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ABSTRACT: This study established a new system for potato transformation using toxoflavin as selection agent and toxoflavin lyase (tflA) as selectable marker gene. Potato plants expressing tflA was successfully transformed on toxoflavin medium with 27% efficiency, similar to that for the hygromycin/hpt selection system. The transgenic potato expressing tflA also showed resistance to Burkholderia glumea infection.
Bioscience Biotechnology and Biochemistry 12/2012; · 1.28 Impact Factor
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ABSTRACT: Transgenic potato plants (SS2 and SS4) that overexpressed a chloroplastic copper/zinc superoxide dismutase lily gene were
utilized as an H2O2-inducible system in order to study the role of H2O2 as a signaling molecule in the biosynthesis of ethylene. SS2 and SS4 plants grown in vitro under sealed microenvironment
(SME) conditions displayed anomalous phenotypes including reduction of stem elongation, radial stem growth, and promotion
of root hair formation in the generated root, which were similar to ethylene-induced responses. In addition, SS4 plants showed
severe vitrification in developing leaves and elevated ethylene production under SME conditions. After the ethylene action
inhibitor AgNO3, 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) inhibitor CoCl2, and ACC synthase inhibitor l-aminoethoxyvinylglycine were added to the growth media, the anomalous phenotypes in SS4 plants reverted to their normal phenotype
with a concurrent decrease in ethylene production. Northern blot analysis showed that ACO transcripts in SS4 plants were constantly at high levels under normal and SME conditions, indicating that a high level of
H2O2 in SS4 plants up-regulates ACO transcripts. Moreover, the direct treatment of H2O2 in potato plants confirmed the elevated expression of the ACO gene. Taken together, these data suggest that the high concentration of H2O2 in transgenic potato plants stimulates ethylene biosynthesis by activating ACO gene expression.
Plant Cell Reports 04/2012; 27(6):973-983. · 2.27 Impact Factor
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ABSTRACT: Comprehensive metabolite fingerprinting of transgenic potatoes that constitutively express human beta amyloid, curdlan synthase
(CRDS), and glycogen synthase (glgA); and of wild-type potatoes was carried out using FT-IR and 1H NMR spectroscopy in combination with multivariate analyses. Comparison of metabolic patterns between transgenic and wild-type
potatoes revealed that there were neither quantitative nor qualitative differences in metabolites between transgenic potatoes
expressing human beta amyloid, CRDS or glgA, and non-transformed control potatoes. However, there were metabolic differences between two control potato lines — one
that was fresh and the other stored. After 1 week of storage, comprehensive metabolite patterns were significantly modified.
Although the differences between CRDS and glgA transgenic and control potato lines were small, PCA analysis of FT-IR and 1H NMR spectral data identified two distinct control lines. These results suggest that the comprehensive metabolite changes
in control potato lines, which occurred after 1 week of storage, were greater than the differences between CRDS and glgA transgenic and wild-type potato lines. Thus, the combination of FT-IR and 1H NMR spectral data and multivariate analysis was valuable for the detection of comprehensive differences in metabolic profiles
between transgenic and non-transformed control plants, even though peak-signal overlap prevented assignment of pure compounds.
The combination of FT-IR and 1H NMR spectral data and multivariate analysis is a simple and rapid method for evaluation of the metabolic equivalence of
GM crops.
Biotechnology and Bioprocess Engineering 04/2012; 14(6):738-747. · 1.28 Impact Factor
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ABSTRACT: Elaeocarpus petiolatus is known to exert active oxygen scavenging, anti-aging, and whitening actions. However, the biological effects of E. petiolatus on inflammation and the underlying mechanisms are yet to be established. In the present study, we investigated the anti-inflammatory effects of the ethanol extract from E. petiolatus (EPE) bark in murine Raw264.7 macrophages stimulated with lipopolysaccharide (LPS). EPE inhibited the production of PGE(2), TNF-α, and IL-1β in a dose-dependent manner in Raw264.7 cells stimulated with LPS. The decrease in PGE(2) production was correlated with reduced COX-2 expression. Furthermore, EPE suppressed the phosphorylation of extracellular signal-related kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 as well as translocation of the NF-κB p65 subunit from the cytosol to nucleus. Our results suggest that EPE exerts anti-inflammatory activity through inhibition of inflammatory mediators, such as PGE(2), TNF-α, and IL-1β, and downregulation of COX-2 via suppression of NF-κB translocation and phosphorylation of ERK, JNK, and p38 in LPS-stimulated Raw264.7 cells.
