Activity of the δ-Opioid Receptor Is Partially Reduced, Whereas Activity of the κ-Receptor Is Maintained in Mice Lacking the μ-Receptor

Previous pharmacological studies have indicated the possible existence of functional interactions between μ-, δ- and κ-opioid receptors in the CNS. We have investigated this issue using a genetic approach. Here we describe in vitro and in vivo functional activity of δ- and κ-opioid receptors in mice lacking the μ-opioid receptor (MOR). Measurements of agonist-induced [35S]GTPγS binding and adenylyl cyclase inhibition showed that functional coupling of δ- and κ-receptors to G-proteins is preserved in the brain of mutant mice. In the mouse vas deferens bioassay, deltorphin II and cyclic[d-penicillamine2,d-penicillamine5] enkephalin exhibited similar potency to inhibit smooth muscle contraction in both wild-type and MOR −/− mice. δ-Analgesia induced by deltorphin II was slightly diminished in mutant mice, when the tail flick test was used. Deltorphin II strongly reduced the respiratory frequency in wild-type mice but not in MOR −/− mice. Analgesic and respiratory responses produced by the selective κ-agonist U-50,488H were unchanged in MOR-deficient mice. In conclusion, the preservation of δ- and κ-receptor signaling properties in mice lacking μ-receptors provides no evidence for opioid receptor cross-talk at the cellular level. Intact antinociceptive and respiratory responses to the κ-agonist further suggest that the κ-receptor mainly acts independently from the μ-receptor in vivo. Reduced δ-analgesia and the absence of δ-respiratory depression in MOR-deficient mice together indicate that functional interactions may take place between μ-receptors and central δ-receptors in specific neuronal pathways.

Plasticity of lifespan: a reaction norm perspective.

It is a well-appreciated fact that in many organisms the process of ageing reacts highly plastically, so that lifespan increases or decreases when the environment changes. The perhaps best-known example of such lifespan plasticity is dietary restriction (DR), a phenomenon whereby reduced food intake without malnutrition extends lifespan (typically at the expense of reduced fecundity) and which has been documented in numerous species, from invertebrates to mammals. For the evolutionary biologist, DR and other cases of lifespan plasticity are examples of a more general phenomenon called phenotypic plasticity, the ability of a single genotype to produce different phenotypes (e.g. lifespan) in response to changes in the environment (e.g. changes in diet). To analyse phenotypic plasticity, evolutionary biologists (and epidemiologists) often use a conceptual and statistical framework based on reaction norms (genotype-specific response curves) and genotype × environment interactions (G × E; differences in the plastic response among genotypes), concepts that biologists who are working on molecular aspects of ageing are usually not familiar with. Here I briefly discuss what has been learned about lifespan plasticity or, more generally, about plasticity of somatic maintenance and survival ability. In particular, I argue that adopting the conceptual framework of reaction norms and G × E interactions, as used by evolutionary biologists, is crucially important for our understanding of the mechanisms underlying DR and other forms of lifespan or survival plasticity.

Factors that cause genotype by environment interaction and use of a multiple-trait herd-cluster model for milk yield of Holstein cattle from Brazil and Colombia

Descriptive herd variables (DVHE) were used to explain genotype by environment interactions (G x E) for milk yield (MY) in Brazilian and Colombian production environments and to develop a herd-cluster model to estimate covariance components and genetic parameters for each herd environment group. Data consisted of 180,522 lactation records of 94,558 Holstein cows from 937 Brazilian and 400 Colombian herds. Herds in both countries were jointly grouped in thirds according to 8 DVHE: production level, phenotypic variability, age at first calving, calving interval, percentage of imported semen, lactation length, and herd size. For each DVHE, REML bivariate animal model analyses were used to estimate genetic correlations for MY between upper and lower thirds of the data. Based on estimates of genetic correlations, weights were assigned to each DVHE to group herds in a cluster analysis using the FASTCLUS procedure in SAS. Three clusters were defined, and genetic and residual variance components were heterogeneous among herd clusters. Estimates of heritability in clusters 1 and 3 were 0.28 and 0.29, respectively, but the estimate was larger (0.39) in Cluster 2. The genetic correlations of MY from different clusters ranged from 0.89 to 0.97. The herd-cluster model based on DVHE properly takes into account G x E by grouping similar environments accordingly and seems to be an alternative to simply considering country borders to distinguish between environments.

