• Applied Biological Chemistry
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• v.30 no.4
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• pp.293-299
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• 1987

Bradyrhizobium japonicum spheroplasts were prepared by culturing cells in the presence of glycine, follwed by treatment with lysozyme. The cells were examined by electron microscopy during the formation of spheroplast. Then Ti plasmid from Agrobacterium tumefaciens 15955 was introduced into Bradyrhizobium japonicum by glycine-lysozyme induced spheroplast transformation. After cell wall regeneration, transformants were selected by the ability of utilization of octopine. Transformation were received at a frequency of $1{\times}10^{-7}$. The transformants obtained from spheroplast transformation harbored the introduced Ti plasmid, which was identified by agarose gel electrophoresis. Furthermore, the differences in their gene products were observed between the transformant and the recipient cell by two-dimensional polyacrylamide gel electrophoresis. The transformants which still possessed the same ability nodulate soybean (Glycine max.) as that of the original host strain, acquired the ability to induce tumor on Petunia hybrida like Agrobacterium, but formed the small crown galls in size compared to those of Agrobacterium tumefaciens.

• BMB Reports
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• v.39 no.2
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• pp.171-177
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• 2006

Nonsteroidal anti-inflammatory drugs such as indomethacin (IN) can exert anti-colorectal cancer (CRC) activity through cyclooxygenase independent mechanism, but the exactly biological mechanism is not completely known. Here we use proteomic tools to investigate the molecular mechanism of this action. First, nude mice bearing tumors derived from subcutaneous injection with human CRC cell line HCT116 were randomly allocated to groups treated with or without indomethacin. Later, tumor lumps were incised and then total proteins extracted. After separated with two-dimensional electrophoresis, thirty-one differently expressed spots were found between IN-treated and non-IN-treated groups, of which 25 spots decreased and 6 spots increased in abundance in IN-treated group. Through matrix-assisted laser desorption ionization time of flight mass spectrometry and then NCBInr and SWISS-PROT databases searching, 12 protein spots were finally identified including galectin-1, annexin A1, annexin IV, trancription factor BTF3A, calreticulin. Most of the identified proteins are correlated with tumor's biological prosperities of proliferation, invasion, apoptosis and immunity, or take part in cell's signal transduction. From above we thought that indomethacin can exert its effect on colorectal cancer through regulating several proteins' expression directly or indirectly. Further study of these proteins may be helpful in founding new targets of drugs for cancer chemotherapy.

• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.911-920
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• 2006

[ $CD8^+$ ] T Iymphocytes with the cytotoxic activity and capability to release various cytokines are the major players in immune responses against viral infection and cancer. To identify the proteins specific to resting or activated human CD8$^+$ T cells, human CD8$^+$ T cells were activated with anti-CD3+anti-CD28 mAb in the presence of IL-2. The solubilized proteins from resting and activated human CD8$^+$ T cells were separated by high-resolution two-dimensional polyacrylamide gel electrophoresis, and their proteomes were analyzed. Proteomic analysis of resting and activated T cells resulted in identification of 35 proteins with the altered expression. Mass spectrometry coupled with Profound and SWISS-PROT database analysis revealed that these identified proteins are to be functionally associated with cell proliferation, metabolic pathways, antigen presentation, and intracellular signal transduction pathways. We also identified six unknown proteins predicted from genomic DNA sequences specific to resting or activated CD8$^+$ T cells. Protein network studies and functional characterization of these novel proteins may provide new insight into the signaling transduction pathway of CD8$^+$ T cell activation.

• Korean Journal of Organic Agriculture
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• v.22 no.1
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• pp.167-182
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• 2014

Korean Panax ginseng C. A. Meyer (P. ginseng) is a well-known and one of the most important tonic herbs used in traditional Korean medicine. The pharmacological effects of P. ginseng have been reported by many researchers. Nevertheless, little is known between the mechanism of action and the active compounds. In this study, we performed a comprehensive proteomic analysis and protein categorization in order to understand the physiological characteristics of the major components in the adventitious roots of P. ginseng. Whole proteins extracted from the cultured adventitious roots of P. ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). Among the 1000 spots which were detected by silver staining, 113 spots were labeled and identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). Our results showed that 40 proteins were identified among the 113 spots, with a hit ratio of 35.3%. A number of proteins identified on the 2-DE gels (30%; 16 spots) were involved in energy metabolism. These proteomic data will be helpful to better understand the physiological and pharmacological effects of P. ginseng.

