Agrobacterium tumefaciens KU12로부터 Octopine형 Ti 및 잠재 플라스미드의 제거에 의한 숙주 개발

하운환,이용욱,문혜연,심웅섭,Ha, Un-Hwan,Lee, Yong-Woog,Moon, Hye-Yeon,Sim, Woong-Seop 한국미생물학회 1994 미생물학회지 Vol.32 No.1

Agrobacterium tumefaciens KU12내에 존재하는 240kb 크기으 octopine형 Ti 플라스미드인 pTiKU12와 45kb 크기의 잠재 플라스미드인 pTi12를 제거하여 무독성의 A. tumefaciens 균주를 제조하였다. Octopine형 Ti 플라스미드인 pTiKU12는 고온(37${\circ}C$)에서의 배양과 ethidium bromide가 첨가되어 있는 배지에서의 배양을 각각 실시하여 제거하였으며, 잠재 플라스미드인 pTi12는 pTi12의 복제기원이 클로닝되어 잇는 재조합 플라스믿인 pYWXP와의 비화합성을 이용하여 제거하였다. pYWXP는 ethidium bromide가 첨가되어 있는 배지에서 고온(37${\circ}C$)으로 배양하여 제거하였다. Agrobacterium tumefaciens KU12 contains pTiKU12 (240kb) of the octopine type Ti plamsid and pTi12 (45 kb) of the cryptic plasmid. To make the avirulent A. tumefaciens, the octopine type Ti plasmid, pTiKU12, was cured with elevated temperature (37${\circ}C$) and ethidium bromide (EtBr), respectively. Also the cryptic plasmid, pTi12, was cured by the introduction of recombinant plasmid, pYWXP, made by pTi12 replication origin and pUC19. pYWXP was cured by elevated temperature (37${\circ}C$) and EtBr simultaneously.

각종 Ti 플라스미드의 Vir 유전자 발현에 미치는 페놀화합물과 숙주의 영향

하운환(Un Hwan Ha),이용욱(Yong Woog Lee),음진성(Jin Seong Eum),심웅섭(Woong Seop Sim) 한국식물학회 1995 Journal of Plant Biology Vol.38 No.1

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단백질 생합성에 관여하는 신장요인과 Phenylalanyl - tRNA Synthetase 및 tRNA 의 활성에 미치는 Polyamine 의 효과

하운환(Un Hwan Ha),한성수(Seong Su Han),심웅섭(Woong Seop Sim) 한국식물학회 1994 Journal of Plant Biology Vol.37 No.3

The effects of polyamines on the activities of elongation factors EF-1 and EF-2, phenylalanyl-tRNA synthetase, and tRNA were investigated. The activities of EF-1 and EF-2 were mostly stimulated by spermidine among three kinds of polyamines. The activities of EF-1 and EF-2 were iincreased in the presence of spermidine by 230 and 181%, respectively. The activity of Phenylalanyl-tRNA synthetase was slightly increased in the presence of polyamines. The effect of spermine on the synthetase was higher than that of the other polyamines. The tRNA activity in the presence of polyamines was increased by 206% with spermidine, by 144% with spermine, and by 114% with putrescine. According to these results, it is concluded that polyamines in higher plants stimulate the protein biosynthesis by promoting the activities of elongation factors EF-1 and EF-2, aminoacyl-tRNA synthetases, and tRNAs, but the effects of polyamines on the various components for protein biosynthesis are different in according to the kind of polyamines.

Effects of Cell-free Culture Fluids for the Expression of Putative Acyltransferase in Corynebacterium glutamicum

김용재,이흥식,하운환,Kim, Yong-Jae,Lee, Heung-Shick,Ha, Un-Hwan The Microbiological Society of Korea 2012 미생물학회지 Vol.48 No.3

