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Extracellular Release (extracellular + release)
Selected AbstractsConstitutive secretion of the granule chymase mouse mast cell protease-1 and the chemokine, CCL2, by mucosal mast cell homologuesCLINICAL & EXPERIMENTAL ALLERGY, Issue 1 2003J. K. Brown Summary Background The mucosal mast cell (MMC) granule-specific ,-chymase, mouse mast cell protease-1 (mMCP-1), is released systemically into the bloodstream early in nematode infection before parasite-specific IgE responses develop and TGF-,1 induces constitutive release of mMCP-1 by homologues of MMC in vitro. Intraepithelial MMC may also express the chemokine CCL2 (monocyte chemotactic protein-1) during nematode infection but the expression of this chemokine by MMC homologues has not been investigated. Objective To investigate the expression and to compare the mechanisms of constitutive release of the chymase, mMCP-1, and the chemokine, CCL2. Methods MMC homologues were generated by culturing bone marrow cells in the presence of TGF-,1, IL-3, IL-9 and stem cell factor (SCF). The intracellular distribution of mMCP-1 and CCL2 was examined by confocal microscopy. The involvement of the Golgi complex and of protein synthesis in the constitutive release of mMCP-1 and CCL2 was investigated using the Golgi-disrupting agent brefeldin A and cycloheximide to block protein synthesis. Secreted analytes were quantified by ELISA. Results mMCP-1 colocalized with Golgi matrix protein 130 but was most abundant in the granules, whereas CCL2 was not found in the granules but appeared to be located uniquely in the Golgi complex. Extracellular release of mMCP-1 was significantly inhibited (, 40%) by cycloheximide and by the Golgi-disrupting agent brefeldin A, indicating both continuous protein synthesis and transportation via the Golgi complex are required for optimal mMCP-1 secretion. A similar but more marked inhibitory effect with both compounds was demonstrated on the constitutive secretion of CCL2. Conclusion The culture conditions that promote mMCP-1 expression and release by MMC homologues also promote the expression and release of CCL2. Constitutive release involves de novo protein synthesis and requires a functional Golgi complex, suggesting that similar mechanisms of extracellular secretion operate for both mediators. [source] Intracellular HMGB1 transactivates the human IL1B gene promoter through association with an Ets transcription factor PU.1EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2008Fumihiko Mouri Abstract High mobility group box 1 protein (HMGB1), originally described as a non-histone, DNA binding protein, was recently identified as a late mediator of inflammation via its extracellular release from activated macrophages/monocytes. In the present study, we report that intracellular HMGB1 synergizes with a macrophage/monocyte-specific E26 transformation-specific sequence (Ets) transcription factor PU.1 to transactivate the promoter of the IL1B gene coding a 31-kDa proIL-1, protein. The ,131 to +12 IL1B promoter, which possesses a PU.1 binding motif essential for its transactivation, was induced when HMGB1 expression vector was transfected into murine RAW264.7 macrophage cells. Our glutathione S -transferase-pulldown and coimmunoprecipitation assays demonstrated direct physical interaction of HMGB1 with PU.1. Deletion of the PU.1 winged helix-turn-helix DNA-binding domain inhibited the association of the two proteins. In electrophoretic mobility shift assay using recombinant PU.1 protein, a ternary complex of PU.1, HMGB1 and PU.1-binding element within the IL1B promoter was generated. The importance of PU.1 was further supported by our observation that induction of the IL1B promoter was obtained only after PU.1 expression in PU.1-deficient murine EL4 thymoma cells. Thus, our data raise the possibility of a novel mechanism which sustains and amplifies inflammatory reactions through physical interaction of PU.1 with intracellular HMGB1 in macrophages/monocytes. [source] Role of MAPK phosphorylation in cytoprotection by pro-vitamin C against oxidative stress-induced injuries in cultured cardiomyoblasts and perfused rat heartJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2003Masahiro Eguchi Abstract The reactive oxygen species (ROS) are known to be generated upon post-ischemic reperfusion (I/R) of