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Parasitology Research Sep 2008Leishmania infantum belongs to the Kinetoplastidae that is characterized by a specific mitochondrial DNA, the kinetoplast. This parasite is responsible for both benign...
Leishmania infantum belongs to the Kinetoplastidae that is characterized by a specific mitochondrial DNA, the kinetoplast. This parasite is responsible for both benign cutaneous leishmaniasis and severe visceral leishmaniasis in humans. Molecular determinants of such differences in pathogenesis are not well understood, and the parasites as well as their hosts may contribute to the disease phenotype. Factors that help parasite to adapt its metabolism to nutritional conditions encountered in different location might play pivotal roles in controlling parasite development in these various host environments. Thus, we have decided to initiate studies aimed to compare the mitochondrial protein content of L. infantum. To avoid the drawback caused by the most abundant proteins such as tubulin and proteins of the cytoskeleton present in whole cell extract, we have decided to fractionate the subcellular components of the cells. Using both cytosolic and mitochondrial markers, we have improved a protein pre-fractionation protocol using digitonin that allowed us to generate an enriched mitochondrial fraction.
Topics: Animals; Chemical Fractionation; Digitonin; Leishmania infantum; Mitochondrial Proteins; Proteome
PubMed: 18575891
DOI: 10.1007/s00436-008-1062-9 -
The Histochemical Journal May 1978The influence of several fixation and dehydration procedures on the retention of free cholesterol and cholesterol esters was studied in filter paper preparations. The...
The influence of several fixation and dehydration procedures on the retention of free cholesterol and cholesterol esters was studied in filter paper preparations. The retention of free cholesterol by the filter paper proved to be decreased by the addition of digitonin to the aldehyde fixative (aqueous phase) and was only slightly enhanced by partial dehydration (alcoholic phase, up to 70% ethanol). Furthermore, digitonin or the presumably formed cholesterol-digitonide complex bound hardly any osmium oxides in glass-fibre paper. Up to 26% of the cholesterol esters was mobilized during the aqueous phase when digitonin was added to the aldehyde fixative. When the glass-fibre papers containing the digitonin cholesterol-ester-osmate complexes were stored in distilled water after fixation, the fluid became turbid. Particulate material isolated from this turbid solution showed ultrastructurally a close resemblance to the 'whorls' observed by several authors in tissue fixed by a digitonin-containing aldehyde fixative. Digitonin also changed the ultrastructural appearance of liposomes, containing lecithin: cholesterol: phosphatidic acid; in a molar ratio 7:2:1. Our observations lead to the conclusion that the use of digitonin-containing fixatives should be abandoned, because they give results which cannot be interpreted. By the use of K4 [Fe(CN)6] containing OSO4 in the post-fixation step were able to demonstrate an increase in the visualization of membranous structures (liposomes).
Topics: Cholesterol; Cholesterol Esters; Desiccation; Digitalis Glycosides; Digitonin; Fixatives; Liposomes; Osmium
PubMed: 649442
DOI: 10.1007/BF01007560 -
Biochimica Et Biophysica Acta Nov 1975The effect of digitonin on glucose uptake by isolated fat cells in the presence and absence of insulin has been studied. At low concentrations of digitonin, the...
The effect of digitonin on glucose uptake by isolated fat cells in the presence and absence of insulin has been studied. At low concentrations of digitonin, the stimulation of glucose uptake by insulin was inhibited without severe cell damage as estimated by the leakage of lactate dehydrogenase from the cells. The inhibition of the insulin effect was not reversed by washing the cells or by the addition of cholesterol or lecithin-cholesterol liposomes to the incubation medium of the cells after treatment with digitonin. Cholesterol was shown to be present in the fat cells and it is suggested that the inhibition of the insulin effect is a consequence of the formation of digitonin-cholesterol complexes in the fat cell plasma membrane. Possible ways in which this may results in inhibition of the effect of insulin are discussed.
Topics: Adipose Tissue; Animals; Biological Transport, Active; Cholesterol; Digitalis Glycosides; Digitonin; Glucose; In Vitro Techniques; Insulin; L-Lactate Dehydrogenase; Male; Rats
PubMed: 1182202
DOI: 10.1016/0304-4165(75)90282-2 -
Molecular and Cellular Endocrinology Jul 1999Non-genomic actions of progesterone have been described in the ovary, and luteal membranes of several species have been shown to possess specific binding sites for...
