• The Korean Journal of Physiology and Pharmacology
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• 제10권5호
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• pp.243-249
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• 2006

The effect of genistein, widely used as a specific tyrosine kinase inhibitor, on rat brain Kv1.5 channels which were stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Genistein inhibited Kv1.5 currents at +50 mV in a concentration-dependent manner, with an $IC_{50}$ of $54.7{\pm}8.2\;{\mu}M$ and a Hill coefficient of $1.1{\pm}0.2$. Pretreatment of Kv1.5 with protein tyrosine kinase inhibitors ($10\;{\mu}M$ lavendustin A and $100\;{\mu}M$ AG1296) and a tyrosine phosphatase inhibitor ($500\;{\mu}M$ sodium orthovanadate) did not block the inhibitory effect of genistein. The inhibition of Kv1.5 by genistein showed voltage-independence over the full activation voltage range positive to 0 mV. The activation (at +50 mV) kinetics was significantly delayed by genistein: time constant for an activation of $1.4{\pm}0.2$ msec under control conditions and $10.0{\pm}1.5$ msec in the presence of $60\;{\mu}M$ genistein. Genistein also slowed the deactivation of the tail currents, resulting in a crossover phenomenon: a time constant of $11.4{\pm}1.3$ msec and $40.0{\pm}4.2$ msec under control conditions and in the presence of $60\;{\mu}M$ genistein, respectively. Inhibition was reversed by the application of repetitive depolarizing pulses, especially during the early part of the activating pulse. These results suggest that genistein directly inhibits Kv1.5 channels, independent of phosphotyrosine-signaling pathway.

• Restorative Dentistry and Endodontics
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• 제44권2호
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• pp.17.1-17.10
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• 2019

Objectives: Root resorption is an unexpected complication after replantation procedures. Combining anti-osteoclastic medicaments with retrograde root filling materials may avert this resorptive activity. The purpose of this study was to assess effects of a cathepsin K inhibitor with calcium silicate-based cements on osteoclastic activity. Methods: MC3T3-E1 cells were cultured for biocompatibility analyses. RAW 264.7 cells were cultured in the presence of the receptor activator of nuclear factor-kappa B and lipopolysaccharide, followed by treatment with Biodentine (BIOD) or ProRoot MTA with or without medicaments (Odanacatib [ODN], a cathepsin inhibitor and alendronate, a bisphosphonate). After drug treatment, the cell counting kit-8 assay and Alizarin red staining were performed to evaluate biocompatibility in MC3T3-E1 cells. Reverse-transcription polymerase chain reaction, tartrate-resistant acid phosphatase (TRAP) staining and enzyme-linked immunosorbent assays were performed in RAW 264.7 cells to determine the expression levels of inflammatory cytokines, interleukin $(IL)-1{\beta}$, IL-6, tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$) and prostaglandin E2 (PGE2). Data were analyzed by one-way analysis of variance and Tukey's post hoc test (p < 0.05). Results: Biocompatibility results showed that there were no significant differences among any of the groups. RAW 264.7 cells treated with BIOD and ODN showed the lowest levels of $TNF-{\alpha}$ and PGE2. Treatments with BIOD + ODN were more potent suppressors of inflammatory cytokine expression (p < 0.05). Conclusion: The cathepsin K inhibitor with calcium silicate-based cement inhibits osteoclastic activity. This may have clinical application in preventing inflammatory root resorption in replanted teeth.

• The Korean Journal of Physiology and Pharmacology
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• 제17권5호
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• pp.447-453
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• 2013

Osteoblastic activity of nectandrin A was examined in C2C12 cells. Nectandrin A enhances the BMP-induced osteoblastic differentiation and mineralization, manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and increased calcium contents. In C2C12 cells co-transfected with expression vector encoding Smad4 and Id1-Luc reporter, nectandrin A increased Id1 luciferase activity in a concentration-dependent manner, when compared to that in BMP-2 treated cells, indicating that Smad signaling pathway is associated with nectandrin A-enhanced osteoblastic differentiation in C2C12 cells. In addition, nectandrin A activated p38 mitogen-activated protein kinase (MAPK) in time- and concentration-dependent manners, and phosphorylated form of pSmad1/5/8 and alkaline phosphatase activity were both decreased when the cells were pretreated with SB203580, a p38 MAPK inhibitor, suggesting that p38 MAPK might be an upstream kinase for Smad signaling pathway. Taken together, nectandrin A enhances the BMP-induced osteoblastic differentiation and mineralization of C2C12 cells via activation of p38 MAPK-Smad signaling pathway, and it has a therapeutic potential for osteoporosis by promoting bone formation.

