Biomedical subjects

F Schuber

Publications and source records attributed to F Schuber.

At least 37 records · Page 2Linked to original sources

PubMed1997

Antitumoral activity of liposomes and immunoliposomes containing 5-fluorouridine prodrugs.

Liposomes and immunoliposomes containing cytotoxic agents may be highly efficacious in intracavity therapy of malignancies confined principally to the peritoneal cavity. To assess the feasibility of this locoregional treatment, we prepared two derivatives of 5-fluorouridine (5-FUR), a highly cytotoxic metabolite of 5-fluorouracile, and incorporated them into REV liposomes, prepared with the reverse phase evaporation method. Encapsulation efficiency, drug leakage, and stability were determined, and size analysis and differential scanning calorimetry were carried out to evaluate the drug delivery potential of liposomes containing 5'-palmitoyl-5-FUR, 5'-succinyl-5-FUR, or the parent drug 5-FUR. The most suitable drug for encapsulation, in terms of minimum leakage and encapsulation efficiency, was 5'-palmitoyl-5-FUR, which differential scanning calorimetry indicated as being firmly anchored to the lipid bilayer. Thus, 5'-palmitoyl-5-FUR was chosen to prepare a chemotherapeutic liposome-monoclonal antibody conjugate (immunoliposome). The covalent linkage between antibody and liposome was realized by coupling the thiolated monoclonal antibody AR-3 with REV liposomes, containing N-[4-(p-maleimidophenyl)butyryl]phosphatidylethanolamine. The cytotoxic activity of drug-bearing liposomes and immunoliposomes was evaluated on the HT-29 human colon adenocarcinoma cell line; the immunoliposomes had higher cytotoxicity than liposomes or 5-FUR. To explore the potential of these drug formulations in anticancer therapy, we ip injected liposomes or immunoliposomes into athymic mice ip grafted with human HT-29 cell line. In this mouse model, the immunoliposome containing 5'-palmitoyl-5-FUR displayed the best antitumoral activity, since on day 27 postgraft only 5% of residual tumor mass was present, compared to control mice; there was a close relationship between exposure time of tumor tissue to the drug and antitumor potency.

Animals↗

PubMed1996

Photodependent inhibition of bovine spleen NAD+ glycohydrolase by 8-azido carbocyclic analogs of NAD+.

Carba-NAD+ and pseudocarba-NAD+, and their 8-azidoadenosyl derivatives, were found to be good competitive inhibitors of calf spleen NAD+ glycohydrolase. The 8-azido compounds, tested as photoaffinity labels, inhibited the enzyme in a light- and time-dependent manner; this inhibition could be prevented by 3-aminopyridine adenine dinucleotide (n3PdAD+), a competitive inhibitor of NAD+ glycohydrolase. Irradiation in the presence of the [3H]-labeled 8-azido-carba-NAD+ derivative resulted in an irreversible incorporation of the radioactivity into the enzyme that could be largely prevented by addition of n3PdAD+. These results indicate that carbocyclic-analogs of NAD+ will be useful in identifying the substrate binding site of NAD+ glycohydrolase.

Affinity Labels↗

PubMed1996

Mechanism of cyclization of pyridine nucleotides by bovine spleen NAD+ glycohydrolase.

