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Non-sense-mediated mRNA decay (NMD) is a mechanism of translation-dependent mRNA surveillance in eukaryotes: it degrades mRNAs with premature termination codons (PTCs) and contributes to cellular homeostasis by downregulating a number of physiologically important mRNAs. In the NMD pathway, Upf proteins, a set of conserved factors of which Upf1 is the central regulator, recruit decay enzymes to promote RNA cleavage. In mammals, the degradation of PTC-containing mRNAs is triggered by the exon-junction complex (EJC) through binding of its constituents Upf2 and Upf3 to Upf1. The complex formed eventually induces translational repression and recruitment of decay enzymes. Mechanisms by which physiological mRNAs are targeted by the NMD machinery in the absence of an EJC have been described but still are discussed controversially. Here, we report that the DEAD box proteins Ddx5/p68 and its paralog Ddx17/p72 also bind the Upf complex by physical interaction with Upf3, thereby interfering with the binding of EJC. By activating the NMD machinery, Ddx5 is shown to regulate the expression of its own, Ddx17 and Smg5 mRNAs. For NMD triggering, the adenosine triphosphate-binding activity of Ddx5 and the 3'-untranslated region of substrate mRNAs are essential.

To investigate correlates for the well-known impaired response of haemodialysis-patients to a variety of recommended vaccinations, the induction of antigen-specific cellular and humoral immunity was characterised after influenza-vaccination in two following seasons where the identical vaccine-composition was used. Influenza-specific T-cells were flow-cytometrically characterised from whole blood of 24 healthy controls and 26 haemodialysis-patients by proliferation-assays, induction of IFN-γ and TNF-α, and maturation markers. Antibody-titres were quantified using ELISA and hemagglutination-inhibition test. Influenza-specific CD4 T-cells were recently activated CD45RO+/CD27+ Th1-cells. Specific T-cell frequencies significantly increased 1-2 weeks after the first vaccination in both controls (mean increase by 0.50±0.64%, max: 3.01%) and haemodialysis-patients (by 0.55±0.71%, max: 3.44%). Thereafter, T-cell levels continuously decreased to pre-vaccination levels within approximately 7 weeks, whereas antibody-titres were more stable over time. By 6 months, haemodialysis-patients had significantly lower precursor-frequencies of proliferating influenza-specific memory T-cells (p=0.006). In the following season, memory-maintenance in immunocompetent individuals led to a significantly less pronounced increase in cellular immunity after re-vaccination (by only 0.12±0.09%, p=0.003), whereas the vaccine induced a strong increase in a second group of vaccination-naïve controls. Of note, haemodialysis-patients responded like vaccination-naïve individuals, as they showed a strong increase in cellular immunity after re-vaccination that was as pronounced as in the year before. In conclusion, the less pronounced T-cell increase after re-vaccination in controls may indicate maintenance of sufficient immunological memory. In contrast, the more rapid loss of proliferating cells in haemodialysis-patients may represent a sign of relative immunodeficiency and contribute to an increased incidence of recurrent infectious complications., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Antigen-specific antibodies are well characterized after vaccination with pandemic H1N1 or seasonal influenza vaccines. However, knowledge on cellular immunity toward pandemic H1N1 after vaccination and infection and cross-reactivities toward seasonal antigens is limited. Nineteen individuals were vaccinated with the pandemic H1N1 vaccine. Among those, ten had been prevaccinated against seasonal influenza. CD4(+) T cells specific for pandemic H1N1 and for seasonal vaccine, and antibodies were monitored using flow cytometry and ELISA/neutralization assays, respectively. In addition, seven patients with acute pandemic influenza infection were analyzed. Pandemic H1N1 vaccination induced a strong 4.63-fold (IQR 4.16) increase in antigen-specific CD4(+) T cells that was more pronounced in individuals not prevaccinated with seasonal influenza (p = 0.01). T-cell levels toward seasonal vaccine concomitantly rose by 2.71-fold (IQR 2.26). Likewise, prevaccination with seasonal influenza induced a less pronounced increase in specific antibodies. Influenza-specific T cells in vaccinees had a Th1 phenotype mainly coexpressing IFN-γ and IL-2, whereas patients with active pandemic influenza showed a shift toward cells predominantly expressing IFN-γ. In conclusion, T cells toward seasonal influenza antigens cross-react with pandemic H1N1 antigens and affect induction of specific T cells after pandemic influenza vaccination. In addition, the cytokine patterns of specific T cells during acute H1N1 infection and after vaccination differ, and the predominantly dual-positive cytokine profile of vaccine-induced T cells suggests sufficient functionality to confer successful virus control., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mitochondrial cytochromes P450 are essential for biosynthesis of steroid hormones, vitamin D and bile acids. In mammals, the electrons needed for these reactions are provided via adrenodoxin and adrenodoxin reductase (AdR). Recently, Schizosaccharomyces pombe was introduced as a new host for the functional expression of human mitochondrial steroid hydroxylases without the coexpression of their natural redox partners. This fact qualifies S. pombe for the biotechnological production of steroids and for application as inhibitor test organism of heterologously expressed cytochromes P450. In this paper, we present evidence that the S. pombe ferredoxin reductase, arh1, and ferredoxin, etp1fd provide mammalian class I cytochromes P450 with reduction equivalents. The recombinant reductase showed an unusual weak binding of flavin adenine dinucleotide (FAD), which was mastered by modifying the FAD-binding region by site-directed mutagenesis yielding a stable holoprotein. The modified reductase arh1_A18G displayed spectroscopic characteristics similar to AdR and was shown to be capable of accepting electrons with no evident preference for NADH or NADPH, respectively. Arh1_A18G can substitute for AdR by interacting not only with its natural redox partner etp1fd but also with the mammalian homolog adrenodoxin. Cytochrome P450-dependent substrate conversion with all combinations of the mammalian and yeast redox proteins was evaluated in a reconstituted system.

The simian virus 40 large tumour-antigen (T antigen) DNA helicase is a hexameric structure; it has been proposed that, in viral DNA replication, two of these hexamers are combined to form a bipartite holoenzyme that acts concurrently at both forks of a replication bubble. In a search for structural components of this helicase complex, we have identified nucleolin as a specific binding protein for the T-antigen hexamer. We show that nucleolin, in co-operation with human topoisomerase I, mediates the cohesion of the T-antigen helicase holoenzyme during plasmid unwinding. Our results provide biochemical evidence for a direct role of nucleolin in DNA replication, in addition to its known function in ribosome biogenesis. The data presented here suggest that nucleolin enables the formation of a functional 'helicase-swivelase' complex at the replication fork.

p68 and p72 are two highly related DEAD box proteins with similar biochemical activities in the nucleus of vertebrate cells; it is unknown whether they have redundant or differential in vivo functions. We report on a third member of this subfamily that is alternatively expressed from p72 mRNA. A detailed analysis of HeLa p72 mRNA was performed. It has an overall length of more than 5 kb and contains a 0.75-kb 5'-untranslated region and a 3'-untranslated region of 2.5 kb. Its open reading frame extends to nucleotide -243 upstream of the first in-frame AUG (A in the AUG triplet is +1) which serves as the p72 translation initiator codon. We provide evidence that alternative translation at a non-AUG within the extra coding region of this mRNA yields an 82-kDa protein (p82). Immunological studies substantiate that p82 is a naturally existing p72 variant and that both proteins are expressed at similar concentrations. p82 purified from HeLa cells is an ATP-dependent RNA helicase with biochemical properties almost identical to those of p72.