Über die Blattausscheidungen des Wermuts und ihre Wirkung auf Andere Pflanzen

Ohne ZusammenfassungMit 5 Textabbildungen (7 Einzelbildern).     

Steroid biosynthesis in prokaryotes: identification of myxobacterial steroids and cloning of the first bacterial 2,3(S)-oxidosqualene cyclase from the myxobacterium Stigmatella aurantiaca

Steroids, such as cholesterol, are synthesized in almost all eukaryotic cells, which use these triterpenoid lipids to control the fluidity and flexibility of their cell membranes. Bacteria rarely synthesize such tetracyclic compounds but frequently replace them with a different class of triterpenoids, the pentacyclic hopanoids. The intriguing mechanisms involved in triterpene biosynthesis have attracted much attention, resulting in extensive studies of squalene-hopene cyclase in bacteria and (S)-2,3-oxidosqualene cyclases in eukarya. Nevertheless, almost nothing is known about steroid biosynthesis in bacteria. Only three steroid-synthesizing bacterial species have been identified before this study. Here, we report on a variety of sterol-producing myxobacteria. Stigmatella aurantiaca is shown to produce cycloartenol, the well-known first cyclization product of steroid biosynthesis in plants and algae. Additionally, we describe the cloning of the first bacterial steroid biosynthesis gene, cas, encoding the cycloartenol synthase (Cas) of S. aurantiaca. Mutants of cas generated via site-directed mutagenesis do not produce the compound. They show neither growth retardation in comparison with wild type nor any increase in ethanol sensitivity. The protein encoded by cas is most similar to the Cas proteins from several plant species, indicating a close evolutionary relationship between myxobacterial and eukaryotic steroid biosynthesis.     

A G1‐like state allows HIV‐1 to bypass SAMHD1 restriction in macrophages

An unresolved question is how HIV‐1 achieves efficient replication in terminally differentiated macrophages despite the restriction factor SAMHD1. We reveal inducible changes in expression of cell cycle‐associated proteins including MCM2 and cyclins A, E, D1/D3 in macrophages, without evidence for DNA synthesis or mitosis. These changes are induced by activation of the Raf/MEK/ERK kinase cascade, culminating in upregulation of CDK1 with subsequent SAMHD1 T592 phosphorylation and deactivation of its antiviral activity. HIV infection is limited to these G1‐like phase macrophages at the single‐cell level. Depletion of SAMHD1 in macrophages decouples the association between infection and expression of cell cycle‐associated proteins, with terminally differentiated macrophages becoming highly susceptible to HIV‐1. We observe both embryo‐derived and monocyte‐derived tissue‐resident macrophages in a G1‐like phase at frequencies approaching 20%, suggesting how macrophages sustain HIV‐1 replication in vivo. Finally, we reveal a SAMHD1‐dependent antiretroviral activity of histone deacetylase inhibitors acting via p53 activation. These data provide a basis for host‐directed therapeutic approaches aimed at limiting HIV‐1 burden in macrophages that may contribute to curative interventions.     

Confirmation of quantitative trait loci affecting fatness in chickens

In this report we describe the analysis of an advanced intercross line (AIL) to confirm the quantitative trait locus (QTL) regions found for fatness traits in a previous study. QTL analysis was performed on chromosomes 1, 3, 4, 15, 18, and 27. The AIL was created by random intercrossing in each generation from generation 2 (G₂) onwards until generation 9 (G₉) was reached. QTL for abdominal fat weight (AFW) and/or percentage abdominal fat (AF%) on chromosomes 1, 3 and 27 were confirmed in the G₉ population. In addition, evidence for QTL for body weight at the age of 5 (BW5) and 7 (BW7) weeks and for the percentage of intramuscular fat (IF%) were found on chromosomes 1, 3, 15, and 27. Significant evidence for QTL was detected on chromosome 1 for BW5 and BW7. Suggestive evidence was found on chromosome 1 for AFW, AF% and IF%, on chromosome 15 for BW5, and on chromosome 27 for AF% and IF%. Furthermore, evidence on the chromosome-wise level was found on chromosome 3 for AFW, AF%, and BW7 and on chromosome 27 for BW5. For chromosomes 4 and 18, test statistics did not exceed the significance threshold.     

