• IMMUNE NETWORK
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• v.4 no.1
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• pp.23-30
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• 2004

Background: A human orthologue of mouse S100A6-binding protein (CacyBP), Siah-1-interacting protein (SIP) had been shown to be a component of novel ubiquitinylation pathway regulating $\beta$-catenin degradation. The role of the protein seems to be important in cell proliferation and cancer evolution but the expression pattern of SIP in actively dividing cancer tissues has not been known. For the elucidation of the role of SIP protein in carcinogenesis, it is essential to produce monoclonal antibodies specific to the protein. Methods: cDNA sequence coding for ORF region of human SIP gene was amplified and cloned into an expression vector to produce His-tag fusion protein. Recombinant SIP protein and monoclonal antibody to the protein were produced. The N-terminal specificity of anti-SIP monoclonal antibody was conformed by immunoblot analysis and enzyme linked immunosorbent assay (ELISA). To study the relation between SIP and colon carcinogenesis, the presence of SIP protein in colon carcinoma tissues was visualized by immunostaining using the monoclonal antibody produced in this study. Results: His-tag-SIP (NSIP) recombinant protein was produced and purified. A monoclonal antibody (Korea patent pending; #2003-45296) to the protein was produced and employed to analyze the expression pattern of SIP in colon carcinoma tissues. Conclusion: The data suggested that anti-SIP monoclonal antibody produced here was valuable for the diagnosis of colon carcinoma and elucidation of the mechanism of colon carcinogenesis.

• The Korean Journal of Physiology
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• v.27 no.2
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• pp.175-183
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• 1993

There are many report suggesting that influx and intracellular calcium concentration $([Ca^{2+}]_i)$ are related to cell signalling in various cells. However, it has not been reported that calcium channel activation is affected by the substances involved in signal transduction pathways in the mouse eggs. In this study, the effects of isoprenaline (ISP) and cyclic AMP on calcium influx through calcium channels were investigated to show their relationship with the signal transduction process in unfertilized mouse eggs. Using whole cell voltage clamp techniques, calcium currents, elicited by the depolarizing pulses of 300 ms duration (from -50 mV to 50 mV in 10 mV increments) from a holding potential of -80 mV, were recorded. The current-voltage (I-V) relation of calcium currents was shown to be bell-shaped; the current began to activate at -50 mV and reached its maximum $(-1.33{\pm}0.16\;nA:\;mean{\pm}S.E.,\;n=7)$ at -10 mV, then decayed at around 50 mV. Calcium currents were fully activated within $7\;ms{\sim}20\;ms$ and completely inactivated 200 ms after onset of the step pulse. ISP within the concentration ranges of $10^{-8}\;M{\sim}10^{-4}\;M$ dose-dependently increased the amplitude calcium current. The permeable cyclic AMP analogue,8-bromocyclic AMP, also increased its maximal amplitude by 46ft at $10^{-5}\;M$, while protein kinase inhibitor (PKI), which is known to inhibit 0.02 phosphorylating units of cyclic AMP-dependent protein kinase (PKA) per microgram decreased calcium currents. Currents recorded in the presence of PKI were resistant to increase by the application of $10^{-5}\;M$. Also, PKI inhibited the calcium current increase elicited by ISP treatment. These results suggest that $\beta-adrenergic$ regulation of the calcium channel is mediated by the cAMP-dependent protein kinase. This signal transduction pathway might play a role in regulating $[Ca^{2+}]_i$, level due to the increase of calcium influx in mouse eggs.

• BMB Reports
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• v.45 no.3
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• pp.183-188
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• 2012

Participates in actin remodeling through Rac and receptor endocytosis via Rab5. Here, we used yeast two-hybrid system with Eps8 as bait to screen a human brain cDNA library. ITSN2 was identified as the novel binding factor of Eps8. The interaction between ITSN2 and Eps8 was demonstrated by the in vivo co-immunoprecipitation and colocalization assays and the in vitro GST pull-down assays. Furthermore, we mapped the interaction domains to the region between amino acids 260-306 of Eps8 and the coiled-coil domain of ITSN2. In addition, protein stability assays and immunofluorescence analysis showed ITSN2 overexpression induced the degradation of Eps8 proteins, which was markedly alleviated with the lysosome inhibitor NH4Cl treatment. Taken together, our results suggested ITSN2 interacts with Eps8 and stimulates the degradation of Eps8 proteins.

