Estimating the Growth Rate of a Bacterial Species in a Complex Mixture by Hybridization of Genomic DNA

Abstract Advances in molecular techniques have enabled new approaches to identifying bacteria. However, once identified, there is no quantitative information on the in situ growth rate of the species, mainly because the technology has not been available. The quantitative incorporation of [methyl-³H]thymidine into dividing bacteria is coupled with a molecular (hybridization) method, to determine the growth rate of bacterial species in situ. The basis of this molecular method is a reverse gene probe—natural populations are labeled in situ with [methyl-³H]thymidine. The probe (³H-Tdr-DNA) is captured, using a hybridization procedure, on a positively charged nylon membrane on which is attached non-labeled target DNA. Two bacterial species, Bacillus cereus and Zoogloea ramigera, were used to demonstrate the principle in laboratory cultures and in a municipal activate sludge treatment process. The DNA of the dividing bacteria in activated sludge was radioactively labeled with [methyl-³H]thymidine, and the DNA of Z. ramigera was recovered using a DNA hybridization method. The recovered radioactively-labeled DNA was used to estimate the growth rate (0.03 × 10⁹ cells · ml⁻¹· h⁻¹) of Z. ramigera in situ. Simultaneously applying these two powerful molecular-based methods could potentially be used to study bacterial population dynamics in situ. Received: 10 September 1997; Accepted: 5 January 1998     

α-DIFLUOROMETHYL DOPA, A NEW ENZYME-ACTIVATED IRREVERSIBLE INHIBITOR OF AROMATIC L-AMINO ACID DECARBOXYLASE

DL-x-Difluoromethyl DOPA (DFMD, RMI 71801), an enzyme-activated irreversible inhibitor of aromatic L-amino acid decarboxylase in vitro, produces a rapid, long-lasting and dose-dependent inhibition of aromatic L-amino acid decarboxylase in peripheral tissues of mice when administered i.p. or orally. Doses of 500 mg/kg i.p. produce only very slight inhibition of the enzyme activity in mouse brain whilst inhibiting the enzyme activity of peripheral tissues by more than 90%. With L-[³H]-DOPA co-administration brain concentrations of L-[³H]DOPA and ³H-catecholamines are increased 3- to 8-fold concomitant with a decrease in the peripheral decarboxylation of L-[³H]DOPA. Under these conditions it is clear that the slight inhibition of enzyme activity in the brain is totally inadequate to inhibit the decarboxylation of L-DOPA in this organ. Similarly, the decarboxylation of exogenously supplied 5-hydroxytryptophan is inhibited peripherally with a consequent increase in brain serotonin concentrations. DFMD is another example of an enzyme-activated irreversible inhibitor which due to its novel and specific mechanism of action, may offer advantages over existing decarboxylase inhibitors.     

Characteristics of Protein Carboxyl Methylation in the Rat Hypothalamus

Abstract: The formation of methyl-labeled S-adenosylmethionine (AdoMet) and methyl esters of endogenous methyl-acceptor proteins (MAPs) was studied in a synaptosomal preparation from the rat hypothalamus labeled with L-[methyl-³H]methionine. Incubation of synaptosomes with l -[methyl-³H]methi-onine resulted in a rapid labeling of the AdoMet pool and a less rapid formation of ³H-methyl-MAPs. Accumulation of ³H-methyl-MAPs was linear over a 30-min period. The effects of various inhibitors of AdoMet-dependent trans-methylation reactions on the formation of carboxylmethylated MAPs were examined. When hypothalamic synaptosomes were preincubated with l -[methyl-³H]methionine and subsequently incubated for 30 min in the presence of S-adenosyl-l -homocysteine (AdoHcy, 100 μm ), ³H-methyl-MAP formation was inhibited by approximately 70%. 100 μm -l -homocysteine thiolactone (HTL) as well as 100 μm -3-deazaadenosine (c³Ado) also caused a 60–70% inhibition of ³H-methyl-MAP formation; the combination of both c³Ado and HTL produced a slightly but not significantly greater inhibition than either agent alone. 10 μm -adenosine or 10 μm -HTL each produced an approximately 40% inhibition of ³H-methyl-MAP formation: the inhibitory effect of the two agents in combination was additive. Sinefungin and A9145C, potent inhibitors of bovine adrenomedullary protein carboxyl methylase, had no effect on ³H-methyl-MAP formation in hypothalamic synaptosomes at concentrations up to 1 mM. However, these compounds were potent inhibitors of ³H-methyl-MAP formation in lysed synaptosomes incubated with [³H-methyl]AdoMet. These results demonstrate that hypothalamic synaptosomes are capable of methio-nine activation and protein carboxyl methylation.     

