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Dependent Mechanism (dependent + mechanism)
Selected AbstractsArachidonic acid as a retrograde signal controlling growth and dynamics of retinotectal arborsDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2008B.H. Leu Abstract In the developing visual system, correlated presynaptic activity between neighboring retinal ganglion cells (RGC) stabilizes retinotopic synapses via a postsynaptic NMDAR (N -methyl- D -aspartate receptor)-dependent mechanism. Blocking NMDARs makes individual axonal arbors larger, which underlies an unsharpened map, and also increases branch turnover, as if a stabilizing factor from the postsynaptic partner is no longer released. Arachidonic acid (AA), a candidate retrograde stabilizing factor, is released by cytoplasmic phospholipase A2 (cPLA2) after Ca2+ entry through activated NMDARs, and can activate presynaptic protein kinase C to phosphorylate various substrates such as GAP43 to regulate cytoskeletal dynamics. To test the role of cPLA2 in the retinotectal system of developing zebrafish, we first used PED6, a fluorescent reporter of cPLA2 activity, to show that 1,3 min of strobe flashes activated tectal cPLA2 by an NMDAR-dependent mechanism. Second, we imaged the dynamic growth of retinal arbors during both local inhibition of tectal cPLA2 by a pharmacological inhibitor, arachidonic tri-fluoromethylketone, and its suppression by antisense oligonucleotides (both injected intraventricularly). Both methods produced larger arbors and faster branch dynamics as occurs with blocking NMDARs. In contrast, intraocular suppression of retinal cPLA2 with large doses of antisense oligos produced none of the effects of tectal cPLA2 inhibition. Finally, if AA is the retrograde messenger, the application of exogenous AA to the tectum should reverse the increased branch turnover caused by blocking either NMDARs or cPLA2. In both cases, intraventricular injection of AA stabilized the overall branch dynamics, bringing rates down below the normal values. The results suggest that AA generated postsynaptically by cPLA2 downstream of Ca2+ entry through NMDARs acts as a retrograde signal to regulate the dynamic growth of retinal arbors. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2008. [source] Effects of prolactin on intracellular calcium concentration and cell proliferation in human glioma cellsGLIA, Issue 3 2002Thomas Ducret Abstract Prolactin (PRL) has several physiological effects on peripheral tissues and the brain. This hormone acts via its membrane receptor (PRL-R) to induce cell differentiation or proliferation. Using reverse transcription,polymerase chain reaction (RT-PCR) combined with Southern blot analysis, we detected PRL-R transcripts in a human glioma cell line (U87-MG) and in primary cultured human glioblastoma cells. These transcripts were deleted or not in their extracellular domains. We examined the effects of PRL on intracellular free Ca2+ concentration ([Ca2+]i) in these cells in order to improve our understanding of the PRL transduction mechanism, which is still poorly documented. [Ca2+]i was measured by microspectrofluorimetry using indo-1 as the Ca2+ fluorescent probe. Spatiotemporal aspects of PRL-induced Ca2+ signals were investigated using high-speed fluo-3 confocal imaging. We found that physiological concentrations (0.4,4 nM) of PRL-stimulated Ca2+ entry and intracellular Ca2+ mobilization via a tyrosine kinase,dependent mechanism. The two types of Ca2+ responses observed were distinguishable by their kinetics: one showing a slow (type I) and the other a fast (type II) increase in [Ca2+]i. The amplitude of PRL-induced Ca2+ increases may be sufficient to provoke several physiological responses, such as stimulating proliferation. Furthermore, PRL induced a dose-dependent increase in [3H]thymidine incorporation levels and in cellular growth and survival, detected by the MTT method. These data indicate that PRL induced mitogenesis of human glioma cells. GLIA 38:200,214, 2002. © 2002 Wiley-Liss, Inc. [source] Roles of AKT and sphingosine kinase in the antiapoptotic effects of bile duct ligation in mouse liver,HEPATOLOGY, Issue 6 2005Yosuke Osawa Tumor necrosis factor (TNF) receptor, and Fas-mediated apoptosis are major death processes of hepatocytes in liver disease. Although antiapoptotic effects in the injured liver promote chronic hepatitis and carcinogenesis, scant information is known about these mechanisms. To explore this issue, we compared acute liver injury after TNF-, or anti-Fas antibody (Jo2) between livers from sham-operated mice and chronic injured liver via bile duct ligation (BDL). BDL inhibited hepatocyte apoptosis induced by TNF-, but not by Jo2. On the other hand, BDL inhibited the massive hemorrhage seen in livers treated with either TNF-, or Jo2. Inactivation of AKT blocked the antiapoptotic effect of BDL. Sphingosine kinase knockout mice also lost the antihemorrhagic effect of BDL and attenuated the antiapoptotic effects of BDL. In bile duct,ligated livers, hepatic stellate cells (HSCs) were activated and produced tissue inhibitor of metalloproteinase 1 in a sphingosine kinase (SphK)-1,dependent mechanism. In conclusion, BDL exerts antiapoptotic effects that appear to require activation of AKT in hepatocytes and SphK in HSCs.(HEPATOLOGY 2005;42:1320,1328.) [source] Nuclear translocation of UDCA by the glucocorticoid receptor is required to reduce TGF-,1,induced apoptosis in rat hepatocytes,HEPATOLOGY, Issue 4 2005Susana Solá Ursodeoxycholic acid (UDCA) inhibits classical mitochondrial pathways of apoptosis by either directly stabilizing mitochondrial membranes or modulating specific upstream targets. Furthermore, UDCA regulates apoptosis-related genes from transforming growth factor ,1 (TGF-,1),induced hepatocyte apoptosis by a nuclear steroid receptor (NSR),dependent mechanism. In this study, we further investigated the potential role of the glucocorticoid receptor (GR) in the antiapoptotic function of UDCA. Our results with short interference RNA (siRNA) technology confirmed that UDCA significantly reduces TGF-,1,induced apoptosis of primary rat hepatocytes through a GR-dependent effect. Immunoprecipitation assays and confocal microscopy showed that UDCA enhanced free GR levels with subsequent GR nuclear translocation. Interestingly, when a carboxy-terminus deleted form of GR was used, UDCA no longer increased free GR and/or GR translocation, nor did it protect against TGF-,1,induced apoptosis. In