PubMed Health⌕ Search

Biomedical subjects

J Davoust

Publications and source records attributed to J Davoust.

69 records · Page 4Linked to original sources
PubMed1988

Rapid lateral diffusion of the variant surface glycoprotein in the coat of Trypanosoma brucei.

The membrane form of the variant surface glycoprotein (mfVSG) is anchored in the plasma membrane of Trypanosoma brucei by a dimyristoylphosphatidylinositol residue connected via a glycan to the COOH-terminal amino acid. The glycoprotein molecules are tightly packed, forming a coat that is impenetrable to lytic serum components. Lateral diffusion of mfVSG was measured by the fluorescence recovery after photobleaching technique. mfVSG labeled on the cell surface with rhodamine-conjugated anti-VSG Fab fragments showed a diffusion coefficient of 1 X 10(-10) cm2/s at 37 degrees C and of 0.7 X 10(-10) cm2/s at 27 degrees C. About 80% of the molecules were mobile. Affinity-purified mfVSG molecules implanted into the plasma membrane of baby hamster kidney cells exhibited a similar mobility to that found in the trypanosome coat [D = (0.4-0.7) X 10(-10) cm2/s at 4 degrees C]. Phospholipid mobility in the plasma membrane of trypanosomes was characterized by a diffusion coefficient of 2.2 X 10(-9) cm2/s at 37 degrees C. It is concluded that mfVSG mobility in the surface coat of the parasite is rapid and comparable to that of other membrane-bound glycoproteins but slower than that of phospholipids.

Animals↗
PubMed1987

Two threshold values of low pH block endocytosis at different stages.

The influence of low extracellular pH on endocytosis was studied in baby hamster kidney cells. When the extracellular medium was adjusted to pH 5.7, the intracellular pH decreased within 2 min to pH 6.2 and the endocytosis of horseradish peroxidase (HRP) in the fluid phase dropped to an undetectable level. With an external pH of 6.3, the internal pH dropped to pH 6.8 and HRP was internalized at a normal rate for 5 min but accumulation during longer incubation times did not occur. Morphologically, HRP was visualized in the lumen of a subpopulation of tubular and vesicular endosomes. These observations were confirmed by subcellular fractionation studies using free flow electrophoresis. Low extracellular pH also had an effect on the endocytosis of the membrane-spanning glycoprotein G of vesicular stomatitis virus which was implanted into the plasma membrane. The internalization of G-protein was quantitated by a surface fluoroimmunoassay. The endocytosis of G-protein was not affected when the external pH was dropped to 6.3, but was reduced at an external pH of 5.7. The intracellular ATP was not depleted and the reduction of endocytosis was reversible upon return to physiological pH. Clathrin coated pits were detected by electron microscopy at the plasma membrane of the low-pH-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1986

Asymmetric lateral mobility of phospholipids in the human erythrocyte membrane.

The fluorescent phospholipid 1-acyl-2-[12-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminododecanoyl]phosphatidylcholine (NBD-phosphatidylcholine) and the corresponding aminophospholipid derivatives (NBD-phosphatidylethanolamine and NBD-phosphatidylserine) were introduced in the human erythrocyte membrane by a nonspecific phospholipid exchange protein purified from corn. The lateral mobility of the fluorescent phospholipids was measured by using an extension of the classical photobleaching recovery technique that takes advantage of a modulated fringe pattern and provides a high sensitivity. In intact erythrocytes and in ghosts resealed in the presence of ATP, the fluorescence-contrast curves after photobleaching decayed biexponentially corresponding to two lateral diffusion constants. With NBD-phosphatidylcholine, the majority of the signal corresponded to a "slow" component (1.08 X 10(-9) cm2/sec at 20 degrees C), whereas with the amino derivatives the majority of the signal corresponded to a "fast" component (5.14 X 10(-9) cm2/sec at 20 degrees C). If the ghosts were resealed without ATP, the fast component of the aminophospholipids disappeared. We interpret these results as follows: (i) Provided the cells or the ghosts contain ATP, the three fluorescent phospholipids distribute spontaneously between inner and outer leaflets as endogenous phospholipids, namely NBD-phosphatidylcholine is located in the outer leaflet, while both aminophospholipids are preferentially located in the inner leaflet. (ii) The viscosity of the inner leaflet of human erythrocyte membranes is lower than that of the outer leaflet.

