• Journal of Microbiology and Biotechnology
• /
• 제19권1호
• /
• pp.108-112
• /
• 2009

A simplified method for the purification of cholera toxin was developed. The 569B strain of Vibrio cholerae, a recognized hyper-producer of cholera toxin, was propagated in a bioreactor under conditions that promote the production of the toxin. The toxin was separated from the bacterial cells using 0.2-${\mu}m$ crossflow microfiltration, the clarified toxin was passed through the membrane into the permeate, and the bacterial cells were retained in the retentate. The 0.2-${\mu}m$ permeate was then concentrated 3-fold and diafiltered against 10 mM phosphate buffer, pH 7.6, using 30-kDa crossflow ultrafiltration. The concentrated toxin was loaded onto a cation exchange column, the toxin was bound to the column, and most of the impurities were passed unimpeded through the column. The toxin was eluted with a salt gradient of phosphate buffer, pH 7.0, containing 1.0 M NaCl. The peak containing the toxin was assayed for cholera toxin and protein and the purity was determined to be 92%. The toxin peak had a low endotoxin level of $3.1\;EU/{\mu}g$ of toxin. The purified toxin was used to prepare antiserum against whole toxin, which was used in a $G_{M1}$ ganglioside-binding ELISA to determine residual levels of toxin in an oral inactivated whole-cell cholera vaccine. The $G_{M1}$ ganglioside-binding ELISA was shown to be very sensitive and capable of detecting as little as 1 ng/ml of cholera toxin.

• 한국식품과학회지
• /
• 제26권5호
• /
• pp.585-589
• /
• 1994

시험관 내에서 T-2 toxin이 병아리 비장세포의 blastogenesis에 미치는 영향을 살펴본 결과, B-cell mitogen인 lipopolysaccharide 및 T-cell mitogen인 concanavalin A 자극에 대해 T-2 toxin의 농도가 증가함에 따라 억제정도가 증가하는 경향을 나타내었다. 노출시기를 달리하여 T-2 toxin을 투여한 병아리의 비장세포에서 mitogen 자극에 내한 반응을 안아 본 결과, 부화하기 전과 후에 계속 T-2 toxin에 노출시킨 실험군은 가장 영향을 많이 받은 것으로 나타났고 부화전 혹은 부화후 어느 한 시기에만 T-2 toxin에 노출된 실험군은 비교적 영향을 적게 받는 것으로 나타났다.

• Journal of Microbiology and Biotechnology
• /
• 제29권10호
• /
• pp.1675-1681
• /
• 2019

Clostridium difficile toxin A is known to cause colonic epithelial cell apoptosis, which is considered the main causative event that triggers inflammatory responses in the colon, reflecting the concept that the essential role of epithelial cells in the colon is to form a physical barrier in the gut. We previously showed that toxin A-induced colonocyte apoptosis and subsequent inflammation were dependent on prostaglandin E2 ($PGE_2$) produced in response to toxin A stimulation. However, the molecular mechanism by which $PGE_2$ mediates cell apoptosis in toxin A-exposed colonocytes has remained unclear. Here, we sought to identify the signaling pathway involved in toxin A-induced, $PGE_2$-mediated colonocyte apoptosis. In non-transformed NCM460 human colonocytes, toxin A exposure strongly upregulated expression of Bak, which is known to form mitochondrial outer membrane pores, resulting in apoptosis. RT-PCR analyses revealed that this increase in Bak expression was attributable to toxin A-induced transcriptional upregulation. We also found that toxin A upregulation of Bak expression was dependent on $PGE_2$ production, and further showed that this effect was recapitulated by an Prostaglandin E2(PGE2) receptor-1 receptor agonist, but not by agonists of other EP receptors. Collectively, these results suggest that toxin A-induced cell apoptosis involves $PGE_2$-upregulation of Bak through the EP1 receptor.