Inflammation 05/2011; 35(2):535-44. · 1.75 Impact Factor
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ABSTRACT: The ability of potato-derived major surface antigen of hepatitis B virus (P-HBsAg) to elicit antibody responses to different dosages of P-HBsAg ranging from 0.02 to 30 μg administered orally in mice was examined. All immunized groups produced specific serum IgG and fecal IgA antibodies against P-HBsAg, even at low levels (<5 μg), after administration of a 0.5-μg yeast-derived HBsAg (Y-HBsAg; LG Life Sciences, Republic of Korea) booster.
Clinical and vaccine immunology: CVI 10/2010; 17(12):2029-32. · 2.37 Impact Factor
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ABSTRACT: Plastid transformation has to date been applied to the expression of heterologous genes involved in agronomic traits and to the production of useful recombinant proteins. Here, we report a feasibility study for producing the human β-site APP cleaving enzyme (BACE) via transformation of tobacco chloroplasts. Stable integration of human BACE into the plastome was confirmed by PCR. Genomic Southern blot analysis detected the presence of the tobacco aadA and human BACE genes between trnI and trnA in the plastome. Northern blot analysis revealed that the aadA and BACE genes were both properly transcribed into a dicistronic transcriptional unit. Human BACE protein expression in transplastomic tobacco was determined by western blot analysis. ELISA analysis revealed that, based on a dilution series of E. coli-derived BACE as a standard, transplastomic lines accumulated BACE to levels of 2.0% of total soluble proteins. When mice were gavaged with the transplastomic tobacco extracts, they showed an immune response against the BACE antigen. The successful production of plastid-based BACE protein has the potential for developing a plant-based vaccine against Alzheimer disease.
Transgenic Research 03/2010; 19(6):1099-108. · 2.75 Impact Factor
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ABSTRACT: An acireductone dioxygenase (ARD) gene of potatoes was isolated from the expressed sequence tags (ESTs) of potato post-suberization cDNA libraries. The highest expression levels of the StARD gene and the protein appeared 36 h after suberization. An approximate 9-fold increase in ARD activity was detected at 36 h after wounding. Real-time reverse transcription-PCR (RT-PCR) analysis and immunolocalization studies revealed that StARD transcripts increase at the wound surface of potato tubers. The polyamine (PA) contents increased significantly after wounding at the wound surface. The increased PA content and ARD activity may play an important role in wound periderm formation.
Plant and Cell Physiology 10/2008; 49(10):1627-32. · 4.70 Impact Factor
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ABSTRACT: Human β-amyloid (Aβ) is believed to be one of the main components of Alzheimer’s disease, so reduction of Aβ is considered
a key therapeutic target. Using Agrobacterium-mediated nuclear transformation, we generated transgenic tomatoes for Aβ with tandem repeats. Integration of the human Aβ
gene into the tomato genome and its transcription were detected by PCR and Northern blot, respectively. Expression of the
Aβ protein was confirmed by western blot and ELISA, and then the transgenic tomato line expressing the highest protein level
was selected for vaccination. Mice immunized orally with total soluble extracts from the transgenic tomato plants elicited
an immune response after receiving a booster. The results indicate that tomato plants may provide a useful system for the
production of human Aβ antigen.
Biotechnology Letters 09/2008; 30(10):1839-1845. · 1.68 Impact Factor
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ABSTRACT: Ethylene-responsive factors (ERFs) are plant-specific transcription factors, many of which have been linked to plant defense responses. However, little is known about the functional significance of ERF genes in potato plants compared to the model plant species Arabidopsis. We show here that overexpression of CaPF1, an ERF/AP2-type pepper transcription factor gene, effectively increased tolerance to freezing, heat, heavy metal, and oxidative stress in potatoes. Interestingly, CaPF1 was involved in tuber formation in potato plants. The time course of microtuber formation was significantly retarded in potato plants that overexpressed CaPF1 compared with wild-type potato plants. Overall, the results of the present study indicate that the pepper transcription factor gene, CaPF1, is involved in promotion of multiple stress tolerance and retardation of in vitro tuberization in potato plants.
