Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells

Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system.     

Tyrosine phosphorylation enhances RAD52-mediated annealing by modulating its DNA binding

RAD52 protein has an important role in homology-directed DNA repair by mediating RAD51 nucleoprotein filament formation on single-stranded DNA (ssDNA) protected by replication protein-A (RPA) and annealing of RPA-coated ssDNA. In human, cellular response to DNA damage includes phosphorylation of RAD52 by c-ABL kinase at tyrosine 104. To address how this phosphorylation modulates RAD52 function, we used an amber suppressor technology to substitute tyrosine 104 with chemically stable phosphotyrosine analogue (p-Carboxymethyl-L-phenylalanine, pCMF). The RAD52(Y104pCMF) retained ssDNA-binding activity characteristic of unmodified RAD52 but showed lower affinity for double-stranded DNA (dsDNA) binding. Single-molecule analyses revealed that RAD52(Y104pCMF) specifically targets and wraps ssDNA. While RAD52(Y104pCMF) is confined to ssDNA region, unmodified RAD52 readily diffuses into dsDNA region. The Y104pCMF substitution also increased the ssDNA annealing rate and allowed overcoming the inhibitory effect of dsDNA. We propose that phosphorylation at Y104 enhances ssDNA annealing activity of RAD52 by attenuating dsDNA binding. Implications of phosphorylation-mediated activation of RAD52 annealing activity are discussed.     

4-1BB signaling synergizes with programmed death ligand 1 blockade to augment CD8 T cell responses during chronic viral infection

Previous studies have identified the inhibitory role that the programmed death 1 (PD-1) pathway plays during chronic infection. Blockade of this pathway results in rescue of viral-specific CD8 T cells, as well as reduction of viral loads in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). We tested the effect of combining PD ligand 1 (PD-L1) blockade with an agonistic regimen that induces 4-1BB costimulation during chronic LCMV infection. There is a boosting effect in the rescue of LCMV-specific CD8 T cell responses after dual treatment with PD-L1 blockade and 4-1BB agonistic Abs when the amount and timing of 4-1BB costimulation are carefully controlled. When PD-L1-blocking Abs are given together with a single low dose of anti-4-1BB agonistic Abs, there is an enhanced and stable expansion of viral-specific CD8 T cells. Conversely, when blocking Abs to PD-L1 are given with a repetitive high dose of anti-4-1BB, there is an initial synergistic expansion of viral-specific CD8 T cells by day 7, followed by dramatic apoptosis by day 14. Viral control paralleled CD8 T cell kinetics after dual treatment. By day 7 posttreatment, viral titers were lower in both of the combined regimens (compared with PD-L1 blockade alone). However, whereas the high dose of anti-4-1BB plus PD-L1 blockade resulted in rebound of viral titers to original levels, the low dose of anti-4-1BB plus PD-L1 blockade resulted in a stable reduction of viral loads. These findings demonstrate the importance of carefully manipulating the balance between activating and inhibitory signals to enhance T cell responses during chronic infection.     

Crystal structures of Enoyl-ACP reductases I (FabI) and III (FabL) from B. subtilis

Enoyl-[acyl carrier protein] (ACP) reductase (ENR) is a key enzyme in type II fatty acid synthesis that catalyzes the last step in each elongation cycle. Therefore, it has been considered as a target for antibiotics. However, recent studies indicate that some pathogens have more than one ENR; in particular, Bacillus subtilis has two ENRs, FabI and FabL. The crystal structures of the ternary complexes of BsFaBI and BsFabL are found as a homotetramer showing the same overall structure despite a sequence identity of only 24%. The positions of the catalytic dyad of Tyr-(Xaa)₆-Lys in FabL are almost identical to that of FabI, but a detailed structural analysis shows that FabL shares more structural similarities with FabG and other members of the SDR (short-chain alcohol dehydrogenase/reductase) family. The apo FabL structure shows significantly different conformations at the cofactor and the substrate-binding regions, and this resulted in a totally different tetrameric arrangement reflecting the flexibility of these regions in the absence of the cofactor and substrate/inhibitor.     

In vivo and in vitro analyses of toxic mutants of HET-s: FTIR antiparallel signature correlates with amyloid toxicity

The folding and interactions of amyloid proteins are at the heart of the debate as to how these proteins may or may not become toxic to their host. Although little is known about this issue, the structure seems to be clearly involved with effects on molecular events. To understand how an amyloid may be toxic, we previously generated a yeast toxic amyloid (mutant 8) from the nontoxic HET-s_(218-289) prion domain of Podospora anserina. Here, we performed a comprehensive structure-toxicity study by mutating individually each of the 10 mutations found in mutant 8. The study of the library of new mutants generated allowed us to establish a clear link between Fourier transform infrared antiparallel signature and amyloid toxicity. All of the mutants that form parallel β-sheets are not toxic. Double mutations may be sufficient to shift a parallel structure to antiparallel amyloids, which are toxic to yeast. Our findings also suggest that the toxicity of antiparallel structured mutants may be linked to interaction with membranes.     

