3,653 research outputs found
Hypoxia activates IKK-NF-κB and the immune response in <em>Drosophila melanogaster</em>
No full textHypoxia, or low oxygen availability, is an important physiological and pathological stimulus for multicellular organisms. Molecularly, hypoxia activates a transcriptional programme directed at restoration of oxygen homoeostasis and cellular survival. In mammalian cells, hypoxia not only activates the HIF (hypoxia-inducible factor) family, but also additional transcription factors such as NF-?B (nuclear factor ?B). Here we show that hypoxia activates the IKK–NF-?B [I?B (inhibitor of nuclear factor ?B)–NF-?B] pathway and the immune response in Drosophila melanogaster. We show that NF-?B activation is required for organism survival in hypoxia. Finally, we identify a role for the tumour suppressor Cyld, as a negative regulator of NF-?B in response to hypoxia in Drosophila. The results indicate that hypoxia activation of the IKK–NF-?B pathway and the immune response is an important and evolutionary conserved response.</p
NF-κB translocation assay ImageJ macro
No full textThis is ImageJ macro used to determine the degree of NF-κB translocation in cells stained for NF-κB and nuclear signal. It is a part of publication entitled "Plasma extracellular vesicles signal spleen fibroblasts facilitating Plasmodium vivax adherence". Please see the publication for full author details.</p
Deletion of vitamin D receptor leads to premature emphysema/COPD by increased matrix metalloproteinases and lymphoid aggregates formation
No full textDeficiency of vitamin D is associated with accelerated decline in lung function. Vitamin D is a ligand for nuclear hormone vitamin D receptor (VDR), and upon binding it modulates various cellular functions. The level of VDR is reduced in lungs of patients with chronic obstructive pulmonary disease (COPD) which led us to hypothesize that deficiency of VDR leads to significant alterations in lung phenotype that are characteristics of COPD/emphysema associated with increased inflammatory response. We found that VDR knock-out (VDR(-/-)) mice had increased influx of inflammatory cells, phospho-acetylation of nuclear factor-kappaB (NF-κB) associated with increased proinflammatory mediators, and up-regulation of matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MMP-12 in the lung. This was associated with emphysema and decline in lung function associated with lymphoid aggregates formation compared to WT mice. These findings suggest that deficiency of VDR in mouse lung can lead to an early onset of emphysema/COPD because of chronic inflammation, immune dysregulation, and lung destruction
ROTATIONAL LINE INTENSITIES IN THE SYSTEM OF THE NF MOLECULE
No full text A. E. Douglas and W. E. Jones, Can, J. Phys. 44, 2251 (1966). J. K. G. Watson, Can. J. Phys. 46, 1637 (1968). P. H. Tennyson, A. Fontijn, and M. A. A. Clyne, Chem. Phys. 62, 171 (1981). F. D. Wayne and E. A. Colbourn, Mol. Phys. 34, 1141 (1977); 51, 531 (1984). S. J. Havriliak and D. R. Yarkony, J. Chem. Phys. 83, 1168 (1985). Address of Vervloet and Watson: Herzberg Institute of Astrophysics, National Research Council of Canada, Ottawa, Ontario, Canada KIA OR6.Author Institution:The 0-0 band of the electronic transition of the NF has been recorded with a Bomem Fourier transform spectrometer and the rotational line intensities have been fitted to theoretical involving both parallel and perpendicular transition moments. The value obtained for the ratio is 0.261, with standard deviation 0.004. This result, which corrects previous misstatements in Refs. 1 and 2 that the perpendicular component is stronger, can be combined with measured radiative to give experimental values of and that will be compared with ab initio
Current concepts on oxidative/carbonyl stress, inflammation and epigenetics in pathogenesis of chronic obstructive pulmonary disease
