• YAKHAK HOEJI
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• v.41 no.6
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• pp.749-755
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• 1997

It has been reported that dose-dependent $Ca^{2+}$ increase by menadione in platelets could be measured by fluorescent dye, quin-2. The problems will be described here rel ating to measuring $Ca^{2+}$ in menadione-exposed platelets using fura-2 and fluo-3, widely used fluorescent indicators. Additions of menadione to fura-2 loaded platelets and their lysates resulted in marked reduction in fluorescence intensity at both 340nm ($Ca^{2+}$-unbound form) 380nm ($Ca^{2+}$-undbound form) excitation wavelengths. Fura-2 excitation spectra were overlapped with UV-visible absorption spectra of menadione, suggesting that light absorption by menadione itself could quench fluorescence generated by fura-2. Next approach was to use fluo-3 which has the higher wavelength (490nm) of excitation. Previous work demonstrated that treatment with probenecid to platelets was required to prevent fluo-3 dye leakage. However, probenecid itself was proven to be inadequate to measure the concentration of intracellular $Ca^{2+}$; by reducing menadione-induced cytotoxicity in platelets. Our results suggest that it is not feasible to measure $Ca^{2+}$ in platelets by using fura-2 and fluo-3 in the presence of probenecid, and cautions should be taken to measure changes of intracellular $Ca^{2+}$ levels by fluorescent dyes following chemical exposure.

• Journal of the Optical Society of Korea
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• v.19 no.3
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• pp.228-236
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• 2015

Fluorescence recovery after photobleaching (FRAP) is a well-established method commonly used to measure the diffusion of fluorescent solutes and biomolecules in living cells or tissues. Here a fiber-optic-based FRAP (f-FRAP) system was developed, and validated using macromolecules in water and agarose gels of different concentrations. We applied f-FRAP to measure the site-specific diffusion of fluorescein (NaFluo) in peritoneal membranes (PMs) on the liver, cecum, and kidney of a living rat during peritoneal dialysis. Diffusion of fluorescein in PM varied in a time-dependent manner according to the type of organ ($D_{PM\;on\;Liver}/D_{NaFluo}=0.199{\pm}0.085$, $D_{PM\;on\;Cecum}/D_{NaFluo}=0.292{\pm}0.151$, $D_{PM\;on\;Kidney}/D_{NaFluo}=0.218{\pm}0.110$). The proposed method allows direct quantitative measurement of the three-dimensional diffusion in local PM in vivo, which was previously inaccessible by peritoneal function test methods such as peritoneal equilibration test (PET) and standardized PM assessment (SPA). f-FRAP is promising for local and dynamic assessments of peritoneal pathophysiology and the mass transport properties of PMs, presumed to be affected by variation of tissue structures over different organs and functional changes of the PM with years of peritoneal dialysis.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.5
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• pp.291-298
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• 2005

To characterize cytosolic $Ca^{2+}$ fluctuations under metabolic inhibition, rat ventricular myocytes were exposed to $200{\mu}M$ 2,4-dinitrophenol (DNP), and mitochondrial $Ca^{2+}$, mitochondrial membrane potential (${\Delta}{\Psi}m$), and cytosolic $Ca^{2+}$ were measured, using Rhod-2 AM, TMRE, and Fluo-4 AM fluorescent dyes, respectively, by Laser Scanning Confocal Microscopy (LSCM). Furthermore, the role of sarcolemmal $Na^+$/$Ca^{2+}$ exchange (NCX) in cytosolic $Ca^{2+}$ efflux was studied in KB-R7943 and $Na^+$-free normal Tyrode's solution (143 mM LiCl ). When DNP was applied to cells loaded with Fluo-4 AM, Fluo-4 AM fluorescence intensity initially increased by $70{\pm}10$% within $70{\pm}10$ s, and later by $400{\pm}200$% at $850{\pm}45$ s. Fluorescence intensity of both Rhod-2 AM and TMRE were initially decreased by DNP, coincident with the initial increase of Fluo-4 AM fluorescence intensity. When sarcoplasmic reticulum (SR) $Ca^{2+}$ was depleted by $1{\mu}M thapsigargin plus $10{\mu}M ryanodine, the initial increase of Fluo-4 AM fluorescence intensity was unaffected, however, the subsequent progressive increase was abolished. KB-R7943 delayed both the first and the second phases of cytosolic $Ca^{2+}$ overload, while $Na^+$-free solution accelerated the second. The above results suggest that: 1) the initial rise in cytosolic $Ca^{2+}$ under DNP results from mitochondrial depolarization; 2) the secondary increase is caused by progressive $Ca^{2+}$ release from SR; 3) NCX plays an important role in transient cytosolic $Ca^{2+}$ shifts under metabolic inhibition with DNP.

