Search

Epidemiologic studies have demonstrated an association between acetaminophen (APAP) use and the development of asthma symptoms. However, few studies have examined relationships between APAP-induced signaling pathways associated with the development of asthma symptoms. We tested the hypothesis that acute APAP exposure causes airway hyper-responsiveness (AHR) in human airways. Precision cut lung slice (PCLS) airways from humans and mice were used to determine the effects of APAP on airway bronchoconstriction and bronchodilation and to assess APAP metabolism in lungs. APAP did not promote AHR in normal or asthmatic human airways ex vivo. Rather, high concentrations mildly bronchodilated airways pre-constricted with carbachol (CCh), histamine (His), or immunoglobulin E (IgE) cross-linking. Further, the addition of APAP prior to bronchoconstrictors protected the airways from constriction. Similarly, in vivo treatment of mice with APAP (200 mg/kg IP) resulted in reduced bronchoconstrictor responses in PCLS airways ex vivo. Finally, in both mouse and human PCLS airways, exposure to APAP generated only low amounts of APAP-protein adducts, indicating minimal drug metabolic activity in the tissues. These findings indicate that acute exposure to APAP does not initiate AHR, that high-dose APAP is protective against bronchoconstriction, and that APAP is a mild bronchodilator. [ABSTRACT FROM AUTHOR]

To determine the importance of tumour necrosis factor receptor 1 in hepatocyte regeneration in acetaminophen toxicity, wild type and tumour necrosis factor receptor 1 knock-out mice were dosed with acetaminophen (300 mg/kg intraperitoneally) and sacrificed at 4, 24, 48, 72, and 96 hr. Biochemical parameters (alanine aminotransferase, ALT) and histologic evidence of hepatocellular injury were comparable in the two groups of mice. To examine the effects of tumour necrosis factor receptor 1 on hepatocyte regeneration, immunohistochemical staining with proliferating cell nuclear antigen was performed. Immunohistochemical staining for proliferating cell nuclear antigen was significantly reduced at multiple time points in the knock-out mice and did not normalize until 96 hr. To evaluate the effect of tumour necrosis factor receptor 1 depletion on cytokines known to be involved in regeneration, levels of macrophage inhibitory protein 2, interferon-γ-inducible protein-10 and monocyte chemoattractant protein 1 were compared in the two groups of mice. Significant elevation of all cytokines was observed in both groups of mice; however, higher levels were present in the knock-out mice. Depletion of tumour necrosis factor receptor 1 has long-lasting effects on hepatocyte regeneration in acetaminophen toxicity but multiple other factors appear to orchestrate eventual recovery in these mice. [ABSTRACT FROM AUTHOR]

Wound healing is a response to injury that is initiated to reconstruct damaged tissue. In skin, reepithelialization involves both epithelial cells and fibroblasts and contributes to the reformation of a barrier between the external environment and internal milieu. Growth factors including epidermal growth factor (EGF) play important roles in promoting this process. In the present studies we employed CV-1 fibroblasts in a tissue culture model of reepithelialization to develop strategies for optimizing wound closure stimulated by EGF. We found that EGF enhanced cell motility within 6-8 h of EGF treatment in serum-free medium but wounds failed to close within 24 h. However, if medium on these cultures was exchanged for medium containing serum, ceils pretreated with EGF closed new scrape wounds more rapidly than did cells that were not pretreated. These results indicate that serum factors work in concert with EGF to coordinate cell motility for efficient wound closure. Indeed, EGF enhanced the rate of wound closure in the presence of serum, and this effect also persisted for at least 24 h after EGF was removed. This coordination of EGF-induced cell motility was accompanied by an increase in the transient phosphorylation of ERK1 and ERK2. The persistent effects of EGF were blocked by transient exposure to reversible inhibitors of transcription and translation, indicating that the expression of new proteins mediated this response. We propose that EGF-stimulated CV-1 fibrublast motility is coordinated by a serum component that induces cell-cell adhesive properties consistent with an epithelial phenotype, thereby enhancing the reepitheliafization process. [ABSTRACT FROM AUTHOR]

A variety of receptors have been analyzed in sufficient detail to identify sorting motifs. Initial studies focused on the identification of sequences in the cytoplasmic tails of the LDL and transferrin receptors that mediated their internalization. These motifs have since been found in the cytoplasmic domains of a wide variety of receptors and provide for numerous sorting functions. This review will outline the early studies on LDL and transferrin receptors and will then focus on two classes of signaling receptors, receptor tyrosine kinases (EGF and the insulin receptors) and heterotrimeric G-protein coupled receptors (β2-adrenergic receptors). The identification of sorting motifs and proteins that bind these motifs will be discussed. Importantly, the studies identify a variety of potential targets for modulating the sorting and hence activity of these medically important receptors. [Copyright &y& Elsevier]

Determines the amounts of fatty acids available for incorporation into membranes by the activities of enzymes involved in fatty acid biosynthesis and metabolism. Indications of a hydropathy path; Method used in the analysis of gene expression in human tissues; Benefit from site-directed mutagenesis in rat desaturase; Effects of the overexpression of MLD.

