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P Bertrand

Publications and source records attributed to P Bertrand.

At least 19 recordsLinked to original sources
PubMed1999

Evidence of simple intramolecular rearrangement at polymer end groups in secondary ion mass spectrometry.

Polystyrenes with various end groups were analyzed by time-of-flight secondary ion mass spectrometry (TOF-SIMS). These end groups were obtained by termination of the active anionic group by sulfonate or chlorosilane derivatives. Characteristic end group fragments for each sulfonate derivative were observed. On the one hand, for PS capped by methyl sulfonate, or -(CH(2))(4)-O-SO(2)-CH(3), almost complete end group fragment is observed at m/z 95 and a [O-SO(2)-CH(3)](-) molecular structure. On the other hand, when PS is terminated by silyl methyl sulfonate, or -Si(CH(3))(2)-(CH(2))(3)-O-SO(2)-CH(3), the most characteristic fragment in the fingerprint secondary ion mass spectrum is located at m/z 153 with [Si(CH(3))(2)-O-SO(2)-CH(3)](+) and the complete end group peak, [Si(CH(3))(2)-(CH(2))(3)-O-SO(2)-CH(3)](+), at m/z 201, is absent. According to the molecular structure, characteristic end group secondary ions can be emitted as complete or rearranged fragments. Various silylalkyl alcohol or sulfonate functionalities are analyzed and fragmentation pathways are discussed. To our knowledge, this is the first time that such rearrangement at silyl functions has been observed and demonstrated in fingerprint secondary ion mass spectra. Copyright 1999 John Wiley & Sons, Ltd.

Journal Article↗
PubMed1999

Human POMp75 is identified as the pro-oncoprotein TLS/FUS: both POMp75 and POMp100 DNA homologous pairing activities are associated to cell proliferation.

We have previously developed an assay to measure DNA homologous pairing activities in crude extracts: The POM blot. In mammalian nuclear extracts, we detected two major DNA homologous pairing activities: POMp100 and POMp75. Here, we present the purification and identification of POMp75 as the pro-oncoprotein TLS/FUS. Because of the pro-oncogene status of TLS/FUS, we studied in addition, the relationships between cell proliferation and POM activities. We show that transformation of human fibroblasts by SV40 large T antigen results in a strong increase of both POMpl00 and TLS/POMp75 activities. Although detectable levels of both POMp100 and TLS/POMp75 are observed in non-immortalized fibroblasts or lymphocytes, fibroblasts at mid confluence or lymphocytes stimulated by phytohaemaglutinin, show higher levels of POM activities. Moreover, induction of differentiation of mouse F9 line by retinoic acid leads to the inhibition of both POMp100 and TLS/POMp75 activities. Comparison of POM activity of TLS/FUS with the amount of TLS protein detected by Western blot, suggests that the POM activity could be regulated by post-translation modification. Taken together, these results indicate that POMp100 and TLS/POMp75 activities are present in normal cells but are connected to cell proliferation. Possible relationship between cell proliferation, response to DNA damage and DNA homologous pairing activity of the pro-oncoprotein TLS/FUS are discussed.

Amino Acid Sequence↗
PubMed1999

Cannabinoid penetration into mouse brain as determined by ex vivo binding.

We have used an ex vivo binding assay in the mouse to evaluate the brain penetration of cannabinoid receptor ligands. After intraperitoneal or oral administration, the pharmacological activity linked to the compound was assessed by using by [3H]WIN 55212-2 binding on cerebellar membranes. The brain penetration was high for compounds like methanandamide or delta9-tetrahydrocannabinol but poor for synthetic agonists such as (cis)-3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-(trans)-4-(3-hydr oxypropyl)cyclohexanol (CP 55940) or, R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-d e]-1,4-benzoxazin-6-yl)(1-napthalenyl)methanone monomethane-sulfonate (WIN 55212-2). After oral administration the duration of action of delta9-tetrahydrocannabinol, methanandamide and WIN 55212-2 is limited and decreased 4 h after administration. The cannabinoid CB1 receptor antagonist: N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-met hyl-1 H-pyrazole-3-carboxamide hydrochloride (SR141716A) exhibited a good brain penetration and a long duration of action.