the heart, and to injure cardiac muscle cells. The hydrogen peroxide-induced mortality of rat cardiomyoblasts H2c9 was markedly inhibited by previous administration with auto-oxidation-resistant pro-vitamin C, the 2- O -phosphorylated derivative (Asc2P) of ascorbic acid (Asc). The cytoprotection was partially counteracted by an inhibitor of MAPK (mitogen-activated protein kinase) kinase (MEK) as shown by DNA strand cleavage assay and mitochondrial dehydrogenase assay. Immunostains indicated that phosphorylated MAPK increased in the hydrogen peroxide-treated cardiomyoblasts, and that this action was moderately inhibited by Asc2P and restored nearly to the initial, pretreatment level by combined administration of the MEK inhibitor and Asc2P. The I/R-induced cell injuries in perfused rat hearts as estimated by extracellular release of the cardiac enzyme CPK were inhibited by 2- O -,-glucosylascorbic acid (Asc2G) and Asc, whereas the observed cytoprotection for the cardiomyoblasts was partially counteracted by the MEK inhibitor. The increase in phosphorylated MAPK in I/R-operated hearts was moderately inhibited by pro-vitamin C, but restored nearly to the normal non-operated level by combined administration with the MEK inhibitor. This is in contrast to no alteration in levels of non-phosphorylated MAPK for all the cases examined as shown by Western blots, consistent with results of immunostains for the cardiomyoblasts. The inhibitory effect of the MEK inhibitor on MAPK phosphorylation was, therefore, suggested to counteract the cytoprotective effects of pro-vitamin C via a thorough interruption of the phosphorylated MAPK signaling pathway. This was not true of ROS-related events; the scavenging effects of Asc2G and Asc on hydroxyl radicals generated from I/R-operated heart were not affected by combined administration with the MEK inhibitor, as shown by the spin-trapping DMPO-based ESR method. J. Cell. Biochem. 90: 219,226, 2003. © 2003 Wiley-Liss, Inc. [source] Nipah virus RNA synthesis in cultured pig and human cellsJOURNAL OF MEDICAL VIROLOGY, Issue 8 2006Li-Yen Chang Abstract Nipah virus infection of porcine stable kidney cells (PS), human neuronal cells (SK-N-MC), human lung fibroblasts cells (MRC-5), and human monocytes (THP-1) were examined. Rapid progression of cytopathic effects (CPE) and cell death were noted in PS cell cultures treated with Nipah virus, followed by MRC-5, SK-N-MC, and THP-1 cell cultures, in descending order of rapidity. Significant increase in the intracellular Nipah virus RNA occurred beginning at 24 hr PI in all the infected cells. Whereas, the extracellular release of Nipah virus RNA increased significantly beginning at 48 and 72 hr PI for the infected MRC-5 cells and PS cells, respectively. No significant release of extracellular Nipah virus RNA was detected from the Nipah virus-infected SK-N-MC and THP-1 cells. At its peak, approximately 6.6 log PFU/µl of extracellular Nipah virus RNA was released from the Nipah virus-infected PS cells, with at least a 100-fold less virus RNA was recorded in the Nipah virus-infected SK-N-MC and THP-1. Approximately 15.2% (±0.1%) of the released virus from the infected PS cell cultures was infectious in contrast to approximately 5.5% (±0.7%) from the infected SK-N-MC cells. The findings suggest that there are no differences in the capacity to support Nipah virus replication between pigs and humans in fully susceptible PS and MRC-5 cells. However, there are differences between these cells and human neuronal cells and monocytes in the ability to support Nipah virus replication and virus release. J. Med. Virol. 78:1105,1112, 2006. © 2006 Wiley-Liss, Inc. [source] Interleukin-1, enhances nucleotide-induced and ,-secretase-dependent amyloid precursor protein processing in rat primary cortical neurons via up-regulation of the P2Y2 receptorJOURNAL OF NEUROCHEMISTRY, Issue 5 2009Qiongman Kong Abstract The heterologous expression and activation of the human P2Y2 nucleotide receptor (P2Y2R) in human 1321N1 astrocytoma cells stimulates ,-secretase-dependent cleavage of the amyloid precursor protein (APP), causing extracellular release of the non-amyloidogenic protein secreted amyloid precursor protein (sAPP,). To determine whether a similar response occurs in a neuronal cell, we analyzed whether P2Y2R-mediated production of sAPP, occurs in rat primary