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M Véron

Publications and source records attributed to M Véron.

At least 73 records · Page 4Linked to original sources
PubMed1992

Nucleoside diphosphate kinase does not directly interact with tubulin nor microtubules.

Nucleoside diphosphate kinase has been shown to play a role in proliferation and development. Microtubules have been evoked as a possible target of NDP kinase action; in particular it was proposed that NDP kinase could regulate the cellular pool of polymerizable GTP-tubulin by direct phosphorylation of tubulin bound GDP. We show that this reaction does not occur in vitro and also that NDP kinase does not bind to microtubules both in the presence and absence of MAPs. Thus, any possible physiological effect of NDP kinase on microtubule dynamics is exerted only by modulating the concentrations of free guanine nucleotides in the vicinity of microtubules.

Animals↗
PubMed1992

A Pro/Ser substitution in nucleoside diphosphate kinase of Drosophila melanogaster (mutation killer of prune) affects stability but not catalytic efficiency of the enzyme.

Nucleoside diphosphate kinase of Drosophila, recently identified as the product of the awd gene, is essential for larval development. The conditional lethal mutation Killer of prune maps to the same gene. We purified the nucleoside diphosphate kinases from wild-type and mutant larvae by a simple procedure involving affinity chromatography on blue Sepharose. Both proteins are purified as hexamers in their native state. The mutant protein, which carries a serine instead of proline at position 97, has structural properties and catalytic efficiency that are very similar to the wild-type protein. However, the mutant protein has a much lower stability to denaturation by heat and urea. Following dilution of urea with buffer the urea-denaturated mutant nucleoside diphosphate kinase accumulates as folded monomers and cannot recover its quaternary structure and enzymatic activity. In contrast, the wild-type enzyme recovers hexameric structure and activity. This suggests that the mutation affects the folding/assembly pathway without affecting the function of the mature protein once folded and assembled into the mature hexameric structure.

Amino Acids↗
PubMed1992

Serine/threonine protein phosphatases in Dictyostelium discoideum: no evidence for type I activity.

Extracts from Dictyostelium discoideum contain type 2A and 2C serine/threonine-specific protein phosphatases with properties very similar to those from mammals according to their sensitivity to okadaic acid and to their dependence for divalent cations. In contrast, no type 1 protein phosphatase is found at any time of development, neither in the cytosolic nor in the particulate fraction, using glycogen phosphorylase a, casein, histone or the non-proteinous 4-Methylumbelliferyl phosphate as substrates. Both type 2A and 2C protein phosphatase activities remain constant throughout the development cycle.

Animals↗
PubMed1992

Thioredoxins from Dictyostelium discoideum are a developmentally regulated multigene family.

Thioredoxins are low molecular weight proteins which serve as hydrogen donors in a wide variety of redox reactions via reversible formation of a disulfide bridge between two neighboring cysteins. We present data demonstrating that in Dictyostelium discoideum thioredoxins constitute a highly conserved multigene family. We have isolated cDNA clones coding for three different Dictyostelium thioredoxins which show 80% mutual identity. Analysis of genomic Southern blots suggests the presence of additional genes. Except for the active site (Trp-Cys-Gly-Pro-Cys), there are only a few amino acid identities with thioredoxins from other organisms. Identity scores do not exceed 43%, the value found with the human lymphocyte protein. DdTRX1 was expressed in Escherichia coli, purified, and shown to have thioredoxin activity, as judged by its capacity to activate the NADP-malate dehydrogenase. Due to its life cycle, during which individual amoebae form a multicellular fruiting body, Dictyostelium is used to study developmental processes such as cell-type differentiation and regulation of gene expression. Transcript levels of Dictyostelium thioredoxins were regulated during the developmental cycle. Low levels of mRNAs could be detected during growth. After the onset of development, where essentially no cell divisions take place, message levels increased with maximal expression during aggregation. In later multicellular stages, RNA levels declined again. The same expression pattern could be seen for all cloned thioredoxins. Protein levels paralleled this time course with a delay of several hours as judged by Western blot and activity measurements.

Amino Acid Sequence↗
PubMed1992

Nucleoside diphosphate kinase/NM23 expression in breast cancer: lack of correlation with lymph-node metastasis.

The product of the nm23-H1 gene, reported to be a metastatic suppressor gene, was recently identified as the nucleoside diphosphate (NDP) kinase A, and was found to be overexpressed in several types of malignant tumors as compared with normal tissues. In order to determine whether NDP-kinase expression serves as a marker for metastatic potential and whether hyperproliferation of neoplastic cells would correlate with expression, we analyzed NDP-kinase levels and activity by immunohistochemical staining and by an enzymatic assay in 13 benign and 98 malignant breast-tissue specimens. Our results confirm that NDP-kinase expression increases in malignant cells of breast carcinomas, but cannot be considered as a biological marker of metastatic dissemination. No correlation was found between NDP-kinase activity and S phase, taken as an index of cell proliferation. Moreover, no correlation was observed between NDP-kinase activity and tumor size, histoprognostic index, estrogen receptors or progesterone receptors. The mechanism of over-expression of NDP in malignant cells and its role in tumor progression remain to be determined.

