131 research outputs found
Pathways of airway oxidant formation by house dust mite allergens and viral RNA converge through myosin motors, pannexons and Toll-like receptor 4.
Introduction
Intracellular reactive oxidant species (ROS) are generated in human airway epithelial cells by the prothrombinase action of Group 1 house dust mite (HDM) allergens and by ligation of viral RNA sensor Toll‐like receptors (TLRs). We explored signaling convergence between HDM allergens and TLRs in ROS generation because epithelial cells form the primary barrier against inhaled substances and dictate host responses to allergens and viruses.
Methods
ROS formation by Calu‐3 human airway cells was studied by measuring dihydrorhodamine 123 oxidation after activation by polyinosinic:polycytidylic acid (to activate TLR3), CL097 (to activate TLR7), a natural mixture of HDM allergens, or BzATP.
Results
TLR4 activation was identified as an indispensable response element for all stimuli, operating downstream from myosin motor activation, pannexon gating for ATP release and the endogenous activation of prothrombin. Exogenous prothrombin activation by HDM allergens was prevented by SGUL 1733, a novel inhibitor of the proteolytic activity of Group 1 HDM allergens, which thus prevented TLR4 from being activated at source.
Conclusions
Our data identify for the first time that endogenously‐generated prothrombin and TLR4 form a shared effector mechanism essential to intracellular ROS generation activated by a group 1 HDM allergen (itself a prothrombinase) or by ligation of viral RNA‐sensing TLRs. These stimuli operate a confluent signaling pathway in which myosin motors, gating of pannexons, and ADAM 10 lead to prothrombin‐dependent activation of TLR4 with a recycling activation of pannexons
Controlled laser texturing of titanium results in reliable osteointegration
We have developed a laser-textured superhydrophilic Ti-6Al-4V surface with unique surface chemistry and topography that substantially promotes osteoblast adhesion in culture. Here we investigate the osteointegration of laser-textured implants in an ovine model. Our hypothesis was that laser-textured implants, without any surface coating (LT), would encourage comparable amounts of bone-implant contact and interfacial strength when compared with widely accepted hydroxyapatite (HA) coated implants. Additionally, we hypothesized that LT would significantly increase bony integration compared with machine-finished (MF) and grit-blasted (GB) implants. Forty-eight tapered transcortical pins were implanted into six sheep. Four experimental groups (LT, HA, MF, and GB) were investigated (n = 12) and implants remained in vivo for 6 weeks. Bone apposition rates, interfacial shear strength, and bone-implant contact (BIC) were quantified. The interfacial strength of LT and HA implants were found to be significantly greater than GB (p = 0.032 and p = 0.004) and MF (p = 0.004 and p = 0.004, respectively), but no significant difference between LT and HA implants was observed. Significantly increased BIC was measured adjacent to HA implants when compared with both LT and GB implant surfaces (p = 0.022 and p = 0.006, respectively). No significant difference was found when LT and GB implants were compared. However, all surface finishes encouraged significantly increased BIC when compared with the MF surface. Maximizing implant fixation to host bone is vital for its long-term success. The production of an LT surface is a simple and cheap manufacturing process and this study demonstrated that laser-textured implants are a very promising technical development that warrants further research. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res
Inattentive Consumers in Markets for Services
In an experiment on markets for services, we find that consumers are likely to stick to default tariffs and achieve suboptimal outcomes. We find that inattention to the task of choosing a better tariff is likely to be a substantial problem in addition to any task and tariff complexity effect. The institutional setup on which we primarily model our experiment is the UK electricity and gas markets, and our conclusion is that the new measures by the UK regulator Ofgem to improve consumer outcomes are likely to be of limited impact
Epidermal growth factor suppresses induction by progestin of the adhesion protein desmoplakin in T47D breast cancer cells
INTRODUCTION: Although the effects of progesterone on cell cycle progression are well known, its role in spreading and adhesion of breast cancer cells has not attracted much attention until recently. Indeed, by controlling cell adhesion proteins, progesterone may play a direct role in breast cancer invasion and metastasis. Progesterone has also been shown to modulate epidermal growth factor (EGF) effects in neoplasia, although EGF effects on progesterone pathways and targets are less well understood. In the present study we identify an effect of EGF on a progesterone target, namely desmoplakin. METHODS: Initially flow cytometry was used to establish the growing conditions and demonstrate that the T47D breast cancer cell line was responding to progesterone and EGF in a classical manner. Differential display RT-PCR was employed to identify differentially expressed genes affected by progesterone and EGF. Western and Northern blotting were used to verify interactions between EGF and progesterone in three breast cancer cell lines: T47D, MCF-7, and ZR-75. RESULTS: We found the cell adhesion protein desmoplakin to be upregulated by progesterone – a process that was suppressed by EGF. This appears to be a general but not universal effect in breast cancer cell lines. CONCLUSION: Our findings suggest that progesterone and EGF may play opposing roles in metastasis. They also suggest that desmoplakin may be a useful biomarker for mechanistic studies designed to analyze the crosstalk between EGF and progesterone dependent events. Our work may help to bridge the fields of metastasis and differentiation, and the mechanisms of steroid action