• 미생물학회지
• /
• 제36권2호
• /
• pp.91-96
• /
• 2000

불와전 균류인 Fusarium s^g pp.를 이용하여 여러 가지 배양조건과 생리적 영향에 따른 균주의 성장 및 T-2 toxin의 생성에 관하여 고찰하였다. T-2 toxin 의 검출방법은 thin layer chromatography (TCL) 법과 미생물학적 검출방법을 사용하였다. 고체 배지의 경우 횐옥수수 가루(Quaker사 제품)베지에서 다른 곡물보다 많은 양의 T-2 toxin이 생성되었으며,비교적 깨끗한 T-2 toxin이 정제되었다. 이 경우 배지 100g당 약 700 mg의 T-2 toxin이 생성되었으며, 그중 약 30%정도가 깨끗한 결정으로 정제되었다. 고온(20-$25^{\circ}C$)에서는 생장은 많았으나, T-2 toxin의 생성은 적었으며, 저온(10-$15^{\circ}C$)에서는 비교적 생장이 적었지만, T-2 toxin의 생성이 많았고, 젖당, 글리세롤, 솔비톨의 경우는 적었다. 유일 탄소원으로 구연산과 초산은 이용하지 못하였으며, 녹발의 경우 생장은 많았으나 T-2 toxin의 생성양은 적었다. 질소원의 경우 $NaNO_2$를 제외하고는 $(NH_4)_2NO_4$, $NH_4Cl_3$, $NH_4NO_3$, $KNO_3$ 를 거의 동일하게 이용하였다. 초기 pH값에 생성과 균주의 성장은 pH4.0-5.0일 경우 최적을 나타냈으며 ph6.0이상에서는 성장도 저하되고, T-2 toxin생성도 적었다. 회전속도에 따른 T-2 toxin 생성과 균주의 성장을 보면 회전속도가 속돠 증가함에 따라 균주의 생장과 T-2 toxin 생성량이 모두 증가하였다. $15^{\circ}C$에서 7일간 배양 후, $25^{\circ}C$로 옮겨 7일간 배양하여, toxin의 생성을 보면, $15^{\circ}C$에 7일간 배양했을 때보다 T-2 toxin양이 적었다. 이는 생성되었던 T-2 toxin이 분해되었음을 보여주는 것이다. 이상의 결과를 볼 때 T-2 toxin 대사 경로는 온도에 의한 효소 억제 또는 효소 유지 시스템에 의해 조절되는 것이라고 생각할 수 있다.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제4권1호
• /
• pp.43-49
• /
• 2002

We have cloned cDNAs encoding toxin from the spider, Araneus ventricosus, and constructed a recombinant baculovirus expressing the insecticidal toxin. The cDNAs encoding toxin were cloned from the cDNA library of A. ventricosus. Sequence analysis of the cDNAs encoding the toxin of A. ventricosus revealed that the 240 bp cDNA for AvTox-1 and 192 bp cDNA for AvTox-2 have an open reading frame of 80 and 64 amino acid residues, respectively. The deduced protein sequence of the toxin genes of AvTox-1 and AvTox-2 was aligned to that of the snack Anemonia sulcata and scorpion Centruroides limpidus limpidus, respectively. Northern blot analysis indicated that AvTox-2 toxin gene showed a fat body-spe-cific expression pattern at the transcriptional level. Furthermore, we have explored the possibility of improving baculovirus by incorporating the A. vontricosus toxin gene into Bombyx mori nuclear polyhedrosis virus genome under the control of polyhedrin promoter, The AvTox-2 toxin gene was expressed as approximately 5.8 kDa band in the recombinant baculovirus-injected silkworm larvae. Bioassays with the recombinant virus expressing AvTox-2 on 5th instar silkworm larvae demonstrated a decrease in the time to kill $(LT_{50} days)$ compared to wild-type BmNPV-Kl $(LT_{50} 6.72 days)$ in the injection of 10 viruses. These results indicate that A. ventricosus toxin is a novel member of the spider toxin family, suggesting that the toxin gene can be used in recombinant baculoviruses to reduce insect feeding damage and increase the speed of insect kill.

• 한국균학회지
• /
• 제16권1호
• /
• pp.9-15
• /
• 1988

H. sativum 배양액에서 두가지 종류의 식물독소물질이 검출되었으며, 이들을 M-toxin과 D-toxin으로 명명하였다. Lettuce 식물반응결과는 D-toxin 보다 뿌리성장의 저해작용이 적었다. 화학분석결과 M-toxin은 특별히 작은 peptid로 되어 있었다. D-toxin는 Helrninthosporal와 화학적 성질이 비슷하였으나 같지는 않았다. 이는 UV, proton NMR와 질량분석기의 기본 자료인 결과이며, D-toxin은 적어도 두개의 isomers로 되어 있었다.