• Journal of Environmental Science International
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• v.29 no.1
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• pp.55-68
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• 2020

The purpose of this study is to investigate the relationship of fine dust PM₁₀ and heavy metals in PM₁₀ in Asian dust flowing into Gwangju from 2013 to 2018. The migration pathways of Asian dust was analyzed by backward trajectory analysis using HYSPLIT (Hybrid Single Particle Lagrangian Integrated Trajectory) model, and the change of heavy metal concentration and heavy metal content per 1 ㎍/㎥ of fine dust PM₁₀ in Gwangju area were analyzed. Also, the characteristics of the heavy metals were analyzed using the correlation between the heavy metals in PM₁₀. As a result of analyzing Asian dust entering the Gwangju region for 6 years, the average concentration of PM₁₀ measured in Asian dust was 148 ㎍/㎥, which was about 4.5 times higher than in non-Asian dust, 33 ㎍/㎥. A total of 13 Asian dust flowed into the Gwangju during 6 years, and high concentration of PM10 and heavy metals in that were analyzed in the C path flowing through the Gobi/Loess Plateau-Korean Peninsula. As a result of the correlation analysis, in case of Asian dust, there was a high correlation between soil components in heavy metals, so Asian dust seems to have a large external inflow. On the other hand, in case of non-Asian dust, the correlation between find dust PM₁₀ and artificial heavy metal components was high, indicating that the influence of industrial activities in Gwangju area was high.

• Toxicological Research
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• v.23 no.1
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• pp.55-63
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• 2007

Diethylnitrosamine (DEN) is a nitrosamine compound that can induce a variety of liver lesions including hepatic carcinoma, forming DNA-carcinogen adducts. In the present study, microarray analyses were performed with Affymetrix Murine Genome 430A Array in order to identify the gene-expression profiles for DEN and to provide valuable information for the evaluation of potential hepatotoxicity. C57BL/6NCrj mice were orally administered once with DEN at doses of 0, 3, 7 and 20 mg/kg. Liver from each animal was removed 2, 4, 8 and 24 hrs after the administration. The histopathological analysis and serum biochemical analysis showed no significant difference in DEN-treated groups compared to control group. Conversely, the principal component analysis (PCA) profiles demonstrated that a specific normal gene expression profile in control groups differed clearly from the expression profiles of DEN-treated groups. Within groups, a little variance was found between individuals. Student's t-test on the results obtained from triplicate hybridizations was performed to identify those genes with statistically significant changes in the expression. Statistical analysis revealed that 11 genes were significantly downregulated and 28 genes were upregulated in all three animals after 2 h treatment at 20 mg/kg. The upregulated group included genes encoding Gdf15, JunD1, and Mdm2, while the genes including Sox6, Shmt2, and SIc6a6 were largely down regulated. Hierarchical clustering of gene expression also allowed the identification of functionally related clusters that encode proteins related to metabolism, and MAPK signaling pathway. Taken together, this study suggests that match with a toxicant signature can assign a putative mechanism of action to the test compound if is established a database containing response patterns to various toxic compounds.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.6
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• pp.661-670
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• 2018

