• BMB Reports
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• v.47 no.10
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• pp.552-557
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• 2014

The main purpose of this study was to investigate whether type 3 muscarinic acetylcholine receptor (M3R) dysfunction induced vascular hyperpermeability. Transwell system analysis showed that M3R inhibition by selective antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and small interfering RNA both increased endothelial permeability. Using coimmunoprecipitation and Western blot assay, we found that M3R inhibition increased VE-cadherin and ${\beta}$-catenin tyrosine phosphorylation without affecting their expression. Using PTP1B siRNA, we found that PTP1B was required for maintaining VE-cadherin and ${\beta}$-catenin protein dephosphorylation. In addition, 4-DAMP suppressed PTP1B activity by reducing cyclic adenosine monophosphate (cAMP), but not protein kinase $C{\alpha}$ ($PKC{\alpha}$). These data indicate that M3R preserves the endothelial barrier function through a mechanism potentially maintaining PTP1B activity, keeping the adherens junction proteins (AJPs) dephosphorylation.

• BMB Reports
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• v.45 no.3
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• pp.141-146
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• 2012

Protein tyrosine phosphatase 1B (PTP1B) is important in the regulation of metabolic diseases and has emerged as a promising signaling target. Previously, we reported the PTP1B inhibitory activity of Rheum undulatum (RU). In the present study, we investigated the metabolic regulatory effects of RU in a high-fat diet (HFD) model. RU treatment significantly blocked body weight gain, which was accompanied by a reduction of feed efficiency. In addition, it led to a reduction of liver weight mediated by overexpression of PPAR${\alpha}$ and CPT1 in the liver, and an increase in the expression of adiponectin, aP2, and UCP3 in adipose tissue responsible for the reduction of total and LDL-cholesterol levels. Chrysophanol and physcion from RU significantly inhibited PTP1B activity and strongly enhanced insulin sensitivity. Altogether, our findings strongly suggest that 2 compounds are novel PTP1B inhibitors and might be considered as anti-obesity agents that are effective for suppressing body weight gain and improving lipid homeostasis.

• Journal of Life Science
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• v.31 no.2
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• pp.183-191
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• 2021

Euglena gracilis is a microalga of great biotechnological interest that can create high levels of bioactive compounds, such as tocopherol, paramylon, and folic acid. The objective of this study was to investigate the biological activities of extracts from E. gracilis, especially those focused on immunological activity. E. gracilis biomass was extracted with hot water (HWE) and the remaining pellet was continuously extracted with methanol (HWME). First, we examined the effect of two extracts from E. gracilis on the production of nitric oxide (NO) and the expression of pro-inflammation cytokines, including IL-1β, IL-6, and TNF-α in murine macrophage RAW 264.7 cells. HWE treatment dose-dependently increased the production of IL-1β and TNF-α. On the other hand, treatment with HWME significantly decreased the generation of NO and pro-inflammatory cytokines (IL-6 and TNF-α) in lipopolysaccharide (LPS)-stimulated macrophage cells. In addition, other biological activities of the extracts were further analyzed: α-glucosidase inhibition, protein tyrosine phosphatase (PTP1B) inhibition, tyrosinase inhibition, xanthine oxidase (XO) inhibition, and angiotensin-converting enzyme (ACE) inhibition. Analysis of these biological activities showed that HWE has more inhibitory effects than HWME against α-glucosidase, tyrosinase, and XO agents. However, the inhibition of PTP1B and ACE with HWME were higher than with HWE. Taken together, the results suggested that E. gracilis possesses various biological activities―especially immunological capabilities―through regulation of cytokine production. Therefore, E. gracilis extract may be potentially useful for food material with immune-regulating effects.

• Bulletin of the Korean Chemical Society
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• v.27 no.10
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• pp.1567-1571
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• 2006

Synthetic color additives approved for general food use are sixteen in European Union, seven in U. S. A. and twelve in Japan. Twelve food dyes were examined for their inhibitory potency against human protein tyrosine phosphatases (PTPases). Half of the food colorants inhibited PTPases significantly and three of them were potent inhibitors with low micromolar IC50 values. Also examined were the synthetic dyes structurally similar but not allowed in food. Some of them were potent inhibitors of PTPases. Considering the importance of PTPases in cellular signal transduction, inhibition of PTPases by food colorants might cause harmful effects in human health.

