Biomedical subjects

G Villani

Publications and source records attributed to G Villani.

At least 37 records · Page 2Linked to original sources

PubMed1998

Herpes simplex virus type-1 single-strand DNA-binding protein (ICP8) enhances the ability of the viral DNA helicase-primase to unwind cisplatin-modified DNA.

The herpes simplex virus type-1 UL5, UL8, and UL52 genes encode an essential heterotrimeric DNA helicase-primase that is responsible for concomitant DNA unwinding and primer synthesis at the viral DNA replication fork. The viral single-strand DNA-binding protein (ICP8) can stimulate DNA unwinding by the helicase-primase as a result of a physical interaction that is mediated by the UL8 subunit. In this study, we investigated the ability of the helicase-primase to unwind a fork-like substrate that contains an intrastrand d(GpG) DNA cross-link produced by the antitumor drug cisplatin. We also examined the ability of ICP8 to modulate the effect of the cisplatin lesion. The data show that the lesion inhibited the helicase-primase when located on the DNA strand along which it translocates. However, the lesion did not represent a permanent obstacle to its progression. In contrast, the adduct did not affect the helicase-primase when located on the opposite DNA strand. ICP8 specifically stimulated DNA unwinding by the helicase-primase. Coating concentrations of ICP8 were necessary for optimal unwinding of damaged DNA. Addition of competitor DNA to helicase reactions led to substantial reduction of DNA unwinding by the helicase-primase, suggesting that the enzyme is distributive. ICP8 did not abolish the competition, indicating that it did not stimulate the helicase by increasing its processivity. Rather, ICP8 may stimulate DNA unwinding and enable bypass of cisplatin damaged DNA by recruiting the helicase-primase to the DNA.

Base Sequence↗

PubMed1998

Cooperative coupling and role of heme a in the proton pump of heme-copper oxidases.

In the last few years, evidence has accumulated supporting the applicability of the cooperative model of proton pumps in cytochrome systems, vectorial Bohr mechanisms, to heme-copper oxidases. The vectorial Bohr mechanism is based on short- and long-range protonmotive cooperative effects linked to redox transitions of the metal centers. The crystal structure of oxidized and reduced bovine-heart cytochrome c oxidase reveals, upon reduction, the occurrence of long-range conformational changes in subunit I of the oxidase. Analysis of the crystal structure of cytochrome c oxidase shows the existence of hydrogen-bonded networks of amino acid residues which could undergo redox-linked pK shifts resulting in transmembrane proton translocation. Our group has identified four proteolytic groups undergoing reversible redox-linked pK shifts. Two groups result in being linked to redox transitions of heme a3. One group is apparently linked to CuB. The fourth group is linked to oxido-reduction of heme a. We have shown that the proton transfer resulting from the redox Bohr effects linked to heme a and CuB in the bovine oxidase displays membrane vectorial asymmetry, i.e., protons are taken up from the inner aqueous space (N), upon reduction, and released in the external space (P), upon oxidation of the metals. This direction of proton uptake and release is just what is expected from the vectorial Bohr mechanism. The group linked to heme a, which can transfer up to 0.9 H+/e- at pHs around neutrality, can provide the major contribution to the proton pump. It is proposed that translocation of pumped protons, linked to electron flow through heme a, utilizes a channel (channel D) which extends from a conserved aspartate at the N entrance to a conserved glutamate located between heme a and the binuclear center. The carboxylic group of this glutamic acid, after having delivered, upon electron flow through heme a, pumped protons towards the P phase, once reprotonated from the N phase, moves to deliver, subsequently, to the binuclear center chemical protons consumed in the conversion of the peroxy to ferryl and of the latter to the oxy intermediate in the redox cycle. Site-directed mutagenesis of protolytic residues in subunit I of the aa3-600 quinol oxidase of Bacillus subtilis to non-polar residues revealed that the conserved Lys 304 is critical for the proton pumping activity of the oxidase. Crystal structures of cytochrome c oxidase show that this lysine is at the N entrance of a channel which translocates the protons consumed for the production of the peroxy intermediate. Inhibition of this pathway, by replacement of the lysine, short-circuits protons from channel D to the binuclear center, where they are utilized in the chemistry of oxygen reduction.

Animals↗

PubMed1997

HMG1 protein inhibits the translesion synthesis of the major DNA cisplatin adduct by cell extracts.