A SENSITIVE MICROASSAY FOR TRYPTOPHAN HYDROXYLASE IN BRAIN

—A specific and sensitive, radioisotopic microassay for tryptophan hydroxylase (EC 1.14.36) is described, which is capable of determining enzymatic activity in as little as 5 μg of crude brainstem homogenate. 5-Hydroxytryptophan, the immediate product of hydroxylation of tryptophan is enzymatically converted to N-acetylserotonin. A radioisotopic label is then introduced by the enzymatic methylation of N-acetylserotonin in the presence of [³H]methyl-S-adenosyl-methionine. The [³H]-melatonin thus formed is isolated by extraction and counted. With this assay, the activity in individual hypothalamic nuclei (arcuate nucleus, median eminence, suprachiasmatic nucleus, and medial forebrain bundle) has been measured.     

MECHANISMS FOR THE EFFLUX OF [14C]DOPA AND [14C]DOPAMINE FROM THE CSF OF RHESUS MONKEYS

—Clearance of [¹⁴C]DOPA and [¹⁴C]dopamine from CSF was investigated in anaesthetized rhesus monkeys (M. Mulatta) subjected to ventriculocisternal perfusion. The efflux coefficients, k_VE, at tracer concentrations (3–5 m ) in the perfusate were 0.0487 ml/min and 0.0325 ml/min for [¹⁴C]DOPA and [¹⁴C]dopamine, respectively. Carrier DOPA (10 mm ) in the perfusate decreased the efflux of [¹⁴C]DOPAsignificantly, but carrier dopamine had no appreciable effect on the clearance of [¹⁴C]dopamine. These findings suggest that DOPA is cleared from CSF in part by a saturable mechanism which may be located in the choroid plexus, whereas dopamine leaves the ventricular system by passive diffusion. Radioactivity in the caudate nucleus immediately adjacent to the perfused ventricle averaged 15.5 % and 12.6% of the radioactivity in the perfusates with [¹⁴C]DOPA or [¹⁴C]dopamine, respectively. These distribution percentages were similar to those found for various extracellular indicators after ventriculocisternal perfusion and may indicate that the efflux of intraventricularly-administered exogenous DOPA and dopamine occurs in part through extracellular channels.     

Determination of unchanged [F]dopamine in human and non-human primate plasma during positron emission tomography studies: a new solid-phase extraction method comparable to radio-thin-layer chromatography analysis

Routine determination of [¹⁸F]DOPA and its metabolites in plasma is essential for assessment and quantification of presynaptic dopamine function in vivo using a modeling approach with positron emission tomography (PET). The determination of unchanged [¹⁸F]DOPA from human and non-human primate plasma using solid-phase extraction (SPE) with Sep-Pak cartridges during PET dopaminergic studies is described here. The results from the studies showed that this new approach in comparsion to a method such as thin-layer chromatography (TLC) possessed a simplicity, rapidity and accuracy as well as good correlation between the two techniques (p<0.0001). A proposed procedure involving radio-analysis on alumina plates (Al₂O₃) was also developed with an excellent correlation compared to the conventional C₁₈ plates (r=0.96). Thus it could be concluded that the SPE on either C₁₈ or alumina cartridges (Waters) compared to radio-TLC analysis on C₁₈ and alumina systems, appears to be a useful analytical method suitable for correcting the input arterial function in routine clinical PET neurotransmission studies.     

Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

ABSTRACT. The fate of the [methyl-¹⁴C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-¹⁴C]methionine, [U-¹⁴C]methionine, S-[methyl-¹⁴C]AdoMet or [³⁵S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-¹⁴C] and [U-¹⁴C]methionine to [³⁵S]methionine. After 4-h incubation with [U-¹⁴C]methionine, [methyl-¹⁴C]methionine or [³⁵S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-¹⁴C] or [methyl-¹⁴C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [³⁵S]methionine and cycloheximide. [Methyl-¹⁴C] and [U-¹⁴C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-¹⁴C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-¹⁴C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-¹⁴C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.     

PREFERENTIAL LABELING OF RAT LYMPHOCYTES WITH A RAPID RATE OF TURNOVER BY TRITIATED DEOXYCYTIDINE

Lymphocytes in thymic cortex and germinal centers of lymphoid tissues are labeled intensely with generally labeled tritiated deoxycytidine [G-³H]dCyd whereas they are weakly labeled with methyl tritiated deoxythymidine [methyl-³H]dThd of the same specific activity, not only by single injection but also by an intensive injection schedule. [G-³H]dCyd can be used to label short-lived lymphocytes strongly, although not specifically. The distribution patterns of labeled lymphocytes were different depending on the injection schedules of [G-³H]dCyd. [G-³H]dCyd can be used as a precursor molecule for cytosine and also thymine found in DNA. The ratios of radioactive thymine to cytosine measured biochemically on DNA extracted from radioactive lymphocytes labeled by the various schedules indicate strongly that short- and long-lived lymphocyte populations have different abilities to utilize pyrimidine nucleosides for DNA synthesis.     

Influence of distamycin A on the methylation,extraction, and formation of UV-inducible DNA-protein cross links of histone H1 in the interphase nuclei of rat liver cells

The incubation in vitro of rat liver nuclei in the presence of S-adenosyl[methyl-³H]methionine ([³H] SAM) leads to the incorporation of a radioactive label not only into core histones H3 and H4, but also into linker histone H1. The addition of distamycin A to the incubation medium stimulates label incorporation into histone H1 by approximately six times and into histone H3 by around two times. The presence of distamycin facilitates histone H1 extraction by polyglutamic acid (poly(Glu)) and decreases UV-induced DNA—histone cross-link formation. These effects give evidence that the weakening H1—chromatin interaction by distamycin may be the result of a histone H1 position change relative toward the nucleosome and (or) a disturbance of the histone H1–H3 interactions, as these histones are exposed to additional methylation.     

A UV-sensitive human clonal cell line,RSa, which has low repair activity

The repair activity of a human transformed cell line, RSa, which was found to be highly sensitive to the lethal effects of 254 nm far-ultraviolet radiation, was compared with that of HeLa cells by evaluating the range of UV-induced incorporation of [methyl-³H]thymidine ([³H]dThd) or 5-[6-³H]bromodeoxyuridine ([³H]BrdUrd) into deoxyribonucleic acid. Direct scintillation counting was used for measuring the extent of unscheduled DNA synthesis (UDS) in UV-irradiated cells, which were treated with hydroxyurea or with arginine deprivation. More quantitative measurements were made by using the density labeling and equilibrium centrifugation method for assaying repair replication. All the amounts of UDS and repair replication in RSa cells were markedly below those in HeLa cells. The possible relationships of the low repair activity to abnormally high UV sensitivity in RSa cells are discussed.     

