• Journal of Microbiology and Biotechnology
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• v.27 no.6
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• pp.1128-1137
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• 2017

The aim of this study was to explore the accuracy and feasibility of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying bacteria from environmental sources, as compared with rpoA gene sequencing, and to evaluate the occurrence of bacteria of the genus Enterococcus in wild birds. In addition, a phyloproteomic analysis of certain Enterococcus species with spectral relationships was performed. The enterococci were isolated from 25 species of wild birds in central Europe (Poland). Proteomic (MALDI-TOF MS) and genomic (rpoA gene sequencing) methods were used to identify all the isolates. Using MALDI-TOF MS, all 54 (100%) isolates were identified as Enterococcus spp. Among these, 51 (94.4%) isolates were identified to the species level (log(score) ${\geq}2.0$), and three isolates (5.6%) were identified at a level of probable genus identification (log(score) 1.88-1.927). Phylogenetic analysis based on rpoA sequences confirmed that all enterococci had been correctly identified. Enterococcus faecalis was the most prevalent enterococcal species (50%) and Enterococcus faecium (33.3%) the second most frequent species, followed by Enterococcus hirae (9.3%), Enterococcus durans (3.7%), and Enterococcus casseliflavus (3.7%). The phyloproteomic analysis of the spectral profiles of the isolates showed that MALDI-TOF MS is able to differentiate among similar species of the genus Enterococcus.

• Journal of Pharmacopuncture
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• v.12 no.3
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• pp.49-59
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• 2009

Background : Anticancer effects of herbal medicine have been reported in various types of cancer, but the systematic approaches to explain molecular mechanism(s) are not established yet. Objective : The purpose of this study is to investigate the apoptotic cell death by Egg White combined Chalcanthite in NCI-H460 human lung cancer cells. Methods : Inhibitory effects were estimated by the MTT-assay. Cancer cells were stained with DAPI and showed condensed and fragmented nuclei. The expression of cleaved caspase-3, bcl-2, and bax was detected by western blotting. To establish a basis of understanding for anti-cancer mechanism, whole proteins have been obtained from NCI-H460 harvested at 24 hrs after the treatment of Egg White combined Chalcanthite, protein expression has been profiled by 2DE-based proteomic approach. Results : NCI-H460 human lung cancer cells were treated by three samples of IS3, IS4 and IS5. IS4 inhibited most effectively the growth of NCI-H460 human lung cancer cells. The expression of cleaved caspase-3 increased in IS4 in a concentration-dependent manner. Various changes of the protein expression have been monitored, and most frequent dysregulation was found in Vimentin, Lamin-A/C. Conclusion : Egg White combined-Chacanthite inhibited the growth of NCI-H460 human lung cancer cells by inducing the apoptotic cell death via caspase-3 activation. Based upon the present findings, the further study will focus on monitoring various cancer survival factors after artificial regulation of the proteins identified, and it would be the basis for the understanding of the Chacabthite anticancer effect(s) at the molecular level.

• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1744-1754
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• 2014

The hepatitis C virus (HCV) RNA genome is replicated by an RNA replicase complex (RC) consisting of cellular proteins and viral nonstructural (NS) proteins, including NS5B, an RNA-dependent RNA polymerase (RdRp) and key enzyme for viral RNA genome replication. The HCV RC is known to be associated with an intracellular membrane structure, but the cellular components of the RC and their roles in the formation of the HCV RC have not been well characterized. In this study, we took a proteomic approach to identify stomatin, a member of the integral proteins of lipid rafts, as a cellular protein interacting with HCV NS5B. Co-immunoprecipitation and co-localization studies confirmed the interaction between stomatin and NS5B. We demonstrated that the subcellular fraction containing viral NS proteins and stomatin displays RdRp activity. Membrane flotation assays with the HCV genome replication-competent subcellular fraction revealed that the HCV RdRp and stomatin are associated with the lipid raft-like domain of membranous structures. Stomatin silencing by RNA interference led to the release of NS5B from the detergent-resistant membrane, thereby inhibiting HCV replication in both HCV subgenomic replicon-harboring cells and HCV-infected cells. Our results identify stomatin as a cellular protein that plays a role in the formation of an enzymatically active HCV RC on a detergent-resistant membrane structure.

