Arachidonylethanolamide (anandamide), a candidate endogenous cannabinoid ligand, has recently been isolated from porcine brain and displayed cannabinoid-like binding activity to synaptosomal membrane preparations and mimicked cannabinoid-induced inhibition of the twitch response in isolated murine vas deferens. In this study, anandamide and several congeners were evaluated as cannabinoid agonists by examining their ability to bind to the cloned cannabinoid receptor, inhibit forskolin-stimulated cAMP accumulation, inhibit N-type calcium channels, and stimulate one or more functional second messenger responses. Synthetic anandamide, and all but one congener, competed for [3H]CP55,940 binding to plasma membranes prepared from L cells expressing the rat cannabinoid receptor. The ability of anandamide to activate receptor-mediated signal transduction was evaluated in Chinese hamster ovary (CHO) cells expressing the human cannabinoid receptor (HCR, termed CHO-HCR cells) and compared to control CHO cells expressing the muscarinic m5 receptor (CHOm5 cells). Anandamide inhibited forskolin-stimulated cAMP accumulation in CHO-HCR cells, but not in CHOm5 cells, and this response was blocked with pertussis toxin. N-type calcium channels were inhibited by anandamide and several active congeners in N18 neuroblastoma cells. Anandamide stimulated arachidonic acid and intracellular calcium release in both CHOm5 and CHO-HCR cells and had no effect on the release of inositol phosphates or phosphatidylethanol, generated after activation of phospholipase C and D, respectively. Anandamide appears to exhibit the essential criteria required to be classified as a cannabinoid/anandamide receptor agonist and shares similar nonreceptor effects on arachidonic acid and intracellular calcium release as other cannabinoid agonists.Both the psychoactive and medicinal properties of marijuana have been known for centuries, but not until the last decade has a clear mechanism of action been ascribed to A9-tetrahydrocannabinol (THC), the active principle of marijuana. It is now known that THC and other more potent synthetic cannabinoid agonists bind to specific cannabinoid receptors and couple functionally to inhibit adenylate cyclase (1, 2) and inhibit N-type calcium channels via a pertussis toxin-sensitive guanine nucleotide binding protein (G protein) (3,4). The existence of the cannabinoid receptor was corroborated with the cloning of a cannabinoid receptor gene from both rat and human (5, 6). To date only a single cannabinoid receptor gene has been identified and its nucleotide sequence indicates that it belongs to the superfamily of G-protein-coupled receptors. Expression studies indicateThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.that the cloned receptor and the native receptor display similar binding and functional coupling to the inhibition of adenylate cyclase (5-7). The ...
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A method to extract peptides and low molecular weight proteins from serum under denaturing conditions using acetonitrile containing 0.1% trifluoroacetic acid has been developed. The extraction procedure precipitates large, abundant proteins to simplify subsequent mass spectral analysis. This sample preparation method provides an efficient way to extract serum peptides, enabling them to be compared and identified using different mass spectrometry approaches. Surface-enhanced laser desorption/ionization-time of flight mass spectrometry analysis of mouse blood serum samples prepared by this method allowed detection of two markers which were significantly reduced in mice with B cell lymphoma tumor. One of these markers has been identified as apolipoprotein A-II.
The mutant beta1,4-galactosyltransferase (beta4Gal-T1), beta4Gal-T1-Y289L, in contrast to wild-type beta4Gal-T1, can transfer GalNAc from the sugar donor UDP-GalNAc to the acceptor, GlcNAc, with efficiency as good as that of galactose from UDP-Gal. Furthermore, the mutant can also transfer a modified sugar, C2 keto galactose, from its UDP derivative to O-GlcNAc modification on proteins that provided a functional handle for developing a highly sensitive chemoenzymatic method for detecting O-GlcNAc post-translational modification on proteins. We report herein that the modified sugar, C2 keto galactose, can be transferred to free GlcNAc residues on N-linked glycoproteins, such as ovalbumin or asialo-agalacto IgG1. The transfer is strictly dependent on the presence of both the mutant enzyme and the ketone derivative of the galactose. Moreover, the PNGase F treatment of the glycoproteins, which cleaves the N-linked oligosaccharide chain, shows that the modified sugar has been transferred to the N-glycan chains of the glycoproteins and not to the protein portion. The application of the mutant galactosyltransferase, beta4Gal-T1-Y289L, to produce glycoconjugates carrying sugar moieties with reactive groups, is demonstrated. We envision a broad potential for this technology such as the possibilities to link cargo molecules to glycoproteins, such as monoclonal antibodies, via glycan chains, thereby assisting in the glycotargeting of drugs to the site of action or used as biological probes.
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