Biochemical and strain properties of CJD prions: complexity versus simplicity

Prions, the agents responsible for transmissible spongiform encephalopathies, are infectious proteins consisting primarily of scrapie prion protein (PrP(Sc)), a misfolded, β-sheet enriched and aggregated form of the host-encoded cellular prion protein (PrP(C)). Their propagation is based on an autocatalytic PrP conversion process. Despite the lack of a nucleic acid genome, different prion strains have been isolated from animal diseases. Increasing evidence supports the view that strain-specific properties may be enciphered within conformational variations of PrP(Sc). In humans, sporadic Creutzfeldt-Jakob disease (sCJD) is the most frequent form of prion diseases and has demonstrated a wide phenotypic and molecular spectrum. In contrast, variant Creutzfeldt-Jakob disease (vCJD), which results from oral exposure to the agent of bovine spongiform encephalopathy, is a highly stereotyped disease, that, until now, has only occurred in patients who are methionine homozygous at codon 129 of the PrP gene. Recent research has provided consistent evidence of strain diversity in sCJD and also, unexpectedly enough, in vCJD. Here, we discuss the puzzling biochemical/pathological diversity of human prion disorders and the relationship of that diversity to the biological properties of the agent as demonstrated by strain typing in experimental models.     

GENETIC POPULATION STRUCTURE AND MATING SYSTEM IN CHONDRUS CRISPUS (RHODOPHYTA)1

Chondrus crispus Stackh. has been intensely studied, yet no study to date has elucidated its population structure or mating system despite many populations in which there was a haploid bias and lack of male gametophytes. Therefore, 12 nuclear microsatellite loci were identified in this red alga. Microsatellite markers were developed and tested against a panel of specimens collected from two shore levels at two sites in Brittany, France: Pointe de Primel and Pointe de la Jument, Concarneau. Single locus genetic determinism was verified at eight polymorphic loci, as only one band was observed for haploid genotypes, whereas one or two bands were observed for diploids. These markers enabled the detection of unique genotypes within sampled populations, indicating that very few fronds shared the same multilocus genotype. This finding suggests that asexual reproduction was not the prevailing mode of reproduction. In addition, we explored the hierarchical population structure showing that gene flow is restricted at small spatial scales (<50 m) between upper and lower Chondrus‐range populations within a shore. Sexual reproduction predominated in the populations of C. crispus studied, but probably due to fine‐scale spatial substructuring, inbreeding was also significant. In conclusion, this study reveals that fine‐scale genetic variation is of major importance in C. crispus, suggesting that differences between microhabitats should be essential in understanding evolutionary processes in this species.     

Identification of a novel neuropathogenic Theiler's murine encephalomyelitis virus

Theiler's murine encephalitis viruses (TMEV) are divided into two subgroups based on their neurovirulence. Persistent strains resemble Theiler's original viruses (referred to as the TO subgroup), which largely induce a subclinical polioencephalomyelitis during the acute phase of the disease and can persist in the spinal cord of susceptible animals, inducing a chronic demyelinating disease. In contrast, members of the neurovirulent subgroup cause an acute encephalitis characterized by the rapid onset of paralysis and death within days following intracranial inoculation. We report herein the characterization of a novel neurovirulent strain of TMEV, identified using pyrosequencing technology and referred to as NIHE. Complete coverage of the NIHE viral genome was obtained, and it shares <90% nucleotide sequence identity to known TMEV strains irrespective of subgroup, with the greatest sequence variability being observed in genes encoding the leader and capsid proteins. The histopathological analysis of infected brain and spinal cord demonstrate inflammatory lesions and neuronal necrosis during acute infection with no evidence of viral persistence or chronic disease. Intriguingly, genetic analysis indicates the putative expression of the L protein, considered a hallmark of strains within the persistent subgroup. Thus, the identification and characterization of a novel neurovirulent TMEV strain sharing features previously associated with both subgroups will lead to a deeper understanding of the evolution of TMEV strains and new insights into the determinants of neurovirulence.     

Genotoxic effects of silver nanoparticles stimulated by oxidative stress in human normal bronchial epithelial (BEAS-2B) cells

Many classes of silver nanoparticles (Ag-NPs) have been synthesized and widely applied, but the genotoxicity of Ag-NPs and the factors leading to genotoxicity remain unknown. Therefore, the purpose of this study is to elucidate the genotoxic effects of Ag-NPs in lung and the role of oxidative stress on the genotoxic effects of Ag-NPs. For this, Ag-NPs were completely dispersed in medium by sonication and filtration. The Ag-NPs dispersed in medium were 43-260nm in size. We observed distinct uptake of Ag-NPs into BEAS-2B cells. The Ag-NPs aggregates were wrapped with an endocytic vesicle within the cytoplasm and nucleus of BEAS-2B cells. In the comet assay and micronucleus (MN) assay for BEAS-2B cells, Ag-NPs stimulated DNA breakage and MN formation in a dose-dependent manner. The genotoxic effect of Ag-NPs was partially blocked by scavengers. In particular, of the scavengers tested, superoxide dismutase most significantly blocked the genotoxic effects in both the cytokinesis-block MN assay and the comet assay. In the modified comet assay, Ag-NPs induced a significant increase in oxidative DNA damage. Furthermore, in the oxidative stress assay, Ag-NPs significantly increased the reactive oxygen radicals. These results suggest that Ag-NPs have genotoxic effects in BEAS-2B cells and that oxidative stress stimulated by Ag-NPs may be an important factor in their genotoxic effects.