Cloning and expression pattern of a putative octopamine/tyramine receptor in antennae of the noctuid moth <Emphasis Type="Italic">Mamestra brassicae</Emphasis>

In insects, biogenic amines have been shown to play an important role in olfactory plasticity. In a first attempt to decipher the underlying molecular mechanisms, we report the molecular cloning and precise expression pattern of a newly identified octopamine/tyramine-receptor-encoding gene in the antennae of the noctuid moth Mamestra brassicae (MbraOAR/TAR). A full-length cDNA has been obtained through homology cloning in combination with rapid amplification of cDNA ends/polymerase chain reaction; the deduced protein exhibits high identities with previously identified octopamine/tyramine receptors in other moths. In situ hybridization within the antennae has revealed that MbraOAR/TAR is expressed at the bases of both pheromone-sensitive and non-sensitive olfactory sensilla and in cells with a neurone-like shape. In accordance with previous physiological studies that have revealed a role of biogenic amines in the electrical activity of the receptor neurones, our results suggest that biogenic amines (either octopamine or tyramine) target olfactory receptor neurones to modulate olfactory coding as early as the antennal level.     

Conferring a template-dependent polymerase activity to terminal deoxynucleotidyltransferase by mutations in the Loop1 region

Terminal deoxynucleotidyltransferase (Tdt) and DNA polymerase μ (pol μ) are two eukaryotic highly similar proteins involved in DNA processing and repair. Despite their high sequence identity, they differ widely in their activity: pol μ has a templated polymerase activity, whereas Tdt has a non-templated one. Loop1, first described when the Tdt structure was solved, has been invoked as the major structural determinant of this difference. Here we describe attempts to transform Tdt into pol μ with the minimal number of mutations in and around Loop1. First we describe the effect of mutations on six different positions chosen to destabilize Tdt Loop1 structure, either by alanine substitution or by deletion; they result at most in a reduction of Tdt activity, but adding Co⁺⁺ restores most of this Tdt activity. However, a deletion of the entire Loop1 as in pol λ does confer a limited template-dependent polymerase behavior to Tdt while a chimera bearing an extended pol μ Loop1 reproduces pol μ behavior. Finally, 16 additional substitutions are reported, targeted at the two so-called ‘sequence determinant’ regions located just after Loop1 or underneath. Among them, the single-point mutant F401A displays a sequence-specific replicative polymerase phenotype that is stable upon Co⁺⁺ addition. These results are discussed in light of the available crystal structures.     

Inhibition of astroglial glutamate transport by polyunsaturated fatty acids: Evidence for a signalling role of docosahexaenoic acid

Brain cells are especially rich in polyunsaturated fatty acids (PUFA), mainly the n-3 PUFA docosahexaenoic acid (DHA) and the n-6 PUFA arachidonic acid (AA). They are released from membranes by PLA2 during neurotransmission, and may regulate glutamate uptake by astroglia, involved in controlling glutamatergic transmission. AA has been shown to inhibit glutamate transport in several model systems, but the contribution of DHA is less clear and has not been evaluated in astrocytes. Because the high DHA content of brain membranes is essential for brain function, we investigated the role of DHA in the regulation of astroglial glutamate transport.We evaluated the actions of DHA and AA using cultured rat astrocytes and suspensions of rat brain membranes (P1 fractions). DHA reduced d-[³H]aspartate uptake by cultured astrocytes and cortical membrane suspensions, while AA did not. This also occurred in astrocytes enriched with α-tocopherol, indicating that it was not due to peroxidation products. The reduction of d-[³H]aspartate uptake by DHA did not involve any change in the concentrations of membrane-associated astroglial glutamate transporters (GLAST and GLT-1), suggesting that DHA reduced the activity of the transporters. In contrast with the inhibition induced by free-DHA, we found no effect of membrane-bound DHA on d-[³H]aspartate uptake. Indeed, the uptake was similar in astrocytes with varying amount of DHA in their membrane (induced by long-term supplementation with DHA or AA). Therefore, DHA reduces glutamate uptake through a signal-like effect but not through changes in the PUFA composition of the astrocyte membranes. Also, reactive astrocytes, induced by a medium supplement (G5), were insensitive to DHA. This suggests that DHA regulates synaptic glutamate under basal condition but does not impair glutamate scavenging under reactive conditions.These results indicate that DHA slows astroglial glutamate transport via a specific signal-like effect, and may thus be a physiological synaptic regulator.     

