Quinone reductase inhibitors block SAPK/JNK and NFkappaB pathways and potentiate apoptosis.
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A variety of environmental stresses stimulate the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEKK) > stress-activated protein kinase (SAPK)-ERK kinase (SEK) > SAPK/c-Jun NH(2)-terminal kinase (JNK) stress-activated protein kinase cascade and coordinately activate the transcription factor NFkappaB. Mechanisms of stress activation upstream of MEKK1 have not been precisely determined. Redox mechanisms involving sulfhydryls are likely because N-acetyl-cysteine at millimolar concentrations blocks stress signals. Because intracellular sulfhydryl concentrations can be regulated through redox cycling involving reactive quinones (1), we tested the ability of quinone reductase inhibitors to alter stress signaling. Several quinone reductases are inhibited by dicoumarol, a coumarin derivative. Dicoumarol prevented SAPK activation in vivo by chemical cell stressors and also prevented SAPK activation induced by expression of the tumor necrosis factor alpha (TNFalpha) receptor-associated protein TRAF2 but not by expression of truncated active MEKK1. Other coumarin derivatives failed to block SAPK activation, but other inhibitors of quinone reductases, particularly menadione, similarly blocked SAPK activation. Cells deficient in a major quinone reductase, NQO1, displayed hypersensitivity to dicoumarol stress inhibition, whereas SAPK in cells reconstituted with the NQO1 gene displayed relative dicoumarol resistance. Consistent with the proposed role of overlapping upstream signaling cascades in activation of NFkappaB, dicoumarol also blocked NFkappaB activation in primary macrophages stimulated with either lipopolysaccharide or TNFalpha. In addition, dicoumarol strongly potentiated TNFalpha-induced apoptosis in HeLa cells, probably by blocking the anti-apoptotic effect of NFkappaB. The ability of dicoumarol to simultaneously inhibit SAPK and NFkappaB activation and to potentiate apoptotic cell death suggests that SAPK is not an obligate participant in apoptosis. Dicoumarol, currently in clinical use as an oral anticoagulant, represents a potential therapeutic inhibitor of the SAPK and NFkappaB response. (+info)
Transcription-independent phosphorylation of the RNA polymerase II C-terminal domain (CTD) involves ERK kinases (MEK1/2).
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The largest subunit of the mammalian RNA polymerase II possesses a C-terminal domain (CTD) consisting of 52 repeats of the consensus sequence, Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7). Phosphorylation of the CTD is known to play a key role in gene expression. We now show that treatments such as osmotic and oxidative shocks or serum stimulation generate a new type of phosphorylated subunit, the IIm form. This IIm form might be generated in vivo by ERK-type MAP kinase phosphorylation as: (i) ERK1/2 are major CTD kinases found in cell extracts; (ii) the immunoreactivity of the IIm form against a panel of monoclonal antibodies indicates that the CTD is exclusively phosphorylated on Ser-5 in the repeats, like RNA polymerase II phosphorylated in vitro by an ERK1/2; and (iii) the IIm form does not appear when ERK activation is prevented by treating cells with low concentrations of highly specific inhibitors of MEK1/2. Since the IIm subunit is not affected by inhibition of transcription and is not bound to chromatin, it does not participate in transcription. (+info)
Effect of various ions, pH, and osmotic pressure on oxidation of elemental sulfur by Thiobacillus thiooxidans.
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The oxidation of elemental sulfur by Thiobacillus thiooxidans was studied at pH 2.3, 4.5, and 7.0 in the presence of different concentrations of various anions (sulfate, phosphate, chloride, nitrate, and fluoride) and cations (potassium, sodium, lithium, rubidium, and cesium). The results agree with the expected response of this acidophilic bacterium to charge neutralization of colloids by ions, pH-dependent membrane permeability of ions, and osmotic pressure. (+info)
Release of bFGF, an endothelial cell survival factor, by osmotic shock.
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PURPOSE: To test the effects of osmotic change on basic fibroblast growth factor (bFGF) release from cultured endothelial cells (ECs). METHODS: Bovine aortic and bovine retinal ECs were exposed to hypoosmotic shock for 2 minutes, were allowed to recover for 15 minutes, and had bFGF release assayed. The role of bFGF in cell recovery was assessed by including neutralizing antibody against bFGF or the addition of exogenous bFGF. Cell number and viability were determined under varying conditions. Apoptosis was assessed by immunoperoxidase detection of digoxigenin-labeled DNA. RESULTS: After shock and recovery, both ECs released significantly greater amounts of bFGF than untreated control. bFGF release after shock for 2 minutes was lower than release after shock and recovery. Bovine retinal endothelial (BRE) cell number was reduced at 48 hours after shock, recovery, and removal of released bFGF compared with cells left in the presence of released bFGF. Cell number was significantly lower when BRE cells were shocked and recovered in the presence of a neutralizing anti-bFGF antibody (P<0.05). Exogenous bFGF reversed this effect. Apoptosis was significantly increased in BRE cells shocked and recovered or in the presence of bFGF antibody (P<0.001). CONCLUSIONS: bFGF is released by cultured ECs in response to osmotically induced cell injury. These results support the concept of bFGF as a "wound" hormone and survival factor for ECs. In further compromised tissue, release of bFGF in this manner may play a role in the pathogenesis of disease. (+info)
Unitary exocytotic and endocytotic events in guard-cell protoplasts during osmotically driven volume changes.