Nouvelles stratégies d’imageries afin de faciliter l’étude des interactions entre les récepteurs couplés aux protéines G

L’étude de l’assemblage et de l’acheminement à la membrane plasmique des complexes de signalisation des RCPGs va jouer un rôle crucial dans le développement de nouveaux médicaments ayant moins d’effets secondaires. Des outils permettant l’étude de ces phénomènes existent déjà, mais un outil polyvalent qui permettrait d’étudier plusieurs aspects pourrait grandement faciliter et accélérer ces études. L’étiquette SNAP est un candidat intéressant puisqu’avec cette étiquette il est possible de marquer une seule construction avec une variété de différents substrats. Une construction encodant pour le récepteur β2AR avec une étiquette SNAP à son extrémité C-terminale a été ingénérée. Cette construction est apte à lier son ligand, à être acheminée à la membrane plasmique et à homodimériser. La protéine exprimée a été marquée avec le fluorophore BG-430. De la fluorescence non spécifique a été détectée dans la cellule (même en absence de l’étiquette SNAP sur le récepteur). Un essai BRET a été développé, utilisant la construction HA-β2AR-SNAP en tant qu’accepteur et est fonctionnel. L’étiquette SNAP, comme utilisée ici, ne présente pas un aussi bon candidat qu’attendu, puisque le substrat n’ayant pas réagi demeure coincé dans la cellule. Le facteur activateur de plaquette (PAF) et son récepteur (PAFR) jouent un rôle critique dans plusieurs réponses inflammatoires et le récepteur FP est impliqué dans l’accouchement prématuré. Une meilleure compréhension des complexes de signalisation associés à ces récepteurs pourrait être la première étape dans la compréhension de leur signalisation dans des situations normales ou de maladies. Des expériences de BRET étudiant des interactions de bases entre les récepteurs et leurs partenaires d’interactions connus ont été réalisées. Elles ont permis de déterminer que : les récepteurs PAF et FP homodimérisent, que les récepteurs PAF et FP hétérodimérisent, que les protéines Gβγ interagissent de manière constitutive avec ces récepteurs et qu’aucun signal de BRET n’a été détecté avec la protéine Gα et ce, en présence ou en absence de stimulation par un agoniste (suggérant qu’il est nécessaire d’optimiser le système présentement utilisé).

Interactions of G-quadruplex binders and Topoisomerase I inhibitors in cancer cells

Top1-DNA cleavage complexes (Top1ccs) trigger an accumulation of antisense RNAPII transcripts specifically at active divergent CpG-island promoters in a replication independent and Top1 dependent manner, leading to transcription-dependent genome instability and altered transcription regulation. Using different cancer cell lines of colon and osteo origins, we show that they display different sensitivity to CPT and G4 binder that is independent from Top1 level. To look at the interactions between Top1 and G4, we show that co-treatment with G4 binders potentiate the cell cytotoxicity of CPT regardless of the treatment sequences. Potentiation is indicated by a reduced inhibition concentration (IC50) with a more profound cytotoxicity in CPT-resistant cell lines, HCT15 and U2OS, hence, indicating an interaction between Top1inhibitor and G4 binders. Moreover, computational analysis confirmed the present of G4 motifs in genes with CPT-induced antisense transcription. G4 motifs are present mostly 5000 bp upstream from transcription start site and notably lower in genes. Comparisons between genes with no antisense transcription and genes with antisense transcription show that G4 motifs in this region are notably lower in the genes with antisense transcripts. Since CPT increases negative supercoils at promoters of intermediate activity, the formation of G4 is also increased in CPT-treated cells. Suprisingly, formation of G4 is regulated in parallel to the transient stabilization of R-loops, indicating a role in response to CPT-induced stress. G4 formation is highly elevated in Pyridostatin treated cells, which previous study shows increased formation of γH2Ax foci. This effect is also seen in the CPT-resistant cell lines, HCT15, indicating that the formation is a general event in response to CPT. We also show that R-loop formation is greatly increased in Pyridostatin treated cells. In order to study the role of R-loops and G4 structures in Top1cc-dependant repair pathway, we inhibited tyrosyl-phosphodiestrase 1 (TDP-1) using a TDP-1 inhibitor.

