• Journal of Microbiology
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• v.43 no.3
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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.

• BMB Reports
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• v.39 no.3
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• pp.284-291
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• 2006

In this study, the cDNA of a new peptide from the venom of the scorpion, Buthotus saulcyi, was cloned and sequenced. It codes for a 64 residues peptide (Bsaul1) which shares high sequence similarity with depressant insect toxins of scorpions. The differences between them mainly appear in the loop1 which connects the $\beta$-strand1 to the $\alpha$-helix and seems to be functionally important in long chain scorpion neurotoxins. This loop is three amino acids longer in Bsaul1 compared to other depressant toxins. A comparative amino acid sequence analysis done on Bsaul1 and some of $\alpha$-, $\beta$-, excitatory and depressant toxins of scorpions showed that Bsaul1 contains all the residues which are highly conserved among long chain scorpion neurotoxins. Structural model of Bsaul1 was generated using Ts1 (a $\beta$-toxin that competes with the depressant insect toxins for binding to $Na^+$ channels) as template. According to the molecular model of Bsaul1, the folding of the polypeptide chain is being composed of an anti-parallel three-stranded $\beta$-sheet and a stretch of $\alpha$-helix, tightly bound by a set of four disulfide bridges. A striking similarity in the spatial arrangement of some critical residues was shown by superposition of the backbone conformation of Bsaul1 and Ts1.

• Journal of Physiology & Pathology in Korean Medicine
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• v.21 no.3
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• pp.721-727
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• 2007

Scorpion (SCP) has been clinically used for the treatment of endogenous wind to relieve convulsion, clearing away toxins, resolving hard masses and removing obstruction in the collaterals to relieve pain. Recent studies showed that scorpion toxins that affect the activating mechanism of sodium channels and indian black scorpion venom induced anti-proliferative and apoptogenic activity against human leukemic cell lines U937 and K562. There is lack of studies regarding the effects of SCP on the immunological activities. The present study was conducted to evaluate the effect of SCP on the regulatory effects of cytokines and nitric oxide (NO) for the immunological activities in Raw 264.7 cells. After the treatment of SCP MeOH extract dissolved in media for 1 h prior to the addition of lipopolysaccharide (LPS: 1 ${\mu}$g/ml), cell viability was measured by MTT assay, NO production was monitored by measuring the nitrite content in culture medium. Inducible nitric oxide synthase (iNOS) was determined by immunoblot analysis, and levels of cytokine were analyzed by sandwich immunoassays. As results, SCP inhibited the production of nitrite and nitrate (0.3 and 1.0 mg/ml), iNOS and p-$I_KB_{\alpha}$ protein, tumor necrosis factor-${\alpha}$ (0.3 and 1.0 mg/ml), interleukin-1${\beta}$ (0.3 and 1.0 mg/ml) and interleukin-6 (1.0mg/ml) in Raw 264.7 cells activated with LPS. These findings suggest that SCP can produce anti-inflammatory effect, which may play a role in adjunctive therapy in Gram-negative bacterial infections.

• BMB Reports
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• v.37 no.3
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• pp.292-297
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• 2004

The current model for the mechanism of action of the Bacillus thuringiensis Cry $\delta$-endotoxins involves the penetration of the ${\alpha}4-{\alpha}5$ hairpin into the target midgut epithelial cell membranes, followed by pore formation. In this study, PCR-based mutagenesis was employed to identify a critical residue within the ${\alpha}4-{\alpha}5$ loop of the 130-kDa Cry4A mosquito-larvicidal protein. Alanine-substitutions of two charged (Asp-198 and Asp-200) and four polar (Asn-190, Asn-195, Tyr-201 and Tyr-202) residues in the ${\alpha}4-{\alpha}5$ loop were performed. Like the wild-type, all of the mutant toxins were over-expressed as inclusion bodies in Escherichia coli. When E. coli cells expressing each mutant toxin were bioassayed against Aedes aegypti larvae, larvicidal activity was completely abolished for the substitution of only Tyr-202, while replacements at the other positions still retained a high level of toxicity. Further replacement of Tyr-202 with an aromatic side chain, phenylalanine, did not affect the toxicity. These results revealed a crucial role in toxin activity for the conserved aromatic residue at the 202 position within the ${\alpha}4-{\alpha}5$ loop of the Cry4A toxin.

