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Biomedical subjects

P Roussel

Publications and source records attributed to P Roussel.

At least 19 recordsLinked to original sources
PubMed2000

In vivo release of mitotic silencing of ribosomal gene transcription does not give rise to precursor ribosomal RNA processing.

Nuclear RNA transcription is repressed when eukaryotic cells enter mitosis. Here, we found that the derepression of ribosomal gene (rDNA) transcription that normally takes place in telophase may be induced in prometaphase, metaphase, and anaphase mitotic HeLa cells, and therefore appears not to be dependent on completion of mitosis. We demonstrate for the first time that in vivo inhibition of the cdc2- cyclin B kinase activity is sufficient to give rise to okadaic acid-sensitive dephosphorylation of the mitotically phosphorylated forms of components of the rDNA transcription machinery, and consequently to restore rDNA transcription in mitotic cells. These results, showing that during mitosis the rDNA transcription machinery is maintained repressed by the cdc2-cyclin B kinase activity, provide an in vivo demonstration of the cell cycle-dependent regulation of rDNA transcription. Interestingly in mitotic cells, the newly synthesized 47S precursor ribosomal RNA (pre-rRNA) is not processed into the mature rRNAs, indicating that rDNA transcription and pre-rRNA processing may be uncoupled. Moreover this suggests that inhibition of the cdc2- cyclin B kinase is not sufficient to activate the 47S pre-rRNA processing machinery and/or to induce its relocalization at the level of newly synthesized 47S pre-rRNA. This in vivo approach provides new possibilities to investigate the correlation between pre-rRNA synthesis and pre-rRNA processing when the nucleolus reforms.

CDC2 Protein Kinase↗
PubMed2000

The AgNOR proteins: qualitative and quantitative changes during the cell cycle.

AgNOR proteins are a set of argyrophilic nucleolar proteins that accumulate in highly proliferating cells whereas their expression is very low in non-proliferating cells. Some of these proteins remain associated with the nucleolar organizer regions (NORs) during mitosis. In situ, the expression of AgNOR proteins is measured globally by quantification of the level of silver staining using morphometry and image analysis. To go deeper into the understanding of the relationship between the cell cycle and quantity of AgNOR proteins, it was necessary to determine the phases of cell cycle during which expression of AgNOR varies and what are the most variable proteins in each phase. To answer these questions, we set up the protocol permitting to detect and quantify AgNOR proteins on protein samples electrophoresed and transferred onto nitrocellulose membranes. This approach makes it possible to quantitatively evaluate individual AgNOR proteins and identify them, using nucleolar, nuclear and whole interphasic cell extracts, and chromosome-associated protein extracts. By this means, we identified nucleolin and protein B23 as the two major AgNOR proteins in the nucleolus during interphase and subunits of RNA polymerase I and transcription factor UBF as AgNOR proteins remaining associated with NORs during mitosis. We also observed that the increase in the level of nucleolin and protein B23 in rat liver seems to be linked with the cell cycle and not exclusively with stimulation of ribosomal gene (rDNA) transcription. Similarly in synchronized cells, the amount of nucleolin rapidly increases when cells enter the S phase (1.6-fold of the value of serum-deprived cells at 9 h, and 2.35-fold at 12 h after refeeding). The amount of protein B23 exhibits a lower and progressive increase with a maximum when the percentage of cells in G2 phase increased, i.e. after 24 h of cell cycle stimulation. We consider that the amount of AgNOR proteins can be a marker of proliferation, because this amount is related to cell cycle phases, schematically low for G1 phase and high for S-G2 phase. Thus, it is a measure of the relative proportion of cells in each phase, and consequently of the timing of each phase. The higher value indicates that the major part of the cells are in the S-G2 phase and correlatively few are in the G1 phase, and this characterizes a rapid cell cycle.

Animals↗
PubMed2000

Involvement of the PP2C-like phosphatase Ptc2p in the DNA checkpoint pathways of Saccharomyces cerevisiae.

