• Title/Summary/Keyword: 1-2 toxin

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Molecular Cloning of Two cDNAs Encoding an Insecticidal Toxin from the Spider, Araneus ventricosus, and Construction of a Recombinant Baculovirus Expressing a Spider Toxin

  • Chung, Eun-Hwa;Lee, Kwang-Sik;Han, Ji-Hee;Je, Yeon-Ho;Chang, Jin-Hee;Roh, Jong-Yul
    • International Journal of Industrial Entomology and Biomaterials
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    • v.4 no.1
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    • pp.43-49
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    • 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.

Effect of Diphtheria Toxin on the Phospholipase D activity and Free Fatty Acid Release in HepG2 Cells (HepG2 세포의 포스포리파제 D 활성과 자유 지방산 방출에 대한 디프테리아 독소의 영향)

  • Koh, Eun-Hie
    • Journal of the Korean Chemical Society
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    • v.59 no.1
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    • pp.22-30
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    • 2015
  • The effect of diphtheria toxin on cell membrane lipids was studied by examining the phospholipase D (PLD) activity and free fatty acids (FFA) release in HepG2 cells. The diphtheria toxin effects on lipid alteration show apparently maximal at pH 5.1, stimulating PLD activity nearly 3.5 fold and enhancing FFA release approximately 5 fold over the control. These results indicate that the membrane is perturbed and its lipid component is rearranged during the diphtheria toxin translocation. Digitonin, a random membrane perturbing detergent, exhibit about four-fold higher perturbation effect over the diphtheria toxin at neutral pH. This observation suggests that the membrane perturbation induced by diphtheria toxin appears to be rather selective. To investigate the cause of the membrane perturbation, Cibacron blue, an inhibitor of membrane pore formation, and hemagglutinin, an influenza virus with fusion peptide, were tested for their effects on diphtheria toxin action. Cibacron blue decreased the diphtheria toxin effect by almost 50%, but the lipid alteration induced by hemagglutinin was similar to the diphtheria toxin effect. These observations imply that the membrane perturbation induced by diphtheria toxin may be caused by a combination of pore formation and insertion of hydrophobic peptide of toxin to the membrane as well. Additionally, we found that the diphtheria toxin increased the HepG2 cells permeability but the cells viability was maintained at high level at the same time. DNA fragmentation which is related to apoptosis was not induced by the toxin. Under these conditions, we could demonstrate that the lipid alteration of HepG2 cells was brought about by diphtheria toxin at acidic pH.

Purification of Diphtheia Toxin and the Production of Detoxificated Toxoid Vaccine (디프테리아 toxin 정제와 무독화 toxoid 백신 생산)

  • Cho, Min;Ryu, Yeon-Woo
    • KSBB Journal
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    • v.14 no.2
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    • pp.248-254
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    • 1999
  • Adverse reactions after injection of diphtheria vaccine are induced by impurities present in crude toxoids that cannot be removed completely by purification of toxoids after formalization. To increase toxoid purity, toxin purification was tried before formalization. Crude toxin was purified with ultrafiltration and ion-exchange chromatography. Purified toxin purity was improved 2.9 times higher than crude toxin, and purity was 2,560 Lf/mg PN. Purified toxin was detoxified with formalin and lysine, and potency test were performed. Toxoid, prepared from toxin treated with formalin and lysine, did not show reversion to toxin and purity was higher than the toxoid purified after formalization. Therefore, we concluded that the use of toxoid vaccine prepared from toxin purified is a useful method of minimize adverse reaction after injection of diphtheria vaccine.