Animals↗
PubMed1999

Modulation of apolipoprotein D and apolipoprotein E expression in rat hippocampus after entorhinal cortex lesion.

Apolipoprotein (apo) D is a member of the lipocalin family of proteins. Although its physiological function is unknown, apoD is thought to transport one or more small hydrophobic ligands. A second apolipoprotein, apoE is known to play an important role in lipid transport, and apoE genetic polymorphism has been shown to be associated with susceptibility to Alzheimer's disease. Both apoD and apoE are expressed in the central nervous system (CNS) and both proteins accumulate at sites of peripheral nerve injury due to increased local synthesis. The two proteins may have overlapping or complementary functions within nervous tissue. In order to define the role of apoD within the CNS, we have studied the regional distribution of apoD and apoE mRNA and protein within the normal rat brain and the changes in apoD and apoE expression in the hippocampus of rats after entorhinal cortex lesion (EC lesion). Within the brains of normal rats, apoD expression in the hippocampus was as high as 180-fold that of the liver. ApoD mRNA levels in other areas of the rat brain ranged from 40 to 120 times the hepatic levels. The distribution of apoE gene expression within the brain was similar to that of apoD, but was much lower than hepatic apoE expression. When rats were subjected to EC lesion, the apoD message increased by 54% at 4 days post lesion (DPL) in the ipsilateral region of hippocampus while apoE mRNA levels (ipsilateral and contralateral) decreased by 43%. At 6 to 8 DPL apoD mRNA in the ipsilateral hippocampus remained elevated (42% above controls) whereas the apoE mRNA levels increased to about 15% above those of controls. At 14 and 31 DPL, both apoD and apoE expression was similar to controls. The increase in immunoreactive apoD in hippocampal extracts was more dramatic. At 1 DPL, immunoreactive apoD levels were already 16-fold higher than those in extracts of non-lesioned animals and, at 31 DPL, levels were still 8-fold higher than those of control animals. Finally, we have demonstrated that the levels of apoD in the brains of apoE-deficient mice are 50-fold those of wildtype control mice. ApoD clearly has an important function within the CNS in both normal and pathological situations.

Animals↗
PubMed1999

Fibronectin-pluronic coadsorption on a polystyrene surface with increasing hydrophobicity: relationship to cell adhesion.

Recently, patterned polystyrene surfaces containing hydrophobic (PS) and more hydrophilic (PSox) areas have been shown to be capable of directing cellular growth, which is mainly due to the competitive adsorption of adhesive and antiadhesive molecules. In this article, the competitive adsorption between a pluronic surfactant and fibronectin was studied on homogeneous PS or PSox substrates conditioned with mixtures containing increasing concentrations of one of the two molecules. Radiolabeling and X-ray photoelectron spectroscopy techniques showed that fibronectin adsorption increased on both surfaces if the fibronectin concentrations increased in the conditioning mixture. In contrast, fibronectin adsorption decreased on PSox and did not occur on PS surfaces when pluronic concentrations increased in the coating mixture. A comparison of these data with pheochromocytoma and Schwann cells cultured on patterned surfaces showed that the direction of cell growth on PSox areas depended first on the relative concentrations of the two components in the mixtures, and second, on their ratio; the best concentration ratio probably depends on the cell's ability to recondition its support.

Adsorption↗
PubMed1999

Analysis of intrachromosomal homologous recombination in mammalian cell, using tandem repeat sequences.

In all the organisms, homologous recombination (HR) is involved in fundamental processes such as genome diversification and DNA repair. Several strategies can be devised to measure homologous recombination in mammalian cells. We present here the interest of using intrachromosomal tandem repeat sequences to measure HR in mammalian cells and we discuss the differences with the ectopic plasmids recombination. The present review focuses on the molecular mechanisms of HR between tandem repeats in mammalian cells. The possibility to use two different orientations of tandem repeats (direct or inverted repeats) in parallel constitutes also an advantage. While inverted repeats measure only events arising by strand exchange (gene conversion and crossing over), direct repeats monitor strand exchange events and also non-conservative processes such as single strand annealing or replication slippage. In yeast, these processes depend on different pathways, most of them also existing in mammalian cells. These data permit to devise substrates adapted to specific questions about HR in mammalian cells. The effect of substrate structures (heterologies, insertions/deletions, GT repeats, transcription) and consequences of DNA double strand breaks induced by ionizing radiation or endonuclease (especially the rare-cutting endonuclease ISce-I) on HR are discussed. Finally, transgenic mouse models using tandem repeats are briefly presented.

