• BMB Reports
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• 제39권3호
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• pp.270-277
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• 2006

Helices 4 and 5 of the Bacillus thuringiensis Cry4Ba $\delta$-endotoxin have been shown to be important determinants for mosquito-larvicidal activity, likely being involved in membrane-pore formation. In this study, the Cry4Ba mutant protein containing an additional engineered tryptic cleavage site was used to produce the $\alpha4$-$\alpha5$ hairpin peptide by an efficient alternative strategy. Upon solubilization of toxin inclusions expressed in Escherichia coli and subsequent digestion with trypsin, the 130-kDa mutant protoxin was processed to protease-resistant fragments of ca. 47, 10 and 7 kDa. The 7-kDa fragment was identified as the $\alpha4$-loop-$\alpha5$ hairpin via N-terminal sequencing and mass spectrometry, and was successfully purified by size-exclusion FPLC and reversed-phase HPLC. Using circular dichroism spectroscopy, the 7-kDa peptide was found to exist predominantly as an $\alpha$-helical structure. Membrane perturbation studies by using fluorimetric calcein-release assays revealed that the 7-kDa helical hairpin is highly active against unilamellar liposomes compared with the 65-kDa activated full-length toxin. These results directly support the role of the $\alpha4$-loop-$\alpha5$ hairpin in membrane perturbation and pore formation of the full-length Cry4Ba toxin.

• The Korean Journal of Physiology and Pharmacology
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• 제1권5호
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• pp.485-493
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• 1997

We investigated the effect of ${\alpha}-adrenergic$ and cholinergic receptor agonists on $Ca^{2+}$ current in adult rat trigeminal ganglion neurons using whole-cell patch clamp methods. The application of acetylcholine, carbachol, and oxotremorine ($50\;{\mu}M\;each$) produced a rapid and reversible reduction of the $Ca^{2+}$ current by $17{\pm}6%,\;19{\pm}3%,\;and\;18{\pm}4%$, respectively. Atropine, a muscarinic antagonist, blocked carbachol- induced $Ca^{2+}$ current inhibition to $3{\pm}1%$. Norepinephrine ($50\;{\mu}M$) reduced $Ca^{2+}$ current by $18{\pm}2%$, while clonidine ($50\;{\mu}M$), an ${\alpha}2-adrenergic$ receptor agonist, inhibited $Ca^{2+}$ current by only $4{\pm}1%$. Yohimbine, an ${\alpha}2-adrenergic$ receptor antagonist, did not block the inhibitory effect of norepinephrine on $Ca^{2+}$ current, whereas prazosin, an ${\alpha}1-adrenergic$ receptor antagonist, attenuated the inhibitory effect of norepinephrine on $Ca^{2+}$ current to $6{\pm}1%$. This pharmacology contrasts with ${\alpha}2-adrenergic$ receptor modulation of $Ca^{2+}$ channels in rat sympathetic neurons, which is sensitive to clonidine and blocked by yohimbine. Our data suggest that the modulation of voltage dependent $Ca^{2+}$ channel by norepinephrine is mediated via an α1-adrenergic receptor. Pretreatment with pertussis toxin (250 ng/ml) for 16 h greatly reduced norepinephrine- and carbachol-induced $Ca^{2+}$ current inhibition from $17{\pm}3%\;and\;18{\pm}3%\;to\;2{\pm}1%\;and\;2{\pm}1%$, respectively. These results demonstrate that norepinephrine, through an ${\alpha}1-adrenergic$ receptor, and carbachol, through a muscarinic receptor, inhibit $Ca^{2+}$ currents in adult rat trigeminal ganglion neurons via pertussis toxin sensitive GTP-binding proteins.