No full textChronic obstructive pulmonary disease (COPD) is a global health problem. The current therapies for COPD are poorly effective and the mainstays of pharmacotherapy are bronchodilators. A better understanding of the pathobiology of COPD is critical for the development of novel therapies. In the present review, we have discussed the roles of oxidative/aldehyde stress, inflammation/immunity, and chromatin remodeling in the pathogenesis of COPD. An imbalance of oxidants/antioxidants caused by cigarette smoke and other pollutants/biomass fuels plays an important role in the pathogenesis of COPD by regulating redox-sensitive transcription factors (e.g., NF-κB), autophagy and unfolded protein response leading to chronic lung inflammatory response. Cigarette smoke also activates canonical/alternative NF-κB pathways and their upstream kinases leading to sustained inflammatory response in lungs. Recently, epigenetic regulation has been shown to be critical for the development of COPD because the expression/activity of enzymes that regulate these epigenetic modifications have been reported to be abnormal in airways of COPD patients. Hence, the significant advances made in understanding the pathophysiology of COPD as described herein will identify novel therapeutic targets for intervention in COPD
Efeito citotóxico do óxido nítrico e do taxol em células linfoblásticas leucêmicas: envolvimento do fator de transcrição NF-kB e papel da glutationa
No full textTese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Farmacologia.O presente trabalho avaliou, através de técnicas moleculares, bioquímicas e farmacológicas, o efeito citotóxico do óxido nítrico (NO) e do taxol sobre células linfoblásticas leucêmicas. Foi investigado o envolvimento do NF-kB e a importância da glutationa na citotoxicidade causada pelo NO e pelo taxol. Os resultados obtidos, demonstraram que o efeito citotóxico do NO e do taxol sobre células leucêmicas linfoblásticas parecem atuar em vias muito semelhantes, ou seja, ambos apresentaram ações independentes de alterações no citoesqueleto, inibiram a atividade do NF-kB e depletaram os níveis de glutationa (GSH). Além disso, quando associados, apresentaram efeito citotóxico sinérgico. Sendo assim, concluímos que o NO e o taxol, além de diminuírem os níveis de glutationa intracelular, o que sensibiliza as células aos danos oxidativos e nitrosativos, também impedem o restabelecimento dos níveis normais de GSH intracelular, através da inibição da atividade do NF-kB
Pathogenetics mechanisms in celiac disease
No full textLa tesi di Dottorato, svolta presso il Dipartimento di Farmacologia Sperimentale, redatta in lingua inglese ed intitolata “PATHOGENETICS MECHANISMS IN CELIAC DISEASE” ha riguardato lo studio dei meccanismi molecolari coinvolti nella patogenesi della Celiachia.
I risultati della ricerca, condotta nel primo anno, ci hanno permesso di dimostrare che il fattore di trascrizione nuclear factor-B (NF-B) è attivato nella mucosa intestinale di pazienti celiaci indicando un ruolo per IB- nel regolare la persistente attivazione di NF-B in questa patologia. Le cellule epiteliali delle cripte e le cellule mononucleate della lamina propria di pazienti celiaci risultavano positive per p65 rispetto ai pazienti celiaci in remissione e di controllo. Inoltre, l'attivazione di NF-B era correlata con l'espressione della monossido d’azoto sintasi inducibile (iNOS) e della cicloossigenasi-2 (COX-2), enzimi che catalizzano la sintesi di monossido d’azoto (NO) e prostaglandine pro-infiammatorie.
Queste osservazioni potrebbero essere di una certa rilevanza clinica poichè una sostenuta attivazione di NF-B nella mucosa intestinale di pazienti celiaci conduce ad una prolungata induzione dell'espressione di geni pro-infiammatori perpetuando in tal modo il processo infiammatorio cronico.
