Increased risk of suicide in schizophrenia patients with linkage to chromosome 13q.

We link schizophrenia in families from the genetically isolated South African Afrikaner population to chromosome 13q (n =51), 1p (n =23) and combined 13q & 1p (n =18). Patients with linkages to chromosome 13q were 4.16 times more likely to meet diagnostic criteria for schizoaffective disorder compared to patients with linkage to 1p. A third of patients with linkage to both 13q &1p met diagnostic criteria for SAD. There was a significant positive relationship between suicidality and a diagnosis of schizoaffective disorder. Identifying linkage to chromosome 13q may be informative in identifying suicide risk early and prevent morbidity and mortality in schizophrenia patients.

Phenotypic features of patients with schizophrenia carrying de novo gene mutations: a pilot study.

Genome-wide scans have revealed a significant role for de novo copy number variants (CNVs) and Single Nucleotide variants (SNVs) in the genetic architecture of schizophrenia. The present study attempts to parse schizophrenia based on the presence of such de novo mutations and attempts genotype-phenotype correlation. We examined phenotypic variables across three broad categories: clinical presentation, premorbid function, disease course and functional outcome and compared them in individuals with schizophrenia carrying either a de novo CNV, a de novo SNV, or no de novo mutation. Work skills were worst affected in patients carrying de novo CNVs. More learning disabilities were found in subjects carrying de novo SNVs. Patients with either mutation had older parents at birth and worse functional outcome as measured by SLOF scores. We found no relation between treatment resistance and the presence of de novo mutations. The combined consideration of the functional outcome scores and early deviant behaviours was found to have higher predictive value for underlying genetic vulnerability. Due to the rare nature of the de novo mutations the sample sizes studied here were small. Despite this, valuable phenotypic characteristics were identified in schizophrenia patients carrying de novo mutations and studying larger samples will be of interest.

Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic glutamate receptor 7.

BACKGROUND AND PURPOSE: The mGlu(7) receptors are strategically located at the site of vesicle fusion where they modulate the release of the main excitatory and inhibitory neurotransmitters. Consequently, they are implicated in the underlying pathophysiology of CNS diseases such as epilepsy and stress-related psychiatric disorders. Here, we characterized a selective, potent and functional anti-mGlu(7) monoclonal antibody, MAB1/28, that triggers receptor internalization. EXPERIMENTAL APPROACH: MAB1/28's activity was investigated using Western blot and direct immunofluorescence on live cells, in vitro pharmacology by functional cAMP and [(35) S]-GTPγ binding assays, the kinetics of IgG-induced internalization by image analysis, and the activation of the ERK1/2 by elisa. KEY RESULTS: mGlu(7) /mGlu(6) chimeric studies located the MAB1/28 binding site at the extracellular amino-terminus of mGlu(7) . MAB1/28 potently antagonized both orthosteric and allosteric agonist-induced inhibition of cAMP accumulation. The potency of the antagonistic actions was similar to the potency in triggering receptor internalization. The internalization mechanism occurred via a pertussis toxin-insensitive pathway and did not require Gα(i) protein activation. MAB1/28 activated ERK1/2 with potency similar to that for receptor internalization. The requirement of a bivalent receptor binding mode for receptor internalizations suggests that MAB1/28 modulates mGlu(7) dimers. CONCLUSIONS AND IMPLICATIONS: We obtained evidence for an allosteric-biased agonist activity triggered by MAB1/28, which activates a novel IgG-mediated GPCR internalization pathway that is not utilized by small molecule, orthosteric or allosteric agonists. Thus, MAB1/28 provides an invaluable biological tool for probing mGlu(7) function and selective activation of its intracellular trafficking.

Characterization of RO4583298 as a novel potent, dual antagonist with in vivo activity at tachykinin NK1 and NK3 receptors.

