ABC drug transporter, MDR1, is a drug flippase that moves a variety of hydrophobic molecules from the inner to the outer leaflet of the plasma membrane. We have previously reported that MDR1 can function as a glycolipid flippase, being one of the mechanisms responsible for the translocation of glucosylceramide into the Golgi for neutral, but not acidic, glycosphingolipids (GSLs) synthesis. The interplay between GSLs and MDR1 could provide a whole new spectrum of innovative therapeutic options. We found that cell surface MDR1 partially co-localized with globotriaosyl ceramide (Gb3) in MDR1 transfected cells. Inhibition of GSL biosynthesis results in the loss of drug resistance and of cell surface MDR1. We speculated that an association of MDR1 and cell surface GSLs, in particular Gb3, may be functional at the cell surface, as MDR1 partitions into plasma membrane lipid rafts regulating MDR1 function. We therefore tested adamantyl Gb3 (adaGb3), a water soluble analog of Gb3, on MDR1 functions. AdaGb3 was able to inhibit MDR1-mediated rhodamine 123 drug efflux from MDR1 expressing cells, like cyclosporin A (CsA), a classical MDR1 inhibitor. AdaGb3 was also able to reverse vinblastine drug resistance in cell culture, whereas adamantyl galactosylceramide had no effect on drug resistance. The strong MDR1 reversal effects of adaGb3, as well as its favourable in vivo features make it a possible choice for inhibition of MDR1 to increase bioavailability of drugs across the intestinal epithelium (De Rosa et al., 2008). Thus, specific GSL analogs provide a new approach to MDR reversal. We have previously shown that MDR1 inhibitor CsA depletes Fabry cell lines of Gb3, the characteristic GSL accumulated in this disease, by preventing its de novo synthesis, and can also deplete Gaucher lymphoid cell lines of accumulated GlcCer (Mattocks et al., 2006). Liver and heart sections of Fabry mice treated with third generation MDR1 inhibitors showed significantly less Gb3 than liver and heart sections of untreated Fabry mice. Thus, MDR1 inhibition offers a potential alternative therapeutic approach not only for Fabry disease given the extraordinary cost of conventional enzyme replacement therapy, but also for other neutral GSL storage diseases, such as Gaucher disease.

The endothelium plays an essential role in the regulation of vascular homeostasis and a state of endothelial dysfunction, which develops in the presence of cardiovascular risk factors, may contribute to the development and progression of cardiovascular disease. As such, the measurement of endothelial function, beyond being an experimental tool, may serve as an important tool to complement current risk assessment algorithms in the identification of high-risk patients. Flow-mediated dilation (FMD) is a non-invasive measure of peripheral conduit artery endothelial function that holds great promise. Presently, FMD suffers from methodological heterogeneity and a poor understanding of the various biological components involved in eliciting the dilatory response to a given shear stimulus. We compared both traditional and alternative methods of arterial diameter characterization with regards to their repeatability, nitric oxide-dependency, and their sensitivity in distinguishing between normal and dysfunctional endothelial responses. Our findings emphasize the importance of continuous arterial diameter measurement and suggest that the time to peak FMD is not a useful adjunctive measure of the FMD response. Given that endothelial dysfunction may be of clinical importance, strategies to correct it or prevent it from occurring may be of benefit. The 3-hydroxy-3-methylglutaryl coenzyme A inhibitors are agents that have demonstrated marked cholesterol-independent, endothelial-protective effects. We investigated the ability of rosuvastatin and atorvastatin to protect against endothelial dysfunction associated with ischemia and reperfusion (IR) injury, and chronic nitrate therapy. Using the FMD technique, we demonstrated, for the first time in humans, that acute rosuvastatin administration protects against IR-induced conduit artery endothelial dysfunction. Additionally, we demonstrated that this effect likely occurred by a cyclooxygenase-2-dependent mechanism, which may provide mechanistic insight into the observed cardio-toxicity with cyclooxygenase-2 inhibitors. In contrast, we observed that this endothelial-protective effect was lost upon sustained rosuvastatin administration, which may have important implications regarding the generation of sustained cardioprotective phenotypes. Finally, we demonstrated that atorvastatin co-administration prevented the development of tolerance and endothelial dysfunction associated with continuous transdermal nitroglycerin therapy in humans, likely through an antioxidant mechanism. Future studies are needed in disease patients to determine whether the concept of nitrate tolerance needs reconsideration in the presence of vascular-protective agents.

