Fatal Mycobacterium peregrinum pneumonia in refractory polymyositis treated with infliximab.
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Aged↗
Biomedical subjects
F Roussel
Publications and source records attributed to F Roussel.
[The script concordance test: a new evaluation method of both clinical reasoning and skills in internal medicine].
OBJECTIVE: The script concordance test is designed to evaluate knowledge organization, which constitutes a crucial parameter of clinical skills. The objective of the present study was to assess the value of a new written evaluation tool to measure clinical skills in Internal Medicine. MATERIALS AND METHODS: A 95-item examination was completed by a group of medical students (N =17), a group of residents in Family practice (N =9), a group of residents in Internal Medicine (N =5), and a group of experienced physicians in Internal Medicine (N =7). The scores obtained were compared by analysis of variance. The reliability of the test was studied by calculating Cronbach's coefficient alpha. RESULTS: The mean score was 220.3 +/-41.7 for medical students, 230.5 +/-31.7 for residents in Family practice, 274.2 +/-32.2 for residents in Internal Medicine, and 352.1 +/-22.9 for experienced physicians in Internal Medicine. The differences observed between the scores for the various groups were significant (P <0.0001). Moreover, the value of Cronbach's coefficient alpha was 0.81 in the whole examination. CONCLUSION: Our data indicate that the script concordance test may easily allow to differentiate various levels of clinical skills in Internal Medicine. Moreover, because of Cronbach's coefficient alpha as high as 0.81, our findings suggest the validity of this test in Internal Medicine.
Diagnostic Tests, Routine↗
Conducting polymers as driving electrodes for Polymer-Dispersed Liquid-Crystals display devices: on the electro-optical efficiency.
Intrinsically conducting polymer (ICP) thin films are used as driving electrodes for Polymer-Dispersed Liquid-Crystals (PDLC) display devices. In order to investigate the electro-optical efficiency of these organic electrodes, three different kinds of conducting polymers, i.e. polyaniline doped with 10-camphorsulfonic acid (PANI(HCSA)), polypyrrole doped with dodecylbenzenesulfonic acid (PPY(DBSA)), and polyethylenedioxythiophene doped with polystyrenesulfonate (PEDOT(PSS)), were prepared or purchased, and coated either on glass or plastic substrates. Optical absorption studies in the UV-Vis range of the conducting polymer-coated substrates were first performed showing the presence of conducting species for the three types of polymers. The electrical characteristics of the resulting films were measured with the four-probes technique. PANI(HCSA) exhibits a higher conductivity sigma approximately 122 S x cm(-1) (RS=1.2x10(3) Omega x (-1)) compared to PPY(DBSA) sigma approximately 2.6 S x cm(-1) (RS=150.7x10(3) Omega x (-1)), and PEDOT(PSS) sigma approximately 1.6 S x cm(-1) (RS=637.3x10(3) Omega x (-1)). It is also shown that for a given conducting polymer, its electrical conductivity decreases when a plastic substrate is used. These observations have been related to significant morphological changes observed by scanning electron microscopy (SEM). A mixture of Norland Optical Adhesive 65 and nematic liquid-crystal E7 in the weight ratio (35:65) was used as precursor of the PDLC material. Better electro-optical responses (transmission properties, drive voltages and switching times) of PDLC films were obtained for devices prepared with (PPY(DBSA))-based electrodes. The electro-optical performances of the PDLC display devices also depend on the nature of the ICP substrate used.
Journal Article↗
Thermophysical properties of fluorinated acrylate homopolymers: mixing and phase separation.
The thermophysical properties of fluorinated acrylate homopolymers are investigated by differential scanning calorimetry (DSC) and optical microscopy and discussed in terms of relative lengths of the fluorinated chain and the hydrocarbon spacer between the acrylate moiety and the fluorinated chain. These compounds exhibit an intrinsic microphase-separation (Isotropic+Isotropic morphology) occurring between the fluorinated chains and the acrylate polymer backbone. It is shown that the enthalpy of mixing is a function of the length of the lateral fluorocarbon chains. The thermophysical behaviour of these materials may be regarded as demixed systems exhibiting an Upper Critical Solution Temperature. The photopolymerization process of one of the monomer is studied by isothermal photocalorimetry. High acrylate double-bond conversion and fast curing rates were obtained thus demonstrating the promising use of these materials for coating and film processing applications using UV-curing techniques.
