• Korean Chemical Engineering Research
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• v.59 no.1
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• pp.100-105
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• 2021

This paper proposes the polydimethylsiloxane (PDMS) blade coating method for fabrication of paper-based analytical device (PAD) that is able to monitor the disease diagnosis and progress without special analytical equipment. The mold that has PAD design is easily modified by using laser cutting technique. And the fabricated mold is used for hydrophobic barrier formation by blade coating. We have optimized the stable formation of PDMS hydrophobic barrier as blade coating condition, which is established by analyzing the structure of the PDMS hydrophobic barrier and change of hydrophilic channel size as thickness of the ink and contact time with the chromatography paper. Based on optimal condition, we demonstrate that PAD as biosensor can apply to detect protein, glucose, and metal ion without special analysis equipment.

• KSBB Journal
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• v.22 no.6
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• pp.443-446
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• 2007

A prognostic indicator of coronary heart disease, C-reactive protein, was tried to be determined by a batch-type quartz crystal microbalance immunosensor. The sensor was operated by direct-binding mode and the optimum concentration for the corresponding antibody for immobilization was $50{\mu}g/ml$. The reaction buffer for the system was 0.1 M sodium phosphate (pH 7.0) and system operation was performed in the order of baseline stabilization, analyte addition and measurement, and regeneration of the sensor chip with 10 mM NaOH. When plotted in double-logarithmic scale, the sensor showed a linear detection range of 0.27-106.00 nM for rat C-reactive protein with the limit of detection of 0.53 nM. It also showed a good reusability.

• Journal of Life Science
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• v.26 no.7
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• pp.826-834
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• 2016

The present investigation was undertaken to examine the effectiveness of the combination treatment of an Hsp90 inhibitor and a SIRT1 inhibitor on suppressing the growth of chemo-resistant human cancer cells. We showed that inhibition of SIRT1 effectively potentiated the cytotoxicity of 17-allylamino-17-demethoxygeldanamycin (17-AAG) and reversed Hsp90 inhibitor resistance in multidrug-resistant (MDR) human ovarian HeyA8-MDR cells. Amurensin G, a potent natural SIRT1 inhibitor, enhanced Hsp90 inhibitor-mediated abrogation of the Hsp90 chaperone function and accelerated degradation of mutated p53 (mut p53), an Hsp90 client protein, by up-regulation of ubiquitin ligase CHIP. Knock-down of CHIP significantly attenuated amurensin G-induced mut p53 degradation. Down-regulation of mut p53 reduced the expression of heat shock factor1 (HSF1)/heat shock proteins (Hsps), a major cause of Hsp90 inhibitor resistance, which led to sensitization of the MDR cells to the Hsp90 inhibitor by the SIRT1 inhibitor. Amurensin G potentiated cytotoxicity of the Hsp90 inhibitor in HeyA8-MDR cells through suppression of 17-AAG-induced Hsp70 and Hsp27 induction via down-regulation of mut p53/HSF1, and it caused activation of PARP and inhibition of Bcl-2. Our data suggests that SIRT1 inhibitors could be used to sensitize MDR cells to Hsp90 inhibitors, possibly through suppression of the mut p53/HSF1-dependent pathway, and a novel mut p53-directed action of SIRT1 inhibition could effectively prevent mut p53 accumulation in MDR cells.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.2
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• pp.173-177
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• 2006

In this study, a novel strategy was developed for the highly selective immobilization of proteins, using the polyhydroxyalkanoate (PHA) depolymerase substrate binding domain (SBD) as an active binding domain. In order to determine the appropriacy of this method for immunodiagnostic assays, the single-chain antibody (ScFv) against the hepatitis B virus (HBV) preS2 surface protein and the severe acute respiratory syndrome coronavirus (SARS-CoV) envelope protein (SCVe) were fused to the SBD, then directly immobilized on PH A-coated slides via microspotting. The fluorescence-labeled HBV antigen and the antibody against SCVe were then utilized to examine specific interactions on the PHA-coated surfaces. Fluorescence signals were detected only at the spotted positions, thereby indicating a high degree of affinity and selectivity for their corresponding antigens/antibodies. Furthermore, we detected small amounts of ScFv-SBD (2.7 ng/mL) and SCVe-SBD fusion proteins (0.6ng/mL). Therefore, this microarray platform technology, using PHA and SBD, appears generally appropriate for immunodiagnosis, with no special requirements with regard to synthetic or chemical modification of the biomolecules or the solid surface.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.6
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• pp.1393-1403
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• 2003