Identification of Brevibacteriaceae by Multilocus Sequence Typing and Comparative Genomic Hybridization Analyses

Multilocus sequence typing with nine selected genes is shown to be a promising new tool for accurate identifications of Brevibacteriaceae at the species level. A developed microarray also allows intraspecific diversity investigations of Brevibacterium aurantiacum showing that 13% to 15% of the genes of strain ATCC 9174 were absent or divergent in strain BL2 or ATCC 9175.Brevibacteriaceae play a major part in the cheese smear community (6, 11). The classification and typing of cheese-related Brevibacteriaceae have been based mainly on molecular methods such as amplified ribosomal DNA restriction enzyme analysis, pulsed-field gel electrophoresis, and ribotyping (8, 10, 12). Recently, the original Brevibacterium linens group was split into two species on the basis of their physiological and biochemical characteristics, the sugar and polyol composition of their teichoic acids, and their 16S rRNA sequence and DNA-DNA hybridization levels. One species remains B. linens and is represented by type strain ATCC 9172. The other, represented by type strain ATCC 9175, has been renamed Brevibacterium aurantiacum. Regarding this new classification, the taxonomic position of cheese-related isolates has to be revisited and potential relationships between phylogenetic affiliation and the potential occurrence of given metabolic characteristics redefined (7). The unfinished genome sequence of B. aurantiacum ATCC 9174 has recently been released by the Joint Genome Institute (http://genome.jgi-psf.org/draft_microbes/breli/breli.home.html). The development of focused phylogenetic approaches using multiple markers in conjunction with whole-genome screening techniques such as comparative genomic hybridization (CGH) has proven to be useful for the detailed characterization of pathogenic species, including food pathogens (3, 5, 9). However, only a few technological species have been investigated at an intraspecies level (2). Our intention was thus to develop modern tools to facilitate the typing of strains of technological interest, for which Brevibacteriaceae could be used as a case study.     

A new stage 3 millennial climatic variability record from a SW France speleothem

We present a new high resolution speleothem stable isotope record from the Villars Cave (SW-France) that covers part of marine isotope stage (MIS) 3. The Vil14 stalagmite grew between ~ 52 and 29 ka. The δ¹³C profile is used as a palaeoclimate proxy and clearly shows the interstadial substages 13, 12 and 11. The new results complement and corroborate previously published stalagmite records Vil9 and Vil27 from the same site. The Vil14 stalagmite chronology is based on 12 Th-U dating by MC-ICP-MS and 3 by TIMS. A correction for detrital contamination was done using the ²³⁰Th/²³²Th activity ratio measured on clay collected in Villars Cave. The Vil14 results reveal that the onset of Dansgaard–Oeschger (DO) events 13 and 12 occurred at ~ 49.8 ka and ~ 47.8 ka, respectively. Within uncertainties, this is coherent with the latest NorthGRIP time scale (GICC05-60 ka) and with speleothem records from Central Alps. Our data show an abrupt δ¹³C increase at the end of DO events 14 to 12 which coincides with a petrographical discontinuity probably due to a rapid cooling. As observed for Vil9 and Vil27, Vil14 growth significantly slowed down after ~ 42 ka and finally stopped ~ 29 ka ago where the δ¹³C increase suggests a strong climate deterioration that coincides with both North Atlantic sea level and sea surface temperature drop.     

Mapping the semantic space for the subjective experience of emotional responses to odors

Two studies were conducted to examine the nature of the verbal labels that describe emotional effects elicited by odors. In Study 1, a list of terms selected for their relevance to describe affective feelings induced by odors was assessed while participants were exposed to a set of odorant samples. The data were submitted to a series of exploratory factor analyses to 1) reduce the set of variables to a smaller set of summary scales and 2) get a preliminary sense of the differentiation of affective feelings elicited by odors. The goal of Study 2 was to replicate the findings of Study 1 with a larger sample of odorant samples and participants and to validate the preliminary model obtained in Study 1 by using confirmatory factor analysis. Overall, the findings point to a structure of affective responses to odors that differs from the classical taxonomies of emotion such as posited by discrete or bidimensional emotion theories. These findings suggest that the subjective affective experiences or feelings induced by odors are structured around a small group of dimensions that reflect the role of olfaction in well-being, social interaction, danger prevention, arousal or relaxation sensations, and conscious recollection of emotional memories.     

FpvA bound to non-cognate pyoverdines: molecular basis of siderophore recognition by an iron transporter

The first step in the specific uptake of iron via siderophores in Gram-negative bacteria is the recognition and binding of a ferric siderophore by its cognate receptor. We investigated the molecular basis of this event through structural and biochemical approaches. FpvA, the pyoverdine–Fe transporter from Pseudomonas aeruginosa ATCC 15692 (PAO1 strain), is able to transport ferric–pyoverdines originating from other species, whereas most fluorescent pseudomonads are only able to use the one they produce among the more than 100 known different pyoverdines. We solved the structure of FpvA bound to non-cognate pyoverdines of high- or low-affinity and found a close correlation between receptor–ligand structure and the measured affinities. The structure of the first amino acid residues of the pyoverdine chain distinguished the high- and low-affinity binders while the C-terminal portion of the pyoverdines, often cyclic, does not appear to contribute extensively to the interaction between the siderophore and its transporter. The specificity of the ferric–pyoverdine binding site of FpvA is conferred by the structural elements common to all ferric–pyoverdines, i.e. the chromophore, iron, and its chelating groups.