Biomedical subjects

M Vandenbranden

Publications and source records attributed to M Vandenbranden.

At least 19 recordsLinked to original sources

PubMed2000

Biophysical and structural properties of DNA.diC(14)-amidine complexes. Influence of the DNA/lipid ratio.

Cationic liposomes are used as vectors for gene delivery both in vitro and in vivo. Comprehension of both DNA/liposome interactions on a molecular level and a description of structural modifications involved, are prerequisites to an optimization of the transfection protocol and, thus, successful application in therapy. Formation and stability of a DNA/cationic liposome complex were investigated here at different DNA:lipid molar ratios (rho). Isothermal titration calorimetry (ITC) of cationic liposomes with plasmid DNA was used to characterize the DNA-lipid interaction. Two processes were shown to be involved in the complex formation. A fast exothermic process was attributed to the electrostatic binding of DNA to the liposome surface. A subsequent slower endothermic reaction is likely to be caused by the fusion of the two components and their rearrangement into a new structure. Fluorescence and differential scanning calorimetry confirmed this interpretation. A kinetic model analyzes the ITC profile in terms of DNA/cationic liposome interactions.

Amidines↗

PubMed2000

Tissue distribution and interindividual variation in human UDP-glucuronosyltransferase activity: relationship between UGT1A1 promoter genotype and variability in a liver bank.

The variability in a liver bank and tissue distribution of three probe UDP-glucuronosyltransferase (UGT) activities were determined as a means to predict interindividual differences in expression and the contribution of extrahepatic metabolism to presystemic and systemic clearance. Formation rates of acetaminophen-O-glucuronide (APAPG), morphine-3-glucuronide (M3G), and oestradiol-3-glucuronide (E3G) as probes for UGT1A6, 2B7, and 1A1, respectively, were determined in human kidney, liver, and lung microsomes, and in microsomes from intestinal mucosa corresponding to duodenum, jejunum and ileum. While formation of E3G and APAPG were detectable in human kidney microsomes, M3G formation rates from kidney microsomes approached the levels seen in liver, indicating significant expression of UGT2B7. Interestingly, rates of E3G formation in human intestine exceeded the hepatic rates by several fold, while APAPG and M3G formation rates were low. The intestinal apparent Km value for E3G formation was essentially identical to that seen in liver, consistent with intestinal UGT1A1 expression. No UGT activities were observed in lung. Variability in APAPG and M3G activity across a bank of 20 human livers was modest (< or = 7-fold), compared to E3G formation, which varied approximately 30-fold. The E3G formation rates were found to segregate by UGT1A1 promoter genotype, with wild-type (TA)6 rates significantly greater than homozygous mutant (TA)7 individuals. Kinetic analyses were performed to demonstrate that the promoter mutation altered apparent Vmax without significantly affecting apparent Km. These results suggest that glucuronidation, and specifically UGT1A1 activity, can profoundly contribute to intestinal first pass metabolism and interindividual variability due to the expression of common allelic variants.

Acetaminophen↗

PubMed1999

Occurrence of an HIV-1 gp160 endoproteolytic activity in low-density vesicles and evidence for a distinct density distribution from endogenously expressed furin and PC7/LPC convertases.

Human immunodeficiency virus (HIV) glycoprotein (gp) 160 processing by host cell proteinases is an essential step for viral fusion and infectivity. We have identified a rat liver subcellular fraction which specifically processes gp160 into gp120 and gp41. Using equilibration of microsomes in sucrose gradients, the gp160 cleavage activity was associated with particles equilibrating at low densities, well-separated from the endoplasmic reticulum, cis-Golgi network, Golgi stacks, lysosomes and plasma membrane. Its density distribution was compatible with light secretory vesicles derived from the trans-Golgi network (TGN) or to endosomes, but association with endosomes was not supported by free flow electrophoresis. Although furin and pro-protein convertase (PC) 7/LPC have been proposed as the major gp160 processing convertases, the rat liver microsomal gp160 processing activity was essentially resolved from furin and only partially overlapped PC7/LPC. These data suggest that proteinase(s) other than furin and PC7/LPC, presumably located in TGN-derived vesicles, may participate in the gp160 processing into gp120 and gp41.

