• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.854-861
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• 2014

The diagnosis of Brucella abortus is mainly based on serological methods using antibody against LPS, which has diagnostic problems. Therefore, to solve this problem, we evaluated two proteins of B. abortus, Cu/Zn superoxide dismutase (SodC) and outer membrane proteins 2b porin (Omp2b). The genes were cloned and expressed in a pMAL system, and the recombinant proteins, rOmp2b and rSodC, were purified as fusion forms with maltose-binding protein. The identity of the proteins was confirmed by SDS-PAGE and Western blot analysis with sera of mice infected with B. abortus. Production of cytokines and nitric oxide (NO) was investigated in RAW 264.7 cells and mouse splenocytes after stimulation with the proteins. Moreover, cellular and humoral immune responses were investigated in BALB/c mice after immunization with the proteins. TNF-${\alpha}$, IL-6, and NO were significantly inducible in RAW 264.7 cells. Splenocytes of naive mice produced IFN-${\gamma}$ and IL-4 significantly by stimulation. Moreover, number of IgG, IFN-${\gamma}$, and IL-4 producing cells were increased in immunized mice with the two proteins. Production of IgG and IgM with rOmp2b was higher than those with rSodC in immunized mice. These results suggest that the two recombinant proteins of B. abortus may be potential LPS-free proteins for diagnosis.

• Applied Microscopy
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• 제30권3호
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• pp.241-248
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• 2000

전자현미경을 이용한 단백질 구조분석은 약 30여년간 발전되어 왔다. Bacteriorhodopsin과 porin 등의 near atomic resolution의 구조분석은 전자현미경의 발전과 진행을 보여주는 하나의 예로 설명될 수 있다. 전자현미경을 이용한 거대분자의 3차원 구조를 규명하기 위해 필요한 기본과정 즉, 시료준비, 자료수집과 자료처리 등에 대하여 토론하였다.

• 생명과학회지
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• 제18권11호
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• pp.1606-1611
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• 2008

Salmoenella를 포함한 그람 음성 세균의 외막 단백질들은 외부 환경 조건에 따라 그 발현에 변화가 생기는 경우가 많으며, 이렇게 발현된 외막 단백질들은 수송에 관여하는 통로, 외부 물질의 인지, 병원체의 부착인자 등 다양한 기능을 가진다. 본 연구에서는 이러한 외막 단백질 중 소수성 porin으로 알려진 OmpW 단백질에 주목하였다. ompW 유전자가 결손된 돌연변이주를 구축하여 CK10으로 명명하였으며, 야생주와 그 표현형적 특징을 비교한 결과 SDS에 대한 내성이 다소 증가함을 확인할 수 있었다. OmpW 특이적인 다클론성 항체를 조제하여 OmpW 단백질의 발현연구에 사용하였다. NaCl의 농도가 높아질수록 OmpW 단백질의 발현이 감소하였으며, OmpW의 발현이 최대인 조건은 NaCl이 배지에 첨가되지 않았을 경우이다. 따라서 OmpW는 Salmonella 균이 삼투환경에 대응하는데 관여할 것으로 예상되어 일차적으로 균의 생육을 조사한 결과, 다양한 삼투환경 하에서 Salmonella의 생육에는 OmpW 단백질의 결손이 큰 영향을 미치지는 않는 것을 확인하였다. 삼투환경 변화에 따른 OmpW 발현의 변화가 지니는 생물학적 의미는 앞으로 연구해야 할 숙제로 남는다.

• 한국미생물·생명공학회지
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• 제41권1호
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• pp.128-134
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• 2013

Salmonella enterica serovar typhi, a Gram-negative food-borne pathogen, causes typhoid fever in humans. OmpC is an outer membrane porin of S. typhi expressed throughout the infection period. OmpC is potentially an attractive antigen for multivalent vaccines and diagnostic kit designs. In this study we combined in silico, in vitro and in vivo approaches to analyze various aspects of OmpC's antigenic properties. The conserved region, in addition to secondary and tertiary structures, and linear B cell epitopes, were predicted. A number of results obtained from in silico analyses were validated by experimental studies. OmpC was amplified, cloned and then expressed, with the recombinant protein then being purified. BALB/c mice were immunized by purified denatured OmpC. The titer of antibody was raised. Results of challenges with the pathogen revealed that the immunity is non-protective. Most of the theoretical and experimental results were in consensus. Introduced linear B cell epitopes can be employed for the design of diagnostic kits based on antigen-antibody interactions.