Initiation of smoking mainly occurs during adolescence. Adolescents experience more stressful life events; therefore, stress may be a factor that contributes to this high risk of smoking initiation. The current study examines the effects of three different stressors (yohimbine, intermittent footshock and social defeat) on nicotine self-administration (NSA) in adolescent and adult rats. The effects of yohimbine and footshock were examined after the establishment of NSA behavior, while the effect of social defeat was tested on the initiation of NSA behavior. Yohimbine increased NSA, but the other two stressors did not. The increase in NSA induced by yohimbine tended to be higher in adults than in adolescents. No marked age differences in response to the other two stressors were observed. These results suggest that stress increases NSA in a stressor-specific manner, and that adolescents do not show enhanced vulnerability to the effect of stress on NSA.

The impact of CYP2A6 and CYP2B6 genetic variation on nicotine metabolism, tobacco use behaviours, and nicotine biomarkers was investigated in a group of Alaska Natives (n = 400). CYP2A6 and CYP2B6 allele frequencies were unique and associations of CYP2A6 genotype and CYP2A6 activity (plasma and urine trans 3’-hydroxycotinine/cotinine (3HC/COT) ratios) were robust. Notably, this population possessed a more rapid rate of CYP2A6 activity (higher plasma 3HC/COT) when compared to CYP2A6 wild-type individuals in other ethnic groups (ANOVA P < 0.001). Also demonstrated was a significant difference in urine total nicotine equivalents by CYP2A6 activity median split (t-test P < 0.01), the first evidence of nicotine titration by CYP2A6 activity within a light smoking population. Overall, this population possessed a distinctive pattern of CYP2A6 and CYP2B6 variant frequencies and a faster rate of nicotine metabolism, which may in part explain higher levels of tobacco use prevalence and tobacco-related disease risk.

Kinesin-1 transports various cargos along the axon, while the light chain subunits play a role in selecting the types of cargos to transport. However, the mechanisms of cargo recognition and interaction have yet to be characterized. Both c-Jun kinase-interacting protein-1 (JIP1) and alcadein-1 (ALC1) are kinesin-1 cargos and compete with each other for the axonal transport machinery. I identified two polar patches of KLC1 that play a role in the interactions with JIP1 and ALC1, respectively. The main components of these two polar patches are asparagine “clamps” surrounded by positively charged lysines. Consistent with this finding, negatively charged residues of JIP1 and ALC1 are required to interact with KLC1. By structural modeling, I narrowed down the possible key residues of KLC1 that are required for interaction with c-Jun kinase interacting protein-3 (JIP3). Together, these findings reveal the versatility of KLC in the mode of interaction with many different cargos.

This study used the D2-preferring dopamine antagonist, haloperidol (3mg) and D1-D2 antagonist, fluphenazine (3mg) to investigate the roles of D1 and D2 receptors in d-amphetamine (20-mg) reinforcement in humans with (9 M; 7 F) and without (12 M; 4 F) an addictive disorder, in a placebo-controlled, between-within counterbalanced design. To preclude neurotoxicity, pathological gamblers served to evaluate effects of addiction status. Incentive motivation (e.g., Desire to Gamble), hedonic impact (e.g., Liking) and risky decision-making were assessed. Haloperidol reduced Desire to Gamble in controls, whereas fluphenazine reduced Desire in gamblers. Both antagonists reduced hedonic impact in both groups, with fluphenazine exhibiting stronger effects in gamblers. Both antagonists decreased risky decisions in controls but increased risky decisions in gamblers. Results suggest that D1 mediates amphetamine-induced motivation to gamble; D2 mediates amphetamine’s hedonic effects; D1 function is deficient in gamblers; and D2 blockade may reverse a restorative effect of amphetamine in addicted individuals.