co-transfection experiments with GR response element reporter and overexpression constructs, UDCA did not enhance the transactivation of GR with TGF-,1. Finally, using a flourescently labeled UDCA molecule, the bile acid appeared diffuse in the cytosol but was aggregated in the nucleus of hepatocytes. Both siRNA assays and transfection experiments with either wild-type or mutant forms of GR showed that nuclear trafficking occurs through a GR-dependent mechanism. In conclusion, these results further clarify the antiapoptotic mechanism(s) of UDCA and suggest that GR is crucial for the nuclear translocation of this bile acid for reducing apoptosis. (HEPATOLOGY 2005;42:925,934.) [source] Butyrate inhibits leukocyte adhesion to endothelial cells via modulation of VCAM-1INFLAMMATORY BOWEL DISEASES, Issue 2 2004Thomas Menzel MD Abstract Background Leukocyte recruitment to areas of inflammation depends on Integrin-VCAM/ICAM interaction. Blocking the vascular cell adhesion molecule (VCAM-1) and the intracellular adhesion molecule (ICAM-1) may have therapeutic benefit for the inflammatory component of bowel disease. Notably, the induction of ICAM and VCAM is mediated by a nuclear factor kappaB (NF-,B)-dependent mechanism. We investigated whether the anti-inflammatory properties of butyrate are mediated via the modulation of VCAM and ICAM on human endothelial cells. Methods VCAM-1 and ICAM-1 expression on human endothelial cells upon tumor necrosis factor-, (TNF-,) stimulation was assessd by FACS analysis. A monocyte adhesion assay was performed to evaluate the relevance of a modulated CAM-expression. Electrophoretic mobility shift assays were applied to investigate NF-,B activation. Results The observed butyrate-associated inhibition of monocyte adhesion to endothelial cells is associated with an inhibition of NF-,B activation in human endothelial cells. In this context, the observed suppression of the TNF-, induced VCAM-1 expression is likely to play an essential role. Conclusions Butyrate inhibits VCAM-1 mediated leukocyte adhesion to human endothelial cells. This inhibition may contribute to the anti-inflammatory effects of butyrate in patients with distal ulcerative colitis. [source] Inhibition of connective tissue growth factor/CCN2 expression in human dermal fibroblasts by interleukin-1, and ,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2010D. Nowinski Abstract Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)-, and intimately involved with tissue repair and overexpressed in various fibrotic conditions. We previously showed that keratinocytes in vitro downregulate TGF-,-induced expression of CTGF in fibroblasts by an interleukin (IL)-1 ,-dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL-1, and ,. Human dermal fibroblasts and NIH 3T3 cells were treated with IL-1, or , in presence or absence of TGF-,1. IL-1 suppressed basal and TGF-,-induced CTGF mRNA and protein expression. IL-1, and , inhibited TGF-,-stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3-binding CAGA elements. Furthermore, IL-1, and , inhibited TGF-,-stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition, RNA interference suggested that TGF-, activated kinase1 (TAK1) is necessary for IL-1 inhibition of TGF-,-stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin. J. Cell. Biochem. 110: 1226,1233, 2010. Published 2010 Wiley-Liss, Inc. [source] The effect of sildenafil, a phosphodiesterase-5 inhibitor, on acetic acid-induced colonic inflammation in the ratJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 6 2009Sevgin Ozlem Iseri Abstract Background and Aim:, Sildenafil, a selective and potent inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase (PDE)5, has a relaxant effect on the smooth muscle cells of the arterioles supplying the human corpus cavernosum acting via nitric oxide (NO)-dependent mechanism. This study aimed to investigate the possible protective effect of sildenafil citrate on the extent of tissue integrity, oxidant-antioxidant status and neutrophil infiltration to the inflamed organ in a rat model of acetic acid-induced colitis. Methods:, Colitis was induced by intrarectal administration of 1 mL of 5% acetic acid to Sprague-Dawley rats (200,250 g; n = 7,8/group). Control rats received an equal volume of saline intrarectally. In treatment groups, the rats were treated with either sildenafil citrate (5 mg/kg/day; subcutaneously) or saline for 3 days. After decapitation, distal colon was weighed and scored macroscopically and microscopically. Tissue samples were used for the measurement of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity, and oxidant production. Trunk blood was collected for the assessment of serum tumor necrosis factor (TNF)-, and interleukin (IL)-1, levels. Results:, In the colitis group, the colonic tissue was characterized by lesions, increased lipid peroxidation with a concomitant reduction in GSH content, increased MPO activity and oxidant production. Serum TNF-, and IL-1, levels were higher in the colitis group compared to control values. Sildenafil reversed these inflammatory parameters nearly back to control values. Conclusions:, Sildenafil citrate administration to rats with acetic acid-induced colitis seems to be beneficial via prevention of lipid peroxidation, oxidant generation, cytokine production and neutrophil accumulation. [source] Abnormal post-translational and extracellular processing of brevican in plaque-bearing mice over-expressing APPswJOURNAL OF NEUROCHEMISTRY, Issue 3 2010Joanne M. Ajmo J. Neurochem. (2010) 113, 784,795. Abstract Aggregation of amyloid-, (A,) in the forebrain of Alzheimer's disease (AD) subjects may disturb the molecular organization of the extracellular microenvironment that modulates neural and synaptic plasticity. Proteoglycans are major components of this extracellular environment. To test the hypothesis that A,, or another amyloid precursor protein (APP) dependent mechanism modifies the accumulation and/or turnover of extracellular proteoglycans, we examined whether the expression and processing of brevican, an abundant extracellular, chondroitin sulfate (CS)-bearing proteoglycan, were altered in brains of A,-depositing transgenic mice (APPsw , APP gene bearing the Swedish mutation) as a model of AD. The molecular size of CS chains attached to brevican was smaller in hippocampal tissue from APPsw mice bearing A, deposits compared to non-transgenic mice, likely because of changes in the CS chains. Also, the abundance of the major