• Asian Pacific Journal of Cancer Prevention
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• v.15 no.4
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• pp.1551-1556
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• 2014

Background: Transient receptor potential melastatin 8 (TRPM8), a principle membrane receptor involved in calcium ion influx and cell signal transduction, has been found to be up-regulated in some cancer types, including melanomas. Efficiency of menthol, an agonist of TRPM8, in killing melanoma cancer cells has been reported previously, but the mechanisms remain unclear. We here determined whether in vitro cytotoxic effects of menthol on A-375 human malignant melanoma cells might be related to TRPM8 transcript expression. Materials and Methods: The $PrestoBlue^{(R)}$ cell viability assay was used to assess the in vitro cytotoxic effect of menthol after 24h of treatment. RT-PCR was used to quantify TRPM8 transcript expression levels in normal and menthol-treated cells. Cell morphology was observed under inverted phase contrast light microscopy. Results: TRPM8 transcript expression was found at low levels in A-375 cells and down-regulated in a potentially dose-dependent manner by menthol. Menthol exerted in vitro cytotoxic effects on A-375 cells with an $IC_{50}$ value of 11.8 ${\mu}M$, which was at least as effective as 5-fluorouracil ($IC_{50}=120{\mu}M$), a commonly applied chemotherapeutic drug. Menthol showed no dose-dependent cytotoxicity on HeLa cells, a TRPM8 non-expressing cell line. Conclusions: The cytotoxic effects on A-375 cells caused by menthol might be related to reduction of the TRPM8 transcript level. This suggests that menthol might activate TRPM8 to increase cytosolic $Ca^{2+}$ levels, which leads to cytosolic $Ca^{2+}$ imbalance and triggers cell death.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.1
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• pp.109-117
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• 1998

Role of $Ca^{2+}$/calmodulin complex in intracellular $Ca^{2+}$ mobilization in neutrophils has not been clearly elucidated. In this study, effects of chlorpromazine, trifluoperazine and imipramine on the intracellular $Ca^{2+}$ mobilization, including $Ca^{2+}$ influx, in C5a-activated neutrophils were investigated. Complement C5a- stimulated superoxide production and myeloperoxidase release in neutrophils were inhibited by chlorpromazine, trifluoperazine and imipramine, except no effect of imipramine on myeloperoxidase release. A C5a-elicited elevation of [$Ca^{2+}$]i in neutrophils was inhibited by chlopromazine, trifluoperazine, imipramine, staurosporine, genistein, EGTA, and verapamil but not affected by pertussis toxin. The intracellular $Ca^{2+}$ release in C5a-activated neutrophils was not affected by chlorpromazine and imipramine. Chlorpromazine and imipramine inhibited $Mn^{2+}$ influx by C5a-activated neutrophils. Thapsigargin-evoked $Ca^{2+}$ entry was inhibited by chlorpromazine, trifluoperazine, imipramine, genistein, EGTA and verapamil, while the effect of staurosporine was not detected. The results suggest that $Ca^{2+}$/calmodulin complex is involved in the activation process of neutrophils. The depressive action of calmodulin inhibitors on the elevation of cytosolic $Ca^{2+}$ level in C5a-activated neutrophils appears to be accomplished by inhibition of $Ca^{2+}$ influx from the extracellular medium.

• Journal of Ginseng Research
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• v.32 no.2
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• pp.107-113
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• 2008

