Eun Jin Park

Electro-gene therapy of collagen-induced arthritis by using an expression plasmid for the soluble p75 tumor necrosis factor receptor-Fc fusion protein.

Tumor necrosis factor (TNF) is a proinflammatory cytokine involved in the pathogenesis of rheumatoid arthritis, and antagonism of TNF may reduce the activity of the disease. Among a number of techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive, and safe. In this study, we attempted to treat collagen-induced arthritis (CIA) with anti-TNF gene therapy by transferring the plasmid encoding soluble p75 TNF receptor linked to the Fc portion of human IgG1 (sTNFR:Fc) using in vivo electroporation. DBA/1 mice were immunized with bovine type II collagen and boosted with the same antigen. At 2 days after boosting, the plasmid vector containing cDNA for the sTNFR:Fc was injected into one selected site in the gastrocnemius muscle followed by electroporation. Serum levels of sTNFR:Fc reached 2.3u2009ng/ml on day 5 when gene expression reached its peak. Macroscopic analysis of paws for redness, swelling and deformities showed that the onset of moderate-to-severe CIA in mice treated with sTNFR:Fc was prevented on a significant level compared with the control mice (P<0.05). The beneficial effect of sTNFR:Fc DNA transfer lasted for at least 18 days following treatment. In addition, both the synovitis and the erosion of cartilage in the knee joints were dramatically reduced in mice treated with sTNFR:Fc (P<0.05). The expression of IL-1β and IL-12 in the paw was also decreased by sTNFR:Fc treatment (P<0.01) while there was little change in the levels of IL-17 and vWF. These data showed that sTNFR:Fc expression plasmid was effective in the prevention of CIA, and in vivo electroporation-mediated gene transfer may provide a new approach to cytokine therapy in autoimmune arthritis.

Protection against collagen-induced arthritis by intramuscular gene therapy with an expression plasmid for the interleukin-1 receptor antagonist

The interleukin-1 receptor antagonist (IL-1Ra) is an endogenous protein that can prevent the binding of IL-1 to its cell-surface receptors. Among a number of techniques for gene transfer in vivo, the direct injection of naked DNA into muscle is simple, inexpensive and safe. In this study, we evaluated the potential of intramuscular gene therapy with plasmid DNA containing the cDNA for IL-1Ra in the prevention of murine collagen-induced arthritis (CIA). DBA/1 mice were immunized with bovine type II collagen. At 4 weeks after the initial immunization, expression plasmid for IL-1Ra was injected into four selected sites in the thigh and calf muscles of DBA/1 mice. Control mice received the same plasmid, but lacking the IL-1Ra coding sequence. Macroscopic analysis of paws for redness, swelling and deformities showed that the onset of moderate to severe CIA in the paws of mice injected with IL-1Ra DNA was significantly prevented (P<0.05). In addition, both the synovitis and the cartilage erosion in knee joints were dramatically reduced in mice treated with IL-1Ra DNA (P<0.05). The expression of IL-1β was significantly decreased in the ankle joints of mice treated with IL-1Ra (P<0.01). Interestingly, the levels of IL-1Ra in sera and joints after intramuscular injection of IL-1Ra DNA were significantly lower than when protein had been used in previous reports, suggesting that the therapeutic effect may be achieved by an alternative mechanism(s) rather than by systemic elevation of IL-1Ra. These observations provide the first evidence that direct intramuscular injection of expression plasmid for IL-1Ra may effectively suppress the inflammatory pathology in arthritis.

Dehydrodiconiferyl Alcohol Isolated from Cucurbita moschata Shows Anti-adipogenic and Anti-lipogenic Effects in 3T3-L1 Cells and Primary Mouse Embryonic Fibroblasts

