Radiolabeling of the rat P2X4 purinoceptor: evidence for allosteric interactions of purinoceptor antagonists and monovalent cations with P2X purinoceptors

The rat recombinant P2X4 purinoceptor was expressed in CHO-K1 cells, and binding studies were performed using the radioligand [35S]adenosine-5'-O-(3-thio)triphosphate ([35S]ATPgammaS). In 50 mM Tris/1 mM EDTA assay buffer, pH 7.4 at 4 degrees, [35S]ATPgammaS bound with high affinity to the P2X4 purinoceptor (KD = 0.13 nM, Bmax = 151 pmol/mg of protein). The purinoceptor agonists ATP and 2-methylthioadenosine triphosphate possessed nanomolar affinity for the P2X4 purinoceptor, whereas the antagonist suramin possessed much lower affinity (IC50 = 0.5 mM). Cibacron blue was more potent than suramin but produced a biphasic competition curve, whereas d-tubocurarine potentiated binding at concentrations in excess of 10 microM. The complex effects of cibacron blue and d-tubocurarine seemed to be due to an allosteric interaction with the P2X4 purinoceptor because these compounds affected radioligand dissociation, measured after isotopic dilution with unlabeled ATPgammaS. Cibacron blue (1-100 microM) and d-tubocurarine (0.1-1 mM) produced rapid (10 sec to 5 min) decreases or increases, respectively, in the level of [35S]ATPgammaS binding measured immediately after initiation of the dissociation reaction. However, the subsequent rates of radioligand dissociation were not markedly different from those measured in their absence. Monovalent cations produced similar affects on the P2X4 purinoceptor and, like d-tubocurarine, increased [35S]ATPgammaS binding. The actions of d-tubocurarine and sodium were not additive. The findings from this study indicate that [35S]ATPgammaS can be used to label the P2X4 purinoceptor and suggest that this binding can be enhanced by monovalent cations and d-tubocurarine and may be subject to negative allosteric modulation to varying degrees by different purinoceptor antagonists.     

4-Diazinyl- and 4-pyridinylimidazoles: potent angiotensin II antagonists. A study of their activity and computational characterization

A series of N-[biphenylyl(tetrazolyl)methyl]-2-butylimidazoles containing variously substituted diazine or pyridine moieties either as their free bases or N-oxide derivatives attached to the 4-position of the imidazole ring was synthesized and tested for interaction with the AT1 receptors of rat adrenal cortex membranes (receptor binding assay). Some compounds were then chosen for further evaluation in vivo in the A II-induced pressor response in conscious normotensive rats. The most potent in the AT1 binding assay were found to be compounds in which the diazine or pyridine ring nitrogen is adjacent to the point of attachment between the two heteroaromatic rings such as 2-butyl-4-(3,6-dimethylpyrazin-2-yl)-1-[[2'-(1H-tetrazol-5-y l)-biphenyl-4- yl]methyl]-1H-imidazole (3b) or 2-butyl-4-[5-(methoxycarbonyl)pyrid-2-yl]-1-[[2'-(1H-tetrazol++ +-5- yl)biphenyl-4-yl]methyl]-1H-imidazole (6c). The binding affinities and oral activities of the pyridine N-oxide imidazoles in which a stabilizing group ortho to the pyridine ring nitrogen is present were markedly improved as in 2-butyl-4-[(3-methoxycarbonyl)-6-methyl-N-oxopyridin-2-yl]-1-[[2'- (1H- tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-imidazole 31b. Molecular modeling studies were carried out to determine the molecular electrostatic potential values of related model systems and to correlate their receptor interaction energies with the observed activities of our compounds.     

Exonuclease enhances hybridization efficiency: improved direct cycle sequencing and point mutation detection

The title compounds (6-9) were prepared and evaluated for serotonin 5-HT4 agonistic activity in in vitro tests. Introducing a propyl or allyl group at the 3-position of benzamide caused only a slight enhancement of agonistic activity. Construction of the benzo[b]furan skeleton and 2,3-dihydrobenzo[b]furan skeleton caused a significant enhancement of the activity. 4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2-methylbenzo[b]furan-7-carboxamide (7b) hemifumarate was as potent as cisapride. 4-Amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2,3-dihydro-2-methylbenzo[b]furan-7-carboxamide (8a) hemifumarate, 4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2,3-dihydro-2-ethylbenzo[b]furan-7-carboxamide (8c) hemifumarate, and 4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2,3-dimethylbenzo[b]furan-7-carboxamide (8d) hemifumarate were more potent than cisapride. Furthermore, 8a hemifumarate was free from dopamine D1, D2, serotonin 5-HT1, 5-HT2 and muscarine M1, M2 receptor binding activity in the in in vitro tests. On the other hand, construction of the indole skeleton caused a remarkable decrease in activity.     

