• Journal of Microbiology and Biotechnology
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• v.34 no.4
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• pp.783-794
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• 2024

The antifungal activity of fisetin against Candida albicans is explored, elucidating a mechanism centered on membrane permeabilization and ensuing disruption of pH homeostasis. The Minimum Inhibitory Concentration (MIC) of fisetin, indicative of its interaction with the fungal membrane, increases in the presence of ergosterol. Hoechst 33342 and propidium-iodide staining reveal substantial propidium-iodide accumulation in fisetin-treated C. albicans cells at their MIC, with crystal violet uptake assays confirming fisetin-induced membrane permeabilization. Leakage analysis demonstrates a significant release of DNA and proteins in fisetin-treated cells compared to controls, underscoring the antifungal effect through membrane disruption. Green fluorescence, evident in both the cytoplasm and vacuoles of fisetin-treated cells under BCECF, AM staining, stands in contrast to controls where only acidic vacuoles exhibit staining. Ratiometric pH measurements using BCECF, AM reveal a noteworthy reduction in intracellular pH in fisetin-treated cells, emphasizing its impact on pH homeostasis. DiBAC4(3) uptake assays demonstrate membrane hyperpolarization in fisetintreated cells, suggesting potential disruptions in ion flux and cellular homeostasis. These results provide comprehensive insights into the antifungal mechanisms of fisetin, positioning it as a promising therapeutic agent against Candida infections.

• IMMUNE NETWORK
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• v.3 no.4
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• pp.268-275
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• 2003

Background: Hemopoietic cells require the constant presence of growth factors for survival in vitro and in vivo. Caspases have been known as central executors of apoptotic cell death. We have, therefore, investigated the pathways that regulate caspase activity and apoptosis using the $CD34^+$ cell line, TF-1 which requires GM-CSF for survival. Methods: Apoptosis was measured by annexin V staining and mitochondrial membrane potential was measured by DiOC6 labelling. Intracellular pH was measured using pH sensitive fluorochrome, BCECF or SNARF-1, followed by flow cytometry analysis. Caspase activation was analyzed by PARP cleavage using anti-PARP antibody. Results: Removal of GM-CSF induceed PARP cleavage, a hallmark of caspase activity, concomitant with pHi acidification and a drop in mitochondrial potential. Treatment with ZVAD, a competitive inhibitor of caspases, partially rescued cell death without affecting pHi acidification and the reduction of mitochondrial potential, suggesting that both these events act upstream of caspases. Overexpression of Bcl-2 prevented cell death induced by GM-CSF deprivation as well as pHi acidification and the reduction in mitochondrial membrane potential. In parental cells maintained with GM-CSF, EIPA, a competitive inhibitor of $Na^+/H^+$ antiporter induced apoptosis, accompanied by a drastic reduction in mitochondrial potential. In contrast, EIPA induced apoptosis in Bcl-2 transfectants without causing mitochondrial membrane depolarization. Conclusion: Taken together, our results suggest that the regulation of $H^+$fluxes, either through a mitochondriondependent or independent pathway, is central to caspase activation and apoptosis.

• International Journal of Oral Biology
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• v.33 no.4
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• pp.143-147
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• 2008

The sodium bicarbonate cotransporter (NBC) protein is functionally expressed in salivary glands. In this experiment, we examined the role of NBC in $HCO_3^-$ formation in human parotid gland acinar cells. Intracellular pH (pHi) was measured in 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded cells. Acetazolamide (0.1 mM) and 4,4'-diisothio cyanatostilbene-2,2'-disulphonic acid (DIDS, 0.5 mM) were used as specific inhibitors of carbonic anhydrase and NBC, respectively. The degree of inhibition was assessed by measuring the pHi recovery rate (${\Delta}pHi$/min) after cell acidification using an ammonium prepulse technique. In control experiments, ${\Delta}pHi$/min was $1.40{\pm}0.06$. Treatment of cells with 0.5 mM DIDS or 0.1 mM acetazolamide significantly reduced ${\Delta}pHi$/min to $1.14{\pm}0.14$ and $0.74{\pm}0.15$, respectively. Simultaneous application of DIDS and acetazolamide further reduced ${\Delta}pHi$/min to $0.47{\pm}0.10$. Therefore, DIDS and acetazolamide reduced ${\Delta}pHi$/min by 19% and 47%, respectively, while simultaneous application of both DIDS and acetazolamide caused a reduction in ${\Delta}pHi$/min of 67%. These results suggest that in addition to carbonic anhydrase, NBC also partially contributes to $HCO_3^-$ formation in human parotid gland acinar cells.