We have shown that bovine spleen NAD+ glycohydrolase (EC), purified to homogeneity, is a multifunctional enzyme. A time-dependent formation of cADPR from NAD+ that did not exceed 1.5-2% of the reaction products was measurable. The cyclase activity of this enzyme was, however, best evidenced by its transformation of NGD+ into cyclic GDP-ribose (cGDPR). The formation of the cyclic compound could be monitored spectroscopically (UV and fluorescence) and by high-performance liquid chromatography; the product ratio of cGDPR/GDP-ribose was 2:1. Bovine spleen NAD+ glycohydrolase is also able to hydrolyze cADPR (Muller-Steffner et al. (1994) Biochem. Biophys. Res. Commun. 204, 1279-1285); the kinetic parameters (V/Km) measured exclude, however, the possibility that cADPR is a kinetically competent reaction intermediate in the transformation of NAD+ into ADP-ribose. Experimental data indicating that NAD+ glycohydrolase-catalyzed hydrolysis and methanolysis of NA(G)D+ occurred at the expense of the formation of the cyclic compounds are in favor of a reaction mechanism involving the partitioning of a common oxocarbenium reaction intermediate between the different acceptors. Thus E.A(G)DP-ribosyl oxocarbenium intermediate can react according to i) intramolecular processes with the positions N-1 of adenine and N-7 of guanine to give cA(G)DPR as reaction products, and ii) intermolecular reactions with water (formation of A(G)DP-ribose) and methanol (formation of methyl A(G)DP-ribose). We attribute the marked difference in yield of cADPR and cGDPR to the intrinsic reactivity (nucleophilicity and positioning) of the purine N-positions that are involved in the cyclization reactions within the E.A(G)DP-ribosyl oxocarbenium complexes.

Adenosine Diphosphate Ribose↗

PubMed1996

Immunogenicity of new heterobifunctional cross-linking reagents used in the conjugation of synthetic peptides to liposomes.

We have investigated the immunogenicity of six thiol-reactive heterobifunctional cross-linking reagents that permit the conjugation of cysteine carrying peptides to the surface of liposome containing monophosphoryl lipid A. Such constructs elicit an immune response against short synthetic peptides and our aim was to find the least immunogenic linkers to limit potential carrier-induced epitopic suppression. For that purpose the properties of three new polyoxyethylene linkers of different lengths and thiol-reactive moieties (maleimide, bromoacetyl, dithiopyridine) were compared to known derivatives obtained by reacting the classical reagents SMPB and SPDP or N-succinimidyl bromoacetate with phosphatidylethanolamine. The least immunogenic linkers were the bromoacetate derivatives whereas those containing a maleimide group evoked a significant anti-linker immune response. In addition, using IRGERA as a model peptide, we found that all six liposomal constructs strongly elicited the production of anti-peptide IgG antibodies. This immune response was therefore independent of the length of the linkers (ranging between 0.3 and 1.6 nm) and of the nature of the linkage. between the peptide and the thiol-reactive moieties of the cross-linkers, i.e. stable thioether or bio-reducible disulfide bonds.

Amino Acid Sequence↗

PubMed1995

Inhibition of 2,3-oxidosqualene cyclase and sterol biosynthesis by 10- and 19-azasqualene derivatives.

The inhibition of 2,3-oxidosqualene-lanosterol cyclase (EC 5.4.99.7) (OSC) by new azasqualene derivatives, mimicking the proC-8 and proC-20 carbocationic high-energy intermediates of the cyclization of 2,3-oxidosqualene to lanosterol, was studied using pig liver microsomes, partially purified preparations of OSC, and yeast microsomes. The azasqualene derivatives tested were: 6E- and 6Z-10aza-10,11-dihydrosqualene-2,3-epoxide 17 and 18, 19-aza-18,19,22,23-tetrahydrosqualene-2,3-epoxide 19 and its corresponding N-oxide 20, and 19-aza-18,19,22,23-tetrahydrosqualene 21. The compounds 17 and 19 (i.e. the derivatives bearing the 2,3-epoxide ring and the same geometrical configuration as the OSC substrate) were effective inhibitors, as shown by the Ki obtained using partially purified OSC: 2.67 microM and 2.14 microM, respectively. Compound 18, having an incorrect configuration and the 19-aza derivative 21, lacking the 2,3-epoxide ring, were poor inhibitors, with IC50 of 44 microM and 70 microM, respectively. Compound 21 was a competitive inhibitor of OSC, whereas 17 and 19 were noncompetitive inhibitors, and showed a biphasic time-dependent inactivation of OSC, their apparent binding constants being 250 microM and 213 microM, respectively. The inhibition of sterol biosynthesis was studied using human hepatoma HepG2 cells. The incorporation of [14C] acetate in the C27 sterols was reduced by 50% by 0.55 microM 17, 0.22 microM 19, and 0.45 microM 21, whereas 2 microM 18 did not affect sterol biosynthesis. In the presence of 17, 19 and 21, only the intermediate metabolites 2,3-oxidosqualene and 2,3,22,23-dioxidosqualene accumulated, demonstrating a very specific inhibition of OSC.

