• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.946-951
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• 2006

To study the effect of iron on Saccharomyces cerevisiae, whole-cell proteins of Saccharomyces cerevisiae were extracted and subjected to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and differentially expressed proteins were identified. The proteins separated were further identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and were compared with a protein database. Of more than 300 spots separated by molecular weight and isoelectric points, 27 differentially expressed spots were identified. Ten proteins were found to be differentially expressed at high iron concentration. Triosephosphate isomerase (TPI), YDR533C hypothetical protein, superoxide dismutase (SOD), 60 kDa heat-shock protein (HSP60), pyruvate dehydrogenase beta subunit 1 (PDB1), and old yellow enzyme 2 (OYE2) were upregulated, whereas thiol-specific antioxidant (TSA), regulatory particle non-ATPase subunit 8 (RPN8), thiol-specific peroxiredoxin 1 (AHP1), and fructose-1, 6-bisphosphate adolase (FBA) were downregulated by iron. Based on the result, we propose that SOD upregulated by iron would protect the yeast from oxidative stress by iron, and that TSA downregulated by iron would render cells hypersensitive to oxidative stress.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.12
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• pp.6435-6439
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• 2012

Chemoresistance to cancer therapy is a major obstacle to the effective treatment of human cancers with cisplatin (DDP), but the mechanisms of cisplatin-resistance are not clear. In this study, we established a cisplatin-resistant human ovarian cancer cell line (COC1/DDP) and identified differentially expressed proteins related to cisplatin resistance. The proteomic expression profiles in COC1 before and after DDP treatment were examined using 2-dimensional electrophoresis technology. Differentially expressed proteins were identified using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and high performance liquid chromatography-electrospray tandem MS (NanoUPLC-ESI-MS/MS). 5 protein spots, for cytokeratin 9, keratin 1, deoxyuridine triphosphatase (dUTPase), aarF domain containing kinase 4 (ADCK 4) and cofilin1, were identified to be significantly changed in COC1/DDP compared with its parental cells. The expression of these five proteins was further validated by quantitative PCR and Western blotting, confirming the results of proteomic analysis. Further research on these proteins may help to identify novel resistant biomarkers or reveal the mechanism of cisplatin-resistance in human ovarian cancers.

• Food Science and Biotechnology
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• v.15 no.3
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• pp.447-452
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• 2006

To determine the functions of specific cell wall polysaccharides, polysaccharides of three mutants, mur3-1, mur3-2, and mur3-3, obtained from Arabidopsis wild type, underwent structural characterization. Upon sequential separation of pectins (RG-I and RG-II) and cross-linking glycans (xyloglucan, XG), only XG was affected by the mud mutation. Wild-type XG contained a considerable amount of fucose, whereas the fucose level in mur3 XGs was less than 20% that of wild type. Further analysis of XGs by matrix-assisted laser-induced/ionization time-of-flight (MALDI-TOF) mass spectrometry indicated that mud lines considerably or completely lost the fucosylated XG oligosaccharides such as XXFG and XLFG and the double-galactosylated oligosaccharide XLLG $^1H$-NMR spectroscopic analyses of the XG oligosaccharides from mur3-3 plant revealed the absence of fucose and a galactose level in the galactosylated side chain that was reduced by 40% compared to that of Arabidopsis wild-type plant. In contrast, 85% less fucose and a slight loss of galactose were observed in the mur3-1 and mur3-2 lines which show normal growth habit. Of the three Arabidopsis mur3 lines studied here, mur3-3 is disrupted by a T-DNA insertion in the exon of MUR3 which encodes XG-specific galactosyltransferase, and exhibits slight dwarfism. These results indicated that the T-DNA insertion at the MUR3 locus did not induce the complete loss of galactose in XG, and that galactose, rather than fucose, in the XG side chains made a major contribution to overall wall strength.

• The Korean Journal of Mycology
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• v.46 no.4
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• pp.511-518
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• 2018

Initiation of spore germination in filamentous fungi such as Aspergillus nidulans and Botrytis cinerea requires the presence of nutrients. In this study, involvement of sugar sensing machinery was suggested in the germination of A. nidulans spores. Germination did not occur when the spores of A. nidulans were incubated in distilled water, whereas they were successfully germinated in the presence of 5% glucose with a germination rate of over 98% after 6hr incubation. Similar results were obtained when the spores were incubated in the presence of various sugars such as fructose, sucrose, and starch. Interestingly, spore germination was not observed in the presence of D-arabinose, whereas L-arabinose could induce germination as determined by the formation of germ tubes, indicating the presence of sugar sensing machinery that distinguish between the enantiomers of sugars. This inference was further supported by a decrease in germination rate (less than 25%) upon treatment of spores with trypsin. Subsequent MALDI-TOF mass spectrometry analysis of the surface proteins of spores identified ten proteins among which eight were involved in sugar metabolism. Taken together, our results suggest that spore germination in A. nidulans is initiated by the interaction of sugars with sugar binding proteins on the surface of spores.

