• The Plant Pathology Journal
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• 제35권4호
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• pp.313-320
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• 2019

Rye was used here to dissect molecular mechanisms of resistance to Fusarium head blight (FHB) and to go deeper with our understanding of that process in cereals. F. culmorum-damaged kernels of two lines different in their potential of resistance to FHB were analyzed using two-dimensional gel electrophoresis and mass spectrometry to identify resistance markers. The proteome profiling was accompanied by measurements of ${\alpha}-$ and ${\beta}-amylase$ activities and mycotoxin content. The proteomic studies indicated a total of 18 spots with clear differences in protein abundance between the more resistant and more susceptible rye lines after infection. Eight proteins were involved in carbohydrate metabolism of which six proteins showed a significantly higher abundance in the resistant line. The other proteins recognized here were involved in stress response and redox homeostasis. Three remaining proteins were associated with protease inhibition/resistance and lignin biosynthesis, revealing higher accumulation levels in the susceptible rye line. After inoculation, the activities of ${\alpha}-$ and ${\beta}-amylases$, higher in the susceptible line, were probably responsible for a higher level of starch decomposition after infection and a higher susceptibility to FHB. The presented results could be a good reference for further research to improve crop resistance to FHB.

• BMB Reports
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• 제39권3호
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• pp.270-277
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• 2006

Helices 4 and 5 of the Bacillus thuringiensis Cry4Ba $\delta$-endotoxin have been shown to be important determinants for mosquito-larvicidal activity, likely being involved in membrane-pore formation. In this study, the Cry4Ba mutant protein containing an additional engineered tryptic cleavage site was used to produce the $\alpha4$-$\alpha5$ hairpin peptide by an efficient alternative strategy. Upon solubilization of toxin inclusions expressed in Escherichia coli and subsequent digestion with trypsin, the 130-kDa mutant protoxin was processed to protease-resistant fragments of ca. 47, 10 and 7 kDa. The 7-kDa fragment was identified as the $\alpha4$-loop-$\alpha5$ hairpin via N-terminal sequencing and mass spectrometry, and was successfully purified by size-exclusion FPLC and reversed-phase HPLC. Using circular dichroism spectroscopy, the 7-kDa peptide was found to exist predominantly as an $\alpha$-helical structure. Membrane perturbation studies by using fluorimetric calcein-release assays revealed that the 7-kDa helical hairpin is highly active against unilamellar liposomes compared with the 65-kDa activated full-length toxin. These results directly support the role of the $\alpha4$-loop-$\alpha5$ hairpin in membrane perturbation and pore formation of the full-length Cry4Ba toxin.

• 한국미생물·생명공학회지
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• 제43권2호
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• pp.91-96
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• 2015

Prion은 양의 scrapie, 소의 bovine spongiform encephalopathy와 사람의 CJD와 같은 다양한 신경 퇴행성 질환을 유발시키는 단백질 병원체이다. 정상 prion 단백질인 PrP^C가 병원성 PrP^Sc로 바뀌는 과정에 대해서는 많은 연구가 진행되었고, PrP^Sc로의 단백질 구조 변화가 다양한 환경적 요소에 의해서 영향 받는 것으로 추측된다. 바다조류로부터 분리된 MAAs는 다양한 스트레스 환경에서 조류를 보호해주는 것으로 알려져 있다. 이와 같은 사실에 기초하여 mycosporineglycine, porphyra-334와 shinorine 3종의 MAAs로 처리한 prion 감염 신경세포 주에서 prion 단백질 축적의 변화를 평가하였다. PK 저항성을 갖는 PrP^Sc를 western blot 방법으로 확인한 결과, MAA에 의해서 PrP^Sc 단백질의 증식을 관찰하였다.

• 한국양식학회지
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• 제11권4호
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• pp.495-503
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• 1998

