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1.  Does phytoestrogen supplementation affect cognition differentially in males and females? 
Brain research  2013;1514:123-127.
Phytoestrogens are plant-derived compounds found mainly in soy with known estrogenic properties and a potential for benefits to human health. Increased intake in phytoestrogens stemmed from the search for safe alternatives to hormone replacement therapies. Based on epidemiologic evidence comparing Western and Asian population and clinical studies, phytoestrogens show promise to improve health and brain function. This review will focus on the effects of phytoestrogens on cognition by examining clinical and animals studies, with special attention placed on (1) a window of therapeutic opportunity which may explains the discrepancy amongst studies, and (2) whether a sex/gender difference exists in response to phytoestrogen intake and what the possible underlying mechanisms may be.
doi:10.1016/j.brainres.2013.02.013
PMCID: PMC3677816  PMID: 23415935
2.  The HIV Antiretroviral Drug Efavirenz has LSD-Like Properties 
Neuropsychopharmacology  2013;38(12):2373-2384.
Anecdotal reports have surfaced concerning misuse of the HIV antiretroviral medication efavirenz ((4S)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoromethyl)-2,4-dihydro-1H-3,1-benzoxazin-2-one) by HIV patients and non-infected teens who crush the pills and smoke the powder for its psychoactive effects. Molecular profiling of the receptor pharmacology of efavirenz pinpointed interactions with multiple established sites of action for other known drugs of abuse including catecholamine and indolamine transporters, and GABAA and 5-HT2A receptors. In rodents, interaction with the 5-HT2A receptor, a primary site of action of lysergic acid diethylamine (LSD), appears to dominate efavirenz's behavioral profile. Both LSD and efavirenz reduce ambulation in a novel open-field environment. Efavirenz occasions drug-lever responding in rats discriminating LSD from saline, and this effect is abolished by selective blockade of the 5-HT2A receptor. Similar to LSD, efavirenz induces head-twitch responses in wild-type, but not in 5-HT2A-knockout, mice. Despite having GABAA-potentiating effects (like benzodiazepines and barbiturates), and interactions with dopamine transporter, serotonin transporter, and vesicular monoamine transporter 2 (like cocaine and methamphetamine), efavirenz fails to maintain responding in rats that self-administer cocaine, and it fails to produce a conditioned place preference. Although its molecular pharmacology is multifarious, efavirenz's prevailing behavioral effect in rodents is consistent with LSD-like activity mediated via the 5-HT2A receptor. This finding correlates, in part, with the subjective experiences in humans who abuse efavirenz and with specific dose-dependent adverse neuropsychiatric events, such as hallucinations and night terrors, reported by HIV patients taking it as a medication.
doi:10.1038/npp.2013.135
PMCID: PMC3799056  PMID: 23702798
Addiction & Substance Abuse; adverse neuropsychiatric events; antiretroviral drug; Behavioral Science; hallucinogen; HIV; Neuropharmacology; Receptor Pharmacology; side effects; hallucinogen; adverse neuropsychiatric events; side effects; AIDS
3.  Transient focal cerebral ischemia induces long-term cognitive function deficit in an experimental ischemic stroke model 
Neurobiology of disease  2013;59:18-25.
Vascular dementia ranks as the second leading cause of dementia in the United States. However, its underlying pathophysiological mechanism is not fully understood and no effective treatment is available. The purpose of the current study was to evaluate long-term cognitive deficits induced by transient middle cerebral artery occlusion (tMCAO) in rats and to investigate the underlying mechanism. Sprague-Dawley rats were subjected to tMCAO or sham surgery. Behavior tests for locomotor activity and cognitive function were conducted at 7 or 30 days after stroke. Hippocampal long term potentiation (LTP) and involvement of GABAergic neurotransmission were evaluated at 30 days after sham surgery or stroke. Immunohistochemistry and Western blot analyses were conducted to determine the effect of tMCAO on cell signaling in the hippocampus. Transient MCAO induced a progressive deficiency in spatial performance. At 30 days after stroke, no neuron loss or synaptic marker change in the hippocampus were observed. LTP in both sides of the hippocampus was reduced at 30 days after stroke. This LTP impairment was prevented by blocking GABAA receptors. In addition, ERK activity was significantly reduced in both sides of the hippocampus. In summary, we identified a progressive decline in spatial learning and memory after ischemic stroke that correlates with suppression of hippocampal LTP, elevation of GABAergic neurotransmission, and inhibition of ERK activation. Our results indicate that the attenuation of GABAergic activity or enhancement of ERK/MAPK activation in the hippocampus might be potential therapeutic approaches to prevent or attenuate cognitive impairment after ischemic stroke.
doi:10.1016/j.nbd.2013.06.014
PMCID: PMC3790570  PMID: 23845275
stroke; long term potentiation; hippocampus; vascular dementia; cognition; GABA; ERK
4.  Locomotor Stimulant and Discriminative Stimulus Effects of “Bath Salt” Cathinones 
Behavioural pharmacology  2013;24(0):437-447.
