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Biochemical and Molecular Medicine Apr 1995For quantitative elucidation of maximal mitochondrial oxidation capacities in human mononuclear cells, cultured human skin fibroblasts and human thrombocytes the optimal... (Comparative Study)
Comparative Study
Oxygraphic evaluation of mitochondrial function in digitonin-permeabilized mononuclear cells and cultured skin fibroblasts of patients with chronic progressive external ophthalmoplegia.
For quantitative elucidation of maximal mitochondrial oxidation capacities in human mononuclear cells, cultured human skin fibroblasts and human thrombocytes the optimal amount of digitonin for plasma membrane permeabilization was determined to be 5, 10, and 0.1 micrograms/10(6) cells, respectively. Using these concentrations the rate of respiration of permeabilized cells with the mitochondrial substrates succinate (+ rotenone) or glutamate + malate can be stimulated between two- and fourfold by ADP and inhibited by carboxyatractyloside. The maximal respiratory activities of well-characterized preparations of permeabilized mononuclear cells of five patients with chronic progressive external ophthalmoplegia were compared to healthy controls and a 30 to 50% decrease of the ADP-stimulated respiration rates with glutamate + malate and succinate + rotenone was detected. This is an indication for the presence of the mitochondrial defect in respiratory active blood cells. Additionally, for two of these patients the mitochondrial defects were proven to be detectable by the determination of maximal oxygen consumption rates of digitonin-permeabilized cultured skin fibroblasts. Therefore, the determination of maximal oxidation capacities of a well-defined cell population using strictly standardized conditions of digitonin permeabilization is judged as a useful and sensitive method for the elucidation of mitochondrial function in extramuscular tissue.
Topics: Adolescent; Adult; Cells, Cultured; Digitonin; Female; Fibroblasts; Glutamates; Humans; Leukocytes, Mononuclear; Malates; Middle Aged; Mitochondria; Muscle, Skeletal; Ophthalmoplegia, Chronic Progressive External; Oxygen; Permeability; Skin; Succinates
PubMed: 8581354
DOI: 10.1006/bmme.1995.1015 -
Vision Research Oct 1994The visual pigment of the main rhabdom of the crayfish (P533) is unstable in digitonin. While slowly hydrolyzing to N-retinylidene opsin, a portion passes through a...
The visual pigment of the main rhabdom of the crayfish (P533) is unstable in digitonin. While slowly hydrolyzing to N-retinylidene opsin, a portion passes through a long-lived intermediate (P'505) with absorption similar to metarhodopsin but with the retinal still in the cis configuration. Crayfish metarhodopsin (M515) is similarly unstable in digitonin, and a portion converts to M'508 while bleaching slowly in the dark. Both P'505 and M'508 are light sensitive and bleach through an intermediate absorbing at still shorter wavelengths, M'460. The photobleaching of M'508 is likely a two-photon process, possibly involving P'505 as an intermediate. The persistence of these altered forms of the pigment with lambda max near 510 nm has compromised earlier efforts to analyze extracts of crayfish rhodopsin by partial bleaching. First, because of the incomplete decay of M515 (a portion of which liners as M'508), the difference spectrum for a red light exposure followed by dark decay has lambda max at 562 nm, but this difference spectrum does not describe a pigment. Because of the photosensitivity of M'508, a second bleaching exposure reveals the presence of a pigment with lambda max near 510 nm, but it is not a visual pigment and it is not present in the extract initially.
Topics: Animals; Astacoidea; Chromatography, High Pressure Liquid; Digitonin; Hot Temperature; In Vitro Techniques; Light; Photoreceptor Cells, Invertebrate; Rhodopsin; Spectrophotometry
PubMed: 7975305
DOI: 10.1016/0042-6989(94)90224-0 -
The Journal of Biological Chemistry Nov 1977
Topics: Adenosine Triphosphatases; Animals; Biological Transport, Active; Digitalis Glycosides; Digitonin; Dogs; Kidney; Kinetics; Macromolecular Substances; Phenanthrolines; Potassium; Sodium
PubMed: 144136
DOI: No ID Found -
The Cornell Veterinarian Jan 1989A study was undertaken to evaluate effectiveness of a digitonin disk inhibition test to discriminate between Acholeplasma laidlawii and Mycoplasma sp. isolated from...
A study was undertaken to evaluate effectiveness of a digitonin disk inhibition test to discriminate between Acholeplasma laidlawii and Mycoplasma sp. isolated from bovine milk. The test measured zone diameters of growth inhibition surrounding a digitonin-containing disk on solid medium. Zones of inhibition for 20 isolates of A. laidlawii, ranging from 8-14 mm, did not overlap those of 261 isolates of Mycoplasma sp., ranging from 16 to 38 mm. Examination of variation in zone diameters for M. bovis found that inhibition was not appreciably affected by agar dehydration. Zones of inhibition increased with increasing dilutions of stock culture and decreased with increasing incubation time. Analysis of variance and Fisher's least significant difference test of logn zone diameters revealed that differences in mean logn zone diameters were different at the 0.01 level of significance between some of the six species of mycoplasma examined, indicating that growth among some species of mycoplasma was effected differently by digitonin. The digitonin test was found to clearly discriminate between A. laidlawii and Mycoplasma sp. indicating that the test would be useful as a practical screening test of individual-cow and bulk tank milk for mycoplasmas.
