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Biomedical subjects

M Leng

Publications and source records attributed to M Leng.

At least 55 records · Page 3Linked to original sources
PubMed1994

Base sequence-independent distorsions induced by interstrand cross-links in cis-diamminedichloroplatinum (II)-modified DNA.

Physico-chemical and immunological studies have been done in order to further characterize the distorsions induced in DNA by the interstrand cross-links formed between the antitumor drug cis-diamminedichloroplatinum (II) (cis-DDP) and two guanines on the opposite strands of DNA at the d(GC/GC) sites. Bending (45 degrees) and unwinding (79 +/- 4 degrees) were determined from the electrophoretic mobility of multimers of 21- 24-base pairs double-stranded oligonucleotides containing an interstrand cross-link in the central sequence d(TGCT/AGCA). The distorsions induced by the interstrand cross-link in the three 22-base pairs oligonucleotides d(TGCT/AGCA), d(AGCT/AGCT) and d(CGCT/AGCG) were compared by means of gel electrophoresis, circular dichroism, phenanthroline-copper footprinting and antibodies specifically directed against cis-DDP interstrand cross-links. The four different technical approaches indicate that the distorsions are independent of the chemical nature of the base pairs adjacent to the interstrand cross-link. The general conclusion is that the interstrand cross-link induces a bending and in particular an unwinding larger than other platinum adducts and the distorsions are independent of the nature of the bases (purine or pyrimidine) adjacent to the d(GC/GC) site.

Base Sequence↗
PubMed1994

DNA double helix promotes a linkage isomerization reaction in trans-diamminedichloroplatinum(II)-modified DNA.

In the reaction between trans-diamminedichloroplatinum(II) and a single-stranded pyrimidin-rich oligodeoxyribonucleotide (22-mer) containing the central sequence TGAGT, the 1,3-trans-[Pt(NH3)2[d(GAG)]] cross-link is formed. The 1,3-intrastrand cross-link is inert within the single-stranded oligonucleotide. In contrast, it rearranges to an interstrand cross-link when the platinated oligonucleotide is paired with its complementary deoxyribo- or ribonucleotide strand. The half-life of the 1,3-intrastrand cross-link, approximately 6 h at 37 degrees C, is independent of the nature and concentration of the salt (NaCl or NaClO4). It is not dramatically affected when the intervening adenine residue between the chelated guanine residues is replaced by a cytosine or a thymine residue or when the T.A base pair adjacent to the 5' or 3' side of the adduct is replaced by a C.G base pair. On the other hand, a mismatch on the 3' or 5' side of the adduct prevents the rearrangement. We propose that the linkage isomerization reaction results from a direct nucleophilic attack of the cytosine residue complementary to the platinated 5' guanine residue on the platinum residue. Among others, the potential use of the DNA.RNA-promoted reaction is discussed in the context of the antisense strategy to irreversibly cross-link the antisense oligonucleotides to their targets.

Base Sequence↗
PubMed1994

Gradual and oriented B-Z transition in 5'-untranscribed region of mouse ribosomal DNA.

The structural transition of an alternating purine-pyrimidine sequence (CG)5(TG)28) from the 5'-untranscribed region of the mouse ribosomal DNA was analyzed by two-dimensional gel electrophoresis and chemical probes. The repeat undergoes a supercoil-dependent gradual and oriented B-Z transition. At a threshold level of negative supercoiling, a limited region of the repeat encompassing the (CG)5 motif converts cooperatively into Z-DNA. As the superhelical stress increases, the Z-structure propagates along the remaining part of the repeat by successive transitions until the full-length sequence is converted. By in situ OsO4 probing experiments, we show also that this (TG)n-containing repeat adopts the Z-structure in Escherichia coli.

Acetaldehyde↗
PubMed1994

DNase I footprinting of cis- or trans-diamminedichloroplatinum(II)-modified DNA.

