• 셀메드
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• 제4권1호
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• pp.6.1-6.12
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• 2014

Anthrax is the deadly disease for human being caused by Bacillus anthracis. Instantaneous research work on the mode of infection of the organism revealed that different proteases are involved in different steps of pathogenesis. Present study reports the in silico characterization and the detection of pathogenic proteases involved in anthrax infection through protein-protein interaction. A total of 13 acid, 9 neutral, and 1 alkaline protease of Bacillus anthracis were selected for analysing the physicochemical parameter, the protein superfamily and family search, multiple sequence alignment, phylogenetic tree construction, protein-protein interactions and motif finding. Among the 13 acid proteases, 10 were found as extracellular enzymes that interact with immune inhibitor A (InhA) and help the organism to cross the blood brain barrier during the process of infection. Multiple sequence alignment of above acid proteases revealed the position 368, 489, and 498-contained 100% conserved amino acids which could be used to deactivate the protease. Among the groups analyzed, only acid protease were found to interact with InhA, which indicated that metalloproteases of acid protease group have the capability to develop pathogenesis during B. anthracis infection. Deactivation of conserved amino acid position of germination protease can stop the sporulation and germination of B anthracis cell. The detailed interaction study of neutral and alkaline proteases could also be helpful to design the interaction network for the better understanding of anthrax disease.

• 한국광과학회:학술대회논문집
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• 한국광과학회 2003년도 학술대회지
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• pp.58-68
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• 2003

• 치위생과학회지
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• 제11권1호
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• pp.55-61
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• 2011

최근에 Odontogenic ameloblast-associated protein (ODAM)은 MMP-20의 발현을 조절하여 법랑모세포 분화와 법랑질의 석회화에 중요한 역할을 한다고 보고되었다. 그러나 이에 대한 명확한 기전은 알려져 있지 않다. 그러므로 이 연구의 목적은 법랑모세포 분화와 법랑질의 석회화 과정에서의 ODAM의 생물학적 기능과 신호 전달 경로를 찾고자 하였다. Ameloblast-lineage cells (ALCs)를 이용하여 ODAM 재조합 단백질을 생성하고 ODAM 과발현 (ODAM overexpressing) 또는 ODAM 억제(ODAM silencing) 세포주를 만들었다. 세포들은 2주 동안 분화 배지에서 ODAM 재조합 단백질을 처리한 군과 처리하지 않는 군으로 나누어 배양하였다. ODAM의 신호 전달 경로를 확인하기 위하여, ALCs에 BMP2와 BMP receptor 1B (BMPR-1B) 억제제인 BAMBI 재조합 단백질을 처리하였고, 또한 BMPR-1B siRNA 이용하여 BMPR-1B의 발현을 억제하였다. 단백질 발현은 western blot 이용하여 분석하였다. 석회화는 sense ODAM 과발현 세포와 ODAM 재조합 단백질을 첨가한 법랑모세포 세포주에서 증진되었다. 또한 ALP 활성화는 sense ODAM 과발현 세포와 ODAM 정제된 단백질를 첨가한 법랑모세포 세포주에서 뚜렷하게 증진되었다. 기관발생과 관련이 있는 BMPR-IB와 석회화 과정과 관련된 CBP2는 ODAM 과발현을 유도한 경우에는 발현이 증가되었으나, ODAM 발현을 억제시킨 경우에는 발현이 현저히 감소하였다. 이상 실험의 결과는 법랑질 형성과정에서 ODAM이 법랑질 석회화를 증진시킬 수 있음을 시사한다.

