Biomedical subjects

P Bigey

Publications and source records attributed to P Bigey.

12 recordsLinked to original sources

PubMed2006

A comparative study on intra-articular versus systemic gene electrotransfer in experimental arthritis.

BACKGROUND: Electric pulse mediated gene transfer has been applied successfully in vivo for increasing naked DNA administration in various tissues. To achieve non-viral gene transfer into arthritic joint tissue, we investigated the use of electrotransfer (ET). Because anti-inflammatory cytokine strategies have proven efficient in experimental models of arthritis, we compared the therapeutic efficiency of local versus systemic delivery of the interleukin-10 (IL-10) using in vivo ET. METHODS: A plasmid vector expressing IL-10 was transferred into DBA/1 mouse knee joints by ET with 12 pulses of variable duration and voltage. The kinetics of transgene expression were analyzed by specific enzyme-linked immunosorbent assay (ELISA) in sera and knees. Optimal conditions were then used to deliver increasing amounts of IL-10 plasmid intra-articularly (i.a.) in the collagen-induced arthritis (CIA) mouse model. The therapeutic efficiency was compared with the potency of intra-muscular (i.m.) ET. RESULTS: Following i.a. ET, local IL-10 secretion peaked on day 7 and dropped 2 weeks after. A second ET produced the same kinetics without enhancing gene transfer efficiency, while transgene was still detected in injected muscles 4 weeks after ET. Only the i.m. ET of 25 microg of IL-10 significantly inhibited all the clinical and biological features of arthritis. The i.a. ET only showed mild improvement of arthritis when 100 microg of IL-10 plasmid were electrotransfered weekly from day 18 following arthritis induction. CONCLUSIONS: The present results suggest that gene transfer into arthritic joints by ET is an effective means to deliver anti-inflammatory cytokines. However, short duration of transgene expression impedes a significant effect for the treatment of arthritis, making i.m. ET more potent than i.a. ET for clinical benefit in CIA.

Animals↗

PubMed2006

Electrotransfer into skeletal muscle for protein expression.

An efficient and safe method to deliver DNA in vivo is a requirement for several purposes, such as study of gene function and gene therapy applications. Among the different non-viral delivery methods currently under investigation, in vivo DNA electrotransfer has proven to be one of the most efficient and simple. This technique is a physical method of gene delivery consisting in local application of electric pulses after DNA injection. Although this technique can be applied to almost any tissue of a living animal, including tumors, skin, liver, kidney, artery, retina, cornea or even brain, this review will focus on electrotransfer of plasmid DNA into skeletal muscle and its possible uses in gene therapy, vaccination, or functional studies. Skeletal muscle is a good target for electrotransfer of DNA as it is: a large volume easily accessible, an endocrine organ capable of expressing several local and systemic factors, and muscle fibres as post-mitotic cells have a long lifespan that allows long-term gene expression. In this review, we describe the mechanism of DNA electrotransfer, we assess toxicity and safety considerations related to this technique, and we focus on important therapeutic applications of electrotransfer demonstrated in animal models in recent years.

Animals↗

PubMed2005

Plasmid electrotransfer of eye ciliary muscle: principles and therapeutic efficacy using hTNF-alpha soluble receptor in uveitis.

Due to its small size and particular isolating barriers, the eye is an ideal target for local therapy. Recombinant protein ocular delivery requires invasive and painful repeated injections. Alternatively, a transfected tissue might be used as a local producer of transgene-encoded therapeutic protein. We have developed a nondamaging electrically mediated plasmid delivery technique (electrotransfer) targeted to the ciliary muscle, which is used as a reservoir tissue for the long-lasting expression and secretion of therapeutic proteins. High and long-lasting reporter gene expression was observed, which was restricted to the ciliary muscle. Chimeric TNF-alpha soluble receptor (hTNFR-Is) electrotransfer led to elevated protein secretion in aqueous humor and to drastic inhibition of clinical and histological inflammation scores in rats with endotoxin-induced uveitis. No hTNFR-Is was detected in the serum, demonstrating the local delivery of proteins using this method. Plasmid electrotransfer to the ciliary muscle, as performed in this study, did not induce any ocular pathology or structural damage. Local and sustained therapeutic protein production through ciliary muscle electrotransfer is a promising alternative to repeated intraocular protein administration for a large number of inflammatory, degenerative, or angiogenic diseases.

