Seung-Jin Lee

Proteomic profiling of endothelial cells in human lung cancer.

Genomic and proteomic analysis of normal and diseased tissues have yielded an abundance of molecular information for diagnostic and potential therapeutic targets. Changing the target of analysis from poorly accessible cells within tissues to easily accessible vascular endothelium has theoretical advantages in tissue-specific targeting. In this study, we sought to map a large-scale proteome of microvascular endothelium in human non-small cell lung cancer (NSCLC) and normal lung tissues, and identify lung cancer-related endothelial cell (EC)-selective proteins. Endothelial cells were isolated within NSCLC tissues and adjacent-normal lung tissue of lung cancer patients by using CD31-immunomagnetic beads. The complex proteins from the ECs were separated by one-dimensional gel electrophoresis, and the proteins in each gel band were digested by trypsin. Peptides were separated by online reverse-phase liquid-chromatography and analyzed by electrospray ionization (ESI) ion trap tandem mass spectrometry. Approximately 600-1000 proteins were identified in each individual sample. Five patient cases of paired individual data, extracted from the protein identification data sets of both normal- and cancer-derived ECs, were analyzed by subtractive proteomics. An average of 300 proteins was specifically identified from each lung cancer-derived EC isolate, compared to normal lung-derived ECs. With the use of several comparative analyses, we identified among those 300 proteins, 16 common candidate proteins that were detected in at least 3 of 5 cases specific to lung cancer-derived ECs. Proteins selectively identified in cancer-derived ECs, including coatomer protein complex, subunit gamma (COPG), and peroxiredoxin 4 (PRDX4), were validated by Western blot analysis. In an additional experiment in which 16 cancer samples were analyzed by immunohistochemistry, PRDX4, thymopoietin (TMPO), and COPG were confirmed to be abundantly expressed in lung cancer-derived ECs and in cancerous lung cells. Further ongoing analysis of these 16 candidate proteins will determine their potential applicability to NSCLC-specific diagnosis and therapeutics.

Mutant-prevention concentration and mechanism of resistance in clinical isolates and enrofloxacin/marbofloxacin-selected mutants of Escherichia coli of canine origin.

The antibacterial activity and selection of resistant bacteria, along with mechanisms of fluoroquinolone resistance, were investigated by integrating the static [MIC or mutant-prevention concentration (MPC)] and in vitro dynamic model approaches using Escherichia coli isolates from diseased dogs. Using the dynamic models, selected E. coli strains and enrofloxacin and marbofloxacin at a range of simulated area under concentration-time curve over a 24 h interval (AUC(24 h))/MIC ratios were investigated. Our results indicated increasing losses in susceptibility of E. coli upon continuous exposure to enrofloxacin and marbofloxacin in vitro. This effect was transferable to other fluoroquinolones, as well as to structurally unrelated drugs. Our results also confirmed an AUC(24 h)/MIC (AUC(24 h)/MPC)-dependent antibacterial activity and selection of resistant E. coli mutants, in which maximum losses in fluoroquinolone susceptibility occurred at simulated AUC(24 h)/MIC ratios of 40-60. AUC(24 h)/MPC ratios of 39 (enrofloxacin) and 32 (marbofloxacin) were considered protective against the selection of resistant mutants of E. coli. Integrating our MIC and MPC data with published pharmacokinetic information in dogs revealed a better effect of the conventional dosing regimen of marbofloxacin than that of enrofloxacin in restricting the selection of resistant mutants of E. coli. Target mutations, especially at codon 83 (serine to leucine) of gyrA, and overexpression of efflux pumps contributed to resistance development in both clinically resistant and in vitro-selected mutants of E. coli. We also report here a previously undescribed mutation at codon 116 of parC in two laboratory-derived resistant mutants of E. coli. Additional studies would determine the exact role of this mutation in fluoroquinolone susceptibility, as well as establish the importance of our findings in the clinical setting.

Differential gene expression in young and senescent endothelial cells under static and laminar shear stress conditions.