When situated in a fork-like synthetic DNA replication substrate, the 1,2-intrastrand crosslink at the d(GpG) site, the most frequent adduct formed in the reaction between DNA and the anticancer drug cisplatin (cis-diamminedichloroplatinum (II)), is efficiently bypassed by eukaryotic cell extracts. We show here that the rat high-mobility-group protein 1 (HMG1) binds preferentially to the platinated fork-like synthetic DNA and inhibits the translesion synthesis. The same protein, but without the acidic tail, inhibits also the translesion synthesis. These results suggest that HMG proteins might contribute to the sensitivity of cells to cisplatin by directly affecting DNA replication.

Animals↗

PubMed1997

Effects of phorbol ester on carbachol-induced contraction in bovine ciliary muscle: possible involvement of protein kinase C.

The aim of the research was to characterize muscarinic receptors of bovine ciliary muscle and to investigate the desensitization process. The role of protein kinase C was analyzed. The results show that muscarinic receptors of bovine ciliary muscle have the pharmacological characteristics of the M3 subtype. Acute exposure to phorbol esters (1 microM phorbol 12,13-dibutyrate, PDB, or 0.1 microM phorbol 12-myristate 13-acetate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensitization and loss of phorbol ester-induced desensitization. Staurosporine (3 microM) and H7 [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 microM), protein kinase C inhibitors, produced a significant potentiation of the contractile effect of carbachol, reduced the desensitization produced by repeated addition of carbachol and suppressed that induced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [3H]quinuclidinyl benzilate. In vitro incubation with PDB and PMA produced, as expected, a significant translocation of protein kinase C from the cytosol to the membrane. The incubation of the ciliary muscle with carbachol, using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ciliary muscle is correlated, at least partially, to the translocation of protein kinase C from the cytosol to the membrane. The desensitization by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the density and affinity of muscarinic receptors is not involved.

Animals↗

PubMed1997

In vivo control of respiration by cytochrome c oxidase in wild-type and mitochondrial DNA mutation-carrying human cells.

The metabolic control of respiration is still poorly understood, due mainly to the lack of suitable approaches for studying it in vivo. Experiments on isolated mammalian mitochondria have indicated that a relatively small fraction of each of several components of the electron transport chain is sufficient to sustain a normal O2 consumption rate. These experiments, however, may not reflect accurately the in vivo situation, due to the lack in the mitochondrial fraction of essential cytosolic components and to the use of excess of substrates in the in vitro assays. An approach is described here whereby the control of respiration by cytochrome c oxidase (COX; EC 1.9.3.1) was analyzed in intact cultured human osteosarcoma 143B.TK- cells and other wild-type cells and in mitochondrial DNA mutation-carrying human cell lines. Surprisingly, in wild-type cells, only a slightly higher COX capacity was detected than required to support the endogenous respiration rate, pointing to a tighter in vivo control of respiration by COX than generally assumed. Cell lines carrying the MERRF mitochondrial tRNA(Lys) gene mutation, which causes a pronounced decrease in mitochondrial protein synthesis and respiration rates, revealed, in comparison, a significantly greater COX capacity relative to the residual endogenous respiration rate, and, correspondingly, a higher COX inhibition threshold above which the overall respiratory flux was affected. The observed relationship between COX respiratory threshold and relative COX capacity and the potential extension of the present analysis to other respiratory complexes have significant general implications for understanding the pathogenetic role of mutations in mtDNA-linked diseases and the tissue specificity of the mutation-associated phenotype.

Antimycin A↗

PubMed1997

Phase II study of continuous-infusion high-dose ifosfamide in advanced and/or metastatic pretreated soft tissue sarcomas.

BACKGROUND: Ifosfamide has important activity in pretreated soft tissue sarcomas (STS), and recent data support a clinically significant dose-response relationship for this agent. Administration by continuous infusion and hematopoietic support have rendered dose intensification regimens possible by reducing both hematologic and non-hematologic toxicities. The optimal dose and schedule of ifosfamide when given at high doses remain to be defined. In a previous phase I study, we demonstrated the feasibility of a continuous infusion (c.i.) high-dose ifosfamide (HDI) regimen in the ambulatory setting for patients with advanced solid tumors. The objective of the present phase II study was to assess the antitumor activity and toxicity of such a schedule in patients with advanced pretreated STS. PATIENTS AND METHODS: Thirty-eight patients with advanced and/or metastatic STS, all pretreated with an anthracycline with or without standard-dose ifosfamide, were treated. Ifosfamide was given by c.i. at a dose of 3.5 g/m2/day over four consecutive days, with equidose mesna uroprotection over five days. G-CSF was added at a dose of 200 micrograms/m2/day subcutaneously from day 6 to day 12. Cycles were repeated every three weeks in the outpatient setting. RESULTS: A total of 159 cycles of therapy were given (median 4 per patient, range 3-6). Treatment compliance was generally satisfactory. The major toxicity was hematologic, with six febrile neutropenic episodes requiring hospitalisation and parenteral antibiotics. Acute renal failure occurred in one patient after three cycles of therapy; central nervous system toxicity was mild. An overall response rate of 39% was observed (95% confidence interval, 26% to 55%), with one complete and 14 partial remissions. All but one of the responder patients had previously received standard-dose ifosfamide. The median response duration was nine months (range 5-21+ months), and the overall median survival ranged from 6-30+ months (median 13 months). CONCLUSIONS: High-dose ifosfamide is an active regimen in anthracycline-pretreated STS. Future clinical trials should be aimed at evaluating the impact of different administration schedules on clinical response and outcome. The potential role of HDI as front-line chemotherapy as well as in the adjuvant treatment of STS needs to be investigated in randomized trials.

