PubMed Health⌕ Search

Biomedical subjects

C Malvy

Publications and source records attributed to C Malvy.

At least 55 records · Page 3Linked to original sources
PubMed1991

Molecular modelling of 9-aminoellipticine interactions with abasic oligonucleotides.

We have used molecular mechanics to study the insertion of the DNA intercalating agent 9-aminoellipticine (9AE) into single and double stranded abasic oligonucleotides containing abasic sites in the aldose or furanose conformations. 9AE-abasic oligonucleotide complexes have been considered with 9AE bound at abasic sites as a covalent complex, a reversible complex or a Schiff base. Results are in good agreement with experimental data available on abasic oligonucleotides (melting temperature measurement, NMR results) and allow an analysis of different possible structures for 9AE-abasic oligonucleotide complexes. Hypotheses concerning the role of 9AE-abasic site complexes in enzymatic inhibition are formulated from these data.

Base Sequence↗
PubMed1991

Comparison of anti-RNA properties of normal and ellipticine functionalized alpha and beta-oligonucleotides.

RNA is not cleaved as a consequence of the binding of RNase H to the duplex between RNA and a complementary alpha-oligodeoxyribonucleotide (oligo). In consequence targets have been selected which do not a priori require the action of RNase H to inhibit genetic expression. Two models have been used: The Friend Murine Leukemia Virus (F-MuLV) and the synthesis of rabbit beta globin.alpha-oligos trigger specific inhibitions in both systems. The functionalisation in 5' with the intercalating agent 9-NH2-ellipticine renders the oligos resistant to degradation and allows a direct action on cells.

Animals↗
PubMed1990

Structural features associated with alpha- anomeric in oligo-alpha-thymidylates.

Comparison between gel electrophoresis migrations of oligo-alpha-thymidylates and oligo-beta-thymidylates indicates that the migration of alpha-oligonucleotides under native conditions is different from the migration of beta-oligonucleotides when the number of thymines, part of the sequence, is higher than 5. Such difference disappears when the gels are run under denaturing conditions. This, together with UV spectra, indicates that the structure of alpha-oligonucleotides is more organized than the structure of beta-oligonucleotides and that such an organisation appears for a length higher than 5 monomeric units.

Electrophoresis, Polyacrylamide Gel↗
PubMed1990

Low concentrations of acridine dimers inhibit micrococcus AP endonuclease through interaction with apurinic sites in DNA.

The effect of dimeric DNA intercalating compounds was assayed on a purified AP endonuclease from Microccoccus luteus using apurinic supercoiled PM2 DNA as a substrate. Binding on apurinic sites was estimated through the competition with the intercalating compound, 9-NH2-ellipticine, which displays great specificity for apurinic sites. An acridine dimer with a spermine linker is at 0.1 microM the best inhibitor of cleavage at the apurinic site induced either by the AP endonuclease or by 9-NH2-ellipticine. Bisintercalating agents are more effective inhibitors of AP endonuclease than monointercalating ones. Most effective inhibitors among dimers have acridine residues.

Acridines↗
PubMed1990

9-amino-ellipticine inhibits the apurinic site-dependent base excision-repair pathway.

The aromatic amine 9-amino-ellipticine is a synthetic DNA intercalating compound derived from the antitumor agent ellipticine, which cleaves at very low doses DNA containing apurinic sites by beta-elimination through formation of a Schiff base. This compound has been shown to potentiate the cytotoxic effect of alkylating drugs, such as dimethyl sulfate, in E. coli through a mechanism involving apurinic sites. We have studied the ability of 9-amino-ellipticine to inhibit an enzymatic repair system mimicking base-excision repair, in which E. coli exonuclease III only presents an endonuclease for apurinic/apyrimidinic site activity. 10 microM of 9-amino-ellipticine inhibits 70% of apurinic site repair. Other intercalating agents with similar affinities for DNA do not induce any inhibition. In another system designed for the direct assay of the exonuclease III-induced incisions 5' to AP sites 10 microM of 9-amino-ellipticine inhibits 65% of the endonuclease for apurinic/apyrimidinic site activity of E. coli exonuclease III. The 9-amino-ellipticine-induced formation of a 2',3'-unsaturated deoxyribose and cleavage at the 3' side of the apurinic site, and possible creation of an adduct, as suggested by Bertrand and coworkers (1989), on the 3' position of the deoxyribose seem to strongly inhibit the endonuclease for apurinic/apyrimidinic site activity. 9-Amino-ellipticine appears therefore to be the first small ligand which can inhibit, by an irreversible modification of the substrate, the repair of apurinic sites through the base excision-repair pathway at a pharmacological concentration.

