seen after introduction of the HSVtk gene in vitro. Co-expression of the HSVtk and Cx37 genes potentiates HSVtk/GCV gene therapy through the bystander effect. Conclusion : These results indicated that the increase of GJIC using Cx37 have potentiated the bystander effect of HSVtk/GCV therapy, and may be a new approach to improve response in suicidal cancer gene therapy.

• Clinical and Experimental Pediatrics
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• v.45 no.3
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• pp.354-361
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• 2002

Purpose : Selective introduction of genes conferring chemosensitivity into proliferating tumor cells may be used to treat cancer. We first investigated the bystander effect of retrovirus-mediated gene transfer of herpes simplex virus thymidine kinase(HSV-TK) gene to murine neuroblstoma cell line(neuro-2a) in vitro and in vivo. Second, we examined the mechanism and its enhancement of the bystander effect in murine neuroblastoma. Methods : To investigate the bystander effect, we studied tumor growth and survival time after HSV-TK/ganciclovir(GCV) treatment in a syngenic A/J mouse neuroblastoma model by mixing various ratios of HSV-TK-expressing neuro-2a cells with wild type neuro-2a cells followed by GCV treatment. To investigate the mechanism of the bystander effect in murine neuroblastoma, immunohistochemistry using connexin 43, CD4 and CD8-specific monoclonal antibodies was analyzed. We studied whether IL-2-secreting neuro-2a cells(neuro-2a/IL-2) would potentiate the bystander effect. Results : A strong bystander effect was observed in vitro and in vivo. The bystander effect in murine neuroblastoma was dependent on the immune response rather than connexin-mediated gap junction. Neuro-2a/IL-2 treatment enhanced the bystander effect in the HSV-TK/GCV system in murine neuroblastoma model. Conclusion : We conclude that the bystander effect in murine neuroblastoma depends on immune response and is enhanced by neuro-2a/IL-2.

• Bulletin of the Korean Chemical Society
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• v.28 no.12
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• pp.2449-2453
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• 2007

L-[18F]FMAU ([18F]1b) was prepared from the precursor 2-O-[(trifluoromethyl)-sulfonyl]-1,3,5-tri-Obenzoyl- α-L-ribofuranose, by coupling the radioactive fluoro-sugar with the corresponding silylated thymine in 4 steps. The final products, including the α and β anomers, were purified using reverse phase HPLC with an appropriate solvent (5% CH3CN/H2O) at a flow rate of 3.0 mL/min. The total elapsed time of synthesis was about 180-200 min from EOB. The α/β anomeric ratio of the compounds was about 1:9, and the radiochemical purity of the product (β-form) was >98% with decay-corrected yields of 25-35%. All radioactive samples were confirmed using co-injection with pure non-radioactive analogues in every step. In the cellular uptake in vitro test of herpes simplex virus-thymidine kinase (HSV1-TK) gene expressed cells, the percent uptake of injected dose (%ID) of L- and D-FMAU was 37.28 and 65.86, respectively after 240 min incubation. However, the relative uptake (MCA-TK/MCA cellular uptake ratio) of L-FMAU was higher than that of D-FMAU (%ID of L-FMAU, 0.36 and D-FMAU, 0.93 after 240 min incubation in MCA cells). This means that L-FMAU will show better specific HSV1-TK gene expressed cell uptake for selective HSV1-TK gene imaging.