A 2-oxoglutarate-dependent dioxygenase from Ruta graveolens L. exhibits p-coumaroyl CoA 2'-hydroxylase activity (C2'H): a missing step in the synthesis of umbelliferone in plants

Coumarins are important compounds that contribute to the adaptation of plants to biotic or abiotic stresses. Among coumarins, umbelliferone occupies a pivotal position in the plant phenylpropanoid network. Previous studies indicated that umbelliferone is derived from the ortho‐hydroxylation of p‐coumaric acid by an unknown biochemical step to yield 2,4‐dihydroxycinnamic acid, which then undergoes spontaneous lactonization. Based on a recent report of a gene encoding a 2‐oxoglutarate‐dependent dioxygenase from Arabidopsis thaliana that exhibited feruloyl CoA 6′‐hydroxylase activity ( Bourgaud et al., 2006 ), we combined a bioinformatic approach and a cDNA library screen to identify an orthologous ORF (Genbank accession number JF799117 ) from Ruta graveolens L. This ORF shares 59% amino acid identity with feruloyl CoA 6′‐hydroxylase, was functionally expressed in Escherichia coli, and converted feruloyl CoA into scopoletin and p‐coumaroyl CoA into umbelliferone with equal activity. Its bi‐functionality was further confirmed in planta: transient expression of JF799117 in Nicotiana benthamiana yielded plants with leaves containing high levels of umbelliferone and scopoletin when compared to control plants, which contained barely detectable traces of these compounds. The expression of JF799117 was also tightly correlated to the amount of umbelliferone that was found in UV‐elicited R. graveolens leaves. Therefore, JF799117 encodes a p‐coumaroyl CoA 2′‐hydroxylase in R. graveolens, which represents a previously uncharacterized step in the synthesis of umbelliferone in plants. Psoralen, which is an important furanocoumarin in R. graveolens, was found to be a competitive inhibitor of the enzyme, and it may exert this effect through negative feedback on the enzyme at an upstream position in the pathway.     

Monothiol Glutaredoxin-BolA Interactions： Redox Control of Arabidopsis thaliana BolA2 and SufE1

A functional relationship between monothiol glutaredoxins and BolAs has been unraveled by genomic analyses and in several high-throughput studies. Phylogenetic analyses coupled to transient expression of green fluo- rescent protein （GFP） fusions indicated that, in addition to the sulfurtransferase SufE1, which contains a C-terminal BolA domain, three BolA isoforms exist in Arabidopsis thaliana, BolA1 being plastidial, BolA2 nucleo-cytoplasmic, and BolA4 dual-targeted to mitochondria and plastids. Binary yeast two-hybrid experiments demonstrated that all BolAs and SufE 1, via its BolA domain, can interact with all monothiol glutaredoxins. Most interactions between protein couples of the same subcellular compartment have been confirmed by bimolecular fluorescence complementation. In vitro experiments indicated that monothiol glutaredoxins could regulate the redox state of BolA2 and SufE1, both proteins possessing a single conserved reactive cysteine. Indeed, a glutathionylated form of SufE1 lost its capacity to activate the cysteine desuifurase, Nfs2, but it is reactivated by plastidial glutaredoxins. Besides, a monomeric glutathionyiated form and a dimeric disulfide-bridged form of BolA2 can be preferentially reduced by the nucleo-cytoplasmic GrxS17. These results indicate that the glutaredoxin-BolA interaction occurs in several subcellular compartments and suggest that a redox regulation mechanism, disconnected from their capacity to form iron-sulfur cluster-bridged heterodimers, may be physiologically relevant for BolA2 and SufE1.     

Discovery and pharmacological characterization of a novel rodent-active CCR2 antagonist, INCB3344