RAD53 encodes a conserved protein kinase that acts as a central transducer in the DNA damage and the DNA replication checkpoint pathways in Saccharomyces cerevisiae. To identify new elements of these pathways acting with or downstream of RAD53, we searched for genes whose overexpression suppressed the toxicity of a dominant-lethal form of RAD53 and identified PTC2, which encodes a protein phosphatase of the PP2C family. PTC2 overexpression induces hypersensitivity to genotoxic agents in wild-type cells and is lethal to rad53, mec1, and dun1 mutants with low ribonucleotide reductase activity. Deleting PTC2 specifically suppresses the hydroxyurea hypersensitivity of mec1 mutants and the lethality of mec1Delta. PTC2 is thus implicated in one or several functions related to RAD53, MEC1, and the DNA checkpoint pathways.

Alleles↗
PubMed2000

Symmetry and twins in the monophosphate tungsten bronze series (P02)4(W03)2m (2 < or = m < or = 14)

Monophosphate tungsten bronze with pentagonal tunnels (PO2)4(WO3)2m are low-dimensional materials with charge density wave (CDW)-type electron instabilities. Two forms of the structure can thus be expected for all the members of the series: a low-temperature form (LT) corresponding to the CDW state and a high-temperature form (HT) corresponding to a normal metallic state. The HT form is described here for m = 9 and compared with that of the m = 5 and m = 7 counterparts. It is shown that a systematic twin phenomenon must be taken into account for HT members because of two possible configurations of the tilting mode of WO6 octahedra, The structure is also compared with that of m = 10, which exhibits the modulated CDW-LT form at room temperature. Owing to two possible polarization directions of the segments built of m WO6 octahedra, a twin phenomenon is also encountered in the LT forms. A review of all the structures known at present (m = 2, 4, 5, 6, 7, 8, 9, 10, 12) leads us to propose a structural law based on the building mode of W06 octahedra in W03-type slabs to explain the symmetry changes observed between even and odd members of the series.

Journal Article↗
PubMed1999

Cluster configurations in modulated EuVxMo8+/-yO14 crystals.

Three orthorhombic crystals of chemical formula Eu(x)V(y)Mo(8+/-z)O(14) were investigated by X-ray diffraction (Mo Kalpha radiation, lambda = 0.71073 Å). They have nearly the same lattice parameters (a approximately 11.3, b approximately 10.0, c approximately 9.2 Å), display one-dimensional incommensurate modulations of wavevector q* = gammac* and are characterized by the same superspace group Cmca(00gamma)s00. The crystals differ both in their compositions (namely Eu(0.976(6))V(1.13(5))Mo(7.10(5))O(14), Eu(0.986(4))V(1.10(3))Mo(7.30(1))O(14) and EuMo(7.96(1))O(14)) and in their gamma components [0.195 (2), 0.245 (2) and 0.286 (3), respectively]. The average structures of these crystals appear closely related to the structures of LaMo(7.7)O(14) (not modulated) and LaMo(8)O(14) (modulated); however, two main differences are outlined: first, the modulation direction is c in the Eu-containing crystals but b in the modulated La-containing crystal [q* = (1/3)b*], second, the Eu-containing crystals have centrosymmetric structures while the La-containing crystals have polar structures (space group C2ca). The Mo (or Mo and V) atoms are stacked to form (001) layers of metallic clusters. The density modulation of these structures implies the existence of the new types of clusters Mo(9), Mo(10), Mo(6)V(4), Mo(7)V(3) and Mo(8)V(2) besides the clusters M(8) (Mo(8), Mo(6)V(2) and Mo(7)V) and M(7) (Mo(7) and Mo(6)V) which are already known. Mo(8) units with cis and trans configurations and Mo(6)V(2) units with a trans configuration appear as the main cluster types in these crystals. The nature of the metallic clusters changes along c, but inside one (001) layer it is likely that only one cluster type with a given configuration is present. The main structural result is the formation, in some unit cells, of strong intercluster Mo-Mo, Mo-V or V-V bonds with distances close to 2.6 Å within a layer as well as between two neighbouring layers.

Journal Article↗
PubMed1999

Sequential biosynthesis of sulfated and/or sialylated Lewis x determinants by transferases of the human bronchial mucosa.

