• The Korean Journal of Physiology and Pharmacology
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• 제3권2호
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• pp.157-164
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• 1999

To determine molecular mechanisms of Aquaporin-CD (AQP2) gene regulation, the promoter region of the AQP2 gene was examined by transiently transfecting a promoter-luciferase reporter fusion gene into mouse renal collecting duct cell lines such as mIMCD-3, mIMCD-K2, and M-1 cells, and NIH3T3 mouse embryo fibroblast cells. PCR-Southern analysis reveals that mIMCD-3 and mIMCD-K2 cells express AQP2, but M-1 and NIH3T3 cells do not, and that the treatment with cpt-cAMP $(400\;{\mu}M)$) or forskolin/isobutylmethylxanthine (IBMX) increased the AQP2 expression in IMCD cells. In both IMCD and NIH3T3 cells, the constructs containing the promoter of AQP2 gene showed promoter activities, indicating lack of tissue-specific element in the 1.4 kb 5'-flanking region of the mouse AQP2 gene. Luciferase activity in the IMCD cells transfected with the construct containing 5-flanking region showed responsiveness to cpt-cAMP, indicating that the 1.4 kb 5'-flanking region contains the element necessary for the regulatory mechanism by cAMP. The promoter-luciferase constructs which do not have a cAMP-responsible element (CRE) still showed the cAMP responsiveness in IMCD cells, but not in NIH3T3 cells. Increase in medium osmolarity did not affect AQP2 promoter activity in mIMCD-K2 cells. These results demonstrate that AQP2 gene transcription is increased with cAMP treatment through multiple motifs including CRE in the 5'-flanking region of the gene in vitro, and the regulatory mechanism may be important for in vivo regulation of AQP2 expression.

• Journal of Microbiology and Biotechnology
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• 제9권4호
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• pp.482-487
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• 1999

The Pseudomonas sp. strain NGK1 (NCIM 5120) was immobilized in calcium alginate, agar, and polyacrylamide gel matrices. The salicylic acid-producing capacity of freely suspended cells was compared with immobilized cells in batches with a shake culture and continuous culture system in a packed bed reactor. Freely suspended cells ($4\times10^{10}cfu/ml$) produced 12 mM of salicylic acid, whereas cells immobilized in calcium alginate ($1.8\times10^{11}$cfu/g beads), agar ($1.8\times10^{11}$cfu/g beads), and polyacrylamide ($1.6\times10^{11}$cfu/g beads) produced 15, 11, and 16mM of salicylic acid, respectively, from naphthalene at an initial concentration of 25 mM. The continuous production of salicylic acid from naphthalene was investigated in a continuous packed bed reactor with two different cell populations. The longevity of the salicylic acid-producing activity of the immobilized cells from naphthalene was also studied in semi continuous fermentations. The immobilized cells could be reused 18, 13, and more than 20 times without losing salicylic acid-producing activity in calcium alginate-,agar-, and polyacrylamide-entrapped cells, respectively. The study reveals a more efficient utilization of naphthalene and salicylic acid production by the immobilized Pseudomonas sp. strain NGK1 as compared to the free cells.

• 대한본초학회지
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• 제26권4호
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• pp.1-7
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• 2011

Objectives : In this study, we investigated the effect of modified-Okbyungpoongsan (mOP) on mast cell-mediated allergic response in basophilic leukemia cell line, RBL-2H3 mast cells. Methods : Cells were stimulated with anti-DNP-IgE after the treatment of DNP-HSA (AI/D), and then incubated with different concentrations of mOP (0.1, 0.2, 0.5 and 1 mg/$m{\ell}$) in RBL-2H3 cells. Cell toxicity was determined by WST-1 assay. The degranulation of mast cells was observed by microscope with toluidine blue staining and also the levels of beta-hexosaminidase, histamine and TNF-alpha were measured in culture supernatants by enzyme-linked immunosorbant assay. Results : mOP inhibited anti-DNP-IgE-imduced degranulation of mast cells in RBL-2H3 cells. mOP also significantly decreased the levels of histamine and inflammatory cytokine, TNF-alpha in RBL-2H3 cells, but slightly decreased the level of beta-hexosaminidase. Conclusions : These results indicate that mOP, an oriental prescription could be inhibit the allergic response through suppressing the mast cell activation.

