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Elucidating the key signal transduction pathways essential for both antipsychotic efficacy and side-effect profiles is essential for developing safer and more effective therapies. Recent work has highlighted noncanonical modes of dopamine D 2 receptor (D 2 R) signaling via β-arrestins as being important for the therapeutic actions of both antipsychotic and antimanic agents. We thus sought to create unique D 2 R agonists that display signaling bias via β-arrestin–ergic signaling. Through a robust diversity-oriented modification of the scaffold represented by aripiprazole (1), we discovered UNC9975 (2), UNC0006 (3), and UNC9994 (4) as unprecedented β-arrestin–biased D 2 R ligands. These compounds also represent unprecedented β-arrestin–biased ligands for a G i -coupled G protein–coupled receptor (GPCR). Significantly, UNC9975, UNC0006, and UNC9994 are simultaneously antagonists of G i -regulated cAMP production and partial agonists for D 2 R/β-arrestin-2 interactions. Importantly, UNC9975 displayed potent antipsychotic-like activity without inducing motoric side effects in inbred C57BL/6 mice in vivo. Genetic deletion of β-arrestin-2 simultaneously attenuated the antipsychotic actions of UNC9975 and transformed it into a typical antipsychotic drug with a high propensity to induce catalepsy. Similarly, the antipsychotic-like activity displayed by UNC9994, an extremely β-arrestin–biased D 2 R agonist, in wild-type mice was completely abolished in β-arrestin-2 knockout mice. Taken together, our results suggest that β-arrestin signaling and recruitment can be simultaneously a significant contributor to antipsychotic efficacy and protective against motoric side effects. These functionally selective, β-arrestin–biased D 2 R ligands represent valuable chemical probes for further investigations of D 2 R signaling in health and disease.

The clinical efficacy and safety of a drug is determined by its activity profile across many proteins in the proteome. However, designing drugs with a specific multi-target profile is both complex and difficult. Therefore methods to design drugs rationally a priori against profiles of several proteins would have immense value in drug discovery. Here we describe a new approach for the automated design of ligands against profiles of multiple drug targets. The method is demonstrated by the evolution of an approved acetylcholinesterase inhibitor drug into brain-penetrable ligands with either specific polypharmacology or exquisite selectivity profiles for G-protein-coupled receptors. Overall, 800 ligand-target predictions of prospectively designed ligands were tested experimentally, of which 75% were confirmed to be correct. We also demonstrate target engagement in vivo. The approach can be a useful source of drug leads when multi-target profiles are required to achieve either selectivity over other drug targets or a desired polypharmacology.

A variety of the title chromenoisoxazole derivatives (VI) is synthesized via intramolecular 1,3-dipolar cycloaddition of a nitrone derived from thioether (III) as the key reaction.

Background— Cardiac cell therapy for older patients who experience a myocardial infarction may require highly regenerative cells from young, healthy (allogeneic) donors. Bone marrow mesenchymal stem cells (MSCs) are currently under clinical investigation because they can induce cardiac repair and may also be immunoprivileged (suitable for allogeneic applications). However, it is unclear whether allogeneic MSCs retain their immunoprivilege or functional efficacy late after myocardial implantation. We evaluated the effects of MSC differentiation on the immune characteristics of cells in vitro and in vivo and monitored cardiac function for 6 months after post–myocardial infarction MSC therapy. Methods and Results— In the in vitro experiments, inducing MSCs to acquire myogenic, endothelial, or smooth muscle characteristics (via 5-azacytidine or cytokine treatment) increased major histocompatibility complex-Ia and -II (immunogenic) expression and reduced major histocompatibility complex-Ib (immunosuppressive) expression, in association with increased cytotoxicity in coculture with allogeneic leukocytes. In the in vivo experiments, we implanted allogeneic or syngeneic MSCs into infarcted rat myocardia. We measured cell differentiation and survival (immunohistochemistry, real-time polymerase chain reaction) and cardiac function (echocardiography, pressure-volume catheter) for 6 months. MSCs (versus media) significantly improved ventricular function for at least 3 months after implantation. Allogeneic (but not syngeneic) cells were eliminated from the heart by 5 weeks after implantation, and their functional benefits were lost within 5 months. Conclusions— The long-term ability of allogeneic MSCs to preserve function in the infarcted heart is limited by a biphasic immune response whereby they transition from an immunoprivileged to an immunogenic state after differentiation, which is associated with an alteration in major histocompatibility complex–immune antigen profile.