Background: A water-soluble extract, prepared from Cucurbita moschata, has potent anti-obesity activities. Results: Dehydrodiconiferyl alcohol (DHCA), isolated from the extract, inhibited the mitotic clonal expansion by suppressing the DNA binding activity of C/EBPβ and directly inhibited the expression of the regulators of lipogenesis in 3T3-L1 and primary embryonic fibroblasts. Conclusion: DHCA may contain the anti-adipogenesis as well as anti-lipogenesis. Significance: DHCA might have potential as an anti-obesity therapeutic. A water-soluble extract from the stems of Cucurbita moschata, code named PG105, was previously found to contain strong anti-obesity activities in a high fat diet-induced obesity mouse model. One of its biological characteristics is that it inhibits 3T3-L1 adipocyte differentiation. To isolate the biologically active compound(s), conventional solvent fractionation was performed, and the various fractions were tested for anti-adipogenic activity using Oil Red O staining method. A single spot on thin layer chromatography of the chloroform fraction showed a potent anti-adipogenic activity. When purified, the structure of its major component was resolved as dehydrodiconiferyl alcohol (DHCA), a lignan, by NMR and mass spectrometry analysis. In 3T3-L1 cells, synthesized DHCA significantly reduced the expression of several adipocyte marker genes, including peroxisome proliferator-activated receptor γ (Pparg), CCAAT/enhancer-binding protein α (Cebpa), fatty acid-binding protein 4 (Fabp4), sterol response element-binding protein-1c (Srebp1c), and stearoyl-coenzyme A desaturase-1 (Scd), and decreased lipid accumulation without affecting cell viability. DHCA also suppressed the mitotic clonal expansion of preadipocytes (an early event of adipogenesis), probably by suppressing the DNA binding activity of C/EBPβ, and lowered the production level of cyclinA and cyclin-dependent kinase 2 (Cdk2), coinciding with the decrease in DNA synthesis and cell division. In addition, DHCA directly inhibited the expression of SREBP-1c and SCD-1. Similar observations were made, using primary mouse embryonic fibroblasts. Taken together, our data indicate that DHCA may contain dual activities, affecting both adipogenesis and lipogenesis.

Highly selective and sensitive detection of neurotransmitters using receptor-modified single-walled carbon nanotube sensors

We present receptor-modified carbon nanotube sensors for the highly selective and sensitive detection of acetylcholine (ACh), one kind of neurotransmitter. Here, we successfully expressed the M1 muscarinic acetylcholine receptor (M1 mAChR), a family of G protein-coupled receptors (GPCRs), in E. coli and coated single-walled carbon nanotube (swCNT)-field effect transistors (FETs) with lipid membrane including the receptor, enabling highly selective and sensitive ACh detection. Using this sensor, we could detect ACh at 100 pM concentration. Moreover, we showed that this sensor could selectively detect ACh among other neurotransmitters. This is the first demonstration of the real-time detection of ACh using specific binding between ACh and M1 mAChR, and it may lead to breakthroughs for various applications such as disease diagnosis and drug screening.

Covalent attachment of biomacromolecules to plasma-patterned and functionalized carbon nanotube-based devices for electrochemical biosensing.

The interface between biomacromolecules and carbon nanotubes (CNTs) is of critical importance in developing effective techniques that provide CNTs with both biomolecular recognition and signal transduction through immobilization. However, the chemical inertness of CNT surfaces poses an obstacle to wider implementation of CNTs in bioanalytical applications. In this paper, we present a review of our recent research activities related to the covalent attachment of biomacromolecules to plasma-patterned and functionalized carbon nanotube films and their application to the fabrication of electrochemical biosensing devices. The SWCNT films were spray-deposited onto a miniaturized three-electrode system on a glass substrate and activated using highly purified atomic oxygen generated in radiofrequency plasma; this introduced oxygen-containing functional groups into the SWCNT surface without fatal loss of the original physicochemical properties of the CNTs. The carboxylated SWCNT electrodes were then selectively modified via amidation or esterification for covalent immobilization of the biomacromolecules. The plasma-treated SWCNT-based sensing electrode had an approximately six times larger effective area than the untreated SWCNT-based electrode, which significantly amplified the amperometric electrochemical signal. Finally, the efficacy of plasma-functionalized SWCNT (pf-SWCNT) as a biointerface was examined by immobilizing glucose oxidase, Legionella pneumophila ( L. pneumophila)-specific antibodies, L. pneumophila-originated DNAs, and thrombin-specific aptamers on the pf-SWCNT-based three-electrode devices. The pf-SWCNT films were found to support direct covalent immobilization of the above-listed biomacromolecules on the films and to thereby overcome the many drawbacks typically associated with simple physisorption. Thus, pf-SWCNT sensing electrodes on which biomacromolecules were covalently immobilized were found to be chemically stable and have a long lifetime.

Reusable Floating-Electrode Sensor for the Quantitative Electrophysiological Monitoring of a Nonadherent Cell

We report a reusable floating-electrode sensor based on aligned semiconducting single-walled carbon nanotubes for the quantitative monitoring of the electrophysiological responses from a nonadherent cell. This method allowed us to monitor and distinguish the real-time responses from normal and small-cell lung cancer (SCLC) cells to the addition of nicotine. The difference was attributed to the overexpressed nicotinic acetylcholine receptors (nAChRs) in the SCLC cells. The sensor was also utilized to monitor the effect of various drugs on the cells. The treatment with inhibitors such as genistin or daidzein was found to reduce Ca(2+) influx in SCLC cells. Moreover, tamoxifen, though known as the antiestrogen compound, was found to only partly block the binding of daidzein to nAChRs. Significantly, the activities of multiple individual cells could be measured repeatedly using a single sensor device, enabling statistically meaningful measurements without errors from the device-to-device variations of the sensor characteristics. This capability of the quantitative monitoring of nonadherent cells should be a major breakthrough for electrophysiology research and various biomedical applications such as drug screening and therapeutic monitoring.

