[Technic and results of radioguided transcutaneous puncture-biopsy of the dorsal spine].
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Biopsy, Needle↗
Biomedical subjects
G Leblanc
Publications and source records attributed to G Leblanc.
Relationships between the Na+-H+ antiport activity and the components of the electrochemical proton gradient in Escherichia coli membrane vesicles.
The kinetics of Na+ efflux from Escherichia coli RA 11 membrane vesicles taking place along a favorable Na+ concentration gradient are strongly dependent on the generation of an electrochemical proton gradient. An energy-dependent acceleration of the Na+ efflux rate is observed at all external pHs between 5.5 and 7.5 and is prevented by uncoupling agents. The contributions of the electrical potential (delta psi) and chemical potential (delta pH) of H+ to the mechanism of Na+ efflux acceleration have been studied by determining the effects of (a) selective dissipation of delta psi and delta pH in respiring membrane vesicles with valinomycin or nigericin and (b) imposition of outwardly directed K+ diffusion gradients (imposed delta psi, interior negative) or acetate diffusion gradients (imposed delta pH, interior alkaline). The data indicate that, at pH 6.6 and 7.5, delta pH and delta psi individually and concurrently accelerate the downhill Na+ efflux rate. At pH 5.5, the Na+ efflux rate is enhanced by delta pH only when the imposed delta pH exceeds a threshold delta pH value; moreover, an imposed delta psi which per se does not enhance the Na+ efflux rate does contribute to the acceleration of Na+ efflux when imposed simultaneously with a delta pH higher than the threshold delta pH value. The results strongly suggest that the Na+-H+ antiport mechanism catalyzes the downhill Na+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)
Carrier Proteins↗
Use of the pH sensitive fluorescence probe pyranine to monitor internal pH changes in Escherichia coli membrane vesicles.
Measurements of the fluorescent properties of 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine) enclosed within the internal space of Escherichia coli membrane vesicles enable recordings and quantitative analysis of: (i) changes in intravesicular pH taking place during oxidation of electron donors by the membrane respiratory chain; (ii) transient alkalization of the internal aqueous space resulting from the creation of outwardly directed acetate diffusion gradients across the vesicular membrane. Quantitation of the fluorescence variations recorded during the creation of transmembrane acetate gradients shows a close correspondence between the measured shifts in internal pH value and those expected from the amplitude of the imposed acetate gradients.
Acetates↗
Characterization of antisera to a partially purified prolactin receptor: effect on prolactin binding in different target tissues.
Prolactin (PRL) receptors have been purified by affinity chromatography using a lactogenic hormone (human growth hormone, hGH) coupled to Affigel-10. The mean binding capacity of 3 separate purifications was 1.18 +/- 0.26 nmoles prolactin per mg protein, representing a 836-fold purification over crude microsomes and 4000-5000-fold over mammary gland homogenates and 16% purity. The receptor was characterized by HPLC using a TSK-SW-4000 and 3000 column connected in series and eluted in 0.1 M borate buffer containing 0.2 M NaCl and 0.1% Triton. A single peak of [125I]hGH binding activity with a retention time of 45.2 min (22.6 ml), representing an apparent molecular weight of 133000, was observed. A single peak of activity was also observed following polyacrylamide gel electrophoresis of the purified receptor in 0.1% Triton coincident with the Coomassie-stained protein band. Antibodies to the partially purified receptor preparations were prepared in sheep, goats and guinea pigs. Antisera to the prolactin receptor prepared in all three species were capable of inhibiting the binding of 125I-labeled ovine prolactin to receptors from rabbit mammary glands. Significant inhibition of binding was observed at antisera dilution of 1:10 000 with the sheep antiserum being the most potent (half-maximal inhibition (IM50) = 1:5700). All three antisera were able to inhibit PRL binding completely, but failed to affect labeled insulin or hCG binding and had very little effect on bGH binding. The specificity of the sheep antiserum was tested in rabbit mammary glands, ovary, adrenal, pig mammary glands and 8 rat tissues which contain PRL receptors. The antiserum was able to inhibit the binding of labeled PRL in all tissues, with the inhibition curves for the rat tissues being non-parallel when compared to rabbit mammary gland, suggesting a homology but not a complete identity between PRl receptors in various tissues and animal species. These studies demonstrate that prolactin receptors can be purified from rabbit mammary tissue and that antisera can be produced in several species. In addition, the binding studies suggest that in the various tissues the receptor molecule is more or less exposed to interaction with the antisera, or that the receptor protein differs somewhat between species.
