The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.     

MinC Protein Shortens FtsZ Protofilaments by Preferentially Interacting with GDP-bound Subunits

The interaction of MinC with FtsZ and its effects on FtsZ polymerization were studied under close to physiological conditions by a combination of biophysical methods. The Min system is a widely conserved mechanism in bacteria that ensures the correct placement of the division machinery at midcell. MinC is the component of this system that effectively interacts with FtsZ and inhibits the formation of the Z-ring. Here we report that MinC produces a concentration-dependent reduction in the size of GTP-induced FtsZ protofilaments (FtsZ-GTP) as demonstrated by analytical ultracentrifugation, dynamic light scattering, fluorescence correlation spectroscopy, and electron microscopy. Our experiments show that, despite being shorter, FtsZ protofilaments maintain their narrow distribution in size in the presence of MinC. The protein had the same effect regardless of its addition prior to or after FtsZ polymerization. Fluorescence anisotropy measurements indicated that MinC bound to FtsZ-GDP with a moderate affinity (apparent K_D ∼10 μm at 100 mm KCl and pH 7.5) very close to the MinC concentration corresponding to the midpoint of the inhibition of FtsZ assembly. Only marginal binding of MinC to FtsZ-GTP protofilaments was observed by analytical ultracentrifugation and fluorescence correlation spectroscopy. Remarkably, MinC effects on FtsZ-GTP protofilaments and binding affinity to FtsZ-GDP were strongly dependent on ionic strength, being severely reduced at 500 mm KCl compared with 100 mm KCl. Our results support a mechanism in which MinC interacts with FtsZ-GDP, resulting in smaller protofilaments of defined size and having the same effect on both preassembled and growing FtsZ protofilaments.     

Evolution of nesting behaviour and kleptoparasitism in a selected group of osmiine bees (Hymenoptera: Megachilidae)

The construction of nests to rear offspring is restricted to vertebrates and few insect taxa, such as termites, wasps, and bees. Among bees, species of the family Megachilidae are characterized by a particularly high diversity in nest construction behaviour. Many megachilid bees nest in excavated burrows in the ground, others place their brood cells in a variety of above‐ground cavities or attach them to the surface of a substrate, and yet others have adopted a kleptoparasitic habit. Evolutionary transitions between the different nesting sites and between conventional nesting and kleptoparasitism in bees are poorly understood. In the present study, we traced the evolution of nesting site selection and kleptoparasitism in the Annosmia–Hoplitis group (Osmiini), which displays an exceptionally high diversity in nesting behaviour. We found that the evolution of nesting behaviour proceeded unidirectionally from nesting in excavated burrows in the ground to nesting in rock depressions and cavities, followed by the colonization of snail shells and insect borings in dead wood or hollow stems. Kleptoparasitism evolved once and the kleptoparasitic species have derived from the same lineage as their hosts. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 108 , 349–360.     

Occurrence of alternariol and alternariol monomethyl ether in beverages from the Entre Rios Province market, Argentina

One hundred and eighty five samples of red, white and rosé wines and different juices purchased in Entre Rios, Argentina, were analyzed for the Alternaria mycotoxins alternariol (AOH) and alternariol methyl ether (AME). White wines were analyzed after removal of alcohol by a nitrogen stream and concentrated. AOH in red wines was cleaned up by solid-phase extraction columns in series (octadecyl and amino propyl modified silica) and AME quantified directly on the sample. The juices were filtered and concentrated, and then all sample extracts were quantified by high performance liquid chromatography with photodiode array detector that allows confirmation through UV spectra. Method validation revealed a good sensitivity with adequate LOD and LOQ for AME and less sensitivity for AOH (i.e. white wine: AME 0.8 and 1.4 ng/mL, AOH 2 and 3.3 ng/mL; red wine: AME 0.1 and 0.2 ng/mL, AOH 4.5 and 7.5 ng/mL; apple juice: AME 1.7 and 2.8 ng/mL, AOH 5 and 9 ng/mL; other juices: AME 2.0 and 3.1 ng/mL, AOH 6 and 10 ng/mL). Recoveries in all cases were greater than 80 %. Four of 53 white wine samples were contaminated with AOH with a maximum level of 18 ng/mL, 6 of 56 samples of red wine had a maximum of 13 ng/mL, and 3 of 68 samples of juices had traces of AOH. AME was less frequently detected than AOH, and the LOD and LOQ for AME are smaller than for AOH. Only three samples of white wine and one of red wine were contaminated, but in only one white wine sample (AME 225 ng/mL) did the toxin level exceed the LOQ.     

