1-Aminocyclopropane-1-Carboxylate Deaminase from Pseudomonas fluorescens Promoting the Growth of Chinese Cabbage and Its Polyclonal Antibody

( Byoung Yul Soh ),( Gun Woong Lee ),( Eun Byeul Go ),( Byeo Ri Kim ),( Kui Jae Lee ),( Jong Chan Chae ) 한국미생물 · 생명공학회 2014 Journal of microbiology and biotechnology Vol.24 No.5

Bacterial 1-aminocyclopropane-1-carboxlyate (ACC) deaminase (AcdS) is an enzyme that cleaves ACC, a precursor of the plant hormone ethylene, into α-ketobutyrate and ammonia. The acdS gene was cloned from Pseudomonas fluorescens, which was capable of improving the seedling of Chinese cabbage under salinity condition. The recombinant AcdS (rAcdS) exhibited optimal activity at pH 8.5 and 30oC. Strong activity was sustained at up to 100 mM NaCl. The polyclonal anti-P. fluorescens AcdS antibody was produced in a rabbit that had been immunized with the purified rAcdS. This antibody successfully recognized the homologous antigens derived from the total proteins of isolated plant growth-promoting microorganisms. A statistically significant correlation was observed between the intensity of hybridization signal and AcdS activity measured by a biochemical method, suggesting its application as a useful indicator for active deaminases.

수은화합물 및 셀레늄이 EMT-6 세포에 미치는 면역독성학적 영향

소병율,고대하,염정호 大韓産業醫學會 1997 대한직업환경의학회지 Vol.9 No.3

The effects of selenium on the productions of ATP and nitrite which are inhibited by mercurial compounds, were examined in a cell culture system of EMT-6 cells. The cells were cultured in DMEM with cytokines, IL-1 and IFN-γ for 24 hours. The NO₂production of EMT-6 cells at the end of culture was significantly decreased by HgCl₂or CH₃HgCl added into the media in dose-dependent manner, and the viability of EMT-6 cells were decreased lower than 90% in the concentrations≥2.0μM HgCl₂or ≥1.0 μM CH₃HgCl. Simultaneous addition of the equimolar dose of selenium significantly increased synthesis of NO₂and ATP which were inhibited by mercurial compounds. But the single addition of selenium influenced neither the viability of cells nor the productions of NO₂and ATP. These results suggest that the disorder of cell mediated immunity by mercurials could be related to the inhibition of nitric oxide synthesis which seems to be caused by the inhibition of ATP synthesis. Administration of selenium with either HgCl₂or CH₃HgCl protected the cells against the cytotoxicity of mercurials. The protective effect of selenium on cytotoxicity of mercury is due to the formation of inactive complex of Se-Hg.

Antimalarial Effect of N-acetyl-L-Leucyl-L-leucyl-L-norleucinal by the Inhibition of Plasmodium falciparum Calpain

Suk-Yul Jung,Hyun Park,Bing Zheng,Yun-Young Choi,Byoung Yul Soh,김성연,박기인 대한약학회 2009 Archives of Pharmacal Research Vol.32 No.6

The biological understanding of malaria parasites has increased considerably over the past two decades with the discovery of many potential targets for the development of new antimalarial drugs. Calpain, a cysteine protease of Plasmodium falciparum, is believed to be a central mediator essential for parasitic activity. However, the utility of calpain as a potential anti-malarial target in P. falciparum has not been fully determined. In the present study, we determined the effect of N-acetyl-L-Leucyl-L-leucyl-L-norleucinal (ALLN)-treatment on the expression of calpain in erythrocytic stages of P. falciparum and its usefulness as an antimalarial chemotherapeutic agent. ALLN was shown to have low toxicity to HeLa cells but high toxicity to malaria. ALLN inhibited the expression of calpain in ring, trophozoite and schizont stages when treated for 48 h. Also, after 48 h, samples were characterized by 6.15% and 0% parasitemia without ALLN treatment and with ALLN treatment, respectively. Brightfield and confocal microscopy revealed that ALLN treatment affects merozoite maturation. As ALLN concentration increased from 1 μM to 100 μM, ring stage parasites did not mature into the schizont stage. When ALLN treatment was continued for 48 h, it also significantly inhibited the maturation of ring-stage parasites into trophozoite or schizont stages and survival of malarial parasites. Taken together, these findings suggest that ALLN inhibit the maturation and survival of P. falciparum and calpain expression, and thus has potential utility as an antimalarial chemotherapeutic agent.