• BMB Reports
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• v.38 no.4
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• pp.386-390
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• 2005

Based on the reported cDNA sequences of $BmK{\alpha}Txs$, the genes encoding toxin $BmK{\alpha}Tx11$ and $BmK{\alpha}Tx15$ were amplified by PCR from the Chinese scorpion Buthus martensii Karsch genomic DNA employing synthetic oligonucleotides. Sequences analysis of nucleotide showed that an intron about 500 bp length interrupts signal peptide coding regions of $BmK{\alpha}Tx11$ and $BmK{\alpha}Tx15$. Using cDNA sequence of $BmK{\alpha}Tx11$ as probe, southern hybridization of BmK genome total DNA was performed. The result indicates that $BmK{\alpha}Tx11$ is multicopy genes or belongs to multiple gene family with high homology genes. The similarity of $BmK{\alpha}$-toxin gene sequences and southern hybridization revealed the evolution trace of $BmK{\alpha}$-toxins: $BmK{\alpha}$-toxin genes evolve from a common progenitor, and the genes diversity is associated with a process of locus duplication and gene divergence.

• Journal of Food Hygiene and Safety
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• v.15 no.4
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• pp.287-292
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• 2000

The toxicity and toxin composition between blue mussel, Mytilus edulis and oyster, Crassostrea gigas collected at Woepori in Ko je island in South Coast of Korea in 1996 and 1997 were compared. The highest toxicity score was about 10 times higher in blue mussel than oyster (blue mussel, 8,670 $\mu\textrm{g}$; oyster, 860$\mu\textrm{g}$ in 1996, blue mussel, 5,657 $\mu\textrm{g}$/100g in 1997). The blue mussel also retained its toxicity for slightly longer period than oyster. In the both shellfish, PSP was composed almost exclusively of C toxicity (Cl and C2, 20~65%) and gonyautoxins (GTXl, 2, 3, and 4, 38~78%). In the early period of toxin accumulation, the ratio of 11$\beta$-epimer toxins (C2, GTX4) whose amount was 25~56 mole% (5th March to 12th April in 1996) and 25~80 mole% (18th March to 7th April in 1997), were higher than that of 11-epimer toxins (Cl, GTX2) whose amount was 41~57 mol%(27th May to 3rd June in 1996) and 25~56 mole% (29th April to 12th May in 1997), became higher than that of 11-epimer toxins. The toxin compositions in the both samples changed on a daily basis, presumably owing to metabolism of the toxin in the bivalves.

• Microbiology and Biotechnology Letters
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• v.25 no.5
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• pp.448-453
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• 1997

Enzyme activities, production of killer toxin and some functionality of forty seven yeasts isolated from traditional Meju were investigated in culture broth and cell free extracts. Activities of $\alpha$-galactosidase, invertase and inulinase were detected in cell free extracts of 38 strains, 43 strains and 45 strains, respectively and acidic and neutral protease activities also were detected in culture broth of all the strains, $\beta$-Galactosidase activity was detected in cell free extracts of OE-20 and S-14 strains. Killer toxins were produced by OE-12, S-8 (Candida spp.), OE-19 (Zygosaccharomyces spp.) and S-3 (Saccharomyces spp.). Culture broth of OE-23 and S-9 showed 61.3% and 59.2% of antioxidant activity to $\alpha$, $\alpha$-diphenyl-$\beta$-picrylhydrazyl(DPPH), but nitrite-scavenging ability as well as inhibition of tyrosinase and polyphenol oxidase were not appeared in all the strains.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.6
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• pp.511-519
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• 2001