Animals↗

PubMed1995

Stereocontrolled hydrolysis of the linoleic acid monoepoxide regioisomers catalyzed by soybean epoxide hydrolase.

Soybean fatty acid epoxide hydrolase (EC 3.3.2.3) was found to possess remarkable and unique stereochemical features. After complete hydrolysis, this enzyme converts racemic or enantiomerically enriched cis-9,10-epoxy-12(Z)-octadecenoic and cis-12,13-epoxyocta-9(Z)-decenoic acids, i.e. the two regioisomers of linoleic acid monoepoxides, into their corresponding 9R, 10R- and 12R, 13R-dihydrodiols with a high enantiomeric excess (> 90%). A straightforward chiral-phase HPLC technique was developed that gives an easy access to the stereochemistry of these reaction products. These results are discussed in terms of a possible model for the substrate binding site of this enzyme.

Epoxide Hydrolases↗

PubMed1995

Efficient gene delivery with neutral complexes of lipospermine and thiol-reactive phospholipids.

The presence of thiol-reactive phospholipid derivatives, such as N-4-(p-maleimidophenyl)butyryl) dipalmitoylphosphatidylethanolamine (MPB-DPPE), in electrically neutral lipospermine/DNA particles results in more than a 100-fold increased transfection efficiency of human hepatoma HepG2 cells and murine 3T3 fibroblasts. These effects could be ascribed to the presence of thiol-reactive functions, such as maleimide, bromoacetamide and dithiopyridyl linkage, on the transfecting particles. We propose that such particles react with thiol groups present at the surface of the cells, leading to their covalent anchoring, a process that is probably followed by an endocytosis of the complex.

3T3 Cells↗

PubMed1995

Retention of the label during the conversion of [3-3H] squalene into (3S)-2,3-oxidosqualene catalyzed by mammalian squalene oxidase.

Squalene epoxidase is the only known flavoprotein that catalyzes the epoxidation of an olefin. In order to test the possibility of a catalytic non-heme metal-based mechanism, the conversion of chemically synthesized [3-3H]squalene into [3H]2,3-oxidosqualene, by partially purified pig liver squalene epoxidase, was studied. No exchange of the labeled hydrogen could be observed, ruling out a mechanism involving, e.g., an iron carbene type species at C-3.

Animals↗

PubMed1995

Targeted gene transfer into hepatoma cells with lipopolyamine-condensed DNA particles presenting galactose ligands: a stage toward artificial viruses.

Optimal in vitro gene delivery with cationic lipids requires an excess of cationic charges with respect to DNA phosphates. In these conditions, in vivo delivery will be hampered by interference from cationic lipid-binding macromolecules either circulating or in the extracellular matrix. To overcome this problem, we are developing a modular transfection system based on lipid-coated DNA particles reminiscent of enveloped viruses. The particle core consists of the lipopolyamine-condensed nucleic acid in an electrically neutral ratio to which other synthetic lipids with key viral properties are hydrophobically adsorbed. As a first result, we have found that a good transfection level can be achieved simply with the neutral core particle, provided a zwitterionic lipid (dioleoyl phosphatidylethanolamine) is added to completely coat the DNA. Addition of lipids bearing a fusogenic or a nuclear localization peptide head group to the particles does not significantly improve an already efficient system, in contrast to polylysine-based gene transfer methods that rely on lysosomotropic or fusogenic agents to be effective. This emphasizes the distinctive properties of the lipopolyamines, including cell membrane destabilization, endosome buffering capacity, and possibly nuclear tropism. Most importantly, addition of lipids with a triantennary galactosyl residue drives the neutral nucleolipidic particles to the asialoglycoprotein receptor of human hepatoma HepG2 cells: Transfection increases approximately 1000-fold with 25% galactolipid. This receptor-mediated process is saturable and slightly less efficient than receptor-independent transfection obtained in vitro with a large excess of cationic lipid alone. Yet, electrically silent particles may provide an attractive solution for gene transfer in vivo where their external saccharide coat should allow them to diffuse within the organism and reach their target cells.

