Zee-Won Lee's scientific contributions

Publications (27)

Publications citing this author (488)

    • RhoGTPases were activated using calpeptin, EGF and transfection with constitutively active RhoGTPase mutants. Calpeptin (CP) is a cell permeable calpain inhibitor known to activate Rho leading to stress fiber formation [24,25]. Treatment with calpeptin and EGF did not influence the transgene expression or polyplex uptake.
    [Show abstract] [Hide abstract] ABSTRACT: Although it is well accepted that the constituents of the cellular microenvironment modulate a myriad of cellular processes, including cell morphology, cytoskeletal dynamics and uptake pathways, the underlying mechanism of how these pathways influence non-viral gene transfer have not been studied. Transgene expression is increased on fibronectin (Fn) coated surfaces as a consequence of increased proliferation, cell spreading and active engagement of clathrin endocytosis pathway. RhoGTPases mediate the crosstalk between the cell and Fn, and regulate cellular processes involving filamentous actin, in-response to cellular interaction with Fn. Here the role of RhoGTPases specifically Rho, Rac and Cdc42 in modulation of non-viral gene transfer in mouse mesenchymal stem (mMSCs) plated in a fibronectin microenvironment was studied. More than 90% decrease in transgene expression was observed after inactivation of RhoGTPases using difficile toxin B (TcdB) and C3 transferase. Expression of dominant negative RhoA (RhoAT19N), Rac1(Rac1T17N) and Cdc42 (Cdc42T17N) also significantly reduced polyplex uptake and transgene expression. Interactions of cells with Fn lead to activation of RhoGTPases. However, further activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes (RhoAQ63L, Rac1Q61L and Cdc42Q61L) did not further enhance transgene expression in mMSCs, when plated on Fn. In contrast, activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes for cells plated on collagen I, which by itself did not increase RhoGTPase activation, resulted in enhanced transgene expression. Our study shows that RhoGTPases regulate internalization and effective intracellular processing of polyplexes that results in efficient gene transfer.
    Full-text · Article · Apr 2012
    • Stabilization of MTs by Taxol is sufficient to induce growth cone turning (Buck and Zheng, 2002 ), and MT-stabilizing proteins like MAP1B have been shown to influence guidance (Mack et al., 2000; Hahn et al., 2005 ). Many +TIPs show high expression in the developing nervous system and especially in outgrowing nerves (Park et al., 2012). Indeed we found that XCLASP1 mRNA localization in spinal cord nerves was high during axon outgrowth but disappeared in later stages (Figure 1, A–C).
    [Show abstract] [Hide abstract] ABSTRACT: Dynamic microtubules (MTs) are required for neuronal guidance, where axons extend directionally toward their target tissues. We found that depletion of the MT-binding protein Xenopus CLASP1 (XCLASP1) or treatment with the MT drug taxol reduced axon outgrowth in spinal cord neurons. To quantify the dynamic distribution of MTs in axons, we developed an automated algorithm to detect and track MT plus-ends that have been fluorescently labeled by end-binding protein 3 (EB3). XCLASP1 depletion reduced MT advance rates in neuronal growth cones very similar to treatment with taxol, demonstrating a potential link between MT dynamics in the growth cone and axon extension.Automatic tracking of EB3 comets in different compartments revealed that MTs increasingly slowed as they passed from the axon shaft into the growth cone and filopodia. We used speckle microscopy to demonstrate that MTs experience retrograde flow at the leading edge. Microtubule advance in growth cone and filopodia was strongly reduced in XCLASP1-depleted axons as compared with control axons, but actin retrograde flow remained unchanged. Instead, we found that XCLASP1-depleted growth cones lacked lamellipodial actin organization characteristic of protrusion. Lamellipodial architecture depended on XCLASP1 and its capacity to associate with MTs, highlighting the importance of XCLASP1 in actin-microtubule interactions.
