Distribution and conservation of mobile elements in the genus Drosophila

Essentially nothing is known of the origin, mode of transmission, and evolution of mobile elements within the genus Drosophila. To better understand the evolutionary history of these mobile elements, we examined the distribution and conservation of homologues to the P, I, gypsy, copia, and F elements in 34 Drosophila species from three subgenera. Probes specific for each element were prepared from D. melanogaster and hybridized to genomic DNA. Filters were washed under conditions of increasing stringency to estimate the similarity between D. melanogaster sequences and their homologues in other species. The I element homologues show the most limited distribution of all elements tested, being restricted to the melanogaster species group. The P elements are found in many members of the subgenus Sophophora but, with the notable exception of D. nasuta, are not found in the other two subgenera. Copia-, gypsy-, and F-element homologues are widespread in the genus, but their similarity to the D. melanogaster probe differs markedly between species. The distribution of copia and P elements and the conservation of the gypsy and P elements is inconsistent with a model that postulates a single ancient origin for each type of element followed by mating-dependent transmission. The data can be explained by horizontal transmission of mobile elements between reproductively isolated species.      

Interactions between Calmodulin, Adenosine A2A, and Dopamine D2 Receptors

The Ca²⁺-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D₂ receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D₂ receptor, within an Arg-rich epitope that is also involved in the binding to G_i/o proteins and to the adenosine A_2A receptor, with the formation of A_2A-D₂ receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A_2A receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A_2A-D₂ receptor oligomerization. BRET competition experiments indicated that, in the A_2A-D₂ receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A_2A receptor. Furthermore, Ca²⁺ was found to induce conformational changes in the CaM-A_2A-D₂ receptor oligomer and to selectively modulate A_2A and D₂ receptor-mediated MAPK signaling in the A_2A-D₂ receptor heteromer. These results may have implications for basal ganglia disorders, since A_2A-D₂ receptor heteromers are being considered as a target for anti-parkinsonian agents.G-protein-coupled receptors are able to form homo- and hetero-oligomers with unique biochemical and functional characteristics (1–7), and they are easily detected in vitro by using biophysical techniques (8–10). Heteromers of adenosine A_2A and dopamine D₂ receptors were one of the first G-protein-coupled receptor heteromers to be described (11). A close physical interaction between both receptors was shown using co-immunoprecipitation and co-localization assays (11) and fluorescence and bioluminescence resonance energy transfer (FRET² or BRET) techniques (12–14). At the biochemical level, two types of antagonistic A_2A-D₂ receptor interactions have been discovered that may explain the A_2A-D₂ receptor interactions described both at the neuronal and behavioral level (11, 15–18). First, by means of an allosteric interaction in the receptor heteromer, stimulation of A_2A receptor decreases the affinity of D₂ receptor for their agonists (12). Second, the stimulation of the G_i/o-protein-coupled D₂ receptor inhibits the cAMP accumulation induced by the stimulation of the G_s/olf-protein-coupled A_2A receptor (11, 17, 18). In view of the well known role of dopamine in Parkinson disease, schizophrenia, and drug addiction, it has been suggested that the A_2A-D₂ receptor interactions in the central nervous system may provide new therapeutic approaches to combat these disorders (16, 19).An epitope-epitope electrostatic interaction between an Arg-rich epitope of the N terminus of the third intracellular loop (3IL) of the D₂ receptor and an epitope containing a phosphorylated Ser localized in the distal part of the C terminus of the A_2A receptor is involved in A_2A-D₂ receptor heteromer interface (14, 20, 21). The same Arg-rich epitope of the D₂ receptor is able to interact with CaM (22–25). In the absence of phosphorylated residues, adjacent aspartates or glutamates, which are abundant in CaM, may also form non-covalent complexes with Arg-rich epitopes (26). Therefore, CaM can potentially convey a Ca²⁺ signal to the D₂ receptor through direct binding to the 3IL of the D₂ receptor (22). Mass spectrometry data have shown that bovine CaM can form multiple non-covalent complexes with an Arg-rich peptide corresponding to the N-terminal region of the 3IL of the D₂ receptor (VLRRRRKRVN) (24) as well as a peptide from the proximal C terminus of the A_2A receptor (24). This epitope, whose sequence is ²⁹¹RIREFRQTFR³⁰⁰ in the human A_2A receptor, also contains several Arg residues. Since the suspected interaction between the A_2A receptor and CaM was awaiting confirmation by assays using complete proteins, the present study was undertaken to demonstrate the existence of interactions between the A_2A receptor and CaM both in a recombinant protein expression cell system and in the brain. A proteomics approach was used for the discovery of protein-protein interactions between the A_2A receptor and CaM in rat brain, whereas BRET in transfected cells demonstrated a direct interaction between CaM and this receptor. Furthermore, by using BRET and sequential resonance energy transfer (SRET) techniques and analyzing MAPK signaling in transfected cells, evidence was obtained for CaM-A_2A-D₂ receptor oligomerization and a selective Ca²⁺-mediated modulation of A_2A and D₂ receptor function in the A_2A-D₂ receptor heteromer.     