Animals↗

PubMed1995

Versatile synthesis of bi- and tri-antennary galactose ligands: interaction with the Gal/GalNAc receptor of human hepatoma cells.

We have synthesized bi- and tri-antennary galactose ligands by coupling 1-thio-beta-D-galactose derivatives to the alpha- and epsilon-amino groups of L-lysine and L-lysyl-L-lysine via highly flexible hydrophilic spacer arms that allow variation of their intergalactose distances. The interaction of these ligands with the Gal/GalNAc receptor of HepG2 cells showed a binding affinity that was: (i) in agreement with the clustering effect known to occur with more complex oligomeric structures, i.e. tri- > bi-antennary; ii) dependent on the intergalactose distances (optimal interactions were observed for the tri-antennary structures with distances > 2 nm). These ligands, that can be easily conjugated to bioactive (macro) molecule carrier systems, could be useful for their targeting to hepatocytes.

Animals↗

PubMed1994

Mechanistic implications of cyclic ADP-ribose hydrolysis and methanolysis catalyzed by calf spleen NAD+glycohydrolase.

Calf spleen NAD+glycohydrolase, besides its well known reactions, was shown to catalyze hydrolysis and methanolysis (with retention of configuration) of cyclic ADP-ribose, indicating that this classical NADase also belongs to the class of cyclic ADP-ribose hydrolases. No formation of cyclic ADP-ribose could be detected during the hydrolysis of beta-NAD+; moreover, the kinetic parameters of cyclic ADP-ribose seem to rule out that it is a kinetically competent reaction intermediate in the conversion of NAD+ into ADP-ribose by this enzyme.

Adenosine Diphosphate Ribose↗

PubMed1994

Effects of a 2,3-oxidosqualene-lanosterol cyclase inhibitor 2,3:22,23-dioxidosqualene and 24,25-epoxycholesterol on the regulation of cholesterol biosynthesis in human hepatoma cell line HepG2.

N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol (8-azadecalin 1), a high-energy intermediate analogue for the 2,3-oxidosqualene-lanosterol cyclase, was found to be a powerful (IC50 approximately 0.1 microM) inhibitor of cholesterol biosynthesis in human hepatoma HepG2 cells. In analogy with other mammalian cells grown in the presence of cyclase inhibitors, the decrease in C27-sterol formation was accompanied by an accumulation of 2,3-oxidosqualene, 2,3:22, 23-dioxidosqualene, and by the formation of a compound characterized as 24,25-epoxycholesterol, a repressor of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase activity. In order to assess the cyclase as a potential pharmacological target for the design of hypocholesterolemic drugs, it is important to test whether inhibitors of this enzyme are able to act synergistically on the biosynthesis of cholesterol, i.e. by decreasing the amount of lanosterol formed and by repressing the regulatory HMG-CoA reductase via the formation of regulatory oxysterols. The accumulation of 24,25-epoxycholesterol in relationship to the decrease of C27-sterol biosynthesis and of HMG-CoA reductase activity showed only a partial correlation: e.g. at [1] = 100 x IC50 only a 50% reduction in enzyme activity could be attained. In contrast, when HepG2 cells were treated with 2,3:22,23-dioxidosqualene or 24,25-epoxycholesterol, excellent correlations were found between the inhibition of C27-sterol biosynthesis and the repression of HMG-CoA reductase activity, which was almost complete at the highest concentrations of these epoxides (10(-5) M). Altogether, our results suggest that treatment of HepG2 cells with a cyclase inhibitor such as 8-azadecalin (1) does not lead to an intracellular accumulation of repressor molecules high enough to fully trigger a regulatory pathway resulting in a complete down-regulation of HMG-CoA reductase. At intermediary concentrations of cyclase inhibitors (IC50), however, a synergistic mode of action of these inhibitors seems plausible.

