• 대한약리학회지
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• 제18권2호
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• pp.1-14
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• 1982

중추신경계의 혈관폐쇄 또는 충격손상에 의한 허혈병소에서 진행되는 병리적 변화에 산소유리라디칼이 중요한 역할을 할 것으로 시사되고 있다. 저자는 산소유리라디칼이 뇌조직에 미치는 영향 중 특히 신경세포 정지막전위 유지에 중요한 $Na^+-K^+-ATPase$의 활성도에 미치는 영향을 관찰하기 위하여 산틴산화효소 반응계와 뇌조직미크로좀을 이용하여 본 연구를 시행하였다. 미크로좀분획(microsomal fraction)을 산틴과 산틴산화효소와 함께 반응시켰을 때, 분획의 $Na^+-K^+-ATPase$의 활성도는 현저한 불활성화를 보인 반면, $Mg^{++}-ATPase$의 활성도는 별로 영향을 받지 않았다. 이 불활성화는 산틴과 신틴산화효소 중 어느 한 물질이라도 반응계에 존재하지 않는 경우에는 나타나지 않았고, 두 물질이 같이 반응계에 존재할 때 나타났다. 산틴과 산틴산화효소의 반응에서 생성되는 산소유리라디칼들 중, 어떤 것이 $Na^+-K^+-ATPase$불활성화에 관계하고 있는가를 알아보기 위하여, 산수유리라디칼 각각에 대하여 제독작용을 가진 효소나 화학물질을 사용하여 불활성화의 저해유무를 관찰하였다. $O_{\bar{2}}{\cdot}$의 제독효소인 superoxide dismutase, $H_2O_2$의 제독효소인 catalase와 $^1O_2$의 제독물질인 1,4-diazabixyclo(2,2,2)octane을 각각 사용하였을 때, 이들 물질들이 농도에 비례하여 $Na^+-K^+-ATPase$의 불활성화가 저해되었다. 그러나 $OH{\cdot}$의 제독물질인 mannitol은 뚜렷한 효과를 보이지 못했다. 이상의 결과는 산소유리라디칼들 중 $O_{\bar{2}}{\cdot},\;H_2O_2$ 및 $^1O_2$가 $Na^+-K^+-ATPase$의 불활성화에 관계하고 있고, $OH{\cdot}$는 거의 관계하지 않는다는 것을 시사하여 주었다. 이로 미루어, 산소유리라디칼에 의한 뇌조직 $Na^+-K^+-ATPase$의 불활성화는 뇌 허혈병소에서 관찰되는 신경세로의 기능적 장해를 유발시키는 한 요인으로 사료되었다.

• Journal of Ginseng Research
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• 제23권2호
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• pp.105-114
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• 1999

본 실험은 백서의 간세포막에서 transferrin receptor(TfR)의 발현에 미치는 인삼 및 3'-methyl-4-dimethylaminobenzene(3'-Me-DAB)의 영향을 연구하기 위하여, 인삼, 3'-Me-DAB 또는 3'-Me-DAB처치후 인삼을 투여한 백서를 부분 간절제한 후 간조직에서 $[^{3}H]thymidine$및 transferrin수용체의 유전자 발현 실험, 그리고 간세포막에서$[^{3}H]thymidine$ 결합 실험을 시행하여 다음과 같은 결과를 얻었다. 1. 8주간 3'-Me-DAB를 투여한 백서에서 간조직의 조직학적 소견은 림프구의 침윤, 담관세포의 증식,교량성괴사 및 이상세포의 증식 소견을 보였다. 그러나 정상 또는 3'-Me-DAB를 투여한 백서에 인삼을 투여하였을 때 간조직의 조직학적 소견은 각각의 대조군에 비해 크게 변화되지 않았다. 2. 인삼 또는 3'-Me-DAB를 투여한 백서에서의 $[^{3}H]thymidine$ uptake는 대조군에서와 비슷하였으나, 부분간절제 수술 후 1일째 간에서는 수술 전보다 현저하게 증가되었다. 특히 3'-Me-DAB투여군의 간조직에서 더 증가되었으며, 이는 인삼 투여에 의해 억제되는 경향을 보였다. 3. 간세포막에서의 transferrin 결합량(Bmax) 및 Kd는 정상 백서에서 각각 6.87pmole/mg protein 및 10.64nM이었으며, 인삼 투여에 의해 크게 변동되지 않았다 그러나 3'-Me-DAB를 투여한 백서에서는 Bmax 및 Kd가 증가되었고, 이 증가는 인삼투여에 의해 감소되었다. 부분 간절제 수술 후 간세포막에서의 Bmax 및 Kd는 모든 그룹에서 수술 후 3일째에는 증가, 수술 후 5일째에는 감소되는 양상을 나타냈다. 그러나 3'-Me-DAB투여 후 간절제 수술군에서의 증가가 가장 컸으며, 이 증가효과는 인삼 투여에 의하여 억제되었다. 4. 부문 간절제 수술 후 간조직에서의 TfR mRrfA 발현은 수술 후 24시간에 최대로 증가되었으며, 이는 3'-Me-DAB를 투여한 백서에서 더 현저하였다. 그리고 인삼은 3'-Me-DAB투여 후 간절제 수술군에서 증가된 발현양을 약간 감소시켰다. 이상의 결과로 백서에서 부분 간절제 수술 및 발암 물질에 의해 간세포막 transffirin수용체는 친화력이 저하된 TfR이 증가될 수 있으며, 이는 일부 이 수용체의유전자 수준에서 조절될 수 있는 것으로 생각된다. 그리고 인삼은 발암물질 투여등으로 간세포의 증식이 촉진되었을 때, 간세포내 핵산 합성 및 TfR mRNA 발현 조절을 통하여 세포막의 TfR출현 및 세포증식을 억제시킬 수 있을 것으로 추측된다.