A number of psychostimulant-like cathinone compounds are being sold as “legal” alternatives to methamphetamine or cocaine. The purpose of these experiments was to determine whether cathinone compounds stimulate motor activity and have discriminative stimulus effects similar to cocaine and/or methamphetamine. 3,4-Methylenedioxypyrovalerone (MDPV), methylone, mephedrone, naphyrone, flephedrone and butylone were tested for locomotor stimulant effects in mice and subsequently for substitution in rats trained to discriminate cocaine (10 mg/kg, i.p.) or methamphetamine (1 mg/kg, i.p.) from saline. All compounds fully substituted for the discriminative stimulus effects of cocaine and methamphetamine. Several commonly marketed cathinones produce discriminative stimulus effects comparable to those of cocaine and methamphetamine, which suggests that these compounds are likely to have similar abuse liability. MDPV and naphyrone produced locomotor stimulant effects that lasted much longer than cocaine or methamphetamine and therefore may be of particular concern, particularly since MDPV is one of the most commonly found substances associated with emergency room visits due to adverse effects from taking “bath salts”.
doi:10.1097/FBP.0b013e328364166d
PMCID: PMC4183201  PMID: 23839026
cathinones; drug discrimination; locomotor activity; abuse liability; mouse; rat
5.  Homer-1a immediate early gene expression correlates with better cognitive performance in aging 
Age  2012;35(5):1799-1808.
The molecular mechanisms underlying cognitive decline during healthy aging remain largely unknown. Utilizing aged wild-type C57BL/6 mice as a model for normal aging, we tested the hypothesis that cognitive performance, memory, and learning as assessed in established behavioral testing paradigms are correlated with the differential expression of isoforms of the Homer family of synaptic scaffolding proteins. Here we describe a loss of cognitive and motor function that occurs when Homer-1a/Vesl-1S protein levels drop during aging. Our data describe a novel mechanism of age-related synaptic changes contributing to loss of biological function, spatial learning, and memory formation as well as motor coordination, with the dominant negative uncoupler of synaptic protein clustering, Homer-1a/Vesl-1S, as a potential target for the prophylaxis and treatment of age-related cognitive decline.
Electronic supplementary material
The online version of this article (doi:10.1007/s11357-012-9479-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s11357-012-9479-6
PMCID: PMC3776093  PMID: 23054826
Behavior; Cognition; Cognitive aging; Learning; Memory; Synapse
6.  Coenzyme Q10 supplementation reverses age-related impairments in spatial learning and lowers protein oxidation 
Age  2012;35(5):1821-1834.
Coenzyme Q10 (CoQ) is widely available as a dietary supplement and remains under consideration as a treatment for age-associated neurodegenerative conditions. However, no studies have determined if supplementation, initiated relatively late in life, could have beneficial effects on mild functional impairments associated with normal brain aging. Accordingly, the current study assessed the effect of CoQ intake in older mice for which cognitive and psychomotor impairments were already evident. Separate groups of young (3.5 months) and relatively old mice (17.5 months) were fed a control diet or a diet supplemented with low (0.72 mg/g) or high (2.81 mg/g) concentrations of CoQ for 15 weeks. After 6 weeks, the mice were given tests for spatial learning (Morris water maze), spontaneous locomotor activity, motor coordination, and startle reflex. Age-related impairments in cognitive and psychomotor functions were evident in the 17.5-month-old mice fed the control diet, and the low-CoQ diet failed to affect any aspect of the impaired performance. However, in the Morris water maze test, old mice on the high-CoQ diet swam to the safe platform with greater efficiency than the mice on the control diet. The old mice supplemented with the high-CoQ diet did not show improvement when spatial performance was measured using probe trials and failed to show improvement in other tests of behavioral performance. Protein oxidative damage was decreased in the mitochondria from the heart, liver, and skeletal muscle of the high-CoQ-supplemented mice and, to some extent, in the brain mitochondria. Contrasting with the deleterious effect of long-term CoQ supplementation initiated during young adulthood previously published, this study suggests that CoQ improves spatial learning and attenuates oxidative damage when administered in relatively high doses and delayed until early senescence, after age-related declines have occurred. Thus, in individuals with age-associated symptoms of cognitive decline, high-CoQ intake may be beneficial.
doi:10.1007/s11357-012-9484-9
PMCID: PMC3776107  PMID: 23138632
Aging; Coenzyme Q10; Ubiquinone; Ubidecarenone; C57BL/6J; Oxidative damage; Mitochondria
7.  Discriminative Stimulus Effects of N,N-Diisopropyltryptamine 
Psychopharmacology  2012;226(2):241-246.
Rationale
Serotonergic hallucinogens such as (+)-lysergic acid diethylamide (LSD) and dimethyltryptamine (DMT) produce distinctive visual effects, whereas the synthetic hallucinogen N,N-diisopropyltryptamine (DiPT) is known for its production of auditory distortions. Objective: This study compares the discriminative stimulus effects of DiPT to those of visual hallucinogens.
Methods
Adult male rats were trained to discriminate DiPT (5 mg/kg, 15 min) from saline under a FR10 schedule. A dose-effect and time course of DiPT’s discriminative stimulus effects were established. DMT, (−)-2,5-dimethoxy-4-methylamphetamine (DOM), LSD, (±)-methylenedioxymethamphetamine (MDMA) and (+)-methamphetamine were tested for cross-substitution in DiPT-trained animals.
