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Surfaces suitable for rock art at the base of Mont Bego, in the south-western Alps, gave rise to one of the most important concentrations of rock art in Western Europe. The open-air rock art site features some 20,000 figurative engravings pecked on 4200 rocks. The Merveilles sector was in use since the Early Neolithic, and perhaps even earlier, with the beginning of Holocene.

PNAs conjugated to carrier peptides have been employed for the targeting of miRNA precursor, with the aim to develop molecules able to interfere in the pre-miRNA processing. The capability of the molecules to bind pre-miRNA has been tested in vitro by fluorescence assayes on Thiazole Orange labeled molecules and in vivo, in K562 cells, evaluating the amount of miRNA produced after treatment of cells with two amounts of PNAs.

Peptide nucleic acids (PNAs) are oligonucleotide mimics in which the sugar‐phosphate backbone has been replaced by a pseudo‐peptide backbone. Among PNA‐based molecules, PNA‐DNA conjugates characterized by tracts of DNA bound to N and/or C terminus of PNA are very soluble in aqueous media, are able to recognize exclusively single strands of DNA and RNA in antiparallel fashion, activate RNAse H, bind to transcription factors and are more stable than DNA to nucleases degradation. Very little information is available on chimeras constituted of alternating monomers of PNA and DNA. In this article, we describe a simple fully automated strategy for the synthesis of 6‐mer and 10‐mer alternate PNA‐DNA chimeras consisting of polythymine oligomers, stability assays in fetal calf serum, UV and CD studies of the single strand alternate chimeras and of alternate chimera/DNA and alternate chimera/RNA duplexes. Evidences supporting the formation of duplex hybrids were found. Furthermore, the ability of forming Hoogsteen base pairing with duplex DNA was investigated. Finally, we tested the ability of the PNA‐DNA alternates in (a) interfering with reverse transcription of eukaryotic mRNA and (b) inhibiting DNA‐protein interactions. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 815–822, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

The DNA binding properties and the effects on protein/DNA interactions of a series of four hybrids prepared combining oligopyrrole minor groove binders and pyrrolo [2,1-c][1,4] benzodiazepine (PBD) were studied. In addition, the effects on in vitro and ex vivo transcription directed by the long terminal repeat (LTR) of the human immunodeficiency type 1 virus (HIV-1) were analysed and structure-activity relationships developed. These hybrids (compounds 2–5) contain from one to four pyrrole units, respectively. To investigate sequence-selectivity and stability of drugs/DNA complexes, DNase I foothyphen-printing, filter binding, and arrested polymerase-chain reaction (PCR) were performed on HIV-1 LTR gene sequences. The antiproliferative activity of the hybrids was tested in vitro on T-lymphoid Jurkat cell lines and compared to antiproliferative effects of the natural product distamycin A 1 and pyrrolo [2,1-c][1,4] benzodiazepine (PBD 6). The results obtained demonstrate that hybrids 2–5 exhibit different DNA-binding activity with respect to both distamycin A 1 and PBD 6. A direct relationship was found between number of pyrrole rings present in the hybrids 2–5 and stability of drugs/DNA complexes. With respect to antiproliferative effects, it was found that the increase in the length of the polypyrrole backbone leads to an increase of in vitro antiproliferative effects, i.e., the hybrid 5, containing the four pyrroles distamycin analogs, is more active than 2, 3 and 4 against the Jurkat cell lines. With respect to inhibition of HIV-1 LTR driven transcription, it was found that the hybrid 5 containing the four pyrroles distamycin analogs, is more active than 2, 3 and 4. Interestingly, the present results suggest that the hybrid 3 exhibits low antiproliferative activity on Jurkat cell lines, being still active in inhibiting HIV-1 LTR driven transcription both in vitro and ex vivo. Drug Dev. Res. 60:173–185, 2003. © 2003 Wiley-Liss, Inc.