Planta 09/2008; 228(4):701-8. · 3.00 Impact Factor
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ABSTRACT: Transgenic potato plants (SS2 and SS4) that overexpressed a chloroplastic copper/zinc superoxide dismutase lily gene were utilized as an H(2)O(2)-inducible system in order to study the role of H(2)O(2) as a signaling molecule in the biosynthesis of ethylene. SS2 and SS4 plants grown in vitro under sealed microenvironment (SME) conditions displayed anomalous phenotypes including reduction of stem elongation, radial stem growth, and promotion of root hair formation in the generated root, which were similar to ethylene-induced responses. In addition, SS4 plants showed severe vitrification in developing leaves and elevated ethylene production under SME conditions. After the ethylene action inhibitor AgNO(3), 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) inhibitor CoCl(2), and ACC synthase inhibitor L -aminoethoxyvinylglycine were added to the growth media, the anomalous phenotypes in SS4 plants reverted to their normal phenotype with a concurrent decrease in ethylene production. Northern blot analysis showed that ACO transcripts in SS4 plants were constantly at high levels under normal and SME conditions, indicating that a high level of H(2)O(2) in SS4 plants up-regulates ACO transcripts. Moreover, the direct treatment of H(2)O(2) in potato plants confirmed the elevated expression of the ACO gene. Taken together, these data suggest that the high concentration of H(2)O(2) in transgenic potato plants stimulates ethylene biosynthesis by activating ACO gene expression.
Plant Cell Reports 07/2008; 27(6):973-83. · 2.27 Impact Factor
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ABSTRACT: Many therapeutic glycoproteins have been successfully generated in plants. Plants have advantages regarding practical and economic concerns, and safety of protein production over other existing systems. However, plants are not ideal expression systems for the production of biopharmaceutical proteins, due to the fact that they are incapable of the authentic human N-glycosylation process. The majority of therapeutic proteins are glycoproteins which harbor N-glycans, which are often essential for their stability, folding, and biological activity. Thus, several glyco-engineering strategies have emerged for the tailor-making of N-glycosylation in plants, including glycoprotein subcellular targeting, the inhibition of plant specific glycosyltranferases, or the addition of human specific glycosyltransferases. This article focuses on plant N-glycosylation structure, glycosylation variation in plant cell, plant expression system of glycoproteins, and impact of glycosylation on immunological function. Furthermore, plant glyco-engineering techniques currently being developed to overcome the limitations of plant expression systems in the production of therapeutic glycoproteins will be discussed in this review.
Molecules and Cells 07/2008; 25(4):494-503. · 2.18 Impact Factor
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ABSTRACT: Patatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers. Potato tubers have recently emerged as bioreactors for the production of human therapeutic glycoproteins (vaccines). Increasing the yield of recombinant proteins, targeting the produced proteins to specific cellular compartments, and diminishing expensive protein purification steps are important research goals in plant biotechnology. In the present study, potato patatins were eliminated almost completely via RNA interference (RNAi) technology to develop potato tubers as a more efficient protein expression system. The gene silencing effect of patatins in the transgenic potato plants was examined at individual isoform levels.
Based upon the sequence similarity within the multi-gene family of patatins, a highly conserved target sequence (635 nts) of patatin gene pat3-k1 [GenBank accession no. DQ114421] in potato plants (Solanum tuberosum L.) was amplified for the construction of a patatin-specific hairpin RNAi (hpRNAi) vector. The CaMV 35S promoter-driven patatin hpRNAi vector was transformed into the potato cultivar Desiree by Agrobacterium-mediated transformation. Ten transgenic potato lines bearing patatin hpRNA were generated. The effects of RNA interference were characterized at both the protein and mRNA levels using 1D and 2D SDS/PAGE and quantitative real-time RT-PCR analysis. Dependent upon the patatin hpRNAi line, patatins decreased by approximately 99% at both the protein and mRNA levels. However, the phenotype (e.g. the number and size of potato tuber, average tuber weight, growth pattern, etc.) of hpRNAi lines was not distinguishable from wild-type potato plants under both in vitro and ex vitro growth conditions. During glycoprotein purification, patatin-knockdown potato tubers allowed rapid purification of other potato glycoproteins with less contamination of patatins.
Patatin-specific hpRNAi effectively suppressed the expression of a majority of patatin variants in potato tubers via the specific degradation of individual mRNAs of the patatin multi-gene family. More importantly, patatin-knockdown potato tubers appear to be an ideal host for the production of human therapeutic glycoproteins, because they eventually allow fast, easy purification of recombinant proteins, with less contamination from potato glycoprotein patatins.
