• The Journal of Korean Medicine
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• v.26 no.4
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• pp.12-21
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• 2005

Objectives: In the present study, we investigated whether an aqueous extract of Armeniacae semen induces apoptotic neuronal cell death upon mouse N2a neuroblastoma cells. Methods: 1. Cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTI) assay. 2. For in situ detection of apoptotic cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, 4,6-diamidino-2-phenylindole (DAPI) staining. 3. The fraction of cells was revealed by flow cytometric analysis used that. 4. For detection of apoptotic DNA cleavage, DNA fragmentation assay was performed. 5. For detection of bax and bcl-2, Western blot analysis was performed. 6. Caspase enzyme activity was measured using caspase-3 assay. Results: From the present results, N2a neuroblastoma cells treated with Armeniacae semen extract exhibited several characteristics of apoptosis. A treatment of Armeniacae semen extract was shown to increase the expression of Bax, a proapoptotic protein, and the treatment decreased the expression of Blc2, an anti-apoptotic protein. In addition, Armeniacae semen extract increased the caspase-3 enzyme activity. Conclusions: The present results show that Armeniacae semen extract induces apoptotic cell death in mouse N2a neuroblastoma cells.

• Korean Journal of Microbiology
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• v.21 no.2
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• pp.61-70
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• 1983

Synchronized cat kidney cells chronically infected with feline leukemia virus (FeLV) were used to study virus production, the synthesis of group specific antigen (gag) and envelope (env) proteins, the expression of env protein on the cell surface during the cell cycle, and the stability of viral RNA. As detecting method, we developed the radioimmunoassay (RIA) system using beta-emission of $^{131}I$ and demonstrated the validity of this system by comparison with routine RIA system using gamma-emission of $^{125}I$. The produced virus was analysed by developed RIA interval was determined by measuring reverse transcriptase activity. The results show that infected cells produce the complete virus particle containing products of gag, env and pol genes of FeLV, and maximum virus production occurs during mitosis of synchronized cells. Labeling of the cell surface of synchronized cells with $^{131}I$ shows that the amount of $gp70^{env}$ on the cell surface parallels cellular gorwth. Therefore, the cell cycle-dependent release of virus is not petition RIA of synchronized cells with $^{131}I$ labeled viral proteins synthesis during the cell cycle. The rate of synthesis of gag protein shows three peaks, corresponding to the $G_1,\;late\;S\;and\;late\;G_2$ phases of cell cycle. But the rate of synthesis of env protein dose not change, suggesting that in these cells the synthesis of these two gene products in controlled seperately. In Actionomycin D treated cells, the synthesis of viral proteins decreased sharply from 8 hours after treatment, and the late S and $G_2$ peaks of gag protein synthesis were disappeared. This shows the stability of viral RNA for about 6 hours in the absence of continuing viral RNA synthesis.

• Proceedings of the Korean Society of Near Infrared Spectroscopy Conference
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• 2001.06a
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• pp.1511-1511
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• 2001

Energy content, expressed as calories per gram, is an important part of the evaluation and marketing of foods in developed countries. Currently accepted methods of measurement of energy by U.S. food labeling legislation include measurement of gross calories by bomb calorimetry with an adjustment for undigested protein and by calculation using specific factors for the energy values of protein, carbohydrate less the amount of insoluble dietary fiber, and total fat. The ability of NIRS to predict the energy value of diverse, processed and unprocessed cereal food products was investigated. NIR spectra of cereal products were obtained with an NIR Systems monochromator and the wavelength range used for analysis was 1104-2494 nm. Gross energy of the foods was measured by oxygen bomb calorimetry (Parr Manual No. 120) and expressed as calories per gram (CPGI, range 4.05-5.49 cal/g). Energy value was adjusted for undigested protein (CPG2, range 3.99-5.38 cal/g) and undigested protein and insoluble dietary fiber (CPG3, range 2.42-5.35 cal/g). Using a multivariate analysis software package (ISI International, Inc.) partial least squares models were developed for the prediction of energy content. The standard error of cross validation and multiple coefficient of determination for CPGI using modified partial least squares regression (n=127) was 0.060 cal/g and 0.95, respectively, and the standard error of performance, coefficient of determination, bias and slope using an independent validation set (n=59) were 0.057 cal/g, 0.98, -0.027 cal/g and 1.05 respectively. The PLS loading for factor 1 (Pearson correlation coefficient 0.92) had significant absorption peaks correlated to C-H stretch groups in lipid at 1722/1764 nm and 2304/2346 nm and O-H groups in carbohydrate at 1434 and 2076 nm. Thus the model appeared to be predominantly influenced by lipid and carbohydrate. Models for CPG2 and CPG3 showed similar trends with standard errors of performance, using the independent validation set, of 0.058 and 0.088 cal/g, respectively, and coefficients of determination of 0.96. Thus NIRS provides a rapid and efficient method of predicting energy content of diverse cereal foods.