Clozapine, by virtue of its absence of extrapyramidal side effects and greater efficacy, revolutionized the treatment of schizophrenia, although the mechanisms underlying this exceptional activity remain controversial. Combining an unbiased cheminformatics and physical screening approach, we evaluated clozapine's activity at2350 distinct molecular targets. Clozapine, and the closely related atypical antipsychotic drug olanzapine, interacted potently with a unique spectrum of molecular targets. This distinct pattern, which was not shared with the typical antipsychotic drug haloperidol, suggested that the serotonergic neuronal system was a key determinant of clozapine's actions. To test this hypothesis, we used pet1(-/-) mice, which are deficient in serotonergic presynaptic markers. We discovered that the antipsychotic-like properties of the atypical antipsychotic drugs clozapine and olanzapine were abolished in a pharmacological model that mimics NMDA-receptor hypofunction in pet1(-/-) mice, whereas haloperidol's efficacy was unaffected. These results show that clozapine's ability to normalize NMDA-receptor hypofunction, which is characteristic of schizophrenia, depends on an intact presynaptic serotonergic neuronal system.

Injectable scaffolds made of biodegradable biomaterials can stabilize a myocardial infarct and promote cardiac repair. Here, we describe the synthesis of a new, temperature-sensitive, aliphatic polyester hydrogel (HG) conjugated with vascular endothelial growth factor (VEGF) and evaluate its effects on cardiac recovery after a myocardial infarction (MI). Seven days after coronary ligation in rats, PBS, HG, or HG mixed or conjugated with VEGF (HG + VEGF or HG-VEGF, respectively) was injected around the infarct (n = 8-11/group). Function was evaluated by echocardiography at multiple time points. Pressure-volume measurements were taken and infarct morphometry and blood vessel density were assessed at 35 days after injection. HG-VEGF provided localized, sustained VEGF function. Compared with outcomes in the PBS group, fractional shortening, ventricular volumes, preload recruitable stroke work, and end-systolic elastance were all preserved (p < 0.05) in the HG and HG + VEGF groups, and further preserved in the HG-VEGF group. Conjugated VEGF also produced the highest blood vessel density (p < 0.05). The infarct thinned and dilated after PBS injection, but was smaller and thicker in hearts treated with HG (p < 0.05). Our temperature-sensitive HG attenuated adverse cardiac remodeling and improved ventricular function when injected after an MI. VEGF delivery enhanced these effects when the VEGF was conjugated to the HG.

The human Ether-a-go-go related gene (hERG) potassium channel is responsible for the rapid delayed rectifier potassium current that plays a critical role in the repolarization of cardiomyocytes during the cardiac action potential. In humans, inhibition of hERG by drugs can prolong the electrocardiographic QT interval, which, in rare instance, leads to ventricular arrhythmia and sudden cardiac death. As such, several medications that block hERG channels in vitro have been withdrawn from the market due to QT prolongation and arrhythmias. The current FDA guidelines recommend that drug candidates destined for human use be evaluated for potential hERG activity ( www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm074963.pdf ). Here, we employed automated planar patch clamp (APPC), high-throughput fluorescent Tl(+) flux, and moderate-throughput [³H]dofetilide competition binding assays to characterize a panel of 49 drugs for their activities at the hERG channel. Notably, we used the same HEK293-hERG cell line for all assays, facilitating comparisons of hERG potencies across screening platforms. In general, hERG inhibitors were most potent in APPC assays, intermediate potent in [³H]dofetilide binding assays, and least potent in Tl(+) flux assays. Binding affinity constants (pK(i) values) and Tl(+) flux potencies (pEC₅₀ values) correlated well with APPC pEC₅₀ values. Further, the inhibitory potencies of many known hERG inhibitors in APPC matched literature values from manual and/or automated patch clamp systems. We also developed a novel fluorescent Tl(+) flux assays to measure the effects of drugs that modulate hERG trafficking and surface expression.

Some antipsychotic drugs are known to cause valvular heart disease by activating serotonin 5-HT(2B) receptors. We have developed and validated binary classification QSAR models capable of predicting potential 5-HT(2B) actives. The classification accuracies of the models built to discriminate 5-HT(2B) actives from the inactives were as high as 80% for the external test set. These models were used to screen in silico 59,000 compounds included in the World Drug Index, and 122 compounds were predicted as actives with high confidence. Ten of them were tested in radioligand binding assays and nine were found active, suggesting a success rate of 90%. All validated actives were then tested in functional assays, and one compound was identified as a true 5-HT(2B) agonist. We suggest that the QSAR models developed in this study could be used as reliable predictors to flag drug candidates that are likely to cause valvulopathy.