Adult↗

PubMed1997

The Bacillus subtilis genome from gerBC (311 degrees) to licR (334 degrees).

As part of the international project to sequence the Bacillus subtilis genome, the DNA region located between gerBC (311 degrees) and licR (334 degrees) was assigned to the institut Pasteur. In this paper, the cloning and sequencing of 176 kb of DNA and the analysis of the sequence of the entire 271 kb region (6.5% of the B. subtilis chromosome) is described; 273 putative coding sequences were identified. Although the complete genome sequences of seven other organisms (five bacteria, one archaeon and the yeast Saccharomyces cerevisiae) are available in public database, 65 genes from this region of the B. subtilis chromosome encode proteins without significant similarities to other known protein sequences. Among the 208 other genes, 115 have paralogues in the currently known B. subtilis DNA sequences and the products of 178 genes were found to display similarities to protein sequences from public databases for which a function is known. Classification of these genes shows a high proportion of them to be involved in the adaptation to various growth conditions (non-essential cell wall constituents, catabolic and bioenergetic pathways); a small number of the genes are essential or encode anabolic enzymes.

Amino Acid Sequence↗

PubMed1997

Screening of gestational diabetes in Tuscany: results in 2000 cases.

According to the guidelines of the "Third international workshop conference on GDM", we have examined 2000 pregnant women. The glucose challenge test (GCT) was positive in 408 cases (20.4%) and negative in 1592 (79.6%). The OGTT (Carpenter and Coustan's criteria) was performed in 647 pregnant women. GDM and IGGT prevalence was of 6.25% and 5.5% respectively and normal glucose tolerance (NGT) 88.25%. The GCT effectiveness for GDM and IGGT diagnosis is: sensibility 75.1%, specificity 44%, positive predictive value 46.4% and negative predictive value 74%. GDM and IGGT compared with NGT women were significantly older (p < 0.05) and prepregnancy BMI was higher (p < 0.01); the prevalence of previous macrosomia (p < 0.01), previous gestational diabetes (p < 0.01) and family history for diabetes mellitus (p < 0.05) was greater in GDM and IGCT. The prevalence of preterm delivery was higher in both GDM and IGCT (GDM 12.5% and IGGT 15.4% vs NGT 6%; p < 0.01), as well as the prevalence of cesarean sections (GDM 31.6% vs IGGT 23.5% and NGT 20.3%; p < 0.02), and the occurrence of macrosomia (GDM 27.6%, IGGT 16.6% and NGT 16.2%). In addition a higher prevalence (p < 0.01) of hyperbilirubinaemia, hypoglycemia and hypertrophy cardiomyopathy was observed in newborns from GDM women. Our data show that: GCT has a good specificity for GDM diagnosis, prevalence of GDM in our population is about 6%, GDM is still correlated to an elevated maternal and neonatal morbility.

Adult↗

PubMed1996

Fork-like DNA templates support bypass replication of lesions that block DNA synthesis on single-stranded templates.

DNA replication is an asymmetric process involving concurrent DNA synthesis on leading and lagging strands. Leading strand synthesis proceeds concomitantly with fork opening, whereas synthesis of the lagging strand essentially takes place on a single-stranded template. The effect of this duality on DNA damage processing by the cellular replication machinery was tested using eukaryotic cell extracts and model DNA substrates containing site-specific DNA adducts formed by the anticancer drug cisplatin or by the carcinogen N-2-acetylaminofluorene. Bypass of both lesions was observed only with fork-like substrates, whereas complete inhibition of DNA synthesis occurred on damaged single-stranded DNA substrates. These results suggest a role for additional accessory factors that permit DNA polymerases to bypass lesions when present in fork-like DNA.

2-Acetylaminofluorene↗

PubMed1996

The UL8 subunit of the herpes simplex virus type-1 DNA helicase-primase optimizes utilization of DNA templates covered by the homologous single-strand DNA-binding protein ICP8.

