• Journal of the Korea Academia-Industrial cooperation Society
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• v.16 no.3
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• pp.1964-1971
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• 2015

Human bone marrow or human umbilical cord vein derived-mesenchymal stem cells (hBM-MSCs or hUC-MSCs) have known as a potentially useful cell type for clinical therapeutic applications. We investigated two-pore domain potassium (K2P) channels in these cells. K2P channels play a major role in setting the resting membrane potential in many cell types. Among them, TREK1 is targets of hydrogen, hypoxia, polyunsaturated fatty acids, antidepressant, and neurotransmitters. We investigated whether hBM-MSCs and hUC-MSCs express functional TREK1 channel using RT-PCR analysis and patch clamp technique. Potassium channel with a single channel conductance of 100 pS was found in hUC-MSCs and BM-MSCs and the channel was activated by membrane stretch (-5 mmHg ~ -15 mmHg), arachidonic acid ($10{\mu}M$) and intracellular acidosis (pH 6.0). These electrophysiological properties were similar to those of TREK1. Our results suggest that TREK1 is functionally present in hBM-MSCs and hUC-MSCs, where they contribute to its resting membrane potential.

• The Journal of Korean Physical Therapy
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• v.20 no.1
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• pp.57-65
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• 2008

Purpose: Heat shock proteins (HSPs) are a group of proteins that are activated when cells are exposed to a variety of environmental stresses, such as infection, inflammation, exposure to toxins, starvation, hypoxia, brain injury, or water deprivation. The activation of HSPs by environmental stress plays a key role in signal transduction, including cytoprotection, molecular chaperone, anti-apoptotic effect, and anti-aging effects. However, the precise mechanism for the action of small HSPs, such as HSP27 and mitogen-activated protein kinases (MAPKs: extracellular-regulated protein kinase 1/2 (ERK1/2), p38MAPK, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), is not completely understood, particularly in application of cell stimulators including platelet-derived growth factor (PDGF), angiotensin II (AngII), tumor necrosis factor $\alpha$ (TNF$\alpha$), and $H_2O_2$. This study examined the relationship between stimulators-induced enzymatic activity of HSP27 and MAPKs from rat smooth and skeletal muscles. Methods: 2-dimensional electrophoresis (2DE) and matrix assisted laser desorption ionizationtime-of-flight/time-of-flight (MALDI-TOF/TOF) analysis were used to identify HSP27 from the intact vascular smooth and skeletal muscles. Three isoforms of HSP27 were detected on silver-stained gels of the whole protein extracts from the rat aortic smooth and skeletal muscle strips. Results: The expression of PDGF, AngII, TNF$\alpha$, and $H_2O_2$-induced activation of HSP27, p38MAPK, ERK1/2, and SAPK/JNK was higher in the smooth muscle cells than the control. SB203580 (30${\mu}$M), a p38MAPK inhibitor, increased the level of HSP27 phosphorylation induced by stimulators in smooth muscle cells. Furthermore, the age-related and starvation-induced activation of HSP27 was higher in skeletal muscle cells (L6 myoblast cell lines) and muscle strips than the control. Conclusion: These results suggest, in part, that the activity of HSP27 and MAPKs affect stressors, such as PDGF, AngII, TNF$\alpha$, $H_2O_2$, and starvation in rat smooth and skeletal muscles. However, more systemic research will be needed into physical therapy, including thermotherapy, electrotherapy, radiotherapy and others.

• Clinical and Experimental Pediatrics
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• v.51 no.10
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• pp.1112-1117
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• 2008

Purpose : We intended to observe cell death and apoptotic changes in neurons in organotypic hippocampal slice cultures following oxygen-glucose deprivation (OGD), using propidium iodide (PI) uptake, Fluoro-Jade (FJ) staining, TUNEL staining and immunofluorescent staining for caspase-3. Methods : The hippocampus of 7-day-old rats was cut into $350{\mu}m$ slices. The slices were cultured for 10 d (date in vitro, DIV 10) and and exposed to OGD for 60 min at DIV 10. They were then incubated for reperfusion under normoxic conditions for an additional 48 h. Fluorescence of PI uptake was observed at predetermined intervals, and the cell death percentage was recorded. At 24 h following OGD, the slices were Cryo-cut into $15{\mu}m$ thicknesses, and Fluoro-Jade staining, TUNEL staining, and immunofluorescence staining for caspase-3 were performed. Results : 1) PI uptake was restricted to the pyramidal cell layer and DG in the slices after OGD. The fluorescent intensities of PI increased from 6 to 48 h during the reperfusion stage. The cell death percentage significantly increased time-dependently in CA1 and DG following OGD (P<0.05). 2) At 24 h after OGD, many FJ positive cells were detected in CA1 and DG. Some neurons had distinct nuclei and processes while others had fragmented nuclei and disrupted processes in CA1. TUNEL and immunofluorescent staining for caspase-3 showed increased expression of TUNEL labeling and caspase-3 in CA1 and DG at 24 h after OGD. Conclusion : The numerous dead cells in the slice cultures after OGD tended to display apoptotic changes mediated by the activation of caspase-3.

