• Journal of Life Science
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• v.19 no.5
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• pp.573-580
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• 2009

It has been previously reported that the carboxyl terminal residues 276 and 277 of ${\alpha}$-tropomyosin are important for actin affinity. In order to investigate actin affinities of these two residues of skeletal (HA) and smooth (QT) muscle ${\alpha}$-tropomyosins, a series of mutant tropomyosins were constructed in which residues at either 276 or 277 were individually replaced with a cysteine residue for chemical modification. These mutants were overexpressed in E. coli as unacetylated and Ala-Ser (AS) dipeptide fusion forms. While actin affinities of unacetylated tropomyosins were considerably low, those of AS/TMs were remarkably higher than those of corresponding unacetylated tropomyosins. However, actin affinities of AS/TM24 (QC) and AS/TM29 (HC) were dramatically lower than those of other AS/TMs and were close to those of unacetylated tropomyosins. In addition, actin affinities of unacetylated TM24 (QC) and TM29 (HC) failed to be restored in the presence of troponin, unlike unacetylated TM10 (HA) and TM23 (CA). These results indicated that the presence of a cysteine residue at 277 caused a drastic decrease in actin affinity, and also that the residue 277 is important for actin affinity of ${\alpha}$-tropomyosin. Since TM23 (CA) showed high actin affinity, it may serve as a valuable tool for chemical modification studies for investigating the interaction of the carboxyl terminal residues of ${\alpha}$-tropomyosin with actin and/or troponin.

• Herbal Formula Science
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• v.26 no.3
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• pp.183-193
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• 2018

Objectives : In Traditional Korean Medicine, Epimedium koreanum Nakai has diverse pharmacological activities to treat impotence, forgetfulness, cataract and exophthalmos. Present study investigated anti-fibrogenic effects of E. koreanum water extract (EKE) in hepatic stellate cells (HSCs). Methods : To study anti-fibrogenic effects of EKE, LX-2 cells, a human immortalized HSCs, were pre-treated with $3-300{\mu}g/mL$ of EKE, and then subsequently exposed to 5 ng/mL of transforming growth $factor-{\beta}1$ ($TGF-{\beta}1$). Expression level of ${\alpha}-smooth$ muscle actin was determined by immunoblot analysis. Phosphorylation of Smad, transactivation of Smad, and expression of plasminogen activator inhibitor-1 (PAI-1) were monitored to investigate the effect of EKE on $TGF-{\beta}1-mediated$ signaling pathway. Results : Up to $100{\mu}g/mL$, EKE did not show any cytotoxicity on LX-2 cells. Pre-treatment of EKE ($100{\mu}g/mL$) significantly inhibited ${\alpha}-smooth$ muscle actin expression induced by $TGF-{\beta}1$. In addition, EKE significantly decreased Smad2 and Smad3 phosphorylations, Smad binding element-driven luciferase activity and PAI-1 expression by $TGF-{\beta}1$. Of three flavonoid compounds found in EKE, only quercertin ($30{\mu}M$) attenuated $TGF-{\beta}1-mediated$ PAI-1 expression. Conclusion : These results suggest that EKE has an ability to suppress fibrogenic process in HSCs via inhibition of $TGF-{\beta}1/Smad$ signaling pathway.

• Journal of Ginseng Research
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• v.37 no.1
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• pp.45-53
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• 2013

Korean red ginseng, the processed root of Panax ginseng Meyer, has been frequently used for various therapeutic purposes in oriental medicine. The present study investigated the possible effect of Korean red ginseng extract (RGE) for the treatment of liver fibrosis in mice injected with carbon tetrachloride ($CCl_4$) for 4 wk. Liver injuries were assessed by blood biochemistry and histopathology in mice treated with $CCl_4$ alone or $CCl_4$+ RGE (30, 100, and 300 mg/kg). Concomitant treatment with RGE and $CCl_4$ (three times/wk for 4 wk) effectively inhibited liver fibrosis as evidenced by decreases in plasma alanine and aspartate aminotransferases, as well as by the percentages of degenerative regions, numbers of degenerative hepatocytes, and collagen accumulation in hepatic parenchyma. Treatment with $CCl_4$ for 4 wk increased mRNA levels of transforming growth factor ${\beta}1$ and plasminogen activator inhibitor 1 in fibrogenic liver, whereas RGE (30, 100, and 300 mg/kg) significantly blocked the induction of fibrogenic genes by $CCl_4$. Similarly, RGE also prevented transforming growth factor ${\beta}1$-mediated induction of fibrogenic genes in human hepatic stellate cell lines. More importantly, RGE markedly reduced the number of ${\alpha}$-smooth muscle actin-positive cells in liver tissue. This study implies that RGE efficaciously protects against the liver fibrosis induced by chronic $CCl_4$ treatment, and may therefore have potential to treat liver disease.

