• Title/Summary/Keyword: MALDI- TOF

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Monoclonal antibody production for CP4 EPSPS detection assays (CP4 EPSPS 검출을 위한 단클론 항체 생산)

  • A-Mi Yoon;Il Ryong Kim;Wonkyun Choi
    • Korean Journal of Environmental Biology
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    • v.39 no.4
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    • pp.445-451
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    • 2021
  • In this study, we described the production of an antibody to living modified organisms (LMOs) containing the gene encoding for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium tumefaciens strain CP4 EPSPS provides resistance to the herbicide glyphosate (N- (phosphonomethyl) glycine). These LMOs were approved and have recently been used in the feed, food production, and processing industries in South Korea. Highly efficient monoclonal antibody (mAb) production is crucial for developing assays that enable the proper detection and quantification of the CP4 EPSPS protein in LMOs. This study describes the purification and characterization of recombinant CP4 EPSPS protein in E. coli BL21 (DE3) based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrixassisted laser desorption/ionization time-of-flight mass spectrometry. The production of mAbs was undertaken based on the standard operating procedure of Abclon, Inc.(South Korea), and the purity of the mAbs was assessed using SDS-PAGE. The following five mAb clones were produced: 2F2, 4B9, 6C11, 10A9, and 10G9. To verify the efficiency and specificity of the five developed mAbs, we performed Western blotting analysis using the LM (living modified) cotton crude extracts. All mAbs could detect the CP4 EPSPS protein in the LM cotton traits MON1445 and MON88913 with high specificity, but not in any other LM cottons or non-LM cottons. These data indicate that these five mAbs to CP4 EPSPS could be successfully used for the further development of antibody-based detection methods to target CP4 EPSPS protein in LMOs.

Characterization of Bacteriocin Produced from Isolated Strain of Bacillus sp. (Bacillus 속 분리주가 생산하는 박테리오신의 특성 조사)

  • Ham, Seung-Hee;Choi, Nack-Shick;Moon, Ja-Young;Baek, Sun-Hwa;Lee, Song-Min;Kang, Dae-Ook
    • Journal of Life Science
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    • v.27 no.2
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    • pp.202-210
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    • 2017
  • As an effort to find a potential biopreservative, we isolated bacterial strains producing bacteriocin from fermented foods. A strain was finally selected and characteristics of the bacteriocin were investigated. The selected strain was identified as Bacillus subtilis E9-1 based on the 16S rRNA gene analysis. The culture supernatant of B. subtilis E9-1 showed antimicrobial activity against Gram-positive bacteria. Subtilisin A, ${\alpha}$-chymotrypsin, trypsin and proteinase K inactivated the antimicrobial activity, which means its proteinaceous nature, a bacteriocin. The bacteriocin activity was fully retained at the pH range from 2.0 to 8.0 and stable at up to $100^{\circ}C$ for 60 min. Solvents such as ethanol, isopropanol and methanol had no effect on the antimicrobial activity at the concentration of 100% but acetone and acetonitrile reduced the activity at up to 100% concentration. Cell growth of four indicator strains was dramatically decreased in dose-dependent manner. Listeria monocytogenes was the most sensitive, but Enterococcus faecium was the most resistant. Bacillus cereus and Staphylococcus aureus showed the medium sensitivity. The bacteriocin showed its antimicrobial activity against B. cereus and L. monocytogenes via bactericidal action. The number of viable cells of L. monocytogenes started to reduce after addition of bacteriocin to the minced beef. The bacteriocin was purified through acetone concentration, gel filtration chromatography and RP-HPLC. The whole purification step led to a 6.82 fold increase in the specific activity and 6% yield of bacteriocin activity. The molecular weight of the purified bacteriocin was determined to be 3.3 kDa by MALDI-TOF/TOF mass spectrometry.