This report describes the characterization of INCB3344, a novel, potent and selective small molecule antagonist of the mouse CCR2 receptor. The lack of rodent cross-reactivity inherent in the small molecule CCR2 antagonists discovered to date has precluded pharmacological studies of antagonists of this receptor and its therapeutic relevance. In vitro, INCB3344 inhibits the binding of CCL2 to mouse monocytes with nanomolar potency (IC(50) = 10 nM) and displays dose-dependent inhibition of CCL2-mediated functional responses such as ERK phosphorylation and chemotaxis with similar potency. Against a panel of G protein-coupled receptors that includes other CC chemokine receptors, INCB3344 is at least 100-fold selective for CCR2. INCB3344 possesses good oral bioavailability and systemic exposure in rodents that allows in vivo pharmacological studies. INCB3344 treatment results in a dose-dependent inhibition of macrophage influx in a mouse model of delayed-type hypersensitivity. The histopathological analysis of tissues from the delayed-type hypersensitivity model demonstrates that inhibition of CCR2 leads to a substantial reduction in tissue inflammation, suggesting that macrophages play an orchestrating role in immune-based inflammatory reactions. These results led to the investigation of INCB3344 in inflammatory disease models. We demonstrate that therapeutic dosing of INCB3344 significantly reduces disease in mice subjected to experimental autoimmune encephalomyelitis, a model of multiple sclerosis, as well as a rat model of inflammatory arthritis. In summary, we present the first report on the pharmacological characterization of a selective, potent and rodent-active small molecule CCR2 antagonist. These data support targeting this receptor for the treatment of chronic inflammatory diseases.     

Wiping behavior, skin resistance, and the metabolic response to dehydration in the arboreal frog Phyllomedusa hypochondrialis

Several species of arboreal frogs secrete lipids from cutaneous glands and wipe these secretions over the body surfaces to reduce evaporative water losses. Following wiping, frogs become immobile in water-conserving postures, and some have suggested they are torpid. Here we report wiping behaviors and the physiological correlates of immobile postures in the arboreal monkey frog Phyllomedusa hypochondrialis. Skin resistance to water loss was comparatively high, and rates of evaporation were as low as 4% of that from a free water surface. Standard rates of metabolism (SMR) varied from 89 microL O2 h(-1) at 18 degrees C to 316 microL O2 h(-1) at 34 degrees C and were sensitive to both temperature (T) and body mass (W; mL O2 h(-1) = 0.016W0.642 x 10(0.030T)). The mean SMR did not change significantly during four consecutive days of dehydration when animals lost 19%-34% of body mass. Therefore, it appears these frogs do not routinely depress metabolic rates following wiping. However, some individuals that lost higher percentages of body water exhibited trends of decreasing oxygen consumption, suggesting that suppression of metabolic rates might occur at greater levels of body water deficit or perhaps during a slower course of dehydration than imposed by our experiments (e.g., individuals that are secluded during periods of drought).     

Crystal structure and activity of Kunjin virus NS3 helicase; protease and helicase domain assembly in the full length NS3 protein

Flaviviral NS3 is a multifunctional protein displaying N-terminal protease activity in addition to C-terminal helicase, nucleoside 5'-triphosphatase (NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to support the separation of RNA daughter and template strands during viral replication. In addition, NS3 assists the initiation of replication by unwinding the RNA secondary structure in the 3' non-translated region (NTR). We report here the three-dimensional structure (at 3.1 A resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin virus, an Australian variant of the West Nile virus. As for homologous helicases, NS3:186-619 is composed of three domains, two of which are structurally related and held to host the NTPase and RTPase active sites. The third domain (C-terminal) is involved in RNA binding/recognition. The NS3:186-619 construct occurs as a dimer in solution and in the crystals. We show that NS3:186-619 displays both ATPase and RTPase activities, that it can unwind a double-stranded RNA substrate, being however inactive on a double-stranded DNA substrate. Analysis of different constructs shows that full length NS3 displays increased helicase activity, suggesting that the protease domain plays an assisting role in the RNA unwinding process. The structural interaction between the helicase and protease domain has been assessed using small angle X-ray scattering on full length NS3, disclosing that the protease and helicase domains build a rather elongated molecular assembly differing from that observed in the NS3 protein from hepatitis C virus.     

RAT BRAIN SYNAPTIC VESICLES: UPTAKE SPECIFICITIES OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN IN PREPARATIONS FROM WHOLE BRAIN AND BRAIN REGIONS1