cortical neurons (rPCNs). In rPCNs, P2Y2R mRNA and receptor activity were virtually absent in quiescent cells, whereas overnight treatment with the pro-inflammatory cytokine interleukin-1, (IL-1,) up-regulated both P2Y2R mRNA expression and receptor activity by four-fold. The up-regulation of the P2Y2R was abrogated by pre-incubation with Bay 11-7085, an I,B-, phosphorylation inhibitor, which suggests that P2Y2R mRNA transcript levels are regulated through nuclear factor-,-B (NF,B) signaling. Furthermore, the P2Y2R agonist Uridine-5,-triphosphate (UTP) enhanced the release of sAPP, in rPCNs treated with IL-1, or transfected with P2Y2R cDNA. UTP-induced release of sAPP, from rPCNs was completely inhibited by pre-treatment of the cells with the metalloproteinase inhibitor TACE inhibitor (TAPI-2) or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, and was partially inhibited by the MAPK/extracellular signal-regulated kinase inhibitor U0126 and the protein kinase C inhibitor GF109203. These data suggest that P2Y2R-mediated release of sAPP, from cortical neurons is directly dependent on a disintegrin and metalloproteinase (ADAM) 10/17 and PI3K activity, whereas extracellular signal-regulated kinase 1/2 and PI3K activity may indirectly regulate APP processing. These results demonstrate that elevated levels of pro-inflammatory cytokines associated with neurodegenerative diseases, such as IL-1,, can enhance non-amyloidogenic APP processing through up-regulation of the P2Y2R in neurons. [source] GAT-1 regulates both tonic and phasic GABAA receptor-mediated inhibition in the cerebral cortexJOURNAL OF NEUROCHEMISTRY, Issue 5 2008Luca Bragina Abstract ,-Aminobutyric acid 1 (GAT-1) is the most copiously expressed GABA transporter; we studied its role in phasic and tonic inhibition in the neocortex using GAT-1 knockout (KO) mice. Immunoblotting and immunocytochemical studies showed that GAT-2 and GAT-3 levels in KOs were unchanged and that GAT-3 was not redistributed in KOs. Moreover, the expression of GAD65/67 was increased, whereas that of GABA or VGAT was unchanged. Microdialysis studies showed that in KOs spontaneous extracellular release of GABA and glutamate was comparable in WT and KO mice, whereas KCl-evoked output of GABA, but not of glutamate, was significantly increased in KOs. Recordings from layer II/III pyramids revealed a significant increase in GABAAR-mediated tonic conductance in KO mice. The frequency, amplitude and kinetics of spontaneous inhibitory post-synaptic currents (IPSCs) were unchanged, whereas the decay time of evoked IPSCs was significantly prolonged in KO mice. In KO mice, high frequency stimulation of GABAergic terminals induced large GABAAR-mediated inward currents associated with a reduction in amplitude and decay time of IPSCs evoked immediately after the train. The recovery process was slower in KO than in WT mice. These studies show that in the cerebral cortex of GAT-1 KO mice GAT-3 is not redistributed and GADs are adaptively changed and indicate that GAT-1 has a prominent role in both tonic and phasic GABAAR-mediated inhibition, in particular during sustained neuronal activity. [source] Neutrophils in chronic and aggressive periodontitis in interaction with Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitansJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2009A. Guentsch Background and Objective:, This study analyzed the interaction of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 with peripheral blood polymorphonuclear neutrophils taken from patients with aggressive periodontitis and chronic periodontitis. Material and Methods:, Peripheral blood polymorphonuclear neutrophils obtained from 12 patients with chronic periodontitis, six patients with aggressive periodontitis and 12 healthy controls were exposed to P. gingivalis and A. actinomycetemcomitans following opsonization of the bacteria using the patient's own serum. Serum immunoglobulin G (IgG) levels against both periodontopathogens were measured. Phagocytosis and killing of the bacteria, as well as the extracellular human neutrophil elastase activity, were quantified. The total amount and the extracellular release of reactive oxygen species were measured using luminol-dependent and isoluminol-dependent chemiluminescence. Results:, Polymorphonuclear neutrophils from patients with chronic (62.16 ± 19.39%) and