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Langmuir : the ACS Journal of Surfaces... Apr 2020OSW-1, a unique steroidal saponin isolated from the bulbs of , has potent cell-growth inhibition activity. In this study, we conducted fluorescence measurements and...
OSW-1, a unique steroidal saponin isolated from the bulbs of , has potent cell-growth inhibition activity. In this study, we conducted fluorescence measurements and microscopic observations using palmitoyloleoylphosphatidylcholine (POPC)-cholesterol (Chol) bilayers to evaluate the membrane-binding affinity of OSW-1 in comparison with another steroidal saponin, digitonin, and the triterpenoid saponin, soyasaponin Bb(I). The membrane activities of these saponins were evaluated using calcein leakage assays and fitted to the binding isotherm by changing the ratios of saponin-lipids. Digitonin showed the highest binding affinity for the POPC-Chol membrane ( = 0.38 μM) and the strongest membrane disruptivity in the bound saponin-lipid ratio at the point of 50% calcein leakage ( = 0.47) occurrence. OSW-1 showed slightly lower activity ( = 0.31 μM; = 0.78), and the soyasaponin was the lowest in the membrane affinity and the calcein leakage activity ( = 0.017 μM; = 1.66). The effect of OSW-1 was further assessed using confocal microscopy in an experiment utilizing DiI and rhodamine 6G as the fluorescence probes. The addition of 30 μM OSW-1 induced inward membrane curvature in some giant unilamellar vesicles (GUVs). At the higher OSW-1 concentration (58 μM, = 0.78) where the 50% calcein leakage was observed, the morphology of some GUVs became elongated. With digitonin at the corresponding concentration (35 μM, = 0.47), membrane disruption and formation of large aggregates in aqueous solution were observed, probably due to a detergent-type mechanism. These saponins, including OSW-1, required Chol to exhibit their potent membrane activity although their mechanisms are thought to be different. At the effective concentration, OSW-1 preferably binds to the bilayers without prominent disruption of vesicles and exerts its activity through the formation of saponin-Chol complexes, probably resulting in membrane permeabilization.
Topics: Cholestenones; Digitonin; Lipid Bilayers; Saponins
PubMed: 32160747
DOI: 10.1021/acs.langmuir.9b03957 -
Spectrochimica Acta. Part A, Molecular... Oct 2022The concentration of potassium ion is an important indicator for human health, and its abnormality is often accompanied by various diseases. However, most tools...
The concentration of potassium ion is an important indicator for human health, and its abnormality is often accompanied by various diseases. However, most tools currently used to study potassium ion transport are low throughput. Herein, we reported a new K fluorescent nanoprobe CP1-KS with high selectivity and sensitivity to K (fluorescence enhanced factor was up to 9.91 at 20 mM K). The polymeric fluorescent probe CP1-KS was composed of the small-molecular K indicator KS and amphiphilic copolymer CP1. This sensor can be easily and uniformly dispersed in cell culture medium and is suitable for high throughput analysis. To assess the utility of the probe CP1-KS in biological field, this probe was employed as an extracellular fluorescent probe to monitor the efflux of K from cells (E coli, B. Subtilis 168, Hela and MCF-7 cells) under various stimulation including lysozyme, nigericin, digitonin, and ATP. Results demonstrated that CP1-KS is an effective analysis tool for extracellular K concentration. We believe that the nanoprobe has great potential in antibacterial drug screening, K ionophore function, K channel activity, cell membrane permeability analysis or other K related field in the future.
Topics: Biological Assay; Escherichia coli; Fluorescent Dyes; Humans; Ionophores; Ions; Potassium
PubMed: 35653810
DOI: 10.1016/j.saa.2022.121435 -
Methods in Molecular Biology (Clifton,... 2022Autophagy is deregulated in cancer cells and often activated as a cellular stress response to anticancer therapies. Flow cytometry-based assays enable detection and...
Autophagy is deregulated in cancer cells and often activated as a cellular stress response to anticancer therapies. Flow cytometry-based assays enable detection and quantification of various cellular markers in live or fixed cells. Here, a flow cytometry-based assay to characterize autophagy across the cell cycle is described. This method is based on selective plasma membrane permeabilization with digitonin and extraction of membrane-unbound LC3 protein followed by staining of the autophagosome-bound LC3 protein with antibody and labeling of DNA with propidium iodide. Staining with the LC3 antibody described here can be also combined with the staining of other cellular markers, allowing to quantitatively assess autophagy in relation to different cellular processes by flow cytometry.