• Korean Journal of Soil Science and Fertilizer
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• v.49 no.6
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• pp.689-698
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• 2016

Specific metabolic network responses to mineral starvation are not well-defined. We examined a detailed broad-scale identification of metabolic responses of tomato leaf and root to N, P or K starvation. Tomato plants were grown hydroponically under optimal (5 mM N, 0.5 mM P, or 5 mM K) and starved (0.5 mM N, 0.05 mM P, or 0.5 mM K) conditions and metabolites were measured by LC-MS and GC-MS. Overall, the levels of metabolites (lipids, nucleotides, peptides and secondary metabolites) presented in this paper largely showed mineral- and tissue-specific responses. Most strikingly, G3P (glycerol-3-P), GPC (glycerol-P-choline) and choline phosphate responded differently to a type of mineral; an increase in N or K starvation and a decrease in P starvation. A dramatic increase in the levels of secondary metabolites, in particular, rutin and chlorogenate in both tomato tissues during N starvation were observed. Based on these data, it is necessary to clearly elucidate an unknown event taking place in a variety of abiotic impacts, and we are now studying to expand our knowledge on metabolic- and proteomic-responses using GS-MS and LC-MS.

• Korean Journal of Microbiology
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• v.48 no.2
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• pp.87-92
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• 2012

The purpose of this study was to examine the cellular response of Salmonella typhimurium exposed to tea polyphenols (TPP) extracted from Korean green tea (Camellia sinensis L.). TPP showed a dose-dependent bactericidal effect on S. typhimurium. Analysis of cell membrane fatty acids of S. typhimurium cultures treated with TPP identified unique changes in saturated and unsaturated fatty acids, while scanning electron microscopic analysis demonstrated the presence of perforations and irregular rod forms with wrinkled surfaces in cells treated with TPP. Two-dimensional polyacrylamide gel electrophoresis of soluble protein fractions from S. typhimurium cultures showed 16 protein spots increased by TPP. These up-regulated proteins including proteins involved in antioxidants and chaperons, transcript and binding proteins, energy and DNA metabolism were identified by peptide mass fingerprinting using MALDI-TOF. These results provide clues for understanding the mechanism of TPP induced stress and cytotoxicity on S. typhimurium.

• Bulletin of the Korean Chemical Society
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• v.28 no.7
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• pp.1187-1194
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• 2007

The three major active isoflavonoids (calycosin-7-O-β -glucoside, isomucronulatol 7-O-β-glucoside, formononetin) and two main saponins (astragaloside I, astragaloside IV) in an extract of Radix Astragali were determined using rapid, sensitive, reliable HPLC/UV and LC-ESI-MS/MS methods. The separation conditions employed for HPLC/UV were optimized using a phenyl-hexyl column (4.6 × 150 mm, 5 μm) with the gradient elution of acetonitrile and water as the mobile phase at a flow rate of 1.0 mL/min and a detection wavelength of 230 nm. The specificity of the peaks was determined using a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source that was operated in multiple reaction monitoring (MRM) in the positive mode. These methods were fully validated with respect to the linearity, accuracy, precision, recovery and robustness. The HPLC/UV method was applied successfully to the quantification of three major isoflavonoids in the extract of Radix Astragali. The results indicate that the established HPLC/UV and LC-ESI-MS/MS methods are suitable for the quantitative analysis and quality control of multi-components in Radix Astragali.

• BMB Reports
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• v.40 no.6
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• pp.853-860
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• 2007

Resistance to anticancer drugs is a major obstacle in the effective treatment of tumors. To understand the mechanisms responsible for multidrug resistance (MDR), a proteomic approach was used to identify proteins that were expressed in different levels by the adriamycinresistant human gastric cancer cell line, SGC7901/ADR, and its parental cell line, SGC7901. Two-dimensional gel electrophoresis (2-DE) and image analysis was used to determine which protein spots were expressed in different levels by the two cell lines. These spots were then partially identified using ESI-Q-TOF mass spectrometry, and the differential expressional levels of the partially identified proteins were then determined by western blot analysis and real-time RT-PCR. Additionally, the association of Nucleophosmin (NPM1), a protein that was highly expressed by SGC7901/ADR, with MDR was analyzed using siRNA. As a result of this study, well-resolved, reproducible 2-DE patterns of SGC7901/ADR and SGC7901 were established, and 16 proteins that may playa role in the development of thermo resistance were identified. Additionally, suppression of NPMl expression was found to enhance adriamycin chemosensitivity in SGC7901/ADR. These results provide a fundamental basis for the elucidation of the molecular mechanism of MDR, which may assist in the treatment of gastric cancer.

