An improved anion-exchange HPLC method for the detection and purification of adenoviral particles.

We have developed an anion-exchange high-performance liquid chromatography (HPLC) method using Q Sepharose XL (Amersham Pharmacia Biotech) as adsorbent to analyze samples containing adenovirus. This method has several major advantages over the HPLC method previously described for quantitating particles, namely (1) a >10-fold improvement in the detection limit of adenovirus in crude preparations; (2) absence of interferences originating from nucleic acids and proteins which usually contaminate crude samples; (3) unprecedented sharpness and symmetry of adenovirus peak, rendering the identification of the viral peak unambiguous, even in extremely crude and dilute preparations; and (4) no enzymatic treatment required even for crude samples. This assay was used to quantitate particles in samples taken at the transfection and amplification stages of production of various recombinant adenovirus, and in cultures of wild-type adenovirus of different serotypes.

4,10-Dihydro-4-oxo-4H-imidazo[1,2-a]indeno[1,2-e]pyrazin-2-carboxylic acid derivatives: highly potent and selective AMPA receptors antagonists with in vivo activity.

A novel series of 2-substituted-4,5-dihydro-4-oxo-4H-imidazo[1,2-a]indeno[1,2-e]pyrazine derivatives was synthesised. One of them, 4e-a highly water soluble compound exhibited a nanomolar affinity and demonstrated competitive antagonist properties at the ionotropic AMPA receptors. This compound also displayed potent anticonvulsant properties against electrically or sound-induced convulsions in mice after systemic administration, thus suggesting adequate brain penetration.

Microbiological high throughput screening: an opportunity for the lead discovery process.

Microbial HTS has been implemented at Rhône-Poulenc Rorer through the development of a dedicated robotic platform. This robot (Turbo) has been designed with the aim of fully integrating microbial HTS into the lead discovery processes. Innovative solutions have been found to reach high throughput as well as flexibility.

Cancer gene therapy mediated by CTS1, a p53 derivative: advantage over wild-type p53 in growth inhibition of human tumors overexpressing MDM2.

Recently, a new p53 derivative has been designed, namely chimeric tumor suppressor 1 (CTS1), in which the p53 domains that are known to mediate p53 inactivation have been replaced. In this study, the antitumoral activity of CTS1 mediated by adenovirus vector has been evaluated in comparison with a p53 adenovirus vector in various human tumor cell lines. In vitro, in terms of cell growth inhibition, the CTS1 vector was significantly (P < .

Importance of association between permeabilization and electrophoretic forces for intramuscular DNA electrotransfer.

Gene transfer using electrical pulses is a rapidly expanding field. Many studies have been performed in vitro to elucidate the mechanism of DNA electrotransfer. In vivo, the use of efficient procedures for DNA electrotransfer in tissues is recent, and the question of the implied mechanisms is largely open.

8-Methylureido-4,5-dihydro-4-oxo-10H-imidazo[1,2-a]indeno[1,2-e]pyrazines: highly potent in vivo AMPA antagonists.

A novel series of readily water soluble 8-methylureido-4,5-dihydro-4-oxo-10H-imidazo[1,2-a]indeno[1,2-e]++ +pyrazines were synthesized. The -10-yl acetic acid ((+)-3) and -10-carboxylidene (4) derivatives exhibit potent affinities (IC50=4 and 19 nM, respectively) and antagonist properties (IC50 = 2 and 3 nM, respectively) at the ionotropic AMPA receptor. These compounds also display anticonvulsant properties against both electrically and sound-induced convulsions in mice after ip, sc and iv administration with ED50 values between 0.

The protective effect of riluzole in the MPTP model of Parkinson's disease in mice is not due to a decrease in MPP(+) accumulation.

Riluzole, has previously been shown to be protective in animal models of Parkinson's disease in vivo. In the present study the effects of riluzole on the intrastriatal formation and accumulation of MPP(+), after i.p.

RPR112378 and RPR115781: two representatives of a new family of microtubule assembly inhibitors.

A screening program aimed at the discovery of new antimicrotubule agents yielded RPR112378 and RPR115781, two natural compounds extracted from the Indian plant Ottelia alismoides. We report their isolation, structural determination, and mechanisms of action. RPR112378 is an efficient inhibitor of tubulin polymerization (IC(50) = 1.

Novel method for covalent fluorescent labeling of plasmid DNA that maintains structural integrity of the plasmid.

We have developed a chemical strategy for covalent coupling of fluorophores to plasmid DNA. A p-azido-tetrafluoro-benzyl-lissamine conjugate was synthesized and purified. This conjugate was used to covalently associate fluorescent molecules to plasmid DNA by photoactivation.

Preclinical in vitro cardiac electrophysiology: a method of predicting arrhythmogenic potential of antihistamines in humans?

The cardiac action potential results from a dynamic balance between inward depolarising Na+ and Ca2+ currents and outward K+ repolarising currents. During a cardiac cycle, the resultant of repolarisation phase from all ventricular cells is represented by the QT interval of the surface ECG. Congenital long QT syndrome (LQTS) is characterised by polymorphic ventricular tachycardia sometimes with twisting QRS morphology (torsade de pointes) which, although usually self-limiting, can result in sudden cardiac death.

Intracellular fate and nuclear targeting of plasmid DNA.

One of the major steps limiting nonviral gene transfer efficiency is the entry of plasmid DNA from the cytoplasm into the nucleus of the transfected cells. The nuclear localization signal (NLS) of the SV40 large T antigen is known to efficiently induce nuclear targeting of proteins. We have developed two chemical strategies for covalent coupling of NLS peptides to plasmid DNA.

