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T Rabilloud

Publications and source records attributed to T Rabilloud.

At least 55 records · Page 3Linked to original sources
PubMed1995

Identification of major proteins associated with Dictyostelium discoideum endocytic vesicles.

Magnetic isolation of endocytic vesicles from Dictyostelium discoideum was accomplished after feeding the amoebae with iron oxide particles. Proteins associated with the endocytic vesicles were resolved by SDS-PAGE and digested 'in-gel' with endoproteinase Lys-C or Asp-N to generate peptides for amino acid sequencing. This strategy allowed the identification of the major protein constituents of the vesicles: namely, the A, B, D, E and 110 kDa subunits of a vacuolar type H(+)-ATPase, actin, a Rab 7-like GTPase, a p34 protein corresponding to a new cysteine proteinase and the 25 kDa product of a recently sequenced D. discoideum open reading frame.

Amino Acid Sequence↗
PubMed1995

Induction of stathmin expression during erythropoietic differentiation.

Stathmin is a M(r) 19,000 cytosolic protein proposed to act as a relay for signal-activating pathways regulating cell proliferation and differentiation. In a study of erythropoietic differentiation, stathmin was detected as a protein that is induced during the early stages of differentiation in several cellular model systems. The unphosphorylated form of stathmin was most prominently induced, which suggests that this form does not only play a role in pathways oriented toward cell proliferation, as is the case for lymphocytic systems, but may also play a role in various differentiation pathways.

Amino Acid Sequence↗
PubMed1995

Non-detergent sulphobetaines: a new class of molecules that facilitate in vitro protein renaturation.

Backgound. Attempts to renature proteins often yield aggregates rather than native protein. To minimize aggregation, low protein concentrations and/or solubilizing agents are used. Here, we test new solubilizing molecules, non-detergent sulphobetaines, to improve the renaturation of two very different enzymes, hen egg white lysozyme and bacterial beta-D-galactosidase. Results. The renaturation was conducted in the presence of five different sulphobetaines and the yield of active enzyme was measured. The five sulphobetaines improved the yield of native lysozyme up to 12-fold. Some sulphobetaines improved the yield of galactosidase up to 80-fold, but one reduced it 100-fold. Conclusion. Non-detergent sulphobetaines strongly affect the balance between aggregation and folding. Their effect depends on their structure and on their interactions with folding intermediates. These results should serve as a basis for designing more efficient sulphobetaines; for designing improved renaturation protocols using existing sulphobetaines; and for characterizing folding intermediates that interact with sulphobetaines.

Journal Article↗
PubMed1994

A new additive for protein crystallization.

The potential usefulness of the new zwitterionic solubilizing agent, dimethyl ethylammonium propane sulfonate (NDSB195), in protein crystallization was shown using hen egg-white lysozyme. In the presence of this agent, highly diffracting crystals were obtained using ammonium sulphate as a precipitant, whereas in its absence only amorphous precipitates were obtained. The crystals possess a triclinic unit cell not previously described and diffract to a resolution of 2 A. To ascertain that the new reagent had not produced significant changes in the protein fold the structure was determined to a resolution of 2.6 A. Only minor differences were observed (notably in regions of crystal contacts) with the known tetragonal lysozyme structure (Brookhaven Protein Data Bank entry 1HEL).

Crystallization↗
PubMed1994

Relationship between core histone acetylation and histone H1(0) gene activity.

In this study we show a striking correlation between histone H1(0) gene expression and histone acetylation. Trichostatin A, a highly specific inhibitor of histone deacetylase, efficiently induces H1(0) gene expression. Moreover, using a cell line sensitive to trichostatin A (FM3A) and a derived cell line selected for its resistance to this inhibitor (TR303), it is shown that the level of H1(0) gene expression is related to the extent of chromatin acetylation. After showing the S-phase-dependent activation of H1(0) gene expression, we demonstrate that hyperacetylation has a dominant effect on H1(0) gene expression, since it enhances the expression of the gene independent of the position of cells in the cell cycle. This response to deacetylase inhibitors is specific to H1(0), since it is not shared by other cell-cycle-dependent histone genes (H1 and H4). Finally, by transfection of trichostatin-A-resistant and trichostatin-A-sensitive cells with a plasmid containing a H1(0) promoter, we show that the exogenous H1(0) promoter is also highly sensitive to trichostatin A treatment and that activation of transcription follows exactly the same pattern as activation of the endogenous gene. These data show that histone acetylation may be used to modulate H1(0) gene activity and offers insight into a possible mechanism in which the developmentally regulated chromatin acetylation acts to potentiate H1(0) gene expression.