• BMB Reports
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• v.42 no.6
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• pp.344-349
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• 2009

Angiogenesis is crucial for solid tumor growth. By secreting angiogenic factors, tumor cells induce angiogenesis. However, targeting these angiogenic factors for cancer therapy is not always successful, suggesting that other factors may be involved in tumor angiogenesis. This work shows that 25 protein spots were differentially expressed by two-dimensional gel electrophoretic analysis when HepG2 cells induced endothelial cell differentiation to tube in vitro, and most of them were upregulated. Twenty-one proteins were identified with MALDITOF-MS, and the other four were identified by LTQ-MS/MS. Keratins were identified as one class of these upregulated proteins. Further study indicated that the expression of keratin 17 in cultured endothelial cells is likely microenvironment regulated, because its expression can be induced by HepG2 cells and bFGF as well as serum in culture media. Increased expression of keratins in endothelial cells, such as keratin 17, may contribute to the angiogenesis induced by HepG2 cells.

• BMB Reports
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• v.53 no.11
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• pp.576-581
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• 2020

Dimethylation of the histone H3 protein at lysine residue 9 (H3K9) is mediated by euchromatin histone methyltransferase II (EHMT2) and results in transcriptional repression of target genes. Recently, chemical inhibition of EHMT2 was shown to induce various physiological outcomes, including endoplasmic reticulum stress-associated genes transcription in cancer cells. To identify genes that are transcriptionally repressed by EHMT2 during apoptosis, and cell stress responses, we screened genes that are upregulated by BIX-01294, a chemical inhibitor of EHMT2. RNA sequencing analyses revealed 77 genes that were upregulated by BIX-01294 in all four hepatic cell carcinoma (HCC) cell lines. These included genes that have been implicated in apoptosis, the unfolded protein response (UPR), and others. Among these genes, the one encoding the stress-response protein Ras-related GTPase C (RRAGC) was upregulated in all BIX-01294-treated HCC cell lines. We confirmed the regulatory roles of EHMT2 in RRAGC expression in HCC cell lines using proteomic analyses, chromatin immune precipitation (ChIP) assay, and small guide RNA-mediated loss-of-function experiments. Upregulation of RRAGC was limited by the reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC), suggesting that ROS are involved in EHMT2-mediated transcriptional regulation of stress-response genes in HCC cells. Finally, combined treatment of cells with BIX-01294 and 5-Aza-cytidine induced greater upregulation of RRAGC protein expression. These findings suggest that EHMT2 suppresses expression of the RRAGC gene in a ROS-dependent manner and imply that EHMT2 is a key regulator of stress-responsive gene expression in liver cancer cells.

• Molecules and Cells
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• v.40 no.9
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• pp.685-695
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• 2017

Obesity and the metabolic disorders that constitute metabolic syndrome are a primary cause of morbidity and mortality in the world. Nonetheless, the changes in the proteins and the underlying molecular pathways involved in the relevant pathogenesis are poorly understood. In this study a proteomic analysis of the visceral adipose tissue isolated from metabolically healthy and unhealthy obese patients was used to identify presence of altered pathway(s) leading to metabolic dysfunction. Samples were obtained from 18 obese patients undergoing bariatric surgery and were subdivided into two groups based on the presence or absence of comorbidities as defined by the International Diabetes Federation. Two dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was carried out. A total of 28 proteins were identified with a statistically significant difference in abundance and a 1.5-fold change (ANOVA, $p{\leq}0.05$) between the groups. 11 proteins showed increased abundance while 17 proteins were decreased in the metabolically unhealthy obese compared to the healthy obese. The differentially expressed proteins belonged broadly to three functional categories: (i) protein and lipid metabolism (ii) cytoskeleton and (iii) regulation of other metabolic processes. Network analysis by Ingenuity pathway analysis identified the $NF{\kappa}B$, IRK/MAPK and PKC as the nodes with the highest connections within the connectivity map. The top network pathway identified in our protein data set related to cellular movement, hematological system development and function, and immune cell trafficking. The VAT proteome between the two groups differed substantially between the groups which could potentially be the reason for metabolic dysfunction.