Jai-Heon Lee

Molecular cloning and characterization of the soybean DEAD-box RNA helicase gene induced by low temperature and high salinity stress.

A novel gene encoding a DEAD-box RNA helicase designated as GmRH was isolated from soybean. Amino acid sequence alignment and phylogenetic tree analysis revealed a close relationship between GmRH and other orthologous DEAD-box RNA helicases from other plant species. Structural motif analysis revealed that the bipartite lysine rich nuclear localization signal (NLS) is present in the N-terminal variable region of GmRH and that there are ten conserved motifs found in DEAD-box RNA helicase proteins. Southern blot analysis revealed the presence of 2 copies of GmRH in the soybean genome. Northern blot analysis demonstrated that the RNA expression of the GmRH was induced during low temperature or high salinity stress, but not by the exogenous application of abscisic acid or drought stress. Subcellular localization studies showed that GmRH((1-355))-GFP is localized in the nucleus, whereas GmRH((130-355))-GFP is localized both in the cytoplasm and in the nucleus. This provides the evidence that the N-terminal region predicted as NLS is essential for nuclear targeting of the GmRH protein in the plant cell. Purified GST-GmRH recombinant protein was shown to unwind dsRNA independent of ATP in vitro. Here, we propose that GmRH plays an important role in RNA processing during low temperature and high salinity stresses in plants.

Genome-wide analysis and molecular characterization of heat shock transcription factor family in Glycine max.

Heat shock transcription factors (Hsfs) play an essential role on the increased tolerance against heat stress by regulating the expression of heat-responsive genes. In this study, a genome-wide analysis was performed to identify all of the soybean (Glycine max) GmHsf genes based on the latest soybean genome sequence. Chromosomal location, protein domain, motif organization, and phylogenetic relationships of 26 non-redundant GmHsf genes were analyzed compared with AtHsfs (Arabidopsis thaliana Hsfs). According to their structural features, the predicted members were divided into the previously defined classes A-C, as described for AtHsfs. Transcript levels and subcellular localization of five GmHsfs responsive to abiotic stresses were analyzed by real-time RT-PCR. These results provide a fundamental clue for understanding the complexity of the soybean GmHsf gene family and cloning the functional genes in future studies.

Ganoderma lucidum ethanol extract inhibits the inflammatory response by suppressing the NF-κB and toll-like receptor pathways in lipopolysaccharide-stimulated BV2 microglial cells

Ganoderma lucidum is a traditional Oriental medicine that has been widely used as a tonic to promote longevity and health in Korea and other Asian countries. Although a great deal of work has been carried out on the therapeutic potential of this mushroom, the pharmacological mechanisms of its anti-inflammatory actions remain unclear. In this study, we evaluated the inhibitory effects of G. lucidum ethanol extract (EGL) on the production of inflammatory mediators and cytokines in lipopolysaccharide (LPS)-stimulated murine BV2 microglia. We also investigated the effects of EGL on the LPS-induced activation of nuclear factor kappaB (NF-κB) and upregulation of toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). Elevated levels of nitric oxide (NO), prostaglandin E2 (PGE2) and pro-inflammatory cytokine production were detected in BV2 microglia following LPS stimulation. We identifed that EGL significantly inhibits the excessive production of NO, PGE2 and pro-inflammatory cytokines, including interleukin (IL)-1β and tumor necrosis factor-α in a concentration-dependent manner without causing cytotoxicity. In addition, EGL suppressed NF-κB translocation and transcriptional activity by blocking IκB degradation and inhibiting TLR4 and MyD88 expression in LPS-stimulated BV2 cells. Our results indicate that the inhibitory effects of EGL on LPS-stimulated inflammatory responses in BV2 microglia are associated with the suppression of the NF-κB and TLR signaling pathways. Therefore, EGL may be useful in the treatment of neurodegenerative diseases by inhibiting inflammatory mediator responses in activated microglia.

Overexpression of VrUBC1, a Mung Bean E2 Ubiquitin-Conjugating Enzyme, Enhances Osmotic Stress Tolerance in Arabidopsis

The ubiquitin conjugating enzyme E2 (UBC E2) mediates selective ubiquitination, acting with E1 and E3 enzymes to designate specific proteins for subsequent degradation. In the present study, we characterized the function of the mung bean VrUBC1 gene (Vigna radiata UBC 1). RNA gel-blot analysis showed that VrUBC1 mRNA expression was induced by either dehydration, high salinity or by the exogenous abscisic acid (ABA), but not by low temperature or wounding. Biochemical studies of VrUBC1 recombinant protein and complementation of yeast ubc4/5 by VrUBC1 revealed that VrUBC1 encodes a functional UBC E2. To understand the function of this gene in development and plant responses to osmotic stresses, we overexpressed VrUBC1 in Arabidopsis (Arabidopsis thaliana). The VrUBC1-overexpressing plants displayed highly sensitive responses to ABA and osmotic stress during germination, enhanced ABA- or salt-induced stomatal closing, and increased drought stress tolerance. The expression levels of a number of key ABA signaling genes were increased in VrUBC1-overexpressing plants compared to the wild-type plants. Yeast two-hybrid and bimolecular fluorescence complementation demonstrated that VrUBC1 interacts with AtVBP1 (A. thaliana VrUBC1 Binding Partner 1), a C3HC4-type RING E3 ligase. Overall, these results demonstrate that VrUBC1 plays a positive role in osmotic stress tolerance through transcriptional regulation of ABA-related genes and possibly through interaction with a novel RING E3 ligase.