Phase determination of a homogentisate dioxygenase from Comamonas sp. strain P19

Suk-Youl Park,Byoung Yul Soh,Jong-Chan Chae,Jeong-Sun Kim 한국구조생물학회 2017 Biodesign Vol.5 No.3

In Comamonas sp. strain P19, homogentisate 1,2-dioxygenase (HGO) catalyzes the conversion of homogentisate to 4-maleylacetoacetate by aromatic ring scission, in the breakdown of tyrosine and phenylalanine. To determine the molecular background of the enzymatic mechanism of HGO in this zinc-resistant organism, hmgA encoding HGO of Comamonas sp. strain P19 was cloned, and the expressed protein was purified. The protein was crystallized in solutions I [25% (w/v) polyethylene glycol 3350 and 0.1 M trisodium citrate at pH 5.6] and II [1.4 M ammonium tartrate and 0.1 M bisTris at pH 5.5]. X-ray diffraction data were collected to 1.8 Å resolution using synchrotron radiation. The crystal belongs to the orthorhombic space group P2 1 2 1 2 1 , with unit cell dimensions of a = 73.2 Å, b = 100.0 Å, and c = 134.9 Å. A traceable electron density map was calculated using anomalous diffraction data obtained from a crystal soaked in zinc ions.

RAW264.7 세포주를 이용한 수은화합물의 세포독성기전에 관한 고찰

김공호,고대하,소병율 大韓産業醫學會 1996 대한직업환경의학회지 Vol.8 No.3

Balb/c 마우스의 복강내에 Abelson leukemia virus(A-MuLV)를 주입하여 발생시킨 종양의 복수에서 기원한 RAW264.7 cell line을 배양하는 조건에 여러농도의 수은을 첨가하여 nitrite와 ATP 생성의 변화를 관찰한 결과는 다음과 같다. 수은화합물을 첨가한 배양조건에서 RAW264.7세포의 생존률은 mercury chloride의 경우는 0.8μM이하, methyl mercury chloride의 경우 0.4μM이하의 농도에서는 95% 이상으로 유지되었으며, 그 이상의 농도에서는 생존률이 현저히 감소되었다. 이때 ATP 및 nitrite의 생성능은 수은화합물의 농도증가에 따라 용량의존적으로 감소되었다. 배양액(DMEM)에 4.5g/ℓ의 glucose를 1배에서 5배를 추가하여 농도를 강화시킨 조건에서 RAW264.7 세포를 48시간동안 배양하면 ATP 및 nitrite 생성량은 전반적으로 1배 추가시 가장 많은 양이 생성되었고 배양액의 pH는 1배 추가시 6.7-6.8의 범위로 감소하였다가 glucose 농도 증가에 따라 대조군과 비슷한 7.2까지 증가하였다. 이때 세포의 생존률은 모든 군에서 95%이상으로 유지되었다. 그리고 배양액의 glucose 농도를 2배로 강화시킨 실험조건(배양액내 glucose 기준농도의 1배를 더 첨가한 경우)에서는 mercury chloride 및 methylmercury chloride의 농도를 증가시켰음에도 불구하고 ATP 및 nitrite 생성량은 감소되지 않고 대조군과 비슷한 값으로 유지되었다. 이상의 결과는 수은이 면역세포의 nitric oxide 생성을 저해하는 것은 세포내 ATP생성과 관련한 대사과정, 특히 구연산회로(citric acid cycle)의 억제를 통해 발휘된다는 것을 시사하고 있다. The effects of glucose on the productions of ATP and nitrite which are inhibited by mercury compounds, were examined in a cell culture system of RAW 264.7 cells. The cells were cultured in Dulbecco's Modified Eagle's medium(DMEM) with cytokines, IL-1 and TNF for 24 hours. The viability of RAW 264.7 cells at the end of culture was significantly decreased by mercury chloride or methylmercury chloride added into the media in dose-dependent manner, however the viability of RAW 264.7 cells were influenced in the concentrations less than 0.8μM of mercury chloride or 0.4μM of methylmercury chloride. The addition of 4.5g/l glucose to normal DMEM lowered the pH of media to the range of 6.7-6.8 after 48 hours of culture, but not for the cell survivals. This supplement of glucose to the media also prevented the inhibitions of ATP and nitrite syntheses which were caused by mercurial compounds. These results suggest that the disorder of cell mediated immunity by mercurials could be related to the inhibition of nitric oxide synthesis which seems to be caused by the inhibition of ATP synthesis, especially related to the citric acid cycle.

Rapid degradation of Pseudomonas fluorescens 1-aminocyclopropane-1-carboxylic acid deaminase proteins expressed in transgenic Arabidopsis.