Drug-induced valvular heart disease (VHD) is a serious side effect of a few medications, including some that are on the market. Pharmacological studies of VHD-associated medications (e.g., fenfluramine, pergolide, methysergide, and cabergoline) have revealed that they and/or their metabolites are potent 5-hydroxytryptamine(2B) (5-HT(2B)) receptor agonists. We have shown that activation of 5-HT(2B) receptors on human heart valve interstitial cells in vitro induces a proliferative response reminiscent of the fibrosis that typifies VHD. To identify current or future drugs that might induce VHD, we screened approximately 2200 U.S. Food and Drug Administration (FDA)-approved or investigational medications to identify 5-HT(2B) receptor agonists, using calcium-based high-throughput screening. Of these 2200 compounds, 27 were 5-HT(2B) receptor agonists (hits); 14 of these had previously been identified as 5-HT(2B) receptor agonists, including seven bona fide valvulopathogens. Six of the hits (guanfacine, quinidine, xylometazoline, oxymetazoline, fenoldopam, and ropinirole) are approved medications. Twenty-three of the hits were then "functionally profiled" (i.e., assayed in parallel for 5-HT(2B) receptor agonism using multiple readouts to test for functional selectivity). In these assays, the known valvulopathogens were efficacious at concentrations as low as 30 nM, whereas the other compounds were less so. Hierarchical clustering analysis of the pEC(50) data revealed that ropinirole (which is not associated with valvulopathy) was clearly segregated from known valvulopathogens. Taken together, our data demonstrate that patterns of 5-HT(2B) receptor functional selectivity might be useful for identifying compounds likely to induce valvular heart disease.

Iron chelators are effective at removing iron from the body in iron overload, but little is known about the handling of iron chelates by the kidney. We studied the transport of deferoxamine, deferasirox, and three hydroxypyridones, and their iron chelates, in polarized renal epithelial MDCK cells growing on Transwell inserts. Directional iron efflux was also studied in (59)Fe-loaded cells. The chelators were transported at comparable rates in the apical and basolateral directions and moved faster than their corresponding chelates, except for deferoxamine, which did not move from the basolateral to the apical side. In contrast, the chelates were transported faster in the apical-to-basolateral direction. More permeable chelators were more efficient at removing iron from iron-loaded cells compared with deferoxamine. Iron is preferentially removed from the basolateral side, and kinetic modeling suggests facilitated diffusion of chelates in some cases. Basolateral iron efflux is temperature-dependent and partially sensitive to ATP depletion. Polarized transport of chelates suggests the kidney may be involved in reabsorption of iron bound to chelators, with a temperature-sensitive facilitated removal of some iron complexes from the basolateral side. Further studies are warranted to determine if these processes may contribute to the observed nephrotoxicity of some iron chelators.

Amisulpride is approved for clinical use in treating schizophrenia in a number of European countries and also for treating dysthymia, a mild form of depression, in Italy. Amisulpride has also been demonstrated to be an antidepressant for patients with major depression in many clinical trials. In part because of the selective D(2)/D(3) receptor antagonist properties of amisulpride, it has long been widely assumed that dopaminergic modulation is the proximal event responsible for mediating its antidepressant and antipsychotic properties.The purpose of these studies was to determine if amisulpride's antidepressant actions are mediated by off-target interactions with other receptors.We performed experiments that: (1) examined the pharmacological profile of amisulpride at a large number of central nervous system (CNS) molecular targets and, (2) after finding high potency antagonist affinity for human 5-HT(7a) serotonin receptors, characterized the actions of amisulpride as an antidepressant in wild-type and 5-HT(7) receptor knockout mice.We discovered that amisulpride was a potent competitive antagonist at 5-HT(7a) receptors and that interactions with no other molecular target investigated in this paper could explain its antidepressant actions in vivo. Significantly, and in contrast to their wild-type littermates, 5-HT(7) receptor knockout mice did not respond to amisulpride in two widely used rodent models of depression, the tail suspension test and the forced swim test.These results indicate that 5-HT(7a) receptor antagonism, and not D(2)/D(3) receptor antagonism, likely underlies the antidepressant actions of amisulpride.