BMC Biotechnology 02/2008; 8:36. · 2.35 Impact Factor
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ABSTRACT: A higher concentration of H2O2 was detected in the sense transgenic potato plant (SS4) with the lily chCu,ZnSOD sequence, whereas higher levels of O2(-) was detected in the antisense transgenic plant (SA1) than the WT plant. The elongation growth in SA1 was significantly inhibited by treatment with diphenyleneiodonium, an inhibitor of O2(-) generation, and promoted in the SS4 on treatment with herbicide methyl viologen, a generator of apoplastic O2(-) . Higher concentrations of GAs were detected during plant growth and the early stage of tuberization in SA1. Complete recovery of the above elongation growth and microtuberization pattern in transgenic plants following treatment of GA(3) or an inhibitor of gibberellin synthesis, paclobutrazol, indicate that these changes were mainly caused by active GA levels. In conclusion, a specific ROS (O2(-) ) acts as a signal transducer via GA biosynthetic pathways for the regulation of plant growth and tuber development of potato.
Plant Cell Reports 11/2007; 26(10):1717-25. · 2.27 Impact Factor
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ABSTRACT: Overexpression of a chloroplast-localized Cu/Zn superoxide dismutase (chCu/ZnSOD) obtained from lily significantly affects
the growth and shape of potato tubers from anin vitro culture system (Kim et al., 2007). Here, we further characterized the sense and antisense transgenic potatoes grown and pots
and the greenhouse to investigate the potential for more practical field applications of such phenotypic manipulations. Underin vitro conditions, antisense transgenic plants showed increased shoot growth, delayed tuberization, and altered tuber shapes. When
antisense plants were treated with paclobutrazol, an inhibitor of GA biosynthesis, tuberization efficiency and tuber shape
were recovered to a status very similar to that ofin vitro- grown wild-type plants. Our results strongly support the idea that potato tuberization and shape is mediated by SOD-catalyzed
reactive oxygen species, possibly via the GA biosynthesis pathway.
Journal of Plant Biology 07/2007; 50(4):490-495. · 1.07 Impact Factor
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ABSTRACT: In this study, two daphnane diterpene esters isolated from the flower buds of Daphne genkwa, genkwadaphnin (1) and yuanhuacine (2), were assessed with regard to their apoptotic activity in human promyelocytic HL-60 cells. Both 1 and 2 were demonstrated to activate the apoptotic process, including DNA fragmentation, chromatin condensation, and sub-G1 hypodiploidy. In our immunoblotting analysis, treatment with compounds 1 and 2 resulted in the cleavage of procaspase-3 and poly(ADP-ribose)polymerase (PARP) into active forms, and the expression of Bcl-2 proteins was shifted toward apoptosis; the expression of the pro-apoptotic protein, Bax, was increased, and the expression of Bcl-2 and Bcl-XL, both anti-apoptotic proteins, were suppressed in a dose-dependent manner. The administration (ip) of the compounds to Lewis lung carcinoma (LLC)-inoculated mice evidenced a significant inhibition of tumor growth (volume), with reductions of 47.9% and 63.1% (1), and 24.2% and 45.8% (2) at concentrations of 0.1 mg/kg and 0.5 mg/kg, as compared with the control mice. These results indicate that compounds 1 and 2 are potent apoptotic constituents of Daphne genkwa, and might be potent as anti-tumoric agents.
Journal of Ethnopharmacology 06/2007; 111(3):496-503. · 3.01 Impact Factor
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ABSTRACT: A new coumarin, hydroxylomatin (1), was isolated from the CHCl(3)-soluble fraction of the roots of Angelica purpuraefolia, along with one ferulate (2) and three other known coumarins (3-5) including khellactone (3). The structure of hydroxylomatin (1) was determined to be 3'beta,5'-dihydroxy-3',4'-dihydroseselin (1) by spectroscopic means including 2D-NMR. The modified Mosher's method was used to determine the chiral center at C-1 of compound 2. Khellactone (3) is a major compound of the roots of A. purpuraefolia. This study also examined the antitumor activity of khellactone (3) using a LLC mouse lung carcinoma in the BDF-1 mice and a NCI-H460 human lung carcinoma in a human tumor xenograft model in nude mice. This compound (3) inhibited LLC tumor growth with a T/C (mean value of treated group/mean value of control group) value of 12.9% at a dose of 5 mg/kg and 33.2% at a dose of 10 mg/kg, respectively, in a dose-dependent manner. In addition, it suppressed the growth of NCI-H460 tumor cells, accounting for 81.4% at a dose of 10 mg/kg in nude mice.