Preparation and steric structure elucidation of saturated or partially saturated isoindolo[2,1-a][3,1]benzoxazinones, -[1,2-b][2,4]benzoxazepinones, cyclopentane- and -cyclohexane[b]pyrrolo[2,1-a][3,1]-benzoxazines

The reactions of 2-carboxybenzaldehyde (1) with 1,3- or 1,4-aminoalcohols (2a-i, 3a,b) were used to prepare partially or fully saturated tetra- and pentacyclic compounds containing a condensed 1,3-oxazino- or oxazepinoisoindolone moiety and one terminal saturated carbocycle. Isoindolo[2,1-a][3,1]benzoxazinones (4a-d, 6, 7), stereoisomeric isoindolo[1,2-b][2,4]benzoxazepinones (5a-c) hexahydrocyclopentane[b]pyrrolo[1,2-a][3,1]-benzoxazinone (10a,b), octahydroindolo[1,2-b]- and decahydroindolo[1,2-a]benzoxazinone (11a,b and 12a,b) and related pentacyclic derivatives (4e-g) were prepared. The diastereomers 5a-c differ in the ring annelation or in the position of the NCHO hydrogens and annelational hydrogens. The stereostructures of these compounds were elucidated by means of 1H and 13C NMR spectroscopy, including DNOE, DEPT, 2D-HSC measurements and X-ray analysis.     

Ranitidine effervescent and famotidine wafer in the relief of episodic symptoms of gastro-oesophageal reflux disease

Radioligands that specifically target dopamine uptake sites can provide a means of determining dopamine fiber loss at intrastriatal mesencephalic grafts in Parkinsonian patients, using Positron Emission Tomography (PET). The BTCP derivative, 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-(2-hydroxyethyl)-piperazine, shows in vitro high affinity and selectivity for the dopamine transporter. To evaluate the potential of such a compound as a potential dopaminergic PET tracer the positron-emitting analogues, 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-(2-[18F]fluoroethyl)-piperazine and 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-[11C]methylpiperazine, were synthesized. Radiofluorination was carried out by the reaction of 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-(2-chloroethyl)-piperazine with cyclotron-produced n.c.a. 18F-(half life 109.9 min) obtained by the (p,n) reaction on 18O-enriched water. Labelling with carbon-11 (half life 20.4 min) was achieved by 11C methylation of 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-piperazine with [11C]methyl iodide. After intravenous administration to rats these two compounds enter the brain, but despite their high in vitro affinity they display a high non specific binding in vivo which greatly limits their use as PET radioligands.     

2-Phenylbenzo[b]thiophene-based antiestrogens with mammary tumor inhibiting activity

In this study we extended our studies on heterocyclic antiestrogens to 2-phenylbenzo[b]thiophenes which can be considered as isosteric to the 2-phenylindole system. We synthesized a number of 6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophenes with carbamoyl and amino functions in the side chain at carbon-3 and analyzed their biological properties. The binding affinities for the estrogen receptor are mainly influenced by the chain length whereas the hormonal profile depends on the nature of the functional group. From this study 3-[10-(2,2,3,3,4,4,4-heptafluorobutyl-methylcarbamoyl) decyl]-6-hydroxy-2-(4-hydroxyphenyl)benzo-[b]thiophene (6e) emerged as an antiestrogen with all the characteristics of a pure antagonist. It did not stimulate gene expression in HeLa cells cotransfected with the expression vector for the human estrogen receptor HEG0 and the luciferase reporter plasmid EREwtc luc nor did it show any estrogenic activity in the mouse uterus weight test. In the latter assay, it completely abrogated the stimulatory effect of estrone. Due to its antiestrogenic potency it strongly inhibited the growth of estrogen-sensitive human MCF-7 breast cancer cells with an IC50 value of 5 nM. These data suggest that an amide function in combination with the fluorination of the terminal carbon atoms is an appropriate modification to abolish the estrogenic action of the 2-phenylbenzothiophene system.     

