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.

Cloning and analysis of structural genes from Streptomyces pristinaespiralis encoding enzymes involved in the conversion of pristinamycin IIB to pristinamycin IIA (PIIA): PIIA synthase and NADH:riboflavin 5'-phosphate oxidoreductase.

In Streptomyces pristinaespiralis, two enzymes are necessary for conversion of pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA), the major component of pristinamycin (D. Thibaut, N. Ratet, D.

Purification of the two-enzyme system catalyzing the oxidation of the D-proline residue of pristinamycin IIB during the last step of pristinamycin IIA biosynthesis.

High levels of conversion of 14C-labelled pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA) were obtained in vivo in Streptomyces pristinaespiralis and in some other streptogramin A producers. This established that PIIB was an intermediate on the pathway to PIIA. In addition, in vitro studies with cell-free protein preparations demonstrated that the oxidation of PIIB to PIIA is a complex process requiring NADH, riboflavin 5'-phosphate (FMN), and molecular oxygen.

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.

Aliphatic nitrilase from a soil-isolated Comamonas testosteroni sp.: gene cloning and overexpression, purification and primary structure.

An aliphatic nitrilase, active on adiponitrile and cyanovaleric acid, was identified and purified from Comamonas testosteroni sp. (Ct). Oligodeoxyribonucleotide probes were designed from limited amino acid (aa) sequence information and used to clone the corresponding gene, named nitA.

Effects of two distamycin-ellipticine hybrid molecules on topoisomerase I and II mediated DNA cleavage: relation to cytotoxicity.

Two distamycin-ellipticine conjugates were examined for their ability to modulate topoisomerase I and topoisomerase II-DNA cleavable complex formation in vitro. Hybrid molecules Distel (1+) and Distel (2+) both contain a DNA-intercalating chromophore and a tris-pyrrole element capable of binding within the minor groove of DNA. The two drugs differ only in the nature of the side chain attached to the distamycin moiety.

Methamphetamine and dopamine neurotoxicity: differential effects of agents interfering with glutamatergic transmission.

The effects of riluzole and lamotrigine, two agents which interfere with the release of glutamate (GLU), and MK-801, a blocker of N-methyl-D-aspartate (NMDA) receptors, were compared in the model of methamphetamine-induced depletion of dopamine (DA) levels in mice. Repeated injections with methamphetamine (4 x 5 mg/kg i.p.

Electrographic studies of the effects of RP 60180, a novel kappa agonist, on the photosensitive baboon Papio papio.

1. The authors describe here the effects of intravenous administration of RP 60180, a novel kappa agonist, on conscious baboons of the species Papio papio, which spontaneously present photically induced epileptic responses. 2.

Unusual transformation of the 3-hydroxy-picolinoyl residue of pristinamycin IA.

Pristinamycin IA was modified in a two-step procedure to give original derivatives possessing a tricyclic nucleus (8a, 8b, 8c) or a substituted pyrrole ring (10a, 10b) in place of the natural exocyclic 3-hydroxy-picolinoyl residue. This transformation involved firstly preparation of pyridinium betaines 5 from pristinamycin IA and secondly a 1-3 dipolar cycloaddition between 5 and N-substituted maleimides or diethyl acetylenedicarboxylate. The compounds obtained were evaluated as antibacterial agents alone and in association with pristinamycin IIA.

Docetaxel (Taxotere): a review of preclinical and clinical experience. Part I: Preclinical experience.

Docetaxel is a taxoid which is currently in phase II/III clinical trials in Europe, the US and Japan. It was found to promote tubulin assembly in microtubules and to inhibit their depolymerization. In vitro, the docetaxel concentrations required to reduce murine and human cell survival by 50% ranged from 4 to 35 ng/ml and the cytotoxic effects were greater on proliferating than on non-proliferating cells.

A 700-bp fragment of the human antithrombin III promoter is sufficient to confer high, tissue-specific expression on human apolipoprotein A-II in transgenic mice.

The human antithrombin III-encoding gene (hAT-III) promoter (phAT-III) was used to generate transgenic mice producing a human hepatic apolipoprotein, apolipoprotein A-II (hApoA-II). Integration of the transgene into the mouse genome resulted in the efficient production of hApoA-II in plasma, reaching up to 0.40 g/l in two transgenic lines.

A role for Grb2 in apoptosis?