Animals↗

PubMed2000

The DNMT1 target recognition domain resides in the N terminus.

DNA-cytosine-5-methyltransferase 1 (DNMT1) is the enzyme believed to be responsible for maintaining the epigenetic information encoded by DNA methylation patterns. The target recognition domain of DNMT1, the domain responsible for recognizing hemimethylated CGs, is unknown. However, based on homology with bacterial cytosine DNA methyltransferases it has been postulated that the entire catalytic domain, including the target recognition domain, is localized to 500 amino acids at the C terminus of the protein. The N-terminal domain has been postulated to have a regulatory role, and it has been suggested that the mammalian DNMT1 is a fusion of a prokaryotic methyltransferase and a mammalian DNA-binding protein. Using a combination of in vitro translation of different DNMT1 deletion mutant peptides and a solid-state hemimethylated substrate, we show that the target recognition domain of DNMT1 resides in the N terminus (amino acids 122-417) in proximity to the proliferating cell nuclear antigen binding site. Hemimethylated CGs were not recognized specifically by the postulated catalytic domain. We have previously shown that the hemimethylated substrates utilized here act as DNMT1 antagonists and inhibit DNA replication. Our results now indicate that the DNMT1-PCNA interaction can be disrupted by substrate binding to the DNMT1 N terminus. These results point toward new directions in our understanding of the structure-function of DNMT1.

Base Sequence↗

PubMed2000

Inhibition of DNA methyltransferase inhibits DNA replication.

Ectopic expression of DNA methyltransferase transforms vertebrate cells, and inhibition of DNA methyltransferase reverses the transformed phenotype by an unknown mechanism. We tested the hypothesis that the presence of an active DNA methyltransferase is required for DNA replication in human non-small cell lung carcinoma A549 cells. We show that the inhibition of DNA methyltransferase by two novel mechanisms negatively affects DNA synthesis and progression through the cell cycle. Competitive polymerase chain reaction of newly synthesized DNA shows decreased origin activity at three previously characterized origins of replication following DNA methyltransferase inhibition. We suggest that the requirement of an active DNA methyltransferase for the functioning of the replication machinery has evolved to coordinate DNA replication and inheritance of the DNA methylation pattern.

Animals↗

PubMed2000

Transcriptional regulation of the human DNA Methyltransferase (dnmt1) gene.

DNA methylation is an important component of the epigenetic control of genome functions. Understanding the regulation of the DNA Methyltransferase (dnmt1) gene expression is critical for comprehending how DNA methylation is coordinated with other critical biological processes. In this paper, we investigate the transcriptional regulatory region of the human dnmt1 gene using a combination of RACE, RNase protection analysis and CAT assays. We identified one major and three minor transcription initiation sites in vivo (P1-P4), which are regulated by independent enhancers and promoter sequences. The minimal promoter elements of P1, P2 and P4 are mapped within 256 bp upstream of their respective transcription initiation sites. P1 is nested within a CG-rich area, similar to other housekeeping genes, whereas P2-P4 are found in CG-poor areas. Three c-Jun-dependent enhancers are located downstream to P1 and upstream to P2-P4, thus providing a molecular explanation for the responsiveness of dnmt1 to oncogenic signals that are mediated by the Ras-c-Jun oncogenic signaling pathway.

Alternative Splicing↗

PubMed1999

Identification of initiation sites for DNA replication in the human dnmt1 (DNA-methyltransferase) locus.

Vertebrates have developed multiple mechanisms to coordinate the replication of epigenetic and genetic information. Dnmt1 encodes the maintenance enzyme DNA-methyltransferase, which is responsible for propagating the DNA methylation pattern and the epigenetic information that it encodes during replication. Direct sequence analysis and bisulfite mapping of the 5' region of DNA-methyltransferase 1 (dnmt1) have indicated the presence of many sequence elements associated with previously characterized origins of DNA replication. This study tests the hypothesis that the dnmt1 region containing these elements is an origin of replication in human cells. First, we demonstrate that a vector containing this dnmt1 sequence is able to support autonomous replication when transfected into HeLa cells. Second, using a gel retardation assay, we show that it contains a site for binding of origin-rich sequences binding activity, a recently purified replication protein. Finally, using competitive polymerase chain reaction, we show that replication initiates in this region in vivo. Based on these lines of evidence, we propose that initiation sites for DNA replication are located between the first intron and exon 7 of the human dnmt1 locus.

