Epithelial Uptake of Flagella Initiates Proinflammatory Signaling

The airway epithelium serves multiple roles in the defense of the lung. Not only does it act as a physical barrier, it acts as a distal extension of the innate immune system. We investigated the role of the airway epithelium in the interaction with flagella, an important virulence factor of the pathogen Pseudomonas aeruginosa, a cause of ventilator associated pneumonia and significant morbidity and mortality in patients with cystic fibrosis. Flagella were required for transmigration across polarized airway epithelial cells and this was a direct consequence of motility, and not a signaling effect. Purified flagella did not alter the barrier properties of the epithelium but were observed to be rapidly endocytosed inside epithelial cells. Neither flagella nor intact P. aeruginosa stimulated epithelial inflammasome signaling. Flagella-dependent signaling required dynamin-based uptake as well as TLR5 and primarily led to the induction of proinflammatory (Tnf, Il6) as well as neutrophil (Cxcl1, Cxcl2, Ccl3) and macrophage (Ccl20) chemokines. Although flagella are important in invasion across the epithelial barrier their shedding in the airway lumen results in epithelial uptake and signaling that has a major role in the initial recruitment of immune cells in the lung.     

Conformational aspect of polypeptide structure. XLIV. Conformational transitions of poly(N-methyl-alanines) induced by trifluoroacetic acid

The synthesis of poly(N-methyl-L -alanine) and poly (N-methyl-DL -alanine) are described. The polymers were examined by 220 MHz high-resolution nuclear magnetic resonance (nmr) and circular dichroism (CD). The results demonstrate that poly(N-methyl-L -alanine) exists as an ordered helical structure with all the amide bonds in the trans configuration in appropriate solvents. As trifluoroacetic acid (TFA) is added to the solutions of the polymer in helix-supporting solvents, resonances corresponding to both trans and cis amide conformations of N-methyl, C-methyl, and α-CH are observed. The presence of both the trans and the cis peptide bonds in a polymer chain disrupts the ordered structures. Our conclusions from CD data are in agreement with the nmr results. Ultracentrifugation shows that degradation of the polymer chain does not occur during the TFA treatment.     

Oxonol dyes as monitors of membrane potential. Their behavior in photosynthetic bacteria

The responses of oxonol dyes to single and multiple single turnovers of the photosynthetic apparatus of photosynthetic bacteria have been studied, and compared with the responses of the endogenous carotenoid pigments. The absorbance changes of the oxonols can be conveniently measured at 587 nm, because this is an isosbestic point in the ‘light-minus-dark’ difference spectrum of the chromatophores.The oxonols appear to respond to the light-induced ‘energization’ by shifting their absorption maxima. In the presence of K⁺, valinomycin abolished and nigericin enhanced such shifts, suggesting that the dyes respond to the light-induced membrane potential. Since the dyes are anions at neutral pH values, they probably distribute across the membrane in accordance with the potential, which is positive inside the chromatophores. The accumulation of dye, which is indicated by a decrease in the carotenoid bandshift, poises the dye-membrane equilibrium in favor of increased dye binding and this might be the cause of the spectral shift.The dye response has an apparent second-order rate constant of approx. 2 · 10⁶ M^?1 · s^?1 and so is always slower than the carotenoid bandshift. Thus the dyes cannot be used to monitor membrane potential on submillisecond timescales. Nevertheless, on a timescale of seconds the logarithm of the absorbance change at 587 nm is linear with respect to the membrane potential calibrated with the carotenoid bandshift. This suggests that under appropriate conditions the dyes can be used with confidence as indicators of membrane potential in energy-transducing membranes that do not posses intrinsic probes of potential.     

β-Catenin loss in hepatocytes promotes hepatocellular cancer after diethylnitrosamine and phenobarbital administration to mice

Hepatocellular Carcinoma (HCC) is the fifth most common cancer worldwide. β-Catenin, the central orchestrator of the canonical Wnt pathway and a known oncogene is paramount in HCC pathogenesis. Administration of phenobarbital (PB) containing water (0.05% w/v) as tumor promoter following initial injected intraperitoneal (IP) diethylnitrosamine (DEN) injection (5 μg/gm body weight) as a tumor inducer is commonly used model to study HCC in mice. Herein, nine fifteen-day male β-catenin knockout mice (KO) and fifteen wild-type littermate controls (WT) underwent DEN/PB treatment and were examined for hepatic tumorigenesis at eight months. Paradoxically, a significantly higher tumor burden was observed in KO (p<0.05). Tumors in KO were β-catenin and glutamine synthetase negative and HGF/Met, EGFR & IGFR signaling was unremarkable. A significant increase in PDGFRα and its ligand PDGF-CC leading to increased phosphotyrosine-720-PDGFRα was observed in tumor-bearing KO mice (p<0.05). Simultaneously, these livers displayed increased cell death, stellate cell activation, hepatic fibrosis and cell proliferation. Further, PDGF-CC significantly induced hepatoma cell proliferation especially following β-catenin suppression. Our studies also demonstrate that the utilized DEN/PB protocol in the WT C57BL/6 mice did not select for β-catenin gene mutations during hepatocarcinogenesis. Thus, DEN/PB enhanced HCC in mice lacking β-catenin in the liver may be due to their ineptness at regulating cell survival, leading to enhanced fibrosis and regeneration through PDGFRα activation. β-Catenin downregulation also made hepatoma cells more sensitive to receptor tyrosine kinases and thus may be exploited for therapeutics.     

