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121.
122.
Arachidonic acid mediates dual effect of TNF-alpha on Ca2+ transients and contraction of adult rat cardiomyocytes 总被引:1,自引:0,他引:1
Amadou A Nawrocki A Best-Belpomme M Pavoine C Pecker F 《American journal of physiology. Cell physiology》2002,282(6):C1339-C1347
Tumor necrosisfactor (TNF)- has a biphasic effect on heart contractility andstimulates phospholipase A2 (PLA2) incardiomyocytes. Because arachidonic acid (AA) exerts a dual effect onintracellular Ca2+ concentration([Ca2+]i) transients, we investigated thepossible role of AA as a mediator of TNF- on[Ca2+]i transients and contraction withelectrically stimulated adult rat cardiac myocytes. At a lowconcentration (10 ng/ml) TNF- produced a 40% increase in theamplitude of both [Ca2+]i transients andcontraction within 40 min. At a high concentration (50 ng/ml) TNF-evoked a biphasic effect comprising an initial positive effect peakingat 5 min, followed by a sustained negative effect leading to50-40% decreases in [Ca2+]i transientsand contraction after 30 min. Both the positive and negative effects ofTNF- were reproduced by AA and blocked by arachidonyltrifluoromethylketone (AACOCF3), an inhibitor of cytosolic PLA2.Lipoxygenase and cyclooxygenase inhibitors reproduced the high-doseeffects of TNF- and AA. The negative effects of TNF- and AA werealso reproduced by sphingosine and were abrogated by the ceramidaseinhibitor n-oleoylethanolamine. These results point out thekey role of the cytosolic PLA2/AA pathway in mediating thecontractile effects of TNF-. 相似文献
123.
Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum 总被引:1,自引:0,他引:1
A large cellulosomal gene cluster was identified in the recently sequenced genome of Clostridium acetobutylicum ATCC 824. Sequence analysis revealed that this cluster contains the genes for the scaffolding protein CipA, the processive endocellulase Cel48A, several endoglucanases of families 5 and 9, the mannanase Man5G, and a hydrophobic protein, OrfXp. Surprisingly, genetic organization of this large cluster is very similar to that of Clostridium cellulolyticum, the model of mesophilic clostridial cellulosomes. As C. acetobutylicum is unable to grow on cellulosic substrates, the existence of a cellulosomal gene cluster in the genome raises questions about its expression, function and evolution. Biochemical evidence for the expression of a cellulosomal protein complex was investigated. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing and Western blotting with antibodies against specific components of the C. cellulolyticum cellulosome suggest that at least four major cellulosomal proteins are present. In addition, despite the fact that no cellulolytic activities were detected, we report here the evidence for the production of a high molecular mass cellulosomal complex in C. acetobutylicum. 相似文献
124.
Interaction between two ubiquitin-protein isopeptide ligases of different classes,CBLC and AIP4/ITCH
Courbard JR Fiore F Adélaïde J Borg JP Birnbaum D Ollendorff V 《The Journal of biological chemistry》2002,277(47):45267-45275
In metazoans, CBL proteins are RING finger type ubiquitin-protein isopeptide (E3) ligases involved in the down-regulation of epidermal growth factor tyrosine kinase receptors (EGFR). Among the three CBL proteins described in humans, CBLC (CBL3) remains poorly studied. By screening in parallel a human and a Caenorhabditis elegans library using the two-hybrid procedure in yeast, we found a novel interaction between Hsa-CBLC and Hsa-AIP4 or its C. elegans counterpart Cel-WWP1. Hsa-AIP4 and Cel-WWP1 are also ubiquitin E3 ligases. They contain a HECT (homologous to E6-AP C terminus) catalytic domain and four WW domains known to bind proline-rich regions. We confirmed the interaction between Hsa-CBLC and Hsa-AIP4 by a combination of glutathione S-transferase pull-down, co-immunoprecipitation, and colocalization experiments. We show that these two E3 ligases are involved in EGFR signaling because both become phosphorylated on tyrosine following epidermal growth factor stimulation. In addition, we observed that CBLC increases the ubiquitination of EGFR, and that coexpressing the WW domains of AIP4 exerts a dominant negative effect on EGFR ubiquitination. Finally, coexpressing CBLC and AIP4 induces a down-regulation of EGFR signaling. In conclusion, our data demonstrate that two E3 ligases of different classes can interact and cooperate to down-regulate EGFR signaling. 相似文献
125.
Maïsterrena B Fiaty K Charcosset C Perrin B Couturier R West IC 《Progress in biophysics and molecular biology》2002,80(3):2288-137
Coupled interrelations occurring between a phosphatase/kinase reaction sequence acting in unstirred layers and on both sides of a charged biomembrane pore structure are presented as a plausible kinetic model for the primary active transport of phosphorylated molecules. Simulations conducted at the cell level and with credible numerical values demonstrate that the enzymes positions strongly regulate the membrane permeability for the transported substrate. Depending on both the enzymes positions (more or less far from the membrane) and the membrane charges, the membrane may appear either impervious, either permeable or able to actively transport a phosphorylated substrate. Globally all happens as if, in function of the enzymes positions, a permanent pore may be regulated, changing from a more closed to a more open conformation. 相似文献
126.