BACKGROUND AND PURPOSE: Clinical results of osanetant and talnetant (selective-NK3 antagonists) indicate that blocking the NK3 receptor could be beneficial for the treatment of schizophrenia. The objective of this study was to characterize the in vitro and in vivo properties of a novel dual NK1/NK3 antagonist, RO4583298 (2-phenyl-N-(pyridin-3-yl)-N-methylisobutyramide derivative). EXPERIMENTAL APPROACH: RO4583298 in vitro pharmacology was investigated using radioligand binding ([³H]-SP, [³H]-osanetant, [³H]-senktide), [³H]-inositol-phosphate accumulation Schild analysis (SP- or [MePhe7]-NKB-induced) and electrophysiological studies in guinea-pig substantia nigra pars compacta (SNpc). The in vivo activity of RO4583298 was assessed using reversal of GR73632-induced foot tapping in gerbils (GFT; NK1) and senktide-induced tail whips in mice (MTW; NK3). KEY RESULTS: RO4583298 has a high-affinity for NK1 (human and gerbil) and NK3 (human, cynomolgus monkey, gerbil and guinea-pig) receptors and behaves as a pseudo-irreversible antagonist. Unusually it binds with high-affinity to mouse and rat NK3, yet with a partial non-competitive mode of antagonism. In guinea-pig SNpc, RO4583298 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurones with an apparent non-competitive mechanism of action. RO4583298 (p.o.) robustly blocked the GFT response, and inhibited the MTW. CONCLUSIONS AND IMPLICATIONS: RO4583298 is a high-affinity, non-competitive, long-acting in vivo NK1/NK3 antagonist; hence providing a useful in vitro and in vivo pharmacological tool to investigate the roles of NK1 and NK3 receptors in psychiatric disorders.

Biochemical and behavioural characterization of EMPA, a novel high-affinity, selective antagonist for the OX(2) receptor.

BACKGROUND AND PURPOSE: The OX(2) receptor is a G-protein-coupled receptor that is abundantly found in the tuberomammillary nucleus, an important site for the regulation of the sleep-wake state. Herein, we describe the in vitro and in vivo properties of a selective OX(2) receptor antagonist, N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulphonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA). EXPERIMENTAL APPROACH: The affinity of [(3)H]EMPA was assessed in membranes from HEK293-hOX(2)-cells using saturation and binding kinetics. The antagonist properties of EMPA were determined by Schild analysis using the orexin-A- or orexin-B-induced accumulation of [(3)H]inositol phosphates (IP). Quantitative autoradiography was used to determine the distribution and abundance of OX(2) receptors in rat brain. The in vivo activity of EMPA was assessed by reversal of [Ala(11),D-Leu(15)]orexin-B-induced hyperlocomotion during the resting phase in mice and the reduction of spontaneous locomotor activity (LMA) during the active phase in rats. KEY RESULTS: [(3)H]EMPA bound to human and rat OX(2)-HEK293 membranes with K(D) values of 1.1 and 1.4 nmol x L(-1) respectively. EMPA competitively antagonized orexin-A- and orexin-B-evoked accumulation of [(3)H]IP at hOX(2) receptors with pA(2) values of 8.6 and 8.8 respectively. Autoradiography of rat brain confirmed the selectivity of [(3)H]EMPA for OX(2) receptors. EMPA significantly reversed [Ala(11),D-Leu(15)]orexin-B-induced hyperlocomotion dose-dependently during the resting phase in mice. EMPA, injected i.p. in rats during the active phase, reduced LMA dose-dependently. EMPA did not impair performance of rats in the rotarod procedure. CONCLUSIONS AND IMPLICATIONS: EMPA is a high-affinity, reversible and selective OX(2) receptor antagonist, active in vivo, which should prove useful for analysis of OX(2) receptor function.

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.

[Blood transfusion safety: current progress]. / Sécurité transfusionnelle: contribution des progrès réalisés.

For the past ten years, a real improvement in knowledge and methods concerning blood transfusion safety has been made. In this observation, concerning a polytraumatism patient who received massive blood transfusion with no immunologic nor infectious complications occurring one year later, brings evidence of real progress on blood transfusion safety for improvement in short and long term prognosis for polytransfused patients.

[Curarization for contributing symptomatic treatment of myoclonic jerks induced by chloralose poisoning]. / Curarisation en traitement symptomatique d'appoint des myoclonies induites par une intoxication par le chloralose.

Chloralose poisoning especially leads to consciousness failure and hyperexcitability with typical myoclonic jerks. In an observation of chloralose poisoning by ingestion of synthetic substance against moles, curarization by cistracurium besylate was proposed to contribute in the symptomatic treatment of myoclonus syndrome induced by chloralose poisoning and resistant to a large dose of diazepam.

Pharmacological characterization of Ro 63-1908 (1-[2-(4-hydroxy-phenoxy)-ethyl]-4-(4-methyl-benzyl)-piperidin-4-ol), a novel subtype-selective N-methyl-D-aspartate antagonist.

