• The Korean Journal of Physiology and Pharmacology
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• v.16 no.1
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• pp.11-16
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• 2012

Cancer stem cells (CSCs) are often characterized by the elevated expression of drug-resistance related stem-cell surface markers, such as CD133 and ABCG2. Recently, we reported that CSCs have a high level of expression of the IL-6 receptor (IL-6R). The purpose of this study was to investigate the effect of anticancer drugs on the expression of the drug resistance-related cancer stem cell markers, ABCG2, IL-6R, and CD133 in non-small cell lung cancer (NSCLC) cell lines. A549, H460, and H23 NSCLC cell lines were treated with the anticancer drugs 5-fluorouracil (5-FU; $25{\mu}g/ml$) and methotrexate (MTX; $50{\mu}g/ml$), and the expression of putative CSC markers was analyzed by fluorescent activated cell sorter (FACS) and the gene expression level of abcg2, il-6r and cd133 by reverse transcriptase-polymerase chain reaction (RT-PCR). We found that the fraction of ABCG2-positive(+) cells was significantly increased by treatment with both 5-FU and MTX in NSCLC cells, and the elevation of abcg2, il-6r and cd133 expressions in response to these drugs was also confirmed using RT-PCR. Also, the number of IL-6R(+) cells was increased by MTX in the 3 cell lines mentioned and increased by 5-FU in the H460 cell line. The number of CD133(+) cells was also significantly increased by both 5-FU and MTX treatment in all of the cell lines tested. These results indicate that 5-FU and MTX considerably enhance the expression of drug-resistance related CSC markers in NSCLC cell lines. Thus, we suggest that antimetabolite cancer drugs, such as 5-FU and MTX, can lead to the propagation of CSCs through altering the expression of CSC markers.

• Journal of Korean Neurosurgical Society
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• v.55 no.6
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• pp.307-312
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• 2014

Objective : We investigated the expression of hippocampal heat shock protein 70 (HSP-70) infarction volume after different durations of experimental ischemic stroke in mice. Methods : Focal cerebral ischemia was induced in mice by occluding the middle cerebral artery with the modified intraluminal filament technique. Twenty-four hours after ischemia induction, both hippocampi were extracted for HSP-70 protein analyses. Slices from each hemisphere were stained with 2,3,5-triphenyltetrazolium chloride (2%), and infarction volumes were calculated. HSP-70 levels were evaluated using western blot and enzyme-linked immunosorbent assay (ELISA). HSP-70 subtype (hsp70.1, hspa1a, hspa1b) mRNA levels in the hippocampus were measured using reverse transcription-polymerase chain reaction (RT-PCR). Results : Cerebral infarctions were found ipsilateral to the occlusion in 10 mice exposed to transient ischemia (5 each in the 30-min and 60-min occlusion groups), whereas no focal infarctions were noted in any of the sham mice. The average infarct volumes of the 2 ischemic groups were $22.28{\pm}7.31mm^3$ [30-min group${\times}$standard deviation (SD)] and $38.06{\pm}9.53mm^3$ (60-min group${\times}$SD). Western blot analyses and ELISA showed that HSP-70 in hippocampal tissues increased in the infarction groups than in the sham group. However, differences in HSP-70 levels between the 2 infarction groups were statistically insignificant. Moreover, RT-PCR results demonstrated no relationship between the mRNA expression of HSP-70 subtypes and occlusion time or infarction volume. Conclusion : Our results indicated no significant difference in HSP-70 expression between the 30- and 60-min occlusion groups despite the statistical difference in infarction volumes. Furthermore, HSP-70 subtype mRNA expression was independent of both occlusion duration and cerebral infarction volume.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.87-93
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• 2001