In this study, we have investigated the effect of panaxatriol (PT) on phosphoinositides (PIS) breakdown and $Ca^{2+}$-elevation in thrombin-induced platelet aggregation. Thrombin (5U/ml), a potent platelet agonist which activates phospholipase $C_{\beta}$ via protease activated receptor (PAR), hydrolyzed PIS in platelet membrane. The phosphatidylinositol 4, 5-bisphosphate $(PIP_2)$ was hydrolyzed after 10 sec of the thrombin-stimulation, and both the phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol (PI) were brokendown after 30 sec of the thrombin-stimulation. However, PT inhibited the thrombin-stimulated hydrolysis of $PIP_2$, PIP, and PI. On the other hand, thrombin increased the level of phosphatidic acid (PA) which is phosphorylated from diacylglycerol (DG) generated by PIS-hydrolysis. However, Pr inhibited the thrombin-increased PA level non-significantly. Thrombin increased cytosolic free $Ca^{2+}([Ca^{2+}])_i$) up to 72% as compared with control $(30.8{\pm}0.9 nM)$ in intact platelet. However, PT (100 ${\mu}g/ml$) inhibited the thrombin-elevated $[Ca^{2+}]_i$ to 100%. These results suggest that PT may have a beneficial effect on platelet aggregation-mediated thrombotic disease by inhibiting thrombin-induced platelet aggregation via suppression of the $[Ca^{2+}]_i$ level and PIS breakdown.

• Biomolecules & Therapeutics
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• v.17 no.2
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• pp.125-132
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• 2009

Calbindin-$D_{9k}$ (CaBP-9k), a cytosolic calcium-binding protein, is expressed in a variety of tissues, i.e., the duodenum, uterus, placenta, kidney and pituitary gland. Duodenal CaBP-9k is involved in intestinal calcium absorption, and is regulated at transcriptional and post-transcriptional levels by 1,25-dihydroxyvitamin D3, the hormonal form of vitamin D, and glucocorticoids (GCs). Uterine CaBP-9k has been implicated in the regulation of myometrial action(s) through modulation of intracellular calcium, and steroid hormones appear to be the main regulators in its uterine and placental regulation. Because phenotypes of CaBP-9k-null mice appear to be normal, other calcium-transporter genes may compensate for its gene deletion and physiological function in knockout mice. Previous studies indicate that CaBP-9k may be controlled in a tissue-specific fashion. In this review, we summarize the current information on calcium homeostasis related to CaBP-9k gene regulation by GCs, vitamin D and its receptors, and its molecular regulatory mechanism. In addition, we present related data from our current research.

• Clinical and Experimental Pediatrics
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• v.57 no.10
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• pp.445-450
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• 2014

Purpose: Familial hypokalemic periodic paralysis (HOKPP) is an autosomal dominant channelopathy characterized by episodic attacks of muscle weakness and hypokalemia. Mutations in the calcium channel gene, CACNA1S, or the sodium channel gene, SCN4A, have been found to be responsible for HOKPP; however, the mechanism that causes hypokalemia remains to be determined. The aim of this study was to improve the understanding of this mechanism by investigating the expression of calcium-activated potassium ($K_{Ca}$) channel genes in HOKPP patients. Methods: We measured the intracellular calcium concentration with fura-2-acetoxymethyl ester in skeletal muscle cells of HOKPP patients and healthy individuals. We examined the mRNA and protein expression of KCa channel genes (KCNMA1, KCNN1, KCNN2, KCNN3, and KCNN4) in both cell types. Results: Patient cells exhibited higher cytosolic calcium levels than normal cells. Quantitative reverse transcription polymerase chain reaction analysis showed that the mRNA levels of the $K_{Ca}$ channel genes did not significantly differ between patient and normal cells. However, western blot analysis showed that protein levels of the KCNMA1 gene, which encodes $K_{Ca}$1.1 channels (also called big potassium channels), were significantly lower in the membrane fraction and higher in the cytosolic fraction of patient cells than normal cells. When patient cells were exposed to 50 mM potassium buffer, which was used to induce depolarization, the altered subcellular distribution of BK channels remained unchanged. Conclusion: These findings suggest a novel mechanism for the development of hypokalemia and paralysis in HOKPP and demonstrate a connection between disease-associated mutations in calcium/sodium channels and pathogenic changes in nonmutant potassium channels.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.319-327
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• 2011

