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Methods (San Diego, Calif.) Nov 2008Among the many unsolved problems of calcium signalling, the role of calcium elevations in apoptotic and necrotic cell death has been a focus of research in recent years.... (Review)
Review
Among the many unsolved problems of calcium signalling, the role of calcium elevations in apoptotic and necrotic cell death has been a focus of research in recent years. Evidence has been presented that calcium oscillations can effectively trigger apoptosis under certain conditions and that dysregulation of calcium signalling is a common cause of cell death. These effects are regularly mediated through calcium signal propagation to the mitochondria and the ensuing mitochondrial membrane permeabilization and release of pro-apoptotic factors from mitochondria to the cytoplasm. The progress in this area depended on the development of (1) fluorescent/luminescent probes, including fluorescent proteins that can be genetically targeted to different intracellular locations and (2) the digital imaging technology, fluorescence-activated cell sorting and fluorescent high throughput approaches, which allowed dynamic measurements of both [Ca2+] in the intracellular compartments of interest and the downstream processes. Fluorescence single cell imaging has been the only possible approach to resolve the cell-to-cell heterogeneity and the complex subcellular spatiotemporal organization of the cytoplasmic and mitochondrial calcium signals and downstream events. We outline here fluorometric and fluorescence imaging protocols that we set up for the study of calcium in the context of apoptosis.
Topics: Animals; Apoptosis; Calcium; Calcium Signaling; Caspases; Cell Death; Cell Line; Digitonin; Fluorescent Dyes; Green Fluorescent Proteins; Humans; Microscopy, Fluorescence; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Necrosis; Permeability; Rats
PubMed: 18948203
DOI: 10.1016/j.ymeth.2008.09.024 -
Drug Delivery and Translational Research Sep 2022State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial...
State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial interference. Digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI), facilitates reliable proliferation quantification of native cell populations and the extraction of morphological features in a fast and label- and interference-free manner by biophysical parameters. DHM therefore has been identified as versatile tool for cytotoxicity testing in biomedical nanotechnology. In a comparative study performed at two collaborating laboratories, we investigated the interlaboratory variability and performance of DHM in nanomaterial toxicity testing, utilizing complementary standard operating procedures (SOPs). Two identical custom-built off-axis DHM systems, developed for usage in biomedical laboratories, equipped with stage-top incubation chambers were applied at different locations in Europe. Temporal dry mass development, 12-h dry mass increments and morphology changes of A549 human lung epithelial cell populations upon incubation with two variants of poly(alkyl cyanoacrylate) (PACA) nanoparticles were observed in comparison to digitonin and cell culture medium controls. Digitonin as cytotoxicity control, as well as empty and cabazitaxel-loaded PACA nanocarriers, similarly impacted 12-h dry mass development and increments as well as morphology of A549 cells at both participating laboratories. The obtained DHM data reflected the cytotoxic potential of the tested nanomaterials and are in agreement with corresponding literature on biophysical and chemical assays. Our results confirm DHM as label-free cytotoxicity assay for polymeric nanocarriers as well as the repeatability and reproducibility of the technology. In summary, the evaluated DHM assay could be efficiently implemented at different locations and facilitates interlaboratory in vitro toxicity testing of nanoparticles with prospects for application in regulatory science.
Topics: Digitonin; Holography; Humans; In Vitro Techniques; Microscopy; Reproducibility of Results
PubMed: 35799027
DOI: 10.1007/s13346-022-01207-5 -
Cellular and Molecular Neurobiology Sep 19881. Calcium-dependent exocytosis of catecholamines from intact and digitonin-permeabilized bovine adrenal chromaffin cells was investigated. 2. 45Ca2+ uptake and... (Review)
Review
1. Calcium-dependent exocytosis of catecholamines from intact and digitonin-permeabilized bovine adrenal chromaffin cells was investigated. 2. 45Ca2+ uptake and secretion induced by nicotinic stimulation or depolarization in intact cells were closely correlated. The results provide strong support for Ca2+ entry being the trigger for exocytosis. 3. Experiments in which the H+ electrochemical gradient across the intracellular secretory granule (chromaffin granule) membrane was altered indicated that the gradient does not play an important role in exocytosis. 4. Ca2+ entry into the cells is associated with activation of phospholiphase C and a rapid translocation of protein kinase C to membranes. 5. The plasma membrane of chromaffin cells was rendered permeable to Ca2+, ATP, and proteins by the detergent digitonin without disruption of the intracellular secretory granules. In this system in which the intracellular milieu can be controlled, micromolar Ca2+ directly stimulated catecholamine secretion. 6. Treatment of the cells with phorbol esters and diglyceride, which activate protein kinase C, enhanced phosphorylation and subsequent Ca2+-dependent secretion in digitonin-treated cells. 7. Phorbol ester-induced secretion could be specifically inhibited by trypsin. The experiments indicate that protein kinase C modulates but is not necessary for Ca2+-dependent secretion.
