• Journal of Microbiology and Biotechnology
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• v.33 no.12
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• pp.1576-1586
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• 2023

Vaccination is the most effective method for preventing the spread of the influenza virus. Cell-based influenza vaccines have been developed to overcome the disadvantages of egg-based vaccines and their production efficiency has been previously discussed. In this study, we investigated whether treatment with forskolin (FSK), an adenylyl cyclase activator, affected the output of a cell-based influenza vaccine. We found that FSK increased the propagation of three influenza virus subtypes (A/H1N1/California/4/09, A/H3N2/Mississippi/1/85, and B/Shandong/7/97) in Madin-Darby canine kidney (MDCK) cells. Interestingly, FSK suppressed the growth of MDCK cells. This effect could be a result of protein kinase A (PKA)-Src axis activation, which downregulates extracellular signal-regulated kinase (ERK)1/2 activity and delays cell cycle progression from G1 to S. This delay in cell growth might benefit the binding and entry of the influenza virus in the early stages of viral replication. In contrast, FSK dramatically upregulated ERK1/2 activity via the cAMP-PKA-Raf-1 axis at a late stage of viral replication. Thus, increased ERK1/2 activity might contribute to increased viral ribonucleoprotein export and influenza virus propagation. The increase in viral titer induced by FSK could be explained by the action of cAMP in assisting the entry and binding of the influenza virus. Therefore, FSK addition to cell culture systems could help increase the production efficiency of cell-based vaccines against the influenza virus.

• Environmental Mutagens and Carcinogens
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• v.19 no.1
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• pp.28-33
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• 1999

The SNF2 family includes proteins from a variety of species with roles I cellular processes such as transcriptional regulation, recombination and various types of DNA repair. Several proteins with unknown function are also included in this family. Here, we report the cloning and characterization of hrp 2+ gene (helicase related gene from S. pombe) which was isolated by PCR amplication using the conserved domain of SNF2 motifs within the ERCC6 gene which encodes a protein involved in DNA excision repair. The hrp2+ gene was isolated by screening with yeast S. pombe genomic library. The isolated cloned contained 6.5 kb insert DNA. Southern blot analysis confirmed that S. pombe chromosome contains the same DNA as hrp2+ gene and this gene exists as a single copy in S. pombe genome. The 4.7 kb transcript of mRNA was identified by Northern blot. To examined the transcriptional regulation of hrp2+ gene, DNA damaging agents were treated. These results indicated that the hrp2+ gene may not be directly involved in DNA replication, but may be involved in damage response pathway.

• Journal of Microbiology and Biotechnology
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• v.28 no.10
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• pp.1716-1722
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• 2018

Immunosuppressive drugs are used to make the body less likely to reject transplanted organs or to treat autoimmune diseases. In this study, five immunosuppressive drugs including two glucocorticoids (dexamethasone and prednisolone), one calcineurin inhibitor (cyclosporin A), one non-steroid anti-inflammatory drug (aspirin), and one antimetabolite (methotrexate) were tested for their effects on viral proliferation using feline foamy virus (FFV). The five drugs had different cytotoxic effects on the Crandell-Ress feline kidney (CRFK) cells, the natural host cell of FFV. Dexamethasone-pretreated CRFK cells were susceptible to FFV infection, but pretreatment with prednisolone, cyclosporin A, aspirin, and methotrexate showed obvious inhibitory effects on FFV proliferation, by reducing viral production to 29.8-83.8% of that of an untreated control. These results were supported by western blot, which detected viral Gag structural protein in the infected cell lysate. As our results showed a correlation between immunosuppressive drugs and susceptibility to viral infections, it is proposed that immune-compromised individuals who are using immune-suppressive drugs may be especially vulnerable to viral infection originated from pets.

• PNF and Movement
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• v.6 no.1
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• pp.41-51
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• 2008

Purpose : This paper reviews evidence supporting adaptive plasticity in skeletal muscle fibers induced by various exercise training and neuromuscular activity. Result : Skeletal muscle fiber demonstrates a remarkable adaptability and can adjust its physiologic and contractile makeup in response to alterations in functional demands. This adaptive plasticity results from the ability of muscle fibers to adjust their molecular, functional, and contractile properties in response to altered physiological demands, such as changes in exercise patterns and mechanical loading. The process of activity-dependent plasticity in skeletal muscle involves a multitude of signalling mechanisms initiating replication of specific genetic sequences, enabling subsequent translation of the genetic message and ultimately generating a series of myosin heavy chain isoform. Conclusions : Knowledge of the mechanisms and interaction of activity-dependent adaptive pathways in skeletal muscle is important for our understanding of the synthesis of muscle myosin protein, maintenance of metabolic and functional capacity with physical activity, and therapeutic intervention for functional improvement.

