Biomedical subjects
Publications and source records attributed to J Crouzet.
Cationic lipids are widely used for in vitro gene transfer due to their efficiency. The major challenges for the improvement of in vivo cationic lipid-mediated gene delivery reside in the design of more biocompatible lipoplexes mimicking viral-mediated gene delivery and in understanding the fate of the lipoplexes within the cells.
We report two cases of discitis due to Propionibacterium acnes and review previously published cases of bone and joint infections in which this organism was recovered as a pure culture. P. acnes is an anaerobic organism usually considered a normal inhabitant of the skin but capable of producing a variety of infections including discitis, osteitis, arthritis, and chest wall osteitis. Most patients were immunocompetent. A few infections occurred spontaneously, whereas others were secondary to a break in the skin or to implantation of foreign material into the body for instance during internal fixation of a fracture or arthroplasty. Cases of P. acnes chest wall infection have been reported in patients with palmoplantar pustulosis or chronic or multifocal osteitis, supporting a role for P. acnes in SAPHO syndrome.
Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 A, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains.
Triple-helix-forming oligodeoxynucleotides are receiving considerable attention due to their potential applications for the inhibition of specific genes in vivo. However, their development is impaired by the lack of triple helix formation under physiological conditions. It is thus crucial to be able to quantitatively assay triple helix formation of various oligodeoxynucleotides on different target sequences. Usual methods to detect triple helix formation are restricted under the experimental conditions that can be studied. In addition, quantitative techniques are limited. We present a novel method for rapid detection and quantification of triple helix formation between an oligodeoxynucleotide and a plasmid carrying a target sequence. The oligodeoxynucleotide was radiolabeled and, after incubation with the target plasmid, the unbound oligodeoxynucleotide was separated from the mixture of plasmids and plasmid-bound oligodeoxynucleotides by rapid gel filtration spun columns. The formation of a triple helix between a target plasmid and several oligodeoxynucleotides was demonstrated and compared. Temperature, sequence and ionic dependencies, and kinetics of association were analyzed. This new technique can be used under a variety of conditions and should allow the rapid determination of optimal conditions required for triple helix formation, as well as the easy selection of an oligodeoxynucleotide that specifically binds with the highest affinity to a target double-stranded sequence.
OBJECTIVES: The aim of this study was to determine the epidemiologic features of bone and joint tuberculosis in the Paris area from 1980 to 1994. METHODS: This retrospective study included 206 cases of confirmed bone and joint tuberculosis (in situ specimen) diagnosed from 1980 to 1994 in 7 hospital units (mainly rheumatology clinics) in Paris and suburban areas. RESULTS: The number of cases increased by a mean 12 per year from 1980 to 1989 then by 17 cases per year from 1990 to 1994. In both 1993 and 1994 there were 21 cases per year. The percentage of immigrant patients was 53% from 1980 to 1989 then reached 74% for 1990-1994, predominantly in subjects from black Africa. Only one patient in the series had human immunodeficiency virus (HIV) infection. A favoring factor was found in 23% of the immigrant and in 45% of the native French patients. Mean age was 37 years in immigrants and 58 years in French patients. Compared with French patients, immigrants had a higher rate of multifocal bone and joint tuberculosis, peripheral osteitis and spondylitus. CONCLUSION: Our findings suggest that the incidence of bone and joint tuberculosis has increased in Paris in 1993 and 1994. Two populations are especially susceptible: the elderly and/or immunodepressed French population and immigrants coming from endemic zones. The role or HIV infection was minimal in this predominantly rheumatology series.
