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C Nicolau

Publications and source records attributed to C Nicolau.

At least 73 records · Page 4Linked to original sources
PubMed1987

Fusion of negatively charged liposomes with clathrin-uncoated vesicles.

The interaction of lipid vesicles with uncoated vesicles from bovine brain has been studied by fluorescence energy transfer between fluorescent lipid analogs (NBD-PE, Rh-DOPE), by loss of fluorescence self-quenching (NBD-PE, carboxyfluorescein) and by freeze-fracture electron microscopy. The fluorescence techniques monitor the mixing of membranous lipids and the induced release of encapsulated material. The results demonstrate a mixing of the negatively charged lipid (PA, PS) vesicles with the uncoated vesicles. In parallel with the lipid mixing a release of intravesicularly encapsulated material takes place. Lipid vesicles composed of zwitterionic lipids (PC, DOPC, PC:PE) do not specifically interact with uncoated vesicles. The electron micrographs reveal single fusion events. Studies on the kinetics are consistent with a fusional mechanism of the negatively charged lipid vesicles with uncoated vesicles.

Animals↗
PubMed1987

Interaction of negatively charged liposomes with nuclear membranes: adsorption, lipid mixing and lysis of the vesicles.

Fluorescence energy transfer studies reveal that negatively charged lipid vesicles interact with nuclei from mouse liver cells. This interaction was observed with charged lipid vesicles composed of PA or PS but not with the uncharged PC or PE:PC vesicles. The vesicles were prepared by bath sonication and contained either a fluorescent marker in the lipid bilayer or in the vesicular interior. The negatively charged vesicles showed an adsorption to the nuclear membrane visible by fluorescence microscopy. The results obtained by resonance energy transfer experiments are interpreted in terms of a mixing of the lipids from the vesicles with the nuclear membrane. Encapsulation studies documented a staining of the nuclei only if the dye molecules of high or low molecular weight were encapsulated inside negatively charged vesicles. As consequence of the vesicle-nuclei interaction morphological changes on the nuclear surface became visible.

Adsorption↗
PubMed1987

Low pH fusion of mouse liver nuclei with liposomes bearing covalently bound lysozyme.

Lysozyme covalently bound to liposomes induces the fusion of liposomes with isolated mouse liver nuclei. The fusion behavior is very similar to the case of erythrocyte ghosts (Arvinte, T., Hildenbrand, K., Wahl, P. and Nicolau, C. (1986) Proc. Natl. Acad. Sci. USA 83, 962-966). Kinetic studies showed that membrane lipid mixing was completed within 15 min, as indicated from the resonance energy transfer (RET) measurements. For the resonance energy transfer kinetic measurements the liposomes contained L-alpha-dipalmitoylphosphatidylethanolamine (DPPE), labeled at the free amino group with the energy donor 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or with the energy acceptor tetramethylrhodamine. The lipid mixing at equilibrium was studied by the fluorescence recovery after photobleaching technique (FRAP). Liposomes (with/without lysozyme) containing Rh-labeled DPPE in their membranes were incubated with nuclei at 37 degrees C, pH 5.2, for 30 min. After washing of nuclei by three centrifugations, 60-70% of the initial amount of labeled DPPE was associated with the nuclei in the case of liposomes bearing lysozyme and only 7-10% in the case of liposomes without lysozyme. For the nuclei incubated with liposomes having lysozyme, about 70% of the total Rh-labeled lipids present in the nuclei diffused in the nuclear membrane(s) (lateral diffusion constant of D = (1.4 +/- 0.5) X 10(-9) cm2/s). By encapsulating fluorescein isothiocyanate-labeled dextran of 150 kDa molecular mass into the liposomes and using a microfluorimetric method, it was shown that after the fusion a part of the liposome contents is found in the nuclei interior. In this lysozyme-induced fusion process between liposomes and nuclei or erythrocyte ghosts, the binding of lysozyme to the glycoconjugates contained in the biomembranes at acidic pH seems to be the determining step which explains the high fusogenic property of the liposomes bearing lysozyme.

Animals↗
PubMed1987

Resonance energy-transfer and fluorescence intensity studies of the transport of liposome-encapsulated molecules into isolated mouse liver nuclei.

