• 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.

• 한국환경과학회지
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• 제5권5호
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• pp.555-560
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• 1996

미생물 세포벽의 독성 물질 (내독소)을 incubator, centrifuge와 UV-Vis spectrophotometer를 이용하여 추출한 후 지방산 조성을 Gas Chromatography로 분석하였으며 독성 물질의 치사 독성과 발열 활성을 측정, 그 결과를 서로 비교하였다. 지방산 조성 분석 결과 V. vulnificus의 독성 물질 (내독소)에 대해 tetradecanoic acid가, E. coli게 대 해서는 dodecanoic acid, 그리고 S. typhimurium에 대해서는 decanoic acid가 주 지방산이었으며 이들 세 지방산이 세가지 독성 물질에서 대부분을 차지하였다 (70% 이상). 지방산 조성에서 특이한 점은 tetra-decanoic acid가 V. vulnificus에 대해 주 지방산이라는 점과 세가지 독성 물질 모두 hexadecanoic acid의 양이 매우 적다는 것이다 (2% 이하). V. vulnificus의 독성 물질의 mice 에서의 치사 독성 (LD50이 52.5 mg/kg)은 E. soli(56.5mg/kg)와 비슷하였으나 S. typhimunum (37.5mg/kg) 보다는 약하였으며 V. vulnificus의 독성 물질의 rabbit에서의 발열 활성은 E. coli보다 강하였으나 5S typhimurium 보다는 약하였다.

• 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.

• 한국산업보건학회지
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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.

• 한국잠사곤충학회지
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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.

• 대한화장품학회지
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• 제32권2호
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• pp.93-97
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• 2006

황색포도상구균이 정상인 피부의 경우 $2{\sim}25%$에서 검출되는 반면 아토피 피부염이 있는 소아와 어른에 있어 $78{\sim}100%$의 높은 비율로 검출되며, 황색포도상구균과 초항원으로 작용하는 독소가 아토피 피부염의 악화 및 발병기간에 중요한 역할을 하는 것으로 알려져 있다. 본 연구는 아토피 피부염 환자 피부의 황색포도상구균의 분석과 함께 아토피 피부염 환자의 연령, 성별, 발병 연령과 투병기간, 악화되는 계절 및 진물과 황색포도상구균과의 관계에 대하여 알아보고자 실시하였다. 아토피 피부염 환자 대부분(84%)에서 병변부에 S. aureus가 집락을 형성하고 있는 것으로 나타났으며, 대부분의 아토피 피부염 환자의 피부에 S. aureus가 분포하고 있음을 알 수 있었고 아토피 피부염 병변부의 진물과 S. aureus의 밀접한 관계를 확인할 수 있었다. 따라서 아토피 피부염 환자의 피부에서 황색포도상구균 및 초항원독소를 차단한다면 아토피 피부염의 개선에 많은 도움이 될 것으로 사료된다.

• 한국응용곤충학회지
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• 제32권4호
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• pp.426-432
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• 1993

경기도 일원의 양잠 농가의 먼지에서 채취한 45개의 샘플중에서 내독소 단백질 결정체를 생산하는 13개의 Bacillus thuringiensis를 분리하였다. 이 중 2개 균주는 파리목에 독성을 나타냈으며, 특히 독성검정에서 NT0423균주의 $LC_{50}$수치는 나비목의 배추좀나방이 최소 1.30$\times$$10^{6}$ CFU/ml이며, 파리목인 빨간 집모기에는 2.88$\times$$10^{5}$ CFU/ml로 나타났다. 새로 분리된 NT0423균주가 생산하는 내독소 단백질 결정체는 주사 전자현미경사진에서 전형적인 이중 피라미드모양을 보였다. 그리고 이 내독소 단백질 결정체의 SDS-PAGE 분석에서는 주요한 130kDa의 polypeptide을 나타내었다. 또한 NT0423균주의 총 플라스미드 DNA분석에서는 9개의 플라스미드를 갖고 있어 기존의 유사한 독성을 나타내는 균주들과 다른 패턴을 보였다.