• IMMUNE NETWORK
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• v.2 no.1
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• pp.25-34
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• 2002

Background: Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia and joint destruction. The synovial fibroblasts express cell adhesion molecules and have a role in adhesive interation with inflammatory cells in synovial tissue. It has been suggested that hypoxic conditioins are thought to exist in arthritic joints, and several studies indicate that reactive oxygen species (ROS) produced in hypoxic condition can initiate events that lead to pro-adhesive changes via increased expression of adhesion molecules. So, this study wsa designed to examine whether antioxidant can inhibit hypoxia-induced expression of ICAM-1 in cultured human synovial fibroblasts. Methods: Synovial fibroblasts were isolated from synovial tissue in patients with RA and cultured at hypoxic condition. Antioxidant, PDTC (pyrrolidine dithiocarbamate) were pre-treated for an hour before the hypoxic culture and synovial fibroblasts were harvested at 0, 6, 12, 24, 48 hours time points. Cell surface ICAM-1 expression in synovial fibroblasts was examined by the flow cytometric analysis. To analyse the expression of ICAM-1 mRNA, reverse-transcriptase polymerase chain reaction (RT-PCR) was performed. The levels of cytokines in culture supernatants were measured by ELISA, and activation of NF-${\kappa}B$ was analysed by electrophoretic mobility shift assay. The adhesive reaction between synovial fibroblasts and lymphocytes was assayed by measurement of fluorescent intensity of BCECF-AM in lymphocytes. Results: Hypoxic stimuli up-regulated the ICAM-1 expression as well as the adhesive interaction of human synvial fibroblasts to lymphocytes in a time-dependent manner, and PDTC inhibited hpyoxia-induced ICAM-1 expression and cell-cell interaction. PDTC also inhibited the hypoxia-induced activation of intracellular transcription factor, NF-${\kappa}B$. PDTC decreased the amount of hypoxia-induced production of IL-$1{\beta}$ and TNF-${\alpha}$. Conclusion: These studies demonstrate that PDTC inhibit the hypoxia-induced expression of the adhesion molecule, ICAM-1 and activation of NF-${\kappa}B$ in cultured human synovial fibroblasts.

• Journal of the korean academy of Pediatric Dentistry
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• v.25 no.2
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• pp.352-367
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• 1998

