Human DNA helicase IV is nucleolin, an RNA helicase modulated by phosphorylation

The cDNA encoding human DNA helicase IV (HDH IV), a 100-kDa protein which unwinds DNA in the 5′ to 3′ direction with respect to the bound strand, was cloned and sequenced. It was found to be identical to the human cDNA encoding nucleolin, a ubiquitous eukaryotic protein essential for pre-ribosome assembly. HDH IV/nucleolin can unwind RNA-RNA duplexes, as well as DNA-DNA and DNA-RNA duplexes. Phosphorylation of HDH IV/nucleolin by cdc2 kinase and casein kinase II enhanced its unwinding activity in an additive way. The Gly-rich C-terminal domain possesses a limited ATP-dependent duplex-unwinding activity which contributes to the helicase activity of HDH IV/nucleolin.     

The effect of seasonality on oxidative metabolism in Nacella (Patinigera) magellanica

We studied the seasonal variation on aerobic metabolism and the response of oxidative stress parameters in the digestive glands of the subpolar limpet Nacella (P.) magellanica. Sampling was carried out from July (winter) 2002 to July 2003 in Beagle Channel, Tierra del Fuego, Argentina. Whole animal respiration rates increased in early spring as the animals spawned and remained elevated throughout summer and fall (winter: 0.09 ± 0.02 μmol O₂ h^− 1 g^− 1; summer: 0.31 ± 0.06 μmol O₂ h^− 1 g^− 1). Oxidative stress was assessed at the hydrophilic level as the ascorbyl radical content / ascorbate content ratio (A / AH⁻). The A / AH⁻ ratio showed minimum values in winter (3.7 ± 0.2 10^− 5 AU) and increased in summer (18 ± 5 10^− 5 AU). A similar pattern was observed for lipid radical content (122 ± 29 pmol mg^− 1 fresh mass [FW] in winter and 314 ± 45 pmol mg^− 1 FW in summer), iron content (0.99 ± 0.07 and 2.7 ± 0.6 nmol mg^− 1 FW in winter and summer, respectively) and catalase activity (2.9 ± 0.2 and 7 ± 1 U mg^− 1 FW in winter and summer, respectively). Since nitrogen derived radicals are thought to be critically involved in oxidative metabolism in cells, nitric oxide content was measured and a significant difference in the content of the Fe–MGD–NO adduct in digestive glands from winter and summer animals was observed. Together, the data indicate that both oxygen and nitrogen radical generation rates in N. (P.) magellanica are strongly dependent on season.     

The NatA Acetyltransferase Couples Sup35 Prion Complexes to the [PSI+] Phenotype

Protein-only (prion) epigenetic elements confer unique phenotypes by adopting alternate conformations that specify new traits. Given the conformational flexibility of prion proteins, protein-only inheritance requires efficient self-replication of the underlying conformation. To explore the cellular regulation of conformational self-replication and its phenotypic effects, we analyzed genetic interactions between [PSI⁺], a prion form of the S. cerevisiae Sup35 protein (Sup35^[PSI+]), and the three N^α-acetyltransferases, NatA, NatB, and NatC, which collectively modify ~50% of yeast proteins. Although prion propagation proceeds normally in the absence of NatB or NatC, the [PSI⁺] phenotype is reversed in strains lacking NatA. Despite this change in phenotype, [PSI⁺] NatA mutants continue to propagate heritable Sup35^[PSI+]. This uncoupling of protein state and phenotype does not arise through a decrease in the number or activity of prion templates (propagons) or through an increase in soluble Sup35. Rather, NatA null strains are specifically impaired in establishing the translation termination defect that normally accompanies Sup35 incorporation into prion complexes. The NatA effect cannot be explained by the modification of known components of the [PSI⁺] prion cycle including Sup35; thus, novel acetylated cellular factors must act to establish and maintain the tight link between Sup35^[PSI+] complexes and their phenotypic effects.     

A duplex real-time PCR assay for detection of drug-induced mitochondrial DNA depletion in HepG2 cells

Screening of drug-induced mitochondrial DNA (mtDNA) depletion during early preclinical drug development is of major interest. Here we describe the establishment of a novel duplex calibrator-normalized real-time polymerase chain reaction (PCR) assay for rapid and reliable quantification of mtDNA in HepG2 cells. This assay involves quantification of an mtDNA target gene (cytochrome b) relative to a nuclear DNA (nDNA) reference gene (β-actin) in one tube. The assay was evaluated for its precision, linearity, and reproducibility, and reliable detection of mtDNA depletion was demonstrated. Using this novel real-time PCR assay, drug-induced mtDNA depletion could be accurately detected.     