Results
Rats learned to discriminate DiPT from saline in an average of 60 training sessions (30 drug and 30 saline). DiPT (0.5 – 5 mg/kg) produced dose-dependent increases in drug-appropriate responding (DAR) to 99% (ED50 = 2.47 mg/kg). Onset of the discriminative stimulus effects was within 5 minutes and the effects dissipated within 4 hours. Full substitution for the discriminative stimulus effects of DiPT occurred with LSD, DOM and MDMA. DMT only partially substituted for DiPT (65% DAR), whereas (+)-methamphetamine failed to substitute for DiPT (29% DAR).
Conclusions
The discriminative stimulus effects of DiPT were similar those of a number of synthetic hallucinogens, only partially similar to those of DMT, but not similar to (+)-methamphetamine. The putative DiPT-induced auditory distortions do not lead to discriminative stimulus effects distinguishable from other hallucinogens.
doi:10.1007/s00213-012-2891-x
PMCID: PMC3577941  PMID: 23070023
N,N-Diisopropyltryptamine; hallucinogens; drug discrimination; rat
8.  Effects of Ethanol on Cocaine Discrimination in Rats 
Ethanol and cocaine are frequently abused in combination, but little is known about how the subjective effects of the two drugs interact. The ability of ethanol and other GABAA-active compounds to alter the discriminative stimulus effects of cocaine was tested. Male Sprague-Dawley rats were trained to discriminate cocaine (10 mg/kg, i.p.) from saline using either single-dose or cumulative dosing methods. In single-dose testing, ethanol (0.1 to 0.5 g/kg) dose-dependently decreased cocaine-appropriate responding following the training dose of cocaine. Ethanol (0.5 g/kg) produced a rightward shift in the cocaine cumulative dose-effect curve. Ethanol (0.1 to 1.0 g/kg) failed to substitute for the discriminative stimulus effects of cocaine and the higher doses (1 to 2 g/kg) completely suppressed responding. Indirect GABAA agonists diazepam (benzodiazepine site) and pentobarbital (barbiturate site) did not block the discriminative stimulus effects of cumulative doses of cocaine. The GABAA antagonist pentylenetetrazol (10 to 40 mg/kg) did not substitute for cocaine. These findings suggest that ethanol can modulate the discriminative stimulus effects of cocaine, and that these effects may not be mediated by the actions of ethanol at the GABAA receptor.
PMCID: PMC3878080  PMID: 12957226
Cocaine; ethanol; drug discrimination; GABAA receptor; rat
9.  Effects of Monoamine Oxidase Inhibitors on Cocaine Discrimination in Rats 
Behavioural pharmacology  2006;17(2):10.1097/01.fbp.0000197459.08892.b5.
This study tested the time course of the discriminative stimulus effects of inhibitors of monoamine oxidase (MAO) alone or in combination with cocaine. Male Sprague-Dawley rats were trained to discriminate cocaine (10 mg/kg, i.p.) from saline using a two-lever choice methodology. The non-selective MAO inhibitors tranylcypromine (0.01 to 5 mg/kg) and phenelzine (1 to 25 mg/kg), the MAO-A selective compound clorgyline (1 to 25 mg/kg), and the MAO-B selective compounds pargyline (0.005 to 50 mg/kg) and selegiline (1 to 25 mg/kg) were tested for substitution 15 min or 24 hr following administration, and in combination with 10 mg/kg of cocaine 24 and 48 hr after administration. At 15 min, selegiline fully substituted for the discriminative stimulus effects of cocaine, whereas all other compounds partially substituted. At 24 hr, substitution of cocaine was diminished for all compounds except phenelzine, which produced a greater amount of substitution at 24 hr than at 15 min. When cocaine was administered 24 hr following clorgyline, selegiline, pargyline, and phenelzine, cocaine-appropriate responding was attenuated at intermediate doses of these drugs, whereas the highest doses did not alter cocaine-lever responding. All compounds except selegiline substantially decreased response rate and produced various adverse effects. At 48 hr, the effects of all compounds except phenelzine were markedly reduced. Selectivity for MAO-A or B did not predict the ability to substitute for or attenuate the subjective effects of cocaine. These findings suggest that MAO inhibitors can modulate the discriminative stimulus effects of cocaine for at least 24 hr, and may be useful for treatment of cocaine abuse.
doi:10.1097/01.fbp.0000197459.08892.b5
PMCID: PMC3867205  PMID: 16495723
Cocaine; drug discrimination; dopamine receptors; monoamine oxidase inhibitors; rat
10.  Reversible inactivation of dihydrolipoamide dehydrogenase by mitochondrial hydrogen peroxide 
Free radical research  2012;47(2):123-133.