• Journal of Chest Surgery
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• v.43 no.4
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• pp.345-355
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• 2010

Background: Extracellular and intracellular pH ($pH_o$ and $pH_i$), which can be changed in various pathological conditions such as hypoxia, affects vascular contractility. To elucidate the mechanism to alter vascular contractility by pH, the effects of pH on reactivity to vasocontracting agents, intracellular $Ca^{2+}$ influx, and $Ca^{2+}$ sensitivity in vascular smooth muscle were examined. Material and Method: Isometric contractions in rat superior mesenteric arteries (SMA) were observed. Intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) was recorded by microfluorometer using Fura-2/acetoxylmethyl ester in muscle cells. $pH_o$ was increased from 7.4 to 7.8 or decreased to 6.9 or 6.4. $pH_i$ was decreased by applying $NH_4^+$ or propionic acid or modulated by changing $pH_o$ after increasing membrane permeability using $\beta$-escin. Result: Decreases in $pH_o$ from 7.4 to 6.9 or 6.4 shifted concentration-response curve by norepinephrine (NE) or serotonin (SE) to the right and significantly increased half maximal effective concentration (EC50) to NE or SE. Increase in $pH_o$ from 7.4 to 7.8 shifted concentration-response curve by norepinephrine (NE) or serotonin (SE) to the left and significantly reduced EC50 to NE or SE. NE increased $[Ca^{2+}]_i$ in cultured smooth muscle cells from SMA and the increased $[Ca^{2+}]_i$ was reduced by decreases in $pH_o$. NE-induced contraction was inhibited by $NH_4^+$, whereas the resting tension was increased by $NH_4^+$ or propionic acid. When the cell membrane of SMA was permeabilized using ${\beta}$-escin, SMA was contracted by increasing extracellular $Ca^{2+}$ concentration from 0 to $10{\mu}M$ and the magnitude of contraction was decreased by a decrease in $pH_o$ and vice versa. Conclusion: From these results, it can be concluded that a decrease in $pH_o$ might inhibit vascular contraction by reducing the reactivity of vascular smooth muscle to vasoactive agents, $Ca^{2+}$ influx and the sensitivity of vascular smooth muscle to $Ca^{2+}$.

• Radiation Oncology Journal
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• v.22 no.4
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• pp.288-297
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• 2004

Puporse: The aims of this study were to evaluate the change of $[^18F]fluoromisonidazole$($[^18F]FMISO$) uptake in C3H mouse squamous cell carcinoma-VII (SCC-VII) treated with mild hyperthermia ($42^{circ}C$) and nicotinamide and to assess the biodistribution of the markers in normal tissues under similar conditions. Methods and Materials: $[^18F]FMISO$ was producedby our hospital. Female C3H mice with a C3H SCC-VII tumor grown on their extremities were used. Tumors were size matched. Non-anaesthetized, tumor-bearing mice underwent control or mild hyperthermia at $42^{circ}C$ for 60 min with nicotinamide (50 mg/kg i.p. injected) and were examined by gamma counter, autoradiography and animal PET scan 3 hours after tracer i.v. injected with breathing room air, The biodistribution of these agents were obtained at 3 h after $[^18F]FMISO$ injection. Blood, tumor, muscle, heart, lung, liver, kidney, brain, bone, spleen, and intestine were removed, counted for radioactivity and weighed. The tumor and liver were frozen and cut with a cryomicrotome into 10- um sections. The spatial distribution of radioactivity from the tissue sections was determined with digital autoradiography. Results: The mild hyperthermia with nicotinamide treatment had only slight effects on the biodistribution of either marker in normal tissues. We observed that the whole tumor radioactivity uptake ratios were higher in the control mice than in the mild hyperthermia with nicotinamide treated mice for $[^18F]FMISO$ ($1.56{\pm}1.03$ vs. $0.67{\pm}0.30$; p=0.063). In addition, autoradiography and animal PET scan demonstrated that the area and intensity of $[^18F]FMISO$ uptake was significantly decreased. Conclusion: Mild hyperthermla and nicotinamide significantly improved tumor hypoxia using $[^18F]FMISO$ and this uptake reflected tumor hypoxic status.