Topics: Adrenal Medulla; Animals; Calcium; Catecholamines; Cattle; Digitonin; Exocytosis; Phorbol Esters; Phosphorylation
PubMed: 3066486
DOI: 10.1007/BF00711168 -
PloS One Jan 2010Most medicinal plants contain a mixture of bioactive compounds, including chemicals that interact with intracellular targets and others that can act as adjuvants to...
BACKGROUND
Most medicinal plants contain a mixture of bioactive compounds, including chemicals that interact with intracellular targets and others that can act as adjuvants to facilitate absorption of polar agents across cellular membranes. However, little is known about synergistic effects between such potential drug candidates and adjuvants. To probe for such effects, we tested the green tea compound epigallocatechin gallate (EGCG) and the membrane permeabilising digitonin on Plasmodium sporozoite motility and viability.
METHODOLOGY/PRINCIPAL FINDINGS
Green fluorescent P. berghei sporozoites were imaged using a recently developed visual screening methodology. Motility and viability parameters were automatically analyzed and IC50 values were calculated, and the synergism of drug and adjuvant was assessed by the fractional inhibitory concentration index. Validating our visual screening procedure, we showed that sporozoite motility and liver cell infection is inhibited by EGCG at nontoxic concentrations. Digitonin synergistically increases the cytotoxicity of EGCG on sporozoite survival, but shows an additive effect on sporozoite motility.
CONCLUSIONS/SIGNIFICANCE
We proved the feasibility of performing highly reliable visual screens for compounds against Plasmodium sporozoites. We thereby could show an advantage of administering mixtures of plant metabolites on inhibition of cell motility and survival. Although the effective concentration of both drugs is too high for use in malaria prophylaxis, the demonstration of a synergistic effect between two plant compounds could lead to new avenues in drug discovery.
Topics: Animals; Catechin; Digitonin; Drug Synergism; Liver; Plasmodium berghei
PubMed: 20072627
DOI: 10.1371/journal.pone.0008682 -
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 -
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 -
Proceedings of the National Academy of... May 2015The four-component intramembrane protease γ-secretase is intricately linked to the development of Alzheimer's disease. Despite recent structural advances, the...
The four-component intramembrane protease γ-secretase is intricately linked to the development of Alzheimer's disease. Despite recent structural advances, the transmembrane segments (TMs) of γ-secretase remain to be specifically assigned. Here we report a 3D structure of human γ-secretase at 4.32-Å resolution, determined by single-particle, electron cryomicroscopy in the presence of digitonin and with a T4 lysozyme fused to the amino terminus of presenilin 1 (PS1). The overall structure of this human γ-secretase is very similar to that of wild-type γ-secretase determined in the presence of amphipols. The 20 TMs are unambiguously assigned to the four components, revealing principles of subunit assembly. Within the transmembrane region, PS1 is centrally located, with its amino-terminal fragment (NTF) packing against Pen-2 and its carboxyl-terminal fragment (CTF) interacting with Aph-1. The only TM of nicastrin associates with Aph-1 at the thick end of the TM horseshoe, and the extracellular domain of nicastrin directly binds Pen-2 at the thin end. TM6 and TM7 in PS1, which harbor the catalytic aspartate residues, are located on the convex side of the TM horseshoe. This structure serves as an important framework for understanding the function and mechanism of γ-secretase.
Topics: Alzheimer Disease; Amyloid Precursor Protein Secretases; Catalysis; Cell Membrane; Cryoelectron Microscopy; Detergents; Digitonin; Endopeptidases; HEK293 Cells; Humans; Image Processing, Computer-Assisted; Membrane Glycoproteins; Membrane Proteins; Peptide Hydrolases; Presenilin-1; Protein Binding; Protein Structure, Secondary
PubMed: 25918421
DOI: 10.1073/pnas.1506242112 -
Gut Feb 1974Moderate unconjugated hyperbilirubinaemia decreasing after pneumatic dilatation of the gastrooesophageal sphincter, so permitting a normal amount of food to be taken was...
