• Bulletin of the Korean Chemical Society
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• v.34 no.9
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• pp.2725-2730
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• 2013

Single C-reactive protein (CRP) molecules, which are non-specific acute phase markers and products of the innate immune system, were quantitatively detected on a gold-nanopatterned biochip using evanescent field-enhanced fluorescence imaging. The $4{\times}5$ gold-nanopatterned biochip (spot diameter of 500 nm) was fabricated by electron beam nanolithography. Unlabeled CRP molecules in human serum were identified with single-molecule sandwich immunoassay by detecting secondary fluorescence generated by total internal reflection fluorescence (TIRF) microscopy. With decreased standard CRP concentrations, relative fluorescence intensities reduced in the range of 33.3 zM-800 pM. To enhance fluorescence intensities in TIRF images, the distance between biochip surface and CRP molecules was optimally adjusted by considering the quenching effect of gold and the evanescent field intensity. As a result, TIRF only detected one single-CRP molecule on the biochip the first time.

• Fisheries and Aquatic Sciences
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• v.7 no.3
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• pp.99-108
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• 2004

Vitellogenin (VTG) was purified from serum of $estradiol-l7{\beta}-treated rockfish$(Sebastes schlegeli) by precipitation with $EDTA-Mg^{2+}$ and ammonium sulfate and two step chromatography (anion exchange chromatography and gel permeation chromatography) was performed on FPLC system. Rockfish VTG (rfVTG) was characterized and its properties were determined. The monomers have apparent, molecular mass of about 188 kDa as indicated by SDS-PAGE. Amino acid composition analysis of rfVTG was similar to VTG from other oviparous teleosts. Cysteine and lysine were present at relatively high level. Leucine was present at relatively lower level than in other species. The N-terminal amino acid sequence was evaluated to identify rfVTG. Western blot analysis using an antibody against the purified VTG showed that the antibody reacted with both plasma of $estradiol-l7{\beta}-treated rockfish$ treated male and purified VTG, whereas there was no reaction with male serum of the control. An ELISA was developed using monoclonal and polyclonal antibodies against rfVTG. The assay range was 3.2 ng/mL and 1,000 ng/mL and the value of the intra and inter assay variations were within $9.7{\%}$ and $11.2{\%}$, respectively. Recovery rate was $96.8{\%}$. The sandwich ELISA could be useful for the detection of VTG and could be good for screening of estrogenic compounds.

• Bulletin of the Korean Chemical Society
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• v.34 no.11
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• pp.3265-3268
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• 2013

The multi-hydrogen bonded systems with the solvated electrons are investigated at the B3LYP/6-311++$G^{**}$ basis set level. The symmetrical linear geometrical characteristic is common for the dimer systems, while for the tetramer system, the tetrahedron configuration is generated. The NBO charge analyses demonstrate that the multi-hydrogen-multi-electron (mH-ne) coupling exist in these anion systems, as is supported by the electrostatic potential and the molecular orbital analyses. The positive chemical shift value of the central hydrogen ($H_c$) and the negative chemical shift value of the terminal hydrogen ($H_t$) indicate that the $H_c$ is electronegative while the $H_t$ is electropositive, respectively. Strong coupling between two central hydrogen atoms is demonstrated by the large spin-spin coupling constants. The solvated electron donates significant contributions for the stability of these systems.

• Journal of Food Hygiene and Safety
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• v.34 no.2
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• pp.135-139
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• 2019

The objective of this research was to compare loop-mediated isothermal amplification (LAMP) with real-time polymerase chain reaction (PCR) for the rapid detection of pathogens in foods. In this study, the limits of detection (LODs) for Salmonella Typhimurium, Listeria monocytogenes, and Cronobacter sakazakii were evaluated in various foods. Among 11 samples tested for S. Typhimurium, LAMP and real-time PCR had the same LODs in beef and duck meat whereas real-time PCR was more sensitive than the LAMP in 8 samples. However, S. Typhimurium in chocolate samples was not detected by real-time PCR. The sensitivity of real-time PCR was high in all samples inoculated with L. monocytogenes and C. sakazakii whereas LAMP was more sensitive than real-time PCR in oil-rich foods. Therefore, LAMP can be shown as an easrer, more convenient method, as well as effective analytical method for testing difficult samples when employed in PCR.

