[show abstract][hide abstract] ABSTRACT: 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.
International Journal of Oncology 12/2013; 43(6):1943-50. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recent studies have shown that an intron is not merely “junk”, but something that plays important roles in many biological processes such as gene expression regulation and alternative splicing. For purposes of studying intron structures and predicting consensus splice motifs, a total of 102 legume species were used to isolate introns across the family. Of 196 gene-targeted PCR primer pairs, we successfully amplified 118 intron-containing genes (60.2 %) and obtained a total of 1,870 introns with an average size of 143 nucleotides, ranging from 61 to 1,036. Species-based compilation of 5′- and 3′-splicing motifs showed, to some extent, lineage-specific conservation in each splicing motif. Compilation of the entire intron set permitted prediction of the consensus sequences of splicing signal motifs in legumes, AYGWGTABABGH and TVNC/TAGGHTV for the 5′SS and 3′SS, respectively. Interestingly, these consensus motifs are very similar to the corresponding genome-wide splicing signals of two model systems, Arabidopsis and rice. This result suggests conservation of pre-mRNA splicing mechanism occurring in higher plants. Multiple alignments of CALTL introns demonstrated that the BP-3′SS region was relatively more conserved than the 5′SS-BP region. We speculate that length of the BP-3′SS region needs to be retained for the interaction with U2AF protein. Phylogenetic analysis demonstrates that each of three splicing motifs is not only phylogenetically informative, but also relevant to evolutionary divergence of species. This result suggests that the splice signal sequences would be a useful tool for the molecular phylogenetic analysis. We also anticipate that gene-targeted amplification in multiple genomes, described in this study, would facilitate studies on intron-located functional elements involved in gene expression regulations.
[show abstract][hide abstract] ABSTRACT: Two cDNA clones encoding RNA binding proteins (RBPs) were isolated from a cDNA library constructed from salt-treated leaf tissues of wild radish (Raphanus sativus var. hortensis for raphanistroide). The deduced amino acid sequence of either RsRBP1 or RsGRP1 contains an RNA-recognition motif (RRM) at the carboxy or amino terminal. Comparative sequence analysis of RsRBP1 reveals extensive homology (63–84%) to known RBPs from other plants. RsGRP1 was shown to be most homologous to AtGRP7 (93%) out of eight members of Arabidopsis glycine-rich RBPs. Transcript levels of RsRBP1 was up-regulated slowly and reached its maximum at 9 h during salt stress. On the other hand, RNA expression of RsGRP1 was up-regulated rapidly but significantly was reduced at 9 h after salt stress. The RsRBP1 and RsGRP1 proteins were detected in the nucleus and cytoplasm. Characterization of the transgenic Arabidopsis plants overexpressing RsRBP1 and RsGRP1 revealed that both transgenic lines displayed enhanced growth under the osmotic stress conditions. Overexpression of RsGRP1 resulted in delayed germination rates under the osmotic stress conditions, whereas RsRBP1 overexpression Arabidopsis did not display any difference in germination rates during osmotic stress. These results suggest that RsRBP1 and RsGRP1 may be involved in the responses to osmotic stress in plant.
[show abstract][hide abstract] ABSTRACT: Heat stress severely affects plant growth and development causing crop loss worldwide. Classical type I DnaJ proteins (also called as J-proteins, J-domain proteins or HSP40 proteins) function as molecular co-chaperones for the HSP70 proteins. In this study, we have cloned and characterized a novel gene GmDjp1 (G
protein 1) encoding a type III J-protein of which function has not been identified in plant. Deduced amino acid sequences of GmDjp1 show the highest homology with a J-protein from Medicago truncatula legume plant (83 %) and with Arabidopsis thaliana type III J-class proteins, atDjC53 (77 %) and atDjC32 (50 %). DNA blot analysis revealed that GmDjp1 exists as a 2-copy gene in soybean genome. GmDjp1 mRNA was induced by a broad spectrum of abiotic stresses, including wounding, heat-shock, dehydration, cold or high-salinity stress, suggesting its role in the signaling events in the abiotic stress-related defense response. Subcellular localization studies demonstrated that the GmDjp1-GFP fusion protein was localized in the nucleus. Differential RNA expression of GmDjp1 by heat-shock stress inspired us to test heat-shock tolerance of GmDjp1in E. coli. Heterologous expression of GmDjp1 conferred tolerance to high temperature stress in E. coli. This report provides strong evidence that GmDjp1 may play a critical role during heat-shock stress in cell.