• Archives of Pharmacal Research
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• 제29권8호
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• pp.645-650
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• 2006

Tributyltin chloride (TBTC) at concentrations of $0.5-1.0\;{\mu}M$ inhibits dopamine biosynthesis in PC12 cells. In this study, the effects of TBTC on L-3,4-dihydroxyphenylalanine (L-DOPA)-induced cytotoxicity in PC12 cells were investigated. TBTC at concentrations up to $1.0\;{\mu}M$ neither affected cell viability, nor induced apoptosis after 24 or 48 h in PC12 cells. However, TBTC at concentrations higher than $2.0\;{\mu}M$ caused cytotoxicity through an apoptotic process. In addition, exposure of PC12 cells to non-cytotoxic (0.5 and $1.0\;{\mu}M$) or cytotoxic $(2.0\;{\mu}M)$ concentrations of TBTC in combination with L-DOPA (20, 50 and $100\;{\mu}M$) resulted in a significant increase in cell loss and the percentage of apoptotic cells after 24 or 48 h compared with TBTC or L-DOPA alone. The enhancing effects of TBTC on L-DOPA-induced cytotoxicity were concentration- and treatment time-dependent. These data demonstrate that TBTC enhances L-DOPA-induced cytotoxicity in PC 12 cells.

• 대한약침학회지
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• 제22권4호
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• pp.253-259
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• 2019

Objectives: It is reported that tumor-associated macrophages (TAMs) contribute to cancer progression by promoting tumor growth and metastasis. The purpose of this study is to investigate the effect of different fractions of Adenophora triphylla var. japonica (AT) on the polarization of macrophages into the M2 phenotype, a major phenotype of TAMs. Methods: We isolated hexane, ethyl acetate, and butanol fractions from crude ethanol extract of AT. The cytotoxicity of AT in RAW264.7 cells was examined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RAW264.7 cells were polarized into the M2 phenotype by treatment with interleukin (IL)-4 and IL-13. The expression of M2 macrophage marker genes was detected by reverse transcription polymerase chain reaction (RT-PCR). The phosphorylation level of signal transducer and activator of transcription 6 (STAT6) was investigated by western blot analysis. The migration of Lewis lung carcinoma (LLC) cells was examined by transwell migration assay using conditioned media (CM) collected from RAW264.7 cells as a chemoattractant. Results: Among various fractions of AT, the ethyl acetate fraction of AT (EAT) showed the most significant suppressive effect on the mRNA expression of M2 macrophage markers, including arginase-1, interleukin (IL)-10 and mannose receptor C type 1 (MRC-1), up-regulated by treatment of IL-4 and IL-13. In addition, EAT suppressed the phosphorylation of STAT6, a critical regulator of IL-4 and IL-13-induced M2 macrophage polarization. Finally, the increased migration of Lewis lung carcinoma (LLC) cells by CM from M2-polarized RAW264.7 cells was reduced by CM from RAW264.7 cells co-treated with EAT and M2 polarization inducers. Conclusion: We demonstrated that EAT attenuated cancer cell migration through suppression of macrophage polarization toward the M2 phenotype. Additional preclinical or clinical researches are needed to evaluate its regulatory effects on macrophage polarization and anti-cancer activities.

• 대한한방내과학회지
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• 제22권4호
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• pp.589-599
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• 2001

Objective: This study was carried out to investigate the effects of Hyangsayukgunja-tang extract on indomethacin-induced gastric mucosal lesions of mice. Methods: To evaluate the effects of Hyangsayukgunja-tang extract and Misoprostol, the morphology of gastric mucosa, and the distribution of mucose cells, PNA(Peanut Agglutinin), ICAM(intercellular adhesion molecule), and apoptotic cells were observed. Hyangsayukgunja-tang extract and Misoprostol were intragastric injected to the test groups at hour 72 before and just before indomethacin treatment(HYT-J, HYT-72, M-J, M-72), while the INDO group was injected only with indomethacin and the control group was subcutaneously injected only with saline. Results: The gastric mucosal lesions incresed in the fundus and body of INDO group, but softened in HYT group and M group, the effects were more excellent in the HYT-72, M-72 groups than the HYT-J, M-J groups and in the HYT group than M group. The disappearance of surface and neck mucose cells were shown in INDO group, but softened in HYT group and M group. The mucosal configuration of HYT-72 group was the same as control group. The numerical increase of PNA positive reaction in cytoplasm of perietal cells were appeared in INDO group. The PNA positive reaction in HYT group and Miso-group were shown in surface mucous cells and microvilli of apical surface in chief cells as control group, and were the same as control group in all mucosa of HYT-72 group. The distribution of ICAM positive cells, increased in INDO group, but decreased in M-72 group, and were the same as control group in HYT-72 group. The apoptotic cells, increased noticeably in gastric mucosa of INDO group, decreased in HYT group and M group, and decreased noticeably in HYT-72 group. Conclusions: Hyangsayukgunja-tang extract had excellent effects on indomethacin-induced gastric mucosal lesions.