Animals↗
PubMed1999

Activation and inhibition of human cancer cell hyaluronidase by proteins.

Results regarding hyaluronidase activity in tumor extracts or cell lines are subject to variations according to the method used for the assay and, sometimes, within an assay. Hyaluronidase was assayed at pH 3.8 in the culture medium of the human cancer-derived cell lines SA87 and H460M by several techniques: HPLC, Reissig technique, ELSA technique, and zymographic analysis. The optimal pH was between 3.3 and 4 in solutions at constant 150 mM sodium concentration. The enzyme was reversibly inhibited by sodium concentration over 200 mM. The activity of purified hyaluronidase increased in the presence of low concentrations of the specific HA-binding glycoprotein hyaluronectin, or of bovine serum albumin or immunoglobulins, or of human albumin, transferrin, or hemoglobin, showing that proteins cooperate in enzyme activity. The ELSA technique showed that optimal pH was slightly lower in the presence of HN than that with BSA. The optimal BSA concentration was determined with the ELSA technique at 0.1 g/liter, and excess of either protein inhibited hyaluronidase. When measured with the Reissig technique, the activity of purified enzyme in the presence of 0.1 g/liter BSA was up to fourfold that without BSA. The cooperative effect of BSA was visualized by zymography. We conclude that the total protein content of hyaluronidase solutions must be considered to correctly interpret quantitation of the enzyme in sera or tissue extracts because protein concentrations above 200 microg/liter lead to underestimation of the enzyme.

Animals↗
PubMed1999

EPR spectroscopy: a powerful technique for the structural and functional investigation of metalloproteins.

Numerous metal centers in proteins can be prepared in a redox state in which their ground state is paramagnetic. Complementary data provided by EPR, Mössbauer, electron nuclear double resonance, magnetic circular dichroism, and NMR spectroscopies have therefore played a major role in the elucidation of the structure and function of these centers. Among those techniques the most commonly used is certainly EPR spectroscopy. In this article various aspects of the current applications of EPR to the structural and functional study of metalloproteins are presented. They are illustrated by recent studies carried out in our laboratory in the field of metalloenzymes and electron transfer systems. The power of numerical simulation techniques is emphasized throughout this work.

Agaricales↗
PubMed1999

Hyaluronectin secretion by monocytes: downregulation by IL-4 and IL-13, upregulation by IL-10.

Hyaluronectin (HN) is a component of the extracellular matrix of connective tissue and is particularly associated with tumour inflammatory and connective stroma reaction, where it co-localizes with hyaluronic acid (HA). The HN/HA ratio has been suggested to be involved in tumour aggressivity and in the atherosclerosis process. IL-10 has also been described in atherosclerotic lesions and in cancer. HN production was therefore investigated in vitro in peripheral blood monocyte cell (PBMC) cultures, with and without bacterial lipolysaccharide (LPS) or interleukins (ILs) in the medium. HN was characterized in monocytic cell cytoplasm and in culture supernatants. Anti-IL-10 antibody suppressed the LPS-stimulating effect on HN production. HN synthesis rate was greatly increased in IL-10-activated cultures while IL-4 and IL-13, two other anti-inflammatory ILs, decreased HN release. In the presence of IL-10, the IL-4 or Il-13 inhibitory effect on HN synthesis was reversed. The results support the view that intratumoral release of IL-10 by monocytes may induce local production of HN. In conjunction with the known ability of HN to bind to HA, which is a cell migration and tumour invasion facilitating factor, and to inhibit HA-induced angiogenesis, our findings suggest that HN may modulate the effect of HA on atherosclerosis, angiogenesis and cancer development.

Antibodies↗
PubMed1999

Long-term outcome in patients under 40 years after revascularization for chronic lower limb ischaemia.