Ro 63-1908, 1-[2-(4-hydroxy-phenoxy)-ethyl]-4-(4-methyl-benzyl)-piperidin-4-ol, is a novel subtype-selective N-methyl-D-aspartate (NMDA) antagonist that has been characterized in vitro and in vivo. Ro 63-1908 inhibited [(3)H]dizocilpine ((3)H-MK-801) binding in a biphasic manner with IC(50) values of 0.002 and 97 microM for the high- and low-affinity sites, respectively. Ro 63-1908 selectively blocked recombinant receptors expressed in Xenopus oocytes containing NR1C + NR2B subunits with an IC(50) of 0.003 microM and those containing NR1C + NR2A subunits with an IC(50) of >100 microM, thus demonstrating greater than 20,000-fold selectivity for the recombinant receptors expressing NR1C + NR2B. Ro 63-1908 blocked these NMDA NR2B-subtype receptors in an activity-dependent manner. Ro 63-1908 was neuroprotective against glutamate-induced toxicity and against oxygen/glucose deprivation-induced toxicity in vitro with IC(50) values of 0.68 and 0.06 microM, respectively. Thus, the in vitro pharmacological characterization demonstrated that Ro 63-1908 was a potent and highly selective antagonist of the NR2B subtype of NMDA receptors. Ro 63-1908 was active against sound-induced seizures (ED(50) = 4.5 mg/kg i.p. when administered 30 min beforehand) in DBA/2 mice. The dose required to give a full anticonvulsant effect did not produce a deficit in the Rotarod test. NMDA-induced seizures were also inhibited by Ro 63-1908 with an ED(50) of 2.31 mg/kg i.v. when administered 15 min before testing. Ro 63-1908 gave a dose-related neuroprotective effect against cortical damage in a model of permanent focal ischemia. Maximum protection of 39% was seen at a plasma concentration of 450 ng/ml. There were, however, no adverse cardiovascular or CNS side-effects seen at this dosing level.

Positive allosteric modulators of metabotropic glutamate 1 receptor: characterization, mechanism of action, and binding site.

We have identified two chemical series of compounds acting as selective positive allosteric modulators (enhancers) of native and recombinant metabotropic glutamate 1 (mGlu1) receptors. These compounds did not directly activate mGlu1 receptors but markedly potentiated agonist-stimulated responses, increasing potency and maximum efficacy. Binding of these compounds increased the affinity of a radiolabeled glutamate-site agonist at its extracellular N-terminal binding site. Chimeric and mutated receptors were used to localize amino acids in the receptor transmembrane region critical for these enhancing properties. Finally, the compounds potentiated synaptically evoked mGlu1 receptor responses in rat brain slices. The discovery of selective positive allosteric modulators of mGlu1 receptors opens up the possibility to develop a similar class of compounds for other family 3 G protein-coupled receptors.

Identification of essential residues involved in the glutamate binding pocket of the group II metabotropic glutamate receptor.

Metabotropic glutamate (mGlu) receptors are a family of G-protein-coupled receptors that play central roles as modulators of both glutamatergic and other major neurotransmitter systems in CNS. Using molecular modeling, site-directed mutagenesis, [(3)H]LY354740 binding, [(35)S]GTPgammaS binding, and activation of GIRK current, we have been able to identify residues crucial for the binding of LY354740 and glutamate to rat mGlu2 receptors. Several of the crucial residues located in the binding site (Arg-57, Tyr-144, Tyr-216, Asp-295) have not been identified previously. We propose that the gamma-carboxyl group of LY354740 forms H-bonds to Arg-57, whereas the alpha-carboxyl group forms an H-bond with the hydroxyl group of Ser-145. The alpha-amino group of LY354740 forms H-bonds to Asp-295 and to the side-chain hydroxyl group of Thr-168. In addition, Tyr-144 may establish a hydrophobic (C-H/pi)-interaction with the bicyclo-hexane ring of LY354740. Furthermore, the mutation of residues Ser-148 and Arg-183, which are too remote for a direct interaction, affected the ligand affinity dramatically. These results suggest that Ser-148 and Arg-183 may be important for the 3D structure and/or are involved in closure of the domain. Finally, Asp-146, which is also remote from the binding site, was shown to be involved in the differential binding affinity of [(3)H]LY354740 for mGlu2 versus mGlu3 receptors. All the mGlu receptors except mGlu2 are activated by Ca(2+) and have serine instead of aspartic acid at this position, which suggests a critical role of this aspartic acid residue in the binding properties of this unique receptor.