Topics: Autophagosomes; Autophagy; Cell Cycle; Flow Cytometry; Microtubule-Associated Proteins
PubMed: 34972986
DOI: 10.1007/978-1-0716-2071-7_5 -
International Journal of Biological... Jan 2022Previously we have shown that lactoferrin (LTF), a protein of secondary neutrophilic granules, can be efficiently modified by hypohalous acids (HOCl and HOBr), which are...
Previously we have shown that lactoferrin (LTF), a protein of secondary neutrophilic granules, can be efficiently modified by hypohalous acids (HOCl and HOBr), which are produced at high concentrations during inflammation and oxidative/halogenative stress by myeloperoxidase, an enzyme of azurophilic neutrophilic granules. Here we compared the effects of recombinant human lactoferrin (rhLTF) and its halogenated derivatives (rhLTF-Cl and rhLTF-Br) on functional responses of neutrophils. Our results demonstrated that after halogenative modification, rhLTF lost its ability to induce mobilization of intracellular calcium, actin cytoskeleton reorganization, and morphological changes in human neutrophils. Moreover, both forms of the halogenated rhLTF prevented binding of N-acetylglucosamine-specific plant lectin Triticum vulgaris agglutinin (WGA) to neutrophils and, in contrast to native rhLTF, inhibited respiratory burst of neutrophils induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine and by two plant lectins (WGA and PHA-L). However, we observed no differences between the effects of rhLTF, rhLTF-Cl, and rhLTF-Br on respiratory burst of neutrophils induced by phorbol 12-myristate 13-acetate (PMA), digitonin, and number of plant lectins with different glycan-binding specificity. Furthermore, all rhLTF forms interfered with PMA- and ionomycin-induced formation of neutrophil extracellular traps. Thus, halogenative modification of LTF is one of the mechanisms involved in modulating a variety of signaling pathways in neutrophils to control their pro-inflammatory activity.
Topics: Acetylglucosamine; Actin Cytoskeleton; Bromates; Calcium; Digitonin; Humans; Hypochlorous Acid; Ionomycin; Lactoferrin; Neutrophils; Recombinant Proteins; Tetradecanoylphorbol Acetate; Triticum; Wheat Germ Agglutinins
PubMed: 34863835
DOI: 10.1016/j.ijbiomac.2021.11.165 -
Bio-protocol Jun 2022Damage to the plasma membrane and loss of membrane integrity are detrimental to eukaryotic cells. It is, therefore, essential that cells possess an efficient membrane...
Damage to the plasma membrane and loss of membrane integrity are detrimental to eukaryotic cells. It is, therefore, essential that cells possess an efficient membrane repair system to survive. However, the different cellular and molecular mechanisms behind plasma membrane repair have not been fully elucidated. Here, we present three complementary methods for plasma membrane wounding, and measurement of membrane repair and integrity. The first protocol is based on real time imaging of cell membrane repair kinetics in response to laser-induced injury. The second and third protocols are end point assays that provide a population-based measure of membrane integrity, after either mechanical injury by vortex mixing with glass beads, or by detergent-induced injury by digitonin in sublytic concentrations. The protocols can be applied to most adherent eukaryotic cells in culture, as well as cells in suspension.
PubMed: 35799909
DOI: 10.21769/BioProtoc.4437 -
Journal of Pharmaceutical and... Sep 2022Membrane proteins constitute around 20-30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets....
Membrane proteins constitute around 20-30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets. However, the isolation of membrane proteins is challenging because of their partial hydrophobicity, requiring detergents to extract them from cell membranes and stabilize them in solution. Many commercial kits use this principle, but they are expensive, and their chemical composition is not known. In this work, we propose a fast, detergent-based protocol for the purification of membrane proteins from murine and human cells. This protocol is based on three steps: cell washing to remove cell culture medium proteins, cells permeabilization using digitonin to remove the intracellular components, and cell membranes disruption using Triton X-100 to solubilize membrane proteins and keep them in solution. We measured the total protein yield using our protocol with two different detergent concentrations and compared it to a commercial kit. We further assessed membrane protein enrichment by comparing markers for specific cellular components using SDS-PAGE/western blot and identifying specific proteins by qualitative mass spectrometry. Our protocol led to a final protein yield analogous to the commercial kit and similar membrane protein purity, while resulting significantly cheaper compared to the commercial kit. Furthermore, this process can be applied to a different number and types of cells, resulting scalable, versatile, and robust. The possibility to perform downstream mass spectrometry analysis is of particular importance since it enables the use of "omics" techniques for protein discovery and characterization. Our approach could be used as a starting point for the isolation of membrane proteins for pharmacological and biochemical studies, or for the discovery of new druggable or prognostic markers.