• KSBB Journal
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• v.18 no.3
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• pp.190-196
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• 2003

An analytical system detecting DNA particularly utilizing a concept of membrane strip chromatography initially applied to home-version tests for, such as, pregnancy and ovulation has been developed. We have chosen S. typhimurium as model analyte among food-contaminating microorganisms that occurred in high frequencies, and invA gene, as a detection target, specific to Salmonella species. This gene was able to be amplified by PCR under optimal conditions employing newly designed primers in our laboratory. The PCR product was specifically measured via hybridization between the analyte and a DNA probe, which was a totally different feature from the conventional gel electrophoresis detecting the products based only on the molecular size. It is notable thar the DNA probe sequence was specially designed such that no separation of excess primers present after PCR was required. This was immobilized on a nitrocellulose (NC) membrane via streptavidin-biotin linkage minimizing a steric effect when the hybridization with the amplified DNA took place. The analyrical system detected the microorganism in a concentration of minimum $10^3$ cfu/mL (i.e., 10 cells per system), estimated from the standard curve, 20 to 40 minutes after adding the sample. This sneitivity was approximately 10 times higher than that of gel electrophoresis as an analytical tool conventionally used. Furthermore, the assay was able to be run at room temperature, which would ofter an extra advantage to users.

• Journal of Life Science
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• v.9 no.3
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• pp.253-261
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• 1999

Leptin gene, an obesity gene, has been known to involve in the regulation of food intake and body weight. It is also thought to be related to the glucose metabolism, insulin secretion and type II diabetes mellitus. Recently, the production of recombinant leptin protein has been attempted for the application in the treatment of obesity and the correction of hereditary obesity and type II diabetes. In the present study, leptin cDNA was cloned from mouse fat cells by RT-PCR and prokaryotic expression of leptin was attempted in order ot prepare a leptin-specific antigen. Immunization of a rabbit with the leptin-specific antigen into a rabbit resulted in the generation of leptin-specific antiserum that could be useful in the detection of leption expressed in various tissues. The sequence of leptin cDNA prepared in the present study wa identical to the previously reported one. Transformation of E. coli(DH5a) cells with the leptin cDNA-inserted translation vector, pGEX-4T-3-leptin followed by treatment with IPTG (0.1mM) resulted in the expression of a large amount of GST-leptin fusion protein with a molecular weight of 44 KDa as an inclusion body. Denaturation of the insoluble fusion protein by 8M urea, 6M guanidium-HCI or 0.1% 2-mercaptoethanol followed by a slow oxidation could not solubilize the inclusion body. The cell extract was subjected to SDS-PAGE and GST-leptin protein electroeluted from the gel was then injected into a rabbit subcutaneously for the immunization. Anti-GST-leptin rabbit antiserum which had a cross reactivity to the GST-leptin protein was generated. Leptin protein expressed in mouse brain and fat tissues was detected by Western blot immunodetection system using the antiserum generated in the present study.

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.11
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• pp.1610-1616
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• 2016

This study aimed to establish an optimal extraction process and high performance liquid chromatography-ultraviolet (HPLC-UV) analytical method for determination of 3,5-dicaffeoylquinic acid (3,5-DCQA) as a part of materials standardization for the development of a xanthine oxidase inhibitor as a health functional food. The quantitative determination method of 3,5-DCQA as a marker compound was optimized by HPLC analysis using a Luna RP-18 column, and the correlation coefficient for the calibration curve showed good linearity of more than 0.9999 using a gradient eluent of water (1% acetic acid) and methanol as the mobile phase at a flow rate of 1.0 mL/min and a detection wavelength of 320 nm. The HPLC-UV method was applied successfully to quantification of the marker compound (3,5-DCQA) in Aster glehni extracts after validation of the method with linearity, accuracy, and precision. Ethanolic extracts of A. glehni (AGEs) were evaluated by reflux extraction at 70 and $80^{\circ}C$ with 30, 50, 70, and 80% ethanol for 3, 4, 5, and 6 h, respectively. Among AGEs, 70% AGE at $70^{\circ}C$ showed the highest content of 3,5-DCQA of $52.59{\pm}3.45mg/100g$ A. glehni. Furthermore, AGEs were analyzed for their inhibitory activities on uric acid production by the xanthine/xanthine oxidase system. The 70% AGE at $70^{\circ}C$ showed the most potent inhibitory activity with $IC_{50}$ values of $77.01{\pm}3.13{\sim}89.96{\pm}3.08{\mu}g/mL$. The results suggest that standardization of 3,5-DCQA in AGEs using HPLC-UV analysis would be an acceptable method for the development of health functional foods.

• BMB Reports
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• v.40 no.4
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• pp.459-466
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• 2007

The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7$\cdot}$Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.

