• Journal of Ginseng Research
• /
• v.43 no.2
• /
• pp.236-241
• /
• 2019

Background: Thromboxane A2 ($TXA_2$) induces platelet aggregation and promotes thrombus formation. Although ginsenoside Ro (G-Ro) from Panax ginseng is known to exhibit a $Ca^{2+}-antagonistic$ antiplatelet effect, whether it inhibits $Ca^{2+}-dependent$ cytosolic phospholipase $A_2$ ($cPLA_{2{\alpha}}$) activity to prevent the release of arachidonic acid (AA), a $TXA_2$ precursor, is unknown. In this study, we attempted to identify the mechanism underlying G-Ro-mediated $TXA_2$ inhibition. Methods: We investigated whether G-Ro attenuates $TXA_2$ production and its associated molecules, such as cyclooxygenase-1 (COX-1), $TXA_2$ synthase (TXAS), $cPLA_{2{\alpha}}$, mitogen-activated protein kinases, and AA. To assay COX-1 and TXAS, we used microsomal fraction of platelets. Results: G-Ro reduced $TXA_2$ production by inhibiting AA release. It acted by decreasing the phosphorylation of $cPLA_{2{\alpha}}$, p38-mitogen-activated protein kinase, and c-Jun N-terminal kinase1, rather than by inhibiting COX-1 and TXAS in thrombin-activated human platelets. Conclusion: G-Ro inhibits AA release to attenuate $TXA_2$ production, which may counteract $TXA_2-associated$ thrombosis.

• BMB Reports
• /
• v.29 no.6
• /
• pp.555-563
• /
• 1996

A membrane-bound phosphatidylinositol 4-kinase (PI 4-kinase) was separated in a sucrose gradient and solubilized with 1% Triton X-100 from mouse brain. The enzyme was purified 2,952-fold by various chromatographic techniques including DEAE-cellulose, PI-Sepharose and Sephacryl S-200 gel filtration. The molecular weight of PI 4-kinase was approximately 76 kDa by gel filtration and 70.8 kDa by SDS-polyacrylamide gel electrophoresis. The purified enzyme exhibited specific activity of 11.2 nmol/min/mg protein and pi value of 4.7. Kinetic analysis of the PI 4-kinase indicated apparent $K_m$, values of 190 ${\mu}M$ and 120 ${\mu}M$ for phosphatidylinositol and ATP, respectively. The maximal activity of this purified enzyme was observed at pH 7.4 at an incubation temperature of $37^{\circ}C$. The enzyme activity was significantly activated by $Mg^{2+}$, $Mn^{2+}$ and $Fe^{2+}$, and inhibited severely by $Ca^{2+}$. PI 4-kinase was proved to be pure in its immunoblot test by polyclonal antibody prepared from immunized rabbit sera. By this test, we were able to detect the existence of the same type of PI 4-kinase from other mouse organ tissues, such as liver, heart, kidney and spleen. Furthermore, similar immunoblot analysis with the same antisera recognized the different epitopes of PI 4-kinase proteins from various organs of rabbit, chinese hamster and rat.