Reports on the identification of a protein, sorting nexin-1 (SNX1) in a human cell line bound to a region of the epidermal growth factor receptor (EGFR) containing the lysosomal targeting code. Effect of the overexpression of SNX1 on the amount of EGFR on the cell surface; Role of SNX1 in sorting EGFR to lysosomes.

Intracellular bacterial pathogens often subvert apoptosis signalling to regulate survival of their host cell, allowing propagation of the bacterial population. Coxiella burnetii, the intracellular agent of human Q fever, inhibits host cell apoptosis through several mechanisms, including prevention of mitochondrial cytochrome c release, triggering of an anti-apoptotic transcriptional programme, and activation of pro-survival kinases. To control host cell survival, C. burnetii delivers effector proteins to the eukaryotic cytosol using a specialized Dot/ Icm type IV secretion system ( T4 SS). Effectors are predicted to regulate activity of pro-survival host signalling proteins, such as Akt and cAMP-dependent protein kinase ( PKA), to control infection. Here, we show that host PKA activity is required for C. burnetii inhibition of macrophage apoptosis. PKA is activated during infection and inhibits activity of the pro-apoptotic protein Bad via phosphorylation. Bad is also phosphorylated at an Akt-specific residue, indicating C. burnetii uses two kinases to fully inactivate Bad. Additionally, Bad and the tethering protein 14-3-3β colocalize at the C. burnetii parasitophorous vacuole ( PV) membrane during infection, an event predicted to alter Bad promotion of apoptosis. Inhibiting PKA activity prevents Bad recruitment to the PV, but the protein is retained at the membrane during induction of apoptosis. Finally, PKA regulatory subunit I ( RI) traffics to the PV membrane in a T4 SS-dependent manner, suggesting a C. burnetii effector(s) regulates PKA-dependent activities. This study is the first to demonstrate subversion of host PKA activity by an intracellular bacterial pathogen to prevent apoptosis and survive within macrophages. [ABSTRACT FROM AUTHOR]

The role of mitochondrial permeability transition (MPT) and oxidative stress in chloroform toxicity was determined in freshly isolated female B6C3F1 mouse hepatocytes. Incubation of chloroform (12 mM) with hepatocytes resulted in cell death (alanine aminotransferase release and propidium iodide fluorescence). Chloroform had volatilized from the incubation and glutathione was depleted by 1 h; however, toxicity was not significantly different between control and chloroform-incubated cells. Hepatocytes were washed and reincubated in fresh media at 1 h. Subsequent reincubation of chloroform-treated hepatocytes resulted in significant toxicity at 3-5 h. Inclusion of the MPT inhibitor cyclosporine A or the antioxidant N-acetylcysteine (NAC) in the reincubation media at 1 h prevented toxicity. Confocal microscopy studies with the dye calcein AM indicated MPT that was blocked by cyclosporine A or NAC. Fluorescence microscopy studies utilizing JC-1 indicated loss of mitochondrial membrane potential, which was also blocked by cyclosporine A or NAC. Dichlorofluorescein fluorescence increased during the reincubation phase, indicating increased oxidative stress, and the increase was blocked by cyclosporine A. Since oxidative stress may occur by peroxynitrite, its role in toxicity was examined. Either of the nitric oxide synthase inhibitors NG-methyl-L-arginine (L-NMMA) and 7-nitroindazole (7-NI) at 1 h blocked toxicity. Western blot analysis of hepatocytes for 3-nitrotyrosine in proteins, a biomarker of peroxynitrite, indicated one major nitrated protein at 81 kD. Nitration of this protein was inhibited by cyclosporine A, L-NMMA, 7-NI, or NAC. The data indicate that chloroform-induced cell death occurs in two phases: a metabolic phase characterized by glutathione depletion, and an oxidative phase characterized by MPT and protein nitration. [ABSTRACT FROM AUTHOR]

