• Toxicological Research
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• 제13권4호
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• pp.417-421
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• 1997

Synaptobrevin is a kind of vesicle associated membrane proteins (VAMPs) which plays a secretary role in the neuronal synapse and was recently known as the biochemical target of botulinum neurotoxin type B. The structural gene of the synaptobrevin was cloned from mouse brain using RT-PCR technique and was seqrtenced. The deduced amino acid sequence showed that the synaptobrevin protein from mouse brain is exactly the same with that of the rat brain in the amino acid level. The synaptobrevin gene was subcloned into pET3a vector and expressed in E. coli. The molecular weight of the recombinant protein was 19 kDa as expected. Moreover, when the recombinant synaptobrevin protein was incubated with the native neurotoxin of Clostridium botulinum type B, it was cleaved by the toxin in a time dependent manner. This implies that the recombinant synaptobrevin protein and the native toxin are reacted in the same way as the native synaptobrevin did in the neuronal cells.

• BMB Reports
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• 제28권3호
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• pp.210-215
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• 1995

Effects of staurosporine, genistein and pertussis toxin on PMA-induced superoxide generation and degranulation in neutrophils were investigated. Their effects were also examined in C5a-stimulated superoxide generation. PMA-induced superoxide generation was inhibited by staurosporine but was not affected by pertussis toxin. Genistein enhanced the stimulatory effect of PMA in a dose dependent fashion. C5a-induced superoxide generation was inhibited by staurosporine, genistein and pertussis toxin. An NADPH oxidase system of resting neutrophils was activated by PMA, and the stimulatory effect of PMA was inhibited by staurosporine but was not affected genistein and pertussis toxin. The activity of NADPH oxidase in the membrane fraction of PMA-activated neutrophils was not affected by staurosporine and genistein. PMA-induced acid phosphatase release was inhibited by staurosporine and genistein, whereas the effect of pertussis toxin was not detected. These results suggest' that the role of protein tyrosine kinase in neutrophil activation mediated by direct activation of protein kinase C may be different from receptor-mediated activation. The action of protein kinase C on the respiratory burst might be affected by the change of protein tyrosine kinase activity.

• 생명과학회지
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• 제28권9호
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• pp.1016-1021
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• 2018

C. difficile 톡신A에 의한 대장상피세포 자살과정은 위막성대장염(Pseudomembranous colitis)의 주요 원인으로 고려되고 있다. 톡신A는 활성산소 를 증가시켜 세포자살 신호를 유도한다. 또한 톡신A는 미세섬유나 미세소관과 같은 세포골격계 형성을 저해함으로써 자살을 유도한다고 알려져 있다. 하지만 톡신A가 야기하는 소화기 상피세포 자살경로는 아직 불분명하다. 본 연구에서는 소화관 상피세포의 성장과 분화 그리고 기능에 중요하다고 알려져 온 Wnt 신호경로에 대한 톡신A의 영향을 확인해보았다. 이를 위해 비암화-인간대장세포주(NCM460)에 톡신A를 처치하고 Wnt 신호 분자들의 변화를 추적하였다. 또한 톡신A를 주입한 생쥐의 회장 상피세포 속 Wnt 신호경로 변화도 평가하였다. 인간 대장상피세포에서 톡신A는 Wnt 경로의 핵심 신호분자인 ${\beta}$-catenin 단백질의 양을 빠르게 감소시켰다. 이 현상은 생쥐 회장 상피세포에서도 동일하게 확인되었다. 연구자 등은 톡신A가 $GSK3{\beta}$ 활성형 인산화(Thr390)를 증가시킴도 확인하였다. 이는 톡신A가 $GSK3{\beta}$의 활성을 높여서 ${\beta}$-catenin의 인산화시키고 이를 통해 단백질 분해 과정이 촉진되었음을 보여준다. 이 결과들을 종합하면, 톡신A에 의한 소화관 상피세포 자살과정이 상피세포의 성장과 자살을 조절하는 Wnt 신호경로 차단과 밀접하게 연관되어 있음을 보여준다.

