Activation with CpG-A and CpG-B oligonucleotides reveals two distinct regulatory pathways of type I IFN synthesis in human plasmacytoid dendritic cells

Two different CpG oligonucleotides (ODN) were used to study the regulation of type I IFN in human plasmacytoid dendritic cells (PDC): ODN 2216, a CpG-A ODN, known to induce high amounts of IFN-alpha in PDC, and ODN 2006, a CpG-B ODN, which is potent at stimulating B cells. CpG-A ODN showed higher and prolonged kinetics of type I IFN production compared with that of CpG-B ODN. In contrast, CpG-B ODN was more active than CpG-A ODN in stimulating IL-8 production and increasing costimulatory and Ag-presenting molecules, suggesting that CpG-A and CpG-B trigger distinct regulatory pathways in PDC. Indeed, CpG-A ODN, but not CpG-B ODN, activated the type I IFNR-mediated autocrine feedback loop. PDC were found to express high constitutive levels of IFN regulatory factor (IRF)7. IRF7 and STAT1, but not IRF3, were equally up-regulated by both CpG-A and CpG-B. CD40 ligand synergistically increased CpG-B-induced IFN-alpha independent of the IFNR but did not affect CpG-B-induced IFN-beta. In conclusion, our studies provide evidence for the existence of two distinct regulatory pathways of type I IFN synthesis in human PDC, one dependent on and one independent of the IFNR-mediated feedback loop. The alternate use of these pathways is based on the type of stimulus rather than the quantity of IFN-alphabeta available to trigger the IFNR. Constitutive expression of IRF7 and the ability to produce considerable amounts of IFN-alpha independent of the IFNR seem to represent characteristic features of PDC.     

The fastest global events in RNA folding: electrostatic relaxation and tertiary collapse of the Tetrahymena ribozyme

Large RNAs can collapse into compact conformations well before the stable formation of the tertiary contacts that define their final folds. This study identifies likely physical mechanisms driving these early compaction events in RNA folding. We have employed time-resolved small-angle X-ray scattering to monitor the fastest global shape changes of the Tetrahymena ribozyme under different ionic conditions and with RNA mutations that remove long-range tertiary contacts. A partial collapse in each of the folding time-courses occurs within tens of milliseconds with either monovalent or divalent cations. Combined with comparison to predictions from structural models, this observation suggests a relaxation of the RNA to a more compact but denatured conformational ensemble in response to enhanced electrostatic screening at higher ionic concentrations. Further, the results provide evidence against counterion-correlation-mediated attraction between RNA double helices, a recently proposed model for early collapse. A previous study revealed a second 100 ms phase of collapse to a globular state. Surprisingly, we find that progression to this second early folding intermediate requires RNA sequence motifs that eventually mediate native long-range tertiary interactions, even though these regions of the RNA were observed to be solvent-accessible in previous footprinting studies under similar conditions. These results help delineate an analogy between the early conformational changes in RNA folding and the "burst phase" changes and molten globule formation in protein folding.     

Immunogenicity of peptide-vaccine candidates predicted by molecular dynamics simulations

We present an in silico, structure-based approach for design and evaluation of conformationally restricted peptide-vaccines. In particular, we designed four cyclic peptides of ten or 11 residues mimicking the crystallographically observed beta-turn conformation of a predicted immunodominant loop of PorA from Neisseria meningitidis. Conformational correctness and stability of the peptide designs, as evaluated by molecular dynamics simulations, correctly predicted the immunogenicity of the peptides. We observed a peptide-induced functional antibody response that, remarkably, exceeded the response induced by the native protein in outer membrane vesicles, without losing specificity for related strains. The presented approach offers tools for a priori design and selection of peptide-vaccine candidates with full biological activity. This approach could be widely applicable: to outer membrane proteins of Gram-negative bacteria, and to other epitopes in a large range of pathogens.     