Breast Neoplasms↗
PubMed1992

X-ray structure of nucleoside diphosphate kinase.

The X-ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta-sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro----Ser substitution in Killer of prune (K-pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three-dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.

Amino Acid Sequence↗
PubMed1992

[Ultrastructural immunocytochemical localization of diphosphate kinase/Nm23 in human cancer cells].

Nucleoside diphosphate (NDP) kinase/Nm23 is highly expressed in certain malignant tissues, as compared with the rate found in normal or hyperplastic tissues. The potential role of this overexpression in tumor progression and the mechanisms involved in it remain to be determined. We studied the ultrastructural localisation of the NDP kinase, and in particular looked for an association of this enzyme with the microtubules or cytoplasmic membrane. Using immunocytochemical methods with an antiserum raised against NDP kinase A, we analysed tissue sections of breast carcinomas and cells in culture derived from a cervical cancer. In malignant cells, a strong labeling of the cytoplasm, related to ribosomes, was observed. No labeling of microtubules, or other intracytoplasmic components was found. No labeling of the nucleus was noted. In contrast, a strong labeling of the cytoplasmic membrane of most malignant cells was observed. In the cytoplasm of non-malignant stromal cells, a slight labeling of ribosomes was observed. These results must be taken into account with regard to the different existing hypotheses relative to the role of the NDP kinase in tumor progression, and in particular relative to its activation function on the GTP-binding proteins involved in membrane signal transduction.

Breast Neoplasms↗
PubMed1991

Crystallization and preliminary X-ray diffraction studies of nucleoside diphosphate kinase from Dictyostelium discoideum.

Nucleoside diphosphate kinase from the slime mold Dictyostelium discoideum is highly homologous to gene products that are involved in development in Drosophila and in oncogenesis in human cells. The cloned protein expressed in Escherichia coli has been purified and crystallized in a hexagonal space group with a = b = 74.9 A, c = 211.4 A. The asymmetric unit contains either one or two 17,000 Mr subunits of the hexamer.

Crystallography↗
PubMed1991

Overexpression of nucleoside diphosphate kinase (Nm23) in solid tumours.

The product of the nm23-H1 gene, reported to be related to the metastatic potential of tumour cells, was recently identified as the nucleoside diphosphate (NDP) kinase A (Gilles et al., 1991, J Biol Chem, 266, 8784-8789). An analysis of the enzyme by activity measurement and immunological techniques using polyclonal antibodies raised against the NDP kinase A purified from human erythrocytes, was performed on 39 human tissue specimens. Markedly increased activity and higher level of the protein were observed in extracts of solid tumours as compared to the corresponding normal tissues (P less than 0.01). An intense immunolabelling of tumoral cells was observed in sections of the malignant tumours and of some but not all benign neoplasia. The staining is observed in noninvasive and invasive ductal breast carcinomas with or without lymph node involvement as well as in colon and cervix carcinomas and in a case of metastatic melanoma. Therefore, NDP kinase A level is increased in neoplastic tissues but no correlation with metastatic potential could be demonstrated.

Blotting, Western↗
PubMed1991

Outbreak of gut colonization by Pseudomonas aeruginosa in immunocompromised children undergoing total digestive decontamination: analysis by pulsed-field electrophoresis.

We analyzed an outbreak of gut colonization by Pseudomonas aeruginosa occurring in an intensive care hematology unit by using conventional typing methods and pulsed-field electrophoresis. In October and November 1989, the feces of four immunocompromised children undergoing total digestive decontamination were colonized by P. aeruginosa. Ten isolates were obtained from the gut flora in pure culture. Retrospective investigations found that one P. aeruginosa isolate from stools of one of the patients was already present at high counts 6 months before the outbreak. This patient had been discharged from the unit in May 1989 and had been readmitted concomitantly with the outbreak. Only pulsed-field electrophoresis could demonstrate that a single epidemic strain was present in the fecal flora of the children. This strain had probably been brought into the unit by the patient with chronic fecal carriage.

Anti-Bacterial Agents↗
PubMed1990

Functional cloning of a nucleoside diphosphate kinase from Dictyostelium discoideum.