• Proceedings of the SCSK Conference
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• 2003.09a
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• pp.13-34
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• 2003

Fructose-1, 6-diphosphate (FOP), a glycolytic metabolite is reported to ameliorate inflammation and inhibit the nitric oxide production in murine macrophages stimulated with endotoxin. It is also reported that FOP has cytoprotective effects against hypoxia or ischemia/reperfusion injury in brain and heart. In this study, we examined whether FDP has protective effects on UV-induced oxidative damage in skin cell culture system and human skin in vivo. FDP had a protective role in UVB-induced LDH release and ROS accumulation in HaCaT although it did not show direct radical scavenging effect in the experiment using 1, 1-diphenyl-2-picrylhydrazyl (DPPH). FDP also preserved cellular GSH content after UV irradiation in HaCaT and normal human fibroblast culture system. Cellular oxidative stress induces multiple downstream signaling pathways that regulate expression of multiple gene including MMP-1 and collagen, we examined the effects of FDP on UV-induced alteration of these protein expression in fibroblast culture and human skin in vivo. The increased MMP-1 expression in fibroblast and human skin by UV irradiation was significantly decreased by FDP. FDP also prevented the UV-induced decrease of collagen expression in fibroblast and human skin. Moreover, the decreasing the intracellular levels of reducing equivalents in human fibroblast by glutathione (GSH) depletion lowered the UVA dose threshold for reduction of procollagen expression, indicating that the differences of glutathione contents define the susceptibility of fibroblasts towards UV-induced reduction of procollagen expression. FDP also preserved cellular GSH content after UV irradiation, indicating that FDP has protective effects on UV-induced reduction of procollagen expression, which are possibly through maintaining intracellular reducing equivalent. Based on these premises, we examined the effect of daily use of a moisturizer containing FDP on facial wrinkle in comparison with vehicle moisturizer lacking FDP. In the clinical study, FDP significantly decreased facial wrinkle compared with vehicle alone after 6 months of use. Our results suggest that FDP has anti-aging effects in skin by increasing cellular antioxidant system and preventing oxidative signal and inflammatory reaction. Therefore FDP may be useful anti-aging agent for cosmetic purpose.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.3
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• pp.1715-1720
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• 2013

Background: The discovery that microRNAs (miRNAs) regulate proliferation, invasion and metastasis provides a principal molecular basis of tumor heterogeneity. Microvessel distribution is an important characteristic of solid tumors, with significant hypoxia occurring in the center of tumors with low blood flow. The distribution of miR-374a in breast tumors was examined as a factor likely to be important in breast cancer progression. Methods: Breast tissue samples from 40 patients with breast cancer were classified into two groups: a highly invasive and metastatic group (HIMG) and a low-invasive and metastatic Group (LIMG). Samples were collected from the center and edge of each tumor. In each group, six specimens were examined by microRNA array, and the remaining 14 specimens were used for real-time RT-qPCR, Western blot and immunohistochemical analyses. Correlation analysis was performed for the miRNAs and target proteins. Follow-up was carried out during 28 months to 68 months after surgery, and survival data were analyzed. Results: In the LIMG, the relative content of miR-374a was lower in the center of the tumor than at its edge; in the HIMG, it was lower at the edge of the tumor, and miR-374a levels were lower in breast cancer tissues than in normal tissues. There was no difference between VEGF-A and VCAM-1 mRNA levels at the edge and center of the tumor; however, we observed a significant difference between VEGF-A and VCAM-1 protein expression levels in these two regions. There was a negative correlation between miR-374a and target protein levels. The microvessel density (MVD) was lower in the center of the tumor than at its edge in HIMG, but the LIMG vessels were uniformly distributed. There was a significant positive correlation between MVD and the number of lymph node metastases (Pearson correlation, r=0.912, P<0.01). The median follow-up time was 48.5 months. LIMG had higher rate of disease-free survival (100%, P=0.013) and longer median survival time (66 months) than HIMG, which had a lower rate of 75% and shorter median survival time (54 months). Conclusions: Our data demonstrated miR-374a to be differentially distributed in breast cancer; VEGF-A and VCAM-1 mRNA had coincident distribution, and the distribution of teh respective proteins was uneven and opposite to that for the miR-374a. These data might explain the differences in the distribution of MVD in breast cancer and variation in breast cancer prognosis.