Journal Article↗
Solid-phase synthesis of a branched hexasaccharide using a highly efficient synthetic strategy.
The solid-phase synthesis of branched lacto-N-neohexaose derivative 1 occurring in human milk is described. The new building block of lactose 3 bearing the orthogonal temporary hydroxy protecting groups 9-fluorenylmethyloxycarbonyl (Fmoc) and levulinoyl (Lev) has been prepared. Its use, together with that of lactosamine donor 4, glucosamine donor 5, and O-galactosyl trichloroacetimidate 6, has enabled the preparation of hexasaccharide 22 following two different approaches in excellent overall yield (43%, 90% per step over eight steps). An additional key feature of this work is the successful use of newly prepared ester-type linker 2, having a benzylic spacer connected to the anomeric oxygen. This linker presents the advantage of producing a benzylic anomeric moiety after cleavage from the polymer support, which could be easily removed to obtain the unprotected oligosaccharide 1.
Carbohydrate Sequence↗
Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition site residues and effect on substrate oxidation and cooperativity.
Phenylalanine and/or tryptophan scanning mutagenesis was performed at 15 sites within CYP3A4 proposed to be involved in substrate specificity or cooperativity. The sites were chosen on the basis of previous studies or from a comparison with the structure of P450(eryF) containing two molecules of androstenedione. The function of the 25 mutants was assessed in a reconstituted system using progesterone, testosterone, 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and alpha-naphthoflavone (ANF) as substrates. CYP3A4 wild type displayed sigmoidal kinetics of ANF 5,6-oxide formation and 7-BFC debenzylation. Analysis of 12 mutants with significant steroid hydroxylase activity showed a lack of positive correlation between ANF oxidation and stimulation of progesterone 6beta-hydroxylation by ANF, indicating that ANF binds at two sites within CYP3A4. 7-BFC debenzylation was stimulated by progesterone and ANF, and 7-BFC did not inhibit testosterone or progesterone 6beta-hydroxylation. Correlational analysis showed no relationship between 7-BFC debenzylation and either progesterone or testosterone 6beta-hydroxylation. These data are difficult to explain with a two-site model of CYP3A4 but suggest that three subpockets exist within the active site. Interestingly, classification of the mutants according to their ability to oxidize the four substrates utilized in this study suggested that substrates do bind at preferred locations in the CYP3A4 binding pocket.
Amino Acid Sequence↗
Importance of amino acid residue 474 for substrate specificity of canine and human cytochrome p450 3A enzymes.
Canine cytochromes P450 3A12 and 3A26 are identical in sequence at 481 of 503 amino-acid positions but exhibit different substrate specificities. A recent study utilizing chimeric enzymes and site-directed mutagenesis identified three residues (187, 368, and 369) that contribute to differences in steroid hydroxylation and also indicated the presence of additional determinants of specificity among the 44 carboxyl terminal residues. Therefore, three 3A26 multiple mutants (I187T-S368P-V369I-S467P, I187T-S368P-V369I-S474P, and I187T-S368P-V369I-R476K-I477L-T479A-R480Q) were constructed. Insertion of 3A12 residue Pro-474 into 3A26 I187T-S368P-V369I resulted in metabolite profiles with testosterone, androstenedione, and progesterone very similar to 3A12. Substitution of Pro-474 with Ser in P450 3A12 or human 3A4 significantly increased 2beta-hydroxylase activity with all three steroids. Residue 474 was also found to be an important contributor to diazepam metabolism by the canine and human enzymes. The results provide further evidence for the role of steric constraints exerted by the enzyme in P450 3A-mediated oxidations.
Amino Acid Substitution↗
O-Glycosyl trichloroacetimidates bearing Fmoc as temporary hydroxy protecting group: a new access to solid-phase oligosaccharide synthesis.
Different O-glycosyl trichloroacetimidates bearing base sensitive Fmoc protected hydroxy groups were efficiently prepared with CCl(3)CN using a catalytic amount of sodium hydride. The resulting glycosyl donors were engaged in glycosylation reactions both in solution and on solid support with a new ester-type linker with good results. In both approaches, Fmoc groups were afterward quantitatively cleaved using mild basic conditions.