• Journal of Periodontal and Implant Science
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• 제32권4호
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• pp.865-873
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• 2002

The primary cause of tooth loss after 30 years of age is periodontal disease. Destruction of alveolar bone by periodontal disease is done by bone resorbing activity of osteoclasts. Understanding differentiation and activation mechanism of osteoclasts is essential for controling periodontal disease. The purpose of this study is to identify the possible effects of Vitamin D and cytokines affecting osteoclasts and its precursor cells. Four to six week-old mice were killed and humerus, radius, tibia and femur were removed aseptically and washed two times with Hank's solution containing penicillin-streptomycin and then soft tissue were removed. Bone marrow cells were collected by 22 gauge needle. Cells were cultured in Hank's solution containing 1 mg/ml type II collagenase, 0.05% trypsin, 41mM EDTA. Supernatant solution was removed 5 times after 15 minutes of digestion with above mentioned enzyme solution, and remained bone particles were maintained in alpha-MEM for 15 minutes and $4^{\circ}C$ temperature. Bone particles were agitated for 1 minute and supernatant solution containing osteoclast precursor cells were filtrated with cell stainer. These separated osteoclast precursor cells were dispensed with 100-mm culture dish by $1{\times}10^7$ cells unit and cultured in ${\alpha}$- MEM containing 20 ng/ml recombinant human M-CSF, 30 ng/ml recombinant human soluble osteoclast differentiation factor and 10% fetal calf serum for 2 and 7 days. Total RNA of osteoclast precursor cells were extracted using RNeasy kit. One ${\mu}g$ of total RNA was reverse transcribed in $42^{\circ}C$ for 30 minutes using SuperScriptII reverse transcriptase. Expression of transcribed receptors of each hormone and cytokine were traced with 1 ${\mu}l$ of cDNA solution by PCR amplification. Vitamin D receptor WAS found in cells cultured for 7 days. TNF-${\alpha}$ receptor was found in cells cultured for 2 days and amount of receptors were increased by 7 days. IL-1 type I receptor was not found in cells cultured 2 and 7 days. But, IL-1 receptor type II was found in cells cultured for 2 days. TGF-${\alpha},{\beta}$type I receptor was found in cells cultured 2 and 7 days, and amount of receptors were increased by 7 days of culture. These results implies Vitamin D and cytokines can affect osteoclasts directly, and affecting period in differentiation cycle of osteoclasts is different by Vitamin D and cytokines.

• The Korean Journal of Physiology and Pharmacology
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• 제16권1호
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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.

• The Korean Journal of Physiology and Pharmacology
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• 제8권4호
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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.

