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Biomedical subjects

J Crouzet

Publications and source records attributed to J Crouzet.

At least 19 recordsLinked to original sources
PubMed2001

Changes of volatile compounds during heating of bacuri pulp.

The formation of volatile compounds from precursors or through chemical rearrangement during heat treatment of bacuri pulp at fruit natural pH were studied using simultaneous distillation/extraction (SDE) technique. An increase of the quantities of oxygenated and hydrocarbon terpenes and, to a lesser degree, aldehydes, was observed after SDE at pH 3, relative to the other extraction methods used, SDE at neutral pH and solid phase extraction (SPE). More particularly, linalool, linalool furanoxides, alpha-terpineol, hotrienol, nerol oxide, nerol, and geraniol were isolated in more important quantities after the first treatment than after the others. These results can be partially explained by the hydrolysis of glycosidically bound compounds previously identified in bacuri. Other pathways such as polyol rearrangements were also involved. The formation of linalool and alpha-terpineol was probably the result of the rearrangement of 2,6-dimethyloct-1-ene-3,7-diol. Moreover, it was assumed that oxidation reactions occurred during SDE at pH 3; more particularly, linalool pyranoxides partially resulted from nonenzymatic oxidation of linalool. When SDE was performed at pH 3, an increase of furfural and 4-methoxy-2,5-dimethyl-3(2H)-furanone was noticed. The modifications of the concentration of aliphatic aldehydes, known as lipid oxidation compounds, and of fatty acid esters were in good agreement with the observed decrease of palmitic and linoleic acid concentrations during this treatment. Moreover, important amounts of 2-acetyl-1-pyrroline were found in the SDE extract recovered at pH 7.

Aldehydes↗
PubMed2001

Degenerated pIX-IVa2 adenoviral vector sequences lowers reacquisition of the E1 genes during virus amplification in 293 cells.

A critical issue for E1-deleted adenoviral vectors manufactured from 293 cells is the emergence of replication-competent adenovirus (RCA). These contaminants arise through homologous recombination between identical sequences framing the E1 locus displayed by 293 cells, and the vector backbones. Modified recombinogenic sequences (syngen) were thus introduced within the vector backbone, and virus viability and RCA emergence were assessed. Syngen#1 is a synthetic sequence displaying silent point mutations in the pIX and IVa2 coding regions. A side by side comparison of Ad5CMV/p53 (E1-deleted adenovirus expressing the p53 tumor suppressor gene) and AVdeltaE1#1CMV/p53 (with syngen#1 in place of wild-type sequences) demonstrated a normal productivity for the modified construct. The altered sequences did not impair p53-mediated apoptosis in a model tumor cell line. Most importantly, a statistically significant decrease in terms of RCA occurrence could also be demonstrated. Degenerescence of the recombinogenic sequences could be further accentuated by modifying noncoding pIX region (syngen #2), with no effect on virus productivity and stability. We concluded that these vector modifications constitute a feasible strategy to reduce RCA emergence during amplification in 293 cells. This approach could also be applied to decrease reincorporation of the E1 genes during amplification of deltaE1deltaE4 vectors in 293/E4-trans-complementing cells.

Adenoviridae↗
PubMed2001

Aroma compounds-proteins interaction using headspace techniques.

Interactions between volatile compounds and proteins in aqueous solution, were studied using static and dynamic headspace techniques. The decrease of aroma compounds concentration in the headspace determined in static mode indicates a retention by beta-lactoglobulin, 2.4% (w/w), pH 3.4, 25 degrees C, varying from 8 to 60% for most of the compounds tested. However 2-hexanone and isoamyl acetate are not significantly retained by the protein and a salting-out effect is noticed for limonene. On the basis of these preliminary results the intensity of interactions of methyl ketones (C7 to C9), ethyl esters (C6 to C9), limonene and mgamma rcene and beta-lactoglobulin for different pH values (2.0 to 11.0) were estimated by the determination of the infinite dilution activity coefficients (gamma i) by exponential dilution. For a constant pH value, the relative activity coefficient in the presence of beta-lactoglobulin (gamma r) of methyl ketones decreases significantly with the hydrophobicity of the volatiles whereas the relative activity coefficient value reaches a minimum for ethyl octanoate in the ester series. For limonene and myrcene an increase of gamma r is generally noticed whereas a decrease occurs in the presence of sodium caseinate and bovine serum albumin. The variations of relative activity coefficient according to the pH of the medium can be related to structural modifications of the beta-lactoglobulin.

