Acetyl-CoA cleavage pathway in a syntrophic propionate oxidizing bacterium growing on fumarate in the absence of methanogens

Abstract In an ompF'-'lacZ fusion system carried by the open reading frame vector pORF1 in a supE mutant of Escherichia coli K12, read-through of an amber codon was decreased at temperatures higher than 40°C. This effect of temperature was dependent on the nucleotide sequence surrounding the amber codon, which was inserted into a site between the ompF and lacZ cistrons. Upon a temperature shift-up from 30 to 42°C, β-galactosidase synthesis directed by this fusion showed a transient arrest.     

Spring phenology at different altitudes is becoming more uniform under global warming in Europe

Under current global warming, high‐elevation regions are expected to experience faster warming than low‐elevation regions. However, due to the lack of studies based on long‐term large‐scale data, the relationship between tree spring phenology and the elevation‐dependent warming is unclear. Using 652k records of leaf unfolding of five temperate tree species monitored during 1951–2013 in situ in Europe, we discovered a nonlinear trend in the altitudinal sensitivity (S_A, shifted days per 100 m in altitude) in spring phenology. A delayed leaf unfolding (2.7 ± 0.6 days per decade) was observed at high elevations possibly due to decreased spring forcing between 1951 and 1980. The delayed leaf unfolding at high‐elevation regions was companied by a simultaneous advancing of leaf unfolding at low elevations. These divergent trends contributed to a significant increase in the S_A (0.36 ± 0.07 days 100/m per decade) during 1951–1980. Since 1980, the S_A started to decline with a rate of ?0.32 ± 0.07 days 100/m per decade, possibly due to reduced chilling at low elevations and improved efficiency of spring forcing in advancing the leaf unfolding at high elevations, the latter being caused by increased chilling. Our results suggest that due to both different temperature changes at the different altitudes, and the different tree responses to these changes, the tree phenology has shifted at different rates leading to a more uniform phenology at different altitudes during recent decades.     

Nest box revealed habitat preferences of arboreal mammals in box‐ironbark forest

Habitat preferences need to be understood if species are to be adequately managed or conserved. Habitat preferences are presumed to reflect requirements for food, shelter and breeding, as well as interactions with predators and competitors. However, one or more of these requirements may dominate. Tree‐cavity‐dependent wildlife species are one example where shelter or breeding site requirements may dominate. We installed 120 nest boxes across 40 sites to target the vulnerable Brush‐tailed Phascogale (Phascogale tapoatafa) and the non‐threatened Sugar Glider (Petaurus breviceps). The provision of shelter sites where few of quality are available may enable better resolution of habitat preferences. Over three years, we observed the Brush‐tailed Phascogale at 17 sites, whereas the Sugar Glider was observed at 39 sites. We tested four broad hypotheses (H1–H4) relating to habitat that may influence occupancy by these species. There was no influence of hollow (cavity) abundance (H1) on either species suggesting our nest boxes had satisfied their shelter requirements. There was no influence of habitat structure (canopy and tree proximity) (H2) immediately around the nest box trees. We found no influence of distance to the forest edge (H3). Variables at and away from the nest box site that appear to reflect foraging substrates (H4) were influential on the Brush‐tailed Phascogale. Sugar Glider occupancy was only influenced by a single variable at the nest box site. The lack of influence of any other variables is consistent with the very high occupancy observed, suggesting most of the forest habitat is suitable when shelter sites are available. We found no evidence that the Sugar Glider reduced site use by the Brush‐tailed Phascogale.     

Mapping DNA damage‐dependent genetic interactions in yeast via party mating and barcode fusion genetics

Condition‐dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State‐of‐the‐art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double‐mutant strains, does not scale readily to multi‐condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG‐GI), by which double‐mutant strains generated via en masse “party” mating can also be monitored en masse for growth to detect genetic interactions. By using site‐specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG‐GI enables multiplexed quantitative tracking of double mutants via next‐generation sequencing. We applied BFG‐GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4‐nitroquinoline 1‐oxide (4NQO), bleomycin, zeocin, and three other DNA‐damaging environments. BFG‐GI recapitulated known genetic interactions and yielded new condition‐dependent genetic interactions. We validated and further explored a subnetwork of condition‐dependent genetic interactions involving MAG1, SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53.     

Src Regulates Sequence‐Dependent Beta‐2 Adrenergic Receptor Recycling via Cortactin Phosphorylation

The recycling of internalized signaling receptors, which has direct functional consequences, is subject to multiple sequence and biochemical requirements. Why signaling receptors recycle via a specialized pathway, unlike many other proteins that recycle by bulk, is a fundamental unanswered question. Here, we show that these specialized pathways allow selective control of signaling receptor recycling by heterologous signaling. Using assays to visualize receptor recycling in living cells, we show that the recycling of the beta‐2 adrenergic receptor (B2AR), a prototypic signaling receptor, is regulated by Src family kinases. The target of Src is cortactin, an essential factor for B2AR sorting into specialized recycling microdomains on the endosome. Phosphorylation of a single cortactin residue, Y466, regulates the rate of fission of B2AR recycling vesicles from these microdomains and, therefore, the rate of delivery of B2AR to the cell surface. Together, our results indicate that actin‐stabilized microdomains that mediate signaling receptor recycling can serve as a functional point of convergence for crosstalk between signaling pathways.      