Carbohydrate Sequence↗
The importance of SRS-1 residues in catalytic specificity of human cytochrome P450 3A4.
The structural basis for the regioselective hydroxylation of Delta-4-3-ketosteroids by human CYP3A4 was investigated. Prior studies had suggested that the chemical reactivity of the allylic 6beta-position might have a greater influence than steric constraints by the enzyme. Six highly conserved CYP3A residues from substrate recognition site 1 were examined by site-directed mutagenesis. F102A and A117L showed no spectrally detectable P450. V101G and T103A exhibited a wild-type progesterone metabolite profile. Of five mutants at residue N104, only N104D yielded holoenzyme and exhibited the same steroid metabolite profile as wild-type. Of four mutants at position S119 (A, L, T, V), the three hydrophobic ones produced 2beta-OH rather than 6beta-OH progesterone or testosterone as the major metabolite. Kinetic analysis showed S(50) values similar to wild-type for S119A (progesterone) and S119V (testosterone), whereas the V(max) values for 2beta-hydroxysteroid formation were increased in both cases. All four mutants exhibited an altered product profile for 7-hexoxycoumarin side-chain hydroxylation, whereas the stimulation of steroid hydroxylation by alpha-naphthoflavone was similar to the wild-type. The results indicate that the highly conserved residue S119 is a key determinant of CYP3A4 specificity and reveal an important role of the active site topology in steroid 6beta-hydroxylation.
Amino Acid Sequence↗
Phase diagrams and morphology of polymer dispersed liquid crystals based on nematic-liquid-crystal-monofunctional-acrylate mixtures
Phase diagrams of unpolymerized and UV-polymerized 2-ethyl hexyl acrylate (EHA) mixtures with the liquid crystal E7 are established using optical microscopy and differential scanning calorimetry. Both diagrams show upper critical solution temperature behavior. From 50 to 90 wt % liquid crystal (LC), the (I+I) phase located between the (N+I) and (I) phases was clearly shown. The nematic phase inside the droplets exhibits a twisted radial structure indicating that homeotropic anchoring occurs at the polymer interface. The experimental phase diagrams were successfully analyzed using a model based on the Flory-Huggins theory of isotropic mixing supplemented with the Maier-Saupe theory of nematic order. The LC solubility limit in the polymer matrix and the fractional amount of LC contained in the droplets were deduced from the calorimetric measurements. For the specific composition EHA/E7 (50:50), the scattering and morphological properties of the films were studied as a function of time elapsed after UV exposure. Drastic changes in the size, shape, spatial distribution, and number density of nematic droplets were observed and analyzed in terms of coalescence/diffusion phenomena.
Journal Article↗
Structure-function analysis of human cytochrome P-450 2B6 using a novel substrate, site-directed mutagenesis, and molecular modeling.
The structural basis for functional differences between human cytochrome P-450 2B6 and rat 2B1 was investigated. An amino acid sequence alignment predicted the location of 2B6 substrate recognition site (SRS) residues. Ten residues within these SRSs unique to 2B6 compared with 2B1, 2B4, and 2B11 were chosen for mutagenesis. Two additional sites that differ between 2B6 and 2B1 and are known to have a role in 2B1 substrate specificity were also mutated. The 2B6 mutants were expressed in Spodoptera frugiperda cells and characterized using the 2B6-specific substrate RP 73401 [3-cyclopentyloxy-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide], the 2B1-selective substrate androstenedione, and the common substrate 7-ethoxy-4-trifluoromethylcoumarin. Mutants F107I and L363V exhibited decreased RP 73401 hydroxylation but retained most of the wild-type level of 2B6 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity. In addition, SRS exchanges were studied in which the amino acid sequence of 2B6 SRSs was converted to the sequence of 2B1. Each of these constructs, having two to seven substitutions, expressed at levels similar to 2B6 but did not acquire significant androstenedione hydroxylase activity. Docking of RP 73401 into the active site of a 2B6 homology model suggested a direct interaction with residue L363 but not with F107. Findings from this study suggest that 1) residues F107 and L363 are necessary for 2B6 RP 73401 hydroxylase activity, 2) 2B6 is able to tolerate multiple SRS substitutions without compromising protein expression levels or protein stability, and 3) conferring androstenedione hydroxylase function to cytochrome P-450 2B6 is more complex than altering a single SRS.