이 연구의 목적은 코리네형 균주(Corynebacterium glutamicum)에 존재하는 acyltransferase 유전인자의 발현에 관여하는 autoinducer에 대한 정보를 얻기 위해 다양한 종류의 균주에서 얻은 세포배양액(cell-free culture fluids)을 처리하여 발현에 미치는 효과를 조사하는 것이다. 다양한 배양시간동안 얻은 Agrobacterium tumefaciens의 세포배양액은 acyltransferase의 발현을 전혀 증가시키지 못하는 것으로 관찰되었다. 반면 AI-1과 AI-2를 분비하는 것으로 알려진 Vibrio harveyi BB120($AI-1^+$, $AI-2^+$)을 지수성장기까지 배양시켜 얻은 세포배양액은 acyltransferase의 발현을 증가시키는 것으로 관찰되었다. 이들 autoinducer가 미치는 효과를 조사하기 위하여 돌연변이 균주인 MM77 ($AI-1^-$, $AI-2^-$)과 BB152 ($AI-1^-$, $AI-2^+$)를 사용하여 조사한 결과, 이들 균주에서 얻은 세포배양액은 BB120와는 달리 acyltransferase의 발현을 증가시키지 않는 것으로 나타났다. 이러한 결과로 보아 acyltransferase의 발현은 AI-1에 의한 효과로 보이며, 이를 확인하기 위해 V. harveyi BB152와 같이 AI-2만을 분비하는 것으로 알려진 Escherichia coli ($AI-1^-$, $AI-2^+$)를 사용하였다. 하지만 E. coli 세포배양액은 acyltransferase의 발현을 증가시키는 것으로 관찰되었다. 이들 결과는 코리네형 균주에 존재하는 autoinducer는 기존에 알려진 형태의 신호분자와 차이가 있을 것이며, 코리네형 균주외에 V. harveyi와 E. coli에도 존재하여 종간 상호작용(interspecies communication)에 관여할 것으로 보인다. Autoinduction is mediated by signaling molecules known as autoinducers (AIs) that are produced, released and detected by bacterium itself. We recently reported that Corynebacterium glutamicum possesses an autoinduction system which secretes autoinducers during the stationary-phase of growth, triggering the expression of acyltransferase gene. However, it is still not clear what may act as autoinducers for the autoinduction in C. glutamicum. In this study, we compared the inducing effects of cell-free culture fluids obtained from a number of microbes including Agrobacterium tumefaciens, Vibrio harveyi, and Escherichia coli. Fluids from A. tumefaciens did not increase the expression of acyltransferase, whereas fluids from V. harveyi BB120 ($AI-1^+$, $AI-2^+$) did. Interestingly, the expression was increased by the fluids obtained from the early exponential-phase culture of BB120. Furthermore, this induction was not observed by the fluids from autoinducer mutants of V. harveyi MM77 ($AI-1^-$, $AI-2^-$) and BB152 ($AI-1^-$, $AI-2^+$). Unlike the effect shown by BB152, fluids from E. coli ($AI-1^-$, $AI-2^+$) still induced the acyltransferase expression. Taken together, these results suggest that C. glutamicum autoinducers seem to be unidentified molecules which do not belong to AI-1 or AI-2.

Bradykinin Receptor의 발현에 미치는 녹농균유래 Nucleoside Diphosphate Kinase 및 Flagellin의 효과

김용재,신희성,하운환,Kim, Yong-Jae,Shin, Hee-Sung,Jin, Shouguang,Ha, Un-Hwan 한국미생물학회 2014 미생물학회지 Vol.50 No.4

Immune defense responses against Pseudomonas aeruginosa infection play an important role in maintaining homeostasis in the human body. Previously, we reported that expression of the bradykinin receptor (BR) is induced in response to P. aeruginosa infection. However, the factors responsible for the induction was uncertain. Here, we found that the type III secretion system (T3SS) is responsible for the induction of BR expression, and nucleoside diphosphate kinase (Ndk), as a novel T3SS effector, mediates the upregulation. The Ndk-mediated expression of BR was not induced by fliC mutant treatment, indicating the involvement of flagellin, one of the well-known pathogen-associated molecular patterns (PAMPs). Taken together, this study demonstrated that Ndk cooperates with flagella in the development of defense responses against P. aeruginosa infection. 병원성 균주인 녹농균(Pseudomonas aeruginosa) 감염에 대응하여 나타나는 면역반응은 인체의 항상성 유지에 중요하다. 선행 연구에서 녹농균의 감염에 대응하여 bradykinin receptor (BR)의 발현이 증가됨을 보고하였지만, 발현유도에 관여하는 녹농균 유래인자에 대해서는 보고한 바가 없었다. 이번 연구에서는 녹농균에 의한 BR의 발현은 Type III secretion system (T3SS)이 관여하지만, 기존에 알려진 T3SS인자가 아닌 nucleoside diphosphate kinase (Ndk)에 의한 것으로 조사되었다. 하지만 pDNNDK를 이용한 transfection 실험 결과, Ndk 만으로는 BR의 발현이 유도되지 않았으며, Ndk와 함께 flagella가 필요함을 발견하였다. 이러한 결과는 기존에 보고된 주요 pathogen-associated molecular patterns (PAMPs)인 flagella와 더불어 감염대응에 관여하는 Ndk를 발굴한 의미가 있으며, 녹농균에 의한 질병기전을 이해하는데 도움을 줄 수 있다.

Pneumolysin-Mediated Expression of β-Defensin 2 Is Coordinated by p38 MAP Kinase-MKP1 in Human Airway Cells

김용재,신희성,이정훈,정용우,김영배,하운환 한국미생물학회 2013 The journal of microbiology Vol.51 No.2

Antimicrobial peptides act as important innate immune defense mediators against invading microbes such as Streptococcus pneumoniae. Among a number of antimicrobial peptides, β-defensin 2 (BD2) has strong antimicrobial activity against S. pneumoniae. However, little is known about the molecular signaling mechanisms leading to the BD2expression. Here, we report that BD2 is strongly induced by S. pneumoniae in human airway cells including human middle-ear cells. Among diverse pneumococcal virulence factors, pneumolysin is required for inducing BD2 whose expression is under the control of p38 mitogen-activated protein kinase (MAPK). Pneumolysin also selectively regulates the expression of MAPK phosphatase 1 (MKP1), which inhibits the p38 signaling pathway, thereby leading to upregulation of BD2 to mount an effective defense against S. pneumoniae infection. These results provide novel insights into the molecular mechanisms underlying the coordinative regulation of BD2 expression via p38-MKP1 in the pathogenesis of airway infectious diseases.