• 대한핵의학회지
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• 제33권1호
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• pp.1-10
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• 1999

Cancer cells are known to show increased rates of glycolysis metabolism. Based on this, PET studies using F-18-fluorodeoxyglucose have been used for the detection of primary and metastatic tumors. To account for this increased glucose uptake, a variety of mechanisms has been proposed. Glucose influx across the cell membrane is mediated by a family of structurally related proteins known as glucose transporters (Gluts). Among 6 isoforms of Gluts, Glut-1 and/or Glut-3 have been reported to show increased expression in various tumors. Increased level of Glut mRNA transcription is supposed to be the basic mechanism of Glut overexpression at the protein level. Some oncogens such as src or ras intensely stimulate Glut-1 by means of increased Glut-1 mRNA levels. Hexokinase activity is another important factor in glucose uptake in cancer cells. Especially hexokinase type II is considered to be involved in glycolysis of cancer cells. Much of the hexokinase of tumor cells is bound to outer membrane of mitochondria by the porin, a hexokinase receptor. Through this interaction, hexokinase may gain preferred access to ATP synthesized via oxidative phosphorylation in the inner mitochondria compartment. Other biologic factors such as tumor blood flow, blood volume, hypoxia, and infiltrating cells in tumor tissue are involved. Relative hypoxia may activate the anaerobic glycotytic pathway. Surrounding macrophages and newly formed granulation tissue in tumor showed greater glucose uptake than did viable cancer cells. To expand the application of FDG PET in oncology, it is important for nuclear medicine physicians to understand the related mechanisms of glucose uptake in cancer tissue.

• Molecules and Cells
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• 제27권3호
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• pp.321-327
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• 2009

Voltage-dependent anion channels (VDACs) are reported to be porin-type, ${\beta}$-barrel diffusion pores. They are prominently localized in the outer mitochondrial membrane and are involved in metabolite exchange between the organelle and the cytosol. In this study, we have investigated a family of VDAC isoforms in Arabidopsis thaliana (AtVDAC). We have shown that the heterologous expression of AtVDAC proteins can functionally complement a yeast mutant lacking the endogenous mitochondrial VDAC gene. AtVDACs tagged with GFP were localized to mitochondria in both yeast and plant cells. We also looked at the response of AtVDACs to biotic and abiotic stresses and found that four AtVDAC transcripts were rapidly up-regulated in response to a bacterial pathogen.

• Journal of Microbiology and Biotechnology
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• 제22권4호
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• pp.470-478
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• 2012

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane ${\beta}$-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.

• 약학회지
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• 제40권1호
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• pp.102-111
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• 1996

LB10522 is a new parenteral broad spectrum cephalosporin with a catechol moiety at C-7 position of beta-lactam ring. This compound can utilize tonB-dependent iron transp ort system in addition to porin proteins to enter bacterial periplasmic space and access to penicillin-binding proteins (PBPs) which are the lethal targets of ${\beta}$-lactam antibiotics. The chelating activity of LB10522 to metal iron was measured by spectrophotometrically scanning the absorbance from 200 to 900nm. When $FeCl_3$ was added, optical density was increased between 450 and 800nm. LB10522 was more active against gram-negative strains in iron-depleted media than in iron-replete media. This is due to the increased expression of iron transport channels in iron-depleted condition. LB10522 showed a similar activity against E. coli DC2 (permeability mutant) and E. coli DCO (wild type strain) in both iron-depleted and iron-replete media, indicating a minimal permeaility barrier for LB10522 uptake. LB10522 had high affinities to PBP 3 and PBP 1A, 1B of E. coli. By blocking these proteins, LB10522 caused inhibition of cell division and the eventual death of cells. This result was correlated well with the morphological changes in E. coli exposed to LB10522. Although the in vitro MIC of LB10522 against P. aeruginosa 1912E mutant (tonB) was 8-times higher than that of the P. aeruginosa 1912E parent strain, LB10522 showed a similar in vivo protection efficacy against both strains in the mouse systemic infection model. This result suggested that tonB mutant, which requires a high level of iron for normal growth, might have a difficulty in surviving in their host with an iron-limited environment.

