1,721,010 research outputs found
2-AG-mediated control of GABAergic signaling is impaired in a model of epilepsy
No full textRepeated seizures result in a persistent maladaptation of endocannabinoid (eCB) signaling, mediated part by anandamide signaling deficiency in the basolateral amygdala (BLA) that manifests as aberrant synaptic function and altered emotional behavior. Here, we determined the effect of repeated seizures (kindling) on 2-arachidonoylglycerol (2-AG) signaling on GABA transmission by directly measuring tonic and phasic eCB-mediated retrograde signaling in an in vitro BLA slice preparation from male rats. We report that both activity-dependent and muscarinic acetylcholine receptor (mAChR)-mediated depression of GABA synaptic transmission was reduced following repeated seizure activity. These effects were recapitulated in sham rats by preincubating slices with the 2-AG synthesizing enzyme inhibitor DO34. Conversely, preincubating slices with the 2-AG degrading enzyme inhibitor KML29 rescued activity-dependent 2-AG signaling, but not mAChR-mediated synaptic depression, over GABA transmission in kindled rats. These effects were not attributable to a change in cannabinoid type 1 (CB1) receptor sensitivity or altered 2-AG tonic signaling since the application of the highly selective CB1 receptor agonist CP55,940 provoked a similar reduction in GABA synaptic activity in both sham and kindled rats, while no effect of either DO34 or of the CB1 inverse agonist AM251 was observed on frequency and amplitude of spontaneous IPSCs in either sham or kindled rats. Collectively, these data provide evidence that repeated amygdala seizures persistently alter phasic 2-AG-mediated retrograde signaling at BLA GABAergic synapses, probably by impairing stimulus-dependent 2-AG synthesis/release, which contributes to the enduring aberrant synaptic plasticity associated with seizure activity.SIGNIFICANCE STATEMENT The plastic reorganization of endocannabinoid (eCB) signaling after seizures and during epileptogenesis may contribute to the negative neurobiological consequences associated with seizure activity. Therefore, a deeper understanding of the molecular basis underlying the pathologic long-term eCB signaling remodeling following seizure activity will be crucial to the development of novel therapies for epilepsy that not only target seizure activity, but, most importantly, the epileptogenesis and the comorbid conditions associated with epilepsy
Design and Synthesis of Fluorescent Probes for Cannabinoid Receptor Type 1
No full textDer G-Protein-gekoppelte Cannabinoid-Rezeptor Typ 1 (CB1R) ist ein zentrales Signalelement des Endocannabinoid-Systems und an neurodegenerativen, kardiovaskulären, entzündlichen und fibrotischen Erkrankungen sowie an Angststörungen, Depressionen, Adipositas und Schmerzprozessen beteiligt. Damit ist CB1R ein relevantes Ziel für die Wirkstoffentwicklung. Fluoreszenzbasierte Studien eignen sich besonders, um Expression, Verteilung, Lokalisierung und Pharmakologie von CB1R mit hoher räumlich-zeitlicher Auflösung zu untersuchen. Derzeit fehlen jedoch geeignete chemische Fluoreszenz-Werkzeuge. Diese Dissertation behandelt daher Design und Synthese fluoreszierender Liganden als niedermolekulare Sonden zur Untersuchung von CB1R. Im ersten Teil wurde eine modulare, amidbasierte Synthesestrategie für CB1R-Sonden entwickelt. Unter Verwendung von α,α-Diethylglyzin als zentralem Strukturelement wurden Kombinationen mit CB1R-Erkennungsmotiven, Linkern und Fluorophoren systematisch untersucht. Ergebnis war eine reversible, CB1R-selektive, invers-agonistische fluoreszierende Sonde. Diese und weitere Vertreter erwiesen sich als nützliche Werkzeuge in CB1R-Fluoreszenzmikroskopie- und Target-Engagement-Studien. Der modulare Ansatz ermöglichte so den Zugang zu vielseitigen chemischen Fluoreszenz-Sonden zur Erforschung der Lokalisierung und Pharmakologie von CB1R. Im zweiten Teil wurde die Strategie auf „liganden-vermittelte“ Markierungssonden übertragen. Erstmals wurde eine fluoreszierende Sonde entwickelt, die den kovalenten Transfer eines fluorogenen Rhodamin-Farbstoffs auf CB1R ermöglichte. Zeitaufgelöste Förster-Resonanz-Energietransferstudien