We present evidence that liposomes (composed of egg yolk L-alpha-phosphatidylcholine/phosphatidylethanolamine/cholesterol, in a molar ratio of 4:5:1) fuse with isolated mouse liver nuclei at low pH. Using the resonance energy-transfer assay, we determined the rate and extent of liposome and nuclear membrane lipid mixing. Fusion was substantial when the pH was below 5. The half-time of lipid mixing decreased by acidification of the solvent, reaching about 2 min at pH 4.5. In order to study the transport of the liposome-aqueous contents to the interior of the nuclei during the process, we developed fluorescence assays in which fluorescein isothiocyanate labeled dextrans of 150 kDa molecular mass (FITC-D150) were encapsulated in liposomes. These liposomes also included in their bilayers the fluorescent lipid N-tetramethylrhodamine-L-alpha-dipalmitoylphosphatidylethanolamine (N-Rh-DPPE). After incubation of these liposomes with mouse liver nuclei (pH 4.5, 37 degrees C, 30 min), we measured the fluorescence spectra of a suspension of washed nuclei and of nuclei treated by the detergent Triton X-100 (membrane-denuded nuclei). These Triton X-100 treated nuclei had no N-Rh-DPPE fluorescence while they showed a FITC-D150 fluorescence which amounted to 20% of that of the intact nuclei. In another assay, a laser beam was focused on single nuclei by a microscope epiexcitation device. The variation of the N-Rh-DPPE and FITC-D150 fluorescence with the nuclear radius was determined with the microphotometric attachment of the microscope.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Long-term physiological effects of enhanced O2 release by inositol hexaphosphate-loaded erythrocytes.

A continuous lysing and resealing procedure with erythrocytes permitted incorporation in these cells of inositol hexaphosphate (InsP6), a strong allosteric effector of Hb. This leads to significant rightward shifts of the HbO2 dissociation curves with in vitro P50 (partial pressure of O2 at 50% Hb saturation), values increasing from 32.2 +/- 1.8 torr for control erythrocytes to 86 +/- 60 torr (pH 7.40; PCO2 40 torr at 37 degrees C; 1 torr = 1.333 X 10(2) Pa). The shape of the dissociation curve was still sigmoidal, although the Hill coefficient was decreased. The life span of InsP6-loaded erythrocytes equaled that of control erythrocytes. The long-term physiological effects of the InsP6-loaded erythrocytes on piglets were increased O2 release and reduced cardiac output. The reduced O2 affinity of the InsP6-loaded erythrocytes was still effective 20 days after transfusion in awake piglets. The electrolyte concentration appeared stable over the 5-day observation period except for a transient, but significant, hyperkalemia immediately after transfusion. The reductions in the O2 affinity of Hb reported here are large compared with previously reported values. Introduction of InsP6 into viable erythrocytes improves tissue oxygenation when, for any reason, normal blood flow is impaired.

Animals↗
PubMed1987

Oxygen transport to tissue modified by entrapment of an allosteric effector of haemoglobin in erythrocytes.

Oxygen affinity of haemoglobin is modulated by several parameters such as the allosteric effector 2-3 DPG for most mammalians. Inositol hexaphosphate (I.H.P.) exerts the same effect on haemoglobin. A previously developed new methodology for the entrapment of drugs into erythrocytes has been adapted to I.H.P.; it is based on a reversible osmotic shock. I.H.P. loaded red blood cells have characteristics very similar to those of native cells. The decrease in oxygen affinity is related to the dose of encapsulated I.H.P. In piglets, transfusion of such cells has led to an increase of oxygen extraction from haemoglobin. Normal anesthetized animals regulate their oxygen consumption by reduction of cardiac output.

2,3-Diphosphoglycerate↗
PubMed1986

Biologically active, recombinant DNA in clathrin-coated vesicles isolated from rat livers after in vivo injection of liposome-encapsulated DNA.

DNA entrapped in liposomes containing lactosylceramide in the bilayers is found to be associated with clathrin-coated vesicles isolated from the rat livers after intravenous injection of these liposomes. The presence of the exogenous DNA in the coated vesicles was detected by Southern blotting. The amount of DNA present in the coated vesicles does not appear to vary up to 4 h after injection of the liposomes into the animals. The recognition of the lactosyl group present in the liposome by the galactose receptor present on the surface of the different liver cells may lead to their internalization in a way analogous to receptor-mediated endocytosis of various macromolecules. DNA present in the lumen of the coated vesicles is found to be biologically active as evidenced by its replication in bacterial cells and mouse fibroblasts.

Animals↗
PubMed1986

Interaction of intravenously injected liposomes with mouse liver mitochondria. A fluorescence and electron microscopy study.

