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D Georgescauld

Publications and source records attributed to D Georgescauld.

At least 19 recordsLinked to original sources
PubMed1995

Spatial and temporal distribution of [Ca2+]i in normal human myotubes. A fura-2 imaging study.

The spatio-temporal distribution of intracellular, free calcium ions, [Ca2+]i, induced in human myotubes by electrical stimulation typically showed a relatively large increase of [Ca2+]i in the vicinity of the plasmalemma. The similarity of this distribution, with that observed after the application of caffeine, and the lack of any effect of lanthanum, strongly suggest that the main source of Ca2+ participating in the electrically induced transient is the sarcoplasmic reticulum. Aneurally cultured human myotubes therefore display a 'skeletal muscle type' coupling between membrane depolarization and calcium release. However, the relatively slow time course of the electrically induced transients compared to rat and mouse myotubes, together with the inability of Ca2+ released from the sarcoplasmic reticulum to activate the contractile machinery, implies that aneurally cultured human myotubes achieve only a limited degree of differentiation. The relevance this may have to an apparent delay between the electrically induced rise in intranuclear Ca2+ relative to cytosolic Ca2+ remains to be determined but, at this stage of differentiation, there appears to be some form of barrier to free diffusion between the two cellular compartments.

Caffeine↗
PubMed1992

Specific interactions of mercury chloride with membranes and other ligands as revealed by mercury-NMR.

High resolution mercury nuclear magnetic resonance (199Hg-NMR) experiments have been performed in order to monitor mercury chemical speciation when HgCl2 is added to water solutions and follow mercury binding properties towards biomembranes or other ligands. Variations of 199Hg chemical shifts by several hundred ppm depending upon pH and/or pCl changes or upon ligand or membrane addition afforded to determine the thermodynamic parameters which describe the equilibria between the various species in solution. By comparison to an external reference, the decrease in concentration of mercury species in solution allowed to estimate the amount as well as the thermodynamic parameters of unlabile mercury-ligand or mercury-membrane complexes. Hence, some buffer molecules can be classified in a scale of increasing complexing power towards Hg(II): EGTA greater than Tris greater than HEPES. In contrast, MOPS, Borax, phosphates and acetates show little complexation properties for mercury, in our experimental conditions. Evidence for complexation with phosphatidylethanolamine (PE), phosphatidylserine (PS) and human erythrocyte membranes has been found. Hg(II) does not form complexes with egg phosphatidylcholine membranes. Interaction with PE and PS model membranes can be described by the presence of two mercury sites, one labile, the other unlabile, in the NMR time scale. In the labile site Hg(PE) and Hg(PS)2 would be formed whereas in the unlabile site Hg(II) would establish bridges between three PE or PS molecules. Calculated thermodynamic data clearly indicate that PE is a better complexing agent than PS. Evidence is also found that complexation with lipids uses at first the HgCl2 species. Interestingly, mercury complexation with ligands or membranes can be completely reversed by addition of decimolar NaCl solutions. Minute mechanisms for mercury complexation with the primary amine of PE or PS membrane head groups are discussed.

Buffers↗
PubMed1991

Free calcium and calpain I activity.

Activation of purified calpain I proceeds through a Ca(2+)-induced autolysis from the 80 kDa catalytic subunit to a 76 kDa form via an intermediate 78 kDa form, and from a 30 kDa form to a 18 kDa form as the result of two autocatalytic processes (intra and intermolecular). The minimum Ca2+ requirements for autolysis and proteolysis have been determined by physico-chemical and electrophoretic methods in the presence or absence of a digestible substrate. According to our results the activation process needs less free Ca2+ than the proteolysis of a digestible substrate, which means that proteolysis is really subsequent to activation. For very low Ca2+ levels, a digestible substrate does not initiate the calpain I activation process. In the presence of phospholipid vesicles, such as PI, PS or a mixture of PI (20%), PS (20%) and PC (60%), the apparent kinetic constants of activation are greatly increased without any change in the initial velocity of the substrate proteolysis. Thus, enzyme activation and substrate proteolysis are observed as independent phenomena. These results obtained from experiments using low free Ca2+ concentrations enable us to propose a hypothesis for the mechanism of regulation by which the enzyme could be activated in the living cell.

Autolysis↗
PubMed1991

Membrane fusion without cytoplasmic fusion (hemi-fusion) in erythrocytes that are subjected to electrical breakdown.