    Full-text · Article · Mar 2013
    • One of our objectives while designing the D-264 prodrug, was to impart additional antioxidant activity by conjugating L-cysteine moiety. Antioxidants such as L-cysteine and its analogues have been demonstrated by numerous studies [31][32][33][34][35]to reduce the oxidative stress associated with PD and increase the endogenous GSH and other antioxidant concentrations in PD patients [18]. It is known that free radicals cause auto-oxidation of unsaturated lipids which in turn produce many harmful toxic substances [36].
    [Show abstract] [Hide abstract] ABSTRACT: Parkinson’s disease (PD) is one of the major debilitating neurodegenerative disorders affecting millions of people worldwide. Progressive loss of dopamine neurons resulting in development of motor dysfunction and other related non-motor symptoms is the hallmark of PD. Previously, we have reported on the neuroprotective property of a potent D3 preferring agonist D-264. In our goal to increase the bioavailability of D-264 in the brain, we have synthesized a modified cysteine based prodrug of D-264 and evaluated its potential in crossing the blood–brain barrier. Herein, we report the synthesis of a novel modified cysteine conjugated prodrug of potent neuroprotective D3 preferring agonist D-264 and systematic evaluation of the hydrolysis pattern of the prodrug to yield D-264 at different time intervals in rat plasma and brain homogenates using HPLC analysis. Furthermore, we have also performed in vivo experiments with the prodrug to evaluate its enhanced brain penetration ability.
    Full-text · Article · Mar 2016 · Sensors
    • The resultant supernatants were assayed for protein and β-galactosidase activity. Luciferase activity was assayed in 10-μl samples of extract; the luciferase luminescence was counted in luminometer (Turner Design, TD-20/20) and normalized to the co-transfected β-galactosidase activity, as described elsewhere (Woo et al., 2000b; Chung et al., 2005; Shin et al., 2007). Transfection experiments were performed in triplicate with two independently isolated sets of cells, and the results were averaged.
    [Show abstract] [Hide abstract] ABSTRACT: Up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the lung airway epithelium is associated with the epithelium-leukocyte interaction, critical for the pathogenesis of various lung airway inflammatory diseases such as asthma. However, little is known about how ICAM-1 is up-regulated in human airway epithelial cells. In this study, we show that tumor TNF-α induces monocyte adhesion to A549 human lung airway epithelium and also up-regulation of ICAM-1 expression. These effects were significantly diminished by pre-treatment with diphenyliodonium (DPI), an inhibitor of NADPH oxidase-like flavoenzyme. In addition, the level of reactive oxygen species (ROS) was increased in response to TNF-α in A549 cells, suggesting a potential role of ROS in the TNF-α-induced signaling to ICAM-1 expression and monocyte adhesion to airway epithelium. Further, we found out that expression of RacN17, a dominant negative mutant of Rac1, suppressed TNF-α-induced ROS generation, ICAM-1 expression, and monocyte adhesion to airway epithelium. These findings suggest that Rac1 lies upstream of ROS generation in the TNF-α-induced signaling to ICAM-1 expression in airway epithelium. Finally, pretreatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, reduced TNF-α-induced ICAM-1 expression and both DPI and RacN17 significantly diminished NF-κB activation in response to TNF-α. Together, we propose that Rac1-ROS-linked cascade mediate TNF-α-induced ICAM-1 up-regulation in the airway epithelium via NF-κB-dependent manner.
    Full-text · Article · Apr 2008
    • These surfaces are quite promising for cell cultivation and sensing, however, are somewhat complex to prepare and may not be suitable for all applications. Other reported approaches involved co-assembly of two different silane molecules (acrylateand thiol-or amine-terminated silanes), one bearing gel anchoring and another protein immobilization chemistries [18,19]. This approach has been used by us recently to create hydrogel microwells for capturing immune cells and detecting secreted cytokines [19].
