MUC4 involvement in ErbB2/ErbB3 phosphorylation and signaling in response to airway cell mechanical injury

The receptor tyrosine kinases ErbB2 and ErbB3 are phosphorylated in response to injury of the airway epithelium. Since we have shown that the membrane mucin MUC4 can act as a ligand/modulator for ErbB2, affecting its localization in polarized epithelial cells and its phosphorylation, we questioned whether Muc4 was involved, along with ErbB2 and ErbB3, in the damage response of airway epithelia. To test this hypothesis, we first examined the localization of MUC4 in human airway samples. Both immunocytochemistry and immunofluorescence showed a co‐localization of MUC4 and ErbB2 at the airway luminal surface. Sequential immunoprecipitation and immunoblotting from airway cells demonstrated that the MUC4 and ErbB2 are present as a complex in airway epithelial cells. To assess the participation of MUC4 in the damage response, cultures of NCI‐H292 or airway cells were scratch‐wounded, then analyzed for association of phospho‐ErbB2 and ‐ErbB3 with MUC4 by sequential immunoprecipitation and immunoblotting. Wounded cultures exhibited increased phosphorylation of both receptors in complex with MUC4. Scratch wounding also increased activation of the downstream pathway through Akt, as predicted from our previous studies on Muc4 effects on ErbB2 and ErbB3. The participation of MUC4 in the phosphorylation response was also indicated by siRNA repression of MUC4 expression, which resulted in diminution of the phosphorylation of ErbB2 and ErbB3. These studies provide a new model for the airway epithelial damage response, in which the MUC4–ErbB2 complex is a key element in the sensor mechanism and phosphorylation of the receptors. J. Cell. Biochem. 107: 112–122, 2009. © 2009 Wiley‐Liss, Inc.     

Pulmonary neuroendocrine cells sense succinate to stimulate myoepithelial cell contraction

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Short hairpin RNAs against eotaxin or interleukin‐5 decrease airway eosinophilia and hyper‐responsiveness in a murine model of asthma

Background

Eosinophilia plays the major role in the pathogenesis of asthma and correlates with the up‐regulation of eotaxin, which, together with interleukin (IL)‐5, is important for differentiation, chemo‐attraction, degranulation, and survival of eosinophils in local tissue. In a previous study, we found that administration of lentivirus‐delivered short hairpin RNA (shRNA) to suppress the expression of IL‐5 inhibited airway inflammation. The present study aimed to investigate the role of eotaxin shRNA and the synergistic effect of eotaxin and IL‐5 shRNAs on airway inflammation in an ovalbumin (OVA)‐induced murine model of asthma.

Methods

Lentivirus‐delivered shRNAs were used to suppress the expression of eotaxin and/or IL‐5 in local tissue in an OVA‐induced murine asthma model.

Results

Intra‐tracheal administration of lentivirus containing eotaxin shRNA expressing cassette (eoSEC3.3) efficiently moderated the characteristics of asthma, including airway hyper‐responsiveness, cellular infiltration of lung tissues, and eotaxin and IL‐5 levels in bronchio‐alveolar lavage fluid. Administration of lentiviruses expressing IL‐5 or eotaxin shRNAs (IL5SEC4 + eoSEC3.3) also moderated the symptoms of asthma in a mouse model.

Conclusions

Local delivery of lentiviruses expressing IL‐5 and eotaxin shRNAs provides a potential tool in moderating airway inflammation and also has the potential for developing clinical therapy based on the application of shRNAs of chemokines and cytokines involved in T helper 2 cell inflammation and eosinophilia. Copyright © 2008 John Wiley & Sons, Ltd.     

咽喉部高频喷射通气调节各参数对狗肺通气效率的影响

用相关和回归处理方法,研究了8条正常狗咽喉部高频喷射通气时,调节驱动压、呼吸比和频率对喷气量、吸入气氧浓度、动脉血气及气道内压的作用。结果显示,驱动压和呼吸此对各观察指标几乎有同等重要的作用,频率的影响很小,喷气量与吸入气氧浓度、动脉血气、气道内压间存在显著的正相关关系。说明调节参数的意义主要在于改变了喷气量。     

