Phospholipase C activity affinity purifies with the Torpedo nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors mediate fast synaptic transmission by fluxing ions across the membrane in response to neurotransmitter binding. We show here that during affinity purification of the nicotinic acetylcholine receptor from Torpedo, phosphatidic acid, but not other anionic or zwitterionic phospholipids, is hydrolyzed to diacylglycerol. The phospholipase C activity elutes with the acetylcholine receptor and is inhibited by a lipid phosphate phosphohydrolase inhibitor, sodium vanadate, but not a phosphatidate phosphohydrolase inhibitor, N-ethylmaleimide. Further, the hydrolysis product of phosphatidic acid, diacylglycerol, enhances the functional capabilities of the acetylcholine receptor in the presence of anionic lipids. We conclude that a phospholipase C activity, which appears to be specific for phosphatidic acid, is associated with the nicotinic acetylcholine receptor. The acetylcholine receptor may directly or indirectly influence lipid metabolism in a manner that enhances its own function.

Structural characterization and agonist binding to human α4ß2 nicotinic receptors.

The Cys-loop receptor super-family of neurotransmitter-gated ion channels mediates fast synaptic transmission throughout the human nervous system. These receptors exhibit widely varying pharmacologies, yet their structural characterization has relied heavily on their homology with the naturally abundant muscle-type Torpedo nicotinic acetylcholine receptor. Here we examine for the first time the structure of a human α4ß2 neuronal nicotinic acetylcholine receptor. We show that human α4ß2 nicotinic receptors adopt a secondary/tertiary fold similar to that of the Torpedo nicotinic receptor with a large proportion of both α-helix and ß-sheet, but exhibit a substantially increased thermal stability. Both receptors bind agonist, but with different patterns of agonist recognition - particularly in the nature of the interactions between aromatic residues and the agonist quaternary amine functional group. By comparing α4ß2 and Torpedo receptors, we begin to delineate their structural similarities and differences.

Total selenium and selenomethionine in pharmaceutical yeast tablets: assessment of the state of the art of measurement capabilities through international intercomparison CCQM-P86.

Results of an international intercomparison study (CCQM-P86) to assess the analytical capabilities of national metrology institutes (NMIs) and selected expert laboratories worldwide to accurately quantitate the mass fraction of selenomethionine (SeMet) and total Se in pharmaceutical tablets of selenised-yeast supplements (produced by Pharma Nord, Denmark) are presented. The study, jointly coordinated by LGC Ltd., UK, and the Institute for National Measurement Standards, National Research Council of Canada (NRCC), was conducted under the auspices of the Comité Consultatif pour la Quantité de Matière (CCQM) Inorganic Analysis Working Group and involved 15 laboratories (from 12 countries), of which ten were NMIs. Apart from a protocol for determination of moisture content and the provision of the certified reference material (CRM) SELM-1 to be used as the quality control sample, no sample preparation/extraction method was prescribed. A variety of approaches was thus used, including single-step and multiple-step enzymatic hydrolysis, enzymatic probe sonication and hydrolysis with methanesulfonic acid for SeMet, as well as microwave-assisted acid digestion and enzymatic probe sonication for total Se. For total Se, detection techniques included inductively coupled plasma (ICP) mass spectrometry (MS) with external calibration, standard additions or isotope dilution MS (IDMS), inductively coupled plasma optical emission spectrometry , flame atomic absorption spectrometry and instrumental neutron activation analysis. For determination of SeMet in the tablets, five NMIs and three academic/institute laboratories (of a total of five) relied upon measurements using IDMS. For species-specific IDMS measurements, an isotopically enriched standard of SeMet (76Se-enriched SeMet) was made available. A novel aspect of this study relies on the approach used to distinguish any errors which arise during analysis of a SeMet calibration solution from those which occur during analysis of the matrix. To help those participants undertaking SeMet analysis to do this, a blind sample in the form of a standard solution of natural abundance SeMet in 0.1 M HCl (with an expected value of 956 mg kg(-1) SeMet) was provided. Both high-performance liquid chromatography (HPLC)-ICP-MS or gas chromatography (GC)-ICP-MS and GC-MS techniques were used for quantitation of SeMet. Several advances in analytical methods for determination of SeMet were identified, including the combined use of double IDMS with HPLC-ICP-MS following extraction with methanesulfonic acid and simplified two-step enzymatic hydrolysis with protease/lipase/driselase followed by HPLC-ICP-IDMS, both using a species-specific IDMS approach. Overall, satisfactory agreement amongst participants was achieved; results averaged 337.6 mg kg(-1) (n = 13, with a standard deviation of 9.7 mg kg(-1)) and 561.5 mg kg(-1) (n = 11, with a standard deviation of 44.3 mg kg(-1)) with median values of 337.6 and 575.0 mg kg(-1) for total Se and SeMet, respectively. Recovery of SeMet from SELM-1 averaged 95.0% (n = 9). The ability of NMIs and expert laboratories worldwide to deliver accurate results for total Se and SeMet in such materials (selensied-yeast tablets containing approximately 300 mg kg(-1) Se) with 10% expanded uncertainty was demonstrated. The problems addressed in achieving accurate quantitation of SeMet in this product are representative of those encountered with a wide range of organometallic species in a number of common matrices.

