Large-scale identification of mammalian proteins localized to nuclear sub-compartments.

Many nuclear components participating in related pathways appear concentrated in specific areas of the mammalian nucleus. The importance of this organization is attested to by the dysfunction that correlates with mis-localization of nuclear proteins in human disease and cancer. Determining the sub-nuclear localization of proteins is therefore important for understanding genome regulation and function, and it also provides clues to function for novel proteins. However, the complexity of proteins in the mammalian nucleus is too large to tackle this on a protein by protein basis. Large-scale approaches to determining protein function and sub-cellular localization are required. We have used a visual gene trap screen to identify more than 100 proteins, many of which are normal, located within compartments of the mouse nucleus. The most common discrete localizations detected are at the nucleolus and the splicing speckles and on chromosomes. Proteins at the nuclear periphery, or in other nuclear foci, have also been identified. Several of the proteins have been implicated in human disease or cancer, e.g. ATRX, HMGI-C, NBS1 and EWS, and the gene-trapped proteins provide a route into further understanding their function. We find that sequence motifs are often shared amongst proteins co-localized within the same sub-nuclear compartment. Conversely, some generally abundant motifs are lacking from the proteins concentrated in specific areas of the nucleus. This suggests that we may be able to predict sub-nuclear localization for proteins in databases based on their sequence.

Absorption rate of methylxanthines following capsules, cola and chocolate.

OBJECTIVE: To compare caffeine and theobromine absorption after oral administration of capsules, cola beverage and chocolate candy. METHODS: Three males and four females who abstained from methylxanthines received five methylxanthine-containing treatments: caffeine in capsules (72 mg), administered twice; theobromine in capsules (370 mg); cola beverage (72 mg caffeine) and chocolate candy (72 mg caffeine and 370 mg theobromine). Plasma methylxanthine levels were assayed from samples collected before and 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, and 3.0 h after caffeine capsule and cola treatments and, additionally, at 4.0 and 6.0 h after theobromine capsule and chocolate treatments. RESULTS: Caffeine plasma concentrations increased rapidly and peaked at approximately 30 min following both capsule treatments 1 (Cmax: 1.93 micrograms.ml-1); and 2 (Cmax: 2.05 micrograms.ml-1). Relative to capsules, caffeine absorption from cola and chocolate was delayed and produced lower maximum caffeine plasma concentrations which peaked 1.5-2.0 h after treatment (For cola, Cmax: 1.57 micrograms.ml-1); and for chocolate, Cmax: 1.50 micrograms.ml-1. Theobromine plasma concentrations peaked approximately 3 h after capsule administration (Cmax: 6.72 micrograms.ml-1). Relative to capsules, theobromine absorption from chocolate was more rapid and produced higher maximum theobromine plasma concentrations which peaked approximately 2 h after treatment (Cmax: 8.05 micrograms.ml-1). CONCLUSIONS: The results suggest that a usual dietary portion of the cola or chocolate used in this study would produce behaviorally discriminable plasma levels of caffeine in most subjects and of theobromine in at least one subject.

Alprazolam absorption kinetics affects abuse liability.

OBJECTIVE: To evaluate the behavioral, subjective, and reinforcing effects of immediate-release (IR) alprazolam and extended-release (XR) alprazolam to assess the effect of release rate on laboratory measures of abuse liability. METHODS: Fourteen healthy men with histories of sedative abuse participated as subjects in a double-blind crossover study. All subjects received placebo, 1 and 2 mg immediate-release alprazolam, and 2 and 3 mg extended-release alprazolam in random order. Behavioral performance, subjective effects, and alprazolam plasma concentrations were assessed repeatedly 1/2 hour before and 1/2, 1, 3, 5, 7, 9, 12, and 24 hours after drug administration. RESULTS: Mean peak alprazolam plasma concentrations occurred 1.7 and 9.2 hours after immediate-release alprazolam and extended-release alprazolam, respectively. Compared to placebo, 2 mg immediate-release alprazolam impaired all measures of psychomotor and cognitive performance (Digit Symbol Substitution Test), motor coordination (circular lights and balance), and memory (digit entry and recall); 2 mg extended-release alprazolam did not affect any of these measures and 3 mg extended-release alprazolam impaired circular lights only. Immediate-release alprazolam, 2 mg, increased all six measures of positive drug effects (e.g., ratings of liking or good effects); none of these measures were increased by 2 mg extended-release alprazolam and only three of the six measures were increased by 3 mg extended-release alprazolam. A drug versus money multiple-choice procedure designed to assess the relative reinforcing effects of each condition was administered 24 hour after the drug. The amount of money subjects were willing to "pay" to take the drug was significantly greater than placebo for both doses of immediate-release alprazolam but for neither dose of extended-release alprazolam. CONCLUSIONS: These data indicate that extended-release alprazolam has less potential for abuse than immediate-release alprazolam.

