Exocytotic insertion of TRPC6 channel into the plasma membrane upon Gq protein-coupled receptor activation

TRPC proteins are the mammalian homologues of the Drosophila transient receptor potential channel and are involved in calcium entry after agonist stimulation of non-excitable cells. Seven mammalian TRPCs have been cloned, and their mechanisms of activation and regulation are still the subject of intense research. TRPC proteins interact with the inositol 1,4,5-trisphosphate receptor, and the conformational coupling plays a critical role in the activation of calcium entry. Some evidence also supports an exocytotic mechanism as part of the activation of calcium entry. To investigate the possible involvement of exocytosis in TRPC6 activation, we evaluated the location of TRPC6 at the plasma membrane by biotinylation labeling of cell surface proteins and by indirect immunofluorescence marking of TRPC6 in stably transfected HEK 293 cells. We showed that when the muscarinic receptor was stimulated or the thapsigargin-induced intracellular calcium pool was depleted the level of TRPC6 at the plasma membrane increased. The carbachol concentration at which TRPC6 externalization occurred was lower than the concentration required to activate TRPC6. Externalization occurred within the first 30 s of stimulation, and TRPC6 remained at the plasma membrane as long as the stimulus was present. These results indicate that an exocytotic mechanism is involved in the activation of TRPC6.     

Identification of two domains involved in the assembly of transient receptor potential canonical channels

Transient receptor potential canonical (TRPC) channels are associated with calcium entry activity in nonexcitable cells. TRPCs can form homo- or heterotetrameric channels, in which case they can assemble together within a subfamily groups. TRPC1, 4, and 5 represent one group, and TRPC3, 6, and 7 represent the other. The molecular determinants involved in promoting subunit tetramerization are not known. To identify them, we generated chimeras by swapping the different domains of TRPC4 with the same regions in TRPC6. We showed that TRPC4 coimmunoprecipitated with the chimeras containing the ankyrin repeats and coiled-coil domains of TRPC4 into TRPC6. However, chimeras containing only the ankyrin repeats or only the coiled-coil domain of TRPC4 did not coimmunoprecipitate with TRPC4. We also showed that a second domain of interaction composed of the pore region and the C-terminal tail is involved in the oligomerization of TRPC4. However, chimeras containing only the pore region or only the C-terminal tail of TRPC4 did not coimmunoprecipitate with TRPC4. Furthermore, we showed that the N terminus of TRPC6 coimmunoprecipitated with the C terminus of TRPC6. Overexpression in HEK293T cells of chimeras that contained an N terminus and a C terminus from different subfamily groups increased intracellular calcium entry subsequent to stimulation of G(q) protein-coupled receptors. These results suggest that two types of interactions are involved in the assembly of the four subunits of the TRPC channel. The first interaction occurs between the N termini and involves two regions. The second interaction occurs between the N terminus and the C terminus and does not appear to be necessary for the activity of TRPCs.     

Comparison of leucine enkephalin and adrenocorticotrophin effects on adrenal function in fetal and adult sheep

At Day 120-125 of gestation equimolar amounts of ACTH and leu-enkephalin injected in vivo provoked similar rises in plasma cortisol concentrations in chronically catheterized fetuses. There was no concomitant change in plasma DHEA concentrations, or in maternal cortisol concentrations. At term (Days 135-140) 2 out of 5 animals responded similarly to both leu-enkephalin and ACTH injections with a rise in plasma cortisol concentrations, but the other 3 animals, in which basal cortisol concentrations had already risen, showed no response to either agonist. In adult sheep, ACTH provoked a significant increase in the plasma cortisol concentrations, but equimolar amounts of leu-enkephalin were without effect. There was a significant output of cortisol in response to ACTH administration by collagenase-dispersed adrenal cells from term sheep fetuses in vitro. Leu-enkephalin had no effect on cortisol output from dispersed adrenal cells when added by itself, or with ACTH. We conclude that leu-enkephalin is able to function as a stimulator of pituitary-adrenal function during fetal life. The lack of effect of leu-enkephalin on adrenal cells implies that its action is exerted not directly at the adrenal gland, but indirectly at the level of the hypothalamus or pituitary through stimulation of the release of other corticotrophic substances.     