Objectives: The purpose of this study was to evaluate effects of heparin-binding epidermal growth factor (HB-EGF) on the rate of blastocyst formation and hatching in the mouse embryos and the expression of matrix metalloproteinase-9 (MMP-9) and ATPase ${\gamma}$-subunit mRNA. Methods: Late 2-cell mouse embryos was cultured for 72 hours in RTF medium containing with 1, 10, and 100 ng/ml HB-EGF. The mRNA expression level of MMP-9 and ATPase ${\gamma}$-subunit was detected by reverse transcription-polymerase chain reaction (RT-PCR). Results: The rate of hatching was significantly higher (p<0.05) in group containing with 1 ng/ml HB-EGF than other groups. Also, the rate of hatched blastocyst was significantly higher (p<0.05) in 10 ng/ml. The mRNA expression level of MMP-9 mRNA was not shown any difference among groups, but ATPase ${\gamma}$-subunit was higher than other groups. Conclusions: Taken together these results suggest that HB-EGF has the positive effect to promote the blastocyst formation and hatching process and influences the blastocoel expansion by promoting the ATPase mRNA expression in the mouse embryos.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.3
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• pp.217-222
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• 2003

Objective: To investigate the association of genetic background between MTHFR C677T genotype and infertile females with polycystic ovarian syndrome. Materials and Methods: We compared 86 infertile females with polycystic ovarian syndrome (PCOS) with 100 healthy fertile females with one or more offspring. Pyrosequencing analysis for MTHFR C677T variation was performed on polymerase chain reaction (PCR) product of study group. To validate pyrosequencing data of C677T variation for randomly selected 50 samples, we compared the pyrosequencing result with the PCR-RFLP (Restriction Fragment Length Polymorphism) result of MTHFR C677T genotype. Results: The prevalence of the C677T mutant homozygous (TT) was significantly lower (p=0.0085) in females with PCOS (8.14%) than in fertile females (21.00%). MTHFR 677 TT genotype had a decreased risk (3.7-fold) of PCOS compared with wild type (MTHFR 677 CC). Conclusion: Our data support a role for MTHFR mutant homozygous (677 TT) genotype in reducing risk in Korean infertile females with Polycystic ovarian syndrome.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.2
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• pp.277-290
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• 2008

The process of wound repair involves an ordered sequence of events such as overlapping biochemical and cellular events that, in the best of circumstances, result in the restoration of both the structural and functional integrity of the damaged tissue. An important event during wound healing is the contraction of newly formed connective tissues by fibroblasts. The polypeptide growth factors, like transforming growth factor-${\beta}$(TGF-${\beta}$, insulin-like growth factor I (IGF- I) and epidermal growth factor (EGF), play very important mediator roles in the process of wound contraction. Deer antlers, as models of mammalian regeneration, are cranial appendages that develop after birth as extensions of a permanent protuberance (pedicle) on the frontal bone. Antlers contain various growth factors which stimulate dermal fibroblast growth. They are involved in digestion and respiration and are necessary for normal wound healing and skin health. In order to investigate and evaluate the effects of red deer antlers on skin wound site, the speed of full-thickness skin wound healing and the expression of IGF-I, TGF-${\beta}$ and EGF in skin wounds, three groups of skin full-thickness rat models with a high concentration of antler ointment, a low concentration of antler ointment and without antler ointment were compared. At post-injury days 0, 2, 4, 8, 16, 20, 32, 40 and 60, the skin wound area was measured, the expressions of IGF-I, TGF- ${\beta}$ and EGF mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and collagen formation by sirius red dye and the localization of IGF-I, TGF-${\beta}$ and EGF peptides were inspected by histological immunohistochemical techniques. Wound healing was significantly more rapid in antler treated skins. In addition, the wound treated with a high concentration antler ointment, a low concentration antler ointment, and the control closed completely at post-injury day 40, day 44 and day 60, respectively. Via RT-PCR, the expressions of IGF-I (day 8 and day 16), TGF-${\beta}$(day 8, day 16 and day 20) and EGF (day 4, day 8, day 16, and day 32) were obviously up-regulated in high concentration antler-treated skins compared to control skins. Similar results could be seen in the histological detection of collagen dye and immunohistochemical methods using the corresponding polyclone antibodies of IGF-I, TGF-${\beta}$ and EGF. These results illustrate that antlers stimulate and accelerate the repair of cutaneous wounds.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.3
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• pp.424-435
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• 2020

