Complete Chloroplast Genome of the Wollemi Pine (Wollemia nobilis): Structure and Evolution

The Wollemi pine (Wollemia nobilis) is a rare Southern conifer with striking morphological similarity to fossil pines. A small population of W. nobilis was discovered in 1994 in a remote canyon system in the Wollemi National Park (near Sydney, Australia). This population contains fewer than 100 individuals and is critically endangered. Previous genetic studies of the Wollemi pine have investigated its evolutionary relationship with other pines in the family Araucariaceae, and have suggested that the Wollemi pine genome contains little or no variation. However, these studies were performed prior to the widespread use of genome sequencing, and their conclusions were based on a limited fraction of the Wollemi pine genome. In this study, we address this problem by determining the entire sequence of the W. nobilis chloroplast genome. A detailed analysis of the structure of the genome is presented, and the evolution of the genome is inferred by comparison with the chloroplast sequences of other members of the Araucariaceae and the related family Podocarpaceae. Pairwise alignments of whole genome sequences, and the presence of unique pseudogenes, gene duplications and insertions in W. nobilis and Araucariaceae, indicate that the W. nobilis chloroplast genome is most similar to that of its sister taxon Agathis. However, the W. nobilis genome contains an unusually high number of repetitive sequences, and these could be used in future studies to investigate and conserve any remnant genetic diversity in the Wollemi pine.     

Crystal structures of the Pyrococcus abyssi Sm core and its complex with RNA. Common features of RNA binding in archaea and eukarya

The Sm proteins are conserved in all three domains of life and are always associated with U-rich RNA sequences. Their proposed function is to mediate RNA-RNA interactions. We present here the crystal structures of Pyrococcus abyssi Sm protein (PA-Sm1) and its complex with a uridine heptamer. The overall structure of the protein complex, a heptameric ring with a central cavity, is similar to that proposed for the eukaryotic Sm core complex and found for other archaeal Sm proteins. RNA molecules bind to the protein at two different sites. They interact specifically inside the ring with three highly conserved residues, defining the uridine-binding pocket. In addition, nucleotides also interact on the surface formed by the N-terminal alpha-helix as well as a conserved aromatic residue in beta-strand 2 of the PA-Sm1 protein. The mutation of this conserved aromatic residue shows the importance of this second site for the discrimination between RNA sequences. Given the high structural homology between archaeal and eukaryotic Sm proteins, the PA-Sm1.RNA complex provides a model for how the small nuclear RNA contacts the Sm proteins in the Sm core. In addition, it suggests how Sm proteins might exert their function as modulators of RNA-RNA interactions.     

Oscillations of phospholipase C activity triggered by depolarization and Ca2+ influx in insulin-secreting cells

Phospholipase C (PLC) is a ubiquitous enzyme involved in the regulation of a variety of cellular processes. Its dependence on Ca2+ is well recognized, but it is not known how PLC activity is affected by physiological variations of the cytoplasmic Ca2+ concentration ([Ca2+](i)). Here, we applied evanescent wave microscopy to monitor PLC activity in parallel with [Ca2+](i) in individual insulin-secreting INS-1 cells using the phosphatidylinositol 4,5-bisphosphate- and inositol 1,4,5-trisphosphate-binding pleckstrin homology domain from PLCdelta(1) fused to green fluorescent protein (PH(PLCdelta1)-GFP) and the Ca2+ indicator fura red. In resting cells, PH(PLCdelta1)-GFP was located predominantly at the plasma membrane. Activation of PLC by muscarinic or purinergic receptor stimulation resulted in PH(PLCdelta1)-GFP translocation from the plasma membrane to the cytoplasm, detected as a decrease in evanescent wave-excited PH(PLCdelta1)-GFP fluorescence. Using this translocation as a measure of PLC activity, we found that depolarization by raising extracellular [K+] triggered activation of the enzyme. This effect could be attributed both to a rise of [Ca2+](i) and to depolarization per se, because some translocation persisted during depolarization in a Ca2+-deficient medium containing the Ca2+ chelator EGTA. Moreover, oscillations of [Ca2+](i) resulting from depolarization with Ca2+ influx evoked concentration-dependent periodic activation of PLC. We conclude that PLC activity is under tight dynamic control of [Ca2+](i). In insulin-secreting beta-cells, this mechanism provides a link between Ca2+ influx and release from intracellular stores that may be important in the regulation of insulin secretion.     