Fimasartan, a new angiotensin II receptor antagonist, reduces myocyte damage and stabilizes atherosclerotic plaque through its anti-inflammatory effect in animal studies. We investigated the protective effects of pretreatment with fimasartan on ischemia-reperfusion injury (IRI) in a mouse model of ischemic renal damage. C57BL/6 mice were pretreated with or without 5 (IR-F5) or 10 (IR-F10) mg/kg/day fimasartan for 3 days. Renal ischemia was induced by clamping bilateral renal vascular pedicles for 30 min. Histology, pro-inflammatory cytokines, and apoptosis assays were evaluated 24 h after IRI. Compared to the untreated group, blood urea nitrogen and serum creatinine levels were significantly lower in the IR-F10 group. IR-F10 kidneys showed less tubular necrosis and interstitial fibrosis than untreated kidneys. The expression of F4/80, a macrophage infiltration marker, and tumor necrosis factor $(TNF)-{\alpha}$, decreased in the IR-F10 group. High-dose fimasartan treatment attenuated the upregulation of $TNF-{\alpha}$, interleukin $(IL)-1{\beta}$, and IL-6 in ischemic kidneys. Fewer TUNEL positive cells were observed in IR-F10 compared to control mice. Fimasartan caused a significant decrease in caspase-3 activity and the level of Bax, and increased the Bcl-2 level. Fimasartan preserved renal function and tubular architecture from IRI in a mouse ischemic renal injury model. Fimasartan also attenuated upregulation of inflammatory cytokines and decreased apoptosis of renal tubular cells. Our results suggest that fimasartan inhibited the process of tubular injury by preventing apoptosis induced by the inflammatory pathway.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.6
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• pp.349-355
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• 2003

We previously shown that LES contraction depends on $M_3$ receptors linked to PTX insensitive $G_q$ protein and activation of PLC. This results in production of $IP_3$, which mediates calcium release, and contraction through a CaM dependent pathway. In the esophagus ACh activates $M_2$ receptors linked to PTX sensitive $G_{i3}$ protein, resulting in activation of PLD, presumably, production of DAG. We investigated the role of PLC isozymes which can be activated by $G_q$ or $G{\beta}$ protein on ACh-induced contraction in LES and esophagus. Immunoblot analysis showed the presence of 3 types of PLC isozymes, $PLC-{\beta}1$, $PLC-{\beta}3$, and $PLC-{\gamma}1$, but not $PLC-{\beta}2$, $PLC-{\beta}4$, $PLC-{\gamma}2$, $PLC-{\delta}1$, and $PLC-{\delta}2$ from both LES and esophageal muscle. ACh produced contraction in a dose dependent manner in LES and esophageal muscle cells obtained by enzymatic digestion with collagenase. $PLC-{\beta}1$ or $PLC-{\beta}3$ antibody incubation reduced contraction in response to ACh in LES but not in esophageal permeabilized cells, but $PLC-{\gamma}1$ antibody incubation did not have an inhibitory effect. The inhibition by $PLC-{\beta}1$ or $PLC-{\beta}3$ antibody on Ach-induced contraction was antibody concentration dependent. The combination with $PLC-{\beta}_1$ and $PLC-{\beta}_3$ antibody completely abolished the contraction, suggesting that $PLC-{\beta}1$ and $PLC-{\beta}3$ have a synergism to inhibit the contraction in LES. $PLC-{\beta}1$, -${\beta}3$ or -${\gamma}1$ antibody did not reduce the contraction of LES cells in response to DAG ($10^{-6}$ M), suggesting that this isozyme of PLC may not activate PKC. When $G_{q/11}$ antibody was incubated, the inhibitory effect of the incubation of PLC ${\beta}3$, but not of PLC ${\beta}_1$ was additive (Fig. 6). In contrast, when $G_{\beta}$ antibody was incubated, the inhibitory effect of the incubation of PLC ${\beta}_1$, but not of PLC ${\beta}_3$ was additive. This data suggest that $G_{q/11}$/11 or $G{\beta}$ may activate cooperatively different PLC isozyme, $PLC{\beta}_1$ or $PLC{\beta}_3$ respectively.

