• Archives of Pharmacal Research
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• v.29 no.3
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• pp.224-234
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• 2006

We employed human SK-MEL-28 cells as a model system to identify cellular proteins that accompany N-(4-methyl)phenyl-O-(4-methoxy)phenyl-thionocarbamate (MMTC)-induced apoptosis based on a proteomic approach. Cell viability tests revealed that SK-MEL-28 skin cancer cells underwent more cell death than normal HaCaT cells in a dose-dependent manner after treatment with MMTC. Two-dimensional electrophoresis in conjunction with matrixassisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis or computer matching with a protein database further revealed that the MMTC-induced apoptosis is accompanied by increased levels of caspase-1, checkpoint suppressor-1, caspase-4, NF-kB inhibitor, AP-2, c-Jun-N-terminal kinase, melanoma inhibitor, granzyme K, G1/S specific cyclin D3, cystein rich protein, Ras-related protein Rab-37 or Ras-related protein Rab-13, and reduced levels of EMS (oncogene), ATP synthase, tyrosine-phosphatase, Cdc25c, 14-3-3 protein or specific structure of nuclear receptor. The migration suppressing effect of MMTC on SK-MEL-28 cell was tested. MMTC suppressed the metastasis of SK-MEL-8 cells. It was also identified that MMTC had little angiogenic effect because it did not suppress the proliferation of HUVEC cell line. These results suggest that MMTC is a novel chemotherapeutic and metastatic agents against the SK-MEL-28 human melanoma cell line.

• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.83-91
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• 2005

Somatic cell nuclear transfer in cattle has limited efficiency in terms of production of live offspring due to high incidence of fetal failure after embryo transfer to recipients. Such low efficiency of cloning could possibly arise from abnormal and poorly developed placenta. In the present study the placental proteome in late pregnancy established from in vitro fertilization (IVF) and nuclear transfer (NT) was analysed. Proteome alternation was tested using two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF). Comparing placenta from NT embryos to those from IVF counterparts, significant changes in expression level were found in 18 proteins. Of these proteins 12 were not expressed in NT placenta but expressed in IVF counterpart, whereas the expression of the other 6 proteins was limited only in NT placenta. Among these proteins, cytokeratin 8 and vimentin are considered to be involved in regulation of post-implantation development. In particular, cytokeratin 8 and vimentin may be used as makers for placental development during pregnancy because their expression levels changed considerably in NT placental tissue compared with its IVF counterpart. Data from 2-DE suggest that protein expression was disorientated in late pregnancy from NT, but this distortion was eliminated with progression of pregnancy. These findings demonstrate abnormal placental development during late pregnancy from NT and suggest that alterations of specific placental protein expression may be involved in abnormal function of placenta.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.40-44
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• 2003

Soil-borne infectious disease including Pythium aphanidermatum and Rhizoctonia solani causes severe damage to plants, such as cucumber. This soil-borne infectious disease was not controlled effectively by chemical pesticide. Since these diseases spread through the soil, chemical agents are usually ineffective. Instead, biological control, including antagonistic microbe can be used as a preferred control method. An efficient method was developed to select an antagonistic strain to be used as a biological control agent strain. In this new method, surface tension reduction potential of an isolate was included in the ‘decision factor’ in addition to the other factors, such as growth rate, and pathogen inhibition rate. Considering these 3 decision factors by a statistical method, an isolate from soil was selected and was identified as Bacillus sp. GB16. In the pot test, this strain showed the best performance among the isolated strains. The lowest disease incidence rate and fastest seed growth was observed when Bacillus sp. GB16 was used. Therefore this strain was considered as plant growth promoting rhizobacteria (PGPR). The action of surface tension reducing component was deduced as the enhancement of wetting, spreading, and residing of antagonistic strain in the rhizosphere. This result showed that new selection method was significantly effective in selecting the best antagonistic strain for biological control of soil-borne infectious plant pathogen. The antifungal substances against P. aphanidermatum and R. solani were partially purified from the culture filtrates of Bacillus sp. GB16. In this study, lipopeptide possessing antifungal activity was isolated from Bacillus sp. GB16 cultures by various purification procedures and was identified as a surfactin-like lipopeptide based on the Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), high performance liquid chromatography mass spectroscopy (HPLC-MS), and quadrupole time-of-flight (Q-TOF) ESI-MS/MS data. The lipopeptide, named GB16-BS, completely inhibited the growth of Pythium aphanidermatum, Rhizoctonia solani, Penicillium sp., and Botrytis cineria at concentrations of 10 and 50 mg/L, respectively. A novel method to prevent the foaming and to provide oxygen was developed. During the production of surface active agent, such as lipopeptide (surfactin), large amount of foam was produced by aeration. This resulted in the carryover of cells to the outside of the fermentor, which leads to the significant loss of cells. Instead of using cell-toxic antifoaming agents, low amount of hydrogen peroxide was added. Catalase produced by cells converted hydrogen peroxide into oxygen and water. Also addition of corn oil as an oxygen vector as well as antifoaming agent was attempted. In addition, Ca-stearate, a metal soap, was added to enhance the antifoam activity of com oil. These methods could prevent the foaming significantly and maintained high dissolved oxygen in spite of lower aeration and agitation. Using these methods, high cell density, could be achieved with increased lipopeptide productivity. In conclusion to produce an effective biological control agent for soil-borne infectious disease, following strategies were attempted i) effective screening of antagonist by including surface tension as an important decision factor ii) identification of antifungal compound produced from the isolated strain iii) novel oxygenation by $H_2O_2-catalase$ with vegetable oil for antifungal lipopeptide production.

