BBA - Molecular Cell Research (v.1495, #2)
Inhibition of phosphatidylinositol 4,5-bisphosphate-stimulated phospholipase D2 activity by Ser/Thr phosphorylation 1 This work was supported in part by research grants from the Ministry of Education, Science, Sports and Culture, Japan. 1 by Hiroshi Watanabe; Yasunori Kanaho (121-124).
Treatment of HeLa cells overexpressing PLD2 with the Ser/Thr-specific protein phosphatase inhibitor, okadaic acid, augmented spontaneous phosphorylation of PLD2 with concomitant inhibition of phosphatidylinositol 4,5-bisphosphate (PIP2)-stimulated PLD2 activity. Dephosphorylation of the immunoprecipitated, spontaneously phosphorylated PLD2 in COS-7 cells by catalytic subunit of protein phosphatase 1γ1 resulted in the stimulation of the PLD2 catalytic activity. These observations suggest that Ser/Thr phosphorylation regulates PLD2 activity.
Keywords: Phospholipase D; Phosphorylation; Okadaic acid;
A calcium-inhibited Drosophila adenylyl cyclase by Vadim Iourgenko; Lonny R Levin (125-139).
Mammals possess a family of transmembrane, G-protein-responsive adenylyl cyclase isoforms (tmACs) encoded by distinct genes differing in their patterns of expression and modes of biochemical regulation. Our previous work confirmed that Drosophila melanogaster also possesses a family of tmAC isoforms defining the fly as a suitable genetic model for discerning mammalian tmAC function. We now describe a Drosophila tmAC, DAC39E, which employs a novel means for regulating its expression; differential exon utilization results in a developmental switch in DAC39E protein. DAC39E protein sequence is most closely related to mammalian type III AC, and it is predominantly expressed in the central nervous system (CNS) and olfactory organs, suggesting a role in processing sensory signaling inputs. DAC39E catalytic activity is inhibited by micromolar concentrations of calcium; therefore, DAC39E is oppositely regulated by calcium compared to the only other tmAC shown to be expressed in the Drosophila CNS, Rutabaga AC. The presence of both positively and negatively regulated tmACs suggests a complex mode of cross-talk between cAMP and calcium signal transduction pathways in the fly CNS.
Keywords: G-protein; Alternative splicing; Transmembrane adenylyl cyclase;
Reconstitution of Listeria motility: implications for the mechanism of force transduction by Donald J Olbris; Judith Herzfeld (140-149).
Listeria monocytogenes and some other infectious bacteria polymerize their host cell’s actin into tails that propel the bacteria through the cytoplasm. Here we show that reconstitution of this behavior in simpler media resolves two aspects of the mechanism of force transduction. First, since dilute reconstitution media have no cytoskeleton, we consider what keeps the tail from being pushed backward rather than the bacterium being propelled forward. The dependence of the partitioning of motion on the friction coefficient of the tail is derived. Consistent with experiments, we find that the resistance of the tail to motion is sensitive to its length. That even small tails are stationary in intact cells is attributed to anchoring to the cytoskeleton. Second, the comparatively low viscosity of some reconstitution media magnifies the effects of diffusion, such that a large gap will develop between the bacterium and its tail if they are unattached. At the viscosities of diluted platelet extracts, steady-state gaps of several bacterium lengths are predicted. Since such gaps are not observed, we conclude that Listeria must be attached to their tails. We consider what purposes such attachments might serve under physiological conditions. The implications for related pathogens and amoeboid locomotion are also discussed.
Keywords: Listeria; Motility; Actin; Shigella; Ascaris;
Effects of hypoxia and glutathione depletion on hemoglobin- and myoglobin-mediated oxidative stress toward endothelium 1 The opinions and assertions contained herein are the scientific views of the authors and are not to be construed as policy of the United States Food and Drug Administration or the United States Army. 1 by Felice D’Agnillo; Francine Wood; Carlos Porras; Victor W. Macdonald; Abdu I. Alayash (150-159).
We investigated the toxicity of hemoglobin/myoglobin on endothelial cells under oxidative stress conditions that include cellular hypoxia and reduced antioxidant capacity. Bovine aorta endothelial cells (BAECs), grown on microcarrier beads, were subjected to cycles of hypoxia and reoxygenation in a small volume of medium, and endothelial cell monolayers were depleted of their intracellular glutathione (GSH) by treatment with buthionine sulfoximine. Incubation of diaspirin cross-linked hemoglobin (DBBF-Hb) or horse skeletal myoglobin (Mb) with BAECs subjected to 3 h of hypoxia caused transient oxidation of the hemoproteins to the ferryl form (Fe4+). Formation of the ferryl intermediate was decreased in a concentration-dependent manner by the addition of L-arginine, a substrate of NO synthase, after 3 h of hypoxia. Optimal inhibition of ferryl formation, possibly due to the antioxidant action of NO, was achieved with 900 μM L-arginine. Addition of hydrogen peroxide to GSH-depleted cells in the presence of DBBF-Hb or Mb significantly decreased cell viability. Ferryl Mb, but not ferryl DBBF-Hb, was observed in samples analyzed at the end of treatment, which may explain the greater toxicity observed with Mb as opposed to DBBF-Hb. This model may be utilized to identify causative agent(s) associated with hemoprotein cytotoxicity and in designing strategies to suppress or control heme-mediated injury under physiologically relevant conditions.
