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Find video protocols related to scientific articles indexed in Pubmed.
StAR enhances transcription of genes encoding the mitochondrial proteases involved in its own degradation.
Mol. Endocrinol.
PUBLISHED: 01-16-2014
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Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR.
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Infarct-induced steroidogenic acute regulatory protein: a survival role in cardiac fibroblasts.
Mol. Endocrinol.
PUBLISHED: 07-05-2013
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Steroidogenic acute regulatory protein (StAR) is indispensable for steroid hormone synthesis in the adrenal cortex and the gonadal tissues. This study reveals that StAR is also expressed at high levels in nonsteroidogenic cardiac fibroblasts confined to the left ventricle of mouse heart examined 3 days after permanent ligation of the left anterior descending coronary artery. Unlike StAR, CYP11A1 and 3?-hydroxysteroid dehydrogenase proteins were not observed in the postinfarction heart, suggesting an apparent lack of de novo cardiac steroidogenesis. Work with primary cultures of rat heart cells revealed that StAR is induced in fibroblasts responding to proapoptotic treatments with hydrogen peroxide or the kinase inhibitor staurosporine (STS). Such induction of StAR in culture was noted before spontaneous differentiation of the fibroblasts to myofibroblasts. STS induction of StAR in the cardiac fibroblasts conferred a marked resistance to apoptotic cell death. Consistent with that finding, down-regulation of StAR by RNA interference proportionally increased the number of STS-treated apoptotic cells. StAR down-regulation also resulted in a marked increase of BAX activation in the mitochondria, an event known to associate with the onset of apoptosis. Last, STS treatment of HeLa cells showed that apoptotic demise characterized by mitochondrial fission, cytochrome c release, and nuclear fragmentation is arrested in individual HeLa cells overexpressing StAR. Collectively, our in vivo and ex vivo evidence suggests that postinfarction expression of nonsteroidogenic StAR in cardiac fibroblasts has novel antiapoptotic activity, allowing myofibroblast precursor cells to survive the traumatized event, probably to differentiate and function in tissue repair at the infarction site.
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Real-time sensing of enteropathogenic E. coli-induced effects on epithelial host cell height, cell-substrate interactions, and endocytic processes by infrared surface plasmon spectroscopy.
PLoS ONE
PUBLISHED: 01-01-2013
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Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cells height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity.
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Cell autonomous and cell-type specific circadian rhythms in Arabidopsis.
Plant J.
PUBLISHED: 09-26-2011
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The circadian system of plants regulates a wide range of rhythmic physiological and cellular output processes with a period of about 24?h. The rhythms are generated by an oscillator mechanism that, in Arabidopsis, consists of interlocking feedback loops of several components including CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1) and CCA1 HIKING EXPEDITION (CHE). Over recent years, researchers have gained a detailed picture of the clock mechanism at the resolution of the whole plant and several tissue types, but little information is known about the specificities of the clock mechanism at the level of individual cells. In this paper we have addressed the question of cell-type-specific differences in circadian systems. Using transgenic Arabidopsis plants with fluorescence-tagged CCA1 to measure rhythmicity in individual leaf cells in intact living plants, we showed that stomatal guard cells have a different period from surrounding epidermal and mesophyll leaf cells. By comparing transcript levels in guard cells with whole plants, we identified differences in the expression of some oscillator genes that may underlie cell-specific differences in clock properties. In addition, we demonstrated that the oscillators of individual cells in the leaf are robust, but become partially desynchronized in constant conditions. Taken together our results suggest that, at the level of individual cells, there are differences in the canonical oscillator mechanism that has been described for plants.
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Activation of the alternative NF?B pathway improves disease symptoms in a model of Sjogrens syndrome.
PLoS ONE
PUBLISHED: 08-25-2011
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The purpose of our study was to understand if Toll-like receptor 9 (TLR9) activation could contribute to the control of inflammation in Sjogrens syndrome. To this end, we manipulated TLR9 signaling in non-obese diabetic (NOD) and TLR9(-/-) mice using agonistic CpG oligonucleotide aptamers, TLR9 inhibitors, and the in-house oligonucleotide BL-7040. We then measured salivation, inflammatory response markers, and expression of proteins downstream to NF-?B activation pathways. Finally, we labeled proteins of interest in salivary gland biopsies from Sjogrens syndrome patients, compared to Sicca syndrome controls. We show that in NOD mice BL-7040 activates TLR9 to induce an alternative NF-?B activation mode resulting in increased salivation, elevated anti-inflammatory response in salivary glands, and reduced peripheral AChE activity. These effects were more prominent and also suppressible by TLR9 inhibitors in NOD mice, but TLR9(-/-) mice were resistant to the salivation-promoting effects of CpG oligonucleotides and BL-7040. Last, salivary glands from Sjogrens disease patients showed increased inflammatory and decreased anti-inflammatory biomarkers, in addition to decreased levels of alternative NF-?B pathway proteins. In summary, we have demonstrated that activation of TLR9 by BL-7040 leads to non-canonical activation of NF-?B, promoting salivary functioning and down-regulating inflammation. We propose that BL-7040 could be beneficial in treating Sjogrens syndrome and may be applicable to additional autoimmune syndromes.
