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D Scherman

Publications and source records attributed to D Scherman.

At least 73 records · Page 4Linked to original sources
PubMed1997

A new DNA vehicle for nonviral gene delivery: supercoiled minicircle.

Plasmids currently used for nonviral gene transfer have the disadvantage of carrying a bacterial origin of replication and an antibiotic resistance gene. There is, therefore, a risk of uncontrolled dissemination of the therapeutic gene and the antibiotic resistance gene. Minicircles are new DNA delivery vehicles which do not have such elements and are consequently safer as they exhibit a high level of biological containment. They are obtained in E. coli by att site-specific recombination mediated by the phage lambda integrase. The desired eukaryotic expression cassette, bounded by the lambda attP and attB sites was cloned on a recombinant plasmid. The expression cassette was excised in vivo after thermoinduction of the integrase gene leading to the formation of two supercoiled molecules the minicircle and the starting plasmid lacking the expression cassette. In various cell lines, purified minicircles exhibited a two- to 10-fold higher luciferase reporter gene activity than the unrecombined plasmid. This could be due to either the removal of unnecessary plasmid sequences, which could affect gene expression or the smaller size of mini-circle which may confer better extracellular and intracellular bioavailability and result in improved gene delivery properties.

Cell Line↗
PubMed1996

Synthesis of glucose-chlorambucil derivatives and their recognition by the human GLUT1 glucose transporter.

A limitation of the use of chemotherapeutic agents against intracerebral tumors lies on their poor uptake into the central nervous system. An approach to enhance brain delivery is to design agents that are transported into the brain by one of the saturable nutrient carriers of the blood-brain barrier, the highly efficient brain and erythrocyte glucose transporter isoform GLUT1. Since the GLUT1 hexose transporter of the blood-brain barrier is also present on erythrocytes, new compounds designed to be transported by the GLUT1 transporter were studied on human erythrocytes, which represent unique, easily accessible human GLUT1 expressing cells. In this paper we describe the synthesis of four glucose-chlorambucil derivatives, namely methyl 6-O-4[bis(2-chloroethyl)amino]benzenebut anoyl-beta-D-glucopyranosi de (3), 6-O-4-[bis(2-chloroethyl)amino]benzenebu tanoyl-D-glucopyranose (6), methyl 6-[4-[bis(2-chloroethyl)amino]benzenebut anoylamido]-6-deoxy-beta-D-glucopyranoside (9) and 6-[4-[bis(2-chloroethyl)amino]benzenebut anoyl amido]-6-deoxy-D-glucopyranose (10), and the study of their interactions with the GLUT1 transporter of the human erythrocytes. All four compounds were able to inhibit [14C]glucose uptake in a concentration-dependent manner. One of them, compound 6, exhibited an approximately 160-fold higher inhibition of [14C]glucose uptake by the GLUT1 transporter than glucose itself. Compound 6 was also able to inhibit [3H]cytochalasin B binding to erythrocytes with approximately 1000-fold higher efficacy than does glucose. The inhibition of glucose uptake was entirely reversible, indicating that it was not due to alkylation of a nucleophilic group of the hexose transporter. The above results suggested specific interactions of compound 6 with the hexose transporter protein. Uptake studies of [14C]compound 6 indicated, in addition, some non-specific interactions with intact and open erythrocyte membranes: only a small amount of the bound [14C]compound 6 can be displaced by cytochalasin B. Collectively, these findings led us to conclude that the interactions of compound 6 with GLUT1 are presumably that of a non-transported inhibitor.

Antineoplastic Agents, Alkylating↗
PubMed1996

Hexose keto-C-glycoside conjugates: design, synthesis, cytotoxicity, and evaluation of their affinity for the glucose transporter Glut-1.