Topics: Animals; Detergents; Electrophoresis, Polyacrylamide Gel; Humans; Hydrophobic and Hydrophilic Interactions; Mammals; Membrane Proteins; Mice; Octoxynol
PubMed: 35839578
DOI: 10.1016/j.jpba.2022.114926 -
Journal of Immunological Methods Feb 2021Lymph nodes (LNs) are essential secondary immune organs where the adaptive immune response is generated against most infections and vaccines. We recently described the...
Lymph nodes (LNs) are essential secondary immune organs where the adaptive immune response is generated against most infections and vaccines. We recently described the use of live ex vivo LN slices to study the dynamics of adaptive immunity. However, when working with reactive lymph nodes from vaccinated animals, the tissues frequently became dislodged from the supportive agarose matrix during slicing, leading to damage that prevented downstream analysis. Because reactive lymph nodes expand into the surrounding adipose tissue, we hypothesized that dislodging was a result of excess lipids on the collagen capsule of the LN, and that a brief wash with a mild detergent would improve LN interaction with the agarose without damaging tissue viability or function. Therefore, we tested the use of digitonin on improving slicing of vaccinated LNs. Prior to embedding, LNs were quickly dipped into a digitonin solution and washed in saline. Lipid droplets were visibly removed by this procedure. A digitonin wash step prior to slicing significantly reduced the loss of LN during slicing from 13 to 75% to 0-25%, without substantial impact on viability. Capture of fluorescent microparticles, uptake and processing of protein antigen, and cytokine secretion in response to a vaccine adjuvant, R848, were all unaffected by the detergent wash. This novel approach will enable ex vivo analysis of the generation of adaptive immune response in LNs in response to vaccinations and other immunotherapies.
Topics: Animals; Antigens; Cytokines; Detergents; Digitonin; Lymph Nodes; Mice; Mice, Inbred C57BL; Vaccination
PubMed: 33333059
DOI: 10.1016/j.jim.2020.112943 -
Acta Pharmacologica Sinica Oct 2023Peroxisome proliferator-activated receptor alpha (PPARα) activation-induced hepatomegaly is accompanied by hepatocyte hypertrophy around the central vein (CV) area and...
Peroxisome proliferator-activated receptor alpha (PPARα) activation-induced hepatomegaly is accompanied by hepatocyte hypertrophy around the central vein (CV) area and hepatocyte proliferation around the portal vein (PV) area. However, the molecular mechanisms underlying this spatial change of hepatocytes remains unclear. In this study, we examined the characteristics and possible reasons for the zonation distinction of hypertrophy and proliferation during PPARα activation-induced mouse liver enlargement. Mice were injected with corn oil or a typical mouse PPARα agonist WY-14643 (100 mg·kg·d, i.p.) for 1, 2, 3, 5 or 10 days. At each time point, the mice were sacrificed after the final dose, and liver tissues and serum were harvested for analysis. We showed that PPARα activation induced zonal changes in hepatocyte hypertrophy and proliferation in the mice. In order to determine the zonal expression of proteins related to hepatocyte hypertrophy and proliferation in PPARα-induced liver enlargement, we performed digitonin liver perfusion to separately destroy the hepatocytes around the CV or PV areas, and found that PPARα activation-induced increase magnitude of its downstream targets such as cytochrome P450 (CYP) 4 A and acyl-coenzyme A oxidase 1 (ACOX1) levels around the CV area were higher compared with those around the PV area. Upregulation of proliferation-related proteins such as cell nuclear antigen (PCNA) and cyclin A1 (CCNA1) after WY-14643-induced PPARα activation mainly occurred around the PV area. This study reveals that the zonal expression of PPARα targets and proliferation-related proteins is responsible for the spatial change of hepatocyte hypertrophy and proliferation after PPARα activation. These findings provide a new insight into the understanding of PPARα activation-induced liver enlargement and regeneration.