Behavioral effects of chronic phencyclidine administration in rats.

The development of tolerance to phencyclidine (PCP) was examined in rats using behavioral rating scales with simultaneous measurements of locomotor activity, stereotyped behaviors, and ataxia. Significant tolerance to the stereotyped behaviors and ataxia induced by 5 or 10 mg/kg PCP was found on day 5 of chronic drug treatment. Because ataxia interferes with PCP-induced locomotor activity (Sturgeon et al. 1979), tolerance to PCP-induced ataxia produced an increase in locomotor activity on day 5. Tolerance to the ataxia, but not to the stereotyped behaviors induced by PCP, was more prominent after day 15 of PCP administration than after day 5. Administration of PCP for 15 days resulted in significant decrease in locomotor activity for the 5 mg/kg group but not for the 10 mg/kg group. These results suggest that behavioral tolerance, rather than supersensitivity, develops after chronic PCP administration. The effects of PCP returned to baseline over a 14-day withdrawal period for rats treated with 5 mg/kg PCP for 15 days. Rats treated with 10 mg/kg PCP for 15 days still had not returned to baseline when tested 28 days after cessation of PCP treatment.

Effects of lithium on behaviour induced by phencyclidine and amphetamine in rats.

d-Amphetamine and phencyclidine (PCP) have both been reported to produce manic-like sequela in humans, effects that are reportedly antagonized by lithium. To test the hypothesis that the acute effects of these drugs in rats may serve as models of mania, the behaviors induced by d-amphetamine (3 mg/kg) or PCP (5 mg/kg) were quantified on behavioral rating scales subsequent to chronic dietary pretreatment with lithium carbonate or control diet. On day 14 of pretreatment, PCP-induced stereotyped behaviors and ataxia were potentiated in rats receiving lithium (plasma levels 1.0 +/- 0.23 mEq/l). PCP-induced locomotor activity was not affected by lithium pretreatment. Stereotypies and locomotion induced by d-amphetamine were also not significantly affected by lithium pretreatment. These results suggest that neither PCP nor amphetamine administered acutely to rats will be useful models to explore the manic-like symptoms produced by these drugs in humans.

Behavioral rating scales for assessing phencyclidine-induced locomotor activity, stereotyped behavior and ataxia in rats.

Behavioral rating scales were developed for quantification of phencyclidine (PCP)-induced locomotor activity, stereotyped behavior and ataxia in rats. The dose-response relationship for PCP-induced locomotor activity was found to be an inverted U-shaped function over the first 25 min after injection while over the last 30 min of the experiment the function was highly linear. A linear dose-response relationship was found for ratings of stereotyped behavior and ataxia throughout the 90 min period of observation. The ratings of these two behaviors were found to be closely parallel. The effects of PCP on locomotor activity were found to be greatest during those intervals when stereotyped behavior and ataxia were at moderate levels. Ratings of locomotor activity may be confounded by ataxia when PCP is administered alone or in combination with other drugs.