Adenosine Triphosphate↗
PubMed1985

Parameters affecting low-pH-mediated fusion of liposomes with the plasma membrane of cells infected with influenza virus.

Unilamellar liposomes can be fused at low pH with the plasma membrane of cells that express the hemagglutinin glycoprotein of influenza virus on their surface [van Meer, G., & Simons, K. (1983) J. Cell Biol. 97, 1365-1374]. Here, we have resolved this fusion process into two kinetically distinct steps. The first and more rapid step converts the bound liposome to a form that can no longer be released by neuraminidase. The second step is the actual membrane fusion as measured by the loss of resonance energy transfer between two liposomal fluorescent phospholipids, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dioleoylphosphatidylethanolami ne (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine (N-Rh-PE). In contrast to the first step, the rate of the second one was highly dependent on the liposomal lipid composition and the cell type used. The replacement of 50% of the phosphatidylcholine (PC) in egg PC-cholesterol liposomes by unsaturated phosphatidylethanolamine (PE) species increased the rate of fusion at least 2-fold. Of the PE-containing liposomes that were associated with Madin-Darby canine kidney (MDCK) cells after 30 s of fusion, 80% had actually fused with the plasma membrane. Fringe pattern fluorescence photobleaching experiments showed that after fusion a fraction of the cell-associated N-Rh-PE diffused laterally in the plasma membrane. Without fusion, the N-Rh-PE was completely immobile. Under optimal conditions, the mobile fractions were 65% on MDCK cells and 78% on baby hamster kidney cells. The mobility was acquired simultaneously with the dilution of the fluorescent phospholipids as measured from the loss of resonance energy transfer. The mobile fraction of N-Rh-PE on the cell surface can therefore be used as a second independent measure of actual membrane fusion. Finally, we observed that upon fusion up to 80% of the nonexchangeable liposome markers cholesterol [14C]oleate and glycerol tri[14C]oleate became accessible to cellular hydrolases. The results showed that this hydrolysis assay can also be used to monitor the second step of the fusion process.

Animals↗
PubMed1984

Recycling of transferrin receptors in A431 cells is inhibited during mitosis.

There is a marked reduction in the number of surface transferrin receptors as A431 cells enter mitosis which persists until telophase when receptors reappear to a level that exceeds the original interphase value. This is most simply explained by assuming that recycling of receptors back to the cell surface is inhibited as cells enter mitosis but that internalisation continues for a short while, causing surface receptor depletion. In telophase recycling would resume before internalisation giving a temporary excess of surface transferrin receptors.

Carcinoma, Squamous Cell↗
PubMed1983

Collisions between nitrogen-14 and nitrogen-15 spin-labels. 1. Lipid-lipid interactions in model membranes.

A new method is described to evaluate collision frequencies between spin-labeled molecules in membranes. This method utilizes a mixture of 14N and 15N nitroxides. The extent of spin-spin interaction between the two different isotopes can be determined by comparing experimental ESR spectra with simulated spectra. The latter are calculated by using modified Bloch equations and by assuming that the spin exchange process is the dominant broadening mechanism. A more approximate analysis is also described; it is based on the evaluation of the line broadening from the measurement of peak heights. Both analyses show that relatively low concentrations of label can be utilized: 1-5% labeled lipids in a membrane give a broadening sufficient to allow quantitation. Collision frequencies equal to or larger than 0.1 MHz can be evaluated by this method. Because of the limited overlap between 15N and 14N spectra, this method allows the investigation of the collisions between unlike molecules of very different concentration. From the collision frequencies, the topological domain explored by a probe can be inferred as well as its lateral diffusion constant. In this paper, it is shown that in spite of the low order of the chain residues near their methyl terminal, a probe linked at the omega - 2 position of an ionized stearic acid does not interact with a probe positioned near the polar region. Nevertheless the former probe, deeply embedded in the membrane, explores a larger volume. It is also shown that if the nitroxide is attached to a fatty ester, the probe "sees" evenly the whole hydrophobic region of the membrane.