Amino Acid Sequence↗

PubMed1999

Intracellular visualization of BrdU-labeled plasmid DNA/cationic liposome complexes.

Difficulties in specific detection of transfected DNA in cells represent an important limitation in the study of the gene transfer process. We studied the cellular entry and fate of a plasmid DNA complexed with a cationic lipid, Vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine) in BHK21 cells. To facilitate its detection inside the cells, bromodeoxyuridine (BrdU) was incorporated into plasmid DNA under conditions that minimize plasmid alteration. BrdU was localized in cells incubated with Vectamidine/BrdU-labeled plasmid DNA complexes by immunogold labeling and electron microscopy (EM). Labeling was predominantly associated with aggregated liposome structures at the surface of and inside the cells. EM observations of cells transfected with Vectamidine/DNA complexes showed that the liposome/DNA aggregates accumulate in large vesicles in the cell cytosol. On the other hand, using rhodamine-labeled Vectamidine and revealing BrdU with FITC-conjugated antibodies permitted simultaneous detection in the cells of both components of the complexes with confocal laser scanning microscopy. The DNA and lipids co-localized at the surface of and inside the cells, indicating that the complex is internalized as a whole. Our results show that the BrdU-labeled plasmid DNA detection system can be a useful tool to visualize exogenous DNA entry into cells by a combination of electron and confocal microscopy.

Amidines↗

PubMed1999

Three-dimensional quantitative structure activity relationship analyses of substrates for CYP2B6.

To begin to build an understanding of the interactions of CYP2B6 with substrates, two different 3-dimensional quantitative structure activity relationship (3D-QSAR) models were constructed using 16 substrates of B-lymphoblastoid expressed CYP2B6. A pharmacophore model was built using the program Catalyst, which was compared with a partial least-squares (PLS) model using molecular surface-weighted holistic invariant molecular (MS-WHIM) descriptors. The Catalyst model yielded a 3-dimensional model of the common structural features of CYP2B6 substrates, whereas PLS MS-WHIM generated a model based on statistical analyses of molecular descriptors for size and shape of the substrate. The pharmacophore model obtained with Catalyst consisted of three hydrophobes and one hydrogen bond acceptor region. The cross-validated PLS MS-WHIM model gave a good q2 value of 0.607. Size, positive electrostatic potential, hydrogen bonding acceptor capacity, and hydrophobicity were found to be the most relevant descriptors for the model. These models were then used to predict the Km (apparent) values of a test set of structurally diverse substrates for CYP2B6 not included in the model building, specifically lidocaine, amitriptyline, bupropion, arteether, and verapamil. Overall, both 3D-QSAR methods yielded satisfactory Km (apparent) value predictions for the majority of the molecules in the test set. However, PLS MS-WHIM was unable to reliably predict the Km (apparent) value for verapamil, whereas Catalyst did not predict the Km (apparent) value for lidocaine. In both of these cases the residual of the Km (apparent) value was greater than one log unit. The strengths and limitations of both of these 3D-QSAR approaches are discussed.

Aryl Hydrocarbon Hydroxylases↗

PubMed1998

Furin and proprotein convertase 7 (PC7)/lymphoma PC endogenously expressed in rat liver can be resolved into distinct post-Golgi compartments.

The intracellular compartmentalization in rat liver of the membrane-associated convertases furin and proprotein convertase 7 (PC7)/lymphoma PC (LPC) was investigated by analytical subcellular fractionation. In control animals, both enzymes were found to localize in fractions depleted of endoplasmic reticulum, cis-Golgi and lysosomal markers, but to co-distribute with the Golgi marker galactosyltransferase and the trans-Golgi network (TGN) marker TGN38. After overloading Golgi-derived vesicles with very-low-density lipoproteins (VLDL) by feeding rats with ethanol, the distribution of PC7/LPC was shifted markedly towards lower densities, in contrast with those of furin and the TGN marker. This provides support for the TGN localization of endogenously expressed furin and indicates that, at steady state, a considerable proportion of PC7/LPC may be associated with vesicles derived from the TGN.