A comparison of the effects of neuroleptics on phencyclidine-induced behaviors in the rat.

The dose-response effects of neuroleptic pretreatment on phencyclidine (PCP; 3 or 5 mg/kg)-induced locomotor activity, stereotyped behaviors and ataxia were quantified in groups of male rats using rating scales recently developed in this laboratory. Three butyrophenone neuroleptics consistently produced dose-dependent antagonism of the behavioral effects of PCP administration. Fluphenazine antagonized the behavioral effects produced by 3 mg/kg PCP but not those produced by 5 mg/kg PCP. Each of the other neuroleptics examined (chlorpromazine, thioridazine, mesoridazine, triflupromazine, cis-flupenthixol) had no consistent antagonistic effect or actually enhanced one or more of the behavioral effects of PCP. Some neuroleptics slightly reduced PCP locomotion or stereotypies at high doses, but these effects were probably a non-specific consequence of the synergistic ataxia-producing properties of these drugs. In a second set of experiments, atropine sulfate pretreatment increased PCP-induced locomotor activity and stereotyped behaviors but had no effect on ataxia; pretreatment with physostigmine produced opposite effects. Combined pretreatment with haloperidol and atropine sulfate significantly reduced only haloperidol antagonism of PCP-induced ataxia, thus suggesting that non-dopoaminergic effects of neuroleptics may interfere with their ability to antagonize PCP.

The graphite furnace and its role in atomic spectroscopy.

The diversity of applications of the graphite furnace is extraordinary, encompassing the fields of physics, thermochemistry, spectroscopy and analytical chemistry. In this respect, the graphite furnace has been used on a continuous basis as a research tool for nearly a century. Following its introduction as an atomization source for atomic absorption spectrometry by L'vov in 1959, its role in atomic spectrometry expanded considerably to encompass analytical applications in emision, fluorescence, absorption and mass spectrometry. In addition to its conspicuous use as an atomization source in these areas, it is frequently employed as a vaporizer when used in the format of combined and tandem sources with other instrumentation. The unique physico-chemical micro-environment which can be attained within the graphite furnace has also been used to advantage in a number of investigations, including the determination of gas- and solid-phase diffusion coefficients of high-temperature metal vapours, the heats of sublimation of refractory metals, fundamental optical constants and the measurement of the heats of desorption of adatoms from high-temperature surfaces. The range of such applications remains to be more fully explored. The attractive features of this source, viz., the high atomization/vaporization efficiency, comparatively long atomic vapour residence times, controllable chemical and thermal environment and its ability to handle high dissolved solids content samples (

Determination of total chromium in Marine Sediment Reference Material BCSS-1.

The determination of Cr in National Research Council of Canada Marine Sediment Reference Material BCSS-1 is addressed. Mixed acid digestions utilizing HF, HClO(4) and HNO(3) were investigated. Single microwave assisted digestions in closed vessels at medium pressures (8 bar) were inappropriate (80% recovery). Double digestion at moderate pressure and digestions at high pressure (70 bar) or lengthy open-beaker hot plate dissolutions resulted in 93+/-5% recovery of certified Cr content. Flame atomic absorption and inductively coupled plasma atomic emission spectrometry were used for quantitation. In all cases, the method of standard additions was necessary to eliminate the approximately 10% enhancement in re- sponce relative to unmatched standards. Compared to a certified content of 123+/-7 microg/g (mean and standard deviation), values of 116+/-6 were obtained using an open beaker digestion; 114+/-5 using a double microwave digestion at 8 bar; 113+/-2 for microwave digestion at high pressure and 111+/-4 at high pressure (81 bar) with triple microwave digestion in a Parr bomb. No acid dissolution procedure is adequate for Cr in this sample although recovery of total Cr is complete from National Institute of Standards and Technology Buffalo River Sediment (SRM 2704).

Electronic absorption spectra and protolytic equilibria of doxorubicin: direct spectrophotometric determination of microconstants.

The ground- and excited-state dissociation constants and the electronic absorption and fluorescence spectra of doxorubicin were investigated by spectrophotometry. A general method for the direct calculation of individual microscopic dissociation constants was derived using the spectrophotometric data obtained. It was concluded that the protonated amino sugar group is slightly more acidic than the phenolic group. The spectrophotometric data were analyzed, and the macro- and microconstants for the various equilibria was reported.