Acetylation↗
PubMed1994

Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension.

We describe a modification in the sample application mode for isoelectric focusing with immobilized pH gradients. Instead of being applied at the surface of the gel in a sample cup, the sample is introduced into the gel during the immobilized pH gradient strip rehydration step. This modification implies the use of low percentage gels (below 3.5% T) and specially designed, but simple, rehydration chambers. The main advantages are a uniform resolution without side effects and the possibility of handling large sample volumes (500 microL for a standard 3 x 160 x 0.5 mm strip), allowing micropreparative work (milligram samples) with a simple experimental design.

Animals↗
PubMed1994

Two-dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte-pH gradients.

A modified procedure for the two-dimensional electrophoretic analysis of basic polypeptides is described. This method uses isoelectric focusing with carrier ampholytes in the first dimension, and sodium dodecyl sulfate-electrophoresis in the second dimension. Counteraction of the cathodic drift is achieved by glass tube treatment (silanization), electrolyte modification (use of weak bases and acids), protection of the catholyte from carbon dioxide, and the addition of glycerol to the gel mix. Better resolution and reproducibility are obtained than with nonequilibrium pH gradient electrophoresis, since quasi equilibrium focusing can be obtained.

Alkalies↗
PubMed1994

Computer analysis of two-dimensional electrophoresis gels as a tool in cell biology: study of the protein expression of human keratinocytes from normal to tumor cells.

The usefulness of computer analysis of two-dimensional electrophoresis gels has been investigated on the example of human keratinocytes transformation. For this purpose, the protein expression of various keratinocytes strains from normal to tumor cells has been analysed by two-dimensional electrophoresis. The resulting gels have been submitted to computer analysis, including various data analysis techniques allowing to select spots on the gels or to classify the gels themselves. The latter techniques appeared very useful, since they demonstrated that the major transition in words of variation of the protein expression lies at the normal cell/transformed cell transition rather than at the transformed cell/tumorigenic cell transition.

Animals↗
PubMed1994

Silver-staining of proteins in polyacrylamide gels: a general overview.

On the basis of the physico-chemical principles underlying silver-staining of proteins, which are recalled in this paper, several methods of silver-staining of proteins after SDS electrophoresis in polyacrylamide gels or isoelectric focusing were tested. The most valuable protocols are presented in this report, including standard methods for unsupported gels and new methods devised for thin (0.5 mm) supported gels for SDS electrophoresis or isoelectric focusing and for staining of small peptides. Generally speaking, the most rapid methods were found to be less sensitive and less reproducible than more time-consuming ones. Among the long methods, those using silver-diammine complex gave the most uniform sensitivity. They require however special home-made gels and cannot be applied to several electrophoretic systems (e.g. systems using tricine or bicine as the trailing ion, or isoelectric focusing in immobilized pH gradients). For these reasons, protocols based on silver nitrate are of a more general use and might be favored. Future trends for silver-staining will also be discussed.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1993

Interaction of two DNA-binding factors expressed in B- and T-lymphocyte precursors.

Two DNA-binding factors detected in pre-B and pre-T cells and absent from mature lymphocytes are described. Factor A displayed no appreciable sequence selectivity but bound only to DNA fragments longer than 120 base pairs. The minimal size of a binding site was lower on an intrinsically curved DNA, suggesting formation of tertiary structures on DNA. Factor B interacted with sequences, other than consensus recombination signals, present in the vicinity of unrearranged immunoglobulin genes. Binding of factor B inhibited the interaction of factor A with the same DNA fragment. The presence of the factor-B-binding site in an episomal V(D)J recombinase substrate lowered the frequency of recombination in vivo. We propose that the two factors described here may function as accessory proteins in V(D)J recombination, possibly modulating accessibility of genes to the recombinase.

B-Lymphocytes↗
PubMed1992

Plasmodium chabaudi p68 serine protease activity required for merozoite entry into mouse erythrocytes.

