Biomedical subjects

E Dufour

Publications and source records attributed to E Dufour.

At least 19 recordsLinked to original sources

PubMed2005

The effect of raw milk microbial flora on the sensory characteristics of Salers-type cheeses.

The sensory characteristics of Salers Protected Denomination of Origin raw-milk cheeses are linked to the biochemical composition of the raw material (milk) and to the resultant microbial community. To evaluate the influence of the microbial community on sensory characteristics, Salers-type cheeses were manufactured with the same pasteurized milk, reinoculated with 3 different microbial communities from 3 different filtrates from microfiltered milks. Each cheese was subjected to microbial counts (on selective media), biochemical tests, and volatile and sensory component analyses at different times of ripening. Adding different microbial communities to specimens of the same (biochemically identical) pasteurized milk lead to different sensory characteristics of the cheeses. Cheeses with fresh cream, hazelnut, and caramel attributes were opposed to those with fermented cream, chemical, and garlic flavors. The aromatic compounds identified (esters, acids, alcohols, and aldehydes) in these cheeses were quite similar. Nevertheless, one milk was distinguished by a higher content of acetoin, and lower 2-butanone and 3-methylpentanone concentrations. Over the production period of 1 mo, the different cheeses were characterized by the same balance of the microbial population assessed by microbial counts on different media. This was associated with the stability of some sensory attributes describing these cheeses. Nevertheless, there was no linear correlation between microbial flora data and sensory characteristics as measured in this study.

Acetoin↗

PubMed2003

Composition and properties of the soluble organic matrix of the otolith of a marine fish: Gadus morhua Linne, 1758 (Teleostei, Gadidae).

The soluble matrix of the sagittal otolith of the cod Gadus morhua was analyzed using UV and IR spectroscopy, liquid chromatography and electrophoresis. This matrix is a complex mixture of proteins and glycoproteins, with a large range of molecular weights. High weights (>1000 kDa) are shown for the first time in water-soluble matrix of otolith. However, the 2D denaturing electrophoresis and large range of sorting used in high performance liquid chromatography columns do not separate the soluble matrix to well-defined molecular weights. The IR data indicate that several conformations are present and the main part of the sugars is not sulfated. Additionally, electrophoresis data show that the acidity of the sugar components is higher than that of the proteins. Despite the relative scarcity of literature data, our study of G. morhua suggests that the chemical composition of otolith soluble organic matrix may differ among species.

Animals↗

PubMed2003

Evaluation of standardized tasks for primary care physicians using the MOXXI electronic prescribing and integrated drug management system.

The Medical Office for the Twenty First Century (MOXXI) is a research project testing the potential benefits of an electronic prescription and drug management system for primary care physicians. This system includes a dynamic electronic pad for prescription entry with fields for treatment indications; a drug profiler with a graphic representation of the list of prescription medications purchased in the last year; a refill compliance calculator; dates of emergency room visits and hospital admissions; cost of drugs dispensed; and an alert system that detects interactions among drugs, treatment duplications, and contraindications with certain allergies or specific diseases. One concern expressed by physicians that could influence uptake and acceptability is the increased time that may be required to use the system. User abilities are a factor in this process, as well as user interface, user training and system speed.

Clinical Pharmacy Information Systems↗

PubMed2001

Experimental discovery of charge-exchange-caused dips in spectral lines from laser-produced plasmas.

We report the first experimental observation of charge-exchange-caused dips (also called x dips) in spectral lines of multicharged ions in laser-produced plasmas. Specifically, in the process of a laser irradiation of targets made out of aluminum carbide, we observed two x dips in the Ly(gamma) line of Al XIII perturbed by fully stripped carbon. From the practical point of view, this opens up a way to experimentally produce not-yet-available fundamental data on charge exchange between multicharged ions, virtually inaccessible by other experimental methods. From the theoretical viewpoint, the results are important because the x dips are the only one signature of charge exchange in profiles of spectral lines emitted by plasmas and they are the only one quasimolecular phenomenon that could be observed at relatively "low" densities of laser-produced plasmas.

Journal Article↗

PubMed2001

The Composition of the Milk Fat Globule Surface Alters the Structural Characteristics of the Coagulum.