The DNA binding properties and the effects on protein/DNA interactions of a series of four hybrids prepared combining oligopyrrole minor groove binders and pyrrolo [2,1‐c][1,4] benzodiazepine (PBD) were studied. In addition, the effects on in vitro and ex vivo transcription directed by the long terminal repeat (LTR) of the human immunodeficiency type 1 virus (HIV‐1) were analysed and structure‐activity relationships developed. These hybrids (compounds 2–5) contain from one to four pyrrole units, respectively. To investigate sequence‐selectivity and stability of drugs/DNA complexes, DNase I foothyphen‐printing, filter binding, and arrested polymerase‐chain reaction (PCR) were performed on HIV‐1 LTR gene sequences. The antiproliferative activity of the hybrids was tested in vitro on T‐lymphoid Jurkat cell lines and compared to antiproliferative effects of the natural product distamycin A 1and pyrrolo [2,1‐c][1,4] benzodiazepine (PBD 6). The results obtained demonstrate that hybrids 2–5exhibit different DNA‐binding activity with respect to both distamycin A 1and PBD 6. A direct relationship was found between number of pyrrole rings present in the hybrids 2–5and stability of drugs/DNA complexes. With respect to antiproliferative effects, it was found that the increase in the length of the polypyrrole backbone leads to an increase of in vitro antiproliferative effects, i.e., the hybrid 5, containing the four pyrroles distamycin analogs, is more active than 2, 3and 4against the Jurkat cell lines. With respect to inhibition of HIV‐1 LTR driven transcription, it was found that the hybrid 5containing the four pyrroles distamycin analogs, is more active than 2, 3and 4. Interestingly, the present results suggest that the hybrid 3exhibits low antiproliferative activity on Jurkat cell lines, being still active in inhibiting HIV‐1 LTR driven transcription both in vitro and ex vivo. Drug Dev. Res. 60:173–185, 2003. © 2003 Wiley‐Liss, Inc.

We report in this paper that the DNA-binding drug mithramycin is a potent inducer of γ-globin mRNA accumulation and fetal hemoglobin (HbF) production in erythroid cells from healthy human subjects and β-thalassemia patients. Erythroid precursors derived from peripheral blood were grown in 2-phase liquid culture. In this procedure, early erythroid progenitors proliferate and differentiate during phase 1 (in the absence of erythropoietin) into late progenitors. In phase 2, in the presence of erythropoietin, the latter cells continue their proliferation and mature into Hb-containing orthochromatic normoblasts. Compounds were added on days 4 to 5 of phase 2 (when cells started to synthesize Hb), and cells were harvested on day 12. Accumulation of mRNAs for γ-globin, β-globin, α-globin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin were measured by real-time quantitative reverse transcription–polymerase chain reaction (RT-PCR); induction of HbF was analyzed by high-performance liquid chromatography (HPLC) and, at cellular level, by flow cytometry. We demonstrated that mithramycin was able to up-regulate preferentially γ-globin mRNA production and to increase HbF accumulation, the percentage of HbF-containing cells, and their HbF content. Mithramycin was more effective than hydroxyurea, being, in addition, not cytotoxic. This was shown by the lack of cytotoxicity on erythroid and myeloid in vitro primary cell cultures treated with mithramycin at concentrations effective for HbF induction. These results are of potential clinical significance because an increase of HbF alleviates the symptoms underlying β-thalassemia and sickle cell anemia. The results of this report suggest that mithramycin and its analogs warrant further evaluation as potential therapeutic drugs.