Sphingolipids represent a minor, but highly dynamic subclass of lipids in all eukaryotic cells. They are involved in functions that range from structural protection to signal transduction and protein sorting, and participate in lipid raft assembly. In polarized epithelial cells, which display an asymmetric apical and basolateral membrane surface, rafts have been proposed as a sorting principle for apical resident proteins, following their biosynthesis. However, raft-mediated trafficking is ubiquitous in cells. Also, sphingolipids per se, which are strongly enriched in the apical domain, are subject to sorting in polarity development. Next to the trans Golgi network, a subapical compartment called SAC or common endosome appears instrumental in regulating these sorting events. 相似文献
127.
Na self inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases
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The regulation of the open probability of the epithelial Na(+) channel (ENaC) by the extracellular concentration of Na(+), a phenomenon called "Na(+) self inhibition," has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na(+) self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na(+) concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q(10)act = 1.5) activation rate and a strongly temperature-dependant (Q(10)inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na(+) self inhibition depended only on the extracellular Na(+) concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na(+) as well as a reduction of Na(+) outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na(+) self inhibition is an intrinsic property of sodium channels resulting from the expression of the alpha, beta, and gamma subunits of human ENaC in Xenopus oocyte. The extracellular Na(+)-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases. 相似文献
128.
Relationship between SU subdomains that regulate the receptor-mediated transition from the native (fusion-inhibited) to the fusion-active conformation of the murine leukemia virus glycoprotein
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![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Envelope glycoproteins (Env) of retroviruses are trimers of SU (surface) and TM (transmembrane) heterodimers and are expressed on virions in fusion-competent forms that are likely to be metastable. Activation of the viral receptor-binding domain (RBD) via its interaction with a cell surface receptor is thought to initiate a cascade of events that lead to refolding of the Env glycoprotein into its stable fusion-active conformation. While the fusion-active conformation of the TM subunit has been described in detail for several retroviruses, little is known about the fusion-competent structure of the retroviral glycoproteins or the molecular events that mediate the transition between the two conformations. By characterizing Env chimeras between the ecotropic and amphotropic murine leukemia virus (MLV) SUs as well as a set of point mutants, we show that alterations of the conformation of the SU glycoprotein strongly elevate Env fusogenicity by disrupting the stability of the Env complex. Compensatory mutations that restored both Env stability and fusion control were also identified, allowing definition of interactions within the Env complex that maintain the stability of the native Env complex. We show that, in the receptor-unbound form, structural interactions between the N terminus of the viral RBD (NTR domain), the proline-rich region (PRR), and the distal part of the C-terminal domain of the SU subunit maintain a conformation of the glycoprotein that is fusion inhibitory. Additionally, we identified mutations that disrupt this fusion-inhibitory conformation and allow fusion activation in the absence of viral receptors, provided that receptor-activated RBD fragments are added in trans during infection. Other mutations were identified that allow fusion activation in the absence of receptors for both the viral glycoprotein and the trans-acting RBD. Finally, we found mutations of the SU that bypass in cis the requirement for the NTR domain in fusion activation. All these different mutations call for a critical role of the PRR in mediating conformational changes of the Env glycoprotein during fusion activation. Our results suggest a model of MLV Env fusion activation in which unlocking of the fusion-inhibitory conformation is initiated by receptor binding of the viral RBD, which, upon disruption of the PRR, allows the NTR domain to promote further events in Env fusion activation. This involves a second type of interaction, in cis or in trans, between the receptor-activated RBD and a median segment of the freed C-terminal domain. 相似文献
129.
Gontier E Cario-André M Lepreux S Vergnes P Bizik J Surlève-Bazeille JE Taïeb A 《Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society》2002,15(1):41-48
Congenital nevi are composed of pigment cells bearing common features with melanocytes but showing altered differentiation which leads to nesting and dermal involvement. Using a dead de-epidermized dermis seeded with a combination of keratinocytes and various sources of pigment cells (normal melanocytes, dermal nevus cells from congenital nevi, Bowes melanoma cells), we have studied the formation of nests and the dermal migration of pigment cells together with their secretion profiles of matrix metalloproteinases (MMP). Dermal fibroblasts were also used as control cells in epidermal reconstructs. Besides their morphologic features, the absence of pigment donation to keratinocytes was the major characteristic of dermal nevus cells. A positive correlation was established between the increasing percentage of seeded nevus cells and the patchy pigmentation of reconstructs, as well as the clustering of cells in junctional nests. However, the presence of nevus cells in the dermis of reconstructs was never detected, whereas melanoma cells and dermal fibroblasts could invade the dermis during the time span of the experiments. MMP9 was never expressed in congenital dermal nevus cells but pro-MMP2 was constitutively expressed by all strains of congenital nevus cells and dermal fibroblasts. Melanocytes produced comparable amounts of both pro-MMP2 and pro-MMP9, and Bowes melanoma cells secreted a marginal level of pro-MMP2. In view of their three-dimensional behaviour and secretion of MMPs, we propose that dermal congenital nevus cells correspond to an intermediate status of differentiation between normal melanocytes and melanoma cells. Activation of MMPs by a cofactor or the activation of another signalling pathway seems necessary to induce the dermal passage of nevus cells. 相似文献
130.