Topics: Acholeplasma laidlawii; Animals; Digitonin; Evaluation Studies as Topic; Microbial Sensitivity Tests; Milk; Mycoplasma
PubMed: 2912675
DOI: No ID Found -
European Journal of Biochemistry Nov 1983Use of a digitonin-permeabilized rat adipocyte preparation overcomes inherent problems which occur when currently used broken cell systems are utilized for studying the...
Use of a digitonin-permeabilized rat adipocyte preparation overcomes inherent problems which occur when currently used broken cell systems are utilized for studying the regulation of hormone-sensitive lipase. The effect of digitonin on plasma membrane permeability was concentration-dependent being nearly maximum at 20 micrograms/ml as assessed by (a) leakage of 85% cellular lactate dehydrogenase after 30 min, (b) the efflux of 72% preloaded cellular (86Rb) rubidium within 10 min and (c) immediate inhibition of glucose oxidation. Hormone-modulated rates of lipolysis were preserved in this preparation. Following maximal activation of lipolysis in adipocytes with catecholamines, the rate of lipolysis in intact cells and digitonin-treated cells was elevated 26-fold and 20-fold respectively, while the rate in homogenates from these cells was elevated only 2.8-fold. Insulin suppressed catecholamine-dependent activation of lipolysis by at least 90% when subsequently measured in intact cells and digitonin-treated cells. Insulin suppression was only 56% when measured in homogenates. The hormone-sensitive lipase in permeabilized cells, as opposed to intact cells and homogenates, was activated by cyclic AMP to a degree that approached activation by catecholamines. In homogenates, cyclic AMP (1.0 mM) plus ATP (0.25 mM) activated the lipase only 36%, while neither alone had any effect. In digitonin-permeabilized cells, however, exogenous cyclic AMP alone activated lipolysis in a concentration-dependent manner with 1 microM, 30 microM and 1.0 mM cyclic AMP activating lipolysis by 41%, 250% and 1300% respectively. In contrast, lipolysis in intact cells was activated by 0%, 25% and 250% by 1 microM, 30 microM and 1.0 mM cyclic AMP. Also in digitonin-treated preparations, ATP alone activated lipolysis 40%, but ATP plus cyclic AMP activated lipolysis to only 74% of the level due to cyclic AMP alone. These studies indicate that the permeabilized adipocyte preparation is an excellent system for investigating the mechanism of regulation of the hormone-sensitive lipase by permitting manipulation of the intracellular environment while preserving the physiological response of the lipase.
Topics: Adenosine Triphosphate; Adipose Tissue; Animals; Cell Membrane Permeability; Chemical Phenomena; Chemistry; Cyclic AMP; Digitonin; Hormones; Lipolysis; Male; Rats; Rats, Inbred Strains; Sterol Esterase
PubMed: 6315436
DOI: 10.1111/j.1432-1033.1983.tb07783.x -
The Journal of Biological Chemistry Sep 1992Secretion of catecholamines from individual bovine adrenal medullary cells grown in primary culture has been investigated with a carbon-fiber microelectrode placed...
Secretion of catecholamines from individual bovine adrenal medullary cells grown in primary culture has been investigated with a carbon-fiber microelectrode placed adjacent to the cells. Oxidation of catecholamines at the electrode surface results in changes in current, which give a real-time measure of catecholamine secretion. Chemical agents are introduced to the individual cells by pressure ejection from micropipettes. When incubated in Ca(2+)-containing buffers, secretion is not observed. However, permeabilization of the cell by exposure to 20 microM digitonin for approximately 15 s results in a Ca(2+)-dependent secretion, and the contents of individual vesicles are detected in the form of sharp spikes. The rate at which spikes occur is a function of the Ca2+ concentration in the external media and reaches a maximum at 19 microM Ca2+. The area of the spikes range from 0.1 to greater than 10 picocoulombs, but the majority are less than 2 picocoulombs, corresponding to less than 6 x 10(6) molecules detected per spike. Histograms of the spike areas are essentially independent of the Ca2+ concentration, indicating that the population of vesicles which undergo exocytosis is the same for all concentrations. Exocytotic secretion can be distinguished from nonexocytotic release by analysis of the shape of the spikes.
