• BMB Reports
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• 제36권3호
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• pp.294-298
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• 2003

The pathological effect of the Bacillus thuringiensis Cry $\delta$-endotoxins on susceptible insect larvae had extensive damage on the midgut epithelial cells. In this study, an ex vivo assay was devised for assessing the insecticidal potency of the cloned Cry4B mosquito-larvicidal protein that is expressed in Escherichia coli. Determination of toxicity was carried out by using a cell viability assay on the midguts that were dissected from 5-day old Aedes aegypti mosquito larvae. After incubation with the toxin proteins, the number of viable epithelial cells was determined photometrically by monitoring the quantity of the bioreduced formazan product at 490 nm. The results showed that the 65-kDa trypsin-activated Cry4B toxin exhibited toxic potency ca. 3.5 times higher than the 130-kDa Cry4B protoxin. However, the trypsin-treated products of the non-bioactive Cry4B mutant (R158A) and the lepidopteran-specific Cry1Aa toxin displayed relatively no ex vivo activity on the mosquito-larval midguts. The ex vivo cytotoxicity studies presented here confirms data that was obtained in bioassays.

• Asian-Australasian Journal of Animal Sciences
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• 제33권7호
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• pp.1087-1095
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• 2020

Objective: The present study aimed to evaluate the effects of γ-aminobutyric acid (GABA)-producing bacteria (GPB) on in vitro rumen fermentation and on the growth performance and meat quality of Hanwoo steers. Methods: The effects of GPB (Lactobacillus brevis YM 3-30)-produced and commercially available GABA were investigated using in vitro rumen fermentation. Using soybean meal as a substrate, either GPB-produced or commercially available GABA were added to the in vitro rumen fermentation bottles, as follows: control, no additive; T1, 2 g/L GPB; T2, 5 g/L GPB; T3, 2 g/L autoclaved GPB; T4, 5 g/L autoclaved GPB; T5, 2 g/L GABA; and T6, 5 g/L GABA. In addition, 27 Hanwoo steers (602.06±10.13 kg) were subjected to a 129-day feeding trial, during which they were fed daily with a commercially available total mixed ration that was supplemented with different amounts of GPB-produced GABA (control, no additive; T1, 2 g/L GPB; T2, 5 g/L GPB). The degree of marbling was assessed using the nine-point beef marbling standard while endotoxin was analyzed using a Chromo-Limulus amebocyte lysate test. Results: In regard to in vitro rumen fermentation, the addition of GPB-produced GABA failed to significantly affect pH or total gas production but did increase the ammonia nitrogen (NH₃-N) concentration (p<0.05) and reduce total biogenic amines (p<0.05). Animals fed the GPB-produced GABA diet exhibited significantly lower levels of blood endotoxins than control animals and yielded comparable average daily gain, feed conversion ratio, and beef marbling scores. Conclusion: The addition of GPB improved in vitro fermentation by reducing biogenic amine production and by increasing both antioxidant activity and NH₃-N production. Moreover, it also reduced the blood endotoxin levels of Hanwoo steers.

• Journal of Microbiology and Biotechnology
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• 제19권7호
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• pp.727-733
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• 2009

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed in E. coli.

• Journal of Environmental Science International
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• 제5권5호
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• pp.555-560
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• 1996

The toxic substances (endotoxins) from the bacterial cell walls were extracted by using incubator, centrifuge, UV-Vis spectrophotometer, and their fatty acid compositions were analyzed by Gas Chromatography. The lethal toxicities and pyrogenic activities of toxic substances were tested and the results were compared each other. The results of fatty acid analyses showed that the major fatty acid of the toxic substance was tetradecanoic acid for Vibrio vulnificus, dodecanoic acid for Escherichia coli, and decanoic acid for Salmonella typhimurium. These three fatty acids were the main fatty acids ofr three toxic substances (more, than 70%). The unique points in the fatty acid compositions were that tetradecanoic acid was composed as important one (37.15%) for V. vulnificus and that the amount of hexadecanoic acid was very small (below 2%) for three toxic substances. The lethal toxicity in ICR mice of toxic substance from V. vulnificus (LD50 was 52.5 mg/kg) was similar to that of E. coli (56.5mg/kg), but weaker than that of S. typhimurium (37.5mg/kg). Toxic substance from V. vulnificus was more pyrogenic in rabbit than that from E. coli, but less than that from S. typhimurium.