Under oxidative stress conditions, mitochondria are the major site for cellular production of reactive oxygen species (ROS) such as superoxide anion and H2O2 that can attack numerous mitochondrial proteins including dihydrolipoamide dehydrogenase (DLDH). While DLDH is known to be vulnerable to oxidative inactivation, the mechanisms have not been clearly elucidated. The present study was therefore designed to investigate the mechanisms of DLDH oxidative inactivation by mitochondrial reactive oxygen species (ROS). Mitochondria, isolated from rat brain, were incubated with mitochondrial respiratory substrates such as pyruvate/malate or succinate in the presence of electron transport chain inhibitors such as rotenone or antimycin A. This is followed by enzyme activity assay and gel-based proteomic analysis. The present study also examined whether ROS-induced DLDH oxidative inactivation could be reversed by reducing reagents such as DTT, cysteine, and glutathione. Results show that DLDH could only be inactivated by complex III- but not complex I-derived ROS; and the accompanying loss of activity due to the inactivation could be restored by cysteine and glutathione, indicating that DLDH oxidative inactivation by complex III-derived ROS was a reversible process. Further studies using catalase indicate that it was H2O2 instead of superoxide anion that was responsible for DLDH inactivation. Moreover, using sulfenic acid-specific labeling techniques in conjunction with two-dimensional Western blot analysis, we show that protein sulfenic acid formation (also known as sulfenation) was associated with the loss of DLDH enzymatic activity observed under our experimental conditions. Additionally, such oxidative modification was shown to be associated with preventing DLDH from further inactivation by the thiol-reactive reagent N-ethylmaleimide. Taken together, the present study provides insights into the mechanisms of DLDH oxidative inactivation by mitochondrial H2O2.
doi:10.3109/10715762.2012.752078
PMCID: PMC3690130  PMID: 23205777
brain; dihydrolipoamide dehydrogenase; H2O2; mitochondria; reactive oxygen species; reversible inactivation; sulfenic acid; sulfenation
11.  Carisoprodol Tolerance and Precipitated Withdrawal 
Drug and Alcohol Dependence  2011;123(1-3):29-34.
Aims
Carisoprodol is a muscle relaxant that acts at the GABAA receptor. Concerns about the abuse liability of carisoprodol are increasing, but evidence that carisoprodol produces tolerance and a significant withdrawal syndrome has yet to be established. The purpose of the current study was to determine if repeated administration of carisoprodol produces tolerance and withdrawal signs in a mouse model.
Methods
Carisoprodol (0, 100, 200, 300, or 500 mg/kg bid, i.p.) was administered to Swiss-Webster mice for 4 days and loss-of-righting reflex was measured 20 to 30 minutes following each administration. On the fourth day, bemegride (20 mg/kg), flumazenil (20 mg/kg), or vehicle was administered following carisoprodol and withdrawal signs were measured. Separate groups of mice receiving the same treatment regimen and dose range were tested for spontaneous withdrawal at 6, 12 and 24 hr after the last dose of carisoprodol.
Results
The righting reflex was dose-dependently impaired following the first administration of carisoprodol. A 75 to 100% decrease in the magnitude of the impairment occurred over the four days of exposure, indicating the development of tolerance to the carisoprodol-elicited loss-of-righting reflex. Withdrawal signs were not observed within 24 hours following spontaneous withdrawal; however, bemegride and flumazenil each precipitated withdrawal within 15 to 30 min of administration.
Conclusions
Carisoprodol treatment resulted in tolerance and antagonist-precipitated withdrawal, suggesting it may have an addiction potential similar to that of other long-acting benzodiazepine or barbiturate compounds.
doi:10.1016/j.drugalcdep.2011.10.010
PMCID: PMC3288484  PMID: 22055010
tolerance; precipitated withdrawal; carisoprodol; GABAA receptor; barbiturate site; benzodiazepine site; bemegride; flumazenil; mouse
12.  Effects of age and caloric restriction on mitochondrial protein oxidative damage in mice 
The hypothesis that life span extension by caloric restriction (CR) is contingent upon the attenuation of macromolecular oxidative damage was tested in two different strains of mice: the C57BL/6, whose life span is extended by CR, and the DBA/2, in which CR has relatively minor or no impact on longevity. Mice were fed ad libitum (AL) or restricted to 40% lesser food, starting at 4 months of age. Protein damage was measured as protein-linked adducts of 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) in skeletal muscle mitochondria at 6- and 23-months of age. Protein-HNE and -MDA content increased with age in C57BL/6 mice and CR significantly attenuated these augmentations. Metalloprotease 1, NADP-dependent mitochondrial malic enzyme (isoform 2) and citrate synthase were identified by mass spectroscopy to contain HNE/MDA adducts. DBA/2 mice exhibited little effect of age or CR on protein HNE/MDA content in skeletal muscle mitochondria. In contrast, protein-HNE levels in liver mitochondria showed a significant increase with age in AL-fed mice of both strains, and CR caused significant attenuation of this damage. Overall, results indicated that the age-related increase in protein oxidative damage and its abatement by CR are genotype- and tissue- specific, and not a universal phenomenon.