This study investigated whether the addition of porcine sperm cytosolic factor (SCF) at fusion/activation affects in vitro development of porcine parthenogenetic(PA) and nuclear transfer (NT) embryos. To determine the optimum concentration of SCF, control group of oocytes was activated with 0.3M mannitol (1.0 mM $CaCl_2{\cdot}2H_2O$), other three groups of oocytes were parthenogentically activated with the fusion medium (0.1mM $CaCl_2{\cdot}2H_2O$) supplemented with 100, 200 or 300 ${\mu}$g/ml SCF, respectively. Matured oocytes were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}$sec. The activated embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. Oocytes activated in the presence of SCF showed a significantly higher blastocyst rate than control (p<0.05). Apoptosis rate was significantly lower in 100 ${\mu}$g/ml SCF group than other groups (p<0.05). Cdc2 kinase activity in control and SCF treatment group of oocytes was determined using MESACUP cdc2 kinase assay kit at 1, 5, 10, 15, 30, 45 and 60 min after activation. Cdc2 kinase activity was significantly decreased (p<0.05) in SCF group than MII oocytes or control within 5 min. For NT embryo production, reconstructed oocytes were fused in the fusion medium supplemented with 0.1 mM $CaCl_2{\cdot}2H_2O$ (T1), 1.0 mM $CaCl_2{\cdot}2H_2O$ (T2) and 0.1 mM $CaCl_2{\cdot}2H_2O$ with 100 ${\mu}$g/ml SCF (T3). Fused embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. Developmental rate to blastocyst stage was significantly higher in T3 than other groups (23.0% vs. 13.5 to 15.2%) (p<0.05). Apoptosis rate was significantly lower in T3 than T1 or T2 (p<0.05). The relative abundance of Bax-${\alpha}$/Bcl-xl was significantly lower in in vivo or SCF group than that of control (p<0.05). Moreover, the expression of p53 and caspase3 mRNA was significantly lower in in vivo or SCF group than that of control (p<0.05). These results indicate that the addition of SCF at fusion/activation might improve in vitro development of porcine NT embryos through regulating cdc2 kinase level and expression of apoptosis related genes.

• Journal of Ginseng Research
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• v.37 no.2
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• pp.176-186
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• 2013

In this study, we have investigated the effects of total saponin from Korean red ginseng (TSKRG) on thrombin-induced platelet aggregation. TSKRG dose-dependently inhibited thrombin-induced platelet aggregation with $IC_{50}$ value of about 81.1 ${\mu}g/mL$. In addition, TSKRG dose-dependently decreased thrombin-elevated the level of cytosolic-free $Ca^{2+}$ ($[Ca^{2+}]_i$), one of aggregation-inducing molecules. Of two $Ca^{2+}$-antagonistic cyclic nucleotides as aggregation-inhibiting molecules, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), TSKRG significantly dose-dependently elevated intracellular level of cAMP, but not cGMP. In addition, TSKRG dose-dependently inhibited thrombin-elevated adenosine triphosphate (ATP) release from platelets. These results suggest that the suppression of $[Ca^{2+}]_i$ elevation, and of ATP release by TSKRG are associated with upregulation of cAMP. TSKRG elevated the phosphorylation of vasodilator-stimulated phosphoprotein (VASP)-$Ser^{157}$, a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-$Ser^{239}$, a cGMP-dependent protein kinase substrate, in thrombin-activated platelets. We demonstrate that TSKRG involves in increase of cAMP level and subsequent elevation of VASP-$Ser^{157}$ phosphorylation through A-kinase activation to inhibit $[Ca^{2+}]_i$ mobilization and ATP release in thrombin-induced platelet aggregation. These results strongly indicate that TSKRG is a beneficial herbal substance elevating cAMP level in thrombin-platelet interaction, which may result in preventing of platelet aggregation-mediated thrombotic diseases.