Chemical Phenomena↗
PubMed1983

Collisions between nitrogen-14 and nitrogen-15 spin-labels. 2. Investigations on the specificity of the lipid environment of rhodopsin.

The method of spin-spin interactions between 15N and 14N spin-labels was used to investigate lipid-protein collision rates in reconstituted vesicles containing rhodopsin from bovine disk membranes and an equimolar mixture of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In each sample, a fraction of one of the three phospholipids was labeled with 14N spin-label while a 15N spin-labeled fatty acid was covalently linked to rhodopsin. The extent of spin-spin interaction between 15N and 14N labels was either calculated by complete spectral simulation or evaluated from the line broadening as deducted from the intensity decrease of the low-field 15N line. It was found that all three spin-labeled phospholipids utilized for these experiments can interact magnetically with the spin-labeled rhodopsin. Above 35 degrees C little difference between the three species can be detected. Calculation of the diffusion constant of the phospholipids at the boundary of rhodopsin proves that the lifetime of the phospholipids at the protein boundary is short and that no long-lived annular lipids are segregated. At temperatures below approximately 30 degrees C the spectra of the samples containing spin-labeled phosphatidylserine depend upon the presence or absence of calcium. The extent of 15N line broadening was found weaker in the presence of Ca2+ than in the presence of ethylenediaminetetraacetate. Thus Ca2+ tends to exclude phosphatidylserine from the lipid environment of rhodopsin. This observation can be attributed to the formation of specific lipid domains within the membrane, induced by Ca2+.

Animals↗
PubMed1982

Fringe pattern photobleaching, a new method for the measurement of transport coefficients of biological macromolecules.

The conventional method of studying mass transport in membranes by spot photobleaching and then following the recovery of fluorescence has disadvantages. Among them, the need for a high density of fluorescent molecules, the measurement of the beam profile, and a knowledge of the photobleaching processes are of a crucial importance. The application of a planar fringe pattern of light both for the bleaching and the monitoring of the fluorescent molecules solves these three major difficulties. Brownian diffusion coefficients and flow velocities can be measured independently and are averaged over the whole fringe pattern volume. These transport coefficients are explored over the wide range of experimentally accessible distances (from an interfringe spacing 0.5-50 micron). The quantification of the mobile and immobile components is further simplified by scanning the fringe pattern and detecting only a modulated fluorescence recovery signal. The fringe pattern photobleaching method is particularly adapted to the measurements of diffusion coefficients and flow velocity of membrane components, as well as of cytoplasmic proteins. The theoretical results and the test experiments with fluorescent bovine serum albumin are described.

Animals↗
PubMed1981

Distinct states of lipid mobility in bovine rod outer segment membranes. Resolution of spin label results.

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0 degrees C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24 degrees C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0 degrees C and a considerable temperature dependence. This component which is not resolved at high temperatures (24--35 degrees C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62--64 G at 0 degrees C, with very little temperature dependence (61--62 G at 35 degrees C), and is attributed to protein aggregation.

Animals↗
PubMed1980

Boundary lipids and protein mobility in rhodopsin-phosphatidylcholine vesicles. Effect of lipid phase transitions.