• The Korean Journal of Physiology and Pharmacology
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• 제19권6호
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• pp.543-547
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• 2015

We investigated the combined moisturizing effect of liposomal serine and a cosmeceutical base selected in this study. Serine is a major amino acid consisting of natural moisturizing factors and keratin, and the hydroxyl group of serine can actively interact with water molecules. Therefore, we hypothesized that serine efficiently delivered to the stratum corneum (SC) of the skin would enhance the moisturizing capability of the skin. We prepared four different cosmeceutical bases (hydrogel, oil-in-water (O/W) essence, O/W cream, and water-in-oil (W/O) cream); their moisturizing abilities were then assessed using a $Corneometer^{(R)}$. The hydrogel was selected as the optimum base for skin moisturization based on the area under the moisture content change-time curves (AUMCC) values used as a parameter for the water hold capacity of the skin. Liposomal serine prepared by a reverse-phase evaporation method was then incorporated in the hydrogel. The liposomal serine-incorporated hydrogel (serine level=1%) showed an approximately 1.62~1.77 times greater moisturizing effect on the skin than those of hydrogel, hydrogel with serine (1%), and hydrogel with blank liposome. However, the AUMCC values were not dependent on the level of serine in liposomal serine-loaded hydrogels. Together, the delivery of serine to the SC of the skin is a promising strategy for moisturizing the skin. This study is expected to be an important step in developing highly effective moisturizing cosmeceutical products.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2001년도 추계학술대회 및 정기총회
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• pp.102-102
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• 2001

Taurine (2-ethaneaminosulfonic acid) is one of the major intracellular ${\beta}$ -amino acids in mammals and is required for a number of biological processes including membrane stabilization, osmoregulation, antioxidation, detoxification, modulation of calcium flux and neurornodulation. The taurine transporter (TAUT) which contains 12 hydrophobic membrane-spanning domains has been cloned from dog kidney, rat brain, mouse brain, human thyroid, placenta and retina. In this study, The TAUT cDNA from the human intestinal epithelial cell, HT-29 was cloned and sequenced. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to amplify partial cDNA encoding human intestinal TAUT. The coding region of the PCR product was 732 bp long. The primers were designed to encode highly conserved amino acid sequences near the transmembrane domains III (IPYFIFLF) and Ⅵ (KYKYNSYR) both in human and mouse. The TAUT cDNA amplified was ligated into the pGEX 4T-1 expression vector. The resulting sequence of human intestinal TAUT cDNA (Accession number of NCBI Genebank is AF346763) was identical to the sequences of the TAUTs previously determined in the human placenta and retina except 3 base pairs from that of the reported human thyroid. TAUT specific antibodies were generated to use them as biological tools in the studies of the biological role of TAUT. Peptides of 149-162 amino acid residue (14 amino acids) of the TAUT were synthesized. The synthetic peptide used in this study was LFQSFQKELPWAHC. This region was chosen not only to avoid putative glycosylation sites but also to exclude regions of known homology with GABA transporters in the extracellular hydrophilic domains. The synthetic peptide, TAUT-1 was conjugated with carrier protein, kehole lympet hemocyanin (KLH) to use as an antigen. When used for immunization on a rabbit to produce polyclonal antiserum, the conjugates elicited high -titered specific anti-TAUT-1 antibodies, which reacted well with the ovalbumin (OVA) conjugated peptides in ELISA. The KLH-conjugated peptide was also used as immunizing antigen in BALB/c mice to produce TAUT specific monoclonal antibodies. From the culture supernatant of the hybridoma, the specificity of anti-TAUT-1 monoclonal antibodies was confirmed by ELISA. Further applications of more tools in TAUT expression analysis will be performed such as western blotting and flow cytometry.

• Asian Pacific Journal of Cancer Prevention
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• 제15권8호
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• pp.3731-3736
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• 2014