proteolytic fragment of brevican was markedly diminished in extracts from several telencephalic regions of APPsw mice compared to non-transgenic mice, yet these immunoreactive fragments appeared to accumulate adjacent to the plaque edge. These results suggest that A, or APP exert inhibitory effects on proteolytic cleavage mechanisms responsible for synthesis and turnover of proteoglycans. As proteoglycans stabilize synaptic structure and inhibit molecular plasticity, defective brevican processing observed in A,-bearing mice and potentially end-stage human AD, may contribute to deficient neural plasticity. [source] Involvement of nuclear factor-kappa B in bcl-xL-induced interleukin 8 expression in glioblastomaJOURNAL OF NEUROCHEMISTRY, Issue 3 2008Chiara Gabellini Abstract We recently reported that bcl-xL regulates interleukin 8 (CXCL8) protein expression and promoter activity in glioblastoma cells. In this paper we demonstrate that CXCL8 induction by bcl-xL is mediated through a nuclear factor-kappa B (NF-kB)-dependent mechanism. Mutational studies on the CXCL8 promoter showed that NF-kB binding site was required for bcl-xL-induced promoter activity and an enhanced nuclear expression of NF-kB subunits p65 and p50 was observed after bcl-xL over-expression. Electrophoretic mobility shift assay showed an increased DNA-binding activity of NF-kB in bcl-xL over-expressing cells and the use of specific antibodies confirmed the involvement of p65 and p50 in NF-kB activity on CXCL8 promoter sequence. NF-kB activity regulation by bcl-xL involved IkB, and IKK complex signaling pathway. In fact, bcl-xL over-expression induced a decrease of cytoplasmic expression of the IkB, protein, paralleled by an increase in the phosphorylation of the same IkB, and IKK,/,. Moreover, the down-regulation of the ectopic or endogenous bcl-xL expression through RNA interference confirmed the ability of bcl-xL to modulate NF-kB pathway, and the transient expression of a degradation-resistant form of the cytoplasmic NF-kB inhibitor IkB, in bcl-xL transfectants confirmed the involvement of that inhibitor in bcl-xL-induced CXCL8 expression and promoter activity. In conclusion, our results demonstrate the role of NF-kB as the mediator of bcl-xL-induced CXCL8 up-regulation in glioblastoma cells. [source] Pregnenolone sulfate induces NMDA receptor dependent release of dopamine from synaptic terminals in the striatumJOURNAL OF NEUROCHEMISTRY, Issue 2 2008Matthew T. Whittaker Abstract Neuromodulators that alter the balance between lower-frequency glutamate-mediated excitatory and higher-frequency GABA-mediated inhibitory synaptic transmission are likely to participate in core mechanisms for CNS function and may contribute to the pathophysiology of neurological disorders such as schizophrenia and Alzheimer's disease. Pregnenolone sulfate (PS) modulates both ionotropic glutamate and GABAA receptor mediated synaptic transmission. The enzymes necessary for PS synthesis and degradation are found in brain tissue of several species including human and rat, and up to 5 nM PS has been detected in extracts of postmortem human brain. Here, we ask whether PS could modulate transmitter release from nerve terminals located in the striatum. Superfusion of a preparation of striatal nerve terminals comprised of mixed synaptosomes and synaptoneurosomes with brief-duration (2 min) pulses of 25 nM PS demonstrates that PS increases the release of newly accumulated [3H]dopamine ([3H]DA), but not [14C]glutamate or [3H]GABA, whereas pregnenolone is without effect. PS does not affect dopamine transporter (DAT) mediated uptake of [3H]DA, demonstrating that it specifically affects the transmitter release mechanism. The PS-induced [3H]DA release occurs via an NMDA receptor (NMDAR) dependent mechanism as it is blocked by D-2-amino-5-phosphonovaleric acid. PS modulates DA release with very high potency, significantly increasing [3H]DA release at PS concentrations as low as 25 pM. This first report of a selective direct enhancement of synaptosomal dopamine release by PS at picomolar concentrations via an NMDAR dependent mechanism raises the possibility that dopaminergic axon terminals may be a site of action for this neurosteroid. [source] Response to Male Odours in Progestin Receptor- and Oestrogen Receptor-Containing Cells in Female Rat BrainJOURNAL OF NEUROENDOCRINOLOGY, Issue 6 2002A. L. Bennett Abstract Sensory cues from male rats, such as odours and vaginal-cervical stimulation (VCS), play a modulatory role in female rat sexual behaviour. For example, exposure to male odours and VCS appears to be at least partially responsible for increases in sexual behaviour following repeated mating of oestradiol-primed female rats. Although there is evidence that VCS influences sexual behaviour via a ligand-independent progestin receptor (PR)-dependent mechanism, the mechanism by which odours influence sexual behaviour is not known. We tested the hypothesis that, similar to VCS, the effects of male odours on sexual behaviour are mediated by progestin receptors. Female rats were injected with the progestin antagonist, RU486, or oil vehicle and were then exposed to male-soiled bedding or clean bedding. Although exposure to male-soiled bedding resulted in higher levels of Fos immunoreactivity in brain areas associated with female sexual behaviour, the progestin antagonist did not reduce this effect. Furthermore, there was minimal coexpression of odour-induced Fos and progestin receptors in brain areas associated with female sexual behaviour. Together, these results suggest that the effects of male odours are not mediated by a PR-dependent mechanism. Therefore, we tested the hypothesis that oestrogen receptor (ER)-containing cells are involved in the effects of olfactory cues. Although there was virtually no coexpression of ER, and odour-induced Fos in brain areas associated with female sexual behaviour, exposure to male odours slightly increased the number of cells coexpressing ER, and odour-induced Fos in the posterodorsal medial amygdala. Although, these results do not support the hypothesis that the effects of odours are mediated by a PR-dependent mechanism, they suggest that integration of male odours and hormonal cues may occur in ER,-containing cells in the posterodorsal medial amygdala. [source] CRTH2 mediates the activation of human Th2 cells in response to PGD2 released from IgE/anti-IgE treated nasal polyp tissueALLERGY, Issue 3 2010C. A. Pérez-Novo To cite this article: Pérez- Novo