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Biomedical subjects

C Nicolau

Publications and source records attributed to C Nicolau.

At least 91 records · Page 5Linked to original sources
PubMed1984

Liposomes for gene transfer and expression in vivo.

A recombinant plasmid encoding rat preproinsulin I was encapsulated in large liposomes and injected intravenously into rats. Glycaemia and blood, splenic and hepatic insulin were assayed from 6 h after inoculation. Control animals received (i) empty liposomes, (ii) liposomes carrying the Escherichia coli pBR 322 plasmid, (iii) the free rat insulin I gene, or (iv) no injection. All controls showed unchanged glucose and insulin levels. Six hours after inoculation the treated rats had 72 +/- 5 mg glucose/100 ml of blood, compared with 107 +/- 2 mg/ml for controls. Radioimmunoassay of blood insulin gave 61 +/- 8 microunits/ml (43 +/- 5 microunits/ml for controls). Spleen and liver values were 242 +/- 22 and 204 +/- 20 microunits/g of tissue, respectively (112 +/- 20 and 87 +/- 15 microunits/g in controls). The kinetics and extent of uptake of liposomes by spleen and liver were studied by external gamma-camera imaging after injection of 111In-labelled liposomes. The results paralleled insulin synthesis in the two organs. The insulin gene was localized in liver cells after injection of liposomes containing the plasmid encoding the gene. Livers were processed 4 h after inoculation for isolation of hepatocytes, Kupffer cells and endothelial cells. DNA was purified and exogenous DNA detected by Southern blotting. Kupffer cells were the primary target for gene incorporation with liposomes consisting of phospholipids and cholesterol. Targeting of liposomes to other liver cells was attempted by including lactosylceramide in the liposomes. This increased the amount of the exogenous gene in hepatocytes and particularly in endothelial cells. The efficiency of liposome-mediated gene transfer in vivo is high, since a few per cent of the transferred DNA is taken up by the liver cells and detected.

Animals↗
PubMed1983

Targeted and nontargeted liposomes for in vivo transfer to rat liver cells of a plasmid containing the preproinsulin I gene.

A plasmid containing the rat preproinsulin I gene was entrapped in large liposomes and intravenously administered to rats. Four hours after inoculation, the livers were processed for the isolation of hepatocytes. Kupffer cells, and endothelial cells, DNA was purified, and the exogenous DNA was detected in the different cell DNA preparations by Southern blotting. By using liposomes consisting of phospholipids and cholesterol, Kupffer cells were shown to be, on a per cell basis, the primary target for gene incorporation. In an attempt to target the liposomes to other liver cells, a glycolipid, lactosylceramide, was included in the lipid bilayer of the liposomes; this resulted in a substantial increase in the proportion of the exogenous gene in the hepatocytes and mainly in the endothelial cells, with a simultaneous decrease of this proportion in the Kupffer cells. Thus, it is shown that inclusion of a specific glycolipid within the bilayer of the liposomes may direct the DNA-containing vesicles to specific cell types in the liver.

Animals↗
PubMed1983

In vivo expression of rat insulin after intravenous administration of the liposome-entrapped gene for rat insulin I.

A recombinant plasmid encoding rat preproinsulin I was encapsulated in large liposomes and intravenously injected in rats. Glycemia and blood, splenic, and hepatic insulin were assayed at various times beginning 6 hr after inoculation. The results were compared with controls that had received (i) empty liposomes, (ii) liposomes carrying the Escherichia coli pBR322 plasmid, (iii) the free rat insulin I gene, and (iv) no injection at all. Whereas all controls showed unchanged glucose and insulin levels, the treated animals had, 6 hr after inoculation, a blood glucose level of 72 +/- 5 mg of glucose/100 ml of blood as compared with 107 +/- 2 mg/ml for controls. Radioimmunoassay of blood insulin gave 61 +/- 8 microunits/ml as compared with 43 +/- 5 microunits/ml for controls and the spleen and liver values were 242 +/- 22 and 204 +/- 20 microunits/g of tissue, respectively, as compared with 112 +/- 20 and 87 +/- 15 microunits/g in controls. External gamma-camera imaging of the organ uptake of 111In-labeled liposomes permitted study of the kinetics and extent of uptake of the liposomes by spleen and liver, results that support the findings concerning insulin synthesis in the two organs.