We report in this paper that the DNA-binding drug mithramycin is a potent inducer of γ-globin mRNA accumulation and fetal hemoglobin (HbF) production in erythroid cells from healthy human subjects and β-thalassemia patients. Erythroid precursors derived from peripheral blood were grown in 2-phase liquid culture. In this procedure, early erythroid progenitors proliferate and differentiate during phase 1 (in the absence of erythropoietin) into late progenitors. In phase 2, in the presence of erythropoietin, the latter cells continue their proliferation and mature into Hb-containing orthochromatic normoblasts. Compounds were added on days 4 to 5 of phase 2 (when cells started to synthesize Hb), and cells were harvested on day 12. Accumulation of mRNAs for γ-globin, β-globin, α-globin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin were measured by real-time quantitative reverse transcription–polymerase chain reaction (RT-PCR); induction of HbF was analyzed by high-performance liquid chromatography (HPLC) and, at cellular level, by flow cytometry. We demonstrated that mithramycin was able to up-regulate preferentially γ-globin mRNA production and to increase HbF accumulation, the percentage of HbF-containing cells, and their HbF content. Mithramycin was more effective than hydroxyurea, being, in addition, not cytotoxic. This was shown by the lack of cytotoxicity on erythroid and myeloid in vitro primary cell cultures treated with mithramycin at concentrations effective for HbF induction. These results are of potential clinical significance because an increase of HbF alleviates the symptoms underlying β-thalassemia and sickle cell anemia. The results of this report suggest that mithramycin and its analogs warrant further evaluation as potential therapeutic drugs.

We have investigated the effects of aromatic polyamidines on HIV-1 transcription. We found a block to Tat-induced HIV-1 transcription assessed by inhibition of CAT activity in HL3T1 cells at a concentration lower than the IC50 value, suggesting that molecules with three (TAPB) and four (TAPP) benzamidine rings could be useful against HIV-1. In contrast, aromatic polyamidines with only two benzamidine rings (DAPP) did not block Tat-induced transcription. We reasoned that this effect could be due to binding of TAPB and TAPP to HIV-1 TAR RNA. By EMSA and filter binding assays, we studied possible interactions of aromatic polyamidines with HIV-1 TAR RNA. Wild-type TAR RNA or TAR RNA with mutations in the stem or bulge sequences, but retaining the stem-loop structure, was used to define the RNA-binding activities of these compounds. Our data suggest that aromatic polyamidines with two (DAPP) and four (TAPP) benzamidine rings, respectively, do not bind to TAR RNA or bind without sequence selectivity. Interestingly, an aromatic polyamidine with three benzamidine rings (TAPB) recognizes the wild-type TAR RNA in a specific manner. Furthermore, we found that introduction of one halogen atom into the benzamidine rings strongly increases the RNA-binding activity of these compounds.

AbstractPeptide nucleic acids (PNA) have recently been proposed as alternative reagents in experiments aimed to the control of gene expression. In PNAs, the pseudopeptide backbone is composed of N-(2-aminoethyl)glycine units and therefore is stable in human serum and cellular extracts. PNAs hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices and giving rise to highly stable (PNA)2-RNA triplexes with RNA targets. Therefore, antisense and antigéne PNAs have been synthetized and characterized.The major issue of the present paper is to describe some computational procedures useful to compare the behaviour of PNA double stranded molecules and PNA/DNA hybrids with the behaviour of regular DNA duplexes in generating complexes with DNA-binding proteins. The performed computational analyses clearly allow to predict that the lack of charged phosphate groups and the different shape of helix play a critical role in the binding efficiency of NF-κB transcription factors. These computational analyses are in agreement with competitive gel shift and UV-cross linking experiments. These experiments demonstrate that NF-κB PNA/PNA hybrids do not interact efficiently with proteins recognizing the NF-KB binding sites in genomic sequences. In addition, the data obtained indicate that the same NF-κB binding proteins recognize both the NF-κB DNA/PNA and DNA/DNA hybrids, but the molecular complexes generated with NF-κB DNA/PNA hybrids are less stable than those generated with NF-κB target DNA/DNA molecules.