The mechanism of action of alemtuzumab (CAMPATH 1H) in chronic lymphocytic leukemia (CLL) is uncertain. We tested the hypothesis that alemtuzumab alone can induce apoptosis in cultured CLL cells. Purified peripheral blood B-lymphocytes from CLL patients were treated in serum free medium (AIM-V). There was minimal spontaneous apoptosis in untreated cells. Alemtuzumab ligation did not alter the membrane distribution of CD52 in single cells but many cells formed transient, small, tightly adherent clusters. Alemtuzumab alone did not induce apoptosis. In contrast, alemtuzumab plus complement was rapidly cytotoxic. We conclude that alemtuzumab does not cause apoptosis in purified CLL B cells cultured in serum free medium. [Copyright &y& Elsevier]

Metabolic derivatives of linoleic acid, both monoepoxides and diols, have been reported to be toxic in humans and multiple animal tissue preparations. A previous electrophysiological study has shown these compounds produce multiple effects on the electrical activity of rat ventricular myocytes. The hydrophobic nature of these compounds suggests the possibility that these effects may be due to nonspecific lipid interactions, i.e., changes in membrane fluidity. This study investigates membrane fluidity as a possible mechanism by which linoleic acid metabolites inhibit Na+/K+ pump current (Ip). This study showed that positional isomers 9,10- and 12,13-epoxy-octadecenoic acid (EOA) and 9,10- and 12,13-dihydroxy-OA (DHOA) inhibit Ip in a dose-dependent manner in N20.1 mouse oligodendrocytes, with greater inhibition produced by EOAs. These compounds, at 10 μM, inhibited Ip by 4.7 ± 1.6, 18.2 ± 0.5, 11.7 ± 0.5, and 25.1 ± 0.9% for 12,13-DHOA, 9,10-DHOA, 12,13-EOA, and 9,10-EOA, respectively, in oligodendrocytes. Fluorescence recovery after photobleaching measurements showed that both DHOA isomers produced a 7–8% increase in diffusion coefficient of the probe at 10 μM, whereas the diffusion coefficient was decreased by 5 and 13% by 9,10-EOA and 12,13-EOA, respectively. There was no apparent correlation between membrane fluidity and inhibition of Ip by these four linoleic acid metabolites. These results indicate that membrane fluidity alone cannot explain the effects of these compounds on Ip and suggest that they have a specific interaction with the Na+/K+ pump. [Copyright &y& Elsevier]

The aim of the present study was to identify the gene for sorting nexin 1 (SNX1) to evaluate the potential for tissue-specific alternative splicing and to analyze the activity of the SNX1 promoter. The coding DNA for SNX1 was divided between 15 exons in 43 kb of genomic DNA located on human chromosome 15q22. Although SNX1 mRNA expression was widespread in human tissues, alternative splicing is thought to generate skipped exons in SNX1 cDNAs. By determination of the SNX1 gene structure and an analysis of the mRNAs in a variety of tissues using RT-PCR, we demonstrated that SNX1 mRNAs are alternatively spliced. Exon-skipped products were less abundant than full-length SNX1 mRNA species, but the ratio of skipped to full-length mRNA indicated that alternative splicing may be developmentally regulated in the liver. Consistent with widespread mRNA expression, the SNX1 promoter was GC rich and lacked a TATA box, features characteristic of housekeeping promoters. The promoter activity was dependent on the presence of proximal sequences that contained initiator elements and predicted binding sites for the transcription factors Sp1 and E2F. These findings indicate that regulation of SNX1 gene expression at the transcriptional level is likely minor. Rather, developmentally specific exon skipping provides a potential mechanism for regulating the activity of SNX1. [ABSTRACT FROM AUTHOR]

