Brain Receptor Mosaics and Their Intramembrane Receptor-Receptor Interactions: Molecular Integration in Transmission and Novel Targets for Drug Development

Abstract The concept of intramembrane receptor-receptor interactions and evidence for their existence was introduced by Agnati and Fuxe in 1980/81 suggesting the existence of heteromerization of receptors. In 1982, they proposed the existence of aggregates of multiple receptors in the plasma membrane and coined the term receptor mosaics (RM). In this way, cell signaling becomes a branched process beginning at the level of receptor recognition at the plasma membrane where receptors can directly modify the ligand recognition and signaling capacity of the receptors within a RM. Receptor-receptor interactions in RM are classified as operating either with classical cooperativity, when consisting of homomers or heteromers of similar receptor subtypes having the same transmitter, or non-classical cooperativity, when consisting of heteromers. It has been shown that information processing within a RM depends not only on its receptor composition, but also on the topology and the order of receptor activation determined by the concentrations of the ligands and the receptor properties. The general function of RM has also been demonstrated to depend on allosteric regulators (e.g., homocysteine) of the receptor subtypes present. RM as integrative nodes for receptor-receptor interactions in conjunction with membrane associated proteins may form horizontal molecular networks in the plasma membrane coordinating the activity of multiple effector systems modulating the excitability and gene expression of the cells. The key role of electrostatic epitope-epitope interactions will be discussed for the formation of the RM. These interactions probably represent a general molecular mechanism for receptor-receptor interactions and, without a doubt, indicate a role for phosphorylation-dephosphorylation events in these interactions. The novel therapeutic aspects given by the RMs will be discussed in the frame of molecular neurology and psychiatry and combined drug therapy appears as the future way to go

Herpes simplex virus as a model vector system for gene therapy in renal disease

Herpes simplex virus as a model vector system for gene therapy in renal disease. The past decade has been marked by significant advances in the application of gene transfer into living cells of animals and humans. These approaches have been tested in a few animal models of inherited and acquired renal diseases, including carbonic anhydrase II deficiency 1 and experimental glomerulonephritis2,3. Gene transfer into proximal tubular cells has been successfully accomplished by intrarenal arterial infusion of a liposomal complex4 or an adenoviral vector5. Tubular cells from the papilla and medulla have been selectively transduced by retrograde infusion into the pelvi-calyceal system of an adenoviral vector containing a reporter for β-galactosidase5. Although the results of these initial studies are promising, further studies to optimize viral vectors, maximize gene delivery, minimize side-effects, and develop cell-specific and long-term regulated gene expression are critical to the success of gene therapy targeted to specific compartments of the kidney. Our recent efforts have focused on defining the cellular pathways responsible for viral entry and infection into renal epithelial cells using herpes simplex virus (HSV) as a model vector. We anticipate that a solid understanding of the basic scientific principles underlying viral entry and gene expression into specific populations of renal cells will facilitate the design of successful therapeutic viral-based gene transfer strategies

Herpes simplex virus triggers activation of calcium-signaling pathways

The cellular pathways required for herpes simplex virus (HSV) invasion have not been defined. To test the hypothesis that HSV entry triggers activation of Ca2+-signaling pathways, the effects on intracellular calcium concentration ([Ca2+]i) after exposure of cells to HSV were examined. Exposure to virus results in a rapid and transient increase in [Ca2+]i. Pretreatment of cells with pharmacological agents that block release of inositol 1,4,5-triphosphate (IP3)–sensitive endoplasmic reticulum stores abrogates the response. Moreover, treatment of cells with these pharmacological agents inhibits HSV infection and prevents focal adhesion kinase (FAK) phosphorylation, which occurs within 5 min after viral infection. Viruses deleted in glycoprotein L or glycoprotein D, which bind but do not penetrate, fail to induce a [Ca2+]i response or trigger FAK phosphorylation. Together, these results support a model for HSV infection that requires activation of IP3-responsive Ca2+-signaling pathways and that is associated with FAK phosphorylation. Defining the pathway of viral invasion may lead to new targets for anti-viral therapy

