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Biomedical subjects

G Villani

Publications and source records attributed to G Villani.

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

Mutagenesis in monkey cells of a vector containing a single d(GPG) cis-diamminedichloroplatinum(II) adduct placed on codon 13 of the human H-ras proto-oncogene.

Cisplatin (cis-[Pt(NH3)2Cl2]) is a widely used antitumor agent whose mutagenic activity raises the possibility of the induction of secondary cancer as a result of treatment. Mutation of the proto-oncogene H-ras is found in more than 30% of all human tumors, where it has been postulated to contribute to the initiation and progression of human cancers. Activating mutations in the H-ras gene are predominantly single-base substitutions, most frequently at codons 12, 13 and 61. In the present work we have studied the mutational spectra induced by a single cis-[Pt(NH3)2d(GpG)] adduct, the most frequent DNA crosslink formed by cisplatin. We have constructed a 25-mer-Pt oligonucleotide singly modified at codon 13 (GGT) within the human H-ras DNA sequence and we have inserted it into a single-stranded SV40-based shuttle vector able to replicate in simian COS7 cells. After replication in the mammalian host, vectors were extracted, amplified in bacteria and DNA from 124 randomly chosen colonies was sequenced. The observed mutation frequency was 21%. Base substitutions were the most frequent modification. 92% of the mutagenic events occurred at one or both of the platinated guanines of codon 13. The single G-->T transversion accounted for 65% of the total mutations scored. All single base substitutions were located at the G in the 3' position showing, for the first time, that the guanine at the 3' side of a cis-[Pt(NH3)2d(GpG)] adduct may be a preferential site for cisplatin induced mutations. The substitution G-->T at this position of the codon 13 of the H-ras proto-oncogene is known to induce the oncogenic properties of the p21ras protein.

Animals↗
PubMed1994

Purification and biochemical characterization of Escherichia coli RecA proteins mutated in the putative DNA binding site.

Escherichia coli RecA protein plays a central role both in DNA repair and in recombination. We report biochemical properties of three new RecA proteins mutated at positions 199 (RecA694), 207 (RecA659), and 211 (RecA611) in the putative DNA binding site. RecA694 had a wild-type phenotype, whereas RecA611 and RecA659 were deficient in promoting both the self-cleavage of LexA repressor and the DNA-strand exchange reaction. In order to determine the origin of this inhibition, we examined the capacity of wild-type and mutant proteins to bind to single-stranded DNA (with and without single-stranded binding protein, SSB), double-stranded DNA, and ATP. DNA strand exchange defects were correlated with the inability of mutant proteins to displace SSB from DNA. For the recA659 mutation this inhibition was reversed by equimolar wild-type protein. In contrast, mixtures of either wild-type/RecA659 or wild-type/RecA611 proteins remained deficient in LexA cleavage, suggesting that the dominant negative phenotype of the mutant proteins may be a consequence of the formation heterologous RecA complexes. Various mutations in the putative DNA binding site of RecA protein altered ATP binding, ATPase activity, displacement of SSB from single-stranded DNA, and protein-protein interactions. These results are consistent with the hypothesis that DNA binding to this site of RecA relays allosteric effects to several functional domains throughout the protein.

Adenosine Triphosphatases↗
PubMed1994

The proton pump of heme-copper oxidases.

Proton pumping heme-copper oxidases represent the terminal, energy-transfer enzymes of respiratory chains in prokaryotes and eukaryotes. The CuB-heme a3 (or heme o) binuclear center, associated with the largest subunit I of cytochrome c and quinol oxidases, is directly involved in the coupling between dioxygen reduction and proton pumping. The role of the other subunits is less clear. The following aspects will be covered in this paper: i) the efficiency of coupling in the mitochondrial aa3 cytochrome c oxidase. In particular, the effect of respiratory rate and protonmotive force on the H+/e- stoichiometry and the role of subunit IV; ii) mutational analysis of the aa3 quinol oxidase of Bacillus subtilis addressed to the role of subunit III, subunit IV and specific residues in subunit I; iii) possible models of the protonmotive catalytic cycle at the binuclear center. The observations available suggest that H+/e- coupling is based on the combination of protonmotive redox catalysis at the binuclear center and co-operative proton transfer in the protein.