Animals↗
The mechanism of Na+-L-lactate cotransport by brush-border membrane vesicles from horse kidney. Analysis by isotopic exchange kinetics of a sequential model and stoichiometry.
The present study determines the characteristics of isotopic Na and lactate exchange under equilibrium conditions in horse kidney brush-border membrane vesicles. The influence of one solute (Na+ or lactate) on the isotopic exchange of the co-transported species (lactate or Na) was analyzed in detail. Analysis of the data suggests that Na and lactate interact sequentially with the carrier. The observed apparent symmetry between the activating effect of low Na concentrations and the inhibiting effect of high Na concentrations on the lactate exchange process suggests that the carrier functions according to a glide symmetry model where the two solutes bind to the carrier according to an iso-ordered Bi Bi process. This conclusion is strengthened by the properties of the 22Na exchange process catalyzed by the lactate carrier. Direct measurement under equilibrium conditions of the coupled Na+/lactate transport indicated a stoichiometry of 2:1. These data, along with those obtained in a previous study (Mengual R., and Sudaka P. (1983) J. Membr. Biol. 71, 163-171) allow us to propose a mechanism for the Na+-lactate cotransport, involving a transport system that functions according to an iso-ordered Bi Bi process where binding and debinding of Na+ occur first, followed by the lactate molecule.
Animals↗
Preferential establishment of a slowly dissociable component in plasma membrane compared to intracellular prolactin receptors.
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Animals↗
Cation transport mechanisms in Mycoplasma mycoides var. Capri cells. Na+-dependent K+ accumulation.
We have studied some features of K+ accumulation by glycolysing Mycoplasma mycoides var. Capri cells. We report that when Na+ is absent from the external medium, K+ accumulates up to the level predicted by the amplitude of the transmembrane electrical potential, delta psi m, measured by Rb+ and methyltriphenylphosphonium cation (TPMP+) distribution. Therefore, under these experimental conditions, the coupling mechanism of K+ uptake consists of a delta psi m-driven uniport. More important, when Na+ is present in the external medium, the level of K+ accumulation by glycolysing Mycoplasma cells is far too steep to be equilibrium with delta psi m (-120 mV for delta muK+ compared with -90mV for delta muRb+ or delta muTPMP+). Our results clearly indicate the presence in Mycoplasma of an active K+-transport system specifically stimulated by Na+. Furthermore, by controlling the amplitude of the energy-dependent delta muH+, we obtain strong evidence that this specific Na+-stimulated K+ transport is modulated by the transmembrane electrical potential. Finally, we show that ATP is consumed when such a transport system is in activity.
Adenosine Triphosphate↗
Cation transport mechanisms in Mycoplasma mycoides var. Capri cells. The nature of the link between K+ and Na+ transport.
We have studied the links between the mechanisms of Na(+), K(+) and H(+) movements in glycolysing Mycoplasma mycoides var. Capri cells. In the light of the results reported in the preceding paper [Benyoucef, Rigaud & Leblanc (1982) Biochem. J.208, 529-538], we investigated certain properties of the membrane-bound ATPase of Mycoplasma cells, with special reference to its ionic requirements and sensitivity to specific inhibitors. Our findings show, first, that, although Na(+) stimulated ATPase activity, K(+) did not affect it, and, secondly, that NN'-dicyclocarboidi-imide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) were potent inhibitors of the basal ATPase activity, which was unaffected by vanadate and ouabain. We also investigated the movements of Na(+) and H(+) under the experimental conditions applied to the study of the K(+) uptake reported in the preceding paper, and found that when ;Na(+)-loaded cells' previously equilibrated with (22)Na(+) were diluted in a sodium-free medium, addition of glucose induced a rapid efflux of (22)Na(+). This energy-dependent efflux was independent of the presence of KCl in the medium. Studies of the changes in internal pH by 9-aminoacridine fluorescence or [(14)C]methylamine distribution indicated that the movement of Na(+) was coupled to that of protons moving in the opposite direction, a finding that supports the presence of an Na(+)/H(+) antiport. When Na(+)-loaded cells are diluted in an Na(+)-rich medium the Na(+)/H(+) antiport is still active, but cannot decrease the intracellular Na(+) concentration. Under such conditions, net (22)Na(+) extrusion is specifically dependent on the presence of K(+) in the medium. The present results and those derived from the study of K(+) accumulation (the preceding paper) can be rationalized by assuming that Mycoplasma mycoides var. Capri cells contain two transport systems for Na(+) extrusion: an Na(+)/H(+) antiport and an ATP-consuming Na(+)/K(+)-exchange system.