Species-diagnostic markers in <Emphasis Type="Italic">Larix</Emphasis> spp. based on RAPDs and nuclear,cpDNA, and mtDNA gene sequences,and their phylogenetic implications

Genetic markers from the nuclear, chloroplast, and mitochondrial genomes were developed to distinguish unambiguously among four larch species [Larix laricina (Du Roi) K. Koch, Larix decidua (Mill.), Larix kaempferi (Lamb.) Sarg., and Larix sibirica (Ledeb.)] used in intensive forestry in eastern North America. Nine random amplified polymorphic DNA (RAPD) fragments had good diagnostic value, and 3 out of 12 nuclear genes were found to harbor fixed interspecific polymorphisms implicating a total of 17 single nucleotide polymorphisms (SNPs) and 2 indels. The sequencing of five mtDNA introns (cox1-intron1, matR-intron1, nad1-intron b/c, nad3-intron1, and nad5-intron1) and four cpDNA regions (matK, trnL-intron, trnT–trnL and trnL–trnF intergenic spacers) resulted in the identification of 14 sites with fixed interspecific differences among the four species. Including the ten Larix species, one polymorphic site per 47 nucleotide sites sampled was observed for nuclear genes, one per 283 sites for cpDNA, and one per 374 sites for mtDNA. The phylogeny of the genus could be estimated from variation among the ten species detected in two cpDNA intergenic regions and four mtDNA introns. There was congruence between cpDNA and mtDNA phylogenies with three large groups delineated: the North American, North Eurasian, and South Asian taxa. The position of L. sibirica differed between organelle genomes. It was regrouped with South Asian species on the cpDNA tree, but with its North Eurasian congenerics on the mtDNA tree. To simplify the detection of diagnostic DNA sequence polymorphisms among the four main Larix species, cleaved amplified polymorphic sequence (CAPS) assays were developed from the polymorphisms identified in the various genomes. Seventeen primer–enzyme combinations were tested, and six were selected for their high level of informativeness. These new species-specific diagnostic markers should be useful for the certification of larch breeding materials and hybrid stocks used in intensive forestry in the northern hemisphere.     

A composite linkage map from two crosses for the species complex Picea mariana × Picea rubens and analysis of synteny with other Pinaceae

Four individual linkage maps were constructed from two crosses for the species complex Picea mariana (Mill.) B.S.P. × Picea rubens Sarg in order to integrate their information into a composite map and to compare with other Pinaceae. For all individual linkage maps, 12 major linkage groups were recovered with 306 markers per map on average. Before building the composite linkage map, the common male parent between the two crosses made it possible to construct a reference linkage map to validate the relative position of homologous markers. The final composite map had a length of 2,319 cM (Haldane) and contained a total of 1,124 positioned markers, including 1,014 AFLPs, 3 RAPDs, 53 SSRs, and 54 ESTPs, assembled into 12 major linkage groups. Marker density of the composite map was statistically homogenous and was much higher (one marker every 2.1 cM) than that of the individual linkage maps (one marker every 5.7 to 7.1 cM). Synteny was well conserved between individual, reference, and composite linkage maps and 94% of homologous markers were colinear between the reference and composite maps. The combined information from the two crosses increased by about 24% the number of anchor markers compared to the information from any single cross. With a total number of 107 anchor markers (SSRs and ESTPs), the composite linkage map is a useful starting point for large-scale genome comparisons at the intergeneric level in the Pinaceae. Comparisons of this map with those in Pinus and Pseudotsuga allowed the identification of one breakdown in synteny where one linkage group homoeologous to both Picea and Pinus corresponded to two linkage groups in Pseudotsuga. Implications for the evolution of the Pinaceae genome are discussed. Electronic Supplementary Material Supplementary material is available for this article at     