    [Show abstract] [Hide abstract] ABSTRACT: Micropatterning strategies often call for attachment of non-fouling biomaterials and immobilization of proteins in order to create biosensing surfaces or to control cell-surface interactions. Our laboratory has made frequent use of hydrogel photolithography - a micropatterning process for immobilizing poly(ethylene glycol) (PEG) hydrogel microstructures on glass surfaces. In the present study we explored the use of thiolsilane as a coupling layer for both covalent anchoring of hydrogel microstructures and covalent immobilization of proteins on glass. These new surfaces were compared to acryl-silane functionalized glass slides that allowed covalent attachment of gels but only physical adsorption of proteins as well as surfaces containing a mixture of both functional groups. We observed comparable attachment and retention of hydrogel microstructures on acryl and thiol-terminated silanes. Ellipsometry studies revealed presence of significantly higher level of proteins on thiol-functionalized glass. Overall, our studies demonstrate that thiol-silane functionalized glass surfaces may be used to create complex micropatterned surfaces comprised of covalently attached hydrogels and proteins. This simple and effective surface modification strategy will be broadly applicable in cellular engineering and biosensing studies employing hydrogel micropatterns.
    Full-text · Article · Apr 2012
    • However, two recent developments that address certain limitations of traditional FRET-based assays deserve mention. First, a report by Lee et al.[9]describes a unique co-translocation assay for visualizing protein interactions and their inhibitors in live cells. The authors exploit the ability of protein kinase C delta (PKCδ) to translocate to the plasma membrane from the cytosol in response to specific stimuli.
    [Show abstract] [Hide abstract] ABSTRACT: In recent years, scientists have expanded their focus from cataloging genes to characterizing the multiple states of their translated products. One anticipated result is a dynamic map of the protein association networks and activities that occur within the cellular environment. While in vitro-derived network maps can illustrate which of a multitude of possible protein-protein associations could exist, they supply a falsely static picture lacking the subtleties of subcellular location (where) or cellular state (when). Generating protein association network maps that are informed by both subcellular location and cell state requires novel approaches that accurately characterize the state of protein associations in living cells and provide precise spatiotemporal resolution. In this review, we highlight recent advances in visualizing protein associations and networks under increasingly native conditions. These advances include second generation protein complementation assays (PCAs), chemical and photo-crosslinking techniques, and proximity-induced ligation approaches. The advances described focus on background reduction, signal optimization, rapid and reversible reporter assembly, decreased cytotoxicity, and minimal functional perturbation. Key breakthroughs have addressed many challenges and should expand the repertoire of tools useful for generating maps of protein interactions resolved in both time and space.
    Full-text · Article · Nov 2011
    • Predicted N-glycosylation site is indicated by closed diamond under the residue.). The glycosylation of Asn 344 is critical for the protein folding, and that of Asn 196 is important for stabilizing the hexamerin state and overall stability (Ryu et al., 2009). There is only one glycosylation site, Asn 750 , in BdAr.
    [Show abstract] [Hide abstract] ABSTRACT: A Bactrocera dorsalis hexamerin (BdAr) cDNA was cloned (GenBank accession no. KF815528), and its transcriptional expression profiles were determined. The complete 2,530-bp cDNA encodes a 780-amino acid protein with a predicted molecular mass of 94.01 kDa. The proportions of phenylalanine (7.8%), tyrosine (11.2%), and methionine (2.6%) in BdAr as well as all other amino acids are reported. BdAr transcripts were detected in the brain, flight muscle, foregut, Malpighian tubules, and fat body. In the larval stage, BdAr transcripts were expressed in the early third instar and increased in the late third instar. In pupae, the highest expression of BdAr mRNA was present on day 1, then declined and persisted through day 2 to day 8. In adult females, the relative expression of BdAr was significantly higher on day 0 and day 1 compared to day 6 to day 10 while it was highest in newly eclosed adult males. The comparison of the BdAr expression between 8–10-day-old males and females showed a higher level in females. Our phylogenetic analysis results suggest to us that BdAr is similar to Drosophila larval serum protein 1γ.
