• Journal of Ginseng Research
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• v.46 no.3
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• pp.387-395
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• 2022

Background: Fermentation may alter the bioavailability of certain compounds, which may affect their efficacy and pharmacological responses. This study investigated the antiplatelet effects of red ginseng extract (RGE) and fermented red ginseng extract (FRG). Methods: A rodent model was used to evaluate the antiplatelet and antithrombotic effects of the extracts. Rats were orally fed with human equivalent doses of the extracts for 1 week and examined for various signaling pathways using standard in vivo and ex vivo techniques. Light transmission aggregometry was performed, and calcium mobilization, dense granule secretion, integrin α_IIbβ₃-mediated signaling molecules, cyclic nucleotide signaling events, and various protein molecules were evaluated ex vivo in collagen-stimulated washed platelets. Furthermore, antithrombotic properties were evaluated using a standard acute pulmonary thromboembolism model, and the effects on hemostasis were investigated using rat and mice models. Results: Both RGE and FRG significantly inhibited platelet aggregation, calcium mobilization, and dense granule secretion along with integrin-mediated fibrinogen binding and fibrinogen adhesion. cAMP levels were found to be elevated in RGE-treated rat platelets. Ginseng extracts did not exert any effect on prothrombin time and activated partial thromboplastin time. RGE-treated mice showed significantly better survival under thrombosis than FRG-treated mice, with no effects on hemostasis, whereas FRG-treated mice exhibited a slight increment in bleeding time. Conclusion: Both extracts, especially RGE, are remarkable supplements to maintain cardiovascular health and are potential candidates for the treatment and prevention of platelet-related cardiovascular disorders.

• International Journal of Oral Biology
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• v.46 no.4
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• pp.176-183
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• 2021

Oral squamous cell carcinoma (OSCC) metastasis is characterized by distant metastasis and local recurrence. Combined chemotherapy with cisplatin and 5-fluorouracil is routinely used to treat patients with OSCC, and the combined use of gefitinib with cytotoxic drugs has been reported to enhance the sensitivity of cancer cells in vitro. However, the development of drug resistance because of prolonged chemotherapy is inevitable, leading to a poor prognosis. Therefore, understanding alterations in signaling pathways and gene expression is crucial for overcoming the development of drug resistance. However, the altered characterization of Ca²⁺ signaling in drug-resistant OSCC cells remains unclear. In this study, we investigated alterations in intracellular Ca²⁺ ([Ca²⁺]_i) mobilization upon the development of gefitinib resistance in human tongue squamous carcinoma cell line (HSC)-3 and HSC-4 using ratiometric analysis. This study demonstrated the presence of altered epidermal growth factor- and purinergic agonist-mediated [Ca²⁺]_i mobilization in gefitinib-resistant OSCC cells. Moreover, Ca²⁺ content in the endoplasmic reticulum, store-operated calcium entry, and lysosomal Ca²⁺ release through the transient receptor potential mucolipin 1, were confirmed to be significantly reduced upon the development of apoptosis resistance. Consistent with [Ca²⁺]_i mobilization, we identified modified expression levels of Ca²⁺ signaling-related genes in gefitinib-resistant cells. Taken together, we propose that the regulation of [Ca²⁺]_i mobilization and related gene expression can be a new strategy to overcome drug resistance in patients with cancer.

• The Korean Journal of Physiology and Pharmacology
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• v.28 no.1
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• pp.93-103
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• 2024

Satellite glial cells (SGCs), a major type of glial cell in the autonomic ganglia, closely envelop the cell body and even the synaptic regions of a single neuron with a very narrow gap. This structurally unique organization suggests that autonomic neurons and SGCs may communicate reciprocally. Glial Ca²⁺ signaling is critical for controlling neural activity. Here, for the first time we identified the machinery of store-operated Ca²⁺ entry (SOCE) which is critical for cellular Ca²⁺ homeostasis in rat sympathetic ganglia under normal and pathological states. Quantitative realtime PCR and immunostaining analyses showed that Orai1 and stromal interaction molecules 1 (STIM1) proteins are the primary components of SOCE machinery in the sympathetic ganglia. When the internal Ca²⁺ stores were depleted in the absence of extracellular Ca²⁺, the number of plasmalemmal Orai1 puncta was increased in neurons and SGCs, suggesting activation of the Ca²⁺ entry channels. Intracellular Ca²⁺ imaging revealed that SOCE was present in SGCs and neurons; however, the magnitude of SOCE was much larger in the SGCs than in the neurons. The SOCE was significantly suppressed by GSK7975A, a selective Orai1 blocker, and Pyr6, a SOCE blocker. Lipopolysaccharide (LPS) upregulated the glial fibrillary acidic protein and Toll-like receptor 4 in the sympathetic ganglia. Importantly, LPS attenuated SOCE via downregulating Orai1 and STIM1 expression. In conclusion, sympathetic SGCs functionally express the SOCE machinery, which is indispensable for intracellular Ca²⁺ signaling. The SOCE is highly susceptible to inflammation, which may affect sympathetic neuronal activity and thereby autonomic output.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.3
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• pp.219-227
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• 2019