A two-step gene replacement procedure was developed that generates infectious adenoviral genomes through homologous recombination in Escherichia coli. As a prerequisite, a human adenovirus serotype 5 (Ad5)-derived genome was first introduced as a PacI restriction fragment into an incP-derived replicon which, in contrast to ColE1-derivatives (e.g., pBR322 or pUC plasmids), is functional in a polA mutant of E. coli. Any modification can be introduced at will following two consecutive homologous recombinations between the incP/Ad5 replicon and the ColE1 plasmid. The overall procedure requires only the in vitro engineering of the ColE1-derivative by flanking the desired modification with small stretches of identical sequences. In the first step, a cointegrate between the tetracycline-resistant incP/Ad5 replicon and the kanamycin-resistant ColE1-derivative is selected by growing the polA host in the presence of both antibiotics. Resolution of this cointegrate is further selected in sucrose growth conditions due to the loss of a conditional suicide marker (the sacB gene of Bacillus subtilis) present in the ColE1 plasmid, leading to unmodified and modified incP/Ad5 replicons that can be differentiated upon restriction analysis. Consecutive rounds of this two-step cloning procedure allowed the introduction of multiple independent modifications within the virus genome, with no requirement for an intermediate virus. The potential of this procedure is demonstrated by the recovery of several E1E3E4-deleted adenoviruses following transfection of the corresponding E. coli-derived genomes in IGRP2 cells.
BACKGROUND: In order to study the biosynthesis of vitamin B12, it is necessary to produce various intermediates along the biosynthetic pathway by enzymic methods. Recently, information on the organisation of the biosynthetic pathway has permitted the selection of the set of enzymes needed to biosynthesise any specific identified intermediate. The aim of the present work was to use recombinant enzymes in reconstituted multi-enzyme systems to biosynthesise particular intermediates. RESULTS: The products of the cobG and cobJ genes from Pseudomonas denitrificans were expressed heterologously in Escherichia coli to afford good levels of activity of the corresponding enzymes, CobG and CobJ. Aerobic incubation of precorrin-3A with the CobG enzyme alone yielded precorrin-3B. When CobJ and S-adenosyl-L-methionine were included in the incubation, the product was precorrin-4. Both precorrin 3B and precorrin-4 are known precursors of vitamin B12 and their availability has allowed new mechanistic studies of enzymic transformations. CONCLUSIONS: Our results show that the expression of the CobG and CobJ enzymes has been successful, thus facilitating the biosynthesis of two precursors of vitamin B12. This lays the foundation for the structure determination of CobG and CobJ as well as future enzymic experiments focusing on later steps of vitamin B12 biosynthesis.
Plasmid DNA used for nonviral therapeutic gene transfer or nucleic acid vaccination has to be highly purified devoid of contaminating components such as bacterial proteins, endotoxins, or bacterial chromosomal DNA. We have developed a new affinity chromatography technique for plasmid DNA purification: triple-helix affinity chromatography (THAC). This technique is based on the sequence-specific interaction of an oligonucleotide forming a triple-helix with plasmid DNA. The oligonucleotide was covalently linked to a chromatographic matrix, thus providing a reusable affinity support. By inserting a suitable homopurine sequence in the plasmid DNA, it is possible to obtain a triple-helix interaction that will only be stable at mild acidic pH and that will dissociate in alkaline conditions. A crude lysate from a recombinant E. coli, or a pre-purified plasmid DNA, is thus applied at acidic pH on to a THAC column. After extensive washing of the column, purified plasmid DNA is eluted using an alkaline buffer. The binding conditions of the plasmid DNA on to the column have been optimized, as well as the hybridization sequence and the linker group between the matrix and the third strand oligonucleotide. The THAC technique makes it possible to purify in one step supercoiled plasmid DNA, and to significantly reduce the level of contaminating RNA, endotoxins and chromosomal DNA. In particular, a 100-fold reduction of chromosomal DNA contamination over that obtained with conventional techniques can be achieved through a single additional THAC step. Further improvements of THAC technology are possible, and we anticipate that this technique can be scaled up for integration into a full commercial-scale DNA production process.
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 lambda integrase. The desired eukaryotic expression cassette, bounded by the lambda 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 mini-circle which may confer better extracellular and intracellular bioavailability and result in improved gene delivery properties.