[show abstract][hide abstract] ABSTRACT: A mungbean low temperature-inducible VrPrx1 encoding 2-Cys peroxiredoxin (2-Cys Prx) was cloned by subtractive suppression hybridization. The deduced VrPrx1 amino acid sequence showed highest sequence homology to 2-Cys Prxs of Phaseolus vulgaris (95%), Pisum sativum (89%), and Arabidopsis thaliana (87%). VrPrx1 RNA and protein levels were increased by low temperature, hydrogen peroxide (H2O2), and wounding but decreased by high salinity, drought, and exogenous abscisic acid. Recombinant His-tagged VrPrx1 recombinant protein protected DNA and glutamine synthetase activity from degradation via the thiol/Fe(III) oxygen mixed-function oxidation system, and exhibited peroxidase activity to H2O2 in the presence of the reducing agent dithiothreitol (DTT) in vitro. The oxidized dimers and oligomers of the VrPrx1 recombinant protein were reduced to monomers by DTT or thioredoxin. Subcellular localization studies confirmed that VrPrx1-GFP was targeted to the plastid. To evaluate the function of VrPrx1
in planta, the antioxidant activities and photosynthetic efficiency were investigated in VrPrx1-overexpressing Arabidopsis plants. VrPrx1 ectopic expression conferred improved photosynthetic efficiency under oxidative stress conditions. Hence, mungbean VrPrx1 may play an important role in protecting the photosynthetic apparatus against oxidative and abiotic stress conditions.
Plant Cell Tissue and Organ Culture 08/2013; 108(3). · 3.63 Impact Factor
[show abstract][hide abstract] ABSTRACT: Flavonoids are ubiquitously present in plants and play important roles in these organisms as well as in the human diet. Flavonol synthase (FLS) is a key enzyme of the flavonoid biosynthetic pathway, acting at the diverging point into the flavonol subclass branch. We isolated and characterized a FLS isoform gene, FtFLS2, from tartary buckwheat (Fagopyrum tataricum). FtFLS2 shares 48% identity and 67% similarity with the previously reported FtFLS1, whereas both genes share 47-65% identity and 65-69% similarity with FLSs from other plant species. Using quantitative real-time PCR and high-performance liquid chromatography (HPLC), the expression of FtFLS1/2 and the production of 3 main flavonols (kaempferol, myricetin and quercetin) was detected in roots, leaves, stems, flowers and different stages of developing seeds. The relationship between the expression of the 2 FLS genes and the accumulation of the 3 basic flavonols was analyzed in 2 tartary buckwheat cultivars. FtFLS1 and FtFLS2 exhibited differential transcriptional levels between the tartary buckwheat cultivars 'Hokkai T10' and 'Hokkai T8'. Generally, higher transcript levels of FtFLS1 and FtFLS2 and a higher amount of flavonols were observed in the 'Hokkai T10' cultivar than 'Hokkai T8'. The content of flavonols showed tissue-specific accumulation between the 2 cultivars. The transcription of FtFLS1 was inhibited by the exogenous application of abscisic acid (ABA), salicylic acid (SA) and sodium chloride (NaCl), while FtFLS2 was not affected by ABA but up-regulated by SA and NaCl. These data indicate that the 2 FtFLS isoforms of buckwheat have different functions in the response of buckwheat to environmental stress.
Journal of plant physiology 07/2013; · 2.50 Impact Factor
[show abstract][hide abstract] ABSTRACT: This study investigated the effect of methyl jasmonate (MeJA) on metabolic profiles and rosmarinic acid (RA) biosynthesis in cell cultures of Agastache rugosa Kuntze. Transcript levels of phenylpropanoid biosynthetic genes, i.e., ArPAL, Ar4CL, and ArC4H, maximally increased 4.5-fold, 3.4-fold, and 3.5-fold, respectively, compared with the untreated controls, and the culture contained relatively high amounts of RA after exposure of cells to 50 mM MeJA. RA levels were 2.1-, 4.7-, and 3.9-fold higher after exposure to 10, 50, and 100 mM MeJA, respectively, than those in untreated controls. In addition, the transcript levels of genes attained maximum levels at different time points after the initial exposure. The transcript levels of ArC4H and Ar4CL were transiently induced by MeJA, and reached a maximum of up to 8-fold at 3 hr and 6 hr, respectively. The relationships between primary metabolites and phenolic acids in cell cultures of A. rugosa treated with MeJA were analyzed by gas chromatography coupled with time-of-flight mass spectrometry. In total, 45 metabolites, including 41 primary metabolites and 4 phenolic acids, were identified from A. rugosa. Metabolite profiles were subjected to partial least square-discriminate analysis to evaluate the effects of MeJA. The results indicate that both phenolic acids and precursors for the phenylpropanoid biosynthetic pathway, such as aromatic amino acids and shikimate, were induced as a response to MeJA treatment. Therefore, MeJA appears to have an important impact on RA accumulation, and the increased RA accumulation in the treated cells might be due to activation of the phenylpropanoid genes ArPAL, ArC4H, and Ar4CL.