Phytic acid (PA) has been reported to have positive nutritional benefits and prevent cancer formation. This study investigated the anticancer activity of rice bran PA against hepatocellular carcinoma (HepG2) cells. Cytotoxicty of PA (0.5 to 4mM) was examined by MTT and LDH assays after 24 and 48h treatment. Apoptotic activity was evaluated by expression analysis of apoptosis-regulatory genes [i.e. p53, Bcl-2, Bax, Caspase-3 and -9] by reverse transcriptase-PCR and DNA fragmentation assay. The results showed antioxidant activity of PA in Fe3+ reducing power assay ($p{\leq}0.03$). PA inhibited the growth of HepG2 cells in a concentration dependent manner ($p{\leq}0.04$). After 48h treatment, cell viability was recorded 84.7, 74.4, 65.6, 49.6, 36.0 and 23.8% in MTT assay and 92.6, 77.0%, 66.8%, 51.2, 40.3 and 32.3% in LDH assay at concentrations of 1, 1.5, 2.0, 2.5, 3.0, and 3.5mM, respectively. Hence, treatment of PA for 24h, recorded viability of cells 93.5, 88.6, 55.5, 34.6 and 24.4% in MTT assay and 94.2, 86.1%, 59.7%, 42.3 and 31.6%, in LDH assay at concentrations of 1, 2.2, 3.0, 3.6 and 4.0mM, respectively. PA treated HepG2 cells showed up-regulation of p53, Bax, Caspase-3 and -9, and down-regulation of Bcl-2 gene ($p{\leq}0.01$). At the $IC_{50}$ (2.49mM) of PA, the p53, Bax, Caspase-3 and-9 genes were up-regulated by 6.03, 7.37, 19.7 and 14.5 fold respectively. Also, the fragmented genomic DNA in PA treated cells provided evidence of apoptosis. Our study confirmed the biological activity of PA and demonstrated growth inhibition and induction of apoptosis in HepG2 cells with modulation of the expression of apoptosis-regulatory genes.

• The Korean Journal of Physiology and Pharmacology
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• 제20권1호
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• pp.9-14
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• 2016

Adenosine 3',5'-cyclic monophosphate (cAMP) participates in the regulation of numerous cellular functions, including the $Na^+-K^+$-ATPase (sodium pump). Ouabain, used in the treatment of several heart diseases, is known to increase cAMP levels but its effects on the atrium are not understood. The aim of the present study was to examine the effect of ouabain on the regulation of atrial cAMP production and its roles in atrial endothelin-1 (ET-1) secretion in isolated perfused beating rabbit atria. Our results showed that ouabain ($3.0{\mu}mol/L$) significantly increased atrial dynamics and cAMP levels during recovery period. The ouabain-increased atrial dynamics was blocked by KB-R7943 ($3.0{\mu}mol/L$), an inhibitor for reverse mode of $Na^+-Ca^{2+}$ exchangers (NCX), but did not by L-type $Ca^{2+}$ channel blocker nifedipine ($1.0{\mu}mol/L$) or protein kinase A (PKA) selective inhibitor H-89 ($3.0{\mu}mol/L$). Ouabain also enhanced atrial intracellular cAMP production in response to forskolin and theophyline ($100.0{\mu}mol/L$), an inhibitor of phosphodiesterase, potentiated the ouabain-induced increase in cAMP. Ouabain and 8-Bromo-cAMP ($0.5{\mu}mol/L$) markedly increased atrial ET-1 secretion, which was blocked by H-89 and by PD98059 ($30{\mu}mol/L$), an inhibitor of extracellular-signal-regulated kinase (ERK) without changing ouabain-induced atrial dynamics. Our results demonstrated that ouabain increases atrial cAMP levels and promotes atrial ET-1 secretion via the mitogen-activated protein kinase (MAPK)/ERK signaling pathway. These findings may explain the development of cardiac hypertrophy in response to digitalis-like compounds.

• The Korean Journal of Physiology and Pharmacology
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• 제18권4호
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• pp.333-339
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• 2014

Exendin-4 (Ex-4), a glucagon-like peptide-1 receptor (GLP-1R) agonist, has been known to reverse hepatic steatosis in ob/ob mice. Although many studies have evaluated molecular targets of Ex-4, its mechanism of action on hepatic steatosis and fibrosis has not fully been determined. In the liver, glucose transporter 4 (GLUT4) is mainly expressed in hepatocytes, endothelial cells and hepatic stellate cells (HSCs). In the present study, the effects of Ex-4 on GLUT4 expression were determined in the liver of ob/ob mice. Ob/ob mice were treated with Ex-4 for 10 weeks. Serum metabolic parameters, hepatic triglyceride levels, and liver tissues were evaluated for hepatic steatosis. The weights of the whole body and liver in ob/ob mice were reduced by long-term Ex-4 treatment. Serum metabolic parameters, hepatic steatosis, and hepatic fibrosis in ob/ob mice were reduced by Ex-4. Particularly, Ex-4 improved hepatic steatosis by enhancing GLUT4 via GLP-1R activation in ob/ob mice. Ex-4 treatment also inhibited hepatic fibrosis by decreasing expression of connective tissue growth factor in HSCs of ob/ob mice. Our data suggest that GLP-1 agonists exert a protective effect on hepatic steatosis and fibrosis in obesity and type 2 diabetes.