• Molecules and Cells
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• 제28권4호
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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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• 제34권4호
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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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• 제25권6호
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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.

• 동의생리병리학회지
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• 제23권4호
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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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• 제30권6호
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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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• 제27권2호
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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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• 제22권4호
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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.

• 한국식품위생안전성학회지
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• 제39권3호
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• pp.273-280
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• 2024

본 연구에서는 천연 항균제인 광귤 추출물의 항균 활성과 항균 메커니즘을 조사해 즉석섭취식품인 샐러드에서 Salmonella Typhimurium을 제어하기 위한 세척수로써 적용 가능성을 평가하였다. 액체배지희석법으로 S. Typhimurium에 대한 광귤 추출물의 최소 억제 농도(MIC)를 구했다. 그런 다음 다양한 농도(1/16 MIC-2 MIC)에 해당하는 광귤 추출물에 S. Typhimurium을 접종하고 성장곡선을 분석해 대조군과 성장값을 비교하여 항균 활성을 확인하였다. 광귤 추출물을 처리한 후, S. Typhimurium의 세포 내 활성산소종 수준과 막 전위 및 손상도의 변화, 핵산 누출량을 측정하여 광귤 추출물의 항균 메커니즘을 확인하였다. 최종적으로 S. Typhimurium을 인위적으로 접종한 샐러드에 다양한 농도의 광귤 추출물을 다양한 시간 동안 침지 방법으로 항균 처리하여 저감화 효과를 확인했다. S. Typhimurium에 대한 광귤 추출물의 MIC는 195.313 mg/L으로, 1 MIC와 2 MIC의 광귤 추출물은 S. Typhimurium의 성장을 완전히 억제하였다. 광귤 추출물의 처리농도가 높아질수록, 세포 내 ROS 수준과 막 전위, 막 손상도 그리고 핵산 방출량은 증가하였다. 마지막으로, 세척수인 광귤 추출물의 농도가 높고 처리 시간이 길수록 샐러드의 S. Typhimurium의 수가 감소하였다. 따라서 광귤 추출물은 S. Typhimurium를 효과적으로 제어할 수 있음을 입증했다. 광귤 추출물은 차아염소산나트륨과 비교하였을 때 효과적인 항균 활성을 보이며 안전한 샐러드 세척수로 사용될 수 있다. 이는 샐러드와 같은 식품에서 광귤 추출물이 식중독 발생을 미연에 방지하는데 기여할 수 있음을 시사한다.

• Applied Biological Chemistry
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• 제32권1호
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• pp.23-29
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• 1989

soybean hypocotyl에서 분리한 미토콘드리아로부터 submitochondrial particles와 matrix 단백질 추출액을 준비하고 전기분해법으로 제조한 superoxide를 처리하여 malate dehydrogenase 및 cytochrome C oxidase의 불활성화와 mitochondrial membrane의 과산화를 각각 조사하였다. 막에 결합되어 있는 cytochrome C oxidase나 수용액상태의 malate dehydrogenas는 모두 $O^{-}_{2}$에 대해 매우 민감하게 불활성화되었다. 즉 dismutation 반응이 빠르게 진행되어 실질적으로 효소의 불활성화에 기여하게 될 농도는 대단히 낮을 것으로 추정되는 $O^{-}_{2}$의 명목상 처리농도 1.4mM 전후에서 두 효소는 그 활성을 완전히 상실하였다. 한편 malondialdehyde의 생성을 지표로 하여 측정된 membrane 과산화는 인지질로서만 이루어진 liposome의 경우보다는 다소 낮은 수준이었으나 무시할 수는 없는 정도였다. mitochondrial membrane의 과산화가 상대적으로 억제된 것은 막에 결합되어 있는 항산화제 및 단백질들에 의한 $O^{-}_{2}$소거효과에 기인하였으리라 해석된다. 식물 미토콘드리아의 대표적인 대사과정인 TCA cycle과 호흡전자전달반응의 성분효소들인 malate dehydrogenase와 cytochrome C oxidase가 불활성화되었고 membrane이 과산화 되었다는 사실은, 식물의 냉해와 광피해 발현기작에서 공히공통적인 화학적 인자로 인정되는 $O^{-}_{2}$의 과잉생성 및 축적이 그것의 주 생성처인 미토콘드리아의 생화학적 기능과 구조에 미칠수 있는 파괴적 효과를 적절히 지시하는 것이다.

• IMMUNE NETWORK
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• 제9권6호
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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.