Kim, Kangmin,Park, Sung-Hee,Chae, Jong-Chan,Soh, Byoung Yul,Lee, Kui-Jae Published by Elsevier/North Holland on behalf of t 2014 FEMS microbiology letters Vol.355 No.2

<P>1-Aminocyclopropane-1-carboxylate (ACC) deaminase is commonly produced by plant growth-promoting rhizobacteria (PGPR) and has been suggested to facilitate the growth and stress tolerance of hosts via a reduction in levels of ethylene. However, the regulatory mechanism of ACC deaminase (AcdS) protein within host plant cells is largely unknown. Here, we demonstrated beneficial effects and post-translational modification of PGPR-originated AcdS proteins in plants. Compared with the wild-type, transgenic Arabidopsis expressing the Pseudomonas fluorescens acdS (PfacdS) gene displayed increased root elongation and reduced sensitivity to 10 μM exogenous ACC, an ethylene precursor. Arabidopsis expressing PfacdS also showed increased tolerance to high salinity (150 mM NaCl). PfAcdS proteins accumulated in transgenic Arabidopsis were rapidly degraded, which was potentially mediated by the 26S proteasome pathway. The degradation of PfAcdS was alleviated in the presence of exogenous ACC. In conclusion, our data suggest that the plant growth-promoting effects of bacterial AcdS proteins are potentially modulated via protein turnover inside the host plant cells. Such post-translational modification plays a physiological role in the mutualistic interactions between microorganisms and plants in the rhizospheric and/or endospheric niche.</P>

The Mycobacterium avium subsp. Paratuberculosis protein MAP1305 modulates dendritic cell-mediated T cell proliferation through Toll-like receptor-4

( Su Jung Lee ),( Kyung Tae Noh ),( Tae Heung Kang ),( Hee Dong Han ),( Sung Jae Shin ),( Byoung Yul Soh ),( Jung Hee Park ),( Yong Kyoo Shin ),( Han Wool Kim ),( Cheol Heui Yun ),( Won Sun Park ),( I 생화학분자생물학회 2014 BMB Reports Vol.47 No.2

In this study, we show that Mycobacterium avium subsp. paratuberculosis MAP1305 induces the maturation of bone marrow-derived dendritic cells (BMDCs), a representative antigen presenting cell (APC). MAP1305 protein induces DC maturation and the production of pro-inflammatory cytokines (Interleukin (IL)-6), tumor necrosis factor (TNF)-α, and IL-1β) through Toll like receptor-4 (TLR-4) signaling by directly binding with TLR4. MAP1305 activates the phosphorylation of MAPKs, such as ERK, p38MAPK, and JNK, which is essential for DC maturation. Furthermore, MAP1305-treated DCs transform naive T cells to polarized CD4+ and CD8+ T cells, thus indicating a key role for this protein in the Th1 polarization of the resulting immune response. Taken together, M. avium subsp. Paratuber-culosis MAP1305 is important for the regulation of innate immune response through DC-mediated proliferation of CD4+ and CD8+ T cells. [BMB Reports 2014; 47(2): 115-120]

Mycobacterium abscessus D-alanyl-D-alanine dipeptidase induces the maturation of dendritic cells and promotes Th1-biased immunity

( Seung Jun Lee ),( Jong-hwa Jang ),( Gun Young Yoon ),( Da Rae Kang ),( Hee Jo Park ),( Sung Jae Shin ),( Hee Dong Han ),( Tae Heung Kang ),( Won Sun Park ),( Young Kyung Yoon ),( Byoung Yul Soh ),( 생화학분자생물학회(구 한국생화학분자생물학회) 2016 BMB Reports Vol.49 No.10

Mycobacterium abscessus, a member of the group of non-tuberculous mycobacteria, has been identified as an emerging pulmonary pathogen in humans. However, little is known about the protective immune response of antigenpresenting cells, such as dendritic cells (DCs), which guard against M. abscessus infection. The M. abscessus gene MAB1843 encodes D-alanyl-D-alanine dipeptidase, which catalyzes the hydrolysis of D-alanyl-D-alanine dipeptide. We investigated whether MAB1843 is able to interact with DCs to enhance the effectiveness of the host’s immune response. MAB1843 was found to induce DC maturation via toll-like receptor 4 and its downstream signaling pathways, such as the mitogen-activated protein kinase and nuclear factor kappa B pathways. In addition, MAB1843-treated DCs stimulated the proliferation of T cells and promoted Th1 polarization. Our results indicate that MAB1843 could potentially regulate the immune response to M. abscessus, making it important in the development of an effective vaccine against this mycobacterium. [BMB Reports 2016; 49(10): 554-559]