BACKGROUND: In order to find out if surgical or endoluminal treatment changes the long-term results of atherosclerotic occlusive disease in patients of under 40 years of age we reviewed 17 consecutive patients. METHODS: Their mean age was 36.5. Patients with Buerger's disease or inflammatory arteriopathy were excluded. All patients were extremely heavy smokers. The indications for surgical procedures were disabling claudication (less than 100 meters) for 11 patients, rest pain for 4 patients and grangrene of a lower limb for 2 patients. The lesions were aorto-iliac in 12 cases and femoro-popliteal in 5. Ten surgical procedures were performed (5 aorto-femoral bypasses, 1 ilio-femoral bypass associated with an aorto-renal bypass, 2 femoropopliteal bypasses, 1 aorto-iliac endarteriectomy, 1 femoral endarteriectomy). On the other hand there were 7 endoluminal procedures (1 aortic, 4 iliac, 1 femoral and 1 popliteal). RESULTS: The mean follow-up was 97.3+/-50 months (range, from 34 to 216 months). Two patients died by 57 and 132 months respectively. At 5 years the survival rate was 94%; the primary patency rate was 59%; the secondary patency rate was 81% and the limb salvage rate was 94%. At 10 years these rates were respectively 94%, 44%, 54% and 75%. A total of 21 reoperations were performed. During follow-up 11 patients were better, 2 were stable and 4 were worse with 2 limbs lost. CONCLUSIONS: These bad results suggest keeping the surgical and endoluminal indications for patients younger than 40 years with threatened limbs.

Adult↗
PubMed1998

Physical interaction between components of DNA mismatch repair and nucleotide excision repair.

Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as "bait," and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes.

Base Pair Mismatch↗
PubMed1998

Orientation of cell adhesion and growth on patterned heterogeneous polystyrene surface.

Studies of neurite outgrowth or cell migration, two important processes in neuronal networks formation, are facilitated by cell culture models capable of orientating cellular growth and of designing a well-defined cellular pattern. Heterogeneous polystyrene surfaces composed of oxygen plasma-treated stripes (PSox) with a low hydrophobicity separated by non-treated areas (PS) have these properties. In this study, to guide cell growth, we developed a cell culture model using these supports and we identified the molecular factors involved in cellular orientation. When the heterogeneous supports were not coated, proteins from a serum culture medium were required for cells to line up on PSox. On the other hand, cell orientation on coated surfaces was clearly influenced by competitive adsorption of adhesive proteins such as fibronectin or collagen and anti-adhesive molecules as pluronic F68 or albumin. Attachment factors were adsorbed on PSox stripes while adsorption of anti-adhesive molecules on the most hydrophobic PS areas prevented cell adhesion or growth. Thus, we describe the preparation of a cell culture substrate that succeeded in orientating cell growth and that led to a line of cells on adhesive PSox stripes ranging from 2 to 100 microns width.

Animals↗
PubMed1998

[3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis.

The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.

Amino Acid Sequence↗
PubMed1998

Temperature-jump and potentiometric studies on recombinant wild type and Y143F and Y254F mutants of Saccharomyces cerevisiae flavocytochrome b2: role of the driving force in intramolecular electron transfer kinetics.

The kinetics of intramolecular electron transfer between flavin and heme in Saccharomyces cerevisiae flavocytochrome b2 were investigated by performing potentiometric titrations and temperature-jump experiments on the recombinant wild type and Y143F and Y254F mutants. The midpoint potential of heme was determined by monitoring redox titrations spectrophotometrically, and that of semiquinone flavin/reduced flavin (Fsq/Fred) and oxidized flavin (Fox)/Fsq couples by electron paramagnetic resonance experiments at room temperature. The effects of pyruvate on the kinetic and thermodynamic parameters were also investigated. At room temperature, pH 7.0 and I = 0.1 M, the redox potential of the Fsq/Fred, Fox/Fsq, and oxidized heme/reduced heme (Hox/Hred) couples were -135, -45, and -3 mV, respectively, in the wild-type form. Although neither the mutations nor excess pyruvate did appreciably modify the potential of the heme or that of the Fsq/Fred couple, they led to variable positive shifts in the potential of the Fox/Fsq couple, thus modulating the driving force that characterizes the reduction of heme by the semiquinone in the -42 to +88 mV range. The relaxation rates measured at 16 degreesC in temperature-jump experiments were independent of the protein concentrations, with absorbance changes corresponding to the reduction of the heme. Two relaxation processes were clearly resolved in wild-type flavocytochrome b2 (1/tau1 = 1500 s-1, 1/tau2 = 200 +/- 50 s-1) and were assigned to the reactions whereby the heme is reduced by Fred and Fsq, respectively. The rate of the latter reaction was determined in the whole series of proteins. Its variation as a function of the driving force is well described by the expression obtained from electron-transfer theories, which provides evidence that the intramolecular electron transfer is not controlled by the dynamics of the protein.