Laminar shear stress (LSS) caused by blood flow is known to regulate endothelial function and to contribute to vascular health. By way of contrast, endothelial cell senescence seems to increase the incidence of vascular disorders. In an attempt to identify genes associated with vascular health/disease states, this study assessed the differential gene expression of young and senescent human umbilical vein endothelial cells (HUVECs) under static and LSS conditions. Replicative cell senescence was induced by continuous subculture in vitro, and LSS was provided using a cone-and-plate device. Young (p4) and senescent (p18) cells were subjected to LSS at 12 dyn.cm(-2) or maintained under static conditions for 24 h. Total mRNA was subjected to cDNA microarray analysis using the Affymetrix GeneChip. Welch t test at a significance level of p < 0.05 provided 961 LSS-responsive genes, whose expression was altered by LSS in both young and senescent cells, and 529 senescence-responsive genes differentially expressed in young vs senescent cells under both static and LSS conditions. The LSS-responsive and senescence-responsive gene groups included 74 genes held in common; these may prove useful for the study of cellular responses commonly affected by LSS and senescence. Among them, 20 genes whose expression was increased by LSS and simultaneously decreased by cellular senescence are suggested as potential vascular health markers in the sense that LSS is antiatherogenic, whereas senescence is proatherogenic. These genes included argininosuccinate synthetase 1, which was determined to be critical for both basal and LSS-induced NO production in young HUVECs. Furthermore, its diminished expression, and not that of nitric oxide synthase 3, was implicated in the insufficient NO production exhibited by senescent HUVECs under LSS conditions. The genes identified in this study are expected to facilitate improvements in our current level of understanding regarding endothelial physiology in association with age-associated vascular disease.

The suppressive effect of myeloid elf-1-like factor (MEF) in osteogenic differentiation

Myeloid Elf‐1 like factor (MEF) is a member of the Ets transcription factor family. Ets family proteins control the expression of genes that are critical for biological processes such as proliferation, differentiation, and cell death. Some of Ets factors are also known to regulate bone development. In this study, we investigated the role of MEF in osteoblast differentiation. MEF expression was highest early in the differentiation of MC3T3‐E1 osteoblasts and was reduced by treatment with BMP‐2. The expression of MEF suppressed the alkaline phosphatase activity and expression induced by BMP‐2 stimulation and mediated by Runx2. The expression of MEF also reduces osteocalcin mRNA levels, and mineralization in MC3T3‐E1 cells. We found that the MEF‐mediated suppression of osteogenic differentiation was critically related to Runx2 regulation. The MEF and Runx2 proteins physically interact to form a complex, and this interaction interferes with Runx2 binding to the cis‐acting element OSE2 derived from the osteocalcin promoter. Co‐transfection of MEF inhibited the 6xOSE2‐luciferase reporter activity induced by Runx2. In addition, MEF stimulated the transcription of a negative mediator Msx2, and a transcriptional repressor, Mab21L1, and suppressed the transcription of a positive mediator, Dlx5 in osteoblast differentiation. MEF overexpression stimulated C2C12 cell proliferation. Together, our findings suggest that MEF promotes cell proliferation and functions as a negative regulator of osteogenic differentiation by directly interacting with Runx2 and suppressing its transcriptional activity. J. Cell. Physiol. 211: 253–260, 2007.

Putative drug and vaccine target protein identification using comparative genomic analysis of KEGG annotated metabolic pathways of Mycoplasma hyopneumoniae

In the present study, a computational comparative and subtractive genomic/proteomic analysis aimed at the identification of putative therapeutic target and vaccine candidate proteins from Kyoto Encyclopedia of Genes and Genomes (KEGG) annotated metabolic pathways of Mycoplasma hyopneumoniae was performed for drug design and vaccine production pipelines against M.hyopneumoniae. The employed comparative genomic and metabolic pathway analysis with a predefined computational systemic workflow extracted a total of 41 annotated metabolic pathways from KEGG among which five were unique to M. hyopneumoniae. A total of 234 proteins were identified to be involved in these metabolic pathways. Although 125 non homologous and predicted essential proteins were found from the total that could serve as potential drug targets and vaccine candidates, additional prioritizing parameters characterize 21 proteins as vaccine candidate while druggability of each of the identified proteins evaluated by the DrugBank database prioritized 42 proteins suitable for drug targets.