Pharmacological characterization of the novel nonpeptide orphanin FQ/nociceptin receptor agonist Ro 64-6198: rapid and reversible desensitization of the ORL1 receptor in vitro and lack of tolerance in vivo.

The novel nonpeptide orphanin FQ/nociceptin (OFQ/N) ligand [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one] (Ro 64-6198) was characterized in vitro and in vivo for its agonistic potential. Ro 64-6198 was 130- to 3500-fold selective for the OFQ/N receptor (ORL1) compared with opiate receptors. In the cAMP inhibition assay, Ro 64-6198 was a full agonist at the ORL1 and a partial agonist at the mu opiate receptor. When human embryonic kidney 293 cells stably expressing the human ORL1 receptor were pre-exposed (30 min) to either OFQ/N or Ro 64-6198, the ability of both agonists to inhibit forskolin-mediated cAMP accumulation was strongly reduced, indicating a functional desensitization of the second messenger cascade. However, acidic washes of OFQ/N-exposed cells fully restored the sensitivity of the ORL1 receptor for agonists. In contrast, the cAMP response in Ro 64-6198-exposed cells remained impaired after acidic washes, suggesting sustained receptor internalization at 30 min. In agreement with this finding, the number of cell-surface ORL1 receptors was significantly reduced after Ro 64-6198 pre-exposure, and this effect could be blocked with high sucrose concentrations. When Ro 64-6198 was chronically administered to rats, no signs of tolerance to its anxiolytic-like effects were detected following 15 days of daily drug exposure. In agreement with the behavioral results, Ro 64-6198 was able to reduce brain ORL1 binding sites in both acutely and chronically treated rats. Full recovery of ORL1 binding sites was observed 24 h after Ro 64-6198 administration with a t1/2 of approximately 5.5 h. These data show that nonpeptide agonists at the ORL1 receptor have a good clinical potential as anxiolytics without causing tolerance.

Characterization of transgenic mice expressing apolipoprotein E4(C112R) and apolipoprotein E4(L28P; C112R).

Apolipoprotein E (ApoE), which is genetically polymorphic, is a constituent of different lipoproteins. Two variants, ApoE4(C112R) and ApoE4(L28P; C112R) have been linked to the risk of developing Alzheimer's disease. Transgenic mice carrying ApoE4(C112R) (AD71) and ApoE4(L28P; C112R) (AD61) were generated and compared to wild-type mice. The use of glial fibrillary acidic protein as promoter led to transgene expression mainly in glial cells but also in neurons. Transgene protein levels were approximately three-and-a-half-fold that of endogenous ApoE in the glial fibrillary acidic protein-ApoE4(C112R) (AD71) and nearly twofold in the glial fibrillary acidic protein-ApoE4(L28P; C112R) (AD61) mouse lines. Neither transgenic mouse differed from wild-type in cognitive tests at the age of approximately one-and-a-half years. The locomotor activity of AD61 mice was similar to controls, whereas AD71 mice exhibited a clearly reduced level of motor activity. Immunohistological and biochemical brain protein analyses revealed no difference between strains.Thus, in the absence of morphological changes over-expression of ApoE4(C112R) on a background of endogenous mouse ApoE, may result in behavioral deficits while for the ApoE4(L28P; C112R) transgene higher expression might be required or some compensatory mechanisms might protect these animals from the behavioral abnormalities.

Characterization of [(3)H]Quisqualate binding to recombinant rat metabotropic glutamate 1a and 5a receptors and to rat and human brain sections.

We have investigated the binding properties of [(3)H]quisqualate to rat metabotropic glutamate (mGlu) 1a and 5a receptors and to rat and human brain sections. Saturation isotherms gave K:(D) values of 27 +/- 4 and 81 +/- 22 nM: for mGlu1a and mGlu5a receptors, respectively. Several compounds inhibited the binding to mGlu1a and mGlu5a receptors concentration-dependently. (S:)-4-Carboxyphenylglycine, (S:)-4-carboxy-3-hydroxyphenylglycine, and (R,S)-1-aminoindan-1,5-dicarboxylic acid, which completely inhibited [(3)H]quisqualate binding to the mGlu5a receptor, were inactive in a functional assay using this receptor. The distribution and abundance of binding sites in rat and human brain sections were studied by quantitative receptor radioautography and image analysis. Using 10 nM: [(3)H]quisqualate, a high density of binding was detected in various brain regions with the following rank order of increasing levels: medulla, thalamus, olfactory bulb, cerebral cortex, spinal cord dorsal horn, olfactory tubercle, dentate gyrus molecular layer, CA1-3 oriens layer of hippocampus, striatum, and cerebellar molecular layer. The ionotropic component of this binding could be inhibited by 30 microM: kainate, revealing the distribution of mGlu1+5 receptors. The latter were almost completely inhibited by the group I agonist (S:)-3,5-dihydroxyphenylglycine. The binding profile correlated well with the cellular sites of synthesis and regional expression of the respective group I receptor proteins revealed by in situ hybridization histochemistry and immunohistochemistry, respectively.