I risultati della ricerca, condotta nel secondo e terzo anno, ci hanno permesso di approfondire alcuni meccanismi dell’interazione diretta della gliadina (un peptide del glutine) con i macrofagi murini RAW 264.7 stimolati con IFN-. In particolare, la gliadina in associazione con IFN- incrementava la produzione di NO, l’espressione della iNOS, l’attività di legame dei fattori di trascrizione NF-B, IRF-1 (interferon regulatory factor-1) e STAT-1 (signal transducer and activator of transcription-1 al DNA rispetto all’IFN- da solo. Questi effetti erano inibiti significativamente da pirrolidina ditiocarbammato, genisteina e tirfostina B42, rispettivamente, inibitori dell’attivazione di NF-B, IRF-1 e STAT-1. Approfondendo questi studi, è stato possibile accertare che la gliadina era in grado di aumentare i livelli di mRNA e l’attività del promotore del gene della iNOS nei macrofagi RAW 264.7 stimolati con IFN-per 1, 6 e 24 ore Questi effetti erano inibiti significativamente da genisteina e tirfostina B42 a 1 ora e da pirrolidina ditiocarbammato a 6 e 24 ore. E’ interessante osservare che le cinetiche di inibizione dell’espressione del gene della iNOS da parte di pirrolidina ditiocarbammato, genisteina e tirfostina B42 erano correlate con l’induzione dei livelli di mRNA dei fattori di trascrizione NF-B/p65, IRF-1 e STAT-1.
Questi risultati suggeriscono che la gliadina può modulare l’espressione del gene della iNOS come co-segnale con l’IFN- attraverso IRF-1 e STAT-1 nelle fasi precoci e NF-B nelle fasi tardive dell’induzione.
In conclusione, le nostre osservazioni possono contribuire ad una maggiore comprensione dei meccanismi molecolari della patogenesi della celiachia delineando nuove vie per il trattamento di questo disordine
Environmental toxicity, redox signaling and lung inflammation:the role of glutathione
No full textGlutathione (gamma-glutamyl-cysteinyl-glycine, GSH) is the most abundant intracellular antioxidant thiol and is central to redox defense during oxidative stress. GSH metabolism is tightly regulated and has been implicated in redox signaling and also in protection against environmental oxidant-mediated injury. Changes in the ratio of the reduced and disulfide form (GSH/GSSG) can affect signaling pathways that participate in a broad array of physiological responses from cell proliferation, autophagy and apoptosis to gene expression that involve H(2)O(2) as a second messenger. Oxidative stress due to oxidant/antioxidant imbalance and also due to environmental oxidants is an important component during inflammation and respiratory diseases such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, and asthma. It is known to activate multiple stress kinase pathways and redox-sensitive transcription factors such as Nrf2, NF-kappaB and AP-1, which differentially regulate the genes for pro-inflammatory cytokines as well as the protective antioxidant genes. Understanding the regulatory mechanisms for the induction of antioxidants, such as GSH, versus pro-inflammatory mediators at sites of oxidant-directed injuries may allow for the development of novel therapies which will allow pharmacological manipulation of GSH synthesis during inflammation and oxidative injury. This article features the current knowledge about the role of GSH in redox signaling, GSH biosynthesis and particularly the regulation of transcription factor Nrf2 by GSH and downstream signaling during oxidative stress and inflammation in various pulmonary diseases. We also discussed the current therapeutic clinical trials using GSH and other thiol compounds, such as N-acetyl-l-cysteine, fudosteine, carbocysteine, erdosteine in environment-induced airways disease
An investigation into the mechanisms by which bim gene expression is regulated in sympathetic neurons
No full textNeuronal apoptosis plays a critical role in development and disease. Developing
sympathetic neurons require nerve growth factor (NGF) for their survival and die by
apoptosis in its absence. Studies with sympathetic neurons have provided important
insights into the molecular mechanisms of neuronal apoptosis and the signalling
pathways that regulate the cell death programme in neurons. Bim is a BH3-only
member of the Bcl-2 family that increases in level after NGF withdrawal and which is
required for NGF withdrawal-induced death. Regulation of bim expression is complex
and remains incompletely understood.
By analysing the DNA sequence of the rat bim promoter I identified a
conserved inverted CCAAT box (ICB) that is bound by the heterotrimeric transcription
factor NF-Y in vitro and in chromatin. Interestingly, mutational analysis revealed that
the ICB is critical for the induction of a bim-LUC reporter construct following NGF
withdrawal. Use of a well-characterised dominant negative NF-YA mutant (YA13 m29)
showed that NF-Y is required for bim promoter activity and its induction following NGF
withdrawal. Overexpression of YA13 m29 also demonstrated that NF-Y activity is
essential for the expression of the endogenous Bim protein and that NF-Y is important
for apoptosis following NGF withdrawal. Furthermore, I found that the transcriptional
coactivators CBP/p300 are required for the activation of bim-LUC following NGF
withdrawal and that CBP/p300 may interact with NF-Y to enhance bim transcription.