DNase I has been used as an enzymatic probe to visualize the conformational alteration induced in DNA by the binding of either the antitumor drug cis-platinum (cis-DDP) or the therapeutically inactive derivatives, trans-platinum (trans-DDP) and chlorodiethylene-triamineplatinum(II) (dien-Pt). We have constructed double-stranded oligonucleotides (52-mer) containing a single adduct either at the d(GG) site (cis-DDP intrastrand cross-link) or at the d(GC/GC) site (cis-DDP interstrand cross-link) or at the d(G/C) site (trans-DDP interstrand cross-link) or at the d(G) site (dien-Pt adduct). The platinated oligonucleotides are differently recognized by DNase I. As judged by DNase I, the distortions induced in the DNA double helix by the cis-DDP and trans-DDP interstrand cross-links spread over more base-pairs than that induced by the cis-DDP intrastrand cross-link.

Base Sequence↗
PubMed1994

A new approach to the measurement of quality of life. The Patient-Generated Index.

Quality of life has been defined as "the extent to which our hopes and ambitions are matched by experience." To improve a patient's quality of life through medical care would be to "narrow the gap between a patient's hopes and expectations and what actually happens." Using the above definition as a conceptual basis, we produced a self-administered, Patient-Generated Index (PGI) of quality of life. The PGI was completed by 359 patients presenting with low back pain. The validity of the measure was assessed by correlating patients' PGI scores with a well-validated health profile, the Short-Form 36-item Health Survey (SF-36), and with their scores on a clinical back pain questionnaire. Stepwise multiple regression was then used to model the relationship between the PGI score and the SF-36. Patients' PGI scores showed a high correlation with SF-36 scales measuring pain, social functioning, and role limitations attributable to physical problems, and with the clinical questionnaire. Together with whether a person was retired or not, these health variables were able to explain 25% of the variance in PGI scores. Patient generated index scores were significantly lower in patients referred to hospital compared with those managed solely in general practice and tended to reflect the general practitioner's assessment of symptom severity. We conclude that it is possible to construct a questionnaire that quantifies the effect of a medical condition on patients' quality of life in a way that has meaning and relevance in the context of their daily lives. The PGI has considerable potential for routine use in a wide range of clinical conditions for which the measurement of outcome has hitherto proved very difficult.

Activities of Daily Living↗
PubMed1993

Instability of the monofunctional adducts in cis-[Pt(NH3)2(N7-N-methyl-2-diazapyrenium)Cl](2+)-modified DNA: rates of cross-linking reactions in cis-platinum-modified DNA.

Single- and double-stranded oligonucleotides containing a single monofunctional cis-[Pt(NH3)2(dG)(N7-N-methyl-2-diazapyrenium)]3+ adduct have been studied at two NaCl concentrations. In 50 mM and 1 M NaCl, the adducts within the single-stranded oligonucleotides are stable. In contrast, they are unstable within the corresponding double-stranded oligonucleotides. In 50 mM NaCl, the bonds between platinum and guanine or N-methyl-2,7-diazapyrenium residues are cleaved and subsequently, intra- or interstrand cross-links are formed as in the reaction between DNA and cis-DDP. In 1 M NaCl, the main reaction is the replacement of N-methyl-2,7-diazapyrenium residues by chloride which generates double-stranded oligonucleotides containing a single monofunctional cis-[Pt(NH3)2(dG)Cl]+ adduct. The rates of closure of these monofunctional adducts to bifunctional cross-links have been studied in 60 mM NaClO4. Within d(TG.CT/AGCA), d(CG.CT/AGCG) and d(AG.CT/AGCT) (the symbol.indicates the location of the adducts in the central sequences of oligonucleotides), the half-lifes (t1/2) of the cis-[Pt(NH3)2(dG)Cl]+ adducts are respectively 12, 6 and 2.8 hr and the cross-linking reactions occur between guanine residues on the opposite strands. Within d(AG.TC/GACT), d(CG.AT/ATCG) and d(TGTG./CACA) or d(TG.TG/CACA) t1/2 are respectively 1.6, 8 and larger than 20 hr and the intrastrand cross-links are formed at the d(AG), d(GA) and d(GTG) sites, respectively. The conclusion is that the rates of conversion of cis-platinum-DNA monofunctional adducts to minor bifunctional cross-links are dependent on base sequence. The potential use of the instability of cis-[Pt(NH3)2(dG)(N7-N-methyl-2-diazapyrenium)]3+ adducts is discussed in the context of the antisense strategy.