Tolerance in the replacement of the benzhydrylic O atom in 4-[2-(diphenylmethoxy)ethyl]-1-benzylpiperidine derivatives by an N atom: development of new-generation potent and selective N-analogue molecules for the dopamine transporter

The replacement of the benzhydrylic oxygen atom of our previously developed dopamine transporter (DAT)-specific ligands 4-[2-(diphenylmethoxy)ethyl]-1-[(4-fluorophenyl)methyl]piperidine, 1a, and 4-[2-(bis(4-fluorophenyl)methoxy)ethyl]-1-benzylpiperidine, 1b, by a nitrogen atom resulted in the development of the N-analogues 4-[2-((diphenylmethyl)amino)ethyl]-1-[(4-fluorophenyl)methyl]pi peridi ne, 4a, and 4-[2-((bis(4-fluorophenyl)methyl)amino)ethyl]-1-benzylpiperidine, 4b. Biological evaluation of these compounds in rat striatal tissue and in HEK-293 cells expressing the cloned human transporters demonstrated high potency and selectivity of these compounds for the DAT. Thus the potency of the compound 4a for the DAT was 9.4 and 30 nM in rat striatal tissue and in the cloned transporter cells, and its binding selectivity for the DAT compared to the serotonin transporter (SERT) for these two systems was 62 and 195, respectively. The compound 4b similarly exhibited high potency and selectivity for the DAT. Thus, the replacement of the O atom in 1a,b by an N atom in 4a,b only had small effects on potency and selectivity. In comparison with GBR 12909 [1-[2-(bis(4-fluorophenyl)methoxy)ethyl]-4-(3-phenylpropyl)piperazine ] and WIN 35,428 [3beta-(p-fluorophenyl)-2beta-carbomethoxytropane] binding, these two novel N-analogues were slightly more potent and far more selective for the DAT. Thus, these novel N-analogues represent more polar new-generation piperidine congeners of GBR 12909. They might have useful potential application in developing a pharmacotherapy for cocaine dependence.     

Nonpeptide peptidomimetic antagonists of the neuropeptide Y receptor: benextramine analogs with selectivity for the peripheral Y2 receptor

We synthesized a new series of benextramine analogs as neuropeptide Y (NPY) functional group mimetics and tested them for N-[propionyl-3H]NPY ([3]NPY) displacement activity in rat brain membrane homogenates and for NPY receptor antagonist activity in the rat femoral artery. The tetraamine, carbon analog N,N'-bis[6-[N-(2-naphthylmethyl)amino]hexyl]-1,6-hexanediamine (15) was equipotent with benextramine (based on comparison of the relevant IC50's) in a rat brain [3H]NPY displacement assay, suggesting that the disulfide is not a necessary feature of benextramine's [3H]NPY displacement activity, although this analog maintained selectivity for the benextramine-sensitive binding site population. The bis(N,N-dialkylguanyl) disulfide and carbon analogs 14a-c were 3-4 times more potent than their respective controls in displacing [3H]NPY from rat brain membrane homogenates with IC50's ranging from 15 to 18 microM and maintained selectivity for the benextramine-sensitive, Y1 binding site population. However, the activity of the carbon analog N,N'-bis[6-[N-(2-naphthylmethyl)amino]hexyl]-N,N'-(1,6- hexanediyl)diguanidine tetrahydrochloride (14b) showed a different profile in a femoral artery vasoconstriction assay; at 1.0 nM, this analog shifted the concentration-effect curve of the Y2-selective agonist NPY13-36 to the right (pA2 = 9.2; Kd = 0.63 nM) without a significant change in the maximum effect, while even at 1.0 mM it had no effect on the vasoconstrictive activity of the Y1-selective agonist [Leu31,Pro34]NPY. Thus, the bis(N,N-dialkylguanidine) analogs of benextramine are selective, competitive antagonists of the postsynaptic NPY receptor in the femoral artery.     