Desmoglein 3, via an Interaction with E-cadherin, Is Associated with Activation of Src
Desmoglein 3 (Dsg3), a desmosomal adhesion protein, is expressed in basal and immediate suprabasal layers of skin and across the entire stratified squamous epithelium of oral mucosa. However, increasing evidence suggests that the role of Dsg3 may involve more than just cell-cell adhesion.To determine possible additional roles of Dsg3 during epithelial cell adhesion we used overexpression of full-length human Dsg3 cDNA, and RNAi-mediated knockdown of this molecule in various epithelial cell types. Overexpression of Dsg3 resulted in a reduced level of E-cadherin but a colocalisation with the E-cadherin-catenin complex of the adherens junctions. Concomitantly these transfected cells exhibited marked migratory capacity and the formation of filopodial protrusions. These latter events are consistent with Src activation and, indeed, Src-specific inhibition reversed these phenotypes. Moreover Dsg3 knockdown, which also reversed the decreased level of E-cadherin, partially blocked Src phosphorylation.Our data are consistent with the possibility that Dsg3, as an up-stream regulator of Src activity, helps regulate adherens junction formation
DSC3 expression is regulated by p53, and methylation of DSC3 DNA is a prognostic marker in human colorectal cancer
Missense mutations in Desmocollin-2 N-terminus, associated with arrhythmogenic right ventricular cardiomyopathy, affect intracellular localization of desmocollin-2 in vitro
<p>Abstract</p> <p>Background</p> <p>Mutations in genes encoding desmosomal proteins have been reported to cause arrhythmogenic right ventricular cardiomyopathy (ARVC), an autosomal dominant disease characterised by progressive myocardial atrophy with fibro-fatty replacement.</p> <p>We screened 54 ARVC probands for mutations in desmocollin-2 (<it>DSC2</it>), the only desmocollin isoform expressed in cardiac tissue.</p> <p>Methods</p> <p>Mutation screening was performed by denaturing high-performance liquid chromatography and direct sequencing.</p> <p>To evaluate the pathogenic potentials of the <it>DSC2 </it>mutations detected in patients affected with ARVC, full-length wild-type and mutated cDNAs were cloned in eukaryotic expression vectors to obtain a fusion protein with green fluorescence protein (GFP); constructs were transfected in neonatal rat cardiomyocytes and in HL-1 cells.</p> <p>Results</p> <p>We identified two heterozygous mutations (c.304G>A (p.E102K) and c.1034T>C (p.I345T)) in two probands and in four family members. The two mutations p.E102K and p.I345T map to the N-terminal region, relevant to adhesive interactions.</p> <p>In vitro functional studies demonstrated that, unlike wild-type DSC2, the two N-terminal mutants are predominantly localised in the cytoplasm.</p> <p>Conclusion</p> <p>The two missense mutations in the N-terminal domain affect the normal localisation of DSC2, thus suggesting the potential pathogenic effect of the reported mutations. Identification of additional DSC2 mutations associated with ARVC may result in increased diagnostic accuracy with implications for genetic counseling.</p
Plakophilin3 Loss Leads to an Increase in PRL3 Levels Promoting K8 Dephosphorylation, Which Is Required for Transformation and Metastasis
The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis
Comparison of Gene Expression Profiles in Chromate Transformed BEAS-2B Cells
Hexavalent chromium [Cr(VI)] is a potent human carcinogen.
Occupational exposure has been associated with increased risk of respiratory
cancer. Multiple mechanisms have been shown to contribute to Cr(VI) induced
carcinogenesis, including DNA damage, genomic instability, and epigenetic
modulation, however, the molecular mechanism and downstream genes mediating
chromium's carcinogenicity remain to be elucidated.We established chromate transformed cell lines by chronic exposure of normal
human bronchial epithelial BEAS-2B cells to low doses of Cr(VI) followed by
anchorage-independent growth. These transformed cell lines not only
exhibited consistent morphological changes but also acquired altered and
distinct gene expression patterns compared with normal BEAS-2B cells and
control cell lines (untreated) that arose spontaneously in soft agar.
Interestingly, the gene expression profiles of six Cr(VI) transformed cell
lines were remarkably similar to each other yet differed significantly from
that of either control cell lines or normal BEAS-2B cells. A total of 409
differentially expressed genes were identified in Cr(VI) transformed cells
compared to control cells. Genes related to cell-to-cell junction were
upregulated in all Cr(VI) transformed cells, while genes associated with the
interaction between cells and their extracellular matrices were
down-regulated. Additionally, expression of genes involved in cell
proliferation and apoptosis were also changed.This study is the first to report gene expression profiling of Cr(VI)
transformed cells. The gene expression changes across individual chromate
exposed clones were remarkably similar to each other but differed
significantly from the gene expression found in anchorage-independent clones
that arose spontaneously. Our analysis identified many novel gene expression
changes that may contribute to chromate induced cell transformation, and
collectively this type of information will provide a better understanding of
the mechanism underlying chromate carcinogenicity
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