Animals↗
Expression and characterization of canine cytochrome P450 2D15.
CYP2D15 is the canine ortholog of human CYP2D6, the human CYP2D isoform involved in the metabolism of drugs such as antiarhythmics, adrenoceptor antagonists, and tricyclic antidepressants. Similar to human, canine CYP2D15 is expressed in the liver, with detectable levels in several other tissues. Three different CYP2D15 cDNA clones were obtained by RT-PCR from dog liver RNA. Two clones corresponded to variant full-length CYP2D15 cDNAs (termed CYP2D15 WT2 and CYP2D15 V1); the third was identified as a splicing variant missing exon 3 (termed CYP2D15 V2). Recombinant baculoviruses were constructed containing full-length cDNAs and used to express CYP2D15 WT2 and CYP2D15 V1 in Spodoptera frugiperda (Sf9) cells with expression levels of up to 0.14 nmol/mg cell protein. As with human CYP2D6, the recombinant CYP2D15 enzymes exhibited bufuralol 1'-hydroxylaseand dextromethorphan O-demethylase activities whencoexpressed with rabbit NADPH:P450 oxidoreductase. For bufuralol 1'-hydroxylase, apparent Km values were 4.9, 3.7, and 2.5 microM and the Vmax values were 0.14, 0.034, and 0.60 nmol/min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. For dextromethorphan O-demethylase, apparent Km values were 0.6, 0.6, and 2.0 microM and the Vmax values were 0.18, 0.034, and 0.057 nmol/min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. The human CYP2D6-specific inhibitor quinidine and the rat CYP2D1-specific inhibitor quinine were both shown to be inhibitors of bufuralol 1'-hydroxylase activity for dog liver microsomes, CYP2D15 WT2, and the CYP2D15 V1 variant with nearly equal potency. Thus, the dog expresses a CYP2D ortholog possessing enzymatic activities similar to human CYP2D6, but is affected by the inhibitors quinine and quinidine in a manner closer to that of rat CYP2D1.
Animals↗
Biliary tract cryptosporidiosis immunosuppressed rat model.
Rats immunosuppressed by hydrocortisone acetate and a low protein diet were challenged with Cryptosporidium Parvum oocysts and studied on days 10, 35 and 70 post-infection. The biliary tract was found to be a major site of parasite infection. C. parvum was visible in the biliary papillary area in association with a proliferation of highly convoluted tubular glands. The papillary lumen was narrowed, and an upstream dilation with bacterial proliferation was seen. The liver was initially free of lesions, and subsequently exhibited late lesions of cholestasis. Parasites were not found in the pancreatic duct, although pancreatitis was frequently observed. Oocysts were consistently present in the distal portion of the ileum. Both challenged and unchallenged immunosuppressed rats, exhibited widespread focal hepatic infarcts and pyelonephritis. Other organs appeared free of lesions. In addition to the intestine, data identified the biliary tract as a major site of C. parvum infection and as a potential protected reservoir which may sustain a chronic infection.
Animals↗
Respiratory tract cryptosporidiosis in immunosuppressed rat is associated with an epithelial metaplasia.
Cryptosporidium parvum is an opportunistic protozoa that chronically infects the digestive tract of immunocompromised hosts. Respiratory cryptosporidiosis, which was reported in AIDS patients, is an uncommon feature of mammalian cryptosporidiosis models. In this study, we document the respiratory lesion; observed in an immunosuppressed rat model of cryptosporidiosis. Twenty rats were immunosuppressed with corticosteroids and low protein diet. They were challenged intratracheally with 10(6) C. parvum sporozoites. Lungs and ileums were examined on D3, D6, D10, D14. On D10 and D14, C. parvum were present in the respiratory tract of all animals in association with the progressive appearance of an immature malpighian metaplasia. On D14, an intestinal infection was also detected in 2/4 animals. The respiratory tract appears to be a fully permissive area for the protozoa in immunosuppressed rats. Introduction of parasites on the respiratory mucosa seems a requisite to induce respiratory cryptosporidiosis. This experimental protocol yields a low mortality rate, and so modelizes late and/or chronic stages of respiratory cryptosporidiosis.