Topics: Adrenal Medulla; Animals; Calcium; Catecholamines; Cations, Divalent; Cattle; Cells, Cultured; Chromaffin Granules; Digitonin; Electrochemistry; Exocytosis; Immunohistochemistry
PubMed: 1526972
DOI: 10.21236/ada251716 -
Phytomedicine : International Journal... Nov 2012We determined the ability of some phytochemicals, including alkaloids (glaucine, harmine, and sanguinarine), phenolics (EGCG and thymol), and terpenoids (menthol,...
We determined the ability of some phytochemicals, including alkaloids (glaucine, harmine, and sanguinarine), phenolics (EGCG and thymol), and terpenoids (menthol, aromadendrene, β-sitosterol-O-glucoside, and β-carotene), alone or in combination with the saponin digitonin to reverse the relative multi-drug resistance of Caco-2 and CEM/ADR5000 cells to the chemotherapeutical agent doxorubicin. The IC(50) of doxorubicin in Caco-2 and CEM/ADR5000 was 4.22 and 44.08μM, respectively. Combination of non-toxic concentrations of individual secondary metabolite with doxorubicin synergistically sensitized Caco-2 and CEM/ADR5000 cells, and significantly enhanced the cytotoxicity of doxorubicin. Furthermore, three-drug combinations (secondary metabolite+digitonin+doxorubicin) were even more powerful. The best synergist was the benzophenanthridine alkaloid sanguinarine. It reduced the IC(50) value of doxorubicin 17.58-fold in two-drug combinations (sanguinarine+doxorubicin) and even 35.17-fold in three-drug combinations (sanguinarine+digitonin+doxorubicin) in Caco-2 cells. Thus synergistic drug combinations offer the possibility to enhance doxorubicin efficacy in chemotherapy.
Topics: Alkaloids; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Benzophenanthridines; Caco-2 Cells; Digitonin; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Humans; Inhibitory Concentration 50; Isoquinolines; Neoplasms; Phenols; Phytotherapy; Plant Extracts; Plants; Terpenes
PubMed: 23146422
DOI: 10.1016/j.phymed.2012.08.010 -
Cell and Tissue Research Jan 1977Pea aphid primary symbiotes have previously been shown to synthesize cholesterol in vitro. Two electron microscopic techniques were used here to determine whether the...
Pea aphid primary symbiotes have previously been shown to synthesize cholesterol in vitro. Two electron microscopic techniques were used here to determine whether the symbiotes also synthesize cholesterol in vivo and whether this cholesterol is made available to the aphid. We also inquired into a possible role of secondary symbiotes in chosesterol biosynthesis. Treatment of aphids with digitonin resulted in significant alteration of ultrastructural sites in primary and secondary symbiote membranes. We concluded that these sites are areas of high cholesterol concentration in the symbiotes. Electron microscopic autoradiography with 3H-mevalonate precursor indicated that both primary and secondary symbiotes synthesize cholesterol; in both cases, the majority of grains were associated with the symbiote membranes. While the frequency of grains on the symbiotes remained constant, irrespective of incubation time in labelled media, the frequency of grains over surrounding tissues increased exponentially as the time of incubation was increased from 30 min to 8 h, indicating that symbiote cholesterol is transported to other tissues. High voltage electron microscopic autoradiography permitted thick section autoradiography, reducing the time of emulsion exposure from 54 days (thin section) to 12 days (0.5 mum sections).
Topics: Animals; Aphids; Autoradiography; Cholesterol; Digitonin; Microscopy, Electron; Rickettsiaceae
PubMed: 401684
DOI: 10.1007/BF00229461 -
Cellular and Molecular Neurobiology Dec 20041. Stellation of astrocytes in culture involves a complex rearrangement of microfilaments, intermediate filaments, and microtubules, which reflects in part the...
1. Stellation of astrocytes in culture involves a complex rearrangement of microfilaments, intermediate filaments, and microtubules, which reflects in part the plasticity of these cells observed during development or after injury. 2. An astrocytic calcium-binding protein, S100B, has been implicated in the regulation of plasticity due to its ability to interact with cytoskeletal proteins. 3. We used digitonin-permeabilized astrocytes to introduce TRTK-12, a peptide that binds to the C-terminal of S100B and blocks its interaction with cytoskeletal proteins. 4. TRTK-12 was able to block cAMP-induced astrocyte stellation and this effect was dependent on the concentration of the peptide. These results support the idea that S100B has a modulatory role on astrocyte morphology.
Topics: Animals; Astrocytes; CapZ Actin Capping Protein; Cells, Cultured; Cyclic AMP; Cytoskeleton; Digitonin; Dose-Response Relationship, Drug; Nerve Growth Factors; Oligopeptides; Peptide Fragments; Permeability; Rats; Rats, Wistar; S100 Calcium Binding Protein beta Subunit; S100 Proteins
PubMed: 15672683
DOI: 10.1007/s10571-004-6922-y -
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