Vhc1, a novel transporter belonging to the family of electroneutral cation–Cl− cotransporters,participates in the regulation of cation content and morphology of Saccharomyces cerevisiae vacuoles

Cation–Cl^? cotransporters (CCCs) are integral membrane proteins which catalyze the coordinated symport of Cl^? with Na⁺ and/or K⁺ ions in plant and mammalian cells. Here we describe the first Saccharomyces cerevisiae CCC protein, encoded by the YBR235w open reading frame. Subcellular localization studies showed that this yeast CCC is targeted to the vacuolar membrane. Deletion of the YBR235w gene in a salt-sensitive strain (lacking the plasma-membrane cation exporters) resulted in an increased sensitivity to high KCl, altered vacuolar morphology control and decreased survival upon hyperosmotic shock. In addition, deletion of the YBR235w gene in a mutant strain deficient in K⁺ uptake produced a significant growth advantage over the parental strain under K⁺-limiting conditions, and a hypersensitivity to the exogenous K⁺/H⁺ exchanger nigericin. These results strongly suggest that we have identified a novel yeast vacuolar ion transporter mediating a K⁺–Cl^? cotransport and playing a role in vacuolar osmoregulation. Considering its identified function, we propose to refer to the yeast YBR235w gene as VHC1 (vacuolar protein homologous to CCC family 1).     

Design of a novel LOX-1 receptor antagonist mimicking the natural substrate

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the major receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, is overexpressed in atherosclerotic lesions. LOX-1 specific inhibitors, urgently necessary to reduce the rate of atherosclerotic and inflammation processes, are not yet available. We have designed and synthesized a new modified oxidized phospholipid, named PLAzPC, which plays to small scale the ligand-receptor recognition scheme. Molecular docking simulations confirm that PLAzPC disables the hydrophobic component of the ox-LDL recognition domain and allows the interaction of the l-lysine backbone charged groups with the solvent and with the charged/polar residues located around the edges of the LOX-1 hydrophobic tunnel. Binding assays, in a cell model system expressing human LOX-1 receptors, confirm that PLAzPC markedly inhibits ox-LDL binding to LOX-1 with higher efficacy compared to previously identified inhibitors.     

Effete,a Drosophila Chromatin-Associated Ubiquitin-Conjugating Enzyme That Affects Telomeric and Heterochromatic Position Effect Variegation

Drosophila telomeres are elongated by the transposition of telomere-specific retrotransposons rather than telomerase activity. Proximal to the terminal transposon array, Drosophila chromosomes contain several kilobases of a complex satellite DNA termed telomere-associated sequences (TASs). Reporter genes inserted into or next to the TAS are silenced through a mechanism called telomere position effect (TPE). TPE is reminiscent of the position effect variegation (PEV) induced by Drosophila constitutive heterochromatin. However, most genes that modulate PEV have no effect on TPE, and systematic searches for TPE modifiers have so far identified only a few dominant suppressors. Surprisingly, only a few of the genes required to prevent telomere fusion have been tested for their effect on TPE. Here, we show that with the exception of the effete (eff; also called UbcD1) mutant alleles, none of the tested mutations at the other telomere fusion genes affects TPE. We also found that mutations in eff, which encodes a class I ubiquitin-conjugating enzyme, act as suppressors of PEV. Thus, eff is one of the rare genes that can modulate both TPE and PEV. Immunolocalization experiments showed that Eff is a major constituent of polytene chromosomes. Eff is enriched at several euchromatic bands and interbands, the TAS regions, and the chromocenter. Our results suggest that Eff associates with different types of chromatin affecting their abilities to regulate gene expression.     

A melissopalynological study of artisanal honey produced in Catamarca (Argentina)

Spores belonging to 43 taxa of pteridophytes were observed from the ambient air of 11 localities in India. It is suggested that pteridophyte spores could be listed among viable particulate air pollutants, especially in the light of the earlier reports regarding their allergenicity.     

Synthesis and antiplasmodial activity of some 1-azabenzanthrone derivatives

Some synthetic 1-azabenzanthrones (7H-dibenzo[de,h]quinolin-7-ones) are weakly to moderately cytotoxic, suggesting that they might also show antiparasitic activity. We have now tested a small collection of these compounds in vitro against a chloroquine-resistant Plasmodium falciparum strain, comparing their cytotoxicity against normal human fibroblasts. Our results indicate that 5-methoxy-1-azabenzanthrone and its 2,3-dihydro analogue have low micromolar antiplasmodial activities and showed more than 10-fold selectivity against the parasite, indicating that the dihydro compound, in particular, might serve as a lead compound for further development.