AmOct2R: Functional Characterization of a Honeybee Octopamine Receptor Inhibiting Adenylyl Cyclase Activity

The catecholamines norepinephrine and epinephrine are important regulators of vertebrate physiology. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they use the phenolamines tyramine and octopamine for similar physiological functions. These biogenic amines activate specific members of the large protein family of G protein-coupled receptors (GPCRs). Based on molecular and pharmacological data, insect octopamine receptors were classified as either - or -adrenergic-like octopamine receptors. Currently, one - and four -receptors have been molecularly and pharmacologically characterized in the honeybee. Recently, an 2-adrenergic-like octopamine receptor was identified in Drosophila melanogaster (DmOct2R). This receptor is activated by octopamine and other biogenic amines and causes a decrease in intracellular cAMP ([cAMP]i). Here, we show that the orthologous receptor of the honeybee (AmOct2R), phylogenetically groups in a clade closely related to human 2-adrenergic receptors. When heterologously expressed in an eukaryotic cell line, AmOct2R causes a decrease in [cAMP]i. The receptor displays a pronounced preference for octopamine over tyramine. In contrast to DmOct2R, the honeybee receptor is not activated by serotonin. Its activity can be blocked eciently by 5-carboxamidotryptamine and phentolamine. The functional characterization of AmOct2R now adds a sixth member to this subfamily of monoaminergic receptors in the honeybee and is an important step towards understanding the actions of octopamine in honeybee behavior and physiology

El Zonda, portavoz del espacio público sanjuanino

En este trabajo analizamos al semanario El Zonda (San Juan, 1839) como el medio representante de la esfera pública sanjuanina, surgida bajo los auspicios de un grupo de jóvenes intelectuales encabezados por Sarmiento, que intentaron constituirse en intermediarios válidos entre el poder provincial y la sociedad. Motivados por la convicción de que la educación y los periódicos eran los medios eficaces para promover el progreso local, auspiciaron valiosas iniciativas que impactaron de manera dispar en el espacio público cuyano, como la creación de la Sociedad Dramática y Filarmónica, la Sociedad Literaria y el Colegio de Pensionistas de Santa Rosa, además del semanario. Este periódico fue renovador en varios sentidos: presentó una nueva concepción estética periodística, al tiempo que se valió de novedosas estrategias comunicacionales para instalar un discurso “renovador” y captar nuevos lectores, incluyendo a las mujeres. Además asumió un perfil político-pedagógico, pero no partidario, abandonando todo fin económico. Inobjetablemente suscitó controversias no obstante su efímera existencia (sólo seis números) la que estuvo condicionada por la conjunción de varias razones: la exigüidad de suscripciones, que imposibilitaron su sostenimiento económico y, con el correr del tiempo, la disipación de la complacencia del poder político de turno, actitudes que podrían responder a las severas críticas a la sociedad local emitidas desde sus enunciados.Facultad de Periodismo y Comunicación Socia

Religionstrends in der Schweiz

Diese Open-Access-Publikation beschreibt gegenwärtige Entwicklungen in der Religionslandschaft der Schweiz. Sie führt eine Reihe von Studien fort, die seit den 1980er Jahren in regelmässigen Zeitabständen publiziert werden. Die hier versammelten Beiträge basieren auf der Auswertung aktueller statistischer Daten und bearbeiten Fragestellungen aus der Religions- und Kirchensoziologie sowie aus der Politikwissenschaft

Examination of Intracellular GPCR-Mediated Signaling with High Temporal Resolution

The GTP-binding protein-coupled receptors (GPCRs) play important roles in physiology and neuronal signaling. More than a thousand genes, excluding the olfactory receptors, have been identified that encode these integral membrane proteins. Their pharmacological and functional properties make them fascinating targets for drug development, since various disease states can be treated and overcome by pharmacologically addressing these receptors and/or their downstream interacting partners. The activation of the GPCRs typically causes transient changes in the intracellular second messenger concentrations as well as in membrane conductance. In contrast to ion channel-mediated electrical signaling which results in spontaneous cellular responses, the GPCR-mediated metabotropic signals operate at a different time scale. Here we have studied the kinetics of two common GPCR-induced signaling pathways: (a) Ca2+ release from intracellular stores and (b) cyclic adenosine monophosphate (cAMP) production. The latter was monitored via the activation of cyclic nucleotide-gated (CNG) ion channels causing Ca2+ influx into the cell. Genetically modified and stably transfected cell lines were established and used in stopped-flow experiments to uncover the individual steps of the reaction cascades. Using two homologous biogenic amine receptors, either coupling to Go/q or Gs proteins, allowed us to determine the time between receptor activation and signal output. With ~350 ms, the release of Ca2+ from intracellular stores was much faster than cAMP-mediated Ca2+ entry through CNG channels (~6 s). The measurements with caged compounds suggest that this difference is due to turnover numbers of the GPCR downstream effectors rather than the different reaction cascades, per s

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Full rescue of an inactive olfactory receptor mutant by elimination of an allosteric ligand-gating site