• Molecules and Cells
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• v.22 no.1
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• pp.119-125
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• 2006

The rat is an accepted model for studying human psychiatric/neurological disorders. We provide a protocol for total soluble protein extraction using trichloroacetic acid/acetone (TCA/A) from rat (female) whole brain, 10 brain regions and the pituitary gland, and show that two-dimensional gel electrophoresis (2-DGE) using precast immobilized pH (4-7) gradient (IPG) strip gels (13 cm) in the first dimension yields clean silver nitrate stained protein profiles. Though TCA/A precipitation may not be "ideal", the important choice here is the selection of an appropriate lysis buffer (LB) for solubilizing precipitated proteins. Our results reveal enrichment of protein spots by use of individual brain regions rather than whole brain, as well as the presence of differentially expressed spots in their proteomes. Thus individual brain regions provide improved protein coverage and are better suited for differential protein detection. Moreover, using a phosphoprotein-specific dye, ingel detection of phosphoproteins was demonstrated. Representative high-resolution silver nitrate stained proteome profiles of rat whole brain total soluble protein are presented. Shortcomings apart (failure to separate membrane proteins), gel-based proteomics remains a viable option, and 2-DGE is the method of choice for generating high-resolution proteome maps of rat brain and brain regions.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.49 no.4
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• pp.342-348
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• 2004

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the $60\%$ isopropanol extract of soybean(Glycine max [L.] Merr.) seed revealed two abundant proteins with molecular masses of 19 and 10 kDa. Amino acid analysis revealed that the isopropanol-extractable protein fraction was rich in cysteine. Two-dimensional gel electro-phoretic analysis indicated that the 19kDa and 10kDa proteins had pI of 4.2 and 4.0 respectively. Peptide mass fingerprints of trypsin digests of the two proteins obtained using matrix-assisted, laser desorption/ionization-time of flight (MALDI-TOF) mass spectroscopy revealed the 19kDa protein was Kunitz trypsin inhibitor and the 10kDa protein was Bowman-Birk proteinase inhibitor. When resolved under non-denaturing conditions, the isopropanol-extracted proteins inhibited trypsin and chymotrypsin activity. Results presented in this study demonstrate that isopropanol extraction of soybean seed could be used as a simple and rapid method to obtain a protein fraction enriched in Kunitz trypsin and Bowman-Birk proteinase inhibitors. Since proteinase inhibitors are rich in sulfur amino acids and are putative anticarcinogens, this rapid and inexpensive isolation procedure could facilitate efforts in nutrition and cancer research.

• Journal of the Korean Society of Food Science and Nutrition
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• v.28 no.3
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• pp.579-585
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• 1999

Proteins from the loin tissues age ranged from 0 to 24 months of ten Korean cattle were extracted, separated and compared on two dimensional(2 D) gels to identify the proteins whose expression is highly correlated to marbling. We also compared the difference of loin proteins between castrated and non castrated bull cows on two dimensional gels. As the marbling in the loin of the cattle is optimized at 18 to 24 months, eight proteins expressed significantly higher level in 24 month than in 0 or 6 month were selected in terms of isoelectric points(pIs) and molecular weights. Using these values, we searched the Swiss Prot database via the ExPASy molecular biology server with TagIdent program. The proteins with the nearest molecular weights and isoelectric points were selected from the lists. These possible candidates were confirmed by N terminal microsequencing of the eight selected proteins. Three proteins, myoglobin, hemoglobin and ATPase, whose N termini were not blocked could be microsequenced and found to be exactly matched to the selected candidates. It is suggested that the proteins increasingly expressed in marbling periods can be involved in meat color, lipid transport and flavor improvement.

• Korean Journal of Veterinary Research
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• v.42 no.2
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• pp.231-239
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• 2002

Proteomics is an emerging powerful tool in studying protein expression and function. At present study, proteomics was employed to evaluate the antigenicity among Escherichia coli O157:H7 strains using 2-dimensional gel electrophoresis (2-DE) and immunoblotting, SDS-PAGE and immunblotting analysis revealed no big differences among E coli O157:H7 strains. 2-DE analysis, however, revealed common antigens as well as specific antigens. The immunoblotting analysis revealed 20 common antigenic spots among E coli O157:H7 strains. In addition, there were 3 and 13 spots as common antigens between ATCC 43894 and KSC 109, and between ATCC 43894 and ACH 5, respectively. Antigenic spots specific for individual strain were also identified as 15, 8 and 22 for ATCC 43894, ACH 5 and KSC 109, respectively. The common antigens would be useful by employing either vaccine development or diagnosis marker, or both, whereas the specific antigens of individual strains would be applicable for epidemiological study. This study suggest that proteome analysis, representative as 2-DE, is valuable tool in exploring the E. coli antigenicity.

• 대한예방의학회:학술대회논문집
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• 2001.10a
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• pp.335-336
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• 2001

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.186-186
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• 2004

In this study, we generated 40 somatic cell cloned (scNT) piglets. Of these, three displayed congestion in both liver and lung, and died within the first week of life. Two-dimensional gel electrophoresis experiments revealed changes in the responses of several detoxification-related proteins to stress and inflammation. As a result, congestive livers and lungs displayed extensive hepatopneumonic apoptosis.(omitted)