The genetic effects of restraint stress challenge on HPA axis and the therapeutic effect of Boshimgeonbi-Tang on the stress were studied with cDNA microarray analyses on hypothalamus using an immobilization-stress mouse as stress model. Male CD-1 mice were restrained in a tightly fitted and ventilated vinyl holder for 2hours once a day, and this challenge was repeated for seven consecutive days. The body weights of the immobilization-stress mice were diminished about 25 percent degree as compared to normal ones. Seven days later, total RNA was extracted from the organs of the mouse, body-labeled with CyDye/sup TM/ fluorescence dyes (Amersham Bioscience Co., NJ), and then hybridized to cDNA microarray chip. Scanning and analyzing the array slides were carried out using GenePix 4000 series scanner and GenePix Pro/sup TM/ analyzing program, respectively. The expression profiles of 109 genes out of 6000 genes on the chip were significantly modulated in hypothalamus by the immobilization stress. Energy metabolism-, lipid metabolism-, apoptosis- and signal transduction-related genes were transcriptionally activated whereas DNA repair-, protein biosynthesis-, and structure integrity-related genes were down-regulated in hypothalamus. The 58 genes were up-regulated by the mRNA expression folds of 1.5 to 7.9. and the 51 genes were down-regulated by 1.5 - 3.5 fold. The 20 genes among them were selected to confirm the expression profiles by RT-PCR. The mRNA expression levels of Tnfrsf1a (apoptosis), Calm2 (cell cycle), Bag3 (apoptosis), Hspe1 (protein folding), Aatk (apoptosis), Dffa (apoptosis), Itgb1 (cell adhesion), Vcam1 (cell adhesion), Fkbp5 (protein folding), BDNF (neuron survival) were restored to the normal one by the treatment of Boshimgeonbi-Tang.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.02a
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• pp.110-111
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• 2013

FcBP consisting of 13 amino acids specifically binds to Immunoglobulin G Fc domain. Initially, we utilized this peptide for preparation of antibody chip as a PEG composite for enhanced solubility. After then, the peptide conjugate was immobilized on agarose resin, resulting in highly efficient affinity column for antibody purification. The efficiency was comparable to commercial Protein A column. Recently, this peptide was conjugated with cell penetrating peptide (CPP) on a backbone of GFP, affording antibody transducer, which carries antibody into live cells by simple mixing of antibody and the transducer in cell culture media. Antibody transduction into cells was monitored by live cell imaging. More recently, the FcBP was fused to ferritin cage, which consists of 24 ferritin protein molecules. The FcBP-ferritin cage showed greatly increased binding affinity to human IgG. Its binding was analyzed by QCM and SPR analysis. Finally, it was selectively delivered by Herceptin to SKBR3, a breast cancer cell, over MCF10A, non-tumorigenic cells (Fig. 1). Fig. 1. Fluorescent microscopic images of SKBR3 breast cancer cells (A~C) and MCF10A breast cells (D~F) treated with Cy3-trastuzumab/fFcBP-Pf_Fn complexes. Trastuzumab and FcBP-Pf_Fn, which were labeled with Cy3 (Cy3-trastuzumab) and fluorescein (fFcBP-Pf_Fn), respectively, selectively targeted SKBR3 over MCF10A.

• Proceedings of the KIEE Conference
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• 2002.11a
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• pp.60-63
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• 2002

This paper presents bead-based microbiocihps to detect and separate target proteins. Micro beads coated with capture proteins were introduced into a microchamber, and target proteins flowing across the chamber were bound and concentrated. The chip was connected with an external fluid system. Bead surfaces were double-coated with photo-cleavable linkers and capture proteins. The proteins bound on the beads were photo-separated under UV irradiation, and excited to be measured in fluorescence. $38{\sim}50{\mu}m$ sized polystyrene beads were used. SOGs(silicon-on-glass) were used to fabricate the microchip having glasses bonded on both sides. 100 ${\mu}m$ thick silicon channel was formed through silicon deep RIE process. The upper glass cover had holed through to have inlets and outlets fabricated by powder-blastings. In this study, biotin and streptavidin were used as capture proteins and detection proteins, respectively. The protein mixtures of streptavidin, HSA(human serum albumin) and ovalbumin were applied for selective detection test.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.2
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• pp.529-541
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• 2003

