Claude Nicolau

Nanosecond fluorescence anisotropy decays of 1,6-diphenyl-1,3,5-hexatriene in membranes.

Nanosecond decays of the fluorescence anisotropy, r, were studied for the emission of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in a series of mixed multilamellar liposomes containing egg yolk phosphatidylcholine, phosphatidylethanolamine and cholesterol in varying molar ratios, as well as in membranes of intact cells and in virus envelopes. The relative contributions of the fast and the infinitely slow decaying component to the steady-state value r, of the fluorescence anisotropy were very similar for artifical and biological membranes. Angles, theta, of the cone, by which the motion of the fluorescent molecule is limited, were calculated from the intensity of the infinitely slow decaying anisotropy component and compared with steady-state fluorescence anisotropies and with microviscosities, (eta). An increase in (eta) from 1.5 to 5.2 P in our systems was accompanied by a decrease in theta from 49 degrees to 30 degrees while the decrease in the mean motional relaxation times, phi f, of the label molecule was not more than 1 ns and due mainly to changes in the potential, by which the diffusion of DPH in the membrane is restricted. From these observations we conclude that differences in the steady-state fluorescence anisotropy and in microviscosities of cholesterol-containing membranes (r greater than 0.15) represent changes in the degree of static orientational constraint rather than changes in diffusion rates of the label.

Appearance of β-lactamase activity in animal cells upon liposome-mediated gene transfer

Abstract A restriction fragment, 875 bp, which encodes for a β-lactamase activity, was isolated from the Escherichia coli plasmid pBR322 DNA and entrapped in liposomes. The incubation of the DNA-liposomes with avian, murine, and human cultured cells results in the uptake of the DNA with the efficiency of around 2000 molecules per cell. Extracts of the recipient cells show a β-lactamase activity as demonstrated by spectroscopic and microbiological methods. These results indicate the expression of a prokaryotic gene in eukaryotic cells.

Liposome-mediated DNA transfer in eukaryotic cells: Dependence of the transfer efficiency upon the type of liposomes used and the host cell cycle stage

The pBR322 plasmid containing the sequence encoding beta-lactamase, the enzyme conferring resistance to ampicillin, was encapsulated in liposomes of different phospholipid composition and incubated with synchronized cells. In mitotic cells as compared to cells synchronized in G1, twice as many exogeneous DNA molecules were found associated with the cell nuclear DNA, when fluid, neutral liposomes were used. These liposomes are taken up by the cells mainly via endocytosis. When fluid, negatively charged liposomes were used as carriers about the same number of exogenous DNA molecules were found associated with the nuclear DNA both in mitotic and in G1-synchronized cells. The efficiency for gene transfer of liposomes entering the cells by different mechanisms was further studied and expressed both by the fraction of the radioactive plasmid associated with the nuclear DNA and by the level of the beta-lactamase activity detected in the transfected cells. It appears that liposomes entering the cells mainly via an energy-dependent mechanisms are more efficient for this type of DNA transfer.

The distribution of 1,6 diphenyl hexatriene fluorescence in normal human lymphocytes

Abstract A study of the distribution of the fluorescent probe 1,6 diphenyl hexatriene in living intact human tonsil lymphocytes showed that it corresponded to the distribution of the phospholipids, except that the fluorescent intensity of the probe in the plasma membrane was enhanced by about 15% relative to its chemical distribution. The lifetime of the probe at 37° was 9.6 ns in the plasma membrane but only 8.6 ns in the whole cell. After correcting for light scattering depolarization, the mean value for the polarization ratio of the probe in the plasma membrane at 37° was 0.244 ± 0.009 (7) compared to 0.217 ± 0.008 (9) for the living cell, and hence the microviscosity of the plasma membrane was greater.

Molecular events during the interaction of envelopes of myxo- and RNA-tumor viruses with cell membranes. A 270 MHz 1H nuclear magnetic resonance study

Abstract The nuclear magnetic resonance (NMR) spectra of chick embryo cells have been analyzed after exposure to Newcastle disease virus (NDV). Virions that contained the envelope glycoproteins in the cleaved form and, thus, had full biological activity have been compared to virions that had reduced infectivity due to the presence of uncleaved glycoprotein F. After exposure to infectious virus, drastic changes occurred in the signals assigned to choline and the hydrocarbon chains of fatty acids. These observations are interpreted to demonstrate alteration of the fluid lipid bilayer structure of the cell membranes. This is compatible with the concept of membrane fusion as a penetration mechanism for NDV. Virus containing uncleaved F glycoprotein did not alter the NMR spectra. This indicates that infection is blocked at the stage of penetration. Similar, though less pronounced, differences have been observed when the effects of highly infectious influenza virus containing the hemagglutinin in the cleaved form were compared to the effects of virus which had a lower infectivity due to the presence of uncleaved hemagglutinin. Thus, it appears that the hemagglutinin of influenza virus is involved in penetration and that cleavage is necessary for this function. Alterations of the NMR spectra of the membrane lipids have also been observed when susceptible chick embryo cells (C/E) were infected with Rous sarcoma virus of subgroup B. Such alterations did not occur when nonsusceptible cells (C/B) were used. Thus, infection appears to be blocked again at the stage of penetration.

