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Fioretta Rambelli Maurizio Brigotti Simonetta Sperti Lucio Montanaro 《Bioscience reports》1987,7(9):737-743
Diphtheria toxin fragment A interacts with Cibacron blue in solution, although it is not retained by blue Sepharose columns. Difference spectral titration of fragment A with the dye gives a dissociation constant of the order of 10–5 M and a 11 stoichiometry for the complex. In equilibrium dialysis experiments Cibacron blue behaves as a competitive inhibitor of the binding of NAD to diphtheria toxin fragment A. The dye inhibits in a non-competitive way the fragment A-catalysed transfer of ADP-ribose from NAD to elongation factor 2 (EF2). By affinity chromatography on blue Sepharose a binding of EF2 and of ADP-ribosyl-EF2 with the dye is also demonstrated. GDP, GTP and GDP(CH2)P are able to displace EF2 from blue Sepharose. 相似文献
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1. Changes in protein fluorescence have been utilized in determining the stoicheiometry and dissociation constants of the complexes of diphtheria toxin with NADH(2), NAD, NADPH(2) and NADP. 2. The binding stoicheiometry is 2moles of NADH(2) and 1mole of NADPH(2)/mole of diphtheria toxin. The binding sites for NADH(2) appear to be equivalent and independent. 3. The toxin shows a higher affinity for the reduced than for the oxidized forms of the nucleotides. 4. Dissociation constants at 0.01I, pH7 and 25 degrees are 0.7x10(-6)m for NADH(2) and 0.45x10(-6)m for NADPH(2). Dissociation constants increase with increasing ionic strength, indicating that the binding is mainly electrostatic. 5. Bound NADH(2) and NADPH(2) may be activated to fluoresce by the transfer of energy from the excited aromatic amino acids of the toxin. Activation and emission spectra of bound and free nucleotides are compared. 6. Since NAD and NADH(2) are cofactors specifically required for the inhibition of protein synthesis by diphtheria toxin, the possible role of toxin-nucleotide complexes is discussed in this regard. 相似文献
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We studied the effect of heparin on proliferation and signalling in normal NIH/3T3 fibroblasts, and in cells transformed by different oncogenes. Heparin inhibited the proliferation of normal as well as of v-sis and v-erbB transformed fibroblasts in the presence of serum, but failed to inhibit v-erbB-driven proliferation in serum-starved cultures; under these conditions, heparin inhibited by approximately 50% the proliferation of normal and v-sis- transformed cells. Heparin also inhibited PDGF-induced cell proliferation and inositol lipid turnover in v-sis transformants, but it did not affect PDGF mitogenic signalling in NIH/3T3 fibroblasts. 相似文献
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Spontaneous Cell Fusion in Macrophage Cultures Expressing High Levels of the P2Z/P2X7 Receptor 总被引:6,自引:0,他引:6 下载免费PDF全文
Paola Chiozzi Juana M. Sanz Davide Ferrari Simonetta Falzoni Arrigo Aleotti Gary N. Buell Ginetta Collo Francesco Di Virgilio 《The Journal of cell biology》1997,138(3):697-706
Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X7. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X7 receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207– 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X7 receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X7 blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X7 receptor is involved in macrophage fusion. 相似文献
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Summary The evidence that the Cd technique identifies the kinetochore was based on the finding that inactive centromeres are C-positive but Cd-negative. The identity between Cd-positivity and centromere function is now confirmed by the reverse procedure: a stable abnormal chromosome is consistently C-negative but Cd-positive at its single centromeric constriction. This demonstrates that the Cd dots are not a relic of C-banding but identify the active centromere. 相似文献
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Aldo Scarpa Katarzyna Sikora Matteo Fassan Anna Maria Rachiglio Rocco Cappellesso Davide Antonello Eliana Amato Andrea Mafficini Matilde Lambiase Claudia Esposito Emilio Bria Francesca Simonato Maria Scardoni Giona Turri Marco Chilosi Giampaolo Tortora Ambrogio Fassina Nicola Normanno 《PloS one》2013,8(11)
Identification of driver mutations in lung adenocarcinoma has led to development of targeted agents that are already approved for clinical use or are in clinical trials. Therefore, the number of biomarkers that will be needed to assess is expected to rapidly increase. This calls for the implementation of methods probing the mutational status of multiple genes for inoperable cases, for which limited cytological or bioptic material is available. Cytology specimens from 38 lung adenocarcinomas were subjected to the simultaneous assessment of 504 mutational hotspots of 22 lung cancer-associated genes using 10 nanograms of DNA and Ion Torrent PGM next-generation sequencing. Thirty-six cases were successfully sequenced (95%). In 24/36 cases (67%) at least one mutated gene was observed, including EGFR, KRAS, PIK3CA, BRAF, TP53, PTEN, MET, SMAD4, FGFR3, STK11, MAP2K1. EGFR and KRAS mutations, respectively found in 6/36 (16%) and 10/36 (28%) cases, were mutually exclusive. Nine samples (25%) showed concurrent alterations in different genes. The next-generation sequencing test used is superior to current standard methodologies, as it interrogates multiple genes and requires limited amounts of DNA. Its applicability to routine cytology samples might allow a significant increase in the fraction of lung cancer patients eligible for personalized therapy. 相似文献