The recent discovery of sensory (tastant and odorant) G proteincoupled receptors on the smooth muscle of human bronchi suggests unappreciated therapeutic targets in the management of obstructive lung diseases. Here we have characterized the effects of a wide range of volatile odorants on the contractile state of airway smooth muscle (ASM) and uncovered a complex mechanism of odorant-evoked signaling properties that regulate excitation-contraction (E-C) coupling in human ASM cells. Initial studies established multiple odorous molecules capable of increasing intracellular calcium ([Ca2+]i) in ASM cells, some of which were (paradoxically) associated with ASM relaxation. Subsequent studies showed a terpenoid molecule (nerol)-stimulated OR2W3 caused increases in [Ca2+]i and relaxation of ASM cells. Of note, OR2W3-evoked [Ca2+]i mobilization and ASM relaxation required Ca2+ flux through the store-operated calcium entry (SOCE) pathway and accompanied plasma membrane depolarization. This chemosensory odorant receptor response was not mediated by adenylyl cyclase (AC)/cyclic nucleotide-gated (CNG) channels or by protein kinase A (PKA) activity. Instead, ASM olfactory responses to the monoterpene nerol were predominated by the activity of Ca2+-activated chloride channels (TMEM16A), including the cystic fibrosis transmembrane conductance regulator (CFTR) expressed on endo(sarco)plasmic reticulum. These findings demonstrate compartmentalization of Ca2+ signals dictates the odorant receptor OR2W3-induced ASM relaxation and identify a previously unrecognized E-C coupling mechanism that could be exploited in the development of therapeutics to treat obstructive lung diseases. [ABSTRACT FROM AUTHOR]

Keywords: asthma; chromosome 17q21; epithelium; LRRC3C; ORMDL3 EN asthma chromosome 17q21 epithelium LRRC3C ORMDL3 1 5 5 07/29/20 20200801 NES 200801 Abbreviations AHR Airway hyperreactivity ASM Airway smooth muscle ATF6 Activating transcription factor 6 BAL Bronchoalveolar lavage GSDMA Gasdermin A GSDMB Gasdermin B IBD Inflammatory bowel disease LRRC3C Leucine rich repeat containing 3C ORMDL3 ORM1-like-3 PBMC Peripheral blood mononuclear cell SERCA2b Sarco/endoplasmic reticulum Ca2 + ATPase 2b SNP Single nucleotide polymorphism ZPBP2 Zona pellucida-binding protein 2 The chromosome 17q21 region has strong linkage to asthma in genetic association studies. The lung genotyping demonstrated that the rs2872507 GG (risk allele for asthma) was detected in 3/10 (30%) of the asthma lungs and 8/13 (61%) of the control lungs (Figure 1F), none of whose lungs expressed LRRC3C mRNA. [Extracted from the article]

Respiratory disease is a leading cause of mortality in patients with osteogenesis imperfecta (OI), a connective tissue disease that causes severely reduced bone mass and is most commonly caused by dominant mutations in type I collagen genes. Previous studies proposed that impaired respiratory function in OI patients was secondary to skeletal deformities; however, recent evidence suggests the existence of a primary lung defect. Here, we analyzed the lung phenotype of Crtap knockout (KO) mice, a mouse model of recessive OI. While we confirm changes in the lung parenchyma that are reminiscent of emphysema, we show that CrtapKO lung fibroblasts synthesize type I collagen with altered posttranslation modifications consistent with those observed in bone and skin. Unrestrained whole body plethysmography showed a significant decrease in expiratory time, resulting in an increased ratio of inspiratory time over expiratory time and a concomitant increase of the inspiratory duty cycle in CrtapKO compared with WT mice. Closed-chest measurements using the forced oscillation technique showed increased respiratory system elastance, decreased respiratory system compliance, and increased tissue damping and elasticity in CrtapKO mice compared with WT. Pressure-volume curves showed significant differences in lung volumes and in the shape of the curves between CrtapKO mice and WT mice, with and without adjustment for body weight. This is the first evidence that collagen defects in OI cause primary changes in lung parenchyma and several respiratory parameters and thus negatively impact lung function. [ABSTRACT FROM AUTHOR]

Rhinovirus (RV) exposure has been implicated in childhood development of wheeze evoking asthma and exacerbations of underlying airways disease. Studies such as the Copenhagen Prospective Studies on Asthma in Childhood (COPSAC) and Childhood Origins of ASThma (COAST) have identified RV as a pathogen inducing severe respiratory disease. RVs also modulate airway hyperresponsiveness (AHR), a key characteristic of such diseases. Although potential factors underlying mechanisms by which RV induces AHR have been postulated, the precise mechanisms of AHR following RV exposure remain elusive.A challenge to RV-related research stems from inadequate models for study. While human models raise ethical concerns and are relatively difficult in terms of subject recruitment, murine models are limited by susceptibility of infection to the relatively uncommon minor group (RV-B) serotypes, strains that are generally associated with infrequent clinical respiratory virus infections. Although a transgenic mouse strain that has been developed has enhanced susceptibility for infection with the common major group (RV-A) serotypes, few studies have focused on RV in the context of allergic airways disease rather than understanding RV-induced AHR. Recently, the receptor for the virulent RV-C CDHR3, was identified, but a dearth of studies have examined RV-C-induced effects in humans.Currently, the mechanisms by which RV infections modulate airway smooth muscle (ASM) shortening or excitation-contraction coupling remain elusive. Further, only one study has investigated the effects of RV on bronchodilatory mechanisms, with only speculation as to mechanisms underlying RV-mediated modulation of bronchoconstriction. [ABSTRACT FROM AUTHOR]