Animals↗
PubMed1994

A prevalence study of known diabetes mellitus in Tuscany assessed from pharmaceutical prescriptions and other independent sources.

This study evaluates the prevalence of diabetes mellitus (DM) in Pisa (Tuscany, Italy) using four independent data sources. The main source, represented by computerized prescriptions for anti-diabetic agents collected over a 4-month period, was validated using three secondary sources: (a) the list of diabetic patients who receive material of self-care from the National Health Service; (b) the clinical records of diabetic patients obtained from a random sample of family doctors; (c) the clinical records of diabetic patients attending our outpatient clinic. The main source provided 3806 patients, and 697 patients were added from the secondary sources, thus identifying a total number of 4503. The prevalence of known DM in the "Pisa area" exclusively reckoned by the main source, was 2.01%, and the prevalence corrected by the addition of the various sources resulted in 2.4%. The capture-recapture method showed a completeness of ascertainment of the survey of 90.1%, and thus an estimated prevalence of known diabetes of 2.64%. Of these, 141 patients had insulin-dependent diabetes mellitus (IDDM) corresponding to 3.2% of identified diabetic subjects (prevalence 0.07% inhabitants); 4362 patients had non-insulin-dependent diabetes mellitus (NIDDM), 96.8% of identified diabetic subjects (prevalence 2.36%). Of patients with NIDDM 10.5% was treated by diet, 65% with oral hypoglycaemic agents (OHA), 23% with insulin and 1.5% with insulin plus OHA. This study shows that the method used in this survey is suitable for epidemiological studies because it does not demand the cooperation of the diabetic patients, is addressed to the entire diabetic population without age discrimination and singles out the diabetic population in a very reliable way.

Adult↗
PubMed1993

Effects of a single intrastrand d(GpG) platinum adduct on the strand separating activity of the Escherichia coli proteins RecB and RecA.

RecB and RecA proteins play key roles in the process of DNA recombination in Escherichia coli and both possess DNA unwinding activities which can displace short regions of duplex DNA in an ATP-dependent manner in vitro. We have examined the effect of the most abundant DNA adduct caused by the chemotherapeutic agent cis-diamminedichloroplatinum(II) on those activities. For this purpose, we have constructed a partially duplex synthetic oligonucleotide containing the intrastrand d(GpG) crosslink positioned at a specific site. We report here that both the DNA strand separating and DNA-dependent ATPase activities of the RecB protein are inhibited by the d(GpG) cis-DDP adduct. In contrast, neither the unwinding nor the ATPase activities of RecA protein appear to be perturbed by this lesion.

Base Sequence↗
PubMed1993

Resistance to cisplatin in an E. coli B/r NalR mutant.

The effects of various mutations in DNA-repair processes have been reported to either enhance or decrease bacterial sensitivity to cis-diamminedichloroplatinum(II) (cis-DDP). In the search for other mutations affecting bacterial sensitivity to this antitumor compound, we tested the E. coli B/r BS80 mutant, which is resistant to nalidixic acid (NalR). This mutation maps in the topoisomerase II gene (gyrA subunit) and leads to cross-resistance to cis-DDP. The mechanism underlying the resistance phenotype was only partly due to decreased DNA platination. BS80 was cross-resistant to mitomycin C and, to a lesser extent, to UV light, while it was normally sensitive to MNNG. The mechanisms involved in cis-DDP and mitomycin C resistance were independent of uvrA (excision repair) and recA (SOS repair and recombination) gene expression. In contrast, UV resistance was dependent upon recA gene expression. Both the reversion to NalS in BS80 and the transduction of NalR in the parental wild type (F26) did not modify cis-DDP toxicity; in addition, platinated plasmids equally survived in BS80 and F26 strains. Hence, it is possible that selection of the NalR phenotype induced other mutation(s) than gyrA responsible for cis-DDP, mitomycin C and UV resistance and/or that lesions with a different toxic potential were introduced by cis-DDP into the BS80 and F26 chromosomes.

Cisplatin↗
PubMed1992

Characterization and localization of cis-diamminedichloro-platinum(II) adducts on a purified oligonucleotide containing the codons 12 and 13 of H-ras proto-oncogene.

