Chemo-enzymatically induced copolymerization of phenolics with acrylate compounds

Initiation of copolymerization of lignin-like phenolic and acrylic compounds by the phenoloxidase laccase (EC 1.10.3.2) and a peroxide species (t-butylhydroperoxide, t-BHP) was compared to a Fenton-like system (ferrous ion, t-BHP). Initially, the relative activity of laccase towards different phenolic compounds and the optimum pH of some characteristic phenolics were determined. The polymer yield and the average molecular weight (Mw) of chemo-enzymatically produced polymers were dependent both on the type of each phenolic tested and on the phenol/monomer ratio. Furthermore, the success of copolymerization of the phenolics was dependent both on their redox potential and on the type of acrylic monomer applied. The extent of phenol incorporation into the polymer chain was enhanced by the presence of laccase in the reaction mixture and was significantly higher than in polymerization initiated by a Fenton-like reaction.     

Kinetic Competition between RNA Polymerase II and Sen1-Dependent Transcription Termination

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Highlights? Fast Pol II elongation rate leads to Sen1-dependent termination defects ? Deletion of Rrp6 reveals a noncoding RNA termination window sensitive to Pol II rate ? Slow Pol II rate promotes earlier Sen1 termination events, rescues sen1 defects ? Fast Pol II rate promotes later Sen1 termination events, worsens sen1 defects     

Influenza A virus NS1 protein activates the PI3K/Akt pathway to mediate antiapoptotic signaling responses

Recently we have shown that influenza A virus infection leads to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and that this cellular reaction is dependent on the expression of the viral nonstructural protein 1 (NS1). These data also suggested that PI3K activation confers a virus-supporting activity at intermediate stages of the infection cycle. So far it is not known which process is regulated by the kinase that supports virus replication. It is well established that upon infection with influenza A virus, the expression of the viral NS1 keeps the induction of beta interferon and the apoptotic response within a tolerable limit. On a molecular basis, this activity of NS1 has been suggested to preclude the activation of cellular double-stranded RNA receptors as well as impaired modulation of mRNA processing. Here we present a novel mode of action of the NS1 protein to suppress apoptosis induction. NS1 binds to and activates PI3K, which results in the activation of the PI3K effector Akt. This leads to a subsequent inhibition of caspase 9 and glycogen synthase-kinase 3beta and limitation of the virus-induced cell death program. Thus, NS1 not only blocks but also activates signaling pathways to ensure efficient virus replication.     

Processus and recessus adhaerentes: giant adherens cell junction systems connect and attract human mesenchymal stem cells

Substrate-adherent cultured cells derived from human bone marrow or umbilical cord blood (“mesenchymal stem cells”) are of special interest for regenerative medicine. We report that such cells, which can display considerable heterogeneity with respect to their cytoskeletal protein complement, are often interconnected by special tentacle-like cell processes contacting one or several other cells. These processus adhaerentes, studded with many (usually small) puncta adhaerentia and varying greatly in length (up to more than 400 μm long), either contact each other in the intercellular space (“ET touches”) or insert in a tight-fitting manner into deep plasma membrane invaginations (recessus adhaerentes), thus forming a novel kind of long (up to 50 μm) continuous cuff-like junction (manubria adhaerentia). The cell processes contain an actin microfilament core that is stabilized with ezrin, α-actinin, and myosin and accompanied by microtubules, and their adhering junctions are characterized by a molecular complement comprising the transmembrane glycoproteins N-cadherin and cadherin-11, in combination with the cytoplasmic plaque proteins α- and β-catenin, together with p120^ctn, plakoglobin, and afadin. The processes are also highly dynamic and rapidly foreshorten as cell colonies approach a denser state of cell packing. These structures are obviously able to establish cell-cell connections, even over long distances, and can form deep-rooted and tight cell-cell adhesions. The possible relationship to similar cell processes in the embryonic primary mesenchyme and their potential in cell sorting and tissue formation processes in the body are discussed. Patrick Wuchter and Judit Boda-Heggemann contributed equally to this work. This work was supported in part by the Joachim Siebeneicher Foundation, by the Deutsche Forschungsgemeinschaft (grant HO 914/4-1 to A.D.H.), by the Deutsche Krebshilfe (grant 10-2049-Fr 1 to W.W.F), and by a grant from the German Ministry for Research and Technology in the special funding program “Regenerative Medicine” (“START-MSC”, grants to A.D.H. and W.W.F).     

Differential inhibition of CYP17A1 and CYP21A2 activities by the P450 oxidoreductase mutant A287P

P450 oxidoreductase (POR) has a pivotal role in facilitating electron transfer from nicotinamide adenine dinucleotide phosphate to microsomal cytochrome P450 (CYP) enzymes, including the steroidogenic enzymes CYP17A1 and CYP21A2. Mutations in POR have been shown recently to cause congenital adrenal hyperplasia with apparent combined CYP17A1 and CYP21A2 deficiency that comprises a variable clinical phenotype, including glucocorticoid deficiency, ambiguous genitalia, and craniofacial malformations. To dissect structure-function relationships potentially explaining this phenotypic diversity, we investigated whether specific POR mutations have differential effects on CYP17A1 and CYP21A2. We compared the impact of missense mutations encoding for single amino acid changes in three distinct regions of the POR molecule: 1), Y181D and H628P close to the central electron transfer area, 2) S244C located within the hinge close to the flavin adenine dinucleotide and flavin mononucleotide domains of POR, and 3) A287P that is clearly distant from the two other regions. Functional analysis using a yeast microsomal assay with coexpression of human CYP17A1 or CYP21A2 with wild-type or mutant human POR revealed equivalent decreases in CYP17A1 and CYP21A2 activities by Y181D, H628P, and S244C. In contrast, A287P had a differential inhibitory effect, with decreased catalytic efficiency (Vmax/Km) for CYP17A1, whereas CYP21A2 retained near normal activity. In vivo analysis of urinary steroid excretion by gas chromatography/mass spectrometry in 11 patients with POR mutations showed that A287P homozygous patients had the highest corticosterone/cortisol metabolite ratios, further indicative of preferential inhibition of CYP17A1. These findings provide novel mechanistic insights into the redox regulation of human steroidogenesis. Differential interaction of POR with electron-accepting CYP enzymes may explain the phenotypic variability in POR deficiency, with additional implications for hepatic drug metabolism by POR-dependant CYP enzymes.     