• Journal of Ginseng Research
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• v.42 no.2
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• pp.165-174
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• 2018

Background: Extended endoplasmic reticulum (ER) stress may initiate apoptotic pathways in cancer cells, and ER stress has been reported to possibly increase tumor death in cancer therapy. We previously reported that caspase-8 played an important role in compound K-induced apoptosis via activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation in HL-60 human leukemia cells. The mechanisms leading to apoptosis in A549 and SK-MES-1 human lung cancer cells and the role of ER stress have not yet been understood. Methods: The apoptotic effects of compound K were analyzed using flow cytometry, and the changes in protein levels were determined using Western blot analysis. The intracellular calcium levels were monitored by staining with Fura-2/AM and Fluo-3/AM. Results: Compound K-induced ER stress was confirmed through increased phosphorylation of $eIF2{\alpha}$ and protein levels of GRP78/BiP, XBP-1S, and $IRE1{\alpha}$ in human lung cancer cells. Moreover, compound-K led to the accumulation of intracellular calcium and an increase in m-calpain activities that were both significantly inhibited by pretreatment either with BAPTA-AM (an intracellular $Ca^{2+}$ chelator) or dantrolene (an RyR channel antagonist). These results were correlated with the outcome that compound K induced ER stress-related apoptosis through caspase-12, as z-ATAD-fmk (a specific inhibitor of caspase-12) partially ameliorated this effect. Interestingly, 4-PBA (ER stress inhibitor) dramatically improved the compound K-induced apoptosis. Conclusion: Cell survival and intracellular $Ca^{2+}$ homeostasis during ER stress in human lung cancer cells are important factors in the induction of the compound K-induced apoptotic pathway.

• Molecules and Cells
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• v.43 no.4
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• pp.360-372
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• 2020

The basal ganglia network has been implicated in the control of adaptive behavior, possibly by integrating motor learning and motivational processes. Both positive and negative reinforcement appear to shape our behavioral adaptation by modulating the function of the basal ganglia. Here, we examined a transgenic mouse line (G2CT) in which synaptic transmissions onto the medium spiny neurons (MSNs) of the basal ganglia are depressed. We found that the level of collaterals from direct pathway MSNs in the external segment of the globus pallidus (GPe) ('bridging collaterals') was decreased in these mice, and this was accompanied by behavioral inhibition under stress. Furthermore, additional manipulations that could further decrease or restore the level of the bridging collaterals resulted in an increase in behavioral inhibition or active behavior in the G2CT mice, respectively. Collectively, our data indicate that the striatum of the basal ganglia network integrates negative emotions and controls appropriate coping responses in which the bridging collateral connections in the GPe play a critical regulatory role.

• Journal of Food Hygiene and Safety
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• v.13 no.4
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• pp.355-359
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• 1998

Platelet activation is originated by the intracellular or/and extracellular $Ca^{2+}$. Agonist-induced $Ca^{2+}$ entry through a plasma-membrane pathway has been reported repeatedly, but the mechanisms has proven harder to elucidate. Recently, a number of natural products have been isolated from medicinal plants and marine organisms and have proved to be useful chemical tools for resolving the mechanism of cellular functions. In an attempt to understand the mechanism of platelet activation in Bupleuri Radix, we have studied some aspects of the isolation of active components and their dependence of external $Ca^{2+}$> on platelet activation. Acetone extract of Bupleuri Radix has the most activity on platelet activation and it's active components were identified as saikosaponin a and d. Their optimal concentration was respectively $20\;\mu\textrm{g}/ml$ and $5\;\mu\textrm{g}/ml$ and their platelet activation was not dependent on external $Ca^{2+}$>, whereas optimal concentration of each agonist was arachidonic acid ($10\;\mu\textrm{M}$), collagen ($10\;\mu\textrm{M}/ml$), thrombin (0.1 unit/mi), PAF (5 nM), PMA ($5\;\mu\textrm{M}$), ionophore A23187 ($2\;\mu\textrm{g}$) and their dependence of external $Ca^{2+}$> on platelet activation appeared to thrombin > collagen $\geq$ PAF > PMA > arachdonic acid> ionophore A23187. These results suggest that saikosaponin is different from each agonists in the dependence of external $Ca^{2+}$ on platelet activation.