The structural determination of sulfated carbohydrate chains from a cystic fibrosis patient respiratory mucins has shown that sulfation may occur either on the C-3 of the terminal Gal, or on the C-6 of the GlcNAc residue of a terminal N -acetyllactosamine unit. The two enzymes responsible for the transfer of sulfate from PAPS to the C-3 of Gal or to the C-6 of GlcNAc residues have been characterized in human respiratory mucosa. These two enzymes, in conjunction with fucosyl- and sialyltransferases, allow the synthesis of different sulfated epitopes such as 3-sulfo Lewis x (with a 3- O -sulfated Gal), 6-sulfo Lewis x and 6-sulfo-sialyl Lewis x (with a 6- O -sulfated GlcNAc). In the present study, the sequential biosynthesis of these epitopes has been investigated using microsomal fractions from human respiratory mucosa incubated with radiolabeled nucleotide-sugars or PAPS, and oligosaccharide acceptors, mostly prepared from human respiratory mucins. The structures of the radiolabeled products have been determined by their coelution in HPAEC with known oligosaccharidic standards. In the biosynthesis of 6- O -sulfated carbohydrate chains by the human respiratory mucosa, the 6- O -sulfation of a terminal nonreducing GlcNAc residue precedes beta1-4-galactosylation, alpha2-3-sialylation (to generate 6-sulfo-sialyl- N -acetyllactosamine), and alpha1-3-fucosylation (to generate the 6-sulfo-sialyl Lewis x determinant). The 3- O -sulfation of a terminal N -acetyllactosamine may occur if this carbohydrate unit is not substituted. Once an N -acetyllactosamine unit is synthesized, alpha1-3-fucosylation of the GlcNAc residue to generate a Lewis x structure blocks any further substitution. Therefore, the present study defines the pathways for the biosynthesis of Lewis x, sialyl Lewis x, sulfo Lewis x, and 6-sulfo-sialyl Lewis x determinants in the human bronchial mucosa.

Acetylglucosamine↗
PubMed1999

The sialylation of bronchial mucins secreted by patients suffering from cystic fibrosis or from chronic bronchitis is related to the severity of airway infection.

Bronchial mucins were purified from the sputum of 14 patients suffering from cystic fibrosis and 24 patients suffering from chronic bronchitis, using two CsBr density-gradient centrifugations. The presence of DNA in each secretion was used as an index to estimate the severity of infection and allowed to subdivide the mucins into four groups corresponding to infected or noninfected patients with cystic fibrosis, and to infected or noninfected patients with chronic bronchitis. All infected patients suffering from cystic fibrosis were colonized by Pseudomonas aeruginosa. As already observed, the mucins from the patients with cystic fibrosis had a higher sulfate content than the mucins from the patients with chronic bronchitis. However, there was a striking increase in the sialic acid content of the mucins secreted by severely infected patients as compared to noninfected patients. Thirty-six bronchial mucins out of 38 contained the sialyl-Lewis x epitope which was even expressed by subjects phenotyped as Lewis negative, indicating that at least one alpha1,3 fucosyltransferase different from the Lewis enzyme was involved in the biosynthesis of this epitope. Finally, the sialyl-Lewis x determinant was also overexpressed in the mucins from severely infected patients. Altogether these differences in the glycosylation process of mucins from infected and noninfected patients suggest that bacterial infection influences the expression of sialyltransferases and alpha1,3 fucosyltransferases in the human bronchial mucosa.

Bronchi↗
PubMed1999

Pseudomonas aeruginosa binds to neoglycoconjugates bearing mucin carbohydrate determinants and predominantly to sialyl-Lewis x conjugates.

Pseudomonas aeruginosa plays an important role in the colonization of the airways of patients suffering from cystic fibrosis. It binds to the carbohydrate part of respiratory and salivary mucins and its binding to cystic fibrosis mucins is even higher, suggesting that qualitative or/and quantitative modifications of the carbohydrate chains may be involved in this process. In order to find out the best carbohydrate receptors for P.aeruginosa, a flow cytometry technique using a panel of polyacrylamide based glycoconjugates labeled with fluorescein was developed. The neoglycoconjugates contained neutral, sialylated or sulfated chains analogous to carbohydrate determinants found at the periphery of respiratory mucins (Le(a), Le(y), Le(x), sialyl- and 3'-sulfo-Le(x), and blood group A determinants). We used also neoglycoconjugates containing Gal(alpha1-2)Galbeta and sialyl- N -acetyllactosamine determinants. The interaction of these glycoconjugates with the nonpiliated strain of P.aeruginosa, 1244-NP, was saturable except for the glycoconjugates containing blood group A or sialyl- N -acetyllactosamine epitopes. The measure of Kd indicated that strain 1244-NP had a higher affinity for the glycoconjugate bearing the sialyl-Le(x)determinant than for all the other glycoconjugates studied. The role of sialic acid was confirmed by competition assay using mainly sialylated mucin glycopeptides. In order to find out if this behavior was the same for pathological strains as for the 1244-NP mutant, four mucoid strains of P.aeruginosa isolated from cystic fibrosis patients were analyzed with the Le(x)neoglycoconjugate, its sialylated and its sulfated derivatives. Individual variations in the binding of these strains to the three glycoconjugates were observed. However, three strains out of four had a higher affinity for the sialyl-Le(x)than for the 3'-sulfo-Le(x)derivative.