Abecarnil and alprazolam in humans: behavioral, subjective and reinforcing effects.

Abecarnil, a novel beta-carboline, is under development for the treatment of Generalized Anxiety Disorder. This study compared the behavioral, subjective and reinforcing effects of abecarnil to those of the benzodiazepine alprazolam in 14 healthy males with histories of sedative drug abuse. Placebo, abecarnil (10, 20, 40 mg) and alprazolam (1.0, 2.0, 4.0 mg) were administered p.o. in a double-blind, cross-over design. Abecarnil and alprazolam produced comparable dose-dependent decreases in behavioral performance on balance, circular lights, digit-recall and digit-symbol-substitution tasks that peaked 2-4 hr after drug administration. Both drugs produced a profile of sedative effects and were categorized by subjects as predominantly barbiturate- or benzodiazepine-like. However, the high dose of alprazolam increased subject-ratings of sleepy, fatigued and tired; these ratings were significantly different from both placebo and all doses of abecarnil. Abecarnil and alprazolam produced comparable dose-dependent increases in ratings of drug strength. The highest dose of alprazolam produced increases in Next Day ratings of drug liking, good effects, monetary value of the drug, and desire to take the drug again that were significantly greater than placebo and the highest dose of abecarnil. Abecarnil, but not alprazolam, produced increases in Next Day ratings of "bad effects" that were significantly greater than placebo. The highest dose of alprazolam produced increases in a direct measure of drug reinforcement (drug vs. money Multiple-Choice Procedure) that were significantly greater than placebo and all doses of abecarnil. Collectively, these data suggest that abecarnil may have less potential for abuse than alprazolam in a sedative-abusing population.

Caffeine dependence syndrome. Evidence from case histories and experimental evaluations.

OBJECTIVE: The extent to which daily caffeine use is associated with a substance dependence syndrome similar to that associated with other psychoactive drugs is unknown. The purpose of this study was to assess volunteers who reported problems with their use of caffeine for evidence suggesting a diagnosis of caffeine dependence based on the generic criteria for substance dependence from the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). DESIGN: Case-series evaluations. SETTING: An academic research center. PARTICIPANTS: Self-identified adults who believed they were psychologically or physically dependent on caffeine. MAIN OUTCOME MEASURE: Diagnoses made by a psychiatrist using a structured clinical interview that included a section on caffeine dependence based on genetic criteria for DSM-IV substance dependence. SECONDARY OUTCOME MEASURE: Double-blind caffeine-withdrawal evaluation. RESULTS: Ninety-nine subjects were screened for the study, and 16 were identified as having a diagnosis of caffeine dependence. Median daily caffeine intake was 357 mg, and 19% of subjects consumed less than the national (US) daily average of caffeine. Criteria used for making diagnoses (and rates of their prevalence) were as follows: withdrawal (94%), use continued despite knowledge of a persistent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by caffeine use (94%), persistent desire or unsuccessful efforts to cut down or control use (81%), and tolerance (75%). Eleven subjects underwent the double-blind caffeine-withdrawal evaluation portion of the study, and nine (82%) of the 11 showed objective evidence of caffeine withdrawal, including eight of 11 with functional impairment. CONCLUSIONS: These results, together with other experimental evidence, suggest that caffeine exhibits the features of a typical psychoactive substance of dependence. It is valuable to recognize caffeine dependence as a clinical syndrome, since some people feel compelled to continue caffeine use despite desires and recommendations to the contrary.