Objective: The study was conducted to investigate variations in the immunophysiological responses to exercise-induced stress in Jeju and Thoroughbred horses. Methods: Blood samples were collected from the jugular veins of adult Jeju (n = 5) and Thoroughbred (n = 5) horses before and after 30 min of exercise. The hematological, biochemical, and immunological profiles of the blood samples were analyzed. Blood smears were stained and observed under a microscope. The concentration of cell-free (cf) DNA in the plasma was determined using real time polymerase chain reaction (PCR). Peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells were separated using Polymorphprep, and the expression of various stress-related and chemokine receptor genes was measured using reverse transcriptase (RT) and real-time PCR. Results: After exercise, Jeju and Thoroughbred horses displayed stress responses with significantly increased rectal temperatures, cortisol levels, and muscle catabolism-associated metabolites. Red blood cell indices were significantly higher in Thoroughbred horses than in Jeju horses after exercise. In addition, exercise-induced stress triggered the formation of neutrophil extracellular traps (NETs) and reduced platelet counts in Jeju horses but not in Thoroughbred horses. Heat shock protein 72 and heat shock protein family A (Hsp70) member 6 expression is rapidly modulated in response to exercise-induced stress in the PBMCs of Jeju horses. The expression of CXC chemokine receptor 4 in PBMCs was higher in Thoroughbred horses than in Jeju horses after exercise. Conclusion: In summary, the different immunophysiological responses of Jeju and Thoroughbred horses explain the differences in the physiological and anatomical properties of the two breeds. The physiology of Thoroughbred horses makes them suitable for racing as they are less sensitive to exercise-induced stress compared to that of Jeju horses. This study provides a basis for investigating the link between exercise-induced stresses and the physiological alteration of horses. Hence, our findings show that some of assessed parameters could be used to determine the endurance performance of horses.

• Clinical and Experimental Pediatrics
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• v.52 no.2
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• pp.187-193
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• 2009

Purpose : Adiponectin is a molecule that plays an important role in the metabolic syndrome. In addition, its concentration is known to be decreased in obesity, type 2 diabetes, and coronary artery disease. Although a relationship between hypertension and serum adiponectin concentrations has been reported by several authors, such findings continue to be debated. We investigated whether hypoadiponectinemia is related to hypertension in adolescents and studied the associated genetic polymorphism. Methods : Forty hypertensive adolescents (Age 16-17 years old) and twenty normotensive matched subjects were included. Serum adipo-nectin, insulin, renin, aldosterone and angiotensin converting enzyme (ACE) levels were compared. Their carotid intima-media thickness (cIMT) and pulse wave velocity (PWV) were measured. Polymorphisms of the adiponectin I164T gene were investigated using polymerase chain reaction (PCR). Results : The hypertensive adolescents had significantly greater cIMT and PWV. In addition, the serum aldosterone, renin and insulin levels were significantly higher in the hypertensive group. The plasma concentrations of adiponectin did not differ significantly between the two groups. TC genotype was not found in our study subjects; they all had the TT genotype of the adiponectine gene. Conclusion : The results of our study showed that adiponectin levels were not significantly different in adolescents with hypertension. There was no distinctive genetic polymorphism observed in this group of patients. Further large scale studies are needed to clarify the association between genetic variations and adiponectin in hypertensive adolescents.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.4
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• pp.219-225
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• 2004

The pelvic ganglia provide autonomic innervations to the various urogenital organs, such as the urinary bladder, prostate, and penis. It is well established that both sympathetic and parasympathetic synaptic transmissions in autonomic ganglia are mediated mainly by acetylcholine (ACh). Until now, however, the properties of ACh-induced currents and its receptors in pelvic ganglia have not clearly been elucidated. In the present study, biophysical characteristics and molecular nature of nicotinic acetylcholine receptors (nAChRs) were studied in sympathetic and parasympathetic major pelvic ganglion (MPG) neurons. MPG neurons isolated from male rat were enzymatically dissociated, and ionic currents were recorded by using the whole cell variant patch clamp technique. Total RNA from MPG neuron was prepared, and RT-PCR analysis was performed with specific primers for subunits of nAChRs. ACh dose-dependently elicited fast inward currents in both sympathetic and parasympathetic MPG neurons $(EC_{50};\;41.4\;{\mu}M\;and\;64.0\;{\mu}M,\;respectively)$. ACh-induced currents showed a strong inward rectification with a reversal potential near 0 mV in current-voltage relationship. Pharmacologically, mecamylamine as a selective antagonist for ${\alpha}3{\beta}4$ nAChR potently inhibited the ACh-induced currents in sympathetic and parasympathetic neurons $(IC_{50};\;0.53\;{\mu}M\;and\;0.22\;{\mu}M,\;respectively)$. Conversely, ${\alpha}-bungarotoxin$, ${\alpha}-methyllycaconitine$, and $dihydro-{\beta}-erythroidine$, which are known as potent and sensitive blockers for ${\alpha}7$ or ${\alpha}4{\beta}2$ nAChRs, below micromolar concentrations showed negligible effect. RT-PCR analysis revealed that ${\alpha}3$ and ${\beta}4$ subunits were predominantly expressed in MPG neurons. We suggest that MPG neurons have nAChRs containing ${\alpha}3$ and ${\beta}4$ subunits, and that their activation induces fast inward currents, possibly mediating the excitatory synaptic transmission in pelvic autonomic ganglia.