• International Journal of Oral Biology
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• 제42권2호
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• pp.63-70
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• 2017

Selecting an appropriate antigen with optimal immunogenicity and physicochemical properties is a pivotal factor to develop a protein based subunit vaccine. Despite rapid progress in modern molecular cloning and recombinant protein technology, there remains a huge challenge for purifying and using protein antigens rich in hydrophobic domains, such as membrane associated proteins. To overcome current limitations using hydrophobic proteins as vaccine antigens, we adopted in silico analyses which included bioinformatic prediction and sequence-based protein 3D structure modeling, to develop a novel periodontitis subunit vaccine against the outer membrane protein FomA of Fusobacterium nucleatum. To generate an optimal antigen candidate, we predicted hydrophilicity and B cell epitope parameter by querying to web-based databases, and designed a truncated FomA (tFomA) candidate with better solubility and preserved B cell epitopes. The truncated recombinant protein was engineered to expose epitopes on the surface through simulating amino acid sequence-based 3D folding in aqueous environment. The recombinant tFomA was further expressed and purified, and its immunological properties were evaluated. In the mice intranasal vaccination study, tFomA significantly induced antigen-specific IgG and sIgA responses in both systemic and oral-mucosal compartments, respectively. Our results testify that intelligent in silico designing of antigens provide amenable vaccine epitopes from hard-to-manufacture hydrophobic domain rich microbial antigens.

• Journal of Microbiology and Biotechnology
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• 제32권9호
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• pp.1195-1208
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• 2022

Silver nanoparticles (AgNPs) have potential applications in medicine, photocatalysis, agriculture, and cosmetic fields due to their unique physicochemical properties and strong antimicrobial activity. Here, AgNPs were synthesized using actinobacterial SL19 strain, isolated from acidic forest soil in Poland, and confirmed by UV-vis and FTIR spectroscopy, TEM, and zeta potential analysis. The AgNPs were polydispersed, stable, spherical, and small, with an average size of 23 nm. The FTIR study revealed the presence of bonds characteristic of proteins that cover nanoparticles. These proteins were then studied by using liquid chromatography with tandem mass spectrometry (LC-MS/MS) and identified with the highest similarity to hypothetical protein and porin with molecular masses equal to 41 and 38 kDa, respectively. Our AgNPs exhibited remarkable antibacterial activity against Escherichia coli and Pseudomonas aeruginosa. The combined, synergistic action of these synthesized AgNPs with commercial antibiotics (ampicillin, kanamycin, streptomycin, and tetracycline) enabled dose reductions in both components and increased their antimicrobial efficacy, especially in the case of streptomycin and tetracycline. Furthermore, the in vitro activity of the AgNPs on human cancer cell lines (MCF-7, A375, A549, and HepG2) showed cancer-specific sensitivity, while the genotoxic activity was evaluated by Ames assay, which revealed a lack of mutagenicity on the part of nanoparticles in Salmonella Typhimurium TA98 strain. We also studied the impact of the AgNPs on the catalytic and photocatalytic degradation of methyl orange (MO). The decomposition of MO was observed by a decrease in intensity of absorbance within time. The results of our study proved the easy, fast, and efficient synthesis of AgNPs using acidophilic actinomycete SL19 strain and demonstrated the remarkable potential of these AgNPs as anticancer and antibacterial agents. However, the properties and activity of such particles can vary by biosynthesized batch.