• Clinical and Experimental Pediatrics
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• 제46권2호
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• pp.143-153
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• 2003

목 적 : Shiga-like toxin (SLT)을 생산하는 Esherichia coli에 의한 감염은 설사, 출혈성 대장염(hemorrhagic colitis) 및 용혈성 요독 증후군(hemolytic uremic syndrome)을 특징으로 하며, 특히 5세 이하의 소아에게서 심각한 결과를 초래한다. SLT-I의 병인으로는 다양한 숙주 매개인자들이 알려져 있다. 본 연구에서는 E. coli 0157 : H7 (ATCC 43890)로부터 정제한 SLT-I이 포유동물 세포들에 대한 세포독성과 종양괴사인자(tumor necrosis factor-${\alpha}$; TNF-${\alpha}$)의 생산에 미치는 효과를 측정하였으며, SLT-I의 수용체인 glycolipid globotriaosylceramide (Gb3)의 발현과 SLT-I의 세포독성의 관계를 규명하고자 하였다. 방 법 : SLT-I과 SLT-I B를 순수분리 정제하고 SLT-I B-FLTC 접합체를 제조하여 vero 세포, 대식세포 및 수지상세포를 대상으로 세포독성능을 측정하고 세포독성능의 차이가 SLT-I의 수용체인 Gb3의 발현과 상관관계가 있는지를 Flow cyotmetry로 분석하였다. 또한 대식세포의 종양괴사인자 생산능은 ELISA법으로 시행하였다. 결 과 : SLT-I은 대식세포(Raw264.7)로부터 TNF-${\alpha}$의 생산을 증가시켰다. 연구 대상 세포 중 SLT-I에 감수성을 나타낸 Vero 세포와 수지상세포(dendritic cells)는 Gb3 발현이 각각 83%와 68%로 높았으며, 29%의 낮은 Gb3 발현을 보인 Raw264.7 세포는 감수성을 보이지 않았다. 따라서 위의 결과로부터 SLT-I에 감수성을 보이지 않은 Raw264.7 세포를 대상으로 Gb3 발현 정도와 SLT-I의 세포독성의 관계를 규명하고자 Gb3의 발현을 증가 시킨 후 SLT-I의 세포독성을 재차 평가하였다. 이 결과 TNF-${\alpha}$의 처리에 의하여 6 h에 Gb3의 발현이 정점(43.5%)에 이르렀으며 36 h에 정상 수준(25.0%)으로 환원되었다. 그러나, Gb3의 발현이 증가함에도 불구하고 SLT-I의 세포독성에는 변화가 관찰되지 않았다. 따라서, SLT-I에 의한 세포독성은 세포의 종류에 따라서 다르며 또한, Gb3의 발현정도에만 의존적이지는 않을 것으로 생각된다. 결 론 : 이와 같은 결과는 E. coli 0157의 감염증 병인 연구에 있어 SLT-I과 Gb3의 발현의 상관관계에 대한 보다 심도 있는 연구가 필요함을 시사한다.

• 한국동물위생학회지
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• 제25권2호
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• pp.127-133
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• 2002

The case reports for clostridium type A enterotoxemia in Formosan deer have rarely been reported. This paper describes a natural case of type A enterotoxemia in farmed Formosan deer in Cheongwon-gun. A dead, male 10-month-old Formosan deer was submitted to Chungbuk Livestock and Veterinary Research Institute, March 24, 2001 and examined. That deer was fed with assorted grain feed, oak leaves, acorn and bean curd. Grossly there was no visible external change. Despite of the carcass being examined within 12 hours of death, there was a quite degree of posonortem decomposition. There was severe hemorrhage in the serosa of abomasum and small intestine. Much blood tinged and watery contents were contained in those organs. Also there were severe swelling of spleen, some red foci in hepatic parenchyma. Microscopically there were severe congestion and hemorrhage in mucosa submucosa, muscular layer, and serosa of abomasum and small intestine. Also spleen and pancreas showed severe Congestion and hemorrhage. There were multifocal hemorrhage with hepatic necrosis in periportal area and focal mononuclear cell deposition in sinusoid. In bacterial culture for small intestine, Cl perfringens was isolated. By toxin typing for the strain, that had $\alpha$ -toxin belonged to type A. In electronmicroscopy for feces, no vims particle was detected. Considering clinical signs, gross lesions, microscopic lesions, bacterial culture, and toxin typing of the isolate, this case was diagnosed as enterotoxemia by Cl perfringens type A.