• BMB Reports
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• v.28 no.4
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• pp.293-300
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• 1995

The radical centers detected in the reaction of metmyoglobin (MetMb) with hydrogen peroxide ($H_2O_2$) have been studied by using a spin trapping technique. A broad 5-line asymmetric electron spin resonance (ESR) spectrum, with $2A_{max}=4.07\;mT$ and $2A_{min}=2.97\;mT$, obtained after incubation of MetMb with $H_2O_2$ in the presence of a spin trap, 5,5-dimethyl pyrroline-N-oxide (DMPO) was gradually weakened with time and disappeared completely by 6 min after addition of guanidine-HCl (14 M). When a higher concentration (6 M) of the agent was added, the signal disappeared within 40 see and the DMPO/OH signal appeared immediately. Then, a new 8-line signal with similar intensities grew gradually and was fixed by 45 min, coexisting with the DMPO/OH signal. This new signal was found to be composite, consisting of two different radical species. One of the 6-line signals, with $a_N$ 1.49 mT and $a_H$ 0.988 mT, was assigned to the DMPO/phenoxyl radical adduct. The second 6-line signal with $a_N$ 1.55 mT and $a_H$ 2.22 mT was assigned to carbon-centered radical adduct. When 3,3,5,5-tetramethylpyrrolin-N-oxide (TMPO), was employed in the place of DMPO, another broad asymmetric 5-line signal was detected with $2A_{max}=3.99\;mT$ and $2A_{min}=3.04\;mT$, which is virtually identical to that obtained from the DMPO system The shape of the spectrum of the TMPO adduct changed drastically, with lapse of time resulting in a broad singlet after 40 min. The broad singlet was assigned to the porphyrin radical adduct. Incubation of globin with Fenton reagent in the presence of DMPO initially gave a DMPO/OH signal. Then, a new 12-line signal began to grow after one minute and fixed after 15 min. coexisting with the DMPO/OH signal, This 12-line signal was assigned to DMPO/phenoxyl with $a_N$ 1.47 mT, $a_{{\beta}H}$ 0.99 mT and $a_{{\gamma}H}$ 0.13 mT. A minor concentration of carbon-centered radical adduct was also detected. This radical composition is identical to that of guanidine HCl treated MetMb/DMPO/$H_2O_2$ system, indicating that the radical producing conditions are somehow common in both systems. Heme iron can be released by excess $H_2O_2$ in the MetMb/$H_2O_2$ system, providing for Fenton reagent. When MetMb was pretreated with tyrosine blocking agent, $KI_3$ the broad 5.line MetMb-derived signal was not detected in the MetMb/DMPO/$H_2O_2$ system, whereas no such effect was detected on such system of Hb in which the radical center was assigned to cysteine residue not tyrosine, indicating that tyrosine residue is a main radical center produced in the MetMb/$H_2O_2$ system Thus, the present data strongly support the previous indication that the apomyoglobin-derived radical center formed in the reaction of MetMb with $H_2O_2$ is a tyrosine residue.

• The Bulletin of The Korean Astronomical Society
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• v.46 no.2
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• pp.38.2-39
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• 2021

I will presents the analysis of the gas properties of the protoplanetary disk surrounding the young low-mass (about 1.2M_sun) triple star, GG Tau A. This work makes use of ALMA observations of rotational lines of CO (¹²CO, ¹³CO and C¹⁸O) together NOEMA observations of a few dozens of other molecules. While the CO emission gives information on the molecular layer close to the disk atmosphere, its less abundant isotopologues ¹³CO and C¹⁸O bring information much deeper in the molecular layer. I will present the analysis of the morphology and kinematics of the gas disk using the CO isotopologues. A radiative transfer model of the ring in CO isotopologues will also be presented. The subtraction of this model from the original data reveals the weak emission of the molecular gas lying inside the cavity. Thus, I am able to evaluate the properties of the gas inside the cavity, such as the gas dynamics, excitation conditions, and the amount of mass in the cavity. High angular resolution observations of CO reveals sprials induced by embedded planet(s) located near the 3:2:1 mean-motion resonance that help to explain the special morphology of the circumbinary disk. I also discuss some chemical properties of the GG Tau A disk. I report the first detection of H2S and C₂S in a protoplanetary disk. The molecule abundance relative to ¹³CO of about twenties other molecules will also be given. In GG Tau A, the detections of rare molecules such as H₂S and C₂S have been probably possible because the disk is more massive (a factor about 3-5) than other disks where the molecules was searched. Such a large disk mass makes the system suitable to detect rare molecules and to study cold-chemistry in protoplanetary disks.