Discriminative stimulus and subjective effects of theobromine and caffeine in humans.

Theobromine versus placebo discrimination and caffeine versus placebo discrimination were studied in two consecutive experiments in seven volunteers who abstained from methylxanthines. Daily sessions involved PO double-blind ingestion of two sets of capsules sequentially, one of which contained drug and the other placebo. Subjects attempted to identify, and were later informed, which set of capsules contained the drug. In each experiment subjects were exposed to progressively lower doses. Five subjects acquired the theobromine discrimination; the lowest dose discriminated ranged from 100 to 560 mg. All seven subjects acquired the caffeine discrimination; the lowest dose discriminated ranged from 1.8 to 178 mg. A final experiment evaluated subjective effect ratings following 560 mg theobromine, 178 mg caffeine and placebo, which were administered double-blind in capsules once daily, five times each in mixed sequence. Caffeine produced changes in both group and individual ratings (e.g. increased well-being, energy, social disposition and alert). Theobromine did not produce changes in group ratings but changed ratings in some subjects. Across subjects, sensitivity to caffeine discriminative effects in the discrimination experiment correlated significantly with the number and magnitude of caffeine subjective effects in the final experiment. This study documents modest discriminative effects of theobromine in humans, but the basis of the discrimination is unclear. This study suggests that commonly consumed cocoa products contain behaviorally active doses of caffeine and possibly theobromine.

Enhancing caffeine reinforcement by behavioral requirements following drug ingestion.

Each morning eight adults with caffeine versus placebo discrimination histories ingested letter-coded capsules containing 100 mg caffeine or placebo and then engaged in a relaxation or vigilance activity. Subjects were first exposed to caffeine and placebo once each with each activity. Then each day for 10 days subjects made two choices; they chose which compound they would prefer if vigilance were scheduled and which they would prefer if relaxation were scheduled, with the restriction that they could not choose the same compound with both activities; only one choice (randomly selected) was reinforced. Eight of eight subjects always chose caffeine with vigilance. The next choice condition was identical, except that subjects were free to take either compound with both activities. Six of six subjects reliably chose caffeine with vigilance. Four reliably chose placebo with relaxation. In the final condition, each day for 10 days subjects chose between each drug and each of 52 monetary values; those choices were made separately for vigilance and relaxation; only one choice (randomly selected) was reinforced. For six of seven subjects, the maximum dollar value at which subjects chose drug over money was higher for caffeine in vigilance than for placebo in either activity. For five subjects, the maximum value at which subjects chose caffeine over money was higher in vigilance than in relaxation. Overall, this study demonstrates enhanced caffeine reinforcement when a vigilance activity followed drug ingestion.

A procedure for studying the within-session onset of human drug discrimination.

The purpose of the present study was to develop a procedure for measuring the within-session onset of human drug discrimination. During daily sessions, under double-blind conditions, caffeine-abstinent adults ingested a letter-coded capsule containing 178 mg caffeine or placebo. Trials were presented at 30-s intervals, beginning immediately after drug ingestion and continuing for 60 min. On each trial, subjects could guess which of their two letter-coded drugs they had received by pressing a left button (for one drug) or right button (for the other drug); subjects could also press a center "no guess" button instead of guessing. Each trial ended after one button press. After each session, subjects were told which drug they had received. Subjects earned one point (worth $0.10 per point) for each correct guess. Subjects lost either 0, 1, or 10 points for each incorrect guess; the point-loss contingencies were varied in random order across sessions. Discrimination earnings accumulated across all sessions. The point-loss contingencies decreased random responding and delayed the discrimination time course. Overall, this procedure provided an orderly and relatively continuous measure of the within-session onset of drug discrimination and should have a range of applications in understanding the human behavioral pharmacology of drugs.