Exploration for the salt stress tolerance genes from a salt-treated halophyte, Suaeda asparagoides

Salinity stress severely affects plant growth and development causing crop loss worldwide. Suaeda asparagoides is a salt-marsh euhalophyte widely distributed in southwestern foreshore of Korea. To isolate salt tolerance genes from S. asparagoides, we constructed a cDNA library from leaf tissues of S. asparagoides that was treated with 200xa0mM NaCl. A total of 1,056 clones were randomly selected for EST sequencing, and 932 of them produced readable sequence. By sequence analysis, we identified 538 unigenes and registered each in National Center for Biotechnology Information. The 80 salt stress related genes were selected to study their differential expression. Reverse transcription-PCR and Northern blot analysis revealed that 23 genes were differentially expressed under the high salinity stress conditions in S. asparagoides. They are functionally diverse including transport, signal transduction, transcription factor, metabolism and stress associated protein, and unknown function. Among them dehydrin (SaDhn) and RNA binding protein (SaRBP1) were examined for their abiotic stress tolerance in yeast (Saccharomyces cerevisiae). Yeast overexpressing SaDhn and SaRBP1 showed enhanced tolerance to osmotic, freezing and heat shock stresses. This study provides the evidence that SaRBP1 and SaDhn from S. asparagoides exert abiotic stress tolerance in yeast. Information of salt stress related genes from S. asparagoides would contribute for the accumulating genetic resources to improve osmotic tolerance in plants.

Oleifolioside B-mediated autophagy promotes apoptosis in A549 human non-small cell lung cancer cells

The biochemical mechanisms of cell death by oleifolioside B (OB), a cycloartane-type triterpene glycoside isolated from Dendropanax morbifera Leveille, were investigated in A549 human lung carcinoma cells. Our data indicated that exposure to OB led to caspase activation and typical features of apoptosis; however, apoptotic cell death was not prevented by z-VAD-fmk, a pan-caspase inhibitor, demonstrating that OB-induced apoptosis was independent of caspase activation. Subsequently, we found that OB increased autophagy, as indicated by an increase in monodansylcadaverine fluorescent dye-labeled autophagosome formation and in the levels of the autophagic form of microtubule-associated protein 1 light chain 3 and Atg3, an autophagy-specific gene, which is associated with inhibiting phospho-nuclear factor erythroid 2-related factor 2 (Nrf2) expression. However, pretreatment with bafilomycin A1, an autophagy inhibitor, attenuated OB-induced apoptosis and dephosphorylation of Nrf2. The data suggest that OB-induced autophagy functions as a death mechanism in A549 cells and OB has potential as a novel anticancer agent capable of targeting apoptotic and autophagic cell death and the Nrf2 signaling pathway.

JNP3, a new compound, suppresses PMA-induced tumor cell invasion via NF-κB down regulation in MCF-7 breast cancer cells.

The expression of matrix metalloproteinase (MMPs)-9 is critical for cell migration and can lead to invasion and metastasis of cancer cells. In the present study, we examined the inhibitory effects of JNP3, a new compound which was isolated from traditional Chinese medicine, on cell invasion and MMP-9 activation in phorbol myristate acetate (PMA)-induced MCF-7 cells. Treatment with JNP3 significantly and selectively inhibited PMA-induced MMP-9 secretion, mRNA expression and protein levels, and these results led to reduction of cell invasion and migration in PMA-induced MCF-7 cells. The results of MMP-9 promoter assay and EMSA showed that JNP3 specifically inhibited PMA-induced MMP-9 gene expression by blocking NF-κB-dependent transcriptional activity. In addition, PMA-induced phosphorylation of ERK1/2 and JNK were suppressed by JNP3 treatment, whereas the phosphorylation of p38 MAPK was not affected by JNP3. These results suggest that JNP3 can be potential anti-cancer agents through specific inhibition of NF-κB-dependent MMP-9 gene expression.

Antiplasmodial and cytotoxic activity of coumarin derivatives from dried roots of Angelica gigas Nakai in vitro

The butanol-soluble fraction of the dried root of Angelica gigas exhibited significant protection against chloroquine-sensitive strains of Plasmodium falciparum using the parasite lactate dehydrogenase assay method. Using antiplasmodial activity-guided fractionation, five coumarins, marmesinin (1), nodakenin (2), skimmin (3), apiosylskimmin (4), and magnolioside (5), were isolated and evaluated for in vitro antiplasmodial activity, as well as for their cytotoxic potential on SK-OV-3 cancer cell lines. Compounds 1 and 5 showed notable growth inhibitory activity against chloroquine-sensitive strains of P. falciparum with IC50 values of 5.3 and 8.2 μM. The compounds showed no significant cytotoxicity (IC50 > 100 μM) toward the SK-OV-3 cancer cell line. This is the first report on the antiplasmodial activity of these coumarin derivatives from the dried root of A. gigas.

RETRACTED: Inhibitory effects of three oleanolic acid glycosides from Achyranthes japonica on the complement classical pathway

RETRACTED

Inhibitory Effects of Isolated Compounds from Black Coloured Rice Bran on the Complement Classical Pathway

The present study evaluated the anticomplement effects of isolated compounds from black coloured rice bran in the classical pathway of the complement system. Using column chromatography, three compounds: oryzafuran, quercetin and protocatechuic acid, were isolated and evaluated for in vitro anticomplement activity. Oryzafuran showed the most potent inhibitory activity against the complement system, with 50% inhibitory concentration (IC50) values of 126.2u2009µg/mL. This is the first report of anticomplement activity of isolated compounds from black coloured rice bran. Copyright