Evaluation of the effect of closed areas on a unique and shallow water coral reef fish assemblage reveals complex responses

Areas closed to fishing are advocated as both fisheries management and biodiversity conservation tools. However, few studies investigate the responses of suites of both target and non-target fish species within an assemblage, which is an important consideration for ecosystem-based fisheries management approaches. Diver-operated stereo-video was used to assess the abundance and length of coral reef fish across multiple areas both open and closed to fishing at the Houtman Abrolhos Islands, Western Australia. After taking into consideration spatial differences in benthic habitat, the composition of fish assemblages was found to differ between open and closed areas. The target species, Plectropomus leopardus, was approximately two times more abundant in closed areas. Furthermore, 51 % of P. leopardus were larger than the minimum legal length (MLL) for retention in closed areas compared with only 1.8 % in areas open to fishing. Another target species, Choerodon rubescens was surveyed in greater abundance at sizes larger than the MLL in closed areas (64 % >400 mm) in comparison with areas open to fishing (36 %). A number of non-target species were also larger in closed areas (e.g., Kyphosus cornelii, Scarus schlegeli). In contrast, several non-targeted prey species were more abundant in open areas (e.g., Pomacentrus milleri was six times more abundant in open areas). Our results document complex responses of target and non-target species in closed areas at the Houtman Abrolhos Islands.     

The primary structure of NG2, a novel membrane-spanning proteoglycan

The complete primary structure of the core protein of rat NG2, a large, chondroitin sulfate proteoglycan expressed on O2A progenitor cells, has been determined from cDNA clones. These cDNAs hybridize to an mRNA species of 8.9 kbp from rat neural cell lines. The total contiguous cDNA spans 8,071 nucleotides and contains an open reading frame for 2,325 amino acids. The predicted protein is an integral membrane protein with a large extracellular domain (2,224 amino acids), a single transmembrane domain (25 amino acids), and a short cytoplasmic tail (76 amino acids). Based on the deduced amino acid sequence and immunochemical analysis of proteolytic fragments of NG2, the extracellular region can be divided into three domains: an amino terminal cysteine-containing domain which is stabilized by intrachain disulfide bonds, a serine-glycine-containing domain to which chondroitin sulfate chains are attached, and another cysteine-containing domain. Four internal repeats, each consisting of 200 amino acids, are found in the extracellular domain of NG2. These repeats contain a short sequence that resembles the putative Ca(++)-binding region of the cadherins. The sequence of NG2 does not show significant homology with any other known proteins, suggesting that NG2 is a novel species of integral membrane proteoglycan.     

Consequences of molecular engineering enhanced DNA binding in a DNA repair enzyme.

Facilitated one-dimensional diffusion is a general mechanism utilized by several DNA-interactive proteins as they search for their target sites within large domains of nontarget DNA. T4 endonuclease V is a protein which scans DNA in a nonspecifically bound state and processively incises DNA at ultraviolet (UV)-induced pyrimidine dimer sites. An electrostatic contribution to this mechanism of target location has been established. Previous studies indicate that a decrease in the affinity of endonuclease V for nontarget DNA results in a decreased ability to scan DNA and a concomitant decrease in the ability to enhance UV survival in repair-deficient Escherichia coli. This study was designed to question the contrasting effect of an increase in the affinity of endonuclease V for nontarget DNA. With this as a goal, a gradient of increasingly basic amino acid content was created along a proposed endonuclease V-nontarget DNA interface. This incremental increase in positive charge correlated with the stepwise enhancement of nontarget DNA binding, yet inversely correlated with enhanced UV survival in repair-deficient E. coli. Further analysis suggests that the observed reduction in UV survival is consistent with the hypothesis that enhanced nontarget DNA affinity results in reduced pyrimidine dimer-specific recognition and/or binding. The net effect is a reduction in the efficiency of pyrimidine dimer incision.     

Immunoprophylaxis of respiratory syncytial virus infection in the infant ferret.

Infant ferrets can be protected from respiratory syncytical virus challenge at 3 days of age by gestational infection of their mothers. Ferrets acquire their immunity to respiratory syncytial virus postpartum via immunizing products of lactation. The level of protection against viral replication correlates with the maternal serum neutralizing titer or a concomitant factor. Passive administration of adult ferret serum with a neutralizing titer of 1:1024 or greater, either i.p. or orally does not confer immunity. A nonantibody-mediated protective mechanism appears to play an important role in protecting the infant ferret from respiratory syncytial virus replication. Our findings allow the testing of the efficacy of future human vaccines before human clinical trial.     

Pathogenesis of adenovirus type 5 pneumonia in cotton rats (Sigmodon hispidus).

Cotton rats (Sigmodon hispidus) were inoculated intranasally with 10(2.0) to 10(10.0) PFU of human adenovirus type 5. The virus replicated to a high titer in pulmonary tissues, with the peak titer being proportional to the input dose. The 50% lethal dose was 10(9.4) PFU. Histopathologic changes were proportional to the infecting inoculum and included the infiltration of interstitial and intra-alveolar areas, moderate damage to bronchiolar epithelium, and cellular infiltration of peribronchiolar and perivascular regions. These changes could be divided into two phases: an early phase (affecting alveoli, bronchiolar epithelium, and peribronchiolar regions) with an infiltrate consisting primarily of monocytes-macrophages and neutrophils, with occasional lymphocytes, and a later phase (affecting peribronchiolar and perivascular regions) with an infiltrate consisting almost exclusively of lymphocytes. In both phases, the predominant process was the response of the host to infection, rather than direct viral damage to infected cells. An infecting inoculum of 10(8.0) PFU or larger caused severe damage to type II alveolar cells, which were swollen, showed a loss of lamellar bodies, and were surrounded by polymorphonuclear leukocytes and macrophages. No evidence of complete viral replication was found in type II alveolar cells.