doi:10.1016/j.mad.2011.12.001
PMCID: PMC3268905  PMID: 22182424
HNE-protein conjugates; oxidative stress; protein oxidative damage; mitochondrial proteins; food restriction
13.  Genotype and age influence the effect of caloric intake on mortality in mice 
Long-term caloric restriction (CR) has been repeatedly shown to increase life span and delay the onset of age-associated pathologies in laboratory mice and rats. The purpose of the current study was to determine whether the CR-associated increase in life span occurs in all strains of mice or only in some genotypes and whether the effects of CR and ad libitum (AL) feeding on mortality accrue gradually or are rapidly inducible and reversible. In one experiment, groups of male C57BL/6, DBA/2, and B6D2F1 mice were fed AL or CR (60% of AL) diets beginning at 4 months of age until death. In the companion study, separate groups of mice were maintained chronically on AL or CR regimens until 7, 17, or 22–24 months of age, after which, half of each AL and CR group was switched to the opposite regimen for 11 wk. This procedure yielded four experimental groups for each genotype, namely AL→AL, AL→CR, CR→CR, and CR→AL, designated according to long-term and short-term caloric regimen, respectively. Long-term CR resulted in increased median and maximum life span in C57BL/6 and B6D2F1 mice but failed to affect either parameter in the DBA/2 mice. The shift from AL→CR increased mortality in 17- and 24-month-old mice, whereas the shift from CR→AL did not significantly affect mortality of any age group. Such increased risk of mortality following implementation of CR at older ages was evident in all three strains but was most dramatic in DBA/2 mice. Results of this study indicate that CR does not have beneficial effects in all strains of mice, and it increases rather than decreases mortality if initiated in advanced age.
doi:10.1096/fj.02-0533fje
PMCID: PMC2839882  PMID: 12586746
caloric restriction; aging; C57BL/6; DBA/2; B6D2F1
14.  Reversible inactivation of dihydrolipoamide dehydrogenase by Angeli’s salt 
Sheng Wu Wu Li Hsueh Bao  2012;28(4):341-350.
Dihydrolipoamide dehydrogenase (DLDH) is a key component of 3 mitochondrial α-keto acid dehydrogenase complexes including pyruvate dehydrogenase complex, α-ketoglutarate dehydrogenase complex, and branched chain amino acid dehydrogenase complex. It is a pyridine-dependent disulfide oxidoreductase that is very sensitive to oxidative modifications by reactive nitrogen species (RNS) and reactive oxygen species (ROS). The objective of this study was to investigate the mechanisms of DLDH modification by RNS derived from Angeli’s salt. Studies were conducted using isolated rat brain mitochondria that were incubated with varying concentrations of Angeli’s salt followed by spectrophotometric enzyme assays, blue native gel analysis, and 2-dimensional gel-based proteomic approaches. Results show that DLDH could be inactivated by Angeli’s salt in a concentration dependent manner and the inactivation was a targeting rather than a random process as peroxynitrite did not show any detectable inhibitory effect on the enzyme’s activity under the same experimental conditions. Since Angeli’s salt can readily decompose at physiological pH to yield nitroxyl anion (HNO) and nitric oxide, further studies were conducted to determine the actual RNS that was responsible for DLDH inactivation. Results indicate that it was HNO that exerted the effect of Angeli’s salt on DLDH. Finally, two-dimensional Western blot analysis indicates that DLDH inactivation by Angeli’s salt was accompanied by formation of protein s-nitrosothiols, suggesting that s-nitrosylation is likely the cause of loss in enzyme’s activity. Taken together, the present study provides insights into mechanisms of DLDH inactivation induced by HNO derived from Angeli’s salt.
PMCID: PMC3490496  PMID: 23139597
Angeli’s salt; brain; dihydrolipoamide dehydrogenase; mitochondria; nitroxyl anion (HNO); S-nitrosylation
15.  Dissociation of functional status from accrual of CML and RAGE in the aged mouse brain 
Experimental gerontology  2008;43(12):1077-1085.
The objectives of this study were: (i) to identify regions of the aged mouse brain in which advanced glycation end-products (AGEs) were increased, and (ii) assess the functional significance of AGEs by assessing the extent to which they could predict age-related brain dysfunction. Densitometric analyses of immunoblots for N epsilon-(carboxymethyl)lysine (CML), a predominant AGE, and receptor for AGE (RAGE), were performed in different brain regions of mice aged 8 or 25 months. The 25-month-old mice were tested for ability to perform on tests of cognitive and psychomotor function prior to assessment of CML or RAGE, to determine if immunostaining results could predict functional impairment among the older mice. The amounts of CML increased with age in cortex, hippocampus, striatum and midbrain, but were unchanged in the brainstem and cerebellum. Increases in RAGE were evident in all brain regions but the hippocampus, and were not linked to increased amounts of CML. Different statistical approaches each failed to reveal any strong association between the degree of age-related functional impairment among individual mice and amounts of CML or RAGE in any particular region of the brain. The findings from this study suggest that accrual of CML and expression of RAGE in different brain regions are time-related phenomena that do not account for individual differences in brain aging or cognitive decline.
doi:10.1016/j.exger.2008.08.045
PMCID: PMC2698668  PMID: 18783731
Advanced glycation end products; N epsilon-(carboxymethyl) lysine; Receptor for advanced glycation end products; Central nervous system; Aging; Motor function; Behavior
16.  Behavioral and toxicological effects of propofol 
Behavioural pharmacology  2011;22(7):718-722.
