Rho-dependent cell spreading activated by E.coli cytotoxic necrotizing factor 1 hinders apoptosis in epithelial cells

Cell-cell and cell-matrix interactions play a pivotal role in numerous cell functions including cell survival and death. In this work, we report evidence that the Rho-dependent cell spreading activated by a protein toxin from E. coli, the cytotoxic necrotizing factor 1 (CNF1), is capable of hindering apoptosis in HEp-2 cells. In addition to the promotion of cell spreading, CNF1 protects cells from the experimentally-induced rounding up and detachment and improves the ability of cells to adhere to each other and to the extracellular matrix by modulating the expression of proteins related to cell adhesion. In particular, the expression of integrins such as alpha 5, alpha 6 and alpha v, as well as of some heterotypic and homotypic adhesion-related proteins such as the Focal Adhesion Kinase, E-cadherin, alpha and beta catenins were significantly increased in cells exposed to CNF1. Our results suggest, however, that the promotion of Rho-dependent cell spreading is the key mechanism in protecting cells against apoptosis rather than cell adhesion per se. A toxin inducing cell spreading without activating Rho, such as Cytochalasin B, was in fact ineffective in favouring cell survival. These data are of relevance (i) for the understanding of the role of the actin-dependent and especially Rho-dependent cellular activities involved in apoptosis regulation and (ii) in providing some clues to understanding the mechanisms by which bacteria, by controlling cell fate, might exert their pathogenic activity.     

The NO x Reduction Mechanism by H2 under near Isothermal Conditions over Pt–K/Al2O3 Lean NO x Trap Systems

The reduction of NO _x stored over a Pt–K/Al₂O₃ Lean NO _x Trap is analyzed when H₂ is used as reductant. An in series 2-steps process is herein proposed, involving at first the formation of NH₃ upon reaction of nitrates with H₂ (step 1), followed by the reaction of NH₃ with residual nitrates to give N₂ (step 2).     

Dynamics and Selectivity of N<Subscript>2</Subscript>O Formation/Reduction During Regeneration Phase of Pt-Based Catalysts

The formation of N₂O has been studied by means of isothermal lean-rich experiments at 150, 180 and 250 °C over Pt–Ba/Al₂O₃ and Pt/Al₂O₃ catalysts with H₂ and/or C₃H₆ as reductants. This allows to provide further insights on the mechanistic aspects of N₂O formation and on the influence of the storage component. Both gas phase analysis and surface species studies by operando FT-IR spectroscopy were performed. N₂O evolution is observed at both lean-to-rich (primary N₂O) and rich-to-lean (secondary N₂O) transitions. The production of both primary and secondary N₂O decreases by increasing the temperature. The presence of Ba markedly decreases secondary N₂O formation. FT-IR analysis shows the presence of adsorbed ammonia at the end of the rich phase only for Pt/Al₂O₃ catalyst. These results suggest that: (i) primary N₂O is formed when undissociated NO in the gas phase and partially reduced metal sites are present; (ii) secondary N₂O originates from reaction between adsorbed NH₃ and residual NO_x at the beginning of the lean phase. Moreover, N₂O reduction was studied performing temperature programming temperature experiments with H₂, NH₃ and C₃H₆ as reducing agents. The reduction is completely selective to nitrogen and occurs at temperature higher than 250 °C in the case of Pt–Ba/Al₂O₃ catalyst, while lower temperatures are detected for Pt/Al₂O₃ catalyst. The reactivity order of the reductants is the same for the two catalysts, being hydrogen the more efficient and propylene the less one. Having H₂ a high reactivity in the reduction of N₂O, it could react with N₂O when the regeneration front is developing. Moreover, also ammonia present downstream to the H₂ front could react with N₂O, even if the reaction with stored NO_x seems more efficient.     

High performances of Pt-K/Al2O3 versus Pt-Ba/Al2O3 LNT catalysts in the simultaneous removal of NO x and soot

The potentiality of a Pt-K/Al₂O₃ catalyst in the simultaneous removal of particulate matter (soot) and NO _x is investigated in this work by means of Temperature Programmed Oxidation (TPO) experiments and Transient Response Method (TRM), and compared with Pt-Ba/Al₂O₃. The results point out the higher performances of K-based sample in the soot combustion as compared to the Ba-based catalyst, and similar behaviour in the NO _x -storage.     

