• International Journal of Industrial Entomology and Biomaterials
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• 제17권1호
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• pp.137-141
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• 2008

We previously isolated and cloned a cDNA of abaecin from the Bombus ignitus. In an effort to produce a large amount of soluble abaecin at low cost, we successfully expressed the peptide in Escherichia coli that are highly sensitive to its mature form. For this, we fused the peptide encoding 39 amino acids of mature B. ignitus abaecin to the thioredoxin gene together with a C-terminal 6xHis tag. An enterokinase cleavage site was introduced between the 6xHis tag and mature abaecin to allow final release of the recombinant peptide. A high yield of 9.6 mg soluble fusion protein from 200 ml of bacterial culture was purified by $Ni^{2+}$-charged His-Bind resin affinity column, and 1.4 mg of pure active recombinant abaecin was readily obtained by enterokinase cleavage, followed by affinity chromatograph. The molecular mass of recombinant abaecin peptide was determined by Tricin-SDS-PAGE analysis. The recombinant abaecin exhibited antibacterial activity against Gram-negative bacteria.

• 동의생리병리학회지
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• 제34권4호
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• pp.184-190
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• 2020

This study was performed to find antibacterial substances contained in Scutellariae Radix (SR) using a systems pharmacological analysis method and to establish an effective strategy for the prevention and treatment of infectious diseases. Analysis of the main active ingredients of SR was performed using Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database and Analysis Platform. 36 active compounds were screened by the parameter values of Drug-Likeness (DL), Oral Bioavailability (OB), and Caco-2 permeability (Caco-2), which were based on the drug absorption, distribution, metabolism, and excretion indicators. The UniProt database was used to obtain information on 159 genes associated with active compounds. The main active compounds with antibacterial effects were wogonin, β-sitosterol, baicalein, acacetin and oroxylin-A. Target proteins associated with the antibacterial action were chemokine ligand 2, interleukin-6, tumor necrosis factor, caspase-8,9 and mitogen-activated protein kinase 14. In the future, systems pharmacological analysis of traditional medicine will be able to make it easy to find the important mechanism of action of active substances present in natural medicines and to optimize the efficacy of medicinal effects for combinations of major ingredients to help treat certain diseases.

• Journal of Plant Biotechnology
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• 제43권4호
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• pp.450-456
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• 2016

한국형 잔디에서는 다른 병에 비해 진전 속도가 빠르고 주로 뿌리에서부터 발병하여 잔디를 고사시키고 발병 후 구제하기 매우 어려운 라이족토니아잎마름병(라지패취)이 큰 문제로 대두되고 있다. 라이족토니아잎마름병(라지패취)은 Rhizoctonia solani AG2-2 (IV)병원균에 의해 발생하는데, 이 병원균에 강한 내병성 들잔디를 개발하기 위해 식물방어반응에 중요한 역할을 하는 것으로 알려진 PR-Protein 중 하나인 ${\beta}-1,3-glucanase$를 들잔디로부터 클로닝 하였다. ${\beta}-1,3-glucanase$는 바이러스나 균의 감염으로 인해 식물조직이 과민반응을 일으킬 때 세포내에서 생성되고 세포 외로 분비되어 세포 사이 공간에서 주로 병원균 저항성기능을 하는 것으로 알려져 있다. ${\beta}-1,3-glucanase$ 단자엽식물 중 내병성에 대한 연구가 되어있는 옥수수, 밀, 보리, 벼의 염기서열에서 공통으로 보존되어 있는 부분을 이용해 degenerate PCR을 수행하고 얻어낸 sequence를 통해 Full-length의 cDNA를 클로닝 하였다. E.coli overexpression을 수행하여 목표 단백질을 대량 정제하여 in vitro 활성 측정 및 항균테스트를 진행하였다. 또한, ZjGlu1 유전자의 기능을 해석하기 위해 각각의 유전자를 도입한 식물형질전환용 벡터를 제작하여 잔디 형질전환체 제작을 하였다. ZjGlu1 단백질을 이용하여 9개의 균주에 대해 항균활성 테스트를 진행 한 결과 R. cerealis, F. culmorum, R.solani AG-1 (1B), T. atroviride 에서 항균활성을 보였으며, 형질전환체를 이용해 18s 유전자의 발현량을 상대로 한 각 유전자의 기관별 발현량은 크게 차이없이 모든기관에 발현되는 것을 확인할 수 있었다.