Biosynthesis of C9-aldehydes in the moss Physcomitrella patens

After wounding, the moss Physcomitrella patens emits fatty acid derived volatiles like octenal, octenols and (2E)-nonenal. Flowering plants produce nonenal from C18-fatty acids via lipoxygenase and hydroperoxide lyase reactions, but the moss exploits the C20 precursor arachidonic acid for the formation of these oxylipins. We describe the isolation of the first cDNA (PpHPL) encoding a hydroperoxide lyase from a lower eukaryotic organism. The physiological pathway allocation and characterization of a downstream enal-isomerase gives a new picture for the formation of fatty acid derived volatiles from lower plants. Expression of a fusion protein with a yellow fluorescent protein in moss protoplasts showed that PpHPL was found in clusters in membranes of plastids. PpHPL can be classified as an unspecific hydroperoxide lyase having a substrate preference for 9-hydroperoxides of C18-fatty acids but also the predominant substrate 12-hydroperoxy arachidonic acid is accepted. Feeding experiments using arachidonic acid show an increase in the 12-hydroperoxide being metabolized to C8-aldehydes/alcohols and (3Z)-nonenal, which is rapidly isomerized to (2E)-nonenal. PpHPL knock out lines failed to emit (2E)-nonenal while formation of C8-volatiles was not affected indicating that in contrast to flowering plants, PpHPL is only involved in formation of a specific subset of volatiles.     

Design and evaluation of novel bivalent thrombin inhibitors based on amidinophenylalanines.

Two bivalent thrombin inhibitors were synthesized, which consist of a benzamidine-based active-site-blocking segment, a fibrinogen recognition exosite inhibitor and a peptidic linker connecting these fragments. BZA-1 hirulog contains an Nalpha-(2-naphthylsulfonyl)-S-3-amidinophenylalanyl-is onipecotic acid residue connected via the carboxyl group to the linker segment. The active-site-directed moiety of BZA-2 hirulog [Nalpha-(2-naphthylsulfonyl-glutamyl)-R-4-amidinophenylal anyl-piperid ide] was coupled to the linker via the side chain of the glutamic acid. Both BZA-hirulogs contain almost identical linker-exo site inhibitor parts, except for the substitution of a glycine as the first linker residue in BZA-1 hirulog by a gamma-amino butyric acid in BZA-2 hirulog, thus increasing flexibility and linker length by two additional atoms. BZA-1 hirulog showed moderate potency (Ki = 0. 50 +/- 0.14 nM), while BZA-2 hirulog was characterized as a slow, tight binding inhibitor of thrombin (Ki = 0.29 +/- 0.08 pM). The stability in human plasma of both analogs was strongly improved compared with hirulog-1. For BZA-2 hirulog a significantly reduced plasma clearance was observed after intravenous injection in rats compared with BZA-1 hirulog and hirulog-1. The X-ray structure of the BZA-2 hirulog in complex with human alpha-thrombin was solved and confirmed the expected bivalent binding mode.     

Differential effects of CpG DNA on IFN-beta induction and STAT1 activation in murine macrophages versus dendritic cells: alternatively activated STAT1 negatively regulates TLR signaling in macrophages

Classical STAT1 activation in response to TLR agonists occurs by phosphorylation of the Y701 and S727 residues through autocrine type I IFN signaling and p38 MAPK signaling, respectively. In this study, we report that the TLR9 agonist CpG DNA induced Ifn-beta mRNA, as well as downstream type I IFN-dependent genes, in a MyD88-dependent manner in mouse myeloid dendritic cells. This pathway was required for maximal TNF and IL-6 secretion, as well as expression of cell surface costimulatory molecules. By contrast, neither A- nor B-type CpG-containing oligonucleotides induced Ifn-beta in mouse bone marrow-derived macrophages (BMM) and a CpG-B oligonucleotide did not induce IFn-beta in the macrophage-like cell line, J774. In BMM, STAT1 was alternatively activated (phosphorylated on S727, but not Y701), and was retained in the cytoplasm in response to CpG DNA. CpG DNA responses were altered in BMM from STAT1(S727A) mice; Il-12p40 and Cox-2 mRNAs were more highly induced, whereas Tlr4 and Tlr9 mRNAs were more repressed. The data suggest a novel inhibitory function for cytoplasmic STAT1 in response to TLR agonists that activate p38 MAPK but do not elicit type I IFN production. Indeed, the TLR7 agonist, R837, failed to induce Ifn-beta mRNA and consequently triggered STAT1 phosphorylation on S727, but not Y701, in human monocyte-derived macrophages. The differential activation of Ifn-beta and STAT1 by CpG DNA in mouse macrophages vs dendritic cells provides a likely mechanism for their divergent roles in priming the adaptive immune response.