Topics: Animals; Mice; Cell Proliferation; Hepatocytes; Hepatomegaly; Hypertrophy; Liver; Mice, Knockout; PPAR alpha
PubMed: 37193756
DOI: 10.1038/s41401-023-01096-5 -
Cell Reports Jun 2021The sterol regulatory element-binding protein (SREBP) pathway monitors the cellular cholesterol level through sterol-regulated association between the SREBP...
The sterol regulatory element-binding protein (SREBP) pathway monitors the cellular cholesterol level through sterol-regulated association between the SREBP cleavage-activating protein (Scap) and the insulin-induced gene (Insig). Despite structural determination of the Scap and Insig-2 complex bound to 25-hydroxycholesterol, the luminal domains of Scap remain unresolved. In this study, combining cryogenic electron microscopy (cryo-EM) analysis and artificial intelligence-facilitated structural prediction, we report the structure of the human Scap/Insig-2 complex purified in digitonin. The luminal domain loop 1 and a co-folded segment in loop 7 of Scap resemble those of the luminal/extracellular domain in NPC1 and related proteins, providing clues to the cholesterol-regulated interaction of loop 1 and loop 7. An additional luminal interface is observed between Scap and Insig. We also show that Scap(D428A), which inhibits SREBP activation even under sterol depletion, exhibits an identical conformation with the wild-type protein when complexed with Insig-2, and its constitutive suppression of the SREBP pathway may also involve a later step in protein trafficking.
Topics: Digitonin; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Micelles; Models, Molecular; Protein Conformation; Protein Folding; Sterols; Structural Homology, Protein
PubMed: 34192549
DOI: 10.1016/j.celrep.2021.109299 -
Journal of Virology Sep 2023Previously, we developed an infectious hepatitis E virus (HEV) harboring the nanoKAZ gene in the hypervariable region of the open reading frame 1 (ORF1) of the HEV3b...
Previously, we developed an infectious hepatitis E virus (HEV) harboring the nanoKAZ gene in the hypervariable region of the open reading frame 1 (ORF1) of the HEV3b (JE03-1760F/P10) genome and demonstrated the usefulness for screening anti-HEV drugs that inhibit the early infection process. In the present study, we constructed another reporter HEV (HEV3b-HiBiT) by placing a minimized HiBiT tag derived from NanoLuc luciferase at the 3'-end of the viral capsid (ORF2) coding sequence. It replicated efficiently in PLC/PRF/5 cells, produced membrane-associated particles identical to those of the parental virus, and was genetically stable and infectious. The HiBiT tag was fused to both secreted ORF2s (ORF2s-HiBiT) and ORF2c capsid protein (ORF2c-HiBiT). The ORF2c-HiBiT formed membrane-associated HEV particles (eHEV3b-HiBiT). By treating these particles with digitonin, we demonstrated that the HiBiT tag was expressed on the surface of capsid and was present inside the lipid membrane. To simplify the measurement of luciferase activity and provide a more convenient screening platform, we constructed an ORF2s-defective mutant (HEV3b-HiBiT/ΔORF2s) in which the secreted ORF2s are suppressed. We used this system to evaluate the effects of introducing small interfering RNAs and treatment with an inhibitor or accelerator of exosomal release on HEV egress and demonstrated that the effects on virus release can readily be analyzed. Therefore, HEV3b-HiBiT and HEV3b-HiBiT/ΔORF2s reporters may be useful for investigating the virus life cycle and can serve as a more convenient screening platform to search for candidate drugs targeting the late stage of HEV infection such as particle formation and release. IMPORTANCE The construction of recombinant infectious viruses harboring a stable luminescence reporter gene is essential for investigations of the viral life cycle, such as viral replication and pathogenesis, and the development of novel antiviral drugs. However, it is difficult to maintain the stability of a large foreign gene inserted into the viral genome. In the present study, we successfully generated a recombinant HEV harboring the 11-amino acid HiBiT tag in the ORF2 coding region and demonstrated the infectivity, efficient virus growth, particle morphology, and genetic stability, suggesting that this recombinant HEV is useful for assays. Furthermore, this system can serve as a more convenient screening platform for anti-HEV drugs. Thus, an infectious recombinant HEV is a powerful approach not only for elucidating the molecular mechanisms of the viral life cycle but also for the screening and development of novel antiviral agents.
PubMed: 37681960
DOI: 10.1128/jvi.00508-23