• BMB Reports
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• v.51 no.7
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• pp.338-343
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• 2018

Transcription termination factor-1 (TTF-I) is an RNA polymerase 1-mediated transcription terminator and consisting of a C-terminal DNA-binding domain, central domain, and N-terminal regulatory domain. This protein binds to a so-called 'Sal box' composed of an 11-base pair motif. The interaction of TTF-I with the 'Sal box' is important for many cellular events, including efficient termination of RNA polymerase-1 activity involved in pre-rRNA synthesis and formation of a chromatin loop. To further understand the role of TTF-I in human immunodeficiency virus (HIV)-I virus production, we generated various TTF-I mutant forms. Through a series of studies of the over-expression of TTF-I and its derivatives along with co-transfection with either proviral DNA or HIV-I long terminal repeat (LTR)-driven reporter vectors, we determined that wild-type TTF-I downregulates HIV-I LTR activity and virus production, while the TTF-I Myb-like domain alone upregulated virus production, suggesting that wild-type TTF-I inhibits virus production and trans-activation of the LTR sequence; the Myb-like domain of TTF-I increased virus production and trans-activated LTR activity.

• Journal of fish pathology
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• v.35 no.2
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• pp.157-165
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• 2022

Single-cycle viruses generated by reverse genetic technology are replication-incompetent viruses due to the elimination of gene(s) essential for viral replication, which provides a way to overcome the safety problem in attenuated viruses. Infectious hematopoietic necrosis virus (IHNV) is a major pathogen causing severe damage in cultured salmonid species. In the present study, we generated a single-cycle IHNV lacking the transmembrane and cytoplasmic domain in the G gene (rIHNV-GΔTM) and evaluated the prophylactic potential of rIHNV-GΔTM in rainbow trout (Oncorhynchus mykiss). To produce rIHNV-GΔTM, IHNV G protein-expressing Epithelioma papulosum cyprini (EPC) cells were established. However, as the efficiency of rIHNV-GΔTM production in EPC cell clones was not high, fish were immunized with a low-tittered single-cycle virus (1.5 × 10² PFU/fish). Despite the low dose, the single-cycle IHNV induced significant protection in rainbow trout against IHNV infection, suggesting high immunogenicity of rIHNV-GΔTM. No significant difference in serum ELISA titers against IHNV between the rIHNV-GΔTM immunized group and the control group suggests that the immunized dose of rIHNV-GΔTM might be too low to induce significant humoral adaptive immune responses in rainbow trout. The involvement of adaptive cellular immunity or innate immunity in the present significantly higher protection by the immunization with rIHNV-GΔTM should be further investigated to know the protection mechanism.

• Journal of Microbiology and Biotechnology
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• v.31 no.7
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• pp.942-948
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• 2021

Canine influenza virus (CIV) induces acute respiratory disease in dogs. In this study, we aimed to determine the signaling pathways leading to the induction of IFN-β in a canine respiratory epithelial cell line (KU-CBE) infected with the H3N2 subtype of CIV. Small interfering RNAs (siRNAs) specific to pattern recognition receptors (PRRs) and transcription factors were used to block the IFN-β induction signals in H3N2 CIV-infected KU-CBE cells. Among the PRRs, only the TLR3 and RIG-I expression levels significantly (p < 0.001) increased in CIV-infected cells. Following transfection with siRNA specific to TLR3 (siTLR3) or RIG-I (siRIG-I), the mRNA expression levels of IFN-β significantly (p < 0.001) decreased, and the protein expression of IFN-β also decreased in infected cells. In addition, co-transfection with both siTLR3 and siRIG-I significantly reduced IRF3 (p < 0.001) and IFN-β (p < 0.001) mRNA levels. Moreover, the protein concentration of IFN-β was significantly (p < 0.01) lower in cells co-transfected with both siTLR3 and siRIG-I than in cells transfected with either siTLR3 or siRIG-I alone. Also, the antiviral protein MX1 was only expressed in KU-CBE cells infected with CIV or treated with IFN-β or IFN-α. Thus, we speculate that IFN-β further induces MX1 expression, which might suppress CIV replication. Taken together, these data indicate that TLR3 and RIG-I synergistically induce IFN-β expression via the activation of IRF3, and the produced IFN-β further induces the production of MX1, which would suppress CIV replication in CIV-infected cells.