    Article · Jul 2014
    • Levels of the CYP4A subtypes were highly up-regulated in livers of db/db diabetic mice which exhibit steatosis compared with C57BL/6J mice that do not. Inhibition of Cyp4a enzymatic function using HET0016 ((N-Hydroxy-N0-(4-butyl-2-methylphenyl)-formamidine)), a potent pan-CYP4 inhibitor in db/db diabetic mice on a standard diet and wild-type mice on a high-fat diet reduced features of diabetes including hepatic steatosis, oxidative stress, and endoplasmic reticulum stress (Park et al., 2014). Further work is needed to determine if increases in the Cyp4a family members are involved in PFAA-induced steatosis.
    [Show abstract] [Hide abstract] ABSTRACT: Persistent presence of perfluoroalkyl acids (PFAAs) in the environment is due to their extensive use in industrial and consumer products, and their slow decay. Biochemical tests in rodent demonstrated that these chemicals are potent modifiers of lipid metabolism and cause hepatocellular steatosis. However, the molecular mechanism of PFAAs interference with lipid metabolism remains to be elucidated. Currently, two major hypotheses are that PFAAs interfere with mitochondrial beta-oxidation of fatty acids and/or they affect the transcriptional activity of peroxisome proliferator-activated receptor α (PPARα) in liver. To determine the ability of structurally-diverse PFAAs to cause steatosis, as well as to understand the underlying molecular mechanisms, wild-type (WT) and PPARα-null mice were treated with perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), or perfluorohexane sulfonate (PFHxS), by oral gavage for 7 days, and their effects were compared to that of PPARα agonist WY-14643 (WY), which does not cause steatosis. Increases in liver weight and cell size, and decreases in DNA content per mg of liver, were observed for all compounds in WT mice, and were also seen in PPARα-null mice for PFOA, PFNA, and PFHxS, but not for WY. In Oil Red O stained sections, WT liver showed increased lipid accumulation in all treatment groups, whereas in PPARα-null livers, accumulation was observed after PFNA and PFHxS treatment, adding to the burden of steatosis observed in control (untreated) PPARα-null mice. Liver triglyceride (TG) levels were elevated in WT mice by all PFAAs and in PPARα-null mice only by PFNA. In vitro β-oxidation of palmitoyl carnitine by isolated rat liver mitochondria was not inhibited by any of the 7 PFAAs tested. Likewise, neither PFOA nor PFOS inhibited palmitate oxidation by HepG2/C3A human liver cell cultures. Microarray analysis of livers from PFAAs-treated mice indicated that the PFAAs induce the expression of the lipid catabolism genes, as well as those involved in fatty acid and triglyceride synthesis, in WT mice and, to a lesser extent, in PPARα-null mice. These results indicate that most of the PFAAs increase liver TG load and promote steatosis in mice We hypothesize that PFAAs increase steatosis because the balance of fatty acid accumulation/synthesis and oxidation is disrupted to favor accumulation.
    Full-text · Article · Dec 2016
    • The protein with the greatest change in expression during cell culture was identified as calcyclin. Lee et al. [24] used 2DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in basic fibroblast growth factor (bFGF) containing medium; a total of 15 differentially expressed proteins were identified, of which nine of them were upregulated and six downregulated. The expression level of three actin-related proteins, F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2 and myosin regulatory light chain 2, was confirmed by complementary analysis.
    [Show abstract] [Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) are undifferentiated cells with an unlimited capacity for self-renewal and able to differentiate towards specific lineages under appropriate conditions. MSCs are, a priori, a good target for cell therapy and clinical trials as an alternative to embryonic stem cells, avoiding ethical problems and the chance for malignant transformation in the host. However, regarding MSCs, several biological implications must be solved before their application in cell therapy, such as safe ex vivo expansion and manipulation to obtain an extensive cell quantity amplification number for use in the host without risk accumulation of genetic and epigenetic abnormalities. Cell surface markers for direct characterization of MSCs remain unknown, and the precise molecular mechanisms whereby growth factors stimulate their differentiation are still missing. In the last decade, quantitative proteomics has emerged as a promising set of techniques to address these questions, the answers to which will determine whether MSCs retain their potential for use in cell therapy. Proteomics provides tools to globally analyze cellular activity at the protein level. This proteomic profiling allows the elucidation of connections between broad cellular pathways and molecules that were previously impossible to determine using only traditional biochemical analysis. However; thus far, the results obtained must be orthogonally validated with other approaches. This review will focus on how these techniques have been applied in the evaluation of MSCs for their future applications in safe therapies.