Acyclic Monoterpenes↗
PubMed2000

Determination of apparent binding constants for aroma compounds with beta-lactoglobulin by dynamic coupled column liquid chromatography.

Apparent binding constants of aroma compounds limonene, alpha- and beta-ionone, and terpenyl acetate, with beta-lactoglobulin (BLG), were determined, using dynamic coupled column liquid chromatography, for pH values varying from 3 to 11. K(a) values varied from 2.61 to 3.21 x 10(3) M(-1) for limonene, indicating a strong interaction with BLG. Similarly, significant and close apparent binding constants were obtained for alpha- and beta-ionone, 1.7 x 10(2) and 4.5 to 5.4 x 10(2) M(-1), respectively. These data indicated that a similar mechanism is involved for the binding of these two molecules. The weaker values obtained at low pH, for alpha-ionone relative to beta-ionone, can be explained by the existence of steric hindrance. An increase of the apparent binding constant was observed, for all the compounds studied, when the pH was increased from 3 to 9. At this pH, an apparent binding constant was obtained for terpenyl acetate (1.04 x 10(2) M(-1)), whereas this determination was not possible at pH 3 and 6. The apparent binding constant increase was in agreement with the decrease of aroma compound relative activity coefficient in the presence of BLG, previously observed at this pH. It indicated a best accessibility to the same binding site. The binding constants of all the aroma compounds studied decreased at pH 11 as a result of the important release of the BLG structure previously reported.

Chromatography, High Pressure Liquid↗
PubMed2000

Effect of pH on retention of aroma compounds by beta-lactoglobulin.

Interactions between volatile compounds and BLG in aqueous solution were studied using static and dynamic headspace techniques (exponential dilution). The intensity of interactions between methyl ketones (C7-C9), ethyl esters (C6-C9), limonene, myrcene, and beta-lactoglobulin (BLG) were estimated by determination of the relative infinite dilution activity coefficients (gamma(r)). For a constant pH value, the methyl ketones retention by BLG increased significantly with the hydrophobicity of the volatiles, whereas the retention reached a maximum for ethyl octanoate in the ester series, indicating a possible steric hindrance. For limonene and myrcene an unexpected increase in headspace concentration or "salting out" effect was noticed for acid pH. The variations of the retention according to the pH increase of the medium from pH 3 to pH 11 could be related to structural modifications of the BLG. The retention increase observed between pH 3 and pH 9 resulted from the flexibility modification of the protein, allowing better accessibility to the primary or the secondary hydrophobic sites, whereas the dramatic decrease observed at pH 11 was the consequence of the alkaline denaturation of BLG. Electrostatic interactions occurring at pH 7.5 could also explain the observed retention increase.

Acyclic Monoterpenes↗
PubMed2000

Osteoporotic fractures at presentation of Cushing's disease: two case reports and a literature review.

Osteoporosis is a common complication of Cushing's disease/syndrome. Fractures can be the presenting manifestation. We report two cases and review 28 others from the literature. Clinical, laboratory, and absorptiometry data are reviewed; the vertebrae and ribs were the most common fracture sites, and osteoporosis reversal after treatment varied with age, gender, and symptom duration. The pathophysiology, which remains controversial, is discussed. A search for Cushing's disease/syndrome should be part of the routine evaluation of osteoporosis in children and adults, particularly men.

Absorptiometry, Photon↗
PubMed1999

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

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.

3T3 Cells↗
PubMed1999

Structural characteristics of supramolecular assemblies formed by guanidinium-cholesterol reagents for gene transfection.