• Proceedings of the Zoological Society Korea Conference
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• 1994.10a
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• pp.199.1-199
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• 1994

No Abstract, See Full Text

• Nutrition Research and Practice
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• v.12 no.3
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• pp.183-190
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• 2018

BACKGROUND/OBJECTIVE: This study was designed to investigate how a Portulaca oleracea L. extract (POE) stimulates insulin secretion in INS-1 pancreatic ${\beta}-cells$. MATERIALS/METHOD: INS-1 pancreatic ${\beta}-cells$ were incubated in the presence of various glucose concentrations: 1.1 or 5.6, 16.7 mM glucose. The cells were treated with insulin secretagogues or insulin secretion inhibitor for insulin secretion assay using an insulin ELISA kit. In order to quantify intracellular influx of $Ca^{2+}$ caused by POE treatment, the effect of POE on intracellular $Ca^{2+}$ in INS-1 pancreatic ${\beta}-cells$ was examined using Fluo-2 AM dye. RESULTS: POE at 10 to $200{\mu}g/mL$ significantly increased insulin secretion dose-dependently as compared to the control. Experiments at three glucose concentrations (1.1, 5.6, and 16.7 mM) confirmed that POE significantly stimulated insulin secretion on its own as well as in a glucose-dependent manner. POE also exerted synergistic effects on insulin secretion with secretagogues, such as L-alanine, 3-isobutyl-1-methylxanthine, and especially tolbutamide, and at a depolarizing concentration of KCl. The insulin secretion caused by POE was significantly attenuated by treatment with diazoxide, an opener of the $K{^+}_{ATP}$ channel (blocking insulin secretion) and by verapamil (a $Ca^{2+}$ channel blocker). The insulinotropic effect of POE was not observed under $Ca^{2+}$-free conditions in INS-1 pancreatic ${\beta}-cells$. When the cells were preincubated with a $Ca^{2+}$ fluorescent dye, Fluo-2 (acetoxymethyl ester), the cells treated with POE showed changes in fluorescence in red, green, and blue tones, indicating a significant increase in intracellular $Ca^{2+}$, which closely correlated with increases in the levels of insulin secretion. CONCLUSIONS: These findings indicate that POE stimulates insulin secretion via a $K{^+}_{ATP}$ channel-dependent pathway in INS-1 pancreatic ${\beta}-cells$.

• Food Science of Animal Resources
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• v.20 no.3
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• pp.231-235
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• 2000

원유에 존재하는 Ps. fluorescens의 균수를 신속하게 측정하기 위한 Inhibition Enzyme Linked Immunosorbant Assay (ELSIA)를 개발하기 위해 Ps, fluorescens(KCTC 2344)을 산란계에 접종하여 Ps.fluorescens에 대한 anti-Ps. fluorescens IgY 항체를 생산하고 그 항체의 역기를 ELISA로 측정한 결과 32일까지 항체 역가가 증가하였으며, 분리된 IgY 항체의 titer는 1:128,000으로 나타났다. Anti-Ps.fluorescens IgY 항체에대한 교차반응을 조사한 결과 그람양성균 Lactococcus faecalis, Staphylococcus aureus 뿐만 아니라 그람음성 균인 Achrombacter sal-monisida, Escherichia coil와도 교차반응을 거의 하지 않는 것으로 나타났다. Anti-Ps. fluo-rescens IgY 항체를 가지고 Inhibition ELISA 방법으로 Ps. fluorescens 균수를 신속한 측정할 수 있는 표준곡선을 작성한 결과 Ps. fluorescens균을 5.0$\times$$10^4$cfu/ml부터 5.0$\times$$10^{8}$cfu/ml 측정할 수 있는 것으로 나타났다.