Nimopidine, one of dihydropyridine derivatives, has been widely used to pharmacologically identify L-type Ca currents. In this study, it was tested if nimodipine is a selective blocker for L-type Ca currents in sensory neurons and heterologous system. In mouse dorsal root ganglion neurons (DRG), low concentrations of nimodipine $(<10\;{\mu}M),$ mainly targeting L-type Ca currents, blocked high-voltage-activated calcium channel currents by ${\sim}38%.$ Interestingly, high concentrations of nimodipine $(>10\;{\mu}M)$ further reduced the 'residual' currents in DRG neurons from ${\alpha}_{1E}$ knock-out mice, after blocking L-, N- and P/Q-type Ca currents with $10\;{\mu}M$ nimodipine, $1\;{\mu}M\;{\omega}-conotoxin$ GVIA and 200 nM ${\omega-agatoxin$ IVA, indicating inhibitory effects of nimodipine on R-type Ca currents. Nimodipine $(>10\;{\mu}M)$ also produced the inhibition of both low-voltage-activated calcium channel currents in DRG neurons and ${\alpha}_{1B}\;and\;{\alpha}_{1E}$ subunit based Ca channel currents in heterologous system. These results suggest that higher nimodipine $(>10\;{\mu}M)$ is not necessarily selective for L-type Ca currents. While care should be taken in using nimodipine for pharmacologically defining L-type Ca currents from native macroscopic Ca currents, nimodipine $(>10\;{\mu}M)$ could be a useful pharmacological tool for characterizing R-type Ca currents when combined with toxins blocking other types of Ca channels.

• Journal of Ginseng Research
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• v.24 no.2
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• pp.79-82
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• 2000

Saponins, the major component of ginseng root, mediate the pharmacological action of the ginseng. It has been reported that ginseng roots have protective effect against various toxins. In this study, the effects of ginseng total saponin (GTS) on tumor necrosis factor-alpha (TNF-$\alpha$) production induced by bacterial toxin was investigated. TNF-$\alpha$ level in lipopolysaccharides (LPS)-activated serum was remarkably reduced by intraperitoneal administration (50 mg/kg)of ginseng total saponin (GTS) into mice. The inhibitory effect against TNF-$\alpha$ production was not significant when GTS was given after the LPS injection, and by oral administration. These results suggested that ginseng root may have protective activity against liver damage accompanying the overproduction of TNF-$\alpha$ and GTS is the active component of ginseng.

• International Journal of Industrial Entomology and Biomaterials
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• v.21 no.1
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• pp.127-132
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• 2010

We have characterized full-length cDNA encoding Gall-6-tox protein, which was cloned from the fat body of the immunized Galleria mellonella larvae. The cloned cDNA of Gall-6-tox consists of 1301 nucleotides and contained an open reading frame of 891 nucleotides corresponding to a protein of 296 residues that includes a putative 16-residue signal sequence and a 280-residue mature peptide with a calculated mass of 30,707.73 Da. The deduced mature peptide contains conserved tandem repeats of six cysteine-stabilized alpha beta ($Cs{\alpha}{\beta}$) motifs, which was detected in scorpion toxins and insect defensins. In the sequence homology search, mature Gall-6-tox showed 34% and 28% amino acid sequence homology with Bomb-6-tox from Bombyx mori and Spod-11-tox from Spodoptera frugiperda, respectively. Gall-6-tox orthologs were only found in Lepidopteran species, indicating that this new immune-related gene family is specific to this insect order. RT-PCR analysis revealed that Gall-6-tox was expressed primarily in the larval fat bodies, hemocytes, and midgut against invading bacteria into hemocoel. Moreover, the expression time course of Gall-6-tox was examined up to 24 h in the fat bodies and midgut after injection of E. coli. Altogether, these results suggest that Gall-6-tox is derived from defensins and Gall-6-tox may play a critical role in Lepidoptera immune system.