Moderate unconjugated hyperbilirubinaemia decreasing after pneumatic dilatation of the gastrooesophageal sphincter, so permitting a normal amount of food to be taken was observed in two patients with achalasia. Liver biopsy was performed, and hepatic digitonin-activated bilirubin UDP-glucuronyltransferase activity was decreased in both, as is usually found in patients with Gilbert's syndrome. In the patients examined the slight and variable hyperbilirubinaemia associated with Gilbert's syndrome seemed thus to have been aggravated because of the decreased food intake due to achalasia. This situation may be compared to the jaundice sometimes found in neonates with pyloric stenosis or other types of obstruction of the upper gastrointestinal tract. Observations in rats and man favour a complex mechanism for fasting-induced hyperbilirubinaemia.
Topics: Adult; Bilirubin; Deglutition Disorders; Digitonin; Dilatation; Esophageal Achalasia; Fasting; Female; Glucosyltransferases; Humans; Hyperbilirubinemia; Hyperbilirubinemia, Hereditary; Liver; Male; Uracil Nucleotides
PubMed: 4820636
DOI: 10.1136/gut.15.2.121 -
Journal of Biochemistry Oct 1984The interaction of digitonin with membrane cholesterol was studied by using various digitonin analogs, and radioactive desglucodigitonin. The following results were...
The interaction of digitonin with membrane cholesterol was studied by using various digitonin analogs, and radioactive desglucodigitonin. The following results were obtained concerning the effect of digitonin on erythrocytes, granulocytes and liposomes. Digitonin and its analogs showed activity to induce hemolysis, granulocyte activation and liposomal membrane damage. The activity was affected by change of the carbohydrate residue of the molecule; the order of hemolytic activity was digitonin greater than or equal to desglucodigitonin much greater than glucosyl-galactosyl-digitogenin greater than galactosyl-digitogenin, digitogenin. The relative activities of these compounds to induce granulocyte activation and liposomal membrane damage were similar to those observed in the hemolysis. [3H]Desglucodigitonin could bind to cholesterol in liposomes. The binding was stoichiometric and the ratio of desglucodigitonin bound to liposomes/cholesterol in liposomes was close to 1, irrespective of the cholesterol content in liposome. Damage to liposomes was, however, induced by desglucodigitonin only when they contained more than 0.2 molar ratio of cholesterol to phospholipid. Addition of digitonin as well as desglucodigitonin to preformed liposomes deprived of cholesterol affected the anisotropic molecular motion of spin-labeled phosphatidylcholine incorporated into the liposomes, suggesting that the molecules could be inserted into the lipid bilayer free of cholesterol. Molecules of desglucodigitonin in the lipid phase may, however, be equilibrated with those in the aqueous phase, unless they form a complex with cholesterol, since no appreciable amount of [3H]desglucodigitonin could be detected in the liposome fraction after separation by column chromatography. Digitonin decreased the order parameter of spin-labeled phosphatidylcholine when liposomes contained equimolar cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Animals; Calorimetry; Cell Membrane; Chemical Phenomena; Chemistry; Cholesterol; Digitonin; Electron Spin Resonance Spectroscopy; Erythrocyte Membrane; Granulocytes; Guinea Pigs; Hemolysis; Humans; Kinetics; Liposomes; Membrane Lipids; Phosphatidylcholines; Tritium
PubMed: 6097588
DOI: 10.1093/oxfordjournals.jbchem.a134941 -
Methods in Molecular Biology (Clifton,... 2014HIV-1 viral protein R (VpR) is a multifunctional protein that plays specific roles at multiple stages of the HIV-1 viral life cycle and affects anti-HIV functions of the...
HIV-1 viral protein R (VpR) is a multifunctional protein that plays specific roles at multiple stages of the HIV-1 viral life cycle and affects anti-HIV functions of the immune cells. VpR is required for efficient viral replication in nondividing cells such as macrophages, and it promotes, to some extent, viral replication in the proliferating target CD4+ T cells. A number of specific activities that may contribute to these effects of VpR have been proposed. In this chapter, we describe two best characterized activities of VpR, nuclear import of the HIV-1 preintegration complex (PIC) and induction of cell cycle G2 arrest, focusing on the methods used for their demonstration.
Topics: Active Transport, Cell Nucleus; Cell Nucleus; Digitonin; G2 Phase Cell Cycle Checkpoints; HIV-1; HeLa Cells; Humans; Schizosaccharomyces; Virus Integration; vpr Gene Products, Human Immunodeficiency Virus
PubMed: 24158819
DOI: 10.1007/978-1-62703-670-2_11