Impact of the FcgammaII-receptor on quartz uptake and inflammatory response by alveolar macrophages

The inflammatory response following particle inhalation is described as a key event in the development of lung diseases, e.g., fibrosis and cancer. The essential role of alveolar macrophages (AM) in the pathogenicity of particles through their functions in lung clearance and mediation of inflammation is well known. However, the molecular mechanisms and direct consequences of particle uptake are still unclear. Inhibition of different classic phagocytosis receptors by flow cytometry shows a reduction of the dose-dependent quartz particle (DQ12) uptake in the rat AM cell line NR8383. Thereby the strongest inhibitory effect was observed by blocking the FcgammaII-receptor (FcgammaII-R). Fluorescence immunocytochemistry, demonstrating FcgammaII-R clustering at particle binding sites as well as transmission electron microscopy, visualizing zippering mechanism-like morphological changes, confirmed the role of the FcgammaII-R in DQ12 phagocytosis. FcgammaII-R participation in DQ12 uptake was further strengthened by the quartz-induced activation of the Src-kinase Lyn, the phospho-tyrosine kinases Syk (spleen tyrosine kinase) and PI3K (phosphatidylinositol 3-kinase), as shown by Western blotting. Activation of the small GTPases Rac1 and Cdc42, shown by immunoprecipitation, as well as inhibition of tyrosine kinases, GTPases, or Rac1 provided further support for the role of the FcgammaII-R. Consistent with the uptake results, FcgammaII-R activation with its specific ligand caused a similar generation of reactive oxygen species and TNF-alpha release as observed after treatment with DQ12. In conclusion, our results indicate a major role of FcgammaII-R and its downstream signaling cascade in the phagocytosis of quartz particles in AM as well as in the associated generation and release of inflammatory mediators.     

Intraspecific genetic diversity of Pyrenophora tritici-repentis (Died.) Drechs. (Drechslera tritici-repentis[Died.] Shoem.) detected by random amplified polymorphic DNA assays

Abstract

Random amplified polymorphic DNAs (RAPDs) were used to study the genetic variation of Pyrenophora tritici-repentis isolates causing wheat tan spot. Two independent experiments were conducted in 2002 – 2003. In 2002, 40 isolates collected in Russia (Krasnodar region, Bashkiria), Germany, and the Czech Republic were studied and 35 unique RAPD genotypes were identified. Most of the genetic variation (72%) was observed within populations and 28% between them. In 2003, 69 new isolates from Russia (Dagestan, North Osetia, Bashkiria), Germany, and the Czech Republic were studied and 47 unique RAPD genotypes were identified. As in 2002, most of the genetic variation (75%) was observed within populations and 25% between them. Total gene diversity in each group ranged from 0.67 – 1.00 for 2002 and was 1.00 for 2003. The average gene diversity was estimated between 0.13 and 0.20 in 2002 and between 0.07 and 0.18 in 2003. A dendrogramme based on genetic distances between isolates illustrates that the variation is distributed on a small scale (0.3 – 4.0%). Estimated F_ST values and clustering of isolates on dendrogrammes suggest that groups of isolates from Bashkiria and groups of isolates from Dagestan and North Osetia are separated from others and may be considered as different geographical populations. No clear differentiation between isolates from other sites was revealed.     

Residues Arg283, Arg285, and Ile287 in the Nucleotide Binding Pocket of Bovine Viral Diarrhea Virus NS5B RNA Polymerase Affect Catalysis and Fidelity

Residues Arg283, Arg285, and Ile287 are highly conserved amino acids in bovine viral diarrhea virus RNA polymerase (BVDV RdRp) and RdRps from related positive-strand RNA viruses. This motif is an important part of the binding pocket for the nascent RNA base pair during initiation and elongation. We found that replacement of the arginines with alanines or more conserved lysines or replacement of isoleucine with alanine or valine alters the ability of the mutant RdRps to incorporate ribonucleotides efficiently. The reduced RdRp activity stems from both decreased ribonucleotide binding and decreased catalytic efficiency in both primer-dependent and de novo initiation, as shown by kinetic studies. In line with other studies on flaviviral RdRps, our data suggest that Arg283 and Ile287 may be implicated in ribonucleotide binding and positioning of the template base in the active site. Arg285 appears to be involved directly in the selection of cognate nucleotide. The findings for Arg285 and Ile287 mutants also agree with similar data from picornavirus RdRps.     

Continuous production of erythrulose using transketolase in a membrane reactor

Transketolase can be used for synthesis of chiral intermediates and carbohydrates. However the enzyme is strongly deactivated by the educts. This deactivation depends on the reactor employed. An enzyme membrane reactor allows the continuous production of L-erythrulose with high conversion and stable operational points. A productivity (space-time yield) of 45g L d was reached.