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Cell Calcium Jul 1991This paper describes a method to load embryogenic plant cells with Fluo-3 in its cell impermeant form with the aid of digitonin. Attempts to load cells with Fluo-3/AM...
This paper describes a method to load embryogenic plant cells with Fluo-3 in its cell impermeant form with the aid of digitonin. Attempts to load cells with Fluo-3/AM were all unsuccessful. Presumably the indicator is cleaved outside the cells and cannot penetrate in its acidic form. At a low pH, Fluo-3 enters the plant cells but normal Ca2+ homeostasis seems to be disturbed. Successful loading of Fluo-3 was achieved by adding 0.1% digitonin during incubation with the Ca(2+)-indicator. A bright fluorescence was observed in the epidermal layer of heart and torpedo shaped somatic embryos of carrot with confocal scanning laser microscopy. Vacuoles were always without fluorescence which indicates that the dye, after loading, remains in the cytosol and does not leak out. The fluorescence intensity was sensitive to treatments with A23187 and EGTA. We conclude that Fluo-3 can be effectively loaded, with the aid of digitonin, into plant embryogenic cells in liquid culture. Therefore, we expect this technique to be very useful for the study of changes in cytosolic free Ca2+ levels during plant growth and development.
Topics: Aniline Compounds; Calcium; Cells, Cultured; Cytological Techniques; Digitonin; Fluorescent Dyes; Plants; Xanthenes
PubMed: 1934038
DOI: 10.1016/0143-4160(91)90033-b -
Experimentelle Pathologie 1978Treatment of erythrocytes by low concentrations of digitonin results in the formation of elongated, bulged membrane areas free from particles and intramembraneous...
Treatment of erythrocytes by low concentrations of digitonin results in the formation of elongated, bulged membrane areas free from particles and intramembraneous tubular structures emerged from these domains. At higher concentrations the tubular structures are present also outside of the membrane. A second, not bulged type of particle-free areas is more or less rounded, but mostly of rectangular shape. The rate of this type of domains increases with the concentration of digitonin. Breaks in these areas lead to a formation of sheets which results in a breakdown of the membrane structure finally into a brittle mass of many small sheets lying irregularly one upon the other. Already during the first steps of this membrane breakdown hemolysis takes place. Cross-linking of the proteins with glutaraldehyde does not entirely block up the membrane alterations. In fixed erythrocytes the formation fo elongated domains is restricted, no tubular structures are present and no dislocations of particles can be observed. Nevertheless smooth sheets localized also outside of the membrane are formed. However the investigated ghosts are not stabilized by glutaraldehyde against the effects of digitonin. Particle dislocations as well as all the other membrane alterations are present. The implications of the obtained results are: 1. The elongated domains and the tubular structures are probably not digitonin-cholesterol-complexes. 2. The formation of crystalline regions by digitonin-cholesterol-complexes destroys the membrane structure.
Topics: Digitonin; Erythrocyte Membrane; Erythrocytes; Freeze Fracturing; Glutaral; Humans
PubMed: 102519
DOI: 10.1016/s0014-4908(78)80007-6 -
The Journal of Pharmacy and Pharmacology Oct 1988The binding of [3H]mepyramine to histamine H1-receptors solublized from guinea-pig cerebellum by 1% digitonin could be assayed by adsorption of the receptor-bound...
The binding of [3H]mepyramine to histamine H1-receptors solublized from guinea-pig cerebellum by 1% digitonin could be assayed by adsorption of the receptor-bound [3H]ligand to glass-fibre filters pretreated with 0.3% polyethylenimine (PEI). Non-specific binding was higher than in parallel experiments in which the bound and free [3H]ligand was separated by gel-filtration on Sephadex G-25 columns, but the parameters characterizing the inhibition curves were otherwise similar. The PEI-treated filter method could also be used to assay [3H]-(+)-N-methyl-4-methyl-diphenhydramine ([3H]QMDP) binding to solubilized H1-receptors, but the level of non-specific binding was higher and was only satisfactorily defined by icotidine or temelastine. A particular utility of the PEI-treated filter assay will be in measurements of the kinetics of H1-receptor-ligand interactions.
Topics: Animals; Chromatography, Gel; Digitonin; Guinea Pigs; In Vitro Techniques; Male; Polyethyleneimine; Pyrilamine; Receptors, Histamine H1; Time Factors
PubMed: 2907544
DOI: 10.1111/j.2042-7158.1988.tb07006.x -
Biochimica Et Biophysica Acta Jul 1984The morphology of interactions between digitonin and cholesterol has been investigated. When precipitated from ethanolic solutions, digitonin-cholesterol complexes form...