To define the role of malaria parasite enzymes during the process of erythrocyte invasion, we have developed an in vitro serum-free invasion assay of mouse erythrocytes by purified Plasmodium chabaudi merozoites. The sensitivity of a merozoite-specific serine protease (p68) to various inhibitors and the effect of these inhibitors on invasion indicate a crucial role for p68. The substrate specificity of the purified enzyme has been partially defined using fluorogenic peptides. Consistent with this, in vitro incubation of mouse erythrocytes with the merozoite enzyme led to the cleavage of band 3 protein. The possible implication of erythrocyte band 3 truncation for the successful entry of the merozoite into the erythrocyte is discussed.

Animals↗
PubMed1992

Modified silver staining for immobilized pH gradients.

Silver development of gels containing an immobilized pH gradient has proved difficult so far because the bonded buffers (especially the tertiary amino acrylamido derivatives) tend to absorb silver ions with a resultant heavy background of increasing darkness from the anode to the cathode. We report a variant of silver staining in which thiosulfate is used twice: (i) prior to silver impregnation, at the millimolar level, to enhance sensitivity, and (ii) during development, at the micromolar level, to decrease the background.

Hydrogen-Ion Concentration↗
PubMed1992

A comparison between low background silver diammine and silver nitrate protein stains.

Several methods of silver staining of proteins after sodium dodecyl sulfate-electrophoresis in polyacrylamide gels were compared. The most rapid methods were found to be less sensitive than more time-consuming methods. Among the long methods, those using glutaraldehyde treatment of the gel and silver diammine complex as the silvering agent were found to be the most sensitive, at the expense of the use of a modified polyacrylamide matrix and higher silver concentrations.

Ammonia↗
PubMed1991

Stage transitions in B-lymphocyte differentiation correlate with limited variations in nuclear proteins.

Total nuclear proteins extracted from cell lines representing various stages of differentiation of mouse B lymphocytes were studied by computer analysis of two-dimensional gels. Of the 1438 spots present on the gels, 55 varied significantly in intensity during differentiation. The variations occurred most often in steps correlating with those classically defined for B-cell differentiation. Seventeen spots were not detectable in at least one of the stages (qualitative variations) and could represent switching on or off of genes coding for nuclear proteins. Detailed analysis of the 55 variable spots showed that they fall into small sets characterized by similar expression profiles, which argues for a combinatorial, multistep control mechanism of gene expression. In addition, analysis of the expression of all the nuclear proteins resolved on the gels clearly differentiated B-lineage cells from myeloid cells and suggested that the most important transition in B-cell differentiation occurs between the resting B cell and plasmocyte stages.

Animals↗
PubMed1990

Deficiency in catalase activity correlates with the appearance of tumor phenotype in human keratinocytes.

Biopsies isolated from various human stratified epitheliomas and cultures of the associated fibroblasts exhibit a breakdown in catalase activity (70 to 95%). We report here that primary human cultures of keratinocytes and various immortalized keratinocyte cell lines differ from one other in their catalase activity. Moreover, deficient catalase activity appears to be related to the intensity of tumor phenotype expression. Since catalase activity in cell lines transformed in vitro is not as weak as activity observed in cell lines isolated from tumors, catalase deficiency may follow the successive steps of tumor progression.

Acatalasia↗
PubMed1990

Amidosulfobetaines, a family of detergents with improved solubilization properties: application for isoelectric focusing under denaturing conditions.

The synthesis of a family of new detergents and their use for isoelectric focusing are described. These detergents differ from the conventional sulfobetaines by the presence of an amido group bridging the hydrophobic linear tail and the polar head, which increases their water solubility and urea tolerance considerably. Four different linear alkyl hydrophobic tails were tested, together with three different polar heads. The solubilization of red blood cell ghosts by the 12 resulting detergent performances was evaluated both in solution and in isoelectric focusing runs. Six chemicals gave performances equal or better than those of conventional detergents and could replace them for general use in focusing experiments. In addition, the synthesis of a whole range of compounds allowed valuable insights in the empirical understanding of protein-detergent interactions, which are extensively discussed.

Amides↗
PubMed1988

Improvement and simplification of low-background silver staining of proteins by using sodium dithionite.

High sensitivity and low background, the attractive characteristics of the procedure of Blum et al., Electrophoresis 1987, 8, 93-99, for silver staining of proteins in polyacrylamide gels have been improved by sensitizing the gels with sodium dithionite instead of sodium thiosulfate and by equilibration in water after fixation and prior to sensitization. These modifications decrease the background and allow for a longer development period, which in turn increases sensitivity and color contrast. In addition, the colors of the spots are shifted toward colder tones when compared with the original method.

Animals↗