• Archives of Pharmacal Research
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• v.24 no.4
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• pp.327-332
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• 2001

The antifibrotic effects of hot water extract (WEC), intracellular biopolymer (IPC) and extracellular biopolymers (EPC) from mycelial liquid culture of Cordyceps militaris on liver fibrosis were studied. Liver fibrosis was induced by a bile duct ligation and scission (BDL/S) operation, duration of 4 weeks in rats. In BDL/S rats, the levels of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total bilirubin in serum and hydroxyproline content in liver were dramatically increased. The WEC or IPC treatment (30mg/kg/day for 4 weeks, p.o.) in BDL/S rats reduced the serum AST, ALT and ALP levels significantly (p<0.01). The EPC treatment (30 mg/kg /day for 4 weeks, p.o.) reduced the serum ALT, AST and ALP levels significantly (p<0.01). Malondialdehyde contents in liver treated with WEC, IPC or EPC were significantly reduced (p <0.05). But Liver hydroxyproline content was decreased only in EPC treated BDL/S rats to 55% that of BDL/S control rats (p < 0.01). The morphological characteristics and expression of alpha smooth muscle like actin in fibrotic liver, which appeared in BDL/S control group were improved in EPC treated fibrotic liver. These results indicate that IPC (30 mg/kg /day for 4 weeks, p.o.) has an antifibrotic effect on fibrotic rats induced by BDL/S.

• Journal of Life Science
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• v.21 no.2
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• pp.183-193
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• 2011

Migration and differentiation of mesenchymal stem cells are crucial for tissue regeneration in response to injury. Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates a variety of biological processes, including proliferation, survival, differentiation and motility. In the present study, we determined the role of S1P in migration and differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs). S1P stimulated migration of BMSCs in a dose- and time-dependent manner, and pre-incubation of the cells with pertussis toxin completely abrogated S1P-induced migration, suggesting involvement of Gi-coupled receptors in S1P-induced cell migration. S1P elicited elevation of intracellular concentration of $Ca^{2+}$ ($[Ca^{2+}]_i$) and pretreatment with VPC23019, an antagonist of $S1P_1/S1P_3$, blocked S1P-induced migration and increase of $[Ca^{2+}]_i$. Small interfering RNA-mediated knockdown of endogenous $S1P_1$ attenuated S1P-induced migration of BMSCs. Furthermore, S1P treatment induced expression of $\alpha$-smooth muscle actin ($\alpha$-SMA), a smooth muscle marker, and pretreatment with VPC23019 abrogated S1P-induced $\alpha$-SMA expression. S1P induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), and pretreatment of cells with SB202190, an inhibitor of p38 MAPK, or adenoviral overexpression of a dominant-negative mutant of the p38 MAPK blocked S1P-induced cell migration and $\alpha$-SMA expression. Taken together, these results suggest that S1P stimulates migration and smooth muscle differentiation of BMSCs through an $S1P_1$-p38 MAPK-dependent mechanism.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.707-714
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• 2004