Megamitochondria, resulting from cuprizone feeding of Swiss ICR mice, were fluorescent in hepatocytes after the intravenous injection to mice of a liposome-encapsulated acridine orange-DNA complex (AO-DNA). Flow cytofluorimetric analysis of isolated megamitochondria showed that the proportion of liposome-encapsulated AO-DNA which localized in megamitochondria increased from 0.02% of the dose injected per liver cell at 3 min after injection to an average of 0.34% at 1 h after injection. Megamitochondria showed negligible fluorescence by fluorescence activated cell sorting (FACS) analysis when free AO-DNA was intravenously injected. Transmission electron micrographs of mouse liver tissue after intravenous injection of liposomes encapsulating iron dextran showed an association of the liposomes with megamitochondria which appeared identical to liposome association with normal mitochondria. These results support and extend our earlier observation that a fraction of the liposomes injected intravenously into mice associate with mitochondria in the liver, and possibly deliver their aqueous contents there.

Acridine Orange↗
PubMed1986

Lysozyme-induced fusion of liposomes with erythrocyte ghosts at acidic pH.

Lysozyme that was covalently bound to the outer surface of sonicated vesicles induced fusion of the vesicles with human white erythrocyte ghosts. The kinetics of membrane mixing were evaluated by the resonance-energy-transfer method using L-alpha-dipalmitoyl phosphatidylethanolamine labeled at the free amino group with the energy donor 7-nitro-2,1,3-benzoxadiazol-4-yl or with the energy acceptor tetramethylrhodamine. The equilibrium state after fusion was characterized by using fluorescence photobleaching and recovery techniques. Rates and equilibrium percentages of fusion were maximal at the pH optimum of the enzyme, and rates were strongly reduced by the addition of N,N',N''-triacetylchitotriose, a competitive inhibitor of lysozyme. An apparent activation energy of 28 +/- kcal/mol was obtained for the lipid-mixing process. At 37 degrees C, the fusion half-time was 0.5 min. After 30 min at 37 degrees C, 40% of the labeled lipids initially present in the fusion mixture had a lateral diffusion constant, D, of 1.1 +/- 0.5 X 10(-9) cm2 X sec-1 in the ghost membrane. The strong induction of fusion at the lysozyme pH optimum was not observed in the absence of lysozyme or when free lysozyme was added to the solution. Bound lysozyme did not induce fusion of electrically neutral liposomes with each other. These observations indicate that it is the liposome-bound lysozyme that induces fusion between liposomes and erythrocyte ghosts.

Erythrocyte Membrane↗
PubMed1986

An original method for submacroscopic metastases visualization in cases of cancer minimal residual disease.

Scintigraphic imaging due to its sensitivity is in many cases one of the most powerful techniques for demonstrating metastases. Severe limitations still exist in cancer when it is necessary to detect the presence of a few tumour cells in the residual minimal disease. In preliminary experiments it had been observed that an immunomodulator isolated from Nocardia bacteria (Nocardia Soluble Peptidoglycan Derivative: NSPD) electively bound to a model of activated macrophages. An hypothesis has been put forward that the enhanced detection of macrophages that are usually present in the vicinity or inside tumours should represent a polyspecific test for scintigraphy of a variety of metastases. NSPD radiolabelled with 99mTechnetium is not usable when injected intravenously due to its physiochemical properties. It has therefore been encapsulated into liposomes then administered via the respiratory tract as an aerosol. Amphiphilic properties, as well as its low molecular weight allow a rapid diffusion of NSPD in blood. Scintigraphy of metastases was possible from 1.5 to 6 hours after inhalation. The first stage of the study was carried out on 5 patients bearing known metastases (skin, lymph nodes, bone) from malignant melanoma that all were imaged with 99mTc-NSPD. The test was then applied to patients with a high risk of recurrent cancers (melanoma: 6, breast tumour: 7) based on the detection in their plasmas of high Lipid Associated Sialic Acid (LASA) concentrations. The association of these two sensitive techniques has resulted in the detection of very small metastases that were not seen using conventional scintigraphy; they were then confirmed histologically.

Aged↗
PubMed1985

Intracellular fate of liposome-encapsulated DNA in mouse liver. Analysis using electron microscope autoradiography and subcellular fractionation.