We determined whether peptide nucleic acids (PNAs) are able to interact with NF-κB p52 transcription factor. The binding of NF-κB p52 to DNA-DNA, DNA-PNA, PNA-DNA, and PNA-PNA hybrid molecules carrying the NF-κB binding sites of human immunodeficiency type 1 long terminal repeat was studied by (i) biospecific interaction analysis (BIA) using surface plasmon resonance technology, (ii) electrophoretic mobility shift, (iii) DNase I footprinting, and (iv) UV cross-linking assays. Our results demonstrate that NF-κB p52 does not efficiently bind to PNA-PNA hybrids. However, a DNA-PNA hybrid molecule was found to be recognized by NF-κB p52, although the molecular complexes generated exhibited low stability. From the theoretical point of view, our results suggest that binding of NF-κB p52 protein to target DNA motifs is mainly due to contacts with bases; interactions with the DNA backbone are, however, important for stabilization of the protein-DNA complex. From the practical point of view, our results suggest that DNA-PNA hybrid can be recognized by NF-κB p52 protein, although with an efficiency lower than DNA-DNA NF-κB target molecules; therefore, our results should encourage studies on modified PNAs in order to develop potential agents for the decoy approach in gene therapy.

In this paper, we analyse the in vitro sequence-selectivity of two synthetic analogues of U-71184 and adozelesin by polymerase chain reaction (PCR) performed on human genomic DNA. In addition, Dnase footprinting and nucleotide sequence analysis on arrested-PCR products were performed to confirm sequence-selective binding. Finally, the antitumor effects were studied in vitro on human leukemic L1210 cells. The binding activity of the two newly synthesized compounds to human gene sequences was compared with the CC-1065 analogue U-71184, the A+T sequence-selective drug distamycin and the G+C sequence-selective drugs mithramycin and chromomycin. As molecular model systems for in vitro DNA-binding studies we used the human estrogen receptor gene and the Ha-ras oncogene. In some experiments the PCR approach was performed using as target DNA a portion of the long terminal repeat (LTR) of the human immunodeficiency type 1 virus (HIV-1). These genomic regions contain sequences that are different with respect to A+T/G+C ratios, being the upstream sequence of the human estrogen receptor gene A+T rich, while the Ha-ras and HIV-1 LTR sequences contain G+C–rich regions. The first conclusion that can be drawn from the experiments reported in our paper is that the two newly synthetized analogues of U-71184 and adozelesin inhibit PCR-mediated amplification of genomic regions in a sequence-dependent manner. A second conclusion of our experiments is that these compounds are active inhibitors of tumor cell growth in vitro. Drug Dev. Res. 46:96–106, 1999. © 1999 Wiley-Liss, Inc.

In this paper, we analyse the in vitro sequence‐selectivity of two synthetic analogues of U‐71184 and adozelesin by polymerase chain reaction (PCR) performed on human genomic DNA. In addition, Dnase footprinting and nucleotide sequence analysis on arrested‐PCR products were performed to confirm sequence‐selective binding. Finally, the antitumor effects were studied in vitro on human leukemic L1210 cells. The binding activity of the two newly synthesized compounds to human gene sequences was compared with the CC‐1065 analogue U‐71184, the A+T sequence‐selective drug distamycin and the G+C sequence‐selective drugs mithramycin and chromomycin. As molecular model systems for in vitro DNA‐binding studies we used the human estrogen receptor gene and the Ha‐ras oncogene. In some experiments the PCR approach was performed using as target DNA a portion of the long terminal repeat (LTR) of the human immunodeficiency type 1 virus (HIV‐1). These genomic regions contain sequences that are different with respect to A+T/G+C ratios, being the upstream sequence of the human estrogen receptor gene A+T rich, while the Ha‐ras and HIV‐1 LTR sequences contain G+C–rich regions. The first conclusion that can be drawn from the experiments reported in our paper is that the two newly synthetized analogues of U‐71184 and adozelesin inhibit PCR‐mediated amplification of genomic regions in a sequence‐dependent manner. A second conclusion of our experiments is that these compounds are active inhibitors of tumor cell growth in vitro. Drug Dev. Res. 46:96–106, 1999. © 1999 Wiley‐Liss, Inc.