Base Sequence↗
PubMed1993

DNA conformational change produced by the site-specific interstrand cross-link of trans-diamminedichloroplatinum(II).

The DNA distortion produced by the interstrand cross-link of trans-diamminedichloroplatinum-(II) has been described by means of gel electrophoresis, chemical probes, and molecular mechanics modeling. Synthetic double-stranded oligodeoxyribonucleotides of varying lengths (19-22 base pairs) were synthesized that contained a unique site-specific interstrand cross-link within their central sequence d(TGCT)/d(AGCT) between complementary guanine and cytosine residues. We find that the platinated deoxyriboguanosine residue adopts syn conformation. The duplex is distorted on both sides of the cross-link, but the bases are still paired. The distortion introduces some flexibility into the helix. In addition, the double helix is unwound and bent toward the major groove.

Base Sequence↗
PubMed1993

Spectrum of DNA--platinum adduct recognition by prokaryotic and eukaryotic DNA-dependent RNA polymerases.

Double-stranded DNA oligomers were constructed to evaluate the effect of bifunctional and monofunctional platinum(II) complexes at the level of DNA transcription. They contained a single lesion, which is either a cis-[Pt(NH3)2(d(GpTpG))] intrastrand cross-link, a trans-[Pt(NH3)2(d(GpTpG))] intrastrand cross-link, a cis-[Pt(NH3)2(d(GpC/GpC))] interstrand cross-link, or a (diethylenetriamine)-platinum(II)-dG adduct. The synthetic duplexes were multimerized and then used as templates in dinucleotide-primed reactions catalyzed by prokaryotic or eukaryotic RNA polymerases. Reactions were conducted in the presence of a single triphosphate substrate (single-step addition reaction) or of a combination of triphosphate substrates, permitting elongation of the trinucleotide products to longer RNA chains (productive elongation reaction), respectively. In transcription of the platinated strands, none of the DNA adducts provided an absolute block to formation of a single phosphodiester bond by either Escherichia coli RNA polymerase or wheat germ RNA polymerase II. However, the single-step addition reactions were much more impeded from transcription of bifunctional adduct-containing templates as compared to those containing monofunctional lesions. Productive elongation was irreversibly blocked in transcription of the platinated strand of templates containing a cis-d(G*pTpG*) intrastrand cross-link or a cis-d(G*pC/G*pC) interstrand cross-link. In both cases transcription stopped at the level of the lesion. Termination occurred also several nucleotides before the elongation complexes reached the interstrand cross-link. A substantial amount of the RNA polymerase molecules was able of bypassing the trans-d(G*pTpG*) cross-links. In all the cases single-step addition reactions were enhanced on the template strand complementary to that containing the intrastrand cross-links.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

DNA interstrand cross-links of trans-diamminedichloroplatinum(II) are preferentially formed between guanine and complementary cytosine residues.

Bases in the opposite strands of DNA cross-linked by clinically ineffective trans-diamminedichloroplatinum(II) (trans-[Pt(NH3)2Cl2]) have been identified by means of three experimental approaches. These include HPLC analysis of enzymatic digests of synthetic oligonucleotide duplexes containing the interstrand cross-link, footprinting experiments on the interstrand cross-linked oligonucleotide duplexes, and termination of the duplex transcription on trans-[Pt(NH3)2Cl2]-treated fragments of plasmid DNA. The results reveal that deoxyguanine and complementary deoxycytosine residues are preferential binding sites of trans-[Pt(NH3)2Cl2] in the interstrand adducts. The interstrand cross-linking reaction was studied by means of gel electrophoresis for the cis and trans isomers. The rate of formation of interstrand cross-links was lower for the trans isomer; however, trans-[Pt(NH3)2Cl2] formed about twice the amount of interstrand cross-links as compared with the cis isomer after 48 hr. The present results are suggested to be relevant to differences in clinical activity of the two platinum(II) isomers.