Transient expression of liposome-encapsulated DNA in liver after intravenous injection to rats and mice has raised questions concerning the intracellular fate of this DNA. Electron microscope autoradiography shows that at 10 min after injection the highest concentration of liposomal DNA which is taken up by the liver is associated with lysosomes and vesicles. The proportion of DNA associated with the mitochondria steadily increases for 1 h after injection, up to 48% of the exogenous DNA found in the tissue. Part of this DNA follows the subcellular fractionation profile of the mitochondrial matrix marker, malate dehydrogenase. In contrast, 14% of the liposomal DNA taken up by the liver is found in the nuclei at 3 min after injection, and this percentage decreases over a period of 1 h. These results permit us to establish the distribution of liposome-encapsulated DNA among subcellular organelles in liver at different times after injection.

Animals↗
PubMed1985

Physiological effects of high-P50 erythrocyte transfusion on piglets.

Rightward shifts of the O2 dissociation curve (ODC) were experimentally obtained in lysed and resealed erythrocytes following encapsulation of inositol hexaphosphate (IHP). This continuous lysing and resealing procedure led to in vitro P50 (Po2 at 50% hemoglobin saturation) increases up to 80 Torr (pH, 7.40; Pco2, 40 Torr; temp, 37 degrees C) for both human and pig erythrocytes. The Hill number of the transformed blood decreased when IHP was fixed on the hemoglobin, but the sigmoid shape of the ODC was maintained. The O2 hemoglobin binding capacity and the mean corpuscular hemoglobin content were found unchanged by the experimental procedure in human and pig erythrocytes. Isovolumic exchange transfusion of high-P50 erythrocytes in anesthetized and ambient air-ventilated piglets (n = 6) led to substantial in vivo P50 increases (range, 8-19 Torr). The rightward shift of the ODC was concomitant with an increase of the arterial Po2 and of the arteriovenous O2 content difference, 19 and 59% respectively above their control values. The mixed-venous Po2 (PVO2) remained unchanged. The cardiac output was shown to be inversely related to the P50 value. In spite of the O2-transport reduction (37%), O2 consumption was maintained due to enhanced O2 extraction.

Animals↗
PubMed1984

Flow cytofluorometric investigation of the uptake by hepatocytes and spleen cells of targeted and untargeted liposomes injected intravenously into mice.

Untargeted liposomes (composition: PC-PS-cholesterol) and targeted liposomes (composition: PC-PS-cholesterol-lactosylceramide) having encapsulated concentration-quenched carboxyfluorescein were injected intravenously into mice. 1 h after injection, the mice livers were perfused, excised and the hepatocytes were separated from nonparenchymal cells and analysed in a fluorescence-activated cell sorter analyzer. The result was that hepatocytes took up significantly more liposomes when lactosylceramide was inserted in the liposome bilayers, which was in good agreement with observations made on the in vivo uptake of liposome-encapsulated insulin gene (Soriano, P. et al. (1983) Proc. Natl. Acad. Sci. USA, 80, 7128-7133). Cytofluorimetric analysis of the spleen cells showed that approx. 10% of the splenic lymphocytes take up high amounts of lactosylceramide liposomes, whereas most of the phospholipid liposomes are taken up by the phagocytic cells. The flow cytofluorimetric analysis shows, moreover, the internalization of the liposomes by the target cells and allows a quantitation of this uptake. Thus, in vivo targeting of the liposomes to specific liver and splenic cells, by means of glycolipid insertion in the liposome bilayer, is shown to take place with delivery of the liposomal aqueous space marker to these cells.

Animals↗
PubMed1984

Liposomes injected intravenously into mice associate with liver mitochondria.

Liposomes encapsulating uranyl acetate or ferritin were injected intravenously into mice. At periods of 20 min, 1 h and 4 h post-injection, animals were killed, and livers were excised. Transmission electron micrographs of liver tissue showed association of oligolamellar liposomes with mitochondria for each time period. At 1 h post-injection, an average of one out of ten mitochondria was associated with liposomes. In most cases, the liposomes were clearly enclosed in a cytoplasmic vacuole. Phagocytosis by Kupffer cells as well as fusion with primary lysosomes and inclusion in secondary lysosomes was observed. No difference in intracellular fate was observed when lactosylceramide was incorporated in the liposome bilayers, suggesting that the differences observed in biochemical studies are at the level of liposome-plasma membrane interaction. When liposomes containing uranyl acetate were intravenously injected and hepatocytes were isolated by collagenase perfusion one hour later, transmission EM revealed the presence of liposomes in these cells, in cytoplasmic vacuoles in the cytoplasm and in association with mitochondria. A freeze-fracture-etching analysis of liver tissue excised 20 min after injection of liposomes encapsulating ferritin, further supported the observation that liposomes associate with mitochondria in the liver.

Animals↗