• Molecules and Cells
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• v.37 no.8
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• pp.613-619
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• 2014

The optic nerve often suffers regenerative failure after injury, leading to serious visual impairment such as glaucoma. The main inhibitory factors, including Nogo-A, oligodendrocyte myelin glycoprotein, and myelin-associated glycoprotein, exert their inhibitory effects on axonal growth through the same receptor, the Nogo-66 receptor (NgR). Oncomodulin (OM), a calcium-binding protein with a molecular weight of an ~12 kDa, which is secreted from activated macrophages, has been demonstrated to have high and specific affinity for retinal ganglion cells (RGC) and promote greater axonal regeneration than other known polypeptide growth factors. Protamine has been reported to effectively deliver small interference RNA (siRNA) into cells. Accordingly, a fusion protein of OM and truncated protamine (tp) may be used as a vehicle for the delivery of NgR siRNA into RGC for gene therapy. To test this hypothesis, we constructed OM and tp fusion protein (OM/tp) expression vectors. Using the indirect immunofluorescence labeling method, OM/tp fusion proteins were found to have a high affinity for RGC. The gel shift assay showed that the OM/tp fusion proteins retained the capacity to bind to DNA. Using OM/tp fusion proteins as a delivery tool, the siRNA of NgR was effectively transfected into cells and significantly down-regulated NgR expression levels. More importantly, OM/tp-NgR siRNA dramatically promoted axonal growth of RGC compared with the application of OM/tp recombinant protein or NgR siRNA alone in vitro. In addition, OM/tp-NgR siRNA highly elevated intracellular cyclic adenosine monophosphate (cAMP) levels and inhibited activation of the Ras homolog gene family, member A (RhoA). Taken together, our data demonstrated that the recombinant OM/tp fusion proteins retained the functions of both OM and tp, and that OM/tp-NgR siRNA might potentially be used for the treatment of optic nerve injury.

• Archives of Pharmacal Research
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• v.21 no.1
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• pp.46-53
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• 1998

When NIH3T3 cells were exposed to mild heat and recovered at $37^{\circ}C$ for various time intervals, they were thermotolerant and resistant to subsequent stresses including heat, oxidative stresses, and antitumor drug methotrexate which are apoptotic inducers. The induction kinetics of apoptosis by stresses were determined by DNA fragmentation and protein synthesis using $[35^S]$methionine pulse labeling. We investigated the hypothesis that thermotolerant cells were resistant to apoptotic cell death compared to control cells when both cells were exposed to various stresses inducing apoptosis. The cellular changes in thermotolerant cells were examined to determine which components are involved in this resistance. At first, the degree of resistance correlates with the extent of heat shock protein synthesis which were varied depending on the heating times at $45^{\circ}C$ and recovery times at $37^{\circ}C$after heat shock. Secondly, membrane permeability change was observed in thermotolerant cells. When cells prelabeled with $[^{3}H]$thymidine were exposed to various amounts of heat and recovered at $37^{\circ}C$ for 1/2 to 24 h, the permeability of cytosolic $[^{3}H]$thymidine in thermotolerant cells was 4 fold higher than that in control cells. Thirdly, the protein synthesis rates in thermotolerant and control cells were measured after exposing the cells to the same extent of stress. It turned out that thermotolerant cells were less damaged to same amount of stress than control cells, although the recovery rates are very similar to each other. These results demonstrate that an increase of heat shock proteins and membrane changes in thermotolerant cells may protect the cells from the stresses and increase the resistance to apoptotic cell death, even though the exact mechanism should be further studied.

• Journal of Radiopharmaceuticals and Molecular Probes
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• v.5 no.1
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• pp.11-17
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• 2019

Recently, antibody-like scaffold proteins have received a great deal of interest in diagnosis and therapy applications because of their intrinsic features that are often required for tumor imaging and therapy. Intrinsic issues that are associated with therapeutic application of antibody-like scaffold proteins, particularly in cancer treatment, include an efficient and straightforward radiolabeling for understanding in vivo biodistribution and excretion route, and monitoring therapeutic responses. Herein, we report an efficient and straightforward method for radiolabeling of antibody-like scaffold proteins with the $[^{99m}Tc(OH_2)_3(CO)_3]^+$ ($^{99m}Tc$-tricarbonyl) by using a site-specific direct labeling method via hexahistidine-tag, which is a widely used for general purification of recombinant proteins with His-affinity chromatography. Repebody is a new class of antibody-like scaffold protein that consists of highly diverse leucine-rich repeat (LRR) modules. Although all possible biomedical applications with repebody are ongoing, it's in vivo biodistribution and excretion pathway has not yet been explored. In this study, hexahistidine ($His_6$)-tag bearing repebody (rEgH9) was labeled with [$^{99m}Tc$]-tricarbonyl. Repebody protein was radiolabeled with high radiolabeling efficiency (>90%) and radiolabeled compound was more than 99% pure after purification. These results clearly demonstrate that the present radiolabeling method will be useful molecular imaging study.