Four pap genes (papA, papB, papC, papM) were found by sequencing near to snbA, a Streptomyces pristinaespiralis gene which was previously shown to encode one of the pristinamycin I (PI) synthetases. Analysis of the homologies observed from the deduced amino acid sequences suggested that these four genes could be involved in the biosynthesis of the PI precursor 4-dimethylamino-L-phenylalanine (DMPAPA). This was first verified when disruption of papA in S. pristinaespiralis led to a PI- phenotype, which was reversed by the addition of DMPAPA into the culture medium. Further confirmation was obtained when papM was overexpressed in Escherichia coli and the corresponding protein purified to homogeneity. It catalysed the two successive N-methylation steps of 4-amino-L-phenylalanine leading to DMPAPA via 4-methylamino-L-phenylalanine. These results allowed us to assign a function to each of the four pap genes and to propose a biosynthetic pathway for DMPAPA.
Three flavonoids which promoted Escherichia coli topoisomerase IV-dependent DNA cleavage were isolated from cottonseed flour and identified as quercetin 3-O-beta-D-glucose-[1,6]-O-alpha-L-rhamnose (rutin), quercetin 3-O-beta-D-galactose-[1,6]-O-alpha-L-rhamnose, and quercetin 3-O-beta-D-glucose (isoquercitrin). The most active one (rutin) also inhibited topoisomerase IV-dependent decatenation activity (50% inhibitory concentration, 64 microg/ml) and induced the SOS response of a permeable E. coli strain. Derivatives of quercetin glycosylated at position C-3 were shown to induce two site-specific DNA cleavages of pBR322 DNA, which were mapped by DNA sequence analysis to the gene encoding resistance to tetracycline. Cleavage at these sites was hardly detectable in cleavage reactions with quercetin or fluoroquinolones. None of the three flavonoids isolated from cottonseeds had any stimulatory activity on E. coli DNA gyrase-dependent or calf thymus topoisomerase II-dependent DNA cleavage, and they were therefore specific to topoisomerase IV. These results show that selective inhibitors of topoisomerase IV can be derived from the flavone structure. This is the first report on a DNA topoisomerase inhibitor specific for topoisomerase IV.
Streptomyces pristinaespiralis and S. virginiae both produce closely related hexadepsipeptide antibiotics of the streptogramin B family. Pristinamycins I and virginiamycins S differ only in the fifth incorporated precursor, di(mono)methylated amine and phenylalanine, respectively. By using degenerate oligonucleotide probes derived from internal sequences of the purified S. pristinaespiralis SnbD and SnbE proteins, the genes from two streptogramin B producers, S. pristinaespiralis and S. virginiae, encoding the peptide synthetase involved in the activation and incorporation of the last four precursors (proline, 4-dimethylparaaminophenylalanine [for pristinamycin I(A)] or phenylalanine [for virginiamycin S], pipecolic acid, and phenylglycine) were cloned. Analysis of the sequence revealed that SnbD and SnbE are encoded by a unique snbDE gene. SnbDE (4,849 amino acids [aa]) contains four amino acid activation domains, four condensation domains, an N-methylation domain, and a C-terminal thioesterase domain. Comparison of the sequences of 55 amino acid-activating modules from different origins confirmed that these sequences contain enough information for the performance of legitimate predictions of their substrate specificity. Partial sequencing (1,993 aa) of the SnbDE protein of S. virginiae allowed comparison of the proline and aromatic acid activation domains of the two species and the identification of coupled frameshift mutations.