Interleukin-4 and -8 Gene Polymorphisms and Risk of Gastric Cancer in a Population in Southwestern China

  • Pan, Xiong-Fei;Wen, Ying;Loh, Marie;Wen, Yuan-Yuan;Yang, Shu-Juan;Zhao, Zhi-Mei;Tian, Zhi;Huang, He;Lan, Hui;Chen, Feng;Soong, Richie;Yang, Chun-Xia
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.7
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    • pp.2951-2957
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    • 2014
  • Background: Gastric carcinogenesis is a complicated process that involves environmental and genetic factors like interleukin-4 (IL-4) and IL-8. Single nucleotide polymorphisms in their genes are associated with changed levels of gene expression. Here, we investigated the association between IL4-590 C>T and IL8-251T>A and gastric cancer (GC) risk in Sichuan of Southwestern China. Materials and Methods: We surveyed the research subjects using a self-designed questionnaire with questions on demographic factors and putative risk factors. Approximately 2-5ml of whole blood was collected after field survey to analyze IL4-590 C>T and IL8-251T>A genotypes using MALDI-TOF MS. Results: Our study recruited 308 pairs of GC patients and controls, including 224 (72.7%) men and 84 (27.3%) women in each group. There were 99 cardia and 176 noncardia GC patients in the case group. The case and control groups had an average age of $57.7{\pm}10.6$ ($mean{\pm}SD$) and $57.6{\pm}11.1$ years. GC patients reported a significantly greater proportion of family history of cancer (29.9% vs 10.7%, p<0.01) and drinking (54.6% vs 43.2%, p<0.01) than did controls. Variant genotypes of IL-4-590 C>T and IL-8-251 T>A were not associated with overall GC risk (adjusted OR, 0.89; 95%CI, 0.61-1.28 for CT or CC vs TT; adjusted OR, 1.14; 95%CI, 0.86-1.79 for TA or AA vs TT). Stratification analysis of two SNPs for risk by subsites only found that variant IL-8-251 TA or AA genotype was associated with increased noncardia GC risk (adjusted OR, 2.58; 95%CI, 1.19-5.57). We did not observe interactions between the IL-8-251 T>A genotype and smoking (adjusted OR, 0.38; 95%CI, 0.08-1.79) or drinking (adjusted OR, 0.36; 95%CI, 0.08-1.65) for risk of noncardia GC. Conclusions: Our data indicate no association between the two SNPs of IL-4-590 and IL-8-251 with overall GC risk, while the IL-8-251 TA or AA genotype conferred risk of cardia GC. Our findings contribute to the evidence body for risk of SNPs associated with the development of gastric cancer in this region.

Effect of Microbial Flora and Inoculation of Probiotics on Fermenting Characteristics of Naked Barley Grain (Hordeum Vulgare L.) (쌀보리 종실 내 미생물 분포와 생균제 접종이 발효특성에 미치는 영향)

  • Ahn, Hye-Jin;Kim, Ki Hyun;Jo, Eun Seok;Kim, Jo Eun;Kim, Kwang-Sik;Kim, Young Hwa;Song, Tae Hwa;Park, Jong Ho;Kang, Hwan Ku;Jang, Sun Sik;Oh, Young Kyoon;Cheon, Dong Won;Seol, Kuk-Hwan
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.35 no.4
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    • pp.321-326
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    • 2015
  • This study was performed to analyze the resident microbial flora and the effects of probiotic inoculation on the fermentation characteristics of whole grain naked barley (Hordeum Vulgare L.) with the goal of evaluating the possibility of utilization as fermented feedstuff. Naked barley grains were harvested 35 days after heading, and the microbial flora was analyzed using MALDI-TOF mass spectrometer. After inoculation of commercial microbes to the naked barley grain (BT), the pH and number of bacteria, such as aerobic bacteria, lactic acid bacteria, yeast and E. coli, were measured and compared with the non-inoculated control (BC). A total of 122 colonies was isolated from the naked barley grain and the most popular bacteria species was Staphylococcus xylosus (n = 30, 24.59%). The pH value decreased more rapidly in BT than in BC, and was significantly lower after 7 days of fermentation at $4.33{\pm}0.02$ and $4.83{\pm}0.01$, respectively. The number of aerobic bacteria, lactic acid bacteria and yeast showed an increasing trend within the first 7 days of fermentation, however, their numbers decreased at 28 and 42 days of fermentation. The population of lactic acid bacteria in BT was higher than in BC, but there was no significant different at 7 days of fermentation, with respective levels of $9.24{\pm}0.20$ and $9.01{\pm}0.10logCFU/g$ (p>0.05). The initial number of E. coli was very high in the naked barley grain but subsequently decreased significantly. After 7 days of fermentation, E. coli was not detected in either BT or BC samples. From these results, it appears that the fermentation of naked barley grain proceeded adequately after 7 days, and that fermentation contributes to the safety of naked barley grain during storage.