CA, Holtappels G, Vinall SL, Xue L, Zhang N, Bachert C, Pettipher R. CRTH2 mediates the activation of human Th2 cells in response to PGD2 released from IgE/anti-IgE treated nasal polyp tissue. Allergy 2010; 65: 304,310. Abstract Background:, Mast cells release mediators upon stimulation that contribute to the pathogenesis of chronic airway disease, including the recruitment and activation of Th2 lymphocytes. The objective was to determine the involvement of prostaglandin D2 (PGD2) and its receptors in the chemotaxis of Th2 cells, using nasal polyp tissue. Methods:, Tissue explants from ten patients with nasal polyposis were incubated with RPMI alone or RPMI containing IgE/anti-IgE for 30 min. Some samples were treated with diclofenac to inhibit the production of PGD2. Supernatants were assayed for PGD2 content and for their ability to promote human Th2 cell chemotaxis in the presence and absence of a CRTH2 antagonist. Transcript levels of D protanoid receptor type 1 (DP1), chemoattractant receptor-homologous receptor expressed on Th2 cells (CRTH2) and PGD2 synthase were analysed by real time PCR. Results:, Increased release of PGD2 by nasal polyp tissue treated with IgE/anti-IgE was significantly inhibited by preincubation of the tissue with diclofenac. Transcript levels of PGD2 synthase, DP1 and CRTH2 receptors increased after stimulation with IgE/anti-IgE. Supernatants from IgE/anti-IgE-stimulated nasal polyp tissue caused significantly increased chemotaxis of Th2 cells. The levels of PGD2 produced and the degree of Th2 cell chemotaxis were highly correlated. Diclofenac inhibited the production of Th2 cell chemotactic activity, and the chemotactic effect of the supernatant on Th2 cells was inhibited by the CRTH2 antagonist ramatroban. Conclusion:, These data suggest that in immunologically activated nasal polyp tissue, PGD2 produced by mast cells promotes the migration of Th2 cells through a CRTH2 dependent mechanism. [source] Inactivation of the decay pathway initiated at an internal site by RNase E promotes poly(A)-dependent degradation of the rpsO mRNA in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 4 2003Paulo E. Marujo Summary In Escherichia coli, RNA degradation is mediated by endonucleolytic processes, frequently mediated by RNase E, and also by a poly(A)-dependent mechanism. The dominant pathway of decay of the rpsO transcripts is initiated by an RNase E cleavage occurring at a preferential site named M2. We demonstrate that mutations which prevent this cleavage slow down degradation by RNase E. All these mutations reduce the single-stranded character of nucleotides surrounding the cleavage site. Moreover, we identify two other cleavage sites which probably account for the slow RNase E-mediated degradation of the mutated mRNAs. Failure to stabilize the rpsO transcript by appending a 5, hairpin indicates that RNase E is not recruited by the 5, end of mRNA. The fact that nucleotide substitutions which prevent cleavage at M2 facilitate the poly(A)-dependent degradation of the rpsO transcripts suggest an interplay between the two mechanisms of decay. In the discussion, we speculate ,that ,a ,structural ,feature ,located ,in ,the ,vicinity of M2 could be an internal degradosome entry site promoting both RNase E cleavages and poly(A)-dependent degradation of the rpsO mRNA. We also discuss the role of poly(A)-dependent decay in mRNA metabolism. [source] Autonomous contractile activity in the isolated rat bladder is modulated by a TRPV1 dependent mechanism,NEUROUROLOGY AND URODYNAMICS, Issue 3 2007Thomas Gevaert Abstract Aims Resiniferatoxin (RTX), a vanilloid compound and agonist of the transient receptor potential channel 1 (TRPV1), is known for its beneficial effects on neurogenic detrusor overactivity. The mainstream rationale for its use is the desensitization of TRPV1 on sensory bladder afferents. However, recent findings showed that TRPV1 is present in other cell types in the bladder. To eliminate the effects of RTX on spinal and central neural circuits, we investigated autonomous contractility in normal and neurogenic rat bladders after treatment with RTX. Methods Female Wistar rats were made paraplegic at vertebral level T8,T9. Animals were intravesically pre-treated with vehicle (ethanol 5%) or RTX (100 nM) and sacrificed after 72 hr. Each bladder was excised and placed in a heated organ bath, where intravesical pressures were measured. Effects on contractile parameters of intravesical volume load, the non-selective muscarinic receptor agonist carbachol (CA) and electrical stimulation (ES) of nerves were studied in both groups. Results In RTX-treated normal bladders we found shorter contractions with higher amplitude than in control bladders (P,<,0.05). In RTX-treated neurogenic bladders the amplitude and duration of autonomous contractions were increased compared with controls (P,<,0.05). Furthermore RTX induced an increased response to CA and to ES (P,<,0.05). Conclusions RTX significantly affected the properties of autonomous bladder contractile activity. This provides evidence for local effects of RTX on bladder contractile activity, which are not mediated by afferent neural pathways and which may contribute to the beneficial effects on detrusor overactivity. TRPV1 and TRPV1+ cells seem to play an important role in (autonomous) bladder contractility. Neurourol. Urodynam. 26:424,432, 2007. © 2006 Wiley-Liss, Inc. [source] Nebivolol Dilates Human Penile Arteries and Reverses Erectile Dysfunction in Diabetic Rats through Enhancement of Nitric Oxide SignalingTHE JOURNAL OF SEXUAL MEDICINE, Issue 8 2010Javier Angulo PhD ABSTRACT Introduction., Traditional beta-blockers have sometimes been associated with erectile dysfunction (ED). Nebivolol is a cardioselective ,1 -adrenoceptor antagonist that promotes vasodilation through a nitric oxide (NO)-dependent mechanism. Aim., We evaluated the effects of nebivolol on the NO/cyclic guanosine monophosphate (cGMP) signaling pathway, on erectile function and dysfunction, and in human penile vascular tissues. Methods., Erectile response to cavernosal nerve electrical stimulation in control and diabetes-induced ED rats were evaluated, along with serum nitrite/nitrate (NOx) concentration and plasma/tissue cGMP levels. Endothelium-dependent and sildenafil-induced relaxation of isolated human corpus cavernosum (HCC) and human penile resistance arteries (HPRA) were also determined. Main Outcome Measures., The effects of nebivolol on erectile function and