NEUROCHEMICAL PROPERTIES OF ADRENERGIC NERVES REGENERATED AFTER 6-HYDROXYDOPAMINE

Abstract— The uptake-storage properties and synthesis of noradrenaline, and fluorescence morphology of adrenergic nerves which have been allowed to regenerate for 4 weeks after a chemical sympathectomy produced by 6-hydroxydopamine have been investigated in mouse iris and atrium. The regenerated nerve terminals displayed a lower formaldehyde-induced fluorescence intensity whereas the non-terminal axons exhibited a stronger fluorescence intensity and a more beaded appearance compared with mature nerves. The endogenous noradrenaline concentration after 6-hydroxydopamine was 30% in iris and 45% in atrium compared to control values. Recovery of [³H]noradrenaline uptake was found to be more rapid than that of endogenous noradrenaline concentration after the 6-hydroxydopamine treatment. [³H]Noradrenaline uptake in regenerating and adult mature nerves both obeyed Michaelis-Menten kinetics having identical K_m values. There was a close correlation between [³H]noradrenaline uptake and nerve density of adrenergic nerves regenerated after 6-hydroxydopamine. These results show that [³H]noradrenaline uptake is a better index for the number of regenerated nerve terminals than is the endogenous noradrenaline concentration. The retention of [³H]noradrenaline taken up and accumulated in vitro was about the same in regenerated and mature nerves, although a slight tendency to less effective retention was observed in the regenerated nerves. Subcellular distribution studies showed that relatively less [³H]noradrenaline was recovered in the microsomal fraction after 6-hydroxydopamine treatment. The formation of ¹⁴C-labelled catecholamines from [¹⁴C]DOPA was higher in regenerating nerves than indicated by the endogenous noradrenaline concentration but lower than that indicated by the [³H]noradrenaline. It is concluded that the regenerating nerves contain less endogenous noradrenaline than adult mature nerves and that the uptake mechanism develops promptly, whereas the development of the storage mechanism lags behind.     

Oxygen effect and influence of buffer on radiation-induced tritium cleavage from phage-T2-DNA-(methyl-3H) in aqueous solution

Summary The effect of gamma irradiation on tritium release into water (HTO) from phage T2-DNA-(methyl-³H) was studied in diluted aqueous solution. The influence of O₂ and of citrate and phosphate buffer was investigated. In oxygenated solutions an enhancement ratio of 3 was found. From the linear dose yield relationsG values were calculated. In the presence of N₂O tritium release was doubled, whereas tritium release decreased with increasing citrate concentration. It has been concluded that the major precursor of this tritium release is the OH radical. This was substantiated by experiments using thymine-(methyl-³H) and dimethylsulfoxide.     

Origin of the quiescent center in the root of Capsella bursa-pastoris (L.) Medik

The origin of the quiescent center in the embryonic radicle of Capsella bursa-pastoris was investigated by in-situ hybridization to cellular polyadenylic-acid-containing RNA using [³H]polyuridylic acid as a probe. In the globular embryo, autoradiographic silver grains were localized in all cells of the presumptive root apex except in the hypophysis. As the inner cell formed by a transverse division of the hypophysis cut off new cells toward the central procambial cylinder of the embryo, these cells remained characteristically unlabeled, in contrast to the labeled cells of the rest of the embryo. In the embryonic radicles of mature seeds and of seedlings, cells derived from the hypophysis appeared as a nonmeristematic, unlabeled, hemispherical group, bounded by the procambium to the inside and the root epidermis to the outside. When root tips excised from 2-d-old seedlings were incubated in [methyl-³H]thymidine, sectioned, and autoradiographed, cells derived from the inner cell of the hypophysis were found to be unlabeled, thus showing that they constitute the specific cells of the quiescent center. These results present evidence for the single-cell origin of the quiescent center in an angiosperm root and a role for the hypophysis in it.Abbreviations poly(A)⁺RNA polyadenylicacid-containing RNA - [³H]poly(U) [³H]polyuridylic acid - QC quiescent center This work was supported in part by National Science Foundation grants PCM-7902898 and DCB-8709092.     