• Biomolecules & Therapeutics
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• v.31 no.2
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• pp.193-199
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• 2023

In this investigation, we made a study of the efficacy of luteolin (a flavonoid found in plants such as vegetables, herbs and fruits) on vascular contractibility and to elucidate the mechanism underlying the relaxation. Isometric contractions of denuded muscles were stored and combined with western blot analysis which was conducted to assess the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to examine the effect of luteolin on the RhoA/ROCK/CPI-17 pathway. Luteolin significantly alleviated phorbol ester-, fluoride- and thromboxane mimetic-elicited contractions regardless of endothelial nitric oxide synthesis, implying its direct effect on smooth muscle. It also significantly alleviated the fluoride-elicited elevation in pCPI-17 and pMYPT1 levels and phorbol 12,13-dibutyrate-elicited increase in pERK1/2 level, suggesting depression of ROCK and PKC/MEK activity and ensuing phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that luteolin-elicited relaxation includes myosin phosphatase reactivation and calcium desensitization, which seems to be arbitrated by CPI-17 dephosphorylation via ROCK/PKC inhibition.

• Journal of Acupuncture Research
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• v.32 no.3
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• pp.27-40
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• 2015

Objectives : This study was performed to evaluate the effects of water extract of Cervi Parvum Cornu(CPC), Aconiti Lateralis Radix Preparata(ALR), and Yongbu-tang(YBT) on suppression of the receptor activator of nuclear factor kappa-B ligand(RANKL)-induced osteoclast differentiation and bone resorption. Methods : The effects of CPC, ALR, YBT extracts on osteoclast differentiation were determined by culture of bone marrow macrophage(BMM). The mRNA expression levels of the nuclear factor of activated T-cells cytoplasmic 1(NFATc1), c-Fos and tartrate-resistant acid phosphatase(TRAP) in BMMs were analyzed by reverse transcriptase polymerase chain reaction(RT-PCR). Similarly, the protein expression levels of NFATc1, c-Fos, mitogen-activated protein kinase(MAPK)s and ${\beta}$-actin in cell lysates were measured by western blotting. In addition, effects of CPC, ALR and YBT extracts were determined by means of Lipopolysaccharide(LPS)-induced bone-loss with mice. Results : CPC, ALR and YBT extracts showed remarkable inhibition on RANKL-induced osteoclast differentiation without cytotoxicity. CPC and ALR extracts significantly reduced the protein expression level of NFATc1. YBT extract significantly reduced the mRNA expression levels of c-Fos, NFATc1 and the protein expression levels of c-Fos, NFATc1, AKT, p38, c-Jun N-terminal kinase(JNK). Further, YBT extract suppressed degradation of$ I-{\kappa}B$. And ALR extract significantly restored the bone erosion by LPS treatment in mice. Conclusions : YBT extract showed more remarkable inhibition on osteoclast differentiation than CPC and ALR extracts in vitro. ALR extract showed remarkable inhibition on bone resorption in vivo. Thus, YBT extract can be a useful treatment for bone-loss diseases such as osteoporosis.

• BMB Reports
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• v.30 no.2
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• pp.89-94
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• 1997

In this study, during the activation of neutrophil responses by sodium fluoride. involvement of protein tyrosine kinase was studied. Respiratory burst lysosomal enzyme release and elevation of $[Ca^{2+}]_i$stimulated by sodium fluoride in neutrophils were inhibited by protein kinase inhibitors, genistein and tyrphostin. The inhibitory effect of genistein and tyrphostin on superoxide and $H_{2}O_{2}$ production was less than that of protein kinase C inhibitors, staurosporine and H-7. Staurosporine and H-7 had little or no effect on the release of myeloperoxidase and acid phosphatase stimulated by sodium fluoride. EGTA and verapamil inhibited the elevation of $[Ca^{2+}]_i$ evoked by sodium fluoride. The inhibitory effect of staurosporine on the elevation of $[Ca^{2+}]_i$ was less than that of genistein. Phorbol 12-myristate 13-acetate (PMA)-stimulated superoxide production, which is sensitive to staurosporine, was further enhanced by genistein, whereas the stimulatory action of PMA on myeloperoxidase release was inhibited by genistein. A pretreatment of neutrophils with PMA signifcantly attenuated sodium fluoride-evoked elevation of $[Ca^{2+}]_i$ These results suggest that protein tyrosine kinase may be involved in the activation process of neutrophil responses due to direct stimulation of guanine nucleotide regulatory proteins. In neutrophil responses, PMA-stimulated neutrophils appear to show a different type of inhibition of protein tyrosine kinase.