Evidence that nicotinic alpha(7) receptors are not involved in the hyperlocomotor and rewarding effects of nicotine.

Neuronal nicotinic receptors are comprised of combinations of alpha(2-9) and beta(2-4) subunits arranged to form a pentameric receptor. Currently, the principal central nervous system (CNS) subtypes are believed to be alpha(4)beta(2) and a homomeric alpha(7) receptor, although other combinations almost certainly exist. The identity of the nicotinic receptor subtype(s) involved in the rewarding effects of nicotine are unknown. In the present study, using some recently described subtype selective nicotinic agonists and antagonists, we investigated the role of the alpha(7) nicotinic receptor in the mediation of nicotine-induced hyperactivity and self-administration in rats. The alpha(7) receptor agonists AR-R 17779 and DMAC failed to stimulate locomotor activity in both nicotine-nontolerant and -sensitized rats. In contrast, nicotine and the putative alpha(4)beta(2) subtype selective agonist SIB1765F increased activity in both experimental conditions. In nicotine-sensitized rats, the high affinity (including the alpha(4)beta(2) subtype) nicotinic antagonist dihydro-beta-erythroidine (DHbetaE), but not the selective alpha(7) antagonist methyllycaconitine (MLA), antagonized a nicotine-induced hyperactivity. Similarly, DHbetaE, but not MLA, pretreatment reduced nicotine self-administration. Electrophysiology experiments using Xenopus oocytes expressing the human alpha(7) receptor confirmed AR-R 17779 and DMAC to be potent agonists at this site, and further studies demonstrated the ability of systemically administered AR-R 17779 to penetrate into the CNS. Taken together, these results indicate a negligible role of alpha(7) receptors in nicotine-induced hyperlocomotion and reward in the rat, and support the view for an involvement of a member from the high-affinity nicotinic receptor subclass, possibly alpha(4)beta(2). Issues such as drug potency, CNS penetration, and desensitization of the alpha(7) receptor are discussed.

Characterization of [(3)H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using semliki forest virus vectors.

The binding properties of [(3)H]-LY354740 were characterized on rat metabotropic glutamate receptors mGlu2 and mGlu3 expressed in Chinese hamster ovary (CHO) cells using Semliki Forest virus vectors. The saturation isotherm gave K(D) values of 20+/-5 and 53+/-8 nM and B(max) values of 474+/-161 and 667+/-89 fmol/mg protein for mGlu2 and mGlu3 receptors, respectively. NMDA, CaCl(2), DHPG and kainate were inactive up to 1 mM, whereas LY341495, DCG IV and ibotenate inhibited [(3)H]-LY354740 binding with similar potencies on both receptors. L-CCG I, L-AP4, L-AP5, LY354740 and 1S,3R-ACPD were 2- to 4-fold more potent inhibitors of [(3)H]-LY354740 binding to mGlu2 than mGlu3 receptors. However, MPPG and L-AP3 had a 6-fold and DTT a 28-fold preference for mGlu2 over mGlu3. ZnCl(2), at 10 mM, inhibited more than 70% of [(3)H]-LY354740 binding to mGlu2 receptors. At the same concentration it did not affect significantly [(3)H]-LY354740 binding to mGlu3 receptors. On the contrary, glutamate, quisqualate, EGLU and NAAG showed a 3-, 5-, 7- and 12-fold preference for mGlu3 over mGlu2. Finally, GTPgammaS, which partially inhibited the binding on mGlu2 receptors, was inactive to inhibit [(3)H]-LY354740 binding on mGlu3 receptors.

Regional and cellular distribution of bleomycin hydrolase mRNA in human brain: comparison between Alzheimer's diseased and control brains.