Inhibitors of histone deacetylases (HDACIs) have emerged as a new class of drugs for the treatment of human cancers and other diseases because of their effects on cell growth, differentiation, and apoptosis. In this study we have developed several quantitative structure-activity relationship (QSAR) models for 59 chemically diverse histone deacetylase class 1 (HDAC1) inhibitors. The variable selection k nearest neighbor (kNN) and support vector machines (SVM) QSAR modeling approaches using both MolconnZ and MOE chemical descriptors generated from two-dimensional rendering of compounds as chemical graphs have been employed. We have relied on a rigorous model development workflow including the division of the data set into training, test, and external sets and extensive internal and external validation. Highly predictive QSAR models were generated with leave-one-out cross-validated (LOO-CV) q2 and external R2 values as high as 0.80 and 0.87, respectively, using the kNN/MolconnZ approach and 0.93 and 0.87, respectively, using the SVM/MolconnZ approach. All validated QSAR models were employed concurrently for virtual screening (VS) of an in-house compound collection including 9.5 million molecules compiled from the ZINC7.0 database, the World Drug Index (WDI) database, the ASINEX Synergy libraries, and other commercial databases. VS resulted in 45 structurally unique consensus hits that were considered novel putative HDAC1 inhibitors. These computational hits had several novel structural features that were not present in the original data set. Four computational hits with novel scaffolds were tested experimentally, and three of them were confirmed active against HDAC1, with IC50 values for the most active compound of 1.00 microM. The fourth compound was later identified to be a selective inhibitor of HDAC6, a Class II HDAC. Moreover, two of the confirmed hits are marketed drugs, which could potentially facilitate their further development as anticancer agents. This study illustrates the power of the combined QSAR-VS method as a general approach for the effective identification of structurally novel bioactive compounds.

Rationale: Allogeneic bone marrow mesenchymal stem cells (MSC) are currently undergoing clinical trials to test their potential to repair the heart after a myocardial infarction (MI). MSCs can improve function, but neither the host immune responses nor the fate of these cells has been extensively investigated. This study compared the outcomes of allogeneic and syngeneic MSC transplantation after an MI. Methods: Female Lewis rat underwent coronary ligation. Three weeks later, allogeneic or syngeneic MSCs (3×10 6 /rat) from male rats (Wister or Lewis, respectively) were implanted into the infracted region. We evaluated implanted cell survival (real-time PCR to quantify Y-chromosomes), cytokines (RT-PCR) and infiltrating cells (immunostaining) in the heart, and allogeneic antibodies in the blood. Cardiac function was assessed by echocardiography and pressure-volume catheters. Immune antigen expression (RT-PCR, immunostaining and flow cytometry) was characterized in the MSCs before and after myogenic differentiation. Results: At 1 week post implantation, cell survival was similar in the allogeneic and syngeneic groups. Gene expression of 11 cytokines and T-cell infiltration into the cell-implanted region were also similar, but more B-cells were observed in the allogeneic group (p Conclusions: Allogeneic BMSC transplantation produced a robust cardiac functional improvement, but following myogenic differentiation the cells were no longer immunoprivileged. Rejection of the allogeneic cells could limit their long term benefit on cardiac remodeling after an MI.

The 2nd outer loop (o2) of muscarinic acetylcholine receptors (mAChRs) contains a highly conserved cysteine residue that is believed to participate in a disulfide bond and is flanked on either side by epitopes that are critical to the binding of many muscarinic allosteric modulators. We determined the allosteric binding parameters of the modulators gallamine, W84, and tetrahydroaminoacridine (THA) at M2 and M3 mAChRs in which these cysteine residues had been mutated to alanines. THA is known to bind to mAChRs with a strong positive homotropic cooperativity (a Hill slope of approximately 2) that implies that it must interact with multiple allosteric sites. The disulfide cysteine mutations in M2 receptors reduced the allosteric potencies of the tested modulators as if the critical adjacent residue (Tyr177) itself had been mutated. However, in M3 receptors, the disulfide cysteine mutations had no effect on the potencies of gallamine or W84 and even increased the potency of THA. It was most interesting that the strong, positive, homotropic interactions of THA at both M2 and M3 receptors were markedly reduced by the cysteine mutations. In addition, gallamine also displayed positive homotropic cooperativity in its interactions with M3 receptors (but not M2 receptors), and this cooperativity was not evident in the cysteine mutants. Thus, it seems that these cysteine residues play a role in linking cooperating allosteric sites, although it is not currently possible to say whether these multiple sites lie within one receptor or on two linked receptors of a dimer or higher order oligomer.