There is increasing concern about abuse of propofol, a widely-used surgical anesthetic and sedative that is currently not a controlled substance. The purpose of the present study was to establish a rat model of the psychoactive effect of sub-anesthetic doses of propofol that could be useful for confirming abuse liability and studying mechanisms of propofol abuse. Sprague-Dawley rats were trained to discriminate propofol (10 mg/kg, i.p.) from vehicle (2% methylcellulose). Carisoprodol (100 mg/kg), chlordiazepoxide (10 mg/kg) and dizocilpine (0.1 mg/kg) were tested for substitution for the discriminative-stimulus effects of propofol (10 mg/kg), whereas pentylenetetrazol (10 mg/kg) was tested for antagonism of the discriminative-stimulus effects. Propofol (10 mg/kg) was tested for substitution in rats trained to discriminate carisoprodol from vehicle. Carisoprodol produced 59% propofol-appropriate responding, chlordiazepoxide 65%, and dizocilpine 34%. Pentylenetetrazol decreased propofol-appropriate responding to 41%. Propofol produced 52% carisoprodol-appropriate responding. Mortality rate during training of 10 mg/kg propofol was 38%. Post-mortem examination revealed cardiovascular abnormalities similar to those observed in propofol-infusion syndrome in humans. The results demonstrate that propofol can be trained as a discriminative stimulus. Its discriminative-stimulus effects were more similar to compounds promoting GABA-A receptor activity than to a compound inhibiting NMDA receptor activity. Because propofol has discriminative-stimulus effects similar to known drugs of abuse and occasions a high mortality rate, its potential for continued abuse is of particular concern.
doi:10.1097/FBP.0b013e32834aff84
PMCID: PMC3175093  PMID: 21921842
discrimination learning; stimulus generalization; propofol; GABA-A receptor; rat
17.  Association between life span extension by caloric restriction and thiol redox state in two different strains of mice 
Free radical biology & medicine  2011;51(1):225-233.
The hypothesis that the life-extending effect of caloric restriction (CR) is associated with an attenuation of the age-related pro-oxidant shift in the thiol redox state was tested employing a novel experimental design. Amounts of GSH, GSSG and protein mixed disulfides (Pr-SSG) in the skeletal muscle and liver were compared between two strains of mice, which have similar life spans when fed ad libitum (AL), however, under the standard CR regimen, life span of only one strain, C57BL/6, is extended, whereas it remains unaffected in the other strain, DBA/2. Mice were fed AL or 40% less food starting at 4 months and compared at 6 and 24 months of age. The amounts of GSSG and Pr-SSG increased and the GSH:GSSG ratios decreased with age in both strains of AL-fed mice. CR prevented these age-related changes in the C57BL/6, whose life span is extended by CR, but not in the DBA/2 mice, in which it remains unaffected. CR enhanced the activity of glutamate cysteine ligase in the C57BL/6, but not in the DBA/2 mice. Results suggest that longevity extension by CR may be associated with the attenuation of age-related pro-oxidizing shifts in the thiol redox state.
doi:10.1016/j.freeradbiomed.2011.04.006
PMCID: PMC3109181  PMID: 21530646
aging; dietary restriction; redox state; mixed protein disulfides; glutathione
18.  Chemical probes for analysis of carbonylated proteins: a review 
Protein carbonylation is a major form of protein oxidation and is widely used as an indicator of oxidative stress. Carbonyl groups do not have distinguishing UV or visible, spectrophotometric absorbance/fluorescence characteristics and thus their detection and quantification can only be achieved using specific chemical probes. In this paper, we review the advantages and disadvantages of several chemical probes that have been and are still being used for protein carbonyl analysis. These probes include 2, 4-dinitrophenylhydazine (DNPH), tritiated sodium borohydride ([3H]NaBH4), biotin-containing probes, and fluorescence probes. As our discussions lean toward gel-based approaches, utilizations of these probes in 2D gel-based proteomic analysis of carbonylated proteins are illustrated where applicable. Analysis of carbonylated proteins by ELISA, immunofluorescent imaging, near infrared fluorescence detection, and gel-free proteomic approaches are also discussed where appropriate. Additionally, potential applications of blue native gel electrophoresis as a tool for first dimensional separation in 2D gel-based analysis of carbonylated proteins are discussed as well.
doi:10.1016/j.jchromb.2010.08.004
PMCID: PMC2995837  PMID: 20732835
Biotin; carbonylation; carbonyls; carbonylated; chemical probes; infrared fluorescence; oxidative stress; proteomics; tritiated sodium borohydride
19.  Perforated appendicitis masquerading as acute pancreatitis in a morbidly obese patient 
Diagnosis and treatment of common conditions in morbidly obese patients still pose a challenge to physicians and surgeons. Sometimes too much reliance is put on investigations that can lead to a misdiagnosis. This case demonstrates an obese woman admitted under the medical team with a presumed diagnosis of pneumonia, who was later found to have an acute abdomen and raised amylase, which led to an assumed diagnosis of pancreatitis. She died within 24 h of admission and post mortem confirmed the cause of death as systemic sepsis due to perforated appendicitis, with no evidence of pancreatitis. Significantly elevated serum amylase level may occur in non-pancreatitic acute abdomen.