• BMB Reports
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• 제34권4호
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• pp.293-298
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• 2001

Tryptic activation of the 130-kDa Bacillus thuringiensis Cry4B $\delta$-endotoxin produced protease-resistant products of ca. 47 kDa and ca. 21 kDa. The 21-kDa fragment was identified as the N-terminal five-helix bundle (${\alpha}1-{\alpha}5$,) which is a potential candidate for membrane insertion and pore formation. In this study, we constructed the recombinant clone over-expressing this putative pore-forming (PPF) fragment as inclusion bodies in Escherichia coli. The partially purified inclusions were composed of a 23-kDa protein, which cross-reacted with Cry4B antibodies, and whose N-terminus was identical to that of the 130-kDa protein. Dissimilar to protoxin inclusions, the PPF inclusions were only soluble when the carbonate buffer, pH 9.0, was supplemented with 6 M urea. After renaturation via a stepwise dialysis, the refolded PPF protein appeared to exist as an oligomer and was structurally stable upon trypsin treatment. Unlike the 130kDa protoxin, the refolded protein was able to release entrapped glucose from liposomes, and showed comparable activity to the full-length activated toxin, although it lacks larvicidal activity These results, therefore, support the notion that the PPF fragment that consists of ${\alpha}1-{\alpha}5$ of the activated Cry4B toxin is involved in membrane pore-formation.

• BMB Reports
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• 제34권5호
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• pp.402-407
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• 2001

Based on the currently proposed toxicity model for the different Bacillus thuringiensis Cry $\delta$-endotoxins, their pore-forming activity involves the insertion of the ${\alpha}4-{\alpha}5$ helical hairpin into the membrane of the target midgut epithelial cell. In this study, a number of polar or charged residues in helix 4 within domain I of the 65-kDa dipteranactive Cry11A toxin, Lys-123, Tyr-125, Asn-128, Ser-130, Gln-135, Arg-136, Gln-139 and Glu-141, were initially substituted with alanine by using PCR-based directed mutagenesis. All mutant toxins were expressed as cytoplasmic inclusions in Escherichia coli upon induction with IPTG. Similar to the wild-type protoxin inclusion, the solubility of each mutant inclusion in the carbonate buffer, pH 9.0, was relatively low When E. coli cells, expressing each of the mutant proteins, were tested for toxicity against Aedes aegypti mosquito-larvae, toxicity was completely abolished for the alanine substitution of arginine at position 136. However, mutations at the other positions still retained a high level of larvicidal activity Interestingly, further analysis of this critical arginine residue by specific mutagenesis showed that conversions of arginine-136 to aspartate, glutamine, or even to the most conserved residue lysine, also abolished the wild-type activity The results of this study revealed an important determinant in toxin function for the positively charged side chain of arginine-136 in helix 4 of the Cry11A toxin.

• Journal of Korean Society of Occupational and Environmental Hygiene
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• 제15권3호
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• pp.270-276
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• 2005