The design, synthesis, cytotoxicity, and biological evaluation of carbohydrate/C-glycoside conjugates are described. The design concept is predicted on the idea that physiological barriers like the blood brain barrier could be crossed selectively by using glucose or glucose derivative/drug conjugates. The study demonstrates that, (1) carbohydrates and C-glycosides can be bonded at nonanomeric positions by the reaction of carbohydrate triflates with C-glycoside alkoxydes in the presence of DMPU; (2) there is a structure-activity relationship between the cytotoxicity of the conjugate and the nature of the carbohydrate residue; and (3) peracetylated hexose keto-C-glycoside conjugates are the most cytotoxic keto-C-glycosides.

Erythrocytes↗
PubMed1996

Functional expression of P-glycoprotein in an immortalised cell line of rat brain endothelial cells, RBE4.

The presence of P-glycoprotein in the cell plasma membrane limits the penetration of many cytotoxic substances into cells that express the gene product. There is considerable evidence also to indicate that P-glycoprotein is expressed as part of the normal blood-brain barrier in the luminal membranes of the cerebral capillary endothelial cells, where it presumably performs a protective function for the brain. This report describes the functional expression of P-glycoprotein in an immortalised cell line, RBE4, derived from rat cerebral capillary endothelial cells. The expression of P-glycoprotein is demonstrated by western immunoblotting and by immunogold and fluorescent staining with monoclonal antibodies. The cellular accumulation of [3H]colchicine and [3H]-vinblastine is investigated and shown to be enhanced by the presence of azidothymidine, chlorpromazine, verapamil, cyclosporin A, and PSC 833 ([3'-keto-Bmt1]-[Val2]-cyclosporin) at 50 or 100 microM concentration. It is concluded that the RBE4 cell line is a valuable tool for investigating the mechanisms of P-glycoprotein activity both in the blood-brain barrier and in multidrug resistance in general.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Synthesis of novel (N-farnesyl) amino acids and their incorporation into peptides.

N-Alkylation, and particularly N-farnesylation, of bioactive peptides might be of wide interest: first, to increase peptide bioavailability by decreasing their elimination and by favouring their transport through biological membranes; second, to target peptides to cellular membranes; and third, to generate therapeutic double-substrate inhibitors of enzymes such as ras-farnesyltransferase. We report the synthesis of novel N-farnesyl amino acids [(N-Frn) amino acids]. We have synthesized (N-Frn)MetOCH3, (N-Frn)ValOBz and (N-Frn)PheOCH3 by alkylation of the corresponding natural amino acid esters. In order to demonstrate the feasibility of the introduction of (N-Frn) amino acids into peptides, we have synthesized representative dipeptide analogs: Cys-(N-Frn)ValOBz, Phe-(N-Frn)ValOBz, Lys-(N-Frn)ValOBz, Phe-(N-Frn)MetOCH3, Glu-(N-Frn)Me OCH3, Ser-(N-Frn)MetOCH3, Trp-(N-Frn)PheOCH3 and Pro-(N-Frn)PheOCH3. We also describe the synthesis of the model peptide Cys-Val-Phe-(N-Frn)MetOCH3, which is derived from the tetrapeptide CysValPheMet inhibitor of human p21ras-farnesyl transferase.

Amino Acids↗
PubMed1996

Detection of the multidrug resistance of P-glycoprotein in healthy tissues: the example of the blood-brain barrier.

The blood-brain barrier is formed by the cerebral capillary endothelial cells, joined together by tight junctions. These cells express the general endothelial cell markers as well as specific markers found on endothelial cells forming physiological barriers such as gamma-glutamyltranspeptidase, the glucose transporter Glut1 and the neutral amino-acid transporter. Using the monoclonal antibodies C219 and MRK16, we have revealed by Western blot and immuno-histochemistry the expression of the multidrug resistance P-glycoprotein on isolated rat cerebral cortex capillaries. On the other hand, P-glycoprotein was not detectable in brain cortex homogenates. P-glycoprotein thus appears to be a blood-brain barrier endothelium-specific marker which could regulate brain penetration of xenobiotics and thus participate in the neuroprotection of the brain.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Gene transfer by naked DNA into adult mouse brain.