The impact of nitrite and antioxidants on ultraviolet-A-induced cell death of human skin fibroblasts

Nitrite (NO(2)(-)) occurs ubiquitously in biological fluids such as blood and sweat. Ultraviolet A-induced nitric oxide formation via decomposition of cutaneous nitrite, accompanied by the production of reactive oxygen (ROS) or nitrogen species (RNS), represents an important source for NO in human skin physiology. Examining the impact of nitrite and the antioxidants glutathione (GSH), Trolox (TRL), and ascorbic acid (ASC) on UVA-induced toxicity of human skin fibroblasts (FB) we found that NO(2)(-) concentration-dependently enhances the susceptibility of FB to the toxic effects of UVA by a mechanism comprising enhanced induction of lipid peroxidation. While ASC completely protects FB cultures from UVA/NO(2)(-)-induced cell damage, GSH or TRL excessively enhances UVA/NO(2)(-)-induced cell death by a mechanism comprising nitrite concentration-dependent TRL radical formation or GSH-derived oxidative stress. Simultaneously, in the presence of GSH or TRL the mode of UVA/NO(2)(-)-induced cell death changes from apoptosis to necrosis. In summary, during photodecomposition of nitrite, ROS or RNS formation may act as strong toxic insults. Although inhibition of oxidative stress by NO and other antioxidants represents a successful strategy for protection from UVA/NO(2)(-)-induced injuries, GSH and TRL may nitrite-dependently aggravate the injurious impact by TRL or GSH radical formation, respectively.     

Seasonal release from competition explains partial migration in European moose

Partial migration, whereby a proportion of a population migrates between distinct seasonal ranges, is common throughout the animal kingdom. However, studies linking existing theoretical models of migration probability, with empirical data are lacking. The competitive release hypothesis for partial migration predicts that due to density-dependent habitat selection, the proportion of migrants increases as the relative quality and size of the seasonal range increases, but decreases with increasing migration cost and population density. To test this prediction, we developed a quantitative framework to predict the proportion of migrants, using empirical data from 545 individually GPS-marked moose Alces alces from across Fennoscandia, spanning latitudes of 56° to 68°N. Moose contracted their ranges to common and spatially limited winter areas (typically at lower elevation), but expanded them during summer due to an increase in suitable habitat (at highland ranges). As predicted from our model, a better and larger highland range relative to the lowland range corresponded to a higher proportion of migrants in an area. Quantitative predictions coupling the balance of habitat availability of seasonal ranges with the probability of migrating in a large herbivore is a necessary step towards an enhanced understanding of the mechanisms underlying migration at the population level.     

Foxp3+ Regulatory T Cells Delay Expulsion of Intestinal Nematodes by Suppression of IL-9-Driven Mast Cell Activation in BALB/c but Not in C57BL/6 Mice

Accumulating evidence suggests that IL-9-mediated immunity plays a fundamental role in control of intestinal nematode infection. Here we report a different impact of Foxp3⁺ regulatory T cells (Treg) in nematode-induced evasion of IL-9-mediated immunity in BALB/c and C57BL/6 mice. Infection with Strongyloides ratti induced Treg expansion with similar kinetics and phenotype in both strains. Strikingly, Treg depletion reduced parasite burden selectively in BALB/c but not in C57BL/6 mice. Treg function was apparent in both strains as Treg depletion increased nematode-specific humoral and cellular Th2 response in BALB/c and C57BL/6 mice to the same extent. Improved resistance in Treg-depleted BALB/c mice was accompanied by increased production of IL-9 and accelerated degranulation of mast cells. In contrast, IL-9 production was not significantly elevated and kinetics of mast cell degranulation were unaffected by Treg depletion in C57BL/6 mice. By in vivo neutralization, we demonstrate that increased IL-9 production during the first days of infection caused accelerated mast cell degranulation and rapid expulsion of S. ratti adults from the small intestine of Treg-depleted BALB/c mice. In genetically mast cell-deficient (Cpa3-Cre) BALB/c mice, Treg depletion still resulted in increased IL-9 production but resistance to S. ratti infection was lost, suggesting that IL-9-driven mast cell activation mediated accelerated expulsion of S. ratti in Treg-depleted BALB/c mice. This IL-9-driven mast cell degranulation is a central mechanism of S. ratti expulsion in both, BALB/c and C57BL/6 mice, because IL-9 injection reduced and IL-9 neutralization increased parasite burden in the presence of Treg in both strains. Therefore our results suggest that Foxp3⁺ Treg suppress sufficient IL-9 production for subsequent mast cell degranulation during S. ratti infection in a non-redundant manner in BALB/c mice, whereas additional regulatory pathways are functional in Treg-depleted C57BL/6 mice.