Low-sulphated oligosaccharides derived from heparan sulphate inhibit normal angiogenesis

Heparin, with or without the addition of an adrenocorticosteroid,can inhibit normal angiogenesis in the chick embryo chorioallantoicmembrane. Low- or non-sulphated heparin fragments also haveanti-angiogenic effect. Attempts to define a saccharide structureresponsible for the anti-angiogenic effect implicated a -[GlcAß1,4-GlcNAc     

Reduced Introgression of Sex Chromosome Markers in the Mexican Howler Monkey (Alouatta palliata × A. pigra) Hybrid Zone

International Journal of Primatology - Interspecific hybridization allows the introgression or movement of alleles from one genome to another. While some genomic regions freely exchange alleles...     

Biosynthesis of 2-hydroxyisobutyric acid (2-HIBA) from renewable carbon

Nowadays a growing demand for green chemicals and cleantech solutions is motivating the industry to strive for biobased building blocks. We have identified the tertiary carbon atom-containing 2-hydroxyisobutyric acid (2-HIBA) as an interesting building block for polymer synthesis. Starting from this carboxylic acid, practically all compounds possessing the isobutane structure are accessible by simple chemical conversions, e. g. the commodity methacrylic acid as well as isobutylene glycol and oxide. During recent years, biotechnological routes to 2-HIBA acid have been proposed and significant progress in elucidating the underlying biochemistry has been made. Besides biohydrolysis and biooxidation, now a bioisomerization reaction can be employed, converting the common metabolite 3-hydroxybutyric acid to 2-HIBA by a novel cobalamin-dependent CoA-carbonyl mutase. The latter reaction has recently been discovered in the course of elucidating the degradation pathway of the groundwater pollutant methyl tert -butyl ether (MTBE) in the new bacterial species Aquincola tertiaricarbonis. This discovery opens the ground for developing a completely biotechnological process for producing 2-HIBA. The mutase enzyme has to be active in a suitable biological system producing 3-hydroxybutyryl-CoA, which is the precursor of the well-known bacterial bioplastic polyhydroxybutyrate (PHB). This connection to the PHB metabolism is a great advantage as its underlying biochemistry and physiology is well understood and can easily be adopted towards producing 2-HIBA. This review highlights the potential of these discoveries for a large-scale 2-HIBA biosynthesis from renewable carbon, replacing conventional chemistry as synthesis route and petrochemicals as carbon source.     

Synthesis of Short-Chain Diols and Unsaturated Alcohols from Secondary Alcohol Substrates by the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ

The Rieske nonheme mononuclear iron oxygenase MdpJ of the fuel oxygenate-degrading bacterial strain Aquincola tertiaricarbonis L108 has been described to attack short-chain tertiary alcohols via hydroxylation and desaturation reactions. Here, we demonstrate that also short-chain secondary alcohols can be transformed by MdpJ. Wild-type cells of strain L108 converted 2-propanol and 2-butanol to 1,2-propanediol and 3-buten-2-ol, respectively, whereas an mdpJ knockout mutant did not show such activity. In addition, wild-type cells converted 3-methyl-2-butanol and 3-pentanol to the corresponding desaturation products 3-methyl-3-buten-2-ol and 1-penten-3-ol, respectively. The enzymatic hydroxylation of 2-propanol resulted in an enantiomeric excess of about 70% for the (R)-enantiomer, indicating that this reaction was favored. Likewise, desaturation of (R)-2-butanol to 3-buten-2-ol was about 2.3-fold faster than conversion of the (S)-enantiomer. The biotechnological potential of MdpJ for the synthesis of enantiopure short-chain alcohols and diols as building block chemicals is discussed.     

A homogeneous HTRF assay for the identification of inhibitors of the TWEAK-Fn14 protein interaction

The TWEAK-Fn14 pathway is upregulated in models of inflammation, autoimmune diseases, and cancer. Both TWEAK and Fn14 show increased expression also in the CNS in response to different stimuli, particularly astrocytes, microglia, and neurons, leading to activation of NF-κB and release of proinflammatory cytokines. Although neutralizing antibodies against these proteins have been shown to have therapeutic efficacy in animal models of inflammation, no small-molecule therapeutics are yet available. Here, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF)-based screening assay together with several counterassays for the identification of small-molecule inhibitors of this protein-protein interaction. Recombinant HIS-TWEAK and Fn14-Fc proteins as well as FLAG-TWEAK and Fn14-FLAG proteins and an anti-Fn14 antibody were used to establish and validate these assays and to screen a library of 60 000 compounds. Two HTRF counterassays with unrelated proteins in the same assay format, an antiaggregation assay and a redox assay, were applied to filter out potential false-positive compounds. The novel assay and associated screening cascade should be useful for the discovery of small-molecule inhibitors of the TWEAK-Fn14 protein interaction.