PLoS ONE 05/2013; 8(5):e64199.. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Radish (Raphanus sativus) sprouts have received attention as an important dietary vegetable in Asian countries. The flavonoid pathway leading to anthocyanin biosynthesis in radishes is induced by multiple regulatory genes as well as various developmental and environmental factors. We investigated anthocyanin accumulation and the transcript level of associated genes in radish sprouts exposed to light and methyl jasmonate (MeJA). The anthocyanin content of sprouts exposed to light and treated with MeJA was higher than that of sprouts grown under dark conditions without MeJA, and the highest anthocyanin content was observed within 6 to 9 days after sowing (DAS). Transcript levels of almost all genes were increased in radish sprouts grown in light conditions with 100 μM MeJA relative to sprouts grown under dark conditions with or without MeJA treatment, especially at 3 DAS. Our results suggest that light and MeJA treatment applied together during radish seedling development enhance anthocyanin accumulation.
Journal of Agricultural and Food Chemistry 04/2013; · 2.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Genetics and Genomics 03/2013; 40(3):127-35. · 2.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 E(2) (PGE(2)) and pro-inflammatory cytokine production were detected in BV2 microglia following LPS stimulation. We identifed that EGL significantly inhibits the excessive production of NO, PGE(2) 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.
Experimental and therapeutic medicine 03/2013; 5(3):957-963. · 0.34 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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. thalianaVrUBC1 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.
PLoS ONE 01/2013; 8(6):e66056. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Apoptosis, the main type of programmed cell death, plays an essential role in a variety of biological events. Whereas "classical" apoptosis is dependent on caspase activation, caspase-independent death is increasingly recognized as an alternative pathway. To develop new anticancer agents, oleifolioside A was isolated from Dendropanax morbifera Leveille and the biochemical mechanisms of oleifolioside A-induced apoptosis in HeLa cells were investigated. Exposure to oleifolioside A resulted in caspase activation and typical features of apoptosis, although cell death was not prevented by caspase inhibition. Oleifolioside A treatment induced up-regulation of Bad, loss of mitochondrial membrane potential, nuclear relocation of mitochondrial factors, apoptosis-inducing factor (AIF), endonuclease G (EndoG), and apoptosis induction. This is the first report of anticancer activity of oleifolioside A, and nuclear translocation of AIF and EndoG in oleifolioside A-treated HeLa cells might represent an alternative death signaling pathway in the absence of caspase activity.
Journal of Agricultural and Food Chemistry 05/2012; 60(21):5400-6. · 2.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: The present study examined the effect of the king oyster mushroom (Pleurotus eryngii) on insulin resistance and dyslipidemia in db/db mice. Four-week-old db/db mice were fed an AIN-93G diet or a diet containing 5% king oyster mushroom for 7 weeks. The blood glycated hemoglobin and
serum glucose levels in the mushroom group were significantly lower than the control group (p<0.05). Dietary king oyster mushroom significantly reduced the homeostasis model assessment for insulin resistance (HOMA-IR),
total cholesterol, and triglyceride, and increased high density lipoprotein (HDL)-cholesterol. These results indicate that
king oyster mushroom improves insulin sensitivity and exerts anti-hyperglycemic and anti-hyperlipidemic effects in db/db mice.
Keywordsdiabetes mellitus-dyslipidemia-hyperglycemia-insulin resistance-king oyster mushroom
Food science and biotechnology 04/2012; 19(1):239-242. · 0.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hydrolytic conditions of rice hulls by acid and alkaline treatments before enzymatic saccharification were optimized in this
study. Based on the results of single-factor experiments and an orthogonal array experiment, reaction time was found to be
the most important factor for the acidic hydrolysis of rice hulls. Maximum yield of sugars from 1 g of rice hulls by acidic
treatment under optimized conditions was 213.6 mg. The yield of lignin removal from acidic pretreated rice hulls by alkaline
treatment increased with increase in reaction temperature and time. The amount of sugars obtained from 1 g of pretreated rice
hulls by enzymatic saccharification was 307.7 mg, and the conversion rate of sugars from crude fibers in pretreated rice hulls
was about 72%. Instrumental analyses with FTIR and SEM indicated that lignin in rice hulls was partially removed by alkaline
treatment, and the structure of rice hulls became deformed and more fibers were exposed to cellulases after acidic treatment.
Biotechnology and Bioprocess Engineering 04/2012; 14(6):828-834. · 1.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Plant secondary metabolites have always been a focus of study due to their important roles in human medicine and nutrition.