Nonviral gene transfer into the central nervous system could provide a basis for therapeutic uses and fundamental research. We show that naked DNA injected intracerebrally into the mouse brain can provide expression of a reporter protein. Expression is dose dependent, being maximal for 150 mu g DNA injected. We observed less than 5 days expression of the luciferase transgene, which is not improved with plasmid preparations virtually free of lipopolysaccharide. Thus, the adult brain behaves as striated muscle for naked DNA uptake and transcription, albeit at a much lower efficiency. In neither adult brain nor muscle did complexation of DNA with cationic lipid improve transgene expression. Double immunolabeling using cell-specific markers shows that both neurons and glia are transfected by naked DNA gene transfer methodology.

Animals↗
PubMed1995

A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.

Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se--i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its gene-delivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.

3T3 Cells↗
PubMed1995

Subcellular distribution of receptor sites in human brain: differentiation between heavy and light structures of high and low density.

Studies of the subcellular localization of neuroreceptors in the rat brain have shown that most of them are associated with light and low density subcellular fractions. In two human brain areas, quite different subcellular distributions were observed. After fractionation by differential centrifugation of frontal cortex homogenates, benzodiazepine and serotonin 5-HT2 receptors were mainly found in the heavy mitochondrial (M) fraction, whereas mu-opiate and muscarinic cholinergic receptors were mainly concentrated in the microsomal (P) fraction. In human putamen, the presynaptic markers of dopaminergic nerve terminals (neurotensin receptors, dopamine uptake sites and amine vesicular transporter-binding sites), benzodiazepine receptors and serotonin uptake sites were recovered both in the high and low density fractions, whereas the muscarinic, opiate and, to a lesser extent, dopamine D2 receptors were mostly concentrated in the microsomal fraction. In the cerebral cortex, after isopycnic centrifugation in sucrose gradients, neuroreceptors were found in the high density fractions where the peaks of cytochrome oxidase and that of nerve endings, as identified by amine uptake and by means of electron microscopy were also found. A single peak of benzodiazepine receptors was observed in high density (1.15-1.17 g/ml) fractions suggesting that these receptors are much more concentrated in the nerve terminals or dendrites rather than in the dendritic spines or vesicles. The fact that muscarinic and opiate receptors were recovered in the P fraction with plasma membrane constituents and also in M and L fractions, which is confirmed by a bimodal distribution in sucrose gradient, suggests that they are localized in both the nerve terminals or dendrites and in the small vesicles or dendritic spines. In the putamen, much of the specific binding to uptake sites for dopamine and serotonin was recovered in the high density fractions, but the existence of another peak at a lower density indicates the presence of microsomal uptake sites. The results indicate that differential and isopycnic fractionation methods performed on human brain samples, make it possible to separate tissue fractions enriched in nerve endings, dendrites, dendritic spines, plasma membranes or vesicles.

Binding, Competitive↗
PubMed1995

Interaction of pristinamycin IA with P-glycoprotein in human intestinal epithelial cells.

Pristinamycin IA is a cyclo-peptidic macrolactone antibiotic belonging to the streptogramin family. In the present work, the interaction of pristinamycin IA with the multidrug transporter P-glycoprotein was investigated in the differentiated human intestinal epithelial cell line Caco-2. Pristinamycin IA specifically inhibited the efflux of the P-glycoprotein substrate [3H]vinblastine, thus increasing the cellular accumulation of the drug. Pristinamycin IA also reduced by 70% the basolateral to apical secretion of [3H]vinblastine across Caco-2 cell monolayers. The cellular accumulation of [14C]pristinamycin IA was very low and was increased by P-glycoprotein inhibitors (verapamil, chlorpromazine and reserpine). The basolateral to apical transport of [14C]pristinamycin IA was 100-fold higher than apical to basolateral passage. This polarized transport was inhibited by verapamil and by ATP depletion. The results suggest that pristinamycin IA is a substrate for the P-glycoprotein, a finding which may have important consequences for the pharmacokinetics of this drug.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Induction of blood-brain barrier differentiation in a rat brain-derived endothelial cell line.