Adenosine Triphosphatases↗
The electrochemical proton gradient in Mycoplasma cells.
The electrochemical proton gradient, delta mu H+ generated upon glycolysis by Mycoplasma mycoides var. Capri cells has been determined. The components, the transmembrane pH gradient, delta pH, and the membrane potential, delta psi, were measured using several methods. The determination of the delta pH was conducted by measuring the transmembrane distribution of weak acids (acetate and butyrate) and of a weak base (methylamine), using flow dialysis and filtration techniques. The transmembrane electrical potential was determined from the distribution of the lipophilic cation Ph3MeP+ and of Rb+ or K+ in the presence of valinomycin. At extra-cellular pH 7.2, glycolyzing Mycoplasma cells maintain an internal pH more alkaline (0.5 pH unit) than that of the milieu and an electrical potential of - 85 mV, interior negative. The delta mu H+ in M. mycoides var. Capri cells is thus about - 115 mV. When the external pH was altered from 7.7 to 5.7 delta psi decreased from - 90 mV to - 60 mV. On other hand although the internal pH decreased, delta pH was found to increase from 0.2 to 1.0 pH unit. Since the changes in delta psi were largely compensated by the changes in delta pH, delta mu H+ remained practically constant at about - 115 mV throughout the pH range tested. Finally, inhibition of delta pH by N,N'-dicyclohexylcarbodiimide, carbonylcyanide-p-trifluoromethoxyphenylhydrazone or nigericin confirmed that chemiosmotic phenomena contribute to energy transduction across the membranes of M. mycoides var. Capri cells.
Biological Transport, Active↗
Gradation of the magnitude of the electrochemical proton gradient in Mycoplasma cells.
The results presented show that in Mycoplasma mycoides var. Capri, regulation of glucose uptake by its non-metabolizable analogue methyl alpha-D-glucoside, can be used to control intracellular ATP content. This in turn leads to a control of the rate of proton extrusion catalysed by the Mg2+-dependent ATPase (phi (cHxN)2C H+) and the respective amplitudes of the components of delta mu H+. When Mycoplasma cells are incubated with 10 mM methyl alpha-D-glucoside, the amplitude of phi (cHxN)2C H+, of the electrical potential delta psi and of the chemical gradient delta pH become continuous functions of external glucose concentration within the limits of the non-energized and fully energized states. Analysis of the relationships between graduated amplitudes of delta psi, delta pH and phi (cHxN) 2C H+ show that the primary form of energy stored by a delta mu H+ generator is the electrical potential.
Adenosine Triphosphate↗
Double-blind study of glaucine in chronic cough.
Thirty-eight patients, affected by chronic cough and hospitalized in 14 different rooms, entered a double-blind cross-over trial aimed at evaluating efficacy and tolerance of single oral doses of glaucine (eo mg) versus single oral doses of codeine (30 mg) and placebo. Patients occupying the same room were administered on 3 consecutive nights, and an objective evaluation of efficacy was ensured by means of a tape recorded. The mean cough counts during the 8-h interval after drug administration were 269.3 after placebo, 241.8 after glaucine, and 201.9 after codeine (p less than 0.05). The antitussive effects of glaucine and codeine were practically superimposable up to the 6th h, when glaucine effect declined. Treatments were well tolerated by all the patients, they themselves were not able to detect any difference in cough suppression among the three treatments.
Antitussive Agents↗
[Studies of immunological status, following autologous bone marrow transplantation in man (author's transl)].