The effects of the composition of a fat globule surface in reconstituted milks on the properties of rennet-induced coagulums were studied by rheological measurements and by front-face fluorescence spectroscopy in combination with a multivariate statistical method to investigate, at a molecular level, the evolution of the structure during the milk coagulation process. Reconstituted milks used in this study were prepared from different fat-in-water emulsions stabilized by whole skim-milk proteins, beta-casein, or beta-lactoglobulin. Coagulation of milk reconstituted with natural fat globules was also investigated. The study showed that the fat droplet/water interface influences the rheological properties (G' modulus) of the reconstituted milks during the coagulation process. The tryptophan fluorescence emission spectra of proteins were recorded during the kinetics of coagulation. The results of the principal component analysis performed on the spectral data showed a discrimination in the different systems investigated. It was shown that the fluorescence properties of protein tryptophans and, consequently, the structures of the protein networks were different for the investigated systems. The development of fluorescence transfer between protein tryptophans and fat-globule vitamin A during the coagulation kinetics agreed with the interactions between the protein network and fat globules. Copyright 2001 Academic Press.

Journal Article↗

PubMed2001

Assessment of the contaminant concentration variability among Lake Geneva Arctic char using stable isotopic composition (delta15N and delta13C).

Measurements of organochlorine [polychlorinated biphenyls (PCBs) and dichloro-diphenyl-dichloroethylene (DDE)] and Hg concentrations and nitrogen and carbon stable isotopic compositions (delta15N and delta13C) were performed on 63 Arctic char (Salvelinus alpinus) from Lake Geneva. Fish exhibited a high interindividual variablity in pollutant concentrations. Since the accumulation of such persistent contaminants is obtained from food, the co-occurrence of dietary differentiation leading to the contaminant interindividual variability was suspected. delta15N and delta13C were used for assessing trophic position and food source differences among Arctic char. The low ranges of delta15N and delta13C could not explain the interindividual variability in pollutant concentrations. The lack of relation between delta15N and contaminant concentration did not suggest a trophic level biomagnification of PCB, DDE, and Hg. Lake Geneva spatial variability in pollutants may be an important factor of variability within the Arctic char population. The bioaccumulation pattern occurring for Hg was not apparent for PCB and DDE. Organochlorines are hydrophobic contaminants, and their bioaccumulation pattern may be masked by seasonal variations in fish lipid content.

Animals↗

PubMed2001

Overexpression of the alternative oxidase restores senescence and fertility in a long-lived respiration-deficient mutant of Podospora anserina.

Several lines of evidence have implicated reactive oxygen species (ROS) in the pathogenesis of various degenerative diseases and in organismal ageing. Furthermore, it has been shown recently that the alternative pathway respiration present in plants lowers ROS mitochondrial production. An alternative oxidase (AOXp) also occurs in the filamentous fungus Podospora anserina. We show here that overexpression of this oxidase does not decrease ROS production and has no effect on longevity, mitochondrial stability or ageing in this fungus. In the same way, inactivation of the gene has no effect on these parameters. In contrast, overexpression of the alternative oxidase in the long-lived cox5::BLE mutant, deficient in cytochrome c oxidase, considerably increases ROS production of the mutant. It rescues slow growth rate and female sterility, indicating an improved energy level. This overexpression also restores senescence and mitochondrial DNA instability, demonstrating that these parameters are controlled by the energy level and not by the expression level of the alternative oxidase. We also suggest that expression of this oxidase in organisms naturally devoid of it could rescue respiratory defects resulting from cytochrome pathway dysfunctions.

Base Sequence↗

PubMed2000

An aspartic acid residue in TPR-1, a specific region of protein-priming DNA polymerases, is required for the functional interaction with primer terminal protein.