• Journal of Applied Biological Chemistry
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• 제50권2호
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• pp.52-57
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• 2007

Quantitative structure-activity relationships (QSARs) on the inhibition activities by oleanolic acid analogues (1-19) as a potent inhibitor against protein tyrosine phosphatase-1B were studied quantitatively using 2D-QSAR and HQSAR methodologies. The inhibition activity was dependent on the variations of $R_{4-}$substituent, and as shown in 2D-QSAR model ($r^2=0.928$), it has a tendency to increase as the negative Randic Indice (RI) goes up. The size of the molecular fragments used in HQSAR varied from five to eight. The fragment distinctions had the best statistic value, whose predictability is $q^2=0.785$ and correlation coefficient is $r^2=0.970$, on condition of connections. From the atomic contribution maps, the factor that contributes to the inhibition activities is the $C_{15}{\sim}C_{17}$ bond in the D ring. From the analysis result of these two the models, the structural distinctions and descriptors that contribute to the inhibition activities were obtained.

• Bulletin of the Korean Chemical Society
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• 제35권6호
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• pp.1769-1776
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• 2014

Binding modes of a series of catechol derivatives such as protein tyrosine phosphatase 1B (PTP1B) inhibitors were identified by molecular modeling techniques. Docking, molecular dynamics simulations and free energy calculations were employed to determine the modes of these new inhibitors. Binding free energies were calculated by involving different energy components using the Molecular Mechanics-Poisson-Boltzmann Surface Area and Generalized Born Surface Area methods. Relatively larger binding energies were obtained for the catechol derivatives compared to one of the PTP1B inhibitors already in use. The Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) free energy decomposition analysis indicated that the hydroxyl functional groups and biphenyl ring system had favorable interactions with Met258, Tyr46, Gln262 and Phe182 residues of PTP1B. The results of hydrogen bound analysis indicated that catechol derivatives, in addition to hydrogen bonding interactions, Val49, Ile219, Gln266, Asp181 and amino acid residues of PTP1B are responsible for governing the inhibitor potency of the compounds. The information generated from the present study should be useful for the design of more potent PTP1B inhibitors as anti-diabetic agents.

• Journal of Microbiology and Biotechnology
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• 제2권3호
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• pp.166-173
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• 1992

The expression of Bacillus subtilis $\alpha$-amylase from the phoA-amyE fusion gene in recombinant E. coli was investigated under various environmental conditions. The overexpression of cloned $\alpha$-amylase caused retardations in cell growth and synthesis of alkaline phosphatase (AP) from the chromosomal phoA gene. The change of culture temperature from $37^\circ{C}$ to $30^\circ{C}$ increased the specific activities of both $\alpha$-amylase and $\beta$-lactamase by six and two times, respectively, whereas the AP activity remained unchanged. The experiments with chlorampenicol (a translation inhibitor) suggested the enhancement of $\alpha$-amylase activity at $30^\circ{C}$, and this was partly due to the stability of $\alpha$-amylase itself. The further decrease of the temperature to $25^\circ{C}$ slowed down both the cell growth and cloned-gene expression rate. The $\alpha$-amylase activity showed a maximum at pH of 7.4 while alkaline phosphatase was most effectively produced at pH of 8.3.

• International journal of thyroidology
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• 제10권2호
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• pp.71-76
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• 2017

Background and Objectives: The role of thyroid-stimulating hormone (TSH) signaling on osteoblastic differentiation is still undetermined. The aim of this study was to investigate the effects of 5-aza-2'-deoxycytidine (5-azacytidine) on TSH-mediated regulations of osteoblasts. Materials and Methods: MG63, a human osteoblastic cell-line, was treated with 5-azacytidine before inducing osteogenic differentiation using osteogenic medium (OM) containing L-ascorbic acid and ${\beta}$-glyceophosphate. Bovine TSH or monoclonal TSH receptor stimulating antibody (TSAb) was treated. Quantitative real-time PCR analyses or measurement of alkaline phosphatase activities were performed for evaluating osteoblastic differentiation. Results: Studies for osteogenic-related genes or alkaline phosphatase activity demonstrated that treatment of TSH or TSAb alone had no effects on osteoblastic differentiation in MG63 cells. However, treatment of 5-azacytidine, per se, significantly increased osteoblastic differentiation and combination treatment of 5-azacytidine and TSH or TSAb in the condition of OM showed further significant increase of osteoblastic differentiation. Conclusion: Stimulating TSH signaling has little effects on osteoblastic differentiation in vitro. However, in the condition of epigenetic modification using inhibitor of DNA methylation, TSH signaling positively affects osteoblastic differentiation in human osteoblasts.

