Quercetin-induced yeast apoptosis through mitochondrial dysfunction under the accumulation of magnesium in Candida albicans

Latterly, the upsurge in use of antifungal drugs has brought about the emergence of several drug-resistance strains, making it skeptical to continue relying on current therapeutic regime. In the necessity of resistance-free antifungal agent, flavonoids presented possibilities of replacing existing drugs, displaying antifungal activity against pathogenic fungi. Among them, quercetin, one of the most representative flavonoids, exhibited antifungal activity against Candida albicans. To inspect the further understanding regarding quercetin, the antifungal mode of action of quercetin was investigated. In the initial step, the apoptosis was monitored after quercetin treatment. Moreover, intracellular levels of Mg²⁺ was assessed and was determined that Mg²⁺ increase occurred under the influence of quercetin. In addition, several features of mitochondrial dysfunction were monitored. Mitochondrial dysfunction triggers decrease in mitochondrial redox levels and leads to disruption in mitochondrial antioxidant system. Increased intracellular ROS and decreased intracellular redox levels were also displayed, indicating the occurrence of overall disruption in antioxidant systems. Sequentially, DNA fragmentation was observed and this DNA damage in turn induces apoptosis. In analyses, hexaamminecobalt(III) chloride (Cohex) was applied to inhibit Mg²⁺ transport between cytosol and mitochondria. Cohex attenuated the effects induced by quercetin, which demonstrates that the presence of Mg²⁺ is essential in quercetin-induced apoptosis.     

Viability measurements of hybridoma cells in suspension cultures

Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.     

Staining with Fluorescein Diacetate Correlates with Hepatocyte Function

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes. we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R² = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition. the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.     

双乙酸钠对小鼠肠道菌群结构和功能的影响

肠道菌群在功能和代谢方面的研究日益成熟,但针对食品添加剂双乙酸钠对人体肠道菌群作用的研究目前仍鲜少有报道。为了探究其对肠道菌群及人体健康的潜在益处及危害,以小鼠为模型,以双乙酸钠为干预物质,干预剂量为0.3 g/(kg·d),干预1周后,收集实验小鼠和空白对照组小鼠的粪便并用菌群16S rDNA高通量测序进行肠道菌群的物种鉴定和丰度检测。对所得数据进行分析,得到小鼠肠道菌群的多样性与丰度比例等指标。结果显示,实验剂量下的双乙酸钠干预后,小鼠的肠道菌群无论是菌群种类还是丰度都有了显著改变,属水平新增447种,减少142种。根据丰度比例变化最显著的9种菌,如拟杆菌属(Bacteroides)、AKK(Akkermansia)等,并结合菌群代谢功能预测,脂肪酸合成显著上调,碳水化合物代谢、氨基酸代谢、脂代谢等明显下调,推测摄入高剂量的双乙酸钠可能有增加肥胖、过敏、慢性炎症和肠胃炎的风险。     

d-Ribose glycates β2-microglobulin to form aggregates with high cytotoxicity through a ROS-mediated pathway

β₂-Microglobulin (β₂M) modified with advanced glycation end products (AGEs) is a major component of the amyloid deposits in hemodialysis-associated amyloidosis (HAA). However, the effect of glycation on the misfolding and aggregation of β₂M has not been studied so far. Here we examine the molecular mechanism of aggregate formation of HAA-related ribosylated β₂M in vitro. We find that the glycating agent d-ribose interacts with human β₂M to generate AGEs that form aggregates in a time-dependent manner. Ribosylated β₂M molecules are highly oligomerized compared with unglycated β₂M, and have granular morphology. Furthermore, such ribosylated β₂M aggregates show significant cytotoxicity to both human SH-SY5Y neuroblastoma and human foreskin fibroblast FS2 cells and induce intracellular reactive oxygen species (ROS). Presence of the antioxidant N-acetylcysteine (1.0 mM) attenuated intracellular ROS and prevented cell death induction in both SH-SY5Y and FS2 cells, indicating that the cytotoxicity of ribosylated β₂M aggregates depends on a ROS-mediated pathway in both cell lines. In other words, d-ribose reacts with β₂M and induces the ribosylated protein to form granular aggregates with high cytotoxicity through a ROS-mediated pathway. These findings suggest that ribosylated β₂M aggregates could contribute to the dysfunction and death of cells and could play an important role in the pathogenesis of β₂M-associated diseases such as HAA.     

Tumor necrosis factor-α accelerates apoptosis of steatotic hepatocytes from a murine model of non-alcoholic fatty liver disease

Non-alcoholic steatohepatitis (NASH) develops in a subset of patients with non-alcoholic fatty liver disease (NAFLD), but the exact mechanisms involved in the progression of NAFLD to NASH remain poorly understood. We investigated the role of tumor necrosis factor-α (TNF-α) in the apoptosis of hepatocytes that is related to the severity of NASH. We separated primary hepatocytes from the NAFLD liver caused by a high-fat diet. The production of intracellular reactive oxygen species was increased in steatotic hepatocytes, which were also sensitive to TNF-α. This factor induced significant apoptosis through the signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK) pathway. We describe here a novel culture model of steatotic hepatocytes separated from the NAFLD liver, and demonstrate that TNF-α induces their apoptosis in vitro.     

Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism

Previous work from us and others has suggested that cholesterol is an important lipid in the context of the organization of the actin cytoskeleton. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol is rarely addressed in the literature. In this work, we explored the signaling crosstalk between cholesterol and the actin cytoskeleton by using a high-resolution confocal microscopic approach to quantitatively measure changes in F-actin content upon cholesterol depletion. Our results show that F-actin content significantly increases upon chronic cholesterol depletion, but not during acute cholesterol depletion. In addition, utilizing inhibitors targeting the cholesterol biosynthetic pathway at different steps, we show that reorganization of the actin cytoskeleton could occur due to the synergistic effect of multiple pathways, including prenylated Rho GTPases and availability of membrane phosphatidylinositol 4,5-bisphosphate. These results constitute one of the first comprehensive dissections of the mechanistic basis underlying the interplay between cellular actin levels and cholesterol biosynthesis. We envision these results will be relevant for future understating of the remodeling of the actin cytoskeleton in pathological conditions with altered cholesterol.     

ROS-Ca is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis

The surfactin can inhibit proliferation and induce apoptosis in cancer cells. Moreover, surfactin can induce cell death in human breast cancer MCF-7 cells through mitochondrial pathway. However, the molecular mechanism involved in this pathway remains to be elucidated. Here, the reactive oxygen species (ROS) and Ca²⁺ on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis which induced by surfactin were investigated. It is found that surfactin evoked mitochondrial ROS generation, and the surfactin-induced cell death was prevented by N-acetylcysteine (NAC, an inhibitor of ROS). An increasing cytoplasmic Ca²⁺ concentration was detected in surfactin-induced MCF-7 apoptosis, which was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium). In addition, the relationship between ROS generation and the increase of cytoplasm Ca²⁺ was determined. The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential (ΔΨ_m). Then the cytoplasmic Ca²⁺ concentration increased in virtue of the changes of mitochondrial permeability, which was prevented by BAPTA-AM. Besides, cytochrome c (cyt c) was released from mitochondria to cytoplasm through the MPTP and activated caspase-9, eventually induced apoptosis. In summary, surfactin has notable anti-tumor effect on MCF-7 cells, however, there was no obvious cytotoxicity on normal cells.