Non-genomic actions of progesterone have been described in the ovary, and luteal membranes of several species have been shown to possess specific binding sites for [3H]-progesterone. However, binding of radiolabelled progesterone to luteal membranes was demonstrable only in the presence of digitonin. Digitonin is a non-ionic detergent which is thought to act by forming one-to-one complexes with certain sterols. It is also a cardiotonic agent, inhibiting (Na+-K+) ATPase activity by interaction with the extracellular (ouabain/K+) binding site. We therefore investigated which properties of digitonin were responsible for its stimulatory actions on progesterone binding to bovine luteal membranes. A range of compounds with detergent, cardiotonic and or cholesterol-complexing activities were tested for their effects on [3H]-progesterone binding to bovine luteal membrane fractions, and on haemolysis of rat erythrocytes. Stimulation of progesterone binding to luteal membranes was highly specific for digitonin, and a number of ionic and non-ionic detergents, cardenolides, saponins and cholesterol-complexing reagents tested failed either to stimulate [3H]-progesterone binding to bovine luteal membranes in the absence of digitonin, or to inhibit binding specifically in the presence of digitonin. When digitonin was first reacted with excess cholesterol or pregnenolone to form the respective digitonides, stimulatory activity was greatly reduced, suggesting that the ability of digitonin to interact with (an) endogenous steroid(s) may be important in its action. High performance liquid chromatography (HPLC)-mass spectrometry of commercially available digitonin preparations indicated the presence of numerous minor impurities in most commercial digitonin preparations. Three major UV-absorbing peaks were isolated and characterised by mass spectrometry: all stimulated progesterone binding to bovine luteal membrane receptors in a dose-dependent manner, though to differing extents. Our data suggest that the unique action of digitonin on luteal membrane progesterone receptors is not related to its detergent or cardiotonic properties, but appears to be related to its ability to complex with membrane sterols.
Topics: Animals; Cattle; Cell Membrane; Cholesterol; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Digitonin; Female; Hemolysis; Luteal Cells; Mass Spectrometry; Pregnenolone; Progesterone; Rats; Rats, Wistar; Receptors, Progesterone; Tritium
PubMed: 10459854
DOI: 10.1016/s0303-7207(99)00091-x -
Biochimica Et Biophysica Acta Jun 1984The effects of the plant glycosides saponin as well as digitonin on the electrical conductance of black lipid membranes and the effect of these agents on the surface...
The effects of the plant glycosides saponin as well as digitonin on the electrical conductance of black lipid membranes and the effect of these agents on the surface pressure of lipid monofilms was investigated. Both saponin and digitonin induced channel-like fluctuations in planar bilayers made either of diphytanoylphosphatidylcholine ( DPhPC ) or of DPhPC and cholesterol 2: 1 (w/w). In cholesterol-free bilayers the amount needed to induce an increase in conductance was 0.3-1 mg/ml for saponin and about 0.2 mg/ml for digitonin. In contrast, in cholesterol-containing bilayers the concentration needed to induce pores was about 10 micrograms/ml for both saponin and digitonin. In cholesterol-containing membranes the fluctuating pores induced by saponin were about 3-times more permeable to K+ than to Cl- and the macroscopic current showed an ohmic behaviour. Surface pressure experiments demonstrate that both glycosides could penetrate into lipid monofilms of pure DPhPC spread at the air/water interface with an initial surface pressure of 30 mN/m. The increase in surface pressure was considerably enhanced in cholesterol-containing films. It is assumed that the channel-like fluctuations induced by saponin as well as digitonin, in both cholesterol-free and cholesterol-rich bilayers are due to the formation of micellar structures within the lipid lattice. Probably the penetration of the glycosides into the lipid bilayer is considerably enhanced by the presence of cholesterol.
Topics: Animals; Cell Membrane; Cholesterol; Digitonin; Lipid Bilayers; Liposomes; Models, Biological; Phosphatidylcholines; Saponins
PubMed: 6733096
DOI: 10.1016/0005-2736(84)90547-9 -
Journal of Visualized Experiments : JoVE Jul 2021Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of...
Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of cell-specific differences in nuclear pore complex structure, prompting a need to adapt nuclear transport methods for use in neurons. Permeabilized cell assays, in which the plasma membrane is selectively perforated by digitonin, are widely used to study passive and active nuclear transport in immortalized cell lines but have not been applied to neuronal cultures. In our initial attempts, we observed the rapid loss of nuclear membrane integrity in primary mouse cortical neurons exposed to even low concentrations of digitonin. We hypothesized that neuronal nuclear membranes may be uniquely vulnerable to the loss of cytoplasmic support. After testing multiple approaches to improve nuclear stability, we observed optimal nuclear integrity following hypotonic lysis in the presence of a concentrated bovine serum albumin cushion. Neuronal nuclei prepared by this approach reliably import recombinant fluorescent cargo in an energy-dependent manner, facilitating analysis of nuclear import by high content microscopy with automated analysis. We anticipate that this method will be broadly applicable to studies of passive and active nuclear transport in primary neurons.