Yukmijihwang-tang has been widely used as an and-aging herbal medicine for hundred years in Asian countries. Numerous studies show that Yukmijihwangtang has anti-oxidative effect both in vivo and in vitro. It has been reported that Moutan Cortex Radicis extract (MCR) was the most effective herb in Yukmijihwang-tang on undifferentiated PC12 cells upon oxidative-stressed with hydrogen peroxide. The purpose of this study is to; 1) evaluate the recovery of neuronal damage by assessing the anti-oxidant effect of MCR on PC12 cells differentiated with nerve growth factor (NGF), 2) identify candidate genes responsible for anti-oxidative effect on differentiated PC12 cells by oligonucleotide chip microarray. PC12 cells, which were differentiated by treating with NGF, were treated without or with hydrogen peroxide in the presence or absence of various concentration of MCR. Cell survival was determined by using MTS assay. Measurement of intracellular reactive oxygen species (ROS) generation was determined using the H2DCFDA assay The viability of cells treated with MCR was significantly recovered from stressed PC12 cell. In addition, wide rage of concentrations of MCR shows dose-dependent inhibitory effect on ROS production in oxidative-stressed cells. Total RNAs of cells without treatment(Control group), only treated with H₂O₂ (stressed group) and treated with both H₂O₂ and of MCR (MCR group) were isolated, and cDNAs was synthesized using oligoT7(dT) primer. The fragmented cRNAs, synthesized from cDNAs, were applied to Affymetrix GeneChip Rat Neurobiology U34 Array. mRNA of Calcium/calmodulin-dependent protein kinase II delta subunit(CaMKII), neuron glucose transporter (GLUT3) and myelin/oligodendrocyte glycoprotein(MOG) were downregulated in Stressed group comparing to Control group. P2X2-5 receptor (P2X2R-5), P2X2-4 receptor (P2X2R-4), c-fos, 25 kDa synaptosomal attachment protein(SNAP-25a) and GLUT3 were downregulated, whereas A2 adenosine receptor (A2AR), cathechol-O-methyltransferase(COMT), glucose transporter 1 (GLUT1), EST223333, heme oxygenase (HO), VGF, UI-R-CO-ja-a-07-0-Ul.s1 and macrophage migration inhibitory factor (MIF) were upregulated in MCA group comparing to Control group. Expression of Putative potassium channel subunit protein (ACK4), P2X2A-5, P2X2A-4, Interferon-gamma inducing factor isoform alpha precursor (IL-18α), EST199031, P2XR, P2X2 purinoceptor isoform e (P2X2R-e), Precursor interleukin 18 (IL-18) were downregulated, whereas MOO, EST223333, GLUT-1, MIF, Neuronatin alpha, UI-R-C0-ja-a-07-0-Ul.s1, A2. adenosine receptor, COMT, neuron-specific enolase (NSE), HO, VGF, A rat novel protein which is expressed with nerve injury (E12625) were upregulated in MCR group comparing to Stressed group. The results suggest that decreased viability and AOS production of PC12 cell by H₂O₂ may be, at lease, mediated by impaired glucose transporter expression. It is implicated that the MCR treatment protect PC12 cell from oxidative stress via following mechanisms; improving glucose transport into the cell, enhancing expression of anti-oxidative genes and protecting from dopamine cytotoxicity by increment of COMT and MIF expression. The list of differentially expressed genes may implicate further insight on the action and mechanism behind the anti-oxidative effects of herbal extract Moutan Cortex Radicis.

• KSBB Journal
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• v.22 no.6
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• pp.371-377
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• 2007

Polyhydroxyalkanoates (PHAs) are a family of microbial polyesters that can be produced by fermentation from renewable resources. PHAs can be used as completely biodegradable plastics or elastomers. In this paper, novel applications of PHAs in biosensor are described. A general platform technology was developed by using the substrate binding domain (SBD) of PHA depolymerase as a fusion partner to immobilize proteins of interest on PHA surface. It could be shown that the proteins fused to the SBD of PHA depolymerase could be specifically immobilized onto PHA film, PHA microbead, and microcontact printed PHA surface. We review the results obtained for monitoring the specific interaction between the SBO and PHA by using enhanced green fluorescent protein, red fluorescent protein, single chain antibody against hepatitis B virus preS2 surface protein and severe acute respiratory syndrome coronavirus surface antigen as model proteins. Thus, this system can be efficiently used for studying protein-protein and possibly protein-biomolecule interactions for various biotechnological applications.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.10
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• pp.1400-1410
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• 2004

Eight crossbred (75% Holstein Friesian) cows in mid-lactation were randomly assigned to a switchback design with a 2x2 factorial arrangement to evaluate two nonstructural carbohydrate (NSC) sources (corn meal and cassava chips) with different rumen degradability and used at two levels of NSC (55 vs. 75%) with protein source (supplied by urea in the concentrate mix). The treatments were 1) Low degradable low level of corn (55%) 2) Low degradable high level of corn (75%) 3) High degradable low level of cassava (55%) and 4) High degradable high level of cassava (75%). The cows were offered the treatment concentrate at a ratio to milk yield at 1:2. Urea-treated rice straw was offered ad libitum as the roughage and supplement with 1 kg/hd/d cassava hay. The results revealed that total DM intake, BW and digestion coefficients of DM were not affected by either level or source of energy. Rumen fermentation parameters; NH3-N, blood urea nitrogen and milk urea nitrogen were unaffected by source of energy, but were dramatically increased by level of NSC. Rumen microorganism populations were not affected (p>0.05) by source of energy, but fungal zoospores were greater for cassava-based concentrate than corn-based concentrate. Milk production and milk composition were not affected significantly by diets containing either source or level of NSC, however concentrate than corn-based concentrate averaging (4.4 and 4.2, respectively). Likewise, income over feed, as estimated from 3.5% FCM, was higher on cassava-based concentrate than corn-based concentrate averaging (54.0 and 51.4 US$/mo, respectively). These results indicate that feeding diets containing either cassava-based diets and/or a higher of oncentrates up to 75% of DM with NPN (supplied by urea up to 4.5% of DM) can be used in dairy rations without altering rumen ecology or animal performance compared with corn-based concentrate.