There are many reports of hemi-fusion in phospholipid vesicles but few published studies on hemi-fusion in cells. We report evidence from both fluorescence microscopy and freeze-fracture electron microscopy for hemi-fusion in the electrofusion of human erythrocytes. We have also characterised the conditions that favour hemi-fusion as opposed to complete fusion, and discuss the possibility that hemi-fusion might precede complete electrically-induced cell fusion. A membrane probe (DiIC16) and a cytoplasmic probe (6-carboxyfluorescein) were used to investigate the behaviour of doubly-labelled human erythrocytes which were aligned in chains by dielectrophoresis and then exposed to high voltage breakdown pulses. Some of the cells were fused by the pulses, as shown by diffusion of both membrane and cytoplasmic probes from labelled to unlabelled cells. With other cells, the membrane probe diffused into unlabelled cells after the breakdown pulses, without the cytoplasmic probe diffusing into unlabelled cells or leaking into the medium. Membrane fusion (hemi-fusion) thus occurred without cytoplasmic fusion in these erythrocytes. Such cells were irreversibly, but fragilely, attached to their neighbours by the breakdown pulses. There was an inverse relationship between conditions that permit complete fusion and those that favour hemi-fusion, with respect to breakdown pulse length, breakdown voltage and, in particular, osmolarity and temperature. The incidence of hemi-fusion in 250 mM erythritol was twice that in 150 mM erythritol, and hemi-fusion was 5-fold greater at 25 degrees C than at 20 degrees C. Hemi-fused erythrocytes occasionally fused completely on heating to 50 degrees C, demonstrating that hemi-fusion can proceed to complete cell fusion. Freeze fracture electron micrographs of preparations of hemi-fused cells revealed long-lived, complementary depressions and protrusions on the E- and P-fracture faces, respectively, of tightly apposed cells that may mediate hemi-fusion. The possibility that the fusion of closely adjacent human erythrocytes by electrical breakdown pulses may involve an intermediate, shared bilayer structure, which is stable in certain conditions but which can be ruptured by osmotic swelling of the permeabilised cells, is discussed.

Cytoplasm↗
PubMed1991

Fura-2 imaging of spontaneous and electrically induced oscillations of intracellular free Ca2+ in rat myotubes.

Rat myotubes have a resting [Ca2+]i of about 82 nM. Myotubes 3-5 days old (quiescent myotubes) display electrically induced and spontaneous transients in the intracellular concentration of free Ca2+ ions ([Ca2+]i) uncoupled to any detectable contraction. By contrast, 1- to 2-day-old myotubes are insensitive to electrical stimuli and, after 6 days in culture, stimulated myotubes always show [Ca2+]i transients and twitch contractions. The spatial distribution of [Ca2+]i variations in quiescent myotubes is heterogeneous, local increases in [Ca2+]i being mainly observed near the periphery of the cell. The small effect of different external Ca2+ concentrations and of Cd2+ on the amplitude of the [Ca2+]i oscillation indicates that the main source of Ca2+ may be the sarcoplasmic reticulum. This conclusion is supported by the close similarity between electrically induced and caffeine-induced [Ca2+]i maps. These findings suggest that, at an early stage of myotube ontogenesis, a part of the excitation/contraction coupling, as membrane ionic channels, voltage sensors and Ca2+ release and reuptake mechanisms, is functional but, apparently, still uncoupled to the contractile machinery.

Animals↗
PubMed1991

Digital-imaging microscopy analysis of calcium release from sarcoplasmic reticulum in single rat cardiac myocytes.

Digital imaging microscopy of fura-2 fluorescence has allowed us to assess the dynamic patterns of local Ca increase in newly isolated rat myocardial cells. Of the myocytes bathed in a saline solution (1.8 mM Ca2+, 37 degrees C, pH 7.4), 10%-20% exhibited local spontaneous contractions. The resting intracellular free calcium concentration ([Ca2+]i) of these cells was 106 +/- 4 nM versus 77 +/- 3 nM for non-contracting cells. The spontaneous contractile activity appeared to be closely related to internal spontaneous Ca waves that spread across the myoplasm (velocity approximately 50 microns/s, maximal Ca amplitude = 195 +/- 11 nM) along the major axis of the cells. Precise topographical examination of Ca wave propagation indicated a refractory period for internal Ca release. The occurrence of both the generation and propagation of spontaneous Ca increases appeared to be closely dependent on the extent of Ca loading of the cells. Most of our observations were in accordance with the assumption that local Ca overload of the sarcoplasmic reticulum (SR) is the main parameter involved in the spontaneous Ca-release phenomena. Using the same approach, the increase in internal Ca evoked by KCl (50 mM) addition was investigated, and compared with that seen during spontaneous activity. Total [Ca2+]i increase induced by K+ depolarization involved three consecutive local Ca-release patterns: (a) a peripheral Ca enhancement that remained during the total [Ca2+]i increase, (b) subsequent transversal local Ca increases occurring in Z-line regions, (c) longitudinal local Ca increases. In addition, a weak heterogeneous Ca distribution was detected in both peripheral and central parts of resting cardiac cells. Thus, the total Ca increase seemed to result consecutively from a peripheral Ca pool, from junctional SR and from longitudinal structures (possibly longitudinal SR).