Animals↗

PubMed1998

Physico-chemical characterization of a double long-chain cationic amphiphile (Vectamidine) by microelectrophoresis.

We recently synthesized a novel cationic amphiphile (N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine or Vectamidine (previously described as diC14-amidine)) that associates with DNA and RNA and facilitates their entry and expression into eukaryotic cells. Among several parameters that have been shown to influence the transfection process, the surface charge density plays a key role. Quantitative information about that charge density associated to the cationic amphiphiles organized in liposomal structure is not yet available. We provide here evidence by titration and microelectrophoresis measurements that an evaluation of the intrinsic acidity constants, the surface pH and the counterion binding constants allows to determine the charge density at physiological pH of Vectamidine liposomes. The knowledge of this superficial charge is a prerequisite to a molecular understanding of the DNA-cationic amphiphile complex formation. The method described could be extended to any kind of cationic amphiphile.

Cations↗

PubMed1998

Liquid chromatography/nuclear magnetic resonance spectroscopy and liquid chromatography/mass spectrometry identification of novel metabolites of the multidrug resistance modulator LY335979 in rat bile and human liver microsomal incubations.

Compound LY335979 is a P-glycoprotein inhibitor currently entering phase I clinical trials for potential reversal of multidrug resistance to cancer chemotherapy. In early exploratory studies, LY335979 was found to be rapidly transformed in incubations with liver microsomes from rats, dogs, monkeys, and humans. Although the parent compound was completely metabolized, no prominent metabolite peaks were observed. One peak did appear early in the time course, but it did not increase over time. In another preliminary experiment, rats were treated iv with [3H]LY335979 (prepared for pharmacology studies), and urine and bile fractions were collected. Analysis of the urine by reverse-phase HPLC with UV and radioactivity detection revealed that almost all of the material eluted with the solvent front. More than half the radioactivity in bile was accounted for by two peaks eluting earlier than the parent compound (the rest eluted at the solvent front). With both bile and the incubations with microsomes, initial attempts to isolate metabolites were not successful. There was also evidence in both systems of products derived from cleavage of LY335979 (by both further metabolism and degradation). LC/NMR was thus used to analyze materials directly in their respective matrices. An N-oxide metabolite (LY389551) formed by oxidation of the quinoline nitrogen was identified in the microsomal incubations; in bile, three glucuronide metabolites were identified, all of which were conjugates of products formed by oxidation of the quinoline ring of LY335979. There have been few reports in the literature of LC/NMR analysis of bile, which is a more complex matrix than either urine or microsomal suspensions. However, the HPLC techniques developed in this work for the HPLC/UV and LC/MS analyses of LY335979 metabolites in the microsomal matrix and in bile proved readily adaptable for LC/NMR. Using a 500-MHz instrument, basic 1H NMR spectra could be obtained in 2-3 hr with approximately 100 ng of material in the LC/NMR microprobe. With approximately 1.5 microg of material injected onto the column, 1H-1H correlation spectroscopy spectra could be acquired overnight. Along with LC/MS data, the LC/NMR technique facilitated direct identification of a number of metabolites of LY335979 at a point at which their identification by traditional methods would not have been pursued.

Animals↗

PubMed1998

Further characterization of the expression in liver and catalytic activity of CYP2B6.

Previous studies in this laboratory have determined the lack of specificity of several antibody and substrate probes of CYP2B6. The goals of the current study were to examine the expression of CYP2B6 in a bank of human liver microsome (HLM) samples using a new specific monoclonal antibody (MAb 49-10-20) and to further characterize the substrate specificity of CYP2B6. A 100-fold variability in expression of immunodetectable CYP2B6 was demonstrated in a bank of 19 HLM samples (0.7 pmol/mg protein to 71. 1 pmol/mg protein) using MAb 49-10-20. CYP2B6 levels were found to significantly (P < .0001) correlate with S-mephenytoin N-demethylation to nirvanol (r2 = 0.89), 7-hydroxy-4-trifluoromethylcoumarin formation (r2 = 0.81) and several markers of CYP3A levels and activity. The relationships between nirvanol formation and CYP3A levels or activity were found to depend on two HLM samples. Km (apparent) values were generated for benzyloxyresorufin O-deethylation (1.3 microM), benzphetamine N-demethylation (93.4 microM), 3-cyano 7-ethoxycoumarin O-deethylation (71.3 microM), midazolam 1'-hydroxylation (46.1 microM) and 4-chloromethyl-7-ethoxycoumarin O-deethylation (33.7 microM) using expressed CYP2B6. Testosterone 16beta-hydroxylation by expressed CYP2B6 resulted in atypical kinetics characteristic of substrate activation. The data best fit the Hill equation with a Km (apparent) of 50.5 microM and an n of 1.3 (n = number of sites bound by activator). In conclusion, the highly specific MAb 49-10-20 was used to provide further confirmation that S-mephenytoin N-demethylation to nirvanol is a CYP2B6 selective probe. Finally, some, but not all substrates of CYP2B6 demonstrate autoactivation.

