Biomedical subjects

M Veron

Publications and source records attributed to M Veron.

At least 37 records · Page 2Linked to original sources

PubMed1995

Candida albicans nucleoside-diphosphate kinase: purification and characterization.

Soluble nucleoside-diphosphate kinase (NDP kinase) from Candida albicans was purified to electrophoretic homogeneity and a partial sequence was determined. The enzyme was kinetically and physically characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single polypeptide of 17 kDa upon staining, by immunodetection with heterologous and homologous antibodies, and by autoradiography of the phosphorylated enzyme. Furthermore, isoelectric focusing of the purified native enzyme showed a single acidic band (pI 4.5). These results together with a native molecular mass of 98 kDa suggest a hexameric native enzyme composed of identical subunits. Like NDP kinases from other sources, the catalysis involves a phosphoenzyme intermediate that is rapidly formed upon incubation of the enzyme with ATP. The transfer of phosphate from phosphoprotein intermediate to nucleoside diphosphates is equally fast. Kinetic experiments indicated that GTP and ATP had the lowest Km compared to UTP, dTTP, and CTP. GDP acted as a preferred acceptor as assessed by kinetic measurements as well as by competition experiments. Experimental data are presented indicating the existence of a membrane-associated NDP kinase. Preliminary characterization of this enzyme suggests that cytosolic and membrane-associated NDP kinases are similar proteins.

Amino Acid Sequence↗

PubMed1995

High nm23-H1/NDPK-A expression in Ewing tumors: paradoxical immunohistochemical reactivity and lack of prognostic significance.

Expression of nm23-H1/NDPK-A has been reported to correlate inversely with metastasizing potential of rodent experimental cells and some human tumors. In the search for reliable molecular prognostic indicators for Ewing tumors (ET), a group of aggressive presumably neuroectodermal malignancies in children and adolescents, we studied nm23-H1/NDPK-A expression. Northern-blot and RT-PCR analyses were employed to semi-quantificatively measure nm23-H1 mRNA levels in ET cell lines and tissue extracts. A panel of monoclonal antibodies (MAbs) were used to evaluate protein abundance by Western blotting and immunohistochemistry. The nm23-H1/NDPK-A gene was also investigated on the DNA level to define possible genomic alterations. Our results revealed neither nm23-H1 allelic loss nor gene amplification and failed to show any significant variation in nm23-H1 mRNA or NDPK-A protein levels of primary or metastatic ET. NDPK-A protein levels were high and comparable to those of MCF-7 breast-cancer cells and to aggressive stage-IV neuroblastoma cell lines. nm23-H2/NDPK-B expression in ET was slightly more variable but generally lower than in MCF-7 cells. In the immunohistochemical analysis, however, discrepancies in the reactivity patterns with different antibodies were observed. Differential sensitivity to various fixation methods and heat treatment pointed to a structurally polymorphic NDPK-A protein. nm23-H1 expression studies using immunohistochemistry for prognostic counselling should thus be interpreted with caution.

Adolescent↗

PubMed1995

A protein kinase from Dictyostelium discoideum with an unusual acidic repeat domain.

DdKinX codes for 1093 amino acids which are organized in four regions: the N-terminal catalytic domain, a region containing 30% acidic amino acids, tandem repeats of the motif VKVEEPVEE and the C-terminus. Identity with other protein kinases is 25 to 30%. Descendent trees show that DdKinX does not belong to any of the known kinase branches.

Amino Acid Sequence↗

PubMed1995

Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi.