Aspects of soybean crop ecophysiology and management

Esta revisión tiene como objetivo detallar algunos de los factores que determinan el rendimiento del cultivo de soja, centrándose en recientes aportes al conocimiento de la ecofisiología, los procesos de crecimiento y desarrollo, y la generación del rendimiento del cultivo. Busca identificar los factores que definen y limitan los rendimientos de soja, y que pueden ser usados para guiar prácticas tendientes a lograr máximos rendimientos, contribuir a la sustentabilidad del cultivo y disminuir las brechas existentes en la actualidad. También se analizan algunos aspectos del manejo agronómico del cultivo de soja como son: i) fecha de siembra, ii) elección del genotipo, iii) marco de plantación, y iv) manejo nutricional. Se consideraron conocimientos científicos y tecnológicos logrados para la región sojera de Argentina, con énfasis en la región centro sur de la provincia de Córdoba.This review aims to detail some of the factors that determine the soybean crop yield focusing on recent contributions to the knowledge of ecophysiology, growth and development processes, and crop yield generation. It seeks to identify the factors that define and limit soybean yields, and that can be used to guide practices aimed at achieving maximum yields, contributing to the crop sustainability and reducing the yield gaps that currently exist. Some aspects of the agronomic management of soybean cultivation are also analyzed, such as: i) sowing date, ii) choice of genotype, iii) plant population density, and iv) nutritional management. Was considered scientific and technological knowledge achieved for the soybean growing region in Argentina, with emphasis on the south central region of Córdoba province.Fil: Morla, FEDERICO DANIEL. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; ArgentinaFil: Sosa Daniele, María Fernanda. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; ArgentinaFil: Marcellino, Natalia. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; ArgentinaFil: Cerioni, Guillermo Angel. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria; Argentin

Pridopidine selectively occupies sigma-1 rather than dopamine D2 receptors at behaviorally active doses

Dopamine stabilizers have stimulatory actions under low dopamine tone and inhibitory actions under high dopamine tone without eliciting catalepsy. These compounds are dopamine D-2 receptor (D2R) antagonists or weak partial agonists and may have pro-mnemonic and neuroprotective effects. The mechanism underlying their stimulatory and neuroprotective actions is unknown but could involve sigma-1R binding. The present study examined sigma-1R and D2R occupancy by the dopamine stabilizer pridopidine (ACR16) at behaviorally relevant doses in living rats. Rats were administered 3 or 15 mg/kg pridopidine, or saline, before injection of the radiotracer C-11-SA4503 (sigma-1R) or C-11-raclopride (D2R). Some animals received 60 mg/kg pridopidine and were only scanned with C-11-raclopride. Cerebral C-11-SA4503 binding was quantified using metabolite-corrected plasma input data and distribution volume (V (T)) calculated by Logan graphical analysis. C-11-raclopride binding was quantified using striatum-to-cerebellum ratios and binding potentials calculated with a simplified reference tissue model. Cunningham-Lassen plots indicated sigma-1R occupancies of 57 +/- 2 and 85 +/- 2 % after pretreatment of animals with 3 and 15 mg/kg pridopidine. A significant (44-66 %) reduction of C-11-raclopride binding was only observed at 60 mg/kg pridopidine. At doses shown to elicit neurochemical and behavioral effects, pridopidine occupied a large fraction of sigma-1Rs and a negligible fraction of D(2)Rs. Significant D2R occupancy was only observed at a dose 20-fold higher than was required for sigma-1R occupancy. The characteristics of dopamine stabilizers may result from the combination of high sigma-1R and low D2R affinity

Selection of reference genes for expression Study in pulp and seeds of Theobroma grandiflorum (Willd. ex Spreng.) Schum