doi:10.3748/wjg.14.1795
PMCID: PMC2695922  PMID: 18350613
Morbid obesity; Perforated appendicitis; Pneumonia; Serum amylase
20.  Nongradient blue native gel analysis of serum proteins and in-gel detection of serum esterase activities 
The objective of the present study was to analyze serum protein complexes and detect serum esterase activities using nongradient blue native polyacrylamide gel electrophoresis (BN-PAGE). For analysis of potential protein complexes, serum from rat was used. Results demonstrate that a total of 8 gel bands could be clearly distinguished after Coomassie blue staining, and serum albumin could be isolated nearly as a pure protein. Moreover, proteins in these bands were identified by electrospray mass spectrometry and low-energy collision induced dissociation (CID)-MS/MS peptide sequencing and the existence of serum dihydrolipoamide dehydrogenase (DLDH) was confirmed. For studies of in-gel detection of esterase activities, serum from rat, mouse, and human was used. In-gel staining of esterase activity was achieved by the use of either α-naphthylacetate or β-naphthylacetate in the presence of Fast blue BB salt. There were three bands exhibiting esterase activities in the serum of both rat and mouse. In contrast, there was only one band showing esterase activity staining in the human serum. When serum samples were treated with varying concentrations of urea, esterase activity staining was abolished for all the bands except the one containing esterase 1 (Es1) protein that is known to be a single polypeptide enzyme, indicating that majority of these esterases were protein complexes or multimeric proteins. We also identified the human serum esterase as butyrylcholinesterase following isolation and partial purification using ammonium sulfate fractioning and ion exchange column chromatographies. Where applicable, demonstrations of the gel-based method for measuring serum esterase activities under physiological or pathophysiological conditions were illustrated. Results of the present study demonstrate that nongradient BN-PAGE can serve as a feasible analytical tool for proteomic and enzymatic analysis of serum proteins.
doi:10.1016/j.jchromb.2010.12.026
PMCID: PMC3034814  PMID: 21237726
Albumin; blue native polyacrylamide gel electrophoresis; butyrylcholinesterase; dihydrolipoamide dehydrogenase; esterase; serum
21.  Is serum brain-derived neurotrophic factor related to craving for or use of alcohol, cocaine, or methamphetamine? 
Background
Data suggests that brain-derived neurotropic factor (BDNF) plays a neuroadaptive role in addiction. Whether serum BDNF levels are different in alcohol or psychostimulants as a function of craving is unknown. Here, we examined craving and serum BDNF levels in persons with alcohol versus psychostimulant dependence. Our goals were to explore BDNF as an objective biomarker for 1) craving 2) abstinence, and 3) years of chronic substance use.
Methods
An exploratory, cross-sectional study was designed. Men and women between 20–65 years old with alcohol, cocaine, or methamphetamine dependence were eligible. A craving questionnaire was used to measure alcohol, cocaine and methamphetamine cravings. Serum levels of BDNF were measured using enzyme linked immunoassay. Analysis of variance, chi-square, and correlations were performed using a 95% confidence interval and a significance level of P < 0.05.
Results
We found a significant difference in the mean craving score among alcohol, cocaine and methamphetamine dependent subjects. There were no significant influences of race, gender, psychiatric disorder or psychotropic medication on serum BDNF levels. We found that among psychostimulant users BDNF levels were significantly higher in men than in women when the number of abstinent days was statistically controlled. Further, a significant correlation between serum BDNF levels and the number of abstinent days since last psychostimulant use was found.
Conclusion
These data suggest that BDNF may be a biomarker of abstinence in psychostimulant dependent subjects and inform clinicians about treatment initiatives. The results are interpreted with caution due to small sample size and lack of a control group.
doi:10.2147/NDT.S18953
PMCID: PMC3140291  PMID: 21792305
BDNF; alcohol; cocaine; methamphetamine; craving
22.  Comparison of the Discriminative Stimulus Effects of Dimethyltryptamine with Different Classes of Psychoactive Compounds in Rats 
Psychopharmacology  2009;204(4):715-724.
Rationale
There has been increased recreational use of dimethyltryptamine (DMT), but little is known of its discriminative stimulus effects.
Objectives
The present study assessed the similarity of the discriminative stimulus effects of DMT to other types of hallucinogens and to psychostimulants.
Methods
Rats were trained to discriminate DMT from saline. To test the similarity of DMT to known hallucinogens, the ability of (+)-lysergic acid diethylamide (LSD), (−)-2,5-dimethoxy-4-methylamphetamine (DOM), (+)-methamphetamine, or (±)3,4-methylenedioxymethyl-amphetamine (MDMA) to substitute in DMT-trained rats was tested. The ability of DMT to substitute in rats trained to discriminate each of these compounds was also tested. To assess the degree of similarity in discriminative stimulus effects, each of the compounds was tested for substitution in all of the other training groups.
Results
LSD, DOM, and MDMA all fully substituted in DMT-trained rats, whereas DMT fully substituted only in DOM-trained rats. Full cross-substitution occurred between DMT and DOM, LSD and DOM, and (+)-methamphetamine and MDMA. MDMA fully substituted for (+)-methamphetamine, DOM, and DMT, but only partially for LSD. In MDMA-trained rats, LSD and (+)-methamphetamine fully substituted, whereas DMT and DOM did not fully substitute. No cross-substitution was evident between (+)-methamphetamine and DMT, LSD, or DOM.
