Biomedical subjects
Publications and source records attributed to Richard Young.
6-(2-Phenylethyl)nicotine (1b; K(i)=15 nM) was unexpectedly found to bind at alpha4beta2 nicotinic cholinergic (nACh) receptors. Although this compound failed to produce nicotine-like agonist action in several functional assays, 1b antagonized the antinociceptive effects of nicotine (mouse tail-flick assay) in a dose-dependent fashion when administered via an intrathecal route.
Biochemical and genetic studies have determined that retinoblastoma protein (pRB) tumor suppressor family members have overlapping functions. However, these studies have largely failed to distinguish functional differences between the highly related p107 and p130 proteins. Moreover, most studies pertaining to the pRB family and its principal target, the E2F transcription factor, have focused on cells that have reinitiated a cell cycle from quiescence, although recent studies suggest that cycling cells exhibit layers of regulation distinct from mitogenically stimulated cells. Using genome-wide chromatin immunoprecipitation, we show that there are distinct classes of genes directly regulated by unique combinations of E2F4, p107, and p130, including a group of genes specifically regulated in cycling cells. These groups exhibit both distinct histone acetylation signatures and patterns of mammalian Sin3B corepressor recruitment. Our findings suggest that cell cycle-dependent repression results from recruitment of an unexpected array of diverse complexes and reveals specific differences between transcriptional regulation in cycling and quiescent cells. In addition, factor location analyses have, for the first time, allowed the identification of novel and specific targets of the highly related transcriptional regulators p107 and p130, suggesting new and distinct regulatory networks engaged by each protein in continuously cycling cells.
Activation of 5-HT(2A) serotonin receptors represents a novel approach to lowering intraocular pressure. Because 5-HT(2A) serotonin receptor agonists might also produce undesirable central effects should sufficient quantities enter the brain, attempts were made to identify 5-HT(2) serotonin receptor agonists with reduced propensity to penetrate the blood-brain barrier. 1-(4-Bromo-2,5-dimethoxyphenyl)-2-aminopropan-1-ol (6), an analogue of the 5-HT(2) serotonin receptor agonist 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane (DOB; 1a) bearing a benzylic hydroxyl group, was identified as a candidate structure. Of the four optical isomers of 6, the 1R,2R-isomer (6d; K(i) = 0.5 nM) was found to bind at 5-HT(2A) receptors with an affinity similar to that of R(-)DOB (K(i) = 0.2 nM). Like R(-)DOB, 6d behaved as a partial agonist (efficacy ca. 50%) in a 5-HT(2)-mediated calcium mobilization assay. However, in an in vivo test of central action (i.e., stimulus generalization with rats as subjects), 6d was >15 times less potent than R(-)DOB. O-Methylation of 6d (i.e., 7d; 5-HT(2A) K(i) = 0.3 nM) resulted in an agent that behaved as a full (93% efficacy) agonist. Intraocular administration of 300 microg of 6d and 7d to ocular hypertensive monkeys was shown to reduce intraocular pressure by 20-27%. Given the route of administration (i.e., topical), and concentrations necessary to reduce intraocular pressure, compounds such as 6d should demonstrate minimal central effects at potentially useful therapeutic doses and offer useful leads for further development.
Using genome-wide analysis of transcription factor occupancy, we investigated the mechanisms underlying three mammalian growth arrest pathways that require the pRB tumor suppressor family. We found that p130 and E2F4 cooperatively repress a common set of genes under each growth arrest condition and showed that growth arrest is achieved through repression of a core set of genes involved not only in cell cycle control but also mitochondrial biogenesis and metabolism. Motif-finding algorithms predicted the existence of nuclear respiratory factor-1 (NRF1) binding sites in E2F target promoters, and genome-wide factor binding analysis confirmed our predictions. We showed that NRF1, a factor known to regulate expression of genes involved in mitochondrial function, is a coregulator of a large number of E2F target genes. Our studies provide insights into E2F regulatory circuitry, suggest how factor occupancy can predict the expression signature of a given target gene, and reveal pathways deregulated in human tumors.
Although sentinel lymph-node biopsy is accepted as a reliable method of staging of melanoma, it is not without problems to the pathologist. It has been re-emphasised that aggregates of benign naevus cells are not uncommon. Usually these are easily identified by a combination of their benign cytology and location in the fibrous skeleton of lymph nodes. This case represents a combination of an unusual pseudo-malignant pattern in the primary lesion with unusual morphology of the sentinel lymph node. The latter prompted reassessment of the cutaneous lesion as a benign naevus. Confirmation of the diagnosis as cutaneous melanoma by a positive sentinel-node biopsy was averted only by a careful comparison of unusual features of the putative primary and the sentinel lymph node. This case illustrates the need for a rigorous protocol for pathological assessment of sentinel lymph nodes for melanomas to assure detection of all metastases but also to avoid misdiagnosis and over-treatment. It also supports "benign metastases" as the mechanism underlying at least some melanocytes in regional lymph nodes.