Pseudomonas aeruginosa-induced IL-1β Production is Inhibited by Sophora flavescens via the NF-κB/inflammasome Pathways

이정훈,신희성,김용재,백세환,ShouguangJin쇼강진,하운환 한국미생물학회 2014 The journal of microbiology Vol.52 No.12

The proinflammatory cytokine interleukin-1β plays an importantrole in protecting the host against airway infection;however, it can also trigger a massive influx of neutrophilsinto the airways, causing tissue damage. Anti-inflammatorytreatments are particularly in demand for patients sufferingfrom chronic inflammatory diseases. Sophora flavescens is atraditional herbal medicine used to reduce inflammation,but no study has examined its ability to block IL-1β production. Here, we show that S. flavescens reduced the Pseudomonasaeruginosa-induced expression of IL-1β by lung epithelialcells and macrophages. S. flavescens was also effectiveat reducing IL-1β production induced by either Staphylococcusaureus or phorbol 12-myristate 13-acetate, indicatingthat the effect is generalizable to diverse inflammatorystimuli. In addition, S. flavescens blocked the phosphorylationof IKKα/β, key upstream kinases involved in the degradationof IκBα, and the cleavage of caspase-1, a key componentof the inflammasome. Thus, this study demonstratesthat S. flavescens exerts its anti-inflammatory effects by blockingP. aeruginosa-mediated NF-κB/inflammasome activationand the subsequent production of IL-1β.

Decreased Expression of Sphingosine-1-Phosphate Receptor 1 in the Blood Leukocyte of Rheumatoid Arthritis Patients

최현석,김경훈,진서훈,김진현,유인설,백승필,하운환,박태원,최수안,육순홍,강성욱,정용우 대한면역학회 2018 Immune Network Vol.18 No.5

Sphingosine-1-phosphate (S1P) plays an important role in trafficking leukocytes and developing immune disorders including autoimmunity. In the synovium of rheumatoid arthritis (RA) patients, increased expression of S1P was reported, and the interaction between S1P and S1P receptor 1 (S1P1) has been suggested to regulate the expression of inflammatory genes and the proliferation of synovial cells. In this study, we investigated the level of S1P1 mRNA expression in the blood leukocytes of RA patients. In contrast to the previous reports, the expression level of this gene was not correlated to their clinical scores, disease durations and ages. However, S1P1 was transcribed at a significantly lower level in the circulating leukocytes of RA patients when compared to age-, and sex-matched healthy controls. Since these data may suggest the participation of S1P1, further studies are needed to determine the role of this receptor in the pathogenesis of RA.

환경 스트레스 변화에 따른 슬러지내 항생제 내성 유전자 전이모델 개발

김두철 ( Doo Cheol Kim ),김용재 ( Yong Jae Kim ),오준식 ( Jun Sik Oh ),하운환 ( Un Hwan Ha ),윤주환 ( Zu Whan Yun ),김성표 ( Sung Pyo Kim ) 한국물환경학회(구 한국수질보전학회) 2011 한국물환경학회·대한상하수도학회 공동 춘계학술발표회 Vol.2011 No.-

The dose response of biofilm formation and antibiotic resistance gene transfer to the different concentrations of antibiotics

Dennis Espineli Salcedo,Jung Hoon Lee(이정훈),Un Hwan Ha(하운환),Sungpyo Kim(김성표) 한국산학기술학회 2014 한국산학기술학회 학술대회 Vol.- No.-

The environment is endangered by the micro-contaminants that are left untreated found in the aquatic system due to human activities and inadequacy and inefficiency of waste water treatment facilities. This study investigated the effects of low levels of antibiotics to the biofilm formation resulting in enhanced Antibiotic Resistance Gene (ARG) transfer. The biofilm formations of Escherichia coli, Pseudomonas aeruginosa, their combination, and their mixture with ppb levels of antibiotics were investigated. The effects on the conjugation of E. coli with P. aeruginosa in the biofilm structure were evaluated using biofilm assay in 96-well plates and ELISA. At 100 ppb to 1000 ppb of antibiotics, the mixed culture has the highest biofilm biomass and transconjugants, values greater than controls. This experiment shows that ppb levels of tetracycline can alter the transfer rate of pB10 plasmid at rates 2 to 5 times faster than without antibiotics in the biofilm, indicating the facilitated ARG spread by the presence of some microcontaminant residues in the environment.