Cupuassu (Theobroma grandiflorum [Willd. ex Spreng.] Schum) is a species of high economic importance in Brazil with great potential at international level due to the multiple uses of both its seeds and pulp in the industry of sweets and cosmetics. For this reason, the cupuassu breeding program focused on the selection of genotypes with high pulp and seed quality—selection associated with the understanding of the mechanisms involved in fruit formation. Gene expression is one of the most used approaches related to such understanding. In this sense, quantitative real-time PCR (qPCR) is a powerful tool, since it rapidly and reliably quantifies gene expression levels across different experimental conditions. The analysis by qPCR and the correct interpretation of data depend on signal normalization using reference genes, i.e. genes presenting a uniform pattern of expression in the analyzed samples. Here, we selected and analyzed the expression of five genes from cupuassu (ACP, ACT, GAPDH, MDH, TUB) to be used as candidates for reference genes on pulp and seed of young, maturing and mature cupuassu fruits. The evaluation of the gene expression stability was obtained using the NormFinder, geNorm and BestKeeper programs. In general, our results indicated that the GAPDH and MDH genes constituted the best combination as reference genes to analyze the expression of cupuassu samples. To our knowledge, this is the first report of reference gene definition in cupuassu, and these results will support subsequent analysis related to gene expression studies in cupuassu plants subjected to different biotic or abiotic conditions as well as serve as a tool for diversity analysis based on pulp and seed quality. (Résumé d'auteur

Adenosine A2A-dopamine D2 receptor-receptor heteromerization: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer

There is evidence for strong functional antagonistic interactions between adenosine A2A receptors (A2ARs) and dopamine D2 receptors (D2Rs). Although a close physical interaction between both receptors has recently been shown using co-immunoprecipitation and co-localization assays, the existence of a A2AR-D2R protein-protein interaction still had to be demonstrated in intact living cells. In the present work, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques were used to confirm the occurrence of A2AR-D2R interactions in co-transfected cells. The degree of A2AR-D2R heteromerization, measured by BRET, did not vary after receptor activation with selective agonists, alone or in combination. BRET competition experiments were performed using a chimeric D2R-D1R in which helices 5 and 6, the third intracellular loop (I3), and the third extracellular loop (E3) of the D2R were replaced by those of the dopamine D1 receptor (D1R). Although the wild type D2R was able to decrease the BRET signal, the chimera failed to achieve any effect. This suggests that the helix 5-I3-helix 6-E3 portion of D2R holds the site(s) for interaction with A2AR. Modeling of A2AR and D2R using a modified rhodopsin template followed by molecular dynamics and docking simulations gave essentially two different possible modes of interaction between D2R and A2AR. In the most probable one, helix 5 and/or helix 6 and the N-terminal portion of I3 from D2R approached helix 4 and the C-terminal portion of the C-tail from the A2AR, respectively

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 Gi/o proteins and to the adenosine A2A receptor, with the formation of A2A-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 A2A receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A2A-D2 receptor oligomerization. BRET competition experiments indicated that, in the A2A-D2 receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A2A receptor. Furthermore, Ca2+ was found to induce conformational changes in the CaM-A2A-D2 receptor oligomer and to selectively modulate A2A and D2 receptor-mediated MAPK signaling in the A2A-D2 receptor heteromer. These results may have implications for basal ganglia disorders, since A2A-D2 receptor heteromers are being considered as a target for anti-parkinsonian agents

Herpes Simplex Virus Entry Is Associated with Tyrosine Phosphorylation of Cellular Proteins

AbstractThe initial step in herpes simplex virus (HSV) entry is binding of virion glycoprotein (g)C and/or gB to cell surface heparan sulfate. After this initial attachment, gD interacts with cell surface receptor or receptors, and the virion envelope fuses with the cell membrane. Fusion requires viral glycoproteins gB, gD, gL, and gH, but the cellular factors that participate in or the pathways activated by viral entry have not been defined. To determine whether signal transduction pathways are triggered by viral–cell fusion, we examined the association of viral entry with tyrosine phosphorylation of cellular proteins. Using immunoprecipitation and Western blotting, we found that at least three cytoplasmic host cell proteins, designated p80, p104, and p140, become tyrosine phosphorylated within 5–10 min after exposure to HSV-1 or HSV-2. However, no phosphorylation is detected when cells are exposed to a mutant virus deleted in gL that binds but fails to penetrate. Phosphorylation is restored when the gL-deletion virus is grown on a complementing cell line. Viral entry and the phosphorylation of p80, p104, and p140 are inhibited when cells are infected with virus in the presence of protein tyrosine kinase inhibitors. Taken together, these studies suggest that tyrosine phosphorylation of host cellular proteins is triggered by viral entry