New indole and pyridazinoindole analogs--synthesis and study as inhibitors of phosphodiesterases and as inhibitors of blood platelet aggregation

This paper presents the synthesis of new indole, pyridazino[4,5-b]-indole, and pyridazino[4,5-a]indole analogs as well as a study of their "in vitro" activity as inhibitors of different phosphodiesterases isolated from dog cardiac tissue, dog aorta, and bovine platelets; the study of their activity as inhibitors of platelet aggregation in guinea pig whole blood, with ADP and arachidonic acid (AA) as pro-aggregants, is also included. The selected compounds 8-benzyloxy-3,4-dihydro-1-(3,4,5-trimethoxy)benzylideneaminopyridazin o[4,5- b]indole 14g, and 8-benzyloxy-4-[(3,4-dimethyl)pyrazolyl]pyridazino[4,5-b]indo le 20 present an interesting profile as potential inodilators, with a complementary, beneficial activity as inhibitors of the aggregation, activities which could possibly be related to the inhibition of the PDE's. Among the other compounds studied, 8-benzyloxy-3,4-dihydro-1-[4-(methyl)piperazino]acetamidopyrida zino[4,5- b]indol-4-one 16c and 8-benzyloxy-3,4-dihydro-1-[4-(2- methoxyphenyl)piperazino]acetamidopyridazino[4,5-b]indol-4-o ne 16f stood out as inhibitors of platelet aggregation, with a mechanism that could possibly be related to the AA cascade.     

Studies on quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides. 8.1, 2 synthesis and pharmacological evaluation of tricyclic fused quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides as potential alpha1-adrenoceptor antagonists

A series of 2-substituted methyl 2,3-dihydroimidazo[1, 2-c]quinazolin-5(6H)-ones (4), 3-substituted methyl 2, 3-dihydroimidazo[1,2-c]quinazolin-5(6H)-ones (5), 3-substituted methyl 2,3-dihydro-5H-thiazolo[2,3-b]quinazolin-5-ones (15a,b), 3-substituted methyl 2,3-dihydroimidazo[2,1-b]quinazolin-5(1H)-ones (16a,b), 3-substituted methyl 2,3-dihydro-1H-imidazo[1,2-b][1,2, 4]benzothiadiazine 5,5-dioxides (33a,b), 2-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (42-45a,b), 3-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (50-53a,b), 3-substituted methyl 5H-thiazolo[2,3-b]quinazolin-5-ones (55-56a,b), and 3-substituted methyl 5-(methylthio)-2,3-dihydroimidazo[1,2-c]quinazoline (57) were synthesized as compound 1conformational rigid congeners for pharmacological evaluation as potential alpha1-adrenoceptor antagonists. Compounds 4, 5, 33a,b, 44a,b, 45a,b, 52a,b, 53a,b, and 57 were found to possess high affinity for the alpha1-adrenoceptor. Compounds 5 and 57 were the most highly selective and potent alpha1 antagonists with Ki = 0.21 +/- 0.02 and 0.90 +/- 0.08 nM, respectively. The S-enantiomers of these two compounds (Ki = 0.13 +/- 0.01 nM for (S)-(-)-5; Ki = 1.0 +/- 0.2 nM for (S)-(+)-57) were 144-200-fold more potent than the R-enantiomers (Ki = 26 +/- 8 nM for (R)-(+)-5; Ki = 144 +/- 23 nM for (R)-(-)-57). Compound 4 showed 8-fold higher affinity to alpha1A-AR better than alpha1B-AR. These compounds possessed weak to no activity against the 5-HT1A receptor.     

Absence of oxidative stress following paradoxical sleep deprivation in rats

With an aim of creating new, high affinity dopaminergic ligands, six different 3- and 4-substituted 1-[2-[5-(1H-benzimidazole-2-thione)] ethyl]piperidines and nine related heterocyclic congeners were synthesized and evaluated for in vitro binding affinity at D1 and D2 dopamine receptors. Synaptosomal membranes prepared from fresh bovine caudate nuclei were used as a source of the dopamine receptors. Only 4-[bis-(4-fluorophenyl)methylene]-piperidines, compounds 9e, 10d, and 11d, expressed moderate affinity for the D1 receptors, while all other compounds were inactive competitors of [3H]SCH 23390. Compounds 9c, 9d, 10c, 11a, and 11c were inactive in the D2 receptor binding assay, as well. Derivatives of 4-phenylpiperidine (9-11b) and 3-phenylpiperidine (10a) expressed a moderate to low affinity for the D2 receptors. However, racemic (+/-)-1-[2-[5-(1H-benzimidazole-2-thione)] ethyl]-3-phenylpiperidine 9a and its enantiomer (+)-9a behaved as selective, high affinity D2 receptor ligands, the latter being some four times more active than the racemate.     

