Partial purification of HCV replicase and in vitro reconstitution of HCV replication

장태용,명희준,박찬희 한국외국어대학교 외국학종합연구센터 부설 기초과학연구소 1999 기초과학연구 Vol.7 No.-

Hepatitis C virus(HCV)는 수혈에 의한 C형 간염을 일으킨다. HCV는 positive sense의 single stranded RNA를 그 genome으로 가지며, 자신의 고유한 RNA-dependent RNA polymerase를 이용하여 복제한다. 본 연구에서는 HCV의 replicase를 부분적으로 정제하여 in vitro에서 HCV replication을 재구성했다. 이 system은 향후 HCV replicase inhibitor의 screening에 이용될 수 있을 것이다. Hepatitis C virus is an RNA virus that causes blood transmitted chronic hepatitis. The genome encodes one open reading frame(ORF) and replicate using an RNA-dependent RNA polymerase (replicase) in the NS5B region. This replicase is dependent on RNA genome of HCV. In this study, we have partially purified this replicase and reconsitituted HCV replication in vitro. This system will be used to screen various replicase inhibitors in the future.

Molecular Cloning, Purification, and Characterization of an Extracellular Nuclease from Aeromonas hydrophila ATCC14715

NAM, IN-YOUNG,MYUNG, HEEJOON,JOH, KISEONG 한국미생물 · 생명공학회 2004 Journal of microbiology and biotechnology Vol.14 No.1

A gene encoding an extracellular nuclease was cloned from Aeromonas hydrophila strain ATCC147 15. The gene was overexpressed and the enzyme was purified by fusing to maltose binding protein. It was shown that the protein possessed DNase activity on both single-stranded and double-stranded DNAs. It exhibited both endo- and exonuclease activities. It was also shown that the protein had an RNase activity. Possible roles of this extracellular enzyme in the A. hydrophila life cycle are discussed.

Eradication of drug-resistant Acinetobacter baumannii by cell-penetrating peptide fused endolysin

Lim Jeonghyun,Jang Jaeyeon,Myung Heejoon,Song Miryoung 한국미생물학회 2022 The journal of microbiology Vol.60 No.8

Antimicrobial agents targeting peptidoglycan have shown successful results in eliminating bacteria with high selective toxicity. Bacteriophage encoded endolysin as an alternative antibiotics is a peptidoglycan degrading enzyme with a low rate of resistance. Here, the engineered endolysin was developed to defeat multiple drug-resistant (MDR) Acinetobacter baumannii. First, putative endolysin PA90 was predicted by genome analysis of isolated Pseudomonas phage PBPA. The His-tagged PA90 was purified from BL21(DE3) pLysS and tested for the enzymatic activity using Gram-negative pathogens known for having a high antibiotic resistance rate including A. baumannii. Since the measured activity of PA90 was low, probably due to the outer membrane, cell-penetrating peptide (CPP) DS4.3 was introduced at the N-terminus of PA90 to aid access to its substrate. This engineered endolysin, DS-PA90, completely killed A. baumannii at 0.25 μM, at which concentration PA90 could only eliminate less than one log in CFU/ml. Additionally, DS-PA90 has tolerance to NaCl, where the ~50% of activity could be maintained in the presence of 150 mM NaCl, and stable activity was also observed with changes in pH or temperature. Even MDR A. baumannii strains were highly susceptible to DS-PA90 treatment: five out of nine strains were entirely killed and four strains were reduced by 3–4 log in CFU/ml. Consequently, DS-PA90 could protect waxworm from A. baumannii-induced death by ~70% for ATCC 17978 or ~44% for MDR strain 1656-2 infection. Collectively, our data suggest that CPP-fused endolysin can be an effective antibacterial agent against Gramnegative pathogens regardless of antibiotics resistance mechanisms.

SEU Measurements of Memory Chips for the Langmuir Probe on STSAT-2

Kwangsun Ryu,Goo-Hwan Shin,Heejoon Kim,hyung-Myung Kim,민경욱 한국물리학회 2008 THE JOURNAL OF THE KOREAN PHYSICAL SOCIETY Vol.52 No.3

We have measured the single event effect (SEE) of candidate memory chips for a Langmuir probe, one of the secondary payloads of STSAT-2 (Science and Technology Satellite-2). The measurements were performed using the cyclotron proton accelerator at Korea Institute of Radiological and Medical Sciences. An ion chamber detector was used for the calibration of the flux of the proton beam. SEU (single event upset) cross-sections for 3 different kinds of memory chips were derived according to the incident proton energy. The SEU rate at the STSAT-2 orbit environment was estimated from the SEU cross-section and the modeled particle flux. The program memory chip for the flight model was selected from the candidates.