• Journal of Korean Neurosurgical Society
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• v.46 no.4
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• pp.389-396
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• 2009

Objective : Triptolide (TP) has been reported to suppress the expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), of which main function is to inactivate the extracellular signal-regulated kinase-1/2 (ERK-1/2), the p38 MAPK and the c-Jun N-terminal kinase-1/2 (JNK-1/2), and to exert antiproliferative and pro-apoptotic activities. However, the mechanisms underlying antiproliferative and pro-apoptotic activities of TP are not fully understood. The purpose of this study was to examine whether the down-regulation of MKP-1 expression by TP would account for antiproliferative activity of TP in immortalized HT22 hippocampal cells. Methods : MKP-1 expression and MAPK phosphorylation were analyzed by Western blot. Cell proliferation was assessed by $^3H$-thymidine incorporation. Small interfering RNA (siRNA) against MKP-1, vanadate (a phosphatase inhibitor), U0126 (a specific inhibitor for ERK-1/2), SB203580 (a specific inhibitor for p38 MAPK), and SP600125 (a specific inhibitor for JNK-1/2) were employed to evaluate a possible mechanism of antiproliferative action of TP. Results : At its non-cytotoxic dose, TP suppressed MKP-1 expression, reduced cell growth, and induced persistent ERK-1/2 activation. Similar growth inhibition and ERK-1/2 activation were observed when MKP-1 expression was blocked by MKP-1 siRNA and its activity was inhibited by vanadate. The antiproliferative effects of TP, MKP-1 siRNA, and vanadate were significantly abolished by U0126, but not by SB203580 or SP600125. Conclusion : Our findings suggest that TP inhibits the growth of immortalized HT22 hippocampal cells via persistent ERK-1/2 activation by suppressing MKP-1 expression. Additionally, this study provides evidence supporting that MKP-1 may play an important role in regulation of neuronal cell growth.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.34 no.4
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• pp.370-377
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• 2001

Flounder brain cytosol contains protein inhibitors that markedly inhibit the activity of partially purified brain membrane phospholipase D (PLD) which is dependent on phosphatidylinositol 4,5-bisphosphate ($PIP_2$) but insensitive to ADP-ribosylation factor (ARF), The PLD inhibitors have been enriched through several chromatographic steps and characterized with respect to size and mechanism of inhibition. Sequential chromatography of the brain cytosol yielded six inhibitor fractions, Two (IIA and IIB) of six inhibitor fractions showed the $PIP_2$-phosphatase activities. IIA was identified as synaptojanin, a nerve terminal protein that has known to be a member of the inositolpolyphosphate 5-phosphatase family, by immunoblot analysis. IIB showed an apparent molecular mass of 158 kDa by Superose 12 gel filtration chromatography and was immunologically distinct from synaptojanin. IIB hydrolyzed $PIP_2$, yielding only phosphatidylinositol phosphate (PIP) as product, suggesting that IIB hydrolyzes only one phosphate from either the 4- or 5-position of PI (4,5)$P_2$. These studies demonstrate that the existence of multiple $PIP_2$-phosphatases have been implicated in the negative regulation of $PIP_2$-dependent PLD activity within flounder brain.

• Bulletin of the Korean Chemical Society
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• v.29 no.2
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• pp.342-346
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• 2008

Aurintricarboxylic acid (ATA) prevents apoptosis in a wide range of cell types, including PC12 cells. ATA is known to increase the phosphorylation level of IGF-1 receptor (IGF-1R) and downstream signaling proteins. ATA can translocate across the plasma membrane of PC12 cells and inhibit protein tyrosine phosphatases (PTPs) and, therefore, it is not clear whether ATA exerted its antiapoptotic effect through activation of IGF-1R or by inhibition of cytosolic PTPs. When PC12 cells, deprived of serum, were treated with Fab fragment of anti-IGF-1R antibody to prevent the binding of ATA to the extracellular domain of IGF-1R, ATA was found to penetrate into the cytosolic space of the cells. Under these conditions, the survival-promoting effects of ATA were abolished, and the increase of phosphorylation and characteristic cleavage of IGF-1R were not observed. These results indicate that the antiapoptotic effect of ATA in PC12 cells is due to the binding of ATA to the extracellular domain of IGF-1R and subsequent activation of the IGF-1R, not inhibition of cytosolic PTP(s).