Cholesterol↗

PubMed1994

Selective induction of protection against influenza virus infection in mice by a lipid-peptide conjugate delivered in liposomes.

We have previously reported (Muller et al. Vaccine 1990, 8, 308) that two cyclic peptide analogues called D loop and K loop, corresponding to residues 139-147 in site A of the haemagglutinin (HA) of influenza A virus (strain X31), were both able to provide protective immunity to infected OF1 mice when administered in the form of peptide-ovalbumin conjugates. The predicted conformation of the D loop is nearly identical to that of the native loop known from the X-ray structure of HA, while the predicted conformation of the K loop differs significantly from the native one. In this study, the two peptides were conjugated to small unilamellar liposomes, thus creating a chemically defined immunogen, and OF1 mice were immunized with these liposomes containing monophosphoryl lipid A as adjuvant. Compared with protein carrier systems, the liposomal preparations are completely synthetic and avoid the use of Freund's adjuvant. By using liposomes associated with the D loop, we were able to achieve 70% protection of the mice against intranasal challenge with the influenza virus while no protection was obtained with the liposome-associated K loop. The difference in effect between the two liposome and ovalbumin carrier systems may result from the induction of different structures in the peptides when coupled to lipid anchors than when coupled to proteins.

Administration, Intranasal↗

PubMed1993

Regio- and stereoselectivity of cytochrome P-450 and peroxygenase-dependent formation of cis-12,13-epoxy-9(Z)-octadecenoic acid (vernolic acid) in Euphorbia lagascae.

Two oxygenases associated with microsomes prepared from Euphorbia lagascae developing seeds were found to convert linoleic acid into cis-12,13-epoxy-9(Z)-octadecenoic acid (vernolate): a cytochrome P-450 and a peroxygenase. The cytochrome P-450 dependent epoxidation is characterized by a remarkable regio- and enantioselectivity, i.e. only the 12(S),13(R)-enantiomer is formed in the endosperm. In germinating seeds, peroxygenase was active but no cytochrome P-450 epoxidase could be detected. Moreover, because of the very high enantioselectivity of the fatty acid epoxide hydrolase, which is also found in these tissues and which preferentially hydrates the 12(R),13(S)-epoxide enantiomer, 12(S),13(R)-epoxy-9(Z)-octadecenoic acid is the only isomer which can accumulate in E. lagascae.

Biotransformation↗

PubMed1993

Lyophilized liposomes as shelf items for the preparation of immunogenic liposome-peptide conjugates.

The conjugation of peptides to liposomes, while relatively simple, does represent a technical hurdle to some laboratories wishing to use such liposomes as vehicles to render peptides immunogenic. For such laboratories, having liposomes available as shelf items instead of having to prepare liposomes each time they are required may make liposomes more attractive. In this paper we report that small unilamellar liposomes bearing maleimide groups at their surface, and containing the adjuvant monophosphoryl lipid A, can be lyophilized and stored without loss of functionality or biological activity. On rehydration, peptides bearing cysteine residues are readily attached and the resulting conjugates exhibit the same immunogenicity as freshly prepared liposomes. These constructs avoid the use of protein carriers and also the use of Freund's adjuvant for raising antibodies against peptides.

Amino Acid Sequence↗

PubMed1993

Mechanism of reaction of fatty acid hydroperoxides with soybean peroxygenase.