Bacterial Adhesion↗
PubMed1999

The mitotically phosphorylated form of the transcription termination factor TTF-1 is associated with the repressed rDNA transcription machinery.

The transcription termination factor TTF-1 exerts two functions in ribosomal gene (rDNA) transcription: facilitating initiation and mediating termination of transcription. Using HeLa cells, we show that TTF-1 protein is colocalized with the active transcription machinery in the nucleolus and also with the inactive machinery present in certain mitotic nucleolar organizer regions (NORs) when rDNA transcription is repressed. We also show that TTF-1 is specifically phosphorylated during mitosis in a manner dependent on the cdc2-cyclin B kinase pathway and on an okadaic acid-sensitive phosphatase. Interestingly, the mitotically phosphorylated form of TTF-1 appearing at the G(2)/M transition phase was more easily solubilized than was the interphase form. This indicates that the chromatin-binding affinity of TTF-1 appears to be different in mitotic chromosomes compared to the interphase nucleolus. Correlated with this, the other DNA-binding factor, UBF, which interferes with chromatin conformation in the rDNA promoter, was more strongly bound to rDNA during mitosis than at interphase. The reorganization of the mitotic rDNA promoter might be induced by phosphorylation of certain components of the rDNA transcription machinery and participate in silencing of rDNA during mitosis.

Antibody Specificity↗
PubMed1998

O-acetylation of sialic acids.

Sialic acids can be acetylated at the 4, 7, 8 and/or 9 position. Biological roles of these substitutions have been missed until recently because of their low abundance and lability to conventional purification methods. The recent advances in the analysis of sialic acids have allowed to demonstrate that O-acetylation has a selective but widespread distribution. The metabolism of acetylated sialic acids is under the control of two groups of enzymes, O-acetyl transferases and 9-O-acetyl esterases. O-acetyl transferases are difficult to purify, and furthermore, attempts at expression cloning have failed in isolating the true 9-O-acetyl transferase cDNA. This explains that the regulation of the selective expression of O-acetylated sialic acid in not completely understood. Acetylation of sialic acid is expressed on the outer most part of the carbohydrate moiety of membrane and secreted glycoconjugates. This particular location explains why this modification is involved in cell/cell interactions and in the non-immune protection of mucosa.

Acetylation↗
PubMed1998

Identification of urinary oligosaccharides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A new method of urinary oligosaccharides identification by matrix-assisted laser desorption time-of-flight mass spectrometry is presented. The method involves three steps: coupling of the urinary oligosaccharides with 8-aminonaphthalene-1,3,6-trisulfonic acid; fast purification over a porous graphite carbon extraction column; and mass spectrometric analysis. Identification of urinary oligosaccharides is based on the patterns and values of the pseudomolecular ions observed. We report here the patterns in urines from patients with Pompe disease, alpha and beta mannosidoses, galacto-sialidosis, and GM1 gangliosidosis. The protocols described here allowed facile and sensitive identification of the pathognomonic oligosacchariduria present in lysosomal diseases and can be extended to any pathological oligosacchariduria.

Adult↗
PubMed1997

Characterization of an N-acetylglucosamine-6-O-sulfotransferase from human respiratory mucosa active on mucin carbohydrate chains.