Animals↗
PubMed1982

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.

Biological Transport↗
PubMed1980

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

A restriction fragment, 875 bp, which encodes for a beta-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 beta-lactamase activity as demonstrated by spectroscopic and microbiological methods. These results indicate the expression of a prokaryotic gene in eukaryotic cells.

Animals↗
PubMed1979

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.

Cholesterol↗
PubMed1979

Early molecular events in the interaction of enveloped viruses with cells. I. A fluorescence and radioactivity study.

The fluorescence depolarization of 1,6-diphenyl-hexatriene was used to study the dynamic properties of the hydrophobic regions of the lipid envelopes of ortho- and paramyxoviruses as well as of the Rous sarcoma virus and of the membrane lipids of susceptible and nonsusceptible cells. The systems investigated where active and inactive influenza viruses, and NDV virus acting on chick embryo fibroblasts and Rous sarcoma virus acting on susceptible (C/E) and nonsusceptible (C/B) chicken-cell. Polarization degrees and mean rotational correlation times of DPH embedded in viral lipids were significantly higher than those of DPH in the cell membranes, due to a higher rigidity of the virus envelopes. When suspensions of labelled viruses and unlabelled cells or unlabelled viruses and labelled cells were mixed, a characteristic change of the fluorescence polarization degrees with time was observed. This behaviour was ascribed to label transfer from virus to cell membranes or vice versa. While the rate constants of label transfer from virus to cells and cells to virus were about the same for the penetrating viruses the rate constants of label release from inactive virus to cells were much larger than for the migration in the opposite direction.

Animals↗
PubMed1979

Early molecular events during the interaction of enveloped riboviruses with cells. II. A kinetic study.

A kinetic model was constructed and partly solved to describe the migration of the fluorescence label 1,6-diphenylhexatriene (DPH) in both directions when enveloped viruses, labelled with DPH in their envelopes are in contact with unlabelled cells or cell labelled in their membranes are in contact with unlabelled enveloped viruses. The central assumption is that two types of receptor sites exist on the cell surface, i.e., physical adsorption sites (P-sites), available to all the viruses studied in these papers and binding sites (B-sites) available only to the viruses which penetrate into the specific cells. The differential equations for the label migration, for different values of the ratio number of viruses number of sites were numerically solved, assuming different fractions of P- and B-sites. The equations also describe, appropriately the mechanism of rapid label migration in the system and substantiate the magnitude "time of residence" of the nonpenetrating viruses adsorbed on the cell surface. The resulting curves match satisfactorily those for the label release by the viruses and account well for the steady state values of the kinetics of label migration in the virus-cell system.

Adsorption↗
PubMed1979

Incorporation of inositol hexaphosphate into intact red blood cells. I. Fusion of effector-containing lipid vesicles with erythrocytes.

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.

Erythrocytes↗
PubMed1979

Incorporation of inositol into intact red blood cells. II. Enhancement of gas transport in inositol hexaphosphate-loaded red blood cells.

The gas-transport function of red blood cells which have incorporated inositol hexaphosphate is significantly improved by fusion with effector-loaded lipid vesicles. "Right-shifts" of the O2-binding curves of inositol hexaphosphate-loaded red blood cells with half-saturation pressures at 37 degrees C up to 98 mmHg are observed. The transformation of 56% of the intracellular haemoglobin into the low affinity state corresponds to an optimum increase of the O2-release capacity of 270%. The CO2 transport is also correspondingly enhanced. The treated red blood cells show no inositol hexaphosphate depletion during a reasonable time.

Carbon Dioxide↗
PubMed1979

Improvement of the red blood cell O2 release capacity by lipid vesicle-mediated incorporation of inositol hexaphosphate.

Fusion of unilamellar lipid vesicles with red blood cells has been used to deliver entrapped inositol hexaphosphate (IHP) to the cells. Incorporated IHP tightly bound to hemoglobin produces a significant and lasting "right-shift" of the O2-binding curve of red blood cells. The O2 release capacity of red blood cells is increased by this method up to 270% of the normal value. The Bohr effect is increased so that carbon dioxide transport is also enhanced. The fine structure of the erythrocytes does not seem to be altered by the lipid vesicle incorporation.

Erythrocytes↗