Joel Crouzet

Bio: Joel Crouzet is an academic researcher from Rhône-Poulenc. The author has contributed to research in topics: Pseudomonas denitrificans & Plasmid. The author has an hindex of 38, co-authored 70 publications receiving 4225 citations.

Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus.

Abstract: Fluoroquinolone-resistant mutants were obtained in vitro from Staphylococcus aureus RN4220 by stepwise selection on increasing concentrations of ciprofloxacin. Results from sequence analysis of the quinolone resistance-determining region of GyrA and of the corresponding region of GrlA, the DNA topoisomerase IV subunit, showed an alteration of Ser-80 to Tyr (corresponding to Ser-83 of Escherichia coli GyrA) or Glu-84 to Lys in GrlA of both low- and high-level quinolone-resistant mutants. Second-step mutants were found to have, in addition to a mutation in grlA, reduced accumulation of norfloxacin or an alteration in GyrA at Ser-84 to Leu or Glu-88 to Lys. Third-step mutants derived from second-step mutants with reduced accumulation were found to have a mutation in gyrA. The results from this study demonstrated that mutations in gyrA or mutations leading to reduced drug accumulation occur after alteration of GrlA, supporting the previous findings (L. Ferrero, B. Cameron, B. Manse, D. Lagneaux, J. Crouzet, A. Famechon, and F. Blanche, Mol. Microbiol. 13:641-653, 1994) that DNA topoisomerase IV is a primary target of fluoroquinolones in S. aureus.

Synthesis, Activity, and Structure−Activity Relationship Studies of Novel Cationic Lipids for DNA Transfer

Abstract: We have designed and synthesized original cationic lipids for gene delivery. A synthetic method on solid support allowed easy access to unsymmetrically monofunctionalized polyamine building blocks of variable geometries. These polyamine building blocks were introduced into cationic lipids. To optimize the transfection efficiency in the novel series, we have carried out structure-activity relationship studies by introduction of variable-length lipids, of variable-length linkers between lipid and cationic moiety, and of substituted linkers. We introduce the concept of using the linkers within cationic lipids molecules as carriers of side groups harboring various functionalities (side chain entity), as assessed by the introduction of a library composed of cationic entities, additional lipid chains, targeting groups, and finally the molecular probes rhodamine and biotin for cellular traffic studies. The transfection activity of the products was assayed in vitro on Hela carcinoma, on NIH3T3, and on CV1 fibroblasts and in vivo on the Lewis Lung carcinoma model. Products from the series displayed high transfection activities. Results indicated that the introduction of a targeting side chain moiety into the cationic lipid is permitted. A primary physicochemical characterization of the DNA/lipid complexes was demonstrated with this leading compound. Selected products from the series are currently being developed for preclinical studies, and the labeled lipopolyamines can be used to study the intracellular traffic of DNA/cationic lipid complexes.

Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency

Abstract: Clinical applications of gene therapy mainly depend on the development of efficient gene transfer vectors. Large DNA molecules can only be transfected into cells by using synthetic vectors such as cationic lipids and polymers. The present investigation was therefore designed to explore the physicochemical properties of cationic lipid-DNA particles, with plasmids ranging from 900 to 52 500 bp. The colloidal stability of the lipoplexes formed by complexing lipopolyamine micelles with plasmid DNA of various lengths, depending on the charge ratio, resulted in the formation of three domains, respectively corresponding to negatively, neutrally and positively charged lipoplexes. Lipoplex morphology and structure were determined by the physicochemical characteristics of the DNA and of the cationic lipid. Thus, the lamellar spacing of the structure was determined by the cationic lipid and its spherical morphology by the DNA. The main result of this study was that the morphological and structural features of the lipopolyamine-DNA complexes did not depend on plasmid DNA length. On the other hand, their gene transfer capacity was affected by the size of plasmid DNA molecules which were sandwiched between the lipid bilayers. The most effective lipopolyamine-DNA complexes for gene transfer were those containing the shortest plasmid DNA.

A new DNA vehicle for nonviral gene delivery: supercoiled minicircle.

Abstract: Plasmids currently used for nonviral gene transfer have the disadvantage of carrying a bacterial origin of replication and an antibiotic resistance gene. There is, therefore, a risk of uncontrolled dissemination of the therapeutic gene and the antibiotic resistance gene. Minicircles are new DNA delivery vehicles which do not have such elements and are consequently safer as they exhibit a high level of biological containment. They are obtained in E. coli by att site-specific recombination mediated by the phage λ integrase. The desired eukaryotic expression cassette bounded by the λ attP and attB sites was cloned on a recombinant plasmid. The expression cassette was excised in vivo after thermoinduction of the integrase gene leading to the formation of two supercoiled molecules: the minicircle and the starting plasmid lacking the expression cassette. In various cell lines, purified minicircles exhibited a two- to 10-fold higher luciferase reporter gene activity than the unrecombined plasmid. This could be due to either the removal of unnecessary plasmid sequences, which could affect gene expression, or the smaller size of minicircle which may confer better extracellular and intracellular bioavailability and result in improved gene delivery properties.

Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer

Abstract: Minicircles are a new form of supercoiled DNA molecule for nonviral gene transfer which have neither bacterial origin of replication nor antibiotic resistance marker. They are thus smaller and potentially safer than the standard plasmids currently used in gene therapy. They were obtained in E. coli by att site-specific recombination mediated by the phage lambda integrase, which was used to excise the expression cassette from the unwanted plasmid sequences. We produced two minicircles containing the luciferase or beta-galactosidase gene under the control of the strong human cytomegalovirus immediate-early enhancer/promoter. Comparing maximal differences, these minicircles gave 2.5 to 5.5 times more reporter gene activity than the unrecombined plasmid in the NIH3T3 cell line and rabbit smooth muscle cells. Moreover, injection in vivo into mouse cranial tibial muscle, or human head and neck carcinoma grafted in nude mice resulted in 13 to 50 times more reporter gene expression with minicircles than with the unrecombined plasmid or larger plasmids. Histological analysis in muscle showed there were more transfected myofibers with minicircles than with unrecombined plasmid.

A simplified system for generating recombinant adenoviruses

Abstract: Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. This invention describes a strategy which simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, employing homologous recombination in bacteria rather than in eucaryotic cells. Following transfections of such plasmids into a mammalian packaging cell line, viral production can be conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system expedites the process of generating and testing recombinant adenoviruses.

Cell biology and molecular basis of denitrification.

Abstract: Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

Non-viral vectors for gene-based therapy

Abstract: Gene-based therapy is the intentional modulation of gene expression in specific cells to treat pathological conditions This modulation is accomplished by introducing exogenous nucleic acids such as DNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA) or antisense oligonucleotides Given the large size and the negative charge of these macromolecules, their delivery is typically mediated by carriers or vectors In this Review, we introduce the biological barriers to gene delivery in vivo and discuss recent advances in material sciences, nanotechnology and nucleic acid chemistry that have yielded promising non-viral delivery systems, some of which are currently undergoing testing in clinical trials The diversity of these systems highlights the recent progress of gene-based therapy using non-viral approaches