Steric inhibition of conjugation in lowest excited singlet state of 9-anthramide by hydrogen bond donor solvents: role of solvent in chemical structure.

9-Anthramide has electronic absorption and fluorescence spectra that, in water, are similar to those of anthracene. This result is attributed to steric hindrance of the 9-carboxamido group with the peri-hydrogen atoms in the 1- and 8-positions of the anthracene ring. However, in aprotic solvents, although the absorption spectrum of 9-anthramide is anthracene-like, its fluorescence spectrum is red shifted and structureless. This finding is attributed to excited-state rotation of the 9-carboxamido group into coplanarity with the anthracene ring and indicates that, in water, the hydrogen-bonded solvent cage affects the steric inhibition of conjugation in excited 9-anthramide. These findings suggest that studies of structure and reactivity of drugs in nonaqueous or solid matrixes are probably of only limited value, since in the strongly interacting aqueous media the aqueous solvent cage plays a substantial role in determining molecular structure and reactivity.

Improved spectrophotometric determination of glycerol and its comparison with an enzymatic method.

A chemical method for the determination of glycerol was developed and compared to an enzymatic assay for sensitivity and reproducibility. The chemical assay is based on glycerol oxidation to formaldehyde and subsequent reaction with chromotropic acid to yield a colored product. With this method, as little as 5 micrograms of glycerol/ml can be detected. The enzymatic assay is based on enzymatic glycerol phosphorylation followed by glycerol phosphate dehydrogenation by nadide (nicotinamide adenine dinucleotide). The reduced nadide is used to reduce iodonitrotetrazolium violet to its colored formazan product. The enzymatic method can be used to deterine 50 micrograms of glycerol/ml in aqueous samples.

In vitro adsorption of doxorubicin hydrochloride on insoluble calcium phosphate.

The adsorption of doxorubicin hydrochloride, a potent antitumor agent, on solid tribasic calcium phosphate was studied in vitro. A Langmuir adsorption isotherm at pH 7.4 and the maximum adsorption capacity of tribasic calcium phosphate were established. Tribasic calcium phosphate was chosen as a model for solid bone samples, which are stained with doxorubicin in patients who have received long-term doxorubicin therapy.

Spectroscopic approach to estimation of microequilibrium constants of prototropic reactions of aminobenzoic acids.

The microequilibrium constants of protolytic dissociation of diprotic acids, dihydric bases, or ampholytes such as the aminobenzoic acids, with dissimilar ionizing groups, can be estimated by spectrophotometric titration and measurement of the molar absorptivity at the long wavelength absorption maximum of simple alkylated derivatives. The method is applicable when the long wavelength absorption spectral bands of the tautomeric species are well resolved. Compared to the traditional method of estimating microequilibrium constants using the dissociation constants of alkylated derivatives, the proposed method is simpler, faster, and more accurate.

Binding of N-substituted anthracenecarboxamides to double-stranded DNA: an electronic spectral study.

The electronic absorption and fluorescence spectra of two isomeric anthracenecarboxamides substituted with quaternary alkyl ammonium groups were studied as a function of solvent polarity, solvent rigidity, and state of protonation. These relatively simple environmental perturbations of the electronic spectra were employed to interpret spectral shifts caused by complexation with the bases or base pairs of DNA. The exocyclic side chains of the compounds studied have some freedom of movement when the spectroscopic probes are bound to double-stranded DNA.

Effects of phencyclidine and methylphenidate on d-amphetamine-induced behaviors in reserpine pretreated rats.