The objective of this study is to evaluate pseudo-substrates of pantothenate kinase (PanK) for the therapeutic treatment of multidrug resistant bacterial infections of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Pantothenate (Pan) analogs, including N- pentylpantothenamide (N5-Pan) and N-heptylpantothenamide (N7-Pan), hamper bacterial growth by utilizing the PanK enzymes, which normally catalyze the rate determining step of the Coenzyme A biosynthetic pathway. Here we report the structures of SaPanK, Human PanK3 and EcPanK complexed with N7-Pan or N5-Pan, all of which have provided the opportunity to investigate the structural differences of bacterial and human Pan binding sites. The MTT assay showed these analogs to exhibit no apparent cytotoxicity against Human A549 lung adenocarcinoma cells, human HepG2 hepatoma cells and human umbilical vein endothelial cells (HUVEC). The presented structural differences have the potential for aiding the development of species-specific antimicrobial compounds with minimal effects on human cells.

Initiation of smoking mainly occurs during adolescence. Adolescents experience more stressful life events; therefore, stress may be a factor that contributes to this high risk of smoking initiation. The current study examines the effects of three different stressors (yohimbine, intermittent footshock and social defeat) on nicotine self-administration (NSA) in adolescent and adult rats. The effects of yohimbine and footshock were examined after the establishment of NSA behavior, while the effect of social defeat was tested on the initiation of NSA behavior. Yohimbine increased NSA, but the other two stressors did not. The increase in NSA induced by yohimbine tended to be higher in adults than in adolescents. No marked age differences in response to the other two stressors were observed. These results suggest that stress increases NSA in a stressor-specific manner, and that adolescents do not show enhanced vulnerability to the effect of stress on NSA.

The impact of CYP2A6 and CYP2B6 genetic variation on nicotine metabolism, tobacco use behaviours, and nicotine biomarkers was investigated in a group of Alaska Natives (n = 400). CYP2A6 and CYP2B6 allele frequencies were unique and associations of CYP2A6 genotype and CYP2A6 activity (plasma and urine trans 3’-hydroxycotinine/cotinine (3HC/COT) ratios) were robust. Notably, this population possessed a more rapid rate of CYP2A6 activity (higher plasma 3HC/COT) when compared to CYP2A6 wild-type individuals in other ethnic groups (ANOVA P < 0.001). Also demonstrated was a significant difference in urine total nicotine equivalents by CYP2A6 activity median split (t-test P < 0.01), the first evidence of nicotine titration by CYP2A6 activity within a light smoking population. Overall, this population possessed a distinctive pattern of CYP2A6 and CYP2B6 variant frequencies and a faster rate of nicotine metabolism, which may in part explain higher levels of tobacco use prevalence and tobacco-related disease risk.

Kinesin-1 transports various cargos along the axon, while the light chain subunits play a role in selecting the types of cargos to transport. However, the mechanisms of cargo recognition and interaction have yet to be characterized. Both c-Jun kinase-interacting protein-1 (JIP1) and alcadein-1 (ALC1) are kinesin-1 cargos and compete with each other for the axonal transport machinery. I identified two polar patches of KLC1 that play a role in the interactions with JIP1 and ALC1, respectively. The main components of these two polar patches are asparagine “clamps” surrounded by positively charged lysines. Consistent with this finding, negatively charged residues of JIP1 and ALC1 are required to interact with KLC1. By structural modeling, I narrowed down the possible key residues of KLC1 that are required for interaction with c-Jun kinase interacting protein-3 (JIP3). Together, these findings reveal the versatility of KLC in the mode of interaction with many different cargos.