Conclusions
DMT produces discriminative stimulus effects most similar to those of DOM, with some similarity to the discriminative stimulus effects of LSD and MDMA. Like DOM and LSD, DMT seems to produce predominately hallucinogenic-like discriminative stimulus effects and minimal psychostimulant effects, in contrast to MDMA which produced hallucinogen- and psychostimulant-like effects.
doi:10.1007/s00213-009-1501-z
PMCID: PMC2865430  PMID: 19288085
Drug discrimination; cross-substitution; hallucinogen; psychostimulant; (−)-2, 5-dimethoxy-4-methylamphetamine (DOM); lysergic acid diethylamide (LSD); dimethyltryptamine (DMT); 3, 4-methylenedioxymethylamphetamine (MDMA); (+)-methamphetamine; rat
23.  Resolving mitochondrial protein complexes using non-gradient blue native polyacrylamide gel electrophoresis 
Analytical biochemistry  2009;389(2):143-149.
Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful technique for separation and proteomic analysis of high molecular weight protein complexes. It is often performed on gradient gels and is widely used for studying mitochondrial membrane complexes involved in electron transportation and oxidative phosphorylation. In this paper, we present an alternative BN-PAGE method that uses highly porous, non-gradient polyacrylamide gels for separation of rat brain mitochondrial protein complexes. Results demonstrate that this method not only resolves mitochondrial complexes I-V, allowing subsequent analysis by in-gel activity staining and mass spectrometry peptide sequencing, but also identifies Hsp60 polymers and dihydrolipoamide dehydrogenase (DLDH). Moreover, with this new method, it is shown for the first time that complex I and DLDH can be simultaneously detected on a single gel strip by in-gel activity staining. Overall, the method provides a simplified, non-gradient gel electrophoretic approach that should be useful in functional proteomics studies.
doi:10.1016/j.ab.2009.03.043
PMCID: PMC2795571  PMID: 19348780
Blue native polyacrylamide gel electrophoresis; dihydrolipoamide dehydrogenase; mitochondria; protein complexes
24.  Abuse Potential of Soma®: the GABAA Receptor as a Target 
Soma® (carisoprodol) is an increasingly abused, centrally-acting muscle relaxant. Despite the prevalence of carisoprodol abuse, its mechanism of action remains unclear. Its sedative effects, which contribute to its therapeutic and recreational use, are generally attributed to the actions of its primary metabolite, meprobamate, at GABAA receptors (GABAAR). Meprobamate is a controlled substance at the federal level; ironically, carisoprodol is not currently classified as such. Using behavioral and molecular pharmacological approaches, we recently demonstrated carisoprodol, itself, is capable of modulating GABAAR function in a manner similar to central nervous system depressants. Its functional similarities with this highly addictive class of drugs may contribute to the abuse potential of carisoprodol. The site of action of carisoprodol has not been identified; based on our studies, interaction with benzodiazepine or barbiturate sites is unlikely. These recent findings, when coupled with numerous reports in the literature, support the contention that the non-controlled status of carisoprodol should be reevaluated.
PMCID: PMC2858432  PMID: 20419052
Carisoprodol; Discrimination; GABAA receptor; Meprobamate; Muscle relaxant; Substance abuse
25.  CALORIC RESTRICTION PREVENTS AGE-ASSOCIATED ACCRUAL OF OXIDATIVE DAMAGE TO MOUSE SKELETAL MUSCLE MITOCHONDRIA 
Free radical biology & medicine  1998;25(9):1089-1097.
The purpose of this study was to understand the nature of the causes underlying the senescence-related decline in skeletal muscle mass and performance. Protein and lipid oxidative damage to upper hindlimb skeletal muscle mitochondria was compared between mice fed ad libitum and those restricted to 40% fewer calories—a regimen that increases life span by ~30–40% and attenuates the senescence-associated decrement in skeletal muscle mass and function. Oxidative damage to mitochondrial proteins, measured as amounts of protein carbonyls and loss of protein sulfhydryl content, and to mitochondrial lipids, determined as concentration of thiobarbituric acid reactive substances, significantly increased with age in the ad libitum-fed (AL) C57BL/6 mice. The rate of superoxide anion radical generation by submitochondrial particles increased whereas the activities of antioxidative enzymes superoxide dismutase, catalase, and glutathione peroxidase in muscle homogenates remained unaltered with age in the AL group. In calorically-restricted (CR) mice there was no age-associated increase in mitochondrial protein or lipid oxidative damage, or in superoxide anion radical generation. Crossover studies, involving the transfer of 18- to 22-month-old mice fed on the AL regimen to the CR regimen, and vice versa, indicated that the mitochondrial oxidative damage could not be reversed by CR or induced by AL feeding within a time frame of 6 weeks. Results of this study indicate that mitochondria in skeletal muscles accumulate significant amounts of oxidative damage during aging. Although such damage is largely irreversible, it can be prevented by restriction of caloric intake.
PMCID: PMC2840627  PMID: 9870563
Aging; Oxidative stress; Free radicals; Caloric restriction; Skeletal muscle; Mitochondria; Protein oxidation

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