Characterization of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one as a positive allosteric modulator of GABAB receptors.

BACKGROUND AND PURPOSE: As baclofen is active in patients with anxiety disorders, GABAB receptors have been implicated in the modulation of anxiety. To avoid the side effects of baclofen, allosteric enhancers of GABAB receptors have been studied to provide an alternative therapeutic avenue for modulation of GABAB 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 GABAB receptors. EXPERIMENTAL APPROACH: Enhancing properties of rac-BHFF were assessed in the Chinese hamster ovary (CHO)-Galpha16-hGABA(B1a,2a) cells by Fluorometric Imaging Plate Reader and GTPgamma[35S]-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 stress-induced hyperthermia (SIH) models. KEY RESULTS: In GTPgamma[35S]-binding assays, 0.3 microM rac-BHFF or its pure enantiomer (+)-BHFF shifted the GABA concentration-response 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(-1) p.o. rac-BHFF (100 mg kg(-1) 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 GABAB receptors in the central and peripheral nervous systems.

Functional expression of mammalian bitter taste receptors in Caenorhabditis elegans.

Bitter taste has evolved as a central warning signal against the ingestion of potentially toxic substances appearing in the environment. The molecular events in the perception of bitter taste start with the binding of specific water-soluble molecules to G protein-coupled receptors (GPCR) called T2Rs and expressed at the surface of taste receptor cells. The functional characterisation of T2R receptors is far from been completed due to the difficulty to functionally express them in heterologous systems. Taking advantage of the parallelisms between the Caenorhabditis elegans (C. elegans) and mammalian GPCR signalling pathways, we developed a C. elegans-based expression system to express functional human and rodent GPCRs of the T2R family. We generated transgenic worms expressing T2Rs in ASI chemosensory neurons and performed behavioural assays using a variety of bitter tastants. As a proof of the concept, we generated transgenic worms expressing human T2R4 or its mouse ortholog T2R8 receptors, which respond to two bitter tastants previously characterised as their functional ligands, 6-n-propyl-2-thiouracil and denatoniun. As expected, expression of human T2R4 or its mouse ortholog T2R8 in ASI neurons counteracted the water-soluble avoidance to 6-n-propyl-2-thiouracil and denatoniun observed in control wild-type worms. The expression in ASI neurons of human T2R16, the ligand of which, phenyl-beta-d-glucopyranoside, belong to a chemically different group of bitter tastants, also counteracted the water-soluble avoidance to this compound observed in wild-type worms. These results indicate that C. elegans is a suitable heterologous expression system to express functional T2Rs providing a tool to efficiently search for specific taste receptor ligands and to extend our understanding of the molecular basis of gustation.

Identification and characterization of human taste receptor genes belonging to the TAS2R family.

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.

Axolotl MHC architecture and polymorphism.

The MHC of the urodele amphibian Ambystoma mexicanum consists of multiple polymorphic class I loci linked, so far as yet known, to a single class II B locus. This architecture is very different from that of the anuran amphibian Xenopus. The number of class I loci in the axolotl can vary from 6 to 21 according to the haplotypes as shown by cDNA analysis and Southern blot studies in families. These loci can be classified into seven sequence groups with features ranging from the class Ia to the class Ib type. All individuals express genes from at least three of the seven groups, and all individuals possess the class Ia-like type.

CTX, a Xenopus thymocyte receptor, defines a molecular family conserved throughout vertebrates.

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.

The T cell receptor beta genes of Xenopus.

cDNA of the T cell receptor beta (TCRB) have been isolated from the anuran amphibian Xenopus and they show strong structural homology to TCRB sequences of other vertebrates. Ten BV families, two D segments, ten J segments, and a single C region have been defined so far. Each V family consists of one to two members per haploid genome. A unique feature of the Xenopus TCRB constant region is the lack of N-linked carbohydrate glycosylation sites. The recombination signal sequences suggest that the mechanism of rearrangements are identical to those of mammals. The locus is inherited in a diploid manner despite the pseudotetraploidy of the Xenopus laevis and X. gilli used in this study.

CTX, a novel molecule specifically expressed on the surface of cortical thymocytes in Xenopus.

CTX, a novel developmentally regulated type-I transmembrane protein is expressed specifically by a large fraction of cortical thymocytes in the amphibian Xenopus. This apparently monomeric 55-kDa glycoprotein is composed of two immunoglobulin domains, one variable (V) and one constant (C2 type), followed by a transmembrane and a 64-amino acid cytoplasmic domain. The first immunoglobulin domain is a V-J segment that is generated without gene rearrangement. In the genome, the V and C2 domains are both encoded by two half-domain exons. Two CTX loci are found per haploid genome, and they exhibit sequence differences with a high replacing/silent ratio in the CDR1-like region of the V domain, suggesting that these differences were selected. The cytoplasmic domain contains a motif that is highly conserved evolutionarily in several types of proteins, including adenylyl cyclases. Based on its unique tissue distribution, the variability of its V region and the motif of its cytoplasmic domain, CTX is a candidate for a new type of specific molecule involved in thymocyte selection.

