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T Rabilloud

Publications and source records attributed to T Rabilloud.

At least 19 recordsLinked to original sources
PubMed2002

Two-dimensional electrophoresis of proteins discriminates aphid clones of Sitobion avenae differing in BYDV-PAV transmission.

Aset of 39 F1 Sitobion avenae clones was obtained by selfing a poorly efficient BYDV-PAV vector clone. These clones were genetically typed by 11 microsatellite loci, and tested for BYDV-PAV4 transmission to barley. The 39 clones displayed a continuum in transmission percentages, from 0% to 88% with a significant clone effect. From this set, two highly efficient (HEV) and two poorly efficient (PEV) vectoring clones were more precisely characterized for transmission of two other PAV isolates. The molecular bases of the lower transmissibility of BYDV-PAV4 by PEV clones and of the aphid vectoring properties were investigated respectively by comparing the sequences corresponding to structural proteins (CP and RTD) of BYDV, and by using proteomic analysis of aphids in two dimensional electrophoresis (2-DE) with immobilized pH gradients (IPG) after an improved protein extraction. Four residues specific to BYDV-PAV4 located in the CP sequence (A(24) and L(130)) or in the RTD region (M(334) and S(456)) could be responsible for the lower transmissibility of this isolate by PEV clones. Among a total of 2150 well-resoluted spots scored on S. avenae proteinic pattern, only twelve proteins were qualitatively or quantitatively different between clones. Four out of them discriminated HEV and PEV groups.

Animals↗
PubMed2001

A comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels.

A comparison between two fluorescent metal chelates for staining proteins separated by electrophoresis has been carried out. One of these chelates is ruthenium II tris (bathophenanthroline disulfonate) and the other is commercial Sypro Ruby. Both can be efficiently detected either with UV tables or with commercial laser fluorescence scanners. The sensitivity and homogeneity of the stains and the interference with mass spectrometry analysis have been investigated. It appears that both stains perform similarly for protein detection, while ruthenium II tris (bathophenanthroline disulfonate) performs better for mass spectrometry analyses and as cost-effectiveness ratio. However, Sypro Ruby is easier to use as a stain.

ATP Synthetase Complexes↗
PubMed2001

A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis.

We report on a new silver stain especially developed for staining large gels (25 cm x 20 cm) from the Hoefer ISO-DALT system for matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of proteins. The staining protocol can be summarized as follows: the gels are sensitised in tetrathionate/potassium acetate solution and washed several times in distilled water. After impregnation with silver nitrate, the silver is reduced in the presence of potassium carbonate, thiosulphate and formaldehyde. The staining procedure is stopped with Tris/acetate after which the gels are rinsed and stored in water before spot picking for MALDI-TOF analysis is performed. This protocol has several advantages over existing ones. The gels are stained in a new apparatus that reduces gel handling to a minimum thus also reducing the contamination with keratins to a minimum. The development times in potassium carbonate are very long (up to 40 min) thus improving batch-to-batch reproducibility. Only the surface of the proteins is stained and the silver can be oxidized, thereafter MALDI-TOF can be performed with protein loads as little as 100 micrograms per gel.

Cell Line↗
PubMed2001

The mitochondrial antioxidant defence system and its response to oxidative stress.

The antioxidant systems of mitochondria are not well known. Using a proteomics-based approach, we defined these mitochondrial antioxidant systems and analyzed their response to oxidative stress. It appears that the major mitochondrial antioxidant system is made of manganese superoxide dismutase on the one hand, and of peroxiredoxin III, mitochondrial thioredoxin and mitochondrial thioredoxin reductase on the other hand. With the exception of thioredoxin reductase, all these proteins are induced by oxidative stress. In addition, a change in the peroxiredoxin III pattern can also be observed.

Animals↗
PubMed2001

Gene induction during differentiation of human monocytes into dendritic cells: an integrated study at the RNA and protein levels.

Changes in gene expression occurring during differentiation of human monocytes into dendritic cells were studied at the RNA and protein levels. These studies showed the induction of several gene classes corresponding to various biological functions. These functions encompass antigen processing and presentation, cytoskeleton, cell signalling and signal transduction, but also an increase in mitochondrial function and in the protein synthesis machinery, including some, but not all, chaperones. These changes put in perspective the events occurring during this differentiation process. On a more technical point, it appears that the studies carried out at the RNA and protein levels are highly complementary.

Cell Differentiation↗
PubMed2001

Comparison of the PR mutant with the wild-type strain of proteus mirabilis brings insight into peroxide resistance factors and regulation of catalase expression.

