• The Korean Journal of Physiology and Pharmacology
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• v.19 no.3
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• pp.219-228
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• 2015

Excessive microglial activation and subsequent neuroinflammation lead to synaptic loss and dysfunction as well as neuronal cell death, which are involved in the pathogenesis and progression of several neurodegenerative diseases. Thus, the regulation of microglial activation has been evaluated as effective therapeutic strategies. Although dieckol (DEK), one of the phlorotannins isolated from marine brown alga Ecklonia cava, has been previously reported to inhibit microglial activation, the molecular mechanism is still unclear. Therefore, we investigated here molecular mechanism of DEK via extracellular signal-regulated kinase (ERK), Akt and nicotinamide adenine dinuclelotide phosphate (NADPH) oxidase-mediated pathways. In addition, the neuroprotective mechanism of DEK was investigated in microglia-mediated neurotoxicity models such as neuron-microglia co-culture and microglial conditioned media system. Our results demonstrated that treatment of anti-oxidant DEK potently suppressed phosphorylation of ERK in lipopolysaccharide (LPS, $1{\mu}g/ml$)-stimulated BV-2 microglia. In addition, DEK markedly attenuated Akt phosphorylation and increased expression of $gp91^{phox}$, which is the catalytic component of NADPH oxidase complex responsible for microglial reactive oxygen species (ROS) generation. Finally, DEK significantly attenuated neuronal cell death that is induced by treatment of microglial conditioned media containing neurotoxic secretary molecules. These neuroprotective effects of DEK were also confirmed in a neuron-microglia co-culture system using enhanced green fluorescent protein (EGFP)-transfected B35 neuroblastoma cell line. Taken together, these results suggest that DEK suppresses excessive microglial activation and microglia-mediated neuronal cell death via downregulation of ERK, Akt and NADPH oxidase-mediated pathways.

• Journal of Life Science
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• v.20 no.5
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• pp.683-690
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• 2010

The yeast strains of Saccharomyces diastaticus produce one of three isozymes of an extracellular glucoamylase I, II or III, a type of exo-enzyme which can hydrolyse starch to generate glucose molecules from non-reducing ends. These enzymes are encoded by the STA1, STA2 and STA3 genes. Another gene, sporulation-specific glucoamylase (SGA), also exists in the genus Saccharomyces which is very homologous to the STA genes. The SGA has been known to be produced in the cytosol during sporulation. However, we hypothesized that the SGA is capable of being secreted to the extracellular region because of about 20 hydrophobic amino acid residues at the N-terminus which can function as a signal peptide. We expressed the cloned SGA gene in S. diastaticus YIY345. In order to compare the biochemical properties of the extracellular glucoamylase and the SGA, the SGA was purified from the culture supernatant through ammonium sulfate precipitation, DEAE-Sephadex A-50, CM-Sephadex C-50 and Sephadex G-200 chromatography. The molecular weight of the intact SGA was estimated to be about 130 kDa by gel filtration chromatography with high performance liquid chromatography (HPLC) column. Sodium dedecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed it was composed of two heterogeneous subunits, 63 kDa and 68 kDa. The deglycosylation of the SGA generated a new 59 kDa band on the SDS-PAGE analysis, indicating that two subunits are glycosylated but the extent of glycosylation is different between them. The optimum pH and temperature of the SGA were 5.5 and $45^{\circ}C$, respectively, whereas those for the extracellular glucoamylase were 5.0 and $50^{\circ}C$. The SGA were more sensitive to heat and SDS than the extracellular glucoamylase.

• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1629-1635
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• 2012

Previously, we demonstrated that the erythropoietin receptor (EpoR) is present on fibroblasts, where it regulates focal contact. Here, we assessed whether this action of EpoR is involved in the reduced cell adhesion observed in colonocytes exposed to Clostridium difficile toxin A. EpoR was present and functionally active in cells of the human colonic epithelial cell line HT29 and epithelial cells of human colon tissues. Toxin A significantly decreased activating phosphorylations of EpoR and its downstream signaling molecules JAK-2 (Janus kinase 2) and STAT5 (signal transducer and activator of transcription 5). In vitro kinase assays confirmed that toxin A inhibited JAK 2 kinase activity. Pharmacological inhibition of JAK2 (with AG490) abrogated activating phosphorylations of EpoR and also decreased focal contacts in association with inactivation of paxillin, an essential focal adhesion molecule. In addition, AG490 treatment significantly decreased expression of occludin (a tight junction molecule) and tight junction levels. Taken together, these data suggest that inhibition of JAK2 by toxin A in colonocytes causes inactivation of EpoR, thereby enhancing the inhibition of focal contact formation and loss of tight junctions known to be associated with the enzymatic activity of toxin A.

• The Plant Pathology Journal
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• v.26 no.4
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• pp.386-393
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• 2010

In order to study disease resistance mechanisms in rice against the rice blast fungus Magnaporthe grisea, we screened fungal elicitor-responsive genes from rice suspension-cultured cells treated with fungal elicitors employing differential hybridization (DH). By DH screening, 31 distinct rice clones were isolated and a majority of them were full-length cDNAs encoding pathogenesisrelated (PR) genes. Sixteen of the 31 genes were upregulated at 4, 8, and 12 h following fungal elicitor treatment. To elucidate the effect of signal molecules and biotic elicitors on the regulation of rice defense genes, we further characterized the transcriptional expression patterns of representative isolated PR genes; OsGlu1, OsGlu2, OsTLP, OsRLK, and OsPR-10, following treatment with fungal elicitor, phytohormones, cycloheximide, and inhibitors of protein phosphorylation. Jasmonic acid (JA) induced transcriptional expression of OsGlu1, OsTLP, and OsRLK, but not of OsGlu2 and OsPR-10 at any of the tested time points. Salicylic acid (SA) and abscisic acid weakly induced the expression of OsTLP and OsRLK. SA showed an antagonistic effect with fungal elicitor and JA. Cycloheximide suppressed all these genes upon elicitor treatment, except for OsGlu2. Staurosporine only induced the expression of OsRLK. Application of calyculin A strongly induced OsRLK expression, but suppressed the expression of OsGlu2. Our study yielded a number of PR genes that play a role in defense mechanisms against the rice blast fungus, as well as contribute towards the elucidation of crosstalk between phytohormones and other modifications during defense signaling.

• Journal of Life Science
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• v.23 no.2
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• pp.324-331
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• 2013

The addition and removal of N-acetylglucosamine (GlcNAc) molecules on serine or threonine residues of a protein is called O-GlcNAcylation. This post-translational modification occurs on both cytoplasmic and nuclear protein, and is fast and reversible as comparable to phosphorylation. In contrast to the phospho-signaling cycles, this emerging moon-lightening signaling is cycled by only two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The simple machinery is a good evolutionary adaptation of a cell for quick accommodation to continuously fluctuating intra- and extracellular microenvironments. Rather than "switching" on or off a specific proteins - this would be done by phosphorylation where numerous specific kinases and phosphatases are involved - O-GlcNAcylation would play a "rheostat" which would be much more delicately increase or decrease the efficacy of signal transductions in response to cellular nutrient and stress conditions. Interestingly, recent evidence indicates that O-GlcNAc is further modified by phosphorylation. The O-GlcNAc-P will upgrade the modulation efficiency of cellular processes to continuous 'analogue' level. So far, only one protein AP180 was reported to have O-GlcNAc-P on Thr310. But, proteomic data from our laboratory indicate that there are multiple O-GlcNAc-P proteins, constituting "O-GlcNAc-P'om". This will focus on the possibility of existence of "O-GlcNAc-P'om".