Src homology type 2 (SH2) and type 3 (SH3) domains appear to have an important role in signal transduction pathways initiated by tyrosine kinases. SH2 domains allow proteins with signalling functions to interact with tyrosine kinases and tyrosine-phosphorylated proteins at the plasma membrane, whereas SH3 domains allow a distinct type of interaction through binding to proline-rich sequences. The adaptor protein Grb2 consists of one SH2 domain and two SH3 domains and connects tyrosine kinase receptors to activation of the Ras pathway.

[Taxoids: structural and experimental properties].

Paclitaxel (Taxol) and docetaxel (Taxotere) are the first representatives of a new class of antitumor compounds. These two taxoids are clinically active against breast, ovarian and lung cancers. Taxoids are highly complex diterpenoids form natural origin.

Preclinical evaluation of docetaxel (Taxotere).

Progress in cancer chemotherapy has been made owing to the discovery and development of drugs that have new structures, new mechanisms of action, and high levels of experimental antitumor activity. Docetaxel (Taxotere; Rhône-Poulenc Rorer, Antony, France) is prepared by semisynthesis from 10-deacetyl baccatin III, an inactive taxoid precursor extracted from the needles of the European yew Taxus baccata. Docetaxel has been found to promote tubulin assembly in microtubules and to inhibit their depolymerization.

Molecular determinants of the species selectivity of neurokinin type 1 receptor antagonists.

Most nonpeptide neurokinin (NK)1 antagonists display a marked difference in affinity for rat versus human NK1 receptors. The molecular basis for the species selectivity of RP67580 and CP96,345 has been previously addressed [J. Biol.

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.

Cloning, pharmacological characterization, and anatomical distribution of a rat cDNA encoding for a galanin receptor.

We have cloned and expressed a rat cDNA, designated GALR1-rat, that encodes a galanin receptor based on homology, pharmacology, and anatomical criteria. This cDNA was isolated from a rat brain cDNA library. The nucleotide sequence of the cloned receptor revealed an open reading frame encoding a 346-amino-acid protein, showing 90.

Asymmetrically substituted ethylenediamine platinum(II) complexes as antitumor agents: synthesis and structure-activity relationships.

A series of platinum dichloroethylenediamine complexes [PtCl2(R-en)] bearing a side chain on one carbon atom of the ethylenediamine ligand, with or without a functional group on the side chain, have been prepared and investigated for antitumor activity against L1210 leukemia. They were tested both in vitro, with cisplatin-sensitive and resistant cell lines, and in vivo, with cisplatin-sensitive and resistant tumors grafted i.p.

Preclinical pharmacology of docetaxel.

Docetaxel is a taxoid cytotoxic agent which promotes tubulin assembly into microtubles and inhibits their depolymerisation. In vitro, docetaxel reduces murine and human tumour cell survival by 50% at concentrations of 4-35 ng/ml, with a greater cytotoxic effect on proliferating than on non-proliferating cells. In vivo, docetaxel is schedule-independent.

Riluzole and experimental parkinsonism: antagonism of MPTP-induced decrease in central dopamine levels in mice.

We have investigated whether riluzole, a compound that interferes with glutamatergic (GLUergic) transmission, would protect central dopaminergic (DAergic) neurones from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity in the striatum in mice. MPTP decreased DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels. Riluzole protected against the MPTP-induced decrease in DA levels.

Congo red protects against toxicity of beta-amyloid peptides on rat hippocampal neurones.

beta-Amyloid peptides are neurotoxic when applied to primary cultures of hippocampal neurones from the embryonic rat. This neurotoxic effect can be inhibited completely by certain disazo dyestuffs. The most potent of these are Congo Red and Congo Rubin, whilst Direct Garnet and sodium 4-aminonaphthalene-1-sulphonate are inactive.

[Pleiotropic resistance associated with topoisomerases].

There are at least two well-characterized mechanism of resistance to Topo I and II inhibitors: modifications of intracellular accumulation and reduced formation of cleavable complexes. Limited drug accumulation is usually due to P-glycoproteinMDR or to Multidrug Resistance associated Protein (MRP). Reduction of Topo I (or II) cleavable complexes not related to drug transport can either be due to decreased enzyme levels or enzyme mutations.

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.

Use of scanning cytometry in studying bradykinin binding in MRC-5 cells.

Ligand-receptor affinity is classically demonstrated by measuring ligand binding density to a specific site on membrane preparations, and receptor function is studied by measuring calcium flux, cell by cell, using microspectrofluorimetry. In order to study these phenomena in a large cell population, calcium flux was measured in MRC-5 cell line expressing the B2 receptor for bradykinin using an ACAS 570 scanning cytometer. Following incorporation of fluo3/AM, different ligands were studied, singly or in association with bradykinin.