We transferred the isoflavone synthase (IFS) gene into soybean [Glycine max (L.) Merr.] using the Agrobacterium-mediated transformation method in an attempt to produce transformed soybean plants which produced increased levels of the
secondary metabolite, isoflavone. Although the trial to produce transgenic plant failed due to unestablished hygromycin selection,
transformed callus cell lines were obtained. The induction rate and degree of callus were similar among the three cultivars
tested, but light illumination positively influenced the frequency of callus formation, resulting in a callus induction rate
of 74% for Kwangan, 67% for Sojin, and 73% for Duyou. Following seven to eight subcultures on selection media, the isoflavone
content of the transformed callus lines were analyzed by high-performance liquid chromatography. The total amount of isoflavone
in the transformed callus cell lines was three- to sixfold higher than that in control callus or seeds. Given the many positive
effects of isoflavone on human health, it may be possible to adapt our transformed callus lines for industrialization through
an alternative cell culture system to produce high concentrations of isoflavones.
[show abstract][hide abstract] ABSTRACT: Photorespiration reduces carbon fixation rate, but it is an essential process in plant. Photorespiration involves reactions
in chloroplasts, peroxisomes, and mitochondria. In photorepiratory peroxisome, alanine glyoxylate aminotransferase (AGT) catalyzes
the conversion of alanine and glyoxylate into glycine and pyruvate, respectively. We isolated a low temperature-inducible
cDNA encoding AGT from mungbean leaves. The full-length cDNA, designated asMLT92, contains an open reading frame of 1,203 nucleotides coding for a protein of 401 amino acids. Genomic DNA blotting showed
that the mungbean genome has one copy ofMLT92. MLT92 mRNA was induced by low temperature and its RNA expression was not much increased by drought stress. ABA and NaCl did not
induce RNA expression ofMLT92. Based on GFP/RFP targeting experiment, GFP-MLT92 fusion protein and SKL-RFP, a peroxisome marker, were colocalized to peroxisome
in tobacco protoplasts. This suggests that peroxisomalMLT92 is involved in molecular response to low temperature stress.
Key wordsalanine glyoxylate aminotransferase-gene expression-green fluorescent protein-low temperature-mungbean-peroxisome
[show abstract][hide abstract] ABSTRACT: Oryza grandiglumis Chitinase IVa (OgChitIVa) cDNA encoding a class IV chitinase was cloned from wild rice (Oryza grandiglumis). OgChitIVa cDNA contains an open reading frame of 867 nucleotides encoding 288 amino acid residues with a predicted molecular weight
of 30.4kDa and isoelectric point of 8.48. Deduced amino acid sequences of OgChitIVa include the signal peptide and chitin-binding domain in the N-terminal domain and conserved catalytic domain. OgChitIVa showed significant similarity at the amino acid level with related monocotyledonous rice and maize chitinase, but low similarity
with dicotyledoneous chitinase. Southern blot analysis showed that OgChitIVa genes are present as two copies in the wild rice genome. It was shown that RNA expression of OgChitIVa was induced by defense/stress signaling chemicals, such as jasmonic acid, salicylic acid, and ethephon or cantharidin and
endothall or wounding, and yeast extract. It was demonstrated that overexpression of OgChitIVa in Arabidopsis resulted in mild resistance against the fungal pathogen, Botrytis cinerea, by lowering disease rate and necrosis size. RT-PCR analysis showed that PR-1 and PR-2 RNA expression was induced in the transgenic lines. Here, we suggest that a novel OgChitIVa gene may play a role in signal transduction process in defense response against B.cinerea in plants.
[show abstract][hide abstract] ABSTRACT: 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.
Biochemical and Biophysical Research Communications 04/2012; 421(2):190-6. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Oleifolioside A, a new triterpenoid compound isolated from Dendropanax morbifera Leveille (D. morbifera), was shown in this study to have potent inhibitory effects on lipopolysaccharide (LPS-)stimulated nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in RAW 264.7 macrophages. Consistent with these findings, oleifolioside A was further shown to suppress the expression of LPS-stimulated inducible nitric oxide synthase (iNOS) and cyclooxigenase-2 (COX-2) in a dose-dependent manner at both the protein and mRNA levels and to significantly inhibit the DNA-binding activity and transcriptional activity of NF-κB in response to LPS. These results were found to be associated with the inhibition of the degradation and phosphorylation of IκB-α and subsequent translocation of the NF-κB p65 subunit to the nucleus. Inhibition of NF-κB activation by oleifolioside A was also shown to be mediated through the prevention of p38 MAPK and ERK1/2 phosphorylation. Taken together, our results suggest that oleifolioside A has the potential to be a novel anti-inflammatory agent capable of targeting both the NF-κB and MAPK signaling pathways.
Evidence-based Complementary and Alternative Medicine 01/2012; 2012:637512. · 1.72 Impact Factor