• Molecules and Cells
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• v.21 no.1
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• pp.42-51
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• 2006

We investigated the mechanism of contraction induced by S1P in esophageal smooth muscle cells. Western blot analysis demonstrated that $S1P_1$, $S1P_2$, $S1P_3$, and $S1P_5$ receptors existed in the cat esophagus. Only penetration of EDG-5 ($S1P_2$) antibody into permeabilized cells inhibited S1P-induced contraction. Pertussis toxin (PTX) also inhibited contraction, suggesting that it was mediated by $S1P_2$ receptors coupled to a PTXsensitive $G_i$ protein. Specific antibodies to $G_{i2}$, $G_q$ and $G_{\beta}$ inhibited contraction, implying that the S1P-induced contraction depends on PTX-insensitive $G_q$ and $G_{\beta}$ dimers as well as the PTX-sensitive $G_{i2}$. Contraction was not affected by the phospholipase $A_2$ inhibitor DEDA, or the PLD inhibitor ${\rho}$-chloromercuribenzoate, but it was abolished by the PLC inhibitor U73122. Incubation of permeabilized cells with $PLC{\beta}3$ antibody also inhibited contraction. Contraction involved the activation of a PKC pathway since it was affected by GF109203X and chelerythrine. Since $PKC{\varepsilon}$ antibody inhibited contraction, $PKC{\varepsilon}$ may be required. Preincubation of the muscle cells with the MEK inhibitor PD98059 blocked S1P-induced contraction, but the p38 MAP kinase inhibitor SB202190 did not. In addition, co-treatment of cells with GF 109203X and PD98059 did not have a synergistic effect, suggesting that these two kinases are involved in the same signaling pathway. Our data suggest that S1P-induced contraction in esophageal smooth muscle cells is mediated by $S1P_2$ receptors coupled to PTX-sensitive $G_{i2}$ proteins, and PTX-insensitive $G_q$ and $G_{\beta}$ proteins, and that the resulting activation of the $PLC{\beta}3$ and $PKC{\varepsilon}$ pathway leads to activation of a p44/p42 MAPK pathway.

• Molecules and Cells
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• v.40 no.2
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• pp.123-132
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• 2017

Insulin signaling is coordinated by insulin receptor substrates (IRSs). Many insulin responses, especially for blood glucose metabolism, are mediated primarily through Irs-1 and Irs-2. Irs-1 knockout mice show growth retardation and insulin signaling defects, which can be compensated by other IRSs in vivo; however, the underlying mechanism is not clear. Here, we presented an Irs-1 truncated mutated mouse ($Irs-1^{-/-}$) with growth retardation and subcutaneous adipocyte atrophy. $Irs-1^{-/-}$ mice exhibited mild insulin resistance, as demonstrated by the insulin tolerance test. Phosphatidylinositol 3-kinase (PI3K) activity and phosphorylated Protein Kinase B (PKB/AKT) expression were elevated in liver, skeletal muscle, and subcutaneous adipocytes in Irs-1 deficiency. In addition, the expression of IRS-2 and its phosphorylated version were clearly elevated in liver and skeletal muscle. With miRNA microarray analysis, we found miR-33 was down-regulated in bone marrow stromal cells (BMSCs) of $Irs-1^{-/-}$ mice, while its target gene Irs-2 was up-regulated in vitro studies. In addition, miR-33 was down-regulated in the presence of Irs-1 and which was up-regulated in fasting status. What's more, miR-33 restored its expression in re-feeding status. Meanwhile, miR-33 levels decreased and Irs-2 levels increased in liver, skeletal muscle, and subcutaneous adipocytes of $Irs-1^{-/-}$ mice. In primary cultured liver cells transfected with an miR-33 inhibitor, the expression of IRS-2, PI3K, and phosphorylated-AKT (p-AKT) increased while the opposite results were observed in the presence of an miR-33 mimic. Therefore, decreased miR-33 levels can up-regulate IRS-2 expression, which appears to compensate for the defects of the insulin signaling pathway in Irs-1 deficient mice.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.2
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• pp.151-159
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• 2019