• The Korean Journal of Pharmacology
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• v.31 no.2
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• pp.241-250
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• 1995

Protein B23 is one of the major nucleolar phosphoproteins associated with pre-ribosomal particles, and is localized in the granular region of the nucleolus. Recent studies suggest that protein B23 shuttles between nucleus and cytoplasm and also interacts with HIV Rev. These findings indicate that protein B23 is important in nucleocytoplasmic relationship and viral replication. However, the exact function of protein B23 is not clear yet. In acute nucleolar hypertrophy of rat liver, treated with thioacetamide, there was observed an increase of not only protein B23 but also B23-like protein p45 when anti-B23 monoclonal antibody (MAb) was used for identification. On the basis of the large B23 specific epitope structure composed of 68 amino acids, a hypothesis was formulated to examine that p45 is the pre-B23 resulting from excessive production of B23. In an attempt to investigate the precursor of B23, we analyzed the subcellular fractions and microsomal subfractions. Subsequently, we analyzed the finger printings of B23-like proteins using the tryptic peptide mapping. The results are summarized: 1) Using B23 MAb, we observed the presence of B23-like proteins in nucleolar fraction, nucleoplasmic fraction and microsomal fraction. 2) In the further microsomal subfractionation, we could partially purify B23-like protein in 2M layer of sucrose gradient. 3) When ion exchange chromatography was employed, there were protein species 80kDa(p80), 65kDa(p65) and 60kDa(p60). 4) Based on the tryptic map analysis of $^{125}I$ labeled proteins, the similarity between B23 and p80 was found only in 9 out of 14(B23) and 21(p80) peptides, and difference was found in the remaining peptides. p80 and p60 had 18 common peptides, and all the peptides of p60 were similar to those of p80. From these results, it is proposed that p45 is an abnormal metabolite resulting from carcinogenesis by thioacetamide, and it is not the precursor of B23. In addition, we suggest that p80 may be a precursor of p45.

• Molecules and Cells
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• v.42 no.10
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• pp.721-728
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• 2019

Non-structural protein 1 (NS1) of influenza virus has been shown to inhibit the innate immune response by blocking the induction of interferon (IFN). In this study, we isolated two single-stranded RNA aptamers specific to NS1 with $K_d$ values of $1.62{\pm}0.30nM$ and $1.97{\pm}0.27nM$, respectively, using a systematic evolution of ligand by exponential enrichment (SELEX) procedure. The selected aptamers were able to inhibit the interaction of NS1 with tripartite motif-containing protein 25 (TRIM25), and suppression of NS1 enabled retinoic acid inducible gene I (RIG-I) to be ubiquitinated regularly by TRIM25. Additional luciferase reporter assay and quantitative real-time PCR (RT-PCR) experiments demonstrated that suppression of NS1 by the selected aptamers induced IFN production. It is noted that viral replication was also inhibited through IFN induction in the presence of the selected aptamers. These results suggest that the isolated aptamers are strongly expected to be new therapeutic agents against influenza infection.

• Animal cells and systems
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• v.5 no.3
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• pp.195-198
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• 2001

Hepatitis B virus (HBV) polymerase, which possesses the activities of terminal binding, DNA polymerase, reverse transcriptase and RNaseH, has been shown to accomplish viral DNA replication through a pregenomic intermediate. Because the HBV polymerase has not been purified, the expression of HBV polymerase was examined in an E. coli expression system that is under the regulation of arabinose operon. The expressed individual domain containing terminal binding protein, polymerase, or RNaseH turned out to be insoluble. The activities of those domains were not able to be recovered by denaturation and renaturation using urea or guanidine-HCI. The expressed reverse transcriptase containing the polymerase and RNaseH domains became extensively degraded, whereas the proteolysis was reduced in a Ion- mutant. These results indicate that Lon protease proteolyzes the HBV reverse transcriptase expressed in E. coli.