Intracellular pH (pHi) plays an important role in the regulation of cellular processes by influencing the acitivity of various enzymes in cells. Therefore, almost every type of mammalian cell possesses an ability to regulate its pHi. One of the most prominent mechanisms in the regulation of pHi is $Na^+/H^+$ exchanger. This exchanger has been known to be activated when cells are stimulated by the binding of agonist to the muscarinic receptors. Therefore, the aims of this study were to compare the rates of $H^+$ extrusion through $Na^+/H^+$ exchanger before and during muscarinic stimulation and to investigate the possible existence of $HCO_3^-$ transporter which is responsible for the continuous supply of $HCO_3^-$ ion to saliva. Acinar cells were isolated from the rat mandibular salivary glands and loaded with pH-sensitive fluoroprobe, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF), for 30min at room temperature. Cells were attached onto the coverglass in the perfusion chamber and the changes in pHi were measured on the iverted microscope using spectrofluorometer. 1. By switching the perfusate from $HCO_3^-$-free to $HCO_3^-$-buffered solution, pHi decreased by $0.39{\pm}0.02$ pH units followed by a slow increase at an initial rate of $0.04{\pm}0.007$ pH units/min. The rate of pHi increase was reduced to $0.01{\pm}0.002$ pH units/min by the simultaneous addition of 1 mM amiloride and $100{\mu}M$ DIDS. 2. An addition and removal of $NH_4^+$ caused a decrease in pHi which was followed by an increase in pHi. The increase of pHi was almost completely blocked by 1mM amiloride in $HCO_3^-$-free perfusate which implied that the pHi increase was entired dependent on the activation of $Na^+/H^+$ exchanger in $HCO_3^-$-free condition. 3. An addition of $10{\mu}M$ carbachol increased the initial rate of pHi recovery from $0.16{\pm}0.01$ pH units/min to $0.28{\pm}0.03pH$ units/min. 4. The initial rate of pHi decrease induced by 1mM amiloride was also increased by the exposure of the acinar cells to $10{\mu}M$ carbachol ($0.06{\pm}0.008pH$ unit/min) compared with that obtained before carbachol stimulation ($0.03{\pm}0.004pH$ unit/min). 5. The intracellular buffering capacity ${\beta}1$ was $14.31{\pm}1.82$ at pHi 7.2-7.4 and ${\beta}1$ increased as pHi decreased. 6. The rate of $H^+$ extrusion through $Na^+/H^+$ exchanger was greatly enhanced by the stimulation of the cells with $10{\mu}M$ carbachol and there was an alkaline shift in the activity of the exchanger. 7. An intrusion mechanism of $HCO_3^-$ was identified in rat mandibular salivary acinar cells. Taken all together, I observed 3-fold increase in $Na^+/H^+$ exchanger by the stimulation of the acinar cells with $10{\mu}M$ carbachol at pH 7.25. In addition, I have found an additional mechanism for the regulation of pHi which transported $HCO_3^-$ into the cells.

• The Korean Journal of Physiology and Pharmacology
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• v.10 no.4
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• pp.217-222
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• 2006

Atherosclerosis is considered as a chronic inflammatory process. However, the nature of the oxidant signaling that regulates monocyte adhesion and its underlying mechanism is poorly understood. We investigated the role of reactive oxygen species on the vascular cell adhesion molecule-1 (VCAM-1) and monocyte adhesion in the cultured endothelial cells. $TNF-{\alpha}$ at a range of $1{\sim}30\;ng/ml$ induced VCAM-1 expression dose-dependently. BCECF-AM-labeled U937 cells firmly adhered on the surface of endothelial cells when the endothelial cells were incubated with $TNF-{\alpha}$ (15 ng/ml). Ten $\;{\mu}mol/L$ of SB203580, an inhibitor of p38 MAPK, significantly reduced $TNF-{\alpha}-induced$ VCAM-1 expression, compared to the JNK inhibitor ($40\;{\mu}mol/L$ of SP60015) or ERK inhibitor ($40\;{\mu}mol/L$ of U0126). Also, SB203580 significantly inhibited $TNF-{\alpha}-induced$ monocyte adhesion in HUVEC. Superoxide production was minimal in the basal condition, however, treatment of $TNF-{\alpha}$ induced superoxide production in the dihydroethidineloaded endothelial cells. Diphenyleneiodonium (DPI, $10\;{\mu}mol/L$), an inhibitor of NADPH oxidase, and rotenone $(1\;{\mu}mol/L)$, an inhibitor of mitochondrial complex I inhibited $TNF-{\alpha}-induced$ superoxide production, VCAM-1 expression and monocyte adhesion in the endothelial cells. Taken together, our data suggest that NADPH oxidase and mitochondrial ROS were involved in $TNF-{\alpha}-induced$ VCAM-1 and monocyte adhesion in the endothelial cells.