    Full-text · Article · Feb 2014
    • During developmental stages increased levels of TG2 were reported in blood and other regions undergoing active apoptosis (Fesus and Tarcsa 1989; Nagy et al. 1997 ). TG2 has also been implicated in programmed cell death induced in response to cellular stressors such as hypoxia, treatment with N-(4-hy- droxyphenyl) retinamide, tumor necrosis factor-α (TNF-α) or serum deprivation (Fesus and Szondy 2005; Antonyak et al. 2003; Kweon et al. 2004). Paradoxical evidence that TG2 may also support cell growth and cell survival functions came from the authors' laboratory where they observed that drug-resistant breast cancer cells express elevated levels of TG2 (Mehta 1994).
    [Show abstract] [Hide abstract] ABSTRACT: The ability of cancer cells to metastasize represents the most devastating feature of cancer. Currently, there are no specific biomarkers or therapeutic targets that can be used to predict the risk or to treat metastatic cancer. Many recent reports have demonstrated elevated expression of transglutaminase 2 (TG2) in multiple drug-resistant and metastatic cancer cells. TG2 is a multifunctional protein mostly known for catalyzing Ca²⁺-dependent -acyl transferase reaction to form protein crosslinks. Besides this transamidase activity, many Ca²⁺-independent and non-enzymatic activities of TG2 have been identified. Both, the enzymatic and non-enzymatic activities of TG2 have been implicated in diverse pathophysiological processes such as wound healing, cell growth, cell survival, extracellular matrix modification, apoptosis, and autophagy. Tumors have been frequently referred to as ‘wounds that never heal’. Based on the observation that TG2 plays an important role in wound healing and inflammation is known to facilitate cancer growth and progression, we discuss the evidence that TG2 can reprogram inflammatory signaling networks that play fundamental roles in cancer progression. TG2-regulated signaling bestows on cancer cells the ability to proliferate, to resist cell death, to invade, to reprogram glucose metabolism and to metastasize, the attributes that are considered important hallmarks of cancer. Therefore, inhibiting TG2 may offer a novel therapeutic approach for managing and treatment of metastatic cancer. Strategies to inhibit TG2-regulated pathways will also be discussed.
    Full-text · Article · Aug 2016
    • Conformal contact between the stamp and the substrate is facilitated by the elasticity of the PDMS stamp. Self-assembly monolayers with defined localization or protein patterns for highly controlled cell growth can be formed on a substrate by microcontact print- ing [32, 33].
    [Show abstract] [Hide abstract] ABSTRACT: Cell–biomaterial interactions are largely governed by the stiffness and chemical composition of the substrate, although the surface topography has also been shown to play an important role in the adhesion and proliferation of various cell types to different substrates. Actually, the engineering of surfaces to control cell adhesion represents an active area of biomaterials research that aims to meet the requirements of each cell–biomaterial interaction. There are many reported techniques for creating substrates with controlled topography on different materials. Among these, some can be used to create ordered features (e.g. photolithography), while the others are mostly used for creating non-ordered surface patterns (e.g. polymer demixing). We present here what we consider the most relevant methods that were used to generate different micro-and nanotopographical features to study cell–biomaterial interactions.