On the Recovery of [3H]Noradrenaline from Different Metabolic Compartments of Rat Brain with Respect to the Role of Catechol-O-Methyltransferase

Abstract: Rats were treated with reserpine, desmethylimipramine, or carrier, either alone or in combination with tropolone. Either 10 min (t₁) or 1 h (t₂) after intraventricular injection of [³H]noradrenaline, they were decapitated. The total ³H activity and the recovery of [³H]noradrenaline were determined in tissue extracts from various brain regions. Maximum total ³H activity was measured at t₁ in all tropolone-treated rats; the mean sum of these results served as an estimate of the initial tissue concentration of [³H]noradrenaline. At t₁, 40–50% of the sum of [³H]noradrenaline and its metabolites was recovered unchanged in normal rats; reserpine and DMI reduced the recovery to 18–27%. In all groups, the decline of [³H]noradrenaline was retarded after t₁. Inhibition of catechol-O-methyltransferase by tropolone caused consistently elevated [³H]noradrenaline levels, but did not affect the metabolic rate after t₁ when compared with similarly pretreated, but tropolone-free rats. Thus, if catechol-O-methyltransferase was inhibited during the injection of [³H]noradrenaline, a higher percentage of the amine had been taken up into spaces with a slow noradrenaline turnover. The maximum increase was seen when the neuronal uptake, was inhibited by desmethylimipramine. This supported the hypothesis that an additional extraneuronal space exists, in addition to the known intraneuronal and extraneuronal compartments, which has a slow noradrenaline turnover. The tropolone effect on the noradrenaline recovery possibly shows that there might be a saturable “methylating system,” similar to that described for the periphery, in which catechol-O-methyltransferase is linked to the extraneuronal uptake₂. By affecting the access of noradrenaline to non-neuronal cells it might influence the rate of noradrenaline elimination from the intercellular space.     

Factors influencing the pattern of dopamine secretion in Tetrahymena pyriformis

Dopamine production and secretion by the unicellular eukaryote Tetrahymena pyriformis were examined through the use of high performance liquid chromatography (HPLC) with electrochemical detection and through labeling studies with radioactive precursors. Growing cultures maintained a steady state intracellular level of 1.6 ± 0.3 pmol dopamine/10⁶ cells while secreting dopamine into the medium at a rate of 0.2–0.3 pmol/10⁶ cells per min. Incorporation of [¹⁴C]tyrosine and l-[³H]dihydroxyphenylalanine (DOPA) into dopamine was most successful in a basal medium (1.3 mM Tris-HCl, 1 mM citric acid, and 1 mM Ca(OH)₂, (pH 6.5)). A rapid conversion of added l-[³H]DOPA into dopamine confirmed the dynamic pattern of dopamine synthesis and secretion first indicated by the quantitative chromatographic analyses. The intracellular concentration of dopamine dropped sharply after cells were resuspended in the basal medium at 10⁶ cells/ml, so that by approx. 1 h after resuspension, dopamine dropped below the level detectable by HPLC (0.15 pmol/10⁶ cells). Under these conditions, dopamine secretion continued at a high rate for some time, finally leading to a maximal extracellular concentration of 8.71 ± 1.73 pmol/ml by 1 h. At this concentration, the rate of secretion appears to match that of degradation. Pulse chase experiments confirmed the rapid 3urnover of intracellular dopamine. Approx. 90% of [³H]dopamine and l-[³H]DOPA disappeared from l-[³H]DOPA-prelabeled cells during a 5 min chase, with approx. 50% of this being recovered as [³H]dopamine in the cells' medium. Dopamine secretion could be increased by nearly 100-fold by adding high levels (15 nmol/ml) of l-DOPA to the medium. In contrast, NSD-1015, a potent inhibitor of dopamine synthesis, completely blocked dopamine production. 0.15 mM dibucaine and 0.02 mM reserpine reduced dopamine secretion by approx. 65% over a 25-min incubation, but 5 mM EGTA had no noticeable effect.     