The morphology of interactions between digitonin and cholesterol has been investigated. When precipitated from ethanolic solutions, digitonin-cholesterol complexes form in flat lamellar sheets. In contrast, when the complex is formed in a bilayer membrane, the membrane is deformed into corrugations of hemitubules. The polarity of the deformations formed in bilayer membranes is highly correlated with the direction of entry of digitonin into the membrane. We suggest that the morphology of digitonin/cholesterol hemitubules is dependent upon the complex being formed within a bilayer and, in addition, is not correlated with asymmetry of cholesterol concentration across the membrane.
Topics: Animals; Cholesterol; Digitonin; Erythrocyte Membrane; Ethanol; Freeze Fracturing; Intestinal Mucosa; Islets of Langerhans; Lipid Bilayers
PubMed: 6375721
DOI: 10.1016/0005-2736(84)90286-4 -
Molecules (Basel, Switzerland) Nov 2015In the present investigation we studied the molecular mechanisms of the monodesmosidic saponin digitonin on natural and artificial membranes. We measured the hemolytic...
In the present investigation we studied the molecular mechanisms of the monodesmosidic saponin digitonin on natural and artificial membranes. We measured the hemolytic activity of digitonin on red blood cells (RBCs). Also different lipid membrane models (large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs) in the presence and absence of cholesterol were employed. The stability and permeability of the different vesicle systems were studied by using calcein release assay, GUVs membrane permeability assay using confocal microscopy (CM) and fluorescence correlation spectroscopy (FCS) and vesicle size measurement by dynamic light scattering (DLS). The results support the essential role of cholesterol in explaining how digitonin can disintegrate biological and artificial membranes. Digitonin induces membrane permeability or causes membrane rupturing only in the presence of cholesterol in an all-or-none mechanism. This effect depends on the concentrations of both digitonin and cholesterol. At low concentrations, digitonin induces membrane permeability while keeping the membrane intact. When digitonin is combined with other drugs, a synergistic potentiation can be observed because it facilitates their uptake.
Topics: Animals; Cell Membrane; Cell Membrane Permeability; Cholesterol; Digitonin; Erythrocytes; Fluoresceins; Hemolysis; Lipid Bilayers; Saponins; Sheep; Steroids
PubMed: 26569199
DOI: 10.3390/molecules201119682 -
Biosensors & Bioelectronics Sep 2011Functionalized colloidal gold is widely used for qualitative and quantitative detection of specific analytes. We report here a novel modification of gold nanoparticles...
Functionalized colloidal gold is widely used for qualitative and quantitative detection of specific analytes. We report here a novel modification of gold nanoparticles by digitonin, a glycoside used for precipitating membrane cholesterol. The specific molecular recognition of cholesterol by digitonin gold nanoparticles (DGNP), make it an attractive alternative to the existing enzymatic methods for cholesterol sensing. To enable cholesterol binding, we modified mercapto modified GNPs with digitonin, by a simple esterification reaction. The blue shift in the plasmon absorption spectra of DGNP with different cholesterol concentrations accompanied by a decrease in the absorbance is the principle applied here for the estimation. The observed size reduction followed by cholesterol binding is reasoned due to the enhanced hydophobicity of the surface which in turn expels the water layers associated with the particles prior to cholesterol binding. The method exhibited linearity between concentration of cholesterol and the corresponding absorbance of the plasmon peak, in the range of 160-600 ng/mL with a detection limit of 100±9 ng/mL. Other steroids did not show any binding affinity towards DGNP. The method depicted here has potential for development as an enzyme free sensor for cholesterol although many factors need to be addressed to transform it for assaying samples like blood.
Topics: Biosensing Techniques; Cholesterol; Digitonin; Gold; Gold Colloid; Humans; Intracellular Membranes; Metal Nanoparticles; Surface Plasmon Resonance
PubMed: 21752631
DOI: 10.1016/j.bios.2011.06.015 -
FEBS Letters Sep 1998Apoptosis, a naturally occurring programmed cell death or cell 'suicide', has been paid much attention as one of the critical mechanisms for morphogenesis and tissue...
Apoptosis, a naturally occurring programmed cell death or cell 'suicide', has been paid much attention as one of the critical mechanisms for morphogenesis and tissue remodeling. Activation of cysteine aspartases (caspases) is one of the critical steps leading to apoptosis. Although a mitochondria-mediated pathway has been postulated to be one of the activation mechanism of caspase-3, another subcellular compartment might be involved in the activation of the enzyme. The present study shows that the supernatant fraction of digitonin-treated lysosomes strongly activates Ac-DEVD-CHO inhibitable caspase-3-like protease. Activation of caspase-3-like protease by digitonin-treated lysosomal fractions was specifically suppressed by leupeptin and E-64, inhibitors of cysteine protease. These results indicate that leakage of lysosomal cysteine protease(s) into the cytosolic compartment might be involved in the activation of caspase-3-like protease.