• Food Science and Preservation
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• v.30 no.2
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• pp.235-246
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• 2023

Yellowball (Citrus hybrid cv. Yellowball ) is a new citrus hybrid between Haruka (C. tamurana × natsudaidai ) and Kiyomi (C. unshiu × sinensis) and is known to possess strong antioxidant activity. However, detailed information on the antioxidant components of its peel has not yet been reported. This study evaluated the antioxidant activity of the peel and identified the antioxidant components by fractionating a methanolic extract of Yellowball peels using liquid-liquid extraction with n-hexane, ethyl ether (ether), ethyl acetate (EA), butanol, and water. The phenolic contents and antioxidant activities of the n-hexane, ether, and EA fractions were higher than those of the other fractions, and these fractions were further separated by semi-preparative high-performance liquid chromatography (HPLC). Four antioxidant peaks, EA1, EA2, EA3, and He1, were isolated and analyzed using ultra-performance liquid chromatography-quadrupole-time- of-flight mass spectrometry (UPLC-Q-TOF MS). Sinapoyl glucoside and hesperidin were identified in EA2 and EA3, respectively, and a polymethoxylated flavone (PMF) complex (5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone, natsudaidain, tetrameth- oxyflavone, and tangeretin) was identified in He1. A compound in EA1 with m/z 223.0246 [M-H] could not be identified and was named unknown2. The antioxidant activity of unknown2 (IC₅₀=69.17 ㎍/mL) was similar to that of Trolox, which was noted as a major antioxidant in Yellowball peel. Further studies on the antioxidant capacity of Yellowball peel are required; however, these results provide a foundation for using Yellowball peel as an antioxidant.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2005.11a
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• pp.66-67
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• 2005

The domestic chicken (Gallus gallus) is an important model for research in developmental biology because its embryonic development occurs in ovo. To examine the mechanism of embryonic germ cell development, we constructed proteome map of gonadal primordial germ cells (gPGC) from chicken embryonic gonads. Embryonic gonads were collected from 500 embryos at 6 day of incubation, and the gPGC were cultured in vitro until colony formed. After 7-10 days in cultured gPGC colonies were separated from gonadal stroma cells (GSCs). Soluble extracts of cultured gPGCs were then fractionated by two-dimensional gel electrophoresis (pH 4-7). A number of protein spots, including those that displayed significant expression levels, were then identified by use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and LC-MS/MS. Of the 89 gPGC spots examined, 50 yielded mass spectra that matched avian proteins found in on-line databases. Proteome map of thistype will serve as an important reference for germ cell biology and transgenic research.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.263-270
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• 2010

Luteal cells produce progesterone that supports pregnancy. Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism. In the present study, the corpus luteum (CL) in early pregnancy established from luteal phase and pregnant phase was analyzed. The first study determined progesterone changes in the bovine CL at day 19 (early maternal recognition period) and day 90 in mid-pregnancy and compared them to the CL from day 12 of the estrous cycle. CL alternation was tested using two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Comparing CL from luteal phase to those from pregnant phase counterparts, significant changes in expression level were found in 23 proteins. Of these proteins 17 were not expressed in pregnant phase CL but expressed in luteal phase counterpart, whereas, the expression of the other 6 proteins was limited only in pregnant phase CL. Among these proteins, vimentin is considered to be involved in regulation of post-implantation development. In particular, vimentin may be used as marker for CL development during pregnancy because the expression level changed considerably in pregnant phase CL tissue compared with its luteal phase counterpart. Data from 2-DE suggest that protein expression was disorientated in mid pregnancy from luteal phase, but these changes was regulated with progression of pregnancy. These findings demonstrate CL development during mid-pregnancy from luteal phase and suggest that alternations of specific CL protein expression may be involved in maintenance of pregnancy.