A lambda gt11 cDNA library from Dictyostelium discoideum was screened by direct labeling of filter replicas with [35S]guanosine 5'-O-(thiotriphosphate) (GTP gamma S). A positive clone was obtained and used as probe to isolate additional clones from which a complete cDNA sequence was determined. The cDNA hybridizes to a single copy gene that is expressed as a 0.6-kilobase mRNA in vegetatively growing amoeba. The open reading frame encodes a protein of 155 amino acids (calculated Mr 16,775), devoid of cysteine residues. The protein contains most of the short consensus motifs characteristic of the catalytic domain of protein kinases although the overall homology with this class of enzymes is not greater than 20%. Its size and amino acid composition indicated that it could be the monomer of a nucleoside diphosphate (NDP) kinase, an enzyme which catalyzes the phosphate transfer from triphospho- to diphosphonucleotides. Indeed, specific NDP kinase activity was found in extracts of bacteria transformed with a plasmid expressing the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extracts incubated in the presence of [35S]GTP gamma S revealed a single 35S-labeled band of size corresponding to the protein, which likely represents the stable thiophosphorylated reaction intermediate characteristic for the ping-pong reaction mechanism of NDP kinases. The formation of this labeled intermediate probably allowed the detection of the enzyme on the filters during the screening procedure. Although NDP kinases from a great variety of sources have been characterized, the primary structure of the D. discoideum NDP kinase is the first reported for an enzyme of eukaryotic origin.

Amino Acid Sequence↗
PubMed1989

Overproduction of the regulatory subunit of the cAMP-dependent protein kinase blocks the differentiation of Dictyostelium discoideum.

During the aggregation of Dictyostelium discoideum extracellular cAMP is known to act as a chemotractant and as an inducer of cellular differentiation. However, its intracellular role as a second messenger remains obscure. We have constructed a fusion gene consisting of the cDNA encoding the regulatory subunit (R) of the cAMP-dependent protein kinase fused to the promoter and N-terminal-proximal sequences of a Dictyostelium actin gene. Stable transformants, containing multiple copies of this gene, overproduce the R subunit which accumulates prematurely relative to the endogenous protein. These transformants fail to aggregate. Detailed analysis has shown that they are blocked at interphase, the period prior to aggregation, and that they are severely defective in most responses to cAMP including the induction of gene expression. Our observations suggest that intracellular cAMP acts, presumably by activation of the catalytic subunit of the cAMP-dependent protein kinase, to facilitate early development.

Blotting, Western↗
PubMed1988

Expression and properties of the regulatory subunit of Dictyostelium cAMP-dependent protein kinase encoded by lambda gt11 cDNA clones.

lambda gt11 phages harboring five different cDNA fragments for the regulatory (R) subunit of Dictyostelium discoideum cAMP-dependent protein kinase (CAK) directed the synthesis of this protein in Escherichia coli cells. Crude bacterial extracts were probed with an antiserum against the Dictyostelium R subunit. The presence of specific epitopes for the R subunit in a given extract was compared with high-affinity cAMP-binding activity and with the ability to inhibit the catalytic (C) subunit through protein-protein interaction. The expression and the biochemical properties of these proteins were correlated with their cDNA nucleotide sequence. The results show that the Dictyostelium R subunit can be functionally expressed in E. coli cells either as a fusion protein with beta-galactosidase or as a nonfusion protein. In both cases, the products of cDNA clones containing the entire coding sequence retained high-affinity cAMP-binding activity and the capacity to interact with the catalytic subunit. One of the fusions, lacking the 94 N-terminal residues, failed to inhibit catalytic activity, although it bound cAMP with an affinity similar to that of the native R protein from D. discoideum.

Amino Acid Sequence↗
PubMed1988

S-adenosylmethionine, S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase variations during differentiation of Dictyostelium discoideum.

We have analyzed the level of substrate (AdoMet) and products (AdoHcy) of transmethylations throughout the developmental cycle of the primitive eukaryote Dictyostelium discoideum. The ratio AdoMet/AdoHcy varied dramatically during differentiation. The intracellular level of AdoHcy decreased sharply after the beginning of starvation reaching a value of 18% of that in vegative cells within 4 h. In contrast, there was a two-fold transient increase in AdoMet at the time of aggregation. However, these changes were not related to changes in AdoHcy hydrolase since constant levels of both the protein and the activity were found until 16 h of differentiation. In particular, there was no indication of an in vivo inactivation of the enzyme by cAMP at the time of aggregation. These results are discussed with respect to the previously postulated role of AdoHcy hydrolase in the regulation of the AdoMet/AdoHcy ratio in eukaryotic cells.

Adenosylhomocysteinase↗
PubMed1988

Vectors for expression of truncated coding sequences in Escherichia coli.

We describe the construction of vectors for expressing in Escherichia coli DNA fragments obtained by progressive deletions of DNA inserts in single-stranded sequencing vectors as M13 or pTZ according to the methode of Dale et al. (Plasmid 1985, 13, 31-40). These vectors, pIMS1, pIMS5, and pIMS6, harbor all of the elements required for the regulated expression of any open reading frame flanked by EcoRI restriction sites. The encoded peptides contain only a few vector-derived amino acids. A method is described for direct selection of recombinant clones by in situ RNA hybridization. The properties of the expression vector have been analyzed with a DNA deletion series obtained from the cDNA coding for the regulatory subunit of Dictyostelium discoideum cAMP-dependent protein kinase.

Cloning, Molecular↗