aggressive (43.26 ± 26.63%) periodontitis phagocytosed more P. gingivalis than the healthy controls (24.43 ± 19.87%) at the 30-min time point after exposure to the bacteria (p < 0.05). High serum IgG levels against P. gingivalis and A. actinomycetemcomitans were detected in subjects with periodontitis. Polymorphonuclear neutrophils from subjects with chronic and aggressive periodontitis released significantly more reactive oxygen species and demonstrated greater human neutrophil elastase activity in the absence of any stimulus than polymorphonuclear neutrophils from healthy controls (p < 0.05). Polymorphonuclear neutrophils in chronic periodontitis released significantly more reactive oxygen species when exposed to P. gingivalis and A. actinomycetemcomitans than polymorphonuclear neutrophils in aggressive periodontitis. Conclusion:, High serum IgG levels against P. gingivalis and A. actinomycetemcomitans promote phagocytosis in periodontitis. The extracellular release of reactive oxygen species and neutrophil elastase by polymorphonuclear neutrophils may also contribute to damage of the surrounding periodontal tissues. [source] Lipid peroxidation caused by oxygen radicals from Fusobacterium -stimulated neutrophils as a possible model for the emergence of periodontitisORAL DISEASES, Issue 1 2001M Sheikhi OBJECTIVE: The possible contribution of bacteria and polymorphonuclear neutrophils (PMN) to the disease process of periodontitis was evaluated. DESIGN: Fusobacterium nucleatum has been associated with chronic adult periodontitis. Intracellular production and extracellular release of reactive oxygen species (ROS) by PMN stimulated by fusobacteria were evaluated. To estimate the potential extracellular damage that might be caused by the ROS, the lipid peroxidation (LPO) of an exogenous phospholipid, Intralipid, was assayed. METHODS: The ROS production of PMN was studied by the nitroblue tetrazolium and chemiluminescence tests. The levels of malonaldehyde (MDA) and 4-hydroxyalkenals were used to indicate LPO. RESULTS: Fusobacterium nucleatum strains stimulated neutrophils to produce a large amount of ROS, independently of plasma complement factors. The two strains tested induced considerable intracellular, but no extracellular chemiluminescence responses during the first hour, indicating that ROS were released into phagosomes. However an incubation period of 4 h, in the presence of the extracellular lipid resulted in a high degree of LPO, presumably caused by ROS release from the Fusobacterium -stimulated PMN. ROS production and lipid peroxidation could be counteracted by vitamin E. CONCLUSION: In periodontitis local bacteria might stimulate PMN to release ROS, which cause inflammation and destruction. [source] Spontaneous oscillation and mechanically induced calcium waves in chondrocytesCELL BIOCHEMISTRY AND FUNCTION, Issue 2 2006Taisuke Kono Abstract The characteristics of spontaneous calcium (Ca2+) oscillation and mechanically induced Ca2+ waves in articular chondrocytes were studied. In some, but not all, chondrocytes in sliced cartilage and primary cultures, we observed spontaneous oscillation of intracellular Ca2+ that never spread to adjacent cells. In contrast, a mechanical stimulus to a single cell by touching with a glass rod induced an increase of intracellular Ca2+ that spread to neighboring cells in a wave-like manner, even though there was no physical contact between the cells. This indicated the release of some paracrine factor from the mechanically stimulated cells. Application of ultrasonic vibration also induced an oscillation of intracellular Ca2+. The application of a uridine 5,-triphosphate (UTP), UTP, induced a transient increase in intracellular Ca2+ and the release of adenosine 5,-triphosphate (ATP) in cultured chondrocytes. A P2 receptor antagonist (suramin) and blockers of Cl, channels, niflumic acid and 4,4,-diisothiocyanostilbene-2,2,-disulfonic acid (DIDS), reduced the UTP-induced ATP release. The results indicated that Cl, channels were involved in the extracellular release of ATP following mechanical or P2Y receptor stimulation. Thus, ATP stimulation of P2Y receptors elicits an increase in intracellular Ca2+, triggering further release of ATP from adjacent cells, thereby expanding the Ca2+ wave in chondrocytes. Copyright © 2005 John Wiley & Sons, Ltd. [source] | |||||||||||||