Alkaloids↗
PubMed1989

Synthesis, thermal stability and reactivity towards 9-aminoellipticine of double-stranded oligonucleotides containing a true abasic site.

A 13 mers abasic oligonucleotide was synthetized. It was therefore possible to compare thermal stability and reactivity of duplex oligonucleotides either with an apurinic/apyrimidinic site or without any lesion. An important decrease in the melting temperature appeared for duplexes with an abasic site. The chemical reaction of these modified oligonucleotides with the intercalating agent 9-aminoellipticine was studied by gel electrophoresis and by fluorescence. The formation of a Schiff base between 9-aminoellipticine and abasic sites was rapid and complete with duplexes at 11 degrees C. Schiff base related fluorescence and beta-elimination cleavage were more important with the apyrimidinic sites than with the apurinic ones. When compared to previous results obtained with the model d(TprpT) some unexpected behaviours appeared with longer and duplex oligonucleotides. For instance only partial beta-elimination cleavage was observed. It is likely that stacking parameters in the double helix play a great role in the studied reaction.

Alkaloids↗
PubMed1989

Comparative activity of alpha- and beta-anomeric oligonucleotides on rabbit beta globin synthesis: inhibitory effect of cap targeted alpha-oligonucleotides.

Alpha-anomeric oligonucleotides are resistant to nucleases and display parallel annealing to RNA complementary sequences. We compared the effect of alpha- and beta-oligonucleotides targeted against various mRNA regions on the rabbit beta globin in vitro synthesis. In order to determine the role of RNase H, experiments were performed in both rabbit reticulocyte lysate and wheat germ extract. As expected beta-oligonucleotides were found more efficient in wheat germ extract which is rich in RNase H activity and alpha-oligonucleotide targeted against the initiation codon or downstream had no effect because they do not induce mRNA cleavage by RNase H. However, we report, for the first time, a specific translation inhibition by alpha-oligonucleotides. This occurs provided they are targeted against the cap region in 5' of the mRNA.

Animals↗
PubMed1989

Mechanism of cleavage of apurinic sites by 9-aminoellipticine.

We have studied the kinetics of breakage of apurinic (AP) sites by the intercalating agent 9-aminoellipticine using fluorimetric methods with single (ss)- and double (ds)-stranded apurinic DNA. In order to understand the chemical process, high performance liquid chromatography was used to follow the reaction kinetics with the apurinic oligonucleotide model T(AP)T. The unstable intermediate, which is responsible for the beta-elimination step, is a Schiff base resulting from the interaction of the amino group of the aromatic amine with the aldehyde function of the deoxyribose moiety (AP site). Fluorescence occurs simultaneously with the breakage of both ss and ds DNA and of the oligonucleotide and arises from the formation of a conjugated double bond on the Schiff base through the beta-elimination reaction. In optimal conditions, the second order rate constant for the fluorescence build up is 15 x 10(3) min-1 M-1 for ds DNA and 0.105 x 10(3) min-1 M-1 for T(AP)T. The ability of 9-aminoellipticine to induce fluorescence and breakage of ss DNA and T(AP)T shows that intercalation is not essential for this reaction to occur. Nevertheless, the greater rate constant with DNA suggests that stacking is an important parameter for the reaction of the aromatic amine with the AP site.

Alkaloids↗
PubMed1989

Inhibition of Moloney murine leukemia virus reverse transcriptase by alpha-anomeric oligonucleotides.

After parallel hybridization to complementary template RNA, alpha-anomeric oligonucleotides are not primers for Moloney murine leukemia virus reverse transcriptase. As can be expected they are competitors of classical primer oligonucleotides (beta-anomeric). They therefore inhibit the RNA dependent DNA polymerase activity of Moloney murine leukemia virus reverse transcriptase with either homopolymeric or heteropolymeric substrates. Non complementary alpha-oligonucleotides display no inhibitory activity. alpha-Oligonucleotides are therefore potential candidates for inhibition of retroviral reverse transcriptases by interference with the primer binding sites.