• 생명과학회지
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• 제26권2호
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• pp.265-274
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• 2016

Toxin-antitoxin (TA) system은 박테리아와 고세균에서 진화적으로 보존되어 흔히 발견되는 유전적 모듈이다. 기본적으로 이 시스템은 세포 내 toxin과 그들의 억제자로 작용하는 antitoxin으로 구성되어있으며, 현재 총 다섯가지 유형으로 구분된다. 공통적으로 toxin은 스트레스 조건에서 활성화됨으로써 세포 내 다양한 과정을 억제하는 활성을 가지는데 이는 결과적으로 세포 사멸 혹은 가역적인 생장 저해를 일으킨다. Toxin의 이러한 효과들은 유전자 발현의 조절, 성장 조절, programmed cell arrest, programmed cell death, persister cell의 형성, 박테리오파지 방어기작, 가동성 유전인자의 안정화, 플라스미드 유지 기작 등 다양한 생리학적 역할을 나타낸다. 그러므로 TA system은 일반적인 스트레스 반응모듈로서 여겨진다. 하지만 이를 역이용한다면 TA system으로부터 toxin을 활성화 시키는 인자를 개발하여 새로운 항균 물질로 이용할 수 있다. 그뿐만 아니라 TA system은 toxin의 세포 사멸 효과를 이용하여 원하는 타겟 유전자가 존재하는 세포만 선택적으로 살아남도록 하는 효율적인 클로닝 전략에 이용될 수 있다. 또한, toxin의 서열 특이적 리보핵산 가수분해효소 활성을 이용하여 타겟 단백질 이외의 단백질 합성을 막아 효과적인 단일 단백질 대량 생산을 위해서도 이용할 수 있다. 더 나아가 일부 TA system의 toxin은 진핵 세포에서도 세포 독성을 나타내기 때문에 암세포, 바이러스 감염 세포에서 toxin의 발현을 유도하여 세포사멸을 일으킴으로써 인간의 질병 치료로 이어질 수 있다.

• Journal of Photoscience
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• 제9권2호
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• pp.323-325
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• 2002

Bacillus Anthracis is the causative agent of anthrax. The major virulence factors are a poly-D glutamic acid capsule and three-protein component exotoxin, which is collectively known as anthrax toxin, protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa). These three proteins individually have no known toxic activities, but in combination with PA form two toxins (lethal toxin and edema toxin), causing different pathogenic responses in animals and cultured cells. However, it remains to be elucidated for pathogenic mechanism of anthrax toxin. In this study, we constructed toxin component in bacterial overexpression system and purified the native toxin from Bacillus anthracis delta sterne F32 using FPLC system. Recombinant toxin showed high homogeneity and rapid purification processes. Also, this recombinant toxin was comparable to B. anthracis native toxin in terms of cytotoxic effects on cultured cell lines.

• Journal of Microbiology and Biotechnology
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• 제21권9호
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• pp.937-946
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• 2011

Several isolates of Bacillus thuringiensis (Bt) were screened for the vegetative insecticidal protein (Vip) effective against sap-sucking insect pests. Screening results were based on $LC_{50}$ values against cotton aphid (Aphis gossypii), one of the dangerous pests of various crop plants including cotton. Among the isolates, the Bt#BREF24 showed promising results, and upon purification the aphidicidal protein was recognized as a binary toxin. One of the components of this binary toxin was identified by peptide sequencing to be a homolog of Vip2A that has been reported previously in other Bacillus spp. Vip2 belongs to the binary toxin group Vip1-Vip2, and is responsible for the enzymatic activity; and Vip1 is the translocation and receptor binding protein. The two genes encoding the corresponding proteins of the binary toxin, designated as vip2Ae and vip1Ae, were cloned from the Bt#BREF24, sequenced, and heterologously expressed in Escherichia coli. Aphid feeding assay with the recombinant proteins confirmed that these proteins are indeed the two components of the binary toxins, and the presence of both partners is essential for the activity. Aphid specificity of the binary toxin was further verified by ligand blotting experiment, which identified an ~50 kDa receptor in the brush border membrane vesicles of the cotton aphids only, but not in the lepidopteran insects. Our finding holds a promise of its use in future as a candidate gene for developing transgenic crop plants tolerant against sap-sucking insect pests.