Purified rhodopsin from bovine retina has been incorporated into phospholipid bilayers. Dimiristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleylphosphatidylcholine and egg phosphatidylcholine were used as host lipids, with ratio of lipid to protein of 120 : 1 (mol to mol). In order to probe the lipid-protein interface specifically, a spin-labeled fatty acid was covalently bound to rhodopsin via an isocyanate reacting group. A spin-labeled phospholipid was used to probe the bulk lipidic phase while a tightly bound maleimide spin label was used to obtain the protein rotational correlation time by the saturation transfer technique. The following results were obtained: (1) The kinetics of reduction by ascorbate of the spin-labeled fatty acid covalently bound to rhodopsin demonstrate that the alkyl chain attached to the protein is positioned in the membrane in the same way as the alkyl chains of a phospholipid. (2) The EPR spectra of the latter shows two components: a strongly immobilized component and a weakly immobilized component. The ratio of the two depends upon the temperature and on the nature of the phospholipids. (3) The signal of the weakly immobilized component is compared to that obtained in the corresponding pure lipids. The latter signal, assumed to represent non-bounded lipids, indicates a sharp transition at the phospholipid phase transition with dimytristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The former signal (corresponding to the lipid-protein interface) indicates only a broad transition extending over 7 degrees C with dipalmitoylphosphatidylcholine and almost no transition with dimyristoylphosphatidylcholine. (4) In a similar way, the rotational correlation time of the protein only changes progressively when the phase transition occurs. Our interpretation of the data can be summarized as follows: The immobilized component seen by the EPR technique in the hydrophobic environment of this intrinsic protein very probably reflects protein-protein contacts and thus corresponds to hindrance of the labeled chains, when they are trapped between neighbouring proteins. Below the phase transition lipid segregation whould increase the probability of protein contact. However, over a certain range of temperature, the contact with the protein interface probably at the same time prevents the non-segregated phospholipids from feezing. The differences in the results obtained with the various phosphatidylcholines above their transition temperature suggest that the solubility of rhodopsin in bilayers depends not only on the fluidity of the lipids, but also, to some extent, on the phospholipid chain length.

Animals↗
PubMed1979

Physical modifications of rhodopsin boundary lipids in lecithin-rhodopsin complexes: a spin-label study.

The microviscosity of rhodopsin boundary lipids was studied with a spin-labeled fatty acid covalently attached to rhodopsin, in rhodopsin-egg lecithin vesicles. When the lipid-to-protein ratio was high (500:1, mole to mole), only narrow peaks were visible in electron paramagnetic resonance spectrum at 37 degrees C. This enabled us to show that, under these conditions, not more than 10% of the probes have their motion strongly restricted by the proximity of the protein. When the temperature was reduced, a second component characteristic of strong immobilization appeared. It corresponds to 50% of the signal at -5 degrees C. At all temperatures reduction of the lipid-to-protein ratio also resulted in an increase of the amount of immobilized lipid. These results show that the rhodopsin boundary layer under physiological conditions is associated with low microviscosity. However, low temperatures, low lipid-to-protein ratios, or combinations of the two can induce dramatic modifications of the physical state of the boundary lipids, which under these conditions may no longer be representative of the functional biological system. These results are relevant to the general theory of lipid-protein interaction.

Animals↗
PubMedDate not supplied

Differentiation state-dependent surface mobilities of two forms of the neural cell adhesion molecule.

The neural cell adhesion molecule (N-CAM) has been implicated in morphogenetic events during formation of the nervous system. Three forms of N-CAM exist, all glycoprotein chains, of relative molecular masses 180,000 (180K), 140K and 120K (N-CAM180, N-CAM140 and N-CAM120) which are differentially expressed on neural cell types and during development. The three chains are thought to carry similar if not identical amino-acid sequences on their extracellular amino-terminal domains, but differ in the length of their carboxy-terminal cytoplasmic region. They occur in highly sialylated embryonic and less sialylated adult forms. N-CAM180 is selectively expressed in more differentiated neural cells and may play a role in the stabilization of cell contacts. To investigate this, we have studied in the surface membrane of a mouse neuroblastoma cell line N2A the lateral mobility of the two predominant forms of N-CAM, N-CAM180 and N-CAM140, as a function of differentiation. Here we report that as judged by fringe pattern photobleaching, the surface mobility of N-CAM140 is higher than that of N-CAM180, suggesting an association of N-CAM180 with the cytoskeleton or other stabilizing factors. We also show that brain spectrin, a membrane-cytoskeleton linker protein, binds only to N-CAM180. The immobilization of N-CAM in differentiated N2A cells is achieved by a shift in expression from N-CAM140 to N-CAM180.

Animals↗