13(S)-Hydroperoxyoctadeca-9(Z),11(E),15(Z)-trienoic acid (13-HPOT) was used to probe the mechanism of the hydroperoxide O-O bond cleavage catalyzed by solubilized and partially purified soybean peroxygenase. When reacted with this ferrihemoprotein, it was converted to 13(S)-hydroxyoctadeca-9(Z),11(E), 15(Z)-trienoic acid (13-HOT) and a single epoxide regio-isomer, i.e. 15,16-cis-epoxy-13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid (15,16-EHOD). In the absence of co-oxidizable substrates, such as oleic acid or thiobenzamide, this latter compound accounted for about two-thirds of the reaction products. 13-HOT and 15,16-EHOD are products of heterolytic scission of the O-O bond of 13-HPOT; no products arising by homolytic scission could be detected. Therefore, soybean peroxygenase catalyzes hydroperoxide reduction exclusively by a heterolytic mechanism leading to a ferryl-oxo complex analogous to peroxidase compound I. In similar experiments, 13(S)-hydroperoxyoctadeca-9(Z),11(E)-dienoic acid gave 13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid and 9,10 epoxy-13(S)-hydroxyoctadec-11(E)-enoic acid. Experiments with 18O-labeled 13-HPOT indicated that about 83% of the oxygen atom incorporated into the epoxide group of 15,16-EHOD, originated from the hydroperoxide group. Moreover, using mixtures of unlabeled and 18O-labeled 13-HPOT, it was established that this transfer takes place predominantly (about 3:1) by an intramolecular process. In the intermolecular reaction 13-HOT, formed after reduction of the hydroperoxide, diffuses from the active site and, after reassociation, is epoxidized at the 15,16-double bond. A unifying mechanistic scheme, which takes into account all of the reactions catalyzed by the peroxygenase, is proposed.

Chromatography, Gas↗

PubMed1993

Calf spleen NAD+ glycohydrolase: solubilization, purification, and properties of the intact form of the enzyme.

NAD+ glycohydrolase was solubilized from calf spleen microsomes with emulphogene, a nonionic detergent, and purified to apparent homogeneity by ion-exchange chromatographies and by affinity chromatography on Affi-Gel blue gel. In contrast to the hydrosoluble form of the enzyme, which can be obtained by a proteolytic treatment of the microsomes with steapsin, the intact form of NAD+ glycohydrolase is characterized by its high hydrophobicity; i.e., the enzyme interacts very strongly with hydrophobic gels such as octyl-Sepharose and partitions into Triton X-114-rich phases. The apparent M(r) of the intact form of calf spleen NAD+ glycohydrolase is about 30 kDa, as compared to 24 kDa for the hydrosoluble form. This difference in molecular mass could account for the polypeptide moiety which allows the anchoring of the enzyme to the membranes. Both forms of the enzyme are strongly adsorbed by immobilized concanavalin A gels and biospecifically eluted with alpha-methylmannoside; this glycoprotein (mannosylated) nature of NAD+ glycohydrolase is in agreement with the previous demonstration that it is an ectoenzyme.

Animals↗

PubMed1993

Induction of immune response against a short synthetic peptide antigen coupled to small neutral liposomes containing monophosphoryl lipid A.

We have investigated the parameters affecting the immunogenicity of a short synthetic hexapeptide associated with liposomes. The model peptide used had the sequence IRGERA which corresponds to the C-terminal hexapeptide region of histone H3. Immunogenicity was measured by the ability of anti-peptide antibodies to cross-react with the parent protein. By itself, the peptide was not able to induce significant antibody production. However, liposomes were shown to be able to render the peptide immunogenic, nevertheless a number of parameters were important: to be immunogenic the peptide had to be surface bound, rather than entrapped within the liposomes, and an adjuvant, monophosphoryl lipid A (MPLA), had to be present in the same population of liposomes. Additionally, the intensity and duration of the immune response were found to be dependent both on the charge of the liposomes; neutral liposomes yielding a longer lasting response than negatively charged liposomes, and on the immunisation schedule where a long time period between immunisation and boosting yielded a better result than a short time period. To account for these phenomena we propose a model in which surface-bound antigen targets liposomal MPLA to B lymphocytes specific for the antigen. These results demonstrate that liposomes containing the non-toxic adjuvant MPLA can act as carriers to induce a long-lasting IgG response against peptides, eliminating the need of protein carriers and conventional adjuvants. Such an approach may be useful for designing synthetic vaccines.

Adjuvants, Immunologic↗