• Journal of Applied Biological Chemistry
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• v.65 no.4
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• pp.463-469
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• 2022

Cudrania tricuspidata (C. tricuspidata), a medicinal plant widely employed throughout Asia in ethnomedicine, has various bioactive properties, including antidiabetic, antiobesity, antitumor, and anti-inflammatory activities. In addition, the C. tricuspidata root extract reportedly inhibits platelet aggregation. Therefore, we focused on the active substances present in the C. tricuspidata extract. Sanggenon N (SN) is a flavonoid found in the root bark of C. tricuspidata. In the present study, we examined the inhibitory effects of SN on platelet aggregation, phosphoproteins, thromboxane A₂ generation, and integrin αIIbβ3 activity. SN inhibited collagen-induced human platelet aggregation in a dose-dependent manner without cytotoxicity. Furthermore, SN suppressed Ca²⁺ mobilization and influx through associated signaling molecules, such as inositol 1, 4, 5-triphosphate receptor I (Ser¹⁷⁵⁶), and extracellular signal-regulated kinase. In addition, SN inhibited thromboxane A₂ generation and associated signaling molecules, including cytosolic phospholipase A₂ and mitogen-activated protein kinase p38. Finally, SN could inhibit integrin (αIIb/β₃) activity by regulating vasodilator-stimulated phosphoprotein and Akt. Collectively, SN possesses potent antiplatelet effects and is a potential therapeutic drug candidate to prevent platelet-related thrombosis and cardiovascular disease.

• International Journal of Oral Biology
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• v.31 no.3
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• pp.99-105
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• 2006

Von Ebner's glands (vEG) are minor salivary glands associated with circumvallate and foliate papilla. The secretions of vEG consist of microenvironment of the taste buds in the circumvallate and foliate papillae, and thus saliva from vEG plays a role in the perception of taste. The $Ca^{2+}$ signaling system in rat vEG acinar cell was examined using the $Ca^{2+}$-sensitive fluorescent indicator Fura-2. Agonist-induced increase in intracellular $Ca^{2+}\;([Ca^{2+}]_i)$ was stimulated by carbachol (CCh) and substance P (SP), but not by norepinephrine (NE), and recovered to control levels by their receptor antagonists dose-dependently. The effects were also observed in $Ca^{2+}$-free medium, suggesting mobilization from intracellular $Ca^{2+}$ store. These results in the vEG acinar cell indicate that 1) $[Ca^{2+}]_i$ is at least regulated by muscarinic and neurokininergic (NK1) receptors; 2) the increases in $[Ca^{2+}])i$ activated by CCh and SP are mainly mediated by discharge of cytosolic calcium pool.

• Journal of Microbiology and Biotechnology
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• v.33 no.3
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• pp.378-386
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• 2023

The CYP707A family genes encoding ABA 8'-hydroxylase catabolize abscisic acid (ABA), a plant stress hormone that plays an important role in stress condition, such as drought, heat, cold and salinity. Phaseic acid (PA) is a catabolic product of ABA. Recent studies have shown that PA is important for the physiological functions in plants. It is also a neuroprotective molecule that protects against ischemic brain injury in mice. To obtain enzymes for the PA production, four CaCYP707A genes (CaCYP707A1, CaCYP707A2, CaCYP707A3 and CaCYP707A4) were isolated from hot pepper. They were heterologously expressed in Escherichia coli. Among them, CaCYP707A2 showed significantly higher expression levels in both the membrane fraction and the soluble fraction. Preferred redox partners were investigated to improve the efficiency of CaCYP707A2's catalytic reaction, and NADPH-cytochrome P450 reductase (CPR) from hot pepper (CaCPR) was preferred over other redox partners (i.e., rat CPR and ferredoxin reductase/ferredoxin). The production of 8'-hydroxy ABA and PA by ABA hydroxylation activity was confirmed in CaCYP707A2 from both membrane and soluble fractions. Therefore, CaCYP707A2 is the first identified plant CYP protein that is expressed a soluble form in cytosolic fraction having stable activity. Taken together, we propose a new CYP707A protein with industrial applications for PA production without additional modifications in E. coli heterologous expression.