The effects and interactions of phencyclidine (PCP), methylphenidate and d-amphetamine on locomotor activity, stereotyped behavior and ataxia in reserpine- and vehicle-pretreated rats were examined. The behaviors of rats receiving PCP alone or in combination with other drugs were quantified along three dimensions (locomotor activity, stereotyped behavior, and ataxia) on scales developed in this laboratory. The behaviors of groups receiving methylphenidate and/or d-amphetamine in treatment combinations other than those including PCP were quantified using a well known d-amphetamine behavioral rating scale. PCP, methylphenidate and d-amphetamine each induced significant increases in locomotor activity and stereotyped behavior when administered alone. Reserpine was found to antagonize PCP-induced locomotor activity and stereotyped behavior, and methylphenidate-induced stereotyped behavior at a dose which either potentiated or had no significant effect upon d-amphetamine-induced behavior (depending upon the scale used). Reserpine also potentiated PCP-induced ataxia. Whereas PCP potentiated the locomotor activity induced by d-amphetamine in both reserpine- and vehicle-pretreated subjects, methylphenidate marginally antagonized d-amphetamine-induced stereotypy in reserpine-pretreated subjects. PCP-induced ataxia in reserpine pretreated subjects appeared moderately reduced in subjects also receiving d-amphetamine. In general, the behavioral effects of PCP appear to be more similar to those of methylphenidate than to those of d-amphetamine, but differences are also found between PCP and methylphenidate. The results are discussed in relation to a behavioral model recently proposed as a method for differentiating indirect dopamine agonists on the basis of their neurochemical mechanisms of action.

The oxidation of terminal D-galactofuranose residues of a galactan and a glycoprotein by a D-galactose oxidase preparation from Dactylium dendroides.

A galactan, isolated from the unicellular organism Prototheca zopfii, and a glycoprotein from a hyphal cell-wall fraction of the fungus Pithomyces chartarum have been oxidised by a D-galactose oxidase preparation from Dactylium dendroides. The oxidised polymers were subsequently reduced with sodium borotritide. The site of oxidation was identified as C-6 of non-reducing D-galactofuranosyl residues in both polymers.

Affinity chromatography of sialoglycoproteins, utilising the interaction of serotonin with n-acetylneuraminic acid and its derivatives.

Serotonin, immobilised on Sepharose 4B, has been used to study the affinity chromatography of neuraminic acid and its derivatives. Free N-acetylneuraminic acid and oligosaccharides, polysaccharides, and glycoproteins containing that sugar are specifically bound to the columns. Removal of neuraminic acid from sialoglycoconjugates, or modification of the neuraminic acid residues by periodate oxidation, abolishes their ability to bind to the ligand. The presence of the N-acetyl group, but not the N-glycolyl group, and the integrity of the side chain (C-7-C-9) of the neuraminic acid are essential for binding to serotonin.

Purification of some glycoside hydrolases by affinity chromatography.

Two glycoproteins have been isolated from the cell walls of baker's yeast. One is a glucan-protein complex which has been partially characterised as having a branched carbohydrate structure composed of chains of (1 leads to 3)-linked beta-D-glucosyl residues, some of which are attached by (1 leads to 6)-linkages to the main chain. Immobilization of this glycoprotein was achieved by covalent attachment to Sepharose, and the product was used to isolate a number of (1 leads to 3)-beta-D-glucan hydrolases from Helix pomatia, malted barley, and Basidiomycete QM806. The second glycoprotein, a mannan-protein complex, after immobilization, has been used in the purification of an alpha-D-mannosidase from jack-bean meal.

Immobilization of 8-hydroxyquinoline onto silicone tubing for the determination of trace elements in seawater using flow injection ICP-MS.

A rapid and simple on-line method is described for the preconcentration of Mn, Co, Ni, Cu, Zn, Cd and Pb from sea water using 8-hydroxyquinoline immobilized onto silicone tubing (Sil-8-HQ) via the Mannich reaction. Recoveries between 35 and 95% and limits of detection in the ppt range were obtained using a 2 m long Sil-8-HQ tube with a sample flow rate of 1.0 ml min(-1). A tube could be subjected to sample loading and elution cycles over 200 times. The capacity was 1.5 and 1.3 mug cm(-2) for Cu and Mn, respectively. Cu, Cd, Co, Pb, Mn, Zn and Ni were determined in coastal and open ocean seawater using flow injection inductively coupled plasma mass spectrometry (FI-ICP-MS). Good agreement with certified values for the certified reference materials NASS-4 and CASS-3 was demonstrated when quantitation was undertaken by the method of additions.