Gasdermin B (GSDMB) on chromosome 17q21 demonstrates a strong genetic linkage to asthma, but its function in asthma is unknown. Here we identified that GSDMB is highly expressed in lung bronchial epithelium in human asthma. Overexpression of GSDMB in primary human bronchial epithelium increased expression of genes important to both airway remodeling [TGF-β1, 5-lipoxygenase (5-LO)] and airway-hyperresponsiveness (AHR) (5-LO). Interestingly, hGSDMBZp3-Cre mice expressing increased levels of the human GSDMB transgene showed a significant spontaneous increase in AHR and a significant spontaneous increase in airway remodeling, with increased smooth muscle mass and increased fibrosis in the absence of airway inflammation. In addition, hGSDMBZp3-Cre mice showed increases in the same remodeling and AHR mediators (TGF-β1, 5-LO) observed in vitro in GSDMB-overexpressing epithelial cells. GSDMB induces TGF-β1 expression via induction of 5-LO, because knockdown of 5-LO in epithelial cells overexpressing GSDMB inhibited TGF-β1 expression. These studies demonstrate that GSDMB, a gene highly linked to asthma butwhose function in asthma is previously unknown, regulates AHR and airway remodeling without airway inflammation through a previously unrecognized pathway in which GSDMB induces 5-LO to induce TGF-β1 in bronchial epithelium. [ABSTRACT FROM AUTHOR]

Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). C. burnetii manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during C. burnetii infection. Avirulent and virulent C. burnetii triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in C. burnetii-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages. [ABSTRACT FROM AUTHOR]

Background and Purpose: Asthma manifests as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyperresponsiveness (AHR). Although the molecular mechanisms remain unclear, activation of specific PI3K isoforms mediate inflammation and AHR. We aimed to determine whether inhibition of PI3Kδ evokes dilation of airways and to elucidate potential mechanisms.Experimental Approach: Human precision cut lung slices from non-asthma donors and primary human airway smooth muscle (HASM) cells from both non-asthma and asthma donors were utilized. Phosphorylation of Akt, myosin phosphatase target subunit 1 (MYPT1) and myosin light chain (MLC) were assessed in HASM cells following either PI3K inhibitor or siRNA treatment. HASM relaxation was assessed by micro-pattern deformation. Reversal of constriction of airways was assessed following stimulation with PI3K or ROCK inhibitors.Key Results: Soluble inhibitors or PI3Kδ knockdown reversed carbachol-induced constriction of human airways, relaxed agonist-contracted HASM and inhibited pAkt, pMYPT1 and pMLC in HASM. Similarly, inhibition of Rho kinase also dilated human PCLS airways and suppressed pMYPT1 and pMLC. Baseline pMYPT1 was significantly elevated in HASM cells derived from asthma donors in comparison with non-asthma donors. After desensitization of the β2 -adrenoceptors, a PI3Kδ inhibitor remained an effective dilator. In the presence of IL-13, dilation by a β agonist, but not PI3K inhibitor, was attenuated.Conclusion and Implications: PI3Kδ inhibitors act as dilators of human small airways. Taken together, these findings provide alternative approaches to the clinical management of airway obstruction in asthma. [ABSTRACT FROM AUTHOR]

The process of osteoclastic bone resorption is complex and regulated at multiple levels. The role of osteoclast (OCL) fusion and motility in bone resorption are unclear, with the movement of OCL on bone largely unexplored. RANKL (also known asTNFSFH) is a potent stimulator of murine osteoclastogenesis, and activin A (ActA) enhances that stimulation in whole bone marrow. ActA treatment does not induce osteoclastogenesis in stroma-free murine bone marrow macrophage cultures (BMM), but rather inhibits RANKL-induced osteoclastogenesis. We hypothesized that ActA and RANKL differentially regulate osteoclastogenesis by modulating OCL precursors and mature OCL migration. Time-lapse video microscopy measured ActA and RANKL effects on BMM and OCL motility and function. ActA completely inhibited RANKL-stimulated OCL motility, differentiation and bone resorption, through a mechanism mediated by ActA-dependent changes in SMAD2, AKT1 and inhibitor of nuclear factor κB (IκB) signaling. The potent and dominant inhibitory effect of ActA was associated with decreased OCL lifespan because ActA significantly increased activated caspase-3 in mature OCL and OCL precursors. Collectively, these data demonstrate a dual action for ActA on murine OCLs. [ABSTRACT FROM AUTHOR]