Antibodies, Monoclonal↗

PubMed1997

The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids.

We used a 32P-labeled pCMV-CAT plasmid DNA to estimate the DNA uptake efficiency and unlabeled pCMV-CAT plasmid DNA to quantify the CAT activity after transfection of COS cells using each of the three following cationic compounds: [1] vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine, and previously described as diC14-amidine [1]), [2] lipofectin (a 1:1 mixture of N-(1-2,3-dioleyloxypropyl)-N,N,N-triethylammonium (DOTMA) and dioleylphosphatidylethanolamine (DOPE)), and [3] DMRIE-C (a 1:1 mixture of N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammonium bromide (DMRIE) and cholesterol). Surprisingly, a high CAT activity was observed with vectamidine although the DNA uptake efficiency was lower as compared to lipofectin and DMRIE-C. Transmission electron microscopy (TEM) revealed endocytosis as the major pathway of DNA-cationic lipid complex entry into COS cells for the three cationic lipids. However, the endosomal membrane in contact with complexes containing vectamidine or DMRIE-C often exhibited a disrupted morphology. This disruption of endosomes was much less frequently observed with the DNA-lipofectin complexes. This comparison of the three compounds demonstrate that efficient transfection mediated by cationic lipids is not only correlated to their percentage of uptake but also to their ability to destabilize and escape from endosomes.

Amidines↗

PubMed1997

Human immunodeficiency virus type-1-specific immune responses induced by DNA vaccination are greatly enhanced by mannan-coated diC14-amidine.

Use of mannan-coated N-t-butyl-N'-tetradecyl-3-tetradecylamino-propionamidine (diC14-amidine) as an adjuvant for a DNA vaccine encoding glycoprotein 160 of human immunodeficiency virus type-1 (HIV-1) enhanced the antigen-specific immune responses. The role of interferon-gamma (IFN-gamma) and interleukin-12 in the mechanism of adjuvant action was also evaluated. Coating of diC14-amidine with mannan significantly augmented the HIV-specific delayed-type hypersensitivity reaction induced by the immunogenic DNA. HIV-1-specific cytotoxic T lymphocyte activity was also markedly enhanced by the mannan-diC14-amidine cocktail. An immunomodulatory effect of this cocktail was inhibited by treatment with anti-IFN-gamma monoclonal antibody in vivo, which suggests that IFN-gamma plays an important role in inducing cell-mediated immunity by the DNA vaccine containing this adjuvant. The results of both antigen-specific immunoglobulin isotype analysis and cytokine measurement showed that the immunogenic DNA incorporated into mannan-coated diC14-amidine elicits Th1-biased immune responses.

AIDS Vaccines↗

PubMed1996

Double long-chain amidine liposome-mediated self replicating RNA transfection.

We present experimental evidence that a complex made of a double long chain cationic amphiphile and recombinant mRNA facilitates the entry and expression of genetic material into cells. Combining the properties of the self replicating recombinant mRNA driven by the Semliki Forest Virus (SFV) replicon and the transfection potentialities of a new cationic amphiphile (N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine) yields a highly efficient mRNA transfection system conferring up to 100% infectivity. The preparation and characterization of the long chain amidine cationic amphiphile-mRNA complex as well as the influence of the diC14-amidine/RNA ratio on the infective activity are described.