Dihydrobenzofuran analogues of hallucinogens. 3. Models of 4-substituted (2,5-dimethoxyphenyl)alkylamine derivatives with rigidified methoxy groups

Tetrahydrobenzodifuran functionalities were employed as conformationally restricted bioisosteres of the aromatic methoxy groups in prototypical hallucinogenic phenylalkylamines 1 and 2. Thus, a series of 8-substituted 1-(2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b']difuran-4-yl)-2-aminoal kanes (7a-e) were prepared and evaluated for activity in the two-lever drug discrimination paradigm in rats trained to discriminate saline from LSD tartrate (0.08 mg/kg) and for the ability to displace [3H]ketanserin from rat cortical homogenate 5-HT2A receptors and [3H]-8-OH-DPAT from rat hippocampal homogenate 5-HT1A receptors. In addition, 1-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b']difuran-4-yl)-2-am inopropane (7b), which was found to be extremely potent in the rat in vivo assays, was evaluated for its ability to compete with [125I]DOI and [3H]ketanserin binding to cells expressing cloned human 5-HT2A, 5-HT2B, and 5-HT2C receptors. All of the dihydrofuranyl compounds having a hydrophobic substituent para to the alkylamine side chain had activities in both the in vitro and in vivo assays that equaled or surpassed the activity of the analogous conformationally flexible parent compounds. For example, 7b substituted for LSD in the drug discrimination assay with an ED50 of 61 nmol/kg and had Kj values in the nanomolar to subnanomolar range for the displacement of radioligand from rat and human 5-HT2 receptors, making it one of the most potent hallucinogen-like phenylalkylamine derivatives reported to date. The results suggest that the dihydrofuran rings in these new analogues effectively model the active binding conformations of the methoxy groups of the parent compounds 1 and 2. In addition, the results provide information about the topography and relative orientation of residues involved in agonist binding in the serotonin 5-HT2 receptors.     

Covalent coupling of immunoglobulin G to self-assembled monolayers as a method for immobilizing the interfacial-recognition layer of a surface plasmon resonance immunosensor

Protocols have been developed for the random and site-directed covalent coupling of immunoglobulin G [anti-hIgG] [IgG] to silver surfaces modified with a self-assembled monolayer [SAM] of thioctic acid, mercaptopropionic acid [MPA], L-cysteine or 4-aminothiophenol [PATP]. A surface plasmon resonance [SPR] immunosensor fabricated with a more ordered and hydrophilic IgG-SAM-silver interfacial layer, demonstrates an increased ability for performing sensitive and selective assay of human immunoglobulin G [hIgG] compared with a device fabricated with a physically-adsorbed IgG-silver interfacial-layer due to reduced levels of non-specific binding. Detection limits [DL] for hIgG from serum down to 6 micrograms/ml (40 nM) and assay sensitivities up to 0.24 ng hIgG/mm2/nM are reported.     

2-[N-Acylamino(C1-C3)alkyl]indoles as MT1 melatonin receptor partial agonists, antagonists, and putative inverse agonists

The synthesis of several novel indole melatonin analogues substituted at the 2-position with acylaminomethyl (8-11), acylaminoethyl (5a-k), or acylaminopropyl (13) side chains is reported. On the basis of a novel in vitro functional assay (specific binding of [35S]GTPgammaS), which can discriminate agonist from partial agonist, antagonist, and inverse agonist ligands, 5a,g, h,j and 13 were shown to be partial agonists, 5d,e and 8-11 competitive antagonists, and 5b,c,k putative inverse agonists. Binding and functional assays were performed on cloned human MT1 receptor. Structure-activity relationship considerations indicate that N-[1-aryl-2-(4-methoxy-1H-indol-2-yl)(C1-C2)alkyl]alkanamides represent a lead structure for this type of ligands.     