Genetic polymorphism of human bleomycin hydrolase (hBH) has been reported to be associated with the risk of sporadic Alzheimer's disease (AD). The regional and cellular distribution of mRNA encoding hBH in the brain from controls and patients with AD was examined using in situ hybridization. A hybridization signal, in the form of clusters of single cells, was observed in the white matter. Our results indicate a predominantly astrocytic expression of hBH in the investigated human brain regions. Although the signal intensity was generally reduced in AD brains, the large variability among controls rendered this trend statistically insignificant.

Effect of the umami peptides on the ligand binding and function of rat mGlu4a receptor might implicate this receptor in the monosodium glutamate taste transduction.

1. The effect of several metabotropic ligands and di- or tripeptides were tested on the binding of [3H]-L(+)-2-amino-4-phosphonobutyric acid ([3H]-L-AP4) on rat mGlu4 receptor. For selected compounds, the functional activity was determined on this receptor using the guanosine-5'[gamma-35S]-thiotriphosphate [gamma-35S]-GTP binding assay. 2. Using the scintillation proximity assay, [3H]-L-AP4 saturation analysis gave binding parameters K(D) and Bmax values of 150 nM and 9.3 pmoles mg-1 protein, respectively. The specific binding was inhibited concentration-dependently by several mGlu receptor ligands, and their rank order of affinity was established. 3. Several peptides inhibited the [3H]-L-AP4 binding with the following rank order of potency: glutamate-glutamate>glutamate-glutamate-leucine=aspartate - glutamate>>glutamate - glutamate-aspartate>lactoyl-glutamate>>aspartate-aspartate. Aspartate-phenylalanine-methyl ester (aspartame) was inactive up to 1 mM and guanosine-5'-monophosphate and inosine-5'-monophosphate were inactive up to 100 micronM. 4. The [gamma-35S]-GTP binding functional assay was used to determine the agonist activities of the different compounds. For the rat mGlu4 agonists, L-AP4 and L-glutamate, the correlation between their occupancy and activation of the receptor was close to one. The peptides, Glu-Glu, Asp-Glu and Glu-Glu-Asp failed to stimulate the [gamma-35S]-GTP binding at receptor occupancy greater than 80% and Glu-Glu-Leu appeared to be a weak partial agonist. These peptides did not elicit a clear dose-dependent umami perception. However, Glu-lac showed a good correlation between its potency to stimulate the [gamma-35S]-GTP binding and its affinity for displacement of [3H]-L-AP4 binding. These data are in agreement with the peptide taste assessment in human subjects, which showed that the acid derivatives of glutamate had characteristics similar to umami.

cDNA cloning of a novel secreted isoform of the human receptor for advanced glycation end products and characterization of cells co-expressing cell-surface scavenger receptors and Swedish mutant amyloid precursor protein.

The receptor for advanced glycation end products (RAGE) has been proposed as a cell surface receptor that binds amyloid-beta protein (Abeta), thereby triggering its cytotoxic effects [S.D. Yan, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T. Slattery, L. Zhao, M. Nagashima, J. Morser, A. Migheli, P. Nawroth, D. Stern, A.M. Schmidt, RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease, Nature 382 (1996) 685-691.]. A cDNA library of human lung was screened for RAGE with an appropriate hybridization probe. In addition to cell surface RAGE, one clone was found which encodes a new version of RAGE, termed hRAGEsec, which lacks the 19 amino acids of the membrane-spanning region and is therefore secreted. Comparison with the genomic sequence revealed that the synthesis of the secreted isoform requires alternative splicing. The deduced protein sequence of the mature hRAGEsec consists of 321 amino acids with a predicted molecular mass of 35.66 kDa. The pattern of expression of hRAGEsec in human brain was analyzed by in situ hybridization histochemistry. The most intense expression of the gene in contrast to cell surface RAGE was detected in hippocampal CA3 pyramidal cells, dentate gyrus granule cells, cortical neurons as well as glial cells in white matter. To investigate the interaction between Abeta and RAGE and another scavenger receptor, SRA, under physiological conditions, they were co-expressed with human betaAPP(695)-SFAD in a human cell and the level of Abeta in the condition medium was assessed by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA) analysis. A nearly 100% reduction of Abeta from the conditioned medium of hRAGE cells and approximately 40% reduction from the SRA-cells implied that hRAGE could be a prominent cell surface receptor interacting with Abeta.