Base Sequence↗
PubMed1993

Detection of minor adducts in cisplatin-modified DNA by transcription footprinting.

Two DNA restriction fragments containing either a d(GC)5 or a d(TTGCTTGATTAGTTGTGTT) insert were subjected to reaction with cis-diamminedichloroplatinum(II) and were then used as templates for RNA synthesis by T7 RNA polymerase. Within the d(GC)5 insert, interstrand cross-links are preferentially formed. Within the second insert, the reactivity order of the potential binding sites is d(ApG) > d(GpC/GpC) = d(GpA) > d(GpTpG). In the presence of cyanide ions, the adducts are much less stable at the d(GpA) sites than at the d(GpCpG) sites, in double-stranded DNA.

Base Sequence↗
PubMed1992

Sequence-dependent distortions induced in DNA by monofunctional platinum(II) binding.

The effects on thermal stability and conformation of DNA produced by the monofunctional adducts of chlorodiethylenetriamineplatinum(II) chloride ([Pt(dien)Cl]Cl) have been investigated. Oligodeoxyribonucleotide duplexes of varying lengths (9-20 base pairs) and of varying central trinucleotide sequences were prepared and characterized that contained site-specific and unique N(7)-guanine adducts. Included are adducts at the sequences of d(AGC), d(AGT), d(CGA), d(TGA), d(TGC), and d(TGT). All these monofunctional adducts decrease the melting temperature (Tm) of the duplexes. This destabilization effect exhibits a sequence-dependent variability. The highest lowering of Tm is observed for the modified duplexes containing the central sequence of pyrimidine-guanine-pyrimidine. The destabilization effect is reduced with decreasing concentrations of Na+. Polarography, circular dichroism, phenanthroline-copper, and chemical probes reveal conformational distortions spreading over several base pairs around the adduct. The effects of monofunctional platinum(II) adducts on conformational distortions in DNA exhibit a sequence-dependent variability similar to those on thermal stability of DNA. The influence of the monofunctional adduct formed by cis-diamminemonoaquamonochloroplatinum(II) on the stability of the oligonucleotide duplex has been also studied. This lesion decreases thermal stability of DNA in the same way as does the adduct of [Pt(dien)Cl]Cl.

Base Sequence↗
PubMed1992

Lability of monofunctional cis-platinum adducts: role of DNA double helix.

Recently, we have shown that the adduct formed in the reaction between the platinum-triamine complex cis-[Pt(NH3)2(N7-N-methyl-2-diazapyrenium)Cl]2+ and one single-stranded oligonucleotide was stable but became labile as soon as the platinated oligonucleotide was paired with its complementary strand (Gaucheron et al. Proc. Natl. Acad. Sci. USA 88, 3516-3519 (1991)). To generalize this finding we have now studied large DNA fragments containing several adducts. The stability of the adducts within single-stranded DNA is demonstrated by absorption spectrophotometry and by replication mapping experiments. Several approaches are used to prove the lability of the adducts within double-stranded DNA. Replication mapping experiments reveal that an unmodified single-stranded DNA when mixed with double-stranded DNA modified by the platinum-triamine complex behaves as a single-stranded DNA modified by the triamine complex. After double-stranded DNA is modified by the platinum-triamine complex, intrastrand and interstrand cross-links are progressively formed during subsequent incubation as revealed by transcription mapping experiments and gel electrophoresis under denaturing conditions. Finally, replication mapping experiments show that the lability of the adducts within a double-stranded DNA depends upon the nature of the flanking nucleotide residues. All these results support the proposal that the DNA double helix acts as a catalyst in the reaction between DNA, cis-diamminedichloroplatinum(II) and N-methyl-2,7-diazapyrenium.

Bacteriophage T7↗
PubMed1992

Distortions induced in DNA by cis-platinum interstrand adducts.