The intracellular bacterial pathogen Coxiella burnetii is a category B select agent that causes human Q fever. In vivo, C. burnetii targets alveolar macrophages wherein the pathogen replicates in a lysosome-like parasitophorous vacuole ( PV). In vitro, C. burnetii infects a variety of cultured cell lines that have collectively been used to model the pathogen's infectious cycle. However, differences in the cellular response to infection have been observed, and virulent C. burnetii isolate infection of host cells has not been well defined. Because alveolar macrophages are routinely implicated in disease, we established primary human alveolar macrophages ( hAMs) as an in vitro model of C. burnetii-host cell interactions. C. burnetii pathotypes, including acute disease and endocarditis isolates, replicated in hAMs, albeit with unique PV properties. Each isolate replicated in large, typical PV and small, non-fused vacuoles, and lipid droplets were present in avirulent C. burnetii PV. Interestingly, a subset of small vacuoles harboured single organisms undergoing degradation. Prototypical PV formation and bacterial growth in hAMs required a functional type IV secretion system, indicating C. burnetii secretes effector proteins that control macrophage functions. Avirulent C. burnetii promoted sustained activation of Akt and Erk1/2 pro-survival kinases and short-termphosphorylation of stress-related p38. Avirulent organisms also triggered a robust, early pro-inflammatory response characterized by increased secretion of TNF-α and IL-6, while virulent isolates elicited substantially reduced secretion of these cytokines. A corresponding increase in pro- and mature IL-1β occurred in hAMs infected with avirulent C. burnetii, while little accumulation was observed following infection with virulent isolates. Finally, treatment of hAMs with IFN-γ controlled intracellular replication, supporting a role for this antibacterial insult in the host response to C. burnetii. Collectively, the current results demonstrate the hAM model is a human disease-relevant platform for defining novel innate immune responses to C. burnetii. [ABSTRACT FROM AUTHOR]

The β2-AR (β2-adrenergic receptor) is an important target for respiratory and CVD (cardiovascular disease) medications. Clinical studies suggest that N-terminal polymorphisms of β2-AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down-regulation of β2-AR variants following β-agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human β2-AR (designated β2-AR-RE, β2-AR-GE, β2-AR-RQ and β2-AR-GQ) were studied using site-directed mutagenesis and recombinant expression in HEK-293 cells (human embryonic kidney cells). Ligand-binding assays demonstrated that after 24 h exposure to 1 μM isoprenaline, isoforms with Arg16 (β2-AR-RE and β2-AR-RQ) underwent increased down-regulation compared with isoforms with Gly16 (β2-AR-GE and β2-AR-GQ). Consistent with these differences in down-regulation between isoforms, prolonged isoprenaline treatment resulted in diminished cAMP response to subsequent isoprenaline challenge in β2-AR-RE relative to β2-AR-GE. Confocal microscopy revealed that the receptor isoforms had similar co-localization with the early endosomal marker EEA1 following isoprenaline treatment, suggesting that they had similar patterns of internalization. None of the isoforms exhibited significant co-localization with the recycling endosome marker Rab11 in response to isoprenaline treatment. Furthermore, we found that prolonged isoprenaline treatment led to a higher degree of co-localization of β2-AR-RE with the lysosomal marker LAMP1 (lysosome-associated membrane protein 1) compared with that of β2-AR-GE. Taken together, these results indicate that a mechanism responsible for differential responses of these receptor isoforms to the β-agonist involves differences in the efficiency with which agonist-activated receptors are trafficked to the lysosomes for degradation, or differences in degradation in the lysosomes. [ABSTRACT FROM AUTHOR]