Nanovesicle-based platform for the electrophysiological monitoring of aquaporin-4 and the real-time detection of its antibody

Aquaporin-4 (AQP4) water channel protein transports water molecules across cell membranes bidirectionally and involves in a neurological disorder, neuromyelitis optica (NMO) caused by anti-AQP4 antibodies. Here, we developed a platform based on nanovesicle-carbon nanotube hybrid nanostructures for the real-time detection of anti-AQP4 antibodies and the electrophysiological monitoring of AQP4 activities. Using the hybrid device, we could detect anti-AQP4 antibodies with a high sensitivity and estimate the binding constants under different osmotic conditions. The results show AQP4 had a better affinity to anti-AQP4 antibodies under hyper-osmotic conditions than normal conditions. Furthermore, our device can be utilized to study the real-time cellular responses related with AQP4 such as those to different osmotic stresses. This nanovesicle-based platform can be a simple but versatile tool for basic research about AQP4 and related biomedical applications such as disease diagnostics.

Berchemiosides A–C, 2-Acetoxy-ω-phenylpentaene Fatty Acid Triglycosides from the Unripe Fruits of Berchemia berchemiifolia

Three compounds in a new class of 2-acetoxy-ω-phenylpentaene fatty acid triglycosides, berchemiosides A-C (1-3), and a biosynthetically related phenolic glycoside (4) were isolated from the unripe fruits of Berchemia berchemiifolia, along with three flavonoid 5-O-diglycosides (5-7) and three known flavonoids (8-10). Their chemical structures including absolute configurations were determined by spectroscopic analysis in combination with chemical derivatization. The pentaene group of 1 was found to have (6E,8E,10Z,12Z,14E)-geometry, whereas those of 2 and 3 exhibited all-E geometries. The isolated compounds were examined for their cytotoxicity and xanthine oxidase (XO) inhibitory activity; only compound 7 showed weak XO inhibitory activity.

Targeted Isolation of Neuroprotective Dicoumaroyl Neolignans and Lignans from Sageretia theezans Using in Silico Molecular Network Annotation Propagation-Based Dereplication

The integration of LC-MS/MS molecular networking and in silico MS/MS fragmentation is an emerging method for dereplication of natural products. In the present study, a targeted isolation of natural products using a new in silico-based annotation tool named Network Annotation Propagation (NAP) is described. NAP improves accuracy of in silico fragmentation analyses by reranking candidate structures based on the network topology from MS/MS-based molecular networking. Annotation for the MS/MS spectral network of the Sageratia theezans twig extract was performed using NAP, and most molecular families within the network, including the known triterpenoids 1-7, could be putatively annotated, without relying on any previous reports of molecules from this species. Based on the in silico dereplication results, molecules were prioritized for isolation. In total, six dicoumaroyl 8- O-4 neolignans (8-13) and three dicoumaroyl lignans (14-16) were isolated from the twigs of S. theezans and structurally characterized by spectroscopic analyses. Isolates were evaluated for their neuroprotective activity, and compounds 14-16 showed potent protective effects against glutamate-induced oxidative stress in mouse HT22 cells at a concentration of 12.5 μM.

Biointerfacial Property of Plasma-Treated Single-Walled Carbon Nanotube Film Electrodes for Electrochemical Biosensors

The single-walled carbon nanotube (SWCNT)-based thin film was spray-coated on the Pt support and functionalized using O2 plasma. The effects of plasma treatment on the biointerfacial properties of the SWCNT films were analyzed by cyclic voltammogram (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV). The plasma-functionalized (pf) SWCNT electrodes modified with Legionella pneumophila-specific probe DNA strands showed a much higher peak current and a smaller peak separation in differential pulse voltammetry and a lower charge transfer resistance, compared to the untreated samples. These results suggest that the pf-SWCNT films have a better electrocatalytic character and an electron transfer capability faster than the untreated SWCNTs, due to the fact that the oxygen-containing functional groups promote direct electron transfer in the biointerfacial region of the electrocatalytic activity of redox-active biomolecules.