• Molecules and Cells
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• v.28 no.4
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• pp.403-406
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• 2009

Rev is an essential regulatory protein for HIV-1 replication. Rev (11-20) is known as the significant region regarding the function of a nuclear entry inhibitory signal (NIS) of Rev. In this study, anticandidal effects and mechanism of action of Rev (11-20) were investigated. The result exhibited that Rev (11-20) contained candidacidal activities. To understand target site(s) of Rev (11-20), the intracellular localization of the peptide was investigated. The result showed that Rev (11-20) rapidly accumulated in the fungal cell surface. The cell wall regeneration test also indicated that Rev (11-20) exerted its anticandidal activity to fungal plasma membrane rather than cell wall. The fluorescent study using 1,6-diphenyl-1,3,5-hexatriene (DPH) further confirmed the membrane-disruption mechanism(s) of Rev (11-20). The present study suggests that Rev (11-20) possesses significant potential regarding therapeutic agents for treating fungal diseases caused by Candida species in humans.

• 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.

• Journal of Microbiology and Biotechnology
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• v.25 no.6
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• pp.759-764
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• 2015

Antimicrobial peptides (AMPs) are one of the critical components in host innate immune responses to imbalanced and invading microbial pathogens. Although the antimicrobial activity and mechanism of action have been thoroughly investigated for decades, the exact biological properties of AMPs are still elusive. Most AMPs generally exert the antimicrobial effect by targeting the microbial membrane, such as barrel stave, toroidal, and carpet mechanisms. Thus, the mode of action in model membranes and the discrimination of AMPs to discrepant lipid compositions between mammalian cells and microbial pathogens (cell selectivity) have been studied intensively. However, the latest reports suggest that not only AMPs recently isolated but also well-known membrane-disruptive AMPs play a role in intracellular killing, such as apoptosis induction. In this mini-review, we will review some representative AMPs and their antimicrobial mechanisms and provide new insights into the dual mechanism of AMPs.

• Journal of Physiology & Pathology in Korean Medicine
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• v.23 no.4
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• pp.888-894
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• 2009

It has been reported that silibinin, a natural polyphenolic flavonoid, induces cell death in various cancer cell types. However, the underlying mechanisms by which silibinin induces apoptosis in human glioma cells are poorly understood. The present study was therefore undertaken to examine the effect of silibinin on glioma cell apoptosis and to determine its underlying mechanism in human glioma cells. Apoptosis was estimated by FACS analysis. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (${\Psi}m$) were measured using fluorescence dyes DCFH-DA and $DiOC_6$(3), respectively. Cytochrome c release from mitochondria and caspase-3 activation were estimated by Western blot analysis using specific antibodies. Exposure of cells to 30 mM silibinin induced apoptosis starting at 6 h, with increasing effects after 12-48h in a time-dependent manner. Silibinin caused ROS generation and disruption of ym, which were associated with the silibinin-induced apoptosis. The silibinin-induced ROS generation and disruption in ym were prevented by inhibitors of mitochondrial electron transport chain. The hydrogen peroxide scavenger catalase blocked ROS generation and apoptosis induced by silibinin. Silibinin induced cytochrome c release into cytosolic fraction and its effect was prevented by catalase and cyclosporine A. Silibinin treatment caused caspase-3 activation, which was inhibited by DVED-CHO and cyclosporine A. Pretreatment of caspase inhibitors also protected against the silibinin-induced apoptosis. These findings indicate that ROS generation plays a critical role in the initiation of the silibinin-induced apoptotic cascade by mediation of the mitochondrial apoptotic pathway including the disruption of ${\Psi}m$, cytochrome c release, and caspase-3 activation.