Crystallization of the bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase domain of the human trifunctional enzyme

Methylenetetrahydrofolate([H4] folate) dehydrogenase (D) and methenyl[H4] folate cyclohydrolase (C) coexist as a bifunctional enzyme (DC) or as the amino-terminal domain of a trifunctional enzyme (DCS) where the third activity is 10-formyl[H4]folate synthetase (S). Two crystal forms of the DC domain of the human cytosolic DCS enzyme have been grown from polyethyleneglycol solution. The monoclinic P2(1) crystals diffract to 2.8 A with a = 72.5 A, b = 68.5 A, c = 125.2 A, and beta = 91.8 degrees but were found to be twinned. The orthorhombic P2(1)2(1)2(1) crystals diffract to 2.5 A with a = 67.7 A, b = 135.9 A, c = 61.6 A, and contain two molecules per asymmetric unit.     

Estimation of unit risk for coke oven emissions

We are investigating the hypothesis that biotin multimers can be used with streptavidin and monoclonal antibody conjugates in cancer pretargeting protocols to provide a method of increasing the amount of radioactivity bound on cancer cells in patients. As part of that investigation, a series of biotinylated Starburst dendrimers (BSBDs) have been prepared and evaluated in vitro and in vivo. In this study, a new biotinidase-stabilized, water-solubilizing biotinylation reagent was prepared and reacted with Starburst (PAMAM) dendrimers, generations 0, 1, 2, 3, and 4. The reaction conditions employed resulted in perbiotinylation of generation 0 (four biotin moieties conjugated), generation 1 (eight biotin moieties conjugated), generation 2 (16 biotin moieties conjugated), and generation 3 (32 biotin moieties conjugated). With generation 4, incomplete biotinylation was achieved resulting in the largest portion of that BSBD having 51 biotin moieties (of 64 possible) conjugated. The ability of each BSBD to cross-link streptavidin (SAv) was examined in an in vitro assay. In that assay, an assessment was made of the quantity of [125I]SAv bound with polystyrene-bound SAv after treatment with the synthesized BSBDs. All BSBDs cross-linked the polystyrene-bound SAv with [125I]SAv; however, the amount of [125I]SAv bound varied with the different BSBDs. Roughly 1 equiv of [125I]SAv was bound when Starburst dendrimers containing three or four biotin moieties (generation 0) were used. Two equivalents were bound with BSBD generation 1, and 4 equiv were bound with BSBDs generations 2, 3, and 4. To assess the distribution of BSBDs generations 0, 1, and 2 in mice (at 4 h postinjection), a method was developed for radioiodinating them using the NHS ester of p-[125I]iodobenzoate ([125I]PIB). It was found that the radioiodinated BSBDs had low blood concentrations (i.e., 0.13-0.20% ID/g) at the 4 h time point. In fact, most tissues examined had low concentrations of biotinylated dendrimers, except kidney and liver. Kidney had the highest concentration of [125I]-labeled BSBDs, and its concentration increased with increasing size and charge of dendrimer (e.g., 8-48% ID/g). On the basis of the increased radioactivity observed in the in vitro assay and the rapid clearance from blood in mice, additional in vivo studies with perbiotinylated Starburst dendrimer, generation 2, are planned.     

A detergent-solubilized nicotinic acetylcholine receptor of Caenorhabditis elegans

We have used a spin column assay to study the detergent-solubilized levamisole receptor, a nicotinic acetylcholine receptor of the nematode Caenorhabditis elegans. The receptor can be successfully solubilized in detergent solutions of Triton X-100, Lubrol PX, or sodium cholate. Centrifugal gel filtration assay using the tritiated ligand [3H]meta-aminolevamisole ([3H]MAL) provides a greater signal and a better signal-to-noise ratio for soluble levamisole receptor binding than either polyethylene glycol precipitation or DEAE filter assay with the same ligand. As for membrane-bound receptor, the detergent-solubilized levamisole receptor consists of more than one affinity state. Detergent solubilization appears to increase the affinity of all states for [3H]MAL (Kd for the highest affinity solubilized [3H]MAL binding state, 41 +/- 5 pM). Data is presented on the equilibrium binding and the association and dissociation reaction rates of the receptor. The similar relative efficacy with which various compounds inhibit specific [3H]MAL binding and deficiencies in solubilizable high affinity specific [3H]MAL binding in two receptor mutants show that the solubilized receptor is the same nicotinic acetylcholine receptor that is detected by assaying membrane-bound specific [3H]MAL binding. The detergent-solubilized levamisole receptor is stable at 0 degree to 4 degrees C, making receptor purification feasible.     