Following transplant, circulating immunoglobulin levels fell moderately and remained depressed less than 2 months for IgG, and for variable and longer periods of time for IgM and IgA. Repeated quantitative determinations of antibodies against multiple antigens did not show any decrease in the pretransplant levels. Indeed some patients developed herpes and cytomegalovirus infections to which they responded by a sharp increase in antibody titers. In 2 cases, a primary immunization was demonstrated (against CMV and BK virus) with increasing levels of IgM and IgG antibodies. Lymphocyte counts in peripheral blood returned to 500 mm# between day 10 and 29 (median day 18) and to pretransplant values within 6 weeks. Non specific stimulation of lymphocytes by mitogens in the immediate post-transplant period showed a decreased response to PHA and Con A, whereas the responses to pokeweek mitogens and alloantigens were only slightly diminished. The degree of the responses was related to the dose of cryopreserved marrow infused. We conclude that:--although the minimum dose for autologous bone marrow transplantation in man is around 0,5 10(8) nucleated bone marrow cells/Kg, much higher doses should be used to ensure faster and better restoration of immune reactivity.--The similarity of the immunological dysfunction following autologous and allogeneous bone marrow transplantation suggest that, in the immediate post-transplant period, the role of GVHD in cellular immunity depression may be minimal.
Bone Marrow Transplantation↗
Temperature-dependent relationship between K+ influx, Mg2+-ATPase activity, transmembrane potential and membrane lipid composition in mycoplasma.
The temperature-dependent relationship between K+ active influx, Mg2+-ATPase activity, transmembrane potential (delta psi) and the membrane lipid composition has been investigated in mycoplasma PG3. Native organisms were grown in a medium containing 10 microgram/ml cholesterol and either oleic plus palmitic (chol (+), O + P) or elaidic (chol (+), E) acids. Adapted cells were grown in a medium free of exogenous cholesterol and supplemented with elaidic acid (chol (-), E). Arrhenius plots of 42K+ active influx gave a linear relationship for (chol (+), O + P) cells (EA = -9 kcal). On the other hand, when oleic plus palmitic acids are replaced by elaidic acid, an upward discontinuity appears between 28 and 30 degrees C, which is associated with a large increase in the apparent activation energy of the process (t > 30 degrees C, EA = -24 kcal; t < 30 degrees C, EA = -40 kcal). Finally, a biphasic response with a break at approx. 23 degrees C (EA = -7 kcal, t > 23 degrees C; EA = -44 kcal, t < 23 degrees C) is observed for (chol(-), E) organisms. From the lack of correspondence between these effects on the K+ influx and the temperature dependence of both the Mg2+-ATPase activity and delta psi, it is suggested that changes in the membrane lipid composition affect the K+ transport at the level of the K+ carrier itself. Differential scanning calorimetry, steady-state fluorescence polarization of diphenylhexatriene and freeze-fracture electron microscopy experiments further suggest that the effect is largely due to modifications of the membrane microviscosity and that the K+ carrier is associated with the most fluid lipid species present in the membrane.
Adenosine Triphosphatases↗
Effect of membrane cholesterol on action of phospholipase A2 in Mycoplasma mycoides var. Capri. Evidence for lysophospholipase activity.
Cells of Mycoplasma mycoides var. Capri grown in a medium containing 10 micrograms/ml cholesterol (native organisms) or in cholesterol-free medium (adapted organisms) were treated with phospholipase A2. Hydrolysis of polar lipids (phosphatidylglycerol and diphosphatidylglycerol) only occurred in the adapted cells. Cholesterol replenishment of the membranes of these adapted cells in vitro which involves an increase from 7 micrograms to 66 micrograms cholesterol/mg membrane protein, completely abolished hydrolysis of polar lipid pools by phospholipase A2. This suggests that cholesterol incorporated either during growth or under conditions in vitro has an identical disposition and function in the membrane. This observation further indicates that cholesterol incorporation in M. mycoides var. Capri can be explained in terms of a simple physical adsorption process. Polar-lipid breakdown products resulting from phospholipase A2 action on intact cells, isolated membranes and lipids extracted from adapted organisms were analyzed. In experiments with intact cells [14C]oleic lysoderivatives but not [3H]palmitic lysoderivatives are accumulated within the membranes. In membrane preparations, again only [14C]oleic lysoderivatives are accumulated but transiently. Finally, both [14C]oleic and [3H]palmitic lysoderivatives were produced in phosphatidylglycerol-diphosphatidylglycerol liposomal preparations. From these results it can be concluded that: (a) 80% of the phosphatidylglycerol and diphosphatidylglycerol have an unusual positional distribution of their fatty acid (unsaturated oleic acid in position 1) and (b) membranes of M. mycoides var. Capri contain an active lysophospholipase which more efficiently hydrolyzes palmitic-acid-containing lysoderivatives.