How to improve the cell culture method on scaffolds is important in the tissue engineering fileld. In this study, we optimized seeding and culture methods to vascular smooth muscle cells (VSMCs) on biodegradable polymer scaffold. The primary culture of VSMCs obtained from canine external jugular vein was accomplished by applying the explant-derived method. The primary cultured VSMCs were seeded into scaffolds and then cultured by using various different methods; static or dynamic seeding, static or dynamic culture. The difference in proliferative response of VSMCs was analyzed with an alamar blue assay. Cell-polymer construct was examined by histochemical method and scanning electron microscopy. Mesh type scaffold ($10 \times 10 \times0.4 mm$) was made of polyglycolic acid (PGA) suture thread. The PGA mesh type scaffold was 45% in porosity, and 0.03 g in weight. The primary cultured VSMCs were confirmed with immunohistochemical staining using monoclonal anti-$\alpha$-smooth muscle actin. The density and distribution of proliferated VSMCs within the scaffold and cellular adherence on the surface of the scaffold showed better results in the static seeding condition than in the dynamic condition. Under the same condition of seeding method as the static condition, the dynamic culture condition showed enhanced proliferation rates of the VSMCs when compared to the static culture condition. In conclusion, to improve the VSMCs proliferation in vitro, static seeding is better than the dynamic condition. In the culture condition, however, culture under the dynamic status is better than the static condition. This was a pilot study to manufacture artificial vascular vessel by tissue engineering.

• The Journal of Korean Medicine
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• v.24 no.2
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• pp.166-178
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• 2003

Objectives : Nitric oxide (NO) plays an important role in normal and pathophysiological cells as a messenger molecule, neurotransmitter, microbiological agent, or dilator of blood vessels and arteriosclerosis, respectively. This study was undertaken to understand the mechanism of NO production and effect of Hyeolbuchukeo-tang (Xiefuzhuyu-tang) on NO production in cultured vascular smooth muscle cell (VSMC). Methods and Results : VSMC was isolated from aorta and cultured. Cultured primary cells were identified as VSMC with anti--smooth muscle actin antibody. A large amount of NO was produced in cultured VSMC treated with $IFN-{\gamma}$ plus TNF in a time- and dose-dependent manner. $TNF-{\alpha}$ was a more efficient stimulator than $IFN-{\gamma}$ in NO production of cultured VSMC. iNOS protein wasdetected within 3 hrs and it increased up to 12 hrs in a time-dependent manner. However, accumulated NO in cytokine-treated VSMC was not detected within 3 hrs. NO production in cytokine-treated VSMC showed the dose- and time-dependent manner, and increased up to 48 hrs. The activated VSMC produced a large amount of NO (about 60 uM). Hyeolbuchukeo-tang (Xiefuzhuyu-tang) alone did not induceNO production, but it potentiated the effect of $TNF-{\alpha}$ on NO production and increased NO production by about 20%. Hyeolbuchukeo-tang (Xiefuzhuyu-tang) did not affect the transcriptional activity of iNOS gene, but increased the accumulation of iNOS. These results indicate that Hyeolbuchukeo-tang (Xiefuzhuyu-tang) could modulate the translational level of iNOS. PKC did not modulate NO production, but calcium ionophore A23187 decreased NO production. However, Hyeolbuchukeo-tang (Xiefuzhuyu-tang) elevated the decreased NO production in A23187-treated VSMC by modulating the stability of iNOS transcripts. Half-life of the synthesized transcripts appeared to have about 6 hrs. PDTC, an $NF-{\kappa}B$ inhibitor, blocked the accumulation of iNOS mRNA, indicating that $NF-{\kappa}B$ served as an important modulator in the transcriptional regulation of iNOS. As Hyeolbuchukeo-tang (Xiefuzhuyu-tang) potentiated the effect of the $TNF-{\alpha}$ on NO production but had no additional effect on PDTC-modulated NO production, it is suggested that Hyeolbuchukeo-tang (Xiefuzhuyu-tang) enhances the $TNF-{\alpha}-mediated$ NO production of VSMC by modulating the iNOS activity and the stability of iNOS transcripts in activated VSMC having the elevated intracellular calcium ion. Conclusions : This study suggests that Hyeolbuchukeo-tang (Xiefuzhuyu-tang) has a potential capacity for preventing and treating diseases of the circulation system, including arteriosclerosis.