This study was conducted to examine whether a specific synthetic metalworking fluid (MWF), "A", in use for 10 months without replacement, displayed microbial resistance and to identify the additives associated with the control of microbial growth. Three synthetic MWF products ("A", "B", and "C") were studied every week for two months. Microbial deterioration of the fluids was assessed through evaluation by endotoxin, bacteria and fungi levels in the MWFs. In addition, formaldehyde, boron, ethanolamine, and copper levels were also studied to determine whether they influence microbial growth in water-based MWFs. Throughout the entire study in the sump where MWF "A" was used, bacteria counts were lower than 103 CFU/mL, and endotoxins never exceeded 103 EU/mL. These levels were significantly lower than levels observed in sumps badly deteriorated with microbes. Boron levels in MWF "A" ranged from 91.7 to 129.6 ppm, which was significantly higher than boron levels found in other MWF products. The total level of ethanolamine (EA) in MWF "A" ranged from 35,595 to 57,857 ppm (average 40,903 ppm), which was over ten times higher than that found in other MWFs. Monoethanolamine (MEA), diethanolamine (DEA) and triethanolamine (TEA) concentrations in MWF "A" were also significantly higher than seen in other MWFs. However, although EA and boron might improve anti-microbial performance, their abuse can pose a serious risk to workers who handle MWFs. From an industrial hygiene perspective, our study results stress that the positive synergistic effect of boron and EA in reducing microbial activity in MWF must be balanced with the potentially negative health effects of such additives. Our study also addresses the disadvantage of failing to comprehensively report MWF additives on Material Safety Data Sheets (MSDS). Future research in MWF formulation is needed to find the best level of EA and boron for achieving optimal synergistic anti-microbial effects while minimizing employee health hazards.

• Journal of Sericultural and Entomological Science
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• 제40권2호
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• pp.126-130
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• 1998

A noninsecticidal strain, Bacillus thuringiensis NTB-88, isolated from Korean soil, had a typical bipyramidal parasporal inclusion and its serotype is identical to B. thuringiensis subspmorrisoni (H8a8b). To elucidate differences between insecticidal and noninsecticidal strains, we compared strain NTB-88 to other toxic B. thuringiensis subsp. morrisoni strains (HD-12 and PG-14). Restriction endonucleases digested plasmid DNA patterns showed that strain NTB-88 was different from lepidopteran-toxic strain, HD-12, but it was similar to dipteran-toxic strain, PG-14. The gene type of strain NTB-88 was different from those of other insecticidal strains, Furthermore, the NH2-terminal amino acid sequence of crystal protein of strain NTB-88 had no relation to those of the previously known $\delta$-endotoxins in other toxic strains as well as HD-12 and PG-14 strains. Therefore, the noninsecticidal crystal protein in strain NTB-88 is novel and its property is different from insecticidal ones.

• Journal of the Society of Cosmetic Scientists of Korea
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• 제32권2호
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• pp.93-97
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• 2006

Staphylococcus aureus is found on the skin of $78{\sim}100%$ of children and adults with atopic dermatitis (AD) but only on the skin of $2{\sim}25%$ of healthy subjects. It is known that S. aureus and their endotoxins as superantigen have important roles in the exacerbation and prolongation of AD. This study was carried out for the detection of S. aureus in the skin of AD, age, sex, outbreak age of AD, treatment duration, aggravation season, and the relation of ooze and S. aureus. Most patients (84%) with AD show colonization of the skin with S. aureus and there is a correlation between the degree of colonization and the serous exudate. It seems likely that the inhibition of S. aureus is associated with improvement in the skin of AD patients.

• Korean journal of applied entomology
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• 제32권4호
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• pp.426-432
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• 1993

Thirteen isolates of Bacillus thunngiensls producing parasporal mclusions, obtained from 45 samples of dust and soil of sericultural tarms in Kyeong-ki province, were exammed for their toxicity against larvae of Lepidoptera, Dipwra and Coleoptera. Of these isolates, Bacillus f thuringiensis NT0423 was toxic to bath Lepidoptera, and Dipteran larvae. NT0423 showed that the $LC_{50}$ values against the Lepldaptora, Plutella macuhpennis and the Diptera, Culex pipiens were $1.30\times10^6$ CFU/ml, $2.88\times10^5$ CFU/ml, respectively. The tYPlcal bipyramldal crystals produced by NT0423 composed of protoxms of 130-140kDa encoded by one or more plasm mid-horne genes. Also, plasmid DNA analysis indicated Lhat this isolate has 9 plasmids which d differ with reported several B. thuringiensis strains.