A multiple sequence alignment of eukaryotic-type DNA polymerases led to the identification of two regions of amino acid residues that are only present in the group of DNA polymerases that make use of terminal proteins. (TPs) as primers to initiate DNA replication of linear genomes. These amino acid regions (named terminal region (TPR protein-1 and TPR-2) are inserted between the generally conserved motifs Dx(2)SLYP and Kx(3)NSxYG (TPR-1) and motifs Kx(3)NSxYG and YxDTDS (TPR-2) of the eukaryotic-type family of DNA polymerases. We carried out site-directed mutagenesis in two of the most conserved residues of phi29 DNA polymerase TPR-1 to study the possible role of this specific region. Two mutant DNA polymerases, in conserved residues AsP332 and Leu342, were purified and subjected to a detailed biochemical analysis of their enzymatic activities. Both mutant DNA polymerases were essentially normal when assayed for synthetic activities in DNA-primed reactions. However, mutant D332Y was drastically affected in phi29 TP-DNA replication as a consequence of a large reduction in the catalytic efficiency of the protein-primed reactions. The molecular basis of this defect is a non-functional interaction with TP that strongly reduces the activity of the DNA polymerase/TP heterodimer.

Amino Acid Motifs↗

PubMed2000

Gelation by phase separation in a whey protein system: in-situ kinetics of aggregation.

The aggregation and gelation properties of beta-lactoglobulin (BLG), a globular protein from milk, was studied in aqueous ethanol solutions at room temperature. The phase state diagrams as a function of pH and ethanol concentration showed that a gel structure appeared after a period ranging from 1 min to 1 week, depending on the physico-chemical conditions. The in-situ kinetics of aggregation were followed by several methods in order to obtain a better understanding of the building of aggregates by the addition of ethanol. It was shown that the aggregation kinetics highly depended upon the pH, the process being fastest at pH 7. Viscoelasticity and infrared measurements indicated that alcohol-induced gelation would proceed via a two-step mechanism: small aggregates loosely connected between them were first built up; a real network took place in a second step. The coarse and irregular structures formed in aqueous ethanol gels revealed by confocal laser scanning microscopy could be analysed in terms of a phase separation. This observation was supported by a syneresis phenomenon visible in the final gel state. BLG in water-ethanol solution would undergo either an inhibition of the demixing by gelation or a binary phase separation accompanied by an irreversible gelation transition.

Ethanol↗

PubMed2000

A causal link between respiration and senescence in Podospora anserina.

Senescence, a progressive degenerative process leading to age-related increase in mortality, is found in most eukaryotes. However, the molecular events underlying aging remain largely unknown. Understanding how longevity is regulated is a fundamental problem. Here we demonstrate that the respiratory function is a key factor that contributes to shortening lifespan of the filamentous fungus Podospora anserina. In this organism, senescence is systematically associated with mitochondrial DNA instabilities. We show that inactivation of the nuclear COX5 gene encoding subunit V of the cytochrome c oxidase complex leads to the exclusive use of the alternative respiratory pathway and to a decrease in production of reactive oxygen species. This inactivation results in a striking increase of longevity associated with stabilization of the mitochondrial chromosome. Moreover, accumulation of several senescence-specific mitochondrial DNA molecules is prevented in this nuclear mutant. These findings provide direct evidence of a causal link between mitochondrial metabolism and longevity in Podospora anserina.

Base Sequence↗

PubMed2000

Oil/Alkanethiol Layers for the Study of Emulsified Protein Conformation by Surface Plasmon Resonance Using Monoclonal Antibodies.

A method combining surface plasmon resonance and epitope mapping was developed to study the protein conformation at the oil/water interface of an emulsion. The conformation of beta-lactoglobulin stabilizing dodecane/water and miglyol/water interfaces was investigated using five anti-beta-lactoglobulin monoclonal antibodies. The developed method allows us to specifically recognize the emulsified beta-lactoglobulin at the surface of a sensor chip with good repeatability; i.e., standard deviations range between 0.7 and 3.6%. Considering that the monoclonal antibodies, recognizing conformational epitopes, still bind to beta-lactoglobulin at oil/water interfaces, it is concluded that the protein retains a globular conformation. It is shown that the inhibition-binding values of two pairs of Mabs are different for beta-lactoglobulin stabilizing dodecane/water and miglyol/water interfaces. This indicates that the conformations of emulsified beta-lactoglobulin are slightly different according to the nature of the oil phase. Copyright 2000 Academic Press.

Journal Article↗

PubMed1999

Tyrosine 319 in the interdomain B of ZAP-70 is a binding site for the Src homology 2 domain of Lck.