Molecular evidence of the survival of subterranean amphipods (Arthropoda) during Ice Age underneath glaciers in Iceland

Two endemic groundwater arthropod crustacean species, Crangonyx islandicus and Crymostygius thingvallensis, were recently discovered on the mid‐Atlantic volcanic island of Iceland. The extent of morphological differences from closest relatives, endemism, along with the geographic isolation of Iceland and its complete coverage by glaciers 21 000 years ago, suggests that these two species have survived glaciation periods in sub‐glacial refugia. Here we provide strong support for this hypothesis by an analysis of mitochondrial genetic variation within Crangonyx islandicus. Our results show that the species is divided into several distinct monophyletic groups that are found along the volcanic zone in Iceland, which have been separated by 0.5 to around 5 million years. The genetic divergence between groups reflects geographic distances between sampling sites, indicating that divergence occurred after the colonization of Iceland. The genetic patterns, as well as the dependency of genetic variation on distances from the tectonic plate boundary and altitude, points to recent expansion from several refugia within Iceland. This presents the first genetic evidence of multicellular organisms as complex as crustacean amphipods which have survived glaciations beneath an ice sheet. This survival may be explained by geothermal heat linked to volcanic activities, which may have maintained favourable habitats in fissures along the tectonic plate boundary in Iceland during glaciations.     

Identification of dopamine D1-D3 receptor heteromers. Indications for a role of synergistic D1-D3 receptor interactions in the striatum

The function of dopamine D(3) receptors present in the striatum has remained elusive. In the present study evidence is provided for the existence of dopamine D(1)-D(3) receptor heteromers and for an intramembrane D(1)-D(3) receptor cross-talk in living cells and in the striatum. The formation of D(1)-D(3) receptor heteromers was demonstrated by fluorescence resonance energy transfer and bioluminescence resonance energy transfer techniques in transfected mammalian cells. In membrane preparations from these cells, a synergistic D(1)-D(3) intramembrane receptor-receptor interaction was observed, by which D(3) receptor stimulation enhances D(1) receptor agonist affinity, indicating that the D(1)-D(3) intramembrane receptor-receptor interaction is a biochemical characteristic of the D(1)-D(3) receptor heteromer. The same biochemical characteristic was also observed in membrane preparations from brain striatum, demonstrating the striatal co-localization and heteromerization of D(1) and D(3) receptors. According to the synergistic D(1)-D(3) intramembrane receptor-receptor interaction, experiments in reserpinized mice showed that D(3) receptor stimulation potentiates D(1) receptor-mediated behavioral effects by a different mechanism than D(2) receptor stimulation. The present study shows that a main functional significance of the D(3) receptor is to obtain a stronger dopaminergic response in the striatal neurons that co-express the two receptors.     

Voltage-sensitivity at the human dopamine D2S receptor is agonist-specific

Recently, we and others have shown that agonist potencies at some, but not all, G protein-coupled receptors are voltage-sensitive. Several of those studies employed electrophysiology assays in Xenopus oocytes with G protein-coupled potassium channels as a readout. Using this assay, we have now obtained evidence that voltage-sensitivity at the dopamine D_2S receptor is agonist-specific. Whereas the potency of dopamine at the D_2S receptor is decreased by depolarization, the potencies of β-phenethylamine, p- and m-tyramine are voltage-insensitive. Furthermore, both monohydroxylated and non-hydroxylated N,N-dipropyl-2-aminotetralin compounds are voltage-sensitive. Differential activation of G protein subtypes or differential ratios between effector and active G protein do not underlie this agonist-selective voltage-sensitivity. This is the first demonstration of voltage-sensitive and voltage-insensitive behaviour of different agonists acting via the same receptor.     

Chrobisiamone A, a new bischromone from Cassia siamea and a biomimetic transformation of 5-acetonyl-7-hydroxy-2-methylchromone into cassiarin A

A new bischromone, chrobisiamone A (1) with an antiplasmodial activity has been isolated from the leaves of Cassia siamea and the structure was elucidated by 2D NMR analysis. Cyclization of 5-acetonyl-7-hydroxy-2-methylchromone (2) in the presence of ammonium acetate resulted in generation of cassiarin A (3) with an unprecedented tricyclic skeleton, supporting a biogenetic pathway proposed for 3.     