Effects of dietary supplementation of Lactobacillus pentosus PL11 on the growth performance, immune and antioxidant systems of Japanese eel Anguilla japonica challenged with Edwardsiella tarda

The aim of this study was to determine the efficacy of dietary administration of Lactobacillus pentosus PL11 on growth performance and the immune and antioxidant systems in Japanese eel Anguilla japonica challenged with Edwardsiella tarda. A total of 75 Japanese eels (24.63±0.83 g) were grouped into 5 treatment diets which were a control diet (C) without E. tarda and 4 treatment diets with E. tarda challenge, including C for E. tarda challenge (NC), C plus L. pentosus PL11 supplemented diet (10⁸ cfu g⁻¹) (T-PL11), C plus L. pentosus KCCM 40997 supplemented diet (10⁸ cfu g⁻¹) (T-Lp) and C plus Weissella hellenica DS-12 supplemented diet (10⁸ cfu g⁻¹) (T-Wh) for 5 weeks (4 week before and 1 week after challenge). The results showed enhanced growth performance in fish fed the diet containing L. pentosus PL11 compared to others. The growth performance parameters including specific growth rate (SGR) and weight gain (WG), feed intake (FI), feed conversion ratio (FCR) and survival were significantly (P<0.05) higher in fish maintained on L. pentosus PL11 supplemented diet compared to C and NC. T-PL11 group also shows a significant increase in the levels of plasma immunoglobulin M, CAT and SOD activities compared to NC. Hematological parameters and mieloperoxidase were significantly better in fish fed the L. pentosus PL11 supplemented diet than in the control. L. pentosus PL11 supplementation recover the reduced expression of SOD, CAT and heat shock protein 70 genes in liver and intestine in pathogen challenged fishes. In conclusion the result of the current study demonstrated L. pentosus PL11 potential as an alternative to antibiotic supplementation to improve the growth and health performance of Japanese eel (A. japonica).

Prevalence and Risk Factors of Urticaria With a Focus on Chronic Urticaria in Children

Purpose Limited data is available on the prevalence and risk factors of acute and chronic urticaria in children. Our purpose was to determine the prevalence and identify the risk factors of acute and chronic urticaria in Korean children. Methods This population-based study examined 4,076 children (age 4 to 13 years) who were enrolled in the 2015 prospective Seongnam Atopy Project (SAP 2015) in Korea. The parents completed an urticaria questionnaire that included questions regarding the duration, severity, and triggering factors of urticaria. Blood sampling (n=464) was performed to measure vitamin D, total eosinophil count (TEC), and total IgE levels, and skin prick tests (n=503) were done. Results The prevalences of the life-time, acute, and chronic urticaria were 22.5%, 13.9%, and 1.8% (chronic continuous urticaria, 0.7%; and chronic recurrent urticaria, 1.1%), respectively. Acute urticaria was significantly associated with allergic diseases and parental history of allergy (P<0.001), but chronic urticaria was not associated with these clinical factors. There was no significant difference in the 25-hydroxyvitamin D level between subjects with chronic urticaria and controls (P=0.124). Chronic continuous urticaria was associated with living in a new residence (aOR=2.38, 95% CI=1.02-5.54, P=0.044) and belonging to a family with a high income (aOR=4.24, 95% CI=1.24-14.56, P=0.022). Conclusions A total of 1.8% of children were found to have chronic urticaria. Living in a new residence and belonging to a family with a high income increased the risk of chronic continuous urticaria.

Mutant prevention concentration and phenotypic and molecular basis of fluoroquinolone resistance in clinical isolates and in vitro-selected mutants of Escherichia coli from dogs

The antibacterial activity, selection of Escherichia coli (E. coli) mutants and mechanisms of fluoroquinolone resistance were investigated by integrating the minimum inhibitory concentration (MIC), mutant prevention concentration (MPC) and in vitro dynamic model approaches. Difloxacin and orbifloxacin, for which the above information has been scarce, were used. A range of area under curve over a 24h interval (AUC(24h))/MIC ratios and selected E. coli strains were investigated using the dynamic models. Continuous incubation for three days in the presence of difloxacin or orbifloxacin resulted in losses in E. coli susceptibility. An AUC(24h)/MIC (AUC(24h)/MPC)-dependent fluoroquinolone activity and selection of E. coli mutants was confirmed. Maximum losses in susceptibility occurred at AUC(24h)/MIC ratios of 54 (orbifloxacin) and 57.3 (difloxacin). AUC(24h)/MIC ratios of 169.8 (orbifloxacin) and 199.5 (difloxacin) were estimated to be protective against the selection of E. coli mutants, and the corresponding ratios based on AUC(24h)/MPC predictions were 34 (orbifloxacin) and 36.3 (difloxacin). When integrating our in vitro data with pharmacokinetic data in dogs, the conventional clinical doses of both drugs were found to be inadequate to attain the above protective values for 90% of the mutant subpopulation (AUC(24h)/MPC(90)). Both target mutations, esp. at codon 83 (Ser to Leu) of gyrA, and overexpression of efflux pumps contributed to resistance development, with mutants also showing decreased susceptibility to enrofloxacin and marbofloxacin. Additional studies would determine the role of mutations found outside the QRDR, at codon 24 of gyrA, and at codon 116 of parC, and establish the significance of these observations in vivo.