The herpes simplex virus type-1 DNA helicase-primase is a heterotrimer encoded by the UL5, UL8, and UL52 genes. The core enzyme, specified by the UL5 and UL52 genes, retains DNA helicase, DNA-dependent nucleoside triphosphatase, and primase activities. The UL8 subunit has previously been implicated in increasing primer stability and in stimulating primer synthesis by the core enzyme. To further characterize the function of the UL8 subunit, we have examined its effect on the activities of the UL5/52 core enzyme using DNA templates covered by the herpes simplex virus type-1 single-strand DNA-binding protein ICP8. We found that while ICP8 stimulated the DNA helicase activity of the UL5/52 proteins up to 3-fold, maximum stimulation by ICP8 required the presence of UL8 protein. Moreover, UL8 protein was required to reverse the inhibitory effect of ICP8 on the DNA-dependent ATPase and primase activities of the UL5/52 proteins. These observations were specific for ICP8 since the heterologous Escherichia coli single-strand DNA-binding protein could not substitute for ICP8. These data suggest that UL8 protein mediates an interaction between the UL5/52 core enzyme and ICP8 that optimizes the utilization of ICP8-covered DNA templates during DNA replication.

Animals↗

PubMed1996

In vitro bypass replication of the cisplatin-d(GpG) lesion by calf thymus DNA polymerase beta and human immunodeficiency virus type I reverse transcriptase is highly mutagenic.

Eukaryotic DNA polymerase beta and the reverse transcriptases are the most inaccurate of the known DNA polymerases. We report here mutagenic replication in vitro past intrastrand N(7)G-N(7)G chelates of the cis-diamminedichloroplatinum(II), the major DNA adduct of the antitumor agent cisplatin by calf thymus DNA polymerase beta and human immunodeficiency virus type I reverse transcriptase (42% and 26% mutations, respectively). The most frequent modifications generated by both enzymes were one-base frameshift deletions. Only one mutational hot spot opposite the platinated guanines was observed with human immunodeficiency virus type I reverse transcriptase, while two hot spots were generated by DNA polymerase beta, one at the base situated 5' to the lesion and the other situated 4-6 nucleotides 5' to the adduct. An unusual mutagenic event, tandem replication of a 12-base pair sequence, was observed with DNA polymerase beta. The mutational spectra of the two DNA polymerases suggest that template slippage occurred with higher frequency in the presence of the more distributive DNA polymerase beta.

Animals↗

PubMed1996

[Solitary fibrous mesothelioma of the peritoneum. Considerations of a clinical case in our observation].

The authors report a case of so-called solitary fibrous peritoneal mesothelioma which was successfully operated on. Their case is compared with the few cases reported in the literature. Analysis of findings shows the fibrous peritoneal mesothelioma to be a solitary well defined tumor, generally of considerable size at the time of the diagnosis, very similar to the fibrous metothelioma of the pleura. It is believed to originate in the sub-mesothelial connective tissue. Surgery provides a definitive cure.

Adult↗

PubMed1995

Functional analysis of subunits III and IV of Bacillus subtilis aa3-600 quinol oxidase by in vitro mutagenesis and gene replacement.

Using the high efficiency of homologous gene recombination in Bacillus subtilis, a strategy for mutational analysis of the proton pumping aa3-600 quinol oxidase of this organism has been developed. The qox operon with the qoxA, qoxB, qoxC and qoxD genes, coding for the four subunits of this oxidase, was deleted and then replaced with mutated copies in which qoxC (subunit III) or qoxD (subunit IV) genes were deleted. The complete deletion of the qox operon caused disappearance of heme aa3-600 and a slight depression of the overall respiratory activity, compensated by alternative oxidase with no proton pumping activity. Deletion of qoxC probably resulted in a defective assembly of the aa3-600 quinol oxidase. The strain with deletion of qoxD gene expressed normal content of heme aa3-600 but exhibited a reduced respiratory activity and a significantly depressed proton pumping activity. These results show that subunit IV is critical for the activity of the proton pumping aa3-600 quinol oxidase.

Bacillus subtilis↗

PubMed1995

DNA polymerase beta bypasses in vitro a single d(GpG)-cisplatin adduct placed on codon 13 of the HRAS gene.