dysfunction and on NO/cGMP-mediated responses. Results., Treatment with nebivolol significantly potentiated erectile response in control rats, regardless of its effects on blood pressure. Nebivolol increased NOx and plasma cGMP by 3-fold and 2.75-fold, respectively, and significantly augmented the elevation of plasma cGMP produced by sildenafil. Nebivolol enhanced endothelium-dependent and sildenafil-induced relaxations of HCC tissue, and produced endothelium-dependent vasodilation of HPRA. Nebivolol, but not atenolol, significantly improved erectile response in diabetic rats (51.6%, 53.2%, and 87.1% of response at 3 Hz in nondiabetic rats, for vehicle-treated, atenolol-treated, and nebivolol-treated diabetic rats, respectively); after sildenafil administration, ED was completely reversed in nebivolol-treated diabetic rats (69.6% and 112% for diabetic rats treated with sildenafil and nebivolol plus sildenafil, respectively). Accordingly, nebivolol restored systemic NOx levels and cGMP content in penile tissue from these animals. Conclusions., Nebivolol in vivo activated the NO/cGMP pathway, enhanced erectile response and reversed ED in diabetic rats. Moreover, nebivolol in vitro potentiated NO/cGMP-mediated relaxation of human erectile tissues. These effects may account for the low incidence of ED in nebivolol-treated hypertensive patients. Nebivolol therefore may have utility in the treatment of ED, particularly ED associated with diabetes. Angulo J, Wright HM, Cuevas P, González-Corrochano R, Fernández A, Cuevas B, La Fuente JM, Gupta S, and de Tejada IS. Nebivolol dilates human penile arteries and reverses erectile dysfunction in diabetic rats through enhancement of nitric oxide signaling. J Sex Med 2010;7:2681,2697. [source] Role of IFN, in Allograft Tolerance Mediated by CD4+CD25+ Regulatory T Cells by Induction of IDO in Endothelial CellsAMERICAN JOURNAL OF TRANSPLANTATION, Issue 11 2007P. Thebault Regulatory T cells have been described to specifically accumulate at the site of regulation together with effector T cells and antigen-presenting cells, establishing a state of local immune privilege. However the mechanisms of this interplay remain to be defined. We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short-term treatment with a deoxyspergualine analogue, LF15-0195, induces long-term allograft tolerance with a specific expansion of regulatory CD4+CD25+T cells that accumulate within the graft. In this study, we show that following transfer of regulatory CD4+T cells to a secondary irradiated recipient, regulatory CD25+Foxp3+ and CD25+Foxp3, CD4+T cells accumulate at the graft site and induce graft endothelial cell expression of Indoleamine 2, 3-dioxygenase (IDO) by an IFN,-dependent mechanism. Moreover, in vivo transfer of tolerance can be abrogated by blocking IFN, or IDO, and anti-IFN, reduces the survival/expansion of alloantigen-induced regulatory Foxp3+CD4+T cells. Together, our results demonstrate interrelated mechanisms between regulatory CD4+CD25+T cells and the graft endothelial cells in this local immune privilege, and a key role for IFN, and IDO in this process. [source] Nuclear autoantigen CENP-B transactivation of the epidermal growth factor receptor via chemokine receptor 3 in vascular smooth muscle cellsARTHRITIS & RHEUMATISM, Issue 9 2009Genevičve Robitaille Objective We have previously found that the CENP-B nuclear autoantigen, which is specifically targeted by autoantibodies in the limited cutaneous form of systemic sclerosis, behaved as a potent migratory factor for human pulmonary artery smooth muscle cells (PASMCs). Other recent studies have shown that several disease-associated autoantigens induced cell migration by interacting with various chemokine receptors. Prompted by this hypothesis, we undertook this study to determine whether CENP-B interacts with chemokine receptors on the surface of human PASMCs, to explore the relevant signaling pathways, and to characterize the effects of anti,CENP-B binding on SMC stimulation. Methods To demonstrate the expression of specific chemokine receptors by human PASMCs at both the messenger RNA and protein levels, reverse transcription,polymerase chain reaction, immunoblotting, and flow cytometry analyses were performed. Desensitization studies and specific inhibitors were used to further identify the CENP-B target on the surface of human PASMCs. Results Our data strongly suggested that CENP-B used chemokine receptor 3 (CCR3) to mediate human PASMCs signaling. Moreover, several lines of evidence indicated that CENP-B binding subsequently stimulated the cross-talk between CCR3 and epidermal growth factor receptor (EGFR) via a matrix metalloprotease,dependent mechanism that involved the processing of heparin-binding EGF-like growth factor. Transactivation of the EGFR through CCR3 was found to be a critical pathway that elicits MAP kinase activation and secretion of cytokines such as interleukin-8. Finally, anti,CENP-B autoantibodies were found to abolish this signaling pathway, thus preventing CENP-B from transactivating EGFR and exerting its cytokine-like activities toward vascular smooth muscle cells. Conclusion The identification of CENP-B as a CCR3 ligand opens up new perspectives for the study of the pathogenic role of anti,CENP-B autoantibodies. [source] Novel markers of inflammation identified in tumor necrosis factor receptor,associated periodic syndrome (TRAPS) by transcriptomic analysis of effects of TRAPS-associated tumor necrosis factor receptor type I mutations in an endothelial cell lineARTHRITIS & RHEUMATISM, Issue 1 2009Susana L. Rebelo Objective To analyze the effects of tumor necrosis factor receptor,associated periodic syndrome (TRAPS),associated mutant tumor necrosis factor receptor type I (TNFRI) expression in a cell type directly relevant to the inflammation in TRAPS, and to identify novel markers associated with mutant TNFRI expression. Methods Transcriptome analysis on 30,000 human genes was performed on SK-Hep-1 human endothelial cells transfected with either wild-type (WT) or TRAPS-associated mutant TNFRI. Quantitative reverse transcriptase,polymerase chain reaction and protein expression levels measured by enzyme-linked immunosorbent assay verified transcriptional changes for selected genes both in supernatants from cells expressing mutant TNFRI and in patient plasma. Results Cells expressing mutant TNFRI showed up-regulation of multiple proinflammatory genes relative to WT transfectants, including genes for pentraxin 