Amidines↗

PubMed1996

In vitro interaction of the antipsychotic agent olanzapine with human cytochromes P450 CYP2C9, CYP2C19, CYP2D6 and CYP3A.

1. The ability of olanzapine to inhibit the metabolism of marker catalytic activities for the cytochromes P450 CYP3A, CYP2D6, CYP2C9, and CYP2C19 was examined. This inhibitory capability was compared with that obtained with clozapine and known inhibitory compounds for the same cytochromes P450. 2. Olanzapine, clozapine, and ketoconazole were all found to non-competitively inhibit 1'-hydroxy midazolam formation, form selective for CYP3A, yielding Ki values of 491, 99 and 0.11 microM, respectively. The 1'-hydroxylation of bufuralol, form selective for CYP2D6, was competitively inhibited by olanzapine (Ki = 89 microM), clozapine (Ki = 19 microM), and quinidine (Ki = 0.03 microM). Tolbutamide metabolism to 4-hydroxy tolbutamide, form selective for CYP2C9, was competitively inhibited by clozapine and phenytoin (Ki of 31 microM and 17 microM, respectively). Olanzapine non-competitively inhibited tolbutamide metabolism with a Ki of 715 microM. The marker catalytic activity for CYP2C19 mediated metabolism, 4'-hydroxy S-mephenytoin formation, was competitively inhibited by clozapine (Ki = 69 microM) and omeprazole (Ki = 4.1 microM). Non-competitive inhibition of CYP2C19 mediated metabolism was seen with olanzapine with a Ki of 920 microM. 3. The calculated percent inhibition by olanzapine of substrates metabolized by CYP3A, CYP2D6, CYP2C9, and CYP2C19 was modeled assuming a total plasma concentration in the therapeutic range (0.2 microM). Total olanzapine vs unbound olanzapine was used to model the worst case (most conservative) situation. In all cases, the calculated percent inhibition of these cytochromes P450 by olanzapine was < 0.3%, suggesting that there would be little in vivo inhibition of the metabolism of substrates of these enzymes when co-administered with olanzapine.

Antipsychotic Agents↗

PubMed1996

Bimodal distribution of renal cytochrome P450 3A activity in humans.

It has been proposed that excessive intrarenal conversion of cortisol to 6 beta-hydroxycortisol by CYP3A may mediate increased tubular reabsorption of sodium, leading to a state of mild volume expansion and the clinical phenotype of salt-sensitive hypertension. Therefore, we characterized CYP3A activity in a bank of microsomes from human kidneys using the formation of 1'-hydroxymidazolam (1'-OHM) as a prototypical CYP3A-catalyzed reaction. Maximal rates of metabolite formation occurred at midazolam concentrations of 12.5-50 microM; higher concentrations resulted in dramatic substrate inhibition. At 12.5 microM midazolam, 4 of 27 kidneys exhibited relatively high mean +/- standard deviation 1'-OHM formation rate (184.0 +/- 14.4 pmol/hr/mg) compared with the remaining 23 samples, which had a mean formation rate of (10.1 +/- 6.4 pmol/hr/mg). Triacetyloleandomycin and anti-CYP3A antibody inhibited midazolam hydroxylation by 53% and 57%, respectively. The correlation between CYP3A5 content, determined through immunoblotting, and 1'-OHM formation rate was high (r2 = 0.84, 24 experiments). The expressions of mRNA corresponding to CYP3A3, CYP3A4, CYP3A5, and CYP3A7 were determined through polymerase chain reaction with specific oligonucleotides as primers. All kidneys examined (25 experiments) expressed CYP3A5 protein and contained the corresponding CYP3A5 mRNA. CYP3A4 mRNA was detected in 40% of the kidney samples, and 70% of those that contained detectable CYP3A4 mRNA also expressed detectable levels of the corresponding protein. Therefore, in contrast to hepatic tissue, in which CYP3A4 is universally expressed, CYP3A5 is the ubiquitously expressed member of the CYP3A family in renal tissue. The distribution of enzyme activity and protein content suggests bimodality and may represent induction of CYP3A5 in a select population and/or a genetically determined organ-specific pattern of expression.