Incorporation of inositol hexaphosphate into intact red blood cells

Fluid charged lipid vesicles loaded with an inositol hexaphosphate solution were used to transport this allosteric effector into human intact red blood cells. Rate and extent of uptake of the vesicles and of the effector by the red blood cells were measured as changes in the O2 half-saturation pressure and the 31P-NMR spectra of the intracellular inositol hexaphosphate-haemoglobin complex.

Membrane lipid dynamics and density dependent growth control in normal and transformed avian cells.

Abstract Membrane fluidity of normal chick embryo fibroblasts and normal Japanese quail fibroblasts and their Rous sarcoma virus and methylcholanthrene transformed counterparts was investigated using the technique of fluorescence depolarisation of 1,6-diphenylhexatriene incorporated in the whole cells and in their isolated plasma membrane vesicles. Normal cells and isolated plasma membranes of normal cells showed significant changes in fluidity as a function of population density while neither Rous sarcoma virus transformed nor methylcholanthrene tumor cells or their isolated plasma membrane showed this effect. Stimulation of growth by addition of calf serum to cultures of quiescent, density-inhibited normal cells was accompanied by rapid changes in the direction of increased membrane lipid fluidity. Neither sparse normal cells, nor sparse or dense transformed cells showed any significant fluidity change in their membrane lipids upon addition of serum. Enzyme and electron microscopic analysis of the ratios of different membrane types in each cell type showed that this ratio was invariant with respect to cell population density but different between transformed and normal cells. Hence, the fluidity changes observed, measured as the mean rotational correlation time of the fluorescene probe in the membrane lipids, truly reflect organisational differences, occurring as a function of population density in cultures of cells which retain density-dependent growth control.

Expression of A β-lactamase activity in Mycoplasma capricolum transfected with the liposome-encapsulated E. coli pBR 322 plasmid

Liposome-encapsulated pBR 322 plasmid of E. coli was used to transfect Mycoplasma capricolum cells in culture. 12 hours after transfection the M. capricolum cells expressed the enzyme β-lactamase as shown by the hydrolysis of cephalosporin by extracts of the transfected cells. Extracts of cells incubated with empty-liposomes or with free DNA failed to hydrolyse cephalosporin. The expression of this bacterial enzyme and of acquired tetracycline resistance by the M. capricolum cells was evidenced also by plating the cells in the presence sence of tetracycline, indicating a transformation rate of ∼ 10−6. This represents direct evidence of fusion of mycoplasma with liposomes as well as the capacity of the former to express an exogenous, bacterial enzyme.

Influence of gangliosides GM1 and GD1a on structural and thermotropic properties of sonicated small 1,2-dipalmitoyl-l-α-phosphatidylcholine vesicles

Abstract The effect of the gangliosides GM 1 and GD 1a on the structure- and temperature-induced transition behavior of sonicated, small 1,2-dipalmitoylphosphatidylcholine (DPPC) vesicles has been studied by 1 H- and 13 C-NMR, laser light scattering and differential scanning microcalorimetry. It became apparent from comparison of the results obtained by the various methods that great care must be taken when the occurrence of phase transitions is postulated on the basis of methods other than high-sensitivity microcalorimetry. Detection of a temperature-dependent decrease in the half-width of methylene signals, which was reminiscent of a phase transition, was not paralleled by characteristic changes in heat capacity in the microcalorimetric experiments. Probing of the local mobility of the choline head groups in vesicles of mixed composition by 1 H-NMR and 13 C-NMR spin-lattice relaxation time ( T 1 ) measurements suggested a decrease of mobility of the hydrophilic DPPC head groups in the presence of either ganglioside. The decreases in T 1 were identical for both gangliosides irrespective of the differences in the structure of the sugar moieties of GM 1 and GD 1a , whereas the heat capacity vs. temperature curves were rather different. Since also the average dimensions of pure DPPC vesicles and vesicles containing GM 1 or GD 1a were the same, the differences in temperature dependence of heat capacity in the presence of the gangliosides were attributed to differences in interaction of the hydrophobic parts of the molecules. The higher content of C 20 sphingosines in GD 1a as compared to GM 1 was envisaged as a possible source for these differences. The presence of a physiological CaCl 2 concentration (2 mM) did not affect the transition behavior of GD 1a -containing vesicles to any detectable level, as monitored by calorimetry.

1H and 13C nuclear magnetic resonance spectra of the lipids in normal and SV 40 virus-transformed hamster embryo fibroblast membranes

Well resolved 1-H and 13-C NMR spectra were obtained with normal and SV 40-transformed cell membranes. Estimation of the ratio of 13-CT2 values of the normal to transformed cell membranes showed an increased intermolecular motion in the transformed cell membranes. The temperature dependence of the (CH2) line in the 1-H spectra in the temperature range 298-343 degrees K shows an activation energy for the lateral diffusion of the fluid phospholipid regions in the normal cell membranes while the transformed ones show practically no temperature dependence in this temperature range. The fluidity of the phospholipid region in the transformed cell membrane seems to be significantly higher than that observed in the normal cell material. These data support and extend the findings concerning the mobility of the concanavalin A binding/agglutinating sites on the surface of normal and virus-transformed cells and suggest further approaches to the study of the membrane alterations in tumor cells.