Alendronate distributed on bone surfaces inhibits osteoclastic bone resorption in vitro and in experimental hypercalcemia models

Alendronate is an aminobisphosphonate that acts as a potent inhibitor of osteoclastic bone resorption. To understand the mechanism of action of alendronate in vivo, in this study we investigated the relationship between distribution of [14C]-alendronate in rat bone and its effects on bone resorption in vitro or in rat hypercalcemic models. A single IV dose of 0.05 approximately 1.25 mg/kg inhibited the increase in plasma calcium level induced by bovine PTH or 1 alpha(OH)D3. The minimal effective dose of pamidronate (1.25 mg/kg) and etidronate (over 31.25 mg/kg) were at least 5 times and 25 times, respectively, higher than the dose of alendronate in the rat hypercalcemic model prepared by 1 alpha(OH)D3. The relative potencies of compounds in the hypercalcemic rat models reflected those of inhibitory effects on bone resorption in vitro. We conducted the ivory-slice assay under two conditions: (a) addition of a given bisphosphonate after adherence of the osteoclasts; and (b) preincubation of the ivory slices with a given bisphosphonate. The inhibitory IC50 values of alendronate under condition (b) were similar to those under condition (a). To evaluate the interaction between osteoclasts and alendronate in bone, we investigated the localization of [14C]-alendronate in the tibia of growing rats (4-day-old rats). Alendronate did not distribute uniformly in the tibia. At 1 day after injection (0.05 mg SC), dense labeling was seen primarily under osteoclasts. We injected 0.05 mg/kg of [14C]-alendronate (single i.v.) into rats [14C]-alendronate was rapidly eliminated from plasma, and mainly distributed to the bone in rats. These data suggest that alendronate which distributed on bone surface mainly contributed to the antihypercalcemic action in vivo.     

Influence of chronic variable stress (CVS) on the association of glucocorticoid receptor with heat-shock protein (HSP) 90 in rat hippocampus

Brevetoxins are produced by the marine dinoflagellate Ptychodiscus brevis, an organism linked to red tide outbreaks, and the accompanying toxicity to marine animals and to neurotoxic shellfish poisoning in humans. Brevetoxins bind with high affinity to voltage-sensitive sodium channels and cause increased sodium ion conductance and nerve cell depolarization. The brevetoxin competitive binding assay with tritium-labeled brevetoxin 3 (3H-PbTx-3) and rat brain synaptosomes is a sensitive and specific assay for pure brevetoxins. Here we report that extracts of manatee, turtle, fish, and clam tissues contain components that interfere with the assay by cooperative, noncompetitive inhibition of 3H-PbTx-3 specific binding and increased nonspecific binding to synaptosomes. By determining the "apparent" toxin concentration ("[Toxin]") in the extract at several assay concentrations, a reasonable correction for the complex inhibition could be made using a semilog plot to extrapolate [Toxin] to zero extract concentration to obtain [Toxin]0. Spiking 4 extracts with 60 nM PbTx-3 caused [Toxin]0 to increase by 41 +/- 8 nM, indicating that the noncompetitive components did not prevent the assay of toxin but did reduce the accuracy of the result. Fourfold repetition of the assay of 4 samples gave standard deviations of 25 to 60% of the value of [Toxin]0, so the error can be fairly large, especially for samples with little toxin. Purification of an extract with a 1 g sample prep column of C-18 decreased the complex inhibition by about 3-fold but did not eliminate interference in the assay.     

Synthetic peptides related to laminin pentapeptide (YIGSR) fragment

The synthetic laminin pentapeptide amide fragment (LF), Tyr-Ile-Gly-Ser-Arg-NH2 corresponding to a part of B1 chain of the glycoprotein, laminin, and six of its analogues having structural modifications at positions 1, 3 and 4 were synthesized by solid phase method employing mainly 9-fluorenylmethoxycarbonyl-amino acid trichlorophenyl esters as coupling agents and Merrifield resin as the solid support. Their biological activities were studied in vivo by lung tumor colonization assay and in vitro by cell adhesion assay. The activity of synthetic LF was found to correlate with the earlier reported results in both in vivo and in vitro assays. Among the analogues made, [Tyr4] LF and [Thr4]LF were found to inhibit the lung tumor colonies more efficiently than LF itself in the in vivo assay whereas [D- Ser4]LF exhibited almost the same inhibition as LF.