Joseph Kwon

Hydroxycinnamoylmalic acids and their methyl esters from pear (Pyrus pyrifolia Nakai) fruit peel.

Two novel caffeoylmalic acid methyl esters, 2-O-(trans-caffeoyl)malic acid 1-methyl ester (6) and 2-O-(trans-caffeoyl)malic acid 4-methyl ester (7), were isolated from pear (Pyrus pyrifolia Nakai cv. Chuhwangbae) fruit peels. In addition, 5 known hydroxycinnamoylmalic acids and their methyl esters were identified: 2-O-(trans-coumaroyl)malic acid (1), 2-O-(cis-coumaroyl)malic acid (2), 2-O-(cis-coumaroyl)malic acid 1-methyl ester (3), 2-O-(trans-coumaroyl)malic acid 1-methyl ester (4), and 2-O-(trans-caffeoyl)malic acid (phaselic acid, 5). The chemical structures of these compounds were determined by spectroscopic data from ESI MS and NMR. Of all the isolated compounds, five hydroxycinnamoylmalic acids and their methyl esters (2-4, 6, 7) were identified in the pear for the first time.

AM251 suppresses the viability of HepG2 cells through the AMPK (AMP-activated protein kinase)-JNK (c-Jun N-terminal kinase)-ATF3 (activating transcription factor 3) pathway.

AM251, a cannabinoid antagonist, has various biological activities. In this study, we found that AM251 suppressed the viability of hepatoma HepG2 cells and also increased phosphorylation of JNK (c-jun N-terminal kinase) and ATF3 (activating transcription factor 3). In addition, AM251 phosphorylated AMPK (AMP-activated protein kinase) in a time and dose-dependent manner. Inhibition of AMPK blocked AM251-induced JNK/ATF3 phosphorylation. Expression of AMPK or treatment with AICAR (5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside), an AMPK activator, activated the JNK/ATF3 pathways. Together, these results suggest that AM251 may have anti-tumor effects in hepatoma through activation of the AMPK-JNK-ATF3 signal pathway.

Comparative proteomic analysis for the insoluble fractions of colorectal cancer patients

We used label-free quantitative proteomics with the insoluble fractions from colorectal cancer (CRC) patients to gain further insight into the utility of profiling altered protein expression as a potential biomarker for cancer. The insoluble fractions were prepared from paired tumor/normal biopsies from 13 patients diagnosed with CRC (stages I to IV). Fifty-six proteins identified in data pooled from the 13 cases were differentially expressed between the tumor and adjacent normal tissue. The connections between these proteins are involved in reciprocal networks related to tumorigenesis, cancer incidence based on genetic disorder, and skeletal and muscular disorders. To assess their potential utility as biomarkers, the relative expression levels of the proteins were validated using personal proteomics and a heat map to compare five individual CRC samples with five normal tissue samples. Further validation of a panel of proteins (KRT5, JUP, TUBB, and COL6A1) using western blotting confirmed the differential expression. These proteins gave specific network information for CRC, and yielded a panel of novel markers and potential targets for treatment. It is anticipated that the experimental approach described here will increase our understanding of the membrane environment in CRC, which may provide direction for making diagnoses and prognoses through molecular biomarker targeting.

Nontargeted Metabolite Profiling in Compatible Pathogen-Inoculated Tobacco (Nicotiana tabacum L. cv. Wisconsin 38) Using UPLC-Q-TOF/MS

A biphasic reactive oxygen species (ROS) production has previously been observed in tobacco at 1 and 48 h after inoculation with the hemibiotrophic compatible pathogen, Phytophthora parasitica var. nicotianae (Ppn). To characterize the response of tobacco to biphasically produced ROS concerning the propagation of Ppn, ultraperformance liquid chromatography-quadrupole-time of flight/ mass spectrometry (UPLC-Q-TOF/MS) based metabolic profiling combined with multivariate statistical analysis was performed. Among the nonredundant 355 mass ions in ESI+ mode and 345 mass ions in ESI- mode that were selected as significantly changed by Ppn inoculation (|p(corr)| > 0.6 on S-plot of orthogonal partial least-squares discriminant analysis (OPLS-DA), fold-change > 2, and p < 0.05 in the independent two-sample t test), 76 mass ions were identified on the basis of their accurate mass ions and MS/MS spectra. Phenolic amino acids, phenylpropanoids, hydroxycinnamic acid amides, linoleic acid, linolenic acid, lysophospholipids, glycoglycerolipids, and trioxidized phospholipids were identified as having changed after Ppn inoculation. On the basis of their quantitative changes, the metabolic responses occurring at each phase of ROS production after Ppn inoculation were investigated in this study.