    Chapter · Jun 2016 · Sensors
    • The development of NAFLD has been frequently studied in animal models exposed to high fat diets (Begriche et al. 2013; Kakimoto and Kowaltowski 2016). Several global omics-based studies have addressed the molecular aspects of fatty liver development in dietinduced obese rodent models and diabetic mice (Baiges et al., 2010; Xie et al., 2010; Zhang et al. 2010; BondiaPons et al., 2011; Kirpich et al., 2011; Oh et al., 2011; Rubio-Aliaga et al., 2011; Almon et al., 2012; Midha et al., 2012; Kim et al., 2013; Benard et al., 2016; Cheng et al., 2016). This led to an emerging interest in the role of mitochondria in NAFLD and nonalcoholic steatohepatitis (Guo et al., 2013; Thomas et al., 2013; Li et al., 2014; Nesteruk et al., 2014 ).
    [Show abstract] [Hide abstract] ABSTRACT: Nonalcoholic fatty liver disease (NAFLD) is a major health burden in the aging society with an urging medical need for a better understanding of the underlying mechanisms. Mitochondrial fatty acid oxidation and mitochondrial-derived reactive oxygen species (ROS) are considered critical in the development of hepatic steatosis, the hallmark of NAFLD. Our study addressed in C57BL/6J mice the effect of high fat diet feeding and age on liver mitochondria at an early stage of NAFLD development. We therefore analyzed functional characteristics of hepatic mitochondria and associated alterations in the mitochondrial proteome in response to high fat feeding in adolescent, young adult, and middle-aged mice. Susceptibility to diet-induced obesity increased with age. Young adult and middle-aged mice developed fatty liver, but not adolescent mice. Fat accumulation was negatively correlated with an age-related reduction in mitochondrial mass and aggravated by a reduced capacity of fatty acid oxidation in high fat-fed mice. Irrespective of age, high fat diet increased ROS production in hepatic mitochondria associated with a balanced nuclear factor erythroid-derived 2 like 2 (NFE2L2) dependent antioxidative response, most likely triggered by reduced tethering of NFE2L2 to mitochondrial phosphoglycerate mutase 5. Age indirectly influenced mitochondrial function by reducing mitochondrial mass, thus exacerbating diet-induced fat accumulation. Therefore, consideration of age in metabolic studies must be emphasized.
    Full-text · Article · Oct 2016
    • Several probiotic strains have been reported to influence haematological cancers including L. reuteri-enhanced TNF-induced apoptosis in chronic myeloid leukemia humanderived cells by modulation of NF-kB and mitogen activated protein kinase signalling and condensed proteins that mediated cell proliferation (cycline D1 and Cox-2) or inhibited apoptosis (Bcl-2, Bcl-xL) [21]. Hwang et al. [68] also reported that LAB induced apoptosis in gastric cancer cells (KATO3) by inhibiting NF-kB and mTOR-mediated signalling. Cousin et al. [69] proposed another mechanism for probiotic induced apoptosis in their report of the pro-apoptotic potential of P. freudenreichii on HGT-1 gastric cancer cell lines.
    [Show abstract] [Hide abstract] ABSTRACT: Cancers figure among the most important causes of morbidity and mortality worldwide. Cancer and its associated infections are always complicated even when specific cancer regimens are available. It is well proved that Lactobacillus and other probiotic bacteria can modulate-ameliorate specific mechanisms against various infections including cancers. The present systematic review is intended to focus on the ‘cellular and molecular mechanisms’ of probiotic bacteria in the prevention and treatment of various cancers. The clinical and experimental findings of various studies explain the mechanisms such as apoptosis, antioxidant activity, immune response and epigenetics and illustrate the role of probiotics in cancer management and prophylaxis. In addition, the present review also discusses the safety aspects of probiotics when they are used in therapeutic and nutritional diet management. However, further investigations are required to reveal the effectiveness of probiotics in cancer treatment in clinical settings.