Iron that is not specifically chaperoned through its essential functional pathways is damaging to biological systems, in major part by catalyzing the production of reactive oxygen species. Iron serves in several essential roles in the mitochondrion, as an essential cofactor for certain enzymes of electron transport, and through its involvement in the assembly of iron-sulfur clusters and iron-porphyrin (heme) complexes, both processes occurring in the mitochondrion. Therefore, there are mechanisms that deliver iron specifically to mitochondria, although these are not well understood. Under normal circumstances the mitochondrion has levels of stored iron that are higher than other organelles, though lower than in cytosol, while in some disorders of iron metabolism, mitochondrial iron levels exceed those in the cytosol. Under these circumstances of excess iron, protective mechanisms are overwhelmed and mitochondrial damage ensues. This may take the form of acute oxidative stress with structural damage and functional impairment, but also may result in long-term damage to the mitochondrial genome. This review discusses the evidence that mitochondria do indeed accumulate iron in several genetic disorders, and are a direct target for iron toxicity when it is present in excess. We then consider two classes of genetic disorders involving iron and the mitochondrion. The first include defects in genes directly regulating mitochondrial iron metabolism that lead to Friedreich's ataxia and the various sideroblastic anemias, with excessive mitochondrial iron accumulation. Under the second class, we discuss various primary hemochromatoses that lead to direct mitochondrial damage, with reference to mutations in genes encoding HFE, hepcidin, hemojuvelin, transferrin receptor-2, ferroportin, transferrin, and ceruloplasmin.

Caco-2 monolayers were used to contrast the bidirectional transport of iron chelators and their chelates and to estimate fundamental kinetics associated with their intestinal absorption.Bidirectional transport was studied at 37 degrees C and pH 7.4 using 500-microM concentrations. Monolayer integrity was tested via transepithelial electrical resistance and sodium fluorescein permeability. Apical and basolateral analysis provided mass balance evidence. Apparent permeability coefficient (P(app)) served to rank and compare molecules and estimate in vivo bioavailability. Model-dependent rate constants defined cellular influx and efflux.1) P(app) ranked in decreasing order for chelators from directional transport studies were CP363deferiproneICL670CP502deferoxamine (DFO). 2) Fe(CP502)(3), Fe(ICL670)(2), and FeDFO were not measurable in receiving chambers, whereas Fe(deferiprone)(3) and Fe(CP363)(3) were detected in both directions. 3) CP363 was transported significantly faster from the basolateral to the apical direction than the converse. 4) Mass balance of donor and receiver chambers gave approximately 100% recovery in all cases. 5) Kinetic analysis supports the view that the Caco-2 chelator efflux constants are generally greater than their influx constants.Caco-2 cells are useful in screening iron chelators and chelates and estimating bioavailabilities. Structure and distribution coefficients partially predict passive transport through Caco-2 monolayers.

The structurally divergent agents gallamine and hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)ammonium]dibromide (W84) are known to interact competitively at a common allosteric site on muscarinic receptors. Previous studies reported that the M2 selectivity of gallamine depended largely on the EDGE (172-175) sequence in the second outer loop (o2) and on 419Asn near the junction of o3 and the seventh transmembrane domain (TM7), whereas the selectivity of W84 depended on nearby residues 177Tyr and 423Thr. However, it has so far proven difficult to confer the high sensitivity for allosteric modulation of the M2 subtype onto the weakly sensitive M5 subtype by substituting these key residues. We now have found that M2 423Thr, not 419Asn, is the dominant residue in the o3/TM7 region for gallamine's high potency, although 419Asn can substitute for 423Thr in some contexts; in contrast, the presence of 419Asn reduces the potency of W84 in every context we have studied. In addition, the orientation of 177Tyr is crucial to high sensitivity toward W84, and it seems that the proline residue at position 179 in M5 (corresponding to M2 172Glu) may interfere with that orientation. Consistent with these observations, a mutant M5 receptor with these three key mutations, M5P179E, Q184Y, and H478T, showed dramatically increased sensitivity for W84 (100-fold), compared with the wild-type M5 receptor. This same mutant receptor approached M2 sensitivity toward gallamine. Thus, gallamine and W84 derive high potency from the same receptor domains (epitopes in o2 and near the junction between o3 and TM7), even though these allosteric agents have quite different structures.