• Nutrition Research and Practice
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• v.4 no.5
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• pp.351-355
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• 2010

Our previous proteomic study demonstrated that oxidative stress and antioxidant delphinidin regulated the cellular level of $p27^{kip1}$ (referred to as p27) as well as some heat shock proteins in human colon cancer HT 29 cells. Current study was conducted to validate and confirm the regulation of these proteins using both in vitro and in vivo systems. The level of p27 was decreased by hydrogen peroxide in a dose-dependent manner in human colon carcinoma HCT 116 (p53-positive) cells while it was increased upon exposure to hydrogen peroxide in HT 29 (p53-negative) cells. However, high concentration of hydrogen peroxide (100 ${\mu}M)$ downregulated p27 in both cell lines, but delphindin, one of antioxidative anthocyanins, enhanced the level of p27 suppressed by 100 ${\mu}M$ hydrogen peroxide. ICR mice were injected with varying concentrations of hydrogen peroxide, delphinidin and both. Western blot analysis for the mouse large intestinal tissue showed that the expression of p27 was upregulated by 25 mg/kg BW hydrogen peroxide. To investigate the association of p27 regulation with hypoxia-inducible factor 1-beta (HIF-$1{\beta}$), the level of p27 was analyzed in wild-type mouse hepatoma hepa1c1c7 and Aryl Hydrocarbon Nuclear Translocator (arnt, HIF-$1{\beta}$)-defective mutant BPRc1 cells in the absence and presence of hydrogen peroxide and delphinidin. While the level of p27 was responsive to hydrogen peroxide and delphinidin, it remained unchanged in BPRc1, suggesting that the regulation of p27 requires functional HIF-$1{\beta}$. We also found that hydrogen peroxide and delphinidin affected PI3K/Akt/mTOR signaling pathway which is one of upstream regulators of HIFs. In conclusion, hydrogen peroxide and antioxidant delphinidin seem to regulate intracellular level of p27 through regulating HIF-1 level which is, in turn, governed by its upstream regulators comprising of PI3K/Akt/mTOR signaling pathway. The results should also encourage further study for the potential of p27 as a biomarker for intracellular oxidative or antioxidant status.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.6
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• pp.699-705
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• 1999

The effects of iron on gill tissue and metabolic rate represented by oxygen consumption of olive flounder, Paralichthys olivaceus were determined. The effects were further studied by means of survival rate of the fish exposed to a serial concentrations of iron. The olive flounder exposed to iron concentrations over 0.93 mg/$\ell$ showed curvature and terminal clubbing of gill lamellae at 2 weeks post-exposure. In iron concentration 4.89 mg/$\ell$, gill of the fish were seriously damaged just after 2 weeks, showing hyperplasia of filament epithelia, deformation of lamella epithelia, chloride cell damage, and separation of lamella epithelial layer, Gills exposed to 9.78 mg/$\ell$ iron concentration resulted in fusion and necrosis of the lamellae after 2 weeks. Significant decreases of metabolic rate of the fish were observed after 4 weeks at iron concentration 0,93 mg/$\ell$ and after 2 weeks at iron concentrations over 4.89 mg/$\ell$. Survival rate of the olive flounder decreased significantly after 4 weeks at the iron concentration over 4.89 mg/$\ell$. These results lead us to conclude that, as far as the iron effects are concerned, its concentrations should not exceed at least more than 0.93 mg/$\ell$ in the fish farm and coastal waters for normal growth of the olive flounder.

• Radiation Oncology Journal
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• v.12 no.1
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• pp.9-16
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• 1994

Hypothesis that hypoxic tumors should be more responsive to the addition of preferential hypoxic cell cytotoxin SR 4233 (tirapazamine) to fractionated irradiation was tested in the mouse SCCVll carcinoma and RIF-1 sarcoma, Model of hypoxic tumor was established using the tumor bed effect: tumors growing in the preirradiated tissue (preirradiated tumors) were more hypoxic than tumors growing in the unirradiated tissue (unirradiated tumors). When the tumors reached a mean volume of 100 $mm^{3}$, both unirradiated and preirradiated tumors were treated with a fractionated course of 6${\times}$2 Gy in 3 days or 8${\times}$2.5 Gy in 4 days with SR 4233 (0.08 mmol/kg/injection) given 30 minutes before each irradiation or without SR 4233. Compared to the unirradiated tumors, hypoxic preirradiated tumors were approximately 5 times more resistant to fractionated irradiation alone but were approximately 5 times more responsive to SR 4233. Addition of SR 4233 Potentiated the effect of fractionated irradiation in both unirradiated and preirradiated tumors. Potentiation in the preirradiated tumors was morequal to or greater than that in the unirradiated tumors and seemed to be higher for more fractionated treatment. We confirm the hypothesis in a transplantable mouse tumor. Present results suggest that radioresistance of some hypoxic tumors can be overcome with hypoxic cytotoxin.