Enhancement of Immune Activities of Peptides from Asterias amurensis Using a Nano-encapsulation Process (나노 입자 불가사리 펩타이드의 면역 활성 증진)

  • Jeong, Hyang-Suk;Oh, Sung-Ho;Kim, Seoung-Seop;Jeong, Myoung-Hoon;Choi, Woon-Yong;Seo, Yong-Chang;Choi, Geun-Pyo;Kim, Jin-Chul;Lee, Hyeon-Yong
    • Korean Journal of Food Science and Technology
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    • v.42 no.4
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    • pp.424-430
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    • 2010
  • Immuno-modulatory activities of peptides from Asterias amurensis were investigated using a nano-encapsulation process. The molecular weights of the peptides in the range of 5-7 kDa were separated using Sephadex G-75 gel filtration. Eighty-five percent of the nano-particles were in the 300 nm range using dynamic light scattering. The cytotoxicity of the A. amurensis nano-particles against CCD-986sk human dermal fibroblast cells was 11.64% after adding 1.0 mg/mL of the samples, which was lower than that from the control (13.28% collagen). The secretion of $NO^-$ from macrophages was estimated as $40\;{\mu}M$ after adding 1.0 mg/mL of gelatin nano-particles, which was higher than the others. Prostaglandin $E_2$ production from UV-induced human skin cells decreased greatly to 860 pg/mL after adding 1.0 mg/mL of the samples. Confocal microscopy revealed that nano-particles effectively penetrated the cells within 1 hour. From these results, we consider that nano-encapsulation of the peptides from A. amurensis can improve their biological functions.

Effects of Feeding Enzyme-Hydrolyzed Poultry By-Product Meal on Productivity and Blood Biochemical Characteristics in Broilers (효소가수분해 도계부산물의 급여가 육계의 생산성 및 혈액 생화학적 특성에 미치는 영향)

  • Gwak, Min-Geun;Park, Hye-Sung;Kim, Bong-Ki;Park, Hee-Bok;Kim, Ji-Hyuk
    • Korean Journal of Poultry Science
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    • v.48 no.3
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    • pp.133-142
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    • 2021
  • The purpose of this study was to investigate whether enzyme-hydrolyzed poultry by-product meal (EHPBM) is more effective as a protein source than poultry by-product meal (PBM) and soybean meal (SBM) for broiler chickens. A group of 300 one-day-old broiler chicks was randomly allocated to three treatments with five replicates (20 birds/replicate) for five weeks. The treatments consisted of basal diets containing 1) SBM, 2) PBM, and 3) EHPBM. The EHPBM-fed group (1,853 g±125.60) showed the highest final body weight (P<0.05) when compared to the PBM-fed group (1,723 g±76.81) and SBM-fed group (1,545 g±62.31). The feed conversion ratio of the EHPBM treatment group (1.740±0.104) was significantly higher (P<0.05) than those of the SBM (1.653±0.056) and PBM groups (1.674±0.072). It can be speculated that the increased feed intake in the EHPBM group led to higher body weight gain and FCR. There was no significant effect of treatments on internal organ weight except for the bursa of Fabricius. Blood biochemical characteristic analysis showed that aspartate aminotransferase and alkaline phosphatase levels were higher in the EHPBM and PBM groups (P<0.05), probably due to the strained liver caused by the rapid growth of birds. In conclusion, EHPBM may partly replace conventional dietary protein sources such as soybean meal or poultry by-product meal and can be used to improve the productivity of broilers.