The peroxide resistant mutant (PR) of Proteus mirabilis was characterized by an increased constitutive catalase activity concomitant with a large production of specific mRNA. Survival toward hydrogen peroxide during exponential phase was increased by H2O2 pretreatment in the wild type but not in the mutant, although the catalase of both strains was not inducible under these conditions. In the mutant, besides catalase, over-produced proteins comprised two different alkyl hydroperoxide reductase subunit C (AhpC) proteins and a protein homologous to the stationary phase transcription factor SspA of Escherichia coli. Conversely, the flagellin A (FlaA) of P. mirabilis was repressed in the PR mutant. Genomic DNA fragments of 2.9 kb carrying the catalase gene (katA) together with the 5' and 3' flanking regions were isolated from both strains and found to be identical. Upstream of katA, a Fur box-like sequence was found, but surprisingly, restricting iron in the culture medium caused a decrease in catalase production. The PR mutant presents similarities with other peroxide resistant mutants, but the regulation of catalase biosynthesis in P. mirabilis seems somewhat different from other close species such as E. coli.

Adhesins, Bacterial↗
PubMed2000

Membrane proteins and proteomics: un amour impossible?

Proteome analysis implies the ability to separate proteins as a first step prior to characterization. Thus, the overall performance of the analysis strongly depends on the performance of the separation tool, usually two-dimensional electrophoresis. This review shows how two-dimensional electrophoresis performs with membrane proteins from bacteria or animal or vegetable cells and tissues, the recent progress in this field, and it examines future prospects in this area.

Animals↗
PubMed2000

Membrane proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties.

Recent efforts at the proteomic level were employed to describe the protein equipment of the plasma membrane of the model plant Arabidopsis thaliana. These studies had revealed that the plasma membrane is rich in extrinsic proteins but came up against two major problems: (i) few hydrophobic proteins were recovered in two-dimensional electrophoresis gels, and (ii) many plasma membrane proteins had no known function or were unknown in the database despite extensive sequencing of the Arabidopsis genome. In this paper, several methods expected to enrich a membrane sample in hydrophobic proteins were compared. The optimization of solubilization procedures revealed that the detergent to be used depends on the lipid content of the sample. The corresponding proteomes were compared with the statistical model AMMI (additive main effects with multiplicative interaction) that aimed at regrouping proteins according to their solubility and electrophoretic properties. Distinct groups emerged from this analysis and the identification of proteins in each group allowed us to assign specific features to several of them. For instance, two of these groups regrouped very hydrophobic proteins, one group contained V-ATPase subunits, another group contained proteins with one transmembrane domain as well as proteins known to interact with membrane proteins. This study provides methodological tools to study particular classes of plasma membrane proteins and should be applicable to other cellular membranes.

Amino Acid Sequence↗
PubMed2000

Proteomic analysis of the Escherichia coli outer membrane.

Outer membrane proteins (OMPs) of Gram-negative bacteria are key molecules that interface the cell with the environment. Traditional biochemical and genetic approaches have yielded a wealth of knowledge relating to the function of OMPs. Nonetheless, with the completion of the Escherichia coli genome sequencing project there is the opportunity to further expand our understanding of the organization, expression and function of the OMPs in this Gram-negative bacterium. In this report we describe a proteomic approach which provides a platform for parallel analysis of OMPs. We propose a rapid method for isolation of bacterial OMPs using carbonate incubation, purification and protein array by two-dimensional electrophoresis, followed by protein identification using mass spectrometry. Applying this method to examine E. coli K-12 cells grown in minimal media we identified 21 out of 26 (80%) of the predicted integral OMPs that are annotated in SWISS-PROT release 37 and predicted to separate within the range of pH 4-7 and molecular mass 10-80 kDa. Five outer membrane lipoproteins were also identified and only minor contamination by nonmembrane proteins was observed. Importantly, this research readily demonstrates that integral OMPs, commonly missing from 2D gel maps, are amenable to separation by two-dimensional electrophoresis. Two of the identified OMPs (YbiL, YeaF) were previously known only from their ORFs, and their identification confirms the cognate genes are transcribed and translated. Furthermore, we show that like the E. coli iron receptors FhuE and FhuA, the expression of YbiL is markedly increased by iron limitation, suggesting a putative role for this protein in iron transport. In an additional demonstration we show the value of parallel protein analysis to document changes in E. coli OMP expression as influenced by culture temperature.

Bacterial Outer Membrane Proteins↗
PubMed1999

Analysis of membrane proteins by two-dimensional electrophoresis: comparison of the proteins extracted from normal or Plasmodium falciparum-infected erythrocyte ghosts.

Parasite-encoded membrane proteins translocated to the surface of infected erythrocytes or in specialized vesicles underneath (Maurer's clefts) play a key role in the asexual life cycle of Plasmodium falciparum (a malaria-causing protozoan), by mediating key steps such as red blood cell invasion, sequestration of infected cells in microcapillaries, and red blood cell rupture. A large-scale analysis of these membrane proteins would therefore be of great help to gain knowledge of the different stages of the Plasmodium falciparum life cycle. In order to be able to detect and identify parasite-encoded proteins directed to the red blood cell membrane, we first defined the conditions required for optimal extraction and separation of normal red blood cell ghost proteins by two-dimensional gel electrophoresis. These conditions included the use of urea, thiourea and new zwitterionic detergents in the extraction and isoelectric focusing media. The optimized conditions were then applied to analyze normal and P. falciparum-infected red blood cell ghosts. Several protein spots were found only in infected ghosts and are expected to represent parasite-encoded proteins. These proteins are currently under investigation.