• Biomedical Science Letters
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• v.29 no.4
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• pp.211-219
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• 2023

Caffeic acid phenethyl ester (CAPE) is an active component of propolis obtained from honeybee hives. CAPE possesses anti-mitogenic, anti-carcinogenic, anti-inflammatory, and immunomodulatory activities in diverse systems, which know as displays antioxidant activity and inhibits lipoxygenase activities, protein tyrosine kinase, and nuclear factor kappa B (NF-κB) activation. This study aimed to investigate the effect of CAPE on lipopolysaccharide (LPS)-induced human neutrophil phagocytosis. Human neutrophils were cultured with various concentrations of CAPE (1, 10, and 100 µM) with or without LPS. The pro-inflammatory proteins (tumor necrosis factor-alpha [TNF-α], interleukin [IL]-6 and IL-8) levels were measured after 4 h incubation. To investigate the intracellular signaling pathway, we measured the levels of mitogen-activated protein kinases (MAPK), including phosphorylation of p38, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Next, to evaluate the potential phagocytosis, neutrophils were labeled with iron particles of superparamagnetic iron oxide nanoparticles (SPIONs, 40 nm) for 1 h in culture medium containing 5 mg/mL of iron. The labeling efficiency was determined by Prussian blue staining for intracellular iron and 3T-wighted magnetic resonance imaging. CAPE decreased the activation of intracellular signaling pathways, including ERK1/2 and c-Jun, and expression of pro-inflammatory cytokines, including TNF-α and IL-6, but had no effect on the signaling pathways of p38 and cytokine IL-8. Furthermore, images obtained after mannan-coated SPION treatment suggested that CAPE induced significantly higher signal intensities than the control or LPS group. Together, these results suggest that CAPE regulates LPS-mediated activation of human neutrophils to reduce phagocytosis.

• Analytical Science and Technology
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• v.27 no.3
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• pp.121-139
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• 2014

In this review, we will discuss aptamer technologies including in vitro selection, signal transduction mechanisms, and designing aptamers and aptazyme for label-free biosensors and catalysts. Dye-displacement, a typical label-less method, is described here which allows avoiding relatively complex labeling steps and extending this application to any aptamers without specific conformational changes, in a more simple, sensitive and cost effective way. We will also describe most recent and advanced technologies of signaling aptamer and aptazyme for the various analytical and clinical applications. Quantum dot biosensor (QDB) is explained in detail covering designing and adaptations for multiplexed protein detection. Application to aptamer array utilizing self-assembled signaling aptamer DNA tile and the novel methods that can directly select smart aptamer or aptazyme experimentally and computationally will also be finally discussed, respectively.

• BMB Reports
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• v.30 no.2
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• pp.157-161
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• 1997

The present paper reports characteristics and specificity of the inhibitory action of $N^{\alpha}-tosyl-L-lysine-chloromethyl\;ketone$ (TLCK) and $N^{\alpha}-tosyl-L-phenylalanine-chloromethyl\;ketone$ (TPCK) on the glucose6-phosphate transporter of rat liver microsomes. The TLCK-induced inhibition was pH dependent. The inhibition constants for TPCK were determined by following pseudo-Lst order reaction mechanism. The inhibition was protected by preincubation with excess amount of glucose-6-phosphate. The results proved that (a) TLCK inactivates the microsomal glucose-6-phosphate transporter, (b) the inhibition results from the modification of sulfhydryl groups of the transporter.

• Journal of Nutrition and Health
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• v.33 no.7
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• pp.763-773
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• 2000

On the basis of the concept retained in nutition label(NL) the consumer's perception use and demand on NL nutrition knowledge(NH) purchase of nutrient controlled food and dietary modification for health were examined. The subjects were 1203 house wives mainly in the age of 30-40 and self administered questionnaire was employed. The results were as follows. Subjects' demand on nutrition information was greater while the availablity and usefulness of NL was unsatisfactory. The purchase frequencies of nutrient controlled foods were higher compared to NL reading. The use comprehenison reliability of nutrition information were better in high NK group compared to low NK group except the reliability on health claims. The required nutrients for content information were in the order of calorie Ca cholesterol Fe protein and total fat. The demand for nutrient content information was carrelated with intention of subjects' dietary modification but not the use of NL. These results indicate that NL be a good source of nutrition information and the consumers' demand for NL was based on their dietary purpose though the use of NL was unconfirmed.