T-cell antigen receptor-induced signaling requires both ZAP-70 and Lck protein-tyrosine kinases. One essential function of Lck in this process is to phosphorylate ZAP-70 and up-regulate its catalytic activity. We have previously shown that after T-cell antigen receptor stimulation, Lck binds to ZAP-70 via its Src homology 2 (SH2) domain (LckSH2) and, more recently, that Tyr319 of ZAP-70 is phosphorylated in vivo and plays a positive regulatory role. Here, we investigated the possibility that Tyr319 mediates the SH2-dependent interaction between Lck and ZAP-70. We show that a phosphopeptide encompassing the motif harboring Tyr319, YSDP, interacted with LckSH2, although with a lower affinity compared with a phosphopeptide containing the optimal binding motif, YEEI. Moreover, mutation of Tyr319 to phenylalanine prevented the interaction of ZAP-70 with LckSH2. Based on these results, a gain-of-function mutant of ZAP-70 was generated by changing the sequence Y319SDP into Y319EEI. As a result of its increased ability to bind LckSH2, this mutant induced a dramatic increase in NFAT activity in Jurkat T-cells, was hyperphosphorylated, and displayed a higher catalytic activity compared with wild-type ZAP-70. Collectively, our findings indicate that Tyr319-mediated binding of the SH2 domain of Lck is crucial for ZAP-70 activation and consequently for the propagation of the signaling cascade leading to T-cell activation.

Amino Acid Sequence↗

PubMed1999

Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling.

Following T cell antigen receptor (TCR) engagement, the protein tyrosine kinase (PTK) ZAP-70 is rapidly phosphorylated on several tyrosine residues, presumably by two mechanisms: an autophosphorylation and a trans-phosphorylation by the Src-family PTK Lck. These events have been implicated in both positive and negative regulation of ZAP-70 activity and in coupling this PTK to downstream signaling pathways in T cells. We show here that Tyr315 and Tyr319 in the interdomain B of ZAP-70 are autophosphorylated in vitro and become phosphorylated in vivo upon TCR triggering. Moreover, by mutational analysis, we demonstrate that phosphorylation of Tyr319 is required for the positive regulation of ZAP-70 function. Indeed, overexpression in Jurkat cells and in a murine T cell hybridoma of a ZAP-70 mutant in which Tyr319 was replaced by phenylalanine (ZAP-70-Y319F) dramatically impaired anti-TCR-induced activation of the nuclear factor of activated T cells and interleukin-2 production, respectively. Surprisingly, an analogous mutation of Tyr315 had little or no effect. The inhibitory effect of ZAP-70-Y319F correlated with a substantial loss of its activation-induced tyrosine phosphorylation and up-regulation of catalytic activity, as well as with a decreased in vivo capacity to phosphorylate known ZAP-70 substrates, such as SLP-76 and LAT. Collectively, our data reveal the pivotal role of Tyr319 phosphorylation in the positive regulation of ZAP-70 and in TCR-mediated signaling.

Amino Acid Sequence↗

PubMed1999

Cross-linking localization of a HIV-1 reverse transcriptase peptide involved in the binding of primer tRNALys3.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) initiates the synthesis of DNA from the 3' end of its specific primer, tRNALys3. The regions of tRNALys3 in close contact with RT are well known, while a precise knowledge of the RT regions interacting with tRNALys3 is not yet available. To address this question we cross-linked the heterodimeric p66/p51 RT to tRNALys3 using cis-aquahydroxydiammino-platinum. Ribonucleoprotein complexes of molecular masses higher than the p66 subunit were obtained. After RNase A digestion of the RT-tRNA complex, a labeled oligoribonucleotide (ORN) was mainly found associated to the p66 subunit. This labeled p66-ORN complex was then proteolyzed with Staphylococcus aureus V8 protease. A highly purified radioactive peptide was obtained after two chromatographic purification steps. Its N-terminal sequence corresponded with amino acid residues 241VQPI244. Using the crystallographic structure of HIV-1 RT, this peptide was localized at the beta14-sheet end, near to the hairpin formed by beta12 and beta13-sheets ("primer grip") and the alphaH-helix. The so called "VQPI peptide" is in the border of the thumb and the palm subdomains of the p66 subunit. This study palliates the absence of a three- dimensional structure of the RT-tRNA complex and led to a peptide in interaction with tRNALys3 present in all HIV-1 RT isolates.