Sporophytic self-incompatibility in Senecio squalidus L (Asteraceae)--the search for S

Senecio squalidus (Oxford Ragwort) is being used as a model species to study the genetics and molecular genetics of self-incompatibility (SI) in the Asteraceae. S. squalidus has a strong system of sporophytic SI (SSI) and populations within the UK contain very few S alleles probably due to a population bottleneck experienced on its introduction to the UK. The genetic control of SSI in S. squalidus is complex and may involve a second locus epistatic to S. Progress towards identifying the female determinant of SSI in S. squalidus is reviewed here. Research is focused on plants carrying two defined S alleles, S(1) and S(2). S(2) is dominant to S(1) in pollen and stigma. RT-PCR was used to amplify three SRK-like cDNAs from stigmas of S(1)S(2) heterozygotes, but the expression patterns of these cDNAs suggest that they are unlikely to be directly involved in SI or pollen-stigma interactions in contrast to SSI in the Brassicaceae. Stigma-specific proteins associated with the S(1) allele and the S(2) allele have been identified using isoelectric focusing and these proteins have been designated SSP1 (Stigma S-associated Protein 1) and SSP2. SSP1 and SSP2 cDNAs have been cloned by 3' and 5' RACE and shown to be allelic forms of the same gene, SSP. The expression of SSP and its linkage to the S locus are currently being investigated. Initial results show SSP to be expressed exclusively in stigmas and developmentally regulated, with maximal expression occurring at and just before anthesis when SI is fully functional, SSP expression being undetectable in immature buds. Together these data suggest that SSP is a strong candidate for a Senecio S-gene.     

Relationships among murine CD11c(high) dendritic cell subsets as revealed by baseline gene expression patterns

The functional relationships and properties of different subtypes of dendritic cells (DC) remain largely undefined. To better characterize these cells, we used global gene analysis to determine gene expression patterns among murine CD11c(high) DC subsets. CD4(+), CD8alpha(+), and CD8alpha(-) CD4(-) (double negative (DN)) DC were purified from spleens of normal C57/BL6 mice and analyzed using Affymetrix microarrays. The CD4(+) and CD8alpha(+) DC subsets showed distinct basal expression profiles differing by >200 individual genes. These included known DC subset markers as well as previously unrecognized, differentially expressed CD Ags such as CD1d, CD5, CD22, and CD72. Flow cytometric analysis confirmed differential expression in nine of nine cases, thereby validating the microarray analysis. Interestingly, the microarray expression profiles for DN cells strongly resembled those of CD4(+) DC, differing from them by <25 genes. This suggests that CD4(+) and DN DC are closely related phylogenetically, whereas CD8alpha(+) DC represent a more distant lineage, supporting the historical distinction between CD8alpha(+) and CD8alpha(-) DC. However, staining patterns revealed that in contrast to CD4(+) DC, the DN subset is heterogeneous and comprises at least two subpopulations. Gene Ontology and literature mining analyses of genes expressed differentially among DC subsets indicated strong associations with immune response parameters as well as cell differentiation and signaling. Such associations offer clues to possible unique functions of the CD11c(high) DC subsets that to date have been difficult to define as rigid distinctions.     

Expression of death-associated protein kinase and recruitment to the tumor necrosis factor signaling pathway following brief seizures

Death-associated protein (DAP) kinase is calcium-regulated and known to function downstream of death receptors, prompting us to examine its role in the mechanism of seizure-induced neuronal death. Brief seizures were focally evoked in rats, eliciting neuronal death within the CA3 subfield of the hippocampus, and to a lesser extent, cortex. Western blotting confirmed expression of DAP kinase within hippocampus and cortex at the predicted weight of approximately 160 kDa. Immunohistochemistry revealed seizures triggered a significant increase in numbers of DAP kinase-expressing cells within CA3 and cortex, without affecting cell counts within seizure-resistant CA2 or the dentate gyrus. Numbers of DAP kinase-expressing cells were increased in relation to specific patterns of injury-causing seizure activity, electrographically defined. Seizures caused an early increase in DAP kinase binding to actin, and association with calmodulin. Co-immunoprecipitation studies also revealed seizures triggered binding of DAP kinase to the tumor necrosis factor receptor 1 and the Fas-associated death domain protein, commensurate with caspase-8 proteolysis. In contrast, within surviving fields of the hippocampus, DAP kinase interacted with the molecular chaperone 14-3-3. These data suggest DAP kinase is involved in the molecular pathways activated during seizure-induced neuronal death.     

Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh7 cells

Progress toward development of better therapies for the treatment of hepatitis C virus (HCV) infection has been hampered by poor understanding of HCV biology and the lack of biological assays suitable for drug screening. Here we describe a powerful HCV replication system that employs HCV replicons expressing the beta-lactamase reporter (bla replicons) and subpopulations of Huh7 cells that are more permissive (or "enhanced") to HCV replication than na?ve Huh7 cells. Enhanced cells represent a small fraction of permissive cells present among na?ve Huh7 cells that is enriched during selection with replicons expressing the neomycin phosphotransferase gene (neo replicons). The level of permissiveness of cell lines harboring neo replicons can vary greatly, and the enhanced phenotype is usually revealed upon removal of the neo replicon with inhibitors of HCV replication. Replicon removal is responsible for increased permissiveness, since this effect could be reproduced either with alpha interferon or with an HCV NS5B inhibitor. Moreover, adaptive mutations present in the replicon genome used during selection do not influence the permissiveness of the resulting enhanced-cell population, suggesting that the mechanisms governing the permissiveness of enhanced cells are independent from viral adaptation. Because the beta-lactamase reporter allows simultaneous quantitation of replicon-harboring cells and reporter activity, it was possible to investigate the relationship between genome replication activity and the frequency with which transfected genomes can establish persistent replication. Our study demonstrates that differences in the replication potential of the viral genome are manifested primarily in the frequency with which persistent replication is established but modestly affect the number of replicons observed per replicon-harboring cell. Replicon copy number was found to vary over a narrow range that may be defined by a minimal number required for persistent maintenance and a maximum that is limited by the availability of essential host factors.     

Insights into the effects on metal binding of the systematic substitution of five key glutamate ligands in the ferritin of Escherichia coli

Ferritins are nearly ubiquitous iron storage proteins playing a fundamental role in iron metabolism. They are composed of 24 subunits forming a spherical protein shell encompassing a central iron storage cavity. The iron storage mechanism involves the initial binding and subsequent O2-dependent oxidation of two Fe2+ ions located at sites A and B within the highly conserved dinuclear "ferroxidase center" in individual subunits. Unlike animal ferritins and the heme-containing bacterioferritins, the Escherichia coli ferritin possesses an additional iron-binding site (site C) located on the inner surface of the protein shell close to the ferroxidase center. We report the structures of five E. coli ferritin variants and their Fe3+ and Zn2+ (a redox-stable alternative for Fe2+) derivatives. Single carboxyl ligand replacements in sites A, B, and C gave unique effects on metal binding, which explain the observed changes in Fe2+ oxidation rates. Binding of Fe2+ at both A and B sites is clearly essential for rapid Fe2+ oxidation, and the linking of FeB2+ to FeC2+ enables the oxidation of three Fe2+ ions. The transient binding of Fe2+ at one of three newly observed Zn2+ sites may allow the oxidation of four Fe2+ by one dioxygen molecule.     

A novel hypoxia-inducible factor-independent hypoxic response regulating mammalian target of rapamycin and its targets

Hypoxia triggers a reversible inhibition of protein synthesis thought to be important for energy conservation in O2-deficient environments. The mammalian target of rapamycin (mTOR) pathway integrates multiple environmental cues to regulate translation in response to nutrient availability and stress, suggesting it as a candidate for O2 regulation. We show here that hypoxia rapidly and reversibly triggers hypophosphorylation of mTOR and its effectors 4E-BP1, p70S6K, rpS6, and eukaryotic initiation factor 4G. Hypoxic regulation of these translational control proteins is dominant to activation via multiple distinct signaling pathways such as insulin, amino acids, phorbol esters, and serum and is independent of Akt/protein kinase B and AMP-activated protein kinase phosphorylation, ATP levels, ATP:ADP ratios, and hypoxia-inducible factor-1 (HIF-1). Finally, hypoxia appears to repress phosphorylation of translational control proteins in a manner analogous to rapamycin and independent of phosphatase 2A (PP2A) activity. These data demonstrate a new mode of regulation of the mTOR pathway and position this pathway as a powerful point of control by O2 of cellular metabolism and energetics.