• Archives of Pharmacal Research
• /
• 제27권5호
• /
• pp.554-561
• /
• 2004

Trichostatin A, an antifungal antibiotics, and HC-toxin are potent and specific inhibitors of histone deacetylase activity. Histone deacetylase inhibitors are new class of chemotherapeutic drugs able to induce tumor cell apoptosis and/or cell cycle arrest. In this study, the antiproliferative activities of trichostatin A and HC-toxin were compared between estrogen receptor positive human breast cancer cell MCF-7 and estrogen receptor negative human breast cancer cell MDA-MB-468. Trichostatin A and HC-toxin showed potent antiproliferative activity in both MCF-7 and MDA-MB-468 cells. In MCF-7 cells that contain high level estrogen receptor, trichostatin A and HC-toxin brought about three-times more potent cell growth inhibitory effect than estrogen receptor negative MDA-MB-468 cells. Both trichostatin A and HC-toxin showed cell cycle arrest at G$_2$/M phases of MCF-7 and MDA-MB-468 cells in a dose- and time- depen- dent manner. Trichostatin A and HC-toxin also induced apoptosis from MCF-7 and MDA-MB-468 cells in a dose- and time-dependent manner. Results of this study suggested that antipro-liferative effects of trichostatin A and HC-toxin might be involved in estrogen receptor signaling pathway, but cell cycle arrest and apoptosis of trichostatin A and HC-toxin might not be involved in estrogen receptor system of human breast cancer cells.

• 대한수의학회지
• /
• 제39권5호
• /
• pp.1028-1032
• /
• 1999

Alpha toxin of S aureus has cytolytic activity respectively. This antigen has been received the most attention since it is a major virulence factor in pathogenesis of staphylococcal mastitis. Thus, alpha toxin has been focused as potential candidate of vaccine to minimize mastitis in cows. The purpose of this study was to develop a simple, efficient production and purification methods of sufficient amount of alpha toxin antigen from S aureus. Alpha toxin production measured by hemolytic activity was the highest at 18 hrs postinoculation in yeast extract culture medium supplemented with thiamine, nicotinic acid and casamino acid. Alpha toxin was purified by ammonium sulfate precipitation (65%) and ultrafiltration. Molecular weight of the toxin was 33 kDa in the analysis with SDS-PAGE. Conclusionally, when alpha toxin was included in the vaccine, the optimal harvest time of alpha toxin was at 18 hrs after inoculation in yeast extract medium supplemented with thiamine and nicotinic acid.

• Journal of Microbiology and Biotechnology
• /
• 제29권1호
• /
• pp.30-36
• /
• 2019

Numerous studies have reported that enteric neurons involved in controlling neurotransmitter secretion and motility in the gut critically contribute to the progression of gut inflammation. Clostridium difficile toxins, which cause severe colonic inflammation, are also known to affect enteric neurons. Our previous study showed that C. difficile toxin A directly induces neural cell toxicities, such as viability loss and apoptosis. In the current study, we attempted to identify a potent inhibitor of toxin A-induced neural cell toxicity that may aid in managing toxin A-induced gut inflammation. In our recent study, we found that the Korea dung beetle-derived antimicrobial peptide CopA3 completely blocked neural cell apoptosis caused by okadaic acid or 6-OHDA. Here, we examined whether the antimicrobial peptide CopA3 inhibited toxin A-induced neural cell damage. In neuroblastoma SH-SY5Y cells, CopA3 treatment protected against both apoptosis and viability loss caused by toxin A. CopA3 also completely inhibited activation of the pro-apoptotic factor, caspase-3. Additionally, CopA3 rescued toxin A-induced downregulation of neural cell proliferation. However, CopA3 had no effect on signaling through ROS/p38 $MAPK/p27^{kip1}$, suggesting that CopA3 inhibits toxin A-induced neural cell toxicity independent of this well-characterized toxin A pathway. Our data further suggest that ability of CopA3 to rescue toxin A-induced neural cell damage may also ameliorate the gut inflammation caused by toxin A.

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