Evidence suggests that +/- 3,4-methylenedioxymethamphetamine (MDMA) and psychostimulants produce similar but non-identical stimulus effects in animals. To examine this hypothesis, groups of rats were trained to discriminate either MDMA (1.5 mg/kg) or cocaine (8 mg/kg) from saline vehicle using a two-lever operant procedure under a variable interval (VI) 15 s schedule of reinforcement. Once the animals were trained, tests of stimulus generalization were conducted with +/- MDMA, cocaine, S+ MDMA, and R- MDMA. As previously demonstrated, both S+ MDMA and R- MDMA (ED50 = 0.8 and 1.2 mg/kg, respectively) substituted for +/- MDMA. Stimulus generalization also occurred upon administration of cocaine (ED50 = 4.6 mg/kg) to the +/- MDMA-trained animals. In the cocaine-trained animals, however, stimulus generalization did not occur to +/- MDMA, S+ MDMA nor R- MDMA. Receptor binding profiles for MDMA and cocaine were compared in an effort to identify any novel and common receptor-based mechanism(s) to explain stimulus generalization of MDMA-trained animals to the effects of cocaine, but only their actions on neurotransmitter transporters seem applicable. Taken together, the results indicate that stimulus substitution between MDMA and cocaine is asymmetric and suggest that although similarities exist between the stimulus actions of MDMA and cocaine, differences might be explained by their differential effects on increasing synaptic concentrations of serotonin (5-HT), dopamine (DA), and/or norepinephrine (NE).
5-HT(6) serotonin receptors are distributed within some dopamine terminal regions in the brain leading to suggestions that they might influence dopaminergic function. In the present study, the 5-HT(6) antagonist 5-methoxy-N,N-dimethyl-N(1)-benzenesulfonyltryptamine (MS-245) was without effect when administered (3.0-7.5 mg/kg) to rats trained to discriminate (+)amphetamine (1.0 mg/kg) from saline vehicle in a two-lever drug discrimination task. Administered in combination, 0.3 mg/kg (i.e., the ED(50) dose) of (+)amphetamine plus 5.0 mg/kg of MS-245 elicited 95% amphetamine-appropriate responding. Similar studies were conducted using rats trained to discriminate cocaine (8.0 mg/kg) from saline vehicle, but a combination of 2.0 mg/kg (i.e., the ED(50) dose) of cocaine together with relatively low doses of MS-245 resulted in the percent response (approximately 50%) expected from administration of this dose of cocaine or in disruption of the animals' behavior. The present results confirm findings from other laboratories that 5-HT(6) antagonists can modulate amphetamine-induced behavioral actions, and further extend these findings to an example of a different structural class of 5-HT(6) antagonists and to a different behavioral paradigm. Taken together, the data suggest that 5-HT(6) serotonin agents (or at least MS-245) could have potential clinical application in therapies that involve modulation of dopamine neurotransmission.
Two agents gaining popularity on the illicit drug market are the phenylalkylamines 4-MTA and 2C-T-7 [or 1-(4-methylthiophenyl)-2-aminopropane and 2-(2,5-dimethoxy-4-n-propylthiophenyl)-1-aminoethane, respectively]. At this time, there exists a paucity of information on the behavioral actions of these sulfur-containing agents. The present investigation examined these agents, and the N-monomethyl analog of 4-MTA (i.e., 4-MTMA), in tests of stimulus generalization (substitution) using a two-lever drug discrimination task with groups of rats trained to discriminate either the hallucinogen DOM [1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane], the stimulant cocaine, or the empathogen MDMA from vehicle. 4-MTA and its N-monomethyl analog 4-MTMA (ED50 = 0.8 mg/kg in both cases) substituted only for the MDMA stimulus, whereas 2C-T-7 (ED50 = 0.8 mg/kg) substituted only for the DOM stimulus. Thus, at the doses examined, 4-MTA and 4-MTMA appear to be MDMA-like agents, and 2C-T-7 seems best classified as a DOM-like hallucinogen. These results provide additional data that extend the structure-activity relationships of phenylalkylamines and that are consistent with what little is currently known about the action of 4-MTA and 2C-T-7 in humans.
Following short-term signs of weakness, depression, and/or anorexia of less than 24 h, two adult male African green monkeys (Cercopithecus aethiops sabeus) of St. Kitts origin died from complications of cecal volvulus. Gaseous distention was radiologically apparent in one animal. Necropsy of both monkeys revealed cecal volvulus, one at the ileocecal junction and one involving a segment of the distal portion of the ileum and cecum. Congestion and hemorrhage were evident microscopically in the lamina propria of the affected intestine, with variable necrosis.
