Background and purpose: As baclofen is active in patients with anxiety disorders, GABA B receptors have been implicated in the modulation of anxiety. To avoid the side effects of baclofen, allosteric enhancers of GABA B receptors have been studied to provide an alternative therapeutic avenue for modulation of GABA B receptors. The aim of this study was to characterize derivatives of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) as enhancers of GABA B receptors. Experimental approach: Enhancing properties of rac-BHFF were assessed in the Chinese hamster ovary (CHO)-Ga16-hGABA B(1a,2a) cells by Fluorometric Imaging Plate Reader and GTPg[35 S]-binding assays, and in rat hippocampal slices by population spike (PS) recordings. In vivo activities of rac-BHFF were assessed using the loss of righting reflex (LRR) and stressinduced hyperthermia (SIH) models. Key results: In GTPg[35 S]-binding assays, 0.3 mM rac-BHFF or its pure enantiomer ( þ )-BHFF shifted the GABA concentrationresponse curve to the left, an effect that resulted in a large increase in both GABA potency (by 15.3-and 87.3-fold) and efficacy (149% and 181%), respectively. In hippocampal slices, rac-BHFF enhanced baclofen-induced inhibition of PS of CA1 pyramidal cells. In an in vivo mechanism-based model in mice, rac-BHFF increased dose-dependently the LRR induced by baclofen with a minimum effective dose of 3 mg kg À1 p.o. rac-BHFF (100 mg kg À1 p.o.) tested alone had no effect on LRR nor on spontaneous locomotor activity, but exhibited anxiolytic-like activity in the SIH model in mice. Conclusions and implications: rac-BHFF derivatives may serve as valuable pharmacological tools to elucidate the pathophysiological roles played by GABA B receptors in the central and peripheral nervous systems.

Although the Xenopus immunoglobulin heavy chain locus is structurally and functionally similar to mammalian IgH loci, Xenopus antibodies are limited in heterogeneity, and they mature only slightly in affinity during immune responses. During the antibody response of isogenic frogs to DNP-KLH, it and v cDNA sequences using elements of the VH1 family were cloned, sequenced and compared with germline counterparts. There were zero to four mutations per sequence, mostly single base substitutions, in the framework and CDRs 1 and 2 of VH. No mutations were found in JH. Since the point mutation rate was only 4-to 7-fold lower than that calculated for mice, affinity maturation does not seem to be limited by mutant availability. Because of a relatively low ratio of replacement to silent mutations in the CDRs and a very high ratio of GC to AT base pairs altered by mutation, it is suggested that the problem results from the absence of an effective mechanism for selecting mutants, which in turn might be related to the absence of germinal centers in Xenopus.

The orexins and their receptors are involved in the regulation of arousal and sleep-wake cycle. Clinical investigation with almorexant has indicated that this dual OX antagonist is efficacious in inducing and maintaining sleep. Using site-directed mutagenesis, ␤ 2 -adrenergic-based OX 1 7.39 are shared between almorexant and 1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone (SB-674042) binding sites in OX 1 . The nonconserved residue at position 3.33 of orexin receptors was identified as occupying a critical position that must be involved in subtype selectivity and also in differentiating two different antagonists for the same receptor. In summary, despite high similarities in the ligand-binding pockets of OX 1 and OX 2 and numerous aromatic/hydrophobic interactions, the local conformation of helix positions 3.32, 3.33, and 3.36 in transmembrane domain 3 and 45.51 in ECL2b provide the structural basis for pharmacologic selectivity between OX 1 and OX 2 .