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Studies for Reestablishment of Approval Toxin Amount in Paralytic Shellfish Poison-Infested Shellfish -4. Detoxification and Toxin Composition in Paralytic Shellfish Poison-Infested Oyster during Processing-

  • Jeong Hyun-Jeong;Shin Il-Shik;Kim Young-Man
    • Fisheries and Aquatic Sciences
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    • v.2 no.2
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    • pp.155-160
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    • 1999
  • Studies on detoxification of Paralytic Shellfish Poison (PSP)-infested oyster, Crassostrea gigas were carried out using available processing resources. Changes of paralytic shellfish toxin components and specific toxicity during canning process were also investigated with high performance liquid chromatography (HPLC). Toxic oysters collected at Hachong in Koje Bay were used for experimental samples. The toxicity of oysters with range of 185-778 ug/100g was reduced below the quarantine limit of 80 ug/100g or not detected level by the mouse bioassay after canning process. The mole $\%$ of toxin components in the shucked oyster was in the order of 25.1 mole $\%$ of gonyautoxin 1, 19.2 mole $\%$ of gonyautoxin 3, 17.2 mole $\%$ of gonyautoxin 4 and 14.6 mole $\%$ of gonyautoxin 2. This sample had tracing amounts of Cl, C2, saxitoxin and neosaxitoxin. In the case of specific toxicity, the major toxins were consisted of gonyautoxin 1-4. The sum of gonyautoxin 1, 2, 3 and 4 was 80% of total toxicity of oyster. Saxitoxin and decarbamoylsaxitoxin were the more thermostable than any other toxin components.

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Effects of T-2 Toxin, Zeolite and Mycosorb on Antioxidant Systems of Growing Quail

  • Dvorska, J.E.;Surai, P.F.
    • Asian-Australasian Journal of Animal Sciences
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    • v.14 no.12
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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.

Cytotoxicity of T-2 Toxin on Primary Cultures of Rat Hepatocytes

  • Kim, Hwan-Mook;Kim, Byung-Sam;Choe, Suck-Young;Yang, Kyu-Hwan
    • Toxicological Research
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    • v.4 no.1
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    • pp.37-45
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    • 1988
  • Primary cultures of adult rat hepatocytes were used to study in vitro cytotoxic effects of T-2 toxin on liver cells. When T-2 toxin was added to the culture, a significant depression of the hormonal induction of ${\alpha}$-aminoisobutyric acid (AIB) uptake and tyrosine aminotransferase (TAT) activity was observed. However, T-2 toxin did not affect the uptake of ouabain into hepatocytes. Protein synthesis was inhibited by T-2 toxin, but RNA synthesis was not severely affected. The inhibitory effects of T-2 toxin on protein synthesis was diminished rapidly with culture time and the hepatocytes culture maintained control level of protein synthesis within 24 hrs.

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Species classification of the toxic dinoflagellate Alexandrium tamarense and A. catenella based on their paralytic shellfish toxin profiles

  • Kim, Young-Soo;Kim, Chang-Hoon
    • Proceedings of the Korean Aquaculture Society Conference
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    • 2003.10a
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    • pp.128-128
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    • 2003
  • The annually outbreak of paralytic shellfish poisoning (PSP) were caused by toxic dinolagellate A. tamarense and A. catenella in Korea. The purpose of this study were to investigate the distribution of PSP-causative organisms, A. tamarense and A. catenella and their species classification. Sediment (Saemangeum, the south open sea) and water samples (southeastern coast) were sampled to establish clonal isolates in 2003. After isolation and purification, strains were cultured under $17^{\circ}C$, f/2 media, 14:10=L:D cycle. PST analysis and species identification were performed by HPLC-FD method and specific DNA probe, respectively. Thirty-ons strains were isolated from the Saemangeum reclamation, southeastern coast including Jinhae Bay and south open sea. PSTs were detected in all cultured strains. In eight strains from south offshore, major toxin components are GTX5, C1/2 and minors are GTX3/4, dcGTX3, neoSTX. Sixteen strains from south coastal area have GTX1/4, neoSTX, C1/2 as major toxin components and GTX2/3 as minors. Seven strains from the Saemangeum reclamation have GTX5, C1/2 as major toxin components and GTX1/2/3/4 as minors. Thus, among eight south offshore isolates, four A. tamarense have more toxic (38.31~l19.16 fmol.$cell^{-1}$) than A. catenella (3.78~13.13 fmol.$cell^{-1}$). With the previous results of different toxin composition, toxin components and toxin contents, .it is toxin profile that could used to diagnosis of regional toxic population and geographical distribution of both A. tamarense and A. catenella and their toxigenic strains.