• 한국작물학회지
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• 제57권1호
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• pp.60-70
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• 2012

This study was conducted to compare the protein characteristics, dough rheology and bread loaf volume of Korean wheat cultivars and CIMMYT lines produced in diverse environments and to determine the genetic and environmental effects on bread making quality. Protein characteristics, including protein content and SDS-sedimentation volume, mixing properties during dough development and bread loaf volume were primarily influenced by the environment. Wheat cultivated in Jinju exhibited higher SDS-sedimentation volume based on constant protein weight and bread loaf volume than those in Suwon and Iksan. SDS-sedimentation volume based on constant protein weight, mixing time of mixograph and mixing tolerance of mixograph were positively correlated with bread volume. Korean wheat cultivars showed different allelic variations of $Glu-1$ and $Glu-3$ compared to CIMMYT wheat lines. Alchanmil, Keumkangmil and Tapdongmil could be suitable for bread making because these cultivars exhibited a 10 point $Glu-1$ score. However, Korean wheat cultivars should be introduced specific alleles in $Glu-3$ loci, including $Glu-A3b$ or $d$ and $Glu-B3b$, $d$, $f$ or $g$, to improve gluten strength related to increase bread loaf volume.

• BMB Reports
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• 제35권2호
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• pp.206-212
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• 2002

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2B-NS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.

• BMB Reports
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• 제34권4호
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• pp.293-298
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• 2001

Tryptic activation of the 130-kDa Bacillus thuringiensis Cry4B $\delta$-endotoxin produced protease-resistant products of ca. 47 kDa and ca. 21 kDa. The 21-kDa fragment was identified as the N-terminal five-helix bundle (${\alpha}1-{\alpha}5$,) which is a potential candidate for membrane insertion and pore formation. In this study, we constructed the recombinant clone over-expressing this putative pore-forming (PPF) fragment as inclusion bodies in Escherichia coli. The partially purified inclusions were composed of a 23-kDa protein, which cross-reacted with Cry4B antibodies, and whose N-terminus was identical to that of the 130-kDa protein. Dissimilar to protoxin inclusions, the PPF inclusions were only soluble when the carbonate buffer, pH 9.0, was supplemented with 6 M urea. After renaturation via a stepwise dialysis, the refolded PPF protein appeared to exist as an oligomer and was structurally stable upon trypsin treatment. Unlike the 130kDa protoxin, the refolded protein was able to release entrapped glucose from liposomes, and showed comparable activity to the full-length activated toxin, although it lacks larvicidal activity These results, therefore, support the notion that the PPF fragment that consists of ${\alpha}1-{\alpha}5$ of the activated Cry4B toxin is involved in membrane pore-formation.

• The Korean Journal of Physiology and Pharmacology
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• 제6권6호
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• pp.319-325
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• 2002

The induction of interleukin-6 (IL-6) using combined proinflammatory agents $(LPS/IFN-{\gamma}\;or\;TNF-{\alpha}/IFN-{\gamma})$ was studied in relation to p38 mitogen-activated protein kinase (MAPK) and $NF-{\kappa}B$ transcriptional factor in primary neonatal cardiomyocytes. When added to cultures of cardiomyocytes, the combined agents $(LPS/IFN-[\gamma}\;or\;TNF-{\alpha}/IFN-{\gamma})$ had stimulatory effect on the production of IL-6 and the elevation was significantly reduced by SB203580, a specific p38 MAPK inhibitor. SB203580 inhibited protein production and gene expression of IL-6 in a concentration-dependent manner. In this study, $IFN-{\gamma}$ enhancement of $TNF-{\alpha}-induced\;NF-{\kappa}B$ binding affinity as well as p38 MAP kinase activation was observed. However, a specific inhibitor of p38 MAPK, SB203580, had no effect on $TNF-{\alpha}/IFN-{\gamma}\;or\;LPS/IFN-{\gamma}-induced\;NF-{\kappa}B$ activation. This study strongly suggests that these pathways about $TNF-{\alpha}/IFN-{\gamma}$ or $LPS/IFN-{\gamma}-activated$ IL-6 release can be primarily dissociated in primary neonatal cardiomyocytes.