Sequence-selectivity of DNA-binding drugs was recently reported in a number of studies employing footprinting and gel retardation approaches. In this paper we studied sequence-selectivity of the binding of chromomycin and distamycin to DNA by performing DNase I footprinting and analysis of the cleaved fragments by the Pharmacia ALF DNA Sequencing System. As a model system we employed the long terminal repeat of the human immunodeficiency type 1 virus. The main conclusion of our experiments is that automated analysis of DNase I footprinting is a fast and reliable technique to study drugs-DNA interactions. The results obtained suggest that distamycin and chromomycin differentially interact with the long terminal repeat of the human immunodeficiency type 1 virus; this differential binding depends upon the DNA sequences recognized. The data presented are consistent with a preferential binding of distamycin to DNA sequences of the binding sites of nuclear factor kappa B and transcription factor IID. By contrast, distamycin exhibits only weak binding to DNA sequences recognized by the promoter-specific transcription factor Sp1. Unlike distamycin, chromomycin preferentially interacts with the binding sites of the promoter-specific transcription factor Sp1.

We have studied the effects of chromomycin and of a triple-helix-forming oligonucleotide (TFO) that recognizes Sp1 binding sites on protein–DNA interactions and HIV-1 transcription. Molecular interactions between chromomycin, the Sp1 TFO and target DNA sequences were studied by gel retardation, triplex affinity capture using streptavidin-coated magnetic beads and biosensor technology. We also determined whether chromomycin and a TFO recognizing the Sp1 binding sites of the HIV-1 long terminal repeat (LTR) inhibit the activity of restriction enzyme HaeIII, which recognizes a sequence (5′-GGCC-3′) located within these Sp1 binding sites. The effects of chromomycin and the TFO on the interaction between nuclear proteins or purified Sp1 and a double-stranded oligonucleotide containing the Sp1 binding sites of the HIV-1 LTR were studied by gel retardation. The effects of both chromomycin and TFO on transcription were studied by using an HIV-1 LTR-directed in vitro transcription system. Our results indicate that low concentrations of chromomycin potentiate the effects of the Sp1 TFO in inhibiting protein–DNA interactions and HIV-1-LTR-directed transcription. In addition, low concentrations of chromomycin do not affect binding of the TFO to target DNA molecules. The results presented here support the hypothesis that both DNA binding drugs and TFOs can be considered as sequence-selective modifiers of DNA–protein interactions, possibly leading to specific alterations of biological functions. In particular, the combined use of chromomycin and TFOs recognizing Sp1 binding sites could be employed in order to abolish the biological functions of promoters (such as the HIV-1 LTR) whose activity is potentiated by interactions with the promoter-specific transcription factor Sp1.

Infectious diseases are a major source of morbidity and mortality for immunosuppressed transplant recipients and the antimicrobial chemotherapy can be often less effective in these individuals, because the contribution of underlying host defenses is absent.

No relevant conflicts of interest to declare.

Abstract 2055

No relevant conflicts of interest to declare.

Abstract 978

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In this paper we report the characterization of a newly sequenced 5′ upstream region of the human HLA-DRA gene. We performed (i) search for transcription factor motifs, (ii) analysis of CpG display and observed/expected frequency ratios, (III) search for regions homologous to the 5′ upstream sequences of the murine EA gene, (iv) DNase I footprinting experiments and (v) electrophoretic mobility shift assays. Our results demonstrate the existence, in the HLA-DRA gene, of Y′ and X′ boxes highly homologous to the Y and X boxes present in MHC class II genes, but oriented in the opposite direction. These Y′ and X′ boxes have been conserved during the molecular evolution of both human HLA-DRA and murine EA genes. DNase I footprinting and gel retardation experiments suggest that the X′ and Y′ boxes of the HLA-DRA upstream gene region are specifically recognized by nuclear proteins that also bind to the X and Y boxes of the HLA-DRA proximal promoter, respectively.