Ephedrine is a long-studied stimulant available both as a prescription and over-the-counter medication, as well as an ingredient in widely marketed herbal preparations, and is also used as a precursor for the illicit synthesis of methamphetamine. Ephedrine is related to phenylpropanolamine, a decongestant removed from the market place due to concerns that its use increased the risk of hemorrhagic stroke. Standard pharmacology texts emphasize that ephedrine is both a direct and indirect adrenergic agonist, activating adrenergic receptors both by direct agonist activity as well as by releasing norepinephrine via a carrier-mediated exchange mechanism. Chemically, ephedrine possesses two chiral centers. In the present study, we characterized the stereoisomers of ephedrine and the closely related compounds pseudoephedrine, norephedrine, pseudonorephedrine (cathine), methcathinone, and cathinone at biogenic amine transporters and a large battery of cloned human receptors (e.g., "receptorome"). The most potent actions of ephedrine-type compounds were as substrates of the norepinephrine transporter (EC50 values of about 50 nM) followed by substrate activity at the dopamine transporter. Screening the receptorome demonstrated weak affinity at alpha2-adrenergic and 5-hydroxytryptamine7 receptors (Ki values 1-10 microM) and no significant activity at beta-adrenergic or alpha1-adrenergic receptors. Viewed collectively, these data indicate that the pharmacological effects of ephedrine-like phenylpropanolamines are likely mediated by norepinephrine release, and although sharing mechanistic similarities with, they differ in important respects from those of the phenylpropanonamines methcathinone and cathinone and the phenyisopropylamines methamphetamine and amphetamine.
The stimulus effects of bupropion metabolites were examined in a drug discrimination procedure using (-)nicotine- and (+)amphetamine-trained rats. (+)- and (-)threohydrobupropion partially substituted in each group. R,R-hydroxybupropion produced vehicle-appropriate responding in (-)nicotine animals but, when given in combination with the training dose of (-)nicotine, resulted in an attenuated effect. S,S-Hydroxybupropion partially (66%) substituted for (-)nicotine. In (+)amphetamine-trained animals, S,S-hydroxybupropion (ED50=4.4 mg/kg) generalized completely and was similar in potency to bupropion (ED50=5.4 mg/kg). Bupropion and its metabolites lacked affinity for nicotinic acetylcholinergic receptors, but all antagonized (-)nicotine-induced 86Rb+ efflux in cells expressing alpha3beta4 nicotinic cholinergic receptors. S,S-Hydroxybupropion possessed affinity at the dopamine transporter comparable to bupropion, and was also found to bind at the norepinephrine transporter. Although it is unlikely that any metabolite isomer is chiefly responsible for the stimulus actions of bupropion, some probably play a role in the complex actions of this agent.
Several 6-alkyl analogues of nicotine were examined in radioligand binding and in vivo functional assays. Although (-)6-ethylnicotine (3) binds with high affinity at nACh receptors (Ki=5.6 nM) and produces nicotine-like actions, its n-propyl homologue (-)4 (Ki=22 nM) failed to produce such effects. In fact, (-)4 antagonized the antinociceptive effects of (-)nicotine in the tail-flick assay in mice, but not the spontaneous activity or discriminative stimulus effects of (-)nicotine. Compound (-)4 appears to selectively antagonize only one of the three effects examined and is an interesting cholinergic agent for subsequent investigation.
Bupropion is a weakly potent central nervous system (CNS) stimulant that is marketed both as an antidepressant and as an anti-smoking aid. The mechanism(s) by which it produces its effects is not well understood. In the present study, the effect of bupropion was examined in rats trained to discriminate the stimulus effect of 0.60 mg/kg of (-)-nicotine from saline in a two-lever drug discrimination task. In tests of stimulus generalization (substitution), the nicotine (ED(50)=0.17 mg/kg) stimulus completely generalized to bupropion (ED(50)=5.50 mg/kg). In addition, interaction studies were conducted that evaluated the effect of 3.0 mg/kg of bupropion, a dose that when given alone produced saline-appropriate responding, in combination with various doses of nicotine. This application resulted in an enhancement of the potency of nicotine (ED(50)=0.05 mg/kg), as indicated by a leftward shift of the nicotine dose-effect function. In tests of stimulus antagonism, various doses of bupropion were administered prior to the training dose of nicotine and were found to be ineffective as antagonists of the nicotine stimulus. In contrast, the nicotinic acetylcholine receptor (nicotine receptor) antagonist mecamylamine (AD(50)=0.40 mg/kg) completely blocked the stimulus effect of nicotine. Mecamylamine did not attenuate the stimulus generalization of bupropion. The results demonstrated that bupropion can produce a nicotine-like response in nicotine-trained animals, but it does so via a mechanism of action that is unlike that of nicotine. It is speculated that bupropion may be somewhat effective as an anti-smoking treatment in people who are motivated to quit smoking because low doses of bupropion produce a nicotine-like effect(s) that serve as a suitable substitute for nicotine.