• Journal of Veterinary Clinics
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• v.27 no.3
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• pp.232-239
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• 2010

Two cell lines derived from spontaneous canine mammary gland tumors were established and characterized. Mammary gland tumors from 9 years old pug and 9 years old toy-poodle dogs were collected by aseptic surgical resection and primary culture was performed. The histopathologic examination of tumors revealed adenocarcinoma and complex carcinoma and two dogs died from metastasis of the tumors. The tumor cells were subcultured over 60 times for more than 1 year and morphological consistency maintained. Light microscopic examination, growth curve, doubling time calculation, xenotransplantation to female nude mice, immunohistochemistry for wide spectrum keratin, vimentin, $\alpha$-smooth muscle actin and cytokeratin 8 was performed for characterization. The cell lines exhibited polygonal, elongated cell shape and cytoplasmic bridge and doubling time of 47.1 hrs and 18.6 hrs, respectively. Subcutaneous xenotransplantation to nude mice of the cells produced localized palpable mass within 4 weeks in 4 of 5 and 5 of 5 nude mice, respectively. In immunohistochemical examination one cell line showed strong positive against wide spectrum keratin and cytokeratin 8 and the other cell line showed strong positive against smooth muscle actin and cytokeratin 8. Additional characterization would be possible by investigator's needs and the cell lines may be useful for in vivo and in vitro studies of canine mammary tumor and adjuvant therapies.

• Childhood Kidney Diseases
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• v.15 no.2
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• pp.125-137
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• 2011

Purpose : Men are generally more prone to chronic renal disease and progression to end stage renal disease than women. The purpose of this study is to prove the effect of gender and sex hormone on renal fibrosis in mice with unilateral ureteral obstruction (UUO) and to elucidate the specific underlying mechanisms. Methods :We compared the expression of ${\alpha}$-smooth muscle actin (${\alpha}$-SMA) in female and male mice with complete UUO (day 7). After this, we estimated the changes of renal fibrosis in the female mice with oophorectomy and in the female mice with oophorectomy and replacement of $17{\beta}$-estradiol, respectively. Results : The level of ${\alpha}$-SMA in the female kidney with UUO was significantly lower than that in the male kidney with UUO. oophorectomy and replacement of $17{\beta}$-estradiol did not change the expression of angiotensin II type 1 (AT1) receptor in the female kidney with UUO, whereas the expression of angiotensin II type 2 (AT2) receptor was significantly more elevated in the intact female (IF) and the oophorectomized female with estrogen (OF+E) than that in the oophorectomized female (OF). The expressions of inducible nitric oxide synthase (iNOS) in the IF and OF+E mice were significantly more elevated than that in the OF mice, which was similar to the expression of AT2 receptor. Conclusion : The female gender is associated with resistance to renal fibrosis in obstructive uropathy and this gender difference may originate from the existence of $17{\beta}$-estradiol, which has an anti-fibrotic effect via upregulation of the AT2 receptor and iNOS.

• Journal of Korean Ophthalmic Optics Society
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• v.13 no.4
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• pp.161-169
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• 2008

Purpose: To better understand the corneal regeneration after alkali burn regarding the initial clinical progression and the therapy, we investigated the changes of the multi factors following chemical injury in cornea. Methods: This study was performed to observation on the healing process of alkali burned cornea in aspect of immunohistochemistry by immunofluorescence or H-E staining and TUNEL assay. Results: The results showed that although a healing process occurred after alkali burn, apoptosis of epithelial, stromal and endothelial cells in the cornea was continuously observed. Neovascularization and expression of ${\alpha}$-smooth muscle actin (${\alpha}$-SMA) from limbus and from injured cornea, respectively, were observed after 3 days of alkali burn. Formation of collagen III in corneal stroma and increased expression of chondroitin sulfate are coincident with expression of ${\alpha}$-SMA and transforming growth factor-${\beta}$ (TGF-${\beta}$). Conclusions: These data suggest that medical treatment within 3 days of alkali burn will be effective to inhibit neovascularization and formation of collagen III and chondroitin sulfate. This study extends our immumohistochemical understanding of healing process in alkali burned cornea, and the results get in this study will be cornerstones in the development of therapeutic agent for accelerating renewal of chemical damaged cornea.