• Bulletin of the Korean Chemical Society
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• v.23 no.10
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• pp.1451-1489
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• 2002

Sphingosine is known to regulate a wide range of cell physiology including growth, differentiation, and apoptosis. In this study, we examined the effect of sphingosine on the phospholipase D (PLD) activity in rat thymocytes. Sphingosine potently stimulated PLD in the absence of extracellular calcium, while depletion of intracellular calcium by BAPTA/AM treatment completely blocked activation of PLD by sphingosine. Sphingosine-induced increase of the intracellular calcium concentration was confirmed using a fluorescent calcium indicator Fluo-3/AM. A phosphoinositide-specific phospholipase C inhibitor U73122 partially inhibited the stimulation of PLD by sphingosine. When mouse PLD2 gene was transfected into mouse thymoma EL4 cells, which lack intrinsic PLD activity, sphingosine could stimulate PLD2 significantly while overexpression of human PLD1 had no effect. Taken together, the sphingosine-stimulated PLD activity in rat thymocytes is dependent on the mobilization of intracellular calcium and appears to be due to the PLD2 isoform.

• Development and Reproduction
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• v.7 no.2
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• pp.95-103
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• 2003

Intracellular calcium is an important physiological factor in most cells, and ruthenium red and ryanodine play an important role as calcium modulators. Ruthenium red inhibits calcium-induced calcium release(CICR) from the intracellular calcium store. Ryanodine activates calcium release through ryanodine channel. The present experiment was performed to investigate the effects of two modulators on calcium ion metabolism and to determine their dose-dependency in oocyte and early embryo of mouse. Intracellular calcium ion concentration was measured in realtime by using confocal laser scanning microscope(CLSM) after loading of Fluo-3/AM in mouse oocytes and early embryos. Ruthenium red decreased intracellular calcium ion concentration in oocytes and early embryos at its high concentration(30, 300 $\mu$M). Ryanodine increased intracellular calcium ion concentration in oocytes and early embryos in low concentration(0.01 $\mu$M) but decreased that at higher concentrations(1, 10 $\mu$M). These results indicate that two modulators affected calcium ion metabolism in oocyte and early embryo of mouse, and their dose-dependency was different from somatic cell including myocytes.

• Natural Product Sciences
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• v.18 no.1
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• pp.26-31
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• 2012

Actinidia arguta (Actinidiaceae) has been reported to have several pharmacological effects such as anti-inflammatory, anti-allergic, and anti-oxidant activities. The present study investigated the protective activity of an ethanol extract from the leaf and stem of A. arguta against glutamate-induced neurotoxicity using cultured rat cortical neurons. Exposure of cultured cortical neurons to $500{\mu}M$ glutamate for 12 h triggered neuronal cell death. A. arguta inhibited glutamate-induced neuronal death and apoptosis, which were measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining, respectively. The increase of pro-apoptotic proteins, Bax and c-caspase-3, in glutamate-treated neurons was significantly inhibited by treatment with A. arguta. A. arguta also inhibited $500{\mu}M$ glutamate-induced elevation of intracellular calcium concentration ($[Ca^{2+}]_i$) and reactive oxygen species (ROS) generation, which were measured by fluorescent dyes, Fluo-4 AM and $H_2DCF$-DA, respectively. These results suggest that A. arguta may prevent glutamate-induced apoptotic neuronal death by inhibiting $[Ca^{2+}]_i$ elevation and ROS generation and, therefore, may have a therapeutic role for the prevention of neurodegeneration in cerebral ischemic diseases.

• Journal of Microbiology
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• v.40 no.3
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• pp.224-229
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• 2002

The effect of human cytomegalovirus (HCMV) on three human monocyte cell lines at different stages of differentiation was investigated. While the viability of HL-60 cells or U-937 cells was not significantly affected by HCMV infection, the viability of THP-1 cells was reduced. Acridine orange/ethidiurn bromide staining revealed that the reduction of THP-1 cell viability was due to increased apoptotic death following HCMV infection. Apoptosis in HL-60 cells was not affected by HCMV infection, and induction of apoptosis of U-937 cells by HCMV was intermediate between HL-60 and THP-1 cells. Since HL-60 cells are the least differentiated and THP-1 cells are the most differentiated, the induction of apoptosis of human monocytes appears to be related to the degree of cell differentiation. Flow cytometric and confocal microscopic studies using fluorescent calcium indicator Fluo-3 suggested a significant increase in intracellular free calcium concentration (［Ca$\^$2+/］i) in THP-1 cells undergoing apoptosis by HCMV infection. Again ［Ca$\^$2+/］i in HCMV-infected HL-60 cells was not critically altered, and that in HCMV-infected U-937 cells was intermediate between THP-1 cells and HL-60 cells. Calcium influx blockers such as verapamil and nifedipine partially reversed HCMV-induced apoptosis in THP-1 cells.