Immunochemical interactions of peptides with site-directed polyclonal immunoglobulin G

The objective of this study was to investigate the immunochemical nature of the polyclonal immune response to the 14mer peptide TINKEDDESPGLYG and to identify interactions among antibodies to more than one epitope. Two groups of rabbits were immunized with the 14mer peptide and a Keyhole Limpet hemocyanin (KLH) carrier, but with KLH attached either to the 14mer's N- or C-terminus. Two approximate epitopes were mapped by an antibody-capture enzyme-linked immunosorbent assay method using antiserum obtained when KLH was oriented on the C-terminus of the 14mer. A precise mapping of the epitopes performed with inhibition enzyme immunoassays (iEIAs) resulted in an N-terminal 6mer epitope TINKED and a C-terminal 10mer epitope EDDESPGLYG. The epitopes overlapped by two amino acids. IEIAs and iEIAs incorporating antibody-blocking peptides indicated that the two anti-epitope antibody fractions did not interfere with one anothers' epitope binding. It was postulated that the anti-TINKED and anti-EDDESPGLYG antibody fractions individually bind their respective hydrophobic epitope "core" region at the N- or C-terminal of peptide TINKEDDESPGLYG, while sharing the two hydrophilic overlap amino acids. This antibody "lap joint" binding interaction can be accomplished by each of the anti-epitope antibodies binding an opposite side of the epitope overlap region in the shallow periphery of its binding site. ^

Specificity of somatostatin receptor and G -protein interactions as characterized by somatostatin receptor subtype specific antibodies

The neuropeptide somatostatin is a widely distributed general inhibitor of endocrine, exocrine, gastrointestinal and neural functions. The biological actions of somatostatin are initiated by interaction with high affinity, plasma membrane somatostatin receptors (sst receptors). Five sst receptor subtypes have been cloned and sequence analysis shows they are all members of the G protein coupled receptor superfamily. The G proteins play a pivotal role in sst receptor signal transduction and the specificity of somatostatin receptor-G protein coupling defines the possible range of cellular responses. However, the data for endogenous sst receptor and G protein coupling is very limited, and even when it is available, the sst receptor subtypes involved in G protein coupling and signal transduction are unknown due to the expression of multiple sst receptor subtypes in target cell lines or tissues of somatostatin.^ In an effort to characterize each individual sst receptor subtypes, antisera against unique C-terminal regions of different sst receptor subtypes have been developed in our lab. In this report, antisera made against the sst1, sst2A and sst4 receptors are characterized. They are highly specific to their corresponding receptors and efficiently immunoprecipitate the sst receptors. Using these antibodies, the cell lines expressing these sst receptor subtypes were identified with both immunoprecipitation and Western blot methods. The development of sst receptor subtype specific antibodies make it possible to determine the specificity of the sst receptor subtype and G protein coupling in target cells or tissues expressing multiple sst receptors, two questions were addressed by this thesis: (1) whether different cellular environments affect receptor subtype and G protein coupling; (2) whether different sst receptors couple to different G proteins in similar cellular environments.^ Taken together our findings, both sst1 and sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells, G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in GH$\sb4$C$\sb1$ cells. Further, sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in AR4-2J cells while sst4 receptors couple with G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells. Therefore, the G protein coupling of the same sst receptors in different cell lines is basically similar in that they all couple with multiple $\alpha$-subunits of the G$\rm \sb{i}$ proteins, suggesting cellular environment has little effect on receptor and G protein coupling. Moreover, different sst receptors have similar G protein coupling specificities in the same cell line, suggesting components other than receptor and G$\alpha$ subunits in the signal transduction pathways may contribute to specific functions of each sst receptor subtype. This series of experiments represent a novel approach in dissecting signal transduction pathways and may have general application in the field. Furthermore, this is the first systematic study of sst receptor subtype and G protein $\alpha$-subunit interaction in both transfected cells and in normal cell lines. The information generated will be very useful in our understanding of sst receptor signal transduction pathways and in directing future sst receptor research. ^