The use of substrates containing well defined adducts at precise sites, is required to perform a careful analysis of the toxic and mutagenic potential of a lesion. As a first step in this direction the octamer 5'-d(CCGGCGGT), containing the sequence of the codons 12 d(GGC) and 13 d(GGT) of the human H-ras gene, was reacted with the antitumoral drug cis-diamminedichloroplatinum(II). The platinated products have been purified by HPLC. A first set of experiments, including enzymatic digestions with nuclease P1 followed by alkaline phosphatase and acid-catalysed hydrolysis, allowed us to determine which bases were engaged in the cis-DDP lesions. Our results indicate that only guanine residues were chelated with cisplatin to yield bifunctional adducts. Furthermore, by performing enzymatic digestions with phosphodiesterases, we have located the adducts with respect to the 5' end of the octamer. Among the purified and characterized platinated oligonucleotides, three present a particular interest, since we have shown here that the cis-d(GpG) adduct is precisely situated either at the d(GGC) or at the d(GGT) or at both sites of their sequence.

Base Sequence↗
PubMed1992

In vitro DNA synthesis by DNA polymerase I and DNA polymerase alpha on single-stranded DNA containing either purine or pyrimidine monoadducts.

The capacity of the large fragment of DNA polymerase I from Escherichia coli and of DNA polymerase alpha from Drosophila embryo to replicate single-stranded M13mp10 DNA containing either purine or pyrimidine monoadducts was compared. The monoadducts were respectively induced by cisplatinum and by furocoumarin photoaddition. For both types of lesions, it is observed that the eukaryotic enzyme is more inhibited than the prokaryotic one. By mapping the arrest sites produced by furocoumarin monoadducts on the synthesis catalysed by DNA polymerase alpha, we show that, in contrast with the photoreaction observed with double-stranded DNA, these compounds do not show a strong sequence specificity in reacting with single-stranded DNA.

Animals↗
PubMed1992

[Serum osteocalcin and diabetes mellitus. A study of 98 patients].

The goal of this study was to evaluate in 98 diabetic patients the serum levels of osteocalcin (OC) and their relationship with glycosylated hemoglobin levels and with the duration, calculated in years, of the disease. Patients were divided in 3 groups: 17 IDDM patients, 62 NIDDM patients treated with oral hypoglycaemic agents, and 19 NIDDM patients treated with insulin. Results were compared to 2 different control groups. In IDDM patients OC serum levels were significantly lower if compared either to control group and to NIDDM patients. The 2 groups of NIDDM patients showed significantly higher OC values than controls. No significant relationship resulted between OC levels, the duration of diabetes and the glycosylated hemoglobin values. The results of the study indicate a direct correlation between pancreatic function and osteoblastic activity: insulin lack is associated with reduced OC serum levels.

Adolescent↗
PubMed1991

Interaction of cis-diamminedichloroplatinum (II) with single-stranded DNA in the presence or absence of Escherichia coli single-stranded binding protein.

We have compared the mode of fixation in vitro of the antitumor drug cis-diamminedichloroplatinum (II) (cis-DDP) to single-stranded M13mp10 DNA either in the presence or absence of the Escherichia coli single-stranded binding protein (SSB). Platinum binding sites have been identified by taking advantage of their capacity to inhibit DNA replication of primed M13 DNA catalysed by E. coli DNA polymerase I large fragment. We report here that the presence of SSB increases the number of platinum-DNA lesions and alters their distribution. We also present evidence that SSB allows cis-DDP to bind to DNA sequences otherwise less accessible.

Bacterial Proteins↗
PubMed1991

H+/e- stoichiometry of mitochondrial cytochrome complexes reconstituted in liposomes. Rate-dependent changes of the stoichiometry in the cytochrome c oxidase vesicles.

The H+/e- stoichiometry of protonmotive cytochrome c oxidase, isolated from bovine heart mitochondria and reconstituted in liposomes, has been determined by making use of direct spectrophotometric measurements of the initial rates of e- flow and H+ translocation. It is shown that the ----H+/e- ratio for redox-linked proton ejection by the oxidase varies from around 0 to a maximum of 1 as a function of the rate of overall electron flow in the complex.

Animals↗