Two genes involved in the biosynthesis of the depsipeptide antibiotics pristinamycins I (PI) produced by Streptomyces pristinaespiralis were cloned and sequenced. The 1.7-kb snbA gene encodes a 3-hydroxypicolinic acid:AMP ligase, and the 7.7-kb snbC gene encodes PI synthetase 2, responsible for incorporating L-threonine and L-aminobutyric acid in the PI macrocycle. snbA and snbC, which encode the two first structural enzymes of PI synthesis, are not contiguous. Both genes are located in PI-specific transcriptional units, as disruption of one gene or the other led to PI-deficient strains producing normal levels of the polyunsaturated macrolactone antibiotic pristinamycin II, also produced by S. pristinaespiralis. Analysis of the deduced amino acid sequences showed that the SnbA protein is a member of the adenylate-forming enzyme superfamily and that the SnbC protein contains two amino acid-incorporating modules and a C-terminal epimerization domain. A model for the initiation of PI synthesis analogous to the established model of initiation of fatty acid synthesis is proposed.
The Bacillus subtilis genes hemB, hemC and hemD, encoding respectively the enzymes porphobilinogen synthase, hydroxymethylbilane synthase and uroporphyrinogen III synthase, have been expressed in Escherichia coli using a single plasmid construct. An enzyme preparation from this source converts 5-aminolaevulinic acid (ALA) preparatively and in high yield into uroporphyrinogen III. The Pseudomonas denitrificans genes cobA and cobI, encoding respectively the enzymes S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) and S-adenosyl-L-methionine:precorrin-2 methyltransferase (SP2MT), were also expressed in E. coli. When SUMT was combined with the coupled-enzyme system that produces uroporphyrinogen III, precorrin-2 was synthesized from ALA, and when SP2MT was also added the product from the coupling of five enzymes was precorrin-3A. Both of these products are precursors of vitamin B12, and they can be used directly for biosynthetic experiments or isolated as their didehydro octamethyl esters in > 40% overall yield. The enzyme system which produces precorrin-3A is sufficiently stable to allow long incubations on a large scale, affording substantial quantities (15-20 mg) of product.
The 6-O-alpha-L-Arabinopyranosyl-beta-D-glucopyranosides of linalool, benzyl alcohol and 3-methyl-but-2-en-1-ol were isolated from passion fruit (Passiflora edulis) by adsorption chromatography on XAD-2 resin, then further extracted on the same resin after partial enzymic hydrolysis and semi-preparative chromatography on RP-18 phase by HPLC. Their structures were identified by 1H NMR spectroscopy and mass spectral analysis and by methylation analysis of the carbohydrate moieties.
Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.
INTRODUCTION: Regression of osteomalacia after exeresis of a skin tumor is unusual. CASE REPORT: A 62-year-old man had suffered from bone and joint symptoms for several years due to osteomalacia which was confirmed both biologically and histologically. The patient also had a plantar neurinoma. After exeresis of the tumor the biological results returned to normal levels within one week followed by regression of the clinical signs of osteomalacia. DISCUSSION: The neurinoma in this patient was apparently the cause of osteomalacia, since signs of the disease disappeared after exeresis. To date, three cases of neurinoma associated with osteomalacia have been published, including a single case with skin localization. The tumor would secrete a substance which inhibits the synthesis of vitamin D and enhances phosphorus excretion.
An aliphatic nitrilase, active on adiponitrile and cyanovaleric acid, was identified and purified from Comamonas testosteroni sp. (Ct). Oligodeoxyribonucleotide probes were designed from limited amino acid (aa) sequence information and used to clone the corresponding gene, named nitA. High homologies were found at the aa level between Ct nitrilase and the sequences of known nitrilases. Multi-alignment of sequenced nitrilases suggests that Cys163 of Ct plays an essential role in the active site. This hypothesis is strengthened by molecular studies on nitrilases from Alcaligenes faecalis JM3, and Rhodococcus rhodochrous J1 and K22 [Kobayashi et al., Proc. Natl. Acad. Sci. USA 90 (1993) 247-251; J. Biol. Chem. 267 (1992) 20746-20751; Biochemistry 31 (1992) 9000-9007]. Large amounts of an active recombinant enzyme could be produced in Escherichia coli when nitA was overexpressed together with the E. coli groESL genes.