• Journal of Periodontal and Implant Science
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• 제33권2호
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• pp.179-191
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• 2003

Since periodontal infections are suggested as risk factors for the development of cardiovascular diseases, the present study was performed to evaluate the T cell immune responses specific to Pophylomonas gingivalis(P. gingivalis) heat shock protein(hsp) 60 and T-cell and B-cell epitope specificities for P. gingivalis hsp60 in atherosclerosis. Anti-P, gingivalis IgG antibody titers were elevated in all patients. We could establish P. gingivalis hsp-specific T cell lines from the atheroma lesions, a mixture of $CD4^+$ and $CD8^+$ cells producing the cytokines characteristic of both Th1 and Th2 subsets. of 108 overlapping synthetic peptides spanning whole P. gingivalis hsp60 molecule, ten peptides with common epitopes specificities for both T-cell and B-cell were identified. it was concluded that P. gingivalis hsp60 might K involved in the immunoregulatory process of atherosclerotic diseases with epitope specificities.

• The Plant Pathology Journal
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• 제25권4호
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• pp.344-351
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• 2009

A biocontrol bacterium Bacillus licheniformis N1 grown in nutrient broth showed no chitinolytic activity, while its genome contains a gene which encodes a chitinase. The gene for chitinase from B. licheniformis N1 was amplified by PCR and the deduced amino acid sequence analysis revealed that the chitinase exhibited over 95% identity with chitinases from other B. licheniformis strains. Escherichia coli cells carrying the recombinant plasmid displayed chitinase activity as revealed by the formation of a clear zone on chitin containing media, indicating that the gene could be expressed in E. coli cells. Chitinase gene expression in B. licheniformis N1 was not detected by RT-PCR analysis. The protein was over-expressed in E. coli BL21 (DE3) as a glutathione S-transferase fusion protein. The protein could also be produced in B. subtilis 168 strain carrying the chitinase gene of N1 strain. The crude protein extract from E. coli BL21 carrying GST fusion protein or culture supernatant of B. subtilis carrying the chitinase gene exhibited enzyme activity by hydrolyzing chitin analogs, 4-methylumbelliferyl-$\beta$-D-N,N'-diacetylchitobioside and 4-methylumbelliferyl-$\beta$-D-N,N',N"-triacetylchitotrioside. These results indicated that even though the chitinase gene is not expressed in the N1 strain, the coding region is functional and encodes an active chitinase enzyme. Furthermore, B. subtilis 168 transformants expressing the chitinase gene exhibited antifungal activity against Fulvia fulva by suppressing spore germination. Our results suggest that the proper engineering of the expression of the indigenous chitinase gene, which will lead to its expression in the biocontrol strain B. licheniformis N1, may further enhance its biocontrol activity.

• 한국환경농학회지
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• 제22권3호
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• pp.203-209
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• 2003

항진균 활성균주를 선발하는 과정에서 DM3 균주를 대천 바닷가에서 수집된 진흙 시료로부터 분리하였으며 API 50CHB kit를 이용하여 동정한 결과 Bacillus licheniformis로 동정되었다. 이 균주는 12종의 식물병원성 진균에 대해 항균활성을 나타내었다. 감마선($^{60}Co$)을 조사하여 항진균 활성 결핍 돌연변이체(mDMB)를 유도한 후, 이차원전기영동으로 단백질을 분석한 결과 DM3와 mDM3에만 존재하는 각각 4종과 3종의 단 단백질을 확인할 수 있었다. 2-DE 결과 B. licheniformis DM3에서 spot 1은 serine hydroxymethyltransferase(45.0kDa), spot 2는 hypothetical protein(40.7 kDa), spot 3는 NifU protein homolog(15.4 kDa), 그리고 spot 4는 resolvase(12.5 kDa)와 상동성을 지닌 단백질로 동정되었고 B. licheniformis mDM3에서만 발현된 spot 5는 lysozyme(18.1 kDa)과 spot 6, 7은 alkyl hydroperoxide reductase(15.6 kDa)으로 동정되었다. B. licheniformis DM3에서 이들 단백질들의 항진균 활성 관련 여부를 규명하기 위해서 더 연구가 진행되어야 할 것으로 사료된다.