• Archives of Pharmacal Research
• /
• v.27 no.7
• /
• pp.757-762
• /
• 2004

The protective adaptive response to electrophiles and reactive oxygen species is mediated by the induction of phase II detoxifying genes including glutathione S-transferases (GSTs). NF-E2-related factor-2 (Nrf2) phosphorylation by protein kinase C (PKC) is a critical event for its nuclear translocation in response to oxidative stress. Previously, we have shown that peroxynitrite plays a role in activation of Nrf2 and Nrf2 binding to the antioxidant response element (ARE) via the pathway of phosphatidylinositol 3-kinase (PI3-kinase) and that nitric oxide synthase in hepatocytes is required for GSTA2 induction. In view of the importance of PKC and Pl3-kinase in Nrf2-mediated GST induction, we investigated the role of these kinases in peroxynitrite formation for GSTA2 induction by oxidative stress and determined the relationship between PKC and PI3-kinase. Although PKC activation by phorbol 12-myristate-13-acetate (PMA) did not increase the extents of constitutive and inducible GSTA2 expression, either PKC depletion by PMA or PKC inhibition by staurosporine significantly inhibited GSTA2 induction by tert-butylhydroquinone (t-SHa) a prooxidant chemical. Therefore, the basal PKC activity is req- uisite for GSTA2 induction. 3-Morpholinosydnonimine (SIN-1), which decomposes and yields peroxynitrite, induced GSTA2, which was not inhibited by PKC depletion, but slightly enhanced by PKC activation, suggesting that PKC promotes peroxynitrite formation for Nrf2-mediated GSTA2 induction. Treatment of cells with S-nitroso-N-acetyl-penicillamine (SNAP), an exogenous NO donor, in combination with t-BHQ may produce peroxynitrite. GSTA2 induction by SNAP ＋ t-BHQ was not decreased by PKC depletion, but rather enhanced by PKC activation, showing that the activity of PKC might be required for peroxynitrite formation. LY294002 a P13-kinase inhibitor blocked GSTA2 induction by t-BHQ, which was reversed by PMA-induced PKC activation. These results provide evidence that PKC may playa role in formation of peroxynitrite that activates Nrf2 for GSTA2 induction and that PKC may serve an activator for GSTA2 induction downstream of PI3-kinase.

• The Korean Journal of Physiology and Pharmacology
• /
• v.17 no.5
• /
• pp.447-453
• /
• 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.

• BMB Reports
• /
• v.28 no.3
• /
• pp.216-220
• /
• 1995

cDNA for human cholesteryl ester transfer protein (CETP), a potent atherogenic plasma protein that redistributes the neutral lipids among lipoproteins, was expressed in recombinant vaccinia virus-infected cells (CV-1). Two insertion vectors regulated by different promoters were constructed. The vectors were introduced into human thymidine kinase-negative ($TK^-$) 1438 cells infected with wild-type vaccinia virus (WR strain). Recombinant viruses were selected with 5-bromodeoxyuridine (BUdR) and X-gal and identified with DNA dot blot analysis (vSC11-CETP and vTM1-CETP). The CETP cDNA insert in the recombinant vaccinia virus genome was identified by Southern blot analysis. Transcription of CETP cDNA in CV-1 cells infected with recombinant vaccinia virus was monitored by Northern blot analysis using the CETP cDNA as a probe. Positive signals were detected at 1.8 kb in cells infected with vSC11-CETP and at 2.3 kb in cells infected with vTM1-CETP. The recombinant vaccinia virus-infected CV-1 cells were shown to produce functional CETP when the culture medium was subjected to the CETP assay.

• Biomedical Science Letters
• /
• v.18 no.3
• /
• pp.291-298
• /
• 2012

The antiobesity effect of the mixture of black garlic and Garsinia cambogia extracts (BGG) was investigated by measuring the Oil red O staining and the expressions of adipogenic genes during preadipocyte differentiation by real-time PCR in the 3T3-L1 adipocytes. BGG reduced contents of Oil red O dye in the 3T3-L1 adipocytes. mRNA expression levels of SREBP1c, C/EBPa, aP2/FABP4, and $PPAR{\gamma}$ which are adipogenic transcription factor, in cells treated with BGG were also significantly down regulated. Also, the phosphorylation of AMP-activated protein kinase (AMPK) in L6 cells was more increased by BGG. These results indicate that BGG seems to be more attractive compound for application of industry than individual extracts such as black garlic and Garsinia cambogia, considering it has two effects not only inhibit the preadipocyte differentiation but also activate the phosphorylation of AMPK unlike other two compound.