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Expression of Bacillus thringiensis HD-1 gene in rhizobacteria Pseudomonas fluorescens KR164 (근권 길항세균 Pseudomonas fluorescens KR164에 Bacillus thuringiensis HD-1 유전자의 삽입과 발현)

  • Kim, Yeong-Yil;Rhee, Young-Hwan;Kang, Heun-Soo
    • Applied Biological Chemistry
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    • v.35 no.4
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    • pp.227-231
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    • 1992
  • The plasmids pSUPBT and pSUPBTR were constructed with a vector pSUP2021 and the BT toxin gene in the plasmid pES 1. The plasmids constructed were introduced into the antagonistic rhizobacteria P. fluorescens KR164 by conjugation and P. fluorescens having pSUPBT and pSUPBTR were named P. fluorescens KR164(pSUPBT)#2, KR164(pSUPBT)#3, KR164(pSUPBTR)#2 and KR164(pSUPBTR)#3, respectively. The BT toxin gene were identified in all transformants by Southern hybridization and the final product of BT toxin gene was identified only in P. fluorescens KR164(pSUPBTR)#3 by SDS-PAGE. This crystal toxin protein were also observed in electron microscopy.

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Studies for Reestabilishment of Approval Toxin Amount in Paralytic Shellfish Poison-Infested Shellfish 5. Comparison of Toxicity and Toxin Composition of Paralytic Shellfish Poison between Blue mussel, Mytilus edulis and Oyster, Crassostrea gigas

  • Shin, Il-Shik;Kim, Young-Man
    • 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.

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The Correlation between Toxin Genotype and Antibiotic Resistance in Methicillin Resistant Staphylococcus aureus Isolated from Clinical Specimen of Intensive Care Unit (중환자실의 임상검체로부터 분리된 Methicillin 내성 Staphylococcus aureus의 독소유전자형과 항생제내성의 상관관계)

  • Park, Chul;Seong, Chi Nam
    • Korean Journal of Clinical Laboratory Science
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    • v.48 no.3
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    • pp.202-209
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    • 2016
  • This study is aimed to determine the correlation between the toxin gene types and antibiotic resistance from MRSA (methicillin-resistant Staphylococcus aureus). Fifty-two strains of MRSA, between January 2014, and December 2014, were isolated from clinical specimens obtained from 2,664 cases in the intensive care unit of a hospital in Suncheon, Jeonnam, Korea. Genes encoding mecA, enterotoxin (SE), toxic shock syndrome toxin-1 (TSST-1), exfoliative toxin (ET), and Panton-Valentine leukocidin (PVL) were detected by multiplex PCR-mediated amplification using specific primers. Toxin genes (seg and sei) were present in 40 strains (76.9%), followed by tst in 34 strains (65.4%). Other genes (eta, etb, sea, sed, see, seh, sej, and pvl) were not detected. Forty strains (76.9%) of MRSA had 2 or more toxin genes simultaneously; 5 coexistent toxin-genes (seb, sec, seg, sei, tst) were the most common in 28 strains (53.8%), and 6 strains (11.5%) had seg and sei genes. The coexistence of genes were 72.5~100%, showing a high correlation among genes (seb, sec, seg, sei and tst). As strains (seb, sec, tst) that had particular toxin genes (seb, sec, seg, sei, tst) in multiple showed 100% resistance to ciprofloxacin, clindamycin, erythromycin, we were able to find that seb, sec, and tst genes have a close relationship to the aforementioned antibiotics. It showed a higher resistance to ciprofloxacin, clindamycin, erythromycin, and tetracycline compared with strains that had toxin genes independent from multiple toxin genes.