• Biomedical Science Letters
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• v.25 no.1
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• pp.40-53
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• 2019

Of the Acinetobacter spp., A. baumannii (genospecies 2) is the most clinically significant in terms of hospital-acquired infections worldwide. It is difficult to perform Acinetobacter-related taxonomy using phenotypic characteristics and routine laboratory methods owing to clusters of closely related species. The ability to accurately identify Acinetobacter spp. is clinically important because antimicrobial susceptibility and clinical relevance differs significantly among the different genospecies. Based on the medical importance of pathogenic Acinetobacter spp., the distribution and characterization of Acinetobacter spp. isolates from 123 clinical samples was determined in the current study using four typically applied bacterial identification methods; partial rpoB gene sequencing, amplified rRNA gene restriction analysis (ARDRA) of the intergenic transcribed spacer (ITS) region of the 16~23S rRNA, the $VITEK^{(R)}$ 2 system (an automated microbial identification system) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). A. baumannii isolates (74.8%, 92/123) were the most common species, A. nosocomialis (10.6%, 13/123) and A. pittii isolates (7.5%, 9/123) were second and third most common strains of the A. calcoaceticus-A. baumannii (ACB) complex, respectively. A. soli (5.0%, 6/123) was the most common species of the non-ACB complex. RpoB gene sequencing and ARDRA of the ITS region were demonstrated to lead to more accurate species identification than the other methods of analysis used in this study. These results suggest that the use of rpoB genotyping and ARDRA of the ITS region is useful for the species-level identification of Acinetobacter isolates.

• BMB Reports
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• v.53 no.7
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• pp.349-356
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• 2020

Mass spectrometry (MS) is an ideal tool for analyzing multiple types of (bio)molecular information simultaneously in complex biological systems. In addition, MS provides structural information on targets, and can easily discriminate between true analytes and background. Therefore, imaging mass spectrometry (IMS) enables not only visualization of tissues to give positional information on targets but also allows for molecular analysis of targets by affording the molecular weights. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS is particularly effective and is generally used for IMS. However, the requirement for an organic matrix raises several limitations that get in the way of accurate and reliable images and hampers imaging of small molecules such as drugs and their metabolites. To overcome these problems, various organic matrix-free LDI IMS systems have been developed, mostly utilizing nanostructured surfaces and inorganic nanoparticles as an alternative to the organic matrix. This minireview highlights and focuses on the progress in organic matrix-free LDI IMS and briefly discusses the use of other IMS techniques such as desorption electrospray ionization, laser ablation electrospray ionization, and secondary ion mass spectrometry.

• Development and Reproduction
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• v.8 no.1
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• pp.1-9
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• 2004

The potential importance of proteomic approaches has been clearly demonstrated in other fields of human medical research, including liver and heart disease and certain forms of cancer. However, reproductive researches have been applied to proteomics poorly. Proteomics can be defined as the systematic analysis of proteins for their identity, quantity, and function. It could increase the predictability of early drug development and identify non-invasive biomarkers of toxicity or efficacy. Proteome analysis is most commonly accomplished by the combination of two-dimensional gel electrophoresis(2DE) and MALDI-TOF(matrix-assisted laser desorption ionization-time of flight) MS(mass spectrometry) or protein chip array and SELDI-TOF(surface-enhanced laser desorption ionization-time of flight) MS. In addition understanding the possessing knowledge of the developing biomarkers used to assess reproductive biology will also be essential components relevant to the topic of reproduction. The continued integration of proteomic and genomic data will have a fundamental impact on our understanding of the normal functioning of cells and organisms and will give insights into complex cellular processes and disease and provides new opportunities for the development of diagnostics and therapeutics. The challenge to researchers in the field of reproduction is to harness this new technology as well as others that are available to a greater extent than at present as they have considerable potential to greatly improve our understanding of the molecular aspects of reproduction both in health and disease.