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The dynamic nature of amyloid beta (1-40) aggregation.
Phys Chem Chem Phys
PUBLISHED: 07-01-2011
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In this paper, we characterize the dynamic nature of the full amyloid beta (1-40) (A? (1-40)) aggregates. We labeled the peptide with either 5-carboxytetramethylrhodamine (TAMRA) or with fluorescein-isothiocyanate (FITC). The labeled peptides were mixed after separate fibrillization, and the dynamic changes in the structure of the fibrils were imaged using confocal microscopy. Fluorescence resonance energy transfer (FRET) measurements showed that the A? (1-40) peptides detach from and reattach to the fibrils in a biologically relevant timescale (days). With time, the two peptides mix at the molecular level. This process is concentration dependent and occurs primarily in the external parts of the aggregates with a half time between 4 and 7 days. This study shows that the combination of confocal microscopy and FRET analysis is a facile method for studying dynamic processes in supra-molecular aggregates.
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Bundle-forming pilus retraction enhances enteropathogenic Escherichia coli infectivity.
Mol. Biol. Cell
PUBLISHED: 05-25-2011
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Enteropathogenic Escherichia coli (EPEC) is an important human pathogen that causes acute infantile diarrhea. The type IV bundle-forming pili (BFP) of typical EPEC strains are dynamic fibrillar organelles that can extend out and retract into the bacterium. The bfpF gene encodes for BfpF, a protein that promotes pili retraction. The BFP are involved in bacterial autoaggregation and in mediating the initial adherence of the bacterium with its host cell. Importantly, BFP retraction is implicated in virulence in experimental human infection. How pili retraction contributes to EPEC pathogenesis at the cellular level remains largely obscure, however. In this study, an effort has been made to address this question using engineered EPEC strains with induced BFP retraction capacity. We show that the retraction is important for tight-junction disruption and, to a lesser extent, actin-rich pedestal formation by promoting efficient translocation of bacterial protein effectors into the host cells. A model is proposed whereby BFP retraction permits closer apposition between the bacterial and the host cell surfaces, thus enabling timely and effective introduction of bacterial effectors into the host cell via the type III secretion apparatus. Our studies hence suggest novel insights into the involvement of pili retraction in EPEC pathogenesis.
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Role for ADP ribosylation factor 1 in the regulation of hepatitis C virus replication.
J. Virol.
PUBLISHED: 11-10-2010
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We hypothesized that ADP-ribosylation factor 1 (Arf1) plays an important role in the biogenesis and maintenance of infectious hepatitis C virus (HCV). Huh7.5 cells, in which HCV replicates and produces infectious viral particles, were exposed to brefeldin A or golgicide A, pharmacological inhibitors of Arf1 activation. Treatment with these agents caused a reduction in viral RNA levels, the accumulation of infectious particles within the cells, and a reduction in the levels of these particles in the extracellular medium. Fluorescence analyses showed that the viral nonstructural (NS) proteins NS5A and NS3, but not the viral structural protein core, shifted their localization from speckle-like structures in untreated cells to the rims of lipid droplets (LDs) in treated cells. Using pulldown assays, we showed that ectopic overexpression of NS5A in Huh7 cells reduces the levels of GTP-Arf1. Downregulation of Arf1 expression by small interfering RNA (siRNA) decreased both the levels of HCV RNA and the production of infectious viral particles and altered the localization of NS5A to the peripheries of LDs. Together, our data provide novel insights into the role of Arf1 in the regulation of viral RNA replication and the production of infectious HCV.
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Cytoplasmic H2O2 prevents translocation of NPR1 to the nucleus and inhibits the induction of PR genes in Arabidopsis.