We have recently discovered that cationic cholesterol derivatives characterized by guanidinium polar headgroups are very efficient for gene transfection in vitro and in vivo. In spite of being based on some rationale at the molecular level, the development of these new synthetic vectors was nevertheless empirical. Indeed, the factors and processes underlying cationic lipid-mediated gene transfer are still poorly understood. Thus, to get a better insight into the mechanisms involved, we have examined the supramolecular structure of lipid/DNA aggregates obtained when using reagent bis(guanidinium)-tren-cholesterol (BGTC), either alone or as a liposomal formulation with the neutral phospholipid dioleoyl phosphatidylethanolamine (DOPE). We here report the results of cryotransmission electron microscopy studies and small-angle x-ray scattering experiments, indicating the presence of multilamellar domains with a regular spacing of 70 A and 68 A in BGTC/DOPE-DNA and BGTC-DNA aggregates, respectively. In addition, DNA lipoplexes with similar lamellar patterns were detected inside transfected HeLa cells by conventional transmission electron microscopy. These results suggest that DNA condensation by multivalent guanidinium-cholesterol cationic lipids involves the formation of highly ordered multilamellar domains, the DNA molecules being intercalated between the lipid bilayers. These results also invite further investigation of the intracellular fate of the internalized lipid/DNA structures during their trafficking toward the cell nucleus. The identification of the basic features of active complexes should indeed help in the design of improved guanidinium-based vectors.

Cholesterol↗
PubMed1999

Synthetic DNA-compacting peptides derived from human sequence enhance cationic lipid-mediated gene transfer in vitro and in vivo.

Cationic lipids can deliver genes efficiently in vitro, but are generally inhibited by the presence of serum, and their efficiency in vivo is much lower than in vitro. An attractive strategy is to induce strong DNA compaction by its association with proteins, before addition of lipids. However the use of whole proteins might present both production and immunological limitations. We have devised a system in which DNA is associated with short peptides derived from human histone or protamine, before the addition of a cationic lipid or polymer. Peptides strongly associating with DNA confer to such peptide-DNA-lipid particles an enhanced in vitro transfection efficiency over that observed with classical DNA/lipid lipoplexes, and particularly confer the capacity to transfect in the presence of serum. This acquisition of serum resistance is cell type-independent, and observed with all four lipopolyamines tested and polyethylenimine. Precompacting DNA with a histone H1-derived peptide enhances cationic lipid RPR 115335-mediated gene transfer in an in vivo model of Lewis lung carcinoma. Apart from their use in peptide-DNA-lipid association, such peptides could be useful as part of chimeric gene delivery vectors presenting a DNA-binding moiety that can be easily associated with other functional domains.

3T3 Cells↗
PubMed1999

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

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.

3T3 Cells↗
PubMed1999

pCOR: a new design of plasmid vectors for nonviral gene therapy.

A totally redesigned host/vector system with improved properties in terms of safety has been developed. The pCOR plasmids are narrow-host range plasmid vectors for nonviral gene therapy. These plasmids contain a conditional origin of replication and must be propagated in a specifically engineered E. coli host strain, greatly reducing the potential for propagation in the environment or in treated patients. The pCOR backbone has several features that increase safety in terms of dissemination and selection: (1) the origin of replication requires a plasmid-specific initiator protein, pi protein, encoded by the pir gene limiting its host range to bacterial strains that produce this trans-acting protein; (2) the plasmid's selectable marker is not an antibiotic resistance gene but a gene encoding a bacterial suppressor tRNA. Optimized E. coli hosts supporting pCOR replication and selection were constructed. High yields of supercoiled pCOR monomers were obtained (100 mg/l) through fed-batch fermentation. pCOR vectors carrying the luciferase reporter gene gave high levels of luciferase activity when injected into murine skeletal muscle.

Drug Design↗
PubMed1999

Cluster organization of the genes of Streptomyces pristinaespiralis involved in pristinamycin biosynthesis and resistance elucidated by pulsed-field gel electrophoresis.

Streptomyces pristinaespiralis synthesizes pristinamycin, a member of the streptogramin antibiotic family which consists of a mixture of two types of chemically unrelated compounds named pristinamycins I and pristinamycins II. In order to estimate the size of the Strep. pristinaespiralis chromosome and to elucidate the organization of the pristinamycin biosynthetic and resistance genes already identified, it was decided to use the pulsed-field gel electrophoresis technique. Results indicate that the Strep. pristinaespiralis chromosome is linear and about 7580 kb, as previously shown for several other Streptomyces species. By hybridization, it could be shown that the biosynthetic and resistance genes for pristinamycins I and pristinamycins II, except for the multidrug resistance gene ptr, are interspersed and seem to be organized as a single large cluster, covering less than 200 kb corresponding to 2.6% of the total size of the chromosome. The consequences and significance of such a genetic organization are discussed.

Journal Article↗
PubMed1999

Spinal tuberculosis in adults. A study of 103 cases in a developed country, 1980-1994.