Membrane exon sequences of the three Xenopus Ig classes explain the evolutionary origin of mammalian isotypes.

We have cloned and sequenced the genes corresponding to the membrane exons of the three immunoglobulin (Ig) heavy chain isotypes (mu, upsilon and chi) of Xenopus. Among membrane Ig (mIg) polypeptides, the transmembrane domain are the most highly conserved. The transmembrane and cytoplasmic domains of Xenopus mIgM are similar to the corresponding domains of all known vertebrate mIgM molecules, supporting the idea that amphibian mu gene is homologous, not just analogous, to the mu gene of higher vertebrates. The membrane forms of the two other Ig isotypes mIgX and mIgY exhibit the specific structure found in all Ig membrane exons, but are not homologous with any specific mammalian non-mu Ig isotype; they are most similar to Xenopus mIgM. Based on the conserved transmembrane domains of Xenopus mIgX, mIgY, we suggest that first the upsilon and later the chi genes arose by duplication from the original mu gene. The transmembrane and the 37-amino-acid-long cytoplasmic domains of Xenopus mIgY have conserved residues found in avian mIgY and mammalian mIgG and mIgE, suggesting that the modern isotypes might share a common ancestor with amphibian mIgY. However, while the sequence similarity between the membrane exons of avian mIgY and mammalian mIgG and IgE is striking, the overall similarity with Xenopus mIgY is very low. Thus, the genes giving rise to Xenopus mIgY and those eventually leading to avian mIgY and mammalian mIgG and mIgE must have diverged early in evolution, probably at the level of the primitive amphibians or before.

Somatic mutations during an immune response in Xenopus tadpoles.

The tadpole B-cell repertoire is less diverse than that of the adult frog; their antibodies are of lower affinity and are less heterogenous. In order to determine whether this difference is due to a lack of or a reduced rate of somatic hypermutation, we analyzed and compared cDNA sequences utilizing VH1 elements with germline counterparts in isogenic LG7 tadpoles during an immune response. Indeed, tadpole VH1 sequences contained somatic mutations. There were zero to 5 mutations per sequence, all single base-point mutations, with the high ratio of GC to AT base-pair alterations similar to that observed in adult frogs.

What limits affinity maturation of antibodies in Xenopus--the rate of somatic mutation or the ability to select mutants?

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, mu and upsilon 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.

Sequences of C mu and the VH1 family in LG7, a clonable strain of Xenopus, homozygous for the immunoglobulin loci.

Twenty-eight heavy-chain variable (VH1) region genes and the immunoglobulin (IgM) heavy-chain constant region of an isogenic Xenopus hybrid, X. laevis/X. gilli, LG7, have been sequenced. The LG7 clone represents the first Xenopus hybrid that is homozygous for the IgH locus. The VH1 family was specifically investigated because VH1 genes are used by the antibodies produced during the Xenopus antidinitrophenol (DNP) response, These VH1 germ-line sequences establish a so-called "dictionary" that is available for studying somatic hypermutational mechanisms in immunized frogs.

Ribosomal and chromosomal protein cDNA clones of Xenopus laevis thymus isolated with differential screening.

1. Xenopus laevis is an excellent system for the study of the evolution and ontogeny of the immune system. But since only immunoglobulin genes have been isolated from this species, we undertook to isolate other genes expressed in an immunologically important organ, the thymus. 2. We used differential screening of a thymus cDNA library with cDNA probes made from thymus and from erythroblasts. 3. Approximately 50 clones which hybridized to the probe from thymus, but not from erythroblast, were isolated and sequenced from their termini. 4. Several clones were identified in data bank searches by their similarity to previously published sequences, and the partial sequences of these loci are reported here. 5. These include elongation factor 2, ribosomal protein S11, ribosomal protein S13, and the high mobility group protein. 6. Although these genes are not expected to be involved in an immune function, the availability of these sequences will facilitate the study of these loci in this species.

The immunoglobulin M heavy chain constant region gene of the channel catfish, Ictalurus punctatus: an unusual mRNA splice pattern produces the membrane form of the molecule.

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.

Molecular cloning of sheep T cell receptor gamma and delta chain constant regions: unusual primary structure of gamma chain hinge segments.