Recently, bitter taste receptors (TAS2Rs) were found in the lung and act to relax airway smooth muscle (ASM) via intracellular Ca2+ concentration signaling generated from restricted phospholipase C activation. As potential therapy, TAS2R agonists could be add-on treatment when patients fail to achieve adequate bronchodilation with chronic β-agonists. The β2-adrenergic receptor (β2AR) of ASM undergoes extensive functional desensitization. It remains unknown whether this desensitization affects TAS2R function, by cross talk at the receptors or distal common components in the relaxation machinery. We studied intracellular signaling and cell mechanics using isolated human ASM, mouse tracheal responses, and human bronchial responses to characterize TAS2R relaxation in the context of β2AR desensitization. In isolated human ASM, magnetic twisting cytometry revealed >90% loss of isoproterenol-promoted decrease in cell stiffness after 18-h exposure to albuterol. Under these same conditions of β2AR desensitization, the TAS2R agonist chloroquine relaxation response was unaffected. TAS2R-mediated stimulation of intracellular Ca2+ concentration in human ASM was unaltered by albuterol pretreatment, in contrast to cAMP signaling, which was desensitized by >90%. In mouse trachea, β2AR desensitization by β-agonist amounted to 92 ± 6.0% (P < 0.001), while, under these same conditions, TAS2R desensitization was not significant (11 ± 3.5%). In human lung slices, chronic β-agonist exposure culminated in 64 ± 5.7% (P < 0.001) desensitization of β2AR-mediated dilation of carbachol-constricted airways that was reversed by chloroquine. We conclude that there is no evidence for physiologically relevant cross-desensitization of TAS2R-mediated ASM relaxation from chronic β-agonist treatment. These findings portend a favorable therapeutic profile for TAS2R agonists for the treatment of bronchospasm in asthma or chronic obstructive lung disease. [ABSTRACT FROM AUTHOR]

Up-regulation of vascular endothelial growth factor (VEGF) is important to hepatocyte regeneration in the late stages of acetaminophen (APAP) toxicity in the mouse. This study was conducted to examine the relationship of hypoxia-inducible factor 1α (HIF-1α) to VEGF and hepatocyte regeneration in APAP toxicity using an inhibitor of HIF-1α DNA-binding activity, echinomycin (EC). B6C3F1 male mice were treated with APAP (200 mg/kg IP), followed by EC (0.15 mg IP) and killed at 4 hr. Serum alanine aminotransferase (ALT), necrosis, hepatic glutathione (GSH) and APAP protein adducts were comparable in the APAP/EC and the APAP/veh mice at 4 hr. Additional studies showed that high dose EC (0.3 mg) reduced hepatic VEGF but also lowered hepatic GSH. Subsequent studies were performed using the 0.15-mg dose of EC. Although EC 0.15 mg had no effect on hepatic VEGF levels at 8 hr, by 24 hr VEGF levels were decreased by 40%. Toxicity (ALT and histopathology) was comparable in the APAP and APAP/EC groups at 24 and 48 hr. Proliferating cell nuclear antigen expression was reduced by both Western blot analysis and immunohistochemical staining in the APAP/EC mice at 48 hr. The data support the hypothesis that induction of HIF-1α, its binding to DNA and subsequent expression of VEGF are important factors in hepatocyte regeneration in APAP toxicity in the mouse. [ABSTRACT FROM AUTHOR]

Background: The β2-adrenergic receptor (β2AR) is a primary target for medications used to treat asthma. Due to the low abundance of β2AR, very few studies have reported its localization in tissues. However, the intracellular location of β2AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to β-agonist medications. Thus, a method for visualizing β2AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant β2AR in primary cell cultures. Methods: A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat β2AR expressed in HEK 293 cells. Antibodies capable of recognizing rat β2AR were identified and used to localize native β2AR in primary cultures of rat airway smooth muscle and epithelial cells. β2AR expression was confirmed by performing ligand binding assays using the β-adrenergic antagonist [3H] dihydroalprenolol ([3H]DHA). Results: Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human β2AR (Ab-Bethyl) specifically recognized human but not rat β2AR. An antibody developed against the C-terminal domain of the mouse β2AR (Ab-sc570) specifically recognized rat but not human β2AR. An antibody developed against 78 amino acids of the C-terminus of the human β2AR (Ab-13989) was capable of recognizing both rat and human β2ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat β2AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface. Conclusion: Antibodies have been identified that recognize human β2AR, rat β2AR or both rat and human β2AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of β2AR in tissues. [ABSTRACT FROM AUTHOR]