STUDIES OF AMINES IN THE STRIATUM IN MONKEYS WITH NIGRAL LESIONS

The effects of ventromedial tegmental lesions on the biosynthesis and disposition of biogenic amines in the striatum of monkeys were investigated. The concentrations of endogenous dopamine and of the intraventricularly injected [³H]dopamine were distinctly lower in the striatum on the lesion side than on the intact side. The storage of [³H]dopamine in the caudate nucleus was impaired to a much greater extent than the storage of the newly synthesized [³H]norepinephrine. The concentrations of endogenous serotonin and of the intraventricularly injected [¹⁴C]serotonin were lower in the striatum on the lesion side than on the intact side. However following MAO inhibition, the concentration of [¹⁴C]serotonin did not differ significantly on the two sides of the caudate nucleus. The in vivo biosynthesis of dopamine from tyrosine was significantly reduced in the striatum on the lesion side. Tyrosine hydroxylase and DOPA decarboxylase activities were decreased on the lesion side of the striatum as compared with the intact side. Thus, the ventromedial tegmental lesions affect the storage and the synthesis of dopamine and serotonin in the ipsilateral striatum.     

Facile synthesis of carbon-11-labeled cholesterol-based cationic lipids as new potential PET probes for imaging of gene delivery in cancer

Gene therapy based on gene delivery is a promising strategy for the treatment of various human diseases such as cancer. Cationic lipids represent one of the important synthetic gene delivery systems. There is a great interest in imaging of gene therapy using the biomedical imaging technique positron emission tomography (PET). Carbon-11-labeled cholesterol-based cationic lipids were first designed and synthesized as new potential PET probes for imaging of gene delivery in cancer. The [¹¹C-methyl]quaternary amine target tracers, N-[¹¹C]methyl-N-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]pyrrolidinium iodide ([¹¹C]4a), N-[¹¹C]methyl-N′-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]imidazolium iodide ([¹¹C]4b), N-[¹¹C]methyl-N-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]piperidinium iodide ([¹¹C]4c), N-[¹¹C]methyl-N-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]-4-methylpiperidinium iodide ([¹¹C]4d), and N-[¹¹C]methyl-N-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]morpholinium iodide ([¹¹C]4e), were prepared from their corresponding tertiary amine precursors with [¹¹C]methyl iodide ([¹¹C]CH₃I) through N-[¹¹C]methylation and isolated by a simplified solid-phase extraction (SPE) method using a Silica Sep-Pak cartridge in 50-60% radiochemical yields decay corrected to end-of-bombardment (EOB), based on [¹¹C]CO₂, and 111-185 GBq/μmol specific activity at the end of synthesis (EOS).     

Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Abstract: The specific binding of [³H]WAY-100635 {N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [³H]WAY-100635 association (k₊₁ = 0.069 ± 0.015 nM^?1 min^?1) and dissociation (k_?1 = 0.023 ± 0.001 min^?1) followed monoexponential kinetics. Saturation binding isotherms of [³H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (B_max) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [³H]WAY-100635 was ～36% higher compared with that of 8-hydroxy-2-(di-n-[³H]-propylamino)tetralin ([³H]8-OH-DPAT). The binding affinity of [³H]WAY-100635 was significantly lowered by the divalent cations CaCl₂ (2.5-fold; p < 0.02) and MnCl₂ (3.6-fold; p < 0.05), with no effect on B_max. Guanyl nucleotides failed to influence the K_D and B_max parameters of [³H]WAY-100635 binding to 5-HT_1A receptors. The pharmacological binding profile of [³H]WAY-100635 was closely correlated with that of [³H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine_1A (5-HT_1A) sites in rat hippocampus. [³H]WAY-100635 competition curves with 5-HT_1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT_1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites.     

A simplified microassay for serotonin: Modification of the enzymatic isotopic assay

A simplification of the enzymatic isotopic assay for serotonin is described, Serotonin is converted to [³H]melatonin by a two-step reaction: N-acetylation of serotonin using acetic anhydride, followed by O-methylation with the enzyme hydroxyindole O-methyltransferase (EC 2.1,1.4) and S-adenosyl- -[methyl-³H]methionine as methyl donor. The present assay avoids the use of unstable acetylating enzyme, rat liver N-acetyltransferase (EC2.3,1.5). Blank values are lowered considerably and the sensitivity is doubly increased. Two-tenths micromole of serotonin per 30 μl of sample in tissue homogenates can be measured.