A fraction containing neurotransmitter storage vesicles was isolated from rat whole brain and brain regions, and the uptakes of [³H]norepinephrine and [³H]serotonin were determined in vitro. Norepinephrine uptake in vesicle preparations from corpus striatum was higher than in prep arations from cerebral cortex, and uptake in vesicles from the remainder (midbrain + brainstem + cerebellum) was intermediate. The K_m for norepinephrine uptake was the same in the three brain regions, but the regions differed in maximal uptake capacity by factors which paralleled total catecholamine concentration rather than content of norepinephrine alone. Intracisternal administration of 6-hydroxydopamine, but not of 5,6-dihydroxytryptamine, reduced vesicular norepinephrine uptake, and pretreat-ment with desmethylimipramine (which protects specifically norepinephrine neurons but not dopamine neurons from the 6-hydroxydopamine) only partially prevented the loss of vesicular norepinephrine uptake. These studies indicate that uptake of norepinephrine by rat brain vesicle preparations occurs in vesicles from norepinephrine and dopamine neurons, but probably not in vesicles from serotonin neurons. Uptake of serotonin by brain vesicle preparations exhibited time, temperature and ATP-Mg²⁺ requirements nearly identical to those of norepinephrine uptake. The affinity of serotonin uptake matched that of serotonin for inhibition of norepinephrine uptake, and the maximal capacity was the same for serotonin as for norepinephrine. Norepinephrine, dopamine and reserpine inhibited serotonin uptake in a purely competitive fashion, with K_is similar to those for inhibition of norepinephrine uptake. Whereas 5,6-dihydroxytryptamine treatment reduced synaptosomal serotonin uptake but not vesicular serotonin uptake, 6-hydroxydopamine reduced vesicular serotonin uptake in the absence of reductions in synaptosomal serotonin uptake. Thus, in this preparation, serotonin appears to be taken up in vitro into catecholamine vesicles, rather than into serotonin vesicles.     

A risk assessment of aquarium trade introductions of seaweed in European waters

Aquaculture and maritime traffic have been identified as the main vectors for introductions of alien marine species. Except for one notorious case of Caulerpa taxifolia, the role of aquarium trade towards the introduction of alien seaweeds has been largely unassessed. Here, we address the risk of accidental release of seaweed species from the aquarium trade market in European waters. We assessed the importance and diversity of seaweed species in the European online aquarium retail circuit. Our web survey revealed more than 30 genera available for online sale into Europe, including known introduced and invasive species. A second aspect of the study consisted in sampling algal diversity found in aquaria. While allowing direct and accurate identification of the specimens, this approach was targeting not only ornamental species, but also seaweeds that may be accidentally present in the aquarium circuit. By DNA-barcoding we identified no less than 134 taxa, 7 of which are flagged as introduced in Europe and 5 reported as invasive. Climate envelope models show that at least 23 aquarium species have the potential to thrive in European waters. As expected by the tropical conditions in most aquaria, southern Atlantic regions of Europe and the Mediterranean are the most vulnerable towards new introductions. Further predictions show that this risk will increase and shift northwards as global warming proceeds. Overall our data indicate that aquarium trade poses a potential risk of new seaweed introductions, and calls for a cautious approach.     

Neuron-Specific Regulation of Associative Learning and Memory by MAGI-1 in C. elegans

Background

Identifying the molecular mechanisms and neural circuits that control learning and memory are major challenges in neuroscience. Mammalian MAGI/S-SCAM is a multi-PDZ domain synaptic scaffolding protein that interacts with a number of postsynaptic signaling proteins and is thereby thought to regulate synaptic plasticity [1], [2], [3].

Principal Findings

While investigating the behavioral defects of C. elegans nematodes carrying a mutation in the single MAGI ortholog magi-1, we have identified specific neurons that require MAGI-1 function for different aspects of associative learning and memory. Various sensory stimuli and a food deprivation signal are associated in RIA interneurons during learning, while additional expression of MAGI-1 in glutamatergic AVA, AVD and possibly AVE interneurons is required for efficient memory consolidation, i.e. the ability to retain the conditioned changes in behavior over time. During associative learning, MAGI-1 in RIA neurons controls in a cell non-autonomous fashion the dynamic remodeling of AVA, AVD and AVE synapses containing the ionotropic glutamate receptor (iGluR) GLR-1 [4]. During memory consolidation, however, MAGI-1 controls GLR-1 clustering in AVA and AVD interneurons cell-autonomously and depends on the ability to interact with the β-catenin HMP-2.

Significance

Together, these results indicate that different aspects of associative learning and memory in C. elegans are likely carried out by distinct subsets of interneurons. The synaptic scaffolding protein MAGI-1 plays a critical role in these processes in part by regulating the clustering of iGluRs at synapses.