Base Sequence↗
PubMed1988

Alpha-anomeric DNA: beta-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase.

Nuclease-resistant alpha-anomeric DNA:beta-RNA hybrids are inhibitors of Escherichia coli RNase H, and Drosophila embryo RNase H. RNase H activities were measured by polyacrylamide gel electrophoresis, employing a short substrate, (A)12:d[G-G-(T)12-G-G], or by acid-solubility techniques, using a long substrate, poly(A):poly(dT). Strand exchanges which could be responsible for the observed inhibition have been ruled out by S1 nuclease experiments and by using inhibitors which do not allow strand exchange. Our results suggest that RNase H, for which DNA:RNA duplexes are the natural substrates, binds to non-physiological alpha-DNA:RNA hybrids and is consequently inhibited. These hybrids also inhibit the RNA-dependent DNA polymerase activity of M-MLV reverse transcriptase, therefore appearing as potential inhibitors of at least two reverse transcriptase activities. However, the inhibitory effect of these hybrids with respect to M-MLV reverse transcriptase is also observed with the single-stranded alpha-DNA itself. Unexpectedly, polymerase activity is highly stimulated by alpha-oligos, analogous in their sequence to the beta primer used at a concentration unable to generate a detectable synthesis. These results suggest that the inhibition of reverse transcriptase activity with the alpha:beta may occur at different levels.

Animals↗
PubMed1988

Alpha are more stable than beta anomer oligonucleotides in 3T3 cellular extracts.

We have compared the stability of alpha and beta anomeric oligonucleotides in NIH 3T3 cellular extracts. We had already shown that alpha are much more resistant than beta oligonucleotides towards purified nucleases. This result is confirmed when using cellular extracts although the difference is smaller. When alpha molecules are combined with an intercalating agent binding in the 3' position a synergistic increase of resistance to degradation is observed.

Animals↗
PubMed1988

Involvement of apurinic sites in the synergistic action of alkylating and intercalating drugs in Escherichia coli.

The toxicity of the intercalating compounds 9-aminoellipticine (9AE) and isopropyl-oxazolopyridocarbazole (Ipr-OPC) were studied. The inhibitory effect of non-toxic doses of 9AE, which incises DNA at apurinic (AP) sites, or Ipr-OPC, which does not cleave DNA at AP sites, with non-toxic doses of the alkylating agent dimethylsulphate (DMS) on the growth of Escherichia coli strain AB1157, is additive. The same result has been observed with an exonuclease III mutant which has only 10% of the AP endonuclease activity. However, 9AE or Ipr-OPC display a synergistic toxic effect with a DMS concentration which allows 20% of E. coli AB1157 survival. This synergy is increased for 9AE in the AP endonuclease mutant when compared to the wild-type strain. Under identical conditions 9AE and Ipr-OPC have no synergistic effect on a mutant deficient in the enzymes which generate AP sites. Therefore AP sites are involved in the synergistic toxicity of DMS and the studied intercalating agents. However, the precise role of the interaction of intercalating agents with AP sites, either without cleavage (type 1 compounds) or with cleavage (type 2 compounds), in the observed effect remains an open question.

Alkylating Agents↗
PubMed1987

alpha-DNA. VI: Comparative study of alpha- and beta-anomeric oligodeoxyribonucleotides in hybridization to mRNA and in cell free translation inhibition.

alpha and beta-anomeric d(G2T12G2) oligodeoxyribonucleotides were compared for their hybridization to rA12: the observed melting temperatures are 27 degrees C for beta-oligodeoxyribonucleotide/RNA hybrid and 53 degrees C for alpha-oligodeoxyribonucleotide/RNA. alpha-oligonucleotides with the four bases, complementary to natural mRNAs, were synthesized for the first time, labeled at their 5'-end with [32P] and used as probes in Northern blot experiments. In spite of these higher affinities for their target RNA's, they were unable to block translation of natural or synthetic mRNA's in rabbit reticulocyte lysate. We have studied the RNase H activity on model rA12:alpha- or beta-d(G2T12G2) hybrids or on mRNA:alpha- or beta-oligonucleotides hybrids. Specific hybridization protects RNA strech when using alpha-oligonucleotides but not beta-oligonucleotides. Thus, our results show the inability of RNase H to degrade RNA in alpha-oligodeoxyribonucleotides:RNA duplexes.