Two 5-substituted derivatives of nicotine (nicotinic acetylcholine receptor: K(i)=2.4 nM) were synthesized and evaluated: 5-bromonicotine (K(i)=6.9 nM) and 5-methoxynicotine (K(i)=14.3 nM). Despite their high affinity, neither 5-bromonicotine nor 5-methoxynicotine mimicked nicotine in producing antinociceptive (tail-flick, hotplate), hypolocomotor, or hypothermic effects in mice. Neither agent antagonized the hypolocomotor actions of nicotine, whereas 5-methoxynicotine, but not 5-bromonicotine, antagonized the antinociceptive (tail-flick) activity of nicotine in a dose-related manner. In tests of stimulus generalization using rats trained to discriminate 0.6 mg/kg of (-)-nicotine from vehicle, 5-bromonicotine substituted for nicotine. Further evaluation of 5-bromonicotine indicated that it might be a partial agonist at alpha4beta2 receptors (stimulation of Rb(+) efflux; alpha4beta2 receptors expressed in oocytes) and at alpha3-containing nicotinic acetylcholine receptors (synaptosomal dopamine release). Thus, 5-bromonicotine might be acting as a partial agonist at alpha4beta2 receptors and/or some of its effects might be related to interactions with non-alpha4beta2 receptors. Clearly, the effects of 5-bromonicotine and 5-methoxynicotine are different from those of nicotine, and from one another. These actions demonstrate that substitution at the 5-position of nicotine exerts a profound influence on the pharmacological profile as well as agonist/antagonist properties of nicotine.
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This investigation is based on the premise that conformational restriction of abused phenylalkylamines in a tetrahydroisoquinoline conformation alters their pharmacology in such a manner that their original action is lost and that a new action emerges. TDIQ or 5,6,7,8-tetrahydro-1,3-dioxolo[4,5-g]isoquinoline, is a conformationally constrained phenylalkylamine that serves as a discriminative stimulus in animals. Although TDIQ bears structural resemblance to phenylalkylamine stimulants (e.g., amphetamine), hallucinogens (e.g., 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane [DOM]), and designer drugs (e.g., N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane [MDMA], N-methyl-1-(4-methoxyphenyl)-2-aminopropane [PMMA]), the TDIQ stimulus failed to generalize to (+)amphetamine or MDMA. In the present investigation, further evaluations were made of the stimulus nature of TDIQ. Specifically, the stimulus similarities of TDIQ, PMMA, and DOM were examined. In no case was stimulus generalization (substitution) observed. The results confirm that TDIQ produces stimulus effects distinct from those of the abovementioned phenylalkylamines. We also examined the structure-activity relationships of a series of TDIQ analogs, including several that might be viewed as conformationally restricted (CR) analogs of phenylalkylamine hallucinogens, stimulants, and designer drugs. These agents were examined in rats trained to discriminate either DOM (1.0 mg/kg), (+)amphetamine (1.0 mg/kg), MDMA (1.5 mg/kg), or TDIQ (5.0 mg/kg) from saline vehicle. Whereas we have demonstrated that none of these agents retains their respective phenylalkylamine stimulus actions, several of these agents were found to substitute for TDIQ. N-Methylation abolished TDIQ-stimulus action. These results, coupled with previous findings, imply that TDIQ derivatives represent a novel class of phenylalkylamines analogs with unique stimulus properties. Preliminary radioligand binding studies suggest that an alpha2-adrenergic mechanism might underlie the stimulus effects produced by TDIQ.
1-(3,4-Methylenedioxyphenyl)-2-aminopropane (MDA) is a drug of abuse that is known to produce stimulus effects similar to those of the stimulant phenylalkylamine (+)amphetamine and the hallucinogenic phenylalkylamine 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). Earlier, a working model was described to account for the stimulus effects produced by phenylalkylamines. Such agents can produce one or more of three distinct effects: an amphetamine effect, a DOM effect and a third effect that is typified by the agent N-methyl-1-(4-methoxyphenyl)-2-aminopropane (PMMA). Because MDA is known to produce two of the three effects, in the present investigation, we sought to determine if racemic MDA or either of its optical isomers could produce a PMMA-like effect in animals. Administration of S(+)MDA, R(-)MDA and (+/-)MDA to rats trained to discriminate 1.25 mg/kg of PMMA from saline vehicle under a VI 15-s schedule of reinforcement resulted in substitution in each case. (+/-)MDA and S(+)MDA were nearly equipotent and several fold more potent than R(-)MDA. The results are not only consistent with the proposed model but also identify (+/-)MDA as the first phenylalkylamine shown to produce all three types of stimulus effects (i.e., amphetamine-like, DOM-like and PMMA-like) in rats.