Guy Dodin

DNA adsorption onto conducting polypyrrole

Abstract Deoxyribonucleic acid (DNA) adsorption onto chemically synthesized polypyrrole powders was investigated as a function of pH, buffer nature, ionic strength and substrate nature. DNA adsorption was found to be in the range of 0.13–0.55 mg m −2 for the conditions under investigation. Strong DNA adsorption was obtained at low pH and high ionic strength. The decreasing order of adsorption as a function of the native dopant was nitrate, chloride>sulfate. The isotherms were found to be of the Langmuir type or sigmoid, depending on the combination of (at least) substrate and buffer natures. Affinity constants determined using Scatchard and Hill plots were found to be as high as 10 6 M −1 .

XPS, NMR and FTIR structural characterization of polysiloxane-immobilized amine ligand systems

Abstract The polysiloxane-immobilized monoamine ligand systems were made by hydrolytic polycondensation of Si(OEt) 4 and (EtO) 3 Si(CH 2 ) 3 NH 2 . These materials were examined by XPS, FTIR and NMR techniques. The analysis indicates that the amine ligand groups almost exist in the free form in the alkaline media and in the cation form in the acid media. 13 C NMR and FTIR spectra combined with the XPS results shows that there is some leaching of small oligomeric ligand containing groups from the siloxane network.

Induction of mitochondrial dysfunction and apoptosis in HeLa cells by bis-pyridinium oximes, a newly synthesized family of lipophilic biscations☆

When tested on HeLa cells, bis-pyridinium oximes (BPO), a family of newly synthesized molecules whose charged pyridinium moieties are linked by a linear polymethylene chain of variable length (N = 3 to 12) have been shown to possess an inhibitory effect on cell growth and finally to provoke cell death. BPO-affected cells displayed reduced mitochondrial oxygen consumption and ATP stores and were blocked in the G1 phase of the cell cycle. Mitochondrial membrane potential, as assayed with the dye 3,3-diexyloxacarbocyanine iodide [DiOC6(3)], increased in BPO-treated cells with time of exposure. Cell growth inhibition as well mitochondrial dysfunction were observed only with derivatives having a long polymethylene linking chain (N > or = 6). Furthermore, the concentration of the compound eliciting such effects was inversely related to the number of methylene groups in the linking chain. None of the BPO with N = 6 to 12 modified the mitochondrial DNA content, relative to the nuclear DNA content. In BPO (N = 8 and N = 12)-treated cells, chromatin fragmentation and internucleosomal DNA cleavage occurred massively, indicating that the death mode induced by these compounds is apoptosis. The possible pathway of action and the potential pharmacological interest of these compounds are discussed.

A rapid purification by affinity chromatography of orotate phosphoribosyltransferase from Escherichia coli K-12

The current interest in the role played by divalent cations in the orotate phosphoribosyltransferase (OPRTase)-catalyzed synthesis of orotidine-5-monophosphate calls for further investigation to gain insight into the nature of the substrate-metal ions-protein interaction [1,2]. Physical methods such as NMR and ESR spectroscopy have proved to be valuable techniques in achieving this goal [1], but they need large amounts of protein. As the currently available purification procedures (requiring 9 steps) are rather tedious [3,4], we have elaborated a simple and rapid procedure based on affinity chromatography reported herein.

Dynamics of Drug-DNA Interactions: A Comparative Temperature Jump Study of Ellipticinium and 9-Hydroxy Ellipticinium

The temperature-jump method has been used to compare the binding of 2-N methyl ellipticinium (NME) and 2-N methyl 9 hydroxy ellipticinium (NMHE) to three natural DNAs of different AT/GC composition. The relaxation signals, analyzed by the Padé-Laplace method, are characterized by two distinct relaxation times, tau 1 and tau 2, respectively in the 1-4 ms and 20-80 ms range. In the case of the NMHE/DNA interaction, the slower relaxation time tau 2 depends on the DNA composition, as follows: tau 2 (Micrococcus lysodeikticus) greater than tau 2 (Calf thymus) greater than tau 2 (Clostridium perfringens). Contrary to NMHE, NME which does not possess an OH group at the C-9 position, shows no relaxation time dependence upon DNA base composition. The observation of two relaxation times indicates that the binding equilibria are associated with at least two distinct drug/DNA complexes (probably arising from two distinct DNA binding sites). Three kinetic models, involving the formation of a weak intermediate ionic complex, are given to explain the binding reaction between these cationic drugs and the DNA. They allow the determination of the four rate constants associated with the two binding steps and lead to equilibrium association constants in agreement with those obtained from spectroscopic studies. The validity of the models is discussed and it is shown that the best kinetic scheme, for either NMHE or NME, could be that in which the ionic step is not a prerequiste to intercalation. The kinetic results show that the residence time of 9 hydroxy ellipticinium is markedly increased in GC rich DNAs and this could be related to the higher in vitro and in vivo cytotoxic properties of 9 hydroxy substituted ellipticines.