• 대한수의학회지
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• 제42권2호
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• pp.209-217
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• 2002

This study was performed to produce monoclonal antibodies (mAb) specifically reacting with chicken leukocyte surface antigens. Popliteal lymph node cells of BALB/c mice previously immunized through foot-pad with peripheral blood mononuclear cells (PBMC) of chickens separated by Ficoll-Histopaque method. They were fused with P3X63Ag14 mouse myeloma cells. A total of 34 hybridomas secreted antibodies specifically binding to the PBMC. According to the reactivity patterns with PBMC, the mAbs were divided into 4 groups. Group 1 mAbs (IIB3, IIB10, IIE10) specifically reacted with non-adherent lymphocytes but not with adherent cells which were mainly composed of thrombocytes and monocytes in PBMC culture. These mAbs were reactive with 25-59% of thymus cells and 42-64% of spleen cells of chickens. They did not show any significant reactivity with cells in the bursa of Fabricius, T-cell (MDCC-MSB1) and B-cell (LSCC-1104B1) lines. These results indicate that Group I mAbs specifically reacted with T-lymphocyte subpopulation. Monoclonal antibodies in Group II (IC6, IG2-2 and IID9) showed specific reactivity with monocytes but not with thrombocytes or non-adherent cells in PBMC culture. These mAbs, though not reacted with the chicken macrophage cell line, HD11, also bound to macrophages of the spleen and lung in immunohistochemical staining. Five mAbs in Group III showed characteristics of binding to lymphocytes and monocytes, but not to thrombocytes. Twenty-three mAbs in Group IV showed specific reactivity to lymphocytes, monocytes, and thrombocytes. Two mAbs (IC3 and IE9) in Group IV reacted with most of PBMC.

• The Korean Journal of Physiology and Pharmacology
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• 제5권3호
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• pp.231-241
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• 2001

The mammalian cortical collecting duct (CCD) plays a major role in regulating renal NaCl reabsorption, which is important in $Na^+$ and $Cl^-$ homeostasis. The M-1 cell line, derived from the mouse cortical collecting duct, has been used as a mammalian model of the study on the electrolytes transport in CCD. M-1 cells were grown on collagen-coated permeable support and short circuit current $(I_{sc})$ was measured. M-1 cells developed amiloride-sensitive current $5{\sim}7$ days after seeding. Apical and basolateral addition of ATP induced increase in $I_{sc}$ in M-1 cells, which was partly retained in $Na^+-free$ or $Cl^--free$ solution, indicating that ATP increased $Na^+$ absorption and $Cl^-$ secretion in M-1 cells. $Cl^-$ secretion was mediated by the activation of apical cystic fibrosis transmembrane regulator (CFTR) chloride channels and $Ca^{2+}-activated$ chloride channels, but $Na^+$ absorption was not mediated by activation of epithelal sodium channel (ENaC). ATP increased cAMP content in M-1 cells. The RT-PCR analysis demonstrated that M-1 cells express $P2Y_2,\;P2X_3\;and\;P2Y_4$ receptors. These results showed that ATP regulates $Na^+$ and $Cl^-$ transports via multiple P2 purinoceptors on the apical and basolateral membranes in M-1 cells.

• Applied Microscopy
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• 제31권1호
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• pp.101-108
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• 2001