Multiple-choice procedure: an efficient approach for investigating drug reinforcement in humans.

Two experiments demonstrated the efficiency of assessing drug reinforcement in humans by using a novel multiple-choice procedure. The distinguishing characteristic of the procedure is that it arranges intermittent reinforcement for choices between pairs of potential reinforcers. The procedure has three key operations: (1) a subject is exposed to the potential reinforcers; (2) a subject then makes two or more choices on a questionnaire; for each choice, the subject is required to choose one of two potential reinforcers (e.g. drug vs. drug choices and/or drug vs. money choices); and (3) subsequently only one of the choices, randomly selected, is reinforced. In the present experiments, two variations of the multiple-choice procedure were evaluated in twelve male drug abusers. Both experiments assessed the reinforcing effects of three drug conditions (200 and 400) mg/70kg pentobarbital and placebo) which were presented no more often than every to other day. The experiments demonstrated dose-related choice of pentobarbital over money as well as choice of a higher dose of pentobarbital over a lower dose or placebo. Orderly data were generated with a single-session exposure to each drug condition. Multiple-choice procedures should have applicability, not only to the investigation of drug reinforcement, but also to the study of non-drug reinforcement in humans.

Qualitative differences in the discriminative stimulus effects of low and high doses of caffeine in the rat.

Caffeine engenders qualitatively different subjective effects in humans at low and high doses. Low doses of caffeine are mildly reinforcing and produce psychomotor stimulation. High doses of caffeine can produce subjective feelings of anxiety, dysphoria and depression. The present study was designed to model these different subjective states in rats using a discrete trial shock avoidance/escape drug discrimination paradigm. Rats were trained to discriminate between i.p. injections of saline and either 10 or 56 mg/kg of caffeine. Rats trained at 10 mg/kg of caffeine acquired the discrimination in an average of 93 sessions and generalized completely to a variety of xanthine and nonxanthine behavioral stimulants including: d-amphetamine, apomorphine, 7-(beta-chloroethyl)theophylline, 9-chloro-2-(2-furanyl)-5,6-dihydro-1, 2,4-triazolo[1,5-c]quinazolin-5-imine (CGS 15943), cocaine, 1,7-dimethylxanthine, diethylpropion, beta-hydroxyethyltheophylline, methylphenidate, phenidimetrazine and theophylline. Rats trained at 56 mg/kg of caffeine acquired the discrimination in an average of 43 sessions and generalized completely only to theophylline. A variety of drugs representing diverse pharmacologic classifications including: benzodiazepine inverse agonists, pentylenetetrazol, yohimbine, ethylketocyclazocine and phencyclidine, were not generalized from either training dose, demonstrating the pharmacologic specificity of the discrimination. The discriminative effects of 10 mg/kg of caffeine appear to derive from a state of behavioral arousal, possibly mediated by catecholamines, and parallel the subjective effects produced by low doses of caffeine in humans. The discriminative effects of 56 mg/kg of caffeine are qualitatively different from those of 10 mg/kg but cannot be defined further at this time.

Do adenosinergic substrates mediate methylxanthine effects upon reinforcement thresholds for electrical brain stimulation in the rat?

Caffeine and other methylxanthines elevate reinforcement threshold for electrical brain stimulation with an order of potency suggesting that the effect is mediated by antagonism of adenosine A2 receptors. The purpose of this study was to evaluate further the possible mechanism by which caffeine and other methylxanthines elevate reinforcement thresholds for ICSS. Drugs known to affect adenosinergic transmission in predictable ways, adenosine receptor agonists and antagonists and benzodiazepine agonists and inverse agonists, were tested to determine their effect upon reinforcement threshold. Both the selective A1 adenosine agonist, R(-)-PIA, and the non-selective A1/A2 adenosine agonist NECA failed to alter reinforcement thresholds, as did CGS 15943, a potent non-xanthine non-selective adenosine receptor antagonist. Chlordiazepoxide, a benzodiazepine agonist, lowered reinforcement thresholds and FG 7142, a benzodiazepine inverse agonist, elevated reinforcement thresholds, perhaps corresponding to their anxiolytic and anxiogenic subjective effects in humans. However, another benzodiazepine agonist, midazolam and another inverse agonist, beta-CCE, did not alter reinforcement thresholds. These results fail to support a general role for adenosinergic systems in the threshold-elevating effect of methylxanthines.