• 한국식품영양학회지
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• 제27권2호
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• pp.267-273
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• 2014

This study found antibacterial activity of $DMfree^{(R)}$ [green tea extract] on facultative bacteria by direct petri dish method and gene array of obligatory M. leprae infected mesenchymal stem cells (MSC). While DMfree showed DPPH radical scavenging effect and high contents of polyphenol, it did not inhibit growth of facultative bacteria such as E. coli and S. aureus on the petri dish. The result does not exclude a possible antibacterial effect of organic solvent extract of green tea rather than DMfree which comes from the water extract of green tea. Pre-treatment of DMfree appeared to have no effect on copy number of 14 genes compared with control MSC by real-time RT-PCR. However pre-treatment of DMfree on M. leprae infected MSC revealed a significant decrease of anti-inflammatory cytokine (IL-6), (P<0.038) and sharp down-regulation of pro-inflammatory cytokine (IL-1). Enhanced expression of VEGFR-1 mRNA was noted in DMfree pretreated M. leprae infected MSC group (P<0.003). These results show that DMfree would stabilize M. leprae infected MSC from further inflammation by down-regulating anti-inflammatory cytokine (IL-6) and pro-inflammatory cytokine (IL-$1{\beta}$). This is the first report on DMfree inhibition of IL-6 and IL-$1{\beta}$ expression in M. leprae infected MSC. Further experiments that detect protein levels of IL-$1{\beta}$ and IL-6 may support the result of this gene array.

• International Journal of Molecular Medicine
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• 제43권6호
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• pp.2516-2522
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• 2019

Particulate matter (PM), a widespread airborne contaminant, is a complex mixture of solid and liquid particles suspended in the air. Recent studies have demonstrated that PM induces oxidative stress and inflammatory reactions, and may cause certain skin diseases. Afzelin is a flavonoid isolated from Thesium chinense Turcz, which has anti-inflammatory, anticancer and antibacterial properties. Therefore, the present study aimed to investigate if afzelin affected inflammatory responses in human keratinocytes exposed to PM. HaCaT cells were treated with PM (25 ㎍/cm2) in the presence or absence of afzelin (200 µM). Here, standard reference material 1649b was used as PM. Cell viability was assessed using the water-soluble tetrazolium salt-1 assay. The generation of reactive oxygen species (ROS) was measured using the dichloro-dihydro-​fluorescein diacetate assay. Gene and protein expression were investigated using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Levels of secreted inflammatory cytokines were measured using ELISA. The results suggested that afzelin inhibited PM-induced proinflammatory cytokine mRNA expression and protein secretion in HaCaT cells. In addition, afzelin suppressed PM-induced intracellular ROS generation, and p38 mitogen-activated protein kinase and transcription factor activator protein-1 component c-Fos and c-Jun activation. The results indicated that afzelin exerts anti-inflammatory and antioxidant effects in PM-exposed HaCaT. Afzelin may have potential for preventing PM-induced inflammatory skin diseases.

• Journal of Microbiology and Biotechnology
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• 제25권9호
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• pp.1493-1501
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• 2015

A Bacillus species, EMD4, with strong antibacterial activity was isolated from ganjang (soy sauce) and identified as B. subtilis. B. subtilis EMD4 strongly inhibited the growth of B. cereus ATCC14579 and B. thuringiensis ATCC33679. The antibacterial activity was stable at pH 3-9 but inactive at pH 10 and above. The activity was fully retained after 15 min at 80℃ but reduced by 50% after 15 min at 90℃. The activity was completely destroyed by proteinase K and protease treatment, indicating its proteinaceous nature. The bacteriocin (BacEMD4) was partially purified from culture supernatant by ammonium sulfate precipitation, and Q-Sepharose and Sephadex G-50 column chromatographies. The specific activity was increased from 769.2 AU/mg protein to 8,347.8 AU/mg protein and the final yield was 12.6%. The size of BacEMD4 was determined to be 3.5 kDa by Tricine SDS-PAGE. The N-terminal amino acid sequence was similar with that of Subtilosin A. Nucleotide sequencing of the cloned gene confirmed that BacEMD4 was Subtilosin A. BacEMD4 showed bactericidal activity against B. cereus ATCC14579.