Pruritus (itching) is classically defined as an unpleasant cutaneous sensation that leads to scratching behavior. Although the scientific criteria of classification for pruritic diseases are not clear, it can be divided as acute or chronic by duration of symptoms. In this study, we investigated whether skin injury caused by chemical (contact hypersensitivity, CHS) or physical (skin-scratching stimulation, SSS) stimuli causes initial pruritus and analyzed gene expression profiles systemically to determine how changes in skin gene expression in the affected area are related to itching. In both CHS and SSS, we ranked the Gene Ontology Biological Process terms that are generally associated with changes. The factors associated with upregulation were keratinization, inflammatory response and neutrophil chemotaxis. The Kyoto Encyclopedia of Genes and Genomes pathway shows the difference of immune system, cell growth and death, signaling molecules and interactions, and signal transduction pathways. Il1a, Il1b and Il22 were upregulated in the CHS, and Tnf, Tnfrsf1b, Il1b, Il1r1 and Il6 were upregulated in the SSS. Trpc1 channel genes were observed in representative itching-related candidate genes. By comparing and analyzing RNA-sequencing data obtained from the skin tissue of each animal model in these characteristic stages, it is possible to find useful diagnostic markers for the treatment of itching, to diagnose itching causes and to apply customized treatment.

• Korean Journal of Optics and Photonics
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• v.33 no.6
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• pp.331-337
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• 2022

Raman spectroscopy is an optical technique that can identify molecules in a label-free manner, and is therefore heavily investigated in various areas ranging from biomedical engineering to materials science. Probe-based Raman spectroscopy can perform minimally invasive chemical analysis, and thus has potential as a real-time diagnostic tool during surgery. In this study, Raman experimentation was calibrated by examining the Raman shifts with respect to the concentrations of chemical substances. Raman signal characteristics, targeted for normal mice and cerebral tissues of the 5xFAD dementia mutant model with accumulated amyloid beta plaques, were measured and analyzed to explore the possibility of diagnosis of Alzheimer's disease. The application to the diagnosis of dementia was cross-validated by measuring Raman signals of amyloid beta. The results suggest the potential of Raman spectroscopy as a diagnostic tool that may be useful in various areas of application.

• Analytical Science and Technology
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• v.35 no.5
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• pp.205-211
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• 2022

Deuterium (D) is an isotope with one more neutron number than hydrogen (H). Heavy elements rarely change their chemical properties with little effect even if the number of neutrons increases, but low-mass elements change their vibration energy, diffusion rate, and reaction rate because the effect cannot be ignored, which is called an isotope effect. Recently, in the semiconductor and display industries, there is a trend to replace hydrogen gas (H₂) with deuterium gas (D₂) in order to improve process stability and product quality by using the isotope effect. In addition, as the demand for D₂ in industries increases, domestic gas producers are making efforts to produce and supply D₂ on their own. In the case of high purity D₂, most of them are produced by electrolysis of heavy water (D₂O), and among D₂, hydrogen deuteride (HD) molecules are present as isotope impurities. Therefore, in order to maximize the isotope effect of hydrogen in the electronic industry, HD, which is an isotope impurity of D₂ used in the process, should be small amount. To this end, purity analysis of D₂ for industrial processing is essential. In this study, HD quantitative analysis of D₂ for high purity D₂ purity analysis was established and hydrogen isotope RM (Reference material) was developed. Since hydrogen isotopes are difficult to analyze with general gas analysis instrument, they were analyzed using a high-precision mass spectrometer (Gas/MS, Finnigan MAT271). High purity HD gas was injected into Gas/MS, sensitivity was determined by a signal according to pressure, and HD concentrations in two bottles of D₂ were quantified using the corresponding sensitivity. The amount fraction of HD in each D₂ was (4518 ± 275) μmol/mol, (2282 ± 144) μmol/mol. D₂, which quantifies HD amount using the developed quantitative analysis method, will be manufactured with hydrogen isotope RM and distributed for quality management and maintenance of electronic industries and gas producers in the future.