• Journal of Microbiology and Biotechnology
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• v.30 no.6
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• pp.878-884
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• 2020

Penicillium digitatum and P. italicum are the two important postharvest pathogens in citrus, causing about 90% of the total loss of citrus fruit during storage and transportation. Natural fungicides such as essential oils have been widely used instead of chemical fungicides for preventing and controlling postharvest diseases. In this research, p-anisaldehyde exhibited a strong inhibitory effect on P. digitatum and P. italicum, with the minimum inhibitory concentration and minimum fungicidal concentration values of both being 2.00 μl/ml. Additionally, p-anisaldehyde visibly inhibited both the green mold and blue mold development of citrus fruits inoculated with P. digitatum and P. italicum. The mycelia morphologies of these pathogens were greatly altered, and the membrane permeability and cell wall integrity of mycelia were severely disrupted under p-anisaldehyde treatment. These results suggest that the antifungal activity of p-anisaldehyde against P. digitatum and P. italicum can be attributed to the disruption of the cell wall integrity.

• Applied Microscopy
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• v.27 no.2
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• pp.121-130
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• 1997

It has been demonstrated that majority of cells in the mammalian body such as myocytes and epithelial cells of skin and intestine respond to mechanical force or environmental factors and exhibit partial disruption of cell membrane, i. e., cell wounding, even in a physiological condition. Myocardial cells are rather apt to be wounded than other cells since they are definitely exposed to mechanical stress by contraction-relaxation and blood flow. However, the mechanism how myocardial cells protect themselves against cell wounding is not yet clarified. On this background, the present study was performed to elucidate whether albumin leakage is related to cell wounding and to assess whether diltiazem, a potent calcium channel blocker, is beneficial in isoproterenol-induced cell wounding in the heart. Hearts isolated from New Zealand White rabbits ($1.5\sim2.0kg$ body weight, n=20) were perfused with Tyrode solution by Langendorff technique. After stabilization of baseline hemodynamics, the hearts were subjected to bolus administration of isoproterenol and diltiazem as following order: $1.6{\mu}M$ isoproterenol at zero min (the beginning point): $16{\mu}M$ diltiazem at 20min; $1.6{\mu}M$ isoproterenol at 25min; $16{\mu}M$ isoproterenol at 45 min; $160{\mu}M$ diltiazem at 65 min; $16{\mu}M$ isoproterenol at 70 min. During all experiments, the left ventricular function was recorded, albumin leakage in the coronary effluents was analyzed by electrophoresis and Western blot, and myocardial cell membranes were examined by conventional transmission electron microscopy. Data were analyzed by t-test and linear regression test. Isoproterenol significantly increased the inotropic and chronotropic contractions, coronary flow, and frequency of arrhythmia, however, diltiazem did not influence on hemodynamics except decrease in the frequency of arrhythmia and a slight decrease in contractility. Isoproterenol also resulted partial disruption of myocardial cell membrane and inclose in albumin leakage, while diltiazem pretreatment showed number of electron-dense plaques in the cell membrane and a tendency of decrease in albumin leakage. These results indicate that albumin leakage may be an indirect index of cell wounding in the heart and diltiazem nay be beneficial to protect myocardial cells against isoproterenol-induced cell wounding. It is likely that diltiazem promotes resealing process of the cell membrane.

• Toxicological Research
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• v.22 no.4
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• pp.315-322
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• 2006

TALP-32 is highly basic protein with a molecular weight of 32 kDa purified from human term placenta. Some basic proteins such as defensins and cecropins are known to induce cell death by increasing membrane permeability and some of them are under development as an anticancer drug especially targeting multi-drug resistant cancers. Therefore, we investigated cytotoxic effect and mechanism of TALP-32 When HeLa cell was incubated with TALP-32, cytotoxicity was increased in time and dose dependent manner. As time goes by, HeLa cells became round and plasma membrane was ruptured. Increase of plasma membrane permeability was determined with LDH release assay. Also in transmission electron microscopy, typical morphology of necrotic cell death, such as cell swelling and intracellular organelle disruption was observed, but DNA fragmentation and caspase activation was not. And necrotic cell death was determined with Annexin V/Pl staining. The cytotoxicity of TALP-32 was minimal and decreased or RBC and Hep3B respectively. These data suggests that TALP-32 induces necrosis on rapidly growing cells but not on slowly growing cells implicating the possibility of its development of anticancer peptide drug.