Antibodies↗

PubMed1994

A novel cationic amphiphile for transfection of mammalian cells.

We describe here a new cationic amphiphile, N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), which interacts with plasmid DNA and generates hydrophobic stable complexes resistant against DNase I. In partition experiments between two non-miscible phases, DNA was transferred into an organic phase upon complex formation with diC14-amidine-containing vesicles. Finally, vesicles made of a diC14-amidine and phosphatidylethanolamine (PE) (1:1, mol:mol) mixture or pure diC14-amidine were efficient in mediating transfection of adherent (CHO) and suspension (K562) cell lines, using the chloramphenicol acetyltransferase (CAT) gene as reporter.

Amidines↗

PubMed1994

The convertases furin and PC1 can both cleave the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp160 into gp120 (HIV-1 SU) and gp41 (HIV-I TM).

Intracellular proteolytic processing of human immunodeficiency virus envelope glycoprotein precursor (gp160) is an essential step for virus infectivity. Northern blot analysis provided evidence that furin and PC1, but not PC2, are expressed in the CD4+ human lymphoblastoid H9 cell line, suggesting the possible participation of these convertases in human immunodeficiency virus (HIV) gp160 proteolytic processing. Purified PC1 and furin cleaved specifically in vitro gp160 into gp120 (HIV-I SU) and gp41 (HIV-I TM). NH2-terminal sequence analysis of the produced gp41 (HIV-I TM) demonstrated that the cleavage occurred within the sequence Arg-Glu-Lys-Arg decreases Ala-Val-Gly-Ile, which is identical to the bond cleaved in vivo. Transition state analog peptides were designed and tested in vitro for their ability to inhibit the PC1- or furin-mediated gp160 cleavage. The best inhibitor was decanoyl-Arg-Lys-Arg-Arg-psi [CH2NH]-Phe-Leu-Gly-Phe-NH2.

Amino Acid Sequence↗

PubMed1993

Orientation and structure of the NH2-terminal HIV-1 gp41 peptide in fused and aggregated liposomes.

For several retroviruses, the N-terminal hydrophobic sequence of the viral envelope glycoprotein has been shown to play a crucial role in the interaction between the virus and the host cell membrane. We report here on the interaction of a synthetic 16 residues peptide corresponding to the gp41 NH2-terminal sequence of Human Immunodeficiency Virus with the phospholipid bilayer. Fluorescence energy transfer measurements show that this peptide can induce lipid mixing of large unilamellar vesicles (LUV) of various compositions at pH 7.4 and 37 degrees C. LUV undergo fusion, provided they contained phosphatidylethanolamine (PE) in their lipid composition. To provide insight into the mechanism of the fusion event, the peptide secondary structure and orientation in the lipid bilayer were determined using Fourier Transform Infrared Spectroscopy (FTIR). The peptide adopts mainly a beta-sheet conformation in the absence of lipids. After interaction with LUV the beta-sheet is partly converted into alpha-helix. The orientation of the peptide with respect to the lipid acyl chains depends on the presence of PE in the lipid bilayer. The peptide is inserted into the lipid bilayer with the helix axis oriented parallel to the lipid acyl chains in the fused vesicles, whereas it is adsorbed parallel to the lipid/water interface in the aggregated vesicles. The role of the two kinds of orientation during the fusion event is discussed.

Amino Acid Sequence↗

PubMed1993

Secondary structure of gp160 and gp120 envelope glycoproteins of human immunodeficiency virus type 1: a Fourier transform infrared spectroscopic study.

The secondary structure of the precursor (gp160) of the envelope protein of human immunodeficiency virus type 1 (BH10) and its receptor-binding subunit (gp120) was studied by Fourier-transformed attenuated total reflection spectroscopy. A higher alpha-helix/beta-sheet ratio in the gp120 subunit than in the precursor indicates a structural heterogeneity between the two subunits (gp120 and gp41), in agreement with classical secondary-structure predictions. The secondary structure of gp41 was estimated and compared with existing models. The high alpha-helical content in gp41 and the dominant beta-sheet content in gp120 resemble the distribution in influenza virus hemagglutinin subunits.

CD4 Antigens↗