Brief Introduction of Research Progresses in Control and Biocontrol of Clubroot Disease in China

  • He, Yueqiu;Wu, Yixin;He, Pengfei;Li, Xinyu
    • 한국균학회소식:학술대회논문집
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    • 2015.05a
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    • pp.45-46
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    • 2015
  • Clubroot disease of crucifers has occurred since 1957. It has spread to the whole China, especially in the southwest and nourtheast where it causes 30-80% loss in some fields. The disease has being expanded in the recent years as seeds are imported and the floating seedling system practices. For its effective control, the Ministry of Agriculture of China set up a program in 2010 and a research team led by Dr. Yueqiu HE, Yunnan Agricultural University. The team includes 20 main reseachers of 11 universities and 5 institutions. After 5 years, the team has made a lot of progresses in disease occurrence regulation, resources collection, resistance identification and breeding, biological agent exploration, formulation, chemicals evaluation, and control strategy. About 1200 collections of local and commercial crucifers were identified in the field and by artificiall inoculation in the laboratories, 10 resistant cultivars were breeded including 7 Chinese cabbages and 3 cabbages. More than 800 antagostic strains were isolated including bacteria, stretomyces and fungi. Around 100 chemicals were evaluated in the field and greenhouse based on its control effect, among them, 6 showed high control effect, especially fluazinam and cyazofamid could control about 80% the disease. However, fluzinam has negative effect on soil microbes. Clubroot disease could not be controlled by bioagents and chemicals once when the pathogen Plasmodiophora brassicae infected its hosts and set up the parasitic relationship. We found the earlier the pathogent infected its host, the severer the disease was. Therefore, early control was the most effective. For Chinese cabbage, all controlling measures should be taken in the early 30 days because the new infection could not cause severe symptom after 30 days of seeding. For example, a biocontrol agent, Bacillus subtilis Strain XF-1 could control the disease 70%-85% averagely when it mixed with seedling substrate and was drenching 3 times after transplanting, i.e. immediately, 7 days, 14 days. XF-1 has been deeply researched in control mechanisms, its genome, and development and application of biocontrol formulate. It could produce antagonistic protein, enzyme, antibiotics and IAA, which promoted rhizogenesis and growth. Its The genome was sequenced by Illumina/Solexa Genome Analyzer to assembled into 20 scaffolds then the gaps between scaffolds were filled by long fragment PCR amplification to obtain complet genmone with 4,061,186 bp in size. The whole genome was found to have 43.8% GC, 108 tandem repeats with an average of 2.65 copies and 84 transposons. The CDSs were predicted as 3,853 in which 112 CDSs were predicted to secondary metabolite biosynthesis, transport and catabolism. Among those, five NRPS/PKS giant gene clusters being responsible for the biosynthesis of polyketide (pksABCDEFHJLMNRS in size 72.9 kb), surfactin(srfABCD, 26.148 kb, bacilysin(bacABCDE 5.903 kb), bacillibactin(dhbABCEF, 11.774 kb) and fengycin(ppsABCDE, 37.799 kb) have high homolgous to fuction confirmed biosynthesis gene in other strain. Moreover, there are many of key regulatory genes for secondary metabolites from XF-1, such as comABPQKX Z, degQ, sfp, yczE, degU, ycxABCD and ywfG. were also predicted. Therefore, XF-1 has potential of biosynthesis for secondary metabolites surfactin, fengycin, bacillibactin, bacilysin and Bacillaene. Thirty two compounds were detected from cell extracts of XF-1 by MALDI-TOF-MS, including one Macrolactin (m/z 441.06), two fusaricidin (m/z 850.493 and 968.515), one circulocin (m/z 852.509), nine surfactin (m/z 1044.656~1102.652), five iturin (m/z 1096.631~1150.57) and forty fengycin (m/z 1449.79~1543.805). The top three compositions types (contening 56.67% of total extract) are surfactin, iturin and fengycin, in which the most abundant is the surfactin type composition 30.37% of total extract and in second place is the fengycin with 23.28% content with rich diversity of chemical structure, and the smallest one is the iturin with 3.02% content. Moreover, the same main compositions were detected in Bacillus sp.355 which is also a good effects biocontol bacterial for controlling the clubroot of crucifer. Wherefore those compounds surfactin, iturin and fengycin maybe the main active compositions of XF-1 against P. brassicae. Twenty one fengycin type compounds were evaluate by LC-ESI-MS/MS with antifungal activities, including fengycin A $C_{16{\sim}C19}$, fengycin B $C_{14{\sim}C17}$, fengycin C $C_{15{\sim}C18}$, fengycin D $C_{15{\sim}C18}$ and fengycin S $C_{15{\sim}C18}$. Furthermore, one novel compound was identified as Dehydroxyfengycin $C_{17}$ according its MS, 1D and 2D NMR spectral data, which molecular weight is 1488.8480 Da and formula $C_{75}H_{116}N_{12}O_{19}$. The fengycin type compounds (FTCPs $250{\mu}g/mL$) were used to treat the resting spores of P. brassicae ($10^7/mL$) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A260) and at 280 nm (A280) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be lyzed by the FTCPs of XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol. In the five selected medium MOLP, PSA, LB, Landy and LD, the most suitable for growth of strain medium is MOLP, and the least for strains longevity is the Landy sucrose medium. However, the lipopeptide highest yield is in Landy sucrose medium. The lipopeptides in five medium were analyzed with HPLC, and the results showed that lipopeptides component were same, while their contents from B. subtilis XF-1 fermented in five medium were different. We found that it is the lipopeptides content but ingredients of XF-1 could be impacted by medium and lacking of nutrition seems promoting lipopeptides secretion from XF-1. The volatile components with inhibition fungal Cylindrocarpon spp. activity which were collect in sealed vesel were detected with metheds of HS-SPME-GC-MS in eight biocontrol Bacillus species and four positive mutant strains of XF-1 mutagenized with chemical mutagens, respectively. They have same main volatile components including pyrazine, aldehydes, oxazolidinone and sulfide which are composed of 91.62% in XF-1, in which, the most abundant is the pyrazine type composition with 47.03%, and in second place is the aldehydes with 23.84%, and the third place is oxazolidinone with 15.68%, and the smallest ones is the sulfide with 5.07%.