생균(Bacillus polyfermenticus, Bacillus mesentericus, Streptococcus faecalis, Bifidobacterium breve)과 소화 요소(protease, lipase)를 함유한 비스루트의 효과를 알아보기 위해 사료 내 1% 첨가하여 나일틸라피아 치어(19.0${\pm}$0.07g)에게 60일간 공급한 다음 성장률, 체조성 및 면역반응의 효과를 조사하였다. 60일간 사육한 결과 비스루트 첨가구와 대조구 사이에 유의적인 성장 차이를 보이지 않았지만(P>0.05), 보체의 용혈 능력은 비스루트 첨가구가 대조구에 비해 용혈능이 높은 것으로 조사되고 라이소자임의 용균 효과는 대조구와 비슷하였다. 식세포의 식작용 능력과 신장 마크로파지의 respiratory burst activity 는 비스루트 첨가구가 대조구에 비해 활성이 매우 높았다. 따라서 비스루트 사료 내 첨가가, 어류의 비특이적 면역 기능중에서 세포성 면역 기능을 활성화하는데 기여하였다. 병원성 세균 Edwardsiella tarda FSW 910410 균주로 공격 실험결과 비스루트 첨가구가 누적폐사율이 59%, 대조구가 80%로 조사되었고, 그 결과 비스루트 첨가구는 병원성 세균에 대한 저항력을 증가시켰다(P<0.05). Hematocrit, hemoglobin, total protein, glucose, GOT, GPT 와 같은 혈액 성분을 분석한 결과 비스루트 첨가구와 대조구 간의 유의차는 없었지만(P>0.05), glucose, GOT, GPT는 비스루트 첨가구가 대조구에 비해 낮게 나타났다. 이상의 결과로 비스루트는 나일틸라피아의 생리 기능에 문제를 야기시키지 않고, 비특이적 면역 기능 중 세포성 면역 기능을 증강시키는 효과가 있으며, 병원성 세균에 대한 저항력의 증강 효과도 높다. 따라서, 비스루트는 나일틸라피아의 사료 첨가물로서의 역할이 충분히 기대된다.

• 한국잠사곤충학회지
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• 제34권1호
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• pp.41-48
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• 1992

해충의 미생물적 방제를 위하여 Bacillus thuringiensis 살충제(BT제) 개발에 관한 기초자료를 얻고자 3균주의 공시균주인 B. thuringiensis var. kurstaki, B. thuringiensis var. dendrolimus, B. thuringiensis var. aizawai와 3종의 공시충으로 누에, 흰불나방, 배추흰나비의 유충을 이용하고 B. thuringiensis 내독소단백질을 alkalidyddor 또는 공시된 3종의 곤충소화액으로 처리하여 전기영동을 비교하여 아래와 같은 결과를 얻었다. 1. 3균주의 B. thuringiensis에서 생산된 내독소단백질은 alkali 또는 곤충의 소화액 처리에 따라서 변화되는 뚜렷한 차이가 없는 것으로 나타났다. 2. 각 균주의 내독소단백질의 분자량은 B. thuringiensis var. aizawai가 130kDa, B. thuringiensis var. kurstaki가 140kDa, 130kDa, 그리고 B. thuringiensis var. dendrolimus가 140kDa의 부분에 2band로 나타났으며, 이들 내독소단백질은 alkali 용액이나 숙주곤충의 소화액으로 처리시 활성화되어 40-65kDa의 저분자 단백질로 전환되었다. 또한 숙주곤충소화액에 따라 내독소단백질의 활성화 속도에 약간의 차이가 있는 것으로 확인되었다.

• 한국미생물·생명공학회지
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• 제29권1호
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• pp.1-11
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• 2001

Lactic acid bacteria (LAB) are widely used for various food fermentation. With the recent advances in modern biotechnology, a variety of bio-products with the high economic values have been produced using microorganisms. For molecular cloning and expression studies on the gene of interest, E. coli has been widely used mainly because vector systems are fully developed. Most plasmid vectors currently used for E, coli carry antibiotic-resistant markers. As it is generally believed that the antibiotic resistance markers are potentially transferred to other bacteria, application of the plasmid vectors carrying antibiotic resistance genes as selection markers should be avoided, especially for human consump-tion. By contrast, as LAB have some desirable traits such that the they are GRAS(generally recognized as safe), able to secrete gene products out of cell, and their low protease activities, they are regarded as an ideal organism for the genetic manipulation, including cloning and expression of homologous and heterologous genes. However, the vec-tor systems established for LAB are stil insufficient to over-produce gene products, stably, limiting the use of these organisms for industrial applications. For a past decade, the two popular plasmid vectors, pAM$\beta$1 of Streptococcus faecalis and pGK12 theB. subtilis-E. coli shuttle vector derived from pWV01 of Lactococcus lactis ssp. cremoris wg 2, were most widely used to construct efficient chimeric vectors to be stably maintained in many industrial strains of LAB. Currently, non-antibiotic markers such as nisin resistance($Nis^{r}$ ) are explored for selecting recombi-nant clone. In addition, a gene encoding S-layer protein, slp/A, on bacterial cell wall was successfully recombined with the proper LAB vectors LAB vectors for excretion of the heterologous gene product from LAB Many food-grade host vec-tor systems were successfully developed, which allowed stable integration of multiple plasmid copies in the vec-mosome of LAB. More recently, an integration vector system based on the site-specific integration apparatus of temperate lactococcal bacteriophage, containing the integrase gene(int) and phage attachment site(attP), was pub-lished. In conclusion, when various vector system, which are maintain stably and expressed strongly in LAB, are developed, lost of such food products as enzymes, pharmaceuticals, bioactive food ingredients for human consump-tion would be produced at a full scale in LAB.