Ligand-gating has recently been proposed as a novel mechanism to regulate olfactory receptor sensitivity. TAAR13c, the zebrafish olfactory receptor activated by the death-associated odor cadaverine, appears to possess an allosteric binding site for cadaverine, which was assumed to block progress of the ligand towards the internal orthosteric binding-and-activation site. Here we have challenged the suggested gating mechanism by modeling the entry tunnel for the ligand as well as the ligand path inside the receptor. We report an entry tunnel, whose opening is blocked by occupation of the external binding site by cadaverine, confirming the hypothesized gating mechanism. A multistep docking algorithm suggested a plausible path for cadaverine from the allosteric to the orthosteric binding-and-activation site. Furthermore we have combined a gain-of-function gating site mutation and a loss-of-function internal binding site mutation in one recombinant receptor. This receptor had almost wildtype ligand affinities, consistent with modeling results that showed localized effects for each mutation. A novel mutation of the suggested gating site resulted in increased receptor ligand affinity. In summary both the experimental and the modeling results provide further evidence for the proposed gating mechanism, which surprisingly exhibits pronounced similarity to processes described for some metabotropic neurotransmitter receptors

Loss of HCN2 in Dorsal Hippocampus of Young Adult Mice Induces Specific Apoptosis of the CA1 Pyramidal Neuron Layer

Neurons inevitably rely on a proper repertoire and distribution of membrane-bound ion-conducting channels. Among these proteins, the family of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels possesses unique properties giving rise to the corresponding Ih-current that contributes to various aspects of neural signaling. In mammals, four genes (hcn1-4) encode subunits of HCN channels. These subunits can assemble as hetero- or homotetrameric ion-conducting channels. In order to elaborate on the specific role of the HCN2 subunit in shaping electrical properties of neurons, we applied an Adeno-associated virus (AAV)-mediated, RNAi-based knock-down strategy of hcn2 gene expression both in vitro and in vivo. Electrophysiological measurements showed that HCN2 subunit knock-down resulted in specific yet anticipated changes in Ih-current properties in primary hippocampal neurons and, in addition, corroborated that the HCN2 subunit participates in postsynaptic signal integration. To further address the role of the HCN2 subunit in vivo, we injected recombinant (r)AAVs into the dorsal hippocampus of young adult male mice. Behavioral and biochemical analyses were conducted to assess the contribution of HCN2-containing channels in shaping hippocampal network properties. Surprisingly, knock-down of hcn2 expression resulted in a severe degeneration of the CA1 pyramidal cell layer, which did not occur in mice injected with control rAAV constructs. This finding might pinpoint to a vital and yet unknown contribution of HCN2 channels in establishing or maintaining the proper function of CA1 pyramidal neurons of the dorsal hippocampus

Caspase-3 and GFAP as early markers for apoptosis and astrogliosis in shRNA-induced hippocampal cytotoxicity

Genetic manipulation of cells and tissue by RNA interference has significantly contributed to the functional characterization of individual proteins and their role in physiological processes. Despite its versatility, RNA interference can have detrimental side effects, including reduced cell viability. We applied recombinant adeno-associated viruses by stereotaxic injection into the murine hippocampus to express different short hairpin RNA (shRNA) constructs along with eGFP. Tissue responses were assessed immunohistochemically for up to 8 weeks post-infection. Strong hippocampal degeneration and tissue atrophy was observed, most likely induced by high shRNA expression. The effect was entirely absent in mice injected with vectors driving only expression of eGFP. Active caspase-3 (Casp-3) and glial fibrillary acidic protein (GFAP) were identified as molecular markers and early indicators of adverse tissue responses. Our findings also demonstrate that detrimental effects of high shRNA expression in hippocampal tissue can be monitored even before the onset of tissue degeneration

Direct electrochemistry of novel affinity-tag immobilized recombinant horse heart cytochrome c

During the last decade protein electrochemistry at miniaturized electrodes has become important not only for functional studies of the charge transfer properties of redox proteins but also for fostering the development of sensitive biosensor and bioelectronic devices. One of the major challenges in this field is the directed coupling between electronic and biologically active components. A prerequisite for a fast and reversible electron transfer between electrode and protein is that the protein can be bound to the electrode in a favourable orientation. We examined electrostatic and bioaffinity-tag binding strategies for the directed immobilization of horse heart cytochrome c (cyt. c) on gold electrode surfaces to achieve this goal. Horse heart cyt. c was expressed in E. coli either as non-modified or genetically modified, i.e. histidine (his)-tag containing protein. The his-tags were introduced at defined positions at the N- or C-terminus of the polypeptide. It was our aim to generate tagged-versions of cyt. c that facilitate strong electronic coupling between protein and electrode and, at the same time, retain their catalytic and regulatory properties. The combination of different immobilization strategies, e.g. his-tag and electrostatic immobilization also opens new avenues for bivalent immobilization of proteins. This is of interest for molecular bioelectronic and biosensing applications where the proteins are immobilized between two crossing electrodes