• Journal of Korean Neurosurgical Society
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• v.37 no.6
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• pp.443-448
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• 2005

Objective: A variety of genetic alterations in human glioblastoma comprises signal transduction and cell cycle arrest control of cellular processes. Subtractive hybridization is potentially a faster method for identifying differentially expressed genes associated with a particular disease state. Using the technique of subtraction, we isolated novel genes that are overexpressed in glioblastoma tissue as compared to normal brain tissue. Methods: We evaluated the differential expression of genes in each of hybridizing tester and driver cDNAs to digested 130 clones. After sequencing of 130 clones and homology search, this study performed to determine mRNA expression of the unknown gene, "clone 47", in brain tissue, glioblasoma, and several cancer cell lines by reverse transcription-polymerase chain reaction (RT-PCR). To test the time course for Go-phase arrest, serum stimulation and expression at various times for RT-PCR performed. Results: We identified 23 novel genes by BLAST of the digested 130 clones. The expressions of "clone 47" mRNA of glioblastoma and several cancer lines were significantly higher than normal brain tissues and several normal cell lines. We confirmed the mRNA expression of "clone 47" was up-regulation for $0.5{\sim}1hr$ of WI-38 cell differentiation. Conclusion: The novel gene, "Clone 47" is upregulated in glioblastoma tissue and several cancer cell lines. This gene is time dependent activation during time course of serum stimulation. This result suggests that "clone 47" playa role in brain tumorigenesis and the activation of this "clone 47" may be necessary for the development of cancer.

• Journal of Korean Neurosurgical Society
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• v.48 no.1
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• pp.20-30
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• 2010

Objective : We determined whether the expression of GRIM-19 is correlated with pathologic types and malignant grades in gliomas, and determined the function of GRIM-19 in human gliomas. Methods : Tumor tissues were isolated and frozen at $-80^{\circ}C$ just after surgery. The tissues consisted of normal brain tissue (4), astrocytomas (2), anaplastic astrocytomas (2), oligodendrogliomas (13), anaplastic oligodendrogliomas (11), and glioblastomas (16). To profile tumor-related genes, we applied RNA differential display using a $Genefishing^{TM}$ DEG kit, and validated the tumor-related genes by reverse transcription polymerase chain reaction (RT-PCR). A human glioblastoma cell line (U343MG-A) was used for the GRIM-19 functional studies. The morphologic and cytoskeletal changes were examined via light and confocal microscopy. The migratory and invasive abilities were investigated by the simple scratch technique and Matrigel assay. The antiproliferative activity was determined by thiazolyl blue Tetrazolium bromide (MTT) assay and FACS analysis. Results : Based on RT-PCR analysis, the expression of GRIM-19 was higher in astrocytic tumors than oligodendroglial tumors. The expression of GRIM-19 was higher in high-grade tumors than low-grade tumors or normal brain tissue; glioblastomas showed the highest expression. After transfection of GRIM-19 into U343MG-A, the morphology of the sense-transfection cells became larger and more spindly. The antisensetransfection cells became smaller and rounder compared with wild type U343MG-A. The MTT assay showed that the sense-transfection cells were more sensitive to the combination of interferon-$\beta$ and retinoic acid than U343MG-A cells or antisense-transfection cells; the antiproliferative activity was related to apoptosis. Conclusion : GRIM-19 may be one of the gene profiles which regulate cell death via apoptosis in human gliomas.