Role of lipid polymorphism in G protein-membrane interactions: Nonlamellar-prone phospholipids and peripheral protein binding to membranes

Heterotrimeric G proteins (peripheral proteins) conduct signals from membrane receptors (integral proteins) to regulatory proteins localized to various cellular compartments. They are in excess over any G protein-coupled receptor type on the cell membrane, which is necessary for signal amplification. These facts account for the large number of G protein molecules bound to membrane lipids. Thus, the protein-lipid interactions are crucial for their cellular localization, and consequently for signal transduction. In this work, the binding of G protein subunits to model membranes (liposomes), formed with defined membrane lipids, has been studied. It is shown that although G protein α-subunits were able to bind to lipid bilayers, the presence of nonlamellar-prone phospholipids (phosphatidylethanolamines) enhanced their binding to model membranes. This mechanism also appears to be used by other (structurally and functionally unrelated) peripheral proteins, such as protein kinase C and the insect protein apolipophorin III, indicating that it could constitute a general mode of protein-lipid interactions, relevant in the activity and translocation of some peripheral (amphitropic) proteins from soluble to particulate compartments. Other factors, such as the presence of cholesterol or the vesicle surface charge, also modulated the binding of the G protein subunits to lipid bilayers. Conversely, the binding of G protein-coupled receptor kinase 2 and the G protein β-subunit to liposomes was not increased by hexagonally prone lipids. Their distinct interactions with membrane lipids may, in part, explain the different cellular localizations of all of these proteins during the signaling process.

Temperature dependence of the crystal structure and g-values of [(HC(Ph2PO)(3))(2)Cu] (ClO4)(2)center dot 2H(2)O: Influence of dynamic Jahn-Teller coupling and lattice strain interactions

The temperature dependence of the X-ray crystal structure and powder EPR spectrum of [(HC(Ph2PO)(3))(2)CU]-(ClO4)(2)center dot 2H(2)O is reported, and the structure at room temperature confirms that reported previously. Below similar to 100 K, the data imply a geometry with near elongated tetragonal symmetry for the [(HC(Ph2PO)(3))(2)Cu](2+) complex, but on warming the two higher Cu-O bond lengths and g-values progressively converge, and by 340 K the bond lengths correspond to a compressed tetragonal geometry. The data may be interpreted satisfactorily assuming an equilibrium among the energy levels of a Cu-O-6 polyhedron subjected to Jahn-Teller vibronic coupling and a lattice strain. However, agreement with the experiment is obtained only if the orthorhombic component of the lattice strain decreases to a negligible value as the temperature approaches 340 K.

Approche biophysique à l'étude des interactions allostériques entre les récepteurs et les protéines G

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Approche biophysique à l'étude des interactions allostériques entre les récepteurs et les protéines G

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Iis--integrated Interactome System: A Web-based Platform For The Annotation, Analysis And Visualization Of Protein-metabolite-gene-drug Interactions By Integrating A Variety Of Data Sources And Tools.