Phytotherapy Research 06/2007; 21(5):406-9. · 2.09 Impact Factor
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ABSTRACT: The antibodies to preS2 synthetic peptides have been probed to neutralize hepatitis B virus (HBV), and also the addition of preS2 sequence could enhance the antibody response compared with a conventional vaccine in the non- and low responders. Previously, we generated transgenic potatoes expressing middle protein, which contains additional 55 amino acid preS2 region at the N-terminus of the S protein, of HBV to determine the feasibility of developing a plant-delivered HBV vaccine. In this study, we monitored the immune response after induction of immunoglobulin by boosting and assessed the efficacy of the mucosal immune response with regard to generate IgA antibodies. The HBsAg middle protein expressed in our transgenic potatoes was well immunized at low antigenic quantities in mice and the induced anti-S or anti-preS2 antibodies were sustained for the whole period without decrease. Orally delivery of plant-derived HBsAg middle protein to mice resulted in fecal anti-S or anti-preS2 as well as serum IgG. In addition, we used antibodies induced from the immunized mice with the potato-derived rHBsAg in competition assay as competitors to confirm the binding ability of preS2 antibodies to surface antigen of hepatitis virus. Anti-preS2 antibodies induced from immunized mice with transgenic potatoes effectively competed with anti-preS2 murine antibody H8 as expected. From these results, the inclusion of preS2 antigen to HBV plant vaccine may provide additional protective immunity in the HBV prevention.
Vaccine 02/2007; 25(3):577-84. · 3.77 Impact Factor
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ABSTRACT: To determine their tissue-specific expression, three GRX genes were isolated from ESTs of a “6-hr post-suberization” cDNA
library forSolatium tuberosum L cv. Desiree. Transcripts of a CCxC/S type GRX, StGRX1, accumulated to higher levels during the vegetative-growth stage
in the leaves, stem, and roots. In contrast, transcripts of a CxxC/S type GRX, StGRX2, accumulated in the reproductive tissues,
such as the flower and tuber. Differential expression of these three potato StGRXs suggest specific and unique physiological
roles during plant development and in response to environmental cues. Therefore, we propose that StGRX1 is a housekeeping
gene, functioning in the rapid defense against various abiotic and biotic stresses, while StGRX2 and-3 are selectively expressed
during growth and in response to internal and external signals.
Journal of Plant Biology 01/2007; 50(6):663-670. · 1.07 Impact Factor
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Sei-Ryang Oh,
Mee Young Lee,
Kyungseop Ahn,
Bo-Young Park,
Ok-Kyuoung Kwon, Hyouk Joung,
Joongku Lee,
Doo-Young Kim,
Sangku Lee,
Joo Heon Kim,
Hyeong-Kyu Lee
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ABSTRACT: Allergic inflammation of the airways has a critical role in asthma development. We investigated a suppressive effect of verproside (3,4-dihydroxy catalpol) isolated from the extract of Pseudolysimachion longifolium on asthmatic parameters--such as immunoglobulin E (IgE) level, cytokine release, eosinophilia, airway hyperresponsiveness and mucus hypersecretion--in an OVA-sensitized/challenged mouse model. Verproside significantly inhibited the increase of total IgE and the cytokines IL-4 and IL-13 in plasma and bronchoalveolar lavage fluid, and also effectively suppressed airway hyperresponsiveness, eosinophilia and mucus hypersecretion in OVA-induced asthmatic mice. The efficacy of verproside was comparable to montelukast, an anti-asthmatic drug that is currently available. These results suggest that verproside could be a major marker in herbal medicines that are used for asthma treatment, and could also act as a lead for anti-asthmatic drugs.
International Immunopharmacology 07/2006; 6(6):978-86. · 2.38 Impact Factor
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Jung Won Youm,
Hee Kim,
Jee Hye Lo Han,
Chang Hwan Jang,
Hee Jin Ha,
Inhee Mook-Jung,
Jae Heung Jeon,
Cheol Yong Choi,
Young Ho Kim,
Hyun Soon Kim, Hyouk Joung
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ABSTRACT: Beta amyloid (Abeta) is believed one of the major pathogens of Alzheimer's disease (AD), and the reduction of Abeta is considered a primary therapeutic target. Immunization with Abeta can reduce Abeta burden and pathological features in transgenic AD model mice. Transgenic potato plants were made using genes encoding 5 tandem repeats of Abeta1-42 peptides with an ER retention signal. Amyloid precursor protein transgenic mice (Tg2576) fed with transgenic potato tubers with adjuvant showed a primary immune response and a partial reduction of Abeta burden in the brain. Thus, Abeta tandem repeats can be expressed in transgenic potato plants to form immunologically functional Abeta, and these potatoes has a potential to be used for the prevention and treatment of AD.
FEBS Letters 01/2006; 579(30):6737-44. · 3.54 Impact Factor