Polycystic kidney disease 2-like-1 (PKD2L1), polycystin-L or transient receptor potential polycystin 3 (TRPP3) is a TRP superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. Although the calmodulin (CaM) inhibitor, calmidazolium, is an activator of the PKD2L1 channel, the activating mechanism remains unclear. The purpose of this study is to clarify whether CaM takes part in the regulation of the PKD2L1 channel, and if so, how. With patch clamp techniques, we observed the current amplitudes of PKD2L1 significantly reduced when co-expressed with CaM and $CaM{\triangle}N$. This result suggests that the N-lobe of CaM carries a more crucial role in regulating PKD2L1 and guides us into our next question on the different functions of two lobes of CaM. We also identified the predicted CaM binding site, and generated deletion and truncation mutants. The mutants showed significant reduction in currents losing PKD2L1 current-voltage curve, suggesting that the C-terminal region from 590 to 600 is crucial for maintaining the functionality of the PKD2L1 channel. With PKD2L1608Stop mutant showing increased current amplitudes, we further examined the functional importance of EF-hand domain. Along with co-expression of CaM, ${\triangle}EF$-hand mutant also showed significant changes in current amplitudes and potentiation time. Our findings suggest that there is a constitutive inhibition of EF-hand and binding of CaM C-lobe on the channel in low calcium concentration. At higher calcium concentration, calcium ions occupy the N-lobe as well as the EF-hand domain, allowing the two to compete to bind to the channel.

• Molecules and Cells
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• v.24 no.2
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• pp.276-282
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• 2007

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin ($Ca^{2+}/CaM$)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about $Ca^{2+}/CaM$-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative $Ca^{2+}$-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a $Ca^{2+}$-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM $Mn^{2+}$. The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other $Ca^{2+}/CaM$-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and $Ca^{2+}/CaM$-dependent protein kinase), increasing the concentration of calmodulin to more than $3{\mu}M$ suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis $Ca^{2+}/CaM$-dependent protein kinase which is presumably involved in CaM-mediated signaling.

• BMB Reports
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• v.53 no.3
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• pp.154-159
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• 2020

We investigated the effects of physalin A, B, D, and F on osteoclastogenesis induced by receptor activator of nuclear factor κB ligand (RANKL). The biological functions of different physalins were first predicted using an in silico bioinformatic tool (BATMAN-TCM). Afterwards, we tested cell viability and cell apoptosis rate to analyze the cytotoxicity of different physalins. We analyzed the inhibitory effects of physalins on RANKL-induced osteoclastogenesis from mouse bone-marrow macrophages (BMMs) using a tartrate-resistant acid phosphatase (TRAP) stain. We found that physalin D has the best selectivity index (SI) among all analyzed physalins. We then confirmed the inhibitory effects of physalin D on osteoclast maturation and function by immunostaining of F-actin and a pit-formation assay. On the molecular level, physalin D attenuated RANKL-evoked intracellular calcium ([Ca(2+)](i)) oscillation by inhibiting phosphorylation of phospholipase Cγ2 (PLCγ2) and thus blocked the downstream activation of Ca2+/calmodulin-dependent protein kinases (CaMK)IV and cAMP-responsive element-binding protein (CREB). An animal study showed that physalin D treatment rescues bone microarchitecture, prevents bone loss, and restores bone strength in a model of rapid bone loss induced by soluble RANKL. Taken together, these results suggest that physalin D inhibits RANKL-induced osteoclastogenesis and bone loss via suppressing the PLCγ2-CaMK-CREB pathway.

• BMB Reports
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• v.38 no.4
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• pp.432-439
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• 2005

$Ca^{2+}$/calmodulin transduction pathways have been implicated in mediating stress response and tolerance in plants. Here, three genes encoding calmodulin (Cam) members of the EF-hand family of $Ca^{2+}$-binding proteins were identified from Oryza sativa L. databases. Complementary DNA for each of the calmodulin genes, OsCam1, OsCam2, and OsCam3 were sequenced. OsCam1 and OsCam2 encode a conventional 148-amino acid calmodulin protein that contains four characteristic $Ca^{2+}$-binding motifs. OsCam3 encode a similar protein with a 38-amino-acid extension containing a putative prenylation site (CVIL) at the carboxyl terminus. RT-PCR showed that each of the genes is expressed in leaves and roots of 2-week old rice seedlings. By RNA gel blot analysis, OsCam1 mRNA levels strongly increased in response to NaCl, mannitol and wounding treatments. In contrast, OsCam2 mRNA levels were relatively unchanged under all conditions investigated. NaCl treatment and wounding also increased the OsCam3 mRNA level, but in a more transient manner. Our results indicate that although the expression of genes encoding different calmodulin isoforms is ubiquitous, they are differentially regulated by various stress signals. In addition, we have demonstrated that the calcium-channel blocker lanthanum chloride inhibited the induction of OsCam1 gene expression by both NaCl and mannitol treatments. These results suggest that osmotic stress induced expression of OsCam1 gene requires the $[Ca^{2+}]_{cyt}$ elevation that is known to occur in response to these stimuli.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.1
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• pp.51-57
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• 2010