• 구강회복응용과학지
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• 제25권3호
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• pp.293-306
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• 2009

본 연구에서는 알카리 열처리 티타늄 임플란트상에서 백서 태자 두개관 세포의 생물학적 반응을 알아보고자 하여 다음과 같은 결과를 얻었다. 알카리 열처리 티타니움 표면에서 Ti-6Al-4V 합금 표면에서보다 세포증식도가 유의하게 높았다 (p<0.01). 면역효소 분석에서 interleukin $(IL)-1{\beta}$와 IL-6의 합성은 배양 시간이 길수록 증가하였다. Herbimycin으로 전처치한 경우에는 세포 배양 7일 째에 IL-6의 합성이 유의하게 억제되었다(p<0.01). 알카리인산 분해 효소 활성은 알카리 열처리 임플란트에서 타이타늄, Ti-6Al-4V 합금 표면에서 보다 유의하게 더 높았다 (p<0.001). 유전자 분석에서 알카리 열처리 임플란트에서 배양된 세포들의 alkaline phosphatase, bone sialoprotein, receptor activated nuclear factor ligand 유전자의 발현이 증가하였다. Herbimycin과 SB203580으로 전처치시 $IL-1{\beta}-induced$ IL-6 유전자의 발현이 감소하였다. 이상의 결과는 알카리 열처리된 티타늄 임플란트가 조골세포의 분화를 자극하고 골개조를 촉진시킴을 시사한다.

• Biomolecules & Therapeutics
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• 제22권6호
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• pp.510-518
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• 2014

Chronic (>24 h) exposure of arsenite, an environmental toxicant, has shown the decreased nitric oxide (NO) production in endothelial cells (EC) by decreasing endothelial NO synthase (eNOS) expression and/or its phosphorylation at serine 1179 ($eNOS-Ser^{1179}$ in bovine sequence), which is associated with increased risk of vascular diseases. Here, we investigated the acute (<24 h) effect of arsenite on NO production using bovine aortic EC (BAEC). Arsenite acutely increased the phosphorylation of $eNOS-Thr^{497}$, but not of $eNOS-Ser^{116}$ or $eNOS-Ser^{1179}$, which was accompanied by decreased NO production. The level of eNOS expression was unaltered under this condition. Treatment with arsenite also induced reactive oxygen species (ROS) production, and pretreatment with a ROS scavenger N-acetyl-L-cysteine (NAC) completely reversed the observed effect of arsenite on $eNOS-Thr^{497}$ phosphorylation. Although protein kinase C (PKC) and protein phosphatase 1 (PP1) were reported to be involved in $eNOS-Thr^{497}$ phosphorylation, treatment with PKC inhibitor, Ro318425, and overexpression of various PKC isoforms did not affect the arsenite-stimulated $eNOS-Thr^{497}$ phosphorylation. In contrast, treatment with PP1 inhibitor, calyculin A, mimicked the observed effect of arsenite on $eNOS-Thr^{497}$ phosphorylation. Lastly, we found decreased cellular PP1 activity in arsenite-treated cells, which was reversed by NAC. Overall, our study demonstrates firstly that arsenite acutely decreases NO production at least in part by increasing $eNOS-Thr^{497}$ phosphorylation via ROS-PP1 signaling pathway, which provide the molecular mechanism underlying arsenite-induced increase in vascular disease.

• Bulletin of the Korean Chemical Society
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• 제33권5호
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• pp.1505-1508
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• 2012

Protein tyrosine phosphatase 1B (PTP1B) is a key factor in negative regulation of the insulin pathway, and is a promising target for the treatment of type-II diabetes, obesity and cancer. Herein, compound ($\mathbf{4}$) was first observed to have moderate inhibitory activity against PTP1B with an $IC_{50}$ value of $13.72{\pm}1.53{\mu}M$. To obtain more potent PTP1B inhibitors, we synthesized a series of chalcone derivatives using compound ($\mathbf{4}$) as the lead compound. Compound $\mathbf{4l}$ ($IC_{50}=3.12{\pm}0.18{\mu}M$) was 4.4-fold more potent than the lead compound $\mathbf{4}$ ($IC_{50}=13.72{\pm}1.53{\mu}M$), and more potent than the positive control, ursolic acid ($IC_{50}=3.40{\pm}0.21{\mu}M$). These results may help to provide suitable drug-like lead compounds for the design of inhibitors of PTP1B as well as other PTPs.