Toll-like receptor 3 is induced by and mediates antiviral activity against rhinovirus infection of human bronchial epithelial cells

Rhinoviruses (RV) are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. Toll-like receptors (TLRs) are a conserved family of receptors that recognize and respond to a variety of pathogen-associated molecular patterns. TLR3 recognizes double-stranded RNA, an important intermediate of many viral life cycles (including RV). The importance of TLR3 in host responses to virus infection is not known. Using BEAS-2B (a human bronchial epithelial cell-line), we demonstrated that RV replication increased the expression of TLR3 mRNA and TLR3 protein on the cell surface. We observed that blocking TLR3 led to a decrease in interleukin-6, CXCL8, and CCL5 in response to poly(IC) but an increase following RV infection. Finally, we demonstrated that TLR3 mediated the antiviral response. This study demonstrates an important functional requirement for TLR3 in the host response against live virus infection and indicates that poly(IC) is not always a good model for studying the biology of live virus infection.     

The interaction of cationic antimicrobial peptides with vesicles containing synthetic glycolipids as models of the outer membrane of gram-negative bacteria.

Two simple lipid A analogues methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside (GL1) and methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside 4-O-phosphate (GL2) were synthesized and used for preparing mixed phosphocholine vesicles as models of the outer membrane of gram-negative bacteria. The interaction of these model membranes with magainin 2, a representative of the alpha-helical membrane active peptides, and apidaecin Ib and drosocin, two insect Pro-rich peptides which do not act at the level of the cellular membrane, were studied by CD and dye-releasing experiments. The CD spectra of apidaecin Ib and drosocin in the presence of GL1- or GL2-containing vesicles were consistent with largely unordered structures, whereas, according to the CD spectra, magainin 2 adopted an amphipathic alpha-helical conformation, particularly in the presence of negatively charged bilayers. The ability of the peptides to fold into amphipathic conformations was strictly correlated to their ability to bind and to permeabilize phospholipid as well as glycolipid membranes. Apidaecin Ib and drosocin, which are unable to adopt an amphipathic structure, showed negligible dye-leakage activity even in the presence of GL2-containing vesicles. It is reasonable to suppose that, as for the killing mechanism, the two classes of antimicrobial peptides follow different patterns to cross the bacterial outer membrane.     

Effect of semen preparation on casa motility results in cryopreserved bull spermatozoa

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility and kinetic patterns because they provide objective data for thousands of sperm tracks. However, these devices are not ready-to-use and standardization of analytical practices is a fundamental requirement. In this study, we evaluated the effects of some settings, such as frame rate and frames per field, chamber and time of analysis, and samples preparations, including thawing temperature, sperm sample concentration, and media used for dilution, on the kinetic results of bovine frozen-thawed semen using a CASA. In Experiment 1, the frame rate (30-60 frame/s) significantly affected motility parameters, whereas the number of frames per field (30 or 45) did not seem to affect sperm kinetics. In Experiment 2, the thawing protocol affects sperm motility and kinetic parameters. Sperm sample concentration significantly limited the opportunity to perform the analysis and the kinetic results. A concentration of 100 and 50 × 10⁶ sperm/mL limited the device's ability to perform the analysis or gave wrong results, whereas 5, 10, 20, and 30 × 10⁶ sperm/mL concentrations allowed the analysis to be performed, but with different results (Experiment 3). The medium used for the dilution of the sample, which is fundamental for a correct sperm head detection, affects sperm motility results (Experiment 4). In this study, Makler and Leja chambers were used to perform the semen analysis with CASA devices. The chamber used significantly affected motility results (Experiment 5). The time between chamber loading and analysis affected sperm velocities, regardless of chamber used. Based on results recorded in this study, we propose that the CASA evaluation of motility of bovine frozen-thawed semen using Hamilton-Thorne IVOS 12.3 should be performed using a frame rate of 60 frame/s and 30 frames per field. Semen should be diluted at least at 20 × 10⁶ sperm/mL using PBS. Furthermore, it is necessary to consider the type of chamber used and perform the analysis within 1 or 2 min, regardless of the chamber used.     

Expression Profiling of a Genetic Animal Model of Depression Reveals Novel Molecular Pathways Underlying Depressive-Like Behaviours

Background

The Flinders model is a validated genetic rat model of depression that exhibits a number of behavioural, neurochemical and pharmacological features consistent with those observed in human depression.

Principal Findings

In this study we have used genome-wide microarray expression profiling of the hippocampus and prefrontal/frontal cortex of Flinders Depression Sensitive (FSL) and control Flinders Depression Resistant (FRL) lines to understand molecular basis for the differences between the two lines. We profiled two independent cohorts of Flinders animals derived from the same colony six months apart, each cohort statistically powered to allow independent as well as combined analysis. Using this approach, we were able to validate using real-time-PCR a core set of gene expression differences that showed statistical significance in each of the temporally distinct cohorts, representing consistently maintained features of the model. Small but statistically significant increases were confirmed for cholinergic (chrm2, chrna7) and serotonergic receptors (Htr1a, Htr2a) in FSL rats consistent with known neurochemical changes in the model. Much larger gene changes were validated in a number of novel genes as exemplified by TMEM176A, which showed 35-fold enrichment in the cortex and 30-fold enrichment in hippocampus of FRL animals relative to FSL.

Conclusions

These data provide significant insights into the molecular differences underlying the Flinders model, and have potential relevance to broader depression research.     