3, granulocyte,macrophage colony-stimulating factor, granulocyte colony-stimulating factor, CCL2, and CCL5, which were also expressed as proteins. In addition, the expression of most of these markers was increased in the plasma and peripheral blood mononuclear cells from TRAPS patients relative to those from healthy controls. The cysteine mutations (C33Y and C52F), which are associated with a more severe clinical phenotype, induced more genes than the low-penetrance mutation R92Q, which is associated with a milder phenotype. The expression of most genes was induced by a death domain (DD),dependent mechanism, since they were not induced by expression of TNFRI mutants with an inactivated DD. Conclusion TRAPS-associated TNFRI mutants induce the expression of multiple genes encoding inflammatory molecules, cellular receptors, transcription factors, and regulators of apoptosis in endothelial cells that require the cytoplasmic signaling properties of the receptor. Different mutants have specific expression profiles, indicating mutation-specific effects. The expression of some of these markers was also elevated in samples from TRAPS patients. [source] Opsonization of late apoptotic cells by systemic lupus erythematosus autoantibodies inhibits their uptake via an Fc, receptor,dependent mechanismARTHRITIS & RHEUMATISM, Issue 10 2007Esther Reefman Objective Decreased clearance of apoptotic cells is suggested to be a major pathogenic factor in systemic lupus erythematosus (SLE). The aim of this study was to investigate whether the binding of SLE autoantibodies to apoptotic cells influences the phagocytosis of these cells by macrophages. Methods Apoptosis was induced in a human T cell line (Jurkat) and a keratinocyte cell line (HaCaT) by ultraviolet B irradiation. Binding of purified IgG from 26 SLE patients and 15 healthy controls to apoptotic cells was assessed by flow cytometry and Western blotting. Phagocytosis of IgG-opsonized apoptotic cells by monocyte-derived macrophages was assessed by light microscopy. Similar experiments were performed with a monoclonal antibody against SSA/Ro and IgG fractions from 5 patients with Sjögren's syndrome (SS) and 5 patients with rheumatoid arthritis (RA). Results IgG fractions from all 26 SLE patients bound to late apoptotic, but not early apoptotic, cells. IgG fractions isolated from SLE patients with different autoantibody profiles showed comparable levels of binding. IgG fractions from healthy controls did not bind. Opsonization of apoptotic cells with IgG fractions from SLE patients resulted in a significant inhibition of phagocytosis as compared with healthy control IgG fractions. A monoclonal antibody directed against SSA/Ro and IgG isolated from 5 antinuclear antibody (ANA),positive patients with SS were also able to elicit these effects, whereas IgG from 5 ANA-negative patients with RA did not. The inhibitory effect of patient IgG was abolished by blocking either the Fc, receptors (Fc,R) or the constant region of IgG, using a specific Fc-blocking peptide. Conclusion Autoantibodies from SLE patients are able to opsonize apoptotic cells and inhibit their uptake by macrophages via an Fc,R-dependent mechanism. [source] Halofuginone inhibition of COL1A2 promoter activity via a c-Jun,dependent mechanismARTHRITIS & RHEUMATISM, Issue 10 2002Tracy L. McGaha Objective The naturally occurring compound halofuginone has been shown to antagonize collagen synthesis by fibroblasts both in vitro and in vivo. We previously demonstrated that this inhibitory property was related to the ability of halofuginone to disrupt transforming growth factor , signal transduction. The present study further analyzed the ability of halofuginone to affect transcription factors that can regulate type I collagen gene expression by examining its effect on c-Jun, the negative regulator of collagen gene transcription. Methods The phosphorylation state of c-Jun in the presence of halofuginone was examined via direct Western blotting, and the transcriptional activity of the activator protein 1 (AP-1) binding element via electrophoretic mobility shift assay and luciferase reporter assay. We determined whether the effect of halofuginone on collagen synthesis was dependent on the presence of c-Jun by ectopic expression of a wild-type or dominant-negative c-Jun construct in the presence of halofuginone and assaying ,2(I) collagen promoter strength via luciferase reporter assay. The effect of halofuginone on ,2(I) collagen message levels in fibroblasts when wild-type or dominant-negative c-Jun was overexpressed was determined. We also determined whether halofuginone had an effect on the phosphorylation state of c-Jun in the skin of TSK/+ mice via immunohistochemistry. Results Treatment of fibroblasts with 10,8M halofuginone enhanced basal and mitogen-mediated phosphorylation of c-Jun in culture. This elevated phosphorylation of c-Jun correlated with enhanced DNA binding and transcriptional activation of an AP-1 complex consisting of c-Jun and Fos but lacking the c-Jun antagonist JunB. Overexpression of c-Jun enhanced in a dose-dependent manner the ability of halofuginone to inhibit the activity of a luciferase reporter construct under control of the ,3200-bp to +54-bp COL1A2 promoter, whereas the expression of a dominant-negative c-Jun construct abolished this effect. Northern blotting showed that overexpression of c-Jun enhanced the ability of halofuginone to reduce collagen ,2(I) messenger RNA levels in fibroblasts, whereas expression of the dominant-negative c-Jun abolished this effect. Topical administration of a halofuginone-containing cream for 20 days to TSK mice, which spontaneously develop dermal fibrosis, greatly increased the phosphorylated form of c-Jun in the skin; this was followed by a decrease in skin thickness and type I collagen messenger RNA expression. Conclusion Our findings illustrate the powerful down-regulatory property of c-Jun toward type I collagen and establish that halofuginone exerts its effect on collagen synthesis in a c-Jun,dependent manner. [source] Significant cytotoxic activity in vitro of the EGFR tyrosine kinase inhibitor gefitinib in acute myeloblastic leukaemiaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2008Elin Lindhagen Abstract Objectives:, Gefitinib inhibits epidermal growth factor receptor (EGFR) signalling, but may also act by non-EGFR dependent mechanisms. We have investigated the activity of gefitinib in haematological tumour cells, in particular acute myeloblastic