The primary structure of sheep T cell receptor (TcR) gamma and delta chain constant (C) regions has been determined by cDNA cloning. A comparison of the nucleotide and deduced amino acid sequences of the sheep chains with known human and mouse sequences shows that the primary structure of the immunoglobulin, transmembrane and cytoplasmic C gamma domains and all of the C delta region has been substantially conserved. However, the hinge or connector region of sheep gamma chains differs significantly from all known TcR chains. Clones representing two different sheep C gamma genes were isolated and both contain additional sequence in this region, making them the longest TcR chains so far identified. The hinge region of both sheep C gamma sequences contains two additional cysteine residues and a motif of five amino acids (TTESP or TTEPP) which has been triplicated in one of the clones. Other repetitive segments of 13-17 amino acids could also be identified suggesting that, as in the human C gamma 2 gene, this region of the sheep genes could have arisen from an exon duplication or triplication event. Southern blot analysis of sheep DNA confirmed the presence of one C delta gene and at least two C gamma genes. A restriction fragment length polymorphism that is probably associated with allelic sequence variation in the sheep C delta gene was detected in DNA from different animals. Although the essential structure of the gamma/delta TcR appears well conserved through evolution, the marked heterogeneity evident in the hinge region of gamma chains both within and between species, and particularly the presence of additional cysteine residues in the sheep sequences, may be of structural and functional importance.

Class I major histocompatibility complex cDNA clones from sheep thymus: alternative splicing could make a long cytoplasmic tail.

To investigate the class I major histocompatibility complex (MHC) genes expressed in the young sheep thymus, a cDNA library was screened with a human HLA-B7 cDNA probe under conditions of relaxed stringency. Thirteen clones were isolated and found by partial sequences to fall into five classes, requiring the expression of at least three loci. One sequence was found six times, almost half of the total, and may thus represent the major message expressed in the young sheep thymus. One of the clones was found to have failed to excise the intron between cytoplasmic exons 7 and 8, leading to the predicted synthesis of a cytoplasmic domain 23 amino acids longer than the other sheep sequences, and 15 amino acids longer than any cytoplasmic domain previously described. The sequences of all the clones were found to be most similar to bovine, and least similar to mouse class I MHC sequences.

T cell receptor gene expression in sheep: differential usage of TcR1 in the periphery and thymus.

We have isolated cDNA clones for each chain of the sheep T cell receptor (TcR) and used these to analyze TcR gene expression in thymocyte and peripheral T cell subsets. Outer cortical thymocytes expressed a low level of mature message for all TcR chains suggesting that intrathymic precursors for the alpha/beta and gamma/delta lineages occur in this population. Inner cortical and medullary thymocytes expressed high levels of mature alpha/beta transcripts, low levels of mature delta transcripts but no detectable gamma message. Mature alpha/beta transcripts were detected in peripheral CD4+ lymphocytes and these as well as CD8+ cells expressed a surface heterodimer of 85 kDa which resolved into 40- and 50-kDa subunits after reduction. Peripheral CD4-CD8-lymphocytes, which in sheep are marked by the T19 antigen and may account for up to 60% of T cells in blood, expressed a surface heterodimer of 75 kDa. The T19+ cells had high levels of mature delta and truncated beta transcripts in their cytoplasm but did not express the C gamma gene detected in DN thymocytes, although they seem to share V gamma and/or J gamma elements. Both forms of the sheep TcR are associated with CD3 molecules on the cell surface. These results show that (a) in contrast to the situation in rodents and humans, a large proportion of peripheral sheep lymphocytes use TcR gamma/delta; (b) the proportion of T cells in the periphery which use TcR gamma/delta is greater than in thymus; and (c) CD4-CD8- cells in thymus and periphery (T19+) use the same C delta gene but appear to use different C gamma genes, suggesting that in sheep there may be more than one lineage of lymphocytes expressing TcR gamma/delta.

Conservation of structural and functional domains in complement component C3 of Xenopus and mammals.

The cDNA sequence and the deduced amino acid sequence of the Mr 34,000 C-terminal fragment of Xenopus laevis complement component C3 are presented. The sequence of Xenopus C3 has 57% nucleotide identity to the corresponding sequence of human C3 and approximately 49% amino acid identity to C3 from human, mouse, and rabbit. The Xenopus C3 sequence shows clusters of high and of low similarity to the mammalian C3 sequences. One of these regions of high similarity represents the domain of mammalian C3b involved in the binding of properdin, a regulator of the alternative pathway of complement activation. It is not clear whether the other highly conserved regions are involved in binding to other C3 ligands. The Xenopus C3 sequence completely lacks the Arg-Gly-Asp sequence, which has been suggested to be the recognition site of the human complement receptor type 3 on the iC3b fragment of human C3. The Xenopus C3 gene is shown not to be linked to the Xenopus major histocompatibility complex, as is also the case in mammals. Since the gene of the related molecule C4 is MHC-linked in both mammals and Xenopus, the C3 and C4 genes may have separated before Xenopus and mammals speciated.