Inflammatory responses to Mycoplasma hyopneumoniae in murine alveolar macrophage cell lines

Abstract AIM: To investigate the mechanism by which Mycoplasma hyopneumoniae induces inflammatory responses in murine alveolar macrophage (MH-S) cells. METHODS: A pathogenic strain of M. hyopneumoniae cultured in modified Friis medium was used to investigate the inflammatory response in MH-S cell lines. The effect of stimulation by M. hyopneumoniae on the production of nitric oxide (NO) and cytokines in MH-S cells and inhibition of their production, using specific inhibitors of signalling pathways, was investigated using the Griess reaction and ELISA respectively. A Western blot assay was used to confirm activation of the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Nuclear translocation of NF-κB was further confirmed using transient transfection and luciferase gene reporter assay. RESULTS: The results revealed dose-dependent production of NO in MH-S cells stimulated by M. hyopneumoniae. Increased concentrations of the cytokines tumour necrosis factor (TNF)-α and interleukin (IL)-1β and IL-6 were also observed (p<0.05). Using immunoblot analysis, involvement of three MAPK pathways, extracellular signal-regulated kinase I/II (ERK1/2), p38 and Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK) was confirmed. Specific inhibitors of signal pathways also demonstrated their effect on the NO and cytokine responses of MH-S cells. Degradation and phosphorylation of inhibitory kappa B (IκB)-alpha was observed, while the luciferase gene reporter assays revealed activation of NF-κB after stimulation by M. hyopneumoniae. Inhibition of NF-κB by pyrrolidine dithiocarbamate decreased M. hyopneumoniae-induced production of NO and IL-1β (p<0.05), whereas no inhibitory effect was observed on concentrations of TNF-α, and IL-6. CONCLUSION: These findings indicate that M. hyopneumoniae induces NO and pro-inflammatory cytokines, and NF-κB and the three MAPK pathways are involved in the process.

Proteomic Analysis of a PDEF Ets Transcription Factor-Interacting Protein Complex

Ets proteins are a family of transcription factors that share an 85 amino acid conserved DNA binding domain, the ETS domain. The 27 known human Ets transcription factors control multiple biological processes, including cellular proliferation, differentiation, apoptosis, angiogenesis, transformation, and invasion. Overexpression of some Ets genes has been linked to numerous malignancies, including breast cancers. The prostate derived Ets transcription factor (PDEF) is reported to be a breast and prostate tumor-associated Ets factor. To understand the roles of PDEF in breast cancers, we transiently overexpressed PDEF in MDA-MB-231 human breast cancer cells by adenoviral-mediated gene delivery. PDEF binding protein complexes were isolated by immunoprecipitation and PDEF-interacting proteins were analyzed by LC-MS/MS. After subtracting the proteins binding nonspecifically to antibody-bead complexes, we identified 286 proteins in the PDEF-associated protein complex. By comparison to published protein-protein interactions, we selected 121 proteins for further analysis. PDEF interactors distribute not only in the nucleus, but also in the cytoplasm, as well as other subcellular compartments. Our data reveals that PDEF interacts with a variety of proteins involved in cell cycle, DNA repair, cytoskeleton organization, mRNA processing, tRNA biosynthesis, protein folding, and cell signaling. Furthermore, the EGFR1- (Erbb1) and Erbb2- (HER2) related proteins erbin, an ERBB2 interacting protein, catenin delta-1 (which interacts with Erbin), and EGFR (a HER2-homology receptor) were associated with PDEF. These findings indicate that PDEF may be regulated by Erbb2 or EGFR-activated signaling pathways in breast cancer cells. Further analysis of these proteins will identify the roles of PDEF-interacting proteins in breast tumorigenesis.