• 생명과학회지
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• 제17권11호
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• pp.1601-1604
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• 2007

Agarivorans sp. JA-1 유래의 내열성 ${\beta}-agarase$ 유전자의 분비형 과발현과 재조합효소분해산물의 항균효과를 확인하였다. B. subtilis 168, DB104, ISW1214의 3가지 재조합발현 숙주를 비교하여 ISW1214 균주가 LB배지에서 배양 9시간에 총 효소활성 52,460 U과 비활성 201 U/mg의 최대 발현량을 보이는 것을 확인하였다. 이는 E. coli BL21 균주에 비해 총 효소활성은 약 90배, 비활성은 약 100배 증가한 결과이다. 재조합 ${\beta}-agarase$의 기질로 저가의 한천을 사용하여 neoagarobiose, neoagarotetraose 등 의 neoagarooligosaccharide를 생산하였으며, 생산된 neoagarooligosaccharides는 그람양성 세균인 B. subtilis와 그람음성 세균인 E. coli 모두의 성장을 저해하는 항균활성이 있음을 확인하였다. 따라서 본 연구에서 생산된 재조합 ${\beta}-agarase$와 재조합 효소의 한천분해 산물은 식품산업 등에 사용가능한 천연 항균제 생산에 활용이 가능할 것으로 기대된다.

• Molecules and Cells
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• 제27권2호
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• pp.243-250
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• 2009

Recent studies suggest a novel role of $HIF-1{\alpha}$ under nonhypoxic conditions, including antibacterial and antiviral innate immune responses. However, the identity of the pathogen-associated molecular pattern which triggers $HIF-1{\alpha}$ activation during the antiviral response remains to be identified. Here, we demonstrate that cellular administration of double-stranded nucleic acids, the molecular mimics of viral genomes, results in the induction of $HIF-1{\alpha}$ protein level as well as the increase in $HIF-1{\alpha}$ target gene expression. Whole-genome DNA microarray analysis revealed that double-stranded nucleic acid treatment triggers induction of a number of hypoxia-inducible genes, and induction of these genes are compromised upon siRNA-mediated $HIF-1{\alpha}$ knock-down. Interestingly, $HIF-1{\alpha}$ knock-down also resulted in down-regulation of a number of genes involved in antiviral innate immune responses. Our study demonstrates that $HIF-1{\alpha}$ activation upon nucleic acid-triggered antiviral innate immune responses plays an important role in regulation of genes involved in not only hypoxic response, but also immune response.

• 대한의생명과학회지
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• 제21권4호
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• pp.233-240
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• 2015

Some virulence proteins of Helicobacter pylori, such as vacuolating cytotoxic protein A (VacA) and cytotoxin-associated gene protein A (CagA) have been reported to be causative agents of various gastric diseases including chronic gastritis, gastric ulcer or gastric adenocarcinoma. The expression level of these virulence proteins can be regulated when H. pylori is exposed to the antibacterial agent, cyanidin 3-O-glucoside (C3G) as previously reported. In this study, we analyzed the quantitative change of various virulence proteins including CagA and VacA by C3G treatment. We used 2-dimensional electrophoresis (2-DE) to analyze the quantitative change of representative ten proteome components of H. pylori 60190 ($VacA^+/CagA^+$; standard strain of Eastern type). After 2-DE analysis, spot intensities were analyzed using ImageMaster$^{TM}$ 2-DE Platinum software then each spot was identified using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) or peptide sequencing using Finnigan LCQ ion trap mass spectrometer (LC-MS/MS). Next, we selected major virulence proteins of H. pylori among quantitatively meaningful ten spots and confirmed the 2-DE results by Western blot analysis. These results suggest that cyanidin 3-O-glucoside can modulate a variety of H. pylori pathogenic determinants.

• 약학회지
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• 제47권3호
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• pp.184-189
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• 2003

Peptide deformylase (PDF) is essential and unique to bacteria, thus making it an attractive target for the discovery of novel antibacterial drugs. PDF deformylates the N-formylmethionine of newly synthesized polypeptides in prokaryotes. In this study, a pdf gene from Staphylococcus aureus 6538p was cloned in pET-14b vector and PDF protein was over-produced in Escherichia coli BL21 (DE3). NH$_2$-terminal His-tagged PDF protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography. Enzymatic activity of purified 6xHis-tagged PDF was tested on the substrate (formyl-Methionine-Alanine-Serine) by formate dehydrogenase-coupled spectrometric assay of peptide deformylase. For the discovery of new PDF inhibitors from chemical libraries and culture broths of soil bacteria, a target-oriented screening system using a 96-well plate was developed. About 3,000 commercial chemical libraries were tested in this screening system, and 2 chemicals (0.07％) among them showed an inhibitory activity against PDF enzyme. This result showed that a new screening system can be used for the discovery of new PDF inhibitors.