Plant Signal Behav
PUBLISHED: 11-01-2010
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Plants activate a number of defense reactions in response to pathogen attack. One of the major pathways involves biosynthesis of Salicylic Acid (SA), which acts as a signaling molecule that regulates local defense reaction at the infection site and in induction of systemic acquired resistance (SAR). SA is sensed and transduced by NPR1 protein, which is a redox sensitive protein that acts as a central transcription activator of many pathogenesis related and defense related genes. In its uninduced state NPR1 exists as an oligomer in the cytoplasm. Following pathogen attack and SAR induction, cells undergo a biphasic change in cellular redox, resulting in reduction of NPR1 to a monomeric form,which moves to the nucleus. Recently, it was shown that pathogen attack or SA treatment cause S-nitrosylation of NPR1, promoting NPR1 oligomerization and restricting it in the cytoplasm. We used A. thaliana mutants in cytosolic ASCORBATE PEROXIDASE, apx1, and plants expressing antisense CATALASE gene, as well as the CATALASE inhibitor 3-amino-1,2,4-triazole, to examine the effect of H2O2 on the pathogen-triggered translocation of the NPR1 to the nucleus. Our results show that the pathogen-triggered or SA-induced nuclear translocation is prevented by accumulation of H2O2 in the cytosol. Moreover, we show that increased accumulation of cytoplasmic ROS in apx1 mutants reduced the NPR1-dependent gene expression. We suggest that H2O2 has a signaling role in pathogenesis, acting as a negative regulator of NPR1 translocation to the nucleus, limiting the NPR1-dependent gene expression.
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EspM inhibits pedestal formation by enterohaemorrhagic Escherichia coli and enteropathogenic E. coli and disrupts the architecture of a polarized epithelial monolayer.
Cell. Microbiol.
PUBLISHED: 11-12-2009
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Enterohaemorrhagic Escherichia coli and enteropathogenic E. coli are enteropathogens characterized by their ability to induce the host cell to form actin-rich structures, termed pedestals. A type III secretion system, through which the pathogens deliver effector proteins into infected host cells, is essential for their virulence and pedestal formation. Enterohaemorrhagic E. coli encodes two similar effectors, EspM1 and EspM2, which activate the RhoA signalling pathway and induce the formation of stress fibres upon infection of host cells. We confirm these observations and in addition show that EspM inhibits the formation of actin pedestals. Moreover, we show that translocation of EspM into polarized epithelial cells induces dramatic changes in the tight junction localization and in the morphology and architecture of infected polarized monolayers. These changes are manifested by altered localization of the tight junctions and bulging out morphology of the cells. Surprisingly, despite the dramatic changes in their architecture, the cells remain alive and the epithelial monolayer maintains a normal barrier function. Taken together, our results show that the EspM effectors inhibit pedestal formation and induce tight junction mislocalization as well as dramatic changes in the architecture of the polarized monolayer.
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Inhibition of HIV-1 integrase nuclear import and replication by a peptide bearing integrase putative nuclear localization signal.
Retrovirology
PUBLISHED: 09-30-2009
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The integrase (IN) of human immunodeficiency virus type 1 (HIV-1) has been implicated in different steps during viral replication, including nuclear import of the viral pre-integration complex. The exact mechanisms underlying the nuclear import of IN and especially the question of whether it bears a functional nuclear localization signal (NLS) remain controversial.
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NPR1 protein regulates pathogenic and symbiotic interactions between Rhizobium and legumes and non-legumes.
PLoS ONE
PUBLISHED: 09-15-2009
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Legumes are unique in their ability to establish symbiotic interaction with rhizobacteria from Rhizobium genus, which provide them with available nitrogen. Nodulation factors (NFs) produced by Rhizobium initiate legume root hair deformation and curling that entrap the bacteria, and allow it to grow inside the plant. In contrast, legumes and non-legumes activate defense responses when inoculated with pathogenic bacteria. One major defense pathway is mediated by salicylic acid (SA). SA is sensed and transduced to downstream defense components by a redox-regulated protein called NPR1.
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Posttranslational regulation of CIRCADIAN CLOCK ASSOCIATED1 in the circadian oscillator of Arabidopsis.
Plant Physiol.