• 한국식품위생안전성학회지
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• 제15권4호
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• pp.287-292
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• 2000

1976년과 1997년 남해안의 거제도 외포리에서 채취한 진주담치와 굴의 독력 덴 독소성분을 비교 조사하였다. 독성은 진주담치가 굴에 비하여 약 10배 정도 높았으며(1996년, 진주담치, 8,670 $\mu\textrm{g}$, 굴 860$\mu\textrm{g}$; 1997년, 진주담치 5,657 $\mu\textrm{g}$, 굴 531$\mu\textrm{g}$/100g), 독화기간도 진주담치가 굴에 비하여 길었다. 두 종류의 시료 모두 독소 주성분은 Cl 및 C2 (20~65%)와 gonyautoxin 1, 2, 3, 4 (38~78%)이었다. 그리고 독화초기에는 11$\beta$-epimer toxin(C2, GTX4)의 비율이 25~56mo1e%(1996년)와 25~80mo1e% (1997년)로 11$\alpha$-epimer toxin(Cl, GTX2)의 비율도다 높았다. 그러나 독화기간이 지남에 따라 11$\alpha$-epimer toxin의 비율이 41~57mo1e%(1996년)와 25~56mo1e%(1997년)로 11$\beta$-epimer toxin의 비율보다 높게 나타났다. 이와 같은 독소성분 조성의 변화는 패류내에서 독소가 대사되기 때문인 것으로 추측된다.

• Journal of Microbiology
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• 제43권3호
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• pp.266-271
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• 2005

In eukaryotic cells, various proteins are anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). To study the biosynthetic pathways and modifications of GPI, various mutant cells have been isolated from the cells of Chinese hamster ovaries (CHO) supplemented with several exogenous genes involved in GPI biosynthesis using aerolysin, a toxin secreted from gram-negative bacterium Aeromonas hydrophila. Alpha toxin from Gram-positive bacterium Clostridium septicum is homologous to large lobes (LL) of aerolysin, binds GPI-anchored proteins and possesses a cell-destroying mechanism similar to aerolysin. Here, to determine whether alpha toxins can be used as an isolation tool of GPI-mutants, like aerolysin, CHO cells stably transfected with several exogenous genes involved in GPI biosynthesis were chemically mutagenized and cultured in a medium containing alpha toxins. We isolated six mutants highly resistant to alpha toxins and deficient in GPI biosynthesis. By genetic complementation, we determined that one mutant cell was defective of the second subunit of dolichol phosphate mannose synthase (DPM2) and other five cells were of a putative catalytic subunit of inositol acyltransferase (PIG-W). Therefore, C. septicum alpha toxins are a useful screening probe for the isolation of various GPI-mutant cells.

• Journal of Acupuncture Research
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• 제34권1호
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• pp.23-30
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• 2017

Objectives : In this study, the roles of Interleukin (IL)-4 and Signal transducer and activator of transcription 6 (STAT6), which have been reported to play a role in the pathogenesis of inflammation and cancer, were evaluated in snake venom toxin (SVT)-induced apoptosis. Methods : Inflammation was induced in human HaCaT kerationocytes, by lipopolysaccharide (LPS; $1{\mu}g/mL$) or tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), followed by treatment with SVT (0, 1, or $2{\mu}g/mL$). Cell viability was assessed by MTT assays after 24 h, and the expression of levels of IL-4, STAT6, and the apoptosis-related proteins p53, Bax, and Bcl-2 were evaluated by western blotting. Electro mobility shift assays (EMSAs) were performed to evaluate the DNA binding capacity of STAT6. Results : MTT assays showed that inflammation-induced growth of HaCaT cells following LPS or TNF-${\alpha}$ stimulation was inhibited by SVT. Western blot analysis showed that p53 and Bax, which promote apoptosis, were increased, whereas that of Bcl-2, an anti-apoptotic protein, was decreased in a concentration-dependent manner in LPS- or TNF-${\alpha}$-induced HaCaT cells following treatment with SVT. Moreover, following treatment of HaCaT cells with LPS, IL-4 concentrations were increased, and treatment with SVT further increased IL-4 expression in a concentration-dependent manner. Western blotting and EMSAs showed that the phosphorylated form of STAT6 was increased in HaCaT cells in the context of LPS- or TNF-${\alpha}$-induced inflammation in a concentration-dependent manner, concomitant with an increase in the DNA binding activity of STAT6. Conclusion : SVT can effectively promote apoptosis in HaCaT cells in the presence of inflammation through a pathway involving IL-4 and STAT6.

• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1849-1855
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• 2015

Staphylococcus aureus plays an important role in sepsis, septic shock, pneumonia, and wound infections. Here, we demonstrate that Lactobacillus plantarum extracts inhibited S. aureus-induced cell death of a human epithelial cell line, HT-29. In particular, we have shown that S. aureus-induced cell death was abolished by neutralization of α-toxin, indicating that α-toxin is the major mediator of S. aureus-induced cell death. DNA fragmentation experiment and caspase assay revealed that the S. aureus-induced cell death was apoptosis. L. plantarum extracts inhibited the generation of effector caspase-3 and the initiator caspase-9 in S. aureus- or α-toxin-induced cell death. Moreover, expression of Bcl-2, an anti-apoptotic protein, was activated in L. plantarum extract-treated cells as compared with the S. aureus- or α-toxin-treated only cells. Furthermore, S. aureus-induced apoptosis was efficiently inhibited by lipoteichoic acid and peptidoglycan of L. plantarum. Together, our results suggest that L. plantarum extracts can inhibit the S. aureus-mediated apoptosis, which is associated with S. aureus spreading, in intestinal epithelial cells, and may provide a new therapeutic reagent to treat bacterial infections.

• 한국수산과학회지
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• 제56권6호
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• pp.826-832
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• 2023

Clostridium perfringens causes diarrhea and other diseases in humans and animals. We investigated the prevalence, toxin gene profiles, and antimicrobial resistance of C. perfringens isolated from commercial jeotgal sample. C. perfringens was isolated from 11 of 22 commercial jeotgals. All C. perfringens strains were positive for the alpha toxin gene, but not for the beta, epsilon, iota, CPE or NetB toxin genes; therefore, all strains were identified as type A C. perfringens. However, the beta2 toxin gene was identified in 54.5% of isolates. Disk diffusion susceptibility tests showed that most isolates were resistant to kanamycin (90.9%), nalidixic acid (72.7%), oxacillin (54.5%), erythromycin (27.3%), ciprofloxacin (9.1%) and clindamycin (9.1%). However, all strains were susceptible to 14 other antimicrobial including amoxicillin, ampicillin, and chloramphenicol. The average minimum inhibitory concentrations against C. perfringens of clindamycin, kanamycin, and nalidixic acid were 128.0, 128.0, and 54.0 ㎍/mL, respectively. These results provide new insight into the necessity for sanitation of commercial jeotgal, and provide evidence to help reduce the risk of contamination with antimicrobial-resistant bacteria.

• Asian-Australasian Journal of Animal Sciences
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• 제14권12호
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• pp.1752-1757
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• 2001

The present study was conducted to assess the dietary effect of T-2 toxin on the antioxidant systems of the liver in growing quail and to comparatively evaluate the protective properties of two different mycotoxin-adsorbent additives, Mycosorb and zeolite, in preventing inhibition of the antioxidant system. Four groups of 4 day old quail were formed with 20 birds in each group. The birds were maintained on the floor for the course of the study. The three treatment diets consisted of the basal diet with T-2 toxin added in the form of Fusarium sporotrichioides culture (8.1 mg/kg feed), T-2 toxin (8.1 mg/kg) plus zeolite (30 g/kg feed), and T-2 toxin (8.1 mg/kg) plus Mycosorb (1 g/kg feed). After 30 days of feeding (34 days old) all birds were sacrificed and liver samples for biochemical analyses were collected from five quail in each of the four groups. Antioxidant concentrations were evaluated by HPLC-based methods. Inclusion of T-2 toxin in the quail diet was associated with a significant (p<0.05) decrease in concentrations of all forms of antioxidants studied, including ${\alpha}$- and ${\gamma}$-tocopherols, ascorbic acid, retinol and retinyl esters. At the same time, liver susceptibility to lipid peroxidation significantly (p<0.05) increased. Inclusion of zeolite in the quail diet at the level of 3% was ineffective in preventing antioxidant depletion in the liver by mycotoxicosis. In contrast, Mycosorb in the diet at a 0.1% level was able to significantly inhibit liver antioxidant depletion and as a result decreased lipid peroxidation in the liver. Concentrations of all forms of antioxidants studied were significantly higher in the livers of the quails fed the basal and T-2 toxin/Mycosorb combination in comparison to birds fed the basal with T-2 toxin alone.