An immortalized brain capillary endothelial cell line displaying blood-brain barrier characteristics may represent a useful tool for studying blood-brain barrier endothelial cell differentiation and for the in vitro prediction of drug brain penetration. In the present study, we have established a rat cerebral capillary endothelial cell line (CR3) by genomic introduction of the immortalizing SV40 large T gene under the control of the human vimentin promoter. The CR3 cell line displayed endothelial morphological and biochemical characteristics for up to 30 passages. However, the CR3 cell line did not spontaneously express the specific blood-brain barrier markers gamma-glutamyl transpeptidase and mdr P-glycoprotein. However, when the cells were treated with the cell differentiating agent all-trans-retinoic acid, the blood-brain barrier markers were induced. Retinoic acid-treated CR3 cells may thus represent a useful tool for biological and pharmacological research related to the blood-brain barrier.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Functional expression of the P-glycoprotein mdr in primary cultures of bovine cerebral capillary endothelial cells.

The P-glycoprotein mdr is expressed not only in tumoral cells, but also in nontransformed cells, including the specialized endothelial cells of brain capillaries which build up the blood-brain barrier. Since all previously identified blood-brain barrier markers are rapidly lost when cerebral capillary endothelial cells are maintained in primary culture, we have investigated whether P-glycoprotein (P-gp) would follow the same rule, in order to address the influence of the cerebral environment on the specific P-gp expression in the brain endothelium. As compared to freshly isolated purified cerebral capillaries, P-glycoprotein was detected by immunochemistry at a high level in 5-7 day primary cultures. In our culture conditions, P-glycoprotein was immunodetected at a lower molecular weight than that found in freshly isolated capillaries. Enzymatic deglycosylation led to the same 130 kDa protein for both fresh and cultured samples, suggesting that P-gp post-translational modifications were altered in primary cultures. However, studies on the uptake and efflux of the P-gp substrate [3H]vinblastine, and on the effect of various mdr reversing agents on the uptake and efflux, clearly indicated that the efflux pump function of the P-glycoprotein was maintained in primary cultures of bovine cerebral capillary endothelial cells. P-Glycoprotein may thus represent the first blood-brain barrier marker which is maintained in cerebral endothelial cells cultured in the absence of factors originating from the brain parenchyma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Lipospermine-based gene transfer into the newborn mouse brain is optimized by a low lipospermine/DNA charge ratio.

Nonviral, plasmid-based gene transfer into somatic tissues offers the prospect of various simple and safe therapeutic possibilities as well as applications in fundamental research. Although cationic lipids display efficient transfection activities in many in vitro systems, only low success rates using these vectors in vivo have been reported. We succeeded in defining conditions providing high levels of in vivo transfection in the brains of newborn mice. Our hypothesis was that conditions favorable for in vitro transfection (highly positively charged particles) were unlikely to be appropriate for in vivo conditions. When using the cationic lipid dioctadecylamido glycylspermine (Transfectam, DOGS) with a cytomegalovirus (CMV)-luciferase reporter gene, the best levels of transfection were obtained when using a low ratio of positive charges (supplied by the DOGS) to negative charges (carried by the DNA). Moreover, addition of the neutral lipid dioleoylphosphatidyl ethanolamine (DOPE) significantly enhanced transfection. Expression of the transgene diminished over time, independently of lipopolysaccharide content of the plasmid preparation used. This suggests that either a mitotic population of cells was preferentially transfected, or that promoter silencing was occurring. Histological examination of the spatial distribution of a beta-galactosidase-expressing transgene showed numerous groups of transfected cells both within the striatal parenchyma and in the paraventricular area. Thus, DNA-lipid complexes bearing overall charges close to neutrality open promising possibilities for modulating gene expression in the developing central nervous system and for therapy in the brain.