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Osmotically driven swelling and shrinking of guard-cell protoplasts (GCPs) requires adjustment of surface area which is achieved by addition and removal of plasma membrane material. To investigate the mechanism for adaptation of surface area we have used patch-clamp capacitance measurements. The recorded membrane capacitance (C(m)) trace of swelling and shrinking GCPs occasionally revealed discrete upward and downward deflecting capacitance steps, respectively, with a median value of about 2 fF. The observed capacitance steps resulted from the fusion and fission of single vesicles with a diameter of around 300 nm. We conclude that exo- and endocytosis of these vesicles accommodate for osmotically driven surface area changes in GCPs. (+info)
Functional characterization of ARAKIN (ATMEKK1): a possible mediator in an osmotic stress response pathway in higher plants.
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The Arabidopsis thaliana ARAKIN (ATMEKK1) gene shows strong homology to members of the (MAP) mitogen-activated protein kinase family, and was previously shown to functionally complement a mating defect in Saccharomyces cerevisiae at the level of the MEKK kinase ste11. The yeast STE11 is an integral component of two MAP kinase cascades: the mating pheromone pathway and the HOG (high osmolarity glycerol response) pathway. The HOG signal transduction pathway is activated by osmotic stress and causes increased glycerol synthesis. Here, we first demonstrate that ATMEKK1 encodes a protein with kinase activity, examine its properties in yeast MAP kinase cascades, then examine its expression under stress in A. thaliana. Yeast cells expressing the A. thaliana ATMEKK1 survive and grow under high salt (NaCl) stress, conditions that kill wild-type cells. Enhanced glycerol production, observed in non-stressed cells expressing ATMEKK1 is the probable cause of yeast survival. Downstream components of the HOG response pathway, HOG1 and PBS2, are required for ATMEKK1-mediated yeast survival. Because ATMEKK1 functionally complements the sho1/ssk2/ssk22 triple mutant, it appears to function at the level of the MEKK kinase step of the HOG response pathway. In A. thaliana, ATMEKK1 expression is rapidly (within 5 min) induced by osmotic (NaCl) stress. This is the same time frame for osmoticum-induced effects on the electrical properties of A. thaliana cells, both an immediate response and adaptation. Therefore, we propose that the A. thaliana ATMEKK1 may be a part of the signal transduction pathway involved in osmotic stress. (+info)
The coronin-like protein POD-1 is required for anterior-posterior axis formation and cellular architecture in the nematode caenorhabditis elegans.
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Establishment of anterior-posterior (a-p) polarity in the Caenorhabditis elegans embryo depends on filamentous (F-) actin. Previously, we isolated an F-actin-binding protein that was enriched in the anterior cortex of the one-cell embryo and was hypothesized to link developmental polarity to the actin cytoskeleton. Here, we identify this protein, POD-1, as a new member of the coronin family of actin-binding proteins. We have generated a deletion within the pod-1 gene. Elimination of POD-1 from early embryos results in a loss of physical and molecular asymmetries along the a-p axis. For example, PAR-1 and PAR-3, which themselves are polarized and required for a-p polarity, are delocalized in pod-1 mutant embryos. However, unlike loss of PAR proteins, loss of POD-1 gives rise to the formation of abnormal cellular structures, namely large vesicles of endocytic origin, membrane protrusions, unstable cell divisions, a defective eggshell, and deposition of extracellular material. We conclude that, analogous to coronin, POD-1 plays an important role in intracellular trafficking and organizing specific aspects of the actin cytoskeleton. We propose models to explain how the role of POD-1 in basic cellular processes could be linked to the generation of polarity along the embryonic a-p axis. (+info)
1. We used whole-cell patch-clamp recording techniques and noise analysis of whole-cell current to investigate the properties of hyposmotic shock (HOS)-activated Cl- channels in SV40-transformed rabbit non-pigmented ciliary epithelial (NPCE) cells. 2. Under conditions designed to isolate Cl- currents, exposure of cells to hyposmotic external solution reversibly increased the whole-cell conductance. 3. The whole-cell current activated with a slow time course (> 15 min), exhibited outward rectification and was Cl- selective. 4. The disulphonic stilbene derivatives 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 0.5 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS, 0. 5 mM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS, 0.5 mM) produced a voltage-sensitive block of HOS-activated Cl- current at depolarized potentials, whereas niflumic acid produced a voltage-independent block of the current. 5. Under Ca2+-free conditions, HOS stimulation still reversibly activated the Cl- current, but the amplitude of current was reduced and the time course of current activation was slower compared with control (P < 0. 05). 6. The non-specific kinase inhibitor H-7 (100 microM), upregulated HOS-activated Cl- current amplitude in all cells tested (P < 0.05). 7. Noise analysis of whole-cell Cl- current indicated that cell swelling activated a high density of small conductance Cl- channels (< 1 pS). 8. We conclude that HOS primarily activates a high density of volume-sensitive small conductance Cl- channels in rabbit NPCE cells, and that Ca2+ and phosphorylation are involved in channel regulation. (+info)