Animals↗
PubMed1988

[Is muscular acetylcholinesterase activity correlated with the intracellular concentration of free calcium?].

The role of the calcium ion Ca2+ as an agent of intracellular control in a variety of physiological processes is well established. In vertebrate skeletal muscle fibers, Ca2+ is involved in muscle contraction, modulation of membrane permeability and regulation of metabolic activity. Recently it was suggested that ion fluxes through membranes regulate the level of two cholinergic macromolecules, the acetylcholine receptor and the A12 form of acetylcholinesterase (AChE), the presumed synaptic form of the enzyme. Muscle cells paralysed by veratridine, which maintains the Na+ channel in the open state, showed an increase in total AChE and in the levels of the A12 form. The effect of veratridine on AChE was blocked in the presence of agents that block Ca2+ permeability suggesting that Ca2+ is involved in this effect. To understand whether the level of muscle AChE is related in some way to the level of free intracellular Ca2+, we analysed the variations of Ca2+ levels in rat muscle cells treated by agents which modify the ionic permeabilities. This level was determined by spectrofluorimetry using the fluorescent Ca2+ indicator: Quin 2. However no correlation between these parameters was observed in our experimental conditions.

Acetylcholinesterase↗
PubMed1987

Movements of fluorescent probes in the mechanism of cell fusion induced by poly(ethylene glycol).

It has been claimed that purified poly(ethylene glycol) (PEG) is able only to aggregate cells and not to fuse them. In our hands, purified PEG 6000 (recrystallized/dialysed) induces both aggregation and fusion of human erythrocytes, and the mechanism of fusion by the purified polymer has been investigated with fluorescent probes. No movement of a carbocyanine probe or of octadecyl rhodamine B chloride from labelled to unlabelled cells occurred in the absence of PEG or with cells treated with concanavalin A, protamine or spermine. With 40% PEG, however, both probes immediately started to diffuse into the membranes of unlabelled cells. This indicates that continuity between the phospholipid bilayer membranes of adjacent erythrocytes (i.e. membrane fusion) is established within seconds in concentrated solutions of the polymer, and precedes the cell fusion event that is induced by purified PEG. These observations are consistent with the idea that micro-regions of shared phospholipid bilayer may be formed in the membranes of cells when they are forced together as a consequence of the dehydrating action of PEG. Intact erythrocytes were cytoplasmically labelled with 6-carboxyfluorescein to avoid the possibility that loading the cells with a cytoplasmic marker by hypotonic haemolysis might modify their response to PEG. Unlike the lipid probes, carboxyfluorescein did not diffuse from labelled to unlabelled cells in the presence of 40% PEG, and there was little diffusion on subsequent dilution of the polymer solution to 13%. However, after the PEG solution had been replaced by an isotonic buffer, a rapid transfer of the cytoplasmic fluorophore to unlabelled cells often occurred. This is considered to be more consistent with the osmotic rupture of a membranous barrier, such as a shared bilayer, between the labelled and unlabelled cells than with the return of cytoplasmic viscosity to normal when the PEG is removed. Possible reasons are discussed for the reported inability of purified PEG to fuse fibroblasts with hypotonically loaded human erythrocytes.

Cell Fusion↗
PubMed1986

Effect of the action potential on the Raman spectrum of the pike olfactory nerve.

Raman bands due to the C-H stretching vibrations of the phospholipid acyl chains, as well as those due to resonance enhanced vibrations of carotenoid pigments, were used to probe for conformational changes during the passage of the action potential through fibers of the pike unmyelinated olfactory nerve. Our results show that if there are any spectral changes during nerve excitation, these are less than 0.5% for both the phospholipid and the carotenoid bands.

Action Potentials↗
PubMed1985

Raman spectroscopy of nerve fibers. A study of membrane lipids under steady state conditions.

The molecular structures of different nerve fibers kept in good physiological conditions were studied by laser Raman spectroscopy. For myelinated nerves like the rat sciatic nerve, the Raman spectrum is dominated by bands due to the lipid component of the myelin sheath. The temperature dependence of these bands does not reveal any thermotropic phase transition between 0 and 40 degrees C. There is, however, with temperature, a linear increase in the intermolecular disorder that is accompanied by an increase in the number of gauche bonds of the phospholipid acyl chains. For unmyelinated nerves such as the lobster leg nerve, the C-H stretching region of the Raman spectrum is covered by bands arising from the protein component of the axoplasm. However, for the garfish olfactory nerve that has a high density of excitable membranes, phospholipid bands are observed and can be used as intrinsic structural probes of the excitable membranes. The relative intensity of these bands is also temperature dependent.