• 한국임상수의학회지
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• 제17권1호
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• pp.152-158
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• 2000

capsular polysaccharide(CPS), alpha toxin과 재조합 fibronectin binding protein (FnBP)으로 구성된 유방염 백신을 개발하여 야외효능실험을 수행하였다. 이 subunit 백신은 비유우 23두와 처녀우 20두를 대상으로 10개월동안 각각 수행되었다. 비유우는 매 3주간격으로 2회 백신접종을 하였으며 대조군은 PBS를 상유방림조절 주위에 피하주사하였다. 처녀우를 대상으로 한 실험에서는 분만 8주전부터 시작하여 매 3주간격으로 2회 비유우 백신접종부위와 동일하게 접종하였다. 접종후 비유우에서는 황색포도상구균에 의한 총유선내 감염율이 대조균에 비해 유의성있게 감소하였으며 (p<0.001) 처녀우에서도 총유선내 감염이 백신 접종균(1.6%)이 대조군(11.7%)에 비해 다음 비유기동안 유의성있게 낮았다. (p<0.001). 비유기동안 백신접종한 젖소의 체세포수는 변화가 없었으며 처녀우에서는 추가접종후 백신접종군에서 대조군의 체세포수에 비해 낮았지만 통계학적으로 유의성은 없었다(p>0.05). 본 실험결과 황색포도구균에 대한 유방염 아단위 백신은 비유우와 처녀우에서 체세포수를 증가시키지 않고 총 유선내 감염율을 낮추어주었다. 하지만 본 실험은 1군데의 목장을 대상으로 하였기 때문에 향후 대규모 목장을 대상으로 하는 유방염 백신의 야외효능실험이 심도있게 이루어져야 한다고 생각된다.

• BMB Reports
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• 제30권2호
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• pp.144-149
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• 1997

An expression vector was constructed containing the gene encoding diphtheria toxin A (DTA) which was placed after a T7 promoter. Cytoplasmic expression of the DTA gene resulted in the formation of an insoluble inclusion body. The inclusion body was collected after the complete lysis of the cell, and subsequent washing with 0.1% Triton X-100 released 16~30% of DTA protein from the inclusion body along with other contaminating proteins. The released DTA protein was purified by dialysis. The remaining pellet was dissolved in 8 M urea containing 5% ${\beta}-mercaptoethanol$, and the denatured DTA was renatured by the dilution-dialysis method. The total yield was 35%, and about 5 mg DTA was obtained from 1 L culture. The DTA protein has a free sulfhydryl group exposed to the protein surface, and was shown to have a tendency to dimerize through disulfide formation in the absence of ${\beta}-mercaptoethanol$. The utility of the sulfhydryl group was tested for the construction of recombinant toxins.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.170-175
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• 2012

Clostridium difficile toxin A glucosylates Rho family proteins, resulting in actin filament disaggregation and cell rounding in cultured colonocytes. Given that the cellular toxicity of toxin A is dependent on its receptor binding and subsequent entry into the cell, we herein sought to identify additional colonocyte proteins that might bind to toxin A following its internalization. Our results revealed that toxin A interacted with ERK1 and ERK2 in two human colonocyte cell lines (NCM460 and HT29). A GST-pulldown assay also showed that toxin A can directly bind to ERK1 and ERK2. In NCM460 cells exposed to PMA (an ERK1/2 activator), the phosphorylation of ERK1/2 did not affect the interaction between toxin A and ERK1/2. However, an in vitro kinase assay showed that the direct binding of toxin A to ERK1 or ERK2 inhibited their kinase activities. These results suggest a new molecular mechanism for the cellular toxicity seen in cells exposed to toxin A.

• International Journal of Industrial Entomology and Biomaterials
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• 제8권1호
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• pp.89-93
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• 2004

Expression of a fusion protein between B. thuringiensis crystal protein, Cry1Ac1 and a scorpion insect toxin (AaIT, Androctonus australis Hector insect toxin) in acrystalliferous B. thuringiensis strain (Cry-B strain) was examined. The cry 1Ac1 gene was cloned in B. thuringiensis-E coli shuttle vector, pHT3101, under the control of the native cry 1Ac1 gene promoter (pProAc) and a gene encoding AaIT was inserted in XhoI site in the middle of the cry 1Ac1 gene (pProAc-ScoR). B. thuringiensis Cry-B strain carrying pProAc-ScoR (PyoAc-ScoR/CB) produced an inclusion body of irregular shape and the expressed fusion protein is approximately 65 kDa in size. Sporulated cells and spore-crystal mixtures of ProAc-ScoR/CB had insecticidal activity against Plutella xylostella larvae, showing $LT_50$ of ProAc-ScoR/CB (22.59 hrs) lower than that of ProAc/CB (30.06 hrs) at $1{\times}{10^7} {CEU/cm^2}$. These results suggest that the fusion protein including a B. thuringiensis crystal protein and an AaIT may be functionally expressed in B. thupingiensis. Moreover, we verified the additive toxicity of AaIT, which is a new feasible candidate for insect control.