PUBLISHED: 04-01-2009
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As an adaptation to life in a world with predictable daily changes, most eukaryotes and some prokaryotes have endogenous circadian (approximately 24 h) clocks. In plants, the circadian clock regulates a diverse range of cellular and physiological events from gene expression and protein phosphorylation to cellular calcium oscillations, hypocotyl growth, leaf movements, and photoperiod-dependent flowering. In Arabidopsis (Arabidopsis thaliana), as in other model organisms, such as Drosophila (Drosophila melanogaster) and mice, circadian rhythms are generated by molecular oscillators that consist of interlocking feedback loops involving a number of elements. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYLS (LHY) are closely related single myb transcription factors that have been identified as key elements in the Arabidopsis oscillator. Research in other model organisms has shown that posttranslational regulation of oscillator components plays a critical role in the generation of the approximately 24-h cycles. To examine the role of posttranslational regulation of CCA1 and LHY in the Arabidopsis oscillator, we generated transgenic plants with tagged CCA1 and LHY under the control of their own promoters. We have shown that these tagged proteins are functional and can restore normal circadian rhythms to CCA1- and LHY-null plants. Using the tagged proteins, we demonstrate that CCA1 can form both homodimers and heterodimers with LHY. Furthermore, we also show that CCA1 is localized to the nucleus in vivo and that there is no significant delay between the translation of CCA1 and its translocation to the nucleus. We discuss our findings in the context of the functioning of the Arabidopsis oscillator.
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Enteropathogenic Escherichia coli subverts phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon epithelial cell infection.
Mol. Biol. Cell
PUBLISHED: 03-28-2009
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Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] are phosphoinositides (PIs) present in small amounts in the inner leaflet of the plasma membrane (PM) lipid bilayer of host target cells. They are thought to modulate the activity of proteins involved in enteropathogenic Escherichia coli (EPEC) infection. However, the role of PI(4,5)P(2) and PI(3,4,5)P(3) in EPEC pathogenesis remains obscure. Here we show that EPEC induces a transient PI(4,5)P(2) accumulation at bacterial infection sites. Simultaneous actin accumulation, likely involved in the construction of the actin-rich pedestal, is also observed at these sites. Acute PI(4,5)P(2) depletion partially diminishes EPEC adherence to the cell surface and actin pedestal formation. These findings are consistent with a bimodal role, whereby PI(4,5)P(2) contributes to EPEC association with the cell surface and to the maximal induction of actin pedestals. Finally, we show that EPEC induces PI(3,4,5)P(3) clustering at bacterial infection sites, in a translocated intimin receptor (Tir)-dependent manner. Tir phosphorylated on tyrosine 454, but not on tyrosine 474, forms complexes with an active phosphatidylinositol 3-kinase (PI3K), suggesting that PI3K recruited by Tir prompts the production of PI(3,4,5)P(3) beneath EPEC attachment sites. The functional significance of this event may be related to the ability of EPEC to modulate cell death and innate immunity.
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Transcription of steroidogenic acute regulatory protein in the rodent ovary and placenta: alternative modes of cyclic adenosine 3, 5-monophosphate dependent and independent regulation.
Endocrinology
PUBLISHED: 02-21-2009
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Steroid hormone synthesis is a vital function of the adrenal cortex, serves a critical role in gonadal function, and maintains pregnancy if normally executed in the placenta. The substrate for the synthesis of all steroid hormones is cholesterol, and its conversion to the first steroid, pregnenolone, by the cholesterol side-chain cleavage cytochrome P450 (CYP11A1) enzyme complex takes place in the inner mitochondrial membranes. Steroidogenic acute regulatory protein (STAR) facilitates the rate-limiting transfer of cholesterol from the outer mitochondrial membrane to CYP11A1 located in the inner organelle membranes. The current study explored the mechanisms controlling transcription of the Star gene in primary cell cultures of mouse placental trophoblast giant cells and rat ovarian granulosa cells examined throughout the course of their functional differentiation. Our findings show that the cis-elements required for Star transcription in the rodent placenta and the ovary are centered in a relatively small proximal region of the promoter. In placental trophoblast giant cells, cAMP is required for activation of the Star promoter, and the cis-elements mediating a maximal response were defined as cAMP response element 2 and GATA. EMSA studies show that placental cAMP-responsive element binding protein (CREB)-1 and activating transcription factor-2 (ATF2) bind to a -81/-78 sequence, whereas GATA-2 binds to a -66/-61 sequence. In comparison, patterns of Star regulation in the ovary suggested tissue-specific and developmental controlled modes of Star transcription. During the follicular phase, FSH/cAMP induced CREB-1 dependent activity, whereas upon luteinization STAR expression becomes cAMP and CREB independent, a functional shift conferred by FOS-related antigen-2 displacement of CREB-1 binding, and the appearance of a new requirement for CCAAT enhancer-binding protein beta and steroidogenic factor 1 that bind to upstream elements (-117/-95). These findings suggest that during evolution, the promoters of the Star gene acquired nonconsensus sequence elements enabling expression of a single gene in different organs, or allowing dynamic temporal changes corresponding to progressing phases of differentiation in a given cell type.