It was hypothesized that NaF induces calcium sensitization in $Ca^{2+}$-controlled solution in permeabilized rat mesenteric arteries. Rat mesenteric arteries were permeabilized with $\beta$-escin and subjected to tension measurement. NaF potentiated the concentration-response curves to $Ca^{2+}$ (decreased $EC_{50}$ and increased $E_{max}$). Cumulative addition of NaF (4.0, 8.0 and 16 mM) also increased vascular tension in $Ca^{2+}$-controlled solution at pCa 7.0 or pCa 6.5, but not at pCa 8.0. NaF-induced vasocontraction and $GTP{\gamma}S$-induced vasocontraction were not additive. NaF-induced vasocontraction at pCa 7.0 was inhibited by pretreatment with Rho kinase inhibitors H1152 or Y27632 but not with a MLCK inhibitor ML-7 or a PKC inhibitor Ro31-8220. NaF induces calcium sensitization in a $Ca^{2+}$ dependent manner in $\beta$-escin-permeabilized rat mesenteric arteries. These results suggest that NaF is an activator of the Rho kinase signaling pathway during vascular contraction.

• Food Science of Animal Resources
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• v.30 no.5
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• pp.787-794
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• 2010

Calcium plays a role as a signaling molecule in various cellular events. It has been reported that calcium suppresses adipocyte differentiation only in the early phase of adipogenesis. Herein, we demonstrate that treatment of A23187, a mobilizer of intracellular calcium, on day 4 post adipocyte differentiation could still reduce lipid accumulation in differentiating 3T3-L1 cells for 48 h. In addition, luciferase reporter gene and RT-Q-PCR assays demonstrate that A23187 can selectively inhibit transcriptional activities and expression of PPAR$\gamma$ and LXR$\alpha$, suggesting that A23187 may reduce lipid accumulation in the late phase of adipogenesis via downregulation of PPAR$\gamma$ and LXR$\alpha$ expression and transactivation. Moreover, inhibition of HDAC activity by trichostatin A (TSA) partially blocked A23187-mediated downregulation of transcriptional activities of PPAR$\gamma$ and LXR$\alpha$. Together, our data demonstrate that calcium mobilization inhibits expression and transcriptional activities of PPAR$\gamma$ and LXR$\alpha$, resulting in reduced lipid accumulation in differentiating adipocytes, and thus, mobilization of intracellular calcium in adipocytes may serve as a new preventive and therapeutic approach for obesity.

• Journal of Ginseng Research
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• v.22 no.2
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• pp.126-132
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• 1998

In the present study, we examined effects of ginseng saponins (ginsenosides) on pp60c-src protein tyrosine kinase (PTK) activity, intracellular calcium concentration ([$Ca^{2+}$]i), and cell proliferation in NIH3T3 cells. Eight different ginsenosides [ginsenoside-Rb1 (G-$Rb_1$), -$Rb_2$, -Rc, -Rd, -Re, -Rf, -$Rg_1$, -$Rg_2$) and ginseng total saponin (GTS) were used for these experiments. All ginsenosides and GTS tested stimulated the activation of $pp60^{c-src}$ kinase, and especially G-$Rb_1$,-Rd,-$Rg_1$, and -$Rg_1$ showed a higher stimulatory effect than others at 16.7 $\mu\textrm{g}$/ml of ginsenosides with a 18 hr-incubation, increasing the activity by 4.5, 3.5, 3.5, and 3.0-fold, respectively, over that of untreated control. In addition, both G-Rd and -$Rg_2$)Rg2 increased ($Ca^{2+}$), to 202 and 334 nM, respectively, about 2-3-fold above the basal level within 7min at 250 $\mu\textrm{g}$/yml of ginsenosides. The increases of ($Ca^{2+}$), were eliminated by Pretreatment of EGTA, an extracellular calcium chelator, suggtasting that they result from an influx of calcium ion from extracellular medium rather than an efflux from intracellular calcium store, endoplasmic reticulum (ER). All ginsenosides studied enhanced cell proliferation to 1.2-1.4-fold over that of untreated control at 5~250 $\mu\textrm{g}$/ml of concentrations. Interestingly the promotion of cell proliferation by ginsenosides corresponded with the activation of c-src kinase, which is an early step in the mitogenic signaling cascade. Taken together, we suggest that some ginsenosides may lead to cellProliferation via the activation of cellular signal transduction Pathway involving $pp60^{c-src}$ kinase.