Asthma is a disease characterized by reversible airway obstruction. An additional hallmark of chronic asthma is altered wound healing that leads to airway remodeling. Although β-agonists are effective in treating the bronchospasm associated with asthma, their effects on airway wound healing, which are related to airway remodeling, are unknown. It has been demonstrated that β-agonists can alter the signaling of epidermal growth factor (EGF) receptors, which are important in timely wound healing. Therefore, we hypothesized that the β-agonist isoproterenol would affect wound healing. Using an in vitro scrape wound assay, we demonstrated that isoproterenol attenuates EGF-stimulated wound healing in 16HBE airway epithelial cell cultures. Through experiments with forskolin and cells overexpressing β2-adrenergic receptor-yellow fluorescent protein, we show that attenuation is due to the accumulation of cAMP and the involvement of at least one additional pathway. Furthermore, attenuation is not due to a direct effect on the EGF receptor or to an alteration of the ERK/MAPK signaling cascade. Based on these results, we propose that isoproterenol may exert its effects through other MAPK signaling pathways (JNK and/or p38) or through parallel mechanisms. These results also demonstrate a problem of potential therapeutic relevance in which a commonly prescribed medication may alter wound healing and contribute to the remodeling of asthmatic airways. [ABSTRACT FROM AUTHOR]

On binding to its receptor, epidermal growth factor (EGF) initiates a cascade of events leading to cell proliferation or differentiation. In addition, the EGF receptor itself is downregulated to attenuate mitogenic signaling. Downregulation occurs through trafficking of receptors to lysosomes, culminating in proteolytic destruction of both the receptor and ligand; however, endocytic sorting mechanisms that underlie lysosomal targeting remain obscure. The goal of this study was to explore one aspect of the molecular basis for ligandinduced lysosomal targeting and degradation of EGF receptors. In this study, we identify a tyrosine-leucine motif (954YLVI) that is essential for transit of ligand-receptor complexes to lysosomes. When this motif is mutated, HEK 293 cells expressing the mutant receptors demonstrate impaired lysosomal targeting and downregulation compared with wildtype receptors. 954YLVI is highly conserved among EGF receptors from various mammalian and invertebrate species and is critical for receptor downregulation. We propose that 954YLVI works in concert with at least two additional regions within the EGF receptor cytoplasmic domain that are essential for efficiently targeting ligand-receptor complexes to the lysosome. [ABSTRACT FROM AUTHOR]

We have demonstrated the subcellular localization of the human UDP-glucuronosyltransferases (UGTs), UGT2B7 and UGT1A6, in endoplasmic reticulum (ER) and nuclear membrane from human hepatocytes and cell lines, by in situ immunostaining and Western blot. Double immunostaining for UGT2B7 and calnexin, an ER resident protein, showed that UGT2B7 was equally present in ER and nuclear membrane whereas calnexin was present almost exclusively in ER. Immunogold labeling of HK293 cells expressing UGT2B7 established the presence of UGT2B7 in both nuclear membranes. Enzymatic assays with UGT2B7 substrates confirmed the presence of functional UGT2B7 protein in ER, whole nuclei, and both outer and inner nuclear membranes. This study has identified, for the first time, the presence of UGT2B7 and UGT1A6 in the nucleus and of UGT2B7 in the inner and outer nuclear membranes. This localization may play an important functional role within nuclei: protection from toxic compounds and/or control of steady-state concentrations of nuclear receptor ligands. [Copyright &y& Elsevier]

Background: The beta2-adrenergic receptor (beta2AR) is a primary target for medications used to treat asthma. Due to the low abundance of beta2AR, very few studies have reported its localization in tissues. However, the intracellular location of beta2AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to beta-agonist medications. Thus, a method for visualizing beta2AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant beta2AR in primary cell cultures.Methods: A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat beta2AR expressed in HEK 293 cells. Antibodies capable of recognizing rat beta2AR were identified and used to localize native beta2AR in primary cultures of rat airway smooth muscle and epithelial cells. beta2AR expression was confirmed by performing ligand binding assays using the beta-adrenergic antagonist [3H] dihydroalprenolol ([3H]DHA).Results: Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human beta2AR (Ab-Bethyl) specifically recognized human but not rat beta2AR. An antibody developed against the C-terminal domain of the mouse beta2AR (Ab-sc570) specifically recognized rat but not human beta2AR. An antibody developed against 78 amino acids of the C-terminus of the human beta2AR (Ab-13989) was capable of recognizing both rat and human beta2ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat beta2AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface.Conclusion: Antibodies have been identified that recognize human beta2AR, rat beta2AR or both rat and human beta2AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of beta2AR in tissues. [ABSTRACT FROM AUTHOR]