Animals↗
Gene structure and promoter analysis of the rat constitutive CYP2C23 gene.
The CYP2C23 gene is expressed constitutively in the rat liver and kidney. It exhibits different profiles of expression in the two tissues, suggesting that several regulation processes could exist. In this paper, we report the structure of the 5'-flanking region of the CYP2C23 gene; 4.5 kbp were sequenced and analyzed. The CYP2C23 gene is present as a single copy into the rat genome and an unique transcription start site is used in both liver and kidney. Four DNase I hypersensitive sites have been mapped to the distal part of the hepatic promoter and three are detected in the kidney: only one site is present in the two tissues (L3/K1). In the proximal region, one site is specific for the kidney and one is detected in all the tissues tested. Footprint experiments allowed precise identification of the sequence of protected regions: HNF4 and CREB binding motifs are present in the distal liver-specific sites, motifs for AP-1, NF-1, and XRE-Bf are in the distal kidney site, and a Tf-LF1 binding site is localized in the L3/K1 protected site. In the proximal region, a sequence protected in all tissues contains a SP1/NF kappa B motif, whereas a sequence containing a HNF-4 binding motif is exclusively protected by kidney nuclear extracts. Altogether, the data clearly demonstrate that trans-acting factors involved in CYP2C23 gene expression differ in liver and kidney.
Animals↗
An immunosuppressed rat model for evaluation of anti-Cryptosporidium activity of sinefungin.
Cryptosporidium parvum causes life-threatening diarrhoea in immunocompromised, especially AIDS patients and the efficiency of proposed anti-cryptosporidial therapies is limited or doubtful. An immunosuppressed adult rat model of C. parvum infection was developed for screening molecules candidate for curative and preventive activity in human cryptosporidiosis. Among 31 drugs tested, lasalocid (2-10 mg/kg/24 h), and sinefungin (2-10 mg/kg/24 h), exhibited some activity against C. parvum infection. Oral sinefungin therapy resulted in a dose related suppression of oocysts shedding, which correlated with oocyst disappearance from ileum sections and was also efficient in preventing infection. Relapses were observed after discontinuation of curative sinefungin therapy, which suggests that the biliary tract, a major location and parasite reservoir which sustains persisting infection, was not cleared of parasites by the drug. Improved therapeutic procedures with sinefungin (or analogues) will result from current pharmacological studies.
Adenosine↗
Age- and tissue-dependent expression of CYP2C23 in the rat.
A cDNA was isolated from a library prepared with 15-day-old rat liver RNA and was shown to correspond to CYP2C23 with some minor alterations which modified the open reading frame. The expression of CYP2C23 was examined in rat liver, kidney, lung and testis and during ontogenesis. An appreciable amount of CYP2C23 RNA was observed in liver and kidney but the age-dependent expression was quite different between these two tissues. In liver, expression reached its maximal value in early neonates and remained quite stable, while the increase was progressive in kidney before declining after 3 weeks of age. In the liver, classical inducers of cytochrome P-450 decreased the CYP2C23 RNA content. Experimental data confirm the classification of CYP2C23 as a constitutive member of the 2C subfamily and clearly establish its age- and tissue-dependent expression in rat.
Aging↗
Assessment of candidate anticryptosporidial agents in an immunosuppressed rat model.
Cryptosporidium parvum causes life-threatening diarrhea in immunocompromised patients, especially those with AIDS. The efficiency of currently proposed anticryptosporidial therapies is limited or doubtful. In this report, molecular candidates for curative or preventive activity were investigated in an immunocompromised rat model that mimics severe human cryptosporidiosis. No significant anticryptosporidial activity was observed when using sulfadoxine-pyrimethamine, quinacrine, trimethoprim-sulfamethoxazole, bleomycin, elliptinium, daunorubicin, pentamidine, alpha-difluoro-methylornithine, diclazuril or N-methylglucamine. Vitamin A appeared to reduce oocyst shedding. Active agents included sinefungin (2-10 mg/kg/24 h), lasalocid A (2-10 mg/kg/24 h), metronidazole (25-50 mg/kg/24 h), and sulfadimethoxine (10-100 mg/kg/24 h). Sinefungin (10 mg/kg/24 h) and lasalocid A (10 mg/kg/24 h) displayed the highest anticryptosporidial activity.
Animals↗