A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme was purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated NDP kinase was identified which accounts for 30% of total enzymatic activity. Western blot analysis of the soluble NDP kinase revealed a 16.5-kDa monomer recognized by polyclonal antibodies to NDP kinase from Dictyostelium discoideum, Candida albicans or human. Most of the T. cruzi NDP kinase is found in the cell as a hexamer composed of 16.5-kDa monomers. The Km values of the enzyme for ATP, GDP and dTDP were 0.2 +/- 0.008 mM, 0.125 +/- 0.012 mM and 0.4 +/- 0.009 mM, respectively. The parasite enzyme was stable, remained active at 65 degrees C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with [gamma-32P]ATP or thiophosphorylated with [35S]GTP gamma S. The incubation of the 32P-labelled phosphoenzyme with unlabelled nucleoside 5'-diphosphates resulted in the formation of 32P-labelled nucleoside 5'-triphosphates without strict base specificity, indicating that the reaction mechanism of the T. cruzi enzyme is the same as reported for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside 5'-diphosphates, GTP was preferentially formed.

Adenosine Triphosphate↗

PubMed1994

NDP-K/nm23 expression in human breast cancer in relation to relapse, survival, and other prognostic factors: an immunohistochemical study.

The nm23 gene was originally identified by Steeg et al. by screening of cDNA libraries from murine melanoma cell lines of varying metastatic potential. An inverse relationship between metastatic potential and nm23 RNA and/or protein was found in four different metastatic model systems. It was proposed that nm23 may function as a suppressor gene for tumour metastasis. It has recently been found that the sequence of nm23 and NDP-kinase (NDP-K) is identical. Using an immunohistochemical technique and employing a polyclonal antibody to purified NDP-K A, we have determined NDP-K expression in a series of 197 breast carcinomas. One hundred and sixty (81.2 per cent) of these tumours were scored positive for NDP-K and 37 (18.8 per cent) scored negative. No relationship was found between NDP-K/nm23 expression and patient relapse or survival. Furthermore, no relationship was found between NDP-K/nm23 expression and a number of other prognostic factors including tumour grade, oestrogen receptor, progesterone receptor, and p53 expression. Our results contradict the hypothesis concerning the possible role of NDP-K/nm23 as a metastatic suppressor gene in human breast cancer, but further studies using antibodies specific for NDP-K/nm23 subtypes are clearly indicated.

Breast Neoplasms↗

PubMed1993

A conserved helix motif complements the protein kinase core.

Residues 40-300 of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase define a conserved bilobal catalytic core shared by all eukaryotic protein kinases. Contiguous to the core is an extended amphipathic alpha-helix (A helix). Trp30, a prominent feature of this helix, fills a deep hydrophobic pocket between the two lobes on the surface opposite to the active site. The C subunit in Dictyostelium discoideum shows sequence conservation of residues 40-350 with the mouse enzyme but contains an N-terminal extension of 332 residues. A sequence corresponding to the A helix contiguous to the core is absent. However, we have now identified a remote A-helix motif (residues 77-98). When the core of the Dictyostelium C subunit was modeled, based on the mouse C subunit, complementarity between this putative A helix and the surface of the core was found to be conserved. Analysis of other protein kinases reveals that the A-helix motif is not restricted to cAMP-dependent protein kinase. In the Src-related family of protein kinases, for example, an A helix is very likely contiguous to the core, thus serving as a linker between the conserved catalytic core and the Src homology 2 domain. We predict that an A-helix motif complementary to the core will be a conserved feature of most eukaryotic protein kinases.

Amino Acid Sequence↗

PubMed1993

Regulation of Dictyostelium morphogenesis by cAMP-dependent protein kinase.

During formation of the Dictyostelium slug extracellular cAMP signals direct the differentiation of prespore cells and DIF, a chlorinated hexaphenone, induces the differentiation of prestalk cells. At culmination the slug transforms into a fruiting body, composed of a stalk supporting a ball of spores. A dominant inhibitor of cAMP-dependent protein kinase (PKA) expressed under the control of a prestalk-specific promoter blocks the differentiation of prestalk cells into stalk cells. Analysis of a gene specifically expressed in stalk cells suggests that PKA acts to remove a repressor that prevents the premature induction of stalk cell differentiation by DIF during slug migration. PKA is also necessary for the morphogenetic movement of prestalk cells at culmination. Expression of the PKA inhibitor under control of a prespore-specific promoter blocks the accumulation of prespore mRNA sequences and prevents terminal spore cell differentiation. Thus PKA is essential for progression along both pathways of terminal differentiation but with different mechanisms of action. On the stalk cell pathway it acts to regulate the action of DIF while on the spore cell pathway PKA itself seems to act as the inducer of spore cell maturation. Ammonia, the extracellular signal which regulates the entry into culmination, acts by controlling the intracellular concentration of cAMP and thus exerts its effects via PKA. The fact that PKA is necessary for both prespore and spore gene expression leads us to postulate the existence of a signalling mechanism which converts the progressive rise in cAMP concentration during development into discrete, PKA-regulated gene activation events.