Spinal tuberculosis (TB) accounts for about 2% of all cases of TB. New methods of diagnosis such as magnetic resonance imaging (MRI) or percutaneous needle biopsy have emerged. Two distinct patterns of spinal TB can be identified, the classic form, called spondylodiscitis (SPD) in this article, and an increasingly common atypical form characterized by spondylitis without disk involvement (SPwD). We conducted a retrospective study of patients with spinal TB managed in the area of Paris, France, between 1980 and 1994 with the goal of defining the characteristics of spinal TB and comparing SPD to SPwD. The 103 consecutive patients included in our study had TB confirmed by bacteriologic and/or histologic studies of specimens from spinal or paraspinal lesions (93 patients) or from extraspinal skeletal lesions (10 patients). Sixty-eight percent of patients were foreign-born subjects from developing countries. None of our patients was HIV-positive. SPD accounted for 48% of cases and SPwD for 52%. Patients with SPwD were younger and more likely to be foreign-born and to have multiple skeletal TB lesions. Neurologic manifestations were observed in 50% of patients, with no differences between the SPD and SPwD groups. Of the 44 patients investigated by MRI, 6 had normal plain radiographs; MRI was consistently positive and demonstrated epidural involvement in 77% of cases. Bacteriologic and histologic yields were similar for surgical biopsy (n = 16) and for percutaneous needle aspiration and/or biopsy (n = 77). Cultures for Mycobacterium tuberculosis were positive in 83% of patients, and no strains were resistant to rifampin. Median duration of antituberculous chemotherapy was 14 months. Surgical treatment was performed in 24% of patients. There were 2 TB-related deaths. Our data suggest that SPwD may now be the most common pattern of spinal TB in foreign-born subjects in industrialized countries. The reasons for this remain to be elucidated.

Adolescent↗
PubMed1999

Nonsurgical treatment of osteoarticular tuberculosis. A retrospective study in 143 adults.

OBJECTIVE AND METHODS: Data are sparse on nonsurgical treatments currently used for osteoarticular tuberculosis in industrialized countries. We conducted a multicenter retrospective study in the Paris urban area, France, in 206 cases of osteoarticular tuberculosis documented by examination of a local specimen. This article reports our findings in the 143 patients who were followed up at least until treatment completion. RESULTS: Mean follow-up after treatment completion was 16 months. Seventy-five (52%) patients had spondylitis and 68 (48%) did not. The number of antituberculous agents used during the initial treatment phase was four in 65% of cases and three in 35%. In the spondylitis subgroup, mean (+/- SD) antibiotic therapy duration was 14.7 +/- 3.4 months, and 25% of patients required surgery; 3% of patients died, 1% suffered a relapse, and 96% achieved a full recovery with no relapse. In the nonspondylitis subgroup, mean antibiotic therapy duration was 13 +/- 3 months and 29% of patients required surgery. The only HIV-positive patient had osteitis of the calcaneus with a relapse that led to discovery of secondary rifampin resistance. CONCLUSION: Based on our findings and on data from the literature, we believe that 12 months is a reasonable duration for antituberculous therapy in osteoarticular tuberculosis, including tuberculous spondylitis.

Adolescent↗
PubMed1998

Synthesis, activity, and structure--activity relationship studies of novel cationic lipids for DNA transfer.

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.

3T3 Cells↗
PubMed1998

Production of a new DNA vehicle for gene transfer using site-specific recombination.

Supercoiled DNA molecules, minicircles, were produced by in vivo site-specific recombination. They contained exclusively the desired excisable fragment. Recombination was driven by bacteriophage lambda integrase from a plasmid substrate containing the attP and attB recombination sites in the same orientation. Conditions for minicircle production within the lysogen Escherichia coli D1210HP were optimised. Up to 1.5 mg minicircles could be produced per litre bacterial culture, and the remaining, unrecombined plasmid comprised less than about 15% of the minicircle produced. However minicircle multimers were also produced, and comprised up to 30% of all minicircles synthesised. The par ABCDE' locus from plasmid RK2 was introduced into the minicircle fragment, resulting in minicircle dimers being reduced to less than 2% of all minicircles. The par A gene encodes a resolvase that catalyses recombination at the multimer resolution site in the parABCDE' locus. Minicircle multimers were also resolved when par A was introduced downstream from the integrase gene of the lambda pL transcript in D1210HP together with a multimer resolution site carried by the minicircle fragment.

DNA, Circular↗