Animals↗
PubMed1999

Large scale characterization of plant plasma membrane proteins.

After a brief review of the strategies used to date to identify systematically plasma membrane (PM) proteins, emphasis was given to the proteomic approach of PM proteins from the model plant Arabidopsis thaliana. Comparative analysis of two-dimensional gels from PM and cytosolic fractions was used to assess the cellular origin of proteins found in PM fraction. The classification obtained was confirmed by protein sequencing that showed, in addition, that most analyzed proteins were peripheral proteins. A large proportion of these appeared to correspond to PM-constitutive proteins that were present in the PM from different plant organs, but were not uniquely located at the PM depending on the organ. In addition, the presence of organ-specific sets of PM-specific proteins was also demonstrated. Additional procedures were developed to identify integral PM proteins. The combined use of PM washes with alkaline carbonate buffer or Triton X-100/KBr, and of a new detergent to solubilize protein, resulted in improved recovery of hydrophobic proteins on gels. Results are discussed in terms of construction of comprehensive proteomes for PM and other membranes and organelles.

Animals↗
PubMed1999

CK2alpha-protein phosphatase 2A molecular complex: possible interaction with the MAP kinase pathway.

Despite its wide range of known substrates, the signaling function of protein kinase CK2 is still enigmatic. Mounting evidence suggests that CK2alpha, the catalytic subunit of holoenzymic CK2, may exist free of its usual regulatory partner CK2beta, raising the possibility that 'free' CK2alpha has regulation and function distinct from those of the holoenzyme. We previously reported that CK2alpha could bind to the core dimer of protein phosphatase 2A, and indirectly cause down-regulation of the PP2A substrate MEK1, possibly via activation of PP2A and/or targeting of PP2A to some element of the Ras/Raf/MEK pathway. Here, these results are confirmed and extended. By using transfection experiments and immune kinase assays, we show that endogenous PP2Ac and CK2beta are the only major substrates associating with epitope-tagged CK2alpha, and that expression of activated Raf results in disruption of the CK2alpha-PP2A association. Such disruption might be a necessary step for maximal activation of the MAP kinase pathway by Raf. In keeping with this idea, overexpression ofCK2alpha dose-dependently inhibits the mitogen-induced activation of cotransfected, epitope-tagged MAP kinase. We suggest that the CK2beta free form of CK2alpha is both a target and a regulator of Raf/MAPK signaling.

3T3 Cells↗
PubMed1998

Interactions of non-detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation.

Non-detergent sulfobetaines (NDSB) are a family of solubilizing and stabilizing agents for proteins. In a previous study [Goldberg, M. E., Expert-Bezancon, N., Vuillard, L. & Rabilloud, T. (1996) Folding & Design 1, 21-27] we showed that the amount of active protein recovered in in vitro folding experiments could be significantly increased by some NDSBS. In this work we investigated the mechanisms by which these molecules facilitate protein renaturation. Stopped-flow and manual-mixing fluorescence and enzyme activity measurements were used to compare the kinetics of protein folding in the presence and absence of N-phenyl-methyl-N,N-dimethylammonium-propane-sulfonate (NDSB 256). Hen lysozyme and the beta2 subunit of Escherichia coli tryptophan synthase were chosen as model systems since their folding pathways had been previously investigated in detail. It is shown that, massive aggregation of tryptophan synthase occurs within less than 2.5 s after dilution in the renaturation buffer, but can be prevented by NDSB 256; only very early folding phases (such as the formation of a loosely packed hydrophobic core able to bind 8-anilino-1-naphthalenesulphonic acid, and the initial burying of tryptophan 177) are significantly altered by NDSB 256; none of the later phases is affected. Furthermore, NDSB 256 did not significantly affect any of the kinetic phases observed during the refolding of denatured lysozyme retaining intact disulphide bonds. This shows that NDSB 256 only interferes with very early steps in the folding process and acts by limiting the abortive interactions that could lead to the formation of inactive aggregates.

Anilino Naphthalenesulfonates↗
PubMed1998

Non-detergent sulphobetaines enhance the recovery of membrane and/or cytoskeleton-associated proteins and active proteases from erythrocytes infected by Plasmodium falciparum.

A better understanding of the causative agent's biology and the definition of new targets for the development of drugs and/or specific immune responses is necessary to face the spred of drug-resistant malaria in developing countries and the absence of an efficient vaccine against this most important infectious disease. Non-detergent sulphobetaines enhance the recovery and isoelectric focussing of active Plasmodium falciparum proteases, cytoskeleton-associated proteins and Maurer's cleft-associated proteins. This is a significant advantage for the purification of such proteins and might help pinpoint their role for red blood cell rupture and merozoite release.

Animals↗