leukaemia (AML). Methods:, Cytotoxic activity of gefitinib, alone or in combination with standard anti-leukaemic drugs, was assessed by the short-term fluorometric microculture cytotoxicity assay in tumour cells from 117 patients representing five haematological and five non-haematological malignancies. In AML, the EGFR status was analysed by immunochemistry. Gefitinib-induced apoptosis was investigated in a subset of AML samples, as well as in the leukaemia cell line MV-4-11, using a multiparametric high content screening assay. To confirm activation of caspase-3 in cells treated with gefitinib, a blocking test was carried out in which MV4-11 cells were pretreated with the specific caspase inhibitor DEVD-FMK. Results:, Gefitinib showed highest cytotoxic activity in AML (n = 19) with many samples being sensitive at concentrations achievable in clinical practice (<10 ,M), and no difference between previously untreated and relapsed patients. No correlation between the activity of gefitinib and standard antileukaemic drugs (cytarabine, doxorubicin, etoposide) was observed. Combining gefitinib with these drugs resulted in mainly additive or synergistic (etoposide) effects, with no evidence of sequence dependency. The AML cells did not express the EGFR. Gefitinib induced apoptosis, which was at least partly mediated by activation of the caspase-3 pathway. Conclusion:,In vitro, gefitinib has significant cytotoxic activity in AML by inducing apoptosis through non-EGFR dependent pathways. [source] Novel CD8+ Treg suppress EAE by TGF-,- and IFN-,-dependent mechanismsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2009Mei-Ling Chen Abstract Although CD8+ Treg-mediated suppression has been described, CD8+ Treg remain poorly characterized. Here we identify a novel subset of CD8+ Treg that express latency-associated peptide (LAP) on their cell surface (CD8+LAP+ cells) and exhibit regulatory activity in vitro and in vivo. Only a small fraction of CD8+LAP+ cells express Foxp3 or CD25, although the expression levels of Foxp3 for these cells are higher than their LAP, counterparts. In addition to TGF-,, CD8+LAP+ cells produce IFN-,, and these cells suppress EAE that is dependent on both TGF-, and IFN-,. In an adoptive co-transfer model, CD8+LAP+ cells suppress myelin oligodendrocyte glycoprotein (MOG)-specific immune responses by inducing or expanding Foxp3+ cells and by inhibiting proliferation and IFN-, production in vivo. Furthermore, in vivo neutralization of IFN-, and studies with IFN-,-deficient mice demonstrate an important role for IFN-, production in the function of CD8+LAP+ cells. Our findings identify the underlying mechanisms that account for the immunoregulatory activity of CD8+ T cells and suggest that induction or amplification of CD8+LAP+ cells may be a therapeutic strategy to help control autoimmune processes. [source] IL-10 modulates cytokine gene transcription by protein synthesis-independent and dependent mechanisms in lipopolysaccharide-treated neutrophilsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2007Marzia Rossato Abstract We have recently reported that the ability of IL-10 to rapidly exert its anti-inflammatory effects on human neutrophils is dependent upon exposure of these cells to LPS for at least 3,4,h. Here, we demonstrate that, in neutrophils "preconditioned" by LPS, IL-10 primarily targets the transcription of TNF-,, CXCL8 and IL-1ra genes, as revealed by primary transcript real-time RT-PCR. We also show that IL-10-induced transcriptional repression of TNF-, and CXCL8 genes consists of two distinct phases: an early one, occurring rapidly and in a protein synthesis-independent manner, followed by a second phase, more delayed and dependent on protein synthesis. Interestingly, the protein synthesis dependence of the latter phase coincides with a reduced ability of IL-10 to induce STAT3 tyrosine phosphorylation. Importantly, inhibition of IL-10-induced STAT3 activation and IL-10-suppressive action by a prolonged exposure to cycloheximide (CHX) was observed to occur also in human monocytes and was caused by a defective IL-10-mediated activation of Jak1 and Tyk2 kinases. Taken together, our findings suggest that CHX interferes with the IL-10-mediated intracellular signaling pathway by interrupting events upstream of STAT3 activation. These data question the concept of the requirement of an IL-10-induced mediator as the unique mechanism to execute IL-10 anti-inflammatory program. [source] Fas and TNFR1, but not cytolytic granule-dependent mechanisms, mediate clearance of murine liver adenoviral infection,HEPATOLOGY, Issue 1 2005Marwan S. Abougergi After intravenous injection of replication-deficient adenovirus, hepatocytes are transduced and express high levels of adenovirus-encoded genes. However, adenovirally encoded gene expression is ablated rapidly by CD8+ T-cell,dependent mechanisms. Thus, this model is suitable for examining intrahepatic cytotoxic T lymphocyte (CTL) effector mechanisms. In the present studies, recombinant adenoviruses encoding secreted (human apolipoprotein A-I) or intracellular (,-galactosidase) gene products were infused into mice with genetic deficiencies affecting the granule exocytosis-, Fas-, or tumor necrosis factor receptor 1 (TNFR1)-mediated pathways of CTL and natural killer cell effector function; the rates of clearance of adenovirus-encoded gene products were assessed. Clearance of secreted or intracellular adenoviral gene products was not delayed in perforin-deficient mice or dipeptidyl peptidase I-deficient mice, which fail to process and activate granzyme A or granzyme B. TNFR1-deficient mice also exhibited no delay in clearance of adenoviral gene products. However, adenoviral clearance from Fas-deficient mice was delayed, and such delays were much greater in mice deficient in both TNFR1 and Fas. In contrast, chimeric mice lacking both hepatic Fas and lymphocyte perforin function exhibited no greater delay in adenoviral clearance than chimeras deficient only in hepatic Fas expression. In conclusion, Fas-dependent mechanisms are required for efficient clearance of virally infected hepatocytes and, in Fas-deficient animals, TNFR1-dependent mechanisms provide an alternative mechanism for hepatic adenovirus clearance. In contrast, perforin- and granule protease,dependent cytotoxicity mechanisms play no apparent role in clearance of adenovirus from the liver. (HEPATOLOGY 2005;41:97,105.) [source] Role of mitogen-activated protein kinases in tauroursodeoxycholic