A microsomal GlcNAc-6-O-sulfotransferase activity from human bronchial mucosa, able to transfer a sulfate group from adenosine 3'-phosphate 5'-phosphosulfate onto methyl-N-acetylglucosaminides or terminal N-acetylglucosamine residues of carbohydrate chains from human respiratory mucins, has been characterized. The reaction products containing a terminal HO3S-6GlcNAc were identified by high performance anion-exchange chromatography. Using methyl-beta-N-acetylglucosaminide as a substrate, the optimal activity was obtained with 0.1% Triton X-100, 30 mM NaF, 20 mM Mn2+, 5 mM AMP in a 30 mM MOPS (3-(N-morpholino) propanesulfonic acid) buffer at pH 6.7. The apparent Km values for adenosine 3'-phosphate 5'-phosphosulfate and methyl-beta-N-acetylglucosaminide were observed at 9.1 x 10(-6) M and 0.54 x 10(-3) M, respectively. The enzyme had more affinity for carbohydrate chains with a terminal GlcNAc residue than for methyl-beta-N-acetylglucosaminide; it was unable to catalyze the transfer of sulfate to position 6 of the GlcNAc residue contained in a terminal Galbeta1-4GlcNAc sequence. However, oligosaccharides with a nonreducing terminal HO3S-6GlcNAc were substrates for a beta1-4 galactosyltransferase from human bronchial mucosa. These data point out that GlcNAc-6-O-sulfotransferase must act before beta1-4 galactosylation in mucin-type oligosaccharide biosynthesis.

Acetylglucosamine↗
PubMed1997

Mucins secreted by a transformed cell line derived from human tracheal gland cells.

High-molecular-mass glycoconjugates are secreted by the continuous cell line MM-39, which has been obtained from cultured human tracheal gland cells transformed by simian virus 40. They were purified on Sepharose(R) CL-4B and then by two steps of density-gradient centrifugation. High-molecular-mass glycoproteins resistant to digestion by hyaluronidase, chondroitin ABC lyase and heparitinase were obtained, in addition to hyaluronic acid and proteoglycans. They were susceptible to beta-elimination. They contained polylactosaminoglycan chains as well as carbohydrate chains with a terminal sialic acid in the NeuAc alpha2-3 sequence. Most of them have a buoyant density of 1.45 g/ml in CsCl-density-gradient centrifugation, except for MUC1. The MM-39 cells were also characterized by a high expression of MUC1 and MUC4 genes, but they did not express MUC2, MUC3, MUC5B and MUC5AC. Therefore the MM-39 cells synthesized mucin-like glycoproteins as well as lysozyme and mucous proteinase inhibitor [Merten, Kammouni, Renaud, Birg, Mattéi and Figarella (1996) Am. J. Respir. Cell. Mol. Biol. 15, 520-528]; they should be considered as having a mixed, both serous and mucous, phenotype.

Cell Line, Transformed↗
PubMed1997

Amount of the two major Ag-NOR proteins, nucleolin, and protein B23 is cell-cycle dependent.

To know the biological basis allowing the use of Ag-NOR protein expression as proliferation marker in human malignancies, the relationship between cell cycle and amount of Ag-NOR protein was analyzed. The quantification of the two major Ag-NOR proteins, nucleolin and protein B23, was performed in exponentially growing, serum-deprived, and cell-cycle stimulated cells. Expression of nucleolin was low in serum-deprived cells and increased mostly in S phase during cell-cycle stimulation. Conversely, expression of protein B23 was slightly repressed in serum-deprived cells, and increased progressively until G2 phase during cell-cycle stimulation. The accumulation of nucleolin and protein B23 in G2 compared to G1 was demonstrated using sorted phase-specific cells. In G0, cells sorted according to their very low RNA content, and the amount of Ag-NOR proteins was half of that found in G1 cells, nucleolin being only weakly detectable. Therefore, the expression of nucleolin increased between G0-G1 and G1-S phases. These data support the hypothesis that quantification of Ag-NOR proteins is an estimation of the percentage of cells in each cell cycle phase because their amount is high in S-G2 and low in G1 phases.

Animals↗
PubMed1997

[How to develop a quality system].

When principles relating to quality assurance are known and understood, they must still be practically and progressively implemented. Many complementary actions are described for the building of a quality system in a blood bank. The aim is to control the quality organization, operational processes and the ensuing documentary system. Some critical points about the development of a quality system and its timelessness are then discussed.

Blood Banks↗