The influence of spatial pulsed magnetic field application on neuropathic pain after tibial nerve transection in rat

The purpose of the study was to examine the influence of the spatial variable magnetic field (induction: 150–300?µT, 80–150?µT, 20–80?µT; frequency 40?Hz) on neuropathic pain after tibial nerve transection. The experiments were carried out on 64 male Wistar C rats. The exposure of animals to magnetic field was performed 1?d/20?min., 5?d/week, for 28?d. Behavioural tests assessing the intensity of allodynia and sensitivity to mechanical and thermal stimuli were conducted 1?d prior to surgery and 3, 7, 14, 21 and 28?d after the surgery. The extent of autotomy was examined. Histological and immunohistochemical analysis was performed. The use of extremely low-frequency magnetic fields of minimal induction values (20–80?µT/40?Hz) decreased pain in rats after nerve transection. The nociceptive sensitivity of healthy rats was not changed following the exposition to the spatial magnetic field of the low frequency. The results of histological and immunohistochemical investigations confirm those findings. Our results indicate that extremely low-frequency magnetic field may be useful in the neuropathic pain therapy.     

α1-AMP-Activated Protein Kinase Regulates Hypoxia-Induced Na,K-ATPase Endocytosis via Direct Phosphorylation of Protein Kinase Cζ

Hypoxia promotes Na,K-ATPase endocytosis via protein kinase Cζ (PKCζ)-mediated phosphorylation of the Na,K-ATPase α subunit. Here, we report that hypoxia leads to the phosphorylation of 5′-AMP-activated protein kinase (AMPK) at Thr172 in rat alveolar epithelial cells. The overexpression of a dominant-negative AMPK α subunit (AMPK-DN) construct prevented the hypoxia-induced endocytosis of Na,K-ATPase. The overexpression of the reactive oxygen species (ROS) scavenger catalase prevented hypoxia-induced AMPK activation. Moreover, hypoxia failed to activate AMPK in mitochondrion-deficient ρ⁰-A549 cells, suggesting that mitochondrial ROS play an essential role in hypoxia-induced AMPK activation. Hypoxia-induced PKCζ translocation to the plasma membrane and phosphorylation at Thr410 were prevented by the pharmacological inhibition of AMPK or by the overexpression of the AMPK-DN construct. We found that AMPK α phosphorylates PKCζ on residue Thr410 within the PKCζ activation loop. Importantly, the activation of AMPK α was necessary for hypoxia-induced AMPK-PKCζ binding in alveolar epithelial cells. The overexpression of T410A mutant PKCζ prevented hypoxia-induced Na,K-ATPase endocytosis, confirming that PKCζ Thr410 phosphorylation is essential for this process. PKCζ activation by AMPK is isoform specific, as small interfering RNA targeting the α1 but not the α2 catalytic subunit prevented PKCζ activation. Accordingly, we provide the first evidence that hypoxia-generated mitochondrial ROS lead to the activation of the AMPK α1 isoform, which binds and directly phosphorylates PKCζ at Thr410, thereby promoting Na,K-ATPase endocytosis.When exposed to low oxygen levels (hypoxia), cells develop adaptative strategies to maintain adequate levels of ATP (21). These strategies include increasing the efficiency of energy-producing pathways, mostly through anaerobic glycolysis, while decreasing energy-consuming processes such as Na,K-ATPase activity (30). Alveolar hypoxia occurs in many respiratory disorders, and it has been shown to decrease epithelial active Na⁺ transport, leading to impaired fluid reabsorption (37, 41, 42). Active Na⁺ transport and, thus, alveolar fluid reabsortion are effected mostly via apical sodium channels and the basolateral Na,K-ATPase (32, 38, 42). We have reported previously that hypoxia inhibits Na,K-ATPase activity by promoting its endocytosis from the plasma membrane by a mechanism that requires the generation of mitochondrial reactive oxygen species (ROS) and the phosphorylation of the Na,K-ATPase α subunit at Ser18 by protein kinase Cζ (PKCζ) (8, 9).The 5′-AMP-activated protein kinase (AMPK) is a heterotrimeric Ser/Thr kinase composed of a catalytic α subunit and regulatory β and γ subunits. Both isoforms of the AMPK catalytic subunit (α1 and α2) form complexes with noncatalytic subunits. The α1 subunit is ubiquitously expressed, whereas the α2 subunit isoform is expressed predominantly in tissues like the liver, heart, and skeletal muscle (36). The α1 and α2 subunit isoforms have ∼90% homology in their N-terminal catalytic domains and ∼60% homology in their C-terminal domains (36), suggesting that they may have distinct downstream targets (31). AMPK activation requires phosphorylation at Thr172 in the activation loop of the α subunit by upstream kinases (12, 19). Findings from recent studies suggest that AMPK is an important signaling intermediary in coupling ion transport and metabolism (15). Indeed, it has been reported that the pharmacological activation of AMPK inhibits amiloride- and ouabain-sensitive epithelial Na⁺ transport (15). Moreover, the activities of the epithelial Na⁺ channel (ENaC) (2, 17), the Na,K-ATPase (40), and the cystic fibrosis transmembrane conductance regulator (17) have been shown to be inhibited by AMPK. Here, we provide evidence that hypoxia, via mitochondrial ROS, leads to AMPK activation and that AMPK binds to and directly phosphorylates PKCζ in an isoform-specific manner, thus promoting Na,K-ATPase endocytosis in alveolar epithelial cells (AEC).