Spiro-imidazo[1,2-a]indeno[1,2-e]pyrazine-4-one derivatives are mixed AMPA and NMDA glycine-site antagonists active in vivo.

Original spiro-imidazo[1,2-a]indeno[1,2-e]pyrazine-4-one derivatives were synthesised and led to the identification of 3e which showed good affinities for both the AMPA and the NMDA glycine-site receptors, and displayed good anticonvulsant effects after i.p. and i.

pCOR: a new design of plasmid vectors for nonviral gene therapy.

A totally redesigned host/vector system with improved properties in terms of safety has been developed. The pCOR plasmids are narrow-host range plasmid vectors for nonviral gene therapy. These plasmids contain a conditional origin of replication and must be propagated in a specifically engineered E.

Riluzole series. Synthesis and in vivo "antiglutamate" activity of 6-substituted-2-benzothiazolamines and 3-substituted-2-imino-benzothiazolines.

Two series of analogues of riluzole, a blocker of excitatory amino acid mediated neurotransmission, have been synthesized: monosubstituted 2-benzothiazolamines and 3-substituted derivatives. Of all the compounds prepared in the first series, only 2-benzothiazolamines bearing alkyl, polyfluoroalkyl, or polyfluoroalkoxy substituents in the 6-position showed potent anticonvulsant activity against administration of glutamic acid in rats. The most active compounds displaying in vivo "antiglutamate" activity were the 6-OCF(3) (riluzole), 6-OCF(2)CF(3), 6-CF(3), and 6-CF(2)CF(3) substituted derivatives with ED(50) values between 2.

Coupling of a targeting peptide to plasmid DNA by covalent triple helix formation.

The nuclear localization signal (NLS) of the SV40 large T antigen efficiently induces nuclear entry of proteins. We have developed a strategy for covalent coupling of one or a controlled number of NLS peptides to plasmid DNA at a specific site by triple helix formation. A psoralen-oligonucleotide-NLS peptide conjugate was synthesized and characterized by proteolysis with trypsin.

Purification and characterization of human DNA topoisomerase IIIalpha.

Human topoisomerase IIIalpha (hTopo IIIalpha), the recently identified first member of the topoisomerase IA subfamily in humans, has a central domain which is highly homologous to the yeast topoisomerase III, but an overall organization closer to that of Escherichia coli DNA topoisomerase I. In order to determine the properties of hTopo IIIalpha, compared to those of other topoisomerase IA subfamily members, we purified this enzyme to near homogeneity, together with an active site-mutant Y337F. We show that hTopo IIIalpha is able to relax negatively supercoiled DNA in a distributive manner, leading to the total disappearance of the initial substrate and the appearance of intermediate topoisomers.

Definition of a p53 transactivation function-deficient mutant and characterization of two independent p53 transactivation subdomains.

The wild-type protein product of the p53 tumor suppressor gene can activate transcription of genes which are involved in mediating either growth arrest, e.g. WAF1 or apoptotis, e.

Cellular uptake of cationic lipid/DNA complexes by cultured myoblasts and myotubes.

Several cationic lipids which are highly efficient for delivering genes in vitro do not increase gene delivery in vivo after an intramuscular injection. In order to elucidate the origin of this phenomenon, we have studied the cellular uptake and intracellular fate of cationic lipid/DNA complexes in vitro on myogenic mouse cells (myoblasts and myotubes) of the C2 cell line and of primary cultures. We used a cationic lipid with a spermine head group and its fluorescent analog, and a fluorescent plasmid obtained by nick-translation.

Coupling of nuclear localization signals to plasmid DNA and specific interaction of the conjugates with importin alpha.

The nuclear localization signal (NLS) of the SV40 large T antigen efficiently induces nuclear targeting of proteins. We have developed a chemical strategy for covalent coupling of NLS peptides to plasmid DNA. A p-azido-tetrafluoro-benzyl-NLS peptide conjugate was synthesized.

Somatic gene transfer of human ApoA-I inhibits atherosclerosis progression in mouse models.

Background: Apolipoprotein (apo) A-I is the major component of HDL, and it displays antiatherogenic properties.

Methods And Results: The human apoA-I gene has been transferred into different mouse models by use of a recombinant adenovirus under the control of an RSV-LTR promoter (AV RSV apoA-I). Administration of AV RSV apoA-I to C57BL/6 mice resulted in moderate expression of human apoA-I for 3 weeks, leading to a transient elevation (40% at day 11 after injection) of HDL cholesterol concentration.

Ouabain-induced increase in dopamine release from mouse striatal slices is antagonized by riluzole.

We have examined the effects of riluzole, a neuroprotective drug which stabilizes voltage-dependent sodium channels in their inactivated state and inhibits the release of glutamate in-vivo and in-vitro, on the release of newly taken up [3H]dopamine induced by ouabain, a potent and selective inhibitor of Na+/K+-ATPase in mouse striatal slices in-vitro. Riluzole potently (IC50 (concentration resulting in 50% inhibition) = 0.9+/-0.

Influence of depolarization on vasorelaxant potency and efficacy of Ca2+ entry blockers, K+ channel openers, nitrate derivatives, salbutamol and papaverine in rat aortic rings.

We examined the effects of various KCl concentrations on the actions of some vasodilators belonging to different pharmacological classes in rat aortic rings. In some experiments, tissues were precontracted with noradrenaline after blocking voltage-dependent channels to assess the effects of depolarisation unaccompanied by the entry of extracellular Ca2+ into the cytosol. Concentration/response curves for the vasorelaxant effect of calcium entry blockers (e.