Amino Acid Sequence↗

PubMed1999

Structural investigation of beta-lactoglobulin gelation in ethanol/water solutions.

The aggregation and gelation properties of beta-lactoglobulin (BLG), a globular protein from milk, was studied in hydro-ethanolic solutions (50/50% (v/v)) at room temperature. The phase state diagrams as a function of pH and ethanol concentration showed that a gel structure appeared after a period ranging from 1 min to 1 week depending on the physico-chemical conditions. The aggregation kinetics, studied by infrared spectroscopy and dynamical rheological measurements, highly depended upon the pH; the process being the fastest at pH 7. Alcohol-induced aggregation of BLG was characterized by the formation of intermolecular hydrogen bonded beta-sheet structures. Small angle neutron scattering indicated that the aggregates structures in the final gels were similar at pH 7, 8 and 9. Through the data obtained at the molecular and macroscopic levels, it can be concluded that the kinetics of gelation were pH dependent while the spatial arrangements of the aggregates were similar in the final structures. The heterogeneous structures formed in hydro-ethanolic gels could be analysed in terms of a phase separation, the syneresis being the final visible state.

Ethanol↗

PubMed1999

Mitochondrial group II introns, cytochrome c oxidase, and senescence in Podospora anserina.

Podospora anserina is a filamentous fungus with a limited life span. It expresses a degenerative syndrome called senescence, which is always associated with the accumulation of circular molecules (senDNAs) containing specific regions of the mitochondrial chromosome. A mobile group II intron (alpha) has been thought to play a prominent role in this syndrome. Intron alpha is the first intron of the cytochrome c oxidase subunit I gene (COX1). Mitochondrial mutants that escape the senescence process are missing this intron, as well as the first exon of the COX1 gene. We describe here the first mutant of P. anserina that has the alpha sequence precisely deleted and whose cytochrome c oxidase activity is identical to that of wild-type cells. The integration site of the intron is slightly modified, and this change prevents efficient homing of intron alpha. We show here that this mutant displays a senescence syndrome similar to that of the wild type and that its life span is increased about twofold. The introduction of a related group II intron into the mitochondrial genome of the mutant does not restore the wild-type life span. These data clearly demonstrate that intron alpha is not the specific senescence factor but rather an accelerator or amplifier of the senescence process. They emphasize the role that intron alpha plays in the instability of the mitochondrial chromosome and the link between this instability and longevity. Our results strongly support the idea that in Podospora, "immortality" can be acquired not by the absence of intron alpha but rather by the lack of active cytochrome c oxidase.

Aging↗

PubMed1998

Conformation of beta-Lactoglobulin at an Oil/Water Interface as Determined from Proteolysis and Spectroscopic Methods.

The rates of appearance of tryptic peptides following the hydrolysis of beta-lactoglobulin in solution or adsorbed at the oil/water interface of an emulsion were investigated as a function of time. It was also shown using hydrophobic labeling that the region 15-40 of beta-lactoglobulin was in the oil phase. The fluorescence results suggested that the conformation of beta-lactoglobulin was modified upon adsorption at the oil/water interface and that at least one tryptophan in adsorbed beta-lactoglobulin was in a more hydrophobic environment. The data obtained by circular dichroism in the peptidic region indicated that the adsorbed beta-lactoglobulin was characterized by a higher content in alpha-helix than the protein in solution. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

Synthesis of amidrazones using an engineered papain nitrile hydratase.

To demonstrate the usefulness of an engineered papain nitrile hydratase as a biocatalyst, a peptide amidrazone was prepared by incubation of the nitrile MeOCO-Phe-Alanitrile with the Gln19Glu papain mutant in the presence of salicylic hydrazide as a nucleophile. The amidrazone results from nucleophilic attack by salicylic hydrazide at the imino carbon of the thioimidate adduct formed between the enzyme and the peptide nitrile substrate. Compared to wild-type enzyme, the engineered nitrile hydratase causes a better than 4000-fold increase in the rate of amidrazone formation and yields a product of much higher purity. The advantages over other nitrile-hydrolyzing enzymes and current limitations of the papain nitrile hydratase are discussed.

Catalysis↗