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Real-time monitoring of transferrin-induced endocytic vesicle formation by mid-infrared surface plasmon resonance.
Biophys. J.
PUBLISHED: 01-18-2009
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We report on the application of surface plasmon resonance (SPR), based on Fourier transform infrared spectroscopy in the mid-infrared wavelength range, for real-time and label-free sensing of transferrin-induced endocytic processes in human melanoma cells. The evanescent field of the mid-infrared surface plasmon penetrates deep into the cell, allowing highly sensitive SPR measurements of dynamic processes occurring at significant cellular depths. We monitored in real-time, infrared reflectivity spectra in the SPR regime from living cells exposed to human transferrin (Tfn). We show that although fluorescence microscopy measures primarily Tfn accumulation in recycling endosomes located deep in the cells cytoplasm, the SPR technique measures mainly Tfn-mediated formation of early endocytic organelles located in close proximity to the plasma membrane. Our SPR and fluorescence data are very well described by a kinetic model of Tfn endocytosis, suggested previously in similar cell systems. Hence, our SPR data provide further support to the rather controversial ability of Tfn to stimulate its own endocytosis. Our analysis also yields what we believe is novel information on the role of membrane cholesterol in modulating the kinetics of endocytic vesicle biogenesis and consumption.
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Fluoxetine induces vasodilatation of cerebral arterioles by co-modulating NO/muscarinic signalling.
J. Cell. Mol. Med.
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Ischaemic stroke patients treated with Selective Serotonin Reuptake Inhibitors (SSRI) show improved motor, cognitive and executive functions, but the underlying mechanism(s) are incompletely understood. Here, we report that cerebral arterioles in the rat brain superfused with therapeutically effective doses of the SSRI fluoxetine showed consistent, dose-dependent vasodilatation (by 1.2 to 1.6-fold), suppressible by muscarinic and nitric oxide synthase (NOS) antagonists [atropine, NG-nitro-l-arginine methyl ester (l-NAME)] but resistant to nicotinic and serotoninergic antagonists (mecamylamine, methylsergide). Fluoxetine administered 10-30 min. following experimental vascular photo-thrombosis increased arterial diameter (1.3-1.6), inducing partial, but lasting reperfusion of the ischaemic brain. In brain endothelial b.End.3 cells, fluoxetine induced rapid muscarinic receptor-dependent increases in intracellular [Ca(2+) ] and promoted albumin- and eNOS-dependent nitric oxide (NO) production and HSP90 interaction. In vitro, fluoxetine suppressed recombinant human acetylcholinesterase (rhAChE) activity only in the presence of albumin. That fluoxetine induces vasodilatation of cerebral arterioles suggests co-promotion of endothelial muscarinic and nitric oxide signalling, facilitated by albumin-dependent inhibition of serum AChE.
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ROS production during symbiotic infection suppresses pathogenesis-related gene expression.
Plant Signal Behav
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Leguminous plants have exclusive ability to form symbiotic relationship with soil bacteria of the genus Rhizobium. Symbiosis is a complex process that involves multiple molecular signaling activities, such as calcium fluxes, production of reactive oxygen species (ROS) and synthesis of nodulation genes. We analyzed the role of ROS in defense gene expression in Medicago truncatula during symbiosis and pathogenesis. Studies in Arabidopsis thaliana showed that the induction of pathogenesis-related (PR) genes during systemic acquired resistance (SAR) is regulated by NPR1 protein, which resides in the cytoplasm as an oligomer. After oxidative burst and return of reducing conditions, the NPR1 undergoes monomerization and becomes translocated to the nucleus, where it functions in PR genes induction. We show that ROS production is both stronger and longer during symbiotic interactions than during interactions with pathogenic, nonhost or common nonpathogenic soil bacteria. Moreover, root cells inoculated with Sinorhizobium meliloti accumulated ROS in the cytosol but not in vacuoles, as opposed to Pseudomonas putida inoculation or salt stress treatment. Furthermore, increased ROS accumulation by addition of H?O? reduced the PR gene expression, while catalase had an opposite effect, establishing that the PR gene expression is opposite to the level of cytoplasmic ROS. In addition, we show that salicylic acid pretreatment significantly reduced ROS production in root cells during symbiotic interaction.
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