High-throughput screening of physical, genetic and chemical-genetic interactions brings important perspectives in the Systems Biology field, as the analysis of these interactions provides new insights into protein/gene function, cellular metabolic variations and the validation of therapeutic targets and drug design. However, such analysis depends on a pipeline connecting different tools that can automatically integrate data from diverse sources and result in a more comprehensive dataset that can be properly interpreted. We describe here the Integrated Interactome System (IIS), an integrative platform with a web-based interface for the annotation, analysis and visualization of the interaction profiles of proteins/genes, metabolites and drugs of interest. IIS works in four connected modules: (i) Submission module, which receives raw data derived from Sanger sequencing (e.g. two-hybrid system); (ii) Search module, which enables the user to search for the processed reads to be assembled into contigs/singlets, or for lists of proteins/genes, metabolites and drugs of interest, and add them to the project; (iii) Annotation module, which assigns annotations from several databases for the contigs/singlets or lists of proteins/genes, generating tables with automatic annotation that can be manually curated; and (iv) Interactome module, which maps the contigs/singlets or the uploaded lists to entries in our integrated database, building networks that gather novel identified interactions, protein and metabolite expression/concentration levels, subcellular localization and computed topological metrics, GO biological processes and KEGG pathways enrichment. This module generates a XGMML file that can be imported into Cytoscape or be visualized directly on the web. We have developed IIS by the integration of diverse databases following the need of appropriate tools for a systematic analysis of physical, genetic and chemical-genetic interactions. IIS was validated with yeast two-hybrid, proteomics and metabolomics datasets, but it is also extendable to other datasets. IIS is freely available online at: http://www.lge.ibi.unicamp.br/lnbio/IIS/.

Search for Active Neutrino Disappearance Using Neutral-Current Interactions in the MINOS Long-Baseline Experiment

We report the first detailed comparisons of the rates and spectra of neutral-current neutrino interactions at two widely separated locations. A depletion in the rate at the far site would indicate mixing between nu(mu) and a sterile particle. No anomalous depletion in the reconstructed energy spectrum is observed. Assuming oscillations occur at a single mass-squared splitting, a fit to the neutral- and charged-current energy spectra limits the fraction of nu(mu) oscillating to a sterile neutrino to be below 0.68 at 90% confidence level. A less stringent limit due to a possible contribution to the measured neutral-current event rate at the far site from nu(e) appearance at the current experimental limit is also presented.

Active to Sterile Neutrino Mixing Limits from Neutral-Current Interactions in MINOS

Results are reported from a search for active to sterile neutrino oscillations in the MINOS long-baseline experiment, based on the observation of neutral-current neutrino interactions, from an exposure to the NuMI neutrino beam of 7.07 x 10(20) protons on target. A total of 802 neutral-current event candidates is observed in the Far Detector, compared to an expected number of 754 +/- 28(stat) +/- 37(syst) for oscillations among three active flavors. The fraction f(s) of disappearing nu(mu) that may transition to nu(s) is found to be less than 22% at the 90% C.L.

Engineering interactions for quasiperfect transfer of polariton states through nonideal bosonic networks of distinct topologies

We present a scheme for quasiperfect transfer of polariton states from a sender to a spatially separated receiver, both composed of high-quality cavities filled by atomic samples. The sender and the receiver are connected by a nonideal transmission channel -the data bus- modelled by a network of lossy empty cavities. In particular, we analyze the influence of a large class of data-bus topologies on the fidelity and transfer time of the polariton state. Moreover, we also assume dispersive couplings between the polariton fields and the data-bus normal modes in order to achieve a tunneling-like state transfer. Such a tunneling-transfer mechanism, by which the excitation energy of the polariton effectively does not populate the data-bus cavities, is capable of attenuating appreciably the dissipative effects of the data-bus cavities. After deriving a Hamiltonian for the effective coupling between the sender and the receiver, we show that the decay rate of the fidelity is proportional to a cooperativity parameter that weighs the cost of the dissipation rate against the benefit of the effective coupling strength. The increase of the fidelity of the transfer process can be achieved at the expense of longer transfer times. We also show that the dependence of both the fidelity and the transfer time on the network topology is analyzed in detail for distinct regimes of parameters. It follows that the data-bus topology can be explored to control the time of the state-transfer process.