Identifying and understanding drug allergies

Drug hypersensitivity reactions frequently occur in hospitalized and out-patients. Clinical presentations are numerous and heterogeneous, from a mild urticaria to a dramatic anaphylactic shock and an extensive bullous skin disease. Allergic reactions are unpredictable reactions, related to immunologic mechanisms. Some reactions mimic allergic reactions but no drug specific antibody or T cell proliferation can be demonstrated. A true diagnosis is rarely set up and the tools for it are lacking. In this review, we will focus on the available epidemiological data concerning these reactions, including data on incidence and mortality and on the most recent advances in the pathophysiology and allergy diagnosis of drug hypersensitivity reactions.     

Production and characterization of an iminoimidazolidine specific monoclonal antibody using para-aminoclonidine as antigen.

Para-aminoclonidine coupled to hemocyanin was used to produce mouse monoclonal antibodies directed against clonidine. The properties of one of these, called mFE7, secreted by a clone of hybrid myeloma, are described. This antibody displayed total crossreactivity with imidazolidines and no crossreactivity at all with catecholamines or other known naturally occurring substances tested. A liquid phase radioimmunoassay permitted the detection of immunoreactivity in human brain extracts. The mFE7 antibody could be useful for immunopurifying the endogenous ligand of Imidazolines Preferring Receptors (IPR) which are catecholamines insensitive.     

Initial responses of rove and ground beetles (Coleoptera,Staphylinidae, Carabidae) to removal of logging residues following clearcut harvesting in the boreal forest of Quebec,Canada

Increased interest in biomass harvesting for bioenergetic applications has raised questions regarding the potential ecological consequences on forest biodiversity. Here we evaluate the initial changes in the abundance, species richness and community composition of rove (Staphylinidae) and ground beetles (Carabidae), immediately following 1) stem-only harvesting (SOH), in which logging debris (i.e., tree tops and branches) are retained on site, and 2) whole-tree harvesting (WTH), in which stems, tops and branches are removed in mature balsam fir stands in Quebec, Canada. Beetles were collected throughout the summer of 2011, one year following harvesting, using pitfall traps. Overall catch rates were greater in uncut forest (Control) than either stem-only or whole-tree harvested sites. Catch rates in WTH were greater than SOH sites. Uncut stands were characterized primarily by five species: Atheta capsularis, Atheta klagesi, Atheta strigosula, Tachinus fumipennis/frigidus complex (Staphylinidae) and to a lesser extent to Pterostichus punctatissimus(Carabidae). Increased catch rates in WTH sites, where post-harvest biomass was less, were attributable to increased catches of rove beetles Pseudopsis subulata, Quedius labradorensis and to a lesser extent Gabrius brevipennis. We were able to characterize differences in beetle assemblages between harvested and non-harvested plots as well as differences between whole tree (WTH) and stem only (SOH) harvested sites where logging residues had been removed or left following harvest. However, the overall assemblage response was largely a recapitulation of the responses of several abundant species.     

Enzymatic synthesis of unsaturated fatty acid glucoside esters for dermo-cosmetic applications.

Unsaturated fatty acid alpha-butylglucoside esters were prepared by enzymatic esterification of alpha-butylglucoside in nonaqueous media. Conditions were firstly optimized using oleic acid as acyl group. Synthesis was possible in several solvents but the presence of water co-product in the medium limited the reaction to a thermodynamic equilibrium corresponding to a maximal conversion yield of 62%. In pure molten substrates, the removal of water under reduced pressure enabled yields superior to 95% to be obtained. Product profiles depended on enzyme origin : whatever the support, immobilized lipase B from Candida antarctica proved to be far more regioselective for the primary hydroxyl group of glucose than immobilized lipase from Rhizomucor miehei. Results obtained could be easily transposed to the acylation of alpha-butylglucoside with a commercial mixture of unsaturated fatty acids containing more than 60% of linoleic acid. The biocatalyst could be recycled more than ten times without any significant activity loss.