Inhibition of glycoconjugate secretion by colchicine and cytochalasin B

Summary The effects have been analyzed of cytochalasin B and colchicine on the secretion of glycoconjugates by human bronchial expiants labeled in vitro with radioactive glucosamine. Both cytochalasin B and colchicine had no effect on baseline ¹⁴C-labeled glycoconjugate release but caused a dose-dependent (10^–7–10^–4 M) inhibition of ¹⁴C-glycoconjugate release and discharge of labeled macromolecules from mucous and serous cells induced by 5 · 10^–5 M methacholine.Quantitative autoradiographic analyses showed that neither cytochalasin B nor colchicine inhibited ³H-threonine or ³H-glucosamine incorporation into mucous and serous cells of the submucosal glands or goblet cells of the airway epithelium. Colchicine (10^–5 M) but not cytochalasin B significantly reduced the rate at which labeled macromolecules were transported through mucous, serous and goblet cells but this effect was not observed until 4 h after the addition of colchicine. Neither cytochalasin B nor colchicine affected the basal rate of labeled-macromolecule discharge from mucous, serous or goblet cells. At a concentration of 10^–5 M, both agents completely inhibited the increase in labeled-macromolecule discharge induced in mucous and serous cells by methacholine.Our results suggest that in the submucosal gland of human airways microtubules and microfilaments may be important in secretagogue-induced but not in baseline cellular glycoconjugate discharge, implying that the mechanisms of the two processes differ significantly. Furthermore, a role for microtubules is suggested in the transport of secretory granules through mucous, serous and goblet cells.Supported by National Institutes of Health Research Grant 5R01HL22444. The authors gratefully acknowledge the technical assistance of Mr. Tudor Williams, Mr. Eduardo Quintanilla and Ms. Maureen Hayes     

Preservation of Tracheal Mucus by Nonaqueous Fixative

Two nonaqueous fixatives, composed of fluorocarbon solvents with dissolved osmium tetroxide, were used to determine the feasibility of preserving the mucous coat in bovine and rat trachea for light and electron microscopy. Aqueous fixatives, while providing excellent cytological preservation, wash away the mucous lining, precluding ultrastructural analysis. Inclusion of ruthenium red or alcian blue within aqueous fixative improved retention of mucus, but provided incomplete, patchy results. Fixation with nonaqueous fluorocarbon solvent and dissolved osmium tetroxide preserved a continuous mucous epiphase layer above a clear hypophase layer. Subcomponents of the mucus included an electron dense surface layer, interrupted patches of mucus above the surface layer and electron dense membrane-like material within the mucus. This method of fixation will preserve mucus for light, scanning and transmission electron microscopy, using either intratracheal or immersion methods of fixation. The latter would enable use of materials from large animal models, autopsy or an abattoir.     

表皮生长因子对培养的气道上皮细胞抗臭氧损伤的保护作用

本研究观察到臭氧（Ｏ３）对体外培养经３Ｈ－ＵｄＲ标记的免气道上皮细胞有明显细胞毒性作用,且损伤程度与Ｏ３作用时间呈正相关。Ｏ３暴露组细胞内丙二醛（ＭＤＡ）产生增多（Ｐ＜０．０１）,提示Ｏ３损伤细胞的机制与胞膜脂质过氧化有关。表皮生长因子（ＥＧＦ）可明显降低Ｏ３所致的３Ｈ释放率（Ｐ＜０．０１）、降低Ｏ３的细胞毒指数及细胞内ＭＤＡ含量（Ｐ＜０．０１）,证明ＥＧＦ对气道上皮细胞有保护作用。进一步还观察到浓度为５ｎｇ／ｍｌ的ＥＯＦ可以取消Ｏ３所引起的细胞内还原型谷胱甘肽（ＧＳＨ）含量降低（Ｐ＜０．０１）,并增加细胞内谷胱甘肽总含量（Ｐ＜０．０５）,但不能改变Ｏ３所致的氧化型谷胱甘肽（ＧＳＳＧ）含量的增加（Ｐ＞０．０５）,对ＧＳＨ／ＧＳＳＧ比值也无明显提高,这些都提示ＥＧＦ的细胞保护机理可能与其促进细胞内谷胱甘肽合成有关,而对ＧＳＳＧ转化为ＧＳＨ的还原过程影响不明显。     

In silico epitope-based vaccine design against influenza a neuraminidase protein: Computational analysis established on B- and T-cell epitope predictions