We have examined the capacity of calf thymus DNA polymerases alpha, beta, delta, and epsilon to perform in vitro translesion synthesis on a substrate containing a single d(GpG)-cisplatin adduct placed on codon 13 of the human HRAS gene. We found that DNA synthesis catalyzed by DNA polymerases alpha, delta, and epsilon was blocked at the base preceding the lesion. Addition of proliferating cell nuclear antigen to DNA polymerase delta and replication protein A to DNA polymerase alpha did not restore their capacity to elongate past the adduct. On the other hand, DNA polymerase beta efficiently bypassed the cisplatin adduct. Furthermore, we observed that DNA polymerase beta was the only polymerase capable of primer extension of a 3'-OH located opposite the base preceding the lesion. Likewise, DNA polymerase beta was able to elongate the arrested replication products of the other three DNA polymerases, thus showing its capacity to successfully compete with polymerases alpha, delta, and epsilon in the stalled replication complex. Our data suggest (i) a possible mechanism enabling DNA polymerase beta to bypass a d(GpG)-cisplatin adduct in vitro and (ii) a role for this enzyme in processing DNA damage in vivo.

Animals↗

PubMed1995

Transfection of mitochondria: strategy towards a gene therapy of mitochondrial DNA diseases.

Successes in classical gene therapies have been achieved by placing a corrected copy of a defective nuclear gene in cells. A similar gene replacement approach for a mutant mitochondrial genome is invariably linked to the use of a yet unavailable mitochondrial transfection vector. Here we show that DNA coupled covalently to a short mitochondrial leader peptide (chimera) can enter mitochondria via the protein import pathway, opening a new way for gene-, antisense-RNA- or antisense-DNA-delivery in molecular therapies. The import behavior of the purified chimera, composed of the amino-terminal leader peptide of the rat ornithine transcarbamylase (OTC) and a double stranded DNA molecule (17 bp or 322 bp), was tested by incubating with coupled and 'energized' rat liver mitochondria in the presence of reticulocyte lysate. The chimera was translocated with a high efficiency into the matrix of mitochondria utilizing the protein import pathway, independent from the size of its passenger DNA.

Amino Acid Sequence↗

PubMed1995

Replication of DNA containing cisplatin lesions and its mutagenic consequences.

Cisplatin [cis-diamminedichloroplatinum II] is widely used in the treatment of a broad range of tumors. A number of biological and biochemical results indicate that the reaction of cisplatin with DNA is responsible for the cytotoxic action of the drug. However, cisplatin can induce mutagenesis and may be carcinogenic in humans. Error prone replication of damaged DNA must be considered as a possible mechanism of mutagenesis. In this short review, we present data indicating that DNA containing cisplatin lesions can be replicated by prokaryotes and eukaryotes in a mutagenic fashion.

Animals↗

PubMed1994

Role of nuclear-encoded subunits of mitochondrial cytochrome c oxidase in proton pumping revealed by limited enzymatic proteolysis.

The role of supernumerary subunits of bovine heart cytochrome c oxidase has been investigated by examining the effect on the enzymatic activity of limited proteolysis by chymotrypsin, thermolysin, and trypsin. All three proteases, when added to the soluble oxidase, digested subunits III, VIa, and VIb and caused inhibition of electron flow in the oxidase. In addition, trypsin and thermolysin also digested subunit IV. Trypsin cleaved off an N-terminal segment of seven residues; thermolysin cleaved only the first four residues at the N-terminus of subunit IV. Digestion of the soluble oxidase by trypsin but not by thermolysin caused decoupling of redox-linked proton pumping in the oxidase. It is concluded that the sequence V5-V6-K7 of the hydrophilic N-terminus of subunit IV, which protrudes out of the matrix side of the mitochondrial membrane, mediates the access of protons into the transmembrane proton translocating pathway in the oxidase.

Amino Acid Sequence↗

PubMed1994

Effect of the major DNA adduct of the antitumor drug cis-diamminedichloroplatinum (II) on the activity of a helicase essential for DNA replication, the herpes simplex virus type-1 origin-binding protein.

To determine the effect of the major DNA adduct, the intrastrand d(GpG) cross-link, produced by the antitumor drug cis-diamminedichloroplatinum(II) on the activity of a helicase known to be essential for DNA replication, we have examined its interaction with the origin-binding protein (UL9 protein) of herpes simplex virus type-1. We found that the helicase activity of the UL9 protein is inhibited only when the adduct is present on the template strand along which the protein translocates. This effect was paralleled by a comparable inhibition of the UL9 protein's DNA-dependent ATPase activity. The inhibitory effect of the lesion can be reduced by the addition of the herpes simplex virus type-1 single-stranded DNA-binding protein, ICP8. This stimulatory effect is specific for ICP8 and appears to be the result of the functional and physical interaction that is known to exist between the UL9 protein and ICP8, and not due to the preferential interaction of ICP8 with the adduct.

Adenosine Triphosphatases↗