In‐depth analysis of cysteine oxidation by the RBC proteome: Advantage of peroxiredoxin II knockout mice

Peroxiredoxin II (Prdx II, a typical 2‐Cys Prdx) has been originally isolated from erythrocytes, and its structure and peroxidase activity have been adequately studied. Mice lacking Prdx II proteins had heinz bodies in their peripheral blood, and morphologically abnormal cells were detected in the dense red blood cell (RBC) fractions, which contained markedly higher levels of reactive oxygen species (ROS). In this study, a labeling experiment with the thiol‐modifying reagent biotinylated iodoacetamide (BIAM) in Prdx II−/− mice revealed that a variety of RBC proteins were highly oxidized. To identify oxidation‐sensitive proteins in Prdx II−/− mice, we performed RBC comparative proteome analysis in membrane and cytosolic fractions by nano‐UPLC‐MSE shotgun proteomics. We found oxidation‐sensitive 54 proteins from 61 peptides containing cysteine oxidation, and analyzed comparative expression pattern in healthy RBCs of Prdx II+/+ mice, healthy RBCs of Prdx II−/− mice, and abnormal RBCs of Prdx II−/− mice. These proteins belonged to cellular functions related with RBC lifespan maintain, such as cytoskeleton, stress‐induced proteins, metabolic enzymes, signal transduction, and transporters. Furthermore, protein networks among identified oxidation‐sensitive proteins were analyzed to associate with various diseases. Consequently, we expected that RBC proteome might provide clues to understand redox‐imbalanced diseases.

Identification of a new functional target of haloperidol metabolite: Implications for a receptor-independent role of 3-(4-fluorobenzoyl) propionic acid

Haloperidol, a dopamine D2 receptor blocker, is a classical neuroleptic drug that elicits extrapyramidal symptoms. Its metabolites include 3‐(4‐fluorobenzoyl) propionic acid (FBPA) and 4‐(4‐chlorophenyl)‐4‐piperidinol (CPHP). Until now, the biological significance of these metabolites has remained largely unknown. Here, we report that the administration of FBPA to mice effected a suppression of locomotor activity and induced catalepsy in a manner similar to that observed with haloperidol, whereas CPHP had no significant effects. Neither of these two metabolites, however, exhibited any ability to bind to the dopamine D2 receptor. FBPA blocked dopamine‐induced extracellular signal‐regulated kinase 1/2 phosphorylation, and it specifically affected mitogen‐activated protein kinase kinase (MEK)1/2 activity in hippocampal HN33 cells. Moreover, FBPA was capable of direct interaction with MEK1/2, and inhibited its activity in vitro. We demonstrated the generation of haloperidol metabolites within haloperidol‐treated cells by mass spectrometric analyses. Collectively, our results confirm the biological activity of FBPA, and provide initial clues as to the receptor‐independent role of haloperidol.

Metabolic survey of defense responses to a compatible hemibiotroph, Phytophthora parasitica var. nicotianae, in ethylene signaling-impaired tobacco.

Reactive oxygen species (ROS) and ethylene play an important role in determining the resistance or susceptibility of plants to pathogen attack. A previous study of the response of tobacco cultivar ( Nicotiana tabacum L. cv. Wisconsin 38) to a compatible hemibiotroph, Phytophthora parasitica var. nicotianae (Ppn) showed that biphasic bursts of ROS and ethylene are positively associated with disease severity. The levels of ethylene and ROS might influence the susceptibility of plants to pathogens, with changing levels of metabolite related to disease resistance or susceptibility. In this study, to obtain more detailed information on the interaction of ROS and ethylene signaling related to resistance and/or susceptibility of plants to pathogen, Ppn-induced metabolic profiles from wild type (WT) and ethylene signaling-impaired transgenic plants that expressed Ein3 antisense (Ein3-AS) were compared using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Nonredundant mass ions (576 in ESI+ mode and 336 in ESI- mode) were selected, and 56 mass ions were identified on the basis of their accurate mass ions and MS/MS spectra. Two-way hierarchical clustering analysis of the selected mass ions revealed that nicotine and phenylpropanoid-polyamine conjugates, such as caffeoyl-dihydrocaffeoyl-spermidine, dicaffeoyl-spermidine, caffeoyl-feruloyl-spermidine, and two bis(dihydrocaffeoyl)-spermine isomers, and their intermediates, such as arginine and putrecine, were present at lower levels in Ein3-AS transgenic plants during Ppn interaction than in WT, whereas galactolipid and oxidized free fatty acid levels were higher in Ein3-AS transgenic plants. Taken together, these results reveal a function for ethylene signaling in tobacco defense responses during Ppn interaction.