• Biomedical Science Letters
• /
• v.10 no.2
• /
• pp.107-113
• /
• 2004

This study was conducted to investigate an effect of Mori Cortex in the cardiac injury following dermal scald burn in rats. Sprague-Dawley rats were induced scald bum (15％ of total body surface area). Heart was removed at 5 h postburn and examined with biochemical assay, ultrastructural observations and stereological analysis. The activity of serum aspartate aminotransferase and creatinine was increased at 5 h postburn compared with them of control. Administration of heat extracts of Mori Cortex after scald burn inhibited the production of KC (neutrophil chemoattractant cytokine) and increased the activity of protein kinase C (PKC) in heart tissue. The activity of myeloperoxidase (MPO) in heart tissue was decreased both at 5 h postburn and in case of Mori Cortex administration after scald burn. Ultrastructurally, many contraction bands and separation of intercalated disk induced by scald burn were decreased by administration of heat extracts of Mori Cortex. In stereological analysis, administration of Mori Cortex after scald burn resulted the volume densities of myofibril and mitochondria were increased compared with them of burn control. These data suggest that Mori Cortex may be a useful stuff to the range of available treatments for cardiac injury induced by skin burn.

• The Korean Journal of Physiology
• /
• v.29 no.2
• /
• pp.243-250
• /
• 1995

A possible potentiation between cholecystokinin (CCK) and secretin in amylase secretion from isolated rat pancreatic acini was investigated. Combined treatment of acini with secretin and CCK at low concentrations, which are known to be physiological, resulted in enzyme secretion larger than the arithmetic sum of their separate effects. Such a potentiating effect also occurred between secretin and A23187 (Ca ionophore), between forskolin (adenylate cyclase activator) and CCK, and between forskolin and A23187. Staurosporin (protein kinase C inhibitor) and W7 (calmodulin antagonist) inhibited markedly the potentiated amylase release induced by the agonists, but KT5720 (protein kinase A inhibitor) did not affect the potentiated amylase release. Therefore, we concluded that the action of CCK in a physiological concentration is potentiated by secretin in a physiological concentration range and vice versa, and that the intracellular mechanism necessary for the potentiation is associated with $Ca^{2+}$. However, it is uncertain what mechanisms are involved in potentiation of amylase release after CAMP and $Ca^{2+}$.

• Preventive Nutrition and Food Science
• /
• v.7 no.2
• /
• pp.128-132
• /
• 2002

The present study was performed to observe the role of protein kinase C (PKC) inhibitors (H-7, staurosporine) and daunorubicin in the cell death process of MCF7 cells; and examined whether or not the type of induced cell death was apoptosis. The usefulness of the combined therapy of PKC inhibitors and daunorubicin to improve the adverse effect of daunorubicin was also investigated. Cell death was induced by treatment with PKC inhibitors or daunorubicin. Characteristic morphologic features of cell shrinkage, chromatic condensation, and cytoplasmic vacuolization were observed. These treatments also stimulated the cleavage of poly-(ADP-ribose) polymerase (PARP), an early event in apoptosis. With slight differences in the percentage of apoptosis-induced cells, staurosporine, H-7 and daunorubicin effectively induced apoptosis in MCF7 cells. Furthermore, combined treatment of PKC inhibitors and daunorubicin significantly drove the cells into an apoptotic state. Hence, our results revealed the possible therapeutic value of combined therapy for the prevention of drug resistance and adverse side effects.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.17 no.2
• /
• pp.443-446
• /
• 2003

To investigate the protective effect of Bulbus Allii Macrostemi (BAM) on the damage by pulmonary vascular endothelial cells by xanthine oxidase (XO)/hypoxanthine (HX)-induced oxygen free radical, Neutral Red (NR) and protein kinase c (PKC) activity assay were used. The results were obtained as follows ; The viability of vascular endothelial cells treated with XO/HX was decreased. And activation of PKC represented a maximal increase in group treated with XO/HX for 15 mins in vasvular pulmonary endothelial cells. But pretreated groups with BAM extracts were not inhibited the increase of PKC activation by XO/HX in a dose-dependent fashion. These results show that XO/HX elicits toxic effects in cultured pulmonary vascular endothelial cells, and suggest that BAM extract is very effective in the prevention of XO/HX-induced PKC activation.