• 대한의생명과학회지
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• 제19권1호
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• pp.1-8
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• 2013

Parkin is known to be a tumor suppressor protein. Previously, we determined that parkin expression restores susceptibility to TNF-${\alpha}$-induced death of HeLa cells, a human cervical cancer cell line resistant to TNF-${\alpha}$-induced cell death. MMP-3 is a zinc-dependent protease recently reported to activate intracellular apoptotic signaling. In this study we examined the regulation of MMP-3 expression by parkin in TNF-${\alpha}$-treated HeLa cells. Furthermore, we investigated the signaling pathway involved in parkin-induced expression of MMP-3. We found that HeLa cells exhibit low levels of MMP-3 but is induced after introduction of the parkin gene into HeLa cells. Furthermore, MMP-3 expression increased further when parkin expressing cells were treated with TNF-${\alpha}$. Using chemical inhibitors of cell signaling pathways, we found that MEK-1 (PD98059), PI3K (LY294002), p38 MAPK (SB203580), and JNK inhibitors alleviated parkin-induced up-regulation of MMP-3. Finally, we show that TNF-${\alpha}$-induced cell death in parkin expressing cells is inhibited by using a MMP-3 inhibitor. These results suggest that parkin expression induces prolonged expression of MMP-3 via MEK-1, PI3K, MAPK, and JNK pathway in HeLa cells allowing the HeLa cells to become sensitive to TNF-${\alpha}$-induced cell death. These results implicate a role of MMP-3 in parkin-induced cell death in TNF-${\alpha}$ treated HeLa cells.

• 한국응용곤충학회지
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• 제47권4호
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• pp.457-465
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• 2008

본 연구는 모기에 활성을 나타내는 Bacillus thuringiensis subsp. tohokuensis CAB167균주에서 생산된 내독소결정단백질의 특성을 조사하였다. B. thuringiensis subsp. tohokuensis CAB167균주는 위상차 현미경과 주사전자현미경으로 관찰하여 일반적으로 모기에 활성을 나타내는 spherical type의 내독소결정 성단백질을 형성하는 것으로 나타났다. 국내에서 서식하는 지하집모기(Culex pipiens molestus), 빨간집모기(Culex pipiens pallens), 이집트숲모기(Aedes aegyti)에 대한 이 균주의 살충활성검정은 각각 173, 190, 580 ng/ml의 $LC_{50}$ 값으로 각각 활성을 보였다. B. thuringiensis subsp. tohokuensis CAB167균주에서 생산된 내독소결정성단백질의 SDS-PAGE를 통해 135, 80, 49와 28-kDa의 주요한 4개의 밴드를 나타냈다. 내독소결정성단백질의 소화에 영향을 미치는 곤충중장액과 유사한 trypsin효소를 처리하여 72와 63-kDa의 단백질이 새롭게 생성하였다. 파리목 위생해충인 모기에 활성을 나타내는 다른 7개의 B. thuringiensis 균주들의 내독소 결정성단백질과 혈청학적인 비교실험에서 독소단백질이 차이가 있는 것을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제21권8호
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• pp.861-868
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• 2011

A xylanase gene, xyn7c, was cloned from Paenibacillus sp. 12-11, an alkalophilic strain isolated from the alkaline wastewater sludge of a paper mill, and expressed in Escherichia coli. The full-length gene consists of 1,296 bp and encodes a mature protein of 400 residues (excluding the putative signal peptide) that belongs to the glycoside hydrolase family 10. The optimal pH of the purified recombinant XYN7C was found to be 8.0, and the enzyme had good pH adaptability at 6.5-8.5 and stability over a broad pH range of 5.0-11.0. XYN7C exhibited maximum activity at $55^{\circ}C$ and was thermostable at $50^{\circ}C$ and below. Using wheat arabinoxylan as the substrate, XYN7C had a high specific activity of 1,886 U/mg, and the apparent $K_m$ and $V_{max}$ values were 1.18 mg/ml and 1,961 ${\mu}mol$/mg/min, respectively. XYN7C also had substrate specificity towards various xylans, and was highly resistant to neutral proteases. The main hydrolysis products of xylans were xylose and xylobiose. These properties make XYN7C a promising candidate to be used in biobleaching, baking, and cotton scouring processes.