An opaque-2-like transcription factor from pearl millet

Pearl millet (Pennisetum glaucum L.), a species of the Poaceae family, is an important food crop in Africa, Asia and South America. Its nutritional value is due to storage prolamins accumulated in the seeds. In other species of the same family, the expression of the genes coding for storage prolamins is mediated by the regulatory protein opaque-2. In this paper we show that an opaque-2 -like protein is present in pearl millet too and is expressed during the early stages of seed development. The organization of the gene coding for this protein is similar to that of orthologous genes in other Poaceae species, i.e. six exons separated by five introns. A comparison of amino acid homologies with other described opaque-2 proteins is presented.     

APOE ε4 moderates abnormal CSF-abeta-42 levels,while neurocognitive impairment is associated with abnormal CSF tau levels in HIV+ individuals – a cross-sectional observational study

Background

Cerebrospinal fluid (CSF) biomarkers Aβ1-42, t-tau and p-tau have a characteristic pattern in Alzheimer’s Disease (AD). Their roles in HIV-associated neurocognitive disorder (HAND) remains unclear.

Methods

Adults with chronic treated HIV disease were recruited (n = 43, aged 56.7 ± 7.9; 32% aged 60+; median HIV duration 20 years, >95% plasma and CSF HIV RNA <50 cp/mL, on cART for a median 24 months). All underwent standard neuropsychological testing (61% had HAND), APOE genotyping (30.9% carried APOE ε4 and 7.1% were ε4 homozygotes) and a lumbar puncture. Concentrations of Aβ1-42, t-tau and p-tau were assessed in the CSF using commercial ELISAs. Current neurocognitive status was defined using the continuous Global Deficit Score, which grades impairment in clinically relevant categories. History of HAND was recorded. Univariate correlations informed multivariate models, which were corrected for nadir CD4-T cell counts and HIV duration.

Results

Carriage of APOE ε4 predicted markedly lower levels of CSF Aβ1-42 in univariate (r = -.50; p = .001) and multivariate analyses (R² = .25; p < .0003). Greater levels of neurocognitive impairment were associated with higher CSF levels of p-tau in univariate analyses (r = .32; p = .03) and multivariate analyses (R² = .10; p = .03). AD risk prediction cut-offs incorporating all three CSF biomarkers suggested that 12.5% of participants had a high risk for AD. Having a CSF-AD like profile was more frequent in those with current (p = .05) and past HIV-associated dementia (p = .03).

Conclusions

Similarly to larger studies, APOE ε4 genotype was not directly associated with HAND, but moderated CSF levels of Aβ1-42 in a minority of participants. In the majority of participants, increased CSF p-tau levels were associated with current neurocognitive impairment. Combined CSF biomarker risk for AD in the current HIV+ sample is more than 10 times greater than in the Australian population of the same age. Larger prospective studies are warranted.

     

First Microsatellite Markers Developed from Cupuassu ESTs: Application in Diversity Analysis and Cross-Species Transferability to Cacao

The cupuassu tree (Theobroma grandiflorum) (Willd. ex Spreng.) Schum. is a fruitful species from the Amazon with great economical potential, due to the multiple uses of its fruit´s pulp and seeds in the food and cosmetic industries, including the production of cupulate, an alternative to chocolate. In order to support the cupuassu breeding program and to select plants presenting both pulp/seed quality and fungal disease resistance, SSRs from Next Generation Sequencing ESTs were obtained and used in diversity analysis. From 8,330 ESTs, 1,517 contained one or more SSRs (1,899 SSRs identified). The most abundant motifs identified in the EST-SSRs were hepta- and trinucleotides, and they were found with a minimum and maximum of 2 and 19 repeats, respectively. From the 1,517 ESTs containing SSRs, 70 ESTs were selected based on their functional annotation, focusing on pulp and seed quality, as well as resistance to pathogens. The 70 ESTs selected contained 77 SSRs, and among which, 11 were polymorphic in cupuassu genotypes. These EST-SSRs were able to discriminate the cupuassu genotype in relation to resistance/susceptibility to witches’ broom disease, as well as to pulp quality (SST/ATT values). Finally, we showed that these markers were transferable to cacao genotypes, and that genome availability might be used as a predictive tool for polymorphism detection and primer design useful for both Theobroma species. To our knowledge, this is the first report involving EST-SSRs from cupuassu and is also a pioneer in the analysis of marker transferability from cupuassu to cacao. Moreover, these markers might contribute to develop or saturate the cupuassu and cacao genetic maps, respectively.