ObjectiveInfluenza A virus belongs to the most studied virus and its mutant initiates epidemic and pandemics outbreaks. Inoculation is the significant foundation to diminish the risk of infection. To prevent an incidence of influenza from the transmission, various practical approaches require more advancement and progress. More efforts and research must take in front to enhance vaccine efficacy.MethodsThe present research emphasizes the development and expansion of a universal vaccine for the influenza virus. Research focuses on vaccine design with high efficacy. In this study, numerous computational approaches were used, covering a wide range of elements and ideas in bioinformatics methodology. Various B and T-cell epitopic peptides derived from the Neuraminidase protein N1 are recognized by these approaches. With the implementation of numerous obtained databases and bioinformatics tools, the different immune framework methods of the conserved sequences of N1 neuraminidase were analyzed. NCBI databases were employed to retrieve amino acid sequences. The antigenic nature of the neuraminidase sequence was achieved by the VaxiJen server and Kolaskar and Tongaonkar method. After screening of various B and T cell epitopes, one efficient peptide each from B cell epitope and T cell epitopes was assessed for their antigenic determinant vaccine efficacy. Identical two B cell epitopes were recognized from the N1 protein when analyzed using B-cell epitope prediction servers. The detailed examination of amino acid sequences for interpretation of B and T cell epitopes was achieved with the help of the ABCPred and Immune Epitope Database.ResultsComputational immunology via immunoinformatic study exhibited RPNDKTG as having its high conservancy efficiency and demonstrated as a good antigenic, accessible surface hydrophilic B-cell epitope. Among T cell epitope analysis, YVNISNTNF was selected for being a conserved epitope. T cell epitope was also analyzed for its allergenicity and cytotoxicity evaluation. YVNISNTNF epitope was found to be a non-allergen and not toxic for cells as well. This T-cell epitope with maximum world populace coverages was scrutinized for its association with the HLA-DRB1*0401 molecule. Results from docking simulation analyses showed YVNISNTNF having lower binding energy, the radius of gyration (Rg), RMSD values, and RMSE values which make the protein structure more stable and increase its ability to become an epitopic peptide for influenza virus vaccination.ConclusionsWe propose that this epitope analysis may be successfully used as a measurement tool for the robustness of an antigen–antibody reaction between mutant strains in the annual design of the influenza vaccine.     

Gene delivery to respiratory epithelial cells by magnetofection

BACKGROUND: For the topical application of DNA vector complexes to the airways, specific extracellular barriers play a major role. In particular, short contact time of complexes with the cell surface caused by the mucociliary clearance hinders cellular uptake of complexes. The aim of this study was to evaluate the ability of magnetofection, a technique based on the principle of magnetic drug targeting, to overcome these barriers in comparison with conventional nonviral gene transfer methods such as lipofection and polyfection. METHODS: Experiments were carried out on permanent (16HBE14o-) and primary airway epithelial cells (porcine and human), and native porcine airway epithelium ex vivo. Transfection efficiency and dose-response relationship of magnetofection were examined by luciferase reporter gene expression. Sedimentation patterns and uptake of gene transfer complexes were characterized by fluorescence and electron microscopy, respectively. RESULTS: We show that (i) application of a magnetic field allows the magnetofectins to sediment and to enrich at the cell surface within a few minutes, (ii) magnetofection bears an improved dose-response relationship, (iii) magnetofection enhances transfection efficiency in both, permanent and primary airway epithelial cells, and (iv) magnetofection leads to significant transgene expression at very short incubation times in an ex vivo airway epithelium organ model. CONCLUSIONS: Magnetofection provides a potential novel method, which may overcome fundamental limitations of nonviral gene transfer to the airways. Due to the accelerated enrichment at the cell surface it may be of major interest for in vivo applications, where long-term incubation times at the target tissue are hardly achievable.     

Reciprocal influence of connexins and apical junction proteins on their expressions and functions

Membranes of adjacent cells form intercellular junctional complexes to mechanically anchor neighbour cells (anchoring junctions), to seal the paracellular space and to prevent diffusion of integral proteins within the plasma membrane (tight junctions) and to allow cell-to-cell diffusion of small ions and molecules (gap junctions). These different types of specialised plasma membrane microdomains, sharing common adaptor molecules, particularly zonula occludens proteins, frequently present intermingled relationships where the different proteins co-assemble into macromolecular complexes and their expressions are co-ordinately regulated. Proteins forming gap junction channels (connexins, particularly) and proteins fulfilling cell attachment or forming tight junction strands mutually influence expression and functions of one another.