Topics: Active Transport, Cell Nucleus; Animals; Cell Nucleus; Digitonin; HeLa Cells; Humans; Mice; Neurons; Nuclear Envelope; Nuclear Pore
PubMed: 34309603
DOI: 10.3791/62710 -
Analytical Chemistry Aug 2006
Topics: Animals; Digitonin; Electrophoresis, Gel, Two-Dimensional; Humans; Leishmania infantum; Proteome; Protozoan Proteins; Research Personnel; Sensitivity and Specificity
PubMed: 16913062
DOI: 10.1021/ac069428s -
Antimicrobial Agents and Chemotherapy Nov 2015The flagellate Trypanosoma brucei causes sleeping sickness in humans and nagana in animals. Only a few drugs are registered to treat trypanosomiasis, but those drugs...
Combinations of alkaloids affecting different molecular targets with the saponin digitonin can synergistically enhance trypanocidal activity against Trypanosoma brucei brucei.
The flagellate Trypanosoma brucei causes sleeping sickness in humans and nagana in animals. Only a few drugs are registered to treat trypanosomiasis, but those drugs show severe side effects. Also, because some pathogen strains have become resistant, new strategies are urgently needed to combat this parasitic disease. An underexplored possibility is the application of combinations of several trypanocidal agents, which may potentiate their trypanocidal activity in a synergistic fashion. In this study, the potential synergism of mutual combinations of bioactive alkaloids and alkaloids with a membrane-active steroidal saponin, digitonin, was explored with regard to their effect on T. b. brucei. Alkaloids were selected that affect different molecular targets: berberine and chelerythrine (intercalation of DNA), piperine (induction of apoptosis), vinblastine (inhibition of microtubule assembly), emetine (intercalation of DNA, inhibition of protein biosynthesis), homoharringtonine (inhibition of protein biosynthesis), and digitonin (membrane permeabilization and uptake facilitation of polar compounds). Most combinations resulted in an enhanced trypanocidal effect. The addition of digitonin significantly stimulated the activity of almost all alkaloids against trypanosomes. The strongest effect was measured in a combination of digitonin with vinblastine. The highest dose reduction indexes (DRI) were measured in the two-drug combination of digitonin or piperine with vinblastine, where the dose of vinblastine could be reduced 9.07-fold or 7.05-fold, respectively. The synergistic effects of mutual combinations of alkaloids and of alkaloids with digitonin present a new avenue to treat trypanosomiasis but one which needs to be corroborated in future animal experiments.
Topics: Alkaloids; Animals; Benzodioxoles; Benzophenanthridines; Berberine; Digitonin; Drug Combinations; Drug Synergism; Emetine; Harringtonines; Homoharringtonine; Models, Theoretical; Piperidines; Polyunsaturated Alkamides; Trypanocidal Agents; Trypanosoma brucei brucei; Vinblastine
PubMed: 26349826
DOI: 10.1128/AAC.01315-15 -
Methods in Enzymology 2000
Review
Topics: Animals; Cells, Cultured; Chickens; Detergents; Digitonin; Horseradish Peroxidase; Immunohistochemistry; Indicators and Reagents; Microscopy, Electron; Octoxynol; Recombinant Fusion Proteins
PubMed: 11044972
DOI: 10.1016/s0076-6879(00)27265-0 -
FEBS Letters Apr 1991Incubation of digitonin-permeabilized bovine chromaffin cells results in a loss of Ca(2+)-dependent catecholamine secretion. The addition of cytosolic proteins prevents...
Incubation of digitonin-permeabilized bovine chromaffin cells results in a loss of Ca(2+)-dependent catecholamine secretion. The addition of cytosolic proteins prevents this loss of secretory activity. It has been proposed that calpactin might be the protein which is responsible for preventing this loss of activity. The experiments described in this paper show that cytosolic proteins which have been depleted of calpactin are as effective as control cytosolic proteins in preventing the loss of Ca(2+)-dependent secretion. Thus, a cytosolic protein(s) other than calpactin appears to be responsible for preventing this loss of secretory activity.
Topics: Adrenal Glands; Animals; Annexins; Calcium; Calcium-Binding Proteins; Catecholamines; Cattle; Cell Membrane Permeability; Cytosol; Digitonin; Immunoblotting; Proteins
PubMed: 1827411
DOI: 10.1016/0014-5793(91)80476-j