Molecular Mechanism of NFAT Family Proteins for Differential Regulation of the IL-2 and TNF-a Promoters

Ji-Hyun Oum,Juhyun Han,Heejoon Myung,Marija Hleb,Surendra Sharma,Jungchan Park 한국분자세포생물학회 2002 Molecules and cells Vol.13 No.1

Nuclear factor of activated T cells (NFAT) is a family of transcription factors that regulates activationinduced transcription of many immunologically important genes. Although all NFAT family proteins contain a highly conserved DNA-binding domain and also bind cooperatively with AP-1 proteins to the interleukin- 2 (IL-2) promoter NFAT site, each member shows characteristic site preferences to other promoters. Previously, we have shown that NFATc.b, an isoform of NFATc, is different from NFATp in both DNA binding and transactivation of the TNF-a promoter. To further characterize target gene specificity of NFATc and NFATp, we generated deletion mutants as well as mutants swapping the C-terminal region of their DNA binding domains, and analyzed their DNAbinding specificity to different target sites. Our results show that the C-terminal one third of DNA binding domain confers different binding specificity of NFATc and NFATp to an NFAT site in the TNF-a promoters. Transient expression of the mutant NFAT proteins also demonstrates that transcriptional activation of the target promoters is consistent with the DNA binding specificity of the mutant NFATs. These results strongly suggest that a binding site preference and availability of different NFAT proteins may program the temporal expression of distinct cytokine genes. Importantly, the C-terminal region of the DNA binding domain plays an important role in determining the binding site preferences at least of NFATp and NFATc members.

Development of an assay system for helicase and replicase of hepatitis C virus

Kee, Younghoon,Park, Chanhee,Kim, Jinyoung,Myung, Heejoon 한국외국어대학교 기초과학연구소 2001 기초과학연구 Vol.11 No.-

Hepatitis C virus(HCV) infection is a global health problem affecting an estimated 170 million individuals worldwide. Despite increasing knowledge of genome structure and individual viral proteins, studies on viral replication and pathogenesis have been hampered by low efficiencies of currently available cell culture. As a beginning effort to our goal of establishing an efficient cell line system for HCV replication, recombinant replicase and helicase of HCV was purified. Using 5'UTR of HCV RNA as the template, both proteins were incubated with in in vitro translation assay system to measure reporter activity. Combination of both proteins showed a reporter signal. Also, the exact open structure of RNA template for replicase recognition was not found to be necessary. Based on this assay system, an efficient cell-based assay for both helicase and replicase can be developed.

Phage Therapy in Korea: A Prescribers' Survey of Attitudes Amongst Korean Infectious Diseases Specialists Towards Phage Therapy

Lee Shinwon,Lynch Stephanie,Lin Ruby C Y,Myung Heejoon,Iredell Jonathan R 대한감염학회 2024 Infection and Chemotherapy Vol.56 No.1

Background Concerns about the rise in antimicrobial resistance have led to renewed interest in phage therapy worldwide, but perceptions among relevant medical professionals in Korea remain largely unknown. Materials and Methods We conducted a semi-quantitative online survey to evaluate the Korean infectious disease specialists' perception of phage therapy. Results We sent out the link to the questionnaire to 380 subjects and received 91 replies, with 90/91 respondents identifying as Korean infectious diseases specialists or trainees. Ten out of 91 (11.0%) respondents scored themselves as well-informed about phage therapy. The majority (93.4%) of respondents would consider using phage therapy if the safety of the phage formulation is guaranteed, and 80% of respondents would consider participating in clinical trials with phage therapy given adequate support. The biggest concern was uncertainty about safety (73.6%) and efficacy (65.9%). Acinetobacter baumannii was ranked as a high priority for phage therapy research, as were bone and joint infections. Conclusion Korean infectious diseases specialists are receptive to phage therapy, but a better understanding of safety, efficacy and clinical trials are warranted to progress phage therapy within the Korean healthcare system. Background Concerns about the rise in antimicrobial resistance have led to renewed interest in phage therapy worldwide, but perceptions among relevant medical professionals in Korea remain largely unknown. Materials and Methods We conducted a semi-quantitative online survey to evaluate the Korean infectious disease specialists' perception of phage therapy. Results We sent out the link to the questionnaire to 380 subjects and received 91 replies, with 90/91 respondents identifying as Korean infectious diseases specialists or trainees. Ten out of 91 (11.0%) respondents scored themselves as well-informed about phage therapy. The majority (93.4%) of respondents would consider using phage therapy if the safety of the phage formulation is guaranteed, and 80% of respondents would consider participating in clinical trials with phage therapy given adequate support. The biggest concern was uncertainty about safety (73.6%) and efficacy (65.9%). Acinetobacter baumannii was ranked as a high priority for phage therapy research, as were bone and joint infections. Conclusion Korean infectious diseases specialists are receptive to phage therapy, but a better understanding of safety, efficacy and clinical trials are warranted to progress phage therapy within the Korean healthcare system.