acid-induced bile formation in cholestatic rat liverHEPATOLOGY RESEARCH, Issue 7 2008Gerald Ulrich Denk Aim:, Ursodeoxycholic acid exerts anticholestatic effects in various cholestatic disorders and experimental models of cholestasis. Its taurine conjugate (TUDCA) stimulates bile salt secretion in isolated perfused rat livers (IPRL) under physiological, non-cholestatic conditions, in part by mitogen-activated protein kinase (MAPK)-dependent mechanisms. The role of MAPK in the anticholestatic effect of TUDCA, however, is unclear. Therefore, we studied the role of MAPK in the anticholestatic effect of TUDCA in IPRL and isolated rat hepatocytes (IRH) in taurolithocholic acid (TLCA)-induced cholestasis. Methods:, Bile flow, biliary levels of 2,4-dinitrophenyl-S-glutathione (GS-DNP) as a marker of hepatobiliary organic anion secretion and activity of lactate dehydrogenase (LDH) in hepatovenous effluate as a marker of hepatocellular damage in IPRL perfused with TUDCA and/or TLCA were determined in the presence or absence of MAPK inhibitors. In addition, phosphorylation of Erk 1/2 and p38MAPK induced by TUDCA and/or TLCA was studied by Western immunoblot in IPRL and IRH. Results:, TUDCA-induced bile flow was impaired by the Erk 1/2 inhibitor PD98059 in normal livers (,28%), but not in livers made cholestatic by TLCA. GS-DNP secretion was unaffected by PD98059 under both conditions. TUDCA-induced bile formation and organic anion secretion both in the presence and absence of TLCA were unaffected by the p38MAPK inhibitor SB202190. Erk 1/2 phosphorylation in liver tissue was unchanged after bile salt exposure for 70 min, but was transiently enhanced by TUDCA in IRH. Conclusion:, MAPK do not mediate the anticholestatic effects of TUDCA in TLCA-induced cholestasis. [source] Differential phosphorylation of myosin light chain (Thr)18 and (Ser)19 and functional implications in plateletsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2010T. M. GETZ Summary. Background:, Myosin IIA is an essential platelet contractile protein that is regulated by phosphorylation of its regulatory light chain (MLC) on residues (Thr)18 and (Ser)19 via the myosin light chain kinase (MLCK). Objective:, The present study was carried out to elucidate the mechanisms regulating MLC (Ser)19 and (Thr)18 phosphorylation and the functional consequence of each phosphorylation event in platelets. Results:, Induction of 2MeSADP-induced shape change occurs within 5 s along with robust phosphorylation of MLC (Ser)19 with minimal phosphorylation of MLC (Thr)18. Selective activation of G12/13 produces both slow shape change and comparably slow MLC (Thr)18 and (Ser)19 phosphorylation. Stimulation with agonists that trigger ATP secretion caused rapid MLC (Ser)19 phosphorylation while MLC (Thr)18 phosphorylation was coincident with secretion. Platelets treated with p160ROCK inhibitor Y-27632 exhibited a partial inhibition in secretion and had a substantial inhibition in MLC (Thr)18 phosphorylation without effecting MLC (Ser)19 phosphorylation. These data suggest that phosphorylation of MLC (Ser)19 is downstream of Gq/Ca2+ -dependent mechanisms and sufficient for shape change, whereas MLC (Thr)18 phosphorylation is substantially downstream of G12/13 -regulated Rho kinase pathways and necessary, probably in concert with MLC (Ser)19 phosphorylation, for full contractile activity leading to dense granule secretion. Overall, we suggest that the amplitude of the platelet contractile response is differentially regulated by a least two different signaling pathways, which lead to different phosphorylation patterns of the myosin light chain, and this mechanism results in a graded response rather than a simple on/off switch. [source] Cover Picture , Mol.MOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 6 2009Nutr. Regular issues provide a wide range of research and review articles covering all aspects of Molecular Nutrition & Food Research. Selected topics of issue 6 are: Quercetin-induced apoptotic cascade in cancer cells: Antioxidant versus estrogen receptor ,-dependent mechanisms. Bog blueberry anthocyanins alleviate photoaging in ultraviolet-B irradiation-induced human dermal fibroblasts Oxidative stress due to anesthesia and surgical trauma: Importance of early enteral nutrition [source] Regulation of Kv channel expression and neuronal excitability in rat medial nucleus of the trapezoid body maintained in organotypic cultureTHE JOURNAL OF PHYSIOLOGY, Issue 9 2010Huaxia Tong Principal neurons of the medial nucleus of the trapezoid body (MNTB) express a spectrum of voltage-dependent K+ conductances mediated by Kv1,Kv4 channels, which shape action potential (AP) firing and regulate intrinsic excitability. Postsynaptic factors influencing expression of Kv channels were explored using organotypic cultures of brainstem prepared from P9,P12 rats and maintained in either low (5 mm, low-K) or high (25 mm, high-K) [K+]o medium. Whole cell patch-clamp recordings were made after 7,28 days in vitro. MNTB neurons cultured in high-K medium maintained a single AP firing phenotype, while low-K cultures had smaller K+ currents, enhanced excitability and fired multiple APs. The calyx of Held inputs degenerated within 3 days in culture, having lost their major afferent input; this preparation of calyx-free MNTB neurons allowed the effects of postsynaptic depolarisation to be studied with minimal synaptic activity. The depolarization caused by the high-K aCSF only transiently increased spontaneous AP firing (<2 min) and did not measurably increase synaptic activity. Chronic depolarization in high-K cultures raised basal levels of [Ca2+]i, increased Kv3 currents and shortened AP half-widths. These events relied on raised [Ca2+]i, mediated by influx through voltage-gated calcium channels (VGCCs) and release from intracellular stores, causing an increase in cAMP-response element binding protein (CREB) phosphorylation. Block of VGCCs or of CREB function suppressed Kv3 currents, increased AP duration, and reduced Kv3.3 and c- fos expression. Real-time PCR revealed higher Kv3.3 and Kv1.1 mRNA in high-K compared to low-K cultures, although the increased Kv1.1 mRNA was mediated by a CREB-independent mechanism. We conclude that Kv channel expression and hence the intrinsic membrane properties of MNTB neurons are homeostatically regulated by [Ca2+]i -dependent mechanisms and influenced by sustained depolarization of the resting membrane potential. [source] | |||||||||||||||||||||||||