A 22 base pair double-stranded oligonucleotide containing a unique interstrand adduct resulting from chelation of the two guanine residues within the central sequence d(TGCT/AGCA) by a cis-platinum residue has been studied by means of gel electrophoresis, chemical probes, and molecular mechanics. The anomalously slow electrophoretic mobility of the multimers of the platinated and ligated oligomers suggests that the platinated oligonucleotide is bent. The two cytosine residues (complementary to the platinated guanines) are hyperreactive to hydroxylamine, indicating a large exposure of the two bases to the solvent. The adduct does not induce a local denaturation within the flanking sequences since the adenine residues are not reactive with diethyl pyrocarbonate. This is confirmed by the nonreactivity of the complementary T residues with osmium tetraoxide. These results and the molecular mechanics modeling suggest that the interstrand adduct bends the double helix by approximately 55 degrees toward the major groove, that the double helix conserves its average twist angle, and that the distortion induced by the adduct is localized at the platinated sequence d(GC/CG).

Base Sequence↗
PubMed1992

RNA polymerases react differently at d(ApG) and d(GpG) adducts in DNA modified by cis-diamminedichloroplatinum(II).

Two duplexes (20-mers) were constructed containing either a single cis-[Pt(NH3)2[d(GpG)]] or cis-[Pt(NH3)2[d(ApG)]] intrastrand cross-link, the major DNA adducts of the antitumor drug cis-diamminedichloroplatinum(II). These synthetic duplexes were multimerized and the resultant polymers used as templates in single-step addition reactions of condensation of a single nucleoside triphosphate substrate to a dinucleotide primer (abortive elongation reaction) catalyzed by prokaryotic or eukaryotic RNA polymerases. Primer-substrate combinations were selected so as to direct trinucleotide product formation within the platinated bases of the templates. Transcription experiments established that cis-DDP-DNA adducts formed at d(ApG) or d(GpG) sites are not an absolute block to formation of a single phosphodiester bond by either Escherichia coli RNA polymerase or wheat germ RNA polymerase II. Furthermore, the kinetic data indicate that single-step addition reactions are much more impeded at the platinated d(GpG) than at the platinated d(ApG) site and that the mechanisms of inhibition of RNA polymerase activity are different at the two platinated sites. In particular, binding affinity between E. coli RNA polymerase and the d(GpG)-containing platinated template is lowered, as the apparent Km of enzyme for the platinated polymer is increased by a factor of 4-5. In contrast, binding affinity between the RNA polymerase and the d(ApG)-containing template is not affected by modification of the d(ApG) site by cis-diamminedichloroplatinum(II). Similar experiments were carried out with synthetic templates containing the adducts at the d(GpG) sites, in which one of the two platinated dG residues is paired with a dT residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

Biophysical analysis of DNA modified by 1,2-diaminocyclohexane platinum(II) complexes.

Modification of DNA and double-stranded deoxyoligonucleotides with antitumour 1,2-diamino-cyclohexanedinitroplatinum(II) (Pt-dach) complexes was investigated with the aid of physico-chemical methods and chemical probes of nucleic acid conformation. The three Pt-dach complexes were used which differed in isomeric forms of the dach nonleaving ligand-Pt(1R,2R-dach), Pt(1S,2S-dach) and Pt(1R,2S-dach) complexes. The latter complex has lower antitumour activity than the other two Pt-dach complexes. Pt(1R,2S-dach) complex exhibits the slowest kinetics of its binding to DNA and of the conversion of monofunctional binding to bifunctional lesions. The anomalously slow electrophoretic mobility of multimers of the platinated and ligated oligomers suggests that bifunctional binding of Pt-dach complexes to a d(GG) site within double-stranded oligonucleotides induces bending of the oligomer. In addition, chemical probing of double-helical deoxyoligonucleotides modified by the Pt-dach complexes at the d(GG) sites reveals that Pt(1R,2S-dach) complex induces more extensive conformational changes in the oligomer than Pt(1R,2R-dach) and Pt(1S,2S-dach) complexes. It is proposed that different effects of the Pt-dach complexes on DNA observed in this work arise mainly from a steric crowding of the axially oriented cyclohexane ring in the DNA adduct of Pt(1R,2S-dach) complex.

Antineoplastic Agents↗