The sense of taste is a chemosensory system responsible for basic food appraisal. Humans distinguish between five primary tastes: bitter, sweet, sour, salty and umami. The molecular events in the perception of bitter taste are believed to start with the binding of specific water-soluble molecules to G-protein-coupled receptors encoded by the TAS2R/T2R family of taste receptor genes. TAS2R receptors are expressed at the surface of taste receptor cells and are coupled to G proteins and second messenger pathways. We have identified, cloned and characterized 11 new bitter taste receptor genes and four new pseudogenes that belong to the human TAS2R family. Their encoded proteins have between 298 and 333 amino acids and share between 23 and 86% identity with other human TAS2R proteins. Screening of a mono-chromosomal somatic cell hybrid panel to assign the identified bitter taste receptor genes to human chromosomes demonstrated that they are located in chromosomes 7 and 12. Including the 15 sequences identified, the human TAS2R family is composed of 28 full-length genes and 16 pseudogenes. Phylogenetic analyses suggest a classification of the TAS2R genes in five groups that may reflect a specialization in the detection of specific types of bitter chemicals.

The early molecular events in the perception of bitter taste start with the binding of specific water-soluble molecules to G protein-coupled receptors (GPCRs) encoded by the Tas2r family of taste receptor genes. The identification of the complete TAS2R receptor family repertoire in mouse and a comparative study of the Tas2r gene families in mouse and human might help to better understand bitter taste perception. We have identified, cloned, and characterized 13 new mouse Tas2r sequences, 9 of which encode putative functional bitter taste receptors. The encoded proteins are between 293 and 333 amino acids long and share between 18% and 54% sequence identity with other mouse TAS2R proteins. Including the 13 sequences identified, the mouse Tas2r family contains approximately 30% more genes and 60% fewer pseudogenes than the human TAS2R family. Sequence and phylogenetic analyses of the proteins encoded by all mouse and human Tas2r genes indicate that TAS2R proteins present a lower degree of sequence conservation in mouse than in human and suggest a classification in five groups that may reflect a specialization in their functional activity to detect bitter compounds. Tas2r genes are organized in clusters in both mouse and human genomes, and an analysis of these clusters and phylogenetic analyses indicates that the five TAS2R protein groups were present prior to the divergence of the primate and rodent lineages. However, differences in subsequent evolutionary processes, including local duplications, interchromosomal duplications, divergence, and deletions, gave rise to species-specific sequences and shaped the diversity of the current TAS2R receptor families during mouse and human evolution.

The immunoglobulin (IgM) heavy chain constant region gene of the channel catfish, Ictalurus punctatus, has been cloned and characterized. The gene contains four constant region domain-encoding exons (CH1 to CH4) expressed in the secreted form of the immunoglobulin, and two exons encoding the transmembrane (TM) domain utilized in the lymphocyte membrane receptor form of the immunoglobulin. The sequence of a cDNA clone encoding the 3' region of the message for the membrane receptor form of the mu chain indicates that the TM1 exon is spliced directly to the CH3 exon, and not into a site within the CH4 exon, as occurs in the mammals, a shark and an amphibian. This unusual pattern of splicing, which produces a membrane heavy chain that is characteristically smaller than the secreted heavy chain, may be common to all teleost fish.

Recent

CTX, a cortical thymocyte marker in Xenopus, is an immunoglobulin superfamily (Igsf) member comprising one variable and one constant C2-type Igsf domain, a transmembrane segment and a cytoplasmic tail. Although resembling that of the TCR and immunoglobulins, the variable domain is not encoded by somatic rearrangement of the gene but by splicing of two half-domain exons. The C2 domain, also encoded by two exons, has an extra pair of cysteines. The transmembrane segment is free of charged residues, and the cytoplasmic tail (70 amino acids) contains one tyrosine and many glutamic acid residues. ChT1, a chicken homologue of CTX, has the same structural and genetic features, and both molecules are expressed on the thymocyte surface. We cloned new mouse (CTM) and human (CTH) cDNA and genes which are highly homologous to CTX/ChT1 but not lymphocyte specific. Similarity with recently described human cell surface molecules, A33 antigen and CAR (coxsackie and adenovirus 5 receptor), and a number of expressed sequence tags leads us to propose that CTX defines a novel subset of the Igsf, conserved throughout vertebrates and extending beyond the immune system. Strong homologies within vertebrate sequences suggest that the V and C2 CTX domains are scions of a very ancient lineage.