Chromosome Mapping↗

PubMed1999

Modified oligonucleotides as bona fide antagonists of proteins interacting with DNA. Hairpin antagonists of the human DNA methyltransferase.

The study of the biological role of DNA methyltransferase (DNA MeTase) has been impeded by the lack of direct and specific inhibitors. This report describes the design of potent DNA based antagonists of DNA MeTase and their utilization to define the interactions of DNA MeTase with its substrate and to study its biological role. We demonstrate that the size, secondary structure, hemimethylation, and phosphorothioate modification strongly affect the antagonists interaction with DNA MeTase whereas base substitutions do not have a significant effect. To study whether DNA MeTase is critical for cellular transformation, human lung non-small carcinoma cells were treated with the DNA MeTase antagonists. Ex vivo, hairpin inhibitors of DNA MeTase are localized to the cell nucleus in lung cancer cells. They inhibit DNA MeTase, cell growth, and anchorage independent growth (an indicator of tumorigenesis in cell culture) in a dose-dependent manner. The inhibitors developed in this study are the first documented example of direct inhibitors of DNA MeTase in living cells and of modified oligonucleotides as bona fide antagonists of critical cellular proteins.

Base Sequence↗

PubMed1995

Cleavage of double-stranded DNA by 'metalloporphyrin-linker-oligonucleotide' molecules: influence of the linker.

Manganese porphyrin-linker-triple-helix-forming oligonucleotide molecules were prepared and their ability to cleave in vitro a double-stranded DNA target present in the HIV-1 genome was studied. The nature of the linker is a determining factor of the cleavage efficiency. Cleavage yields as high as 80% were observed when the linker was a spermine residue and in the absence of a large excess of free spermine known to stabilize triplex structures. The hydrophobic nature of aliphatic diamine linker modified the cleaver-DNA interactions and reduced the efficiency of DNA cleavage.

Antiviral Agents↗

PubMedDate not supplied

Improvement of porphyrin cellular delivery and activity by conjugation to a carrier peptide.

The chemical nuclease metalloporphyrin (manganese(III) porphyrin) can cleave DNA irreversibly and can thus constitute a potential antitumor drug. However, these molecules show low permeability to cell surface membranes. We report here the conjugation of an amphipathic carrier peptide to improve considerably its cellular delivery. The metalloporphyrin-peptide conjugate can be internalized by cells within only 5 min of incubation with a yield as high as 80%. Furthermore, the metalloporphyrin-peptide conjugate is able to cleave in vitro high or low molecular weight DNA to the same extend as metalloporphyrin alone without affecting the sequence-specific cleaving activity of the porphyrin. The conjugate is 100-fold more efficient at inducing tumor cells death than the free metalloporphyrin via a mechanism involving genomic DNA cleavage. The results are promising for further therapeutic applications with antitumor drugs such as metalloporphyrin, and also with other existing drugs by using a carrier peptide system in order to improve the cellular uptake of such molecules.

Amino Acid Sequence↗

PubMedDate not supplied

DNA binding and cleavage by a cationic manganese porphyrin-peptide nucleic acid conjugate.

A cationic manganese porphyrin-peptide nucleic acid (PNA) conjugate has been prepared and used to cleave a double-stranded DNA target. Cleavage experiments were performed with a 247-base pair restriction DNA fragment containing a 10-base pair homopurine binding target for the PNA. Oxidative activation by this Mn porphyrin-PNA conjugate leads to sequence specific, 3'-staggered cleavage of both DNA strands near the strand displacement junction. Furthermore, the Mn porphyrin-PNA porphyrin conjugates bind over 100-fold better to double-stranded DNA compared to the native PNA.

Binding Sites↗

PubMedDate not supplied

Genomic structure of the human DNA methyltransferase gene.

We determined the genomic structure of the gene encoding human DNA methyltransferase (DNA MTase). Six overlapping human genomic DNA clones which include all of the known cDNA sequence were isolated. Analysis of these clones demonstrates that the human DNA MTase gene consists of at least 40 exons and 39 introns spanning a distance of 60 kilobases. Elucidation of the chromosomal organization of the human DNA MTase gene provides the template for future structure-function analysis of the properties of mammalian DNA MTase.

Cloning, Molecular↗