Helper-dependent adenovirus for the gene therapy of proliferative retinopathies: stable gene transfer, regulated gene expression and therapeutic efficacy

BACKGROUND: Ocular neovascular disorders, such as diabetic retinopathy and age-related macular degeneration, are the principal causes of blindness in developed countries. Current treatments are of limited efficacy, whereas a therapy based on intraocular gene transfer of angiostatic factors represents a promising alternative. For the first time we have explored the potential of helper-dependent adenovirus (HD-Ad), the last generation of Ad vectors, in the therapy of retinal neovascularization. METHODS: We first analyzed efficiency and stability of intraretinal gene transfer following intravitreous injection in mice. A HD-Ad vector expressing green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter (HD-Ad/GFP) was compared with a first-generation (E1/E3-deleted) Ad vector carrying an identical GFP expression cassette (FG-Ad/GFP). We also constructed HD-Ad vectors expressing a soluble form of the VEGF receptor (sFlt-1) in a constitutive (HD-Ad/sFlt-1) or doxycycline (dox)-inducible (HD-Ad/S-M2/sFlt-1) manner and tested their therapeutic efficacy upon intravitreous delivery in a rat model of oxygen-induced retinopathy (OIR). RESULTS: HD-Ad/GFP promoted long-lasting (up to 1 year) transgene expression in retinal Müller cells, in marked contrast with the short-term expression observed with FG-Ad/GFP. Intravitreous injection of HD-Ad vectors expressing sFlt-1 resulted in detectable levels of sFlt-1 and inhibited retinal neovascularization by more than 60% in a rat model of OIR. Notably, the therapeutic efficacy of the inducible vector HD-Ad/S-M2/sFlt-1 was strictly dox-dependent. CONCLUSIONS: HD-Ad vectors enable stable gene transfer and regulated expression of angiostatic factors following intravitreous injection and thus are attractive vehicles for the gene therapy of neovascular diseases of the retina.     

Distinct signalling pathways control Toxoplasma egress and host‐cell invasion

Calcium signalling coordinates motility, cell invasion, and egress by apicomplexan parasites, yet the key mediators that transduce these signals remain largely unknown. One underlying assumption is that invasion into and egress from the host cell depend on highly similar systems to initiate motility. Using a chemical‐genetic approach to specifically inhibit select calcium‐dependent kinases (CDPKs), we instead demonstrate that these pathways are controlled by different kinases: both TgCDPK1 and TgCDPK3 were required during ionophore‐induced egress, but only TgCDPK1 was required during invasion. Similarly, microneme secretion, which is necessary for motility during both invasion and egress, universally depended on TgCDPK1, but only exhibited TgCDPK3 dependence when triggered by certain stimuli. We also demonstrate that egress likely comes under a further level of control by cyclic GMP‐dependent protein kinase and that its activation can induce egress and partially compensate for the inhibition of TgCDPK3. These results demonstrate that separate signalling pathways are integrated to regulate motility in response to the different signals that promote invasion or egress during infection by Toxoplasma gondii.     

Structure of the heme/hemoglobin outer membrane receptor ShuA from Shigella dysenteriae: Heme binding by an induced fit mechanism

Shigella dysentriae and other Gram‐negative human pathogens are able to use iron from heme bound to hemoglobin for growing. We solved at 2.6 Å resolution the 3D structure of the TonB‐dependent heme/hemoglobin outer membrane receptor ShuA from S. dysenteriae. ShuA binds to hemoglobin and transports heme across the outer membrane. The structure consists of a C‐terminal domain that folds into a 22‐stranded transmembrane β‐barrel, which is filled by the N‐terminal plug domain. One distal histidine ligand of heme is located at the apex of the plug, exposed to the solvent. His86 is situated 9.86 Å apart from His420, the second histidine involved in the heme binding. His420 is in the extracellular loop L7. The heme coordination by His86 and His420 involves conformational changes. The comparisons with the hemophore receptor HasR of Serratia marcescens bound to HasA‐Heme suggest an extracellular induced fit mechanism for the heme binding. The loop L7 contains hydrophobic residues which could interact with the hydrophobic porphyring ring of heme. The energy required for the transport by ShuA is derived from the proton motive force after interactions between the periplasmic N‐terminal TonB‐box of ShuA and the inner membrane protein, TonB. In ShuA, the TonB‐box is buried and cannot interact with TonB. The structural comparisons with HasR suggest its conformational change upon the heme binding for interacting with TonB. The signaling of the heme binding could involve a hydrogen bond network going from His86 to the TonB‐box. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Formation of Glucose Isomerase by Streptomyces sp.

Sophoradin (I) [2′,4,4′-trihydroxy-3,3′,5-tris(3-methyl-2-butenyl)chalcone] which had been isolated from “Guang-Dou-Gen” (the root of Sophora subprostrata Chun et T. Chen) was synthesized through Claisen rearrangement. The reaction of p-hydroxybenzaldehyde and 3-chloro-3-methyl-1-butyne (III) gave 4-(1,1-dimethylpropargyloxy)benzaldehyde (VIII), which was catalytically hydrogenated over Lindlar catalyst to afford 4-(1,1-dimethylallyloxy)benzaldehyde (IX). IX was converted to 4-hydroxy-3-(3-methyl-2-butenyl)benzaldehyde (X) by Claisen rearrangement. The reaction of X and III gave 3-(3-methyl-2-butenyl)-4-(1,1-dimethylpropargyloxy)benzaldehyde (XI). Condensation of 2-hydroxy-4-(1,1-dimethylpropargyloxy)acetophenone (IV) and XI in alkaline solution gave a chalcone (XIII), which was catalytically hydrogenated over Lindlar catalyst to give 2′-hydroxy-4,4′-bis(1,-dimethylallyloxy)-3-(3-methyl-2-butenyl)chalcone (XIV). XIV was converted to I by Claisen rearrangement.