• Bulletin of the Korean Chemical Society
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• v.34 no.8
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• pp.2419-2424
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• 2013

Guanosine-5'-diphosphate 3'-diphosphate (ppGpp) serves as alarmone in bacterial stringent responses. In this study, an affinity column was constructed by immobilizing ppGpp to NHS-Sepharose for isolating ppGpp-binding proteins. A novel ppGpp-binding protein, YjgA, was isolated and characterized by MALDI-TOF MS (matrix-assisted laser desorption ionization-time-of-flight mass spectrometry) coupled with two-dimensional gel electrophoresis. YjgA and truncated forms of YjgA were cloned and over-expressed in BL21 (DE3). The binding affinity of YjgA to ppGpp was determined by equilibrium dialysis. The interaction of YjgA with ppGpp was very specific, considering that the dissociation constant of YjgA with ppGpp was measured as $5.2{\pm}2.0{\mu}M$, while the affinities to GTP and GDP were about 60 and 30 times weaker than ppGpp. Expression of yjgA gene in Escherichia coli K-12 MG1655 was examined by reverse transcription polymerase chain reaction (RT-PCR). RT-PCR results revealed that yjgA was expressed from early to late stationary phase. The yjgA deletion mutant exhibited decreased cell number at stationary phase compared to parent strain and the over-expression of YjgA increased the cell number. These results suggested that YjgA might stimulate cell division under stationary phase. In most prokaryotic genome, about half of the protein candidates are hypothetical, that are expected to be expressed but there is no experimental report on their functions. The approach utilized in this study may serve as an effective mean to probe the functions of hypothetical proteins.

• 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.

• Radiation Oncology Journal
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• v.22 no.4
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• pp.298-306
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• 2004

Purpose: The purpose of this study was to identify Radiosensitivity of proteins in tissues with different radiosensitivity. Materials and Methods: C3H/HeJ mice were exposed to 10 Gy. The mice were sacrifiud 8 hrs after radiation. Their spleen and liver tissues were collected and analyzed histologicaly for apoptosis. The expressions of radiosusceptibillty protein were analyzed by 2-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Resilts: The Peak of apoptosis levels were $35.3{\pm}1.7{\%}$ in spleen and $0.6{\pm}0.2{\%}$ in liver at 8 hrs after radiation. Liver, radioresistant tissues, showed that the levels of ROS metabolism related to proteins such as cytochromm c, glutathione S transferase, NADH dehydrogenase, riken cDNA and peroxiredoxin Vl increased after radiation. The expression of cytochrome c increased significantly in spleen and liver tissues after radiation. In spleen, radiosensitivity tissue, the identified proteins showed a significantly quantitative alteration following radiation. It was categorized as signal transduction, apoptosis, cytokine, Ca signal related protein, stress-related protein, cytoskeletal regulation, ROS metabolism, and others. Conclusion: Differences of radiation-induced apoptosis by tissues specifted were coupled with the induction of related radiosensitivity and radioresistant proteins. The result suggests that apoptosis relate protein and redox proteins play important roles in this radiosusceptibility.

• Journal of Life Science
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• v.26 no.2
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• pp.220-225
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• 2016

Coagulase-negative staphylococci (CNS) have recently become the bacteria most frequently found in clinical infections. The aim of this study was to investigate the prevalence, antimicrobial susceptibilities, and molecular characteristics of CNS isolates from dental clinic environments in Busan, Korea. One hundred and fifty-four samples were collected from 10 dental clinics and dental hospitals in Busan from December 2014 to January 2015. Species were identified by matrix-assisted laser desorption/ionization–time-of-flight. Antimicrobial susceptibility was determined by disk diffusion methods. A polymerase chain reaction was performed to detect mecA, mupA gene, and SCCmec types. Of the 154 samples, 10(6.5%) isolates were identified as CNS (5 Staphylococcus epidermidis, 2 Staphylococcus capitis, 2 Staphylococcus, and 1 Staphylococcus haemolyticus). Among the 10 isolates, 6 were resistant to penicillin, 5 were resistant to gentamicin, 3 were resistant to tetracycline, and 2 were resistant to cefoxitin and erythromycin. However, clindamycin, ciprofloxacin, teicoplanin, and trimethoprim-sulfamethoxazole resistant isolates were not present. Genes encoding mecA were detected in 4 (2 S. warneri and 2 S. haemolyticus) isolates, and mupA in 1 (S. epidermidis) isolate. One methicillin-resistant CNS (S. warneri) isolate was determined as being of the SCCmec type I. It is concluded that CNS resistant to various antimicrobial agents was widely distributed in dental clinic environments in Korea.