Escherichia coli cytotoxic necrotizing factor 1 (CNF1), a toxin that activates the Rho GTPase

Cytotoxic necrotizing factor 1 (CNF1), a 110-kDa protein toxin from pathogenic Escherichia coli induces actin reorganization into stress fibers and retraction fibers in human epithelial cultured cells allowing them to spread. CNF1 is acting in the cytosol since microinjection of the toxin into HEp-2 cells mimics the effects of the externally applied CNF1. Incubation in vitro of CNF1 with recombinant small GTPases induces a modification of Rho (but not of Rac, Cdc42, Ras, or Rab6) as demonstrated by a discrete increase in the apparent molecular weight of the molecule. Preincubation of cells with CNF1 impairs the cytotoxic effects of Clostridium difficile toxin B, which inactivates Rho but not those of Clostridium sordellii LT toxin, which inhibits Ras and Rac. As shown for Rho-GTP, CNF1 activates, in a time- and dose-dependent manner, a cytoskeleton-associated phosphatidylinositol 4-phosphate 5-kinase. However, neither the phosphatidylinositol 4,5-bisphosphate (PIP2) nor the phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) or 3,4,5-trisphosphate (PIP3) cellular content were found increased in CNF1 treated HEp-2 cells. Cellular effects of CNF1 were not blocked by LY294002, a stable inhibitor of the phosphoinositide 3-kinase. Incubation of HEp-2 cells with CNF1 induces relocalization of myosin 2 in stress fibers but not in retraction fibers. Altogether, our data indicate that CNF1 is a toxin that selectively activates the Rho GTP-binding protein, thus inducing contractility and cell spreading.     

Crosstalk between β2- and α2-Adrenergic Receptors in the Regulation of B16F10 Melanoma Cell Proliferation

Adrenergic receptors (AR) belong to the G protein-coupled receptor superfamily and regulate migration and proliferation in various cell types. The objective of this study was to evaluate whether β-AR stimulation affects the antiproliferative action of α2-AR agonists on B16F10 cells and, if so, to determine the relative contribution of β-AR subtypes. Using pharmacological approaches, evaluation of Ki-67 expression by flow cytometry and luciferase-based cAMP assay, we found that treatment with isoproterenol, a β-AR agonist, increased cAMP levels in B16F10 melanoma cells without affecting cell proliferation. Propranolol inhibited the cAMP response to isoproterenol. In addition, stimulation of α2-ARs with agonists such as clonidine, a well-known antihypertensive drug, decreased cancer cell proliferation. This effect on cell proliferation was suppressed by treatment with isoproterenol. In turn, the suppressive effects of isoproterenol were abolished by the treatment with either ICI 118,551, a β2-AR antagonist, or propranolol, suggesting that isoproterenol effects are mainly mediated by the β2-AR stimulation. We conclude that the crosstalk between the β2-AR and α2-AR signaling pathways regulates the proliferative activity of B16F10 cells and may therefore represent a therapeutic target for melanoma therapy.     

Effect of potassium on a model soot combustion: Raman and HRTEM evidences

The effect of potassium on the oxidation of a model carbonaceous material (Printex U, namely, soot for brevity) has been investigated under isothermal conditions. For this purpose, Raman spectroscopy, Transmission Electron Microscopy (TEM), and Brunaure, Emmet, Teller surface area characterization have been applied to investigate structural changes occurring during soot oxidation both in the presence and in the absence of potassium. The Raman spectra of the model soot during combustion showed that oxidation preferentially involves the amorphous carbon fraction of the soot and only subsequently it affects the more ordered sp² domains. However, in the K-doped Printex U the oxidation of both the amorphous and more ordered sp² structures occurs concurrently. These findings have been confirmed by TEM analysis and explain the observed higher combustion activity of K-containing sample.

Copyright © 2016 American Association for Aerosol Research     

Photo-electrochemical behavior at different wavelengths of electrochemically obtained TiO<Subscript>2</Subscript> nanotubes

The results of an experimental study on the photo electrochemical behavior of nanotubular TiO₂ structures are presented in this work. TiO₂ samples were prepared by electrochemical anodization of Ti foils and submitted to thermal annealing. The influence of the current transient during the anodization, and of the annealing temperature on the photochemical response of the samples at different wavelengths was studied. Different behavior of the samples was observed, which may be attributed to the distributions of defects and to their different sensitivity to the temperature. The analysis of the performance of the samples in absence or in the presence of glycerol, used as hole scavenger, provided more information on the photo-catalytic properties of these structures.