Cardiolipins↗
Active K+ transport in Mycoplasma mycoides var. Capri. Net and unidirectional K+ movements.
Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K+ concentration (Ki: 200--300 mM) which greatly exceeds that of the growth medium, and a low Na+ concentration (Na+i: 20 mM). Unlike Na+i,K+i varies with cell aging. The K+ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K+ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, K+i decreases and is partially compensated by a gain in Na+. This substitution completely reverses when metabolic substrate is added (K+ reaccumulation process). Kinetic analysis of K+ movement in cells with steady K+ level shows that most of K+ influx is mediated by an autologous K+-K+ exchange mechanism. On the other hand, during K+ reaccumulation by K+-depleted cells, a different mechanism (a K+ uptake mechanism) with higher transport capacity and affinity drives the net K+ influx. Both mechanisms are energy-dependent. Ouabain and anoxia have no effect on K+ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg2+ -dependent ATPase activity.
Biological Transport, Active↗
Active K+ transport in Mycoplasms mycoides var. Capri. Relationships between K+ distribution, electrical potential and ATPase activity.
The addition of 5 . 10(-5) M or less of dicyclohexylcarbodiimide to Mycoplasma mycoides var. Capri preferentially influences K+ influx rather than efflux and reduces by 30--40% the activity of the membrane-bound Mg2+- ATPase. Adding valinomycin to metabolizing cells does not markedly affect K+ distribution but induces a rapid and complete loss of intracellular K+ in non-metabolizing cells. Uncoupling agents such as dinitrophenol, carbonylcyanide p-trifluoromethoxyphenylhydrazone, dissipate the K+ concentration gradient only when combined with valinomycin. Variations in the merocyanine fluorescence intensity indicate that a transmembrane electrical potential (delta psi) is generated on cell energization. This delta psi, not affected by valinomycin or uncouplers when used alone, is collapsed by a mixture of both. No change in fluorescence intensity can be detected when glucose is added to dicyclohexylcarbodiimide treated organisms. These experiments suggest that the membrane-bound Mg-ATPase activity control K+ distribution in these organisms through the generation of a transmembrane electrical potential difference.
Adenosine Triphosphatases↗
Estimation of total prolactin-binding sites after in vitro desaturation.
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Animals↗
Effect of membrane cholesterol on potassium transport in Mycoplasma mycoides var. Capri (PG3).
Relationships between membrane lipid composition and physiological properties, particularly intracellular potassium levels, have been studied at 37 degrees C in Mycoplasma mycoides var. Capri (PG3). Native organisms grown on medium supplemented with either oleic acid plus palmitic acid or elaidic acid have identical growth characteristics, acidification properties and intracellular K content. On the other hand, when the cholesterol normally present in the membrane (20--25% of total lipids) is reduced to less than 2%, we observe: (1) the intracellular K content decreases (20 microgram K/mg cell protein instead of 40) and is independent of the phase of growth; (2) K passive permeability is drastically increased but K distribution remains in equilibrium with the transmembrane potential (delta psi); (3) organisms stop growing at pH 6.5 (instead of 5.2) and acidification is reduced by 40%, suggesting a large increase in H+ permeability, and (4) intracellular Na contents rise from 3 to 9 microgram Na/mg cell protein. Replenishing cholesterol in membranes of depleted cells results in a recovery of the high intracellular K level (35--40 microgram K/mg cell protein) and acidification properties. It is suggested that cholesterol affects the cation content via the increase in proton permeability which in turn controls the value of the delta psi responsible for the value of intracellular K equilibrium. Changes in K passive permeability, although related to the amount of cholesterol present in the plasma membrane, are probably not involved in the control of the intracellular K level.
Biological Transport↗