Animals↗
PubMed1984

Transport of mercury compounds across bimolecular lipid membranes: effect of lipid composition, pH and chloride concentration.

The use of bimolecular lipid membranes (BLM) as model membrane allows the analysis of the transport of mercury compounds across the lipidic barriers of biological membranes. The results of flux measurements show that two mercury compounds--HgCl2 and CH3HgCl--cross the BLM but the overall permeabilities are dependent on the pH of the aqueous media, and are not apparently influenced by the different phospholipid constituents of the bilayers. On the other hand, electrical measurements show that, function of the chemical speciation, the transport of this metal is done essentially in the neutral form.

Biological Transport↗
PubMed1983

[Relation between the axonal membrane microfluidity and excitability].

The olfactory nerve of the garfish, the rabbit vagus nerve and the sciatic nerve of the frog labelled with pyrene or triethylammonium-butyl-pyrene show during the action potential a transient decrease in the Ie/Im ratio which suggest a small transient decrease in nerve membrane fluidity.

Animals↗
PubMed1982

The relationship between the membrane potential of neurosecretory nerve endings, as measured by a voltage-sensitive dye, and the release of neurohypophysial hormones.

The membrane potential of isolated rat neurohypophyses and isolated neurosecretosomes (neurosecretory nerve endings) was monitored with the voltage sensitive fluorescent probe diS-C3-(5). K ions, in contrast to Na or Cl ions, give rise to large changes of the fluorescent signal. The fluorescent response is linearly related to log[K+]0 at values higher than 10 mM, whereas at lower [K+]0 the permeability of the membrane for Na ions has to be taken into account. Veratridine increases the fluorescent signal only in the presence of external sodium; this effect is blocked by tetrodotoxin. After prolonged K-induced depolarisation, addition of veratridine to the medium gives a further change in fluorescence of diS-C3-(5) associated with release of vasopressin. Vasopressin release from isolated neurohypophyses started to increase significantly only above 25 mk [K+]0, while the depolarization of the membrane was linearly related to log[K+]0. The results are consistent with the view that neurosecretory nerve endings have voltage-dependent calcium channels that regulate the amount of hormone released during depolarisation.

Animals↗
PubMed1979

Temperature dependence of the fluorescence of pyrene labeled crab nerve membranes.

A method, using albumin-pyrene complexes, has been developed for labeling, in a controlled manner, crab leg nerves whose excitability was preserved. The excimer-to-monomer fluorescence intensity ratio of pyrene, embedded in nerve membrane lipids and in their crude lipid extracts, is a fluidity parameter which displayed the following features with temperatures. a--a temperature-dependent increase of fluidity b--three breaks (6 degrees, 19 degrees and 37 degrees C) in the physiological medium c--In Ca++-depleted sea water, the 37 degrees characteristic temperature vanished. These breaks may reflect some lateral phase separations of the lipid components of nerve membranes. The calcium dependent temperature break may involve a segregation of acidic phospholipids while the other two breaks (6 degrees and 19 degrees C) may be due to neutral lipids phase separation. The relationship of these findings to nerve function is discussed.

Animals↗
PubMed1979

A spin label study of myelin fluidity with normal and pathological peripheral nerves.

The myelin fluidity in peripheral nerves from normal subjects and patients with neuropathy has been studied. Nerve biopsies were made in subjects without neuropathy and in patients with Guillain-Barré, alcoholic or diabetic polyneuropathies and heredodegenerative diseases. The fluidity was studied using a stearic acid spin label with a nitroxide radical at C12 (12-doxylstearic acid) which was introduced by exchange between spin label-charged BSA and nerve myelin. Spectra was obtained with a VARIAN E 109 ESR spectrometer, at temperatures from 5 to 45 degrees C. The ultrastructure of biopsied nerves was also studied. The electron spin resonance (ESR) spectra were all typical of a partially immobilized nitroxide with a distance between outer hyperfine lines (2T//) of about 50 G, at room temperature. The curve 2T// against temperature shows two critical temperatures: 10 and 30 degrees C for all nerves. No correlation could be established between ultrastructure and fluidity of nerves. However, the myelin of nerves from diabetic patients showed a significant increase in viscosity. This physicochemical modification may represent an initial alteration of the myelin sheath, and demands further investigation. No modification of the myelin fluidity was observed in any of the other nerves studied.

Adolescent↗