아프리카 왕달팽이 (Achatina fulica)의 내장신경절(visceral ganglion)과 우 체 벽신경절(right parietal ganglion)을 투과전자현미경을 통해 관찰한 결과 5종류(type-A, B, C, D 그리고 E)의 신경분비세포(neurosecretory cell)와 그 주위를 둘러싸고 있는 신경망 등이 관찰되었다. A형 세포(직경 $35{\mu}m$)는 두 신경절의 피질부에서 가장 많이 관찰된 삼각형 또는 불규칙한 세포로서, 세포질에는 직경 $1{\mu}m$인 큰 과립과 $0.1{\mu}m$정도인 작은 둥근과립들이 관찰되었다. 또한 작은 과립들은 전자밀도가 높은 과립과 전자밀도가 중등도인 과립 등 두 종류로 구분되었다. B형 세포(직경 $19\times12{\mu}m$)는 두 신경절의 피질부와 수질부의 여러 부위에서 고르게 관찰된 세포로서 A형 신경분비세포와 그 형태가 비슷하였다. 세포질 내에는 $0.1{\mu}m$정도 크기의 전자밀도가 높은 과립들로 가득찬 반면 둥글고 큰 과립(직경 $0.7{\mu}m$정도)들은 드물게 관찰되었다. C형 세포는 크기가 $8\times6{\mu}m$정도인 가장 작은 세포로서, $6\times5{\mu}m$정도인 큰 핵을 소지하고 있었다. 세포질에는 $0.23{\mu}m$정도인 전자밀도가 높은 과립들로 가득차 있었는데, 이들은 $0.03{\mu}m$정도 크기의 작은 과립들이 둥글게 모여 있는 특이한 형태였다. D형 세포는 $28\times20{\mu}m$정도 크기의 중형세포서, 타원형 또는 불규칙한 형태를 보였다. 이들은 두 신경절의 수질부와 피질부 중, 피질부에서 가장 많은 수가 관찰되었다. 세포질은 전자밀도가 높아 어둡게 관찰되고 직경 $1.6{\mu}m$와 $0.6{\mu}m$인 두 종의 둥근 과립들이 관찰되었다. E형 세포는 크기가 $100\times50{\mu}m$정도인 대형세포로서 두 신경절의 상단부와 중앙부위에서 드물게 나타났다. 핵은 $70\times30{\mu}m$정도로 세포질 대비 매우 컸다. 이들은 다양한 크기의 전자밀도가 높은 둥근 과립(직경 $1\sim0.2{\mu}m$)을 소지하고 있었으며, 세포의 표면은 여러 형태의 사상족(filopodia)들을 뻗어 노쇠한 세포들을 포식하였다. 신경망(neuropiles)들은 신경분비세포를 둘러싸고 있었으며, 신경섬유 속에서 다양한 종류의 연접소포들(synaptic vesicles)이 관찰되었는데, 전자밀도, 크기 그리고 모양에 따라 6종류로 분류되었다.

• The Korean Journal of Physiology
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• 제29권1호
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• pp.69-80
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• 1995

Renal proximal tubular hypertrophy and hyperfunction are known to be early manifestations of experimental and human diabetes. As the hypertrophy and hyperfunction have been suggested to be central components in the progression to renal failure, an understanding of their underlying causes is potentially important for the development of therapy. A primary rabbit kidney proximal tubule cell culture system was utilized to evaluate the possibility that the renal proximal tubular hypertrophy and hyperfunction observed in vivo in diabetes mellitus, can be attributed to effects of elevated glucose levels on membrane transport systems. Primary cultures of rabbit proximal tubules, which achieved confluence at 10 days, exhibited brush-border characteristics typical of proximal tubular cells. Northern analysis indicated $2.2{\sim}2.3$ and 2.0 kb Na/glucose cotransporter RNA species appeared in fresh and cultured proximal tubule cells after confluence, repectively. The cultured cells showed reduced Na/glucose cotransporter activity compared to fresh proximal tubules. Primary cultured proximal tubule cells incubated in medium containing 20 mM glucose have reduced ${\alpha}-MG$ transport compared to cells grown in 5 mM glucose. In the proximal tubule cultures incubated in medium containing 5 mM or 20 mM glucose, phlorizin at 0.5 mM inhibited 0.5 mM ${\alpha}-MG$ uptake by 84.35% or 91.85%, respectively. The uptake of 0.5 mM ${\alpha}-MG$ was similarly inhibited by 0.1 mM ouabain (41.97% or 48.03% inhibition was observed, respectively). In addition, ${\alpha}-MG$ uptake was inhibited to a greater extent when $Na^{+}$ was omitted from the uptake buffer (81.86% or 86.73% inhibition was observed, respectively). In cell homogenates derived from the primary cells grown in 5 mM glucose medium, the specific activity of the Na/K-ATPase $(6.17{\pm}1.27\;{\mu}mole\;Pi/mg\;protein/hr)$ was 1.56 fold lower than the values in cell homogenates treated with 360 mg/dl D-glucose, 20 mM $(9.67{\pm}1.22\;{\mu}mole\;Pi/mg\;protein/hr)$. Total $Rb^{+}$ uptake occurred at a significantly higher rate (1.60 fold increase) in primary cultured rabbit kidney proximal tubule cell monolayers incubated in 20 mM glucose medium $(10.48{\pm}2.45\;nM/mg\;protein/min)$ as compared with parallel cultures in 5 mM glucose medium. $Rb^{+}$ uptake rate in 5 mM glucose medium was reduced by 28% when the cultures were incubated with 1 mM ouabain. The increase of the $Rb^{+}$ uptake by rabbit kidney proximal tubule cells in 20 mM glucose could be attributed primarily to an increase in the rate of ouabain-sensitive $Rb^{+}$ uptake $(5\;mM\;to\;20\;mM;\;4.68{\pm}0.85\;to\;8.38{\pm}1.37\;nM/mg\;protein/min)$. In conclusion, the activity of the renal proximal tubular Na,K-ATPase is elevated in high glucose concentration. In contrast, the activity of the Nafglucose cotransport system is inhibited.