Interactions between ellipticine derivatives and the mitochondrial respiratory chain

Abstract The interaction of monomeric and dimeric derivatives of ellipticine (a plant alkaloid) with plant mitochondria was studied by following electron transport and phosphorylative activities. It is shown that these compounds act as powerful inhibitors of the electron transfer in the terminal enzyme, i.e. cytochrome c oxidase, (presumably in the vicinity of cytochromes a-a 3 ) and exhibit uncoupling activities. The possibility of mitochondrial inner membrane being one of the sites of action of ellipticine derivatives is discussed in relation with their well-known pharmacological properties.

Mitochondrial uptake of bridged bis-methylpyridinium aldoximes and induction of the “petite” phenotype in yeast

The 3,3-[omega,omega-alkanediylbis(oxy)]bis[2- (hydroxyimino)methyl]-1-methylpyridinium derivatives bearing a linking chain of 4, 5 and 6 methylene groups are accumulated in mitochondria with increasing efficiency under the effect of the electrical potential. Accumulation does not take place with derivatives carrying a 2 and 3 methylene-long linking chain. The uptake process is saturable. The efficiency of the various derivatives to induce the petite phenotype in yeast reflects the uptake rate observed with purified mitochondria.

Role of magnesium cations in the yeast orotate phosphoribosyltransferase catalyzed reaction. Mechanism of the inhibition by Cu++ and Ni++ ions

The magnesium chelate of the N(3)H tautomer of orotate, L3Mg, is the true substrate in the biosynthesis of orotidine 5-monophosphate (OMP) catalyzed by yeast orotate phosphoribosyltransferase (OPRTase, E.C. 2.4.210) with a Michaelis constant KmL3Mg equal to 12(2) muM. It is postulated that Mg++ cations activate the transport of orotate to the active site by neutralizing the orotate charges; the ligand N(3)H is then exchanged between the incoming cation and the cation bound to the enzyme, thus ensuring the stabilization of the appropriate isomeric structure of orotate. This scheme, together with kinetic and thermodynamic data on orotate complexation by Mg++ and Ca++, accounts for the role of Ca++ cations that neither activate nor inhibit OMP synthesis. Cu++ and Ni++ inhibiting properties arise from the formation of inert complexes of orotate. Ni++ complexes have a poor affinity for the protein, whereas Cu++ complexes have a Michaelis constant similar to that of the L3Mg active species. The inertness of these complexes is tentatively understood in terms of low phosphoribosyl transfer rates as postulated from the kinetic study of the protonation of the complexes in water.

Complexation between oxonic acid and divalent metal cations. Influence of metal salt addition on decarboxylation kinetics under acidic conditions

Abstract The dissociation constants of oxonic acid are determined by UV spectrometry (pKa 1 = 0.8 ± 0.1) and potentiometry (pKa 2 = 6.85 ± 0.06). The N(3)H tautomer seems to be the predominant form of HL 2− , the oxonic acid dianion. This latter species binds with divalent metal ions to form complexes. Their formation constants, K M HL , are determined by UV spectrometry and/or potentiometry for Ca 2+ , Mg 2+ , Co 2+ , Ni 2+ and Cu 2+ . The rate of decarboxylation decreases when metal salt is added to acidic solutions; this is explained by the formation of a complex, H 2 LM + , between a metal ion and the monoanion of oxonic acid. The stability constants are determined for complexes formed with Ca 2+ , Mg 2+ , Co 2+ and Ni 2+ . These experimental results serve as a basis for assumptions concerning enzymatic phosphoribosylation and decarboxylation.

The binding of bridged bis-pyridinium oximes to DNA and its relevance to the induction of mitochondrial dysfunction in yeast*

Bis-pyridium oximes and methoximes from a newly synthesized series are weak DNA binders (K = 3.10(4) M-1 under physiological conditions). From the number of binding sites per phosphate, 0.25, the ionic strength dependence of the binding constant and the negative electric dichroism, it is concluded that monointercalation is the mode of association. In contrast to methoxy compounds, the oxime derivatives are able both to induce the mutated petite phenotype in yeast S. cerevisiae and to cause in vitro extensive condensation of single stranded DNA. This reaction is postulated to be relevant to the mutational process that leads to peptide cells. The absence of nuclear mutation is interpreted in terms of sequestration of the drug in mitochondria under the effect of the organelle inner membrane electrochemical potential.