Bactericidal Effect of Cecropin A Fused Endolysin on Drug-Resistant Gram-Negative Pathogens

Lim, Jeonghyun,Hong, Juyeon,Jung, Yongwon,Ha, Jaewon,Kim, Hwan,Myung, Heejoon,Song, Miryoung The Korean Society for Microbiology and Biotechnol 2022 Journal of microbiology and biotechnology Vol.32 No.6

The rapid spread of superbugs leads to the escalation of infectious diseases, which threatens public health. Endolysins derived from bacteriophages are spotlighted as promising alternative antibiotics against multi-drug resistant bacteria. In this study, we isolated and characterized the novel Salmonella typhimurium phage PBST08. Bioinformatics analysis of the PBST08 genome revealed putative endolysin ST01 with a lysozyme-like domain. Since the lytic activity of the purified ST01 was minor, probably owing to the outer membrane, which blocks accessibility to peptidoglycan, antimicrobial peptide cecropin A (CecA) was fused to the N-terminus of ST01 to disrupt the outer membrane. The resulting CecA::ST01 has been shown to have increased bactericidal activity against gram-negative pathogens including Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Enterobacter cloacae and the most affected target was A. baumannii. In the presence of 0.25 µM CecA::ST01, A. baumannii ATCC 17978 strain was completely killed and CCARM 12026 strain was wiped out by 0.5 µM CecA::ST01, which is a clinical isolate of A. baumannii and resistant to multiple drugs including carbapenem. Moreover, the larvae of Galleria mellonella could be rescued up to 58% or 49% by the administration of CecA::ST01 upon infection by A. baumannii 17978 or CCARM 12026 strain. Finally, the antibacterial activity of CecA::ST01 was verified using 31 strains of five gram-negative pathogens by evaluation of minimal inhibitory concentration. Thus, the results indicate that a fusion of antimicrobial peptide to endolysin can enhance antibacterial activity and the spectrum of endolysin where multi-drug resistant gram-negative pathogens can be efficiently controlled.

Implications of Exonuclease Activity of Bacteriophage P2 Old Protein for Lambda Exclusion

KIM, KWANGHO,PARK, CHANHEE,YEO, HYEONJOO,KEE, YOUNGHOON,PARK, JUNGCHAN,MYUNG, HEEJOON 한국미생물 · 생명공학회 2000 Journal of microbiology and biotechnology Vol.10 No.2

Temperate bacteriophage P2 has a nonessential gene called old (overcoming lysoginization defection). In the presence of old, the growth of the host (Escherichia coli) with recBC^- genotype is inhibited, and another bacteriophage, lambda, cannot superinfect. The Old protein has been shown to possess an exonuclease activity. Three mutant P2s (old 1. old 17, old 49) which did not exhibit lambda exclusion were obtained and each old gene was cloned into expression vectors to produce hexahistidine-tagged proteins. The proteins were affinity-purified and shown to lose its exonuclease activity on both double-stranded and single-stranded DNA substrates. Thus, it was concluded that the lambda exclusion was related to Old's exonuclease activity.

Hepatitis C Virus Replication-Specific Inhibition of MicroRNA Activity with Self-Cleavable Allosteric Ribozyme

Lee, Chang Ho,Kim, Ji Hyun,Kim, Hee Won,Myung, Heejoon,Lee, Seong-Wook Mary Ann Liebert 2012 Nucleic acid therapeutics Vol.22 No.1