Topics: Animals; Apoptosis; Caspase 3; Caspases; Cysteine Endopeptidases; Digitonin; Enzyme Activation; Enzyme Inhibitors; Enzyme Precursors; Indicators and Reagents; Lysosomes; Rats; Rats, Wistar
PubMed: 9762916
DOI: 10.1016/s0014-5793(98)01080-1 -
Journal of Pharmaceutical Sciences Feb 1984Digitoxin was modified by condensation with propylene oxide or with 1,4-butanediol diglycidyl ether in aqueous alkali, yielding products in which some of the CH2OH...
Digitoxin was modified by condensation with propylene oxide or with 1,4-butanediol diglycidyl ether in aqueous alkali, yielding products in which some of the CH2OH groups of digitonin were converted to CH2OCH2CHOHCH3 or to CH2OCH2CHOHCH2O(CH2)4OCH2CHOHCH2OH groups, respectively. These modified digitonins were very soluble in water and chloroform and effectively solubilized lipophilic compounds into aqueous solutions; e.g., 2 mg of vitamin A or 0.6 mg of cholecalciferol could be dissolved per 1 mL of 5% aqueous solutions of modified digitonins. Compared with the toxicity of digitonin (LD50 4 mg/kg iv), the toxicity of modified digitonin was greatly reduced: doses of 500 mg/kg by intravenous infusion were not lethal for mice.
Topics: Adjuvants, Pharmaceutic; Animals; Cells, Cultured; Chemistry, Pharmaceutical; Cholesterol; Dialysis; Digitonin; Female; Mice; Mice, Inbred C57BL; Micelles; Molecular Weight; Solubility
PubMed: 6707892
DOI: 10.1002/jps.2600730224 -
The Biochemical Journal Feb 1985Perfusing a rat liver with digitonin in the concentration range 2-20 mg/ml results in complete decolorization of the organ within 45-250 s. Decolorization progresses...
Perfusing a rat liver with digitonin in the concentration range 2-20 mg/ml results in complete decolorization of the organ within 45-250 s. Decolorization progresses with time in the direction of flow, and it is therefore possible, by collecting the eluate, to obtain material from specific intracellular compartments of hepatocytes in different zones in the microcirculatory unit of the liver. The results demonstrate that cytoplasmic marker enzymes from periportal or perivenous hepatocytes can be collected with as little contamination from the other compartment as is obtained in micro-dissection studies. Furthermore, a fraction enriched in mitochondrial marker enzymes can be achieved with only 10-20% contamination by cytoplasmic material.
Topics: Animals; Cell Compartmentation; Digitonin; Female; Glutamate Dehydrogenase; Intracellular Fluid; L-Lactate Dehydrogenase; Liver; Perfusion; Rats; Rats, Inbred Strains; Subcellular Fractions
PubMed: 3977871
DOI: 10.1042/bj2260289 -
Hepatology (Baltimore, Md.) Dec 1997Morphological and functional heterogeneity of hepatocytes according to their position in the liver lobule has been known for many years. The digitonin-collagenase...
An improved digitonin-collagenase perfusion technique for the isolation of periportal and perivenous hepatocytes from a single rat liver: physiological implications for lobular heterogeneity.
Morphological and functional heterogeneity of hepatocytes according to their position in the liver lobule has been known for many years. The digitonin-collagenase perfusion technique is widely used to study hepatocyte heterogeneity and has yielded reliable data. However, with this procedure, periportal (PP) or perivenous (PV) hepatocytes are isolated from different livers, allowing only comparison between cell populations issued from two separate animals. To overcome this drawback, we have modified this technique by perfusing the two main rat liver lobes of a single animal in succession. The procedure involved alternate clamping of the median and the left lateral lobes, restricting digitonin infusion to one lobe via the portal vein, and to the other via the caudal vena cava. Lobe exclusion during digitonin perfusion, and zonal restriction of digitonin-induced damage, were monitored using macroscopic and histological controls. We compared our results with previous data on PP and PV hepatocytes issued from two different livers using the conventional digitonin-collagenase perfusion technique. First, we found that the cellular sensitivity to angiotensin II, a calcium-mobilizing agonist, was 60% to 80% higher in PV than in PP hepatocytes, whereas, previously, no difference had been recorded. Second, we found that albumin messenger RNAs (mRNAs) were 35% more abundant in PP than in PV hepatocytes, whereas, previously, larger differences had been reported. Our results show that PP and PV hepatocytes may be isolated from a single liver using an improved digitonin-collagenase perfusion technique. Furthermore, we suggest that zonal differences can be artificially masked or amplified when comparing PP and PV cell populations from two different livers, indicating that it is preferable to use a single liver for accurate zonal comparisons between hepatocytes.
Topics: Albumins; Angiotensin II; Animals; Calcium; Cell Separation; Cell Survival; Collagenases; Digitonin; Liver; Microcirculation; Perfusion; RNA, Messenger; Rats; Rats, Wistar
PubMed: 9398003
DOI: 10.1053/jhep.1997.v26.pm0009398003