Amino Acid Substitution↗
PubMed1998

Molybdenum cofactor properties and [Fe-S] cluster coordination in Escherichia coli nitrate reductase A: investigation by site-directed mutagenesis of the conserved his-50 residue in the NarG subunit.

Most of the molybdoenzymes contain, in the amino-terminal region of their catalytic subunits, a conserved Cys group that in some cases binds an [Fe-S] cluster. In dissimilatory nitrate reductases, the first Cys residue of this motif is replaced by a conserved His residue. Site-directed mutagenesis of this residue (His-50) was performed on the NarG subunit from Escherichia coli nitrate reductase A. The results obtained by EPR spectroscopy enable us to exclude the implication of this residue in [Fe-S] binding. Additionally, we showed that the His-50 residue does not coordinate the molybdenum atom, but its substitution by Cys or Ser introduces a perturbation of the hydrogen bonding network around the molybdenum cofactor. From potentiometric studies, it is proposed that the high-pH and the low-pH forms of the Mo(V) are both involved during the redox turnover of the enzyme. Perturbation of the Mo(V) pKV value might be responsible for the low activity reported in the His-50-Cys mutant enzyme. A catalytic model is proposed in which the protonation/deprotonation of the Mo(V) species is an essential step. Thus, one of the two protons involved in the catalytic cycle could be the one coupled to the molybdenum atom in the dissimilatory nitrate reductase of E. coli.

Cell Membrane↗
PubMed1998

Evaluation of clinical criteria for diagnosis of bullous pemphigoid. French Bullous Study Group.

OBJECTIVE: To check the potential usefulness of clinical criteria for the diagnosis of bullous pemphigoid when state-of-the-art techniques such as Western immunoblotting, immunoprecipitation, and indirect immunofluorescence on salt-split skin or direct immunoelectron microscopy are not available. DESIGN: Comparison of the clinical criteria between 2 groups (with and without bullous pemphigoid) as defined by immunoelectron microscopy used as standard criterion, in a prospective study. Multivariate logistic regression analysis was carried out by including all items that were statistically significant (at P < .05 level) in univariate analysis. SETTING: Five dermatology departments in teaching hospitals. PATIENTS: The 231 patients studied had subepidermal autoimmune bullous diseases with linear IgG or C3 deposits in the basement membrane zone (157 with bullous pemphigoid, 33 with cicatricial pemphigoid, 30 with epidermolysis bullous acquisita, 5 with lupus erythematosus, and 6 others). A second set of patients was used to calculate predictive values. RESULTS: The multivariate logistic stepwise analysis resulted in a final set of predictors that included only 4 items: absence of atrophic scars, absence of head and neck involvement, absence of mucosal involvement, and age greater than 70 years. No additional variables met the .05 significance level to enter into the model. If 3 of these 4 characteristics were present, a diagnosis of bullous pemphigoid could be made with a sensitivity of 90% and a specificity of 83%; these predictive values were calculated on a sample of 70 new cases. CONCLUSIONS: With and estimated incidence of bullous pemphigoid among subepidermal autoimmune bullous diseases of 80%, the presence of 3 of the 4 significant criteria allows the diagnosis of bullous pemphigoid, with a positive predictive value of 95%. Our set of clinical criteria thus allows the diagnosis of bullous pemphigoid with good validity for both clinical practice and therapeutic trials.

Aged↗