Animals↗
PubMed1994

Polarized transport of docetaxel and vinblastine mediated by P-glycoprotein in human intestinal epithelial cell monolayers.

The expression of the multidrug transporter P-glycoprotein has been studied in two human intestinal epithelial cell lines. No functional expression of P-glycoprotein was found in the differentiated HT29-18-C1 cell line. The expression of P-glycoprotein in the Caco-2 cell line was very high, as judged by immunoblotting and by active efflux of vinblastine. The polarized transport of vinblastine in the basolateral to apical direction was temperature and energy dependent, and was reduced by P-glycoprotein inhibitors such as verapamil, chlorpromazine and reserpine. This adds further evidence that the polarized transport of vinblastine across Caco-2 monolayers is mediated by P-glycoprotein. The anticancer drug docetaxel (Taxotere) was transported in a polarized manner: basolateral to apical permeability was 20-fold higher than in the reverse direction. This polarized transport was inhibited by verapamil, chlorpromazine and reserpine, thus demonstrating that docetaxel is a substrate of P-glycoprotein. The implications of these results for the pharmacokinetics and toxicity of taxoids are discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Monoamine vesicular uptake sites in patients with Parkinson's disease and Alzheimer's disease, as measured by tritiated dihydrotetrabenazine autoradiography.

The monoaminergic innervation of the caudate nucleus, putamen and ventral striatum was investigated post mortem, in patients with Parkinson's and Alzheimer's disease as compared to control subjects, by autoradiographic detection of tritiated dihydrotetrabenazine (3H-TBZOH), a specific high affinity ligand of the vesicular monoamine transporter. The binding of 3H-TBZOH was specific and saturable (Kd 5.3 nM). In control striatum, the pattern of distribution of 3H-TBZOH binding was heterogeneous, with higher binding levels in the 'matrix' than in the 'striosome' compartment. Changes in ligand binding levels were observed in the pathological brains compared to controls. In Parkinson's disease (PD), characterized by a severe damage of mesostriatal dopaminergic neurons, the density of 3H-TBZOH binding was reduced. A severe decrease in 3H-TBZOH binding was observed in all parts of the striatum (caudate nucleus: -80%, putamen: -86%, ventral striatum: -94%) in PD brains. The data corroborate the deficiency in striatal dopaminergic transmission and suggest that in PD brains dopaminergic terminals have disappeared and/or no longer contain synaptic vesicles. In Alzheimer's disease (AD), 3H-TBZOH binding was significantly reduced by 57% in the ventral striatum and not in the caudate nucleus and putamen. The specific decrease of monoaminergic transporter levels in the ventral striatum confirm that this nucleus is a target area in AD.

Aged↗
PubMed1994

Differentiated intestinal epithelial cell lines as in vitro models for predicting the intestinal absorption of drugs.

The oral absorption of a compound is a critical factor for the future of the compound as a drug. This absorption is mainly controlled by the passage across the intestinal epithelium. Thus, the prediction of the intestinal absorption by means of an in vitro model may represent a powerful tool for the early selection of molecules during the process of drug development. In the present study, the differentiated human intestinal epithelial cell line HT29-18-C1, was grown on permeable filters in dual chambers. These cells formed tight monolayers that were used to measure in vitro the transepithelial permeability coefficient (Pc) of various molecules. The results were compared with in vivo data of oral absorption. A threshold value of in vitro permeability of 2 x 10(-6) cm/s was found. Molecules having a permeability coefficient higher than this value were absorbed orally more than 80%, while drugs with Pc values lower than 2 x 10(-6) cm/s were poorly absorbed. By mathematical simulation, it was found that this Pc value, when extrapolated to the surface area and volume of the small intestine, corresponds to an absorption of 80% for a compound with a transit time through the small intestine of 5 h. This demonstrates the predictive utility of the threshold value of the permeability coefficient derived from the in vitro model of intestinal epithelium.

Cell Line↗