To study the thresholds and potential of malaria transmission by Anopheles anthropophagus in Hubei Province and provide indicators for the disease surveillance, early warning, prevention and control in the locality.From July to August 2001, field investigations on vectors and malaria situation were carried out in the village of Yanjiafan, Suizhou City, where the malaria incidence was high. The entomological investigations included the man-biting rate, the proportion of parous anophelines, the human blood index and the blood preference. The others included malaria incidence and parasite rate in human population, the intervals from episode to treatment of the cases, and collection of data on the mean temperature in the area. Based on the formula of basic reproductive rate, the critical man-biting rate was estimated.92.6% (63/68) of An. anthropophagus were found to have human blood meals, it occupied 91.5% (97/106) of the mosquitoes in human dwellings, its human blood index and vectorial capacity were 12.5 times (0.50/0.04) and 6.5 times (0.9448/0.1449) higher than those of An. sinensis. The critical man-biting rate was 0.2823 and the adjusted man-biting rate was 3.5 times of its critical man-biting rate (0.9892/0.2823). The malaria incidence was 0.65% (12/1844) and the parasite rate in pupils was 0.51%(1/198).A reduction of the adjusted man-biting rate of An. anthropophagus by 71.5% is needed for interrupting malaria transmission by this vector in the study area.

We report the creation of transgenic mice with an inducible, tissue-targeted expression of a constitutively active mutant form (Q227L) of G(alpha)(s). Mice expressing activated G(alpha)(s) in fat tissue, liver, and skeletal muscle displayed normal body mass and blunted glucose metabolism. cAMP accumulation in adipose tissue was increased in the basal state, but far less than would be expected. Marked adaptation to elevated cAMP levels occurred, leading to an increase in total cAMP-specific phosphodiesterase activity, a 50% decline in cAMP-dependent protein kinase (protein kinase A) activity, and an increased expression of G(alpha)(i2). The reduction in kinase activity coincided with50% increase in the expression of RIalpha and RIIalpha regulatory subunits of protein kinase A, with no change in the amount of catalytic subunit. These data demonstrate the existence of adaptive responses of protein kinase A, phosphodiesterase, and G(alpha)(i2) in tissues expressing constitutively active G(alpha)(s) that may act to rectify the impact of increased cAMP accumulation.

The study of gene expression is fundamental to research in development, metabolic regulation, and human disease. Genetics has made great use of a variety of molecular techniques to measure the expression of genes relevant to development. Traditional methods include DNA excess solution hybridization, riboprobe hybridization, and Northern analysis to ascertain the steady state levels of specific RNA molecules, products of genes of interest. Although each of the technologies has specific advantages, all the traditional approaches are labor intensive and designed to explore in great detail a rather limited number of RNAs. The pace or technological advancement in the analysis of gene expression has been unparalleled, affording a rich set of tools with which development research can be accelerated. The Human Genome Project and efforts to identify unique fragments of gene expressed sequence have resulted in an impressive expansion in the amount and quality or molecular probes amenable to aid research in the study of development specifically, and human disease in general, cDNA spotting and DNA oligonucleotide arraying offer powerful, high-throughput capabilities for gene profiling. Differential display, serial analysis of gene expression (SAGE), and subtractive cloning technologies offer alternative approaches.

Magnesium borate ( Mg 2 B 2 O 5) nanorods were synthesized by a two-step process, including solution-chemical technology and a ternary-flux method, using concentrated seawater and H 3 BO 3 as raw materials. X-ray diffraction (XRD) showed that the sample had triclinic structure. Scanning electron microscopy (SEM) and high resolution transmission electron microscopy (HR-TEM) indicated that it consisted of rod-like particles with an average diameter of 100–150 nm and length over 5 μm. Differential thermal analysis (DTA) confirmed that the melting point of the ternary-flux and the formation temperature of Mg 2 B 2 O 5 were lower than single-flux process. The formation of Mg 2 B 2 O 5 nanorods was more efficient by ternary-flux than single-flux. Mechanical property of Mg 2 B 2 O 5 nanorods reinforced Nylon-6 composites showed that KH550 was the optimal coupling agent and made the strength of the composites to be improved to different degrees.