Endoribonucleases↗
PubMed1987

In vivo exposure to four ellipticine derivatives with topoisomerase inhibitory activity results in chromosome clumping and sister chromatid exchange in murine bone marrow cells.

A single dose of 9-hydroxy-ellipticine, 2-N-methyl-9-hydroxy-ellipticine, 9-fluoro-ellipticine, and 9-amino-ellipticine (5 to 10 mg/kg body wt, ip) resulted in murine bone marrow toxicity as shown by chromosome clumping, chromatid aberrations, and micronuclei formation. An increase in sister chromatid exchanges (SCE) was also observed. These effects are most likely directly related to the topoisomerase inhibitory effect of these drugs since topoisomerase II is involved in the separation of intertwined chromosomal DNA molecules during mitosis as well as being a mediator of DNA exchanges. The two antitumor drugs 2-N-methyl-9-hydroxy-ellipticine and 9-hydroxy-ellipticine were most genotoxic with chromosome abnormalities occurring in 33-95% of the cells and SCE on the order of 12.3 to 19.2 events per cell. Both of these drugs show high topoisomerase II inhibitory activity in vitro. In contrast, 9-amino-ellipticine and 9-fluoro-ellipticine were less genotoxic with chromosomal abnormalities occurring in 14-17% of the cells and SCE on the order of 7.1 to 7.7 events per cell. These two derivatives are both inactive toward experimental tumors and show less topoisomerase II inhibitory activity in vitro. Our results suggest that the ellipticine drugs owe at least some of their cytotoxicity to their genotoxic effects, which seem to be mediated through interaction with topoisomerase II.

Alkaloids↗
PubMed1987

alpha-DNA II. Synthesis of unnatural alpha-anomeric oligodeoxyribonucleotides containing the four usual bases and study of their substrate activities for nucleases.

This paper describes for the first time the synthesis of alpha-oligonucleotides containing the four usual bases. Two unnatural hexadeoxyribonucleotides: alpha-[d(CpApTpGpCpG)] and alpha-[d(CpGpCpApTpG)], consisting only of alpha-anomeric nucleotide units, were obtained by an improved phosphotriester method, in solution. Starting material was the four base-protected alpha-deoxyribonucleosides 3a-d. Pyrimidine alpha-deoxynucleosides 3a and 3b were prepared by self-anomerization reactions followed by selective deprotection of sugar hydroxyles, while the two purine alpha-deoxynucleosides 3c and 3d were prepared by glycosylation reactions. In the case of guanine alpha-nucleoside derivative a supplementary base-protecting group: N,N-diphenylcarbamoyl was introduced on O6-position in order to avoid side-reactions during oligonucleotide assembling. The hexadeoxynucleotide alpha-[d(CpApTpGpCpG)] was tested as substrate of selected endo- and exonucleases. In conditions where the natural corresponding beta-hexamer was completely degradated by nuclease S1 and calf spleen phosphodiesterase, the alpha-oligonucleotide remained almost intact.

Deoxyribonucleases↗
PubMed1987

Quantification by fluorescence of apurinic sites in DNA.

Time dependent fluorescence is observed when single or double stranded DNA with apurinic sites are mixed with 9-NH2-ellipticine. A concentration dependent plateau is obtained which is linearly related to the ratio of apurinic sites in DNA. We therefore suggest that it is possible to have a direct measurement of apurinic sites in DNA by fluorescence.

Alkaloids↗
PubMed1986

Differential reactivity of 9-NH2-ellipticine on apurinic and apyrimidinic sites in circular DNA.

Endonucleases for apurinic sites as well as chemical compounds reacting with aldehydes do not generally differentiate between apurinic and apyrimidinic sites. We have studied the effect of the apurinic site reagent, 9-NH2-ellipticine, on apyrimidinic sites enzymatically generated on PBR322 DNA and compared it to its' action on apurinic PM2 and PBR322 DNAs. In conditions where this compound induces breakage of apurinic sites, it does not display any action on apyrimidinic sites.

Alkaloids↗