Animals↗

PubMed1993

P36, a Dictyostelium discoideum protein whose phosphorylation is stimulated by GDP, is homologous to the alpha-subunit of succinyl-CoA synthetase.

Previously, we reported the phosphorylation of a 36 kDa protein, p36, in crude membranes from the amoeba Dictyostelium discoideum (Anschutz, A.L., Howlett, A. and Klein, C. (1989) Proc. Natl. Acad. Sci. USA 86, 3665-3668). Here, we report the purification and identification of p36. The protein was purified approximately 35-40-fold with a yield of 8-10%. This material was then separated on 10% SDS-polyacrylamide gels and the band corresponding to p36 was isolated. Partial peptide sequencing of this band revealed p36 to be homologous to the alpha-subunit of succinyl-CoA synthetase. This identification of the protein was supported by the results of phosphorylation studies which examined the effects of substrates of succinyl-CoA synthetase on p36 phosphorylation. In crude sample preparations, p36 could be phosphorylated by both ATP or GTP and in either case, its phosphorylation was stimulated by low concentrations of GDP. Partially purified p36 retained its ability to be phosphorylated with GTP while exhibiting little or no phosphorylation with ATP. GDP still enhanced the rate of p36 phosphorylation with GTP. Therefore, the stimulation of p36 phosphorylation by GDP is not due to substrate conversion and is best explained by a regulatory mechanism.

Adolescent↗

PubMed1993

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins.

Animals↗

PubMed1993

Coryneform bacteria isolated from middle ear fluid.

Nineteen strains of facultatively anaerobic gram-positive rods isolated in pure culture from middle ear fluids were identified. All effusions were collected by tympanocentesis in children with acute otitis media. Identification of microorganisms to the genus level was done by studying the cell wall composition. Sixteen strains contain meso-diaminopimelic acid and arabinogalactan polymer but lack mycolic acids; therefore, these strains do not belong to a previously described taxon. Because of similarities with Corynebacterium afermentans (Centers for Disease Control group ANF-1), we temporarily classified these mycolateless strains ANF-1 like. Isolation of these microorganisms in pure culture from middle ear fluids collected by tympanocentesis is a strong argument for their involvement in acute otitis media.

Actinomycetales↗

PubMed1993

Value of ELISA using antigen 60 for the diagnosis of tuberculosis in children.

We evaluated the possible value of enzyme-linked immunosorbent assay (ELISA) using antigen 60 (A60) for improved diagnosis of tuberculosis in children. Results obtained in 31 children with active tuberculosis and in 16 patients with tuberculous infection without disease were compared with the results of 198 control subjects with no mycobacterial disease. In control children, anti-A60 IgG increased with age and the optical density (OD) in ELISA assays rose from .079 +/- .053 (OD +/- SD) in children younger than 5 years old to 0.146 +/- 0.082 OD in children older than 5 years. In control subjects younger than 2 years old, IgG OD values were significantly higher in BCG-vaccinated children than in nonvaccinated children. At a chosen specificity of 98 percent, a positive serodiagnosis was observed in 68 percent of children with clinically active tuberculosis. In these children with active disease, smears were positive in only 26 percent of cases and mycobacterial cultures yielded the organism in 45 percent of cases. None of the infected children without disease had high IgG OD values. IgM measurements were also evaluated. Mean values from control and diseased children overlapped, leading to a low sensitivity (19 percent) in children with clinically tuberculosis active. We conclude that anti-A60 IgG measurement is a rapid and low-cost technique that enhances the diagnosis of clinically active tuberculosis in children and may distinguish recent infection without disease from infection with disease.