• Journal of Food Hygiene and Safety
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• v.39 no.3
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• pp.273-280
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• 2024

In this study, the antibacterial activity and mechanisms of bitter orange extract, a natural antibacterial agent, were investigated, with a focus on its potential application in washing water for controlling Salmonella Typhimurium contamination of salad, a ready-to-eat food. The minimum inhibitory concentration (MIC) of bitter orange extract against S. Typhimurium was determined using the broth dilution method. Subsequently, S. Typhimurium was exposed to various concentrations of bitter orange extract (1/16 MIC-2 MIC) and growth curves were measured. Following treatment with bitter orange extract, we investigated its antibacterial mechanism by measuring intracellular reactive oxygen species (ROS) levels, alterations in membrane potential and integrity, and nucleic acid leakage in S. Typhimurium. Additionally, salads artificially contaminated with S. Typhimurium were treated with different concentrations of bitter orange extract using the dipping method for various durations to assess the reduction effect. The MIC of bitter orange extract against S. Typhimurium was 195.313 mg/L, and bacterial growth was completely inhibited at a concentration of 1 MIC. Furthermore, an increase in bitter orange extract concentration correlated with elevated intracellular ROS levels, membrane potential disruption, membrane damage, and nucleic acid release. Importantly, salads treated with bitter orange extract exhibited a significant reduction in S. Typhimurium counts compared to the control, and prolonged treatment times resulted in further reductions in bacterial counts. Bitter orange extract was more effective than sodium hypochlorite and can be used as a safer salad wash. These findings indicate the potential treatment of salads to prevent foodborne illnesses.

• Applied Biological Chemistry
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• v.32 no.1
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• pp.23-29
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• 1989

Biochemical consequence of the accumulation in cells of superoxide $(O^{-}_{2})$ which was proposed to be probably a common chemical factor in the secondary process of the mechanism of chilling injury as well as in the visible light photodamage in cells of higher plants, has been investigated in the present work. Especially focused was the destructive effect of $O^{-}_{2}$ on the biochemical activity of mitochondria, as informations which support the suggestion that mitochondrial inner membrane is the major site of $O^{-}_{2}$ production have been collected. Mitochondria and submitochondrial particles (SMP) were prepared from soybean hypocotyls for this case study. When SMP were treated with the electrolytically produced $O^{-}_{2}$ they suffered not only inhibition of the membrane-bound enzymes as demonstrated by cytochrome c oxidase, but also lipid peroxidation of membrane as proved by malondialdehyde production. Malate dehydrogenase present in the protein extract from mitochondrial matrix was also inhibited by the $O^{-}_{2}$ treatment. These results exhibited the chaotic effect of the overproduction and accumulation of $O^{-}_{2}$ in cells under a certain abnormal circumstance such as environmental stress on the physiological function of mitochondrial; disruption of the cellular metabolic pathways and the structural integrity of membrane.

• IMMUNE NETWORK
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• v.9 no.6
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• pp.274-284
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• 2009

Background: Swiprosin-1 was identified in human CD8+ lymphocytes, mature B cells and non-lymphonoid tissue. We have recently reported that swiprosin-1 is expressed in mast cells and up-regulated in both in vitro and in vivo. Methods: The expression of cytokines and swiprosin-1 were determined by by real time PCR and conventional PCR. Pharmacological inhibitors were treated to investigate potential mechanism of swiprosin-1 in mast cell activation. Actin content was evaluated by confocal microscopy and flow cytometry. Results: The swiprosin-1 augmented PMA/A23187-induced expression of cytokines and release of histamine. However, knock-down of swiprosin-1 showed only a modest effect on PMA/A23187-induced cytokine expression, suggesting that swiprosin-1 has gain-of-function characteristics. Swiprosin-1 was found in microvilli-like membrane protrusions and highly co-localized with F-actin. Importantly, either disruption of actin by cytochalasin B or inhibition of PI3 kinase, an enzyme involved in actin remodeling, by wortmannin blocked cytokine expression only in swiprosin-1-overexpressing cells. Conclusion: These results suggest that swiprosin-1 modulates mast cell activation potentially through actin regulation.