Metabotropic glutamate receptors (mGluRs) are G-protein-coupled glutamate receptors that subserve a number of diverse functions in the central nervous system. The large extracellular amino-terminal domains (ATDs) of mGluRs are homologous to the periplasmic binding proteins in bacteria. In this study, a region in the ATD of the mGluR4 subtype of mGluR postulated to contain the ligand-binding pocket was explored by site-directed mutagenesis using a molecular model of the tertiary structure of the ATD as a guiding tool. Although the conversion of Arg(78), Ser(159), or Thr(182) to Ala did not affect the level of protein expression or cell-surface expression, all three mutations severely impaired the ability of the receptor to bind the agonist L-[(3)H]amino-4-phosphonobutyric acid. Mutation of other residues within or in close proximity to the proposed binding pocket produced either no effect (Ser(157) and Ser(160)) or a relatively modest effect (Ser(181)) on ligand affinity compared with the Arg(78), Ser(159), and Thr(182) mutations. Based on these experimental findings, together with information obtained from the model in which the glutamate analog L-serine O-phosphate (L-SOP) was "docked" into the binding pocket, we suggest that the hydroxyl groups on the side chains of Ser(159) and Thr(182) of mGluR4 form hydrogen bonds with the alpha-carboxyl and alpha-amino groups on L-SOP, respectively, whereas Arg(78) forms an electrostatic interaction with the acidic side chains of L-SOP or glutamate. The conservation of Arg(78), Ser(159), and Thr(182) in all members of the mGluR family indicates that these amino acids may be fundamental recognition motifs for the binding of agonists to this class of receptors.

Background Nor-BNI, GNTI and JDTic induce selective κ opioid antagonism that is delayed and extremely prolonged, but some other effects are of rapid onset and brief duration. The transient effects of these compounds differ, suggesting that some of them may be mediated by other targets. Results In binding assays, the three antagonists showed no detectable affinity (K i≥10 µM) for most non-opioid receptors and transporters (26 of 43 tested). There was no non-opioid target for which all three compounds shared detectable affinity, or for which any two shared sub-micromolar affinity. All three compounds showed low nanomolar affinity for κ opioid receptors, with moderate selectivity over μ and δ (3 to 44-fold). Nor-BNI bound weakly to the α2C-adrenoceptor (K i = 630 nM). GNTI enhanced calcium mobilization by noradrenaline at the α1A-adrenoceptor (EC50 = 41 nM), but did not activate the receptor, displace radioligands, or enhance PI hydrolysis. This suggests that it is a functionally-selective allosteric enhancer. GNTI was also a weak M1 receptor antagonist (K B = 3.7 µM). JDTic bound to the noradrenaline transporter (K i = 54 nM), but only weakly inhibited transport (IC50 = 1.1 µM). JDTic also bound to the opioid-like receptor NOP (K i = 12 nM), but gave little antagonism even at 30 µM. All three compounds exhibited rapid permeation and active efflux across Caco-2 cell monolayers. Conclusions Across 43 non-opioid CNS targets, only GNTI exhibited a potent functional effect (allosteric enhancement of α1A-adrenoceptors). This may contribute to GNTI's severe transient effects. Plasma concentrations of nor-BNI and GNTI may be high enough to affect some peripheral non-opioid targets. Nonetheless, κ opioid antagonism persists for weeks or months after these transient effects dissipate. With an adequate pre-administration interval, our results therefore strengthen the evidence that nor-BNI, GNTI and JDTic are highly selective κ opioid antagonists.

In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as 'designer drugs' and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS)-2-(ethylamino)-2-(3-methoxyphenyl)cyclohexanone) and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenyl)cyclohexanamine) and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenyl)cyclohexyl]piperidine and 1-[1-(4-methoxyphenyl)cyclohexyl]piperidine), were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.

A comparison of the pharmacological and physiological properties of the metabotropic glutamate 1 alpha and 1 beta receptors (mGluR1 alpha and mGluR1 beta) expressed in baby hamster kidney (BHK 570) cells was performed. The mGluR1 beta receptor is an alternatively spliced form of mGluR1 alpha with a modified carboxy terminus. Immunoblots of membranes from the two cell lines probed with receptor-specific antipeptide antibodies showed that mGluR1 alpha migrated with an M(r) = 154,000, whereas mGluR1 beta migrated with an M(r) = 96,000. Immunofluorescence imaging of receptors expressed in BHK 570 cells revealed that the mGluR1 alpha receptor was localized to patches along the plasmalemma and on intracellular membranes surrounding the nucleus, whereas mGluR1 beta was distributed diffusely throughout the cell. Agonist activation of the mGluR1 alpha and the mGluR1 beta receptors stimulated phosphoinositide hydrolysis. At both receptors, glutamate, quisqualate, and ibotenate were full agonists, whereas trans-(+)-1-aminocyclopentane-1,3-dicarboxylate appeared to act as a partial agonist. The stimulation of phosphoinositide hydrolysis by mGluR1 alpha showed pertussis toxin-sensitive and insensitive components, whereas the mGluR1 beta response displayed only the toxin-insensitive component. The mGluR1 alpha and mGluR1 beta receptors also increased intracellular calcium levels by inducing release from intracellular stores. These results indicate that the different carboxy terminal sequences of the two receptors directly influences G protein coupling and subcellular deposition of the receptor polypeptides and suggest that the two receptors may subserve different roles in the nervous system.