Adolescent↗

PubMed1993

[Multifactorial analysis of the phenotypes for beta-lactams of 1044 Escherichia coli strains].

1,044 E. coli strains were randomly collected by the beginning of 1992. Their susceptibility for seven beta-lactam antibiotics: amoxycillin, augmentin, ticarcillin, claventin, cephalothin, cefoxitin and cefotaxime, was studied routinely by the agar diffusion method. The datas were analyzed by the CERIB multifactorial analysis package which yields to homogeneous populations. This analysis showed four well defined populations: 1) 588 strains (56.4%) susceptible to all antibiotics; 2) 410 strains (39.3%) present a penicillinase phenotype; 3) 11 strains (1.05%) are cephalosporinase producer; 4) 7 strains (0.67%) were identified as producing an extended-spectrum beta-lactamase. The remaining strains: 28 (2.68%) had a reduced susceptibility to all antibiotics, which suggests the combination of few resistance mechanisms or other hypothesis.

Anti-Bacterial Agents↗

PubMed1992

Culmination in Dictyostelium is regulated by the cAMP-dependent protein kinase.

We placed a specific inhibitor of cyclic AMP-dependent protein kinase (PKA) under the control of a prestalk-specific promoter. Cells containing this construct form normally patterned slugs, but under environmental conditions that normally trigger immediate culmination, the slugs undergo prolonged migration. Slugs that eventually enter culmination do so normally but arrest as elongated, hairlike structures that contain neither stalk nor spore cells. Mutant cells do not migrate to the stalk entrance when codeveloped with wild-type cells and show greatly reduced inducibility by DIF, the stalk cell morphogen. These results suggest that the activity of PKA is necessary for the altered pattern of movement of prestalk cells at culmination and their differentiation into stalk cells. We propose a model whereby a protein repressor, under the control of PKA, inhibits precocious induction of stalk cell differentiation by DIF and so regulates the choice between slug migration and culmination.

Animals↗

PubMed1992

Mutation of protein kinase A causes heterochronic development of Dictyostelium.

In heterochronic mutants the relative timing of developmental events is altered compared with the wild type. This generally results in a disordered embryo, though heterochronic mutations may also be an important source of evolutionary variation. In the rapidly developing (rde) mutants of Dictyostelium, stalk and spore cells differentiate before morphogenesis is complete. We have traced the lesion in one class of these mutants to the regulatory subunit of cyclic AMP-dependent protein kinase (pk-A). Inactivation of this protein results in the unrestrained activity of the catalytic subunit, so prematurely triggering terminal cell differentiation.

Amino Acid Sequence↗

PubMed1992

Multiple roles for cAMP-dependent protein kinase during Dictyostelium development.

The cAMP-dependent protein kinase (PKA) holoenzyme of Dictyostelium comprises a single regulatory (R) and catalytic (C) subunit, and both proteins increase in concentration during cellular aggregation. In order to determine the role of the kinase, we have constructed mutants of the R subunit that are defective in cAMP binding, in inhibition of the C subunit, or in both functions. Analysis of these mutants suggests that overexpression of the unmutated R subunit, which is known to block development, occurs by direct inactivation of the C subunit rather than by an effect on intracellular cAMP levels. Cells with an inactive C subunit (PKA- cells) are defective in cAMP relay, the production of cAMP in response to extracellular cAMP stimulation. This presumably accounts for their inability to undertake aggregation. When mixed with wild-type cells, PKA- cells migrate toward the signalling centre but remain confined to the periphery of the tight aggregate and are lost from the back of the migratory slug. This suggests that PKA may be required during the late, multicellular stages of development. Consistent with this, we find that a number of postaggregative genes are not expressed in PKA- cells, even when they are allowed to synergise with normal cells.