In addition to this, the prosurvival MEK/ERK pathway has been found to
inhibit bim expression independently of the PI3-K/Akt pathway. 3' RACE and
experiments in sympathetic neurons with a new bim-LUC+3'UTR reporter construct
revealed that this negative regulation is mediated through the bim 3' UTR. Mutational
analysis and RNA stability experiments have been employed to further investigate this
mechanism
A Novel Role for NF-κB in Proximal T Cell Signaling
No full textThe interrogation of T cell signaling over the past fifty years has led to the discovery of amazingly intricate cascade networks and elaborate descriptions of individual proteins\u27 domains and functions. A complex landscape has been rendered in which proteins relay messages from the extracellular ligation of the TCR by a cognate peptide loaded MHC via changes in sub-cellular location, phosphorylation, and binding affinities and partners to enact nuclear localization of three key transcription factors required for cellular effector function and proliferation: AP-1, NF-AT, and NF-κB. Dogma has favored activation of each of these transcription regulating elements to be a linear and parallel activity, thus very little interaction between pathways has been highlighted by previous findings in the molecular immunology community. The focus of this dissertation explores the role of NF-κB in T cell signaling with emphasis on subunits p50, cRel, IκBα, and IKKβ, and with respect to NF-κB’s ability to modulate calcium and NF-AT signaling, proximal TCR phosphorylation, and CRAC and purinergic calcium channel proteins.
The role of NF-κB in T cells can be a difficult thing to establish, as this thirteen member family innervates almost every cellular process from homeostasis to activation, and even functions in the opposing processes of survival and apoptosis. To convolute the investigation further, many family members also fulfill redundant tasks, as a result of their high evolutionarily conserved sequence homology. To this end, we discovered the best way to evaluate the function of NF-κB in the activation of T cells was to knockdown two family members: p50 and cRel. In doing this, we rendered mice that were viable (unlike knockdown of RelA) and fertile, but possessed T cells that were highly unresponsive to strong stimulation (anti CD3/CD28) or foreign antigen (OVA) presented to mice bearing the correct transgenic TCRs (OT-1) by professional antigen presenting cells (APC).
Through in vitro assays, we discovered that in addition to the specific defects in NF-κB activation, NF-AT signaling was also greatly disrupted in these cells, sequela to retarded calcium influx and signaling. This was of great interest, as while several studies have shown that calcium signaling has the ability to amplify and fine tune NF-κB activation, there is a dearth of studies and publications highlighting the effect of an activated NF-κB pathway on calcium influx and signaling leading to the activation of NF- AT. Another fascinating discovery, that explicated the calcium reduction and NF-AT inhibition, was that ablation of p50 and cRel led to decreases in mRNA and protein levels of two additional NF-κB family members: IKKβ and IKKγ. The results presented here suggest that it is the reduction in IKKβ and IKKγ that leads to impaired phosphorylation of the key TCR proximal proteins: Zap70 and PLC&gamma1, and it is the decrease in activated PLCγ1 that renders less IP3 and ultimately abrogates calcium signaling.
Overall, this thesis highlights the ability of IKKβ to enhance general proximal TCR protein phosphorylation (and specifically Zap70) leading to a greater influx of calcium (perhaps aided by IKKβ also augmenting the function of the CRAC protein, STIM1) which leads to superior activation of NF- AT, and amplifies downstream cellular effector functions such as IL-2 production and proliferation. Moreover, this work demonstrates that NF-κB subunits likely form supermolecular clusters, and ablation of certain subunits (i.e. p50 and cRel) can lead to instability and decreased levels of other family members (i.e. IKKβ and IKKγ.
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