Keywords: Hemoglobin; Myoglobin; Hypoxia; Oxidative stress; Glutathione; Endothelium;
Inhibition of hyaluronan synthesis by vesnarinone in cultured human myofibroblasts by Noboru Ueki; Tomohiro Taguchi; Masayuki Takahashi; Masakazu Adachi; Toshihisa Ohkawa; Yoshiki Amuro; Toshikazu Hada; Kazuya Higashino (160-167).
Hyaluronan (HA), which is a major component of the extracellular matrix (ECM), is regulated during myofibroproliferative responses to numerous forms of inflammatory stimuli. It is a key factor involved in cellular migration and adherence. The development of a potent and non-toxic inhibitor of HA synthesis would open up a new avenue for the treatment of fibrocontractive diseases such as pulmonary fibrosis and liver cirrhosis. In this study, the effects of vesnarinone (OPC-8212: 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone) on the secretion of HA in human myofibroblast cell lines (MRC-5 and LI90 cells, referred to as pulmonary and hepatic myofibroblasts, respectively) were examined. Vesnarinone specifically and dose-dependently inhibited HA secretion by myofibroblasts up-regulated by fetal calf serum (FCS). The treatment of vesnarinone did not modify the phenotype of myofibroblast cells in culture. Vesnarinone also potently inhibited the HA secretion by the two myofibroblast cell lines up-regulated by transforming growth factor-β1 (TGF-β1) or tumor necrosis factor-α (TNF-α). The addition of vesnarinone to myofibroblasts resulted in a significant decrease of HA synthase (HAS) activity, with or without the addition of FCS or either cytokine. These findings suggest that vesnarinone inhibits the secretion of HA in myofibroblasts by specifically suppressing HAS activity, and may therefore prove useful for the treatment of chronic inflammation and tissue fibrosis.
Keywords: Vesnarinone; Hyaluronan; Hyaluronan synthase; Transforming growth factor-β; Tumor necrosis factor-α; Myofibroblast;
Biochemical interactions in the wnt pathway by Martin J. Seidensticker; Jürgen Behrens (168-182).
The wnt signal transduction pathway is involved in many differentiation events during embryonic development and can lead to tumor formation after aberrant activation of its components. The cytoplasmic component β-catenin is central to the transmission of wnt signals to the nucleus: in the absence of wnts β-catenin is constitutively degraded in proteasomes, whereas in the presence of wnts β-catenin is stabilized and associates with HMG box transcription factors of the LEF/TCF family. In tumors, β-catenin degradation is blocked by mutations of the tumor suppressor gene APC (adenomatous polyposis coli), or of β-catenin itself. As a consequence, constitutive TCF/β-catenin complexes are formed and activate oncogenic target genes. This review discusses the mechanisms that silence the pathway in cells that do not receive a wnt signal and goes on to describe the regulatory steps involved in the activation of the pathway.
Keywords: Wnt signal transduction; Tumor development; Adenomatous polyposis coli; β-Catenin; TCF family; Gene expression;
H2O2 activity on platelet adhesion to fibrinogen and protein tyrosine phosphorylation by Maria Antonietta Belisario; Simona Tafuri; Carmela Di Domenico; Caterina Squillacioti; Rossella Della Morte; Antonia Lucisano; Norma Staiano (183-193).
Platelets represent a target of reactive oxygen species produced under oxidative stress conditions. Controversial data on the effect of these species on platelet functions have been reported so far. In this study we evaluated the effect of a wide range of H2O2 concentrations on platelet adhesion to immobilized fibrinogen and on pp72syk and pp125FAK tyrosine phosphorylation. Our results demonstrate that: (1) H2O2 does not affect the adhesion of unstimulated or apyrase-treated platelets to immobilized fibrinogen; (2) H2O2 does not affect pp72syk phosphorylation induced by platelet adhesion to fibrinogen-coated dishes; (3) H2O2 reduces, in a dose-dependent fashion, pp125FAK phosphorylation of fibrinogen-adherent platelets; (4) concentrations of H2O2 near to physiological values (10–12 μM) are able to strengthen the subthreshold activation of pp125FAK induced by epinephrine in apyrase-treated platelets; (5) H2O2 doses higher than 0.1 mM inhibit ADP-induced platelet aggregation and dense granule secretion. The ability of H2O2 to modulate pp125FAK phosphorylation suggests a role of this molecule in physiological hemostasis as well as in thrombus generation.
Keywords: H2O2; Platelet adhesion; Protein phosphorylation; pp72syk; pp125FAK;