Animals↗

PubMed1992

Proliferation-related expression of p19/nm23 nucleoside diphosphate kinase.

High level expression of the nm23-H1 gene, which encodes for a nucleoside diphosphate kinase, has been found to correlate with diminished metastasis in some tumors but not in others. We have previously identified the protein product of the nm23-H1 gene in two-dimensional electrophoretic gels and have designated it p19/nm23. In neuroblastoma, higher levels of p19/nm23, which are associated with amplification of the N-myc oncogene, large tumor mass, and metastasis, were observed in advanced stage tumors compared with limited stage disease. Because of the variable expression of nm23-H1 in different tumors, we have investigated the relationship between amounts of the protein and cell proliferation. The levels of p19/nm23 were compared between resting and mitotically stimulated normal human PBLs and in leukemia cells. The amount of p19/nm23 increased in normal lymphocytes in response to mitotic stimulation and paralleled the increase in DNA synthesis. In leukemia cells obtained from patients with different subtypes of acute leukemia, p19/nm23 levels were also increased relative to resting normal lymphocytes. Treatment of mitotically stimulated lymphocytes with cyclosporin, which inhibits proliferation, blocked the increase in p19/nm23; treatment of the leukemia cell line HL-60 with dimethylsulfoxide, which induces terminal differentiation, resulted in diminished levels of p19/nm23. Our data therefore provide evidence that nm23-H1 expression is related to cell proliferative activity.

Flow Cytometry↗

PubMed1992

[Survival of the human immunodeficiency virus (HIV-1) in Triatoma infestans (Klug, 1834)].

Triatoma infestans is the main domestic vector of Trypanosoma cruzi, the parasitic agent of Chagas' disease in South America. We investigated whether Triatoma infestans could shelter the HIV-1 virus. For this purpose, we measured the survival time of the virus in the alimentary tract. Fifth-instar nymphs of the blood-sucking bug were fed through an ad hoc apparatus with venous blood from asymptomatic HIV-1 seropositive patients. We attempted to evidence the virus by cultivating material from the insect gut (wall and content) on lymphocyte co-culture. Retrovirus activity was demonstrated in the culture supernatant by dosing the p24 antigen and the reverse transcriptase activity. The virus has been found alive in the gut content of Triatoma infestans up to the 7th day after the last infectious meal of the insect.

Animals↗

PubMed1990

Dictyostelium nucleoside diphosphate kinase highly homologous to Nm23 and Awd proteins involved in mammalian tumor metastasis and Drosophila development.

Two complementary DNAs (cDNAs) previously isolated, one by functional screening and the other by immunological screening of a Dictyostelium discoideum expression library, encode two proteins, Gip17 and Guk7.2, sharing 71% homology. In the present study, we found that the expression of their messenger RNAs (mRNAs) is developmentally regulated, with a sharp decrease during the first hours of differentiation. The Gip17 protein was purified to homogeneity from D. discoideum amoebas and from recombinant Escherichia coli and was conclusively identified as a nucleoside diphosphate (NDP) kinase. NDP kinases play a major role in synthesis of nucleoside triphosphates and, in many systems, are found associated with guanosine triphosphate (GTP)-binding proteins. We found the Gip17 protein to be 77% homologous to the human Nm23 protein and 75% homologous to the Drosophila melanogaster Awd protein. The levels of murine and human nm23 mRNA and Nm23 protein are significantly reduced in tumor cells of high metastatic potential, suggesting that Nm23 is involved in suppression of mammalian tumor metastasis, and mutants of the awd gene exhibit widespread development abnormalities, suggesting that Awd is involved in D. melanogaster development. The high percentage of homology of the Gip17 and Guk7.2 proteins with the Nm23 and Awd proteins indicates that Nm23 and Awd also have nucleoside diphosphate kinase activity. Possible modulations in the activity of this metabolic enzyme could be related to the altered metabolism of tumor cells and the control of metastatic potential. Our results point to an unexpected role of NDP kinase in development, growth control, and oncogenic transformation.

Amino Acid Sequence↗