    Full-text · Article · Nov 2016
    • Dozens of ginseng accessions have been bred through pure line selection from three local landrace populations (Jakyung, Chungkyung, and Hwangsook) and registered as cultivated varieties (cultivars) in Korea (Kim et al., 2012). These cultivars exhibit different morphological and physiological characters (Kim et al., 2012;Lee et al., 2015), as well as different metabolite accumulation patterns (Ahn et al., 2008;Kim et al., 2009;Lee et al., 2011;Cho et al., 2012). In P. ginseng, ginsenosides are mainly of the dammarane type, which are in turn classified into two types according to the number of hydroxyl groups: protopanaxadiol (PPD)-type ginsenosides with hydroxyl groups at positions C3, C12, and C20 and protopanaxatriol (PPT)-type ginsenosides with hydroxyl groups at positions C3, C6, C12, and C20 (Jung et al., 2014;Kim et al., 2014).
    [Show abstract] [Hide abstract] ABSTRACT: Panax ginseng C.A. Meyer is a traditional medicinal herb that produces bioactive compounds such as ginsenosides. Here, we investigated the diversity of ginsenosides and related genes among five genetically fixed inbred ginseng cultivars (Chunpoong [CP], Cheongsun [CS], Gopoong [GO], Sunhyang [SH], and Sunun [SU]). To focus on the genetic diversity related to ginsenoside biosynthesis, we utilized in vitro cultured adventitious roots from the five cultivars grown under controlled environmental conditions. PCA loading plots based on secondary metabolite composition classified the five cultivars into three groups. We selected three cultivars (CS, SH, and SU) to represent the three groups and conducted further transcriptome and gas chromatography-mass spectrometry analyses to identify genes and intermediates corresponding to the variation in ginsenosides among cultivars. We quantified ginsenoside contents from the three cultivars. SH had more than 12 times the total ginsenoside content of CS, with especially large differences in the levels of panaxadiol-type ginsenosides. The expression levels of genes encoding squalene epoxidase (SQE) and dammarenediol synthase (DDS) were also significantly lower in CS than SH and SU, which is consistent with the low levels of ginsenoside produced in this cultivar. Methyl jasmonate (MeJA) treatment increased the levels of panaxadiol-type ginsenosides up to 4-, 13-, and 31-fold in SH, SU, and CS, respectively. MeJA treatment also greatly increased the quantity of major intermediates and the expression of the underlying genes in the ginsenoside biosynthesis pathway; these intermediates included squalene, 2,3-oxidosqualene, and dammarenediol II, especially in CS, which had the lowest ginsenoside content under normal culture conditions. We conclude that SQE and DDS are the most important genetic factors for ginsenoside biosynthesis with diversity among ginseng cultivars.
    Full-text · Article · Jun 2017
    • The PCR product was digested with NdeI and NcoI, and ligated into the same sites of pET-22b(+) (Novagen, San Diego, CA, USA) to make pET-6HGBP. For the cloning and expression of the 6HGBP-ScFv fusion gene, the DNA sequence encoding ScFv fragment was amplified by PCR using plasmid pET-ScFv-SBD [18] as a template, and P3 (5'-CAAGACCATGGGTGTCGACTGAGGAGTCTGGA-3') and P4 (5'-TCCGCTCGAGACGTTTTATTTCCAGGTAGGT-3') as primers. This PCR product was digested with NcoI and XhoI, and ligated into the same sites of pET-6HGBP to make pET-6HGBP-ScFv.
    [Show abstract] [Hide abstract] ABSTRACT: We have developed a simple electrochemical biosensing strategy for the label-free diagnosis of hepatitis B virus (HBV) on a gold electrode surface. Gold-binding polypeptide (GBP) fused with single-chain antibody (ScFv) against HBV surface antigen (HBsAg), in forms of genetically engineered protein, was utilized. This GBP-ScFv fusion protein can directly bind onto the gold substrate with the strong binding affinity between the GBP and the gold surface, while the recognition site orients toward the sample for target binding at the same time. Furthermore, this one-step immobilization strategy greatly simplifies a fabrication process without any chemical modification as well as maintaining activity of biological recognition elements. This system allows specific immobilization of proteins and sensitive detection of targets, which were verified by surface plasmon resonance analysis and successfully applied to electrochemical cyclic voltammetry and impedance spectroscopy upto 0.14 ng/mL HBsAg.
    Full-text · Article · Dec 2012