Ligand-gated ion channels gated by glutamate constitute the major excitatory neurotransmitter system in the mammalian brain. The functional modulation of GluR6, a kainate-activated glutamate receptor, by adenosine 3',5'-monophosphate-dependent protein kinase A (PKA) was examined with receptors expressed in human embryonic kidney cells. Kainate-evoked currents underwent a rapid desensitization that was blocked by lectins. Kainate currents were potentiated by intracellular perfusion of PKA, and this potentiation was blocked by co-application of an inhibitory peptide. Site-directed mutagenesis was used to identify the site or sites of phosphorylation on GluR6. Although mutagenesis of two serine residues, Ser684 and Ser666, was required for complete abolition of the PKA-induced potentiation, Ser684 may be the preferred site of phosphorylation in native GluR6 receptor complexes. These results indicate that glutamate receptor function can be directly modulated by protein phosphorylation and suggest that a dynamic regulation of excitatory receptors could be associated with some forms of learning and memory in the mammalian brain.

A series of substituted 4-aryl-piperazine-ethyl heteroarylcarboxamides were prepared and tested in in vitro radioligand binding studies. The presence of a quinoxaline has a favourable impact in terms of serotonin 5-HT1A versus dopamine D4.2 receptor selectivity. Compounds with a 3-CF3 group at the distal phenyl ring are the most effective in terms of affinity and selectivity for 5-HT1A versus D4.2 receptors. A 4-phenyl-1,2,3,6-tetrahydropyridine in place of the corresponding 4-phenyl-piperazine side chain is also favourable not only for the affinity for 5-HT1A and D4.2 receptors but also in some cases for α 2A-adrenoceptors.

The primary amino acid sequences of the kainate binding proteins from the amphibian and avian central nervous systems are homologous with the functional alpha-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptors that have been cloned from rat brain. In this study, we have analysed the anatomical and subcellular distribution of the alpha-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptors in the rat hippocampus and cerebellum, using a monoclonal antibody that was raised against a kainate binding protein purified from frog brain. Immunoblots of rat hippocampus and cerebellum, and membranes from COS cells transfected with rat brain alpha-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptor cDNAs (GluR1, GluR2, or GluR3) showed a major immunoreactive band migrating at a relative molecular weight of 107,000. In the cerebellum, an additional immunoreactive protein of approximately 128,000 mol. wt was also seen on immunoblots probed with the antibody. The distribution of this protein is apparently restricted to the cerebellum since the 128,000 mol. wt band was not present in other brain areas examined. The identity of the 128,000 mol. wt cerebellar protein is not known. Immunocytochemical analyses of the hippocampus demonstrated that alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate receptor subunits are present in the cell bodies and dendrites of pyramidal cells. The granule cells were also immunostained. All of the pyramidal cell subfields were heavily labeled. In the pyramidal cell bodies, a high level of immunoreactivity was observed throughout the cytoplasm. In the cerebellum, the Purkinje cell bodies and dendrites also displayed very high levels of immunoreactivity. In addition to the Purkinje neurons, the Bergmann glia and some Golgi neurons were clearly immunostained. Subcellular fractionation and lesioning experiments using the excitotoxin domoic acid indicated that the alpha-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptor subunits were associated with postsynaptic membranes. Direct visualization of the immunoreactivity using electron microscopy confirmed the postsynaptic localization of the staining in the dendritic areas in both the hippocampus and the cerebellum. Thus, unlike the kainate binding proteins, which are found primarily extrasynaptically in the frog and on glial cells in the chicken cerebellum, the GluR1, GluR2, and GluR3 receptor subunits are localized to the postsynaptic membrane in the dendrites of neurons in the rat central nervous system.