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Evaluation of the Radioimmunotherapy Using I-131 labeled Vascular Endothelial Growth Factor Receptor2 Antibody in Melanoma Xenograft Murine Model (흑색종에서의 I-131표지 혈관내피세포성장인자 수용체2항체를 이용한 방사면역치료 평가)

  • Kim, Eun-Mi;Jeong, Hwan-Jeong;Park, Eun-Hye;Cheong, Su-Jin;Lee, Chang-Moon;Jang, Kyu-Yun;Kim, Dong-Wook;Lim, Seok-Tae;Sohn, Myung-Hee
    • Nuclear Medicine and Molecular Imaging
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    • v.42 no.4
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    • pp.307-313
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    • 2008
  • Purpose: Vascular endothelial growth factor (VEGF) and its receptor, fetal liver kinase 1 (Flk-1), play an important role in vascular permeability and tumor angiogenesis. The aim of this study is to evaluate the therapeutic efficacy of $^{131}I$ labeled anti-Flk-1 monoclonal antibody (DC101) on the growth of melanoma tumor, which is known to be very aggressive in vivo. Materials and Methods: Balb/c nude mice were injected subcutaneously with melanoma cells in the right flank. Tumors were allowed to grow up to $200-250\;mm^3$ in volume. Gamma camera imaging and biodistribution studies were performed to identify an uptake of $^{131}I$-DC101 in various organs. Mice with tumor were randomly divided into five groups (10 mice per group) and injected intravenously; control PBS (group 1), $^{131}I$-DC101 $50\;{\mu}g/mouse$ (group 2), non-labeled DC101 $50\;{\mu}g/mouse$ (group 3), $^{131}I$-DC101 $30\;{\mu}g/mouse$ (group 4) and $15\;{\mu}g/mouse$ (group 5) every 3 or 4 days for 20 days. Tumor volume was measured with caliper twice a week. Results: In gamma camera images, the uptake of $^{131}I$-DC101 into tumor and thyroid was increased with time. Biodistribution results showed that the radioactivity of blood and other major organ was gradually decreased with time whereas tumor uptake was increased up to 48 hr and then decreased. After 4th injection of $^{131}I$-DC101, tumor volume of group 2 and 4 was significantly smaller than that group 1. After 5th injection, the tumor volume of group 5 also significantly reduced. Conclusion: These results indicated that delivery of $^{131}I$ to tumor using FlK-1 antibody, DC101, effectively blocks tumor growth in aggressive melanoma xenograft model.