• Korean Journal of Environmental Biology
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• v.21 no.2
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• pp.87-100
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• 2003

It is important to understand the potential human health implications of exposure to environmental chemicals that may act as hormonally active agents. It is necessary to have an understanding of how pharmaceutical and personal care products and other chemicals affect the ecosystem of our planet as well as human health. Endocrine disruption is defined as the ability of a chemical contaminating the workplace or the environment to interfere with homeostasis, development, reproduction, and/or behavior in a living organism or it's offspring. Certain classes of environmentally persistent chemicals such as polychlorinated biphenyls (PCBs), dioxins, furans, and some pesticides can adversely effect the endocrine systems of aquatic life and terrestrial wildlife. Research continues to support the theory of endocrine disruption. However, endocrine disruption researches have been applied to proteomics poorly. Proteomics can be defined as the systematic analysis of proteins for their identity, quantity and function. It could increase the predictability of early drug development and identify non-invasive biomarkers of tonicity or efficacy. Proteome analysis is most commonly accomplished by the combination of two-dimensional gel electrophoresis (2D/E) and MALDI-TOF mass spectrometry (MS) sr protein chip array and SELDI-TOF MS. Proteomics have an opportunity to play an important role in resolving the question of what role endocrine disruptors play in initiating human disease. Proteomics can also play an imfortant role in the evaluation of the risk assessment and use of risk management and risk communication tools required to address public health concerns related to notions of endocrine disruptors. Understanding the need for the proteomics and possessing knowledge of the developing biomakers used to abbess endocrine activity potential will he essential components relevant to the topic of endocrine disruptors.

• Proceedings of the Korean Society of Crop Science Conference
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• 2004.04a
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• pp.12-27
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• 2004

Thanks to spectacular advances in the techniques for identifying proteins separated by two-dimensional electrophoresis and in methods for large-scale analysis of proteome variations, proteomics is becoming an essential methodology in various fields of plant sciences. Plant proteomics would be most useful when combined with other functional genomics tools and approaches. A combination of microarray and proteomics analysis will indicate whether gene regulation is controlled at the level of transcription or translation and protein accumulation. In this review, we described the catalogues of the rice proteome which were constructed in our program, and functional characterization of some of these proteins was discussed. Mass-spectrometry is a most prevalent technique to identify rapidly a large of proteins in proteome analysis. However, the conventional Western blotting/sequencing technique us still used in many laboratories. As a first step to efficiently construct protein data-file in proteome analysis of major cereals, we have analyzed the N-terminal sequences of 100 rice embryo proteins and 70 wheat spike proteins separated by two-dimensional electrophoresis. Edman degradation revealed the N-terminal peptide sequences of only 31 rice proteins and 47 wheat proteins, suggesting that the rest of separated protein spots are N-terminally blocked. To efficiently determine the internal sequence of blocked proteins, we have developed a modified Cleveland peptide mapping method. Using this above method, the internal sequences of all blocked rice proteins (i. e., 69 proteins) were determined. Among these 100 rice proteins, thirty were proteins for which homologous sequence in the rice genome database could be identified. However, the rest of the proteins lacked homologous proteins. This appears to be consistent with the fact that about 30% of total rice cDNA have been deposited in the database. Also, the major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that fumed out to be calreticulin, gibberellin-binding protein, which is ribulose-1,5-bisphosphate carboxylase/oxygenase activate in rice, and leginsulin-binding protein in soybean have functions in the signal transduction pathway. Proteomics is well suited not only to determine interaction between pairs of proteins, but also to identify multisubunit complexes. Currently, a protein-protein interaction database for plant proteins (http://genome .c .kanazawa-u.ac.jp/Y2H)could be a very useful tool for the plant research community. Recently, we are separated proteins from grain filling and seed maturation in rice to perform ESI-Q-TOF/MS and MALDI-TOF/MS. This experiment shows a possibility to easily and rapidly identify a number of 2-DE separated proteins of rice by ESI-Q-TOF/MS and MALDI-TOF/MS. Therefore, the Information thus obtained from the plant proteome would be helpful in predicting the function of the unknown proteins and would be useful in the plant molecular breeding. Also, information from our study could provide a venue to plant breeder and molecular biologist to design their research strategies precisely.

• Journal of Plant Biotechnology
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• v.32 no.4
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• pp.269-273
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• 2005

Toll-like receptors (TLR) are important components of innate immunity in the defense against pathogens. TLRs recognize pathogen-associated common molecular patterns. TLRs are similar to the receptors involved in defense responses in plants. TLR protein is a type 1 membrane protein, consisting of an extracellular domain containing leucine-rich repeats and a cytoplasmic domain. The cytoplasmic domain delivers ligand recognition signals that result in production of anti-microbial agents. The cytoplasmic domain (amino acid 858-1032) of toll-like receptor 9 has been expressed using methylotrophic yeast Pichia pastoris. The protein expression was confirmed by Western-blot, N-terminal sequencing and MALDl-TOF mass spectrometry. The proteins have been purified by nickel affinity, cation exchange and gel-filtration chromatography.