Methylxanthines elevate reinforcement threshold for electrical brain stimulation: role of adenosine receptors and phosphodiesterase inhibition.

Caffeine increases reinforcement threshold in a brain stimulation procedure that allows the rat to self-regulate the intensity of electric current delivered via an electrode in the medial forebrain bundle. In order to determine the generality of that finding, we used this same autotitration procedure to assess the behavioral effects of 5 methylxanthines in addition to caffeine. All of the methylxanthines produced significant dose-dependent elevations of reinforcement threshold with the following order of potency: 7-(beta-chloroethyl)theophylline greater than isobutylmethyl-xanthine greater than 1,7-dimethylxanthine greater than theophylline greater than caffeine greater than 8-chlorotheophylline. This order of potency correlates significantly with the reported order of potency for inhibiting adenosine-stimulated cAMP, an effect mediated by the adenosine A2 receptor. Some of the behavioral effects of methylxanthines have been attributed to antagonism of adenosine receptor binding and inhibition of phosphodiesterase. The nonxanthine phosphodiesterase inhibitors papaverine and Ro 20-1724, had small but significant effects on reinforcement threshold, papaverine increasing it and Ro 20-1724 decreasing it. Thus, as a pharmacologic class, methylxanthines appear to increase reinforcement threshold, an effect which may be mediated via adenosine receptor antagonism but probably not by inhibition of phosphodiesterase.

Caffeine elevates reinforcement threshold for electrical brain stimulation: tolerance and withdrawal changes.

Caffeine dose-dependently increased the reinforcement threshold for electrical self-stimulation of the brain in rats, which is opposite to the effect of other behavioral stimulants. Tolerance to this effect of caffeine developed rapidly with daily drug administration. Abrupt cessation of daily drug treatment was followed by decreases in reinforcement threshold and response rate lasting 24-48 h, changes consistent with a drug withdrawal phenomenon. Because caffeine has the characteristics of a drug of abuse, these results question the generality of hypotheses relating the abuse potential of a drug to its ability to sensitize brain reward systems.

High efficiency coupling of alpha-1 adrenergic receptors to inositol phospholipid metabolism revealed by denervation of rat vas deferens.

The effect of surgical denervation on alpha-1 adrenergic receptor-stimulated inositol phosphate (IP) formation was examined in rat vas deferens. Rings of tissue from acutely reserpinized animals were incubated with [3H]inositol in the presence of lithium to block IP degradation and desmethylimipramine to block neuronal uptake of norepinephrine. Eighteen days after denervation the potency of norepinephrine in stimulating [3H]IP accumulation was increased 10-fold. The potency of epinephrine was increased only 3.5-fold, and the potency of phenylephrine was not altered significantly. The potency of norepinephrine in control tissues incubated with 0.1 microM desmethylimipramine was unaffected by addition of cocaine to further block neuronal uptake; however, addition of pyrogallol and pargyline to block metabolic degradation increased the potency of norepinephrine in these tissues by 6-fold. Two days after denervation there was a similar 5-fold increase in the potency of norepinephrine. In denervated tissues, the potency of norepinephrine in stimulating [3H]IP accumulation was decreased about 40-fold after receptor inactivation with 1 microM phenoxybenzamine. These results suggest that there is a substantial alpha-1 adrenergic receptor reserve for stimulating [3H]IP accumulation in rat vas deferens which is normally obscured by rapid inactivation of norepinephrine. The increase in the potency of norepinephrine after denervation appears to be due to removal of these inactivation mechanisms.