führten zur Entdeckung eines konformationellen Fluoreszenz-Biosensors zur Untersuchung orthosterischer Liganden und allosterischer Modulatoren. Diese neuartige Markierungssonde ermöglichte neue Einblicke in die molekulare Pharmakologie von CB1R.The G protein-coupled cannabinoid receptor type 1 (CB1R) is a key component of the endocannabinoid system. It is involved in neurodegenerative, cardiovascular, inflammatory, and fibrotic diseases, as well as anxiety disorders, depression, obesity, and pain processing. CB1R therefore represents a relevant target for drug development. Fluorescence-based studies are particularly suitable to investigate the expression, distribution, localization, and pharmacology of CB1R with high spatial and temporal resolution. However, suitable chemical fluorescent tools are currently lacking. This thesis addresses the design and synthesis of fluorescent ligands as small-molecule probes for studying CB1R.
In the first part, a modular amide-based synthetic strategy for CB1R probes was developed. Using α,α-diethylglycine as a central element, combinations with drug-like CB1R recognition motifs, linkers, and fluorophores were systematically explored. This led to the development of a reversible, CB1R-selective inverse agonist fluorescent probe. This and related probes proved effective in fluorescence microscopy and target engagement studies. The modular concept thus enabled access to versatile and high-quality fluorescent tools for investigating CB1R localization and pharmacology. In the second part, the strategy was applied to the design of “ligand-directed” labeling probes. For the first time, a fluorescent probe enabling covalent transfer of a fluorogenic rhodamine dye to CB1R was developed. Characterization by time-resolved Förster resonance energy transfer revealed a conformational fluorescent biosensor for studying orthosteric ligands and allosteric modulators. This new labeling probe provided valuable insights into the molecular pharmacology of CB1R
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Oxygenation of Anandamide by Lipoxygenases
Full text linkThe endocannabinoids anandamide and 2-arachidonoylglycerol are not only metabolized by serine hydrolases, such as fatty acid amide hydrolase, monoacylglycerol lipase, and α,β-hydrolases 6 and 12, but they also serve as substrates for cyclooxygenases, cytochrome P450s, and lipoxygenases. These enzymes oxygenate the 1Z,4Z-pentadiene system of the arachidonic acid backbone of endocannabinoids, thereby giving rise to an entirely new array of bioactive lipids. Hereby, a protocol is provided for the enzymatic synthesis, purification, and characterization of various oxygenated metabolites of anandamide generated by lipoxygenases, which enables the biological study and detection of these metabolites
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Development of a multiplexed activity-based protein profiling assay to evaluate activity of endocannabinoid hydrolase inhibitors
Full text linkEndocannabinoids, an important class of signaling lipids involved in health and disease, are predominantly synthesized and metabolized by enzymes of the serine hydrolase superfamily. Activity-based protein profiling (ABPP) using fluorescent probes, such as fluorophosphonate (FP)-TAMRA and β-lactone-based MB064, enables drug discovery activities for serine hydrolases. FP TAMRA and MB064 have distinct, albeit partially overlapping, target profiles, but cannot be used in conjunction due to overlapping excitation/emission spectra. We therefore synthesized a novel FP-probe with a green BODIPY as fluorescent tag and studied its labeling profile in mouse proteomes. Surprisingly, we found that the reporter tag plays an important role in the binding potency and selectivity of the probe. A multiplexed ABPP-assay was developed in which a probe cocktail of FP-BODIPY and MB064 visualized most endocannabinoid serine hydrolases in mouse brain proteomes in a single experiment. The multiplexed ABPP-assay was employed to profile endocannabinoid hydrolase inhibitor activity and selectivity in mouse brain
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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